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  • Topographic Phosphorylation of Neuronal Cytoskeleton is Regulated by Kinases and Phosphatases

    Final Number:

    Jared S Rosenblum MD(1)(3); Brandon C Rosenblum(1)(2); Harish C Pant PhD(1)(2)

    Study Design:
    Laboratory Investigation

    Subject Category:

    Meeting: Congress of Neurological Surgeons 2017 Annual Meeting - Late Breaking Science

    Introduction: Cyclin dependent kinase-5 (Cdk5) is a critical post-mitotically active kinase restricted to neurons by its activator protein, p35(1). Topographic regulation of phosphorylation of neuronal cytoskeletal elements, axonal transport, neurite outgrowth, and cell survival by Cdk5 has been demonstrated(1, 2, 3). Aberrant hyperactivity due to neuronal insult has been shown to lead to pathologic states(4). P5, a modified truncated 24-aa peptide, derived from the Cdk5 activator p35, has been shown to penetrate the blood-brain barrier and selectively inhibit abnormal Cdk5 hyperactivity, significantly rescuing Alzheimer’s Disease (AD) pathology (up to 70–80%) in model mice(5). It has also been shown that Cdk5 plays a central role in glioblastoma/glioma tumorigenesis(6, 7). Utilizing P5, we aimed to demonstrate a balanced topographic regulation of neuronal cytoskeleton by kinases and phosphatases known to cross-talk(8) that would explain the observed compartmentalization of neuronal cytoskeletal phosphorylation.

    Methods: Homogenization and differential centrifugation of extruded axoplasm and giant fibrous lobe (GFL) cell body compartments cleanly separated in loligo pealei model system allowed evaluation of endogenous phosphatase and kinase for comparative study of the effect of concentration variation of P5 on activity of protein phosphatase 2A (PP2A). A colorimetric molybdate-based free-phosphate detection system was employed to evaluate the effect of variation in P5 concentration on phosphatase activity in the axon and cell body compartments.

    Results: The GFL compartment demonstrated elevated baseline PP2A activity compared to the axonal compartment. Dose-dependent inhibition of PP2A activity was seen in both compartments with higher concentrations of P5 showing two-fold inhibition in the GFL compartment compared to the axonal compartment.

    Conclusions: P5 peptide is known to specifically inhibit hyperactivated Cdk5 in situ, which is critical to development of neuropathology. The preliminary study presented shows that P5 also inhibits phosphatase activity at higher concentrations. This elucidates balanced topographic regulation of the neuronal cytoskeleton in compartment specific fashion.

    Patient Care: Study of the P5 peptide in axonal and cell body neuronal compartments further elucidates the pathways involved in neuropathology and may prove a point of intervention to regulate downstream effectors.

    Learning Objectives: By conclusion of this session, participants should be able to: 1) Understand the dynamic nature of phosphorylation in topographic regulation of the cell body and axon 2) Appreciate the importance of the loligo pealei model system in evaluation of cell body and axonal compartments in neuropathology which is not cleanly afforded by mammalian systems. 3) Understand the role of Cdk5 in neuropathology, both neurodegeneration and tumorigenesis, and the inhibitory effect of the P5 peptide on aberrant hyperactivity of Cdk5 in situ 4) Understand the homeostatic topographic regulation of neuronal cytoskeletal elements by kinases and phosphatases in compartment specific fashion and the relationship of this to physiologic regulation and neuropathologic development. 5) Identify the P5 peptide as a specific means of further study of this pathway in glioblastoma/glioma

    References: 1. Kesavapany, S., Li, B.S., Amin, N., Zheng, Y., Grant, P., Pant, H.C. (2004). Neuronal cyclin-dependent kinase 5 : role in nervous system function an its specific inhibition by the Cdk5 inhibitory peptide. Biochimica et Biophysica Acta 1697, 143-153 ? 2. Rosenbum, JS. Cdk5 acts as a surveillance system in the nervous system. In: Yamaguchi M, editor. New developments in signal transduction research. Nova Science Publishers ; 2013 3. Shea, B. T., Zheng, Y., Ortiz, D., Pant, H. C. (2004). Cyclin-Dependent Kinase 5 Increases Perikaryal Neurofilament Phosphorylation and Inhibits Neurofilament Axonal Transport in Response to Oxidative Stress. J. Neurosci. Res. 76, 795-800. ? 4. Cruz, J. C., Tseng, H. C., Goldman, J. A., Shih, H., Tsai, L. H. (2003). Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles. Neuron 40: 471–483. 5. Shukla, V., Zheng, Y. L., Mishra, S. K., Amin, N. D., Steiner, J., Grant, P., Kesavapany, S., Pant, H. C. (2013). A truncated peptide from p35, a Cdk5 activator, prevents Alzheimer's disease phenotypes in model mice. FASEB J. 27(1): 174–186. 6. Yushan, R., Wenjie, C., Suning, H., Yiwu, D., Tengfei, Z., Madushi, W. M., Feifei, L., Changwen, Z., Xin, W., Roodrajeetsing, G., Zuyun, L., Gang, C. (2015). Insights into the clinical value of cyclin-dependent kinase 5 in glioma : a retrospective study. World J Surg Oncol. 13: 223. 7. Xie, Q., Wu, Q., Horbinski, C. M., Flavahan, W. A., Yang, K., Zhou, W., Dombrowski, S. M., Huang, Z., Fang, X., Shi, Y., Ferguson, A. N., Kashatus, D. F., Bao, S., Rich, J. N. (2015). Mitochondrial control by DRP1 in brain tumor initiating cells. Nature Neuroscience. 18 : 501-510 8. Lu, P. J., Wulf, G., Zhou, X. Z., Davies, P., and Lu, K. P. (1999) The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein. Nature 399, 784 –788

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