Potential Transfer of Polyglutamine and CAG-Repeat RNA in Extracellular Vesicles in Huntington’s Disease: Background and Evaluation in Cell Culture

Abstract

In Huntington’s disease (HD) the imperfect expanded CAG repeat in the first exon of the HTT gene leads to the generation of a polyglutamine (polyQ) protein, which has some neuronal toxicity, potentially mollified by formation of aggregates. Accumulated research, reviewed here, implicates both the polyQ protein and the expanded repeat RNA in causing toxicity leading to neurodegeneration in HD. Different theories have emerged as to how the neurodegeneration spreads throughout the brain, with one possibility being the transport of toxic protein and RNA in extracellular vesicles (EVs). Most cell types in the brain release EVs and these have been shown to contain neurodegenerative proteins in the case of prion protein and amyloid-beta peptide. In this study, we used a model culture system with an overexpression of HTT-exon 1 polyQ-GFP constructs in human 293T cells and found that the EVs did incorporate both the polyQ-GFP protein and expanded repeat RNA. Striatal mouse neural cells were able to take up these EVs with a consequent increase in the green fluorescent protein (GFP) and polyQ-GFP RNAs, but with no evidence of uptake of polyQ-GFP protein or any apparent toxicity, at least over a relatively short period of exposure. A differentiated striatal cell line expressing endogenous levels of Hdh mRNA containing the expanded repeat incorporated more of this mRNA into EVs as compared to similar cells expressing this mRNA with a normal repeat length. These findings support the potential of EVs to deliver toxic expanded trinucleotide repeat RNAs from one cell to another, but further work will be needed to evaluate potential EV and cell-type specificity of transfer and effects of long-term exposure. It seems likely that expanded HD-associated repeat RNA may appear in biofluids and may have use as biomarkers of disease state and response to therapy.

Mutant Huntingtin Is Secreted via a Late Endosomal/Lysosomal Unconventional Secretory Pathway

“…The toxicity of secreted mHtt aggregates has been associated with their propensity to seed aggregation of the soluble protein in recipient cells (Chen et al, 2002;Ren et al, 2009;Tan et al, 2015). In light of the potential role of propagation in both HD and other neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS) (Soto, 2012;Jucker and Walker, 2013), further characterizing the cellular mechanisms of protein secretion is critical for better understanding disease pathogenesis. Furthermore, designing preventative strategies in HD may require targeted approaches against the cellular secretion of mHtt.…”

“…Cell cloning was performed by limiting dilution to obtain monoclonal cell population with high Htt expression. Striatal cells STHdh ϩ /Hdh ϩ , STHdh Q111 /Hdh ϩ , and STHdh Q111 /Hdh Q111 were a kind gift from Marcy MacDonald (Trettel et al, 2000). They were grown in DMEM with 10% FCS, penicillin/streptomycin, and 250 g/ml geneticin (Thermo Fisher Scientific).…”

Role of exosomes in the protection of cellular homeostasis

“…48 Interestingly, exosomes may be also involved in the release of toxic RNAs since expanded trinucleotide repeat RNAs, such as the CAG repeats that underlie RNA toxicity in Huntington disease, are released from the cell in exosomes. 49 Other evidences support the relation between RNA degradation and its export to extracellular vesicles. In this regard, proteins involved in RNA processing are abundant in exosomes, and secreted RNAs have almost twice shorter half-life times than intracellular mRNAs.…”

Insulin-like growth factor 2 (IGF2) protects against Huntington’s disease through the extracellular disposal of protein aggregates

“…While the precise mechanisms of mHtt secretion are not completely understood, several possibilities have been suggested, including synaptic vesicle release (41), vesicular transport (42), exosomes/extracellular vesicles release (43,44), exophers (45) and secretory lysosomes (40). Since the initial link connecting IGF2 with mHtt was through the UPR, we evaluated if the polyQ peptide is secreted by the conventional secretory pathway.…”