The Role Of CDK5 In The Pathogenesis Of OPIDN

This thesis comprises three parts. First part is the observation of time course of pathological changes of neuronal bodies and axons in hen's model of organophosphate-induced delayed neurotoxicity, and determined some typical time points. Second part is the temporal and spatial expressing of CDK5, p35/p25 in lumbar spinal cord and their co-exist state in OPIDN. Third part reported the inhibitory effects of amlodipine maleate and CDK5 inhibitor-Roscovitine on OPIDN. These experiments aim to discover if aberrant activation of CDK5 is involved in the pathogenesis of OPIDN.Organophosphate-induced delayed neuropathy (OPIDN) is a neurodegenerative disorder appears about 2 weeks after some organophosphoms esters intoxication, characterized by abnormality of sense and movement, with a concomitant axonal degeneration (mainly large diameter, long axons) and secondary demyelination of central and peripheral axons. TOCP is one of the most familiar organophosphoms (OP) ester associated with neuropathy. A single dose of TOCP may induce delayed neurotoxicity in humans, hens, and other susceptible species (such as cats, dogs, cows). The widespread industrial and commercial use of OP esters as flame retardants, plasticizers, and lubricants extends the possibilities of serendipitous exposure from numerous sources, including everyday household reagents. The chance of OPs poisoning is increasing, and Ops is often used formilitary or terrorism aims.The precise mechanism of OPIDN has not been elucidated and there is no effective treatment for OPIDN.So it has especial means to elucidate the pathogenesis of OPIDN, not only for the purpose of prevention but also for the aim of clinical treatment of OPIDN.Cyclin-dependent kinase 5 (CDK5) is a proline-directed protein kinase that phosphorylates serine and threonine residues. It is a special member of CDK family and functions predominantly in nervous system. CDK5 phosphorylates a diverse list of substrates including KSP protein motif on MAPlb, KSPXK on tau or KSPXX on neurofilament proteins As monomeric CDK5 displays no enzymatic activity, an interaction with either p35 or p39 anchored to membrane is necessary for CDK5 activation. There is evidence that links CDK5 activity associated with extracellular signal and cell cytoskeleton/membrane system to regulation of the cytoskeleton,neuronal migration , axon growth and guidance, axonal transport, synaptic function,dopamine signalling and drug addiction,and is associated with the release of neurotransmitters.Deregulation of CDK5 may induce dysfunction of cytoskeletal components axonal transport ,lesion of axons.Focusing on the links between CDK5 activity and components of the cytoskeletal membrane and adhesion systems has allowed us to postulate a role for CDK5 in directing intracellular traffic in neurons. Microtubule dynamics and axonal transport might be targets for CDK5 through its phosphorylation of the microtubule-associated protein (MAP). Aberrant phosphorylation of the neuronal cytoskeleton by CDK5 could alter cellular trafficking and transport and contribute to neuronal dysfunction and death. So it is hypothesized that p25 (a truncated form of p35) causes aberrant activation of CDK5, leading to the breakdown of cytoskeletal proteins and intracellulartrafficking, which, in turn, contributes to neurodegeneration. Cleavage of p35 to p25, may be a critical step in this pathological cascade, including neurite retraction, microtubule collapse and apoptosis.There exist many similarities between OPIDN and neurodegenerative disorders caused by aberrant activation of CDK5: they both target cytoskeleton proteins serine and threonine residues, cause microtubule collapse and cause axonal transport dysfunction. So the question is whether or not they have common mechanisms. Thus, the aim of this study is to investigate the relationship between aberrant activation of CDK5 and OPIDN and its implications. On this ground, we established OPIDN animal model by injecting hens with TOCP, and firstly observed pathological and temporal changes of OPIDN under light and electronic microscopy.Then we observed expression and coexist state of CDK5 and p35/p25 by immunohistochemistry and western-blot in hens with OPIDN; finally we observed the effect of inhibition of CDK5 on retrograde axonal transport so as to investigate if dysfunction of CDK5 is involved in the pathogenesis of OPIDN.Part one is to investigate the dynamic morphological changes of hens' spinal cords in the model of organophosphate-induced delayed neuropathy (OPIDN) and to analyze the impact of apoptosis on the pathogenesis of OPIDN; adult hens were challenged with triorthocresyl phosphate (TOCP) at a single dose (750mg/kg.). Neuropathologic changes as well as the number of nerve cells and apoptotic cells in the 3rd lumbar spinal cord were assessed by hematoxylin and eosin (H-E) staining, Nissl, immunohistochemistry, terminal-deoxynucleotidyl transferase mediated nickend labeling (TUNEL) and Electron-microscopy methods at 3, 5, 7, 10, 14, 18 days post exposure respectively. The typical OPIDN signs (progressive ataxia and hypotonia)were seen in the TOCP exposed hens at about 9 days after injection. Some nuclei of the large neurons in the anterior horn of the lumbar spinal cord showed apoptotic-like changes in H-E staining at 5 days, but this phenomenon disappeared in 18th day. Nissl method showed that the number of large nerve cells in the anterior horn of lumbar spinal cord declined gradually. And these cells were verified as neurons by immunostaining with neuronal marker NeuN and MAP-2. TUNEL positive cells began to appear 5 days after TOCP injection, and their number reached maximal in 7d group, but disappeared in 18d group. Some neurons showed neurodegenerative and apoptotic ultrastructure profiles in electronic microscopy, such as: swollen axon, disruption of myelin sheath, delamination of myelin sheath, disorganization of cytoskelton components, swollen or ruptured mitochondria, membrane blebbing, nuclear and cytosolic shrinkage and chromatin condensation. These indicated that apoptosis might be involved in the development of OPIDN, and might play a role in the pathogenesis of OPIDN.Through first part study, we determined 5, 9, 14 days post exposure as the typical time points, and found that OPIDN show apoptosis of cell body and degeneration of axon simultaneously. These are similar to the mechanism of CDK5. So part two of this study was to investigate changes of CDK5 and its activator p35/p25 in neurons of spinal cord in hens challenged with triorthocresyl phosphate (TOCP), and the relationship between CDK5 and organophosphate-induced delayed neuropathy (OPIDN). CDK5, p35/p25 protein levels and distribution were evaluated by immunohistochemistry, Laser Scanning Confocal microtechnic and immuno-blotting in lumbar spinal cord. Immunohistochemistry showed p-CDK5 (specific for ser-159 phosphorylated CDK5) and p35 begin to be positive in big neurons in anteriorhorn of spinal cord in groups post 5d. In groups of 7d and 9d, the number of positive cells increased most significant, but in 18d group, the number decreased. Double immunofluorescence labeling of P-CDK5 and p35/p25 showed that p35/p25 was expressed predominantly in phosphorylation of CDK5 immunoreactivity positive neurons, and the co-localization was most evident around 9 days after TOCP exposure. Western-blot showed that the CDK5 and p-CDK5 protein level in spinal cord of OPIDN hens were increased, they reached maximal at the onset of OPIDN, and then decreased gradually. p35/p25 protein levels were significantly increased too, and reached maximal in 9d group. These findings indicate that conversion of p35 to p25 deregulates CDK5 activity and aberrant activation of CDK5 may be involved in the pathogenesis of OPIDN.To further verify that aberrant activation of CDK5 is involve in the pathogenesis of OPIDN, functional inhibit experiments had been done. We observed the effect of Amlodipine maleate(AM)(a calcium channel blocker which inhibit the influx of calcium and activation of calpain so asto inhibit CDK5 activation indirectly ) and roscovitine(CDK5 inhibitor) on axonal transport of OPIDN. AM were given im at the dose of 0.167 mg/kg/day for 4 days beginning 1 day before administration of TOCP (750 mg/kg, po). Additional 5 hens' unilateral common peroneal was treated by roscovitine 3 days after TOCP exposure. Axonal transport of these nerves was faster than that of their opposite side and of those simply treated by TOCP. These inhibitors can ameliorate the clinical signs and histological lesion of nerves during OPIDN in hens too. These findings indicate that inhibition of CDK5 activity is benefical to development of OPIDN and confirmed aberrant activation of CDK5 might be involved in the pathogenesis of OPIDN.