| Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative diseases of central nervous system (CNS), including Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome(GSS), Kuru and fatal familial insomnia (FFI) in humans and bovine spongiform encephalopathy in cattle, scrapie in sheep and goat, chronic wasting disease (CWD) in deer and elk. All kinds of TSE have similar neuropathological changes, including progressive neuronal degeneration, neuronal vacuolation and gliosis. Prion includes two isoforms, which are PrPc and PrPSc, the loss of the normal function of PrPc and the accumulation of the abnormal form in CNS may be the pathogeny.Microtubules are polarized structures and assemble from heterodimers of α-and (3-tubulin in a GTP-dependent fashion. It plays a central role in cellular transport, structural integrity and cellular architecture. Many previously studies have identified that the levels of tubulin decreased in CNS tissues of naturally occurred or experimental human and animal TSEs. Disturbance of microtubule associated transport and dystrophy of axonal structure were occured in GSS patient. Our previous studies have also confirmed that PrP could induce tubulin to form oligomer and restain the formation of microtubule; PrP mutants related with CJD possess stronger inhibitive effect on microtubule assembly through different pathways; PG12with seven extra-repeat insertion in PRNP and Cyto-PrP with a deleted signal peptide and GPI anchor could destroy microtubule structure.The polymerization, stabilization, arrangement of microtubules can be modulated by interactions with a series of microtubule-associated proteins (MAPs). Microtubule associated protein2(MAP2) belongs to a family of heat stable MAPs, which takes part in neuronal morphogenesis, maintaining cellular architecture and internal organization, cell division and cellular processes. In mammalian brain, MAP2isoforms have been divided into high-molecular weight MAP2(HMWMAP2) and low-molecular weight MAP2(LMWMAP2). HMWMAP2consists of MAP2A (Mr.280kDa) and MAP2B (Mr.270kDa,) that are specially expressed in neurons, while LMWMAP2includes MAP2C (Mr.70kDa) and MAP2D (Mr.75kDa) that are present in glial cells. Previous researches suggested that MAP2may take part in the process of neurodegenerative diseases, for example, MAP2monoclonal antibody could mark neurofibrillary tangles(NFTs) and MAP2poloclonal antibody also labled abnormal neurites around senile plaques in AD; the accumulation of Aβ induced the decrease of MAP2in transgenic mice before the formation of plaques; treatment of Aβ1-42on C57BL/6J mouse primary cerebral neurons and human neuroblastoma cells induce a reduction of MAP2; in the brains of Alzheimer’s diseases, the levels of MAP2are usually decreased. However, the situations and roles of MAP2in TSE pathogenesis remain unclear. Recently, by screening the transcriptional diversity in the brains of human prion diseases with a commercial mRNA microarray, we found that the expression level of MAP2is obviously decreased, which suggested that MAP2may have a roll in the process of TSEs. Therefore, in order to study the role of MAP2in Prion deseases and provide new sights in the process of the deseases, we designed a series of experiments in animals and cells, hoping to clarify the variation and change mechanism of MAP2.The main results of our research are as follows:1. The expression level of MAP2was detected in brain tissues of scrapie agent263K-infected hamsters at the terminal stages and on the0th,20th,40th,60th, and80th days post-inoculation.The data indicated that MAP2level in the brains of scrapie experimental rodents was significantly reduced at terminal stages of the disease and it was closely related with prolonged incubation time.2. Levels of MAP2expression in postmortem brain from a G114V gCJD patient and a D178N FFI patient were assessed by Western blots, including frontal lobe, parietal lobe, occipital lobe, temporal lobe, thalamus and cingulated gyrus. The results showed that the MAP2expression level decreased in all tested brain regions compared with control.3. The transcriptional level of MAP2was detcted by RT-PCR in brain tissues of scrapie agent263K-infected hamsters and in different regions of a G114VgCJD patient and a D178N FFI patient. The data indicated that MAP2transcriptional level was declined significantly in contrast to the normal controls.4. SK-N-SH cells were treated with synthesized peptide PrP106-126to mimic the pathological features of PrPSc in vitro as a cellular model. The expression level of MAP2, microtubule and tubulin were detected by Western blot and immunofluorescent assays. The data suggested that cytotoxic peptide PrP106-126induces a decrease of MAP2and a rapid disorganization of cellular microtubule.5. The expression level of Calpain was detected in SK-N-SH cells, in brain tissues of scrapie agent263K-infected hamsters at the terminal stages and on the0th,20th,40th,60th, and80th days post-inoculation.The data indicated that Calpain level in the PrP106-126treated cells and in the brains of scrapie experimental rodents was increased and it was closely related with prolonged incubation time.6. SK-N-SH cells were treated with the inhibition of calpain activity (ALLN) and PrP106-126.The result suggested that inhibition of calpain activity is able to partially reverse PrP106-126induced cytotoxicity and the decrease of MAP2.Taking together, we found that MAP2proteins in the brain tissues of scrapie agent263K-infected rodents and human genetic prion diseases were almost undetectable at the terminal stages.The decline of MAP2in the brains of agent263K-infected hamsters was closely related with prolonged incubation time. The further experiment suggested that the increase of Calpain may be a reason for the decline of MAP2. Meanwhile, the results that the inhibition of calpain activity (ALLN) was able to partially reverse PrP106-126induced cytotoxicity and the decrease of MAP2provided more evidences. |
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