We have demonstrated that phosphorylation of the numerous acceptor sites on such proteins as Tau and neurofilaments (NFs) were tightly regulated topographically and generally confined to the axonal compartment and selectively occurs on proline directed serine (Ser) and threonine (Thr) residues. It was recognized that in neurodegenerative disorders such as Alzheimers disease (AD) and Amyotrophic lateral sclerosis (ALS), the pathology was characterized by an accumulation of aberrantly phosphorylated cytoskeletal proteins in perikarya on these residues, suggesting that topographic regulation had been compromised. The proline directed Ser/Thr residues are stably phosphorylated in the nervous system. The mechanism of this stable phosphorylation is not understood.Recently, studies from our laboratory have shown that peptidyl prolyl isomerase 1 (Pin1) stabilizes the neurofilament phosphorylation in normal and stressed neurons. Pin1 selectively binds to the phosphorylated Ser/Thr-Pro residues and converts the cis isomers to the more stable trans isomers. The multiple SP repeats of NF-M/H are stabilized by Pin1 in a phosphorylation specific manner. Pin1 modulates the excitotoxic and oxidative stress induced perikaryal phosphorylation of NF-M/H. The other factors regulating the topographic phosphorylation of neuronal cytoskeletal proteins are phosphatases. We found that protein phosphatase 2A (PP2A) expression is high in neuronal cell bodies and inhibition of PP2A activity by okadaic acid (OA), microcystin LR and fostriecin leads to perikaryal hyperphosphorylation of neurofilaments.Inhibition of Pin1 by Pin1 siRNA and dominant (DN) Pin1 inhibits okadaic acid (OA) induced aberrant perikaryal phosphorylation of neurofilaments. In order to determine if aberrant hyperphosphorylation of NF in OA-treated neurons in response to Pin1 inhibition is a consequence of NF kinase activation, or a direct effect of PP2A mediated dephosphorylation of NF, we tested the NF phosphorylation with kinase inhibitiors (JNK, ERK and cdk5) prior to the addition of PP2A inhibitor. Non-treated and kinase inhibitor treatment of cortical neurons with JNK inhibitor (SP600125),ERK inhibitor (PD98059) or Cdk5 inhibitor, roscovitine (OA) prior to the addition of OA did not change the levels of phospho-NF accumulation, suggesting that the aberrant phosphorylation of NF mediated by PP2A is not due to the activation of JNK/ERK/Cdk5 kinase pathways. Thus, Pin1 directly modulates PP2A mediated hyperphosphorylation of NF. Phosphorylation has long been considered to regulate neurofilament (NF) interaction and axonal transport, and, in turn, influence axonal stability and their maturation of axons. We transfected GFP-NF-H in primary cortical neurons and monitored transport of GFP-NF-H by quantifying the total levels of GFP-NF-H in distal versus the proximal axons, as well as by real-time monitoring the movement of GFP-tagged structures, before and after treatment with OA. Transfected GFP NF-H is well translocated in to the axonal compartment in normal neurons. When cortical neurons are treated with OA or fostriecin (Fos) for 2 h, NF translocation is disrupted as observed by the GFP-NF-H in the cell bodies and proximal axons only. The NF translocation is disrupted in OA/Fos treated neurons due to the hyperphosphorylation of NF in the cell bodies. Knockdown of Pin1 rescues the NF axonal translocation in OA/Fos treated neurons. These results indicate a possible role of Pin1 in NF dynamics. This study highlights a novel signaling role of PP2A by Pin1 and implicates Pin1 as a therapeutic target to reduce aberrant phosphorylation of neurofilament proteins in neurodegenerative disorders such as AD, PD and ALS.
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