Phosphorylation Of Neuronal Cytoskeleton

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Phosphorylation process plays an important role in the structural organization of neuronal cytoskeleton. Synthesis, transport and assembly of neurofilament (NF) proteins are developmentally and spatially regulated by specific kinases that extensively phosphorylate different motifs such as Lys-Ser-Pro (KSP) repeats in the carboxyl-terminal tail domain of NF- M and NF-H. This phosphorylation stabilizes the NF network in the axon, and to affect the axonal transport and conduction velocity in the neurons. Cyclin dependent kinase-5 (Cdk5) is believed to phosphorylate KSP motifs in NF and tau protein. The later phosphorylation occurs exclusively at the same sites found in the tau protein from Alzheimer?s disease brain. Abnormal NF phosphorylation has also been associated with neuro-degenerative diseases. We have previously reported the generation and characterization of Cdk5 null mice. In order to elucidate the function of Cdk5 in vivo, we have examined neuro-developmental deficiencies that arise as a consequence of disruption of the Cdk5 gene in these null mice. These mice exhibit a novel phenotype with perinatal lethality. The cerebral cortical layering pattern of the Cdk5 null mice is inverted with respect to the birthdate of neurons, and the trajectories of the thalamocortical axons were disordered. Evidently it is the intermediate stage of cortical neuronal migration that are affected. There is an abnormal stratification in the hippocampus, and the cerebellum lacks typical foliation. The brain stem and the spinal cord display ballooning change of the neurons. Aberrant phosphorylation of cytoskeletal elements occurs in neuronal cell bodies of Cdk5 null mice in the spinal cord and the brain stem. Studies with Cdk5 null embryonic stem (ES) cell derived chimeras indicated a cell autonomous effect of Cdk5 on neuronal migration in the cerebellum. Interestingly, p35 null mice also have an inverted cerebral cortical layering pattern and aberrant axonal trajection, but these mice are viable. The cerebellum of the p35 null mice shows subtle phenotypic changes, but the brain stem, spinal cord and peripheral nervous system are unaffected. We also carried out studies to determine whether targeted reconstitution of Cdk5 expression in the nervous system of Cdk5 null mice leads to reversal of the phenotype and perinatal mortality. We also determined the functional significance of Cdk5 in the brain and peripheral organs using a tissue specific expression strategy. Towards these goals, we generated and characterized the transgenic mice overexpressing Cdk5 driven by p35 Promoter (TgCdk5 mice). These mice were crossed with Cdk5 heterozygous mice to obtain mice that only express Cdk5 in the nervous tissue (TgKO). The TgKO mice were born normal and did not exhibit any developmental abnormality. The restoration of Cdk5 expression reversed the aberrant phosphorylation of cytoskeletal elements in the soma of brain stem neurons. This investigation confirms that Cdk5 expression in the nervous system is critical for embryonic development and survival. In order to delineate the role of Cdk5 in neurodegenerative disorders and specially in motor neurons, we have generated the Cdk5 conditional knockouts in which Cdk5 expression is abrogated in the adult brain. For this purpose we utilized loxP system to excise Cdk5 gene postnatally by the targeted expression of Cre recombinase enzyme. These mice exhibit abnormal postures and neuromuscular coordination. Our initial analysis indicates accumulation of NF-H in motor neurons of spinal cord in these mice associated with lack ChAc exoression. We are still carrying out detailed analysis of this phenotype. Members of the N-methyl-D-aspartate (NMDA) class of glutamate receptors are critical for development, synaptic transmission, learning and memory; they are targets of pathological disorders in the central nervous system. NMDA receptors are phosphorylated by both serine/threonine and tyrosine kinases . Here, we demonstrate that cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at serine1295 in vitro and in intact cells. Moreover, we show that roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation (LTP) induction and NMDA-evoked currents in rat CA1 hippocampal neurons. These results suggest that Cdk5 plays a key role in synaptic transmission and plasticity through its up-regulation of NMDA receptors.