Targeting the VCP-binding motif of ataxin-3 improves phenotypes in Drosophila models of Spinocerebellar Ataxia Type 3

Abstract

Of the family of polyglutamine (polyQ) neurodegenerative diseases, Spinocerebellar Ataxia Type 3 (SCA3) is the most common. Like other polyQ diseases, SCA3 stems from abnormal expansions in the CAG triplet repeat of its disease gene resulting in elongated polyQ repeats within its protein, ataxin-3. Various ataxin-3 protein domains contribute to its toxicity, including the valosin-containing protein (VCP)-binding motif (VBM). We previously reported that VCP, a homo-hexameric protein, enhances pathogenic ataxin-3 aggregation and exacerbates its toxicity. These findings led us to explore the impact of targeting the SCA3 protein by utilizing a decoy protein comprising the N-terminus of VCP (N-VCP) that binds ataxin-3’s VBM. The notion was that N-VCP would reduce binding of ataxin-3 to VCP, decreasing its aggregation and toxicity. We found that expression of N-VCP in Drosophila melanogaster models of SCA3 ameliorated various phenotypes, coincident with reduced ataxin-3 aggregation. This protective effect was specific to pathogenic ataxin-3 and depended on its VBM. Increasing the amount of N-VCP resulted in further phenotype improvement. Our work highlights the protective potential of targeting the VCP-ataxin-3 interaction in SCA3, a key finding in the search for therapeutic opportunities for this incurable disorder.

Drosophila as a Model of Unconventional Translation in Spinocerebellar Ataxia Type 3

“…The ATXN3-Q80(CAGCAA) fly line has been employed in our previous studies and no specific protein signal was observed in either the MYC- or V5-tagged reading frames [ 39 , 40 , 41 ]. Upon the transfection of cultured, mammalian HEK-293T cells with either ATXN3-Q80(CAG) or ATXN3-Q80(CAGCAA), we observed no specific protein signal from alternate reading frames with ATXN3-Q80(CAGCAA) and its alternating repeat.…”

“…To investigate SCA3/MJD RAN translation in vivo, we again used Drosophila melanogaster [ 39 , 40 , 41 , 42 , 43 , 45 , 70 , 71 ]. Utilizing the versatility of Drosophila genetics along with the Gal4-UAS binary system of expression, we can express either the homomeric or the alternating polyQ repeat of pathogenic ataxin-3 transgenes individually in specific tissues [ 39 , 40 , 41 , 42 , 43 , 45 , 47 , 51 , 70 , 72 , 73 ]. We focused our investigations on the type of tissue that is primarily impacted in SCA3/MJD, neuronal and glial [ 14 , 15 , 18 , 74 ].…”

“…The ATXN3-Q80(CAGCAA) cDNA was based on the human ATXN3 sequences used in previous publications [ 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ]. ATXN3-Q80(CAG) cDNA and the Met-Null cDNA counterparts to both ATXN3-Q80 constructs were designed from the ATXN3-Q80(CAGCAA) sequence and synthesized using the company Genscript (genscript.com, accessed on January 2, 2019).…”

“…ATXN3-Q80(CAG) cDNA and the Met-Null cDNA counterparts to both ATXN3-Q80 constructs were designed from the ATXN3-Q80(CAGCAA) sequence and synthesized using the company Genscript (genscript.com, accessed on January 2, 2019). The transgenes were subcloned into pWalium-10.moe and transgenic fly lines were generated with the phiC31 integrase system into attP2 on chromosome 3 at Duke University Model Systems [ 38 , 39 , 40 , 41 , 42 , 43 , 45 , 47 , 48 , 49 , 50 ]. All insertions were confirmed and validated with PCR, genomic sequencing and then Western blotting using procedures described in this paper and previous work [ 39 , 40 , 41 , 42 , 43 , 45 , 47 , 51 ].…”

“…The transgenes were subcloned into pWalium-10.moe and transgenic fly lines were generated with the phiC31 integrase system into attP2 on chromosome 3 at Duke University Model Systems [ 38 , 39 , 40 , 41 , 42 , 43 , 45 , 47 , 48 , 49 , 50 ]. All insertions were confirmed and validated with PCR, genomic sequencing and then Western blotting using procedures described in this paper and previous work [ 39 , 40 , 41 , 42 , 43 , 45 , 47 , 51 ].…”