Poster Sessions

Oxi-2

Parvathi Rudrabhatla

P. Rudrabhatla, Y. L. Zheng, A. Niranjana, S. Kesavapany, W. Albers, H. Pant

Pin1 dependent prolyl isomerization modulates oxidative stress induced phosphorylation of NF-H. An implication in neurodegenerative disorders

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS), but the underlying mechanisms are unclear. In normal neurons, the KSP sites in high molecular weight neurofilament protein (NF-H) are phosphorylated within axons. However, in case of AD and ALS, phosphorylated NF-H are found as accumulations in neuronal cell bodies. The precise mechanism for this compartment-specific phosphorylation of neurofilaments is not completely understood. One possibility involves Pin1 modulation of phosphorylation of the proline-directed serine/threonine residues. Pin1, a peptidyl prolyl isomerase, selectively binds to phosphorylated proline-directed serine/threonine (S/T-P) residues in target proteins and isomerizes cis isomers to more stable trans configurations. Rat NF-H has 52 KSP repeats in the tail domain and almost all of them are phosphorylated in vivo. Here, we show that Pin1 modulates oxidative stress-induced phosphorylation of NF-H. In vitro, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline directed kinases, Erk1/2 and Cdk5 in a concentration-dependent manner. In vivo, stimulation of mitogen activated protein kinases (MAPK) by epidermal growth factor (EGF) in HEK293 cells transfected with NF-H substantially increased NF-H phosphorylation. Dominant-negative (DN) Pin1 and Pin1 siRNA inhibited EGF-induced NF-H phosphorylation. Endogenous Pin1 in NF-H transfected HEK293 cells co localizes with p-NF-H. Since oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed primary cortical neurons and HEK293 cells. Both hydrogen peroxide (H2O2) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 siRNA but not control siRNA rescues the effect of stress-induced NF-H phosphorylation. The H2O2 and heat shock induced perikaryal phospho-NF-H accumulations and neuronal apoptosis in primary neurons is rescued by inhibition of Pin1 by Pin1 siRNA and DN Pin1. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and ALS. Thus, Pin1 may be a potential therapeutic target for these diseases.