Collapsin Response Mediator Protein 2 Phosphorylation Research Articles

Drug Treatment Attenuates Retinal Ganglion Cell Death by Inhibiting Collapsin Response Mediator Protein 2 Phosphorylation in Mouse Models of Normal Tension Glaucoma.

Normal tension glaucoma (NTG) is a progressive neurodegenerative disease in glaucoma families. Typical glaucoma develops because of increased intraocular pressure (IOP), whereas NTG develops despite normal IOP. As a subtypeof open-angle glaucoma, NTG is characterized by retinal ganglion cell (RGC) degeneration, gradualloss of axons, andinjury to the optic nerve. The relationship between glutamate excitotoxicity and oxidative stress has elicited great interest in NTG studies. We recently reported that suppressing collapsin response mediator protein 2 (CRMP2) phosphorylation in S522A CRMP2 mutant (CRMP2 KIKI) mice inhibited RGC death in NTG mouse models. This study evaluated the impact of the natural compounds huperzine A (HupA) and naringenin (NAR), which have therapeutic effects against glutamate excitotoxicity and oxidative stress, on inhibiting CMRP2 phosphorylation in mice intravitreally injected with N-methyl-D-aspartate (NMDA) and GLAST mutant mice. Results of the study demonstrated that HupA and NAR significantly reduced RGC degeneration and thinning of the inner retinal layer, and inhibited the elevated CRMP2 phosphorylation. These treatments protected against glutamate excitotoxicity and suppressed oxidative stress, which could provide insight into developing new effective therapeutic strategies for NTG.

Zinc Deficiency Decreases Neurite Extension via CRMP2 Signal Pathway.

Previous reports indicated that zinc deficiency could increase the risk of infectious diseases and developmental retardation in children. In experimental study, it has been reported that zinc deficiency during the embryonic period inhibited fetal growth, and disturbed neural differentiation and higher brain function later in adulthood. Although it has been suggested that zinc deficiency during development can have significant effects on neuronal differentiation and maturation, the molecular mechanisms of the effects of low zinc on neuronal differentiation during development have not been elucidated in detail. This study was performed to determine the effects of low zinc status on neurite outgrowth and collapsin response mediator protein 2 (CRMP2) signal pathway. Low zinc suppressed neurite outgrowth, and caused increase levels of phosphorylated CRMP2 (pCRMP2) relative to CRMP2, and decrease levels of phosphorylated glycogen synthase kinase 3β (pGSK3β) relative to GSK3β in human neuroblastoma cell line (SH-SY5Y) cells on days 1, 2, and 3 of neuronal differentiation induction. Neurite outgrowth inhibited by low zinc was restored by treatment with the GSK3β inhibitor CHIR99021. These results suggested that low zinc causes neurite outgrowth inhibition via phosphorylation of CRMP2 by GSK3β. In conclusion, this study is the first to demonstrate that CRMP signaling is involved in the suppression of neurite outgrowth by low zinc.

Genetic inhibition of collapsin response mediator protein-2 phosphorylation ameliorates retinal ganglion cell death in normal-tension glaucoma models.

Glaucoma is a neurodegenerative disorder caused by the death of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is a cause of glaucoma. However, glaucoma often develops with normal IOP and is known as normal-tension glaucoma (NTG). Glutamate neurotoxicity is considered as one of the significant causes of NTG, resulting in excessive stimulation of retinal neurons via the N-methyl-D-aspartate (NMDA) receptors. The present study examined the phosphorylation of collapsin response mediator protein-2 (CRMP2), a protein that is abundantly expressed in neurons and involved in their development. In two mouse models, NMDA-injection and glutamate/aspartate transporter (GLAST) mutant, CRMP2 phosphorylation at the cyclin-dependent kinase-5 (Cdk5) site was elevated in RGCs. We confirmed that the decrease in the number of RGCs and thickness of the inner retinal layer (IRL) could be suppressed after NMDA administration in CRMP2KI/KI mice with genetically inhibited CRMP2 phosphorylation. Next, we investigated GLAST heterozygotes (GLAST+/-) with CRMP2KI/KI (GLAST+/-;CRMP2KI/KI) and GLAST knockout (GLAST-/-) mice with CRMP2KI/KI (GLAST-/-;CRMP2KI/KI) mice and compared them with GLAST+/- and GLAST-/- mice. pCRMP2 (S522) inhibition significantly reduced RGC loss and IRL thinning. These results suggest that the inhibition of CRMP2 phosphorylation could be a novel strategy for treating NTG.

Hypothiocyanous acid oxidizes collapsin response mediator protein 2 in brain pericytes

AbstractBackgroundPericytes cover brain endothelium to enable maintenance of the blood‐brain barrier (BBB). Decreasing pericyte numbers are reported in Alzheimer’s disease and could lead to BBB dysfunction, triggering vascular inflammation and the recruitment of immune cells. Neutrophils contain high contents of myeloperoxidase, a heme enzyme that produces several oxidising species, including hypothiocyanous acid (HOSCN). HOSCN specifically targets cysteine residues in proteins and low molecular weight thiols, and is more stable than other oxidants in biological systems. A microtubule stabilising protein, collapsin response mediator protein 2 (CRMP2), is oxidised by hydrogen peroxide (H2O2) in some cell types, purportedly through a peroxiredoxin (Prx) relay mechanism. Here, we have tested whether HOSCN can oxidise CRMP2 and lead to disruption of microtubules (MT) in pericytes.MethodMurine pericytes were harvested and cultured, then treated with sub‐lethal concentrations of HOSCN and H2O2. Western blotting was used to observe CRMP2 and Prx oxidation, along with CRMP2 phosphorylation at sites important for MT binding. Fluorescent staining, live‐cell imaging and in vitro scratch assays were used to view cytoskeleton alterations and pericyte migration. Immunoprecipitation and proximity ligation assays were used to investigate physical associations between CRMP2 and Prxs.ResultTreatment of pericytes with HOSCN led to maximal CRMP2 oxidation after 60 min, whereas H2O2 reaches this in 5 min. Oxidation was closely associated with pericyte shrinkage, less growing microtubules and lower pericyte cell migration. CRMP2 oxidation occurred at the same time as Prx2 oxidation, and there was a physical association between these proteins, but it is not possible to conclude a redox relay occurs.ConclusionWe hypothesized that the longer‐lived oxidant HOSCN could oxidize CRMP2 and impair pericyte function at sub‐lethal doses. Indeed, HOSCN led to less dynamic MTs and subsequent lower migration of pericytes, likely lowering their ability to cover empty area on the inflamed vasculature to protect endothelial function. This effect is reversible upon HOSCN removal, nevertheless HOSCN could be a cause of BBB disruption during neuroinflammation. The mechanisms of CRMP2 oxidation are currently being investigated. Strategies that protect this MT regulatory protein from oxidation could have therapeutic benefit in neurological disease.

Involvement of CRMP2 in Regulation of Mitochondrial Morphology and Motility in Huntington's Disease.

Mitochondrial morphology and motility (mitochondrial dynamics) play a major role in the proper functioning of distant synapses. In Huntington’s disease (HD), mitochondria become fragmented and less motile, but the mechanisms leading to these changes are not clear. Here, we found that collapsin response mediator protein 2 (CRMP2) interacted with Drp1 and Miro 2, proteins involved in regulating mitochondrial dynamics. CRMP2 interaction with these proteins inversely correlated with CRMP2 phosphorylation. CRMP2 was hyperphosphorylated in postmortem brain tissues of HD patients, in human neurons derived from induced pluripotent stem cells from HD patients, and in cultured striatal neurons from HD mouse model YAC128. At the same time, CRMP2 interaction with Drp1 and Miro 2 was diminished in HD neurons. The CRMP2 hyperphosphorylation and dissociation from Drp1 and Miro 2 correlated with increased fission and suppressed motility. (S)-lacosamide ((S)-LCM), a small molecule that binds to CRMP2, decreased its phosphorylation at Thr 509/514 and Ser 522 and rescued CRMP2’s interaction with Drp1 and Miro 2. This was accompanied by reduced mitochondrial fission and enhanced mitochondrial motility. Additionally, (S)-LCM exerted a neuroprotective effect in YAC128 cultured neurons. Thus, our data suggest that CRMP2 may regulate mitochondrial dynamics in a phosphorylation-dependent manner and modulate neuronal survival in HD.

CRMP2 as a Candidate Target to Interfere with Lung Cancer Cell Migration.

Collapsin response mediator protein 2 (CRMP2) is an adaptor protein that adds tubulin dimers to the growing tip of a microtubule. First described in neurons, it is now considered a ubiquitous protein that intervenes in processes such as cytoskeletal remodeling, synaptic connection and trafficking of voltage channels. Mounting evidence supports that CRMP2 plays an essential role in neuropathology and, more recently, in cancer. We have previously described a positive correlation between nuclear phosphorylation of CRMP2 and poor prognosis in lung adenocarcinoma patients. In this work, we studied whether this cytoskeleton molding protein is involved in cancer cell migration. To this aim, we evaluated CRMP2 phosphorylation and localization in the extending lamella of lung adenocarcinoma migrating cells using in vitro assays and in vivo confocal microscopy. We demonstrated that constitutive phosphorylation of CRMP2 impaired lamella formation, cell adhesion and oriented migration. In search of a mechanistic explanation of this phenomenon, we discovered that CRMP2 Ser522 phospho-mimetic mutants display unstable tubulin polymers, unable to bind EB1 plus-Tip protein and the cortical actin adaptor IQGAP1. In addition, integrin recycling is defective and invasive structures are less evident in these mutants. Significantly, mouse xenograft tumors of NSCLC expressing CRMP2 phosphorylation mimetic mutants grew significantly less than wild-type tumors. Given the recent development of small molecule inhibitors of CRMP2 phosphorylation to treat neurodegenerative diseases, our results open the door for their use in cancer treatment.

CRMP2 Is Involved in Regulation of Mitochondrial Morphology and Motility in Neurons.

Regulation of mitochondrial morphology and motility is critical for neurons, but the exact mechanisms are unclear. Here, we demonstrate that these mechanisms may involve collapsin response mediator protein 2 (CRMP2). CRMP2 is attached to neuronal mitochondria and binds to dynamin-related protein 1 (Drp1), Miro 2, and Kinesin 1 light chain (KLC1). Treating neurons with okadaic acid (OA), an inhibitor of phosphatases PP1 and PP2A, resulted in increased CRMP2 phosphorylation at Thr509/514, Ser522, and Thr555, and augmented Drp1 phosphorylation at Ser616. The CRMP2-binding small molecule (S)-lacosamide ((S)-LCM) prevented an OA-induced increase in CRMP2 phosphorylation at Thr509/514 and Ser522 but not at Thr555, and also failed to alleviate Drp1 phosphorylation. The increased CRMP2 phosphorylation correlated with decreased CRMP2 binding to Drp1, Miro 2, and KLC1. (S)-LCM rescued CRMP2 binding to Drp1 and Miro 2 but not to KLC1. In parallel with CRMP2 hyperphosphorylation, OA increased mitochondrial fission and suppressed mitochondrial traffic. (S)-LCM prevented OA-induced alterations in mitochondrial morphology and motility. Deletion of CRMP2 with a small interfering RNA (siRNA) resulted in increased mitochondrial fission and diminished mitochondrial traffic. Overall, our data suggest that the CRMP2 expression level and phosphorylation state are involved in regulating mitochondrial morphology and motility in neurons.

Somatostatin Ameliorates β-Amyloid-Induced Cytotoxicity via the Regulation of CRMP2 Phosphorylation and Calcium Homeostasis in SH-SY5Y Cells.

Somatostatin is involved in the regulation of multiple signaling pathways and affords neuroprotection in response to neurotoxins. In the present study, we investigated the role of Somatostatin-14 (SST) in cell viability and the regulation of phosphorylation of Collapsin Response Mediator Protein 2 (CRMP2) (Ser522) via the blockade of Ca2+ accumulation, along with the inhibition of cyclin-dependent kinase 5 (CDK5) and Calpain activation in differentiated SH-SY5Y cells. Cell Viability and Caspase 3/7 assays suggest that the presence of SST ameliorates mitochondrial stability and cell survival pathways while augmenting pro-apoptotic pathways activated by Aβ. SST inhibits the phosphorylation of CRMP2 at Ser522 site, which is primarily activated by CDK5. Furthermore, SST effectively regulates Ca2+ influx in the presence of Aβ, directly affecting the activity of calpain in differentiated SH-SY5Y cells. We also demonstrated that SSTR2 mediates the protective effects of SST. In conclusion, our results highlight the regulatory role of SST in intracellular Ca2+ homeostasis. The neuroprotective role of SST via axonal regeneration and synaptic integrity is corroborated by regulating changes in CRMP2; however, SST-mediated changes in the blockade of Ca2+ influx, calpain expression, and toxicity did not correlate with CDK5 expression and p35/25 accumulation. To summarize, our findings suggest two independent mechanisms by which SST mediates neuroprotection and confirms the therapeutic implications of SST in AD as well as in other neurodegenerative diseases where the effective regulation of calcium homeostasis is required for a better prognosis.

Cortical transcriptome analysis after spinal cord injury reveals the regenerative mechanism of central nervous system in CRMP2 knock-in mice.

Recent studies have shown that mutation at Ser522 causes inhibition of collapsin response mediator protein 2 (CRMP2) phosphorylation and induces axon elongation and partial recovery of the lost sensorimotor function after spinal cord injury (SCI). We aimed to reveal the intracellular mechanism in axotomized neurons in the CRMP2 knock-in (CRMP2KI) mouse model by performing transcriptome analysis in mouse sensorimotor cortex using micro-dissection punching system. Prior to that, we analyzed the structural pathophysiology in axotomized or neighboring neurons after SCI and found that somatic atrophy and dendritic spine reduction in sensorimotor cortex were suppressed in CRMP2KI mice. Further analysis of the transcriptome has aided in the identification of four hemoglobin genes Hba-a1, Hba-a2, Hbb-bs, and Hbb-bt that are significantly upregulated in wild-type mice with concomitant upregulation of genes involved in the oxidative phosphorylation and ribosomal pathways after SCI. However, we observed substantial upregulation in channel activity genes and downregulation of genes regulating vesicles, synaptic function, glial cell differentiation in CRMP2KI mice. Moreover, the transcriptome profile of CRMP2KI mice has been discussed wherein energy metabolism and neuronal pathways were found to be differentially regulated. Our results showed that CRMP2KI mice displayed improved SCI pathophysiology not only via microtubule stabilization in neurons, but also possibly via the whole metabolic system in the central nervous system, response changes in glial cells, and synapses. Taken together, we reveal new insights on SCI pathophysiology and the regenerative mechanism of central nervous system by the inhibition of CRMP2 phosphorylation at Ser522. All these experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Waseda University, Japan (2017-A027 approved on March 21, 2017; 2018-A003 approved on March 25, 2018; 2019-A026 approved on March 25, 2019).

How scars shape the neural landscape: Key molecular mediators of TGF-β1's anti-neuritogenic effects.

Following injury to the peripheral and central nervous systems, tissue levels of transforming growth factor (TGF)-β1 often increase, which is key for wound healing and scarring. However, active wound regions and scars appear to inhibit process outgrowth by regenerating neurons. We recently showed that corneal wound myofibroblasts block corneal nerve regeneration in vivo, and sensory neurite outgrowth in vitro in a manner that relies critically on TGF-β1. In turn, delayed, abnormal re-innervation contributes to long-term sensory dysfunctions of the ocular surface. Here, we exposed morphologically and biochemically-differentiated sensory neurons from the ND7/23 cell line to TGF-β1 to identify the intracellular signals regulating these anti-neuritogenic effects, contrasting them with those of Semaphorin(Sema)3A, a known inhibitor of neurite outgrowth. Neuronal morphology was quantified using phase-contrast imaging. Western blotting and specific inhibitors were then used to identify key molecular mediators. Differentiated ND7/23 cells expressed neuron-specific markers, including those involved in neurite extension and polarization. TGF-β1 increased phosphorylation of collapsin response mediator protein-2 (CRMP2), a molecule that is key for neurite extension. We now show that both glycogen synthase kinase (GSK)-3β and Smad3 modulate phosphorylation of CRMP2 after treatment with TGF-β1. GSK-3β appeared to exert a particularly strong effect, which could be explained by its ability to phosphorylate not only CRMP2, but also Smad3. In conclusion, TGF-β1's inhibition of neurite outgrowth in sensory neurons appears to be regulated through a highly-conserved signaling pathway, which involves the GSK-3β/CRMP-2 loop via both canonical and non-canonical mechanisms. It is hoped that by defining the signaling pathways that control neurite outgrowth in wound environments, it will become possible to identify optimal molecular targets to promote re-innervation following injury.

PKC\u03b3-Mediated Phosphorylation of CRMP2 Regulates Dendritic Outgrowth in Cerebellar Purkinje Cells

The signalling protein PKCγ is a major regulator of Purkinje cell development and synaptic function. We have shown previously that increased PKCγ activity impairs dendritic development of cerebellar Purkinje cells. Mutations in the protein kinase Cγ gene (PRKCG) cause spinocerebellar ataxia type 14 (SCA14). In a transgenic mouse model of SCA14 expressing the human S361G mutation, Purkinje cell dendritic development is impaired in cerebellar slice cultures similar to pharmacological activation of PKC. The mechanisms of PKCγ-driven inhibition of dendritic growth are still unclear. Using immunoprecipitation-coupled mass spectrometry analysis, we have identified collapsin response mediator protein 2 (CRMP2) as a protein interacting with constitutive active PKCγ(S361G) and confirmed the interaction with the Duolink™ proximity ligation assay. We show that in cerebellar slice cultures from PKCγ(S361G)-mice, phosphorylation of CRMP2 at the known PKC target site Thr555 is increased in Purkinje cells confirming phosphorylation of CRMP2 by PKCγ. miRNA-mediated CRMP2 knockdown decreased Purkinje cell dendritic outgrowth in dissociated cerebellar cultures as did the transfection of CRMP2 mutants with a modified Thr555 site. In contrast, dendritic development was normal after wild-type CRMP2 overexpression. In a novel knock-in mouse expressing only the phospho-defective T555A-mutant CRMP2, Purkinje cell dendritic development was reduced in dissociated cultures. This reduction could be rescued by transfecting wild-type CRMP2 but only partially by the phospho-mimetic T555D-mutant. Our findings establish CRMP2 as an important target of PKCγ phosphorylation in Purkinje cells mediating its control of dendritic development. Dynamic regulation of CRMP2 phosphorylation via PKCγ is required for its correct function.

CRMP2 is a therapeutic target that suppresses the aggressiveness of breast cancer cells by stabilizing RECK

Metastatic breast cancer is characterized by high mortality and limited therapeutic target. During tumor metastasis, cytoskeletal reorganization is one of the key steps in the migration and invasion of breast cancer cells. Collapsin response mediator protein 2 (CRMP2) is a cytosolic phosphoprotein that plays an important role in regulating cytoskeletal dynamics. Previous researches have reported that altered CRMP2 expression is associated with breast cancer progression, but the underlying mechanism remains poorly understood. Here, we show that CRMP2 expression is reduced in various subtypes of breast cancers and negatively correlated with lymphatic metastasis. Overexpression of CRMP2 significantly inhibits invasion and stemness in breast cancer cells, while downregulation of CRMP2 promotes cell invasion, which is not required for tubulin polymerization. Mechanistic studies demonstrate that CRMP2 interacts with RECK, prevents RECK degradation, which, in turn, blocks NF-κB and Wnt signaling pathways. Furthermore, we find that phosphorylation of CRMP2 at T514 and S522 remarkably abolishes its functions to bind with RECK and to inhibit cell invasion. Pharmacologic rescue of CRMP2 expression suppressed breast cancer metastasis in vitro and in vivo and stimulated a synergetic effect with FN-1501 that induces CRMP2 dephosphorylation. Collectively, this study highlights the potential of CRMP2 as a therapeutic target in breast cancer metastasis and reveals a distinct mechanism of CRMP2.

Collapsin Response Mediator Protein 2 and Endophilin2 Coordinate Regulation of AMPA Receptor GluA1 Subunit Recycling.

The dynamic trafficking of AMPA receptors (AMPARs), which enables the endocytosis, recycling, and exocytosis of AMPARs, is crucial for synaptic plasticity. Endophilin2, which directly interacts with the GluA1 subunit of AMPARs, plays an important role in AMPAR endocytosis. Collapsin response mediator protein 2 (CRMP2) promotes the maturation of the dendritic spine and can transfer AMPARs to the membrane. Although the mechanisms of AMPAR endocytosis and exocytosis are well known, the exact molecular mechanisms underlying AMPAR recycling remain unclear. Here, we report a unique interaction between CRMP2 and endophilin2. Our results showed that overexpression of CRMP2 and endophilin2 increased the amplitude and frequency of miniature excitatory synaptic currents (mEPSCs) and modestly enhanced AMPAR levels in hippocampal neurons. Furthermore, the CRMP2 and endophilin2 overexpression phenotype failed to occur when the interaction between these two proteins was inhibited. Further analysis revealed that this interaction was regulated by CRMP2 phosphorylation. The phosphorylation of CRMP2 inhibited its interaction with endophilin2; this was mainly affected by the phosphorylation of Thr514 and Ser518 by glycogen synthase kinase (GSK) 3β. CRMP2 phosphorylation increased degradation and inhibited the surface expression of AMPAR GluA1 subunits in cultured hippocampal neurons. However, the dephosphorylation of CRMP2 inhibited degradation and promoted the surface expression of AMPAR GluA1 subunits in cultured hippocampal neurons. Taken together, our data demonstrated that the interaction between CRMP2 and endophilin2 was conductive to the recycling of AMPA receptor GluA1 subunits in hippocampal neurons.

Lanthionine ketimine ester improves outcome in an MPTP-induced mouse model of Parkinson's disease via suppressions of CRMP2 phosphorylation and microglial activation

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Levodopa (L-Dopa), the current main treatment for PD, reduces PD symptoms by partially replacing dopamine, but it does not slow neurodegeneration. Recent studies have evidenced that neuroinflammatory processes contribute to the degeneration of dopaminergic neurons in the SNc under cytopathic conditions, while other lines of inquiry have implicated phosphorylation of collapsin response mediator protein 2 (CRMP2) as a causal factor in axonal retraction after neural injury. We recently reported on the therapeutic effect of lanthionine ketimine ester (LKE) which associates with CRMP2 following axonal injury in the spinal cord.In the present study, we report that LKE protects SNc dopaminergic neurons after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) challenge, a common model for PD, and reduces the number of activated microglia proximal to the damaged SNc. The results also show that MPTP-induced motor impairment was suppressed in LKE treatment. Furthermore, the results show that LKE inhibits the elevation of CRMP2 phosphorylation in dopaminergic neurons in the SNc after MPTP injection. These data suggest that modification of CRMP2 phosphorylation and suppression of microglial activation with LKE administration may represent a novel strategy for slowing progress of pathological processes in PD.

Phosphorylation of CRMP2 is required for migration and positioning of Purkinje cells: Redundant roles of CRMP1 and CRMP4

Proper migration and positioning of Purkinje cells are important for formation of the developing cerebellum. Although several cyclin-dependent kinase 5 (Cdk5) substrates are known to be critical for ordered neuronal migration, there are no reports of mutant mouse-based, in vivo studies on the function of Cdk5-phosphorylation substrates in migration of Purkinje cells.We focused on the analysis of collapsin response mediator protein 2 (CRMP2), one of the Cdk5 substrates, because a previous study reported migration defects of cortical neurons with shRNA-mediated knockdown of CRMP2. However, CRMP2 KI/KI mice, in which Cdk5-phosphorylation is inhibited, showed little defects in Purkinje cell migration and positioning. We hypothesized compensatory redundant functions of the other CRMPs, and analyzed the migration and positioning of Purkinje cells in the cerebellum in every combination of CRMP1 knockout (KO), CRMP2 KI/KI, and CRMP4 KO mice. Severe disturbance of migration and positioning of Purkinje cells were observed in the triple mutant mice. We also found motor coordination defects in the triple CRMPs mutant mice. These results suggest the importance of both, phosphorylation of CRMP2 by Cdk5 and the redundant functions of CRMP1 and CRMP4 in proper migration and positioning of Purkinje cells in developing cerebellum.

Discovery of suppressors of CRMP2 phosphorylation reveals compounds that mimic the behavioral effects of lithium on amphetamine-induced hyperlocomotion

The effective treatment of bipolar disorder (BD) represents a significant unmet medical need. Although lithium remains a mainstay of treatment for BD, limited knowledge regarding how it modulates affective behavior has proven an obstacle to discovering more effective mood stabilizers with fewer adverse side effects. One potential mechanism of action of lithium is through inhibition of the serine/threonine protein kinase GSK3β, however, relevant substrates whose change in phosphorylation may mediate downstream changes in neuroplasticity remain poorly understood. Here, we used human induced pluripotent stem cell (hiPSC)-derived neuronal cells and stable isotope labeling by amino acids in cell culture (SILAC) along with quantitative mass spectrometry to identify global changes in the phosphoproteome upon inhibition of GSK3α/β with the highly selective, ATP-competitive inhibitor CHIR-99021. Comparison of phosphorylation changes to those induced by therapeutically relevant doses of lithium treatment led to the identification of collapsin response mediator protein 2 (CRMP2) as being highly sensitive to both treatments as well as an extended panel of structurally distinct GSK3α/β inhibitors. On this basis, a high-content image-based assay in hiPSC-derived neurons was developed to screen diverse compounds, including FDA-approved drugs, for their ability to mimic lithium’s suppression of CRMP2 phosphorylation without directly inhibiting GSK3β kinase activity. Systemic administration of a subset of these CRMP2-phosphorylation suppressors were found to mimic lithium’s attenuation of amphetamine-induced hyperlocomotion in mice. Taken together, these studies not only provide insights into the neural substrates regulated by lithium, but also provide novel human neuronal assays for supporting the development of mechanism-based therapeutics for BD and related neuropsychiatric disorders.

Tau-tubulin kinase 1 and amyloid-\u03b2 peptide induce phosphorylation of collapsin response mediator protein-2 and enhance neurite degeneration in Alzheimer disease mouse models

The accumulation of phosphorylated tau protein (pTau) in the entorhinal cortex (EC) is the earliest tau pathology in Alzheimer’s disease (AD). Tau tubulin kinase-1 (TTBK1) is a neuron-specific tau kinase and expressed in the EC and hippocampal regions in both human and mouse brains. Here we report that collapsin response mediator protein-2 (CRMP2), a critical mediator of growth cone collapse, is a new downstream target of TTBK1 and is accumulated in the EC region of early stage AD brains. TTBK1 transgenic mice show severe axonal degeneration in the perforant path, which is exacerbated by crossing with Tg2576 mice expressing Swedish familial AD mutant of amyloid precursor protein (APP). TTBK1 mice show accumulation of phosphorylated CRMP2 (pCRMP2), in the EC at 10 months of age, whereas age-matched APP/TTBK1 bigenic mice show pCRMP2 accumulation in both the EC and hippocampal regions. Amyloid-β peptide (Aβ) and TTBK1 suppress the kinetics of microtubule polymerization and TTBK1 reduces the neurite length of primary cultured neurons in Rho kinase-dependent manner in vitro. Silencing of TTBK1 or expression of dominant-negative Rho kinase demonstrates that Aβ induces CRMP2 phosphorylation at threonine 514 in a TTBK1-dependent manner, and TTBK1 enhances Aβ-induced CRMP2 phosphorylation in Rho kinase-dependent manner in vitro. Furthermore, TTBK1 expression induces pCRMP2 complex formation with pTau in vitro, which is enhanced upon Aβ stimulation in vitro. Finally, pCRMP2 forms a complex with pTau in the EC tissue of TTBK1 mice in vivo, which is exacerbated in both the EC and hippocampal tissues in APP/TTBK1 mice. These results suggest that TTBK1 and Aβ induce phosphorylation of CRMP2, which may be causative for the neurite degeneration and somal accumulation of pTau in the EC neurons, indicating critical involvement of TTBK1 and pCRMP2 in the early AD pathology.

Relative D3 vitamin deficiency and consequent cognitive impairment in an animal model of Alzheimer's disease: Potential involvement of collapsin response mediator protein-2

Alzheimer's disease (AD) starts with memory impairments that can be observed before the appearance of significant neuropathology; thus, identifying mechanisms to stop AD progression is an urgent priority. Epidemiological and clinical data show that the consequences of vitamin D deficiency are relevant to disease risk and can be observed in the progression of many diseases, especially AD, whereas higher serum levels of vitamin D are associated with better cognitive test performance. However, the potential therapeutic strategy and underlying mechanisms of vitamin D supplementation against AD still need to be further investigated. In the present study, we found that 3xTg-AD mice with vitamin D supplementation exhibited an increase in serum vitamin D concentrations and improved cognition. We measured serum vitamin D binding protein (VDBP) concentrations and found that serum VDBP levels were increased in 3xTg-AD mice compared to B6129S control mice, but there was no significant difference between control- and vitamin D-treated 3xTg-AD groups. The vitamin D-mediated memory improvement may be accompanied by the suppression of increased hippocampal collapsin response mediator protein-2 (CRMP2) phosphorylation, and the restoration of CRMP2 phosphorylation by okadaic acid (OA) could abolish the beneficial effects of vitamin D. In addition, we found that CRMP2 was associated with NR2B and PSD-95 in 3xTg-AD mice with vitamin D supplementation. This CRMP2-NR2B interaction could be disrupted by a TAT-CBD3 peptide or OA, leading to attenuated memory protection in vitamin D-treated 3xTg-AD mice. Therefore, CRMP2 may be involved in vitamin D-mediated memory improvement in AD.

Genetic inhibition of CRMP2 phosphorylation delays Wallerian degeneration after optic nerve injury

Axonal degeneration occurs in patients with various neurological diseases and traumatic nerve injuries, and Wallerian degeneration is a phenomenon in the prototypical axonal degradation that is observed after injury. Collapsin response mediator protein 2 (CRMP2) is phosphorylated by glycogen synthase kinase 3β (GSK3β), and it is involved in Wallerian degeneration after optic nerve injury. We previously developed a CRMP2 knock-in (CRMP2 KI) mouse line, in which CRMP2 phosphorylation by GSK3β is inhibited; however, Wallerian degeneration in CRMP2 KI mice has not yet been examined.In this study, we examined whether Wallerian degeneration of the optic nerve is suppressed in CRMP2 KI mice. Using one eye removal model, we compared Wallerian degeneration of the optic nerve based on histological and biochemical analyses. Our experimental results indicated that the genetic inhibition of CRMP2 phosphorylation delays Wallerian degeneration after optic nerve injury.

Genetic inhibition of CRMP2 phosphorylation at serine 522 promotes axonal regeneration after optic nerve injury

Axonal degeneration occurs in various neurological diseases and traumatic nerve injury, and axonal regeneration is restricted by inhibitory factors in the central nervous system. Cyclin-dependent kinase 5 and glycogen synthase kinase 3β (GSK3β) are activated by one of those inhibitors, and collapsin response mediator protein 2 (CRMP2) is phosphorylated by both kinases. We previously developed a CRMP2 knock-in (CRMP2 KI) mouse line, in which CRMP2 phosphorylation at Ser 522 is inhibited. Because CRMP2 KI mice showed promotion of axonal regeneration after spinal cord injury, we hypothesized that CRMP2 KI mice would show higher axonal regeneration after optic nerve injury. In this study, we first show that depolymerization of microtubules after optic nerve crush (ONC) injury was suppressed in CRMP2 KI mice. Loss of retinal ganglia cells was also reduced after ONC. We found that protein level of GAP43, a marker of regenerative axons, was higher in the optic nerve from CRMP2KI than that from wild type 4 weeks after of ONC. We further observed increased numbers of axons labeled by tracer in the optic nerve after ONC in CRMP2 KI mice. These results suggest that inhibition of phosphorylation of CRMP2 suppresses axonal degeneration and promotes axonal regeneration after optic nerve injury.