Patent Application
20090010873
The present invention relates to methods and compositions for induction and/or enhancement of endogenous neurogenesis and oligodendrogenesis and for enhancement of engraftment, survival and differentiation of stem cells injected or transplanted into an individual suffering from a disease or disorder associated with the central nervous system (CNS) or peripheral nervous system (PNS). The invention in particular relates to immune-based manipulations in combination with exogenously applied stem cells of different origins for the treatment of injuries, diseases, disorders and conditions of the CNS and PNS.
Abbreviations: Aβ, β-amyloid; ACSF, artificial cerebrospinal fluid; AD, Alzheimer's disease; BDNF, brain-derived neurotrophic factor; BMS, Basso motor score; BrdU, 5-bromo-2′-deoxyuridine; CFA, complete Freund's adjuvant; CNS, central nervous system; DCX, doublecortin; EAE, experimental autoimmune encephalomyelitis; EGF, epidermal growth factor; EPSP, excitatory postsynaptic potential; FCS, fetal calf serum; FGF, fibroblast growth factor; i.c.v., intracerebroventricular; GA, glatiramer acetate; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; IB4, isolectin B4; IFA, incomplete Freund's adjuvant; IGF-I, insulin-like growth factor 1; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; LTP, long-term potentiation; MBP, myelin basic protein; MG, microglia; MHC-II, class II major histocompatibility complex molecules; MOG, myelin oligodendrocyte glycoprotein; MWM, Morris water maze; NeuN, neuronal nuclear antigen; NPC, neural stem/progenitor cell; PBS, phosphate-buffered saline; PDL, poly-D-lysine; SCI, spinal cord injury; SGZ, subgranular zone; TBS, theta-burst stimulation; TNF, tumor necrosis factor
The central nervous system (CNS) is particularly vulnerable to insults that result in cell death or damage in part because cells of the CNS have a limited capacity for repair. Since damaged brain tissue does not regenerate, recovery must come from the remaining intact brain.
Poor recovery from acute insults or chronic degenerative disorders in the CNS has been attributed to lack of neurogenesis, limited regeneration of injured nerves, and extreme vulnerability to degenerative conditions. The absence of neurogenesis was explained by the assumption that soon after birth the CNS reaches a permanently stable state, needed to maintain the equilibrium of the brain's complex tissue network. Research during the last decade showed, however, that the brain is capable of neurogenesis throughout life, albeit to a limited extent (Morshead et al., 1994). In the inflamed brain, neurogenesis is blocked (Ekdahl et al., 2003; Monje et al., 2003). This latter finding strengthened the traditional view that local immune cells in the CNS have an adverse effect on neurogenesis. Likewise, the limited regeneration and excessive vulnerability of CNS neurons under inflammatory conditions or after an acute insult were put down to the poor ability of the CNS to tolerate the immune-derived defensive activity that is often associated with local inflammation and cytotoxicity mediated, for example, by tumor necrosis factor (TNF)-α or nitric oxide (Merrill et al., 1993). More recent studies have shown, however, that although an uncontrolled local immune response indeed impairs neuronal survival and blocks repair processes, a local immune response that is properly controlled can support survival and promote recovery (Hauben and Schwartz, 2003; Schwartz et al., 2003). It was further shown that after an injury to the CNS, a local immune response that is well controlled in time, space, and intensity by peripheral adaptive immune processes (in which CD4+ helper T cells are directed against self-antigens residing at the site of the lesion) is a critical requirement for post-traumatic neuronal survival and repair (Moalem et al., 1999; Butovsky et al., 2001; Schwartz et al., 2003; Shaked et al., 2004). These and other results led our group to formulate the concept of ‘protective autoimmunity’ (Moalem et al., 1999).
Neurogenesis occurs throughout life in adult individuals, albeit to a limited extent. Most of the newly formed cells die within the first 2-3 weeks after proliferation and only a few survive as mature neurons. Little is known about the mechanism(s) underlying the existence of neural stem/progenitor cells (NPCs) in an adult brain and why these cells are restricted in amount and limited to certain areas. Moreover, very little is known about how neurogenesis from an endogenous NPC pool can be physiologically increased. Knowledge of the factors allowing such stem cells to exist, proliferate, and differentiate in the adult individual is a prerequisite for understanding and promoting the conditions conducive to CNS repair. This in turn can be expected to lead to the development of interventions aimed at boosting neural cell renewal from the endogenous stem-cell pool or from exogenously applied stem cells.
Experiments with rat and mouse models in our laboratory have shown that well-controlled implantation of specifically activated blood-borne macrophages (Rapalino et al., 1998) or dendritic cells (Hauben et al., 2003) promotes recovery from spinal cord injury (SCI). Other studies showed that the well-controlled activity of autoimmune T cells reactive to CNS antigens residing in the lesioned site can promote recovery from axonal insults (Hauben et al., 2000; Moalem et al., 1999). It was also shown that neuroprotection, mediated by T cells directed specifically to CNS-related autoantigens, is the body's physiological response to CNS injury (Yoles et al., 2001a, 2001b).
Under normal conditions in the adult brain, new neurons are formed in the neurogenic niches of the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (Kempermann et al., 2004). Under pathological conditions some neurogenesis can also be induced in non-neurogenic brain areas. Several studies have demonstrated, for example, that injury to the CNS in animals is followed by recruitment of endogenous NPCs, which can undergo differentiation to neurons and glia at the injured site (Nakatomi et al., 2002; Imitola et al., 2004a). However, this injury-triggered cell renewal from endogenous progenitors is limited in extent and is not sufficient for full replacement of the damaged tissue. To overcome the deficit, scientists are currently seeking ways to promote recovery by transplanting cultured adult NPCs (aNPCs) (McDonald et al., 2004). Exogenous aNPCs might contribute to recovery by acting as a source of new neurons and glia in the injured CNS (Cummings et al., 2005; Lepore and Fischer, 2005) or by secreting factors that directly or indirectly promote neuroprotection (Lu et al., 2005) and neurogenesis from endogenous stem-cell pools (Enzmann et al., 2005).
A potential approach for treatment of CNS damage includes the use of adult neural stem cells or any type of stem cells. The adult neural stem cells are progenitor cells present in the mature mammalian brain that have the ability of self-renewal and, given the appropriate stimulation, can differentiate into brain neurons, astrocytes and oligodendrocytes. Stem cells (from other tissues) have classically been defined as pluripotent and having the ability to self-renew, to proliferate, and to differentiate into multiple different phenotype lineages. Hematopoietic stem cells are defined as stem cells that can give rise to cells of at least one of the major hematopoietic lineages in addition to producing daughter cells of equivalent potential. Three major lineages of blood cells include the lymphoid lineage, e.g. B-cells and T-cells, the myeloid lineage, e.g. monocytes, granulocytes and megakaryocytes, and the erythroid lineage, e.g. red blood cells. Certain hematopoietic stem cells are capable of differentiating to other cell types, including brain cells.
Separation and cloning of neural stem cell lines from both the murine and human brain have been reported (Gage et al., 1995; Kuhn et al., 1997). Human CNS neural stem cells, like their rodent homologues, when maintained in a mitogen-containing (typically epidermal growth factor or epidermal growth factor plus basic fibroblast growth factor), serum-free culture medium, grow in suspension culture to form aggregates of cells known as neurospheres. Upon removal of the mitogens and provision of a substrate, the stem cells differentiate into neurons, astrocytes and oligodendrocytes. When such stem cells are reintroduced into the developing or mature brain, they can undergo through division, migration and growth processes, and assume neural phenotypes, including expression of neurotransmitters and growth factors normally elaborated by neurons. Thus, use of neural stem cells may be advantageous for CNS damage recovery in at least two ways: (1) by the stem cells partially repopulating dead areas and reestablishing neural connections lost by CNS damage, and (2) by secretion of important neurotransmitters and growth factors required by the brain to rewire after CNS damage. Efforts to promote recovery from brain injury in animals using neural stem cells have been reported (Park et al., 1999).
Recently, a renewable source of neural stem cells was discovered in the adult human brain (Eriksson et al., 1998). These cells may be a candidate for cell-replacement therapy for nervous system disorders. The ability to isolate these cells from the adult human brain raises the possibility of performing autologous neural stem cell transplantation. It has been reported that clinical trials with adult human neural stem cells have been initiated for treatment of Parkinson's disease patients (Fricker et al., 1999). If adult neural stem cells are to be used in clinical trials they must be amenable to expansion into clinically significant quantities. Unfortunately, these cells seem to have a limited life-span in the culture dish (Kukekov et al., 1999) and it remains to be determined whether they are stable at later passages and capable of generating useful numbers of neurons.
The brain has long been viewed as an immune-privileged site. However, autoimmune T cells (controlled with respect to the onset, duration, and intensity of their activity) were recently shown to exert a beneficial effect on neuronal survival after CNS injury (Schwartz et al., 2003), as well as in cases of mental dysfunction (Kipnis et al., 2004). Moreover, in-depth understanding of the mechanisms underlying the beneficial effect of T cells for degenerative neural tissue has pointed out that the T cells instruct the microglia, at the injured area, to acquire a phenotype supportive of neural tissue. In addition, it appeared that several immune-based intervention can boost this protective response, all of which converts to microglial activation (Shaked et al., 2004). The type of damage does not determine the choice of the approach, it is the site which determines it. Some antigens cross-react with numerous antigens and thus can overcome tissue specificity barrier.
According to the present invention, we show that the same manipulation that leads to neuronal survival leads to neurogenesis and oligodendrogenesis. It appears that the same microglia, activated by T cells or by their cytokines, not only support neuronal survival but also support oligodendrogenesis and neurogenesis. These results indicate that T-cell-based manipulation will create conditions in damaged neural tissue that favor cell renewal not only from endogenous stem cell resources but also from exogenously applied stem cells.
Based on the hypothesis that the same immune response that causes cell loss under neurodegenerative conditions also blocks neurogenesis in the adult CNS and that an immune response that protects against cell loss also supports neurogenesis and oligodendrogenesis, we postulated that both neurogenesis and oligo-dendrogenesis can be induced by microglia that encounter well-controlled levels of cytokines associated with adaptive immunity, but are blocked by microglia that encounter endotoxin, which is associated with an uncontrolled local immune response that impairs neuronal survival and blocks repair processes.
In one aspect, the present invention relates to a method for inducing and enhancing neurogenesis and/or oligodendrogenesis from endogenous as well as from exogenously administered stem cells, which comprises administering to an individual in need a neuroprotective agent selected from the group consisting of:
In another aspect, the present invention provides a method of stem cell therapy comprising transplantation of stem cells in combination with a neuroprotective agent to an individual that suffers from an injury, disease, disorder or condition of the central nervous system (CNS) or peripheral nervous system (PNS). The neuroprotective agent is selected from the group consisting of the agents (i) to (x) defined above.
In a further aspect, the invention relates to the use of a neuroprotective agent selected from the group consisting of the agents (i) to (x) defined above for the preparation of a pharmaceutical composition for inducing and enhancing neurogenesis and/or oligodendrogenesis from endogenous as well as from exogenous stem cells administered to a patient.
Research during the last decade has disclosed that the brain is potentially capable of cell renewal throughout life, albeit to a limited extent (Morshead et al., 1994). However, the mechanisms that might restrict or favor the renewal of adult neural cells are not known. Recent studies from the laboratory of the present inventors have shown that after an injury to the CNS a local immune response that is properly controlled in time, space, and intensity by the peripheral adaptive immunity is a pivotal requirement for posttraumatic neuronal survival (Moalem et al., 1999; Butovslky et al., 2001; Schwartz et al., 2003; Shaked et al., 2004). We therefore envisaged the possibility that the lack of neurogenesis and the restricted recovery might be attributable to a common factor, which might in turn be related to the local immune response.
The present invention is based on the assumption that well-regulated adaptive immunity is needed for cell renewal in the brain. We postulated that neurogenesis and oligodendrogenesis are induced and supported by microglia that encounter cytokines associated with adaptive immunity, but are not supported by naïve microglia and are blocked by microglia that encounter endotoxin.
In fact, it is shown herein that certain specifically activated microglia can induce and support neural cell renewal. Thus, both neurogenesis and oligodendrogenesis were induced and supported in NPCs co-cultured with microglia activated by the cytokines IL-4 and IFN-γ, both associated with adaptive immunity. In contrast, microglia exposed to LPS blocked both neurogenesis and oligodendrogenesis, in line with previous reports that MG(LPS) block cell renewal (Monje et al., 2003).
Defense mechanisms in the form of activated microglia are often seen in acute and chronic neurodegenerative conditions, and the CNS is poorly equipped to tolerate them (Dijkstra et al., 1992). As a result, activated microglia have generally been viewed as a uniformly hostile cell population that causes inflammation, interferes with cell survival (Popovich et al., 2002), and blocks cell renewal (Monje et al., 2002, 2003).
Recent studies have shown, however, that the type of activation determines microglial activity, and that just as their effects can be inimical to cell survival in some circumstances, they can be protective in others. Thus, for example, microglia that encountered adaptive immunity (CD4+ T cells) were shown to acquire a protective phenotype (Butovsky et al., 2001). Among the cytokines that are produced by such T cells and can endow microglia with a neuroprotective phenotype are IFN-γ and IL-4, characteristic of Th1 and Th2 cells, respectively. Thus, microglia exposed to activated Th1 cells or to IFN-γ show increased uptake of glutamate, a key player in neurodegenerative disorders (Shaked et al., unpublished observation), while their exposure to IL-4 results in down-regulation of TNF-α, a common player in the destructive microglial phenotype, and up-regulation of insulin-like growth factor (IGF-1) (shown herein in the examples), which promotes differentiation of oligodendrocytes from multipotent adult neural progenitor cells (Hsieh et al., 2004). In addition, IGF-1 prevents the acute destructive effect of glutamate-mediated toxicity on oligodendrocytes in vitro (Ness et al., 2002) and inhibits apoptosis of mature oligodendrocytes during primary demyelination (Mason et al., 2000). These and other findings strongly suggest that the outcome of the local immune response (in terms of its effect on the microglia) in the damaged CNS will be either beneficial or harmful, depending on how the microglia interpret the threat.
In general, tissue repair is a process that is well synchronized in time and space, and in which immune activity is needed to clear the site of the lesion and create the conditions for migration, proliferation, and differentiation of progenitor cells for renewal. In light of the well-known fact that constitutive cell renewal is limited in the CNS, as well as the reported observations that treatment with MG(LPS) causes neuronal loss (Boje et al., 1992) and interferes with the homing and differentiation of NPCs (Monje et al., 2003), and that adaptively activated microglia can support neuronal survival, it is not surprising to discover that immune conditions favoring neuronal survival will also support cell renewal. MG(LPS) produce excessive amounts of NO (causing oxidative stress) and TNF-α, as well as other cytotoxic elements, leading to a spiral of worsening neurotoxicity (Boje et al., 1992). NO was found to act as an important negative regulator of cell proliferation and neurogenesis in the adult mammalian brain (Packer et al., 2003), and TNF-α has an inhibitory effect on oligodendrogenesis (Cammer et al., 1999).
The results of the present invention show that MG(LPS) are indeed detrimental to NPC survival and differentiation, but that when microglia are activated by cells or cytokines possessing adaptive immune function, not only are they not cytotoxic but they even exert a positive effect on NPC proliferation, inducing and supporting their differentiation into neurons or oligodendrocytes. In vivo, injection of MG(IL-4) into rat brain lateral ventricles resulted in no neuronal loss, minimal migration of microglia to the CNS parenchyma, and the appearance of new neurons and oligodendrocytes (indicated by the double-staining of BrdU+ cells with markers of neurons or oligodendrocytes). Staining for microglia revealed significant invasion of the healthy CNS by MG(LPS), with consequent massive tissue loss, unlike in the case of MG(IL-4) or MG(−). Interestingly, in the non-injected hippocampus, resident microglia were found adjacent to the subventricular zone. It is tempting to speculate that these might be the cells responsible for controlling neurogenesis, restraining it when in their resting state (as found in the present work using MG(−)), but inducing and supporting it when suitably activated.
Our findings are supported by the observation that in mice with experimental autoimmune encephalomyelitis (EAE), NPCs migrate to sites of CNS damage (Pluchino et al., 2003). They are also in line with the common experience that cell renewal is favored by injury, since they imply that in the absence of injury the conditions that might favor renewal do not exist.
Renewal of cells and their replenishment by new growth is the common procedure for tissue repair in most tissues of the body. It was thought that in the brain those processes do not occur, and therefore that any loss of neurons, being irreplaceable, results in functional deficits that range from minor to devastating. Since an insult to the CNS, whether acute or chronic, is often followed by the postinjury spread of neuronal damage, much research has been devoted to finding ways to minimize this secondary degeneration by rescuing as many neurons as possible.
The results of the present invention leads us to an intriguing conclusion. First, under pathological conditions (when cell renewal is critical), not only do the microglia not favor cell renewal, but they interfere with it. Secondly, this paradoxical situation can be remedied by well-controlled adaptive immunity, which shapes the microglia in such a way that their activity is not cytotoxic but is both protective and conducive to renewal. This indicates that in those cases in which protective autoimmunity leads to improved recovery, both neurogenesis and gliogenesis are likely to occur. These data can also explain the lack of cell renewal in autoimmune diseases; in such cases, it is likely that the quantity of circulating autoimmune T cells exceeds the threshold above which TNF-α production, due to an excess of IFN-γ, does not allow the microglia to acquire a protective phenotype. They can also explain why steroids are not helpful, as their anti-inflammatory activity masks not only the destructive but also the beneficial adaptive immunity. The therapy of choice for both autoimmune diseases and neurodegenerative conditions would therefore appear to be immunomodulation in which, after the acute phase of disease, the surviving tissue can be maintained by relatively small quantities of T cells.
The findings of the present invention indicate that the limitation of spontaneous, endogenous neurogenesis and oligodendrogenesis in the adult brain is, at least in part, an outcome of the local immune activity, and that harnessing of adaptive immunity rather than immunosuppression is the path to choose in designing ways to promote cell renewal in the CNS.
Cell renewal in the adult mammalian CNS is limited. Recent studies suggest that it is arrested by inflammation. That view is challenged by the findings of the present invention that microglia, depending on environmental stimulation, can either induce and support or block such renewal. In vitro, neurogenesis and oligodendrogenesis from neural progenitor cells were shown herein to be promoted by mouse microglia that encountered T-cell-associated cytokines (IFN-γ, IL-4), but were blocked by microglia that encountered endotoxin. Anti-IGF-1 antibodies neutralized the IL-4 effect, while anti-TNF-α antibodies augmented the effect of IFN-γ. Injection of IL-4-activated microglia into cerebral ventricles of adult rats induced significant hippocampal neurogenesis and cortical oligodendrogenesis, whereas endotoxin-activated microglia caused neuronal loss and blocked neurogenesis and oligodendrogenesis. These results strengthen our assumption that controlled adaptive immunity, unlike uncontrolled (e.g. endotoxin-induced) inflammation, activates microglia to induce and support neuronal and oligodendrocyte survival and renewal. Thus, to promote cell renewal in the CNS, well-controlled immunity is needed and should not be suppressed.
Studies from the inventors' laboratory over the last few years have shown that the controlled activity of T cells directed to auto-antigens in the CNS is needed for postinjury survival and repair (Moalem et al., 1999; Yoles et al., 2001; Kipnis et al., 2002; Schwartz and Kipnis, 2001). These results led us to suspect that a fundamental role of autoimmune T cells, known to be present in healthy individuals, is to help maintain the integrity of the CNS, and that their remedial effect in a neurodegenerative environment is a manifestation of the same restorative role under extreme conditions. Moreover, accumulating evidence attesting to the participation of such autoimmune T cells in postinjury neuronal survival led us to postulate that if they have a similar role in the healthy CNS, it might well have to do with neurogenesis in adult life, possibly by maintaining the conditions needed for such cell renewal.
Severe immune deficiency in mice was shown here to impair three aspects of hippocampal plasticity at adulthood: long-term potentiation (LTP), spatial learning/memory, and adult neurogenesis. Using immune-defficient mice and transgenic mice that express only T cells specific to the abundant brain self-antigen MBP, we show herein that hippocampus-dependent activities (LTP and spatial learning and memory), and adult neurogenesis could be controlled by a single population of T cells recognizing MBP, namely, MBP-specific autoimmune T cells.
The results of the present invention show that T cells directed to a specific CNS self-antigen play a key role in maintaining the functional activity of the hippocampus. Accordingly, we postulated that under non-pathological conditions a primary role of such autoimmune T cells is to maintain the plasticity of the adult brain, and thus the observed beneficial effect of antigen-specific anti-self T cells under degenerative conditions is an extension of their role in the healthy brain.
The present findings that autoimmune T cells (TMBP) in the adult hippocampus affect not only neurogenesis but also other aspects of plasticity (LTP and spatial learning/memory) might suggest that a common mechanism underlies them all. Alternatively, autoimmune T cells might be of direct benefit to neurogenesis only, while their observed effect on LTP and spatial learning and memory might be an indirect result of this neurogenesis. This possibility appears to be in line with accumulating information that some aspects of LTP and hippocampal activities, such as performance of the MWM task, are related to neurogenesis in the dentate gyrus (Zhao et al., 2003).
The T-cell dependence of brain plasticity observed here in SCID mice as well as transgenic mice expressing monospecific T cells, was attributed to autoimmune T cells. The function of such T cells in the wild type is implied by the superior performance of these mice relative to SCID mice and by the established presence of autoimmune T cells in healthy animals. The findings of the present invention thus provide new clues to the nature of the process or processes underlying neuronal cell renewal in adulthood, and might explain the age-related loss of certain cognitive activities in terms of an aging immune system. They might also point to novel ways to maintain brain plasticity, promote neurogenesis, and prevent functional decay in the aging brain by systemic manipulation of the peripheral immune system via a mechanism that allows autoimmunity to be safely promoted without inducing an autoimmune disease.
According to the present invention, we examined how the nature of microglial activation affects neurogenesis in the adult rat hippocampus under physiological and pathological conditions associated with brain inflammation. Transient inflammatory conditions associated with transient accumulation of myelin-specific Th1 cells promoted neurogenesis. Injection of microglia (MG) activated by IFN-γ (MG(IFNγ)) or by IL-4 (MG(IL-4)) into the lateral ventricles of the brains of healthy rats promoted neurogenesis. In rats that developed monophasic (transient) EAE, the induced neurogenesis was further promoted by MG(IL-4). Our results in vitro showed that MG(IFN-γ) supported neurogenesis from adult rat NPCs as long as the IFN-γ concentration was low. The impediment to neurogenesis imposed by high-dose IFN-γ could be counteracted by IL-4. Neurogenesis induced by IL-4 was weaker, however, than that induced by low-dose IFN-γ or by high-dose IFN-γ administered in combination with IL-4.
We also examined whether IL-4, via modulation of microglia both in vitro and in vivo, can overcome the destructive effects of high-dose IFN-γ, known to be associated with EAE. In vitro, a high dose of IFN-γ, but not a low dose, impaired the ability of microglia to support oligodendrogenesis from adult neural stem cells/progenitor cells (NPCs). IL-4 counteracted the interference with oligodendrogenesis caused by high IFN-γ concentrations. When IL-4-activated microglia were stereotaxically injected through the cerebral ventricles into the cerebrospinal fluid (CSF) of rats with acute EAE or of mice with a remitting-relapsing autoimmune disease, the animals demonstrated significantly more oligodendrogenesis and significantly less neurological deficit than did their vehicle-injected diseased controls.
The injection of IL-4-activated microglia into the CSF of rats suffering from acute or chronic EAE caused an increase in the numbers of newly formed microglia. Most of the new microglia expressed MHC-II and IGF-I. Since IGF-I is known to play an important role in the differentiation and survival of oligodendrocytes, and to be beneficial in the treatment of EAE, it seems reasonable to assume that the IGF-I produced by IL-4-activated microglia is responsible, at least in part, for the shift to a Th2 phenotype and thus for the increased number of MHC-II+ microglia as well as of the newly formed BrdU+/MHC-II+ microglia expressing IGF-I. The increased oligodendrogenesis was found to correlate with a higher incidence of newly formed MHC-II+ microglia, suggesting that these microglia exert their effects on oligodendrogenesis by acting as antigen-presenting cells for CD4+ T-helper cells.
The results of the present invention indicate a novel role for microglia in ameliorating EAE and promoting differentiation of oligodendrocytes from adult NPCs (Hsieh et al., 2004), and suggest a link between the known beneficial effect of IL-4 in ameliorating EAE, the role of IGF-I derived from IL-4-activated microglia, and the requirement of viable microglia for remyelination. Our findings thus support a key role for microglia in promoting cell renewal from endogenous progenitors under pathological conditions. Based on the present findings, as well as our previously reported results, we suggest that the cross-talk between T cells and microglia lays the foundation for protection and repair in the adult CNS. We further believe that immunosuppressive treatment, if administered alone, while ameliorating clinical signs at an early stage, could eventually have devastating results. We therefore suggest that rather than suppression, immunomodulation aimed at appropriate and well-controlled activation of microglia might be the approach to adopt in designing ways to promote cell renewal under neurodegenerative conditions.
We have identified herein cellular elements in the CNS that can respond to local environmental changes and needs, and consequently can support the formation of new cells from adult aNPCs. We demonstrated that once the microglia become suitably activated by circulating T cell-derived cytokines, they can induce neuronal and oligodendroglial differentiation from aNPCs. In view of that observation, and our previous demonstration in rodents that a T cell-based vaccination promotes recovery from contusive spinal cord injury (SCI), we postulated that translation of those findings into a therapeutic approach might benefit the repair process by creating a niche-like neurogenic/glidgenic environment at the injured site. Thus, we expected to find that supplementing the vaccination by transplantation of homologous aNPCs would further promote functional recovery after SCI. In the present invention we in fact demonstrated, using a mouse model, synergistic interaction between T cell-based immune activation and transplanted aNPCs in promoting functional motor recovery after contusive injury of the spinal cord.
Previous studies by the present inventors have shown that local and systemic immune activities have multiple tasks; they are spontaneously evoked after a traumatic injury to the CNS, and are crucial for post-traumatic survival and repair (Yoles et al., 2001a). Immunization with an antigen (such as MOG) that resides in the lesion site can enhance this immune-dependent beneficial effect. The relevant autoimmune T cells evoked by the immunization exert their protective effect in part by interacting with resident APCs such as microglia/macrophages. In line with these studies, our present results showed that the T cells needed for synergistic action with aNPCs were specific to a peptide derived from a CNS-myelin protein; no synergistic effect was obtained following a T cell-mediated response to an irrelevant antigen (ovalbumin). It should be noted that immunization with encephalitogenic peptides was utilized here for proof of concept; we do not advocate such immunization for therapeutic purposes as it carries the risk in some individuals or strains (e.g., those susceptible to autoimmune diseases) of inducing an overwhelming inflammatory response that is detrimental for recovery. For purposes of immunization, weak agonists of encephalitogenic antigens or synthetic antigens that weakly cross-react with self-antigens can replace the encephalitogenic peptides. We found that immunization with MOG peptide emulsified in IFA rather than in CFA failed to demonstrate synergism with aNPCs, suggesting that the T-cell response should be biased towards Th1, as we have previously proposed (Kipnis et al., 2002a).
We also demonstrate herein, using a rat model of environmental enrichment (in which neurogenesis in adults is physiologically augmented, see Kempermann et al., 1997), a spatial association between T cells, microglial activity, and hippocampal neurogenesis. We further show that neurogenesis is impaired in immune-deficient mice under both regular and environmental enriched conditions, and can be restored by T cells directed to brain autoantigens but not by T cells directed to irrelevant (nonself) antigens. Similar dependence on T cells was found with respect to spatial learning/memory, an additional aspect of hippocampal plasticity.
The findings of the present invention indicate that T cells affect adult neurogenesis, both in the dentate gyrus and in the SVZ, primarily via their effect on progenitor cell proliferation. The finding that relative to the wild type the TMBP transgenic mice exhibited increased neurogenesis, taken together with the improved spatial-learning abilities in these mice, whereas both neurogenesis and spatial learning were impaired in TOVA mice, suggests that the T cells, in order to function properly in brain plasticity, should become activated by their cognate brain antigens. Thus, the mere presence of T cells is not enough to maintain neurogenesis and learning abilities; these T cells need, in addition, to be directed to CNS antigens such as, but not limited to, MBP, MOG and others.
According to our view, the T cells interact locally with microglia and possibly also with other cellular components (endothelial cells, astrocytes) of the special microenvironment known as the ‘stem-cell niche’. The finding that microglia expressing MHC-II proteins can be seen adjacent to a site of increased neurogenesis (induced by an enriched environment) further supports the notion that. T cells can locally interact with microglia through these proteins. Some of the MIC-II+ microglia were found to express IGF-I, a growth factor known to be associated with cell renewal in the CNS. Co-expression of MHC-II and IGF-I can be observed in the examples herein in microglia that encounter T cell-derived cytokines.
The reduction in neurogenesis observed in TMBP-transgenic mice after treatment with minocycline (which suppresses microglial activation) supports our suggestion that T cells can benefit neurogenesis by activating microglia (or other CNS-resident antigen-presenting cells). This does not argue against reported observations that severe inflammation, whether induced experimentally (by LPS) or as a result of neurodegenerative conditions (e.g., Alzheimer's disease or severe chronic multiple sclerosis), impairs neurogenesis (Ekdahl et al., 2003). On the other hand, our results might explain why pathological conditions such as acute CNS insult and experimental autoimmune diseases might be accompanied by an increase in neurogenesis as well as by induction of neurogenesis in non-neurogenic areas (Pluchino et al., 2003).
It is feasible that ‘resting’ microglia, recently described as highly dynamic surveillants of the brain, function in the dentate gyrus as stand-by cells for purposes not only of housekeeping and repair, but also of support for neurogenesis.
Our observation that environmental enrichment fails to enhance neurogenesis in mice suffering from immune deficiency strongly suggests that the availability of T cells that recognize CNS antigens is a critical requirement for an activity-induced increase in neurogenesis. Presumably these T cells are recruited when neurogenesis is needed. The active participation of CNS-specific T cells in brain-cell renewal does not exclude the possibility that T cells are needed for neurogenesis during development as well.
The results herein might partially explain the age-related loss of certain cognitive activities, by viewing the decline in terms of an aging immune system. They might also explain why conditions of immune compromise (e.g. AIDS) result in cognitive impairment (HIV-associated dementia).
Previous studies by the present inventors have shown that systemic manipulations of the immune system, based on increasing the numbers of T cells directed to weak agonists of autoantigens, beneficially affect neurodegenerative conditions by promoting neuronal survival (Moalem et al., 1999; Hauben et al., 2001; Schwartz and Kipnis, 2002). The same manipulations, for example, T cell-based vaccination, is proposed here for increasing neurogenesis, yielding novel ways to maintain the integrity of the aging brain and the diseased mind.
It thus seems that maintenance and repair of brain cells necessitate a dialog between CNS-autoreactive T cells and brain-resident microglia. This dialog cannot take place, however, unless the microglia are able to act as APCs, presenting the relevant antigens to the homing T cells. We therefore postulated that in order to halt the progression of Alzheimer disease (AD), T cells that recognize CNS-specific antigens other than aggregated amyloid-β (Aβ) must target sites of aggregated Aβ plaques in the brain. On reaching these sites they become activated by the encounter with their specific antigens, presented to them by microglia acting as APCs. Such activation enables these T cells to offset the negative effect of aggregated Aβ on locally resident microglia, thus preventing the latter from becoming cytotoxic to neurons and blocking neurogenesis. We tested this hypothesis by vaccinating AD mice with glatiramer acetate (GA, also known as copolymer 1 or Cop-1), a synthetic copolymer approved by the FDA for treatment of multiple sclerosis, and capable of weakly cross-reacting with a wide range of CNS-resident autoantigens (Kipnis et al., 2000). GA-activated T cells, after infiltrating the CNS, have the potential to become locally activated without risk of the overwhelming proliferation that is likely to cause an autoimmune disease. Studies by the present inventors and others have shown that GA can simulate the protective and reparative effects of autoreactive T cells (Kipnis et al., 2000; Benner et al., 2004).
In the present invention, APP/PS1 double-transgenic AD mice (which coexpress mutated human presenilin 1 and amyloid-β precursor protein) suffering from decline in cognition and accumulation of Aβ plaques, a T cell-based vaccination, by altering the microglial phenotype, ameliorated cognitive performance, reduced plaque formation, rescued cortical and hippocampal neurons, and induced hippocampal neurogenesis.
We show here that vaccination of Tg mice with GA reduced plaque formation, and prevented and even partially reversed cognitive decline, even if the vaccination was given after some loss of cognition and some plaque formation had already occurred. It should be noted that vaccination with GA was effective not only in preventing disease progression but also—when administered after onset of the clinical symptoms of learning/memory loss and pathological appearance of plaques—in promoting tissue repair. The above findings are in line with our observation that in mice deficient in CNS-autoreactive T cells the expression of brain-derived neurotrophic factor (BDNF), known to be associated with both cognitive activity and cell renewal, is impaired. They are also in accord with our observation that T cells are needed for the maintenance of cognitive functioning in the healthy as well as in the diseased brain (Kipnis et al., 2004). Since aggregated Aβ evidently interferes with the ability of microglia to engage in dialog with T cells, its presence in the brain can be expected to cause loss of cognitive ability and impairment of neurogenesis. Homing of CNS-autoreactive T cells to the site of disease or damage in such cases is critical, but will be effective only if those T cells can counterbalance the destructive activity of the aggregated Aβ. Myelin-presenting microglia, with which myelin-specific T cells can readily hold a dialog, are likely to be present in abundance. Myelin-related antigens, or antigens (such as GA) that are weakly cross-reactive with myelin, are therefore likely to be the antigens of choice for the therapeutic vaccination. Myelin-specific T cells will then home to the CNS and, upon encountering their relevant APCs there, will become locally activated to supply the cytokines and growth factors needed for appropriate modulation of harmful microglia like those activated by aggregated Aβ. The resulting synapse between T cells and microglia will create a supportive niche for cell renewal by promoting neurogenesis from the pool of adult stem cells, thereby overcoming the age-related impairment induced in the inflammatory brain.
In another embodiment, we used the immunomodulator PN277, a random synthetic copolymer composed of glutamic acid and tyrosine residues (also known as polyYE and poly-Glu, Tyr), in a model of stroke. Boosting T-cell mediated immune response following insult to the CNS can be done by either vaccinating with a CNS-specific antigen or by abolishing regulatory T cells (Treg) activity (Moalem et al., 1999; Hauben et al., 2000; Schwartz et al., 2003). PN277 was recently shown to down-regulate the inhibitory effects of naturally occurring regulatory T cells and to be capable of reducing Treg suppressive activity and confer neuroprotection (WO 2005/055920). While vaccination induce T-cell response primarily to one antigen, reducing Treg suppressive activity facilitate a broader T-cell response against various antigens residing in the injured site. Thus, following stroke, mostly T cells specific for various CNS-antigens would be propagated in response to PN277 treatment.
The finding of enhanced hippocampal neurogenesis in the PN277-treated rats is consistent with our finding that neurogenesis was enhanced in transgenic mice over-expressing T-cell receptor for MBP. The fact that this effect was seen in both ipsilateral and contralateral sides further imply that elevation in neurogenesis is caused by a systemic event (T-cell response). Such autoimmune T cells can locally interact with microglia, which in turn can recruit NPCs and direct their differentiation. This scenario is supported by the findings herein showing that neurogenesis from aNPCs could be induced by microglia activated by cytokines (IL-4 or IFN-γ) associated with T-helper cells. The effect of PN277 treatment on neurogenesis was even more robust with regard to cortical neurogenesis. In conclusion, the results of this study suggest that proper immune modulation can increase neurogenesis, and that lack of neurogenic permissiveness in brain regions such as the cortex can be changed by the local immune response. These findings introduce a new therapeutic approach for augmenting spontaneously occurring neurogenesis following an ischemic insult.
The present invention thus relates, in one aspect, to a method for inducing and enhancing neurogenesis and/or oligodendrogenesis from endogenous as well as from exogenously administered stem cells, which comprises implanting stem cells into an individual in need, and administering to said individual a neuroprotective agent selected from the group consisting of:
In one embodiment, the present invention relates to a method for inducing and enhancing neurogenesis from endogenous or exogenously applied stem cells, by immune modulation, which comprises administering to an individual in need a neuroprotective agent selected from the group consisting of the agents (i) to (x) defined above.
In another embodiment, the present invention relates to a method for inducing and enhancing oligodendrogenesis from endogenous or exogenously applied stem cells, by immune modulation, which comprises administering to an individual in need a neuroprotective agent selected from the group consisting of the agents (i) to (x) defined above.
The stem cells for use in the present invention are stem cells of any origin used today or to be used in the future in stem cell therapy and include, without limitation, adult stem cells (found in various tissues of an adult organism that remain in an undifferentiated, or unspecialized, state, and can renew themselves and differentiate to yield all the specialized cell types of the tissue from which it originated and other types of cells), embryonic stem cells, umbilical cord blood stem cells, hematopoietic stem cells, peripheral blood stem cells, mesenchimal stem cells, multipotent stem cells, neural stem cells, stromal cells, progenitor cells (cells that can differentiate into a limited number of cell types, but cannot make more stem cells or renew itself), and any other type of stem cells and precursors thereof.
The nervous system (NS)-specific antigens and analogs thereof defined herein as agent (i), the peptides derived from NS-specific antigens or from analogs thereof, and analogs or derivatives of said peptides defined as agent (ii), and T cells activated with (i) or (ii), include the agents described in U.S. patent application Ser. No. 10/810,653 and PCT Publications WO 99/60021 and WO 02/055010 of the applicant, all these applications being herewith incorporated by reference as if fully disclosed herein.
The term “nervous system (NS)-specific antigen” as used herein refers to an antigen of the central or peripheral nervous system that specifically activates T cells such that following activation the activated T cells accumulate at a site of injury or disease in the NS of the patient. In one embodiment, the agent for use in the invention is a NS-antigen of mammal, preferably human, origin, such as, but not limited to, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), myelin-associated glycoprotein (IAG), S-100, β-amyloid, Thy-1, a peripheral myelin protein such as P0, P2 and PMP22, neurotransmitter receptors, the protein Nogo (Nogo-A, Nogo-B and Nogo-C) and the Nogo receptor (NgR), or an analog thereof. This definition also includes analogs of said NS-specific antigens in which one or more amino acid residues of the NS-antigen have been deleted or replaced by different amino acid residues and/or amino acid residues have been added to the antigen sequence.
In another embodiment, the agent is a peptide derived from an NS-specific antigen or from an analog thereof, as defined hereinabove. As used herein, the term “a peptide derived from an NS-specific antigen” relates to a peptide that has a sequence comprised within the NS-antigen sequence. In one embodiment, the peptide is an immunogenic epitope or a cryptic epitope derived from said antigen. Examples of such peptides include the MBP peptides p11-30, p51-70, p91-110, p131-150, and p151-170 of MBP.
In a further embodiment, the agent is an analog of an NS-specific antigen peptide obtained by modification of a self-peptide derived from a CNS-specific antigen, which modification consists in the replacement of one or more amino acid residues of the self-peptide by different amino acid residues or by deletion or addition of one or more amino acid residues, said modified CNS peptide still being capable of recognizing the T-cell receptor recognized by the self-peptide but with less affinity (hereinafter “modified CNS peptide” or “altered peptide”).
The modified CNS peptide preferably is immunogenic but not encephalitogenic. The most suitable peptides for this purpose are those in which an encephalitogenic self-peptide is modified at the T cell receptor (TCR) binding site and not at the MHC binding site(s), so that the immune response is activated but not anergized. The invention also encompasses peptides derived from any encephalitogenic epitopes in which critical amino acids in their TCR binding site but not MHC binding site are altered as long as they are non-encephalitogenic and still recognize the T-cell receptor.
In one embodiment, the modified peptide is derived from the residues 86 to 99 of human MBP by alteration of positions 91, 95 or 97 as disclosed in U.S. Pat. No. 5,948,764. In another embodiment, the modified peptide is a peptide disclosed in WO 02/055010, derived from the residues 87-99 of human MBP, in which the lysine residue 91 is replaced by glycine (G91) or alanine (A91), or the proline residue 96 is replaced by alanine (A96). In a further embodiment, the peptide is derived from the encephalitogenic MOG peptide p35-55 of the SEQ ID NO:1 such as the peptide obtained by deletion of 1 amino acid from either the N- or C-terminus of the truncated peptide pMOG44-54 or the modified MOG peptide of SEQ ID NO:2. In a further embodiment, the modified peptide is an analog of the peptide 95-117 of PLP.
In another embodiment, the agent for use in the invention are T cells activated by a NS-specific antigen, an analog of said antigen, a peptide derived from said antigen or antigen analog or an analog or derivative of said peptide, all as defined above. The T cells can be endogenous and activated in vivo by administration of the antigen or peptide, thereby producing a population of T cells that accumulate at a site of injury or disease of the CNS or PNS.
In one embodiment, the T cells are prepared from T lymphocytes isolated from the blood and then sensitized to the NS-antigen or peptide, preferably to a modified peptide as defined herein. The T cells are preferably autologous, most preferably of the CD4 and/or CD8 phenotypes, but they may also be allogeneic T cells from related donors, e.g., siblings, parents, children, or HLA-matched or partially matched, semi-allogeneic or fully allogeneic donors. Methods for the preparation of said T cells are described in the above-mentioned WO 99/60021.
In another embodiment, the agent is PN277 (Poly-YE), a random copolymer of Tyr and Glu that may contain the amino acids Glu and Tyr in any available ratio such as, for example, poly-(Glu, Tyr) 1:1 and poly(Glu, Tyr) 4:1. The modulation of the immune response and modulation of autoimmunity response by poly-YE is described in U.S. Pat. No. 6,838,711, U.S. application Ser. No. 10/807,414, WO 03/002140, WO 2005/055920, and WO 2005/089787, all these applications being herewith incorporated by reference as if fully disclosed herein. As used herein, the term “poly-YE related peptides” refers to random copolymers of Tyr and Glu with different ratios of Glu and Tyr and/or lower or higher molecular weight and to random peptides containing several residues of Tyr and Glu.
In another embodiment, the antigen-presenting cells (APCs) for use in the invention are APCs that have been pulsed with a NS-specific antigen or an analog thereof, a peptide derived from a NS-specific antigen or from an analog thereof, or an analog or derivative of said peptide, or Poly-YE, as described in WO 03/105750, herewith incorporated by reference as if fully disclosed herein. The APCs for use in the invention are preferably human APCs and are selected from the group consisting of monocytes, macrophages, B cells and, preferably, dendritic cells. The human dendritic cells can be obtained from skin, spleen, thymus, bone marrow, lymph nodes or peripheral blood of an individual, and cultured as described in WO 03/105750, preferably in a medium containing IL-4, GM-CSF, or both IL-4 and GM-CSF.
In a further embodiment, the agent is activated mononuclear phagocytic leukocytes, such as the activated cells described in U.S. Pat. No. 5,800,812, No. 6,117,424 and No. 6,267,955, and WO 03/044037, all these patents and applications being herewith incorporated by reference as if fully disclosed herein. The activated mammalian mononuclear phagocytes are obtained by culturing the cells together with at least one stimulatory tissue such as skin, dermis and a nerve, e.g. peripheral, nerve, segment, or with stimulatory cells derived from skin, dermis, and a nerve segment, or with medium conditioned by skin, dermis, and/or a nerve segment, a medium conditioned by at least one stimulatory tissue or cells, or with medium to which at least one stimulatory biologically active agent has been added. The stimulatory biologically active agent may be neurotrophic factor 3 (NT-3), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and transforming growth factor-β (TGF-β).
In another embodiment the agent is dopamine or a pharmaceutically acceptable salt thereof, optionally in combination with the dopamine precursor levodopa, and further optionally in combination with carbidopa. The D1-R agonist may be selected from the group consisting of SKF-82958, SKF-38393, SKF-77434, SKF-81297, A-77636, fenoldopam and dihydrexidine. The dopamine, dopamine precursor or D1-R agonist may be in combination with a dopamine D2-R antagonist selected from the group consisting of amisulpride, eticlopride, raclopride, remoxipride, sulpride, tropapride, domperidone, iloperidone, risperidone, spiperone, haloperidol, spiroperidol, clozapine, olanzapine, sertindole, mazapertine succinate, zetidoline, CP-96345, LU111995, SDZ-HDC-912, and YM 09151-2. All these agents are as described in WO 2005/055920, herewith incorporated by reference as if fully disclosed herein. These agents cause down-regulation of the suppressive activity of CD4+ CD25+ regulatory T (Treg) cells on CD4+ CD25− effector T cells (Teff) and thus, they modulate the immune response and/or the autoimmune response
In a further embodiment, the agent is microglia, a major glial component of the CNS that play a critical role as resident immunocompetent cells and phagocytic cells in the CNS, wherein the microglia are activated by IFN-γ or IL-4. Administration of the cytokine alone, IL-4 or low dose of IFN-γ, will also cause the activation of endogenous microglia.
In further embodiments, the present invention provides a combination of any two or more of the agents (i) to (ix). In some embodiments, the combination comprises T-cell vaccination by administration of T cells and the NS-antigen to which the cells were sensitized, or administration of T cells and microglia, as defined herein.
The methods of the invention are useful for inducing and enhancing neurogenesis and/or oligodendrogenesis both from endogenous and exogenously administered stem cells and may assist in solving the problems found today with the poor results of stem cell transplantation, particularly in the cases of injuries, diseases, disorders and conditions of the nervous system, both the CNS and PNS.
In one embodiment, the method of the invention is applied to induce and enhance neurogenesis and/or oligodendrogenesis from endogenous pools of neural stem/progenitor cells. Thus, the immunomodulators used in the present invention will by themselves boost endogenous neurogenesis and oligodendrogenesis in damaged tissues, supporting also the survival of the new neurons and oligodendrocytes.
In another embodiment, the method of the invention is applied to induce and enhance neurogenesis and/or oligodendrogenesis from both endogenous and exogenous stem cells administered to the patient. The administration of a neuroprotective/immunomodulator agent (i) to (ix) or a combination thereof will assist to enhance the successful engraftment of the implanted stem cells, cell renewal and differentiation of the stem cells into neurons and/or oligodendrocytes, while at the same time inducing the endogenous neurogenesis and oligodendrogenesis in the damaged tissues and supporting the survival of the new neurons and oligodendrocytes.
The method of cell therapy according to the present invention may be carried out by different routes. In one embodiment, the stem cells are injected/transplanted to the patient, followed by vaccination with the neuroprotective/immunomodulator agent. In another embodiment, a combination of the stem cells with the neuroprotective/immunomodulator agent is injected/transplanted to the patient. In a further embodiment, the stem cells can be cultured in vitro (artificially) with the neuroprotective/immunomodulator agent and differentiated prior to transplantation
In one embodiment, the method of the invention comprises stem cell therapy by transplantation of stem cells in combination with a neuroprotective agent (i) to (x), to an individual that suffers from an injury in the CNS such as spinal cord injury, closed head injury, blunt trauma, penetrating trauma, hemorrhagic stroke, ischemic stroke, cerebral ischemia, optic nerve injury, myocardial infarction and injury caused by tumor excision. The transplanted stem cells will migrate to the region of the injury where cells had died (for example, due to ischaemia) and will differentiate into neurons and/or oligodendrocytes.
In another embodiment, the method of the invention comprises stem cell therapy by administration of stem cells in combination with a neuroprotective agent (i) to (x), to a patient suffering from of a neurodegenerative disease or disorder such as Parkinson's disease and Parkinsonian disorders, Huntington's disease, Alzheimer's disease, multiple sclerosis, or amyotrophic lateral sclerosis (ALS). For ALS, the neuroprotective agent is preferably a peripheral myelin or a peptide derived from a peripheral myelin or an analog thereof.
In another embodiment, the method of the invention comprises stem cell therapy by administration of stem cells in combination with a neuroprotective agent (i) to (x), to a patient suffering from a disease, disorder or condition of the CNS or PNS such as facial nerve (Bell's) palsy, glaucoma, Alper's disease, Batten disease, Cockayne syndrome, Guillain-Barré syndrome, Lewy body disease, Creutzfeldt-Jakob disease, or a peripheral neuropathy such as a mononeuropathy or polyneuropathy selected from the group consisting of adrenomyeloneuropathy, alcoholic neuropathy, amyloid neuropathy or polyneuropathy, axonal neuropathy, chronic sensory ataxic neuropathy associated with Sjogren's syndrome, diabetic neuropathy, an entrapment neuropathy nerve compression syndrome, carpal tunnel syndrome, a nerve root compression that may follow cervical or lumbar intervertebral disc herniation, giant axonal neuropathy, hepatic neuropathy, ischemic neuropathy, nutritional polyneuropathy due to vitamin deficiency, malabsorption syndromes or alcoholism, porphyric polyneuropathy, a toxic neuropathy caused by organophosphates, uremic polyneuropathy, a neuropathy associated with a disease or disorder selected from the group consisting of acromegaly, ataxia telangiectasia, Charcot-Marie-Tooth disease, chronic obstructive pulmonary diseases, Fabry's disease, Friedreich ataxia, Guillain-Barré syndrome, hypoglycemia, IgG or IgA monoclonal gammopathy (non-malignant or associated with multiple myeloma or with osteosclerotic myeloma), lipoproteinemia, polycythemia vera, Refsum's syndrome, Reye's syndrome, and Sjogren-Larsson syndrome, a polyneuropathy associated with various drugs, with hypoglycemia, with infections such as HIV infection, or with cancer. For peripheral neuropathies, the neuroprotective agent is preferably a peripheral myelin or a peptide derived from a peripheral myelin or an analog thereof.
In a further embodiment, the method of the invention comprises stem cell therapy by administration of stem cells in combination with a neuroprotective agent (i) to (x), to a patient suffering from epilepsy, amnesia, anxiety, hyperalgesia, psychosis, seizures, oxidative stress, opiate tolerance and dependence, and for the treatment of a psychosis or psychiatric disorder selected from the group consisting of an anxiety disorder, a mood disorder, schizophrenia or a schizophrenia-related disorder, drug use and dependence and withdrawal, and a memory loss or cognitive disorder.
The stem cells that can be injected or transplanted to an individual according to the method of the invention include, but are not limited to, adult stem cells, neural precursors, neural stem cells, neural adult cells, neural progenitor cells, hematopoietic stem cells, mesenchimal stem cells, embryonic stem cells, stromal stem cells, pluripotent stem cells and any other type of stem cells and precursors thereof, that may be found suitable for the purpose of the present invention. Examples of such cells include the CNS neural stems cells disclosed in U.S. Pat. No. 6,777,233 and U.S. Pat. No. 6,680,198; the neural stem cells and hematopoietic cells disclosed in U.S. Pat. No. 6,749,850 for administration with neural stimulants; and the stromal cells disclosed in U.S. Pat. No. 6,653,134 for treatment of CNS diseases.
As used herein, the term “neural stem cell” is used to describe a single cell derived from tissue of the central nervous system, or the developing nervous system, that can give rise in vitro and/or in vivo to at least one of the following fundamental neural lineages: neurons (of multiple types), oligodendroglia and astroglia as well as new neural stem cells with similar potential. “Multipotent” or “pluripotent” neural stem cells are capable of giving rise to all of the above neural lineages as well as cells of equivalent developmental potential.
In a more preferred embodiment, the neural stem cells are human neural stem cells that can be isolated from both the developing and adult CNS, and can be successfully grown in culture, are self-renewable, and can generate mature neuronal and glial progeny. Embryonic human neural stem cells can be induced to differentiate into specific neuronal phenotypes. Human neural stem cells integrate into the host environment after transplantation into the developing or adult CNS. Human neural stem cells transplanted into animal models of Parkinson's disease and spinal cord injury have induced functional recovery. However, there are still problems with the engraftment of said cells and the present invention will enhance the successful engraftment, survival and further differentiation of the implanted cells. In a most preferred embodiment, the neural stem cells are autologous.
As used herein, the term “hematopoietic stem cells” refer to stem cells that can give rise to cells of at least one of the major hematopoietic lineages in addition to producing daughter cells of equivalent potential. Certain hematopoietic stem cells are capable of giving rise to many other cell types including brain cells.
The stem cells, once isolated, are cultured by methods known in the art, for example as described in U.S. Pat. No. 5,958,767, U.S. Pat. No. 5,270,191, U.S. Pat. No. 5,753,506, all of these patents being herewith incorporated by reference as if fully disclosed herein.
The treatment regimen according to the invention is carried out, in terms of administration mode, timing of the administration, and dosage, depending on the type and severity of the injury, disease or disorder and the age and condition of the patient. The immunomodulator may be administered concomitanly with, before or after the injection or implantation of the cells.
The administration of the cells may be carried out by various methods. In certain embodiments, the cells are preferably administered directly into the stroke cavity, the spinal fluid, e.g., intraventricularly, intrathecally, or intracistemally. The stem cells can be formulated in a pharmaceutically acceptable liquid medium, which can contain the immunomodulator molecule (i) to (iii), (vii) or (ix) or the immunomodulator cells (iv) to (vi) and (wiii) as well. Cells may also be injected into the region of the brain surrounding the areas of damage, and cells may be given systemically, given the ability of certain stem cells to migrate to the appropriate position in the brain.
The invention will now be illustrated by the following non-limiting examples.
Animals. The animals used in the experiments, if not indicated differently, were supplied by the Animal Breeding Center of the Weizmann Institute of Science (Rehovot, Israel). All animals were handled according to the regulations formulated by the Weizmann Institute's Animal Care and Use Committee.
Reagents. Lipopolysaccharide (LPS) (containing<1% contaminating proteins) was obtained from Escherichia coli 0127:B8 (Sigma-Aldrich, St. Louis, Mo.). Recombinant mouse tumor necrosis factor (TNF)-α and insulin-like growth factor (IGF)-I (both containing endotoxin at a concentration below 1 EU per μg of cytokine), recombinant rat and mouse interferon (IFN)-γ and interleukin (IL)-4 (both containing endotoxin at a concentration below 0.1 ng per μg of cytokine), goat anti-mouse neutralizing anti-TNF-α antibodies (ãTNF-α; containing endotoxin at a concentration below 0.001 EU per μg of Ab), and goat anti-mouse/rat neutralizing anti-IGF-I (ãIGF-I; containing endotoxin at a concentration below 0.1 EU per μg of Ab) used in the Examples hereinafter were obtained from R&D Systems (Minneapolis, Minn.).
(i) Neuralprogenitor cell (NPC) culture. Coronal sections (2 mm thick) of tissue containing the subventricular zone of the lateral ventricle were obtained from the brains of adult C57B16/J mice. The tissue was minced and then incubated for digestion at 37° C., 5% CO2 for 45 mine in Earle's balanced salt solution containing 0.94 mg/ml papain (Worthington, Lakewood, N.J.) and 0.18 mg/ml of L-cysteine and EDTA. After centrifugation at 110×g for 15 min at room temperature, the tissue was mechanically dissociated by pipette trituration. Cells obtained from single-cell suspensions were plated (3500 cells/cm2) in 75-cm2 Falcon tissue-culture flasks (BD Biosciences, Franklin Lakes, N.J.), in NPC-culturing medium [Dulbecco's modified Eagles's medium (DMEM)/F12 medium (Gibco/Invitrogen, Carlsbad, Calif.) containing 2 mM L-glutamine, 0.6% glucose, 9.6 μg/ml putrescine, 6.3 ng/ml progesterone, 5.2 ng/ml sodium selenite, 0.02 mg/ml insulin, 0.1 mg/ml transferrin, 2 μg/ml heparin (all from Sigma-Aldrich, Rehovot, Israel), fibroblast growth factor-2 (human recombinant, 20 ng/ml), and epidermal growth factor (human recombinant, 20 ng/ml; both from Peprotech, Rocky Hill, N.J.)]. Spheres were passaged every 4-6 days and replated as single cells. Green fluorescent protein (GFP)-expressing neural progenitor cells (NPCs) were obtained as previously described (Pluchino et al., 2003).
(ii) Primary microglial culture. Brains from neonatal (P0-P1) C57B1/6J mice were stripped of their meninges and minced with scissors under a dissecting microscope (Zeiss, Stemi DV4, Germany) in Leibovitz-15 medium (Biological Industries, Beit Ha-Emek, Israel). After trypsinization (0.5% trypsin, 10 min, 37° C./5% CO2), the tissue was triturated. The cell suspension was washed in culture medium for glial cells [DMEM supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, Rehovot), L-glutamine (1 mM), sodium pyruvate (1 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml)] and cultured at 37° C./5% CO2 in 75-cm2 Falcon tissue-culture flasks (BD Biosciences) coated with poly-D-lysine (PDL) (10 mg/ml; Sigma-Aldrich, Rehovot) in borate buffer (2.37 g borax and 1.55 g boric acid dissolved in 500 ml sterile water, pH 8.4) for 1 h, then rinsed thoroughly with sterile, glass-distilled water. Half of the medium was changed after 6 h in culture and every 2nd day thereafter, starting on day 2, for a total culture time of 10-14 days. Microglia were shaken off the primary mixed brain glial cell cultures (150 rpm, 37° C., 6 h) with maximum yields between days 10 and 14, seeded (105 cells/ml) onto PDL-pretreated 24-well plates (1 ml/well; Corning, Corning, N.Y.), and grown in culture medium for microglia [RPMI-1640 medium (Sigma-Aldrich, Rehovot) supplemented with 10% FCS, L-glutamine (1 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml)]. The cells were allowed to adhere to the surface of a PDL-coated culture flask (1 h, 37° C./5% CO2), and non-adherent cells were rinsed off.
(iii) Co-culturing of neural progenitor cells (NPCs) and mouse microglia. Microglia were treated for 24 h with cytokines (IFN-γ, 20 ng/ml; IL-4, 10 ng/ml) or LPS (100 ng/ml). Cultures of treated or untreated microglia were washed twice with fresh NPC-differentiation medium (same as the culture medium for NPCs but without growth factors and with 2.5% FCS) to remove all traces of the tested reagents, then incubated on ice for 15 min, and shaken at 350 rpm for 20 min at room temperature. Microglia were removed from the flasks and immediately co-cultured (5×104 cells/well) with NPCs (5×104 cells/well) for 5 or 10 days on cover slips coated with Matrigel (BD Biosciences) in 24-well plates, in the presence of NPC differentiation medium, with or without insulin. The cultures were then fixed with 2.5% paraformaldehyde in PBS for 30 min at room temperature and stained for neuronal and glial markers. Cell proliferation rates and cell survival in vitro were determined by staining with 5-bromo-2′-deoxyuridine (BrdU, 2.5 μM; Sigma-Aldrich, St. Louis). For quantification of live and dead cells, live cultures were stained with 1 μg/ml propidium iodide (Molecular Probes, Invitrogen, Carlsbad, Calif.) and 1 μg/ml Hoechst 33342 (Sigma-Aldrich, St. Louis), and cells were counted using Image-Pro (Media Cybernetics, Silver Spring, Md.), as described (Hsieh et al., 2004)
(iv) Immunocytochemistry. Cover slips from co-cultures of NPCs and mouse microglia were washed with PBS, fixed as described above, treated with a permeabilization/blocking solution containing 10% FCS, 2% bovine serum albumin, 1% glycine, and 0.1% Triton X-100 (Sigma-Aldrich, Rehovot) and stained with a combination of the mouse or rabbit anti-tubulin β-III-isoform C-terminus antibodies (β-II-tubulin; 1:500), rabbit anti-NG2 chondroitin sulfate proteoglycan (NG2; 1:500), mouse anti-RIP (RIP; 1:2000), mouse anti-galactocerebroside (GalC; 1:250), mouse anti-glutamic acid decarboxylase 67 (GAD; 1:1000), mouse anti-nestin (Nestin; 1:1000), rat anti-myelin basic protein (MBP; 1:300) (all from Chemicon, Temecula, Calif.), goat anti-double cortin (DCX; 1:400, Santa Cruz Biotechnology, Santa Cruz, Calif.) and mouse anti-glial fibrillary acidic protein (GFAP; 1:100, Sigma-Aldrich, St. Louis). For labeling of microglia we used either rat andi-CD11b (MAC1; 1:50, BD-Pharmingen, NJ) or FITC-conjugated Bandeiraea simplicifolia isolectin B4 (IB4; 1:50, Sigma-Aldrich, Rehovot). Expression of IGF-1 was detected by goat anti-IGF-1 (1:20, R&D Systems).
(v) RNA purification, cDNA synthesis, and reverse-transcription PCR analysis. Cells were lysed with TRI reagent (MRC, Cincinnati, Ohio), and total cellular RNA was purified from lysates using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Residual genomic DNA was removed during the purification process by incubation with RNase-free DNase (Qiagen). RNA was stored in RNase-free water (Qiagen) at −80° C. RNA (1 μg) was converted to cDNA using SuperScript II (Promega, Madison, Wis.), as recommended by the manufacturer. The cDNA mixture was diluted 1:5 with PCR-grade water.
We assayed the expression of specific mRNAs using semi-quantitative reverse transcription PCR(RT-PCR) with selected gene-specific primer pairs, using OLIGO v6.4 (Molecular Biology Insights, Cascade, Colo.).
The primers used were:
The RT-PCR reactions were carried out using 1 μg of cDNA, 35 nmol of each primer, and ReadyMix PCR Master Mix (ABgene, Epsom, UK) in 30-μl reactions. PCR reactions were carried out in an Eppendorf PCR system with cycles (usually 25-30) of 95° C. for 30 s, 60° C. for 1 min, 72° C. for 1 min, and 72° C. for 5 min, and then kept at 4° C. As an internal standard for the amount of cDNA synthesized, we used β-actin mRNA. PCR products were subjected to agarose gel analysis and visualized by ethidium bromide staining. Signals were quantified using a Gel-Pro analyzer 3.1 (Media Cybernetics). In all cases one product was observed with each primer set, and the observed product had an amplicon size that matched the size predicted from published cDNA sequences.
(vi) Quantification. For microscopic analysis we used a Zeiss LSM 510 confocal laser scanning microscope (40× magnification). For experiments in vitro we scanned fields of 0.053 mm2 (n=8-16 from at least two different coverslips) for each experimental group. For each marker, 500-1000 cells were sampled. Cells co-expressing GFP and β-III-tubulin, NG2, RIP, GalC, and GFAP were counted.
(vii) Statistical analysis. The results were analyzed by the Tukey-Kramer multiple comparisons test (ANOVA) and are expressed as means±SD (unless differently indicated).
Adaptive immunity, in the form of a well-controlled Th1 or a Th2 response to a CNS insult, induces microglia (MG) to adopt a phenotype that facilitates neuronal protection and neuronal tissue repair (Butovsky et al., 2001). Here we examined the ability of adaptive immunity, via activation of microglia, to induce or support the differentiation of NPCs. Neurogenesis is reportedly blocked by the inflammation caused by microglia activated with endotoxin (such as lipopolysaccharide, LPS) (Ekdahl et al., 2003). We therefore compared the effects on NPCs of microglia exposed to LPS (MG(LPS)) with the effects of microglia exposed to the low levels of characteristic Th1 (pro-inflammatory) and Th2 (anti-inflammatory) cytokines, IFN-γ (MG(IFN-γ)) and IL-4 (MG(IL-4γ)), respectively, shown herein to be supportive of neural survival. We used NPCs expressing green fluorescent protein (GFP) to verify that any neural cell differentiation seen in the culture was derived from the NPCs rather than from contamination of the primary microglial culture.
Microglia were grown in their optimal growth medium (Zielasek et al., 1992) and were then treated for 24 h with IL-4, IFN-γ (low level), or LPS. Residues of the growth medium and the cytokines were washed off, and each of the treated microglial preparations, as well as a preparation of untreated microglia (MG(−)), was freshly co-cultured with dissociated NPC spheres in the presence of differentiation medium. We examined the effects of both IFN-γ-activated and IL-4-activated microglia. After 5 days in culture, GFP+ cells that expressed the neuronal marker β-III tubulin were identified as neurons.
Since we recently showed that IL-4-activated microglia (MG(IL-4)) produce a high level of IGF-1 (Butovsky et al., 2005), and because IGF-1 is reportedly a key factor in neural cell renewal (O'Kusky et al., 2000), we envisioned a situation in which IGF-1 might be one of the factors in the effect of IL-4-activated microglia. Therefore, the following experiments were carried out both in insulin-free (to allow detection, if exists, of the effect of insulin-related factors secreted by the activated microglia) and in insulin-containing differentiation media.
Quantitative analysis revealed that neurogenesis, in the absence of insulin, was only minimally supported by MG(IFN-γ) and was impaired by MG(LPS), but was almost 3-fold higher in NPCs co-cultured with MG(IL-4) than in controls (
In co-cultures of NPCs with MG(−), however, addition of insulin increased the percentage of GFP+/β-III-tubulin+ cells (
Morphological differences were observed between the newly differentiating neurons in NPCs co-cultured with MG(IFN-γ) and those generated in co-cultures with MG(IL-4) (
Next we examined whether, under the same experimental conditions, microglia would also induce NPCs to differentiate into oligodendrocytes. Under high magnification, we were able to detect newly formed oligodendrocytes. In attempting to detect possible differentiation of NPCs to oligodendrocytes, we first looked for GFP-labeled cells co-expressing oligodendrocyte progenitor marker NG2. Quantitative analysis confirmed that both MG(IL-4) and (to a lesser extent) MG(IFN-γ) induced differentiation of NG2+ cells from co-cultured NPCs (
Addition of insulin to the NPC cultures did not affect the incidence of NG2+ cells in the absence of microglia (control;
In light of the observed early differences between the effects of MG(IL-4) and MG(IFN-γ) on both neurogenesis and oligodendrogenesis, we examined NPCs co-cultured with the cytokine-activated microglia after 10 days in co-culture. As on day 5, few NG2+ cells were seen in the absence of microglia (
In the presence of MG(IL-4), the GFP+/NG2+ cells were more branched at 10 days (
Table 1 records the proliferation of NPCs co-cultured with non-activated, IL-4-activated, or IFN-γ-activated microglia. Cultures of untreated NPCs (control) or NPCs co-cultured with MG(−) or MG(IL-4) or MG(IFN-γ) or MG(LPS), with or without insulin, were analyzed for proliferation and cell death 24, 48, or 72 h after plating. For the proliferation assay, a pulse of BrdU was applied 12 h before each time point. Numbers of BrdU+ cells are expressed as percentages of GFP+ cells (mean±SEM from three independent experiments in duplicate) and analyzed by ANOVA. Cell death with and without insulin was determined by live staining with 1 μg/ml propidium iodide and 1 μg/ml Hoechst 33342 (mean±SEM from two independent experiments in duplicate; *P<0.05; ***P<0.001; ANOVA).
As shown in Table 1, comparisons of proliferation at 24 h and 48 h of culture revealed no differences. After 72 h a slight but non-significant difference was seen between NPCs alone and NPCs co-cultured with MG(−) or MG(IFN-γ), possibly because of decreased proliferation in the culture of NPCs alone rather than any increase in the co-cultures. In the absence of insulin there were no significant differences at any time in culture between NPCs alone and NPCs co-cultured with MG(−) or with MG(IL-4). A reduction in proliferation was observed in NPCs co-cultured with MGLPS, with or without insulin. After 5 days, no proliferation was detectable in any of the co-cultures (data not shown). To identify dead or dying cells we stained live cultures with 1 μg/ml propidium iodide, which stains dead cells, and 1 μg/ml Hoechst 33342, which stains both live and dead cells (Hsieh et al., 2004). Significant cell death was observed in NPCs co-cultured with MG(LPS), both in the absence and in the presence of insulin, whereas in NPCs cultured alone or with MG(IFN-γ) or MG(IL-4) the percentage of cell death was low and did not differ significantly from that seen in cultures of NPCs alone (Table 1). These results suggested that the primary effect of the cytokine-activated microglia on the fate of NPCs in vitro occurs via a mechanism that is instructive rather than selective.
Proliferation and survival of neural progenitor cells (NPCs) in co-cultures with microglia. Cultures of untreated NPCs (control) or NPCs co-cultured with MG(−) or MG(IL-4) or MG(IFN-γ) or MG(LPS), with or without insulin, were analyzed for proliferation and cell death 24, 48, or 72 h after plating. For the proliferation assay a pulse of BrdU was applied 12 h before each time point. Numbers of BrdU+ cells are expressed as percentages of GFP+ cells (mean±SEM from three independent experiments in duplicate) and analyzed by ANOVA. Cell death with and without insulin was determined by live staining with 1 μg/ml propidium iodide and 1 μg/ml Hoechst 33342 (mean±SEM from two independent experiments in duplicate; *P<0.05; ***P<0.001; ANOVA).
Insulin-like growth factor (IGF)-I is reportedly a key factor in neurogenesis and oligodendrogenesis (Aberg et al., 2000; O'Kusky et al., 2000; Hsieh et al., 2004). To determine whether the beneficial effect of the cytokine-activated microglia on the differentiation of NPCs is mediated, at least in part, by the ability of the microglia to produce IGF-I, we added neutralizing antibodies specific to IGF-I (aIGF-I) to the NPCs co-cultured with activated microglia. ãIGF-I blocked the MG(IL-4)-induced effect on oligodendrogenesis (
In light of the observed beneficial effect of ãTNF-α on the outcome of MG(IFN-γ)-induced neurogenesis (
Comparative RT-PCR analyses of microglial mRNA disclosed that in the absence of activation the microglia produced both IGF-I and low levels of TNF-α. Analysis of TNF-α and IGF-I production as a function of time revealed that IFN-γ, unlike IL-4, caused a transient increase in TNF-α production and down-regulation of IGF-I (
The results of this study strongly suggest that certain specifically activated microglia can induce neural cell renewal in the adult CNS. The findings showed that microglia can determine the fate of differentiating adult NPCs. Both neurogenesis and oligodendrogenesis were induced in NPCs co-cultured with MG(IL-4), and MG(IFN-γ), whereas both were blocked by MG(LPS), in line with reports that inflammation associated with LPS blocks adult neurogenesis (Ekdahl et al., 2003; Monje et al., 2003). NPCs co-cultured with MG(IL-4) showed a bias towards oligodendrogenesis, whereas NPCs co-cultured with MG(IFN-γ) were biased towards neurogenesis.
Neurogenesis in the hippocampal dental gyrus occurs throughout life and is reportedly increased by social, mental, or physical challenges and impeded by inflammation. Here we show that hippocampal neurogenesis is promoted by conditions characterized by transient accumulation of proinflammatory T cells (Th1), such as experimental autoimmune encephalomyelitis (EAE). In vitro, we showed that whereas the Th1-associated cytokine IFN-γ at a low concentration was able to endow rat microglia with a phenotype supportive of neurogenesis from adult stems, a high concentration of this cytokine impeded neurogenesis. IL-4, antibodies specific to TNF-α, and IL-4-activated microglia could all counteract the negative effect of microglia activated by high-dose IFN-γ; and the resultant neurogenesis was significantly greater than that shown by microglia encountering IL-4 only. Injection of IL-4- or IFN-γ-activated microglia into the cerebrospinal fluid enhanced hippocampal neurogenesis in healthy rats and in rats with EAE.
(viii) Neural progenitor cell culture was prepared as described in section (i) in Materials and Methods above in Example 1.
(ix) Primary microglial culture was prepared from brains from neonatal (P0-P1) C57B1/6J mice or Lewis rats as described in section (ii) in Materials and Methods above in Example 1.
(x) Co-culturing of mouse neural progenitor cells and mouse microglia was carried out as described in section (iii) in Materials and Methods above in Example 1. Mouse microglia were treated for 24 hours with cytokines (IFN-γ, 10 ng/ml or 100 ng/ml; IL-4, 10 ng/ml), and co-cultured with NPCs for 10 days, without insulin.
(xi) Induction of acute EAE in rats. To induce monophasic EAE, we immunized adult male Lewis rats s.c. in the hind footpad with 50 μg MBP peptide 68-86, emulsified (1:1) in 100 μl of complete Freund's adjuvant (CFA) containing 2 mg of Mycobacterium tuberculosis (strain H37Ra, Difco).
(xii) Stereotaxic injection of activated microglia. Seven days after MBP immunization, Lewis rats with monophasic EAE were injected bilaterally with syngeneic MG(IL-4) (10 ng/ml) or PBS stereotaxically (1×105 cells in 5 μl PBS for 5 min) into the CSF via the brain lateral ventricles (Bregma −0.8, L 1.2, V 4.5).
(xiii) Administration of 5-bromo-2′-deoxyuridine and tissue preparation. The cell-proliferation marker BrdU was dissolved by sonication in PBS and injected i.p. (50 mg/kg body weight) every 12 hours for 2.5 days, starting on day 14 after MBP immunization. One week after the first BrdU injection, the animals were deeply anesthetized and perfused transcardially, first with PBS and then with 4% paraformaldehyde. Their spinal cords were removed, postfixed overnight, and then equilibrated in phosphate-buffered 30% sucrose. Free-floating 30-μm sections were collected on a freezing microtome (Leica SM2000R) and stored at 4° C. prior to immunohistochemistry.
To study the potential effects of injected microglia on proliferation, differentiation, and survival of progenitor cells in vivo, rats in another group received BrdU injections (every 12 hours for 2.5 days). The rats were killed and their dentate gyri were analyzed 1 day or 28 days after the last BrdU injection.
(xiv) Immunocytochemistry and immunohistochemistry. Immuno-cytochemistry was carried out as described in section (iv) in Materials and Methods above in Example 1. The staining was performed with a combination of the mouse anti-tubulin β-III-isoform C-terminus antibodies (β-III-tubulin; 1:500) and goat anti-doublecortin (DCX; 1:400; Santa Cruz Biotechnology, Santa Cruz, Calif.).
For immunohistochemistry, tissue sections were treated with a permeabilization/blocking solution containing 10% FCS, 2% bovine serum albumin, 1% glycine, and 0.05% Triton X-100 (Sigma-Aldrich, St. Louis). Primary antibodies were applied for 1 hour in a humidified chamber at room temperature. For BrdU staining, sections were washed with PBS and incubated in 2N HCl at 37° C. for 30 min. Sections were blocked for 1 hour with blocking solution. The tissue sections were stained overnight with specified combinations of the following primary antibodies: rat anti-BrdU (1:200; Oxford Biotechnology, Kidlington, Oxfordshire, UK), goat anti-DCX (1:400; Santa Cruz Biotechnology), and mouse anti-NeuN (1:200; Chemicon, Temecula, Calif.). For labeling of microglia we used FITC-conjugated Bandeiraea simplicifolia isolectin B4 (IB4, 1:50; Sigma-Aldrich, Rehovot). For labeling of activated microglia stereotaxically injected in naïve rats we used mouse anti-EDI (ED1; 1:250; Oxford, Serotec, UK). To detect expression of cell-surface MHC-II proteins we used anti-MHC-II Abs (mouse, 1:50; IQ Products, Groningen, The Netherlands). Expression of IGF-I was detected by goat anti-IGF-I Abs (1:20; R&D Systems). Secondary antibodies used were FITC-conjugated donkey anti-goat, Cy-3-conjugated donkey anti-mouse and Cy-3- or Cy-5-conjugated donkey anti-rat (1:200; Jackson ImmunoResearch, West Grove, Pa.). Control sections (not treated with primary antibody) were used to distinguish specific staining from staining of non-specific antibodies or autofluorescent components. Sections were then washed with PBS and coverslipped in polyvinyl alcohol with diazabicylo-octane as anti-fading agent.
(xv) Quantification and stereological counting procedure. For microscopic analysis we used a Zeiss LSM 510 confocal laser scanning microscope (40× magnification). For experiments in vitro we scanned fields of 0.053 mm2 (n=8-16 from at least two different coverslips) for each experimental group. For each marker, 500-1000 cells were sampled. Cells co-expressing GFP and β-III-tubulin were counted. For in-vivo experiments we analyzed nine coronal sections (30 μm) throughout the entire dentate gyrus at 300-μm intervals in each rat. Proliferation and neurogenesis in the dentate gyrus was evaluated by automatic counting of BrdU+, BrdU+/DCX+ or BrdU+/NeuN+ cells using Image-Pro Plus 4.5 software (Media Cybernetics). Specificity of BrdU+/NeuN+ co-expression was assayed using the confocal microscope (LSM 510) in optical sections at 1-μm intervals.
(xvi) Statistical analysis. The in-vitro results were analyzed by the Tukey-Kramer multiple comparisons test (ANOVA) and are expressed as means±SD. In-vivo results were analyzed by Student's t-test or one-way ANOVA and are expressed as means±SEM.
To determine whether MG(IL-4) would create conditions conducive to oligodendrogenesis in vivo, we injected MG(LPS), MG(IL-4) or MG(−) into the right lateral ventricle of the rat (SPD rats, 10-12 weeks old) brain and examined whether injected activated microglia have any effect on oligodendrogenesis. Control rats were injected with PBS instead of microglia. Twenty-four hours later rats were injected with BddU intraperitoneally every 12 hours for 3.5 days. After 28 days, the brains were excised and coronal sections taken from the hippocampus were analyzed. Newly formed oligodendrocytes were found mainly in the brain parenchyma adjacent to the injected ventricle (
We showed in Example 1 above that both neurogenesis and oligodendrogenesis of adult NPCs in vitro are blocked by inflammation-associated (endotoxin-activated) microglia but induced by microglia activated by cytokines (IFN-γ or IL-4) associated with Th cells. The same microglia were found, respectively, to be capable of blocking neuronal cell survival and of protecting neurons against degeneration. In the case of microglial activation by IFN-γ, however, the effect was beneficial only over a narrow range of concentrations, beyond which it turned destructive. The observation that inflammation in patients suffering from autoimmune diseases of the CNS is characterized by excessive quantities of Th1, and therefore of IFN-γ, prompted us to examine whether such situations might also be associated with impaired neurogenesis, and if so, whether there are ways to reverse them.
We examine here the effects on NPCs of microglia that were pre-incubated in their optimal growth media for 48 hours in the presence of a low dose (10 ng/ml) or a high dose (100 ng/ml) of IFN-γ. NPCs without microglia were tested as a control. After the growth media and cytokine residues were washed off, each of the treated microglial preparations was freshly co-cultured with dissociated NPC spheres on coverslips coated with Matrigel® in the presence of differentiation medium, with each of the treated microglial preparations as schematically depicted in
Addition of neutralizing anti-TNF-α antibodies (ãTNF-α) to MG(IFN-γ,100 ng) resulted in a significant increase in the number of β-III-tubulin-positive cells in their co-cultured NPCs (
We demonstrate hereinafter that IL-4-activated microglia exhibit neuroprotective features, at least in part via production of IGF-I. Because MG(IFN-γ,100 ng) decreased NPC survival (
The above results prompted us to examine whether MG(IL-4) are beneficial in vivo under pathological conditions of impeded neurogenesis associated with an excess of Th1 cells. The right lateral ventricles of healthy rat brains were injected with rat microglia (MG(−) or MG(IL-4)) (
To determine whether the MG(IL-4)-induced increase in neurogenesis occurs via an effect on proliferation and differentiation or on survival of the newly formed neurons, we repeated the above experiments and also examined the effect of MG(IFN-γ) (
We also examined the distribution of activated microglia (stained with activated microglial marker EDI) adjacent to the site of microglial injection in these brain sections. Analysis of brains in the first set of experiments (
Acute EAE is known to be associated with a transient increase in accumulation of encephalitogenic Th1 cells that recognize myelin antigens. We immunized 16 adult Lewis rats with MBP emulsified in CFA. The resulting transient hindlimb paralysis peaked on day 14 and then gradually resolved. Seven days after the immunization (1 day earlier than the reported average time of clinical onset of induced EAE), we subjected eight rats to bilateral stereotaxic injection of syngeneic MG(IL-4) into the cerebrospinal fluid (CSF) lateral ventricles. The other eight rats were similarly injected with PBS. On day 14 after immunization (7 days after MG(IL-4) injection) the rats were injected intraperitoneally (i.p.) with BrdU every 12 hours for 2.5 days to identify proliferating cells. Seven days after the first BrdU injection the dentate gyrus of each rat was examined immunohistochemically for evidence of newly formed neurons. The dentate gyrus of each rat was subjected to quantitative immunohistochemical analysis for evidence of newly formed neurons, which revealed an increase in proliferating (BrdU+) cells. Differentiation of these NPCs into neurons was indicated by their double labeling with DCX. The number of newly-formed neurons (BrdU+/DCX+ cells) in sections from the dentate gyrus was significantly higher in the MBP-immunized rats than in naïve rats, and was further increased by injection of MG(IL-4) (
To substantiate our suspicion that activated microglia would be found among the newly proliferating cells in the immunized rats, we stained the hippocampal sections for the microglial marker IB4 and for MHC-II (
We concluded that a transient local immune response induced by pro-inflammatory cytokines is capable of supporting neurogenesis.
The results of this study showed that whereas IFN-γ at a low concentration was able to endow rat microglia in vitro with a phenotype supportive of adult neurogenesis from stem-cell pools, a high concentration of this cytokine impeded neurogenesis. IL-4, ãTNF-α, and MG(IL-4) could all counteract the negative effect of high-dose MG(IFNγ) on neurogenesis. It thus became apparent that moderate amounts of IFN-γ activate microglia to create conditions that are more favorable for differentiation of adult NPCs to neurons than those created by IL-4, but that IL-4 or MG(IL-4) can counteract the negative side effect of an overabundance of IFN-γ, at least in vitro. In apparent correlation in vivo, acute EAE promoted adult neurogenesis in the rat hippocampal dentate gyrus. Moreover, in naïve rats or in rats with acute EAE an injection of MG(IL-4) into the CSF increased neurogenesis from stem-cell pools.
The role of activated microglia in demyelinating neurodegenerative diseases is controversial. We show that high levels but not low levels of interferon-γ (IFN-γ, a cytokine associated with inflammatory autoimmune diseases) confer on rodent microglia a phenotype that impedes oligodendrogenesis from adult neural stem/progenitor cells. Interleukin (IL)-4, reversed the impediment, attenuated TNF-α production, and overcame blockage of insulin like growth factor (IGF)-I production caused by IFN-γ. In rodents with acute or chronic experimental autoimmune encephalomyelitis, injection of IL-4-activated microglia into the cerebral spinal fluid resulted in improved clinical symptoms and increased oligodendrogenesis in the spinal cord, spatially associated with microglia expressing MHC-II and IGF-I. These results point to a novel role for microglia in oligodendrogenesis from the endogenous stem cell pool.
Here we examined whether IL-4, via modulation of microglia both in vitro and in vivo, can overcome the destructive effects of high-dose IFN-γ, known to be associated with EAE. In vitro, a high dose of IFN-γ, but not a low dose, impaired the ability of microglia to support oligodendrogenesis from adult neural stem cells/progenitor cells (NPCs). IL-4 counteracted the interference with oligodendrogenesis caused by high IFN-γ concentrations. When IL-4-activated microglia were stereotaxically injected through the cerebral ventricles into the cerebrospinal fluid (CSF) of rats with acute EAE or of mice with a remitting-relapsing autoimmune disease, the animals demonstrated significantly more oligodendrogenesis and significantly less neurological deficit than did their vehicle-injected diseased controls.
(xvii) Neural progenitor cell culture was prepared as described in section (i) in Materials and Methods above in Example 1.
(xviii) Primary microglial culture was prepared from brains from neonatal (P0-P1) C57B1/6J mice or Lewis rats as described in section (ii) in Materials and Methods above in Example 1.
(xix) Co-culturing of mouse neural progenitor cells and mouse microglia was carried out as described in section (iii) in Materials and Methods above in Example 1.
(xx) Induction and evaluation of acute and chronic EAE. To induce chronic EAE we injected adult male C57B1/6J mice s.c. with 200 μg (300 μl) of myelin oligodendrocyte glycoprotein peptide 35-55 (MOG)35-55 (Multiple Peptide System) in incomplete Freund's adjuvant containing 8 mg/ml Mycobacterium tuberculosis (strain H37Ra; Difco). Pertussis toxin (Sigma Aldrich; 500 ng) was injected on the day of the immunization and again 2 days later.
Induction of monophasic EAE in adult male Lewis rats was carried out as described in section (xi) of Example 2 above. Clinical signs were evaluated in a blinded fashion by at least two investigators. Body weight and clinical score were recorded daily (0=healthy; 1=limb tail paralysis; 2=ataxia and/or paresis of hindlimbs; 3=paralysis of hindlimbs and/or paresis of forelimbs; 4=tetraparalysis; 5=moribund state or death).
(xxi) Stereotaxic injection of activated microglia. Seven days after the vaccination, Lewis rats with monophasic EAE were injected bilaterally with syngeneic MG(IL-4) (10 ng/ml) or PBS stereotaxically (1×105 cells in 5 μl PBS for 5 min) into the CSF via the brain lateral ventricles (Bregma −0.8, L 1.2, V 4.5). Ten days after the vaccination, C57BL/6J mice with chronic EAE received bilateral stereotaxic injections of syngeneic MG(IL-4) (10 ng/ml) or PBS (1×105 cells in 3 μl PBS for 3 min) into the CSF via the brain lateral ventricles (Bregma −0.4, L 0.8, V 2.5).
(xxii) Administration of 5-bromo-2′-deoxyuridine and tissue preparation were carried out as described in section (xiii) of Example 2 above. BrdU was injected i.p. starting on day 14 after MBP vaccination in adult male Lewis rats or on day 20 after MOG vaccination in adult male C57BL/6J mice. One week (rats) or 2 weeks (mice) after the first BrdU injection, the animals were deeply anesthetized and perfused transcardially as described.
(xxiii) Immunocytochemistry and immunohistochemistry. For immunocytochemistry, co-cultures of NPCs and mouse microglia treated as described in section (iv) of Example 1 and stained with a combination of the rabbit anti-NG2 chondroitin sulfate proteoglycan (NG2; 1:500) and mouse anti-RIP (RIP; 1:2000). To capture the microglia we used FITC-conjugated Bandeiraea simplicifolia isolectin B4 (IB4; 1:50; Sigma-Aldrich). Expression of IGF-I was detected by goat anti-IGF-I (1:20; R&D Systems).
For immunohistochemistry, tissue sections were treated as described in section (xiv) of Example 2. After blocking the sections for 1 h with blocking solution, the tissue was then stained with rat anti-BrdU (1:200; Oxford Biotechnology) in combination with rabbit anti-NG2 (1:300) and mouse anti-RIP (1:1000) antibodies diluted in PBS containing 0.05% Triton X100, 0.1% Tween 20, and 2% horse serum. For labeling of microglia we used IB4 (1:50). To detect expression of cell-surface MHC-II proteins we used mouse anti MHC-II Abs (1:50; IQ Products). Expression of IGF-I was detected by goat anti IGF-I Abs (1:10-1:100; R&D Systems). Sections were incubated with the primary antibody for 24 h at 4° C., washed with PBS, and incubated with the secondary antibodies in PBS for 1 h at room temperature while protected from light. Secondary antibodies used for both immunocytochemistry and immunohistochemistry were Cy-3-conjugated donkey anti-mouse, Cy-3-conjugated goat anti-rabbit, Cy-5-conjugated goat anti-rat, Cy-2-conjugated goat anti-rat, and Cy-5-conjugated donkey anti-goat. Of Example 1 abovetibodies were purchased from Jackson ImmunoResearch Laboratories and used at a dilution of 1:250-500.
(xxiv) Real-time quantitative PCR analysis. Total cellular RNA purification and cDNA synthesis was performed as described in section (v) of Example 1 above. We assayed the expression of specific mRNAs using real-time quantitative PCR (Q-PCR) with selected gene-specific primer pairs. Q-PCR reactions were performed with a high-speed thermal cycler (LightCycler; Roche Diagnostics), and the product was detected by FastStart Master SYBR Green I (Roche Molecular Biochemicals) according to the manufacturer's instructions. The amplification cycle was 95° C. for 10 s, 60° C. for 5 s, and 72° C. for 10 s. The primers used were:
Melting curve analysis confirmed that only one product was amplified.
(xxv) Quantification and stereological counting procedure. For microscopic analysis we used a Zeiss LSM 510 confocal laser scanning microscope (40× magnification). For experiments in vitro we scanned fields of 0.053 mm2 (n 8-16 from at least two different coverslips) for each experimental group. For each marker, 500-1000 cells were sampled. Cells co-expressing GFP, NG2 and RIP were counted.
For in-vivo experiments with rats and mice with EAE, oligodendrogenesis and proliferation of microglia in the spinal cord were evaluated by counting of cells that were double- or triple-labeled with BrdU and markers of premature oligodendrocytes (NG2) and a pre-ensheathing marker of oligodendrocytes (RIP), microglia (IB4), or antigen-presenting cells (MHC-II) from sagittal longitudinal sections at segment T8-T9 of the spinal cord. The numbers of cells per mm3 were counted at 300-μm intervals in gray and white matter in each rat or mouse (n=8 per group). Specificity of BrdU+/NG2+ or BrdU+/RIP+ or BrdU+/IB4+ or BrdU+/IB4+/MHC-II+ co-expression was assayed using the confocal microscope (LSM 510) in optical sections at 1-μm intervals. Counting was evaluated automatically using Image-Pro Plus 4.5 software (Media Cybernetics).
(xxvi) Statistical analysis. The in-vitro results were analyzed by the Tukey-Kramer multiple comparisons test (ANOVA) and are expressed as means±SD. In-vivo results were analyzed by Student's t-test or one-way ANOVA and are expressed as means±SEM. Significance of the EAE score was analyzed by Mann-Whitney test, two-factor repeated measures (ANOVA).
In the present study we first examined whether microglia that encounter a high dose of IFN-γ adopt a phenotype that interferes with oligodendrogenesis from NPCs, and if so, whether IL-4 can counteract this negative effect.
We first examined the effects on NPCs of microglia that were pre-incubated for 24 h in their optimal growth media, in the presence or absence of the cytokines IL-4 (10 ng/ml), IFN-γ (10 ng/ml or 100 ng/ml), or IL-4 (10 ng/ml) together with IFN-γ (100 ng/ml). After the growth media and cytokine residues were washed off, each of the treated microglial preparations was freshly co-cultured with dissociated NPC spheres on coverslips coated with Matrigel in the presence of differentiation medium. We used NPCs expressing GFP to verify that any differentiation of oligodendrocytes seen in the culture was derived from the NPCs rather than from contamination of the primary microglial culture. After 10 days we could discern both GFP-expressing NPCs co-labeled with the oligodendrocyte-progenitor marker NG2 and RIP-stained mature oligodendrocytes at the pre-ensheathing stage (Hsieh et al., 2004) (
To examine the correlation between the production of IGF-I and the ability of IL-4-activated microglia to overcome the impediment to oligodendrogenesis, and to determine how this relationship is influenced by TNF-α production, we carried out quantitative real-time PCR analyses (Q-PCR) (
The finding that microglia activated by IL-4 in vitro created conditions favoring differentiation of NPCs into oligodendrocytes and overcame the negative effect of MG(IFN-γ,100 ng) on oligodendrogenesis encouraged us to investigate whether MG(IL-4) would promote oligodendrogenesis from endogenous adult NPCs in animals with acute or chronic EAE. To examine this possibility, we first induced acute EAE in adult Lewis rats (n=8) by immunizing them with MBP emulsified in CFA. Seven days later we introduced MG(IL-4) into the CSF of these rats via stereotaxic bilateral injection into their cerebral ventricles. Control rats (n=8) were similarly injected with PBS. Seven days after injection of MG(IL-4) or PBS, the rats were injected i.p. with BrdU every 12 h for 2.5 days to identify proliferating cells, and 21 days after the MBP vaccination their spinal cords were examined for the appearance of newly formed oligodendrocytes.
Although the symptoms of paralysis observed in this model of acute EAE do not result from demyelination, they were beneficially affected by IL-4. We therefore hypothesized that not only oligodendrogenesis but also functional integrity would benefit from IL-4-activated microglia, possibly in part through the IGF-I that these microglia are producing. Follow-up of the clinical manifestations of EAE with and without microglial treatment showed that after injection with MG(IL-4), the onset of disease was delayed and the severity and duration of clinical paralysis (
Because the clinical symptoms of EAE in this model are manifested by tail and hindlimb paralysis, we assessed oligodendrogenesis in the spinal cords of these rats. Spinal cords were excised and longitudinal sections from the T8-T9 region were analyzed for newly formed oligodendrocytes and microglia. In both the untreated and the MG(IL-4)-treated groups of rats with EAE we detected cells that were double-labeled for BrdU (proliferating cells) and NG2 (oligodendrocyte precursors). Some of the proliferating cells in both the gray and the white matter of MG(IL-4)-treated rats were BrdU+/NG2+/RIP+, indicating that they had differentiated into committed oligodendrocytes. In those rats there were significantly more BrdU/NG2+ cells in the gray matter, and significantly more BrdU+/RIP+ cells in the white matter, than in PBS-treated rats (
It should be noted that BrdU in these experiments was injected at the peak of the disease, a stage at which a spontaneous feedback mechanism that limits the proinflammatory response is likely to be operating, with consequent reduction in the numbers of Th1 cells and/or the appearance of Th2 cells. It is therefore possible that the oligodendrogenesis seen in the PBS-injected rats with EAE was an outcome triggered by the Th1 cells whose numbers had declined by the time we injected the BrdU or by the spontaneous increasing numbers of Th2 (Duda et al., 2000).
According to the traditional view, microglia that express MHC-II molecules are the activated microglia that are present in inflammation-associated diseases (Neumann et al., 1998). Recent studies by our group shown herein in the specification and by others showed, however, that not all MHC-II-expressing microglia are destructive (Li et al., 2005). For example, MHC-II-expressing microglia that are activated by low-dose IFN-γ or by IL-4 support cell survival. Analysis of consecutive sections obtained from the rats described above revealed the presence of newly formed microglia in both PBS-treated and MG(IL-4)-treated rats, with the highest accumulation seen in the gray matter of the MG(IL-4)-treated rats (
Because microglia can be induced to express IGF-I, and because the microglia in this study induced oligodendrogenesis in vitro (
The results in vitro suggested that when Th1 cells persist, meaning that IFN-γ concentrations are high for a relatively long time, the result will be impairment of oligodendrogenesis. To verify that this is indeed the case we used the mouse model of chronic EAE to compare mice treated with MG(IL-4) and with PBS. EAE was induced in C57BL/6J mice by immunization with the encephalitogenic myelin oligodendrocyte glycoprotein (MOG) peptide (35-55) emulsified in incomplete Freund's adjuvant containing Mycobacterium tuberculosis and pertussis toxin. Ten days after EAE induction we injected MG(IL-4) or PBS into the CSF, via bilateral stereotaxic injection into the cerebral ventricles. The two groups of injected mice differed significantly both in severity of paralysis (
With the aid of scanning confocal microscopy, co-expression of NG2 and RIP by the newly formed oligodendrocytes was confirmed in the white matter of the MGIL-4-treated mice with EAE (
The results of this study lead us to attribute a novel role for microglia as both supporters and blockers of oligodendrocyte renewal from the endogenous NPC pool in the adult CNS. The in-vitro findings showed that IL-4-activated microglia, in part via production of IGF-I and down-regulation of TNF-α, were remarkably potent in counteracting the impediment to oligodendrogenesis induced by high-dose IFN-γ. In vivo, MG(IL-4) supported oligodendrogenesis and clinical recovery in rats and mice in which severe inflammatory conditions are known to evoke clinical symptoms of transient or chronic EAE.
Recovery from spinal cord injury evidently necessitates a local immune response that is amenable to well-controlled boosting by immunization with T lymphocytes recognizing myelin-associated antigens at the injury site. The relevant T cells can activate local microglia to express a phenotype supportive of neuronal survival and renewal. We show that recovery of mice from spinal contusion is synergistically promoted by T-cell-based vaccination with a myelin-derived peptide and injection of adult neural stem/progenitor cells (aNPCs) into the cerebrospinal fluid. Significantly more aNPCs targeted the lesion site in vaccinated than in nonvaccinated mice. Synergistic interaction between aNPCs and T cells in vitro was critically dependent on T-cell specificity and phenotype. The results suggest that controlled immune activity underlies efficient regulation of the stem-cell niche, and that stem-cell therapy necessitates autologous or histocompatibility-matched donors instead of the immunosuppressive anti-rejection drugs that would eliminate any beneficial effect of immune cells on spinal cord repair.
(xxvii) Antigens. The following peptides were synthesized by the Synthesis Unit at the Weizmann Institute (Rehovot, Israel): MOG, residues 35-55 MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO:1) and an altered MOG peptide (45D) MEVGWYRSPFDRVVHLYRNGK (SEQ ID NO:2), a peptide analog of MOG 35-55 containing a serine to aspartic acid substitution at P6. OVA was purchased from Sigma.
(xxviii) Immunization. Adult mice were immunized with MOG, 45D, or OVA (all 100 μg), each emulsified in an equal volume of CFA (Difco, Detroit, Mich.) containing Mycobacterium tuberculosis (5 mg/ml; Difco), or IFA. The emulsion (total volume 0.15 ml) was injected s.c. at one site in the flank. Control mice were injected with PBS.
(xxix) Spinal cord injury. Mice were anesthetized, their spinal cords were exposed by laminectomy at T12, and a force of 200 kdyn was placed for 1 s on the laminectomized cord using the Infinite Horizon spinal cord impactor (Precision Systems and Instrumentation, Lexington, Ky.), a device shown to inflict a well-calibrated contusive injury of the spinal cord.
(xxx) Assessment of functional recovery from spinal cord contusion. Functional recovery from spinal cord contusion in mice was determined by hindlimb locomotor performance. Recovery was scored by the Basso Mouse Scale (BMS) open-field locomotor rating scale, a scale recently developed specifically for mice, with scores ranging from 0 (complete paralysis) to 9 (normal mobility) (Engesser-Cesar et al., 2005). Blind scoring ensured that observers were not aware of the treatment received by each mouse. Twice a week locomotor activities of mice in an open field were monitored by placing the mouse for 4 min in the center of a circular enclosure (90 cm in diameter, 7 cm wall height) made of molded plastic with a smooth, non-slip floor. Before each evaluation the mice were examined carefully for perineal infection, wounds in the hindlimbs, and tail and foot autophagia.
(xxxi) Stereotaxic injection of neural progenitor cells. Mice were anesthetized and placed in a stereotactic device. The skull was exposed and kept dry and clean. The bregma was identified and marked. The designated point of injection was at a depth of 2 mm from the brain surface, 0.4 mm behind the bregma in the anteroposterior axis, and 1.0 mm lateral to the midline. Neural progenitor cells were applied with a Hamilton syringe (5×105 cells in 3 μl, at a rate of 1 μl/min) and the skin over the wound was sutured.
(xxxii) Neural progenitor cell culture. Cultures of adult neural progenitor cells (aNPCs) were obtained as previously described.
(xxxiii) Co-culturing of neural progenitor cells and T cells. CD4+ T cells were purified from lymph nodes of 8-week-old C57B16/J mice as previously described (Kipnis et al., 2004). T cells were activated in RPMI medium supplemented with L-glutamine (2 mM), 2-mercaptoethanol (5×10−5 M), sodium pyruvate (1 mM), penicillin (100 IU/ml), streptomycin (100 μg/ml), nonessential amino acids (1 ml/100 ml), and autologous serum 2% (v/v) in the presence of mouse recombinant IL-2 (mrIL-2; 5 ng/ml) and soluble anti-CD28 and anti-CD3 antibodies (1 ng/ml). T cells were co-cultured (5×104 cells/well) with aNPCs (5×104 cells/well) for 5 d on cover slips coated with Matrigel (BD Biosciences) in 24-well plates. The cultures were then fixed with 2.5% paraformaldehyde in PBS for 30 min at room temperature and stained for neuronal markers.
(xxxiv) Immunohistochemistry. Mice subjected to SCI were re-anesthetized 14 or 60 days later and perfused with cold PBS. Their spinal cords were removed, postfixed with Bouin's fixative (75% saturated picric acid, 25% formaldehyde, 5% glacial acetic acid; Sigma-Aldrich) for 48 h, and then transferred to 70% EtOH. The tissues were hydrated through a gradient of 70%, 95%, and 100% EtOH in xylene and paraffin, and were then embedded in paraffin. For each stain, five tissue sections, each 6 μm thick, were taken from each mouse. The paraffin was removed by successive rinsing of slides for 15 min with each of the following: xylene, EtOH 100%, 95%, 70%, 50%, and PBS. Exposure of the slides to antigen was maximized by heating them to boiling point in 10 mM sodium citrate pH 6.0 in a microwave oven, then heating them at 20% microwave power for a further 10 min. The slides were blocked with 20% normal horse serum for 60 min prior to overnight incubation at room temperature (GFAP, neurofilaments, and BDNF), or for 48 h at 4° C. (CD3), with the monoclonal antibody in 2% horse serum. We used rabbit anti-mouse GFAP (1:200) (DakoCytomation, Glostrup, Denmark) for GFAP; rabbit anti-neurofilament (1:200), low and high molecular weight (Serotec, Oxford, UK) for neurofilaments; and rat anti-human CD3 (1:50) (Serotec) for CD3. For BDNF we used the monoclonal antibody chicken anti-human BDNF (1:100) (Promega, Madison, Wis.) with 0.05% saponin.
After rinsing, sections were incubated for 1 h at room temperature with the secondary antibody Cy3 donkey anti-rat (1:300) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) (staining for CD3), Cy3 donkey anti-chicken (Jackson ImmunoResearch) (1:250) (staining for BDNF), or Cy3 donkey anti-rabbit (Jackson ImmunoResearch) (1:250) (staining for GFAP and neurofilaments). For IB4 staining, sections were blocked for 1 h with 20% horse serum and then incubated for 1 h at room temperature with Cy2-IB4 (1:50) (Sigma-Aldrich). All sections were stained with Hoechst (1:2000) (Molecular Probes-Invitrogen, Carlsbad, Calif.). They were then prepared for examination under a Nicon E-600 fluorescence light microscope. Results were analyzed by counting the cells (CD3-labeled) in the site of injury, or by determination of the density (IB4-labeled or BDNF-labeled), or by measurement of the unstained area (GFAP, neurofilaments). Each of the parameters was measured by an observer who was blinded to the treatment received by the mice.
Our working hypothesis in this study was that the protective immune response evoked at a site of injury by T-cell based immunization creates a niche that supports not only cell survival but also tissue repair. We further suspected that a local T-cell mediated immune response could attract exogenously delivered aNPCs and support their contribution to recovery. To test this hypothesis we vaccinated C57B1/6J mice, immediately after SCI, with the encephalitogenic peptide pMOG 35-55 (SEQ ID NO: 1) emulsified in CFA containing 0.5% Mycobacterium tuberculosis (MOG/CFA), and 1 week later administered aNPCs via the intracerebroventricular (i.c.v.) route. Mice subjected to this dual treatment protocol (MOG/CFA/aNPC) were compared to a control group of mice that were immunized with the same MOG peptide emulsified in the same adjuvant but were not transplanted with aNPCs and instead were injected i.c.v. with PBS (MOG/CFA/PBS), or to mice that were injected with PBS and CFA (0.5%) and transplanted i.c.v. with aNPCs (PBS/CFA/aNPC) or a control group of mice that were injected with PBS/CFA and then injected i.c.v. with PBS (PBS/CFA/PBS). To assess behavioral outcome after SCI we used the Basso motor score (BMS) rating scale (Engesser-Cesar et al., 2005), in which 0 indicates complete paralysis of the hindlimbs and 9 denotes full mobility. The mean motor recovery (BMS) scores of mice receiving the MOG/CFA/aNPC (4.21±0.45; all values are mean±SEM) were higher than those of mice treated with MOG/CFA/PBS. In mice treated with PBS/CFA/aNPC, recovery was not better than in control mice treated with PBS/CFA/PBS (1.5±0.27). A BMS of 4.21 indicates extensive movement of the ankle and plantar placement of the paw (three animals showed, in addition, occasional weight support and plantar steps), whereas a score of 1.5 indicates ankle movement ranging from slight to extensive. Mice treated with MOG/CFA/PBS scored 2.71±0.5, a, significantly higher score than that of control PBS/CFA/aNPC-treated mice (1.5±0.4) or of control mice treated with PBS/CFA/PBS (
We have previously demonstrated that boosting of the amounts of T cells needed for promoting recovery from SCI does not necessitate the use of encephalitogenic peptides; weak agonists of encephalitogenic peptides are just as effective and do not carry the risk of inducing EAE (Hauben et al., 2000, 2001). To test whether such ‘safe’ vaccination could be utilized in combination with aNPCs transplantation we used a MOG-derived altered peptide ligand (PMOG 35-55 APL; 45D peptide, SEQ ID NO: 2), in which aspartic acid is substituted for serine. Mice were vaccinated with the 45D peptide emulsified in CFA containing 2.5% Mycobacterium tuberculosis. One week later the immunized mice were subjected to contusive SCI, and after another week were transplanted i.c.v. with aNPCs. Increased motor activity (as expressed by the BMS, mean±SEM) was seen in these mice than in control mice treated i.c.v with PBS/CFA/aNPC (4.11±0.27 compared to 1.94±0.22;
To determine the phenotype and specificity of the T cells needed for synergistic interaction with aNPCs we repeated the above experiments using different immunization protocols. Incomplete Freund's adjuvant (IFA), unlike CFA, is free of bacteria and is known to elicit a Th2-like response to encephalitogenic peptides. We found that although immunization with the MOG analog (peptide 45D) emulsified in IFA had some beneficial effect, it showed no synergy with subsequent transplantation of aNPCs (BMS of 3.2±0.76 for MOG/IFA/aNPC-treated mice compared to 2.93±1.03 for MOG/IFA/PBS-treated mice and 2.07±0.53 in the PBS/CFA/PBS-treated mice;
To verify that specificity to CNS-antigens is required for a synergistic effect of vaccination and stem-cell transplantation, mice were immunized with the nonself protein ovalbumin (OVA) emulsified in CFA (containing 2.5% Mycobacterium tuberculosis), and 1 week later were injected i.c.v. with aNPCs or with PBS as control. Immunization with OVA/CFA resulted in a slight, nonsignificant increase in BMS, and implantation of aNPCs did not increase the BMS any further (2.43±1.78 for the OVA/CFA/aNPC-treated mice compared to 2.2±0.68 for OVA/CFA/PBS-treated mice and 1.5±0.29 for PBS/CFA/PBS-treated control group,
Immune activation in the injured CNS, and specifically in the spinal cord, has been a major focus of research attention in recent years (Schwartz and Hauben, 2002). In some of the studies, T cell-based immune responses were shown to be protective only if their intensity and duration were well regulated. Overly strong immunization yielded excessive immune activity, which neutralized the potential benefit of the immune response for the injured spinal cord and even had a detrimental effect. We considered the possibility that if aNPCs home to the site of damage they can offset the negative effect of excessive immune activity and thus contribute to recovery. We therefore set out to determine whether administration of aNPCs can contribute to functional recovery even when the local immune activity is excessive. One week prior to SCI we immunized mice with MOG peptide 35-55 emulsified in CFA containing 2.5% Mycobacterium tuberculosis. Under conditions in which motor recovery from SCI was worse after immunization with MOG/CFA than after injection with PBS/CFA (BMS of 0.35±0.2 and 1.94±0.32, respectively), we found that recovery was improved upon administration of aNPCs (BMS of 2.68±0.51;
In an attempt to gain an insight into the mechanism underlying the apparent synergy between aNPCs and resident immune cells, we examined whether any of the injected aNPCs find their way to the injured spinal cord. We repeated the experiment showed on
One of the morphological features that characterize recovery from SCI is the size of the lesion. To delineate the site of injury we stained longitudinal sections of the spinal cord with antibodies to glial fibrillary acidic protein (GFAP) (Blaugrund et al., 1992). We assessed the lesion size by measuring the areas that were not stained by GFAP. This analysis disclosed that as early as 7 days after aNPCs were transplanted in the MOG/CFA-vaccinated rats, the averaged size of the site of injury was significantly smaller in mice that had received both vaccination and aNPC transplantation than in mice that had only been vaccinated or had received only aNPCs (
Next we examined whether the observed differences in the extent of recovery could be correlated with local immunological changes. Sections of spinal cord tissue were stained for markers of T cells (CD3) and accumulation of activated microglia/macrophages (IB4) (
Both activated T cells and T cell-activated microglia can serve as sources of growth factors such as brain-derived neurotrophic factor (BDNF). BDNF immunoreactivity was more intense in the spinal cords of mice treated with MOG/CFA/aNPC than in the other groups (
Recent studies have shown that noggin, a bone morphogenesis protein (BMP) inhibitor, can induce neuronal differentiation from aNPCs in the injured spinal cord (Setoguchi et al., 2004). This protein was also shown to be needed to provide a neurogenic environment in the subventricular zone. We therefore assayed noggin immunoreactivity in the various experimental groups, and found that it was significantly increased in mice that received the dual treatment protocol, but was unaffected by MOG immunization alone and was slightly decreased by aNPC transplantation alone (
The above results raised an important question: can a T cell-based vaccination, when given in combination with aNPC transplantation, create conditions favorable for neuronal differentiation of endogenous or exogenous aNPCs?. To examine this possibility, we repeated the experiment described in
The above results indicated that cross-talk between immune cells and aNPCs was taking place at the site of injury. We showed hereinabove that microglia pre-activated with the Th1- and Th2-associated cytokines, IFN-γ and IL-4, respectively, can induce neuronal differentiation from aNPCs. We therefore sought to determine whether direct interaction between aNPCs and T cells would also result in an altered pattern of aNPC differentiation. To address this question, we activated CD4+ T cells in vitro by a cognate protocol (with anti-CD3 antibodies, anti-CD28 antibodies, and IL-2) for 24 h and then allowed their activation to continue in co-cultures with aNPCs in a transwell culture system. As controls we used cultures of aNPCs alone (in the presence of anti-CD3 antibodies, anti-CD28 antibodies and IL-2) or aNPCs cultured with CD4+ T cells in a resting state (supplemented with IL-2 only). After 5 days in culture the aNPCs in the lower chamber were fixed and analyzed for the appearance of newly formed neurons. Staining for the early neuronal marker β-III-tubulin revealed a dramatic effect of T cells on neuronal differentiation (
To exclude the possibility that the T cells had affected NPC differentiation as a consequence of encountering NPC-derived compounds, and to further substantiate our finding that the observed effect was caused by T cell-derived soluble substances, we allowed aNPCs to differentiate in the presence of medium conditioned by activated T cells. After 5 days, staining of these cultures for β-III tubulin revealed similar results to those obtained in the co-culture system (
We next sought to determine whether the T cell-induced neurogenesis was mediated by cytokines secreted by activated T cells. aNPCs were cultured in the presence of different concentrations of the characteristic T-cell derived cytokines IFN-γ and IL-4. Analysis revealed that IFN-γ, at concentrations as low as 1 ng/ml, could induce an increase in β-III tubulin expression after 5 days in culture (
These findings suggested that IFN-γ, unlike IL-4, can account in part for the T cell-induced neurogenesis. Even the effect of IFN-γ, however, was limited relative to that of the T cells or to the T cell-derived soluble factors. PCR analysis of expression of the IFN-γ receptor-1 on aNPCs disclosed that this receptor is expressed by aNPCs under all of the conditions examined here (data not shown).
Activation of the Notch pathway is essential for maintenance of aNPCs, and blockage of this pathway and its downstream transcription factors of the Hes gene family underlie the first events in neuronal differentiation. To determine whether T cell-mediated neuronal differentiation induces changes in Notch signaling, we looked for possible changes in expression of Hes genes in aNPCs following their interaction with T cell-derived substances. Real-time PCR disclosed that relative to control cultures, aNPCs cultured for 24 h in the presence of T cell-conditioned medium underwent a five-fold decrease in Hes-5 expression (
Our observation that aNPCs express an IFN-γ receptor, taken together with recent studies showing that these cells express immune-related molecules such as B-7 (Imotola et al., 2004) and CD44 (Pluchino et al., 2003), known to participate in the dialog between T cells and antigen-presenting cells (APCs), prompted us to examine whether aNPCs could affect T-cell function. First we examined the effects of aNPCs on proliferation of CD4+ T cells by assaying [3H]thymidine incorporation by the T cells. Co-culturing of T cells with aNPCs and APCs (lethally irradiated splenocytes) for 3 days resulted in a significant dose-dependent inhibition of T-cell proliferation (
Local interaction between immune cells and aNPCs underlies functional recovery. In the present study we combined two different therapeutic approaches for SCI: T cell-based vaccination and transplantation of neural progenitor cells into the CSF. Each of these approaches has been shown to be potentially capable of promoting functional recovery from SCI; we show here that when combined, they operate in synergy. Our experiments, both in vivo and in vitro, demonstrated that cross-talk between immune cells and aNPCs can take place at the lesion site. The vaccination elicits a local immune response which, if well controlled, provides the cellular and molecular elements needed to attenuate degeneration and promote repair. The same response also plays a role in recruiting aNPCs to the injured site and creating niche-like compartments that support neurogenesis from endogenous aNPCs. The interaction between aNPCs and immune cells was found to be reciprocal: aNPCs could modulate the postinjury immune activity, ensuring functional recovery even under conditions of excessive immune activity (which, in the absence of aNPCs, have a detrimental effect on recovery).
Neurogenesis is known to take place in the adult brain. This work identifies T lymphocytes and microglia as pivotal players in the maintenance of hippocampal neurogenesis and spatial learning abilities in adulthood. Hippocampal neurogenesis was dramatically impaired in three different types of T cell-deficient mice, but could be restored and even boosted by T cells recognizing a specific central nervous system (CNS) antigen. Environmental enrichment did not evoke enhanced neurogenesis in immune-deficient animals, whereas in wild-type animals it led to enhanced hippocampal neurogenesis coupled with recruitment of T cells and activation of microglia. CNS-specific T cells were also found to be required for spatial learning/memory and for expression of brain-derived neurotrophic factor in the dentate gyrus, implying that a common immune-associated mechanism underlies different aspects of hippocampal plasticity and cell renewal in the adult brain.
We show here that adult mice (4-5-month-old) with severe immune deficiency experienced loss of long-term potentiation and impairment of spatial learning/memory and hippocampal neurogenesis. These functions were restored by myelin-associated autoimmune T cells. The results might shed light on age-related cognitive loss and hint at a novel approach to the maintenance of brain plasticity in adulthood.
(xxxv) Animals. Adult male Sprague-Dawley (SPD) rats (12 weeks old) (n=6) were placed together in an enriched environment complex consisted of running wheels, tunnels, nesting material and rubber balls. Handling was limited to once a week when bedding was exchanged. Control rats were housed, three per standard cage (45×22×18 cm), under controlled conditions (23±2° C., lights on 06.00-18.00 h).
Inbred adult male C57B1/6J mice and Balb/c/OLA wildtype and SCID mice, TMBP-transgenic mice, TOVA-transgenic mice, RAG−/− mice, and TMBP-RAG−/− transgenic mice, all aged 4-5 months, and B10.PL TMBP-transgenic mice, and Balb/c/OLA nude and TOVA-transgenic mice, all aged 8-16 weeks, were supplied by the Animal Breeding Center of the Weizmann Institute of Science (Rehovot, Israel). The B10.PL wild-type mice were purchased from The Jackson Laboratory (Bar Harbor, Me.). Mice were housed in an enriched environment complex (6 mice per cage) similar to that of the rats or in standard housing (6 mice per cage).
Both rats and mice were matched for age in each experiment and their cages were placed in a light- and temperature-controlled room.
Inbred adult male C57B1/6J mice and Balb/c/OLA wildtype and SCID mice, TMBP-transgenic mice, TOVA-transgenic mice, RAG−/− mice, and TMBP-RAG−/− transgenic mice, all aged 4-5 months, and B10.PL TMBP-transgenic mice, and Balb/c/OLA nude and TOVA-transgenic mice, all aged 8-16 weeks, were supplied by the Animal Breeding Center of the Weizmann Institute of Science (Rehovot, Israel). The B10.PL wild-type mice were purchased from The Jackson Laboratory (Bar Harbor, Me.). Mice were housed in an enriched environment complex (6 mice per cage) similar to that of the rats or in standard housing (6 mice per cage).
(xxxvi) Administration of 5-bromo-2′-deoxyuridine and tissue preparation. Adult SPD rats were injected i.p. (50 mg/kg body weight), once daily for 5 days, with BrdU Sigma-Aldrich, St. Louis, Mo.). Rats housed under either standard or environmentally enriched conditions began receiving BrdU after being kept for 6 weeks in their respective environments, and continued to live in those environments for 1 week after the last injection. They were then deeply anesthetized and perfused transcardially, first with PBS and then with 4% paraformaldehyde. Their brains were removed, postfixed overnight, and equilibrated in phosphate-buffered 30% sucrose. Free-floating 40-m-thick coronal hippocampal sections were collected on a freezing microtome (Leica SM2000R) and stored at 4° C. prior to immunohistochemistry. Adult mice housed in an enriched environment were injected with BrdU according to the protocol used for the rats.
Under standard conditions, after injection of BrdU in mice (i.p., 50 mg/kg body weight) at 12-h intervals for 2 or 2.5 days (a total of 4 or 5 injections, respectively), mice were deeply anesthetized and euthanized by transcardial perfusion with PBS followed by 2.5% paraformaldehyde. Mice that received four injections were killed 48 h or 7 days after the first injection, whereas mice injected five times were killed 7 or 28 days after the first injection (for experiments presented in
(xxxvii) Immunohistochemistry. Specimens obtained from rats were treated with a permeabilization/blocking solution containing 10% fetal calf serum, 2% bovine serum albumin, 1% glycine, and 0.05% Triton X-100 (Sigma-Aldrich). Primary antibodies were applied for 1 h in a humidified chamber at room temperature. For labeling of microglia we used FITC-conjugated Bandeiraea simplicifolia isolectin B4 (IB-4, 1:50; Sigma-Aldrich). To detect expression of cell-surface MHC-II proteins we used anti-MHC-II Abs (mouse, 1:50; IQ Products, Groningen, The Netherlands). T cells were detected with anti-TCR (α:/β) Abs (mouse, 1:20; Acris Antibodies, Hiddenhausen, Gmbh). Expression of IGF-I was detected by anti-IGF-I Abs (goat, 1:20; R&D Systems, Minneapolis, Minn.).
For BrdU staining, sections were washed with PBS and incubated in 2N HCl at 37° C. for 30 min. Sections were blocked for 1 h with blocking solution. The tissue was then stained with the antibodies anti-BrdU (rat, 1:200; Oxford Biotechnology, UK), anti-NeuN (mouse, 1:200; Chemicon, Temecula, Calif.), and a combination of propidium iodide (5 μg/ml) and anti-MHC-II or FITC-IB-4 diluted in PBS containing 0.05% Triton X100, 0.1% Tween 20, and 2% horse serum. Sections were incubated with the primary antibody for 24 h at 4° C., washed with PBS, and then incubated with the secondary antibodies in PBS for 1 h at room temperature while protected from light.
For BrdU staining of mouse specimens tissue sections were washed with PBS, incubated in 2N HCl at 37° C. for 30 min, and then blocked for 1 h with blocking solution (PBS containing 20% normal horse serum and 0.5% Triton X-100, or PBS containing mouse immunoglobulin blocking reagent obtained from Vector Laboratories, Burlingame, Calif.). The tissue sections were stained overnight with specified combinations of the following primary antibodies: rat anti-BrdU (1:200; Oxford Biotechnology, Kidlington, Oxfordshire, UK), goat anti-DCX (1:400; Santa Cruz Biotechnology, Santa Cruz, Calif.), mouse anti-NeuN (1:200; Chemicon, Temecula, Calif.) and rabbit anti GFAP (1:200 DAKO). Secondary antibodies used for both mouse and rat tissues were FITC-conjugated donkey anti-goat, Cy-3-conjugated donkey anti-mouse, and Cy-3- or Cy-5-conjugated donkey anti-rat (1:200; Jackson ImmunoResearch, West Grove, Pa.). For BDNF staining, tissue sections were washed with PBS and blocked for 1 h with blocking solution (PBS containing 20% normal horse serum and 0.05% saponin). They were then stained overnight at room temperature with a primary antibody solution consisting of chicken anti-BDNF (1:100; Promega, Madison, Wis.) in PBS solution containing 2% normal horse serum and 0.05% saponin. The secondary antibody was Cy-3-conjugated donkey anti-chicken (1:250; Jackson ImmunoResearch).
(xxxviii) Transfer of splenocytes to immune-deficient mice. Mice were killed and their spleens were harvested and mashed. Red blood cells were removed with a lysing buffer. The splenocyte population was depleted of T cells by the use of anti CD90 (Thy-1) microbeads (Mylteni Biotech, Bergisch Gladbach, Germany) in an autoMACS cell sorter. FACS analysis using PE-conjugated anti-CD3 antibodies (PharMingen, Becton-Dickinson, Franklin Lakes, N.J.) confirmed that fewer than 10% of the depleted splenocytes were T cells. SCID or nude mice were injected i.v. with 6×107 splenocytes (nondepleted) or 2×107 splenocytes that were depleted of T cells.
(xxxix) Minocycline administration. Minocycline (Sigma-Aldrich, St. Louis, Mo.) was dissolved in PBS and injected i.p into TMBP-transgenic mice once a day for 14 days. The dosages injected were 50 mg/kg body weight during the first 7 days and 25 mg/kg during the last 7 days. As controls we used TMBP-transgenic mice injected with corresponding volumes of PBS.
(xl) Rotarod test for basal motor ability. Mice were tested for their ability to balance on a rotating rod 7 mm in diameter (Hamilton-Kinder, Poway, Calif.). After a 1-min adaptation period the rod was accelerated by 2 rpm every 30 s, and the time that the mice remained on the rod (fall latency) was recorded. Each mouse was tested five times with a 25-min interval between tests.
(xli) Morris water maze (MWM) behavioral test. Spatial learning/memory was assessed by performance on a hippocampus-dependent visuo-spatial learning task in the MWM. Mice were given four trials per day on 4 consecutive days. In each trial they were required to find a hidden platform located 1.5 cm below the water surface in a pool 1.4 m in diameter. Within the testing room, only distal visuo-spatial cues for location of the submerged platform were available. The escape latency, i.e., the time required by the mouse to find the platform and climb onto it, was recorded for up to 60 s. Each mouse was allowed to remain on the platform for 30 s and was then moved from the maze to its home cage. If the mouse did not find the platform within 60 s, it was placed manually on the platform and returned to its home cage after 30 s. The interval between trials was 300 s. On day 5 the platform was removed from the pool and each mouse was tested by a probe trial for 60 s. On days 6-7 the platform was placed at the quadrant opposite the location on days 1-4, and the mice were then retrained in four sessions per day. Data were recorded using an EthoVision automated tracking system (Noldus).
(xlii) Quantification. For microscopic analysis we used a Zeiss LSM 510 confocal laser scanning microscope (40× magnification) or Nikon E800. Proliferation was assessed by bilateral counting of BrdU+ cells in the subgranular zone (SGZ) of the dentate gyrus (defined as a zone of the hilus, the width of two cell bodies, along the base of the granular layer). Neurogenesis in the dentate gyrus was evaluated by counting of cells that were double-labeled with BrdU and DCX or with BrdU and NeuN. Microglial numbers were obtained by counting of cells that were double-labeled with BrdU and markers of microglia (IB-4) or antigen-presenting cells (MHC-II). We counted the number of labeled cells in nine coronal sections (330 μm apart) per rat brain (6 rats per group) and six coronal sections (370 μm apart) per mouse brain (3-8 mouse brains per group) that were stained and mounted on coded slides. To obtain an estimate of the total number of labeled cells per dentate gyrus, the total number of cells counted in the selected coronal sections from each brain was multiplied by the volume index (the ratio between the volume of the dentate gyrus and the total combined volume of the selected sections).
For quantification of proliferation in the SVZ we used the protocol previously described (Brown et al., 2003); four coronal sections per brain (145 μm apart; 4-5 mouse brains per group), spanning from 0.38 mm anterior to bregma to 0.34 mm posterior to bregma, were stained with BrdU and mounted on coded slides. BrdU+ cells were counted manually in the lateral walls of both lateral ventricles Values obtained from this count were multiplied by the volume index to obtain an estimate of the total number of proliferating cells per lateral ventricle wall BDM immunoreactivity was quantified with Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, Md.) by measuring the intensity per unit surface area at the granule cell layer of the dentate gyrus. At least three hippocampal sections per mouse were used for this analysis.
(xliii) Statistical analysis. A two-tailed unpaired Student's t-test was used for analyses of the experiments presented in
(xliv) Measurements of hippocampal activity and plasticity in vitro. Mice from each group were decapitated and their brains were placed in ice-cold artificial cerebrospinal fluid (ACSF) containing 124 mM NaCl, 2 mM KCl, 1.24 mM KH2PO4, 2 mM MgSO4, 2.5 mM CaCl2, 26 mM NaHCO3, and mM 10 glucose. pH 7.4. After removal of the hippocampi, 350-μm hippocampal slices were obtained with a McIlwain tissue slicer (Mickle Laboratory Engineering. Guildford, Surrey, UK) and incubated for 1.5 h at room temperature in ACSF saturated with 95% O2 and 5% CO2 gas mixture. The slices were then submerged in a perfusion chamber, heated to 30-32° C., and superfused with ACSF at a rate of 2 ml/min. A bipolar stimulating electrode (25-μm nichrome wires) was placed in the stratum radiatum of the CA1 region. Test stimuli (100-μs pulse duration) were delivered at 30-s intervals, with the intensity adjusted such that the evoked responses were approximately 50% of the maximal response. An extracellular recording electrode containing 0.75 M NaCl (2-4 MΩ) was placed in the stratum radiatum of the CA1 region for recording of excitatory postsynaptic potential (EPSP). LTP vas induced by theta-burst stimulation (TBS) of the Schaffer collaterals (10 trains of 4 pulses at 100 Hz, separated by 200-ms inter-train intervals, at the same intensity as the test stimulation). Stimulation and data acquisition were performed using the LTP software (Anderson and Collingridge, 2001).
Our working hypothesis was that both CNS resident and systemic immune cells are active participants in adult hippocampal neurogenesis. With the object all examining whether this is indeed the case, we looked for a model in which neurogenesis is augmented physiologically without any intervention (Kemperman et al., 1997). Mental and physical activities have been shown to promote neurogenesis in adulthood by increasing survival and proliferation, respectively, of newly formed neurons in the dentate gyrus (van Praag et al., 1999). We anticipated that if T cells and activated resident microglia participate in neurogenesis, they would reach detectable levels under enriched conditions.
Three-month-old Sprague-Dawley rats were housed in standard capes (control; three rats per cage) or in an enriched environment (six rats per cage) that provided favorable conditions with regard to space, social interaction, sensory stimuli, and opportunities for physical activity. After 6 weeks each rat received a 5 day course of daily i.p. injections of BrdU to enable detection of newly formed cells in the dentate gyrus. One week later the rats were euthanized and the hippocampal areas of their brains were examined with antibodies against BrdU, the neuronal marker NeuN, and the microglial marker IB-4. As expected, significantly more newly formed neurons (BrdU+/NeuN+) were found in the dentate gyri of rats housed in the enriched environment than in control rats (
Detection of T cells in the hippocampus of adult rats under conditions of activity-induced enhanced neurogenesis raised the question of whether T cells area needed for neurogenesis under normal conditions. To address this question, we first compared neurogenesis in the hippocampal dentate gyri of wild-type adult C57B1/6J mice and mice with the same genetic background but suffering from deficiency in adaptive immunity and thus lacking both T- and B-cell populations (severe combined immune deficiency; SCID). We examined three phases of neurogenesis: cell proliferation, early neuronal differentiation, and long-term survival of newly-formed neurons. For comparison of the numbers of proliferating cells in the SGZ of the dentate gyrus in the two groups, all mice received four BrdU injections (ever, 12 h, i.p.). The mice were euthanized 2 days after the first injection (i.e., 12 h after the last injection), their brains were excised, and sections of the hippocampus were examined immunohistochemically. Significantly fewer BrdU+ cells in the SGZ of the dentate gyrus were observed in the SCID mice than in the wild-type controls (36.4±2.2% fewer,
Next we examined whether deficiency of adaptive immunity also affects the differentiation of newly proliferating cells into neurons. SCID and wild-type mice received five BrdU injections (every 12 h, i.p.) and were euthanized 7 days or 28 days after the first injection. On day 7 their dentate gyri were analyzed immunohistochemically for the presence of proliferating cells (BrdU+) expressing the early neuronal differentiation marker doublecortin (DCX+). Significantly fewer BrdU+/DCX+ cells were found in the dentate gyri of SCID mice than in those of the wild type (65.9±8.1% fewer,
Since most of the new neurons undergo apoptosis within 3-4 weeks of their formation, we examined whether the deficiency in adaptive immunity would also be reflected in the number of surviving new neurons at that time. On day 28, significantly fewer cells double-labeled for BrdU and NeuN were seen in the SCID mice than in the wild type (39.9±7.2% fewer,
The SCID mutation is also characterized by defective DNA-dependent protein kinase activity, with resulting impairment of DNA repair. To exclude the possibility that the impairment of neurogenesis observed here in the SCID mice was caused by this non-immunological defect, and to verify that the impaired neurogenesis in these mice could be attributed specifically to a deficiency of T cells, we compared neurogenesis in SCID mice replenished by intravenous (i.v.) injection of splenocytes from wild-type matched controls (‘normal splenocytes’) to that in SCID mice replenished with splenocytes depleted of T cells. Seventeen days after splenocyte replenishment, the mice were injected i.p. with BrdU (twice daily for 2 days) and 7 days later their hippocampi were analyzed. The T-cell content of each replenished mouse on the day of hippocampal excision was verified by FACS analysis (not shown).
Other than in the dentate gyrus, continuous neurogenesis from neural stem/progenitor cells in the adult CNS occurs in the subventricular zone (SVZ) of the lateral ventricles. We suspected that if immune deficiency causes impairment of progenitor cell proliferation in the dentate gyrus, it would have a similar effect in the SVZ. Quantification of BrdU+ cells in the SVZ on day 2 after BrdU injection disclosed significantly fewer in the SCID mice than in the wild type (60.6±4.8% fewer;
The observed correlation between activated immune cells and enhanced neurogenesis in the dentate gyri of rats kept in an enriched environment (
To further substantiate our contention that the peripheral immune cells participating in adult neurogenesis are T cells, we compared the formation of new neurons in wild-type Balb/c/OLA mice to that in strain-matched nude mice, which are deprived of their mature T-cell population but not of their B cells. As with SCID mice, 7 days after BrdU injection significantly fewer cells double-labeled with BrdU and DCX cells were seen in the dentate gyrus of the nude mice than of the wild type (68±8% fewer;
To further support our contention that the impaired neurogenesis in T cell-deficient nude mice is attributable to the absence of T cells, we transferred splenocytes (containing a normal population of mature T cells) from wild-type mice to syngeneic nude mice and, as in the SCID mice, analyzed the hippocampi 17 days after splenocyte injection. However, preliminary data had suggested that the ability of transferred splenocytes to restore neurogenesis depends on the time lapse after their injection. Therefore, with the object of analyzing the results in the same splenocyte-recipient mice at an additional time point, we used two markers of cell proliferation (BrdU and PCNA—an endogenous marker of proliferating cells). We injected BrdU 10 days after splenocyte injection, and then analyzed the hippocampi (excised on day 17) for both BrdU (which detected proliferation which took place on day 10 after splenocyte injection), and for PCNA (which detected the proliferating cells at the day of excision, day 17).
Using anti-CD3 antibodies, we were able to detect T cells in the brain parenchyma of both wild-type and splenocyte-replenished nude mice. The location of the T cells was not restricted, however, to areas of active adult neurogenesis, T cells could also be seen in the brain parenchyma, mainly around the walls of the ventricles adjacent to the hippocampus.
Previous findings from our group have shown that antigenic specificity to CNS autoantigens, such as certain peptides of MBP, is required for expression of the neuroprotective effect of T cells under traumatic or degenerative conditions (Moalem et al., 1999; Yoles et al., 2001). To determine whether the same specificity rule is also applicable to the T-cell effect on adult neurogenesis, we used transgenic mice possessing a normal B-cell population and a genetically engineered excess of monospecific T cells directed either to MBP (TMBP) or to an irrelevant (nonself) antigen. Mice in the former group express a transgene encoding a T-cell receptor that recognizes an epitope (Ac1-11) of MBP, and therefore approximately 98% of the T-cell pool in these ‘TMBP-transgenic’ mice consists of Ac1-11 TMBP cells. The remaining 2% are endogenous T cells whose regulatory activity is apparently sufficient to prevent the TMBP transgenic mice from spontaneously developing autoimmune encephalomyelitis. As a control for the antigenic specificity we used transgenic mice that possess a normal B-cell population but express a T-cell receptor that recognizes ovalbumin (‘TOVA-transgenic’ mice), and thus bears mainly TOVA cells. We expected that if neurogenesis requires the presence of CNS-specific T cells, such as TMBP cells, the TOVA-transgenic mice would behave like SCID and nude mice.
Because the two types of transgenic mice had different genetic backgrounds, we compared each of them to their matched wild-type controls. Examination of hippocampal dentate gyri from TMBP-transgenic mouse brains that were excised 7 days after the first BrdU injection and then double-stained with BrdU and DCX revealed significantly more BrdU+/DCX+ cells than in their wild-type counterparts (
To verify that the effect of CNS-specific T cells on neurogenesis is exerted via their activation of microglia, we used the antibiotic drug minocycline, which blocks microglial activity in the CNS under pathological inflammatory conditions (Ekdahl et al., 2003). The experiment was carried out with TMBP-transgenic mice because of the likelihood that their higher incidence of CNS-specific T cells and their enhanced neurogenesis relative to the wild type would make it easier to detect any potential effect of the drug. Treatment of the TMBP-transgenic mice with a daily dose of minocycline resulted in a significant decrease in the number of BrdU+/DCX+ cells in the dentate gyrus (
Recent findings point to an association between adult neurogenesis and certain hippocampal activities (Gould et al., 1999), although an association between adult neurogenesis and performance of the hippocampus-dependent task of spatial learning/memory in the MWM is still a matter of debate (Snyder et al., 2005). A study by our group showed that spatial learning/memory, as assessed by MWM performance, is impaired in immune-deficient mice (Kipnis et al., 2004). Those findings, together with the present finding that hippocampal neurogenesis is significantly impaired in mice deficient in CNS-specific T cells, prompted us to examine whether the hippocampus-dependent activity of spatial learning/memory is also dependent on the presence of autoimmune T cells. We therefore compared the MWM performance of TMBP mice and of TOVA mice relative to their respective matched wild-type controls. To ensure that any observed differences in performance in the MWM could be attributed to cognitive activity rather than to motor ability, we first subjected all the mice to a rotarod test. Recordings of the time that each mouse spent balancing on the rotating rod before falling disclosed no significant differences among all tested groups in fall latencies (mean±s.e.m in seconds: TMBP, 39±2.3; B10.PL wild type, 37.6±1.1; TOVA, 40.7±1.9; Balb/c wild type, 40.7±4.2).
When tested in the MWM, the TMBP transgenic mice performed better than their controls in all three phases of the task (
Measurement of the length of the path taken by each mouse to reach the hidden platform showed that, relative to their respective wild-type controls, the path taken by the TMBP mice was shorter (
T cells can provide neurotrophic factors, such as BDNF, and can regulate, via their secreted cytokines, the production of growth factors (e.g. IGF-I) by other CNS-resident cells such as microglia. In the absence of external stimuli (for example, in an enriched environment), MHC-II- or IGF-I-expressing microglia are hardly detectable in the dentate gyrus. We therefore postulated that additional factors might play a role in the constitutive T cell-dependent neurogenesis. BDNF is an essential component of many hippocampal activities, including both spatial learning/memory and adult neurogenesis. We therefore compared the expression of BDNF in the hippocampal dentate gyri of SCID, TMBP transgenic, and TOVA transgenic mice to BDNF expression in their wild-type counterparts. Relative to their respective wild-type controls, BDNF immunoreactivity was significantly lower in both SCID mice and in TOVA transgenic mice (
We also examined whether, in the adult animal, autoimmune T cells specific to MBP (TMBP cells) affect hippocampus-dependent cognitive functions such as LTP and spatial learning/memory, the latter measured by performance of a task in the MWM. LTP provides a model of synaptic plasticity that is assumed to underlie memory formation (Bliss et al., 1993). Before examining the effect of the TMBP cells on these functions, we examined whether LTP is impaired in mice with immune deficiency. This was done by comparing LTP in hippocampal slices taken from adult (12-week-old) C57B1/6J or Balb/c/OLA mice suffering from severe combined immune deficiency (SCID) to that in their respective wild-type controls. LTP was induced by theta-burst stimulation (TBS) of the Schaffer collaterals and was recorded from the CA1 region of the hippocampus. In both strains of SCID mice the post-theta potentiation declined approximately 15 min after TBS, whereas in the wild-type controls it was maintained for the duration of the experiment (approximately 30 min), indicating a capacity for LTP (
To determine whether the observed impairment of LTP in SCID mice can be attributed to the lack of autoimmune T cells, we used transgenic mice expressing only T cells directed to MBP (Lafaille et al., 1994). The TMBP-transgenic mice used in this experiment were on a RAG−/− (immune-deficient) background, so that their entire T cell population was specific only to MBP. As controls for this group we used RAG−/− mice (congenitally devoid of T cells). As with the SCID mice, LTP in the RAG1−/− mice was impaired relative to that in the TMBP-transgenic RAG1−/− mice, in which normal LTP could be generated and sustained under the same experimental conditions (
To study the behavioral manifestation(s) of the above differences, we examined the performance of the mice on a task in the MWVM. The TMBP-transgenic RAG1−/− mice learned to swim towards the hidden escape platform via the shortest route, whereas the ability of mice in the control group (devoid of T cells) to perform this task was significantly impaired (
To exclude the possibility that any T cell population, not only autoimmune T cells, can affect hippocampal activity, we repeated the experiment with transgenic mice possessing monospecific T cells directed against the non-self antigen ovalbumin (TOVA-transgenic mice). Since these transgenic mice are available only on a background of Balb/c/OLA, this was the strain used as a wild-type control for these experiments. We anticipated that if autoimmune T cells specific to myelin proteins are the cells needed for brain cognition, the TOVA-transgenic mice would behave like SCID mice on the same background. Relative to the wild type, performance of the MWM task was significantly impaired in the TOVA mice (
Taken together, these results suggested that autoimmune T cells directed to myelin-specific brain proteins such as MBP can help restore lost hippocampus-dependent LTP and spatial learning/memory in immune-defficient mice.
Several recent findings point to an association between adult neurogenesis and certain hippocampal activities (Shors et al., 2002). To examine the possibility that the role of T cells in adult plasticity is mediated at least in part via their effect on hippocampal neurogenesis in adulthood, we examined three pivotal phases in the process of neurogenesis: cell proliferation, early neuronal differentiation and long term survival of newly-formed neurons. We first compared the proliferation of neural precursor cells in adult (4-5-month old) wild-type mice to that in immune-deficient or in TMBP-expressing mice (both on a wild-type and on a RAG−/− background). After receiving four injections (once every 12-hours) of BrdU, which labels dividing cells, mice were killed (48 h after the first injection), their brains were excised, and sections of the hippocampus were examined for BrdU-positive cells. Significantly fewer labeled cells in the subgranular zone of the hippocampus were found in immune-deficient than in wild-type mice (
To determine the relevance of the autoimmune T cells to neurogenesis in the hippocampus, we examined the dentate gyrus of brains that were excised 7 days after the first BrdU injection and double-stained for BrdU and the early neuronal differentiation marker doublecortin (DCX). Significantly more BrdU/DCX-positive cells were found in the wild type than in the immune-deficient mice (
Next we examined whether the long-term survival of newly formed neurons is also affected by autoimmune T cells. Brain tissues were excised from all four groups of mice 4 weeks after the first BrdU injection, and sections were double-stained for BrdU and NeuN (a marker of mature neurons) in the dentate gyrus. Significantly fewer BrdU/NeuN-positive cells were found in the immune-deficient (SCID) mice than in their wild-type counterparts (
The present work identifies CNS specific autoimmune T cells as pivotal players in adult brain plasticity and shows that their participation occurs, at least in part, via their cross-talk with resident microglia. This novel finding is in line with earlier demonstrations that CNS-specific T cells, provided that the onset, duration, and intensity of their activity are well controlled, exert a beneficial effect on neuronal survival after CNS injury (Moalem et al., 19991 Hauben et al., 2001; Schwartz and Kipnis, 2002).
The findings of this study suggest that T cells affect adult neurogenesis, both in the dentate gyrus and in the SVZ, primarily via their effect on progenitor cell proliferation. We cannot rule out the possibility that T cells play a role not only in the proliferation of progenitor cells but also in their neuronal differentiation, as indicated by the results obtained with nude mice.
Severe immune deficiency in mice was shown here to impair three aspects of hippocampal plasticity at adulthood: LTP, spatial learning/memory, and adult neurogenesis. A single population of T cells recognizing the abundant CNS self-antigen MBP was sufficient to ameliorate the impairment. The results of the present study show that T cells directed to a specific CNS self-antigen play a key role in maintaining the functional activity of the hippocampus. Accordingly, we postulated that under non-pathological conditions a primary role of such autoimmune T cells is to maintain the plasticity of the adult brain. If this is so, it would follow that the observed beneficial effect of antigen-specific anti-self T cells under degenerative conditions is an extension of their role in the healthy brain.
Accumulation of β-amyloid deposition (Aβ), neuronal loss, cognitive decline, and microglial activation, are characteristic features of Alzheimer's disease (AD). Using AD double-transgenic mice expressing mutant human genes encoding presenilin 1 and chimeric mouse/human amyloid precursor protein, we show that vaccination with glatiramer acetate prevented and restored cognitive decline, assessed by performance in a Morris water maze. The vaccination modulated microglial activation, eliminated plaque formation, and induced neuronal survival and neurogenesis. In vitro, Aβ-activated microglia impeded neurogenesis from adult neural stem/progenitor cells. This was counteracted by IL-4, and more so when IFN-γ was added, but not by IFN-γ alone.
(xlv) Animals. Nineteen adult double-transgenic APPK670N, M671L+PS1ΔE9 mice of the B6C3-Tg (APPswe, PSEN1dE9) 85 Dbo/J strain were purchased from The Jackson Laboratory (Bar Harbor, Me.) and were bred and maintained in the Animal Breeding Center of The Weizmann Institute of Science. All animals were handled according to the regulations formulated by the Weizmann Institute's Animal Care and Use Committee. Tg AD mice were produced by co-injection of chimeric mouse/human APPswe (APP695 [humanized Aβ domain] harboring the Swedish [K594M/N595L] mutation) and human PS1dE9 (deletion of exon 9) vectors controlled by independent mouse prion protein promoter (MoPrP) elements, as described (Borchelt et al., 1997).
(xlvi) Reagents. Recombinant mouse IFN-γ and IL-4 (both containing endotoxin at a concentration below 0.1 ng/μg cytokine) were obtained from R&D Systems (Minneapolis, Minn.). β-amyloid peptides [amyloid protein fragment 1-40 and 1-42 (Aβ1-40/1-42)] were purchased from Sigma-Aldrich, St. Louis, Mo. The Aβ peptides were dissolved in endotoxin-free water, and Aβ aggregates were formed by incubation of Aβ, as described (Ishii et al., 2000).
(xlvii) Genotyping. All mice used in this experiment were genotyped for the presence of the transgenes by PCR amplification of genomic DNA extracted from 1-cm tail clippings (Jankowsky et al., 2004). Reactions contained four primers: one anti-sense primer-matching sequence within the vector that is also present in mouse genomic PrP (5′-GTG GAT ACC CCC TCC CCC AGC CTA GAC C) (SEQ ID NO:13); a second sense primer specific for the genomic PrP coding region (which was removed from the MoPrP vector) (5′-CCT CTT TGT GAC TAT GTG GAC TGA TGT CGG) (SEQ ID NO:14); and twvo sense and anti-sense primers specific for the PS1 transgene cDNA (PS1-a: 5′-AAT AGA GAA CGG CAG GAG CA (SEQ ID NO:15), and PS1-b: 5′-GCC ATG AGG GCA CTA ATC AT) (SEQ ID NO:16). All reactions give a 750-bp product of the endogenous PrP gene as a control for DNA integrity and successful amplification; PS1 transgene-positive samples have an additional band at approximately 608 bp.
(xlviii) Glatiramer acetate vaccination. Each mouse was subcutaneously injected five times with a total of 100 μg of glatiramer acetate (GA (TV-5010), MW 13.5-18.5 kDa, average 16 kDa, Teva Pharmaceutical Industries Ltd., Petach Tikva, Israel), emulsified in 200 μl PBS×1, from experimental day 0 until day 24, twice during the first week and once a week thereafter.
(xlix) Behavioral testing. Spatial learning/memory was assessed by performance on a hippocampus-dependent visuo-spatial learning task in the Morris water maze (MWM) (Morris, 1984). Mice were given four trials per day on 4 consecutive days, during which they were required to find a hidden platform located 1.5 cm below the water surface in a pool 1.4 m in diameter. Within the testing room, only distal visuo-spatial cues for location of the submerged platform were available. The escape latency, i.e., the time required by the mouse to find the platform and climb onto it, was recorded for up to 60 s. Each mouse was allowed to remain on the platform for 30 s and was then moved from the maze to its home cage. If the mouse did not find the platform within 60 s, it was placed manually on the platform and returned to its home cage after 30 s. The interval between trials was 300 s. On day 5 the platform was removed from the pool and each mouse was tested by a probe trial for 60 s. On days 6 and 7 the platform was placed at the quadrant opposite the location chosen on days 1-4, and the mice were then retrained in four sessions per day. Data were recorded using an EthoVision automated tracking system (Noldus).
(l) Administration of BrdU and tissue preparation. BrdU was dissolved by sonication in PBS and injected i.p. into each mouse (50 mg/kg body weight; 1.25 mg BrdU in 200 μl PBS×1). Starting from experimental day 22 after the first GA vaccination, BrdU was injected i.p. twice daily, every 12 h for 2.5 days, to label proliferating cells. Three weeks after the first BrdU injection the mice were deeply anesthetized and perfused transcardially, first with PBS and then with 4% paraformaldehyde. The whole brain was removed, postfixed overnight, and then equilibrated in phosphate-buffered 30% sucrose. Free-floating 30-μm sections were collected on a freezing microtome (Leica SM2000R) and stored at 4° C. prior to immunohistochemistry.
(li) Neural progenitor cell culture. Coronal sections (2 mm thick) of tissue containing the subventricular zone of the lateral ventricle were obtained from the brains of adult C57B1/6J mice. The tissue was minced and then incubated for digestion at 37° C., 5% CO2 for 45 min in Earle's balanced salt solution containing 0.94 mg/ml papain (Worthington, Lakewood, N.J.) and 0.18 mg/ml of L-cysteine and EDTA. After centrifugation at 110×g for 15 min at room temperature, the tissue was mechanically dissociated by pipette trituration. Cells obtained from single-cell suspensions were plated (3500 cells/cm2) in 75-cm2 Falcon tissue-culture flasks (BD Biosciences, San Diego, Calif.), in NPC-culturing medium [Dulbecco's modified Eagles's medium (DMEM)/F12 medium (Gibco/Invitrogen, Carlsbad, Calif.) containing 2 mM L-glutamine, 0.6% glucose, 9.6 μg/ml putrescine, 6.3 ng/ml progesterone, 5.2 ng/ml sodium selenite, 0.02 mg/ml insulin, 0.1 mg/ml transferrin, 2 μg/ml heparin (all from Sigma-Aldrich, Rehovot, Israel), fibroblast growth factor-2 (human recombinant, 20 ng/ml), and epidermal growth factor (human recombinant, 20 ng/ml; both from Peprotech, Rocky Hill, N.J.)]. Spheres were passaged every 4-6 days and replated as single cells. Green fluorescent protein (GFP)-expressing NPCs were obtained as previously described (Pluchino et al., 2003).
(lii) Primary microglial culture. Brains from neonatal (P0-P1) C57B1/6J mice were stripped of their meninges and minced with scissors under a dissecting microscope (Zeiss, Stemi DV4, Germany) in Leibovitz-15 medium (Biological Industries, Kibbutz Beit Ha-Emek, Israel). After trypsinization (0.5% trypsin, 10 min, 37° C./5% CO2), the tissue was triturated. The cell suspension was washed in culture medium for glial cells [DMEM supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, Rehovot), L-glutamine (1 mM), sodium pyruvate (1 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml)] and cultured at 37° C./5% CO2 in 75-cm2 Falcon tissue-culture flasks (BD Biosciences) coated with poly-D-lysine (PDL) (10 mg/ml; Sigma-Aldrich, Rehovot) in borate buffer (2.37 g borax and 1.55 g boric acid dissolved in 500 ml sterile water, pH 8.4) for 1 h, then rinsed thoroughly with sterile, glass-distilled water. Half of the medium was changed after 6 h in culture and every 2nd day thereafter, starting on day 2, for a total culture time of 10-14 days. Microglia were shaken off the primary mixed brain glial cell cultures (150 rpm, 37° C., 6 h) with maximum yields between days 10 and 14, seeded (105 cells/ml) onto PDL-pretreated 24-well plates (1 ml/well; Corning), and grown in culture medium for microglia [RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% FCS, L-glutamine (1 mM), sodium pyruvate (1 mM), P-mercaptoethanol (50 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml)]. The cells were allowed to adhere to the surface of a PDL-coated culture flask (30 min, 37° C./5% CO2), and non-adherent cells were rinsed off.
(liii) Co-culturing of mouse neural progenitor cells and mouse microglia. Cultures of treated or untreated microglia were washed twice with fresh NPC-differentiation medium (same as the culture medium for NPCs but without growth factors and with 2.5% FCS) to remove all traces of the tested reagents, then incubated on ice for 15 min, and shaken at 350 rpm for 20 min at room temperature. Microglia were removed from the flasks and immediately co-cultured (5×104 cells/well) with NPCs (5×104 cells/well) for 10 days on cover slips coated with Matrigel™ (BD Biosciences) in 24-well plates, in the presence of NPC differentiation medium. The cultures were then fixed with 2.5% paraformaldehyde in PBS for 30 min at room temperature and stained for neuronal and glial markers.
(liv) Immunocytochemistry and immunohistochemistry. Cover slips from co-cultures of NPCs and mouse microglia were washed with PBS, fixed as described above, treated with a permeabilization/blocking solution containing 10% FCS, 2% bovine serum albumin, 1% glycine, and 0.1% Triton X-100 (Sigma-Aldrich, Rehovot), and stained with a combination of the mouse anti-tubulin β-III-isoform C-terminus antibodies (P-II-tubulin; 1:500; Chemicon, Temecula, Calif.) and CD11b (MAC1; 1:50; BD-Pharmingen, Franklin Lakes, N.J.).
For BrdU staining, sections were washed with PBS and incubated in 2N HCl at 37° C. for 30 min. Sections were blocked for 1 h with blocking solution (PBS containing 20% normal horse serum and 0.1% Triton X-100, or PBS containing mouse immunoglobulin blocking reagent obtained from Vector Laboratories (Burlingame, Calif.)).
For immunohistochemistry, tissue sections were treated with a permeabilization/blocking solution containing 10% FCS, 2% bovine serum albumin, 1% glycine, and 0.05% Triton X-100 (Sigma-Aldrich, St. Louis). Tissue sections were stained overnight at 4° C. with specified combinations of the following primary antibodies: rat anti-BrdU (1:200; Oxford Biotechnology, Kidlington, Oxfordshire, UK), goat anti-DCX [doublecortin] (1:400; Santa Cruz Biotechnology), and mouse anti-NeuN [neuronal nuclear protein] (1:200; Chemicon, Temecula, Calif.). Secondary antibodies were FITC-conjugated donkey anti-goat, Cy-3-conjugated donkey anti-mouse, and Cy-3- or Cy-5-conjugated donkey anti-rat (1:200; Jackson ImmunoResearch, West Grove, Pa.). For labeling of microglia we used either CD11b (MAC1; 1:50; BD-Pharmingen) or FITC-conjugated Bandeiraea simplicifolia isolectin B4 (IB4, 1:50; Sigma-Aldrich, Rehovot). To detect expression of cell-surface MHC-II proteins we used anti-MHC-II Abs (rat, clone IBL-5/22; 1:50; Chemicon, Temecula, Calif.). To detect expression of human Aβ we used anti-Aβ (human amino-acid residues 1-17) (mouse, clone 6E10, Chemicon, Temecula, Calif.). Expression of IGF-I was detected by goat anti-IGF-I Abs (1:20; R&D Systems). Expression of TNF-α was detected by goat anti-TNF-α Abs (1:100; R&D Systems). T cells were detected with anti-CD3 polyclonal Abs (rabbit, 1:100; DakoCytomation, Calif.). Propidium iodide (1 μg/ml; Molecular Probes, Invitrogen, Carlsbad, Calif.) was used for nuclear staining.
Control sections (not treated with primary antibody) were used to distinguish specific staining from staining of nonspecific antibodies or autofluorescent components. Sections were then washed with PBS and coverslipped in polyvinyl alcohol with diazabicylo-octane as anti-fading agent.
(lv) Quantification and stereological counting procedure. For microscopic analysis we used a Zeiss LSM 510 confocal laser scanning microscope (40× magnification). For experiments in vitro we scanned fields of 0.053 mm2 (n=8-16 from at least two different coverslips) for each experimental group. For each marker, 500-1000 cells were sampled. Cells co-expressing GFP and β-III-tubulin were counted.
For in-vivo experiments, the number of Aβ+ plaques and CD11b+/IB-4+ microglia in the hippocampus were counted at 300-μm intervals from 6-8 coronal sections (30 μm) from each mouse. Neurogenesis in the DG was evaluated by counting of premature neurons (DCX+), proliferating cells (BrdU+), and newly formed mature neurons (BrdU+/NeuN+) in six coronal sections (30 μm) from each mouse. Specificity of BrdU+/NeuN+ co-expression was assayed using the confocal microscope (LSM 510) in optical sections at 1-μm intervals/. Cell counts, numbers of Aβ+ plaques, plaque areas, and intensity of NeuN staining per unit area in the DG were evaluated automatically using Image-Pro Plus 4.5 software (Media Cybernetics).
(lvi) Statistical analysis. MWM behavior scores were analyzed using 3-way ANOVA, with treatment group and trial block as sources of variation, was used to evaluate the significance of differences between mean scores during acquisition trial blocksin the MWM. When the P-value obtained was significant, a pairwise Fisher's least-significant-difference multiple comparison test was run to determine which groups were significantly different.
The in-vitro results were analyzed by the Tukey-Kramer multiple comparisons test (ANOVA) and are expressed as means±SEM. In-vivo results were analyzed by Student's t-test or 1-way ANOVA and are expressed as means±SEM.
We examined the effect of GA in AD double-transgenic mice (Tg mice) expressing a mutant human presenilin 1 gene (PS1dE9) and a chimeric mouse/human amyloid precursor protein (APPswe), leading to learning/memory impairment and accumulation of Aβ plaques mainly in the cortex and the hippocampus, both characteristic features of early-onset familial AD (Borchelt et al., 1997). Expression of both transgenes in each mouse was verified by PCR amplification of genomic DNA. APP/PS1 Tg mice aged approximately 8 months were then vaccinated subcutaneously with GA (n=6) twice during the first week and once a week thereafter. Age-matched Tg mice (n=7) and non-Tg littermates that did not carry the transgenes (n=6), were not treated and served as untreated Tg and wild-type non-Tg controls, respectively. Five weeks after the first GA injection all mice were assessed in a Morris water maze (MWM) for cognitive activity, as reflected by performance of a hippocampus-dependent spatial learning/memory task. The MWM performance of the untreated Tg mice was significantly worse, on average, than that of the age-matched non-Tg littermates (
The above results prompted us to examine the possibility that the observed arrest and reversal of cognitive loss was related to reduction of Aβ plaques and survival of neurons in the hippocampus. Staining of brain cryosections from Tg mice with antibodies specific to human Aβ disclosed numerous plaques in the untreated Tg mice but very few in those vaccinated with GA (
Activated microglia are known to play a role in the pathogenesis of AD. We have shown in the Examples hereinbefore that, unlike microglia seen in association with inflammatory and neurodegenerative diseases, the microglia associated with neural tissue survival express MHC-II, produce IGF-I, and express little or no TNF-α. We therefore examined brain sections from GA-vaccinated and untreated Tg mice for the presence of microglia that stain positively for CD 11b or TNF-α (markers of activation associated with a cytotoxic inflammatory phenotype). The presence of plaques was found to be correlated with the appearance of CD 11b+microglia (
In addition, unlike in the untreated Tg mice, in the GA-vaccinated Tg mice numerous T cells (identified by anti-CD3 antibodies) were seen in close proximity to MHC-II+ microglia. Any Aβ-immunoreactivity seen in these mice appeared to be in association with MHC-II+ microglia, creating an immune synapse with CD3+ T cells (
Quantitative analysis confirmed that mice vaccinated with GA showed significantly fewer plaques than untreated Tg mice when examined 6 weeks later (
Because MHC-II-expressing microglia are also associated with neurogenesis in vitro, we examined the same sections for the formation of new neurons in the dentate gyrus (DG) of the hippocampus. This was possible because all mice had been injected with BrdU, a marker of proliferating cells, 3 weeks before tissue excision. Quantitative analysis disclosed significantly more BrdU+ cells in GA-vaccinated Tg mice (
The in-vivo results presented above point to a relationship between arrested neurogenesis, aggregated AD, and the phenotype of the activated microglia. To determine whether aggregated Aβ-activated microglia block neurogenesis, and whether T cell-derived cytokines can counteract the inhibitory effect, we co-cultured GFP-expressing NPCs with microglia that had been pre-incubated for 48 h in their optimal growth medium in the presence or absence of aggregated Aβ peptide 1-40/1-42 (Aβ(1-40/1-42); 5 μM) and subsequently treated with IFN-γ (10 ng/ml), or with IL-4 (10 ng/ml) together with IFN-γ (10 ng/ml), for an additional 48 h. Growth media and cytokine residues were then washed off, and each of the treated microglial preparations was freshly co-cultured with dissociated NPC spheres on coverslips coated with Matrigel™ in the presence of differentiation medium (
In this study of APP/PS1 double-transgenic AD mice suffering from decline in cognition and accumulation of Aβ plaques, a T cell-based vaccination, by altering the microglial phenotype, ameliorated cognitive performance, reduced plaque formation, rescued cortical and hippocampal neurons, and induced hippocampal neurogenesis.
Using a rat model of permanent middle cerebral artery occlusion, we show herein that systemic immune-modulation with pYE enhances hippocampal and cortical neurogenesis from aNPCs following stroke. We show that the sate of neurogenic unpermissiveness in brain regions such as the cortex can be changed by the local immune response. These findings offer a new immune-based therapeutic approach for augmenting cell renewal from endogenous NPCs.
(lvii) Animals. Sprague-Dawley (SPD) male rats, aged 8-12 weeks, were supplied by Harlan Biotech (Jerusalem, Israel) and were handled according to the ARVO resolution on the use of animals in research.
(lviii) Permanent middle cerebral artery occlusion in rats. Rats were anesthetized by i.p. injection of ketamine HCl 100 mg/kg (Fort Dodge Laboratories, Fort Dodge, Iowa) and xylazine 2%, 10 mg/kg (Vitamed, Benyamina, Israel). A temperature control unit was used to maintain the body temperature of the rats at approximately 37° C. during surgery. Following a mid-line longitudinal incision in the cervical area, the right common carotid artery (CCA) was dissected from the surrounding tissue and the right internal carotid artery (ICA) and the right external carotid artery (ECA) were exposed. Branches of the right ECA were occluded by electrocoagulation and the distal portion was ligated. The right CCA and ICA were temporarily occluded by clamping. The tip of a 4-0 nylon filament was blunted by heating near a flame, to form a bead. The filament was then coated with poly-L-lysine solution (0.1% in water, Sigma, St. Louis, Mo.) and dried at 60° C. for 1 h prior to use. The beaded filament was inserted into the right ICA through a puncture in the right ECA and advanced over a distance of 2.0 cm to the right anterior cerebral artery, bypassing and occluding the origin of the right middle cerebral artery (MCA). The protruding tip of the filament was cut, the blood vessels and tissues were returned to their usual positions ands the incision was sutured. After recovering from the anesthesia the rats were returned to their home cages and allowed free access to food and water.
(lix) Treatment with Poly YE. Upon completion of surgery the rats were randomly assigned to different treatment groups. All rats were injected once subcutaneously (s.c.) at the indicated times either with the designated dosages of PN277 (Poly YE, Sigma, St. Louis, Mo.) in a final volume of 1 ml phosphate-buffered saline (PBS) or with PBS only (control group). Rats were weighed before MCAO and twice weekly thereafter over the indicated time period.
(lx) Administration of 5-bromo-2′-deoxyuridine. MCAO was performed as described above, and PN277 (1 mg/rat) was subcutaneously injected 24 h later. The cell proliferation marker BrdU was dissolved by sonication in PBS (6.25 mg/ml), and injected i.p. into rats (50 mg/kg body weight), twice daily from day 7 to day 9 after MCAO. On day 28 the rats were deeply anesthetized and killed by transcardial perfusion with PBS followed by 4% paraformaldehyde. Brains were removed, fixed overnight, and then equilibrated in phosphate-buffered sucrose (30% sucrose). Free-floating 25-μm sections were collected on a freezing microtome (Leica SM2000R) and stored at 4° C. prior to immunohistochemistry. Since BrdU is known to exert cytotoxic effects on proliferating lymphocytes, we chose in our experiments to inject BrdU with a short pulse of 3 days, starting on day 7 after MCAO to coincide with the peak of spontaneous proliferation. Thus the results of our analysis might even underestimate the absolute level of proliferation because of a lack of constantly available BrdU along the experimental period.
(lxi) Immunohistochemistry. For BrdU staining, tissue sections were washed with PBS and incubated for 30 min in 2N HCl at 37° C. Sections were blocked for 1 h with blocking solution (PBS containing 20% normal horse serum and 0.5% Triton X-100). Tissue sections were then stained overnight with the primary antibodies rat anti-BrdU (1:100; Oxford Biotechnology, Kidlington, Oxfordshire, UK), mouse anti nestin (1:1000; Chemicon, Temecula, Calif.), rabit anti MAP-2 (1:200; Chemicon, Temecula, Calif.) and mouse anti-NeuN (1:150; Chemicon, Temecula, Calif.). Secondary antibodies were Cy-2-conjugated donkey anti-mouse, Cy-3-conjugated donkey anti-mouse, and Cy-3- or Cy-5-conjugated donkey anti-rat (1:200; Jackson ImmunoResearch, West Grove, Pa.).
(lxii) Quantification. For analysis of cell proliferation and neurogenesis, six coronal hippocampal sections per rat brain (five rats per group), spanning the entire dentate gyrus, were taken for immunohistochemistry. Proliferation was assessed in a Nikon E800 fluorescent microscope by counting BrdU+ cells in the dentate gyrus. The numbers obtained were multiplied by the distance between each section to obtain an estimate of the total number of proliferating cells per dentate gyrus. Neuronal differentiation in the dentate gyrus was evaluated using confocal microscopy by counting cells that were double-labeled with BrdU and NeuN. Cortical neurogenesis was assessed by staining with BrdU together with MAP-2 or nestin. At least 2 coronal sections from 4 animals per group were included in this analysis. Measurements of intensity per surface area were obtained with ImageProPlus software.
We first examined the effect of PN277 on hippocampal neurogenesis after stroke. Stem-cell proliferation and survival in the hippocampus were assessed by injecting control and PN277-treated rats with the cell proliferation marker BrdU for 3 days starting at day 7 after MCAO. The rats were killed 28 d after MCAO, and neurogenesis was evaluated by staining of coronal hippocampal sections with antibodies against BrdU and NeuN (a marker of post-mitotic neurons). Treatment with PN277 (1 mg/rat) immediately after stroke resulted in a two-fold increase in the number of newly formed neurons in the ipsilateral dentate gyrus relative to PBS treated controls (
Newly formed BrdU+/NeuN+ cells were observed in the granular cell layer of the dentate gyrus in PN277-treated rats (
The ischemic injury imposed by MCAO causes severe damage to striatal and cortical structures. Recent studies demonstrated that such ischemic or traumatic injury can induce neurogenesis in regions of the brain that are normally non-neurogenic (Arvidsson et al., 2002; Nakatomi et al., 2002; Emsley et al., 2005). We next asked to determine whether in our model of stroke neurogenesis was induced in cortical areas that were affected by the stroke. We thus stained coronal sections containing the cortical lesion site for BrdU, the progenitor cell marker Nestin and neuronal markers such as β-III tubulin and MAP-2. A large number of BrdU+ cells and Nestin+ cells could be seen surrounding the lesion site (
We next asked to determine whether treatment with PN277 could enhance cortical neurogenesis. To avoid staining artifacts caused by non-specific staining of myelin debris we choose MAP2 instead of NeuN. We took special care to count only MAP-2+ cells that exhibit the typical morphology of pyramidal neurons in the cortex and in which BrdU appeared in the center of the cell. Brains in which the cortex was not appear to be damaged were excluded from the analysis. Quantitative analysis revealed a 2-fold increase in the number newly formed cortical neurons in the PN277 treated rats relative to PBS-treated rats (
Nestin expressing cells with similar morphology to those detected in the surrounding of the cortical lesion were also detected in the striatum adjacent to the lateral ventricles. We found that the great majority of these Nestin+ cells were also expressing the astrocytic marker GFAP. Interestingly, we found IB-4+microglia/macrophages within the striatum area occupied by the GFAP+/Nestin+ cells (
Boosting T-cell mediated immune response following insult to the CNS can be done by either vaccinating with a CNS-specific antigen or by abolishing regultory T cells (Treg) activity (Moalem et al., 1999; Hauben et al., 2000; Schwartz et al., 2003). We chose to use PN277, an immuno-modulator that was recently shown to be capable of reducing Treg suppressive activity and confer neuroprotection. While vaccination induce T-cell response primarily to one antigen, reducing Treg suppressive activity facilitate a broader T-cell response against various antigens residing in the injured site. Thus, following stroke, mostly T cells specific for various CNS— derived antigens would be propagated in response to PN277 treatment.
The finding of enhanced hippocampal neurogenesis in the PN277 treated rats is consistent with our finding herein that neurogenesis was enhanced in transgenic mice over-expressing T-cell receptor for MBP. The fact that this effect was seen in both ipsilateral and contralateral sides further imply that elevation in neurogenesis is caused by a systemic event (T-cell response). Such autoimmune T cells can locally interact with microglia, which in turn can recruit NPCs and direct their differentiation. This hypothetical scenario is supported by our findings herein showing that neurogenesis from aNPCs could be induced by microglia activated by cytokines (IL-4 or IFN-γ) associated with T-helper cells.
The effect of PN277 treatment on neurogenesis was even more robust with regard to cortical neurogenesis. In conclusion, the results of this study suggest that proper immune modulation can increase neurogenesis, and that lack of neurogenic permissiveness in brain regions such as the cortex can be changed by the local immune response. These findings introduce a new therapeutic approach for augmenting spontaneously occurring neurogenesis following an ischemic insult.
Animals. Transgenic mice overexpressing the defective human mutant SOD1 allele containing the Gly93→Ala (G93A) gene (B6SJL-TgN (SOD1-G93A)1Gur (herein “ALS mice”) were purchased from The Jackson Laboratory (Bar Harbor, Me., USA).
Immunization. Adult mice were immunized with Cop-1, 100 μg in 200 μl PBS s.c.
Neural progenitor cell culture. Cultures of adult neural progenitor cells (aNPCs) were obtained as described in orevious examples
Stereotaxic injection of neural progenitor cells. Mice were anesthetized a week after the first immunization and placed in a stereotactic device. The skull was exposed and kept dry and clean. The bregma was identified and marked. The designated point of injection was at a depth of 2 mm from the brain surface, 0.4 mm behind the bregma in the anteroposterior axis, and 1.0 mm lateral to the midline. Neural progenitor cells were applied with a Hamilton syringe (5×105 cells in 3 μl, at a rate of 1 μl/min) and the skin over the wound was sutured.
Motor dysfunction. Motor dysfunction of the mice was evaluated using the rotarod task twice a week from 60 d of age onward. Animals were placed on a horizontal accelerating rod [accelerating rotarod (Jones and Roberts) for mice 7650] and time it took for each mouse to fall from the rod was recorded. We performed three trials at each time point for each animal and recorded the longest time taken. A cut-off time point was set to 180 sec and mice remaining on the rod for at least 180 sec were deemed asymptomatic. Onset of disease symptoms was determined as a reduction in rotarod performance between weekly time points. Animals were killed by euthanization when no longer able to right themselves within 30 seconds of being placed on their sides.
The animals were treated with Cop-i starting from day 59: in the first two weeks twice a week Cop-1, thereafter they received a weekly injection of Cop-1. The stem cells were given into the CSF: 500,000 cells (single injection of adult neural stem cells).
The experiment was carried out in order to explore whether administration of a combination of Cop-i vaccination and stem cells has beneficial effect in a mice model of ALS (herein “ALS mice”). For this purpose, 59 days old ALS mice were treated as follows: group 1 (
Mice from each of the groups were weighted (twice a week) and examined routinely for vital signs, and for signs of motor dysfunction. The results obtained in
Jankowsky J L, Fadale D J, Anderson J, Xu G M, Gonzales V, Jenkins N A, Copeland N G, Lee M K, Younkin L H, Wagner S L, Younkin S G, Borchelt D R. Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase. Hum Mol. Genet. January 15; 13(2):159-70 (2004).
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IL05/01270 | 11/29/2005 | WO | 00 | 7/3/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60631163 | Nov 2004 | US |
