In previous studies, stimulation of ionotropic AMPA/kainate glutamate receptors on cultured oligodendrocyte cells induced the formation of a signaling complex that includes the AMPA receptor, integrins, calcium-binding proteins, and, surprisingly, the myelin proteolipid protein (PLP). AMPA stimulation of cultured oligodendrocyte progenitor cells (OPCs) also caused an increase in OPC migration. The current studies focused primarily on the formation of the PLP-␣v integrin-AMPA receptor complex in vivo and whether complex formation impacts OPC migration in the brain. We found that in wild-type cerebellum, PLP associates with ␣v integrin and the calciumimpermeable GluR2 subunit of the AMPA receptor, but in mice lacking PLP, ␣v integrin did not associate with GluR2. Live imaging studies of OPC migration in ex vivo cerebellar slices demonstrated altered OPC migratory responses to neurotransmitter stimulation in the absence of PLP and GluR2 or when ␣v integrin levels were reduced. Chemotaxis assays of purified OPCs revealed that AMPA stimulation was neither attractive nor repulsive but clearly increased the migration rate of wild-type but not PLP null OPCs. AMPA receptor stimulation of wild-type OPCs caused decreased cell-surface expression of the GluR2 AMPA receptor subunit and increased intracellular Ca 2ϩ signaling, whereas PLP null OPCs did not reduce GluR2 at the cell surface or increase Ca 2ϩ signaling in response to AMPA treatment. Together, these studies demonstrate that PLP is critical for OPC responses to glutamate signaling and has important implications for OPC responses when levels of glutamate are high in the extracellular space, such as following demyelination.

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Plp1 gene expression occurs very early in development, well before the onset of myelination, creating a conundrum with regard to the function of myelin proteolipid protein (PLP), one of the major proteins in compact myelin. Using PLP-EGFP mice to investigate Plp1 promoter activity, we found that, at very early time points, PLP-EGFP was expressed in Sox2ϩ undifferentiated precursors in the spinal cord ventricular zone (VZ), as well as in the progenitors of both neuronal and glial lineages. As development progressed, most PLP-EGFPexpressing cells gave rise to oligodendrocyte progenitor cells (OPCs). The expression of PLP-EGFP in the spinal cord was quite dynamic during development. PLP-EGFP was highly expressed as cells delaminated from the VZ. Expression was downregulated as cells moved laterally through the cord, and then robustly upregulated as OPCs differentiated into mature myelinating oligodendrocytes. The presence of PLP-EGFP expression in OPCs raises the question of its role in this migratory population. We crossed PLP-EGFP reporter mice into a Plp1-null background to investigate the role of PLP in early OPC development. In the absence of PLP, normal numbers of OPCs were generated and their distribution throughout the spinal cord was unaffected. However, the orientation and length of OPC processes during migration was abnormal in Plp1-null mice, suggesting that PLP plays a role either in the structural integrity of OPC processes or in their response to extracellular cues that orient process outgrowth.

Neurite outgrowth upon extracellular cues is an essential event in early neuronal differentiation. Rac1 and Cdc42, members of Rho family GTPases, are involved in this process as regulators of actin cytoskeletal reorganization. We previously showed that NGF/TrkA signaling drives the cycling of a positive feedback loop comprised of PI3-kinase, Vav2/Vav3, Rac1/Cdc42, and actin cytoskeleton at neurite tips of PC12 cells using FRET imaging and RNAi techniques. In this study, we demonstrate that SHIP2 is a critical component of the negative feedback on PIP 3 in NGF-induced neurite outgrowth. This negative feedback loop has been unexpected previously. Acute inhibition of TrkA transiently decreased Rac1/Cdc42 activity and the PIP 3 level to below basal levels, indicating the presence of NGF-dependent negative regulation. With the help of in silico simulation, we speculated that the activation of PI-5-phosphatase for PIP 3 was involved. In agreement with this model, depletion of SHIP2 by RNA interference attenuated the inhibitor-induced super-suppression of Rac1/Cdc42 activity and the level of PIP 3 . The critical role of SHIP2 in neurite outgrowth of PC12 cells was further supported by the finding that depletion of SHIP2 and PTEN, phosphatases for PIP 3 , markedly potentiated NGF-induced Rac1/ Cdc42 activation and PIP 3 accumulation, and significantly increased the number and the length of neurites. This increase in neurite number is possibly due to that the depletion of SHIP2 and PTEN disturbs the proper localization of PIP 3 and Rac1/ Cdc42 activities. Further refinement of the computational model predicted a negative feedback from Rac1 to SHIP2, which was validated experimentally. We propose that the SHIP2-mediated negative feedback on PIP 3 coordinately works with the PI3kinase-mediated positive feedback to form an initial protrusive pattern determined by Turing's reaction-diffusion system, and later, to punctuate the PIP 3 accumulation to maintain proper neurite outgrowth.

Regeneration in the central nervous system of teleost fish is well documented. A study using epifluorescence microscopy was conducted to correlate regeneration stages of severed axons of the optic nerve in adult zebrafish (Danio rerio) with analysis of changes in gene expression in the retina. Optic nerve injury was performed on wild‐type zebrafish which were then sacrificed at one day intervals for 10 days. DiI neurotrace paste was injected into the eye 24 hours prior to sacrifice. Fish were whole mounted leaving the retina, optic nerve and brain in situ. Regenerating axons were visualized by epifluorescence microscopy. Preliminary results indicate that after 3 days axons have crossed the lesion site and by 7 days have reached the optic tectum of the brain. This work supported by NSF IOB‐0615762.