| Molecular chaperones protect the cells from the deleterious effects of protein misfolding and aggregation. HspB1 is the most inducible sHsp and is observed at elevated levels in the vicinity of senile plaques in Alzheimer's disease (AD). Although it was shown to prevent toxicity of Abeta oligomers, the mechanism was not studied. Here, I looked at the in vitro and in vivo interaction of sHsp with amyloid protein Abeta. In vivo, HspB1 localized to the penumbral areas of plaques in AD and two distinct mouse models of AD indicating the importance of HspB1 in Abeta deposition. In vitro, HspB1 abrogated the toxicity of Abeta oligomers at sub-stoichiometic concentrations upon brief coincubation at room temperature. To further study the mechanism of interaction, fluorescence microscopy, electron microscopy, atomic force microscopy light scatter techniques were employed. Intrinsic and extrinsic fluorescence comparison studies of resultant large aggregates with individual proteins showed absence of a change in the secondary or tertiary structure of the proteins. Each of these techniques suggested the formation of large aggregates of the order of size 200nm. Biochemical assays and fluorescence microscopy confirmed the presence of HspB1 in the large aggregates. HspB1 expression was induced more than six-fold as detected by quantitative RT-PCR in response to an acute challenge of toxic Abeta oligomer. Primary mouse neurons cultured from HspB1-deficient mice were more susceptible to Abeta toxicity in comparison to the WT neurons. Our set of unpublished data suggests uptake of Abeta oligomers and colocalization of induced HspB1 on Abeta oligomer treatment, which emphasizes the significance of sHsps as an arm of the proteostatsis machinery of cells.;In vivo validation of the hypothesis was done by crossing AD mouse model Tg2576 that shows the cognitive decline following 6 months of age and active deposition of plaques post 9 months. In accordance with the hypothesis, I asked the question whether absence of two sHsps (HspB2 & HspB5) would cause early appearance of cognitive failure or would prolong the plaque deposition in comparison with the Tg2576 mice alone. To examine the differences in AD like symptoms, (1) spatial orientation was tested by open field test, (2) sequential organization and associative learning was monitored by fear conditioning and lastly (3) evoked behavioral responses were tested by hot plate test. KO mice overexpressing toxic Abeta showed some impairment in the locomotion and sensory function, but motor reflexes or learning was not affected significantly. Some limitations were imposed by the fact that KO mice develop severe muscle degeneration issues post 40 weeks and therefore all the tests were performed with the age matched groups at 36 weeks. Our results emphasize the significance of sHsps in AD mice as the mice deficient for HspB2 and HspB5 develop pathology and behavior defect not noticed in Tg2576 mice or KO mice alone. |
