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[Preprint]. 2023 Nov 23:rs.3.rs-3592641.
doi: 10.21203/rs.3.rs-3592641/v1.

Progressive degeneration in a new Drosophila model of Spinocerebellar Ataxia type 7

Alyson L Sujkowski  1 , Bedri Ranxhi  1 , Matthew V Prifti  1 , Nadir Alam  1 , Sokol V Todi  1   2 , Wei-Ling Tsou  1
Affiliations

Progressive degeneration in a new Drosophila model of Spinocerebellar Ataxia type 7

Alyson L Sujkowski et al. Res Sq. .

Update in

Abstract

Spinocerebellar ataxia type 7 (SCA7) is a progressive neurodegenerative disorder resulting from abnormal expansion of polyglutamine (polyQ) in its disease protein, ataxin-7 (ATXN7). ATXN7 is part of Spt-Ada-Gcn5 acetyltransferase (SAGA), an evolutionarily conserved transcriptional coactivation complex with critical roles in chromatin remodeling, cell signaling, neurodifferentiation, mitochondrial health and autophagy. SCA7 is dominantly inherited and characterized by genetic anticipation and high repeat-length instability. Patients with SCA7 experience progressive ataxia, atrophy, spasticity, and blindness. There is currently no cure for SCA7, and therapies are aimed at alleviating symptoms to increase quality of life. Here, we report novel Drosophila lines of SCA7 with polyQ repeats in wild-type and human disease patient range. We find that ATXN7 expression has age- and polyQ repeat length-dependent reduction in survival and retinal instability, concomitant with increased ATXN7 protein aggregation. These new lines will provide important insight on disease progression that can be used in the future to identify therapeutic targets for SCA7 patients.

Keywords: Aggregation; Polyglutamine; Proteinopathy; Retinal degeneration.

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Conflict of interest statement

Competing Interests Statement The authors declare that they do not have any conflicts of interest to disclose.

Figures

Figure 1:
Figure 1:. Generation of Drosophila SCA7 models.
(A) Schematic of Myc-tagged ATXN7 Q10 and Q92 including construct-specific polyQ insertions. ATXN7 amino acid sequence, with 5X Myc tag (underlined) and polyQ repeat location (red) is also shown. (B) Top: diagrammatic representation of the cloning strategy used to insert ATXN7 cDNA into pWALIUM10.moe. Bottom: PCR reactions from genomic DNA indicating that the transgene was integrated into the correct site in the proper orientation.
Figure 2:
Figure 2:. Expression of ataxin-7 in flies.
Western blots from control, Q10, and Q92 flies in (A) fly eyes, (B) pan-neuronally, (C) in glia, and (D) ubiquitously. Quantifications are on the right of each blot. Q10 (105 kD) and Q92 (115 kD) indicated by blue arrows. Potential proteolysis products indicated with black arrows. The entire ataxin-7-positive signal was quantified in each case. qRT-PCR quantifications are found directly below Western blot quantifications. 10 fly heads (GMR-Gal4) or 5 whole flies (sqh-Gal4, elav-Gal4, repo-Gal4) were homogenized, depending on experiment. At least 3 biological replicates were used; ‘n’ indicated above panels. Significance determined by student t-test. *p>0.05, ****p<0.0001.
Figure 3:
Figure 3:. Effects of ataxin-7 expression on development and survival.
A summary of experiments performed can be found in (A). Ubiquitous expression of Q10 reduced longevity in female flies but not males, and 92Q expression decreased female longevity (B, E). Pan-neuronal expression of Q92 decreased female longevity (C) and both Q10 and Q92 male flies had reduced survival compared to controls (F). Glial expression of ATXN7 Q10 reduced longevity in females and males, and glial Q92 expression did not (D, G). n>200 flies for all groups, analyzed by log-rank.
Figure 4:
Figure 4:. Effects of ataxin-7 expression on motility.
(A) Both Q10 and Q92 expression reduced mobility in female flies. (B) Female flies expressing Q10 in neurons have slightly better climbing speed than background control flies. (C) Glial expression of Q92 reduces female climbing speed across ages, and Q10 expression impairs climbing further. (D-F) Male flies expressing Q10 or Q92 have lower climbing speed than age-matched controls at days 5 and 10 whether ATXN7 is expressed in all tissues (D), neurons (E), or glia (F). n≥100 per genotype, per sex, analyzed by linear regression.
Figure 5:
Figure 5:. Effects of fly-eye specific ataxin-7 expression.
(A) Pigmentation is normal in control and Q10 flies across ages. Q92 flies have pseudopupil (dotted circles) loss by adult day 56 n≥10. (B) CD8-GFP expression declines slightly with age in control flies (left panels, quantified at right; black histograms). Both Q10 (middle panels, green histograms) and Q92 (right panels, magenta histograms) have lower GFP fluorescence than age-matched control flies, and Q92 flies have lower GFP fluorescence than both control and Q10 flies by day 20 n≥29. (C) 5μM histological sections of control (left), Q10 (middle) and Q92 (right) flies at adult day 1 (upper panels). At adult day 56 (lower panels) Q92 flies have darkly stained aggregates, corneal disruption, loss of ommatidial boundaries (red bracket) and retinal disorganization. n≥6. (D) Western blots of ATXN7 in fly eyes over time. SDS-resistant species are present in the upper portions of the blot in Q92 flies by adult day 35. Lysates from 20 fly heads and at least 2 biological replicates performed per group.
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