Methylene Blue Modulates Huntingtin Aggregation Intermediates and Is Protective in Huntington's Disease Models

Abstract

Huntington’s disease (HD) is a devastating neurodegenerative disorder with no disease modifying treatments available. The disease is caused by expansion of a CAG trinucleotide repeat and manifests with progressive motor abnormalities, psychiatric symptoms and cognitive decline. Expression of an expanded polyglutamine repeat within the Huntingtin (Htt) protein impacts numerous cellular processes, including protein folding and clearance. A hallmark of the disease is the progressive formation of inclusions that represent the culmination of a complex aggregation process. Methylene blue (MB) has been shown to modulate aggregation of amyloidogenic disease proteins. We investigated whether MB could impact mutant Htt-mediated aggregation and neurotoxicity. MB inhibited recombinant protein aggregation in vitro, even when added to preformed oligomers and fibrils. MB also decreased oligomer number and size and decreased accumulation of insoluble mutant Htt in cells. In functional assays, MB increased survival of primary cortical neurons transduced with mutant Htt, reduced neurodegeneration and aggregation in a Drosophila melanogaster model of HD, and reduced disease phenotypes in R6/2 HD modeled mice. Further, MB treatment also promoted an increase in levels of BDNF RNA and protein in vivo. Thus, MB, which is well tolerated and used in humans, has therapeutic potential for HD.

Proteasome-mediated Proteolysis of the Polyglutamine-expanded Androgen Receptor Is a Late Event in Spinal and Bulbar Muscular Atrophy (SBMA) Pathogenesis

“…To further understand the biochemical characteristics of AR aggregation species, we utilized SDS-AGE, a highly sensitive and specific method to evaluate polyglutamine-expanded aggregated species (23,24,33). We demonstrate the formation of two distinct, albeit heterogeneous, aggregation species resolved by SDS-AGE and show that early, soluble aggregation species consist of full-length AR, whereas the insoluble AR aggregation species that are formed at later time points comprise both full-length and proteolyzed AR.…”

“…Samples were electrophoresed in agarose as described previously (23)(24)(25)(26). Briefly, protein lysates were electrophoresed on a 12-cm-long, 8-mm-thick 1% agarose gel containing 0.1% SDS (in 375 mM Tris-HCl, pH 8.8) at 100 V at 4°C.…”

“…To evaluate this prediction, we utilized SDS-AGE, which was previously shown to be a sensitive and specific method for resolving polyglutamine-expanded aggregates (23,24,33). Using SDS-AGE, we detected a heterogeneous population of AR species that are both polyglutamine length-dependent and hormone-dependent (Fig.…”

Exogenous delivery of chaperonin subunit fragment ApiCCT1 modulates mutant Huntingtin cellular phenotypes

“…Accordingly, mHtt oligomers and insoluble Htt species were next evaluated to determine whether exogenous delivery of ApiCCT1 r influences accumulation of these forms. We used SDS agarose gel electrophoresis (AGE) to resolve oligomeric species, as this approach seems to preferentially resolve fibrillar oligomers of mHtt (26)(27)(28)(29). Equivalent amounts of protein from cell lysates were loaded on SDS-AGE gels.…”

“…The analysis of mHtt by SDS-AGE with Western analysis was performed as previously described (26)(27)(28)(29).…”