Patent Application
20120276073
This invention relates to treatment of nerve injuries, in particular, traumatic, acute and/or chronic lesions of the central and peripheral nervous system, and to regeneration of nerves and nerve function. The invention provides a pharmaceutical composition comprising histone, in particular, histone H1. It was surprisingly shown that histones can be used for the preparation of a pharmaceutical composition for treatment and/or prevention of acute and chronic injuries to the central and peripheral nervous system, for promoting growth and/or regrowth of nerves and enhancing formation of synapses and synaptic plasticity, and for neuroprotection.
Polysialic acid (PSA) is known to play a crucial role in nervous system development and function. It has also been associated with regeneration of nerve function. PSA is a long, linear polymer composed entirely of negatively charged sialic acid ('5-N-acetylneuraminic acid) in α2,8 linkage, and belongs to a class of functionally important anionic glycans (Rutishauser et al., 2008). Because of its high negative charge, PSA is highly hydrated and has therefore been suggested to attenuate cellular interactions and increase motility (Mühlenhoff et al., 1998).
In the mammalian brain, PSA is predominantly or probably exclusively attached to the neural cell adhesion molecule NCAM. So far, only neuropilin-2 has been identified in human dendritic cells as another carrier of PSA in mammalia (Curreli et al., 2007), but it retrains to be shown whether neuropilin-2 expressed in brain also carries PSA. PSA is associated with morphogenetic changes during developmental processes such as cell migration, synaptogenesis, axonal growth, and branching, whereas PSA persists in the adult brain only in structures that display a high degree of functional plasticity (Rutishauser et al., 2008; Kleene and Schachner, 2004). During embryonic development, PSA is expressed abundantly on Schwann cells and axons in the peripheral nervous system, while its expression decreases in adulthood. After nerve injury in adult rodents, PSA is up-regulated in axons and skeletal muscle fibres (Olsen et al., 1995; Mehanna et al., 2009a). Additionally, in vitro and in vivo functional assays using PSA mimetics and/or the bacterial homolog of PSA, colominic acid, indicated that PSA is involved in myelination and regeneration of peripheral and central nerves (Jungnickel et al., 2009; Mehanna et al., 2009a and 2009b).
In the adulthood, PSA plays an important role in nervous system function and in synaptic plasticity. The functional and developmental defects observed upon ablation of PSA in mouse mutants lacking the main PSA carrier NCAM or the polysialyltransferases essential for the synthesis of PSA underscore the functional importance of PSA. In addition, the effects observed upon application of endoneuraminidase N which specifically degrades PSA further documented the functional plasticity of PSA. Although much is known about the functions of PSA which have been intensively studied, only a few possible binding partners have been described so far: BDNF (Muller et al., 2000), heparan sulfate proteoglycans (Storms and Rutishauser 1998), AMPA receptors (Vaithianathan et al., 2004), NR2B subunit containing NMDA receptors (Hammond et al., 2006) and estradiol (Garcia-Sequra et al., 1995). BDNF is a neurotrophic factor that shows a direct binding to PSA in vitro (Kanato et al., 2008) and promotes neuronal survival and differentiation during development (Sofroniew et al., 2001). Heterophilic binding of PSA-NCAM to the heparan sulfate proteoglycans has a positive effect on cell adhesion (Storms and Rutishauser 1998). The interaction between PSA and AMPA receptors results in age-dependent potentiation of AMPA receptor, which might modulate signalling via AMPA receptors in immature neurons and thereby affecting their development (Vaithianathan et al., 2004), while the interaction of PSA with NR2B subunit containing NMDA receptors prevents excitotoxicity (Hammond et al., 2006). The interaction of PSA with estradiol appears to participate in the estradiol-induced changes of glial cells with a polygonal shape to process-bearing cells (Garcia-Sequra et al., 1995).
It was recently shown that a PSA-mimicking peptide has positive effects on recovery after femoral nerve and spinal cord lesion and synaptic plasticity in mice (Mehanna et al., 2009a and 2009b; Marino et al., 2009; Papastefanaki et al., 2007; Senkov et al., 2006; Stoenica et al., 2006). This peptide improves myelination but does not influence motoneuron survival and regrowth. Brief low-frequency electrical stimulation of the injured femoral nerve also leads to long-lasting enhancement of axonal regrowth (Al-Majed et al., 2000; Brushart et al., 2005) and functional recovery (Ahlborn et al., 2007).
In light of the state of the art, the present inventors addressed the problem of providing alternative compounds that improve nerve regeneration or that might improve motoneuron regeneration and that may be used in therapy of nerve injury.
The present invention surprisingly identifies histone H1 as novel interaction partner of PSA and shows direct binding of histone H1 to PSA.
The invention therefore provides a pharmaceutical composition comprising a histone, in particular for treatment or prevention of nerve injury or for reducing the risk of nerve injury. The histone may be selected from the group consisting of histone H1, H2A, H2B, H3, and H4. Preferably, the histone is histone H1.
The pharmaceutical composition may comprise one or more different histones or a nucleosome, e.g., in the form of optionally digested chromatin. In the context of this invention “a” is not meant to be limiting to “one” unless explicitly specified, it can thus stand for one, two, three, four, five or more.
In one embodiment, the histone is a human histone, in particular, human histone H1, especially if humans are to be treated. However, alternatively, e.g., bovine histone, rat or murine histone or ape histone, such as chimpanzee histone may be used. There are eleven known mammalian subtypes of linker histone, H1.0, H1.1, H1.2, H1.3, H1.4, H1.5, H1.X, H1.t, H1.t2, HILS and H1oo, as well as reptilian and amphibian H5, which are all referred to herein as H1. Preferably, H1 is selected from H1.1, H1.2, H1.3, H1.4 and H1.5, who all have an amino acid identity of at least 70% to H1.4. There is a high homology between species, e.g., human and murine histone H1 have 94% amino acid identity. Histone H1 preferably is human histone H1 or a variant or derivative thereof comprising a sequence with at least about 70% amino acid identity, at least about 80% amino acid identity, at least about 90% amino acid identity or at least about 95% or 99% amino acid identity to human H1.4. Preferably, the variant or derivative is capable of specifically binding to PSA and/or colominic acid, as tested in the present application e.g., by the ELISA with binding to colominic acid. The histone may be non-phosphorylated, phosphorylated or derivatized, e.g., PEGylated or biotinylated.
The histone or histones may be isolated from biological samples, e.g., from bovine thymus or cell culture, or produced recombinantly, e.g., in insect cells, COS cells, yeast or E. coli. Methods for recombinant production of histone H1 are disclosed, e.g., in US 2003/0078204.
Histone H1 was initially described as a classical nuclear protein that is involved as a linker histone in the organization of nucleosomes which mediate DNA-packing (Doenecke et al., 1997), but it also has been discovered in extranuclear (Parseghian and Lulus, 2006) and even extra-cellular locations. Previously it has been reported that immunohistochemistry revealed a staining of histone H1 at neuronal cell bodies of cultured primary mouse cortical neurons, suggesting that histone H1 was located at the cell surface of neurons (Bolton and Perry, 1997). However, an extracellular matrix localization of histone H1 had not been shown for neuronal cells.
Extracellular histones, among them histone H1, have previously been detected at the surface of human monocytes (Holers and Kotzin, 1985), activated human peripheral blood lymphocytes (Watson et al., 1995) and cultured T-cells (Watson et al., 1995; Watson et al., 1999) as well as a macrophage cell line where it interacts with thyroglobulin and may mediates thyroglobulin endocytosis (Brix et al., 1998). The trafficking of histone proteins from the nucleus to the cytoplasm, cell surface or extracellular environment is not known yet, but since penetration of extracellular histone through the plasma membrane and into the cell has been demonstrated (Hariton-Gazal et al., 2003) it may be possible that a similar pathway may exist for the export of histones.
Extracellular histones including H1 have been shown to bind to cell surface heparan sulfate proteoglycans (Watson et al., 1999) and lipopolysaccharide (LPS) (Hampton et al., 1988; Bolton and Perry, 1997) which upregulate the expression of histones on the cell surface of monocytes (Emlen et al., 1992). In addition, extracellular histones, in particular histone H1, have also been reported to exhibit antibacterial activity (Hirsch, 1958; Hiemstra et al., 1993) which is antagonized by acid polysaccharides like heparin. There are also indications that histone H1 binds to amyloid precursor protein (Potempska et al., 1993) and amyloid-like motifs, such as β-amyloid and α-synuclein (Duce et al., 2006) and it was found associated with amyloid plaques (Duce et al., 2006). It has also been discussed that it binds to the pathogenic scrapie form of the prion protein and it has been shown that histone H1 is upregulated in brains of mice showing clinical sign of scrapie and an increased immunostaining of histone H1 was observed in region of pathology (Bolton et al., 1999). It is noteworthy to mention that prion protein as well as amyloid precursor protein binds to heparan sulfate proteoglycans which play a role in the pathogenesis of Alzheimer's disease, Scrapie or related diseases (van Horssen et al., 2003; Horonchick et al., 2005), and which bind to PSA-NCAM.
Histone H1 has been localized on the plasma membrane or in the extracellular matrix of skeletal muscle cells, and the level of extracellular histone H1 increases during muscle differentiation (Henriquez et al., 2002). The extracellular pool of histone H1 colocalizes with heparan sulfate proteoglycan perlecan in the extracellular matrix of myotube cultures and in regenerating skeletal muscle. In addition, histone H1 was identified as endogenous extracellular ligand for muscle cell heparan sulfate proteoglycans which regulate skeletal muscle differentiation. Extra-cellular histone H1 strongly stimulated myoblast proliferation via a heparan-sulfate-dependent mechanism, suggesting that extracellular histone H1 in conjunction with heparan sulfate proteoglycans plays a functional role during skeletal muscle development and regeneration. The role of extracellular histone H1 in regulating cell proliferation and differentiation is also supported by its anti-proliferative effects on cancer cells (Vani et al., 2006). Interestingly, it has effects on estrogen receptor status of human breast cancer cells (Vani and Devi, 2005); suggesting that, together with PSA, which is a putative binding partner of estradiol, it regulates estrogen-induced effects on proliferation. It has previously been suggested to use Histone H1 as an antimicrobial agent (e.g., U.S. Pat. No. 6,884,423) or for treatment of rheumatoid arthritis (U.S. Pat. No. 6,204,242).
The present inventors showed that histone H1 is additionally present extracellularly at the cell surface of cerebellar neurons and Schwann cells, and that histone H1 has a beneficial effect on neural precursor cell migration, neurite outgrowth as well as Schwann cell proliferation and process elongation; suggesting that extracellular histone H1 plays an important role during development and regenerating of the peripheral and central nervous system and in addition enhances synaptic plasticity. The better functional recovery and preferential motoneuron reinnervation after peripheral nerve injury by histone H1 indicates that it has a functional role in regeneration in vivo and can be used therapeutically.
In one aspect, the pharmaceutical composition of the present invention comprising histone, in particular, histone H1, is for treatment or prevention of nerve injury, such as acute and chronic trauma, central and peripheral nervous system regeneration and neurodegenerative diseases. The pharmaceutical composition may also be for enhancing learning and memory.
The invention also provides the use of a histone for the preparation of the pharmaceutical composition comprising a histone, e.g., histone H1, for the treatment and/or prevention of nerve injury. The invention also provides a method for treating and/or preventing nerve injury, wherein a pharmaceutical composition comprising an effective amount of an isolated histone, e.g., histone H1, is administered to a patient in need thereof. The histone can be in the form of a protein, alternatively, a gene transfer vector for expression of a histone and/or a histone expressing cell may be used in the pharmaceutical composition of the invention. The histone expressing cell preferably is recombinant and overexpresses the histone. The cell preferably comprises a gene transfer vector for expression of a histone. The vector comprises a nucleic acid sequence encoding a histone, wherein expression may be under control of a constitutive or inducible promoter. The nucleic acid sequence may comprise a signal sequence capable of mediating secretion of the histone by a transfected cell.
The nerve injury may be injury in the peripheral and/or central nervous system. The nerve injury may comprise a lesion to the brain, the spinal cord, sensory neurons and/or motoneurons. The nerve injury may be inflicted by an operation, a disease, in particular a degenerative disease and/or trauma.
The composition may be formulated for administration, e.g., by injection or intraoperational administration, preferably locally, e.g., at the site of the lesion. If the injury is inflicted by an operation, or if there is a danger that an operation may inflict an injury to a nerve, the composition is preferably administered intraoperationally, e.g., similar to the administration in the example. It can e.g. be beneficial to administer the pharmaceutical composition of the invention in the context of prostate surgery/prostatectomy to reduce complications due to nerve injury, in the context of surgery near the spinal cord or brain surgery, e.g., due to a brain tumor. When injury due to a trauma is treated by an operation, this also opens the option of intraoperational administration. Other options are infusion into the lumbar spinal cord or into the brain, e.g. by using an Alzet osmotic pump. Alternatively, the pharmaceutical composition of the invention may be administered by injection, e.g., at the site of the lesion or by intravenous injection, or by infusion into the diseased tissue.
In the context of the invention, it is also possible to administer to the patient cells, preferably autologous or syngeneic or histologically compatible cells, e.g., fibroblasts or stem cells, which are genetically engineered for expressing histones, preferably overexpressing histones, e.g., histone H1 and/or the histones described above, or to administer a gene transfer vector e.g. an adenoviral vector or adeno-associated vector engineered to induce expression of said histones. Preferably, in the context of gene therapy of the invention, an extracellular expression of histones is achieved. The skilled person is aware of suitable vectors, promoters, signal sequences for extracellular expression and methods. If appropriate, a promotor suitable for expression in cells of the nervous system may be used.
Similar positive effects on recovery after femoral nerve lesion in mice as with histone Hl were observed for PSA using PSA mimicking peptides (Mehanna et al., 2009a). The time course of improvement is similar for histone H1, with positive effects becoming apparent later, i.e. at 2-3 months after injury and treatment, leading to the question how, e.g., a single intra-operative application of a protein can produce this late-appearing and long-lasting effect of histone Hl. Local availability of a protein is most likely limited to hours or at best days after application due to its degradation. Therefore, without intending to be bound by the hypothesis, it is proposed that histone H1 modulates early cellular and molecular responses to injury such that subsequent regeneration processes are positively influenced over weeks. Based on the in vitro data of enhanced proliferation and process elongation of Schwann cells shown in the Examples, it can be concluded that histone Hl stimulates an early response of these cells to injury in vivo resulting in priming of injured femoral nerve at the lesion site to regenerate.
The pharmaceutical composition of the present invention may thus be administered to the patient once, preferably as soon as possible after the lesion has occurred or has been detected. It may e.g., be administered directly (i.e. within 10 min or within 1 or 2 hours) after this time point, or within 1, 2, 3, 4 or 5 days after this time point. Alternatively, the pharmaceutical composition of the present invention may be administrated constantly over a time period of days or weeks, e.g., using an Alzet osmotic pump, infusion or grafting of histone-expressing cells, e.g stem cells or fibroblasts that are transfected with histone-expressing cell or engrafting histone encoding adeno-associated viral (AAV) vector. Such transfected cells may be allogeneic or syngeneic cells.
The pharmaceutical composition of the invention may also be administered before a lesion is formed or detected, to reduce the risk of nerve injury. Prevention of nerve injury can thus be a reduction of a risk of nerve injury. The invention in one aspect provides a method of reducing the risk of nerve injury, e.g., due to an operation, wherein the composition of the invention is administered at the site of the risk of nerve injury in the context of an operation, e.g., prostate surgery or brain surgery.
The pharmaceutical composition of the invention may also be administered twice, three or four times or more. It may also be formulated as a sustained release formulation. Of course, if there is more than one lesion, the composition may be administered at more than one lesion.
Since neurite outgrowth was promoted by histone H1, it is likely that regeneration of the injured nerve by histone H1 is accompanied by an enhanced axon regrowth in vivo. This notion was confirmed by retrograde-labeling of motoneurons showing that survival of motoneurons, axon regrowth and precision of reinnervation was significantly enhanced.
The diverse and, importantly, long-lasting effects of the histone H1 treatment suggest its involvement in basic signaling mechanisms which are either PSA-dependent or PSA-independent. PSA associated with NCAM acts as a positive modulator of NCAM functions by inhibiting the cis-interactions of NCAM and making more NCAM available for trans-interactions for instance with heparan sulfate proteoglycans which promote axonal growth and synaptic plasticity. On the other hand, histone H1 may bind to PSA-NCAM and trigger its diverse functions via NCAM-mediated signal transduction and/or it may bind to other binding partners such as heparan sulfate proteoglycans and trigger PSA- and NCAM-independent signal transduction pathways. Interaction of histone H1 with NCAM binding partners, such as heparan sulfate proteoglycans and prion protein, may modulate the interaction with PSA and its binding partners such as BDNF and further influence PSA-dependent signaling transduction pathways.
The diverse functions of histone H1 in different neural cell types and its interaction with different binding partners show that it plays a central role in modulating and integrating various cellular functions associated with cell survival, proliferation and regeneration in a PSA-dependent and -independent manner.
The pharmaceutical composition of the present invention thus has been shown to be useful for inducing neurite outgrowth, for promoting process formation of Schwann cells, for promoting proliferation of Schwann cells, for enhancing migration and differentiation of neural progenitor cells (neural stem cells), for promoting survival of neurons, for promoting stem cell functions and/or promoting functional recovery of imperiled and/or injured neurons in acute and chronic injuries (including neurodegeneration) as well as enhancing synapse formation and synaptic plasticity. Histones can thus also be used for enhancing learning and/or memory, e.g., in treatment of diseases associated with deficiencies in learning and/or memory.
The following examples are intended to illustrate the present invention, but not to limit the scope thereof. All publications cited herein are herewith incorporated herein by reference.
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U.S. Pat. No. 6,204,242
U.S. Pat. No. 6,884,423
US 2003/0078204
Rabbit polyclonal antibody against human histone H1 (Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal antibody AE-4 against histone 1-11 (Millipore/Upstate, Schwalbach, Germany), horseradish peroxidase (HRP) conjugated NeutrAvidin (Thermo Scientific/Pierce, Bonn, Germany), HNK-1 antibody 412 (Kruse et al., 1984) and mouse antibody G3G4 against BrdU (Developmental Studies Hybridoma Bank, Iowa City, Iowa, USA) were used, as well as polyclonal rabbit antibodies against human glyceraldehyde-3-phosphate dehydrogenase (Abcam, Cambridge, UK). Mouse monoclonal IgG2a antibody 735 against PSA (Frosch et al., 1985) and endoglycosidase N was a kind gift from Dr. Rita Gerardy-Schahn, (Zentrum Biochemie, Zellulare Chemie, Medizinische Hochschule, Hannover, Germany). Secondary antibodies were purchased from Dianova (Dianova USA Inc. New york, USA). Histone H1 (#14-155, bovine histone H1.4) was purchased from Millipore/Upstate (Millipore) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), colominic acid, chondroitin sulfate A and C and heparin are from Sigma-Aldrich, Steinheim, Germany. For immunohistochemistry, the following antibodies were used: goat anti-choline acetyltransferase (ChAT, Chemicon, Hofheim, Germany;
1:100), rabbit anti-tyrosine hydroxylase (TH, Chemicon, 1:800), rabbit anti-Ki67 antibody (Synaptic Systems, Gottingen, Germany; 1:1000), rat anti-serotonin antibody (5HT, Chemicon, 1:50) and Cy3-conjugated goat anti-rabbit, anti-rat, and donkey anti-goat secondary antibodies (Jackson ImmunoResearch Laboratories, Dianova, Hamburg, Germany).
The “Griffin.1” phage scFv library (Griffiths et al., 1994) consisting of ˜106 individual synthetic human scFv antibodies cloned into the phagemid pHEN2 was a kind gift from Dr. Greg Winter (Medical Research Council Center for Protein Engineering, Cambridge, England). The suppresser E. coli strain TG1 (K12, supE, hsdD5/F′, thi, Δ(lac− pro), traD36, proA+B+, lacl9, lacZΔM15) was infected with the “Griffin.1” phage scFv library and grown in 2TY medium (16 g/L trypton, 10 g/L yeast extract, 5 g/L NaCl) supplemented with 100 μg/ml ampicillin and 1% glucose. When reaching exponential growth (OD600=0.5) 2×1011 M13K07 helper phages (GE Healthcare, Uppsala, Sweden) were applied to 25 ml culture (corresponding to 1×1010 bacteria). After incubation for 30 min at 37° C. the infected bacteria were centrifuged for 10 min at 3300 g and the pellet was incubated in 2YT medium supplemented with 100 μg/ml ampicillin and 25 μg/ml kanamycin overnight at 30° C. The bacteria were pelleted at 10,800 g for 10 min at 4° C. and 0.2 volumes of PEG/NaCl (20% PEG6000 in 2.5 M NaCl) was added to the resulting supernatant containing the phages. After incubation on ice for 1 h, phages were pelleted by centrifugation at 10,800 g for 30 min and the resulting pellet was resuspended in 40 ml H2O and 8 ml PEG/NaCl and incubated for 20 min at 4° C. After centrifugation at 10,800 g for 30 min the phage pellet was resuspended in 5 ml PBS (8.1 mM Na2HPO4, 1.74 mM NaH2PO4, 0.15 M NaCl, pH 7.5).
Selection of PSA Binding scFv Antibodies
PSA antibody 735 (100 μg/ml) dilute in 4 ml PBS was incubated in immunotubes (Maxisorb, Nunc, GmbH & Co. KG, Langenselbold, Germany) overnight at 4° C. After three washes with PBS, the tubes were blocked with 2% skim milk (Frema Reform, DE-VAU-GE, Lüneburg, Germany) in PBS (MPBS) for 2 h at room temperature. In parallel, an immunotube was blocked with 2% MPBS and incubated with 4 ml PBS containing ˜4×1013 phages, 2 mg mouse serum (Sigma-Aldrich) and 24 μg IgG2a (Cymbus Biotechnology, Hampshire, UK) for 1 h at room temperature. After incubation of the tube with PSA antibody, the tube was washed three times with PBS and incubated with the preincubated phage suspension at room temperature for 30 min on a head-over-top rotator and 1.5 h without agitation. After incubation, the tube was washed 20 times with PBS containing 0.1% Tween-20, followed by 20 washes with PBS. Bound phages were eluted with 1 ml 100 mM triethylamine and immediately neutralized with 0.5 ml 1 M Tris/HCl, pH 7.4. The eluted phages were used to infect exponentially growing E. coli TG1 cells, and infected bacteria were grown overnight at 30° C. on TYE plates (15 g/L agar, 10 g/L yeast extract, 8 g/L NaCl) containing 100 μg/ml ampicillin and 1% glucose. After four rounds of selection, individual clones were analyzed by phage ELISA.
Microtiter plates (Maxisorb, Nunc) were coated overnight at 4° C. with 50 μl purified PSA antibody in PBS (100 μg/ml) per well. All following steps were done at room temperature. The plates were blocked with 2% MPBS. As negative controls 50 μl mouse serum (500 μg/ml) and IgG2a (25 μg/ml) were used. After blocking with 2% MPBS for 1 h at room temperature, plates were incubated with bacterial supernatants containing selected phages. After 6 washes with PBS, bound phages were detected using a horse radish peroxidase (HRP)-conjugated monoclonal antibody against M13 phage (GE Healthcare) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. Absorption at 405 nm was measured.
DNA Sequencing and Truncation of the scFv Linker Region
The sequences of selected clones were determined with the primers LMB3 (5′-CAG GAA ACA GCT ATG AC-3′) (SEQ ID NO:10) and fdSeq1 (5′-GAA TTT TCT GTA TGA GG-3′) (SEQ ID NO:11) by the dideoxy chain terminating method using a model 377 DNA sequencer from Applied Biosystems (Foster City, Calif., USA). For truncation of the linker region from 14 amino acids (SSGGGGSGGGGSGG) (SEQ ID NO:1) to 5 amino acids (SSGGG) (SEQ ID NO:2), pHEN2-derived plasmid DNA deriving from selected phages were subjected to PCR using the primers 5′-CAG CCG GCC ATG GCC CAG GTG-3′ (SEQ ID NO:3) and 5′GAG TCA CCG TGC ACT GCC TCC ACC ACT CGA GAC GGT GAC CAG GGT-3′(SEQ ID NO:4). The PCR product and the pHEN2 phage-derived plasmids were cut with NcoI and ApaL1 and ligated.
Production and Purification of scFv Antibodies
The nonsuppressor strain HB2151 (K12, ara, thi/F′, proA+B+, Δ(lac− pro), lacl9, lacZΔM15) was infected with selected phages to produce soluble scFv antibodies due to an amber stop codon in the pHEN2 sequence between the scFv gene and the M13 minor coat protein III gene. Bacteria transformed with selected phages were grown at 37° C. in LB medium containing 0.1% glucose and 100 μg/ml ampicillin. At OD600 0.8-0.9, production of scFv antibodies was induced by addition of isopropyl-β-D-thiogalactopyranoside at a final concentration of 1 mM. After an overnight incubation at 30° C., culture supernatants were concentrated by ultrafiltration and applied to Ni-NTA agarose beads (Qiagen, Hilden, Germany) which were equilibrated with 50 mM phosphate buffer, pH 7.5, 300 mM NaCl, and 10 mM imidazole. After an overnight incubation at 4° C., the beads were centrifuged at 2500 g for 5 min and washed with 50 mM phosphate buffer, pH 7.5, 300 mM NaCl, and 20 mM imidazole. The scFv antibodies were eluted by incubating beads with 50 mM phosphate buffer, pH 7.5, 300 mM NaCl, and 250 mM imidazole for 30 min at 4° C. and the eluted scFv antibodies were concentrated by centrifugation in Centricon-5 tubes (5 kD cut off; Millipore-Amicon) and diluted in PBS.
ELISA using scFv Antibodies
96-well microtiter plates (Mule) were coated with 100 μl purified PSA antibody (100 μg/ml PBS) overnight at 4° C. and blocked with 2% MPBS. Purified scFv antibodies supplemented with 2% skimmed milk were added to the plates and incubated for 1.5 h. For competition, colominic acid, chondroitin sulfate A and C or heparin were preincubated with the coated antibody before addition of scFv antibodies. After washing three times with 0.05% Tween 20 in PBS and three times with PBS, anti-myc tag antibody (Santa Cruz Biotechnology) was added and incubated for 1 h. The wells were washed again and incubated with HRP-conjugated secondary antibody for 1 h. Binding was detected using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. All steps were carried out at room temperature.
The PSA antibody 735 was immobilized on a CM5 sensor chip (Biacore, Uppsala, Sweden). Purified scFv antibodies in PBS were injected and sensorgrams were recorded using a Biacore 3000 instrument. For competition, 5 μM of scFv antibody were mixed with the competitor before injection.
Postnatal (7 days old) or and adult (12-16 weeks old) C57BL/6J mice were decapitated; brains were immediately transferred into a homogenizer (Teflon pestle, 0.1 μm; Wheaton, Millville, USA) and homogenized in 3 ml of homogenization buffer (0.32 M sucrose, 1 mM CaCl2, 1 mM MgCl2, 5 mM Tris/HCl, pH 7.5, and protease inhibitor (Roche, Mannheim, Germany) and applying 10-12 up-and-down strokes. This step and all following steps were carried out on ice or at 4° C. The homogenate was centrifuged for 10 min at 17,000 g. The pellet was re-suspended in homogenization buffer, applied to a 1.2 M, 1.0 M and 0.8 M sucrose step gradient, and centrifuged for 2 h at 100,000 g. The material from the 1.0/1.2 M sucrose interface was collected, diluted and pelleted by centrifugation for 20 min at 17,000 g. The pellet enriched in synaptosomal membranes was re-suspended in PBS containing 1% Triton X-100 and incubated for 30 min. This fraction was taken as a detergent-solubilized synaptosomal membrane subfraction.
For the preparation of membrane-associated proteins, brains were homogenized in 4 ml TNE buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA) using a homogenizer. The homogenates were centrifuged at 1000 g for 10 min and the resulting supernatant was centrifuged at 100,000 g for 30 min. The pellet was resuspended in 2 ml TNE and adjusted to 150 mM NaHCO3, pH 11.5. After incubation for 30 min the sample was subjected to centrifugation at 100,000 g for 30 min. The resulting supernatant was taken as a subfraction enriched in membrane-associated protein.
Affinity Chromatography with scFv Antibodies
The fraction containing membrane-associated proteins was first run over a Sepharose 4B column (GE Healthcare) and then run over a column with 3.2 mg purified scFv antibodies immobilized on 2 ml CNBr-activated Sepharose 4B (GE Healthcare) overnight at 4° C. (0.2 ml/min). After washing the column with 15 ml PBS (0.3 ml/min) bound proteins were eluted with 200 mM glycine/500 mM NaCl, pH 2.7 (0.3 ml/min). The collected elution fractions were neutralized immediately with 1 M Tris/HCl, pH 8.8. After ultrafiltration using Centricon-5 tubes (Millipore-Amicon) the proteins were analyzed by SDS-PAGE, silver staining and mass spectrometry.
Gels were incubated in methanol/acetic acid/water (50:5:45) for 30 min, washed two times with water, incubated in water for 1 h and exposed for 1 min to 0.02% sodium thiosulfate. After washing two times with water, the gels were incubated with pre-cooled 0.1% silver nitrate for 30 min at 4° C. and washed two times with water. Color development was started by addition of 2% sodium carbonate/0.04% formaldehyde and stopped by addition of 2.3 M citric acid.
Postsynaptic densities (PSDs) were prepared according to a slightly modified protocol previously described (Cohen et al., 1977). All steps were carried out at 4° C. or on ice. Brains were homogenized in 3 ml homogenization buffer per brain. The homogenates were centrifuged at 1000 g for 10 min. The supernatant (Si) was saved and the pellet was again homogenized with three strokes. After centrifugation at 1000 g for 10 minutes, the supernatant was pooled with Si and spun at 1000 g. The resulting supernatant was then centrifuged at 17,000 g and the pellets were re-suspended in homogenization buffer and centrifuged at 17,000 g for 10 min. The pellets were re-suspended in solution A (0.32 M sucrose and 1 mM NaHCO3) and applied to a 0.85 M, 1.0 M, and 1.2 M sucrose gradient. The gradients were run for 2 h at 100,000 g. The band between 1.0 and 1.2 M sucrose containing synaptosomes was then re-suspended in 4 volumes of solution A and centrifuged at 48,200 g for 20 min. The supernatant was re-suspended in solution B (0.5% Triton X-100 in 0.16 M sucrose, 6 mM Tris/HCl, pH 8.1) and centrifuged at 48,200 g for 20 min. The pellet was re-suspended in 2.5 ml of solution A, and 2 ml of this material was layered onto gradients composed of 3 ml of 2.0 M sucrose, 3.5 ml of 1.5 M sucrose and 3.5 ml of 1.0 M sucrose all containing 1 mM NaHCO3. The gradients were spun for 2 h at 275,000 g. The PSDs banded at the interphase of 1.5 M and 2.0 M sucrose.
Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using acrylamide slab gels and transferred to nitrocellulose membranes (Protran, Schleicher & Schuell, Dassel, Germany). Membranes were blocked in 5% milk powder in PBS for 1 h at room temperature and incubated with primary antibodies in blocking buffer overnight at 4° C. The appropriate HRP-conjugated secondary antibodies (Dianova) were applied after six washing steps with PBS containing 0.1% Tween-20. After incubation for 1 h at room temperature with secondary antibodies, membranes were washed again with PBS containing 0.1% Tween-20 for six times and blots were developed using enhanced chemiluminescence (Thermo Scientific).
384-well flat bottom micro titer plates (Corning, Lowell, Mass., USA) were coated in triplicates with 25 pi of histone H1 and GAPDH (10 μg/ml in PBS) or PBS alone as control at 4° C. over-night. After blocking for 1 h at room temperature with PBS containing 1% BSA, the wells were washed 3 times with PBS containing 0.05% Tween-20 (PBST). Wells were incubated with increasing concentration of soluble colominic acid for 1 h at room temperature. After wells were washed 5 times with PBST and 2 times with PBS, 25 μl of the PSA antibody 735 (5 μg/ml) were added to each well. After 1 h incubation of wells at room temperature, the plates were washed 6 times with PBST and 2 times with PBS. 25 μl of HRP-conjugated anti-mouse IgG were added to each well and incubated at room temperature for 1 h and then washed again. Antibody binding was visualized by incubating wells with 50 μl of 0.5 mg/ml o-phenylenediamine dihydrochloride (Thermo Scientific) for 2 to 5 min. The reaction was stopped by addition of 50 μl 2.5 M H2SO4 and quantified using an ELISA reader at 490 nm and the software KCjunior (Bio-TEK, Winooski, Vt., USA).
Primary cultures of cerebellar granule cells were seeded at a density of 2×105 cells/ml into 48-well plates (Nunc) and pre-treated with 0.01% poly-L-lysine (PLL) overnight at 4° C., washed twice with water, and air-dried. Histone H1 and laminin were substrate-coated, i.e., coated onto the surfaces overnight at a concentration of 10 μg/ml onto the dried surfaces at 4° C. The plates were washed two times with PBS, and neurons were plated at a density of 105 cells/well in 300 μl of chemically defined serum-free culture medium. For antibody blocking experiments, rabbit polyclonal antibodies against histone H1 or PSA antibody 735 were added to the wells at a concentration of 10 μg/ml or 40 μg/ml one hour after cells had been seeded. At the same time, cells were treated with soluble histone H1 and/or soluble colominic acid (10 μg/ml). Endoglycosidase N was added to the neurons at a concentration of 4.8 μg/ml. After 24 h, cells were fixed by the gentle addition of 25% glutaraldehyde to a final concentration of 2.5% (Agar Scientific, Stansted, UK). After fixation, cultures were stained with 1% methylen blue/toluidine blue (Sigma-Aldrich) in 1% borax. Morphological parameters were quantified with the Axiovert microscope and the AxioVision image analysis system 4.6 (Carl Zeiss Micro Imaging, Gottingen, Germany). For morphometric analysis, only cells without contact with other cells were evaluated. Neurites were defined as those processes with a length of at least one cell body diameter. To determine the total neurite length per cell, 50 cells in each of two wells were analyzed per experiment.
Schwann cells were isolated from dorsal root ganglia (DRG) of 7-day-old C57BL/6J mice. Tissues were removed, washed once with ice-cooled Ham's F-12 (PAA Laboratories, Cölbe, Germany), and then incubated with 0.25% trypsin and 0.1% collagenase (Sigma-Aldrich) at 37° C. for 30 min. After enzymatic digestion, tissues were washed twice with ice-cooled Ham's F-12 medium and then suspended in 1 ml Ham's F-12 medium containing 0.01% DNase (Sigma-Aldrich). Mechanical digestion was performed using fire-polished Pasteur pipettes, and cells were suspended in 5 ml Ham's F-12 medium, added on top of 5 ml of a 4% bovine serum albumin (BSA, fraction V, PAA Laboratories) cushion and centrifuged for 10 min at 4° C. and 500 g. Finally, Schwann cells were suspended in fresh pre-warmed (37° C.) medium and seeded on 48-well plates (Nunc), that were pre-treated with 0.01% poly-L-lysine (PLL) and coated with laminin (10 μg/ml, Sigma-Aldrich) or purified histone H1. The medium used for Schwann cell culture contained DMEM high glucose/Ham's F-12 (1:1) (PAA Laboratories), 60 ng/ml progesterone (Sigma-Aldrich), 16 μg/ml putrescine (Sigma-Aldrich), 5 μg/ml insulin (Sigma-Aldrich), 0.4 μg/ml L-thyroxine (Sigma-Aldrich), 160 ng/ml sodium selenite (Sigma-Aldrich), 10 ng/ml triiodothyronine (Sigma-Aldrich), 38 ng/ml dexamethasone (Sigma-Aldrich), 100 U/ml penicillin (PAA Laboratories), 100 μg/ml streptomycin (PAA Laboratories) and 2 mM L-glutamine (PAA Laboratories). Schwann cells were seeded at a density of 50,000 cells/ml. Cells were treated with soluble histone H1 and/or soluble colominic acid (10 μg/ml) 1 h after cells had been seeded. After incubation for 24 h at 37° C., cells were fixed with 2.5% glutaraldehyde and stained with 1% methylen blue/toluidine blue in 1% borax. The length of processes was measured by using the Axiovert microscope and the AxioVision image analysis system 4.6. For morphometric analysis, only cells without contact with other. cells were evaluated. Processes were defined as those processes with a length of at least one cell body diameter. To determine the total neurite length per cell, 50 cells in each of two wells were analyzed per experiment.
Schwann cells were seeded at a density of 250,000 cells/ml onto 12-well plates (Nunc), containing 12 mm glass coverslips pre-treated with 0.01% poly-L-lysine (PLL) and coated with laminin and purified histone H1 at a concentration of 10 μg/ml. The cells were cultured in the presence of neuroregulin (12 ng/ml, ImmunoTools, Friesoythe, Germany). 20 μM BrdU (Sigma-Aldrich) was added to the culture 4 h after seeding. One hour after BrdU treatment, cells were treated with soluble histone H1 and/or soluble colominic acid at a concentration of 10 μg/ml and then cultured for 48 h. Cells were washed shortly with PBS and fixed with 4% formaldehyde in 0.1 M phosphate buffer, pH 7.3. After incubation for 30 min with 2 N HCl at 37° C., cells were washed, blocked with goat serum and incubated overnight with mouse primary antibodies G3G4 against BrdU (1:100 dilution). Appropriate secondary antibodies coupled to fluorescent dye Cy3 were applied to the cells in the dilution 1:200 for 1 h at room temperature in the dark. The coverslips were finally washed and mounted with Fluoromount-G (Southern Biotech, Birming-ham, UK). To estimate numbers of proliferating cells, 10 photographs per treatment were taken from different areas of the coverslip using an Axiophot 2 microscope (Carl Zeiss Microlmaging, Inc. Thornwood, N.Y. 10594, USA) and a 20× objective. Each area was photographed using phase contrast and epifluorescence. The two digital images were then overlaid using the Image J software (http://rsbweb.nih.gov/ij/download.html) and Image Tool 2.0 software (University of Texas, San Antonia, Tex., USA, http://ddsdx.uthscsa.edu/dig/) was used to count BrdU-positive Schwann cells and total number of Schwann cells. Schwann cells in culture have a long spindle-shaped cell body and two processes in opposite directions, which makes them easily distinguishable from other contaminating cells (DRG neurons and fibroblasts). Approximately 1,000 cells were counted for each experimental value.
2×105 Schwann cells or cerebellar neurons were seeded on 12 mm glass coverslips pre-treated with 0.01% PLL. 24 h after seeding, cells were washed three times with the corresponding culture medium containing 1% BSA. Cells were incubated on ice for 20 min with the primary antibodies against PSA and histone H1. Cells were then washed gently with serum-containing medium and appropriate secondary antibodies coupled to fluorescent dyes Cy3 and Cy 2 were applied to the cells for 20 min at room temperature in the dark. After washing with serum-free culture medium, cells were fixed with 4% formaldehyde for 15 min at room temperature, washed again three times with PBS. Cells were stained with DAPI for 2 min and finally washed three times with PBS and then mounted with Fluoromount-G. Images were taken with an Olympus Fluoview 1000 microscope (Olympus, Hamburg, Germany).
Mouse embryos (E13.5) were decapitated, mid-dissection-embryo brain skin was exposed and skull peeled away. Developing striatum were dissected and transferred to serum-free media and then mechanically dissociated by pipetting up and down with three fire-polished Pasteur pipettes with sequentially smaller diameters. After dissociation, the neurospheres were centrifuged at 200 g for 1 min at room temperature, re-suspended with 0.5 ml of accutase (Invitrogen GmbH, Karlsruhe, Germany) and incubated at 37° C. for 10 min. 0.5 ml of neural stem cell medium was applied to the cells and pipetted up and down for 15 to 20 times. After centrifugation at 200 g for 5 minutes at room temperature, cells were re-suspended in 5 ml of neural stem cell medium and diluted to 105 cells/ml of neural stem cell medium.
Neurospheres were allowed to settle for 10 minutes after culture in neurosphere-formation medium (DMEM/F12 (1:1) without L-glutamine, lx B27 supplement (Invitrogen GmbH,), 0.02% sodium bicarbonate (GIBCO, Invitrogen GmbH), 0.5 M HEPES (Sigma-Aldrich), 16.6 mM glucose (Sigma-Aldrich), 100 mM glutamine (Invitrogen GmbH), 20 ng/ml EGF, 20 ng/ml FGF-2 for 4-5 days. After washing three times in pre-cooled PBS, neurospheres were fixed with 4% formaldehyde in filtered 0.4 M cacodylate buffer, pH 7.3, at 4° C. for 1 h and dehydrated with 30% sucrose in cacodylate buffer at 4° C. for 4 h. The neurospheres were embedded in tissue-tag, frozen at −30° C. and sectioned at 14 μm on a cryostat (Leica CM 3050, Leica instruments GmbH, Nul3loch, Germany). Sections were blocked with 10% goat serum and 0.1% Triton X-100 at room temperature for 1 h before being incubated with primary antibodies against PSA and histone H1 at 4° C. overnight. After washing three times with PBS, appropriate secondary antibodies coupled to the fluorescent dyes Cy3 and Cy 2 were applied to the cells for 1 h at room temperature in the dark. Washed three times with PBS, the sections were labeled with bisbenzimide (Sigma-Aldrich) for nuclei staining and mounted with Fluoromount-G after additional washes in PBS. Images were taken with an Olympus Fluoview 1000 microscope (Olympus).
After 10 to 15 days of culture in vitro in neurosphere-formation medium, the cell migration assay was performed according to Decker et al. (2000). Briefly, the non-adherent neurospheres were seeded in 10 μl droplets on glass coverslips pre-treated with 0.02% PLL followed by laminin and histone H1 as substrates. Neurospheres were incubated at 37° C. for 20 min. The medium was adjusted to 1 ml per well with neurosphere-formation medium (without FGF-2 and EGF) containing soluble colominic acid and/or histone H1. After 24 h, migration was assessed by measurement of the distance from the edge of the neurosphere to all cells having migrated out of the neurosphere using an Axiovert microscope and the AxioVision image analysis system 4.6 (Carl Zeiss Vision GmbH).
Femoral injury and analysis of regeneration were performed according to Simova et al., (2006). Three-month-old female C57BL/6J mice were obtained from the central animal facility of the Universitatsklinikum Hamburg-Eppendorf. All experiments were conducted in accordance with the German and European Community laws on protection of experimental animals. The procedures used were approved by the responsible committee of The State of Hamburg. For surgery, the animals were anaesthetized by intra-peritoneal injections of 0.4 mg kg−1 fentanyl (Fentanyl-Janssen, Janssen-Cilag, Neuss, Germany), 10 mg kg−1 droperidol (Dehydrobenzpe-ridol, OTL Pharma, Paris, France) and 5 mg kg−1 diazepam (Ratiopharm, Ulm, Germany). The right femoral nerve was exposed and nerve transection performed at a distance of approximately 3 mm proximal to the bifurcation of the nerve into motor and sensory branches. A polyethylene tubing (3 mm length, 0.58 mm inner diameter; Becton Dickinson, Heidelberg, Germany) was placed between the two nerve stumps and filled with PBS containing scaffold peptide that forms a gel matrix support (0.5% PuraMatrix Peptide Hydrogel, 3D, BD Biosciences, San Jose, Calif., USA), or PBS/scaffold peptide supplemented with histone H1 (100 μg/ml). The cut ends of the nerve were inserted into the tube and fixed with single epineural 11-0 nylon stitches (Ethicon, Norderstedt, Germany) so that a 2 mm gap was present between the proximal and distal nerve stumps. Finally, the skin wound was closed with 6-0 sutures (Ethicon).
Functional recovery was assessed by single-frame motion analysis (Irintchev et al., 2005; Ahlborn et al., 2007) to evaluate quadriceps muscle function during ground locomotion. Prior to operation, mice were trained to perform a beam-walking test in which the animal walks unforced from one end of a horizontal beam (length 1000 mm, width 38 mm) towards its home cage located at the other end of the beam. A rear view of one walking trial per animal was captured prior to the operation with a high-speed camera (A602fc, Basler, Ahrensburg, Germany) at 100 frames per second and stored on a personal computer in Audio Video Interleaved (AVI) format. The recordings were repeated 1, 2, 4, 8, 12 and 16 weeks after nerve transection. The video sequences were examined using SIMI-Motion 7.0 software (SIMI Reality Motion Systems, Unterschleissheim, Germany). Selected frames in which the animals were seen in defined phases of the step cycle were used for measurements of two parameters as described previously (Irint-chev et al., 2005): the foot-base angle (FBA) and the heels-tail angle (HTA). The analysis was performed with UTHSCSA Image Tool 2.0 software (University of Texas, San Antonia, Tex., USA, http://ddsdx.uthscsa.edu/dig/). Both parameters are directly related to the ability of the quadriceps muscle innervated by the motor branch of the femoral nerve to keep the knee joint extended during contralateral swing phases. As a relative measure of functional recovery at different time-point after nerve injury, we calculated the stance recovery index, which is a mean of the recovery index (RI) for the HTA and the FBA. The index for each angle is calculated in percent as: RI=[(Xreinn−Xden)/(Xpre−Xden)]×100, where Xpre, Xden and Xreinn are values prior to operation, during the state of denervation (7 days after injury), and at any given time-point during reinnervation, respectively. A third parameter, the limb protraction length ratio (PLR), was evaluated from video recordings of voluntary pursuit movements of the mice. The mouse, when held by its tail and allowed to grasp a pencil with its fore paws, tries to catch the object with its hind paws and extends simultaneously both hind limbs. In intact animals the relative length of the two extremities, as estimated by lines connecting the most distal mid-point of the extremity with the anus, is approximately equal and the PLR (ratio of the right to left limb length) is close to 1. After unilateral injury, the denervated limb cannot extend maximally and the PLR increases significantly above 1.
Following the last video recording, mice were re-operated for retrograde labelling. Under fentanyl/droperidol/diazepam anaesthesia, the two nerve branches were transected ˜5 mm distal to the bifurcation, and two fluorescent dyes, Fast Blue (EMS-Chemie, GroBumstadt, Germany) and Fluoro-Gold (Fluorochrome, Denver, Colo., USA), were applied to the motor and sensory branches, respectively. One week later, the animals were perfused with 4% formaldehyde (Sigma-Aldrich) in 0.1 M sodium cacodylate buffer, pH 7.3, and spinal cords and femoral nerves were dissected for morphological analyses. After overnight fixation, the lumbar part of the spinal cord was cut transversely (serial sections of 50 μm thickness) on a Leica vibratome VT1000S (Leica CM 3050,Leica instruments GmbH). The sections were examined under a fluorescence microscope (Axiophot 2, Zeiss) with appropriate filter sets. All cell profiles labeled with one of the dyes or with both tracers are distributed within a stack of 35-45 serial cross-sections. Each section, containing typically 2-5 labeled cell profiles, was examined using a 40× objective by focusing through the section thickness starting from the top surface. All profiles except those visible at the top surfaces of sections were counted (Simova et al., 2006). The application of this simple stereological principle prevents double counting of labelled cells and allows an unbiased evaluation of cell numbers, which does not rely on assumptions or requires corrections. The same sections were used for measurements of soma size using Neurolucida software-controlled computer system (MicroBrightField Europe, Magdeburg, Germany).
After fixation with 4% formaldehyde, femoral nerves were post-fixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer, pH 7.3, for 1 h at room temperature, dehydrated and embedded in resin according to standard protocols. Transverse 1 μm-thick sections from the motor and sensory nerve branches were cut (Ultramicrotome, (Leica CM 3050, Leica instruments GmbH) at a distance of ˜3 mm distal to the bifurcation and stained with 1% toluidine blue/1% borax in distilled water. Axonal and nerve fibre diameters were measured in a random sample from each section using a 100× oil objective and a grid with a line spacing of 30 μm projected into the visual field of an Axioskop microscope (Carl Zeiss Vision GmbH, Munich, Germany) equipped with a motorized stage and NeProlucida software (MicroBrightField Europe). Selection of the reference point (zero coordinates) of the grid was random. For all myelinated axons crossed by or attaching the vertical grid lines through the sections, mean orthogonal diameters of the axon (inside the myelin sheath) and of the nerve fiber (including the myelin sheath) were measured. The mean orthogonal diameter is calculated as a mean of the line connecting the two most distal points of the profile (longest axis) and the line passing through the middle of the longest axis at right angle. The degree of myelination was estimated by the ratio axon to fiber diameter (g-ratio).
For surgery, three to four month old C57/BL6J mice were anesthetized by intraperitoneal injections of ketamin and xylazin (100 mg Ketanest®, Parke-Davis/Pfizer, Karlsruhe, Gerthany, and 5 mg Rompun®, Bayer, Leverkusen, Germany, per kg body weight). Laminectomy was performed at the T7-T9 level with mouse laminectomy forceps (Fine Science Tools, Heidelberg, Germany). A mouse spinal cord compression device was used to elicit compresion injury (Curtis et al., 1993). Compression force (degree of closure of the forceps) and duration were controlled by an electromagnetic device: The spinal cord was maximally compressed [100%, according to the operational definition of Curtis et al] for 1 second by a time-controlled current flow through the electromagnetic device. Muscles and skin were then closed using 6-0 nylon stitches (Ethicon, Norderstedt, Germany). After the operation, mice were kept in a heated room (37° C.) for several hours to prevent hypothermia and thereafter singly housed in a temperature-controlled (22° C.) room with water and standard food provided ad libitum. During the post-operative time-period the bladders of the animals were manually voided twice daily.
Alzet osmotic pumps (model 1002, 14 days infusion, Durect, Cupertino, Calif., USA) were filled with phosphate buffered saline (PBS) alone or with histone H1 (see below). Each pump was connected to a vinyl catheter (Durect). The distal tip of the catheter was stretched by hand upon fire to fit its diameter to the subdural space (<0.4 mm). For subdural insertion of the catheter, the lumbar vertebral column was exposed and a hole between vertebrae L4-L5 was made using a needle with a 0.4 mm outer diameter. Leakage of the cerebrospinal fluid was taken as proof for penetration of the dura. The catheter was then inserted into the hole and fixed to the surrounding tissue with a 6-0 filament (Ethicon). The pump was placed subcutaneously on the left side of the back over the thorax and the skin was closed with 6-0 nylon sutures. In pilot experiments, proper placement of the catheter in the subdural space was confirmed in four mice without spinal cord injury using magnetic resonance imaging (data not shown). After sacrifice of these animals six weeks after pump implantation, the appropriate catheter position was confirmed by microscopic examination. During the 6-week observation period, no adverse effects of the pump implantation, such as infections, paralyses or aberrant behavior, were observed.
The recovery of ground locomotion was evaluated using Basso Mouse Scale (Basso et al., 2006). In addition, we used a more complex and more objective assessment of locomotion, single-frame motion analysis (Apostolova et al., 2006). This approach includes evaluation of 4 parameters in 3 different tests: beam walking (foot-stepping angle, rump-height index), voluntary movements without body weight support (extension-flexion ratio) and inclined ladder climbing (number of correct steps). The foot-stepping angle is defined by a line parallel to the dorsal surface of the hind paw and the horizontal line. The angle is measured with respect to the posterior aspect at the beginning of the stance phase. In intact mice, this phase is well defined and the angle is around 20°. After spinal cord injury and severe loss of locomotor abilities, the mice drag behind their hind limbs with dorsal paw surfaces facing the beam surface. The angle is increased to >150°. The second parameter, the rump-height index, was estimated from the recordings used for measurements of the foot-stepping angle. The parameter is defined as height of the rump, i.e., the vertical distance from the dorsal aspect of the animal's tail base to the beam, normalized to the thickness of the beam measured along the same vertical line. The third parameter, the limb extension-flexion ratio, was evaluated from video recordings of voluntary movements of the mice. An intact mouse, when held by its tail and allowed to grasp a pencil with its forepaws, tries to catch the object with its hind paws and performs cycling flexion-extension movements with the hind limbs. The extension-flexion ratio is a numerical estimate of the animal's ability to initiate and perform voluntary, non-weight bearing movements. Such movements require connectivity of the spinal cord to supraspinal motor control centers but, in the form evaluated here, no coordination or precision. Assessment was performed before and at 1, 3 and 6 weeks after the injury. Values for the left and right extremities were averaged.
Mice were anaesthetized with a 16% solution of sodium pentobarbital (Narcoren, Merial, Hallbergmoos, Germany, 5 μl/g body weight). The animals were transcardially perfused with fixative consisting of 4% formaldehyde and 0.1% CaCl2 in 0.1M cacodylate buffer, pH 7.3, for 15 minutes at room temperature (RT). Following perfusion, the spinal cords were left in situ for 2 hours at RT, after which they were dissected out and post-fixed overnight (18-22 hours) at 4° C. in the same solution used for perfusion. Tissue was then immersed into a 15% sucrose solution in 0.1M cacodylate buffer, pH 7.3, for two days at 4° C., embedded in Tissue Tek (Sakura Finetek, Zoeterwoude, NL), and frozen by a 2 minute immersion into 2-methyl-butane (isopentane) precooled to −80° C. Serial parasagittal sections were cut in a cryostat (Leica CM3050, Leica Instruments, Nussloch, Germany). Sections, 25 μm thick, were collected. on SuperFrost Plus glass slides (Roth, Karlsruhe, Germany). Sampling of sections was always done in a standard sequence so that 6 sections 250 μm apart were present on each slide.
Procedures for immunohistochemistry have been described in detail elsewhere (Jakovcevski et al., 2009). Water-bath antigen de-masking was performed in 0.01M sodium citrate solution, pH 9.0, for 30 minutes at 80° C. for all antigens. Non-specific binding was blocked using 5% normal serum from the species in which the secondary antibody was produced, dissolved in PBS and supplemented with 0.2% Triton X-160, 0.02% sodium azide for 1 hour at room temperature (RT). Incubation with the primary antibody (anti-ChAT, anti-Ki67, anti-serotonin or anti-TH), diluted in PBS containing 0.5% lambda-carrageenan (Sigma) and 0.02% sodium azide, was carried out for 3 days at 4° C. After washing in PBS (3×15 minutes at RT), the appropriate secondary antibody, diluted 1:200 in PBS-carrageenan solution, was applied for 2 hours at RT. After a subsequent wash in PBS, cell nuclei were stained for 10 minutes at RT with bisbenzamide solution (Hoechst 33258 dye, 5 μg/ml in PBS, Sigma). Finally, the sections were washed again, mounted in anti-quenching medium (Fluoromount G, Southern Biotechnology Associates, Biozol, Eching, Germany) and stored in the dark at 4° C. Photographic documentation was made on an LSM 510 confocal microscope (Zeiss, Oberkochen, Germany) or an Axiophot 2 microscope equipped with a digital camera AxioCam HRC and AxioVision software (Zeiss). The images were processed for brightness and contrast using Adobe Photoshop 7.0 software (Adobe Systems Inc., San Jose, Calif.).
Estimations of motoneuron soma areas and perisomatic terminals were performed as described (Apostolova et al., 2006). Longitudinal spinal cord sections stained for ChAT were examined under a fluorescence microscope to select sections that contained motoneuron cell bodies for a distance of at least 500 μm distal from the lesion scar. Stacks of 1 μm thick images were obtained on the LSM 510 confocal microscope using a 63×1.5 oil immersion objective and digital resolution of 1024×1024 pixels. One image (optical slice) per cell at the level of the largest cell body cross-sectional area was used to measure soma area, perimeter and number of perisomatic terminals. Areas and perimeters were measured using the Image Tool 2.0 software program (University of Texas, San Antonio, Tex., USA). Density of ChAT+ terminals was calculated as number of perisomatic terminals per unit length of the cell surface.
Spaced serial 25 μm-thick longitudinal sections 250 μm apart were stained with Cresyl Violet/Luxol Fast Blue and used for estimations of the scar volume and myelin volume using the Cavalieri principle as previously described (Mehanna et al., 2010). Area measurements required for volume estimation were done directly under the microscope using Neurolucida software (Brightfield).
For cultures of astrocytes, brains of 1- to 2-day-old C57BL/6J mice were stripped of meninges, dissected free from brainstem, and cut into small pieces that were subjected to mechanical dissociation by several passages through a Pasteur pipette and successive passage through 200, 150, and 30 μm nylon mesh filters (VWR, Darmstadt, Germany). Poly-L-lysine (0.1 mg/ml) in water was used for coating plastic dishes. Cells were kept for 7 d at 37° C. in a water-saturated atmosphere containing 5% CO2 by replacing the medium every 2-3 days. Cells were seeded on plastic 35 mm dishes or 12-well plates (Greiner BioOne, Frickenhausen, Germany) at a density of 105/cm2 in DMEM medium supplemented with 10% FCS, 0.6% glucose, 25 mM NaHCO3, 200 nM glutamine, and antibiotics (50 U/ml penicillin, 50 μg/ml streptomycin, and 50 μg/ml gentamicin).
Five to seven days later, when astrocytes reached confluence, a denuded area was produced by scratching the inside diameter of the well with a 100 μl pipette tip (Jakovcevski et al., 2007). After washing with Hank's Balanced Salt Solution (H15-010; PAA) once, fresh culture medium DMEM supplemented with 10% FCS, 0.6% glucose, 25 mM NaHCO3, 200 nM glutamine, and antibiotics (50 U/ml penicillin, 50 μg/ml streptomycin, and 50 μg/ml gentamicin was added into each well. Several photographs from each well were taken under Axiovert (Zeiss) microscope immediately after the scratch wound was made (0 h) and at 20 h after the scratch wound assay. To determine the dependency of migration on histone H1 and polysialic acid, astrocytes were treated with either histone H1 or antibody against polysialic acid (to block endogenous polysialic acid). The denuded area was gradually covered by migrating astrocytes in control samples wells but migrations of astrocytes were inhibited in histone H1 treated wells. Using an Axiovert microscope (Zeiss) equipped with Kontron software (Kontron, Munich, Germany), at least 10 random images were captured from each treatment along the wound. The average distance between the edges of scar was measured by diving total cell free area to that of the length of one of the edges of the scar by using the Image J image processing program (http://rsbweb.nih.gov/ij/download.html). The experiment was repeated three times.
Astrocyte cultures were lysed in RIPA buffer (50 mM tris (pH 7.5), 150 mM NaCl, 1% NP40, 1 mM Na4P2O4, 1 mM NaF, 1 mM EDTA, 2 mM Na3VO4 and 1 mM PMSF) for 1 h at 4° C. Samples were analyzed by SDS-PAGE followed by immunoblotting as described elsewhere (Mishra et al., 2010).
All numerical data are presented as group mean values with standard errors of mean (SEM). Parametric or non-parametric tests were used for comparisons, as indicated in the text and figure legends. Analyses were performed using the SYSTAT 9 software package (SPSS, Chicago, Ill., USA). The threshold value for acceptance of differences was 5%.
To identify novel PSA binding partners, a PSA-mimicking anti-idiotypic scFv antibody for immunoaffinity chromatography was generated. First, scFv antibodies that bind to a PSA antibody had to be isolated. Therefore, a phage scFv library was screened for binding to immobilized PSA antibody. After four rounds of selection, ten clones were found to bind to PSA antibody. Four of these clones expressed the sequences LNDGPVTSA (SEQ ID NO:5), five clones expressed the sequence GLPIDS (SEQ ID NO:6) and one clone expressed SPFES (SEQ ID NO:7) in the CDR3 region of the VH region of the scFv antibody. All clones consisted of a VH and VL chain deriving from the VH1 (DP-3) and VL1 (1-d) antibody gene family.
Next, it was checked by ELISA and surface plasmon resonance whether the binding of scFv antibodies deriving from the selected phages is PSA-specific. Since the yield of scFv antibody from the clone with the sequence SPFES (SEQ ID NO:7) in the CDR3 region was extremely low, this clone was not further analyzed. ELISA of the scFv antibody deriving from one of the five clones showing the sequence GLPIDS (SEQ ID NO:6) revealed that the binding of the scFv antibodies to substrate coated PSA antibody was inhibited in a concentration-dependent manner by the bacterial homolog of PSA, colominic acid, while other negatively charged polymers such as chondroitin sulfate A or C or heparin showed no inhibition of the binding (
To identify binding partners, ELISA was performed using purified scFv antibodies and different subfractions from mouse brain as substrate coats. No significant binding of scFv antibodies was observed (data not shown). The lack of binding could have been due to a low affinity of the scFv antibodies. This possibility was tested by increasing the avidity and thus the functional affinity of the scFv antibodies. Since truncation of the linker region between the VH and VL domains of the scFv antibody results in enhanced dimerization and/or oligomerization of scFv monomers and thus in an increased avidity (Holliger et al., 1993), the linker region of the selected phages was truncated (
Identification of Histone H1 as Synaptic Membrane-Associated PSA Binding Partner using a PSA-Mimicking Scfv Antibody
Since the purified scFv antibody with the sequence GLPIDS (SEQ ID NO:6) in the CDR3 region showed binding to brain proteins, it was used for affinity chromatography using different brain subfractions. A ˜30 kDa protein band was detected by silver staining upon separating an eluate obtained after application of a subfraction which was enriched in membrane-associated proteins that were released from the membranes by alkaline treatment (
To verify the direct interaction between histone H1 and PSA, ELISA with purified colominic acid which is a bacterial derived PSA homologue was performed using purified histone H1 from calf thymus as substrate coat and GAPDH a negative control substrate coat. PSA directly binds to histone H1, but not GAPDH, in a concentration dependent manner (
To further confirm that histone H1 is tightly associated with the plasma membrane, the distribution of histone H1 in brain membrane subfractions was analyzed upon detergent treatment. Western blot analysis using a histone H1 specific antibody revealed that histone H1 was detected as ˜30 kDa protein only in the Triton X-100 insoluble subfraction enriched in synaptic membranes or in postsynaptic densities but not in the detergent soluble subfraction enriched in synaptic membranes (
Since PSA is located extracellularly, we investigated whether its binding partner histone H1 is also present extracellularly. To test this, cell surface biotinylation using primary cerebellar neurons was performed and histone H1 was detectable as a biotinylated extracellular cell surface protein with an apparent molecular weight of ˜60 kDa, while it was detectable as a ˜30 kDa protein in the cell lysate (
To further corroborate the notion that histone H1 interacts with PSA extracellularly, it was tested by live immunocytochemical staining whether histone H1 and PSA co-localize at the surface of cultured cerebellar neurons or Schwann cells. Confocal microscopic analysis using a polyclonal histone H1 antibody and a monoclonal PSA antibody showed histone H1 and PSA immuno-reactivity both at the surface of cell bodies and along neurites of neurons (
Next, the functional role of extracellular histone H1 in neural cells was analyzed and it was investigated whether the interaction between PSA and histone H1 has functional consequences. First, we tested whether histone H1 influences neurite outgrowth (
To demonstrate that histone H1 induced neurite outgrowth depends on the presence of PSA on the surface of cerebellar neurons, neurite lengths were measured in the presence of endoglycosidase N which degrades PSA or in the presence of the PSA-specific antibody 735. On PLL substrate-coat, neurite outgrowth in the presence of PSA antibody or endoglycosidase N was not altered in comparison to that observed in the absence of antibody and enzyme. However, the enhanced neurite outgrowth in the presence of histone H1 was reduced nearly to PLL level in the presence of either the PSA-specific antibody or the PSA-degrading enzyme. (
Further data confirms previous experiments. Cerebellar neurons were grown on substrate-coated PLL or PLL with histone H1 or as control with laminin or histone H2A, H2B, H3, and H4. In parallel, neurons were grown on PLL in the absence or presence of soluble histone H1 and/or soluble PSA or as control in the presence of histone H2A, H2B, H3, or H4. Neurite outgrowth on substrate-coated histone H1 was enhanced by approximately threefold when compared with PLL and nearly reached values obtained for the positive control substrate laminin (FIG. 9A,B). Similarly, neurite outgrowth on PLL was increased with soluble histone H1 and soluble PSA (FIG. 9A,C), indicating that soluble PSA and histone H1 both trigger neurite out-growth. Moreover, a concentration-dependent promotion of neurite outgrowth by both substrate-coated and soluble histone H1 was observed (
To investigate whether endogenous extracellular histone H1 is involved in PSA-mediated neurite outgrowth, neurite outgrowth was determined in the absence or presence of soluble PSA and/or a polyclonal histone H1 antibody with the expectation that the antibody binds to endogenous histone H1 and blocks its effect on neurite outgrowth. Addition of soluble histone H1 antibody indeed blocked neurite outgrowth promotion by soluble PSA, whereas the antibody had no significant effect on neurite outgrowth in the absence of PSA (
The inventors showed that histone H1 and PSA co-localize on Schwann cell surface, and it has been shown that a PSA mimicking peptide and over-expression of PSA by Schwann cells improve myelination by Schwann cells in vivo (Mehanna et al., 2009a; Papastefanaki et al., 2007). These findings suggested that histone H1, like PSA, may affect Schwann cell process formation. To test this assumption, Schwann cells from dorsal root ganglia of early postnatal mice were cultured on substrate-coated PLL or histone H1 or in the presence of soluble histone H1 protein and/or soluble PSA, and the lengths of Schwann cell processes were measured. Morphometric analysis of Schwann cell process length revealed an increase of process length on substrate-coated histone H1 relative to PLL substrate and also in the presence of soluble PSA or soluble histone H1 (
Previous studies have suggested that PSA is required for migration of neural precursors (Angata et al., 2007; Chazal et al., 2000; Hu, 2000; Hu et al., 1996; Patrzykat et al., 2001). Based on these studies, it was next tried to investigate whether histone H1 affects neural precursor cell migration. Beforehand, it was investigated by immunostaining whether neural precursor cells in neurospheres express histone H1 and PSA and observed that neural precursor cells not only express both histone H1 and PSA at their cell surface, but also that both protein partially co-localize (
Histone H1 Promotes Functional Recovery after Femoral Nerve Injury in Adult Mice
Since PSA enhances regeneration after injury of the peripheral nervous system of adult mice (Mehanna et al., 2009a), and since the inventors have observed that histone H1 has beneficial effects on Schwann cells and neurons, the question was addressed whether histone H1 also enhances regeneration after femoral nerve injury. The function of the quadriceps muscle was evaluated during a four-month period after injury and after application of histone H1 or PBS.
Femoral nerve injury in mice induces changes in gait which can be quantitatively evaluated by two parameters, the heels-tail angle (HTA) (
To search for structural correlates of the superior functional recovery after application of histone H1, retrograde labeling of regenerated motoneurons was performed in animals after analyzing functional recovery. The number of motoneurons back-labeled through the motor (quadriceps) nerve branch which represent correctly projected regrown motoneurons significantly increased in animals to which histone H1 had been applied relative to PBS treated animals (
Since a significant correlation between recovery index and motoneuron size has been found 3 months after femoral nerve injury (Simova et al., 2006; Ahlborn et al., 2007), soma size of moto-neurons regrowing their axons correctly was measured and a significant increase in soma size of correctly projecting motoneurons in histone H1 treated animals in comparison to PBS treated animals was found, while no difference in soma size of incorrectly projecting motoneurons was observed among the groups (
Regenerated nerves were next analyzed morphometrically to assess the degree of myelination evaluated by the g-ratio (axon- to fiber-diameter ratio), and no significant between frequency distributions of the g-ratio for axons of intact and histone H1 or PBS treated injured nerves was found (
Histone Promotes Regeneration after Spinal Cord Injury
Improved motor recovery after immediate post-traumatic administration of histone H1 To investigate whether histone H1 improves functional recovery after severe spinal cord injury, two experimental groups were designed, in which either histone H1 or PBS (vehicle) as a control were applied during the first two weeks after injury. Spinal cord compression caused severe locomotor disabilities in all mice, as assessed by the BMS scores at 1 week after injury (
Enhanced Monoaminergic and Cholinergic Innervation in the Lumbar Spinal Cord after Histone H1 Treatment
Recovery of locomotor abilities after spinal cord injury depends on the extent of regrowth and/or sprouting of monaminergic axons (Jakovcevski et al., 2007). To assess whether improved motor functions in histone H1 treated mice were related to enhanced monoaminergic innervation of the spinal cord caudal to the injury, numbers of catecholaminergic (TH+) and serotonergic (5HT+) axons projecting beyond an arbitrarily selected border 250 μm caudally to the lesion site in spaced serial (250 μm apart) parasagittal sections were counted 6 weeks after the injury (
As previously reported, the density of large perisomatic cholinergic (ChAT+) boutons, known to form C-type synapses on motoneurons associated with muscarinic type 2 receptors (Davidoff et al., 1986) is strongly reduced in the lumbar spinal cord after thoracic spinal cord compression in mice (Apostolova et al., 2006; Jakovcevski et al., 2007). Importantly, the locomotor performance assessed by the BMS score or the foot-stepping angle positively correlated with the number of these terminals 6 weeks after spinal cord injury (Jakovcevski et al., 2007; Mehanna et al., 2010). Cholinergic synaptic coverage of motoneuron cell bodies was measured in histone H1 treated and PBS control treated animals (
Since it is well documented that PSA has a role in progenitor cell proliferation and is specifically expressed by new born neurons in the adult nervous system (Seki and Arai, 1993; Nacher et al., 2001), progenitor cell proliferation in lumbar spinal cord was analyzed 6 weeks after injury in histone H1 and PBS treated mice, based on Ki67 proliferating cell antigen expression (
Astrocyte Scar Formation is Impaired after Histone H1 Treatment
In order to study the role of histone H1 in astrocyte scar formation, the in vitro wound-healing assay was used: a cell-free zone was created within a confluent monolayer of an astrocyte culture by scratching cells off with a 100 μl pipette tip. The cell-free zone was commonly ˜200-250 gm wide and several millimeters long. To quantify the migratory activity of astrocytes, the area of the cell-free zone under the different experimental conditions was measured. Images were taken at 0 hours or 20 hours after scratching and analyzed. In control conditions, about 90% of the wound area was repopulated within 20 h of wound initiation (
To further demonstrate the contribution of histone H1 to the glial scar, immunoblot analysis was performed with protein extracts from the in vitro astrocyte scratch assay (
The experiments confirm that histone H1 promotes regeneration after spinal cord injury. Beneficial effects of histone H1 extend from the enhanced locomotor recovery after injury, to better cholinergic and monoaminergic reinnervation, to increased cell proliferation in the spinal cord treated with histone H1 compared with control (vehicle) treatment. In previous studies, it was shown that PSA has beneficial effects when applied after spinal cord injury as a glycomimetic peptide (Marino et al., 2009; Mehanna et al., 2010), and in the form of PSA-overexpressing Schwann cells (Papastefanaki et al., 2007). Most prominent effects of PSA application were an enhanced motor performance after injury, enhanced monoaminergic reinnervation, enhanced cholinergic reinnervation (Mehanna et al, 2010) and decreased astrocyte scaring upon injury. Treatment with the histone H1 affected all those parameters in a similar manner. The finding that PSA and histone H1 have similar effects when applied in vivo after spinal cord injury is suggestive for PSA-dependent action of histone H1, but it is also possible that PSA-independent effects of histone H1 are mediated by the same down-stream signalling.
Another notable effect of histone H1 treatment in spinal cord injured mice reported here is an enhancement of the progenitor cell proliferation in the lumbar spinal cord. Two particularly interesting cell types that could contribute to the difference in proliferation rate observed between the histone H1 treated mice and the controls are oligodendrocyte progenitors and neuronal progenitors. As shown above, Schwann cells treated with histone H1 proliferate more than non-treated cells, and PSA was shown to increase proliferation of Schwann cells and oligodendrocytes in vitro and improves remyelination after femoral nerve injury in vivo (Mehanna et al., 2009a). Histone H1 may increase proliferation of oligodendrocytes, like of Schwann cells, considering their similar function. Ependymal cells around the central canal strongly express PSA and were shown to give rise to mature neurons (Marichal et al., 2009). These cells may also get stimulated to proliferate upon histone H1 treatment.
Taken together, the experiments shown confirm that histone, e.g., Histone H1, can be used for treatment of central nervous system injuries such as spinal cord injuries.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09014837.0 | Nov 2009 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP10/07265 | 11/30/2010 | WO | 00 | 7/13/2012 |
