Purification of Insoluble Protein Aggregates from Skeletal Muscle Using a Pre‐Clinical Model of Huntington’s Disease

Abstract

Insoluble protein aggregates are distinctive attributes of several tissues affected by aging and are suggested to play a pathological role in the development of neuromuscular disorders (i.e., Huntington’s Disease). However, the influence of insoluble protein aggregates in skeletal muscle on muscle‐specific function is unclear. This is due, in part, to the absence of standardized protocols for isolating protein aggregates so they can be identified and further investigated in vivo. The overall aim of the present methodological study was to determine the most effective method for isolating insoluble protein aggregates from mouse skeletal muscle. This study was conducted using a pre‐clinical Huntington’s Disease model. Electroporation with plasmids encoding EGFP fused to exon 1 of human huntingtin containing 23 (EGFP‐Q23, control) or 74 polyglutamine repeats (EGFP‐Q74, aggregate‐prone) in contralateral TAs was performed (N = 9 muscles/group). Using three adapted protocols from the literature that combined mild detergents and centrifugation to purify insoluble and soluble fractions (Protocol 1 (1% Triton‐X‐100, pH 7.5), Protocol 2 (1% NP‐40, pH 7.4) and Protocol 3 (0.5% Triton‐X‐100, pH 8.0)), we hypothesized that the most efficient protocol would show aggregate‐prone proteins (i.e., EGFP‐Q74) accompanied by adaptors involved in the degradation of protein aggregates by autophagy (p62, NBR1), enriched in insoluble fractions. Approximately 6‐, 5‐ and 3‐fold increases in EGFP‐Q74 vs. EGFP‐Q23 were observed in the insoluble fractions of Protocols 1, 2 and 3, respectively (P < 0.05). 2.5‐fold increases in adaptor p62 in the same insoluble fractions from Protocols 1 and 2 were observed, while only a 2‐fold increase was observed with Protocol 3. Lastly, we observed trends for NBR1, ubiquitinated proteins and total proteins to be elevated using Protocol 1. These observations indicate that Protocol 1 and 2 might be more effective than Protocol 3 for isolating protein aggregates from skeletal muscle and these findings should be considered in future studies extracting insoluble proteins using this Huntington’s Disease model.Support or Funding InformationThis research is supported by an SDG grant from the American Heart Association (V.A.L.).