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

Abstract

Aggregation of misfolded proteins is characteristic of a number of neurodegenerative diseases, including Huntington disease (HD). The CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring) chaperonin complex can inhibit aggregation and cellular toxicity induced by expanded repeat Huntingtin (mHtt) fragments. The substratebinding apical domain of CCT/TRiC subunit CCT1, ApiCCT1, is sufficient to inhibit aggregation of expanded repeat mHtt fragments in vitro, providing therapeutic promise for HD. However, a key hurdle in considering ApiCCT1 as a potential treatment is in delivery. Because ApiCCT1 has a region of similarity to the HIV Tat protein cell-transduction domain, we tested whether recombinant ApiCCT1 (ApiCCT1 r ) protein could enter cells following exogenous delivery and modulate an established panel of mHtt-mediated cell-based phenotypes. Cell fractionation studies demonstrate that exogenous ApiCCT1 r can penetrate cell membranes and can localize to the nucleus, consistent with a strategy that can target both cytosolic and nuclear pathogenic events in HD. ApiCCT1 r application does indeed modulate HD cellular phenotypes by decreasing formation of visible inclusions, fibrillar oligomers, and insoluble mHtt derived from expression of a truncated mHtt exon 1 fragment. ApiCCT1 r also delays the onset of inclusion body formation as visualized via live imaging. ApiCCT1 r reduces mHtt-mediated toxicity in immortalized striatal cells derived from full-length knock-in HD mice, suggesting that therapeutic benefit may extend beyond effects on aggregation. These studies provide the basis for a potentially robust and unique therapeutic strategy to target mHtt-mediated protein pathogenesis.

Structural Mechanisms of Mutant Huntingtin Aggregation Suppression by the Synthetic Chaperonin-like CCT5 Complex Explained by Cryoelectron Tomography

“…Because most patients develop HD late in life, this slowing may translate to a delay in the age of onset sufficient to allow the disease to bypass a large number of potential patients. The results presented here and reported previously for apiCCT1 (22) highlight the idea that one single subunit or a specific peptide component of TRiC is sufficient for the suppression of huntingtin aggregation. Additionally, a subunit of CCT5 or a specific peptide derived from CCT5 are appropriate sizes for packaging into an adeno-associated virus vector capable of treating the brain.…”

“…A previous genome-wide RNAi screen in Caenorhabditis elegans identified six of the eight subunits of TRiC chaperonin (CCTs 1, 2, and 4 -7) as suppressors of polyglutamine aggregation (21), whereas a co-overexpression assay in yeast identified the bovine TRiC subunits CCT1 and CCT4 as inhibitors of mHTT aggregation (17). In the same vein, the exogenously applied bovine recombinant purified CCT1 apical domain has been shown to penetrate cell membranes and decrease mHTT-Ex1 aggregation in vivo (22). Furthermore, recently, the purified human CCT5 subunit has been shown to form a homo-oligomeric complex and suppress mHTT aggregation in vitro (23).…”

TRiC subunits enhance BDNF axonal transport and rescue striatal atrophy in Huntington’s disease

“…More remarkable, following exogenous delivery, the substrate-binding apical domain of CCT1, ApiCCT1, entered the cytosol and nucleus of cells and suppressed mHTT aggregation (26). In addition, cryoelectron tomography uncovered that another individual subunit, CCT5, caps fibrils and encapsulates oligomers to inhibit mHTT aggregation (27).…”

Vaccinia-Related Kinase 2 Controls the Stability of the Eukaryotic Chaperonin TRiC/CCT by Inhibiting the Deubiquitinating Enzyme USP25

“…Growing evidences reveal that functional TRiC inhibits polyQ aggregation at early stages and alleviates soluble or insoluble toxic speciesmediated harmful effects (10,14). Notably, overexpression of either a specific TRiC subunit or recombinant CCT1 significantly suppresses polyQ aggregation and neuronal cell death (23,24). We coexpressed Flag-USP25 or enzymatically inactive USP25 (C178S) with pathogenic HttQ103-GFP in HEK293A cells.…”