| The PI3-kinase (PI3-K) pathway is critical for peripheral myelination, but its effectors in this context are largely unknown. To get at this issue, we screened myelinating sciatic nerve with an antibody specific for a substrate recognition motif common to a few PI3-K effectors like Akt and Sgk. N-myc downstream regulated gene1 (NDRG1) and Akt Substrate of 160 kDa (AS160) are two of several substrates we identified. Although AS160 has not yet been described in peripheral nerve, we focused on NDRG1, a Schwann cell-specific protein of unknown function, because its mutation causes a human demyelinating disease (CMT-4D). Immunofluoresence and immuno-electron microscopy data indicate that phospho-NDRG1 localizes to the abaxonal compartment of myelinating Schwann cells. Addition of laminin, a known abaxonal matrix signal, is sufficient to induce NDRG1 phosphorylation in Schwann cell-neuron co-cultures in a PI3-K dependent manner. Using in vivo loss-of-function models, we find that the alpha2-laminin signal is transduced to NDRG1 by the beta1 and beta4 integrin subunits and dystroglycan. These results show that laminin indeed drives NDRG1 phosphorylation, suggesting that NDRG1 is principally downstream of abaxonal PI3-K activity. In Neuregulin-1 type III haploinsufficient nerve, a model for defective axon-dependent PI3-K activation, NDRG1 phosphorylation is unchanged, supporting the notion that PI3-K activity does not propagate freely throughout the myelinating Schwann cell. To investigate where PI3-K is acting, we localized its catalytic subunit and phospho-Akt, a readout for its activity, in sciatic nerve. We find that PI3-K is enriched at the glial node and paranode and phospho-Akt is specific to the glial paranode. These data show that the locus of PI3-K activity is spatially restricted and that PI3-K signaling from the axon and matrix are compartmentalized during peripheral myelination. |
