Increased expression of cdk5/p25 in N2a cells leads to hyperphosphorylation and impaired axonal transport of neurofilament proteins.
OBJECTIVE
Alzheimer's disease (AD) is the leading cause of dementia. The increased cdk5 expression and enhanced phosphorylation of tau and NFs have been seen in AD patients. Our study aimed at investigating the effects of increased cdk5 activity on axonal transport of neurofilaments (NFs).


METHODS
In this study, we used a molecular engineering approach to overexpress cdk5/p25 in neuroblastoma N2a cells and investigated the effects on axonal transport with live cell imaging techniques.


RESULTS
In stably transfected cells, there was a 2.5-fold increase in cdk5 activity compared to non-transfected cells, which in turn led to a dramatic increase in phosphorylation of NFs and tau at several phosphorylation sites. Using time-lapse imaging technology, the transport of NFs was captured in the cells overexpressing cdk5/p25, which were also transiently transfected with fluorescence protein linked to the N-terminus of NF-M (EGFP-NFM). The cdk5/p25 cells displayed significantly slower rates of axonal transport of NFs, with accumulation of immobile NF clusters observed in the cell body. Roscovitine, an inhibitor of cdk5, significantly reversed this defect in axonal transport.


CONCLUSIONS
These results suggest that increased cdk5 activity found in AD subjects may be crucially related to the pathogenesis of AD via an underlying mechanism by which it promotes accumulation of excessively phosphorylated cytoskeletal NF proteins, leading to the enduring impairment of axonal transport of NFs.
Zhou
Jie
J
Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Wang
Hongxing
H
Feng
Youmei
Y
Chen
Juan
J
D001370:Axonal Transport / Q000187:drug effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D000077546:Roscovitine; D016875:tau Proteins / Q000378:metabolism
D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D047428:Protein Kinase Inhibitors; D011687:Purines; C118136:TPPP protein, human; D016875:tau Proteins; D000077546:Roscovitine; D051360:Cyclin-Dependent Kinase 5