Role of Pur Alpha in Neuronal Cells

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CNS disorders are a major sequelae of HIV-1 infection occurring in more than 60% of HIV-1 infected individuals. Neuronal cells are particularly vulnerable and their injury is related to the development of cognitive and motor impairment seen in AIDS patients. A brain derived nucleic acid binding protein, Pura has been implicated in diverse biological events including control of the HIV-1 genome and several cellular genes whose expression are affected upon HIV-1 infection of the CNS. The association of Pur(alpha), which is highly expressed in neurons several key regulatory proteins including cyclins and cyclin dependent kinases coupled with its fluctuating levels throughout the cell cycle, position Pur(alpha) as a crucial factor in the control of cell growth and differentiation. Recent studies in our laboratory have revealed the ability of Pur(alpha) to associate with cdk5, a brain- specific isoform of cdc2, which upon interaction with the neuron- specific p35 protein gains catalytic activity in differentiated neurons. A complex of protein containing cdk5 and p35 with kinase activity is essential for neurite outgrowth during neuronal differentiation. Evidently, the cdk5:p35 complex phosphorylates several axonal cytoskeletal proteins including neurofilaments , tau, and the beta- amyloid precursor protein, and plays an important role in the regulation of N-cadherin-mediated cell adhesion during cortical development. Mice with targeted disruptions of either cdk5 or p35 genes exhibit abnormalities in the laminar structure of the cerebral cortex and contain neurofilaments which are hypophoshorylated at their critical C-terminal domains. These mutant mice show neurological defects including spontaneous seizures. Our effort to determine the biological role of Pur(alpha) in mice through gene knockout has yielded a surprising discovery as mice with no (Pur(alpha) -/-) or reduced (Pur(alpha) -/+) level of Pur(alpha) demonstrated similar abnormalities in the cortical lamination, hypophosphorylation of neurofilaments, and spontaneous seizures as the cdk5, p35 mice. In light of earlier data showing the ability of Pur(alpha) to associate with the cdk5 complex, we hypothesize that Pur(alpha) may play a critical role in regulating the activity of cdk5 and its associated factor(s) in neurons and thus may be important for neuronal cell development and the formation of cortical layers. To examine this hypothesis, we will: (i) examine the developmental expression and association of Pur(alpha) with the cdk5 complex, and the ability of Pur(alpha) to modulate cdk5 kinase activity during brain development; (ii) utilize biochemical assays t o map the region within Pur(alpha) which is important for cdk5 binding and its kinase activity, and examine the function of Pur(alpha) mutants in neuronal cell differentiation in a cell culture system; (iii) examine the importance of Pur(alpha) in neuronal cell differentiation and the development of stratification of cortical neurons at various stages of brain development by generating transgenic mice that conditionally inactivate Pur(alpha) function. The results of these studies should provide insight into molecular events involved in neuronal differentiation and further assist us in understanding the neuronal injury and neuropathology seen in several debilitating CNS disorders and NeuroAIDS.