The ability to convert transient stimuli from the extracellular environment into long-term changes in neuronal function is central to an animal's capacity to adapt and learn from its environment. This is mediated through sensory organs which transduce physical and chemical stimuli into precise patterns of neuronal activity that elicit specific changes in the structure and function of the nervous system. Insight into the mechanisms that underlie these activity-dependent changes has been facilitated by the discoveries of many laboratories over the last several decades demonstrating that neurotransmitters released at neuronal synapses drive proteasome dependent protein degradation (J Biol Chem 284, 26655 (2009); Nat Neurosci 6, 231 (2003)). Consistent with a role for neural activity in regulating protein degradation, the proteasome localize to sites of synaptic activity (Nature 441, 1144 (2006)). This regulation is central to the ability of a neuron to appropriately respond to stimuli, as inhibition of protein degradation impairs a host of neuronal functions, ranging from plasticity at the Aplysia sensorimotor synapse to cell migration, neurotransmission, and physiology in the mammalian nervous system (Neuron 32, 1013-1026, 2001; Neuron 52, 239-245, 2006; Cell 89, 115-126, 1997; J Neurosci 26, 11333-11341, 2006) including the maintenance of long-term potentiation, a critical cellular mechanism underlying learning and memory (Neuron 52, 239 (2006); Nat Neurosci 9, 478 (2006)). Moreover, mutations in components of protein degradation machinery cause profound defects in human cognitive function (Biochim Biophys Acta 1843, 13 (2014); Nat Rev Genet 8, 711 (2007)).
However, roles for proteasome function in the nervous system are more complex than they may appear. Proteasome function is required for certain aspects of nervous system function over long timescales (hours to days), such as synaptic remodeling and cell migration (Nat Neurosci 6, 231-242, 2003; Science 302, 1775-1779, 2003). Contrastingly, proteasome function is also required for activity-dependent neuronal processes over very short timescales (seconds to minutes), such as regulating the speed and intensity of neuronal transmission or the maintenance of long-term potentiation (Nature 441, 1144-1148, 2006; Neuroscience 169, 1520-1526, 2010; J Biol Chem 284, 26655-26665, 2009; Learn Mem 15, 335-347, 2008; J Neurosci 26, 4949-4955, 2006; J Neurosci 30, 3157-3166, 2010).
Proteasomes are heterogeneous multisubunit catalytic complexes that consist of a core 20S stacked ring of α/β subunits with a α7β7β7α7 architecture, and can be associated with 19S or 11S regulatory cap-particles to form a 26S proteasome (Ann. Rev Biochem 65, 801-847, 1996). While the natural behavior of 26S capped proteasomes is to mediate ATP-dependent degradation of ubiquitinated proteins, 20S uncapped proteasomes do not require ubiquitin or ATP for their catalytic function (Biomolecules 4, 862-884, 2014; EMBO J 17, 7151-7160, 1998; Proc Natl Acad Sci U S A 95, Proc Natl Acad Sci U S A 95, 2727-2730 2727-2730, 1998) Recent studies have shown that 20S proteasomes may have key biological functions separate from the canonical 26S ubiquitin-proteasome degradation pathway, particularly in clearing unstructured proteins and in degrading proteins during cellular stress (Ben-Nissan and Sharon, 2014). Despite extensive studies on proteasome function in neuronal signaling, the role of the 20S proteasome in the nervous system has remained unknown.
Critically, the functional studies addressing the role for proteasomes in the nervous system have either failed to discriminate between 20S and 26S proteasomes through the use of pan-proteasome inhibitors such as MG-132 or lactacystin, or have focused on the 26S proteasome through altering the ubiquitination pathway. Despite these and other efforts to understand the role of proteasomes in the nervous system, distinct proteasomes that potentially function independent of their proteostatic role to mediate rapid neuronal signaling have not been discovered. Therefore, we considered that taking an unbiased approach to evaluating proteasomes in the nervous system, without bias for 20S or 26S proteasomes, would provide a means to identify unique proteasomes that could possibly have acute signaling functions.
There exists an unmet need for understanding how protein synthesis and protein degradation cooperate in neurons and whether this cooperation is linked to cognitive function and neurological disease. The use of this information in modulation of cognitive function and neurological disease remains undone.
In considering that protein synthesis and protein degradation have independent and opposing effects on the expression level of proteins, it remains to be determined why neuronal activity induces their simultaneous upregulation and co-localization. Indeed, classic studies in the immune system have identified a coordinated and constitutive mechanism of proteasome mediated degradation of newly synthesized proteins, a protein quality control process shown to be critical for proper immune function.
The present inventors hypothesized that in the nervous system coordination of protein synthesis and protein degradation may also alter the turnover of newly synthesized proteins, but unlike the constitutive process in the immune system, may only do so during states of neural activity.
The present inventors' investigation revealed a novel neuronal-specific 20S proteasome complex that was expressed at neuronal plasma membranes and exposed to the extracellular space. It was found that the activity of this novel neural membrane bound proteasome (NMP) converted intracellular proteins into extracellular peptides that rapidly induced neuronal signaling. Specific inhibition of this NMP through a novel membrane-impermeable proteasome inhibitor rapidly attenuated activity-induced neuronal function. These findings identify a new signaling modality in the nervous system and unveil the possibility that the membrane proteasome may be responsible for the previously observed decades of research showing that acute proteasome-mediated effects on nervous system function.
The present inventors monitored the fate of synthesized proteins and found that degradation of proteins by the NMP produced peptides which were directly released into the cell media. Hypothesizing that the NMP may play a role in neuronal activity-dependent mechanisms of nervous system function the inventors found that this release was suppressed when neuronal activity was blocked. Consistent with this finding, the release of these peptides into the media was dramatically enhanced in response to neuronal stimulation. These secreted, neuronal activity-induced, proteasomal peptides (SNAPPs) range in size from about 500 Daltons to about 3000 Daltons. Surprisingly none of these peptides produced by the NMP appear to be those previously known. Moreover, these SNAPPs have stimulatory activity and are heretofore a new class of signaling molecules.
Taken together this discovery defines a new modality of critical neuronal communication through production of biologically meaningful peptides, SNAPPs, that requires the function of a novel neuronal specific transmembrane proteasome, NMP. Changes in the NMP level and possibly activity greatly impact SNAPP production and activity dependent neuronal signaling critical for nervous system function.
In accordance with an embodiment, the present invention provides a method for modulating the NMP in neuronal cells in a subject comprising administering to the subject an effective amount of a NMP stimulator or inhibitor to the subject.
In accordance with another embodiment, the present invention provides a method for modulating an NMP associated disease or disorder of neuronal cells in a subject comprising administering to the subject an effective amount of a NMP stimulator or inhibitor to the subject.
In accordance with a further embodiment, the present invention provides a method for inhibiting neuronal activity or cognitive function in a subject comprising administering to the subject, an effective amount of NMP inhibitor to the subject.
In accordance with a yet another embodiment, the present invention provides a method for stimulating or enhancing neuronal activity or cognitive function in a subject comprising administering to the subject, an effective amount of NMP stimulator to the subject.
In accordance with an embodiment, the present invention provides a method for stimulating or enhancing neuronal activity or cognitive function in a subject comprising administering to the subject, an effective amount of SNAPPs to the subject.
In accordance with a further embodiment, the present invention provides a method for returning neuronal activity or cognitive function to a normal or pre-disease or disorder state in a subject comprising administering to the subject, an effective amount of SNAPPs to the subject.
In accordance with an embodiment, the present invention provides SNAPPs which are covalently linked to one or more biologically active agents.
In accordance with another embodiment, the present invention provides a method for delivery of one or more biologically active agents to activated neurons comprising contacting the activated neurons with SNAPPs which are covalently linked to one or more biologically active agents.
Proteasomes are ubiquitously expressed large multi-subunit catalytic complexes, generally characterized by a uniform cytoplasmic and nuclear distribution. The present inventors have now identified a nervous system-specific proteasome that is bound to the plasma membrane and exposed to the extracellular space. While it is unclear how these proteasomes bind to and orient themselves within neuronal plasma membranes, it has been known for decades through in vitro studies that proteasomes can orient perpendicularly to membranes specifically enriched in phosphatidylinositol (PI), a key signaling phospholipid that is notably elevated in the nervous system over other tissues.
The present inventors have discovered the presence of a 20S proteasome that is tightly associated with the neuronal plasma membrane and exposed to the extracellular space. In this capacity, it can degrade intracellular proteins into bioactive extracellular peptides that induce calcium signaling through NMDA receptors. Without reliance on any particular theory, the preferred model (discussed further below) based on these data are that a 20S proteasome complex is coupled to the plasma membrane by GPM6 glycoproteins, and that the extracellular peptides generated are the means by which the NMP acutely regulates neuronal function.
Identification of the GPM6 glycoprotein family as proteins that interact with proteasomes and are sufficient to induce the expression of proteasomes at the plasma membrane provides some insight into how proteasomes, as hydrophilic protein complexes, could interact so tightly with the hydrophobic plasma membrane. However, we noticed that the magnitude to which GPM6-induced membrane proteasome expression in heterologous cells did not match the magnitude of endogenous membrane proteasome expression in neurons. This suggests that there may in fact be other proteins that mediate the interaction of the NMP with the membrane, an area being actively investigated.
It is presently thought that the GPM6 glycoproteins may form a protein pore, perhaps through oligomeric interactions, which have been proposed previously35,44. In the right conformation, proteasomes binding to pore-containing membrane proteins could give proteasomes a hydrophilic binding surface to the hydrophobic plasma membrane, allowing the proteasome to gain access to the extracellular space. We propose a few models for how GPM6 proteins, or other membrane tethers may localize the proteasome to the plasma membrane (
The inventors made significant attempts to identify NMP interacting partners in an effort to determine whether the NMP was capped by the 19S, 11S, or PA200 subunits. Our data likely preclude the presence of the canonical 19S proteasome cap, or regulatory caps such as 11S or PA2004,45,46. While we identified a few 19S subunits co-fractionating with the NMP by mass spectrometry, we could not identify significant amount of key 19S subunits Rpt5 or S2. We also made the intriguing observation that immunoproteasome subunit PSMB8 uniquely co-fractionated with the NMP. Our finding that the NMP is likely a 20S core proteasome lacking the 19S cap is significant for two primary reasons. First, while a few functions for 20S proteasomes have been ascribed, their function independent of the 19S cap largely remains a mystery, especially in the nervous system46. Second, significant implications come from the idea that 20S proteasomes are primarily tasked with clearing misfolded or unstructured proteins4,47,48. A large source of disordered or unfolded proteins is derived from failed products of protein translation and misfolded or improperly folded proteins. These end-products of proteotoxic stress are hallmarks of many neurodegenerative disorders49,50, a fact which places the NMP at the heart of various disease states.
The present inventors have found that neuronal activity does not simply promote global protein degradation, but rather, it promotes protein degradation exclusively of newly synthesized proteins through the NMP for the express purpose of generating a new class of signaling molecules, SNAPPs.
Unconventional secretion pathways have been implicated in release of cellular protein cargos51,52. Moreover, many groups have demonstrated that inhibition of ubiquitin-dependent proteasome function affects synaptic signaling and transmission. The data of the present invention support a role for the existence of a specialized neuronal membrane proteasome that mediates neuronal function by “inside-out” signaling through the production of extracellular proteasome-derived peptides. While it remains possible, we have not detected any role for secretion pathways or ubiquitin in the release of these peptides (Ramachandran and Margolis, unpublished data).
The SNAPPs of the present invention are a new modality for neuronal communication. In the release experiments described herein, we show that there is some peptide release under non-stimulating conditions that is inhibited by MG-132, a known proteasomal inhibitor. It is thought that this is due to baseline spontaneous network activity causing some baseline degradation of proteins by the NMP, leading to peptides being released into the media. These peptides are different than SNAPPs, as they do not possess the same signaling capacity as SNAPPs.
SNAPPs, which when purified, rapidly and robustly stimulate neurons. Pharmacological dissection of the downstream pathways of peptide signaling revealed that NMP-derived peptides act in part by modulating NMDARs. The signaling through NMDARs only makes up ˜50% of the total activity of the peptides. Other possible targets include: 1) Peptides interact with major histocompatibility immune complexes (MHC) that have recently been shown to play key roles in developmental and experience-dependent mechanisms in the nervous system53,54; 2) peptides modulate metabotropic ion channels, thereby altering calcium-mediated signaling; and/or 3) peptides signal to neuronal or non-neuronal cells such as glial cells through yet to be identified receptors.
It is well-established that NMDARs are critical for neuronal activity-dependent signaling relevant to learning and memory55-57. Given that cytosolic proteasomes have been shown to be regulated by neuronal activity, it is thought that the NMP and the resulting extracellular peptides are also modulated by changes in neuronal activity. It is also unclear how this signaling is specified within the brain, but we postulate that it relies on how the NMP recognizes and targets proteins for degradation. Therefore, it will be critical to identify not only the sequences of the peptides, but also the substrates from which they are derived. These insights into substrate identity and targeting will reveal how the NMP functions, but may begin to link proteostatic failure under pathological conditions to NMP dysfunction.
Of note in some aspects of the present invention, is the role for phosphorylated CamKII in NMP expression. This is particularly intriguing given the role for phosphorylated CaMKII in serving as a scaffold for recruiting the proteasome into dendritic spines, and additionally for its long-known and well-studied role in learning and memory.
The same groups that have demonstrated the role for CaMKII in proteasome recruitment to spines have also shown that rapid inhibition of the proteasome has profound effects on synaptic signaling and transmission. These effects range from changes in transmission at the Drosophila neuromuscular synapse, regulation of activity-dependent spine dynamics, and an essential role in maintenance of LTP. In accordance with the inventive compositions and methods, we see a similar rapid and acute role for the proteasome in mediating SNAPP release (data not shown). It is important to note that pharmacological inhibitors used in previous studies take a substantially longer time to achieve functional inhibition of the cytosolic proteasome, according to data from groups studying the kinetics of proteasome inhibitors in neurons. Given the present findings, it is thought that at least some of the effects on synaptic transmission and function demonstrated by older studies may be due to inhibition of the neuronal membrane proteasome first reported in this study, and not of the cytosolic proteasome.
As used herein, the term “Neuronal Membrane Proteasome (NMP)” means a neuronal-specific 20S proteasome complex that was expressed at neuronal plasma membranes and exposed to the extracellular space. The NMP is unique to the nervous system and produces SNAPPs into the extracellular space.
As used herein, the analysis of proteins which are located on the plasma membrane surface of the neuronal cell, can be performed using many different means known in the art. In an embodiment, the plasma membrane fraction is isolated from neurons by lysing them in either a sucrose buffer or hypotonic lysis buffer. Nuclei were pelleted, and the supernatant containing plasma membranes was then pelleted at high RPM. Once the supernatant (cytosolic fraction) was set aside, the pellet was washed 2× with lysis buffer, and then resuspended in lysis buffer with indicated concentrations of detergent. Following a 15-minute incubation in the buffer, samples were spun down. This was repeated for all indicated concentrations of detergent. Membrane association was determined by classic methods of sodium carbonate extraction. The proteins were visualized by SDS-PAGE methods. Other methods can be used.
As used herein the 20S core proteins associated with the NMP can be identified and analyzed through the use of an antibodies that detect β2, anti-α1-7 proteasome subunit, anti-α5 proteasome subunit, anti-β1 proteasome subunit, anti-β2,5 subunit, anti-β2 proteasome subunit, and anti-Rpt5 proteasome subunit, for example. Other method for identification are known in the art, and include, for example, surface biotinylation methods and mass spectrometry.
In accordance with an embodiment, the present invention provides a composition comprising one or more SNAPPs.
In accordance with an embodiment, the present invention provides a composition comprising secreted, neuronal activity-induced, proteasomal peptides (SNAPPs), in an effective amount, for use in stimulating or enhancing neuronal activity or cognitive function in a subject.
In some embodiments, the SNAPPs have a molecular weight between 500 to 3000 Daltons.
In some embodiments, the SNAPPs are derived from a neuron selected from the group consisting of cortical, hippocampal, cerebellar, motor, sensory,
In some embodiments, the SNAPPs comprise at least one detectable moiety as an imaging agent.
In some embodiments, the SNAPPs comprise at least one detectable moiety as a radionuclide.
In some embodiments, the at least one detectable moiety is covalently attached to the SNAPPs via a biotinylated linker molecule.
In some embodiments, the subject is suffering from Alzheimer's disease or dementia.
In some embodiments, the composition further comprises an effective amount of at least one additional biologically active agent.
As used herein, the term “SNAPP” means proteins and peptides which are secreted extracellularly by a novel neural membrane bound proteasome (NMP) as the result of neural stimulation. Typically, these SNAPPs are secreted extracellularly within a few seconds to minutes after neural stimulation. These SNAPPs range in size from about 500 Daltons to about 3000 Daltons.
The term, “amino acid” includes the residues of the natural α-amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Lys, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as (3-amino acids, synthetic and unnatural amino acids. Many types of amino acid residues are useful in the adipokine polypeptides and the invention is not limited to natural, genetically-encoded amino acids. Examples of amino acids that can be utilized in the peptides described herein can be found, for example, in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the reference cited therein. Another source of a wide array of amino acid residues is provided by the website of RSP Amino Acids LLC.
The term, “peptide,” or “oligopeptide,” as used herein, includes a sequence of from four to sixteen amino acid residues in which the α-carboxyl group of one amino acid is joined by an amide bond to the main chain (α- or β-) amino group of the adjacent amino acid. In some embodiments, peptides provided herein for use in the described and claimed methods and compositions can be cyclic.
The term “imaging agent,” is known in the art. As used herein, the one or more imaging agents can be any small molecule or radionuclide which is capable of being detected. Typically, the imaging agents are covalently linked to the SNAPPs using any known methods in the art. Examples include use of a linker molecule. Other examples include biotinylation and biotin linked dyes.
In accordance with some embodiments the imaging agent is a fluorescent dye. The dyes may be emitters in the visible or near-infrared (NIR) spectrum. Known dyes useful in the present invention include carbocyanine, indocarbocyanine, oxacarbocyanine, thiiicarbocyanine and merocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein, boron-dipyrromethane (BODIPY), CyS, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.
Organic dyes which are active in the NIR region are known in biomedical applications. However, there are only a few NIR dyes that are readily available due to the limitations of conventional dyes, such as poor hydrophilicity and photostability, low quantum yield, insufficient stability and low detection sensitivity in biological system, etc. Significant progress has been made on the recent development of NIR dyes (including cyanine dyes, squaraine, phthalocyanines, porphyrin derivatives and BODIPY (borondipyrromethane) analogues) with much improved chemical and photostability, high fluorescence intensity and long fluorescent life. Examples of NIR dyes include cyanine dyes (also called as polymethine cyanine dyes) are small organic molecules with two aromatic nitrogen-containing heterocycles linked by a polymethine bridge and include Cy5, Cy5.5, Cy7 and their derivatives. Squaraines (often called Squarylium dyes) consist of an oxocyclobutenolate core with aromatic or heterocyclic components at both ends of the molecules, an example is KSQ-4-H. Phthalocyanines, are two-dimensional 18π-electron aromatic porphyrin derivatives, consisting of four bridged pyrrole subunits linked together through nitrogen atoms. BODIPY (borondipyrromethane) dyes have a general structure of 4,4′-difluoro-4-bora-3a, 4a-diaza-s-indacene) and sharp fluorescence with high quantum yield and excellent thermal and photochemical stability.
Other imaging agents which can be attached to the SNAPPs of the present invention include PET and SPECT imaging agents. The most widely used agents include branched chelating agents such as di-ethylene tri-amine penta-acetic acid (DTPA), 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and their analogs. Chelating agents, such as di-amine dithiols, activated mercaptoacetyl-glycyl-glycyl-gylcine (MAG3), and hydrazidonicotinamide (HYNIC), are able to chelate metals like 99mTc and 186Re. Instead of using chelating agents, a prosthetic group such as N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) is necessary for labeling peptides with 18F. In accordance with a preferred embodiment, the chelating agent is DOTA.
In accordance with some embodiments, the present invention provides one or more SNAPPs wherein the imaging agent comprises a metal isotope suitable for imaging. Examples of isotopes useful in the present invention include Tc-94m, Tc-99m, In-111, Ga-67, Ga-68, Y-86, Y-90, Lu-177, Re-186, Re-188, Cu-64, Cu-67, Co-55, Co-57, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, or Dy-i66.
In accordance with some embodiments, the present invention provides a SNAPP wherein the reporter portion comprises 111In labeled DOTA which is known to be suitable for use in SPECT imaging.
In accordance with some other embodiments, the present invention provides SNAPPs wherein the imaging agent comprises Gd3+ labeled DOTA which is known to be suitable for use in MR imaging. It is understood by those of ordinary skill in the art that other suitable radioisotopes can be substituted for 111In and Gd3+ disclosed herein.
In some embodiments, the present invention provides methods for detecting neuronal activity using voltage-sensitive dye, whose optical properties change during changes in electrical activity of neuronal cells. The spatial resolution achieved by this technique is near the single cell level. For example, researchers have used the voltage-sensitive dye merocyanine oxazolone to map cortical function in a monkey model. Blasdel, G. G. and Salama, G., “Voltage Sensitive Dyes Reveal a Modular Organization Monkey Striate Cortex,” Nature 321:579-585, 1986. However, the use of these kinds of dyes would pose too great a risk for use in vivo in view of their toxicity.
It will be understood by those of ordinary skill in the art that the SNAPPs of the present invention have the ability to bind activated neurons, and therefore they can be used as targeting molecules for other therapies. For example, SNAPPs can be conjugated with another small molecule, or biologically active agent, including, drugs, antibodies and the like. In accordance with some embodiments, the SNAPPs can be conjugated or linked with compounds which stimulate or inhibit neuronal activity, or which have some other pharmacological effect.
As used herein, the term “biologically active agent” include any compound, biologics for treating brain-related diseases, e.g. drugs, inhibitors, and proteins. An active agent and a biologically active agent are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and/or physiological effect, wherein the effect may be prophylactic or therapeutic. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms “active agent,” “pharmacologically active agent” and “drug” are used, then, it is to be understood that the invention includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs etc.
In accordance with some embodiments, the SNAPPs can be conjugated or linked with compounds which stimulate or inhibit neuronal activity. Examples of such classes of compounds include, but are not limited to, cholinergic agonists and antagonists, opiate agonists and antagonists, muscarinic agonists and antagonists, GABAergic agonists and antagonists, parasympathomimetics, sympathomimetics, adrenergic agonists and antagonists, general anesthetics, such as inhalation anesthetics, halogenated inhalation anesthetics, intravenous anesthetics, barbiturates, benzodiazepines, antidepressants, heterocyclic antidepressants, monoamine oxidase inhibitors selective serotonin re-uptake inhibitors tricyclic antidepressants, antimanics, anti-psychotics, phenothiazine antipsychotics, anxiolytics, calcium channel blockers, and anti-Parkinson's agents such as bromocriptine, levodopa, carbidopa, and pergolide.
It is understood by those of ordinary skill in the art that the compounds and/or imaging agents can be attached to the SNAPPs by use of linker molecules. For instance linking groups having alkyl, aryl, combination of alkyl and aryl, or alkyl and aryl groups having heteroatoms may be present. For example, the linker can be a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C1-C20 hydroxyalkyl, C1-C20 alkoxy, C1-C20 alkoxy C1-C20 alkyl, C1-C20 alkylamino, di-C1-C20 alkylamino, C1-C20 dialkylamino C1-C20 alkyl, C1-C20 thioalkyl, C2-C20 thioalkenyl, C2-C20 thioalkynyl, C6-C22 aryloxy, C6-C22 arylamino C2-C20 acyloxy, C2-C20 thioacyl, C1-C20 amido, and C1-C20 sulphonamido.
Compounds are assembled by reactions between different components, to form linkages such as ureas (—NRC(O)NR—), thioureas (—NRC(S)NR—), amides (—C(O)NR— or —NRC(O)—), or esters (—C(O)O— or —OC(O)—). Urea linkages may be readily prepared by reaction between an amine and an isocyanate, or between an amine and an activated carbonamide (—NRC(O)—). Thioureas may be readily prepared from reaction of an amine with an isothiocyanate. Amides (—C(O)NR— or —NRC(O)—) may be readily prepared by reactions between amines and activated carboxylic acids or esters, such as an acyl halide or N-hydroxysuccinimide ester. Carboxylic acids may also be activated in situ, for example, with a coupling reagent, such as a carbodiimide, or carbonyldiimidazole (CDI). Esters may be formed by reaction between alcohols and activated carboxylic acids. Triazoles are readily prepared by reaction between an azide and an alkyne, optionally in the presence of a copper (Cu) catalyst.
Protecting groups may be used, if necessary, to protect reactive groups while the compounds are being assembled. Suitable protecting groups, and their removal, will be readily available to one of ordinary skill in the art.
In this way, the compounds may be easily prepared from individual building blocks, such as amines, carboxylic acids, and amino acids.
It is contemplated that any of the SNAPPs of the present invention described above can also encompass a pharmaceutical composition comprising the SNAPPs and a pharmaceutically acceptable carrier.
With respect to the SNAPPs described herein, the carrier can be any of those conventionally used, and is limited only by physico-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the carrier be one which is chemically inert to the active agent(s), and one which has little or no detrimental side effects or toxicity under the conditions of use. Examples of the carriers include soluble carriers such as known buffers which can be physiologically acceptable (e.g., phosphate buffer) as well as solid compositions such as solid-state carriers or latex beads.
The carriers or diluents used herein may be solid carriers or diluents for solid formulations, liquid carriers or diluents for liquid formulations, or mixtures thereof.
Solid carriers or diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
For liquid formulations, pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, or suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic/aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media.
Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Formulations suitable for parenteral administration include, for example, aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
The choice of carrier will be determined, in part, by the particular SNAPP composition, as well as by the particular method used to administer the composition. Accordingly, there are a variety of suitable formulations of the pharmaceutical SNAPP composition of the invention. The following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and interperitoneal administration are exemplary, and are in no way limiting. More than one route can be used to administer the compositions of the present invention, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
Injectable formulations are in accordance with the invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630 (2009)).
For purposes of the invention, the amount or dose of the SNAPPs of the present invention that is administered should be sufficient to effectively target the cell, or population of cells in vivo, such that the stimulation of the neuronal cells can be detected, in the subject over a reasonable time frame. The dose will be determined by the efficacy of the particular SNAPP formulation and the location of the target population of neuronal cells in the subject, as well as the body weight of the subject to be treated.
The dose of the SNAPPs of the present invention also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular SNAPP. Typically, an attending physician will decide the dosage of the SNAPPs with which to treat each individual subject, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound to be administered, route of administration, and the severity of the condition being treated. By way of example, and not intending to limit the invention, the dose of the SNAPPs of the present invention can be about 0.001 to about 1000 mg/kg body weight of the subject being treated, from about 0.01 to about 100 mg/kg body weight, from about 0.1 mg/kg to about 10 mg/kg, and from about 0.5 mg to about 5 mg/kg body weight. In another embodiment, the dose of the SNAPPs of the present invention can be at a concentration from about 1 nM to about 10,000 nM, preferably from about 10 nM to about 5,000 nM, more preferably from about 100 nM to about 500 nM.
In accordance with another embodiment, the present invention provides a method for identifying activated neurons in vitro comprising: a) providing a plurality of in vitro cultures comprising a plurality of neurons in a growth medium; b) stimulating at least one or more of the cultures with a stimulant; c) removing the growth medium of the plurality of in vitro cultures; d) fixing the plurality of in vitro cultures; e) staining the plurality of in vitro cultures with at least one or more SNAPP compositions as described herein; f) quantifying the detectable moiety of the compositions of e) using imaging and/or radiography; g) identifying the activated neurons as those neurons from stimulated in vitro cultures which have a significantly increased amount of detectable signal from the detectable moiety compared to the amount of detectable signal in neurons from in vitro cultures which were not stimulated.
In accordance with another embodiment, the present invention provides a method for identifying activated neurons in vivo comprising: a) administering to the neuronal tissue of a mammal an effective amount of at least one or more SNAPP compositions as described herein, wherein the imaging agent is a SPECT or PET, or magnetic resonance imaging agent; b) imaging the neuronal tissue of the mammal; and c) identifying the activated neurons as those neurons which have a significantly increased amount of detectable signal from the detectable moiety compared to the amount of detectable signal from other neurons in the tissue.
In accordance with a further embodiment, the present invention provides a method for screening for compounds which stimulate NMP and subsequent production of secreted neuronal-activity induced proteasomal peptides (SNAPPs) comprising the steps of: a) administering to a subject a test compound for a period of time sufficient to stimulate NMP and allow production of SNAPPs in the neurons of the subject; b) providing a negative control by administering to at least a second subject for a period of time sufficient with a carrier or vehicle which will not stimulate NMP mediated production of SNAPPs in the neurons of the second subject; c) obtaining a biological sample from a) and b) and performing an isolation step to purify the SNAPPs from the biological samples of b) and c); d) quantifying the amount of SNAPPs isolated in e) from the biological samples of a) and b); and e) determining that the test compound is a stimulator of NMP mediated SNAPP production when the quantity of SNAPPs isolated from the biological samples of a) are significantly increased when compared with the amount of SNAPPs isolated from the biological samples of b).
In accordance with another embodiment, the present invention provides a method for identifying activated neurons in vivo comprising: a) administering to the neuronal tissue of a mammal an effective amount of at least one or more SNAPP compositions as described herein, wherein the imaging agent is a SPECT or PET, or magnetic resonance imaging agent; b) imaging the neuronal tissue of the mammal; and c) identifying the activated neurons as those neurons which have a significantly increased amount of detectable signal from the detectable moiety compared to the amount of detectable signal from other neurons in the tissue.
In some embodiments the present invention employs an electromagnetic radiation (emr) source for uniformly illuminating an area of neurons of interest, and an optical detector capable of detecting and acquiring data relating to one or more optical properties of an area of interest. In a simple form, the apparatus of the present invention may include an optical fiber operably connected to an emr source that illuminates tissue or neuronal cultures in vitro, and another optical fiber operably connected to an optical detector, such as a photodiode, that detects one or more optical properties of the illuminated tissue. The detector is used to obtain control data representing the “normal” or “background” optical properties of an area of interest, and then to obtain subsequent data representing the optical properties of an area of interest during neuronal activity, e.g., stimulation of neuronal tissue, or during a monitoring interval. The subsequent data is compared to the control data to identify changes in optical properties representative of neuronal activity. According to a preferred embodiment, the control, subsequent and comparison data are presented in a visual format as images.
In some embodiments, the present invention provides methods for optically imaging neuronal tissue and the physiological events associated with neuronal activity. The methods of the present invention may be used for optically imaging and mapping functional neuronal activity, differentiating neuronal tissue from non-neuronal tissue, identifying and spatially locating dysfunctional neuronal tissue, and monitoring neuronal tissue to assess viability, function and the like.
Numerous devices for acquiring, processing and displaying data representative of one or more optical properties of an area of interest can be employed. One preferred device is a video camera that acquires control and subsequent images of an area of interest that can be compared to identify areas of neuronal activity or dysfunction. Examination of images provides precise spatial location of areas of neuronal activity or dysfunction. Apparatus suitable for obtaining such images have been described in the patents incorporated herein by reference and are more fully described below. For most surgical and diagnostic uses, the optical detector preferably provides images having a high degree of spatial resolution at a magnification sufficient to detect single neuronal cells or nerve fiber bundles. Several images are preferably acquired over a predetermined time period and combined, such as by averaging, to provide control and subsequent images for comparison.
In some embodiments the video camera is a Charge Coupled Device (CCD). A CCD is a type of optical detector that utilizes a photo-sensitive silicon chip in place of a pickup tube in a video camera.
Various data processing techniques may be advantageously used to assess the data collected in accordance with the present invention. Comparison data may be assessed or presented in a variety of formats. Processing may include averaging or otherwise combining a plurality of data sets to produce control, subsequent or comparison data sets. Images are preferably converted from an analog to a digital form for processing, and back to an analog form for display.
Data processing may also include amplification of certain signals or portions of a data set (e.g., areas of an image) to enhance the contrast seen in data set comparisons, and to thereby identify areas of neuronal activity and/or dysfunction with a high degree of spatial resolution. For example, according to one embodiment, images are processed using a transformation in which image pixel brightness values are remapped to cover a broader dynamic range of values. A “low” value may be selected and mapped to zero, with all pixel brightness values at or below the low value set to zero, and a “high” value may be selected and mapped to a selected value, with all pixel brightness values at or above the high value mapped to the high value. Pixels having an intermediate brightness value, representing the dynamic changes in brightness indicative of neuronal activity, may be mapped to linearly or logarithmically increasing brightness values. This type of processing manipulation is frequently referred to as a “histogram stretch” and can be used according to the present invention to enhance the contrast of data sets, such as images, representing changes in neuronal activity.
In accordance with another embodiment, the present invention provides a method for making SNAPPs comprising the steps of: a) providing an in vitro culture of a plurality of neurons in a growth medium; b) stimulating the neurons for a period of time sufficient to allow secretion of SNAPPs into the growth medium; c) removing at least a portion of the growth medium containing the SNAPPs.
The term “neuron” is used herein to denote a cell that arises from neuroepithelial cell precursors. Mature neurons (i.e., fully differentiated cells from an adult) display several specific antigenic markers.
The term “neuroepithelium” is used herein to denote cells and tissues that arise from the neural epithelium during development; such cells include retinal cells, diencephalon cells and midbrain cells. Neuroepithelium is also defined as neuroectoderm, and more specifically as ectoderm on the dorsal surface of the early vertebrate embryo that gives rise to the cells (neurons and glia) of the nervous system.
As used herein, the term “neuron” means neuronal cells derived from the central nervous system of a subject, including, for example, the brain, spinal cord, as well as the peripheral nervous system, including, for example, sensory and motor neurons. Areas of the brain where these neurons can originate from include, but are not limited to, Cortex (Ctx), Hippocampus (Hip), Olfactory bulb (Olf), Hind Brain (Brn), for example. Neurons can also be cells derived from induced pluripotent stem cell (iPSC) cultures.
The cell culture systems and methods used in the present invention may be used in conjunction with any glass surface (including, for instance, coverslips) that has been coated with an attachment-enhancing substance, such as poly-lysine, Matrigel, laminin, polyornithine, gelatin and/or fibronectin. Feeder cell layers, such as glial feeder layers or embryonic fibroblast feeder layers, may also find use within the methods and compositions provided herein.
Neuronal cells used in the present invention can be placed into any known culture medium capable of supporting cell growth, including MEM, DMEM, RPMI, F-12, and the like, containing supplements which are required for cellular metabolism such as glutamine and other amino acids, vitamins, minerals and useful proteins such as transferrin and the like. Medium may also contain antibiotics to prevent contamination with yeast, bacteria and fungi such as penicillin, streptomycin, gentamicin and the like. In some cases, the medium may contain serum derived from bovine, equine, chicken and the like. A particularly preferable medium for cells is a mixture of Neurobasal and B-27 (catalog #21103049 and 17504044 respectively, Life Technologies, Gaithersburg, Md.).
Conditions for culturing should be close to physiological conditions. The pH of the culture media should be close to physiological pH, preferably between pH 6-8, more preferably close to pH 7, even more particularly about pH 7.4. Cells should be cultured at a temperature close to physiological temperature, preferably between 30° C.-40° C., more preferably between 32° C.-38° C., and most preferably between 35° C.-37° C.
Neuronal cells can be grown in suspension or on a fixed substrate. In the case of propagating (or splitting) suspension cells, flasks are shaken well and the neurospheres allowed to settle on the bottom corner of the flask. The spheres are then transferred to a 50 ml centrifuge tube and centrifuged at low speed. The medium is aspirated, the cells resuspended in a small amount of medium with growth factor, and the cells mechanically dissociated and resuspended in separate aliquots of media.
Cell suspensions in culture medium are supplemented with any growth factor which allows for the proliferation of progenitor cells and seeded in any receptacle capable of sustaining cells, though as set out above, preferably in culture flasks or roller bottles. Cells typically proliferate within 3-4 days in a 37° C. incubator, and proliferation can be reinitiated at any time after that by dissociation of the cells and resuspension in fresh medium containing growth factors.
As used herein, the term “stimulation” means the activation or firing of the neuron when the neuron is stimulated by pressure, heat, light, or chemical information from other cells. The type of stimulation necessary to produce firing depends on the type of neuron. The cytosol inside a neuron is separated from that outside by a polarized cell membrane that contains electrically charged particles known as ions. When a neuron is sufficiently stimulated to reach the neural threshold (a level of stimulation below which the cell does not fire), depolarization, or a change in cell potential, occurs.
In accordance with some embodiments, neurons which produce SNAPPs can be stimulated by the use of a depolarizing buffer. Examples of such buffers include, but are not limited to physiological buffers containing high concentration of KCl (60 mM to 150 mM or more), and can also include additional Ca++ ions (10-20 mM). Other such depolarizing buffers include glutamate or bicuculine and others.
Removal of cell growth medium from cell cultures which have been stimulated can be performed using any known means in the art, e.g., pipetting, filtration, etc.
In accordance with an embodiment, the present invention provides a method for inhibiting secreted neuronal-activity induced proteasomal peptides (SNAPPs) in a neuronal cell or population of cells comprising contacting the cell or population of cells with an effective amount of at least one proteasomal inhibitor for a time sufficient to inhibit secretion of SNAPPs.
In some embodiments, the proteasomal inhibitor can be one known in the art. For example, compounds such as Epoxomicin, Lactacystin, Bortezomib, MG-132, Carfilzomib, MLN9708, Ixazomib, PI-1840, ONX-0914, Oprozomib, CEP-18770, and Gabexate Mesylate are known proteasomal inhibitors.
In accordance with a further embodiment, the present invention provides a method for screening for compounds which stimulate secretion of secreted neuronal-activity induced proteasomal peptides (SNAPPs) comprising the steps of: a) providing a plurality of in vitro cultures comprising a plurality of neurons in a growth medium; b) providing one or more test cultures by contacting the neurons of at least a first culture with a test compound for a period of time sufficient to allow secretion of SNAPPs into the growth medium; c) providing a negative control by contacting the neurons of at least a second culture for a period of time sufficient with a carrier or vehicle which will not stimulate secretion of SNAPPs into the growth medium; d) removing at least a portion of the growth medium of the cultures of b) and c) and performing an isolation step to purify the SNAPPs from the cultures of b) and c); e) quantifying the amount of SNAPPs isolated in e) from the cultures of b) and c); and f) determining that the test compound is a stimulator of SNAPP secretion when the quantity of SNAPPs isolated from b) are significantly increased when compared with the amount of SNAPPs in c).
In accordance with yet another embodiment, the present invention provides a method for screening for compounds which inhibit secretion of secreted neuronal-activity induced proteasomal peptides (SNAPPs) comprising the steps of: a) providing a plurality of in vitro cultures comprising a plurality of neurons in a growth medium; b) providing one or more test cultures by contacting the neurons of at least a first culture with a test compound and with a known neuronal stimulant for a period of time sufficient to allow secretion of SNAPPs into the growth medium; c) providing a negative control by contacting the neurons of at least a second culture for a period of time sufficient with a carrier or vehicle which will not stimulate secretion of SNAPPs into the growth medium; d) providing a positive control by stimulating the neurons of a third culture for a period of time sufficient with a known neuronal stimulant to allow secretion of SNAPPs into the growth medium; e) removing at least a portion of the growth medium of the cultures of b) to d) and performing an isolation step to purify the SNAPPs from the cultures of b) to d); f) quantifying the amount of SNAPPs isolated in e) from the cultures of b) to d); and g) determining that the test compound is a inhibitor of SNAPP secretion when the quantity of SNAPPs isolated from b) are significantly reduced when compared with the amount of SNAPPs in c) and/or d).
The isolation and quantification of SNAPPs can be performed by various methods in the art. In some embodiments the SNAPPs can be isolated various chromatographic methods, including, for example, UHPLC Hydrophilic Interaction Chromatography (HILIC), normal phase, and/or reverse-phase C18 chromatography. These methods can be combined with ultraviolet-visible (UV-vis) spectrophotometry, and other detection methods, to detect the SNAPPs eluting at various times off the different columns.
In accordance with some embodiments, the sequences of SNAPPs can be identified with many known methods. In an embodiment, advanced mass spectrometric techniques after fractionation using matrix assisted laser desorption/ionization after HPLC (LC-MALDI) or fractionation of an HPLC column directly into an electrospray mass spectrometer (LC/MS-ESI) can be used to identify the specific SNAPPs. Other methods, such as Edman degradation and sequencing can be used.
Considering that many neurodegenerative disorders may result from improperly degraded proteins, we have tested whether the NMP is at all dysregulated in mouse models for neurodegeneration. Interestingly, in accordance with some aspects of the present invention, the inventors found that the NMP is significantly perturbed very early in a disease model of Alzheimer's (
As such, in accordance with an embodiment, the present invention provides a method for identifying a neuron or population of neurons as having aberrant or dysregulated NMP function comprising: a) providing at least one first in vitro culture comprising a neuron or population of neurons of interest; b) providing at least one second in vitro normal or control cultures comprising a wild type or standard neuron or population of neurons; c) contacting the neurons of the first and second cultured with a stimulant compound for a period of time sufficient to allow secretion of SNAPPs into the growth medium; c) providing a negative control in vitro culture comprising a wild type or standard neuron or population of neurons by contacting the neurons of the negative control for a period of time sufficient with a carrier or vehicle which will not stimulate secretion of SNAPPs into the growth medium; d) removing at least a portion of the growth medium of the cultures of a) to c) and performing an isolation step to purify the SNAPPs from the cultures of a) to c); e) quantifying the amount of SNAPPs isolated in e) from the cultures of a) to c); and f) determining that the first in vitro culture of interest has dysregulated NMP function when the quantity of SNAPPs isolated from a) are significantly increased or decreased when compared with the amount of SNAPPs in b).
In some embodiments, the above methods can be performed using cysteine or methionine amino acids labeled with 35S added to the culture medium prior to performing the methods of the present invention. Other labeled amino acids known in the art can also be used.
For example, the above methods can be used to compare the NMP function of neurons having known neurodegenerative diseases or models for such diseases to normal neuronal function to determine which neurological diseases or conditions are associated with dysregulated or aberrant NMP function.
In accordance with an embodiment, the present invention provides a method for modulating the NMP in neuronal cells in a subject comprising administering to the subject an effective amount of a NMP stimulator or inhibitor to the subject.
In accordance with another embodiment, the present invention provides a method for modulating an NMP associated disease or disorder of neuronal cells in a subject comprising administering to the subject an effective amount of a NMP stimulator or inhibitor to the subject.
Examples of proteasomal stimulators useful in the inventive methods can include, but are not limited to, PA28, PA200, PA700, arginine-rich histone H3), small molecules (oleuropein, betulinic acid—and derivtives), lipid activators (lysophosphatidylinositol, cardiolipin, ceramides), fatty acids (linoleic, oleic, linolenic acids), synthetic peptidyl alcholos (pnitroanilides, nitriles). (Curr Med Chem. 2009; 16(8):931-939).
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In accordance with a further embodiment, the present invention provides a method for inhibiting neuronal activity or cognitive function in a subject comprising administering to the subject, an effective amount of NMP inhibitor to the subject.
It will be understood by those of skill in the art that by inhibition of the NMP activity on neurons, either through downregulation of expression of NMP or through direct inhibition with an inhibitory agent, the neurons, when stimulated, will release less SNAPPs into their surrounding environment. This can potentially result in lesser post-synaptic stimulation of surrounding neurons and diminished post-synaptic activity as a result of pre-synaptic stimulation. While not being bound to any particular theory, it is thought that downregulation of NMP expression in neurons, or direct inhibition through the use of inhibitory agents such as NMP inhibitors will have an inhibitory effect on basal neural activity. These effects could be useful in neurological diseases where there is a loss of inhibitory neuronal function. Examples of such diseases include, but are not limited to, epilepsy, encephalopathy, seizures due to other conditions, such as brain tumors, chronic pain, Parkinson's disease, Huntington's disease and other muscle spasm disorders.
In accordance with a yet another embodiment, the present invention provides a method for stimulating or enhancing neuronal activity or cognitive function in a subject comprising administering to the subject, an effective amount of NMP stimulator to the subject.
It will be understood by those of skill in the art that by stimulation of the NMP activity on neurons, either through upregulation of expression of NMP or through direct stimulation with an excitory agent, the neurons, when stimulated, will release increased amounts of SNAPPs into their surrounding environment. This can potentially result in greater post-synaptic stimulation of surrounding neurons and increased post-synaptic activity as a result of pre-synaptic stimulation. While not being bound to any particular theory, it is thought that upregulation of NMP expression in neurons, or direct stimulation through the use of stimulatory/agonist agents such as proteasomal stimulators will have a stimulatory effect on basal neural activity. Moreover, it will be understood by those of skill in the art that modulation of SNAPP release or NMP activity can lead to reversal of neuronal disease states. These effects could be useful in neurological diseases or cognitive conditions where there is a loss of excitatory neuronal function. Examples of uses include, but are not limited to psychiatric disorders, epilepsy, multiple sclerosis, autism, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's, aging, dementia, enhancement learning and memory and other neurodegenerative diseases.
In some embodiments, the NMP stimulators or inhibitors are combined with a pharmaceutically acceptable carrier as described herein. Moreover, the proteasomal stimulators or inhibitors can be combined with other biologically active agents.
In accordance with an embodiment, the present invention provides a method for stimulating or enhancing neuronal activity or cognitive function in a subject comprising administering to the subject, an effective amount of SNAPPs to the subject.
These effects could be useful in neurological diseases or cognitive conditions where there is a loss of excitatory neuronal function. Examples of uses include, but are not limited to psychiatric disorders, epilepsy, multiple sclerosis, autism, Alzheimer's disease, Parkinson's disease, am otroplaie lateral sclerosis, Huntington's, aging, dementia, enhancement learning and memory and other neurodegenerative diseases.
In some embodiments, the one or more SNAPPs are combined with a pharmaceutically acceptable carrier as described herein. Moreover, the SNAPPs can be combined with other biologically active agents.
An active agent and a biologically active agent are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and/or physiological effect, wherein the effect may be prophylactic or therapeutic. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms “active agent,” “pharmacologically active agent” and “drug” are used, then, it is to be understood that the invention includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs etc. The active agent can be a biological entity, such as a virus or cell, whether naturally occurring or manipulated, such as transformed.
The biologically active agent may vary widely with the intended purpose for the composition. The term active is art-recognized and refers to any moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of biologically active agents, that may be referred to as “drugs”, are described in well-known literature references such as the Merck Index, the Physicians' Desk Reference, and The Pharmacological Basis of Therapeutics, and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. Various forms of a biologically active agent may be used which are capable of being released the subject composition, for example, into adjacent tissues or fluids upon administration to a subject.
Examples of active agents that can be used with the inventive SNAPPs, and NMP stimulators or inhibitors, and methods include, but are not limited to autonomic agents, such as anticholinergics, antimuscarinic anticholinergics, ergot alkaloids, parasympathomimetics, cholinergic agonist parasympathomimetics, cholinesterase inhibitor parasympathomimetics, sympatholytics, α-blocker sympatholytics, sympatholytics, sympathomimetics, and adrenergic agonist sympathomimetics, anesthetics, such as inhalation anesthetics, halogenated inhalation anesthetics, intravenous anesthetics, barbiturate intravenous anesthetics, benzodiazepine intravenous anesthetics, and opiate agonist intravenous anesthetics, skeletal muscle relaxants, neuromuscular blocker skeletal muscle relaxants, and reverse neuromuscular blocker skeletal muscle relaxants; neurological agents, such as anticonvulsants, barbiturate anticonvulsants, benzodiazepine anticonvulsants, anti-migraine agents, anti-parkinsonian agents, anti-vertigo agents, opiate agonists, and opiate antagonists, psychotropic agents, such as antidepressants, heterocyclic antidepressants, monoamine oxidase inhibitors selective serotonin re-uptake inhibitors tricyclic antidepressants, antimanics, anti-psychotics, phenothiazine antipsychotics, anxiolytics, sedatives, and hypnotics, barbiturate sedatives and hypnotics, benzodiazepine anxiolytics, sedatives, and hypnotics, and psychostimulants.
In another embodiment, the term “administering” means that at least one or more SNAPPs or NMP stimulators or inhibitors of the present invention are introduced into a subject, preferably a subject receiving treatment for a disease, and the at least one or more SNAPPs or NMP stimulators or inhibitors are allowed to come in contact with the one or more disease related cells or population of cells in vivo.
As used herein, the term “treat,” as well as words stemming therefrom, includes diagnostic and preventative as well as disorder remitative treatment.
As used herein, the term “subject” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
Antibodies.
The following were used according to manufacturer's and/or published suggestions for western blotting and immunocytochemistry: anti-α1-7 proteasome subunit (Enzo), anti-β2 proteasome subunit (Cell Signaling), anti-α5 proteasome subunit (Santa Cruz), anti-β1 proteasome subunit (Santa Cruz), anti-β2 proteasome subunit (Santa Cruz), anti-β2 proteasome subunit (Enzo), anti-β2 proteasome subunit (Novus), anti-β2 proteasome subunit (Santa Cruz), anti-β5 proteasome subunit (Santa Cruz), anti-β5 proteasome subunit (Enzo), anti-Rpt5 proteasome subunit (Enzo), anti-calregulin (Santa Cruz), anti-β-Actin (Abcam), anti-Biotin (Cell Signaling), Streptavidin-AF647 (Invitrogen), anti-Tubulin (Milipore), anti-GluR1 (Cell Signaling), anti-Myc (Abcam), anti-Transferrin (Invitrogen), anti-EphB2 (M. Greenberg)58, anti-NGluR1 (R. Huganir), cleaved Caspase-3 (Cell Signaling), anti-Kv1.3 (NeuroMab), anti-S2 (Milipore), anti-PA200 (Novus), anti-11Sα (Cell Signaling), anti-11Sβ (Cell Signaling). Antibodies obtained from commercial vendors were verified for specificity using western blotting, immunofluorescence, or immunoprecipitation. We prioritize those antibodies with a continued record of use in multiple independent studies (Table A). For proteasome antibodies, many antibodies used recognize a single band or set of bands at the known molecular weight. Genetic validation of these antibodies is impossible as all proteasome subunits are essential and no knockout controls can be obtained.
indicates data missing or illegible when filed
Mice.
All animal procedures were performed under protocols compliant and approved by the Institutional Animal Care and Use Committees of The Johns Hopkins University School of Medicine. No difference was observed in experiments performed distinguishing between sexes. As such, both male and female mice were considered for analyses for this study. For all experiments, we use wild-type C57BL/6 mice (stock number 027 from Charles River Laboratories). These are general-use animals that are used by many laboratories in the field. The specific age of animal used is listed in the experimental procedure sections. For the majority of experiments, mice were euthanized with carbon dioxide-induced anoxia and decapitated as a secondary method of euthanasia. For in vivo experiments, animals were anesthetized with isofluorane and then decapitated.
Perfusion.
P30 WT C57B1/6 Mice were anesthetized with Isoflourane and rapidly perfused with phosphate buffer and 0.5% paraformaldehyde/1.0% glutaraldehyde and brains were thin-sectioned for Immuno-EM analysis.
Immuno-Electron Microscopy and Analysis.
Brain slices from perfused mice and neuronal cultures were fixed and processed for Electron Microscopy. EM Grids were incubated in the primary antibody overnight at 4° C. followed by secondary antibodies for 2 hours at room temperature. All grids were viewed with a Phillips CM 120 TEM operating at 80 Kv and images were captured with an XR 80-8 Megapixel CCD camera by AMT. Neuronal cultures were fixed in 1.5% glutaraldehyde (EM grade, Pella) buffered with 70 mM sodium cacodylate containing 3 mM MgCl2 (356 mOsmols pH 7.2), for 1 hour at room temperature. Thin-sectioned fixed brain slices and neuronal cultures were processed using the following protocol. Following a 30 minute buffer rinse (100 mM cacodylate, 3% sucrose, 3 mM MgCl2, 316 mOsmols, pH 7.2), samples were post-fixed in 1.5% potassium ferrocyanide reduced 1% osmium tetroxide in 100 mM cacodylate containing 3 mM MgCl2, for 1 hr in the dark at 4° C. After en-bloc staining with filtered 0.5% uranyl acetate (aq.), neurons were dehydrated through graded series of ethanols and embedded/cured with Eponate 12 (Pella). LR-white procedural staining was used for HEK293 cells as well as neuronal cultures. A metal hole punch was used to remove 5 mm discs from the polymerized plates. Discs were mounted onto epon blanks and trimmed. Sections were cut on a Reichert Ultra cut E with a Diatome diamond knife. 80 nm sections were picked up on formvar coated 200 mesh nickel grids and treated for antigen removal followed by on grid immunolabelling. Grids were floated on 95° C. citrate buffer pH 6.0 in a porcelain staining dish for 25 minutes, and then allowed to cool on the same solution for 20 min. After a brief series of 50 mM TBS rinses, grids were floated on 50 mM NH4Cl in TBS, blocked with 2% horse serum in TBS (no tween) for 20 minutes. Grids were incubated in primary antibody diluted in blocking solution (1-50 Goat, mouse, rabbit antibody). Grids incubated on blocking solutions served as negative controls. Sections were allowed to come to room temperature (1 hour) on antibody solutions and placed on appropriated blocking solutions for 10 min. After further TBS rinses, grids were floated upon 12 nm Au conjugated donkey anti-goat, 12 nm Au conjugated goat anti-rabbit, 12 nm Au conjugated donkey anti-mouse, or Au conjugated streptavidin (Jackson Immunoresearch) at 1-40 dilutions in TBS for 2 hours at room temperature. Grids were then rinsed in TBS, floated upon 1% glutaraldehyde for 5 min, rinsed again and stained with 2% filtered uranyl acetate. All grids were viewed with a Phillips CM 120 TEM operating at 80 Kv and images were captured with an XR 80-8 Megapixel CCD camera by AMT.
Cell Lines
For primary mouse neuronal cultures, pregnant wild-type C57/B6 mice were obtained from Charles River Laboratories, and sacrificed at E17.5. Whole cortices were dissected, processed into a single cell suspension, and plated as previously described58. Primary cell lines isolated in our laboratory from mouse brains are identified by surface markers that are unique to neuronal cells. These approaches have high sensitivity to accurately identify specific cells. Alternatively, for biochemical studies analysis of primary cell lines can be done using western blotting with well-validated antibodies to neuronal specific markers. Human Embryonic Kidney (HEK293) and Neuro-2A neuroblastoma cells were obtained from ATCC and maintained and expanded and frozen down in a series of aliquots. These aliquots are cultured for a limited number of passages (<10). They are regularly tested for any infection. The lab maintains strict guidelines for cell culture and monitoring of cell health in order to minimize biological variability and to prevent cell line cross-contamination during culture. Each cell line is maintained in its own culture medium.
Cell Culture and Transfection.
HEK293 and Neuro2A cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine (Sigma), and penicillin/streptomycin (100 U/mL and 100 μg/mL, respectively; Sigma). Mouse cortical neurons were prepared from E17.5 C57B1/6 mouse embryos as previously described58. Neurons were maintained in Neurobasal Medium (Invitrogen) supplemented with 2% B-27 (Invitrogen), penicillin/streptomycin (100 U/mL and 100 μg/mL, respectively), and 2 mM glutamine. Dissociated neurons were transfected using the Lipofectamine method (Invitrogen) according to the manufacturer's suggestions.
Each cell line is maintained in its own culture medium. Neurons were maintained in Neurobasal Medium (Invitrogen) supplemented with 2% B-27 (Invitrogen), penicillin/streptomycin (100 U/mL and 100 μg/mL, respectively), and 2 mM glutamine. HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine (Sigma), and penicillin/streptomycin (100 U/mL and 100 μg/mL, respectively; Sigma).
For analyzing the expression of immediate-early gene products, unique care was taken to ensure that neurons had reduced activity at baseline as measured by the expression of immediate early genes. After switching 500K neurons/well in 12 well format of cultured cortical neurons into 1 mL Neurobasal/B27 at DIV3, neurons were maintained in that medium, with only one 100 μl media exchange at DIV9. At DIV15, neurons were treated with pharmacological agents as indicated. Great caution was taken to minimize physical perturbation of these cultures so as not to induce any activation of IEG proteins. For example, drugs were resuspended in a small volume of growth media (media in which neurons were growing in) before addition, so cultures did not have to be shaken to treat neurons.
Antibody Feeding and Immunocytochemistry.
Cultured cortical neurons were plated on glass coverslips coated with poly-L lysine overnight. Neurons were allowed to mature to DIV 14 for feeding experiments. DIV 14 cortical neurons were slowly washed twice with cold PBS supplemented with 1 mM CaCl2 and 2 mM MgCl2 to slow recycling and internalization. Care was taken not to shear cell bodies from the neuron, and to maintain neuronal morphology. Cold neurons, while alive, were treated with Chicken anti-MAP2 antibodies (1:100), Goat anti-β5 proteasome subunit antibodies (1:50), and Rabbit anti-GluR1 (1:100) in PBS supplemented with 1 mM CaCl2 and 2 mM MgCl2 for 30 minutes at 4° C. Antibodies were washed off, and neurons were rinsed twice in cold PBS, 1 minute each. Neurons with bound antibodies were fixed in 4% paraformaldehyde/4% sucrose in PBS for 75 seconds, so not to destroy the antibody itself but to maintain neuronal morphology. Samples were visualized using donkey anti-goat AF-488, donkey anti-chicken AF-555, and donkey anti-rabbit AF-647 (1:250 each) in 1×non-permeabilizing GDB (30 mM phosphate buffer pH 7.4 containing 0.2% gelatin, and 0.8 M NaCl) for 1 hour at 25° C. Samples on coverslips were mounted on glass slides using Fluoromount-G (Southern Biotech). Neurons were imaged using a laser scanning Zeiss LSM780 FCS microscope. Images are representative maximal Z projections of multiple optical sections.
Protease Protection Assay.
Cortical neuronal cultures were treated for the indicated times with 1 μg/mL of Proteinase K (NEB) in HBSSM (Hank's Balanced Salt Solution without CaCl2 or phenol red, supplemented with 1 mM MgCl2). Excess Proteinase K was quickly washed away three times in HBSSM, and Proteinase K activity was quenched twice for 3 minutes with 10 μM PMSF in HBSSM at 4° C. Neurons were then fractionated into cytosolic and membrane fractions as described above, and samples were prepared for SDS-PAGE and western analysis.
Surface Biotin-Labeling, Cell Lysis, Streptavidin Pulldown, and Western Blots.
Surface biotin-labeling was performed as previously described26. Whole mouse brains, cultured cells or whole animal tissue were obtained where indicated and each sample was labeled using Sulfo-NHS-LC-Biotin (ThermoFisher). Cultured cells were washed in pH 8.0 PBS (Gibco) with 1 mM CaCl2 and 2 mM MgCl2 (PBSCM) and treated with 1 mg/mL Sulfo-NHS-LC-Biotin dissolved in PBSCM for 20 minutes at 4° C. before the reaction was quenched for 10 minutes in 50 mM glycine in PBSCM. Intact tissue was quickly and manually chopped, following biotinylation for only 10 minutes at 4° C. in 0.5 mg/mL Sulfo-NHS-LC-Biotin prior to quenching the reaction. Whole mouse tissues and cultured neurons were collected and homogenized in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, 5 mM EDTA, complete protease inhibitor cocktail tablet (Roche), 1 mM β-glycerophosphate). Where indicated, the salt concentration in our RIPA lysis buffer was increased up to 300 mM NaCl. Primary, human central nervous system (CNS) tissue, gestational weeks 19-21. were obtained under surgical written consent following protocols approved by the Johns Hopkins Institutional Review Board, based on its designation as biological waste. Tissue was mechanically chopped at 4° C., and immediately processed for surface biotinylatioti. For streptavidin pulldown experiments, lysed cells were incubated with high-capacity streptavidin agarose beads (ThermoFisher) overnight and then washed thrice with RIPA buffer before elution in SDS sample buffer. Western blots were performed using conventional approaches. Gels were run either on 4-15% SDS-PAGE gradient gels (Bio-Rad) or on 10% gels made in the laboratory. Proteins were transferred to nitrocellulose membranes at 100V for 1.5 hours in 20% methanol containing transfer buffer. All antibodies were made up in 5% BSA in 0.1% TBST. Western blots were incubated with appropriate secondary antibodies coupled to Horseradish Peroxidase, extensively washed, and incubated with ECL. Images were exposed on film, and were scanned in and quantified using ImageJ by standard densitometry analysis.
Cellular Fractionation and Integral Membrane Determination
For cellular fractionation experiments to determine the membrane attachment of the proteasome, cultured neurons were lysed in either a sucrose buffer (0.32 M sucrose, 5 mM HEPES, 0.1 mM EDTA, 0.25 mM DTT) or hypotonic lysis buffer (5 mM HEPES, 2 mM ATP, 1 mM MgCl2) collected. Nuclei were pelleted at 800 RPM for 5 minutes, and the supernatant containing membranes was pelleted at 55000 RPM for 1 hour. Pelleted membranes were washed twice by homogenizing in lysis buffer and re-pelleted. Following two washes, membranes were processed for appropriate application. Supernatants containing the cytosolic extracts were concentrated down to the same volume that membranes were eventually resuspended in. Membrane association was determined by classic methods of sodium carbonate extraction. Briefly, purified neuronal membranes were resuspended in 50 mM sodium carbonate, pH 11 and incubated for 10 minutes at 4° C. to strip away membrane-associated proteins. Membranes, along with tightly-associated membrane proteins, were pelleted at 55000 RPM for 1 hour. Incubating membranes with sodium carbonate at high pH is thought to strip peripherally-associated proteins from the membranes, leaving only tightly-associated and integral membrane proteins bound to the membranes. Samples were subsequently prepared for SDS-PAGE analysis. For Digitonin fractionation, samples were lysed in sucrose buffer. Once the supernatant (cytosolic fraction) was set aside, the pellet was washed 2× with sucrose buffer, and then resuspended in sucrose buffer with indicated concentrations of digitonin. Following a 30 minute incubation in the buffer, samples were spun down at 55000 RPM for 1 hour. This was repeated for all indicated concentrations of detergent. For
TX-114 Phase Extraction.
Protocol was adapted from33. Briefly, primary neuronal cultures were treated with 1% precondensed TX-114. Samples were dounce homogenized, spun at 4° C., and incubated at 30° C. Samples were centrifuged for 3 minutes at room temperature. Supernatant was retained as the TX114-free fraction and resulting pellet was kept as the TX114-rich fraction. This approach relies upon a temperature-dependent shift of the critical micellar concentration of TX-114, and provides an approximate determination of the hydrophobicity of proteins.
Concanavalin-A Plasma Membrane Isolation.
Protocol was adapted from31. Briefly, 0.25 mg biotinylated Concanavalin-A (ConA) was first coupled to 1 mL of streptavidin-coated agarose beads. Nuclei were pelleted from hypotonically lysed DIV 16 cultured cortical neurons, as described above, and the supernatant containing plasma membranes and cytosol were applied to 150 ul of ConA beads. After thorough washing in lysis buffer containing 0.025% Nonidet-P40, samples were prepared for SDS-PAGE and western analysis.
DNA Constructs.
The full-length mouse tagged GPM6A, tagged GPM6B, tagged β5 constructs were acquired from Origene. All vectors obtained from commercial sources are verified and tested for the appropriate expression of the inserts using primary antibodies or epitope-tag antibodies against the expressed proteins. While we keep stocks of each validated plasmid, we periodically sequence these plasmids to confirm their authenticity. All plasmids used in this study are amplified and purified using standard kits from commercial vendors.
shRNA Knockdown.
Four unique shRNA constructs were obtained each against GPM6A, GPM6B, and PLP from Origene. These were validated HuSH 29mer shRNA constructs expressing GFP. Each construct was transfected into neurons using previously described and standard protocols. Each construct was transfected at 100 ng and 500 ng/well. In addition, the constructs were co-transfected in combination to knockdown either two, or all three genes.
Human Subjects.
Fetal brain tissue was obtained at Johns Hopkins University. Primary cultures of fetal cortical tissues were prepared. The use of fetal brain tissue was approved by the Johns Hopkins University institutional review board (IRB). Informed consent was obtained from all subjects. The authors did not have access to any identifying personal information.
Co-Immunoprecipitations.
Transfected HEK293 cells were collected and homogenized in IP Buffer (1% NP-40, 2mM MgCl2, 300 mM NaCl, 2 mM CaCl2, 50 mM HEPES, 10% Glycerol) buffer. For immunoprecipitations, lysates were incubated with FLAG-M2 agarose beads (Sigma-Aldrich). Precipitated samples were washed and prepared for SDS-PAGE and immunoblot analysis.
Proteasome Purification and Assessment of Catalytic Activity.
For proteasome purification, cells were treated and then immediately put on ice before purifications were performed as previously described45. Briefly, proteasomes were purified out of neuronal cytosol and detergent-extracted neuronal plasma membranes using the 20S proteasome purification kit (Enzo Life Sciences) or the 26S proteasome purification kit (UBPBio). The first method relies on immunoprecipitating proteasomes using proteasome β2 or β5 subunit antibodies covalently coupled to agarose beads (20S purification matrix). It is important to note that this purification scheme can purify any 20S-containing proteasome complex. As an alternative method, we used a previously described affinity purification that utilizes GST-Ubl binding to the 19S cap and subsequent pulldown on Glutathione-coupled sepharose (26S purification matrix). This method enriches for proteasomes that are capped by the 19S complex. For western blots, samples were denatured at 65° C. for 5 minutes in SDS sample buffer, resolved by SDS PAGE, transferred to nitrocellulose, and immunoblotted. For catalytic activity assays, ⅙th of the bead volume following proteasome purification was resuspended in activity assay buffer (20 mM Tris-HCl, pH8.0, 5 mM ATP, 5 mM MgCl2, 1 mM DTT). 26S Proteasomal activity was assessed by the addition of 10 μM of SUC-LLVY-AMC (Enzo Life Sciences). The contribution of 20S proteasomal activity was assessed by the comparison of 26S proteasome activity to that of total proteasome activity (26S+20S), measured by the activity of samples containing SDS at a final concentration of 0.05%.
Cell Culture Radiolabeling
Cortical neurons were cultured for 12 days in vitro. Radioactive labeling was done in Neurobasal growth media with B-27 supplement and without methionine or cysteine (Life Technologies, special order). 35S methionine/cysteine (EasyTag PerkinElmer) was incorporated during indicated times at 55 mCi in the met/cys free growth medium. Where indicated, MG-132 (25 μM, Cell Signaling) and ATPγS (1 mM, Sigma) was added during the radioactive labeling window. For all labeling experiments, normal growth media on neurons was switched into labeling media supplemented with radioactive label for 10 minutes. Lysates were prepared in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, 5 mM EDTA, complete protease inhibitor cocktail tablet (Roche), 1 mM sodium orthovanadate, 1 mM β-glycerophosphate). SDS sample buffer was added and samples were boiled for 5 minutes prior to loading onto SDS-PAGE gels. Autoradiographs were done by loading samples onto large SDS-PAGE gels, coomassie stained to verify equal loading, and then gels were dried down on a large gel drier onto Whatman filter paper. Dried gels were exposed to phosphorimager screens and scanned with a Typhoon FLA5500 imager. A variety of other manipulations and pharmacological agents were used during the pulse-chase protocol as indicated in supplementary
Peptide (SNAPP) Collection and Quantification.
Following incorporation of radioactive 35S methionine/cysteine, neurons were rapidly washed in PBS and fresh Neurobasal media without phenol red and with 2×B-27 supplement was added. At the two-minute time point, all of the media was collected and then spun through a 10 kDa Amicon filter (Millipore) and the flow through was then spun through a 3 kDa Amicon filter (Millipore). The flow-through from this sequential filtering was then dialyzed using dialysis tubing with a 100-500 Da cutoff (Spectrum Labs) into either 1×PBS (Gibco) or 20 mM Ammonium Bicarbonate (Sigma). Following four days of dialysis, samples were lyophilized and resuspended in MilliQ water for downstream calcium imaging. Quantification of peptides was done by counting the amount of radioactivity in each sample by liquid scintillation (Wallac 1410). Proteinase K control experiments were done by treating the media with 100 μg/mL proteinase K overnight in 2 M Urea and 10 mM BME, prior to re-dialyzing the proteolyzed media into 2 M Urea for two days, and then gradually reducing the Urea concentration down into NaCl and then into Ammonium Bicarbonate. Resuspended peptides were quantified prior to applications using LavaPep Fluorescent Peptide Quantification Kit (LP022010, Gel Company).
Biotin-Epoxomicin.
Biotin-epoxomicin is de-novo synthesized and purchased from Leiden University Institute of Chemistry. They are fully equipped with synthetic capabilities in organic chemistry. Mass spectrometry and NMR verify all batches produced by his lab for quality and purity. All batches used have had >99% purity. To further minimize batch variation, we test all batches in biological experiments (dose-titration for peptide release, NMP inhibition and cell viability responses).
Biotin-epoxomicin was added to neuronal cultures at 25 μM immediately after labeling. Following peptide release assays, treated cells were lysed in a sucrose homogenization buffer (0.32 M sucrose, 5 mM HEPES, 0.1 mM EDTA, 0.25 mM DTT). Membranes were separated from the cytosol by high-speed centrifugation at 55,000 RPM for 1 hour. Fractions were solubilized in SDS sample buffer prior to loading on SDS-PAGE gels for western analysis. EM processing was done after 5 minutes of treatment with Biotin-Epoxomicin.
Calcium Imaging
Calcium imaging was performed as previously described59. Briefly, for the Biotin-Epoxomicin experiments, cultured embryonic cortical neurons were transfected with 1 μg of a mammalian expression construct encoding GCaMP3 at DIV10 and imaged at DIV 12-14. Bicuculline treatment was administered as a 1 μM stimulation in calcium imaging buffer in a perfusion setup. Once the bicuculline stimulation was washed out, biotin-epoxomicin (25 μM) was co-administered with 1 μM Bicuculline in calcium imaging buffer. Each treatment was monitored for three minutes prior to washout. Coverslips were not imaged twice due to Biotin-Epoxomicin being a covalent inhibitor. Cells were ensured to be healthy at the end of the imaging process by stimulating with 55 mM KCl and washing out and assessing for a proper calcium signal. Quantification was done by picking multiple regions of interests in primary and secondary dendrites across multiple coverslips over different imaging days. Data was analyzed using the Time Series Analyzer V3.0 ImageJ plugin and the ROI manager. Data were pooled for all the ROIs to generate a single N value. Brains from P0-P3 mouse pups (Cre-GCaMP3; Nestin-Cre ER) were dissected and plated in Neurobasal-A with B-27 supplement for two weeks. At DIV7, 4-hydroxytamoxifen (4-HT, concentration) was added to induce GCaMP expression. Neurons were imaged in a calcium-imaging buffer (130 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 0.6 mM MgCl2, 10 mM Hepes, 10 mM glucose, 1.2 mM NaHCO3 pH 7.45). Peptides (SNAPPs) were collected, filtered, and dialyzed and then lyophilized prior to resuspension in 1 mL of MilliQ water and addition onto GCaMP-encoding neurons. 5 μl of resuspended peptides were sufficient to induce the described calcium-induced effects. Peptides treated with Proteinase K were spun through a 10 kDa MW cutoff filter prior to addition onto neurons in order to remove Proteinase K. Pharmacological inhibitors were perfused in at the indicated times at the following concentrations: BAPTA (2 μM), Thapsigargin (5 μM), Tetrodotoxin (1 μM), Nifedipine (1 μM), APV (2 μM).
Mass Spectrometry
Mass spectrometry for proteasomes isolated from cytosolic and membrane fractions was performed at MS Bioworks, LLC. Otherwise, the fractionated peptides were analyzed on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer coupled with the UltiMate™ RSLCnano nano-flow liquid chromatography system (Thermo Fisher Scientific). The peptides from each fraction were reconstituted in 0.1% formic acid and loaded on a Acclaim PepMap100 Nano-Trap Column (100 μm×2 cm, Thermo Fisher Scientific) packed with 5 μm C18 particles at a flow rate of 5 μl per minute. Peptides were resolved at 250-nl/min flow rate using a linear gradient of 10% to 35% solvent B (0.1% formic acid in 95% acetonitrile) over 95 minutes on an EASY-Spray column (50 cm×75 μm ID, Thermo Fisher Scientific) packed with 2 μm C18 particles, which was fitted with an EASY-Spray ion source that was operated at a voltage of 2.0 kV.
Mass spectrometry analysis was carried out in a data-dependent manner with a full scan in the mass-to-charge ratio (m/z) range of 350 to 1550 in the “Top Speed” setting, three seconds per cycle. MS1 and MS2 were acquired for the precursor ion detection and peptide fragmentation ion detection, respectively. MS1 scans were measured at a resolution of 120,000 at an m/z of 200. MS2 scan were acquired by fragmenting precursor ions using the higher-energy collisional dissociation (HCD) method and detected at a mass resolution of 50,000, at an m/z of 200. Automatic gain control for MS1 was set to one million ions and for MS2 was set to 0.05 million ions. A maximum ion injection time was set to 50 ms for MS1 and 100 ms for MS2. MS1 was acquired in profile mode and MS2 was acquired in centroid mode. Higher-energy collisional dissociation was set to 35 for MS2. Dynamic exclusion was set to 30 seconds, and singly-charged ions were rejected. Internal calibration was carried out using the lock mass option (m/z 445.1200025) from ambient air.
Statistics
No statistical methods were used to predetermine sample size. The experiments were not randomized. All statistical analyses were performed using Origin Prism and Graphpad software, accounting for appropriate distribution and variance to ensure proper statistical parameters were applied. Experimental sample sizes were chosen according to norms within the field. The observed magnitude of differences, together with the low replicate variance, permits high power of analysis based on the sample size chosen. For quantification of proteasomal localization by EM analysis, images were acquired by an independent assistant in the Johns Hopkins imaging core not involved in the experimentation and counts were then objectively tallied by a second assistant without knowledge of the experimental groups. Statistical methods used are described in figure legends for the respective EM experiments. For remaining experiments investigators were not blinded to allocation during experiments and outcome assessment.
Statistical analysis using Student's t tests, 1-way ANOVAs and the appropriate post hoc tests were performed as described in each figure legend. P values≤0.05 were considered significant. Notable exceptions to this are in the mass spectrometry data.
Antibodies. The following were used according to manufacturer's and/or published suggestions for immunoblotting: anti-β-Actin (Abcam), anti-Biotin (Cell Signaling), Streptavidin-AF647 (Invitrogen), anti-Arc (Gift from P. Worley, Johns Hopkins, verified against knockout), anti-Fos (Cell Signaling), anti-Npas4 (Gift from Y. Lin, MIT, verified against knockout), anti-PSD-95 (Pierce), anti-Ube3A (Sigma, verified against knockout), anti-Ubiquitin (FK2, Enzo), anti-S6 ribosomal subunit (Cell Signaling), standard secondary antibodies were purchased. We attempted to use antibodies that were verified by knockout controls in either our study, or by other groups. We only used antibodies that provided a signal at the appropriate molecular weight, and where minimal nonspecific bands were observed.
Immunoblot Analysis.
Immunoblots were performed using conventional approaches. Tris/Glycine gels were run on either 10% or 12% gels made in the laboratory. Proteins were transferred to nitrocellulose membranes at 100V for 2 hours in 20% methanol containing transfer buffer. All antibodies were made up in 5% BSA in 0.1% TBST, except for Arc antibody which was made up in 5% Milk in 0.1% TBST. Immunoblots were incubated with appropriate secondary antibodies coupled to Horseradish Peroxidase, extensively washed, and incubated with ECL. Blots were exposed on film, and were scanned in and quantified using ImageJ by standard densitometry analysis.
Ribosome Pelleting
Ribosome-nascent chain complexes were isolated according to well established protocols (Brandman et al., 2012; Duttler et al., 2013). Following various treatments and radiolabelling, neurons were lysed in a buffer containing either 100 ug/mL Cycloheximide or Puromycin (25 mM HEPES pH7.5, 10 mM MgCl2, 20 mM KCl, 50 mM NaCl, 2 mM ATP, 10u SuperASE-In, 20 μM MG-132, 1.5% Triton X-100, protease inhibitors). Lysates were cleared by centrifugation at 10,000 RPM for 10 minutes, and the supernatant was layered onto a 1M sucrose cushion. Ribosome-nascent chain complexes or empty ribosomes (following puromycin treatment) were pelleted via centrifugation at 70,000 RPM in a Ti 70.3 rotor. Supernatants were discarded and ribosomal pellets were washed three times with lysis buffer. 1/10 of the ribosomes were counted by liquid scintillation and the remainder was prepared in SDS loading buffer.
2 Dimensional Gels for Nascent Chain Analysis.
2-dimensional gels to analyze the ribosome-nascent chain complex were performed as previously described (Ito et al., 2011). Briefly, following 30 seconds of radiolabel incorporation at room temperature, neurons were lysed in buffers containing either Cycloheximide or Puromycin. Following lysis, RNCs were isolated as described above. Isolated RNC complexes were resuspended in SDS loading buffer, and then loaded onto neutral pH SDS-PAGE gels to minimize in-gel tRNA hydrolysis. Each samples was run with a few microliters of prestained ladder to delineate the lanes. After running in a single dimension, lanes were cut out of the gel and then incubated with 1N NaOH at 80° C. to degraded any RNA in the sample. This treatment hydrolyzes the ester bond linking the tRNA to its nascent polypeptide, generating a population of radiolabeled proteins whose mass is reduced by the weight of the tRNA (˜25 kDa). Following RNA hydrolysis, samples were run in a second dimension, and then transferred onto nitrocellulose membranes. After exposure for autoradiography, membranes were blocked in BSA and immunoblotted using anti-ubiquitin antibodies.
Protein Extraction, Digestion, and Labeling.
After indicated treatments, the cells were lysed by adding in 6 M urea and 2 M thiourea buffer with protease inhibitor cocktail. The lysates were sonicated with 35% amplitude for 1 min. Protein lysates were centrifuged at 16,000 g at 4° C. to exclude cell debris (pelleting at the bottom), and protein concentration was estimated using a SDS-PAGE method. Briefly, protein lysate was loaded with BSA standard ranging from 0.33 μg to 9 μg on a 3-12% NuPAGE gradient gel and separated for about 0.5 cm. The gel was stained with Colloidal Coomassie G-250 followed by destaining with water. The band intensities were measured by ImageJ software. A total of 200 μg of each sample was reduced with 10 mM dithiothreitol at room temperature for one hour and alkylated with 30 mM iodoacetamide for 20 minutes in the dark. The protein samples were digested using endoproteinase LysC (1:100) at 37° C. for 3 hours followed by sequencing-grade trypsin (1:50) at 37° C. overnight. After the digestion, the peptide samples were subjected to desalting and labeling with 10-plex TMT reagents according to the manufacturer's instructions (Thermo Fisher Scientific) and the 9/10 channels (126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C) were used for labeling. The labeling reaction was performed for one hour at room temperature, followed by quenching with 100 mM Tris-HCl (pH 8.0). The digested and labeled peptides from all 10 channels were pooled.
The peptides were fractionated by basic pH reversed-phase liquid chromatography (bRPLC) into 96 fractions, followed by concatenation into 24 fractions by combining every 24th fractions. Briefly, Agilent 1260 offline LC system was used for bRPLC fractionation, which includes a binary pump, VWD detector, an autosampler, and an automatic fraction collector. In brief, lyophilized samples were reconstituted in solvent A (10 mM triethylammonium bicarbonate, pH 8.5) and loaded onto XBridge C18, 5 μm 250×4.6 mm column (Waters, Milford, Mass.). Peptides were resolved using a gradient of 3 to 50% solvent B (10 mM triethylammonium bicarbonate in acetonitrile, pH 8.5) at a flow rate of 1 ml per min over 50 min collecting 96 fractions. Subsequently, the fractions were concatenated into 24 fractions followed by vacuum drying using SpeedVac. The dried peptides were suspended in 0.1% formic acid.
Data Analysis.
Proteome Discoverer (v 2.1; Thermo Scientific) suite was used for quantitation and identification. During the preprocessing of MS/MS spectra, the top 10 peaks in each window of 100 m/z were selected for database search. The tandem mass spectrometry data were then searched using SEQUEST algorithms against mouse RefSeq protein database (version 84) with common contaminant proteins. The search parameters used were as follows: a) trypsin as a proteolytic enzyme (with up to two missed cleavages); b) peptide mass error tolerance of 10 ppm; c) fragment mass error tolerance of 0.02 Da; and d) carbamidomethylation of cysteine (+57.02146 Da) and TMT tags (+229.162932 Da) on lysine residues and peptide N-termini as a fixed modification and oxidation of methionine (+15.99492 Da) as a variable modification. The minimum peptide length was set to 6 amino acids. Peptides and proteins were filtered at a 1% false-discovery rate (FDR) at the PSM level using percolator node and at the protein level using protein FDR validator node, respectively.
The protein quantification was performed with following parameters and methods. The most confident centroid option was used for the integration mode while the reporter ion tolerance was set to 20 ppm. The MS order was set to MS2 and the activation type was set to HCD. Unique and razor peptides both were used for peptide quantification while protein groups were considered for peptide uniqueness. Reporter ion abundance was computed based on signal-to-noise ratio and the missing intensity values were replaced with the minimum value. The quantification value corrections for isobaric tags and data normalization were disabled while the co-isolation threshold was set to 50%. The highest signal-to-noise ratio value from PSMs for a peptide was used to generate a peptide level abundance followed by averaging peptide level signal-to-noise ratio values for a protein to generate a protein level abundance.
Protein grouping was performed with strict parsimony principle to generate the final protein groups. All proteins sharing the same set or subset of identified peptides were grouped while protein groups with no unique peptides were filtered out. The Proteome Discoverer iterated through all spectra and selected PSM with the highest number of unambiguous and unique peptides.
TMT Differential Expression
The list of quantified proteins exported from Proteome Discoverer 2.1 was utilized as the input for our differential expression analysis. The raw values were organized in a matrix where each column represented a sample and each row a protein. To normalize the raw expression values, we began by loge transforming the matrix with a +1 for computation. Then we median polished the log-transformed values by subtracting the row median from each row, followed by the subtraction of the column median from each column. The resulting normalized expression values for each sample appeared normally distributed and was comparable across samples.
For the detection of differential regulation, we followed the recommendation outline in (Kammers et al., 2015). An empirical Bayes method was employed on the normalized matrix to detect differences between the 3 samples of the biotin-epoxomicin treated group compared to the 6 samples of the control and cycloheximide groups. The empirical Bayes method shrinks individual protein's sample variance towards a pooled estimate, and creates a more stable and powerful inference in differential protein abundance detection.
The output of the differential abundance analysis detected 1340 and 408 proteins to be differentially abundant at the 0.05 and 0.01 level respectively. However, due to the large number of proteins tested, we were more interested in q-values that adjust for multiple comparisons. Using a cutoff of q<0.1, which corresponds to a false discovery rate of 10%, we detect 190 proteins to be differentially abundant in the 2 groups that we defined. Of those 190 proteins, 122 were up-regulated.
For the selection of the colors in the heatmap, we carried out feature-scaling of the normalized expression values on a gene-by-gene basis. For each gene, this process assigns the largest expression a value of 1, and the smallest expression a value of 0. The remaining values are scaled between 0 and 1 based on where they are relative to the largest and smallest expression values. For instance, a feature-scaled value of 0.5 represents an expression level that is halfway between the lowest expression and the highest expression observed for a gene. In other words, this sample's expression is 50% of the maximum fold change away from the lowest and the highest expression values at this gene.
Markov Chains to Model Radioisotope Release
To model the radioisotope release curves that were experimentally observed, we employed Markov chain simulations. A given Markov chain simulates the location of a single radioisotope in 1-second increments, starting at the moment of washout until 1800 seconds (30 minutes) after. The transition process and probabilities between states is given in
A free isotope has at each second interval a pBackground chance of diffusing across the cell membrane to become a free isotope extracellularly. In that same second interval, that isotope also has a pLoading chance of coming in contact with a ribosome and becoming a part of a nascent polypeptide. This leaves that for each interval, a free isotope has a 1-pLoading-pBackground chance of remaining as a free isotope.
Once a radioisotope has progressed to the state of a nascent polypeptide, it has some probability pCTD of being released co-translationally. If entering that release path, the time it takes for the release to be realized extracellularly requires a time that is distributed N(8, sd=2)/2.5. The N(8, sd=2) represents that on average cleave sites are every 8 or so amino acids, while 2.5 is the well-established rate which degradation occurs. If not entering this pathway, the nascent chain becomes a folding intermediate. The time required for this is dependent upon the length of the protein that this isotope is being incorporated into, the location at which it is being incorporated, and the rate of translation. To determine the length of the protein, we sampled a protein at random from the list of detected intracellular proteins under full protein degradation inhibited conditions. The probability of sampling each protein is proportional to their relative abundance. Once the protein has been selected, we simulated the point of incorporation of the radioisotope to be uniform along the length of the full protein. The time to progress from a nascent polypeptide to the folding intermediate is determined as the (# of AA in the protein after the incorporation point/5), with 5AA/s being the established rate translation.
Upon becoming a folding intermediate, the radioisotope has a chance pFID of being degraded and released extracellularly. If entering this degradation path, the time before the radioisotope is realized extracellularly is calculated as the # AA in the protein before the incorporation point (recorded from the previous step) divided by the well-established rate of degradation of 2.5 AA/second. If at this point, the radioisotope does not enter the degradation path, it will initiate the process towards a folded protein.
The time it takes for a folding intermediate to become a folded protein is based upon the power law (Lane and Pande, 2013)and is calculated as a random variable following exp(5*log(#AA)−27.7+Norm(0, sd=3)). This corresponds to a folding time of approximately 30 seconds for a 50 kDa protein. Once the protein is folded, it has a probability pFD of entering degradation in any 1-second interval. If it does enter the pathway, we assume the time it takes for the isotope to be released extracellularly is determined mostly by the unfolding time, which we assumed conservatively to be equal to the folding time distribution. Otherwise, the protein remains folded with a probability of 1-pFD. We chose a pFD of 1e-5 for our model because it corresponded to a conservative representation that the half-life of a folded protein existing in a folded state is approximately 20 hours.
Monte Carlo Inference for Model Parameters
With this formulation of the Markov chain, there remains 4 variables that are not based upon previously established results: pLoading, pBackground, pCTD, and pFID. We employed Monte Carlo simulations in a 2-stage process to optimize those parameters to most closely mirror the experimental observed release curves. Experimental release curves were estimated as follows. For each experimental condition, we have observations of released radioisotope at times 0, 60, 120, 300, 600, and 1800 seconds after washout. The value of each time point was divided by the total amount of radioisotope within the cell at 0 seconds after washout to rescale the observations as a proportion. For any point in time between the 5 observed time points, the released proportion was assumed to follow a linear relationship.
We first exploited the assumption that the dominant isotope release pathway should be diffusion (between 0-600 seconds) in an experimental condition where all degradation of proteins is inhibited. We inferred the optimal values of pLoading and pBackground by exploring the parameter space of all pairwise combinations of pLoading between 0.0035 and 0.0075 in 0.0001 increments and pBackground between 0.00001 and 0.0004 in 0.00001 increments. For each of combination of pLoading and pBackground we used Monte Carlo simulations of 2500 Markov chains, each one starting as a free isotope and having transition probabilities given by the pairwise combination of pLoading and pBackground. The proportion of the 2500 initial radioisotopes that is released extracellularly at each second in time was recorded as the simulated release curve. The simulated release curves were compared to the experimental release curve when all protein degradation was inhibited to determine the optimal combination of pLoading and pBackground. The penalty measure is the sum of the squared distance between observed and simulated at each time point between 1 and 600 seconds. We chose not to evaluate the curves beyond 600 seconds because it appeared reasonable that diffusion was the dominant form of isotope release prior to 600 seconds. For the time range between 600-1800 seconds, other release mechanisms like autophagy might confound our efforts. This process revealed pLoading and pBackground to be optimized at 0.0056 and 0.00017 respectively.
After having optimized pLoading and pBackground, we continue on to find the pair of pCTD and pFID that best matches the experimental release curves under control conditions. We used a similar Monte Carlo simulation approach looking at all pairwise combinations of pCTD and pFID both between 0 and 0.7 in 0.001 increments. Using experimental data, we calculated that at the moment of washout, the ratio of free radioisotopes to isotopes in folded protein to isotopes in nascent polypeptides to be 300:20:1. As such, for each pairwise simulation, we initiated the initial state of the Markov chains to reflect that ratio. For each pairwise simulation, we simulated between 15,000-20,000 Markov chains, and tracked the progression of the isotopes for 1800 seconds. The simulated proportion of radioisotopes at any point of time that is extracellular was calculated as our simulated release curve. We searched for the pair of pCTD and pFID that produced the minimum total squared error at each time point from 1-1800 seconds between the simulated curve and the observed control release curve. The optimal values for pCTD and pFID were observed to be 0.047 and 0.0 respectively.
We conducted the same optimization process of pCTD and pFID under KCl stimulation to in a manner that mirrored the above approach. We evaluated a parameter space for pCTD and pFID both between 0 and 0.2 in 0.05 increments. We searched for the pair that produced the minimum total squared error at each time point from 1-600 seconds between the simulated curve and the observed KCl release curve. The optimal values for pCTD and pFID were 0.165 and 0 respectively.
20S proteasome subunits are localized to neuronal plasma membranes.
Previous studies have identified localization as a key feature in determining proteasome function16. Distribution of the 26S proteasome in the nervous system has been measured using fluorescently-tagged 19S cap subunits or electron cryotomography (Cryo-ET). While cryo-ET approaches are theoretically unbiased, the processing methods inherently select for analysis of larger complexes, and therefore are more likely to identify singly- and doubly-capped proteasomes. In order to take a high resolution and unbiased approach to evaluate localization of all proteasomes (20S and 20S-containing) in the nervous system, we performed an immunogold electron microscopy (Immuno-EM) analysis of hippocampal slice preparations using antibodies raised against either the proteasome β2, β5 or β2 subunits. These are core 20S proteasome subunits common to all catalytically active proteasomes.
We first performed western blot analysis of mouse brain lysates to assess the antibodies used for our immuno-EM studies. Brains from P30 mice were lysed and prepared for SDS-PAGE, and then immunoblotted using proteasome β2, β5, and α2 subunit antibodies. Each antibody recognized a single band by western analysis at the appropriate molecular weight (
Extending these findings, we performed immuno-EM analysis from mouse primary neuronal cultures, as these preparations are largely devoid of non-neuronal cell types and can provide higher resolution analysis20,21. No immunogold label was observed in samples treated with secondary gold-conjugated antibodies alone (data not shown). Using proteasome β2 and β5 subunit antibodies in mature cultured neurons, we observed ˜40% of immunogold signal at neuronal PMs (
Neuronal membrane proteasomes are exposed to the extracellular space
Immuno-EM staining with a previously validated antibody raised against the cytoplasmic domain of the voltage-gated potassium channel, Kv1.3, only showed cytosolic labeling and labeling on the intracellular face of the PM as previously described22 (data not shown). By immuno-EM analysis we see 20S proteasome staining on the extracellular face of the PM, which raises the possibility that proteasomes may be exposed to the extracellular space (
To biochemically determine whether proteasomes were surface-exposed, we turned to previously described surface-biotinylation/purification approaches26,27 followed by immunoblotting with antibodies against Actin, GluR1, Rpt5 and 20S proteasome subunits. As expected, in our streptavidin pulldown samples from surface-biotinylated neurons we did not detect cytosolic Actin and did detect GluR1 (
As an orthogonal method of identifying surface exposed proteins, we used a protease protection assay, which relies on the proteolysis of extracellularly exposed epitopes of proteins upon treatment of live cells with an extracellular protease28,29. Cultured cortical neurons were treated with Proteinase K (PK) for varying times and then fractionated into either cytosolic or membrane fractions. By immunoblot analysis, we found that proteasomes fractionated to the membrane, similar to N-GluR1, and were susceptible to proteolysis by extracellular PK (
Neuronal membrane proteasomes are tightly associated with plasma membranes
We wanted to further enhance our biochemical understanding of how proteasomes, as largely hydrophilic complexes, could be localized to the hydrophobic PM. Neuronal membranes were isolated and sequentially extracted with increasing concentrations of digitonin to pull out increasingly hydrophobic proteins. Samples were prepared for western analysis (
We considered there were two primary ways this could be possible: (1) the proteasome itself was hydrophobic in some way or (2) the proteasome was tightly associating with integral membrane proteins. In an attempt to distinguish between these possibilities, we performed Triton X-114 (TX114) phase partitioning of cultured neurons to separate hydrophilic and hydrophobic proteins33. Immunoblotting the TX114-rich and TX114-free fractions, we observed Actin fractionated into the TX114-free phase, multi-pass transmembrane protein GluR1 fractionated into the TX114-rich phase, and EphB2, a single-pass transmembrane protein fractionated into both phases (
Neuronal membrane proteasomes are largely a 20S proteasome and in complex with GPM6 family glycoproteins.
To identify potential auxiliary membrane proteins associated with the NMP we isolated proteasome complexes out of neurons using two different affinity methods34. Cytosolic and membrane-extracted fractions from neuronal cultures were incubated with 20S purification matrix (purifies any 20S-containing proteasome complex) or 26S purification matrix (only purifies 26S cap-containing proteasome complex). Immunoblot analysis revealed that both 20S and 26S affinity purification matrices isolated cytosolic proteasomes, but only the 20S purification matrix was able to purify proteasomes out of the membrane (
Using the 20S-purification matrix, we purified 20S proteasomes from the cytosol and membrane of neurons for in-depth mass spectrometry (MS) analysis. As expected, we identified all of the core 20S proteasome subunits in the purification from both membranes and cytosol (data not shown). While we identified a variety of regulatory cap proteins to co-purify with the cytosolic proteasome, we identified very few to co-purify with the proteasome purified from membranes (data not shown). These findings were validated by extensive western analysis (data not shown).
We sought to identify auxiliary membrane proteins in our MS data sets that may be capable of mediating proteasome association with the plasma membrane. We postulated that such a protein would specifically associate with the NMP compared to the cytosolic proteasome, be highly expressed in the nervous system, and be transmembrane (Supplemental Table 1b). Based on these criteria, we focused our efforts on the neuronal membrane glycoprotein GPM6A, a known member of the Proteolipid Protein family of multi-pass transmembrane glycoproteins35,36. To validate these mass spectrometry data, we turned to HEK293 cells as a non-neuronal heterologous system that does not express the NMP (data not shown). Lysates from HEK293 cells previously transfected with expression plasmids encoding myc-/FLAG-tagged GPM6A and GPM6B (myc/FLAG-GPM6A/B) were immunoprecipitated using an anti-FLAG antibody. Immunoblotting using antibodies against myc and 20S proteasome subunits, we found that endogenous proteasome subunits from HEK293s co-immunoprecipitate with myc/FLAG-GPM6A/B (
GPM6A and GPM6B are primarily expressed in the nervous system37. Consistent with these data, using our surface biotinylation assay in whole mouse tissues, we determined that NMP expression was restricted to mouse neuronal tissues (
Neuronal membrane proteasomes degrade intracellular proteins into extracellular peptides (SNAPPs).
To test whether the NMP was catalytically active, we purified proteasomes from both the cytosol and neuronal plasma membranes using a 20S purification matrix and incubated them with SUC-LLVY-AMC, a substrate that fluoresces upon proteasomal chymotrypsin-like cleavage38. Addition of a low concentration of SDS to the reaction relieves the gating mechanism of the 20S proteasome without denaturing the 20S or 26S proteasome holocomplex4. Addition of SDS greatly stimulated the catalytic activity of membrane proteasomes and had little effect on cytosolic proteasome activity (
We were curious as to the purpose of a surface-exposed catalytically active 20S proteasome in the neuronal plasma membrane. Since the core 20S complex alone is ˜11×15 nm, any orientation of the NMP at the neuronal PM, which is 6-10 nm across, would provide it access to both the intracellular and extracellular space. We hypothesized that in neurons, a catalytically active proteasome in such an orientation would be able to promote proteasome-dependent degradation of intracellular proteins into the extracellular space. To test this hypothesis, we used 35S-methionine/cysteine-radiolabelling approaches to trace the fate of newly synthesized intracellular proteins39 (
Neuronal membrane proteasomes are required for release of extracellular peptides (SNAPPs) and modulate neuronal activity.
To specifically determine the contribution of the NMP in the generation of these extracellular peptides, separately from that of the cytosolic proteasome, we identified a chemical tool that was selective to the NMP. We found that biotinylation of the non-reactive portion of epoxomicin, a highly potent and specific proteasome inhibitor, generates a cell-impermeable compound (biotin-epoxomicin) that maintains target specificity41. This compound covalently modifies the catalytic proteasome β subunits, tagging them with biotin. Cultured neurons acutely treated with biotin-epoxomicin were separated into cytosolic and membranes fractions, and immunoblotted using streptavidin-AF647. Biotin signal was only observed in membranes from neurons treated with biotin-epoxomicin and at a size denoting the covalent modification of the membrane proteasome β subunits (
Furthermore, Immuno-EM analysis of neuronal cultures treated with biotin-epoxomicin showed 92±5% of biotin at plasma membranes (
Using this inhibitor, we sought to separate the role of the NMP from the role of the cytosolic proteasome in regulating extracellular peptide production. Acute application of biotin-epoxomicin to radiolabeled neurons inhibited radioactive peptide release into the extracellular space (
Neuronal membrane proteasome-derived peptides (SNAPPs) are sufficient to induce neuronal signaling.
To systematically test the effects of proteasome-directed peptide signaling, peptides (SNAPPs) were purified and then perfused onto GCaMP3-encoding neurons under various conditions. Neurons were ensured to be healthy at the end of every experiment by stimulating with 55 mM KCl, which consistently induced strong calcium signaling. The proteasome-directed peptides were purified and lyophilized following extensive dialysis into ammonium bicarbonate to remove small molecules and neurotransmitters. The lyophilizate was resuspended in calcium imaging buffer. Peptide concentration was determined to be ˜50 ng/mL and was added back at that concentration. Alone, purified peptides induced a robust degree of calcium signaling in naïve neurons (
To identify which channels were relevant to peptide-induced calcium activity, we used different ion channel inhibitors to pharmacologically identify relevant pathways. Blocking fast voltage-gated sodium channels using Tetrodotoxin did not block the peptide-induced calcium signal, revealing that the influx of calcium was probably not due to action potential-induced signaling, and more likely directly due to effects on calcium channels (
NMP-mediated mechanisms ameliorate the early events of Aβ-induced neurological decline; a first step toward NMP-directed therapeutics in treating Alzheimer's disease.
A prevailing hypothesis in Alzheimer's disease (AD) research is that amyloid beta peptide (Aβ) causes plaque formation in the brain, ultimately giving rise to neurodegeneration observed in the AD patient population. Aβ targeted therapeutics is the leading effort in the medical and pharmaceutical community aimed at ridding the world of Alzheimer's disease.
Based on data already provided we now know that the levels of NMP are significantly reduced in AD human brains, brains from AD mouse models and cultured primary neurons treated with Aβ1-42 peptide. Aβ1-42 peptide has been shown extensively both in vitro and in vivo to cause events that lead to neuronal degeneration and animal decline in behavior and physiology relevant to AD.
Based on our work, the NMP is the only enzyme complex in the nervous system that generates proteasome-derived extracellular signaling peptides. Thus, we considered that downregulated levels of the NMP in AD would lead to reduced extracellular peptide production in these patient brains. How early in the disease this occurs remains unclear. If indeed the NMP and its resulting extracellular peptides played a role in AD, two ensuing mechanisms would be possible: 1) reduced levels of the NMP would no longer turnover a certain set of intracellular proteins important for neuronal health, thereby leading to AD, or 2) reduced levels of the NMP would, by definition, lead to reduced extracellular peptide production and reduction of these peptides would make the neuron more susceptible to AD-relevant events, possibly mediated by the 36 to 42 amino acid Aβ-peptide. We favor the second possibility as we have not yet detected any significant change in the levels of any given protein through the NMP. Thus, we hypothesized that reduced NMP-derived peptide production may contribute to AD. We first considered that this could be in relation to the pathogenicity of Aβ-induced neurotoxicity. The reason for this thinking is that Aβ is an endogenous peptide and may either cooperate or compete with the endogenous NMP-derived peptides in the nervous system. To first test this hypothesis, we incubated primary neuronal cultures with fluorescently labeled Aβ1-42 with or without NMP peptides. The endogenous concentration of these peptides is 250 ng/mL, which is approximately 250 nM. We performed a titration of NMP-derived peptides together with a constant concentration of Alexa Fluor 488-labeled Aβ. This labeled Aβ has been previously shown to interact with neurons in a manner that leads to neurodevelopmental decline. It is a surrogate for investigating how Aβ interacts with neurons, the very first step that leads to cognitive decline in AD. We identified that increasing concentrations of NMP-derived peptides led to a reduction in Aβ binding to neurons, with half-maximal effect observed at the endogenous concentration of NMP-derived peptides (
Because NMP peptides could compete away Aβ binding, we hypothesized that this competition might lead to a reduction of Aβ-induced neurotoxic effects. While there are many measures of Aβ-induced toxicity, we were primarily interested in the widely accepted Aβ-induced effects on signaling which are thought to be initiating stimuli to the onset of neurodegeneration in AD. These include decreased phospho-CREB, elevated phospho-c-Jun, elevated phospho-Erk1/2, and elevated cleaved caspase-3 [Vitolo et al 2002 PNAS, Morishima et al 2001 J Neurosci, Chong et al 2006 JBC]. We replicated all of these effects upon treatment of primary neuronal cultures with Aβ1-42. When neurons were incubated with Aβ1-42 and then treated with NMP-derived peptides, we observed that neurons were insensitive to Aβ effects on intracellular signaling. As a control treatment, we compared samples treated with NMP-derived peptides to samples treated with NMP-derived peptides pre-treated with proteinase K (PK), which destroys the peptides. Consistent with previous data, we did not observe any molecular phenotypes when treating neurons with the reverse Aβ42-1 peptide in comparison to Aβ1-42 (
Collectively, these data support the hypothesis that that NMP peptides can serve as endogenous blockers or inhibitors of Aβ binding to neurons. They are the first data of their kind to demonstrate the existence of an endogenous inhibitor of Aβ binding. Moreover, they provide promise that exogenously elevating NMP-derived peptide levels or in theory, chemically inducing NMP levels to enhance endogenous peptide production, may both be viable therapeutic approaches to reverse molecular phenotypes in AD. It should be noted, that because the NMP is specific to the nervous system, targeted approaches to this system should have minimal off-target effects. We expect that NMP-directed pathways will serve as critical players in the hunt to identify new avenues for reversing AD phenotypes in intact systems, efforts which we are currently undertaking.
Using parameters determined in the above experiments, we constructed Markov process chain models in silico which predicted that the kinetics of this process necessitate coordination of translation and degradation. In a series of biochemical analyses, this predicted coordination was instantiated by NMP-mediated and ubiquitin-independent degradation of ribosome-associated nascent polypeptides. Using in-depth, global, and unbiased mass spectrometry, we identified the nascent protein substrates of the NMP. Among these substrates, we found that immediate-early gene products c-Fos and Npas4 were targeted to the NMP during ongoing activity-dependent protein synthesis, prior to activity-induced transcriptional responses. The following examples provided herein generally define an activity-dependent protein homeostasis program through the NMP that selectively targets nascent polypeptides prior to adopting their final functional conformations.
Neuronal stimulation induces NMP-dependent degradation of newly synthesized proteins into extracellular peptides.
To extend our observed findings in
Our working hypothesis was that the observed stimulation-induced NMP-dependent increase in extracellular peptide production would be reflected in enhanced NMP-mediated degradation of a pool of intracellular protein substrates. To test this, we measured the intracellular pool of proteins made during elevated neuronal activity using SDS-PAGE and autoradiography. Neurons were treated with the radiolabeling protocols described above. All samples were coomassie stained after SDS-PAGE to ensure equal sample loading (
Our experiments thus far monitored the NMP-mediated and activity-dependent turnover of proteins made during stimulation. Given that certain protein populations have been shown to be more susceptible to degradation than others (Ha et al., 2016; McShane et al., 2016; Wheatley et al., 1980), we asked whether the degradation kinetics for proteins synthesized during stimulation were different than those for proteins made prior to or following stimulation. Surprisingly, by changing our radiolabeling protocols, we did not observe the same magnitude of stimulation-induced degradation of proteins from neurons that had been radiolabelled prior to the onset of stimulation, even after sustained stimulation (
Monte Carlo simulation of Markov chains favors degradation of nascent polypeptides as the source for NMP-derived extracellular peptides.
Our understanding of NMP function was that it directly degrades intracellular proteins into peptides in the extracellular space (Ramachandran and Margolis, 2017). This predicts that degradation kinetics of intracellular NMP substrates are directly coupled to the release kinetics of the extracellular peptides (Ramachandran and Margolis, 2017). The data thus far relied on 35S-methionine/cysteine addition to neuronal cultures and tracing the fates of the proteins in which radioactive isotopes were incorporated. Following charging onto a tRNA, isotopes go through two major steps on their way to being incorporated into a folded protein: First, they must be incorporated into the growing nascent polypeptide which is associated with the ribosome during protein synthesis. Subsequently, this polypeptide must go through the complex task of folding before achieving its proper folded conformation, some of which is achieved while still ribosome-associated (Gloge et al., 2014; Hartl et al., 2011; Kramer et al., 2009; Pechmann et al., 2013). Very generally, polypeptides progressing from one stage to the next adopt increasing conformational stability with a corresponding increase in their half-lives (Alberts B, 2002). We sought to understand whether our data revealed any selectivity by which population of polypeptides (i.e. nascent polypeptide, folding intermediate, or folded protein) were being targeted for degradation by the NMP.
To achieve this goal, we constructed a simplified Markov chain model to track the fate of radioisotopes over a time course that mirrors our experimental peptide release data. Each Markov chain follows the trajectory of a single radioisotope that begins as a free radioisotope inside the cell, following 10 minutes of simulated isotope incorporation (
While our model was simple, we attempted to account for as many factors as reasonable using biologically determined parameters. When the model was biased towards turnover of nascent polypeptides, we observed that the shape of the in silico release curve closely mirrors the shape of the experimental release curves (
The shapes of the release curves for co-translational degradation and folding intermediate degradation more closely approximated our experimental data than those for folded protein degradation. To further refine our analysis, we used Monte Carlo simulations to optimize which combinations of the probabilities for co-translational and for folding intermediate degradation best give rise to the observed release data (
Co-translational degradation requires translation elongation (Duttler et al., 2013; Inada, 2017; Kramer et al., 2009; Wheatley et al., 1982). One of the hallmarks of co-translational degradation is its sensitivity to the translation elongation inhibitor puromycin (Nathans, 1964). Puromycin is an aminoacyl-tRNA structural analog that engages into the peptidyl transferase center of the ribosome and covalently modifies the growing polypeptide (
Neuronal stimulation induces NMP-mediated co-translational degradation of ribosome-associated nascent polypeptides.
During translation elongation, nascent polypeptides are bound to a tRNA within the ribosome. This complex is collectively referred to as a ribosome-nascent chain complex (RNC)(Duttler et al., 2013). However, multiple groups have reported conditions where nascent polypeptides are separated from the RNC prior to their completion and are subsequently degraded (Duttler et al., 2013; Shao et al., 2013; Wang et al., 2013). To determine whether the NMP was targeting nascent polypeptides while still associated with the RNC, we performed ribosome pelleting assays to isolate RNCs (Brandman et al., 2012). Briefly, 35S-cysteine/methionine radiolabel was added to neuronal cultures in the presence of proteasome inhibitors for only 30 seconds. This shortened protocol preferentially labels nascent polypeptides before they finish synthesis into full-length proteins (Dunler et al., 2013; Ito et al., 2011). Immediately following radiolabelling, neurons were lysed either in the presence of cycloheximide (CHX) and proteasome inhibitors to freeze translation and degradation, or with puromycin and proteasome inhibitors to release the nascent polypeptide from the ribosome and freeze degradation (
To extend these analyses and specifically monitor nascent polypeptides separately from the RNC complex, we leveraged previously described two-dimensional gel electrophoresis (2D-gel) approaches that separate the nascent polypeptides in the form of peptidyl-tRNA from full-length proteins (Ito et al., 2011). Briefly, pelleted RNCs from neurons radiolabeled for 30 seconds were separated in the first dimension by SDS-PAGE (
Using this approach, we analyzed isolated RNCs from radiolabelled neurons following KCl stimulation. We observed approximately a 40% reduction in radiolabel signal intensity of both the fast- (tRNA-hydrolyzed polypeptide) and slow-migrating bands from KCl-stimulated versus control samples (
Identification of activity-dependent nascent NMP substrates.
During neuronal stimulation, were all nascent polypeptides similarly susceptible to co-translational degradation or was there some selectivity in which nascent polypeptides were being targeted? To specify these principles of co-translational degradation through the NMP in an unbiased manner, we turned to global proteomic analysis. A variety of methods have been developed to analyze newly synthesized polypeptides, typically by introducing chemically modifiable noncanonical or unnatural amino acids (Aakalu et al., 2001; Dieterich et al., 2010; Dieterich et al., 2006; Landgraf et al., 2015). These are typically methionine analogs that are incorporated into newly synthesized polypeptides, and serve as a handle to isolate the polypeptides they modify (Aakalu et al., 2001; Dieterich et al., 2010; Dieterich et al., 2006; Landgraf et al., 2015). While these are powerful tools, two issues confounded our use of such approaches. First, decades of work into the stability of nascent chains and newly synthesized polypeptides has shown that proteins made with non-natural amino acids have a higher propensity to be turned over by the proteasome during or immediately following their synthesis [(Benaroudj et al., 2001; Etlinger and Goldberg, 1977; Goldberg and Dice, 1974; Prouty and Goldberg, 1972; Prouty et al., 1975; Rock et al., 2014; Rock et al., 1994; Wheatley, 2011; Wheatley et al., 1980; Wheatley et al., 1982)]. This method would likely bias our analysis of newly synthesized proteasome substrates, and provide an artificial overestimate of this population. Second, the met-tRNA that charges these amino acids prefers endogenous methionine. Therefore, to induce the incorporation of noncanonical amino acids, cells must be incubated in methionine-free media. Additionally, the charging of noncanonical amino acids on met-tRNA is slower, and the efficiency of chemical modification and purifications are imperfect (Hartman et al., 2006). To overcome these limitations, studies utilizing these techniques usually incubated cells for at least one hour in media containing noncanonical amino acids to maximize labeling. These timescales were incongruent with the timescales at which we were conducting our experiments.
Because of the combination of these variables, we chose not to use noncanonical or unnatural amino acids to identify co-translationally degraded substrates of the NMP. Instead, we leveraged unbiased and high-coverage mass spectrometry-based quantitative proteomic analysis using tandem mass tag (TMT) technology (
In our MS data, we identified NMP substrates that were previously described as ubiquitin-proteasome system (UPS) targets, such as Odcl and Rgs4 (
To independently validate our MS data, we used similar treatment conditions as in our MS analysis and analyzed IEG protein levels by immunoblot analysis. Neurons were stimulated with bicuculline for one hour, and treated with either MG-132 or biotin-epoxomicin for the final 10 minutes. The addition of either MG-132 or biotin-epoxomicin in the presence of bicuculline led to an accumulation of IEG proteins, but no change in the protein levels of UPS targets such as PSD95 or Ube3A (
Taking these experimental data together with the Markov modeling and validation, we hypothesized that the NMP exclusively mediates co-translational degradation of IEGs, and not full-length proteins. The data above demonstrating NMP-mediated IEG protein turnover do not distinguish between co-translational degradation and full-length protein degradation. To monitor turnover only of the full-length protein population, we took advantage of the robust induction of IEG protein expression following two hours of bicuculline stimulation (
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
unconventional secretion of inflammatory mediators. J Innate Immun 5, 471-479, doi:10.1159/000346707 (2013).
This application claims the benefit of U.S. Provisional Patent Application No. 62/464,446, filed Feb. 28, 2017, and U.S. Provisional Patent Application No. 62/470,433, filed on Mar. 13, 2017, both of which are hereby incorporated by reference for all purposes as if fully set forth herein.
This invention was made with government support under grant no. 1R01MH102364, awarded by the National Institutes of Health. The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US2018/020173 | 2/28/2018 | WO | 00 |
Number | Date | Country | |
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62464446 | Feb 2017 | US | |
62470433 | Mar 2017 | US |
Number | Date | Country | |
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Parent | 15550632 | Aug 2017 | US |
Child | 16489506 | US |