Mammalian Central Nervous System Research Articles

SASH1 contributes to glial cell migration in the early development of the central nervous system

SAM and SH3 domain-containing 1 (SASH1), a member of the SLy protein family, is a tumor suppressor gene that has been studied for its association with various cancers. SASH1 is highly expressed in the mammalian central nervous system, particularly in glial cells, and is expressed in the central nervous system during zebrafish embryo development. However, SASH1's role in brain development has rarely been investigated. In this study, Morpholino oligonucleotides (MO) were used to down-regulate sash1a expression in zebrafish to observe morphological changes in the brain. Three transgenic zebrafish lines, Tg(gfap:eGFP), Tg(hb9:eGFP), and Tg(coro1a:eGFP) were selected to observe changes in glial cells, neurons, and immune cells after sash1a knockdown. Our results showed that the number of microglia residing in the developmental brain was reduced, whereas the axonal growth of caudal primary motor neurons was unaffected by sash1a downregulation. And more significantly, the gfap + glia presented abnormal arrangements and disordered orientations in sash1a morphants. The similar phenotype was verified in the mutation induced by the injection of cas9 mRNA and sash1a sgRNA. We further performed behavioral experiments in zebrafish larvae that had been injected with sash1a MO at one-cell stage, and found them exhibiting abnormal behavior trajectories. Moreover, injecting the human SASH1 mRNA rescued these phenomena in sash1a MO zebrafish. In summary, our study revealed that the downregulation of SASH1 leads to malformations in the embryonic brain and disorganization of glial cell marshalling, suggesting that SASH1 plays an important role in the migration of glial cells during embryonic brain development.

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia.

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

Amino-terminal proteolytic fragment of the axon growth inhibitor Nogo-A (Rtn4A) is upregulated by injury and promotes axon regeneration

After adult mammalian central nervous system injury, axon regeneration is extremely limited or absent, resulting in persistent neurological deficits. Axon regeneration failure is due in part to the presence of inhibitory proteins, including NogoA (Rtn4A), from which two inhibitory domains have been defined. When these inhibitory domains are deleted, but an amino-terminal domain is still expressed in a gene trap line, mice show axon regeneration and enhanced recovery from injury. In contrast, when there is no amino-terminal Nogo-A fragment in the setting of inhibitory domain deletion, then axon regeneration and recovery are indistinguishable from WT. These data indicated that an amino-terminal Nogo-A fragment derived from the gene trap might promote axon regeneration, but this had not been tested directly and production of this fragment without gene targeting was unclear. Here, we describe posttranslation production of an amino-terminal fragment of Nogo-A from the intact gene product. This fragment is created by proteolysis near amino acid G214-N215 and levels are enhanced by axotomy. Furthermore, this fragment promotes axon regeneration in vitro and acts cell autonomously in neurons, in contrast to the inhibitory extracellular action of other Nogo-A domains.Proteins interacting with the amino-terminal Nogo-A fragment by immunoprecipitation include HSPA8 (HSC70, HSP7C). Suppression of HSPA8 expression by shRNA decreases axon regeneration from cerebral cortical neurons and overexpression increases axon regeneration. Moreover, the amino-terminal Nogo-A fragment increases HSPA8 chaperone activity. These data provide an explanation for varied results in different gene-targeted Nogo-A mice, as well as revealing an axon regeneration promoting domain of Nogo-A.

Role of kainate receptors in pruriceptive processing in the mouse spinal cord

Pruritus, including neuropathic and psychogenic pruritus, is an unpleasant feeling that causes a desire to scratch, which negatively impacts physical and psychological aspects of daily life. Nonetheless, little is known about the neural mechanisms involved in pruritus. Glutamate is a predominant excitatory neurotransmitter in the mammalian central nervous system and exerts its effects by binding to various glutamate receptors, including kainate (KA) receptors; however, the precise involvement of each glutamate receptor in pruriceptive processing remains unclear, particularly that of KA receptors. Therefore, the roles of KA receptors in histamine-dependent and -independent itch were investigated using CNQX, an AMPA/KA receptors antagonist, UBP310 and UBP302, antagonists of KA receptors, and small interfering (si)RNAs against KA receptor subunits in mice with acute and chronic pruritus. The effects of KA receptor antagonists on histamine-induced c-Fos expression in the spinal cord were also examined. The intrathecal administration of CNQX reduced the number of scratching events induced by histamine and chloroquine. On the other hand, UBP310 or UBP302 and the siRNAs of KA receptor subunits 1–3 significantly inhibited the induction of scratching events in mice treated with histamine, while no significant change was observed in the induction of spontaneous scratching events in mice with chronic pruritus. In addition, antagonists of KA receptors attenuated c-Fos expression in the superficial layers of the dorsal horn induced by histamine. These results indicate that KA receptors are involved in acute pruriceptive processing in the spinal cord induced by histamine, but not chloroquine or chronic itch.

Customization of the translational complex regulates mRNA-specific translation to control CNS regeneration

In the adult mammalian central nervous system (CNS), axons fail to regenerate spontaneously after injury because of a combination of extrinsic and intrinsic factors. Despite recent advances targeting the intrinsic regenerative properties of adult neurons, the molecular mechanisms underlying axon regeneration are not fully understood. Here, we uncover a regulatory mechanism that controls the expression of key proteins involved in regeneration at the translational level. Our results show that mRNA-specific translation is critical for promoting axon regeneration. Indeed, we demonstrate that specific ribosome-interacting proteins, such as the protein Huntingtin (HTT), selectively control the translation of a specific subset of mRNAs. Moreover, modulating the expression of these translationally regulated mRNAs is crucial for promoting axon regeneration. Altogether, our findings highlight that selective translation through the customization of the translational complex is a key mechanism of axon regeneration with major implications in the development of therapeutic strategies for CNS repair.

Synaptophysin Expression by Supraependymal Structures of the Rat Brain

Supraependymal plexus in ventricular system is one of the most cryptic structures in the mammalian central nervous system. Since both the topography of supraependymal elements and their functional role remain unclear, the aim of this research was to study the distribution of supraependymal structures within the ventricular system of the rat brain with synaptic function associated marker, synaptophysin. Serial sections of Wistar rats (4–6 month, n = 6) forebrain were examined using immunohistochemical detection of synaptophysin and tyrosine hydroxylase. It was shown that supraependymal plexus can form on the surface of ependymal cells synaptophysin-immunopostitive discrete structures, which indicates the formation of synaptic contacts. Although catecholaminergic nerve fibers were present on the ventricular surface in all studied zones, it seems that these nerve fibers may not always contain synaptophysin. Thus, it is assumed that the functional purpose of the supraependymal nerve plexus depends on its localization and can be associated whether with the regulation of ependymal cells and cerebrospinal fluid formation, or with the formation of long-range interneuronal connectivities.

Renewal of oligodendrocyte lineage reverses dysmyelination and CNS neurodegeneration through corrected N-acetylaspartate metabolism.

Myelinating oligodendrocytes are essential for neuronal communication and homeostasis of the central nervous system (CNS). One of the most abundant molecules in the mammalian CNS is N-acetylaspartate (NAA), which is catabolized into L-aspartate and acetate by the enzyme aspartoacylase (ASPA) in oligodendrocytes. The resulting acetate moiety is thought to contribute to myelin lipid synthesis. In addition, affected NAA metabolism has been implicated in several neurological disorders, including leukodystrophies and demyelinating diseases such as multiple sclerosis. Genetic disruption of ASPA function causes Canavan disease, which is hallmarked by increased NAA levels, myelin and neuronal loss, large vacuole formation in the CNS, and early death in childhood. Although NAA's direct role in the CNS is inconclusive, in peripheral adipose tissue, NAA-derived acetate has been found to modify histones, a mechanism known to be involved in epigenetic regulation of cell differentiation. We hypothesize that a lack of cellular differentiation in the brain contributes to the disruption of myelination and neurodegeneration in diseases with altered NAA metabolism, such as Canavan disease. Our study demonstrates that loss of functional Aspa in mice disrupts myelination and shifts the transcriptional expression of neuronal and oligodendrocyte markers towards less differentiated stages in a spatiotemporal manner. Upon re-expression of ASPA, these oligodendrocyte and neuronal lineage markers are either improved or normalized, suggesting that NAA breakdown by Aspa plays an essential role in the maturation of neurons and oligodendrocytes. Also, this effect of ASPA re-expression is blunted in old mice, potentially due to limited ability of neuronal, rather than oligodendrocyte, recovery.

Suppression of Fibroblast Growth Factor Receptor-5 (FGFR5) has no Impact on Axon Regeneration after SCI.

One of the most common forms of the mammalian central nervous system (CNS) injuries is spinal cord injury (SCI), and any lesion to the CNS can result in a lifelong functional impairment since CNS axons cannot regenerate. The relative axon regenerating genes following spinal SCI were examined using the regenerative SN, pSN + DC, and non-regenerating DC lesion models. By using qRT-PCR, we discovered that fibroblast growth factor receptor-5 (FGFR5) was 4.2-fold more highly expressed in non-regeneration lesions compared to intact control and regenerating animals. Furthermore, in cultured dorsal root ganglion neurons (DRGN), short interfering RNA (siRNA)-mediated knockdown of FGFR5 had no effect on DRGN neurite outgrowth, indicating that the gene's suppression has no effect on axon regeneration and may play other roles in the CNS besides axon regeneration.

Localization Pattern of Dispatched Homolog 2 (DISP2) in the Central and Enteric Nervous System

Dispatched homolog (DISP) proteins have been implicated in the regulation of hedgehog signaling during embryologic development. Although DISP2 has recently been associated with neuronal development and control of cognitive functions, its localization pattern in the mammalian central and peripheral nervous system has not yet been investigated. In this study, the Disp2 expression profile was assessed in human tissues from publicly available transcriptomic datasets. The DISP2 localization pattern was further characterized in the human and rat central nervous system (CNS), as well as within the colonic enteric nervous system (ENS) using dual-label immunohistochemistry. Colocalization of DISP2 with neuronal and glial markers was additionally analyzed in murine primary ENS culture. At transcriptomic level, DISP2 expression was predominant in neuronal cell types of the CNS and ENS. DISP2 immunoreactivity was mainly located within PGP9.5-positive neurons rather than in S100-positive glial cells throughout the nervous system. Investigation of human and rat brain tissues, colonic specimens, and murine ENS primary cultures revealed that DISP2 was located in neuronal cell somata, as well as along neuronal processes both in the human and murine CNS and ENS. Our results indicate that DISP2 is prominently localized within neuronal cells of the CNS and ENS and support putative functions of DISP2 in these tissues.

Acyl coenzyme A binding protein (ACBP): An aging- and disease-relevant "autophagy checkpoint".

Acyl coenzyme A binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is a phylogenetically ancient protein present in some eubacteria and the entire eukaryotic radiation. In several eukaryotic phyla, ACBP/DBI transcends its intracellular function in fatty acid metabolism because it can be released into the extracellular space. This ACBP/DBI secretion usually occurs in response to nutrient scarcity through an autophagy-dependent pathway. ACBP/DBI and its peptide fragments then act on a range of distinct receptors that diverge among phyla, namely metabotropic G protein-coupled receptor in yeast (and likely in the mammalian central nervous system), a histidine receptor kinase in slime molds, and ionotropic gamma-aminobutyric acid (GABA)A receptors in mammals. Genetic or antibody-mediated inhibition of ACBP/DBI orthologs interferes with nutrient stress-induced adaptations such as sporulation or increased food intake in multiple species, as it enhances lifespan or healthspan in yeast, plant leaves, nematodes, and multiple mouse models. These lifespan and healthspan-extending effects of ACBP/DBI suppression are coupled to the induction of autophagy. Altogether, it appears that neutralization of extracellular ACBP/DBI results in "autophagy checkpoint inhibition" to unleash the anti-aging potential of autophagy. Of note, in humans, ACBP/DBI levels increase in various tissues, as well as in the plasma, in the context of aging, obesity, uncontrolled infection or cardiovascular, inflammatory, neurodegenerative, and malignant diseases.

Computational Investigation of GluN2B Receptor Effectiveness of Octreotide in Nervous Diseases by Using AutoDock Vina

: The N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors that participate in excitatory postsynaptic signaling in the mammalian central nervous system. In the 1980s, octreotide first became available for the treatment of acromegaly. In acromegaly patients and healthy volunteers, somatostatin and its analogs, such as octreotide and lanreotide, dramatically decrease the release of GH. In this study, we investigated the effect of octreotide, which controls blood lipids, on one of the proteins involved in epilepsy. After examining the genes involved in epilepsy by the DisGeNET server, we selected the ionotropic glutamate receptor NMDA type subunit 2B gene with the symbol GRIN2B. The docking and molecular simulation process was done using the AutoDock Vina 1.2.0 algorithm under the Chimera user interface. The analysis of docking results has been done by the PDBsum and Protein-Ligand Interaction Profiler servers. The VMD program prepared the images. The Pocket Drug server detected eighteen packets. Only the molecular docking results of 5 out of 18 pockets were acceptable. The results of docking for eighteen pockets were found in the target protein after the simulations; 13 have fewer than 14 residues, and 5 have more than 14 residues. We selected the best 18 pockets (P35, P64), (P10, P14, P17).

Moving CNS axon growth and regeneration research into human model systems.

Axon regeneration is limited in the adult mammalian central nervous system (CNS) due to both intrinsic and extrinsic factors. Rodent studies have shown that developmental age can drive differences in intrinsic axon growth ability, such that embryonic rodent CNS neurons extend long axons while postnatal and adult CNS neurons do not. In recent decades, scientists have identified several intrinsic developmental regulators in rodents that modulate growth. However, whether this developmentally programmed decline in CNS axon growth is conserved in humans is not yet known. Until recently, there have been limited human neuronal model systems, and even fewer age-specific human models. Human in vitro models range from pluripotent stem cell-derived neurons to directly reprogrammed (transdifferentiated) neurons derived from human somatic cells. In this review, we discuss the advantages and disadvantages of each system, and how studying axon growth in human neurons can provide species-specific knowledge in the field of CNS axon regeneration with the goal of bridging basic science studies to clinical trials. Additionally, with the increased availability and quality of 'omics datasets of human cortical tissue across development and lifespan, scientists can mine these datasets for developmentally regulated pathways and genes. As there has been little research performed in human neurons to study modulators of axon growth, here we provide a summary of approaches to begin to shift the field of CNS axon growth and regeneration into human model systems to uncover novel drivers of axon growth.

Impact of highly deleterious non-synonymous polymorphisms on GRIN2A protein's structure and function.

GRIN2A is a gene that encodes NMDA receptors found in the central nervous system and plays a pivotal role in excitatory synaptic transmission, plasticity and excitotoxicity in the mammalian central nervous system. Changes in this gene have been associated with a spectrum of neurodevelopmental disorders such as epilepsy. Previous studies on GRIN2A suggest that non-synonymous single nucleotide polymorphisms (nsSNPs) can alter the protein's structure and function. To gain a better understanding of the impact of potentially deleterious variants of GRIN2A, a range of bioinformatics tools were employed in this study. Out of 1320 nsSNPs retrieved from the NCBI database, initially 16 were predicted as deleterious by 9 tools. Further assessment of their domain association, conservation profile, homology models, interatomic interaction, and Molecular Dynamic Simulation revealed that the variant I463S is likely to be the most deleterious for the structure and function of the protein. Despite the limitations of computational algorithms, our analyses have provided insights that can be a valuable resource for further in vitro and in vivo research on GRIN2A-associated diseases.

Neuropharmacology of human TERA2.cl.SP12 stem cell-derived neurons in ultra-long-term culture for antiseizure drug discovery.

Modeling the complex and prolonged development of the mammalian central nervous system in vitro remains a profound challenge. Most studies of human stem cell derived neurons are conducted over days to weeks and may or may not include glia. Here we have utilized a single human pluripotent stem cell line, TERA2.cl.SP12 to derive both neurons and glial cells and determined their differentiation and functional maturation over 1 year in culture together with their ability to display epileptiform activity in response to pro-convulsant agents and to detect antiseizure drug actions. Our experiments show that these human stem cells differentiate in vitro into mature neurons and glia cells and form inhibitory and excitatory synapses and integrated neural circuits over 6-8 months, paralleling early human neurogenesis in vivo; these neuroglia cultures display complex electrochemical signaling including high frequency trains of action potentials from single neurons, neural network bursts and highly synchronized, rhythmical firing patterns. Neural activity in our 2D neuron-glia circuits is modulated by a variety of voltage-gated and ligand-gated ion channel acting drugs and these actions were consistent in both young and highly mature neuron cultures. We also show for the first time that spontaneous and epileptiform activity is modulated by first, second and third generation antiseizure agents consistent with animal and human studies. Together, our observations strongly support the value of long-term human stem cell-derived neuroglial cultures in disease modeling and neuropsychiatric drug discovery.

High-resolution structural and functional retinal imaging in the awake behaving mouse

The laboratory mouse has provided tremendous insight to the underpinnings of mammalian central nervous system physiology. In recent years, it has become possible to image single neurons, glia and vascular cells in vivo by using head-fixed preparations combined with cranial windows to study local networks of activity in the living brain. Such approaches have also succeeded without the use of general anesthesia providing insights to the natural behaviors of the central nervous system. However, the same has not yet been developed for the eye, which is constantly in motion. Here we characterize a novel head-fixed preparation that enables high-resolution adaptive optics retinal imaging at the single-cell level in awake-behaving mice. We reveal three new functional attributes of the normal eye that are overlooked by anesthesia: 1) High-frequency, low-amplitude eye motion of the mouse that is only present in the awake state 2) Single-cell blood flow in the mouse retina is reduced under anesthesia and 3) Mouse retinae thicken in response to ketamine/xylazine anesthesia. Here we show key benefits of the awake-behaving preparation that enables study of retinal physiology without anesthesia to study the normal retinal physiology in the mouse.

Comprehensive behavioral analyses of mice with a glycine receptor alpha 4 deficiency

Glycine receptors (GlyRs) are ligand-gated chloride channels comprising alpha (α1–4) and β subunits. The GlyR subunits play major roles in the mammalian central nervous system, ranging from regulating simple sensory information to modulating higher-order brain function. Unlike the other GlyR subunits, GlyR α4 receives relatively little attention because the human ortholog lacks a transmembrane domain and is thus considered a pseudogene. A recent genetic study reported that the GLRA4 pseudogene locus on the X chromosome is potentially involved in cognitive impairment, motor delay and craniofacial anomalies in humans. The physiologic roles of GlyR α4 in mammal behavior and its involvement in disease, however, are not known. Here we examined the temporal and spatial expression profile of GlyR α4 in the mouse brain and subjected Glra4 mutant mice to a comprehensive behavioral analysis to elucidate the role of GlyR α4 in behavior. The GlyR α4 subunit was mainly enriched in the hindbrain and midbrain, and had relatively lower expression in the thalamus, cerebellum, hypothalamus, and olfactory bulb. In addition, expression of the GlyR α4 subunit gradually increased during brain development. Glra4 mutant mice exhibited a decreased amplitude and delayed onset of the startle response compared with wild-type littermates, and increased social interaction in the home cage during the dark period. Glra4 mutants also had a low percentage of entries into open arms in the elevated plus-maze test. Although mice with GlyR α4 deficiency did not show motor and learning abnormalities reported to be associated in human genomics studies, they exhibited behavioral changes in startle response and social and anxiety-like behavior. Our data clarify the spatiotemporal expression pattern of the GlyR α4 subunit and suggest that glycinergic signaling modulates social, startle, and anxiety-like behaviors in mice.

Interrelationships Between Spinal Sympathetic Preganglionic Neurons, Autonomic Systems and Electrical Synapses Formed by Connexin36-containing Gap Junctions

Spinal sympathetic preganglionic neurons (SPNs) are among the many neuronal populations in the mammalian central nervous system (CNS) where there is evidence for electrical coupling between cell pairs linked by gap junctions composed of connexin36 (Cx36). Understanding the organization of this coupling in relation to autonomic functions of spinal sympathetic systems requires knowledge of how these junctions are deployed among SPNs. Here, we document the distribution of immunofluorescence detection of Cx36 among SPNs identified by immunolabelling of their various markers, including choline acetyltransferase, nitric oxide and peripherin in adult and developing mouse and rat. In adult animals, labelling of Cx36 was exclusively punctate and dense concentrations of Cx36-puncta were distributed along the entire length of the spinal thoracic intermediolateral cell column (IML). These puncta were also seen in association with SPN dendritic processes in the lateral funiculus, the intercalated and central autonomic areas and those within and extending medially from the IML. All labelling for Cx36 was absent in spinal cords of Cx36 knockout mice. High densities of Cx36-puncta were already evident among clusters of SPNs in the IML of mouse and rat at postnatal days 10–12. In Cx36BAC::eGFP mice, eGFP reporter was absent in SPNs, thus representing false negative detection, but was localized to some glutamatergic and GABAergic synaptic terminals. Some eGFP+ terminals were found contacting SPN dendrites. These results indicate widespread Cx36 expression in SPNs, further supporting evidence of electrical coupling between these cells, and suggest that SPNs are innervated by neurons that themselves may be electrically coupled.

Glypican-2 defines age-dependent axonal response to chondroitin sulfate

Axons of terminally differentiated neurons in the mammalian central nervous system (CNS) are unable to regenerate after dissection. One of the mechanisms underlying this is the inhibition of axonal regeneration by chondroitin sulfate (CS) and its neuronal receptor, PTPσ. Our previous results demonstrated that the CS–PTPσ axis disrupted autophagy flux by dephosphorylating cortactin, which led to the formation of dystrophic endballs and to the inhibition of axonal regeneration. In contrast, juvenile neurons vigorously extend axons toward their targets during development and maintain regenerative activity for axons even after injury. Although several intrinsic and extrinsic mechanisms have been reported to mediate the differences, the detailed mechanisms are still elusive. Here, we report that Glypican-2, a member of heparan sulfate proteoglycans (HSPG), which are able to antagonize CS–PTPσ by competing with the receptor, is specifically expressed in the axonal tips of embryonic neurons. Glypican-2 overexpression in adult neurons rescues the dystrophic endball back to a healthy growth cone on the CSPG gradient. Consistently, Glypican-2 restored cortactin phosphorylation in the axonal tips of adult neurons on CSPG. Taken together, our results clearly demonstrated Glypican-2's pivotal role in defining the axonal response to CS and provided a new therapeutic target for axonal injury.

Axonal growth inhibitors and their receptors in spinal cord injury: from biology to clinical translation.

Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelin-associated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19 (that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the RhoA/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.

Orthogonal approaches required to measure proteasome composition and activity in mammalian brain tissue

Proteasomes are large macromolecular complexes with multiple distinct catalytic activities that are each vital to human brain health and disease. Despite their importance, standardized approaches to investigate proteasomes have not been universally adapted. Here, we describe pitfalls and define straightforward orthogonal biochemical approaches essential to measure and understand changes in proteasome composition and activity in the mammalian central nervous system. Through our experimentation in the mammalian brain, we determined an abundance of catalytically active proteasomes exist with and without a 19S cap(s), the regulatory particle essential for ubiquitin-dependent degradation. Moreover, we learned that in-cell measurements using activity-based probes (ABPs) are more sensitive in determining the available activity of the 20S proteasome without the 19S cap and in measuring individual catalytic subunit activities of each β subunit within all neuronal proteasomes. Subsequently, applying these tools to human brain samples, we were surprised to find that post-mortem tissue retained little to no 19S-capped proteasome, regardless of age, sex, or disease state. In comparing brain tissues (parahippocampal gyrus) from patients with Alzheimer's disease (AD) and unaffected individuals, the available 20S proteasome activity was significantly elevated in severe cases of AD, an observation not previously noted. Taken together, our study establishes standardized approaches for the comprehensive investigation of proteasomes in mammalian brain tissue, and we reveal new insight into brain proteasome biology.