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Amyotrophic lateral sclerosis (ALS) is an adult-onset neurological disease characterized by the selective degeneration of motor neurons leading to paralysis and immobility. Missense mutations in the gene coding for the Cu2+/Zn2+ superoxide dismutase 1 (SOD1) accounts for 15-20% of familial ALS, and mice overexpressing ALS-linked SOD1 mutants have been frequently used as an animal model for ALS. Degeneration of motor neurons in ALS progresses in a manner called "dying back", in which the degeneration of synapses and axons precedes the loss of cell bodies. Phosphorylation of collapsin response mediator protein 2 (CRMP2) is implicated in the progression of neuronal/axonal degeneration of different etiologies. To evaluate the role of CRMP2 phosphorylation in ALS pathogenesis, we utilized CRMP2 S522A knock-in (CRMP2ki/ki) mice, in which the serine residue 522 was homozygously replaced with alanine and thereby making CRMP2 no longer phosphorylatable by CDK5 or GSK3B. We found that the CRMP2ki/ki/SOD1G93A mice showed delay in the progression of the motor phenotype compared to their SOD1G93-Tg littermates. Histological analysis revealed that the CRMP2ki/ki/SOD1G93A mice retained more intact axons and NMJs than their SOD1G93A-Tg littermates. These results suggest that the phosphorylation of CRMP2 may contribute to the axonal degeneration of motor neurons in ALS.

Pyridoxal, an active form of vitamin B6, is known to inhibit formation of advanced glycation end-products and protect tissues from diabetic complications. Here we identified that pyridoxal is a required component for establishing Schwann cell myelination in our Schwann cell-dorsal root ganglion neuron co-culture system. When the co-culture was maintained without pyridoxal, carboxymethylation of collapsin response mediator protein 2 (CRMP2) became detectable. Carboxymethylation decreased the affinity of CRMP2 to bind with microtubules, indicating that carboxymethylation affected CRMP2 function. These results suggest that carboxymethylation of CRMP2 may be an indicator of dysfunction caused by glycation which is observed in pathological conditions, including diabetic neuropathy.

Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) protein and tau deposition in the brain. Numerous studies have reported a central role of Aβ in the development of AD, but the pathogenesis is not well understood. Collapsin response mediator protein 2 (CRMP2), an intracellular protein mediating a repulsive axon guidance molecule, Semaphorin3A, is also accumulated in neurofibrillary tangles in AD brains. To gain insight into the role of CRMP2 phosphorylation in AD pathogenesis, we investigated the effects of Aβ neurotoxicity in CRMP2 phosphorylation-deficient knock-in (crmp2(ki/ki)) mice, in which the serine residue at 522 was replaced with alanine. Intracerebroventricular (i.c.v.) injection of Aβ₂₅₋₃₅ peptide, a neurotoxic fragment of Aβ protein, to wild-type (wt) mice increased hippocampal phosphorylation of CRMP2. Behavioral assessment revealed that i.c.v. injection of Aβ₂₅₋₃₅ peptide caused impairment of novel object recognition in wt mice, while the same peptide did not in crmp2(ki/ki) mice. In electrophysiological recording, wt and crmp2(ki/ki) mice have similar input-output basal synaptic transmission and paired-pulse ratios. However, long-term potentiation was impaired in hippocampal slices of Aβ₂₅₋₃₅ peptide-treated wt but not those of crmp2(ki/ki). Our findings indicate that CRMP2 phosphorylation is required for Aβ-induced impairment of cognitive memory and synaptic plasticity.

Spinal cord injury (SCI) is a serious condition often affecting young and healthy individuals around the world. Electro-acupuncture (EA) has been proven to contribute towards neurologic and functional recoveries in SCI, but the underlying mechanism remains largely unknown especially regarding neural specific proteins involved in the development of EA. The protein expression profile of spinal cord in both SCI and EA treatment models was analyzed by using two-dimensional electrophoresis-based proteomics. Using a MALDI-TOF/TOF MS and subsequent protein database searching, we identified changes in 15 proteins in the spinal cord following Governor Vessel (GV) EA treatment on SCI. These proteins are involved in inflammation, cell adhesion and migration, signal transduction and apoptosis processes. We selected 2 proteins (ANXA5 and CRMP2) beneficial to neuronal survival and axonal regeneration, and further identified these protein changes using Western blot analysis. Subsequently, Nissl staining and immunofluorescence double labeling approaches were used to explore possible role of the two neural specific proteins in the process of GV-EA treatment on SCI. Our results suggest that ANXA5 and CRMP2 may be neural specific proteins in the process of GV-EA treatment on SCI. This work might contribute to the better understanding of the mechanism involved in EA treatment on SCI at protein levels and provide a new therapeutic strategy for SCI.

A growing tip of neurite, the neuronal growth cone, is a highly motile and adhesive form of cytoarchitecture. The growth cone plays vital roles for navigation, elongation and maintenance of neurites. One major constituent of growth cones, regulated by the intracellular Ca2+ signal, is the actin-based cytoskeleton. In this article, I have summarized four types of Ca(2+)-dependent regulation of the actin-based cytoskeleton in growth cones: gelsolin-actin, myosin II-actin microfilament, myosin II-actin, and Ca(2+)-sensitive alpha-actinin-actin systems. The four examples of Ca(2+)-dependent regulation described here may be involved in growth cone motility. The actin-based membrane skeleton forming a meshwork beneath the cytoplasmic surface of the growth cone membrane is also important for adhesion of growth cones to recognize cue molecules. The actin-based membrane skeleton participates in this recognition process and the adhesion-dependent signal transduction in association with receptors for cell adhesion molecules.

The growth cone plays crucial roles in neural wiring, synapse formation, and axonal regeneration. Continuous rearrangement of cytoskeletal elements and targeting of transported vesicles to the plasma membrane are essential to growth cone motility; however, the proteins directly involved in these processes and their specific functions are not well established. We recently identified 17 proteins as functional marker proteins of the mammalian growth cone and as neuronal growth-associated proteins in rat cortical neurons (nGAPs; Nozumi et al., 2009). To determine whether these 17 proteins are growth cone markers in other neuronal cell types, we examined their expression and function in PC12D cells. We found that all 17 nGAPs were highly concentrated in the growth cones of PC12D cells, and that knockdown of all of them by RNAi reduced or inhibited neurite outgrowth, indicating that all of the 17 nGAPs may be general growth cone markers. Among them, eight proteins were shown to regulate the amount of F-actin in PC12D growth cones. Two of these nGAP that are cytoskeletal proteins, Cap1 and Sept2, increased the mean growth cone area and the mean neurite length by regulating the amount of F-actin; Sept2 also induced filopodial growth. Taken together, our data suggested that some of the nGAPs were generalized markers of the growth cone in multiple neuronal cell types and some of them, such as Cap1 and Sept2, regulated growth cone morphology through rearrangement of F-actin and thereby controlled neurite outgrowth.