Collapsin Response Mediator Protein Research Articles

ROCK2 is a major regulator of axonal degeneration, neuronal death and axonal regeneration in the CNS.

The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increased survival of RGCs after optic nerve axotomy. Moreover, knockdown of ROCK2 attenuated axonal degeneration of the proximal axon after ONC assessed by in vivo live imaging. Mechanistically, we demonstrate here that knockdown of ROCK2 resulted in decreased intraneuronal activity of calpain and caspase 3, whereas levels of pAkt and collapsin response mediator protein 2 and autophagic flux were increased. Taken together, our data characterize ROCK2 as a specific therapeutic target in neurodegenerative diseases and demonstrate new downstream effects of ROCK2 including axonal degeneration, apoptosis and autophagy.

MICAL-Family Proteins: Complex Regulators of the Actin Cytoskeleton

The molecules interacting with CasL (MICAL) family members participate in a multitude of activities, including axonal growth cone repulsion, membrane trafficking, apoptosis, and bristle development in flies. An interesting feature of MICAL proteins is the presence of an N-terminal flavo-mono-oxygenase domain. This mono-oxygenase domain generates redox potential with which MICALs can either oxidize proteins or produce reactive oxygen species (ROS). Actin is one such protein that is affected by MICAL function, leading to dramatic cytoskeletal rearrangements. This review describes the MICAL-family members, and discusses their mechanisms of actin-binding and regulation of actin cytoskeleton organization. Recent studies show that MICALs directly induce oxidation of actin molecules, leading to actin depolymerization. ROS production by MICALs also causes oxidation of collapsin response mediator protein-2, a microtubule assembly promoter, which subsequently undergoes phosphorylation. MICAL proteins oxidize proteins through two mechanisms: either directly by oxidizing methionine residues or indirectly via the production of ROS. It remains unclear whether MICAL proteins employ both mechanisms or whether the activity of MICAL-family proteins might vary with different substrates. The identification of additional substrates oxidized by MICAL will shed new light on MICAL protein function. Additional directions include expanding studies toward the MICAL-like homologs that lack flavin adenine dinucleotide domains and oxidation activity.

Paraneoplastic Hu and CRMP5 antibodies are present in smokers without cancer or neurological disease

We investigated if the paraneoplastic Hu and collapsin response mediator protein 5 (CRMP5) antibodies could be used as early markers for lung cancer in smokers with or without chronic obstructive pulmonary disease (COPD). Hu and CRMP5 antibodies were measured by radioimmunoprecipitation assay (RIPA) in sera from 552 smokers; 379 with and 173 without COPD. Three hundred blood donors served as controls. The positive sera were also tested by indirect immunofluorescence and line blot with recombinant proteins. The 552 smokers were matched with data from the Cancer Registry of Norway, and the hospital medical records from the subjects positive for Hu and CRMP5 antibodies were reviewed. The mean follow-up time was 4.4 years (range 2.5-5.7 years). The RIPA showed that 5/379 (1.3%) smokers with COPD had Hu antibodies and 1/379 (0.3%) smokers with COPD had CRMP5 antibodies. Only the smoker with the highest RIPA index had Hu antibodies also detected by immunofluorescence and line blot. One of 173 (0.6%) smokers without COPD had Hu antibodies, but none had CRMP5 antibodies. None of the 300 controls had Hu antibodies, but 2/300 (0.7%) had CRMP5 antibodies. Hu antibodies remained positive for more than 5 years. No cancer or neurological disease was recorded in the Hu or CRMP5 positive patients. The total cancer frequency in the smokers with and without COPD was 70/552 (13%). Hu and CRMP5 antibodies were not associated with cancer or neurological disease in a large cohort of smokers and are therefore not always paraneoplastic.

Changes in microtubule-related proteins and autophagy in long-term vitamin E-deficient mice

Vitamin E deficiency induces neuronal dysfunction and while oxidative stress is likely to be involved in mediating this process, the detailed mechanisms remain to be elucidated. Previously, we found axonal degeneration in the hippocampal CA1 region in vitamin E-deficient mice of 6 months of age (long-term). However, 3 month-old (short-term) vitamin E-deficient mice did not exhibit axonal degeneration in same region. In order to characterize the mechanisms involved in axonal degeneration in long-term vitamin E-deficient mice, we examined changes in microtubule-related proteins. Long-term vitamin E-deficiency led to significantly increased expression of the phosphorylated form of collapsin response mediator protein (CRMP)-2 compared to short-term deficiency. It is well known that CRMP-2 plays a crucial role in the maintenance of neurite function. Similarly, long-term vitamin E-deficiency significantly decreased the expression of silent mating type information regulation (SIRT)-2 mRNA compared to short-term deficiency. SIRT-2 belongs to a family of class III histone deacetylases (HDACs) and functions in the deacetylation of tubulins. Furthermore, the expression of microtubule-associated protein light chain (MAP-LC)3-2, which is a key autophagy protein was significantly higher in the short-term vitamin E-deficiency than the long-term deficiency. These results indicate that the mechanisms of axonal injury in long-term vitamin E-deficient mice are related to dysfunction in microtubules assembly via alterations in microtubule-related proteins and autophagy.

MMP-2 mediates Purkinje cell morphogenesis and spine development in the mouse cerebellum

Matrix metalloproteinase-2 (MMP-2) is a highly studied proteolytic enzyme, involved in many detrimental and beneficial functions throughout the body, and also active in the central nervous system (CNS). MMP-2 is profoundly expressed in the developing cerebellum and was recently reported to modulate granule cell proliferation by affecting cell cycle kinetics in cerebella of postnatal day 3 mouse pups. In this report, a two-dimensional difference gel electrophoresis proteomics study was implemented at this postnatal stage and revealed 16 differentially expressed proteins between MMP-2-deficient (MMP-2(-/-)) and wild-type cerebella. Among those, collapsin response mediator protein 1 (CRMP1) could be identified as the most significant differential protein between the two genotypes. Western blot experiments confirmed this finding and further disclosed a significant increase in phosphorylated CRMP1 expression in MMP-2(-/-) cerebella. Strikingly, subsequent immunohistochemical and microscopic analyses revealed an aberrant Purkinje cell (PC) dendritogenesis, possibly related to upregulated (phospho-) CRMP1 levels in these neonatal MMP-2(-/-) animals. Further, detailed morphometric analyses showed persistent PC morphological changes in MMP-2(-/-) mice, from the neonatal stage until adulthood. These were characterized by a reduced growth of PC somata, reduced dendritic tree sizes, and a decreased dendritic arborization. During development, the observed defects were accompanied by a temporarily disturbed parallel fiber and climbing fiber synaptic input on the PCs, while in adult MMP-2(-/-) animals, an increased PC spine density and reduced spine lengths were noted. The observed PC abnormalities might contribute to the milddefects in motor performance, i.e. balance and coordination, detected in adult MMP-2(-/-) mice. Overall, these findings indicate the importance of MMP-2 in CNS development and dendritogenesis, and highlight the importance of a correct developmental wiring for adult brain morphology and function.

Localized changes to glycogen synthase kinase-3 and collapsin response mediator protein-2 in the Huntington's disease affected brain

All cases of Huntington's disease (HD) are caused by mutant huntingtin protein (mhtt), yet the molecular mechanisms that link mhtt to disease symptoms are not fully elucidated. Given glycogen synthase kinase-3 (GSK3) is implicated in several neurodegenerative diseases as a molecular mediator of neuronal decline and widely touted as a therapeutic target, we investigated GSK3 in cells expressing mhtt, brains of R6/1 HD mice and post-mortem human brain samples. Consistency in data across the two models and the human brain samples indicate decreased GSK3 signalling contributes to neuronal dysfunction in HD. Inhibitory phosphorylation of GSK3 (pGSK3) was elevated in mhtt cells and this appeared related to an overall energy metabolism deficit as the mhtt cells had less ATP and inhibiting ATP production in control cells expressing non-pathogenic htt with paraquat also increased pGSK3. pGSK3 was increased and ATP levels decreased in the frontal cortex and striatum of R6/1 mice and levels of cortical pGSK3 inversely correlated with cognitive function of the mice. Consistent with decreased GSK3 activity in the R6/1 mouse brain, β-catenin levels were increased and phosphorylation of collapsin response mediator protein-2 (CRMP2) decreased in the frontal cortex where inhibitory phosphorylation of GSK3 was the greatest. pGSK3 was predominantly undetectable in HD and healthy control human brain samples, but levels of total GSK3 were decreased in the HD-affected frontal cortex and this correlated with decreased pCRMP2. Thus, disruptions to cortical GSK3 signalling, possibly due to localized energy metabolism deficits, appear to contribute to the cognitive symptoms of HD.

Collapsin Response Mediator Protein 2 (CRMP2) Interacts with N-Methyl-d-aspartate (NMDA) Receptor and Na+/Ca2+ Exchanger and Regulates Their Functional Activity

Collapsin response mediator protein 2 (CRMP2) is traditionally viewed as an axonal growth protein involved in axon/dendrite specification. Here, we describe novel functions of CRMP2. A 15-amino acid peptide from CRMP2, fused to the TAT cell-penetrating motif of the HIV-1 protein, TAT-CBD3, but not CBD3 without TAT, attenuated N-methyl-d-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca(2+) dysregulation, suggesting the key contribution of CRMP2 in these processes. In addition, TAT-CBD3, but not CBD3 without TAT or TAT-scramble peptide, inhibited increases in cytosolic Ca(2+) mediated by the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) operating in the reverse mode. Co-immunoprecipitation experiments revealed an interaction between CRMP2 and NMDAR as well as NCX3 but not NCX1. TAT-CBD3 disrupted CRMP2-NMDAR interaction without change in NMDAR localization. In contrast, TAT-CBD3 augmented the CRMP2-NCX3 co-immunoprecipitation, indicating increased interaction or stabilization of a complex between these proteins. Immunostaining with an anti-NCX3 antibody revealed that TAT-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. Indeed, the forward mode of NCX, evaluated in experiments with ionomycin-induced Ca(2+) influx into neurons, was strongly suppressed by TAT-CBD3. Knockdown of CRMP2 with short interfering RNA (siRNA) prevented NCX3 internalization in response to TAT-CBD3 exposure. Moreover, CRMP2 down-regulation strongly attenuated TAT-CBD3-induced inhibition of reverse NCX. Overall, our results demonstrate that CRMP2 interacts with NCX and NMDAR and that TAT-CBD3 protects against glutamate-induced Ca(2+) dysregulation most likely via suppression of both NMDAR and NCX activities. Our results further clarify the mechanism of action of TAT-CBD3 and identify a novel regulatory checkpoint for NMDAR and NCX function based on CRMP2 interaction with these proteins.

Poly(Trimethylene Carbonate-co-ε-Caprolactone) Promotes Axonal Growth

Mammalian central nervous system (CNS) neurons do not regenerate after injury due to the inhibitory environment formed by the glial scar, largely constituted by myelin debris. The use of biomaterials to bridge the lesion area and the creation of an environment favoring axonal regeneration is an appealing approach, currently under investigation. This work aimed at assessing the suitability of three candidate polymers – poly(ε-caprolactone), poly(trimethylene carbonate-co-ε-caprolactone) (P(TMC-CL)) (11∶89 mol%) and poly(trimethylene carbonate) - with the final goal of using these materials in the development of conduits to promote spinal cord regeneration. Poly(L-lysine) (PLL) coated polymeric films were tested for neuronal cell adhesion and neurite outgrowth. At similar PLL film area coverage conditions, neuronal polarization and axonal elongation was significantly higher on P(TMC-CL) films. Furthermore, cortical neurons cultured on P(TMC-CL) were able to extend neurites even when seeded onto myelin. This effect was found to be mediated by the glycogen synthase kinase 3β (GSK3β) signaling pathway with impact on the collapsin response mediator protein 4 (CRMP4), suggesting that besides surface topography, nanomechanical properties were implicated in this process. The obtained results indicate P(TMC-CL) as a promising material for CNS regenerative applications as it promotes axonal growth, overcoming myelin inhibition.

Collapsin response mediator proteins: Potential diagnostic and prognostic biomarkers in cancers (Review).

The collapsin response mediator proteins (CRMPs) were originally identified as mediators of semaphorin 3A signaling and neuronal differentiation. The CRMP family consists of five homologous cytosolic proteins, CRMP1-5. Altered expression levels of CRMPs have been observed in several malignant tumors, including lung, breast, colorectal, prostate, pancreatic and neuroendocrine lung cancer. The aim of the current study was to review the recent progress achieved in understanding the association between the different levels of CRMP expression in tumors and their involvement in pathological functions, such as tumor metastasis, disease progression, subtype differentiation and clinical outcome, to address the potential value of CRMPs as biomarkers for the diagnosis and prognosis of cancer patients.

A case of paraneoplastic optic neuropathy and outer retinitis positive for autoantibodies against collapsin response mediator protein-5, recoverin, and α-enolase

BackgroundSpecific cross-reacting autoimmunity against recoverin or collapsin response mediator protein (CRMP)-5 is known to cause cancer-associated retinopathy or paraneoplastic optic neuropathy, respectively. We report a rare case with small cell lung carcinoma developing bilateral neuroretinitis and unilateral focal outer retinitis positive for these antibodies.Case presentationA 67-year-old man developed bilateral neuroretinitis and foveal exudation in the right eye. Optical coherence tomography showed a dome-shaped hyperreflective lesion extending from inner nuclear layer to the photoreceptor layer at the fovea in the right eye. Single-flash electroretinography showed normal a-waves in both eyes and slightly reduced b-wave in the left eye. Results of serological screening tests for infection were within normal limits. The patient’s optic disc swelling and macular exudation rapidly improved after oral administration of prednisolone. Systemic screening detected lung small cell carcinoma and systemic chemotherapy was initiated. Immunoblot analyses using the patient’s serum detected autoantibodies against recoverin, CRMP-5, and α-enolase, but not carbonic anhydrase II. Neuroretinitis once resolved after almost remission of carcinoma on imaging but it recurred following the recurrence of carcinoma.ConclusionsThe development of neuroretinitis in this cancer patient with anti-retinal and anti-optic nerve antibodies depended largely on the cancer activity, suggesting the possible involvement of paraneoplastic mechanisms. Patients with paraneoplastic optic neuropathy and retinopathy are likely to develop autoimmune responses against several antigens, thus leading to various ophthalmic involvements.

Collapsin Response Mediator Protein 5 (CRMP5) Induces Mitophagy, Thereby Regulating Mitochondrion Numbers in Dendrites

Degradation of damaged mitochondria by mitophagy is an essential process to ensure cell homeostasis. Because neurons, which have a high energy demand, are particularly dependent on the mitochondrial dynamics, mitophagy represents a key mechanism to ensure correct neuronal function. Collapsin response mediator proteins 5 (CRMP5) belongs to a family of cytosolic proteins involved in axon guidance and neurite outgrowth signaling during neural development. CRMP5, which is highly expressed during brain development, plays an important role in the regulation of neuronal polarity by inhibiting dendrite outgrowth at early developmental stages. Here, we demonstrated that CRMP5 was present in vivo in brain mitochondria and is targeted to the inner mitochondrial membrane. The mitochondrial localization of CRMP5 induced mitophagy. CRMP5 overexpression triggered a drastic change in mitochondrial morphology, increased the number of lysosomes and double membrane vesicles termed autophagosomes, and enhanced the occurrence of microtubule-associated protein 1 light chain 3 (LC3) at the mitochondrial level. Moreover, the lipidated form of LC3, LC3-II, which triggers autophagy by insertion into autophagosomes, enhanced mitophagy initiation. Lysosomal marker translocates at the mitochondrial level, suggesting autophagosome-lysosome fusion, and induced the reduction of mitochondrial content via lysosomal degradation. We show that during early developmental stages the strong expression of endogenous CRMP5, which inhibits dendrite growth, correlated with a decrease of mitochondrial content. In contrast, the knockdown or a decrease of CRMP5 expression at later stages enhanced mitochondrion numbers in cultured neurons, suggesting that CRMP5 modulated these numbers. Our study elucidates a novel regulatory mechanism that utilizes CRMP5-induced mitophagy to orchestrate proper dendrite outgrowth and neuronal function.

Identification of a Dithiol-disulfide Switch in Collapsin Response Mediator Protein 2 (CRMP2) That Is Toggled in a Model of Neuronal Differentiation

Vertebrate-specific glutaredoxin 2 (Grx2) is expressed in at least two isoforms, mitochondrial Grx2a and cytosolic Grx2c. We have previously shown that cytosolic Grx2 is essential for embryonic development of the brain. In particular, we identified collapsin response mediator protein 2 (CRMP2/DPYSL2), a mediator of the semaphorin-plexin signaling pathway, as redox-regulated target of Grx2c and demonstrated that this regulation is required for normal axonal outgrowth. In this study, we demonstrate the molecular mechanism of this regulation, a specific and reversible intermolecular Cys-504-Cys-504 dithiol-disulfide switch in homotetrameric CRMP2. This switch determines two conformations of the quaternary CRMP2 complex that controls axonal outgrowth and thus neuronal development.

Isoaspartate Accumulation in Mouse Brain Is Associated with Altered Patterns of Protein Phosphorylation and Acetylation, Some of Which Are Highly Sex-Dependent

Isoaspartate (isoAsp) formation is a major source of protein damage that is kept in check by the repair function of protein L-isoaspartyl methyltransferase (PIMT). Mice deficient in PIMT accumulate isoAsp-containing proteins, resulting in cognitive deficits, abnormal neuronal physiology and cytoarchitecture, and fatal epileptic seizures 30–60 days after birth. Synapsins I and II, dynamin-1, collapsin response mediator protein 2 (CRMP2), and α/β-tubulin are major targets of PIMT in brain. To investigate links between isoAsp accumulation and the neurological phenotype of the KO mice, we used Western blotting to compare patterns of in vivo phosphorylation or acetylation of the major PIMT targets listed above. Phosphorylations of synapsins I and II at Ser-9 were increased in female KO vs. WT mice, and acetylation of tubulin at Lys-40 was decreased in male KO vs. WT mice. Average levels of dynamin-1 phosphorylation at Ser-778 and Ser-795 were higher in male KO vs. WT mice, but the statistical significance (P>0.1) was low. No changes in phosphorylation were found in synapsins I and II at Ser-603, in CRMP2 at Ser-522 or Thr-514, in DARPP-32 at Thr-34, or in PDK1 at Ser-241. General levels of phosphorylation assessed with Pro-Q Diamond stain, or an anti-phosphotyrosine antibody, appeared similar in the WT and KO mice. We conclude that isoAsp accumulation is associated with altered functional status of several neuronal proteins that are highly susceptible to this type of damage. We also uncovered unexpected differences in how male and female mice respond to isoAsp accumulation in the brain.

Sustained relief of neuropathic pain by AAV-targeted expression of CBD3 peptide in rat dorsal root ganglion

The Ca2+ channel-binding domain 3 (CBD3) peptide, derived from the collapsin response mediator protein 2 (CRMP-2), is a recently discovered voltage-gated Ca2+ channel (VGCC) blocker with a preference for CaV2.2. Rodent administration of CBD3 conjugated to cell penetrating motif TAT (TAT-CBD3) has been shown to reduce pain behavior in inflammatory and neuropathic pain models. However, TAT-CBD3 analgesia has limitations, including short half-life, lack of cellular specificity and undesired potential off-site effects. We hypothesized that these issues could be addressed by expressing CBD3 encoded by high-expression vectors in primary sensory neurons. We constructed an adeno-associated viral (AAV) vector expressing recombinant fluorescent CBD3 peptide and injected it into lumbar dorsal root ganglia (DRGs) of rats before spared nerve injury (SNI). We show that selective expression of enhanced green fluorescent protein (EGFP)-CBD3 in lumbar 4 (L4) and L5 DRG neurons and their axonal projections results in effective attenuation of nerve injury-induced neuropathic pain in the SNI model. We conclude that AAV-encoded CBD3 delivered to peripheral sensory neurons through DRG injection may be a valuable approach for exploring the role of presynaptic VGCCs and long-term modulation of neurotransmission, and may also be considered for development as a gene therapy strategy to treat chronic neuropathic pain.

Heme Oxygenase 1-Mediated Neurogenesis Is Enhanced by Ginkgo biloba (EGb 761®) After Permanent Ischemic Stroke in Mice

Stroke is the fourth leading cause of death and a major cause of disability in stroke survivors. Studies have underlined the importance of repair mechanisms in the recovery phase of stroke. Neurogenesis in response to brain injury is one of the regeneration processes that, if enhanced, may offer better stroke treatment alternatives. Previously, we have demonstrated antioxidant, neuritogenic, and angiogenic properties of Ginkgo biloba/EGb 761® (EGb 761) in different mouse models of stroke. In the present study, we were interested to study whether EGb 761 could protect mice from permanent middle cerebral artery occlusion (pMCAO) and enhance neurogenesis. EGb 761 pre- and posttreated mice had lower infarct volume and improved motor skills with enhanced proliferation of neuronal stem/progenitor cells (NSPCs) at 24 h and 7 days posttreatment. Netrin-1 and its receptors (DCC and UNC5B) that mediate axonal attraction and repulsion were observed to be overexpressed in NSPCs only, implying that netrin-1 and its receptors might have partly played a role in enhanced neurogenesis. Interestingly, in heme oxygenase 1 knockout mice (HO1(-/-)), neurogenesis was significantly lower than in vehicle-treated mice at day 8. Furthermore, EGb 761 posttreated mice also demonstrated heme oxygenase 1 (HO1)-activated pathway of phosphorylated glycogen synthase kinase 3 α/β (p-GSK-3 α/β), collapsin response mediator protein 2 (CRMP-2), semaphorin3A (SEMA3A), and Wnt, suggesting probable signaling pathways involved in proliferation, differentiation, and migration of NSPCs. Together, these results propose that EGb 761 not only has antioxidant, neuritogenic, and angiogenic properties, but can also augment the repair and regeneration mechanisms following stroke.

Amelioration of β-amyloid-induced cognitive dysfunction and hippocampal axon degeneration by curcumin is associated with suppression of CRMP-2 hyperphosphorylation

The Alzheimer's disease (AD) brain is characterized by β-amyloid deposition, hyperphosphorylation of microtubule-associated proteins, formation of senile plaques and neurofibrillary tangles, and degeneration of specific neuronal populations. Collapsin response mediator protein 2 (CRMP-2) hyperphosphorylation has been implicated in AD-associated neural process regression and neurofibrillary tangle formation. Curcumin is a promising AD drug with incompletely defined therapeutic mechanisms. One possibility is that curcumin prevents β-amyloid-induced CRMP-2 hyperphosphorylation, thereby protecting against axonal regression and (or) promoting axonal regrowth. We examined spatial learning in the Morris water maze, hippocampal expression levels of CRMP-2 and phosphorylated CRMP-2 (p-CRMP-2) by Western blot, and NF-200 (an axon-specific marker) by immunohistochemistry in Sprague-Dawley rats subjected to a single intrahippocampal injection of Aβ1–40 alone or Aβ1–40 followed by curcumin (i.p. daily for 7 days). Compared to controls, spatial learning was significantly impaired in these Aβ1–40-injected AD model rats (P<0.05). In addition, hippocampal expression levels of CRMP-2 and NF-200 were reduced while p-CRMP-2 expression was significantly enhanced (P<0.05 for all). Overexpression of p-CRMP-2 was correlated with NF-200 underexpression (r2=−0.67308, P<0.05), suggesting that Aβ1–40 damaged hippocampal axons. Spatial learning deficits were reversed, CRMP-2 and NF-200 expression levels increased, and p-CRMP-2 expression reduced in curcumin-treated rats (all P<0.05). We propose that curcumin improves spatial learning by inhibiting CRMP-2 hyperphosphorylation, thus protecting against β-amyloid-induced hippocampal damage or promoting regeneration.

Orthostatic hypotension secondary to CRMP-5 paraneoplastic autonomic neuropathy

The collapsin response mediator protein 5 (CRMP-5) autoantibody is one of only several paraneoplastic antibodies associated with autonomic neuropathy. Such paraneoplastic neuropathies manifest with a constellation of autonomic abnormalities. We present a unique case of orthostatic hypotension as the sole feature of a CRMP-5 paraneoplastic autonomic neuropathy in a patient with small cell lung cancer. Given the poor prognosis of paraneoplastic autonomic dysfunction, it is important to accurately diagnose the cause of orthostatic hypotension occurring on a background of malignancy.

Phosphorylation and mRNA Splicing of Collapsin Response Mediator Protein-2 Determine Inhibition of Rho-associated Protein Kinase (ROCK) II Function in Carcinoma Cell Migration and Invasion

The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II interaction is controlled, we further mapped the ROCK II interaction site of CRMP-2 and examined whether phosphorylation states of CRMP-2 affected the interaction. Here, we show that an N-terminal fragment of the long CRMP-2 splice variant (CRMP-2L) alone binds ROCK II and inhibits colon carcinoma cell migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition of PI3K also reduced colocalization of ROCK II and CRMP-2 at the cell periphery in human breast carcinoma cells. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant full-length and catalytic domain of ROCK II. These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2.

A Derivative of the Brain Metabolite Lanthionine Ketimine Improves Cognition and Diminishes Pathology in the 3×Tg-AD Mouse Model of Alzheimer Disease

Lanthionine ketimine ([LK] 3,4-dihydro-2H-1,4-thiazine-3,5-dicarboxylic acid) is the archetype for a family of naturally occurring brain sulfur amino acid metabolites, the physiologic function of which is unknown. Lanthionine ketimine and its synthetic derivatives have recently demonstrated neurotrophic, neuroprotective, and antineuroinflammatory properties in vitro through a proposed mechanism involving the microtubule-associated protein collapsin response mediator protein 2. Therefore, studies were undertaken to test the effects of a bioavailable LK ester in the 3 × Tg-AD mouse model of Alzheimer disease. Lanthionine ketimine ester treatment substantially diminished cognitive decline and brain amyloid-β (Aβ) peptide deposition and phospho-Tau accumulation in 3 × Tg-AD mice and also reduced the density of Iba1-positive microglia. Furthermore, LK ester treatment altered collapsin response mediator protein 2 phosphorylation. These findings suggest that LK may not be a metabolic waste but rather a purposeful neurochemical, the synthetic derivatives of which constitute a new class of experimental therapeutics for Alzheimer disease and related entities.

Detection of autoantibodies against heat shock proteins and collapsin response mediator proteins in autoimmune retinopathy

BackgroundAutoimmune retinopathy (AR) and Cancer-Associated Retinopathy (CAR) are associated with a diverse repertoire of anti-retinal autoantibodies (AAbs) but not all antigenic targets have been characterized. Identification of new AAbs may help with clinical diagnosis and prognosis of retinal dysfunction in AR. The goal was to identify frequently targeted retinal autoantigens within the 60-70-kDa molecular weight range.MethodsHuman retinal proteins were separated by SDS-PAGE and 2D gel electrophoresis (2-DE) and sera from AR patients with and without cancer were used to identify immunoreactive proteins by Western blotting. Proteins were identified following separation by electrophoresis, Coomassie staining using in-gel trypsin digestion and mass spectrometric analysis. Circulating serum hsp60 and anti-hsp60 antibody levels were determined by quantitative ELISA.ResultsRetrospective evaluation of 819 patients with anti-retinal AAbs showed that 29% patients had AAbs targeted proteins between 60-70-kDa. Shotgun mass spectrometry of human retinal proteins present in 1D-gel found 66 species within this range. To identify the immunoreactive proteins, we performed Western blots of 2-DE gels and showed a group of heat shock proteins (hsps), including hsp60 and CRMP proteins that were frequently recognized by AR patient AAbs, irrespective of cancer status. These results were validated by immunostaining of purified hsp60 and CRMP2 proteins. ELISA results revealed that patients with AR and CAR had significantly increased levels of serum anti-hsp60 antibodies compared to control healthy subjects (p < 0.0001). However, circulating hsp60 protein was not significantly elevated in sera of either patient group.ConclusionsDifferent anti-retinal antibodies frequently co-exist in a single patient, creating antibody-arrays related to the syndrome. Hsps and CRMP-2 are newly identified autoantigens in AR. A frequent co-association of anti-hsp antibodies with other anti-retinal AAbs may augment pathogenic processes, leading to retinal degeneration.