Phenotypic defects from the expression of wild-type and pathogenic TATA-binding proteins in new Drosophila models of Spinocerebellar Ataxia Type 17

Abstract Spinocerebellar Ataxia Type 17 (SCA17) is the most recently identified member of the polyglutamine (polyQ) family of disorders, resulting from abnormal CAG/CAA expansion in the TATA box-binding protein (TBP), an initiation factor essential for of all eukaryotic transcription. A largely autosomal dominant inherited disease, SCA17, is unique in both its heterogeneous clinical presentation and low incidence of genetic anticipation, the phenomenon in which subsequent generations inherit longer polyQ expansions that yield earlier and more severe symptom onset. Like other polyQ disease family members, SCA17 patients experience progressive ataxia and dementia, and treatments are limited to preventing symptoms and increasing quality of life. Here, we report 2 new Drosophila models that express human TBP with polyQ repeats in either wild-type or SCA17 patient range. We find that TBP expression has age- and tissue-specific effects on neurodegeneration, with polyQ-expanded SCA17 protein expression generally having more severe effects. In addition, SCA17 model flies accumulate more aggregation-prone TBP, with a greater proportion localizing to the nucleus. These new lines provide a new resource for the biochemical characterization of SCA17 pathology and the future identification of therapeutic targets.


Introduction
The polyglutamine (polyQ) family of progressive neurodegenerative disorders comprises a group of 9 diseases caused by abnormal expansion of the trinucleotide repeat (CAG/A) that encodes glutamine (Zoghbi and Orr 2000;La Spada and Taylor 2003;Todi et al. 2007b;Paulson et al. 2017;Lieberman et al. 2019;Buijsen et al. 2019;Liu et al. 2019).Spinocerebellar Ataxia Type 17 (SCA17) is the most recently discovered member of the polyQ disease family, resulting from CAG/CAA expansion in the gene that encodes the TATA box-binding protein (TBP), an essential player in the basal transcriptional machinery of all eukaryotes (Koide et al. 1999;Nakamura et al. 2001).SCA17 is largely an autosomal dominant inherited disorder (Fujigasaki et al. 2001), but de novo TBP mutations have also been reported (Bech et al. 2010) in patients.
Like other diseases in the polyQ family, SCA17 usually manifests in adulthood with classical symptoms that include ataxia, dystonia, and dementia (Toyoshima and Takahashi 2018;Liu et al. 2019).Juvenile-onset SCA17 has also been reported, characterized by fast progressing symptoms including ataxia, muscle weakness, intellectual decline, growth defects, and early mortality (Koide et al. 1999;Maltecca et al. 2003).Brain imaging is characterized by cerebellar and brainstem atrophy and degeneration (Nakamura et al. 2001).SCA17 is unique, however, due to the extensive variability in both clinical presentation and age of onset, and the unusually small gap between normal and pathological polyQ repeat length (Toyoshima and Takahashi 2018;Gardiner et al. 2019;Liu et al. 2019).Wild-type alleles of TBP have 25-40 CAG/A repeats, while most SCA17 mutations fall within the repeat range of 46-55 (Gao et al. 2008).To date, sporadic and familial SCA17 patients have been documented with CAG/CAA expansions ranging from 41-66 repeats (Magri et al. 2022).There is a weak correlation between repeat length and age of onset, with juvenile forms usually associated with polyQ lengths over 62, but several SCA17 studies find high genetic heterogeneity, even among the same family of patients (Liu et al. 2019).In fact, full disease presentation has been reported in patients with CAG/CAA repeat lengths as low as 41 (Origone et al. 2018).Disease penetrance is variable, with intermediate TBP alleles harboring 41-48 CAG/CAA repeats yielding a 50% reduction in SCA17 presentation (Toyoshima and Takahashi 2018;Magri et al. 2022).Recent investigations indicate a pathogenic interaction with the protein quality control gene STUB1, suggesting that TBP genetic variation and interaction with other genetic factors contribute to incomplete penetrance of SCA17 (Magri et al. 2022).
Abnormal polyQ expansion in disease genes causes protein misfolding and aggregation, forming toxic intraneuronal inclusions that lead to neuronal degeneration and death (Zoghbi andOrr 2000, 2009).In SCA17 patients, polyQ-expanded TBP accumulates in Purkinje neurons in the cerebellum, a finding that is recapitulated in animal models (Friedman et al. 2007;Ren et al. 2011).Studies in animal models have also discovered that polyQ expansion in TBP reduces endogenous protein levels and disrupts its normal function, yielding transcriptional dysregulation that further contributes to SCA17 disease progression (Friedman et al. 2008).Earlier Drosophila models employing expanded and amorphic protein expression have provided important information about TBP function, dysfunction, and interactions during SCA17 disease progression (Ren et al. 2011;Hsu et al. 2014).
To more thoroughly characterize the contribution of TBP variants to SCA17, we report new Drosophila transgenic lines of SCA17 that express HA-tagged, full-length human TBP with CAG/CAA repeats encoding either Q25 (wild-type) or Q63 (SCA17).These new lines leverage both the phiC31 integrase system for construct integration at a conserved location (Groth et al. 2004) and the Drosophila Gal4-UAS GeneSwitch system (Roman and Davis 2002).The use of identical genetic backgrounds allows for comparison between SCA17 models with polyQ expansions in wild-type and SCA17 patient range.We find that global expression of either construct causes developmental arrest or delay, and that adult-restricted, ectopic expression of either Q25 or Q63 reduces survival and mobility whether the expression pattern is ubiquitous, in neurons, or in glia.In general, polyQ-expanded TBP expression is more toxic in adult flies and is associated with increased aggregation propensity of disease protein, particularly in mid-to late-life, compared to its wild-type version.This model adds to the toolkit of genetically isogenous Drosophila polyQ models, making possible rapid, cost-effective investigations and increasing our mechanistic understanding of SCA17 pathology.
Prior to all experiments, fly cultures were maintained at a constant density for at least 2 generations.Twenty to twenty-five virgin females (depending on genotype) and 5 males were mated in 300-mL bottles with 50-mL standard 10% sucrose 10% yeast spiked with 500-μL Penicillin-Streptomycin [10,000 u/mL, 10 mg/ mL in 0.9% sterile NaCl (Sigma-Aldrich)].Adult progeny were synchronized by collecting within 6 hours of eclosion over a 24-hour time period.Groups of 20 age-and sex-matched flies were immediately transferred into narrow polypropylene vials containing 5 mL of standard 10% sucrose 10% yeast (no antibiotic) or RU486 food as indicated by experiment.
Flies were housed in a 25°C incubator on a 12:12-hour light: dark cycle at 40% relative humidity.Control flies for all non-gene-switch Gal4-UAS experiments consisted of heterozygous Gal4 lines backcrossed into y, w; +; attP2 and/or w 1118 flies, depending on experiment.For gene-switch experiments, RU− flies of the same genotype served as the negative control.RU+ group received 100-μM RU486/mifepristone (Cayman Chemical, Ann Arbor, MI, USA), which activates the gene switch (GS) driver, while RU− group received the same volume of vehicle solution (70% ethanol).Adult progeny were synchronized by collecting within 12 hours of eclosion over a 24-hour time period.Groups of 20 age-and sex-matched flies were immediately transferred into narrow polypropylene vials containing 5 mL of 2% gelidium agar, 10% sucrose, and 10% brewer's yeast with appropriate preservatives (for full recipe, see Toivonen et al. 2007).Food vials were changed every second to third day.

Developmental biology
Developmental survival was performed by placing 24 gravid female control, Q25 or Q63 flies in aerated 6-oz bottles capped with grape juice agar plates seeded with yeast for 72 hours, after which adults were removed and embryo counted.Embryo were New Drosophila models of SCA17 | 3 transferred to 6-oz bottles containing standard 10% sucrose/10% yeast media and allowed to develop at 25°C.Viable adults or arrested pharate pupae were phenotypically scored and counted twice per day.
A similar method was used to score pupariation rate, with the day of embryo transfer considered hour 48 (±24 hours after egg laying).Twice daily, bottles from each genotype were scored for pupariation until no more larvae emerged.Following pupariation rate assay, a representative sample of at least 10 pharates per genotype was removed and affixed to a glass slide for imaging and measurement at 2× magnification.All pupae were isolated and photographed on the same day.Images were quantified using Image J. Three individual bottle crosses were performed for each experiment.Statistical analyses were performed in GraphPad Prism (San Diego, CA, USA), specified in figure legends.

Longevity
At least 400 adults were age-matched and separated by sex within 12 hours of eclosion.Flies were transferred to narrow polypropylene vials containing 5 mL of standard 2% agar, 10% sucrose, and 10% yeast food.Flies were transferred and scored for death events every 2-3 days until no flies remained.Survival curves were analyzed by log-rank in GraphPad Prism (San Diego, CA, USA).Longevity experiments were performed in duplicate and in parallel with background controls, with each individual graph depicting a representative biological repetition.

Climbing speed
Mobility was assessed using the Rapid Iterative Negative Geotaxis (RING) assays in groups of at least 100 flies as described in Sujkowski et al. (2022).Briefly, 5 vials of 20 age-and sex-matched flies were briskly tapped down, then measured for climbing distance 2 seconds after inducing the negative geotaxis instinct.For each group of vials, an average of 5 consecutive trials was calculated and batch processed using ImageJ (Bethesda, MD, USA).Flies were longitudinally tested twice per week for 5 weeks or until fewer than 5 flies remained per vial.Between assessments, flies were returned to food vials and housed normally as described above.Negative geotaxis results were analyzed using linear regression comparing differences in slope and y-intercept using GraphPad Prism (San Diego, CA, USA).Slope measures of rate of mobility decline, while y-intercept (arbitrary units, see Sujkowski et al. 2022) represents height climbed in 2 seconds at baseline.Individual y-values depict climbing height in 2 seconds at the age indicated on the x-axis.P-values depicted by a ">" symbol represent comparisons where both slope and intercept are not different.All statistically significant P-values (<0.05) represent differences in y-intercept.Mobility experiments were performed in duplicate, with 1 complete trial shown in each graph with the following exception: For both longevity and motility measurements in Fig. 3, viability of sqh-Gal4 > UAS TBPQ25 was too low for either parametric assessment of survival or regression analysis of mobility.Mixed-effects models were used to estimate significance, and remaining experiments utilized adult-specific expression to circumvent developmental lethality and obtain necessary statistical power.

Eye scoring
Eye scores were represented using the below scoring system, with higher numbers indicating worsening phenotypes as follows:

NMJ histology
Neuromuscular junction (NMJ) dissections and staining were modified from Sidisky and Babcock (2020).Briefly, whole flies were anesthetized with fly nap then submerged for 30 seconds in 70% ethanol to remove wax coating from cuticle before being transferred to a Sylgard coated dissecting dish.Thoraxes were isolated and fixed in 4% paraformaldehyde for 60 minutes.Following fixation, thoraxes were submerged in liquid nitrogen for 10 seconds, bisected with a sharp razor blade under a dissecting scope, and transferred to ice cold PBS.Samples were then blocked for 2 hours before staining overnight with primary antibodies (rabbit TFIID, Santa Cruz, 1:100).The following day, tissues were washed, and stained with fluorescent conjugated primary and secondary antibodies [AlexaFluor (AF)640 HRP, 1:200, AF594 phalloidin, 1:1,000, anti-rabbit AF488, 1:200, DAPI, 1:10,000, ThermoFisher], and imaged using the WSU Department of Physiology Confocal Microscopy Core using a Leica DMI 6000 outfitted with a Photometrics Prime 95B CMOS camera and X-light spinning disc Confocal.Colocalization was quantified using ImageJ using 10 biological replicates per genotype, and statistical analysis (Student t-test) was performed in GraphPad Prism.Colocalization analysis was performed on individual slices by comparing % area of overlap of green and blue channels and dividing by % area of the blue channel.Dots indicate individual biological replicates.

Generation of wild-type and polyQ-expanded SCA17 models
We have generated several Gal4-UAS model lines that express human SCA disease proteins (Tsou et al. 2015(Tsou et al. , 2016;;Johnson et al. 2019;Prifti et al. 2023), leveraging a cloning strategy that yields a single-copy, phiC31-dependent insertion into the same attp2 integration site, in the same orientation, on the third Drosophila chromosome (Groth et al. 2004).This expression system allows us to easily modify protein expression and makes it possible to compare across our Drosophila SCA models under conditions of similar transgene expression levels and identical genetic background (Markstein et al. 2008;Ni et al. 2008;Tsou et al. 2016).As previously mentioned, wild-type TBP alleles range from 25 to 40 polyQ repeats, whereas SCA17 expansions extend from 41 to 66 repeats, with CAA interruptions that may further stabilize disease penetrance (Liu et al. 2019).We used this strategy to generate 2 new Drosophila lines that express HA-tagged human TBP harboring repeat lengths of either Q25 (wild-type) or Q63 (SCA17), CAG/CAA polyQ tracts specifically designed to be within patient range (Toyoshima and Takahashi 2018) (Fig. 1a).Genomic DNA was isolated from founder lines, and the inserted sequences were amplified and sequenced, confirming integration into the third chromosome, in the correct insertion site and in the proper orientation (Fig. 1b).

Developmental toxicity in TBP-expressing flies
TBP is a widely expressed protein.Thus, we first examined the toxicity of each transgene using the constitutive, ubiquitous driver sqh-Gal4.While SCA17 usually presents in adulthood, juvenile-onset is also possible, and patient ages range from 3 to 60 years (Toyoshima and Takahashi 2018).Constitutive, ubiquitous expression of either Q25 or Q63 in Drosophila led to marked developmental lethality compared to control flies (Fig. 2a).Nearly all Q25 flies died at "pharate" pupal stage, without emerging as adults, while ubiquitous Q63 expression caused high percentages of both embryonic and pupal lethality, with fewer than 10% of flies surviving to adulthood (Fig. 2a).Interestingly, more Q63 flies survived to adulthood than those expressing Q25, suggesting a dominant negative effect of exogenous, wild-type TBP expression that may interfere with basal transcriptional machinery critical for development.
Development was delayed in both models compared to background control flies, with Q63 expression extending time to pupariation by approximately 1 day and Q25 expression nearly doubling pupariation time compared to controls (Fig. 2b).Perturbations in maturation timing are often associated with disruptions in organismal growth (Delanoue and Romero 2020), and we observed small pupal size in Q25 flies (Fig. 2c and d), while pupal length-to-width ratio was greater than controls in flies ubiquitously expressing Q63 (Fig. 2c and d).Previous Drosophila lines with exogenous human TBP expression showed disrupted intrinsic TBP transcriptional function (Hsu et al. 2014), supporting a link between dysfunctional TBP and SCA17 pathogenesis.This finding is partially supported by the high amino acid sequence similarity of TBP between flies and humans (∼64% identity; Fig. 2e).Altogether, these results highlight the critical importance of TBP sequence integrity and expression level to normal development, with perturbations from exogenous wild-type TBP expression severely impacting developmental timing and growth in these fly models.

Constitutive TBP expression reduces adult lifespan and mobility and impacts fly eye uniformity
We next assessed longitudinal physiology in our Q25 and Q63 flies that successfully emerged as adults.Expression of human polyQ proteins in Drosophila neurodegeneration models tends to reduce survivorship and impair mobility, phenocopying disease presentation in human patients (Blount et al. 2014;Tsou et al. 2015Tsou et al. , 2016;;Sutton et al. 2017;Ristic et al. 2018;Johnson et al. 2019;Walters et al. 2019;Johnson et al. 2022a;Sujkowski et al. 2022;Prifti et al. 2023).Compared to age-matched background controls, ubiquitous Q25 expression significantly decreased survival in both female and male flies, and survival in Q63 male flies was reduced even further (Fig. 3a).Similarly, mobility was severely impaired across ages in both female (Fig. 3b) and male (Fig. 3c) Q25 flies, while Q63 expression impacted mobility more severely, with most flies not responding to the climbing stimulus and remaining on the bottom of the vial (Fig. 3d).
The Drosophila eye has been used extensively to assess neural toxicity in a variety of neurodegenerative disease models (St Johnston 2002;Matsumoto et al. 2003;Passarella and Goedert 2018;Mishra and Knust 2019;Smylla et al. 2021).We therefore used GMR-Gal4 to drive expression of each construct specifically in eyes, observing for phenotypic neurodegeneration with a scoring system we have used before (Fig. 4a).Neither construct caused detectable anomalies in the external eye in 1-week-old flies (Fig. 4b, upper panels), but progressive defects were observed in both Q25 and Q63 expressing flies by week 5 (middle panels) that worsened by 10 weeks of age (Fig. 4b bottom panels, quantifications in Fig. 4c).Taken together, these results indicate that both wild-type (Q25) and polyQ-expanded (Q63) human TBP expression is toxic in male and female Drosophila, with Q25 expression having pronounced effects on normal development and Q63 expression more severely impacting longevity, motility, and eye morphology.

Adult-specific TBP expression in Drosophila
In order to circumvent the high developmental lethality of our constructs, we next used the inducible Gal4-UAS GeneSwitch system (Roman and Davis 2002) to restrict TBP expression to adult tissues.We and others have shown that polyQ proteins increase in amount and aggregation propensity with age, and that disease protein aggregation is associated with toxicity (Blount et al. 2014;Tsou et al. 2015Tsou et al. , 2016;;Sutton et al. 2017;Ristic et al. 2018;Johnson et al. 2019;Blount et al. 2020;Johnson et al. 2022a;Sujkowski et al. 2022).Flies expressing either Q25 or Q63 TBP in all adult tissues [RU+ (ON), Fig. 5a and d; Supplementary Fig. 1a  and d], had strong TBP expression in weeks 1, 3, and 5 of adult life.To assess aggregation propensity, we resolved both Q25 and Q63 proteins through SDS-PAGE and quantified SDS-soluble vs SDS-resistant species across timepoints.In both female (Fig. 5a  and d) and male (Supplementary Fig. 1a and d) Q63 flies, SDS-resistant protein increased in weeks 3 and 5.In contrast, Q25 expression did not significantly alter SDS-resistant protein species at any age.
We observed similar patterns of TBP accumulation and SDS-resistant migration when expression was restricted to either adult neurons (Fig. 5b and e; Supplementary Fig. 1b and e) or adult glia (Fig. 5c and f; Supplementary Fig. 1c and f).In both female and male flies, Q25 and Q63 expression was strongly detected across ages, and aggregation propensity increased in Q63 flies whether protein expression was restricted to adult neurons or glia, with the greatest increase in SDS-resistant protein accumulation at week 5.Our results indicate that although overall protein levels of either construct do not necessarily increase with age, Q63 protein is more prone to progressive aggregation whether expressed in adult neurons, adult glia, or in all adult tissues.

Tissue-specific adult TBP expression negatively impacts aging
We next assessed tissue-and time-dependent effects of TBP expression on longevity and mobility using the same inducible drivers as above.First, we examined the effects of global, adult-specific expression using Tub5-Gal4 (GS), since TBP is ubiquitously expressed throughout the body (Liu et al. 2019).Adult-specific expression of either Q25 (Fig. 6a and b; Supplementary Fig. 2a and b) or Q63 (Fig. 6c  and d; Supplementary Fig. 2a and b) reduced the lifespan in female flies without significant effects on mobility.
Like other members of the polyQ disease family, neurons are particularly susceptible to degeneration and cell death despite TBP being widely expressed (Liu et al. 2019).Adult-specific, panneural Q25 expression using elav-Gal4 (GS) did not reduce either lifespan (Fig. 7a) or mobility (Fig. 7b) in comparison to uninduced background control flies.To further explore the connection between TBP expression and SCA17 disease phenotypes, we then examined intracellular TBP localization.One common pathological hallmark of polyQ expansion diseases is the formation of neuronal intranuclear inclusions (Lieberman et al. 2019), a finding recapitulated in both cell and animal models of SCA17 (Friedman et al. 2007;Huang et al. 2011).In flies expressing Q25 specifically in adult neurons, histological examination of the adult NMJ showed TBP localization in both the nucleus and cytoplasm of muscle cells, with approximately half of the nuclei positive for TBP (Fig. 7e  and f).
Unlike TBP Q25, expression of TBP Q63 specifically in adult neurons shortened the lifespan in both female and male flies (Fig. 7c; Supplementary Fig. 2c) and significantly reduced mobility compared to age-matched, uninduced controls (Fig. 7d; Supplementary Fig. 2d).In addition, our neural Q63 model had a greater proportion of intranuclear TBP colocalization than Q25 flies (Fig. 7e and f).
We then repeated these studies using Repo-Gal4 (GS) to restrict TBP expression to adult glia.We and others have demonstrated that polyQ expansion in glial cells contributes to progressive neurodegenerative phenotypes in animal models (Furrer et al. 2011;Yang et al. 2017;Johnson et al. 2022a;Schuster et al. 2022) and may also participate in human SCA17 pathogenesis (Yang et al. 2017).Both Q25 and Q63 expression in adult glia decreased longevity (Fig. 8a and c; Supplementary Fig. 2e) and impaired mobility (Fig. 8b and d; Supplementary Fig. 2f), with glial-specific Q63 model females affected most severely (Fig. 8c and d; Supplementary Fig. 2e and f).Flies expressing either Q25 or Q63 in adult glia accumulated TBP intranuclearly, with the highest proportion of colocalization observed in the glial models of Q63 (Fig. 8e and f).
These new Drosophila lines allow for future biochemical characterization of the relative contribution of tissue-specific polyQ expansion to SCA17 disease progression.In SCA17, like other polyQ family members, certain neural populations are more sensitive to dysfunction and death despite disease proteins being widely expressed.These new models enable the identification of disease modifiers that drive specific phenotypes, opening the door for future characterization of protective targets in neural populations most susceptible to neurodegeneration.

Discussion
SCA17 is caused by abnormal expansion of the polyQ tract in TBP, a highly conserved component of the basal cellular machinery essential for eukaryotic transcription initiation, growth, and development of virtually all cells (Burley and Roeder 1996).It is perhaps not surprising that evidence from animal models suggests that TBP mutation contributes to SCA17 progression in 2 ways as follows: (1) through toxic gain-of-function, resulting in protein misfolding and neural cell death, and (2) through loss-of-function of endogenous TBP (Nakamura et al. 2001;Hsu et al. 2014;Toyoshima and Takahashi 2018;Liu et al. 2019).We describe here 2 new Drosophila models that ectopically express human TBP harboring polyQ tracts within either the wild-type (Q25) or SCA17 (Q63) disease range, providing novel tools to dissect the relative contribution of these effects to polyQ neurodegeneration.
Human and Drosophila TBP share approximately 64% amino acid similarity (Fig. 2e), and our data indicate that both our wild-type and polyQ-expanded transgenes perturb normal Drosophila development.Here, global expression of polyQ-expanded TBP confers high embryonic and pupal mortality, with about 10% of flies eclosing as adults.Interestingly, although a greater number of embryos expressing wild-type TBP survive to larval stages, developmental lethality is more severe, with arrest at the "pharate" pupal stage and almost no adult flies emerging.It is formally possible that ectopic wild-type human TBP expression has a dominant negative effect, outcompeting and interfering with endogenous dTbp function essential for normal development.In contrast, the increased aggregation propensity and nuclear accumulation of polyQ-expanded TBP may disrupt its normal transcriptional role, resulting in less developmental dysfunction but more severe adult-specific neurodegenerative effects.Taken together, these findings lend further support to the idea that exogenous TBP overexpression interferes with intrinsic transcriptional control, and that organismal TBP expression is precisely regulated to ensure normal function.
The essential role for TBP in transcription initiation is highlighted by the fact that loss-of-function in either flies or mice is developmentally lethal (Martianov et al. 2002;Hsu et al. 2014).Previous investigations further determined that polyQ-expanded TBP binds more tightly with DNA, disrupting intrinsic TBP function and resulting in compensatory loss of wild-type protein (Friedman et al. 2008;Huang et al. 2015).Our results further support these ideas, underscoring the contribution of dysfunctional TBP to SCA17 neurodegeneration.
We also observe neurodegenerative phenotypes when TBP is expressed specifically in fly eyes, a long-used genetic platform for understanding proteotoxic neurodegenerative diseases (Bonini 1999;Bonini and Fortini 2003;McGurk and Bonini 2012;Tsou et al. 2013;Blount et al. 2014;Burr et al. 2014;Casci and Pandey 2015;Tsou et al. 2015Tsou et al. , 2016)).Here, we notice age-and polyQ repeat length-dependent retinal defects in our Q25 and Q63 flies, allowing for future investigation of genetic or pharmacological modifiers of SCA17 neurodegeneration in a robust and easy-to-use genetic model.A prior model of SCA17 in Drosophila showed robust eye degeneration phenotype that was more severe than our observations (Ren et al. 2011).The difference in severity is likely due to the method use for transgene generation.The prior study utilized random insertion of the SCA17 construct, which can lead to multiple copy insertions.Our approach was to utilize a single-copy insertion, in the same orientation, between the new lines.Differences in expression levels as a result of varying numbers  of inserted copies, as well as perturbations in the genetic environment as a result of random insertion, most likely account for this difference between the studies.
Like other polyQ family disease members, neurons are particularly impacted by TBP misfolding, aggregation, and nuclear inclusion despite the mutant disease gene being broadly expressed.By restricting ectopic TBP expression to specific adult tissues, we observed cell-specific contributions to neurotoxicity in our Drosophila models.Adult-specific whole-body TBP expression reduced survival independent of repeat length without having significant effects on mobility.On the other hand, Q63 expression in either adult neurons or adult glia was consistently more toxic than Q25 expression in the same tissues, causing reductions in both longevity and mobility.These detrimental phenotypes correlated with higher Q63 aggregation (western blotting) and increased intranuclear puncta (histological preparations), in agreement with previous findings in animal models and humans (Yang et al. 2017;Toyoshima and Takahashi 2018;Liu et al. 2019).Although we did not directly measure neuromuscular synapse number or size, neural length and branching between groups appeared morphologically similar.Future work examining potential differences in synaptic complexity may further elucidate the neurodegenerative contribution of polyQ TBP on SCA17 symptomology.Our data suggest some proteotoxicity from both Q25 and Q63 expression in Drosophila, with more pronounced mobility effects when polyQ-expanded TBP is expressed in neurons and glia.Toxicity in flies with Q63 expression specifically in adult glia suggests a cell nonautonomous role of polyQ-expanded TBP that warrants further examination; cell nonautonomous effects for polyQ disease proteins have been described before in SCA7, HD, and SBMA (Todi et al. 2007a;Lieberman et al. 2019;Johnson et al. 2022b).
These new Drosophila lines are an important addition to the collective library of genetic tools for polyQ disease models, allowing efficient examination of the biochemical pathways that contribute to SCA17.TBP is universally essential for eukaryotic transcription, and understanding how its function and dysregulation contribute to neurotoxicity in animal models will aid in our understanding of both neural development and pathophysiology.Previous studies have also implicated mutant TBP dysfunction in other polyQ family disorders (Hsu et al. 2014), suggesting some common pathways of progressive neurodegeneration in this family of diseases.The curation of these tools, with comparable protein expression levels and identical genetic backgrounds, will allow us to precisely, expediently, and cost-effectively explore both disease-specific and shared pathways driving polyQ neurodegeneration.

Fig. 1 .
Fig. 1.Generation of Drosophila SCA17 models.a) Human TBP amino acid sequences used to model wild-type (Q25, upper), and pathogenic (Q63, lower) TBP, with polyQ mutations indicated in red.C-terminal HA tag in bold.b) Top: Schematic representation of the cloning strategy to insert TBP cDNA into pWALIUM10.moe.Bottom: Triplicate PCR from genomic DNA indicating that both transgenes were integrated into the correct insertion site in the proper orientation.

Fig. 2 .
Fig. 2. Developmental impact of ubiquitous TBP expression.a) Percentage embryonic (red) and pupal (blue) lethality, and adult eclosion (green).Data from 3 individual crosses, n-values indicated in panel.b) Pupariation rate for control (black) wild-type (blue) and SCA17 (red) model flies.Data from 3 individual crosses.Statistics: linear regression, n-values indicated in panel.c) Representative image of pharate pupae, quantified in (d).n ≥ 15, statistics: ANOVA with Dunnett's post hoc comparison.e) Human-Drosophila TBP amino acid sequence alignment.Alignment and legend are from Clustal Omega.Asterisk indicates fully conserved amino acids; period indicates conservation between groups of weakly similar properties, and : (colon) indicates conservation between groups of strongly similar properties.

Fig. 3 .
Fig. 3. Effects of ubiquitous TBP expression on Drosophila lifespan and mobility.Impact of constitutive, ubiquitous TBP Q63 (red) or TBP Q25 (blue) expression on survival (a) and climbing speed in female (b) and (c) male flies compared to age-matched controls (black).Open symbols, females, closed symbols, males.d) Representative image of day-1 climbing height for indicated genotypes 2 seconds after climbing induction.Scale bar = 95 mm.Statistics: Survival, log-rank, excluding sqh-Gal4 > UAS TBP (Q25) (low n, see Materials and methods).Motility: Linear regression for slope, intercept.The same flies were used for both mobility and survival.n-Values indicated on panels.A single P-value indicates identical P-values for all comparisons not indicated by brackets.

Fig. 4 .
Fig. 4. Effects of TBP expression in Drosophila eyes.a) Scoring parameters.b) Representative images of adult eyes in control flies (black) and flies expressing either Q25 (blue) or Q63 (red) in eyes at the indicated ages, quantified in (c).Statistics: Fisher exact probability test.Bars indicate mean ± SD, n ≥ 10.

Fig. 5 .
Fig. 5. Impact of aging on TBP aggregation.Representative western blots in uninduced female control [RU− (OFF)] and experimental flies [RU+ (ON)] with adult-specific, pathogenic TBP (Q63, red) or wild-type (Q25, black) TBP expression in weeks 1, 3, and 5 of adulthood, indicated by lane.Expression was induced on adult day 3 in (a) all tissues (quantified in d), neurons (b and e), or glia (c and f) and continued until sample isolation.Black arrows: TBP, red arrows: polyQ-expanded TBP, blue brackets: SDS-resistant TBP, asterisks: nonspecific signal.Quantification and statistics: Kruskal-Wallis test with Dunn's post hoc comparison.Bars indicate mean ± SD, n = 5 biological replicates of 3 flies per lysate.

Fig. 6 .
Fig. 6.Impact of adult-specific, ubiquitous TBP expression on longevity and motility.Representative survival (a and c) and climbing speed (b and d) experiments in male and female flies expressing Q25 (blue, a and b) and Q63 (red, c and d) TBP in all adult tissues compared to age-matched, uninduced control siblings (closed symbols).The same flies were used for both survival and climbing assays and assessed longitudinally.Statistics: survival: log-rank, climbing speed: linear regression.Longevity and motility experiments performed in 2 biological replicates, n ≥ 186.

Fig. 7 .
Fig. 7. Effect of adult-specific neural TBP expression on neuromuscular physiology.Representative survival (a and c) climbing speed (b and d) and experiments in male and female flies expressing Q25 (blue, a and b) and Q63 (red, c and d) TBP in adult neurons compared to age-matched, uninduced control siblings (closed symbols).The same flies were used for both survival and climbing assays and assessed longitudinally.Representative 100× images of adult NMJ for Q25 (e) and Q63 flies (f).Red: actin, yellow: HRP, green: TBP, blue: DAPI.Scale bar = 15 μm.Statistics: survival: log-rank, climbing speed: linear regression.Histology: Student t-test.Longevity and motility experiments performed in 2 biological replicates, n ≥ 184.Histology, n = 10.

Fig. 8 .
Fig. 8. Effect of adult-specific glial TBP expression on neuromuscular physiology.Representative survival (a and c) climbing speed (b and d) and experiments in male and female flies expressing Q25 (blue, a and b) and Q63 (red, c and d) TBP in adult glia compared to age-matched, uninduced control siblings (closed symbols).The same flies were used for both survival and climbing assays and assessed longitudinally.Representative 100× images of adult NMJ for Q25 (e) and Q63 flies (f).Red: actin, yellow: HRP, green: TBP, blue: DAPI.Scale bar = 15 μm.Statistics: survival: log-rank, climbing speed: linear regression.Histology: Student t-test.Longevity and motility experiments performed in 2 biological replicates, n ≥ 173.Histology, n = 10.