Neur opharmacological and Molecular Characterization of Okadaic acid Induced Neuro degeneration in Rat

Abstract

Okadaic acid (OKA) is a selective inhibitor of the serine/threonine phosphatases 1 (PP1) and 2A (PP2A) and considered as a main culprit for memory impairment in the aged population using sea foods dinoflagellates (Helicondria Okadai) containing OKA. The present study is an investigation on the effect of intra cerebro ventricular (ICV) OKA on memory function in rat and associated biochemical, cellular, molecular and histological changes the brain. The major objectives of the study were 1. Development of an experimental model in rodents is suitable to screen memory enhancer (anti-dementia) 2. Understanding of biochemical, cellular and histological changes in rat brain areas following 3. Neuroinflammation in the rat brain following ICV OKA administration 4. Protein phosphorylation and its implication at molecular level leading to neuronal death in the rat brain areas after ICV OKA administration The study was conducted on adult male SD rats. OKA (100 and 200 ng) was administered by ICV route and after 14 days rats were subjected to testing of memory function by Morris water maze. Spontaneous Locomotor Activity (SLA) was recorded by Optovarimex activity meter. After completion of behavioral testing, the rats were sacrificed to remove the brain. The biochemical markers of oxidative stress (MDA, GSH, ROS, nitrite), cholinergic (AChE activity, ACh level), mitochondrial function (mitochondrial [Ca2+]i, mitochondrial membrane potential, ATP) and apoptosis (caspase- 3 and caspase-9) were estimated in rat brain areas (cortex and hippocampus). Molecular changes were studied by RT-PCR and western blotting. Neuroinflammatory mediators cytokines TNF-α and IL-1β were estimated by ELISA method whereas, iNOS and nNOS were estimated by mRNA expression and western blotting. Cholinergic markers (acetylcholinestrase and α7-nicotinic receptor) were estimated by mRNA expression and modulators of phsosphorylated Tau, PP2A, CaMKII, Calpain and GSK-3β were studied in cortex and hippocampus by RT-PCR and western blotting. Histological alterations

were seen by hematoxylin and eosin and cresyl violet staining. The effects of standard anti-dementia drugs donepezil (5 mg/kg, p.o) and memantine (10 mg/kg,po) were investigated on OKA-induced changes in above mentioned parameters.

OKA based experimental model in rodents

After successive trials, control and artificial CSF ICV groups showed significant decrease in latency time and path length suggesting of successful learning and memory while OKA (200 ng, ICV) treated rats did not exhibit any significant decrease in latency time and path length indicating memory impairment tested in rats. Administration of memantine (10mg/kg/day) and donepezil (5mg/kg/day) were effective against OKA induced memory impairment in rats suggesting a high predictive validity of this model to screen anti-dementia (memory improvement) drugs.

Biochemical changes in the rat brain areas (cortex and hippocampus)

 Oxidative stress

OKA (200ng, ICV) caused increase in malondialdehyde (MDA), nitrite level, reactive oxygen species (ROS) generation, mitochondrial calcium ion [Ca2+]i, level and decreased glutathione (GSH) level in the homogenate of rat brain areas, indicating oxidative stress. Administration of memantine and donepezil countered the OKA induced oxidative stress in rat.

 Cholinergic function

Okadaic acid (ICV) caused a significant decrease in acetylcholine level, acetylcholinestrase activity and mRNA expression of acetylcholinestrase and α7-nicotinic receptor in rat brain homogenate of cortex and hippocampus. The decrease in cholinergic markers indicates hypocholinergic activity in the brain areas closely associated with learning and memory. The lowered cholinergic influence in hippocampus can be a major contributory factor for memory impairment. This study also demonstrated that clinically used antidementic drugs were effective in OKA induced cholinergic dysfunction in rat rtex and hippocampus of OKA (200ng ICV) treated rats were used to study mitochondrial functions. There was increase in Ca2+ and reactive oxygen species along with decrease in, SDH activity, mitochondrial membrane potential and ATP. These alterations in mitochondrial preparation point to mitochondrial dysfuntions. Moreover, a significant increase in lipid peroxidation (MDA) and nitrite was also observed in mitochondtrial rich synaptosomal preparations. Memantine and donepezil offered significant prevention against OKA induced mitochondrial dysfunction.  Apopt osis Activity and mRNA expression of Caspase-3 and caspase-9 were also increased in OKA ICV treated rat brain indicating apoptotic cell death. Pretreatment with memantine and donepezil offered significant protection against OKA induced apoptotic cell death in rat brain.  Neuroinflammatory OKA caused increased expression of proinflammatory cytokine TNF-α and IL-1β and total nitrite in homogenates of brain regions hippocampus and cortex. The expression of mRNA and protein of iNOS was increased while; the expressions were decreased in case of nNOS. Thus, Okadaic acid (ICV) induces neuroinflammation is apparent by elevated inflammatory markers. Administration of donepezil and memantine prevented OKA induced neuroinflammatory changes. Protein phosphorylation  OKA-induced decrease in activity and mRNA expression of PP2A in hippocampus and cortex. The increase in protein and mRNA expression of Tau, CaMKII, Calpain, and GSK-3β was found following OKA ICV administration. The results indicate the rise of intracellular Ca2+ as a key factor in the protein hyperphosphorylation that also suggest a possible involvement of NMDA receptors. Therefore, the role of NMDA receptors was determined by using specific NMDA receptor antagonist MK801. OKA induced mRNA and protein hyperphosphorylation related changes were blocked by pretreatment with MK801. Moreover, inhibition of these changes by antidementic drug memantine which is nist further implicated NMDA receptors in OKA induced ezil against OKA induced protein hyperphosphorylation also a NMDA receptor antago toxicity. Effectiveness of donep of Tau may be attributed to decrease entry of Ca2+ into cell. Histopathology  OKA (ICV) treated rats showed vacuoles, shrinkage, cell blebbing and sponginess in cortical and hippocampal areas which indicate neurodegeneration in these areas intimately involved in learning and memory functions. Donepezil and memantine treatment prevents these histological changes. Thus, taken together it may be suggestive that OKA induces memory deficit and neurodegeneration in hippocampus and cortex along with Tau hyperphosphorylation, which are the major features of AD. The major factors appear to be involved in OKA induced neurotoxicity are mitochondrial dysfunction, cholinergic dysfunction, neuroinflammation, oxidative stress and NMDA receptor. Moreover, efficacy of clinically used antidementic drugs against OKA induced changes concretes the validity of OKA induced memory impairment as an experimental model of dementia