Identification and targeting of a unique NaV1.7 domain driving chronic pain.

Small molecules directly targeting the voltage-gated sodium channel (VGSC) NaV1.7 have not been clinically successful. We reported that preventing the addition of a small ubiquitin-like modifier onto the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 function and was antinociceptive in rodent models of neuropathic pain. Here, we discovered a CRMP2 regulatory sequence (CRS) unique to NaV1.7 that is essential for this regulatory coupling. CRMP2 preferentially bound to the NaV1.7 CRS over other NaV isoforms. Substitution of the NaV1.7 CRS with the homologous domains from the other eight VGSC isoforms decreased NaV1.7 currents. A cell-penetrant decoy peptide corresponding to the NaV1.7-CRS reduced NaV1.7 currents and trafficking, decreased presynaptic NaV1.7 expression, reduced spinal CGRP release, and reversed nerve injury-induced mechanical allodynia. Importantly, the NaV1.7-CRS peptide did not produce motor impairment, nor did it alter physiological pain sensation, which is essential for survival. As a proof-of-concept for a NaV1.7 -targeted gene therapy, we packaged a plasmid encoding the NaV1.7-CRS in an AAV virus. Treatment with this virus reduced NaV1.7 function in both rodent and rhesus macaque sensory neurons. This gene therapy reversed and prevented mechanical allodynia in a model of nerve injury and reversed mechanical and cold allodynia in a model of chemotherapy-induced peripheral neuropathy. These findings support the conclusion that the CRS domain is a targetable region for the treatment of chronic neuropathic pain.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Insular cortex stimulation alleviates neuropathic pain through changes in the expression of collapsin response mediator protein 2 involved in synaptic plasticity.

In recent studies, brain stimulation has shown promising potential to alleviate chronic pain. Although studies have shown that stimulation of pain-related brain regions can induce pain-relieving effects, few studies have elucidated the mechanisms of brain stimulation in the insular cortex (IC). The present study was conducted to explore the changes in characteristic molecules involved in pain modulation mechanisms and to identify the changes in synaptic plasticity after IC stimulation (ICS). Following ICS, pain-relieving behaviors and changes in proteomics were explored. Neuronal activity in the IC after ICS was observed by optical imaging. Western blotting was used to validate the proteomics data and identify the changes in the expression of glutamatergic receptors associated with synaptic plasticity. Experimental results showed that ICS effectively relieved mechanical allodynia, and proteomics identified specific changes in collapsin response mediator protein 2 (CRMP2). Neuronal activity in the neuropathic rats was significantly decreased after ICS. Neuropathic rats showed increased expression levels of phosphorylated CRMP2, alpha amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR), and N-methyl-d-aspartate receptor (NMDAR) subunit 2B (NR2B), which were inhibited by ICS. These results indicate that ICS regulates the synaptic plasticity of ICS through pCRMP2, together with AMPAR and NR2B, to induce pain relief.

CRMP2 conditional knockout changes axonal function and ultrastructure of axons in mice corpus callosum.

Collapsin response mediator protein 2 (CRMP2) is a member of a protein family, which is highly involved in neurodevelopment, but most of its members become heavily downregulated in adulthood. CRMP2 is an important factor in neuronal polarization, axonal formation and growth cone collapse. The protein remains expressed in adulthood, but is more region specific. CRMP2 is present in adult corpus callosum (CC) and in plastic areas like prefrontal cortex and hippocampus. CRMP2 has been implicated as one of the risk-genes for Schizophrenia (SZ). Here, a CRMP2 conditional knockout (CRMP2-cKO) mouse was used as a model of SZ to investigate how it could affect the white matter and therefore brain connectivity. Multielectrode electrophysiology (MEA) was used to study the function of corpus callosum showing an increase in conduction velocity (CV) measured as Compound Action Potentials (CAPs) in acute brain slices. Light- and electron-microscopy, specifically Serial Block-face Scanning Electron Microscopy (SBF-SEM), methods were used to study the structure of CC in CRMP2-cKO mice. A decrease in CC volume of CRMP2-cKO mice as compared to controls was observed. No differences were found in numbers nor in the size of CC oligodendrocytes (OLs). Similarly, no differences were found in myelin thickness or in node of Ranvier (NR) structure. In contrast, abnormally smaller axons were measured in the CRMP2-cKO mice. Using these state-of-the-art methods it was possible to shed light on specific parts of the dysconnectivity aspect of deletion of CRMP2 related to SZ and add details to previous findings helping further understanding the disease. This paper substantiates the white matter changes in the absence of CRMP2 and ties it to the role it plays in this complex disorder.

Clinical evidence that a dysregulated master neural network modulator may aid in diagnosing schizophrenia.

There are no validated biomarkers for schizophrenia (SCZ), a disorder linked to neural network dysfunction. We demonstrate that collapsin response mediator protein-2 (CRMP2), a master regulator of cytoskeleton and, hence, neural circuitry, may form the basis for a biomarker because its activity is uniquely imbalanced in SCZ patients. CRMP2's activity depends upon its phosphorylation state. While an equilibrium between inactive (phosphorylated) and active (nonphosphorylated) CRMP2 is present in unaffected individuals, we show that SCZ patients are characterized by excess active CRMP2. We examined CRMP2 levels first in postmortem brains (correlated with neuronal morphometrics) and then, because CRMP2 is expressed in lymphocytes as well, in the peripheral blood of SCZ patients versus age-matched unaffected controls. In the brains and, more starkly, in the lymphocytes of SCZ patients <40 y old, we observed that nonphosphorylated CRMP2 was higher than in controls, while phosphorylated CRMP2 remained unchanged from control. In the brain, these changes were associated with dendritic structural abnormalities. The abundance of active CRMP2 with insufficient opposing inactive p-CRMP2 yielded a unique lowering of the p-CRMP2:CRMP2 ratio in SCZ patients, implying a disruption in the normal equilibrium between active and inactive CRMP2. These clinical data suggest that measuring CRMP2 and p-CRMP2 in peripheral blood might reflect intracerebral processes and suggest a rapid, minimally invasive, sensitive, and specific adjunctive diagnostic aid for early SCZ: increased CRMP2 or a decreased p-CRMP2:CRMP2 ratio may help cinch the diagnosis in a newly presenting young patient suspected of SCZ (versus such mimics as mania in bipolar disorder, where the ratio is high).

Inhibition of ERK1/2 or CRMP2 Disrupts Alcohol Memory Reconsolidation and Prevents Relapse in Rats.

Relapse to alcohol abuse, often caused by cue-induced alcohol craving, is a major challenge in alcohol addiction treatment. Therefore, disrupting the cue-alcohol memories can suppress relapse. Upon retrieval, memories transiently destabilize before they reconsolidate in a process that requires protein synthesis. Evidence suggests that the mammalian target of rapamycin complex 1 (mTORC1), governing the translation of a subset of dendritic proteins, is crucial for memory reconsolidation. Here, we explored the involvement of two regulatory pathways of mTORC1, phosphoinositide 3-kinase (PI3K)-AKT and extracellular regulated kinase 1/2 (ERK1/2), in the reconsolidation process in a rat (Wistar) model of alcohol self-administration. We found that retrieval of alcohol memories using an odor-taste cue increased ERK1/2 activation in the amygdala, while the PI3K-AKT pathway remained unaffected. Importantly, ERK1/2 inhibition after alcohol memory retrieval impaired alcohol-memory reconsolidation and led to long-lasting relapse suppression. Attenuation of relapse was also induced by post-retrieval administration of lacosamide, an inhibitor of collapsin response mediator protein-2 (CRMP2)-a translational product of mTORC1. Together, our findings indicate the crucial role of ERK1/2 and CRMP2 in the reconsolidation of alcohol memories, with their inhibition as potential treatment targets for relapse prevention.

The Nogo-66 Receptors NgR1 and NgR3 Are Required for Commissural Axon Pathfinding.

Nogo-66 receptors (NgR1-3) are glycosylphosphatidyl inositol-linked proteins that belong to the leucine-rich repeat superfamily. Through binding to myelin-associated inhibitors, NgRs contribute to the inhibition of axonal regeneration after spinal cord injury. Their role in limiting synaptic plasticity and axonal outgrowth in the adult CNS has been described previously, but not much is known about their role during the development of the nervous system. Here, we show that NgR1 and NgR3 mRNAs are expressed during spinal cord development of the chicken embryo. In particular, they are expressed in the dI1 subpopulation of commissural neurons during the time when their axons navigate toward and across the floorplate, the ventral midline of the spinal cord. To assess a potential role of NgR1 and NgR3 in axon guidance, we downregulated them using in ovo RNAi and analyzed the trajectory of commissural axons by tracing them in open-book preparations of spinal cords. Our results show that loss of either NgR1 or NgR3 causes axons to stall in the midline area and to interfere with the rostral turn of postcrossing axons. In addition, we also show that NgR1, but not NgR3, requires neuronal PlexinA2 for the regulation of commissural axon guidance.SIGNIFICANCE STATEMENT Over the last decades, many studies have focused on the role of NgRs, particularly NgR1, in axonal regeneration in the injured adult CNS. Here, we show a physiological role of NgRs in guiding commissural axons during early development of the chicken spinal cord in vivo Both NgR1 and NgR3 are required for midline crossing and subsequent turning of postcrossing axons into the longitudinal axis of the spinal cord. NgR1, but not NgR3, forms a receptor complex with PlexinA2 during axon guidance. Overall, these findings provide a link between neural regenerative mechanisms and developmental processes.

OMICS Analyses Unraveling Related Gene and Protein-Driven Molecular Mechanisms Underlying PACAP 38-Induced Neurite Outgrowth in PC12 Cells.

The study aimed to understand mechanism/s of neuronal outgrowth in the rat adrenal-derived pheochromocytoma cell line (PC12) under pituitary adenylate cyclase-activating polypeptide (PACAP) treatment. Neurite projection elongation was suggested to be mediated via Pac1 receptor-mediated dephosphorylation of CRMP2, where GSK-3ß, CDK5, and Rho/ROCK dephosphorylated CRMP2 within 3 h after addition of PACAP, but the dephosphorylation of CRMP2 by PACAP remained unclear. Thus, we attempted to identify the early factors in PACAP-induced neurite projection elongation via omics-based transcriptomic (whole genome DNA microarray) and proteomic (TMT-labeled liquid chromatography-tandem mass spectrometry) analyses of gene and protein expression profiles from 5-120 min after PACAP addition. The results revealed a number of key regulators involved in neurite outgrowth, including known ones, called 'Initial Early Factors', e.g., genes Inhba, Fst, Nr4a1,2,3, FAT4, Axin2, and proteins Mis12, Cdk13, Bcl91, CDC42, including categories of 'serotonergic synapse, neuropeptide and neurogenesis, and axon guidance'. cAMP signaling and PI3K-Akt signaling pathways and a calcium signaling pathway might be involved in CRMP2 dephosphorylation. Cross-referencing previous research, we tried to map these molecular components onto potential pathways, and we may provide important new information on molecular mechanisms of neuronal differentiation induced by PACAP. Gene and protein expression data are publicly available at NCBI GSE223333 and ProteomeXchange, identifier PXD039992.

The Rac-GAP alpha2-Chimaerin Signals via CRMP2 and Stathmins in the Development of the Ocular Motor System.

A precise sequence of axon guidance events is required for the development of the ocular motor system. Three cranial nerves grow toward, and connect with, six extraocular muscles in a stereotyped pattern, to control eye movements. The signaling protein alpha2-chimaerin (α2-CHN) plays a pivotal role in the formation of the ocular motor system; mutations in CHN1, encoding α2-CHN, cause the human eye movement disorder Duane Retraction Syndrome (DRS). Our research has demonstrated that the manipulation of α2-chn signaling in the zebrafish embryo leads to ocular motor axon wiring defects, although the signaling cascades regulated by α2-chn remain poorly understood. Here, we demonstrate that several cytoskeletal regulatory proteins-collapsin response mediator protein 2 (CRMP2; encoded by the gene dpysl2), stathmin1, and stathmin 2-bind to α2-CHN. dpysl2, stathmin1, and especially stathmin2 are expressed by ocular motor neurons. We find that the manipulation of dpysl2 and of stathmins in zebrafish larvae leads to defects in both the axon wiring of the ocular motor system and the optokinetic reflex, impairing horizontal eye movements. Knockdowns of these molecules in zebrafish larvae of either sex caused axon guidance phenotypes that included defasciculation and ectopic branching; in some cases, these phenotypes were reminiscent of DRS. chn1 knock-down phenotypes were rescued by the overexpression of CRMP2 and STMN1, suggesting that these proteins act in the same signaling pathway. These findings suggest that CRMP2 and stathmins signal downstream of α2-CHN to orchestrate ocular motor axon guidance and to control eye movements.SIGNIFICANCE STATEMENT The precise control of eye movements is crucial for the life of vertebrate animals, including humans. In humans, this control depends on the arrangement of nerve wiring of the ocular motor system, composed of three nerves and six muscles, a system that is conserved across vertebrate phyla. Mutations in the protein alpha2-chimaerin have previously been shown to cause eye movement disorders (squint) and axon wiring defects in humans. Our recent work has unraveled how alpha2-chimaerin coordinates axon guidance of the ocular motor system in animal models. In this article, we demonstrate key roles for the proteins CRMP2 and stathmin 1/2 in the signaling pathway orchestrated by alpha2-chimaerin, potentially giving insight into the etiology of eye movement disorders in humans.

CRMP2 modulates mossy fiber sprouting in dentate gyrus of pilocarpine induced rat model of epilepsy.

As a hallmark of epilepsy, mossy fiber sprouting was regarded as an ideal mode to study neural rewiring upon injury. The process of mossy fiber sprouting constitutes key steps for neural circuit formation, including axon collateral formation and outgrowth, reversed pathfinding and synapse connection. The canonical function of CRMP2 is to promote neurite/axon outgrowth via binding to tubulin heterodimers, which is mainly regulated by its phosphorylation state. CRMP2 expression and phosphorylation were reported to change in medial temporal epilepsy patients and animal modes of epilepsy. As a novel anti-epilepsy drug, Lacosamide is able to impair CRMP2 mediated tubulin polymerization. Previous studies suggested possible roles of CRMP2 in mossy fiber sprouting. Here, we provide direct evidence to support the role of CRMP2 in the process of mossy fiber sprouting in an animal model of epilepsy. We found that CRMP2 phosphorylation was downregulated specifically in the hippocampus during latent phase of epileptic rats. In addition, with the reduction of CRMP2 expression levels in dentate gyrus by CRMP2 shRNA, we observed decreased mossy fiber sprouting in these CRMP2 knockdown rats. Our results demonstrated that CRMP2 modulates mossy fiber sprouting in dentate gyrus of pilocarpine induced rat model of epilepsy.

Effects of Lanthionine Ketimine-5-Ethyl Ester on the α-Synucleinopathy Mouse Model.

Potentially druggable mechanisms underlying synaptic deficits seen in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are under intense interrogations. In addition to defective synaptic vesicle trafficking, cytoskeletal disruption, autophagic perturbation, and neuroinflammation, hyperphosphorylation of microtubule-associated protein collapsin response mediator protein 2 (CRMP2, also known as DPYSL2) is newly determined to correlate with synaptic deficits in human DLB. The small molecule experimental therapeutic, lanthionine ketimine-5-ethyl ester (LKE), appears to interact with CRMP2 in a host of neurodegenerative mouse models, normalizing its phosphorylation level while promoting healthful autophagy in cell culture models and suppressing the proinflammatory phenotype of activated microglia. Accordingly, this study examined the effect of LKE on α-synuclein A53T transgenic (Tg) mice which were employed as a DLB model. We found that chronic administration of LKE to A53T mice suppressed (1) the accumulation of LBs, (2) neuroinflammatory activation of microglia, (3) impairment of contextual fear memory, and (4) CRMP2 phosphorylation at Thr509 in A53T Tg mice. These results suggest that CRMP2 phosphorylation by GSK3ß in the hippocampus is related to pathology and memory impairment in DLB, and LKE may have clinical implications in the treatment of α-synucleinopathy.

Cdc42 Promotes Axonogenesis of Primary Hippocampal Neurons by Inhibiting Glycogen Synthase Kinase-3ß.

BACKGROUND: Progressive axon degeneration is a common pathological feature of neurodegenerative diseases. Cdc42 is a member of the Rho GTPase family that participates in axonogenesis. GSK-3ß is a serine/threonine kinase highly implicated in neuronal development and neurodegeneration. This study aimed to examine whether cdc42 promotes axonogenesis by regulating GSK-3ß activity. METHODS: Hippocampal neurons were isolated from neonatal Sprague-Dawley rats and transfected with designated plasmid vectors to alter the activities of cdc42 and GSK-3ß. LiCl treatment was used to inhibit the GSK-3ß activity in primary neurons. GSK-3ß activity was determined by an enzyme activity assay kit. Immunofluorescence staining was used to detect axons stained with anti-Tau-1 antibody and dendrites stained with anti-MAP2 antibody. RESULTS: Transfection with an active cdc42 mutant (cdc42F28L) decreased the activity of GSK-3ß and induced axonogenesis in primary rat hippocampal neurons, while transfection with a negative cdc42 mutant (cdc42N17) resulted an opposite effect. Moreover, transfection with plasmid vectors carrying wild-type GSK-3ß or a constitutively active GSK3ß mutant (GSK-3ß S9A) increased the activity of GSK-3ß and attenuated axonogenesis of primary hippocampal neurons with excessive cdc42 activity, whereas inhibition of GSK-3ß by LiCl abolished the inhibitory effect of the negative cdc42 mutant on axonogenesis. CONCLUSIONS: This study suggests that cdc42 induces axonogenesis of primary rat hippocampal neurons via inhibiting GSK-3ß activity. These findings support further investigation into the mechanisms of cdc42/GSK-3ß-mediated axonogenesis.

Cdc42 Facilitates Axonogenesis by Enhancing Microtubule Stabilization in Primary Hippocampal Neurons.

The establishment of polarity is an essential process in early neuronal development. Cdc42, a GTPase of the Rho family, is a key regulator of cytoskeletal dynamics and neuronal polarity. However, the mechanisms underlying the action of cdc42 in regulating axonogenesis have not been elucidated. Here, we expressed wild-type cdc42, a constitutively active cdc42 mutant (cdc42F28L) and a dominant negative cdc42 mutant (cdc42N17), respectively, in the primary hippocampal neurons to alter the activity of cdc42. We found that cdc42 activities were paralleled with the capacities to promote axonogenesis in the cultured neurons. Cdc42 also enhanced microtubule stability in the cultured neurons. Pharmacologically stabilizing microtubules significantly abrogated the defective axonogenesis induced by cdc42 inhibition. Moreover, cdc42 promoted the dephosphorylation of collapsing response mediator protein-2 (CRMP-2) at Thr514 by increasing GSK-3ß phosphorylation at Ser9 in the cultured neurons. These findings suggest that cdc42 may facilitate axonogenesis by promoting microtubule stabilization in rat primary hippocampal neurons.

Bone marrow-derived mesenchymal stem cells improve post-ischemia neurological function in rats via the PI3K/AKT/GSK-3ß/CRMP-2 pathway.

BACKGROUND: Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) is a potential therapy for cerebral ischemia. However, the underlying protective mechanism remains undetermined. Here, we tested the hypothesis that transplantation of BMSCs via intravenous injection can alleviate neurological functional deficits through activating PI3K/AKT signaling pathway after cerebral ischemia in rats. METHODS: A cerebral ischemic rat model was established by the 2 h middle cerebral artery occlusion (MCAO). Twenty-four hours later, BMSCs (1 × 106 in 1 ml PBS) from SD rats were injected into the tail vein. Neurological function was evaluated by modified neurological severity score (mNSS) and modified adhesive removal test before and on d1, d3, d7, d10 and d14 after MCAO. Protein expressions of AKT, GSK-3ß, CRMP-2 and GAP-43 were detected by Western-bolt. NF-200 was detected by immunofluorescence. RESULTS: BMSCs transplantation did not only significantly improve the mNSS score and the adhesive-removal somatosensory test after MCAO, but also increase the density of NF-200 and the expression of p-AKT, pGSK-3ß and GAP-43, while decrease the expression of pCRMP-2. Meanwhile, these effects can be suppressed by LY294002, a specific inhibitor of PI3K/AKT. CONCLUSION: These data suggest that transplantation of BMSCs could promote axon growth and neurological deficit recovery after MCAO, which was associated with activation of PI3K/AKT /GSK-3ß/CRMP-2 signaling pathway.

Genetic suppression of collapsin response mediator protein 2 phosphorylation improves outcome in methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's model mice.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by slow and progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Levodopa (l-Dopa), the current main treatment for PD, supplies dopamine, but it does not prevent neurodegeneration. There is thus no promising remedy for PD. Recent in vitro study showed the increase in the phosphorylation levels of Collapsin Response Mediator Protein 2 (CRMP2) is involved in dopaminergic axon degeneration. In the present study, we report elevation of CRMP2 phosphorylation in dopaminergic neurons in SNc after challenge with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a common model for PD. Genetic suppression of CRMP2 phosphorylation by mutation of the obligatory Cyclin-dependent kinase 5 (Cdk5)-targeted serine-522 site prevented axonal degradation in the nigrostriatal pathway of transgenic mice. As a result, the degree of MPTP-induced motor impairment in the rotarod test was suppressed. These results suggest that suppression of CRMP2 phosphorylation may be a novel therapeutic target for PD.

Downregulation of hippocampal SIRT6 activates AKT/CRMP2 signaling and ameliorates chronic stress-induced depression-like behavior in mice.

Sirtuin 6 (SIRT6) has been reported to play a key role in cognitive function and mood regulation, yet its role in mood disorders is not completely understood. Here, we confirmed that knockdown of hippocampal SIRT6 alleviated depression-like behaviors induced by chronic unpredictable stress (CUS) in mice. Our in vitro data showed that SIRT6 negatively regulated protein kinase B (AKT) signaling by deacetylating histone 3 at Lys9 and Lys56. Knockdown of SIRT6 significantly increased AKT phosphorylation activity, while decreased collapsin response mediator protein 2 (CRMP2) phosphorylation activity. Furthermore, pharmacologic inhibition of SIRT6 by ferulic acid (FA) (40 or 80 mg· kg-1 per day, i.g.) could activate AKT/CRMP2 pathway in vitro, which has been proved to exert an antidepressant-like effect on CUS-induced depressive models. In conclusion, our study suggested that hippocampal SIRT6 contributes to the performance of depression-like behaviors by suppressing AKT/CRMP2 signaling, and FA ameliorates CUS-induced depression-like behaviors in mice as a potential pharmacologic inhibitor of SIRT6.

Impairment of chaperone-mediated autophagy affects neuronal homeostasis through altered expression of DJ-1 and CRMP-2 proteins.

Chaperone-mediated autophagy (CMA) is a substrate-specific mode of lysosomal proteolysis, with multiple lines of evidence connecting its dysfunction to both ageing and disease. We have recently shown that CMA impairment through knock-down of the lysosomal receptor LAMP2A is detrimental to neuronal viability in vivo; however, it is not clear which subset of proteins regulated by the CMA pathway mediate such changes. In this study, we have manipulated CMA function through alterations of LAMP2A abundance in primary rat cortical neurons, to identify potential changes to the neuronal proteome occurring prior to neurotoxic effects. We have identified a list of proteins with significant, >2-fold change in abundance following our manipulations, of which PARK7/DJ-1 - an anti-oxidant implicated in hereditary forms of Parkinson's Disease (PD), and DPYSL2/CRMP-2 - a microtubule-binding phosphoprotein involved in schizophrenia pathogenesis - were both found to have measurable effects on neuronal homeostasis and phenotype. Taken together, this study describes alterations in the abundance of neuronal proteins involved in neuropsychiatric disorders upon CMA manipulation, and suggests that such alterations may in part be responsible for the neurodegeneration observed upon CMA impairment in vivo.

Probing the lithium-response pathway in hiPSCs implicates the phosphoregulatory set-point for a cytoskeletal modulator in bipolar pathogenesis.

The molecular pathogenesis of bipolar disorder (BPD) is poorly understood. Using human-induced pluripotent stem cells (hiPSCs) to unravel such mechanisms in polygenic diseases is generally challenging. However, hiPSCs from BPD patients responsive to lithium offered unique opportunities to discern lithium's target and hence gain molecular insight into BPD. By profiling the proteomics of BDP-hiPSC-derived neurons, we found that lithium alters the phosphorylation state of collapsin response mediator protein-2 (CRMP2). Active nonphosphorylated CRMP2, which binds cytoskeleton, is present throughout the neuron; inactive phosphorylated CRMP2, which dissociates from cytoskeleton, exits dendritic spines. CRMP2 elimination yields aberrant dendritogenesis with diminished spine density and lost lithium responsiveness (LiR). The "set-point" for the ratio of pCRMP2:CRMP2 is elevated uniquely in hiPSC-derived neurons from LiR BPD patients, but not with other psychiatric (including lithium-nonresponsive BPD) and neurological disorders. Lithium (and other pathway modulators) lowers pCRMP2, increasing spine area and density. Human BPD brains show similarly elevated ratios and diminished spine densities; lithium therapy normalizes the ratios and spines. Consistent with such "spine-opathies," human LiR BPD neurons with abnormal ratios evince abnormally steep slopes for calcium flux; lithium normalizes both. Behaviorally, transgenic mice that reproduce lithium's postulated site-of-action in dephosphorylating CRMP2 emulate LiR in BPD. These data suggest that the "lithium response pathway" in BPD governs CRMP2's phosphorylation, which regulates cytoskeletal organization, particularly in spines, modulating neural networks. Aberrations in the posttranslational regulation of this developmentally critical molecule may underlie LiR BPD pathogenesis. Instructively, examining the proteomic profile in hiPSCs of a functional agent-even one whose mechanism-of-action is unknown-might reveal otherwise inscrutable intracellular pathogenic pathways.

Boosting CNS axon regeneration by harnessing antagonistic effects of GSK3 activity.

Implications of GSK3 activity for axon regeneration are often inconsistent, if not controversial. Sustained GSK3 activity in GSK3S/A knock-in mice reportedly accelerates peripheral nerve regeneration via increased MAP1B phosphorylation and concomitantly reduces microtubule detyrosination. In contrast, the current study shows that lens injury-stimulated optic nerve regeneration was significantly compromised in these knock-in mice. Phosphorylation of MAP1B and CRMP2 was expectedly increased in retinal ganglion cell (RGC) axons upon enhanced GSK3 activity, but, surprisingly, no GSK3-mediated CRMP2 inhibition was detected in sciatic nerves, thus revealing a fundamental difference between central and peripheral axons. Conversely, genetic or shRNA-mediated conditional KO/knockdown of GSK3ß reduced inhibitory phosphorylation of CRMP2 in RGCs and improved optic nerve regeneration. Accordingly, GSK3ß KO-mediated neurite growth promotion and myelin disinhibition were abrogated by CRMP2 inhibition and largely mimicked in WT neurons upon expression of constitutively active CRMP2 (CRMP2T/A). These results underscore the prevalent requirement of active CRMP2 for optic nerve regeneration. Strikingly, expression of CRMP2T/A in GSK3S/A RGCs further boosted optic nerve regeneration, with axons reaching the optic chiasm within 3 wk. Thus, active GSK3 can also markedly promote axonal growth in central nerves if CRMP2 concurrently remains active. Similar to peripheral nerves, GSK3-mediated MAP1B phosphorylation/activation and the reduction of microtubule detyrosination contributed to this effect. Overall, these findings reconcile conflicting data on GSK3-mediated axon regeneration. In addition, the concept of complementary modulation of normally antagonistically targeted GSK3 substrates offers a therapeutically applicable approach to potentiate the regenerative outcome in the injured CNS.

Canonical TGF-ß Signaling Negatively Regulates Neuronal Morphogenesis through TGIF/Smad Complex-Mediated CRMP2 Suppression.

Functional neuronal connectivity requires proper neuronal morphogenesis and its dysregulation causes neurodevelopmental diseases. Transforming growth factor-ß (TGF-ß) family cytokines play pivotal roles in development, but little is known about their contribution to morphological development of neurons. Here we show that the Smad-dependent canonical signaling of TGF-ß family cytokines negatively regulates neuronal morphogenesis during brain development. Mechanistically, activated Smads form a complex with transcriptional repressor TG-interacting factor (TGIF), and downregulate the expression of a neuronal polarity regulator, collapsin response mediator protein 2. We also demonstrate that TGF-ß family signaling inhibits neurite elongation of human induced pluripotent stem cell-derived neurons. Furthermore, the expression of TGF-ß receptor 1, Smad4, or TGIF, which have mutations found in patients with neurodevelopmental disorders, disrupted neuronal morphogenesis in both mouse (male and female) and human (female) neurons. Together, these findings suggest that the regulation of neuronal morphogenesis by an evolutionarily conserved function of TGF-ß signaling is involved in the pathogenesis of neurodevelopmental diseases.SIGNIFICANCE STATEMENT Canonical transforming growth factor-ß (TGF-ß) signaling plays a crucial role in multiple organ development, including brain, and mutations in components of the signaling pathway associated with several human developmental disorders. In this study, we found that Smads/TG-interacting factor-dependent canonical TGF-ß signaling regulates neuronal morphogenesis through the suppression of collapsin response mediator protein-2 (CRMP2) expression during brain development, and that function of this signaling is evolutionarily conserved in the mammalian brain. Mutations in canonical TGF-ß signaling factors identified in patients with neurodevelopmental disorders disrupt the morphological development of neurons. Thus, our results suggest that proper control of TGF-ß/Smads/CRMP2 signaling pathways is critical for the precise execution of neuronal morphogenesis, whose impairment eventually results in neurodevelopmental disorders.