HspB1 protection against amyloid-ß toxicity

Conway, Megan G.(Megan Gabriella) (2012) HspB1 protection against amyloid-ß toxicity. Masters thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (36MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

Alzheimer's disease (AD) is hallmarked by the presence of neurofibrillary tangles and amyloid-β plaques. Amyloid-β plaques consist of the amyloid-β peptide (Aβ) that has been shown to induce toxic effects on neurons through the activation of stress-related signalling and neuronal loss. The small heat shock protein, HspB1 (also referred to as Hsp27 and Hsp25 in mouse), is accumulated in 15% of neocortical amyloid-β plaques in AD brains (Wilhelmus, 2006). Whether this represents a potentially protective response to stress, or is part of the disease process is unknown. We have previously reported that expression of HspB1 not only protects cortical neurons against amyloid toxicity, but also enhances total neurite growth in these neurons(King et al., 2009). -- The amyloid-β peptide is derived from the proteolytic processing of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Mutations in APP alter secretase cleavage sites, resulting in higher production of the toxic Aβ(1-42) peptide that undergoes aggregation more readily. Since HspB1 has been shown to protect neurons against amyloid toxicity, it is conceivable that HspB1 may interact directly with Aβ or APP. Recent studies have demonstrated exogenous HspB1 binding to synthetic Aβ. We have replicated these results in an attempt to determine the role of HspB1 in the inclusion of Aβ aggregates. We hypothesize that HspB1 interacts with Aβ or its precursor APP, to either alter the distribution of Aβ/APP within the cell, or its release from the cell. -- In order to test our hypothesis, we incubated His-tagged HspB1 with synthetic Aβ(1-42), at physiological temperature 37°C overnight. Immunoprecipitation, using agarose protein A/G beads incubated with the monoclonal anti-His primary antibody, was used for our primary analysis of interaction. Western blotting of the nitrocellulose membrane, using the 6E10β-amyloid (1-16) mouse monoclonal antibody, demonstrates that Aβ is immunoprecipitated with His-HspB1. These results point to a direct interaction between HspB1 and Aβ(1-42). We investigated the interaction of HspB1 with APP in HEK293 cell line expressing wild-type APP (APP-wt) or APP-swedish mutation (APP-swe) that predominately yields Aβ(1-42) through immunoprecipitation. His-tagged HspB1 was incubated with the conditioned media from the APP-wt and APP-swe cells overnight at 37°C. Immunoprecipitation was performed using magnetic protein A/G beads incubated with either 6E10β-amyloid (1-16) mouse monoclonal antibody, or anti-HspB1 human (SPA-803)rabbit polyclonal antibody. Subsequent western blotting of the nitrocellulose membrane using the 6E10 and the 803 antibodies demonstrate that APP is immunoprecipitated with His-HspB1. -- These data suggest HspB1, perhaps via its chaperone activity, may be altering production of APP and/or Aβ within the cell preventing secretion into the extracellular environments.

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