dAtaxin-2 Mediates Expanded Ataxin-1-Induced Neurodegeneration in a Drosophila Model of SCA1

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of neurodegenerative disorders sharing atrophy of the cerebellum as a common feature. SCA1 and SCA2 are two ataxias caused by expansion of polyglutamine tracts in Ataxin-1 (ATXN1) and Ataxin-2 (ATXN2), respectively, two proteins that are otherwise unrelated. Here, we use a Drosophila model of SCA1 to unveil molecular mechanisms linking Ataxin-1 with Ataxin-2 during SCA1 pathogenesis. We show that wild-type Drosophila Ataxin-2 (dAtx2) is a major genetic modifier of human expanded Ataxin-1 (Ataxin-1[82Q]) toxicity. Increased dAtx2 levels enhance, and more importantly, decreased dAtx2 levels suppress Ataxin-1[82Q]-induced neurodegeneration, thereby ruling out a pathogenic mechanism by depletion of dAtx2. Although Ataxin-2 is normally cytoplasmic and Ataxin-1 nuclear, we show that both dAtx2 and hAtaxin-2 physically interact with Ataxin-1. Furthermore, we show that expanded Ataxin-1 induces intranuclear accumulation of dAtx2/hAtaxin-2 in both Drosophila and SCA1 postmortem neurons. These observations suggest that nuclear accumulation of Ataxin-2 contributes to expanded Ataxin-1-induced toxicity. We tested this hypothesis engineering dAtx2 transgenes with nuclear localization signal (NLS) and nuclear export signal (NES). We find that NLS-dAtx2, but not NES-dAtx2, mimics the neurodegenerative phenotypes caused by Ataxin-1[82Q], including repression of the proneural factor Senseless. Altogether, these findings reveal a previously unknown functional link between neurodegenerative disorders with common clinical features but different etiology

Comparison of an expanded ataxia interactome with patient medical records reveals a relationship between macular degeneration and ataxia

Spinocerebellar ataxias 6 and 7 (SCA6 and SCA7) are neurodegenerative disorders caused by expansion of CAG repeats encoding polyglutamine (polyQ) tracts in CACNA1A, the alpha1A subunit of the P/Q-type calcium channel, and ataxin-7 (ATXN7), a component of a chromatin-remodeling complex, respectively. We hypothesized that finding new protein partners for ATXN7 and CACNA1A would provide insight into the biology of their respective diseases and their relationship to other ataxia-causing proteins. We identified 118 protein interactions for CACNA1A and ATXN7 linking them to other ataxia-causing proteins and the ataxia network. To begin to understand the biological relevance of these protein interactions within the ataxia network, we used OMIM to identify diseases associated with the expanded ataxia network. We then used Medicare patient records to determine if any of these diseases co-occur with hereditary ataxia. We found that patients with ataxia are at 3.03-fold greater risk of these diseases than Medicare patients overall. One of the diseases comorbid with ataxia is macular degeneration (MD). The ataxia network is significantly (P= 7.37 × 10−5) enriched for proteins that interact with known MD-causing proteins, forming a MD subnetwork. We found that at least two of the proteins in the MD subnetwork have altered expression in the retina of Ataxin-7266Q/+ mice suggesting an in vivo functional relationship with ATXN7. Together these data reveal novel protein interactions and suggest potential pathways that can contribute to the pathophysiology of ataxia, MD, and diseases comorbid with ataxia

Dorsoventral Boundary for Organizing Growth and Planar Polarity in the Drosophila Eye

A fundamental feature of developing tissues and organs is generation of planar polarity of cells in an epithelium with respect to the body axis. The Drosophila compound eye shows two-tier dorsoventral (DV) planar polarity. At the individual ommatidium level, the eight photoreceptors in each unit eye form a dorsoventrally asymmetric cluster. At the level of eye field, hundreds of ommatidia in the upper and lower halves of an eye are uniformly polarized dorsally or ventrally, respectively. This results in DV mirror symmetries about the equator. The uniform orientations of photoreceptor clusters over long distance in the eye field provide an excellent model for studying the genetic basis of long-range planar polarity. Ommatidial DV polarity can be detected in third instar eye imaginal disc during the early stage of retinal differentiation. The foundation for this DV polarity pattern is laid much earlier in undifferentiated eye disc. The eye disc primordium is partitioned into the DV compartments of independent cell lineages. The chapter outlines key genetic events involved in early DV patterning and growth of eye disc, and its potential role in organizing long-range signaling for DV planar polarity during later differentiation of the eye

Bar represses dPax2 and decapentaplegic to regulate cell fate and morphogenetic cell death in Drosophila eye.

The coordinated regulation of cell fate and cell survival is crucial for normal pattern formation in developing organisms. In Drosophila compound eye development, crystalline arrays of hexagonal ommatidia are established by precise assembly of diverse cell types, including the photoreceptor cells, cone cells and interommatidial (IOM) pigment cells. The molecular basis for controlling the number of cone and IOM pigment cells during ommatidial pattern formation is not well understood. Here we present evidence that BarH1 and BarH2 homeobox genes are essential for eye patterning by inhibiting excess cone cell differentiation and promoting programmed death of IOM cells. Specifically, we show that loss of Bar from the undifferentiated retinal precursor cells leads to ectopic expression of Prospero and dPax2, two transcription factors essential for cone cell specification, resulting in excess cone cell differentiation. We also show that loss of Bar causes ectopic expression of the TGFβ homolog Decapentaplegic (Dpp) posterior to the morphogenetic furrow in the larval eye imaginal disc. The ectopic Dpp expression is not responsible for the formation of excess cone cells in Bar loss-of-function mutant eyes. Instead, it causes reduction in IOM cell death in the pupal stage by antagonizing the function of pro-apoptotic gene reaper. Taken together, this study suggests a novel regulatory mechanism in the control of developmental cell death in which the repression of Dpp by Bar in larval eye disc is essential for IOM cell death in pupal retina

Drosophila TRAP230/240 are Essential Coactivators for Atonal in Retinal Neurogenesis

The TRAP (thyroid hormone receptor associated proteins)/Mediator complex serves as a transcriptional coactivator. In Drosophila, Kohtalo (Kto) and Skuld (Skd), homologs of TRAP subunits, TRAP230 and TRAP240, respectively, are necessary for eye development. However, the transcriptional activators that require Kto and Skd have not been identified. Here we provide evidence that Kto and Skd are essential for the function of transcription factor Atonal (Ato) in spatial patterning of proneural clusters in the morphogenetic furrow. In the absence of Kto/Skd, Ato fails to induce its inhibitory target events such as EGFR signaling and Scabrous expression that result in ectopic Ato expression in the space between proneural groups. Kto/Skd are also required for positive Ato functions to induce Ato targets such as Ato itself and Senseless within the proneural clusters. We also show that Skd forms a protein complex with Ato in vivo. These data suggest that Kto/Skd act as essential coactivators for Ato expression during early retinalneurogenesis

Identifying Disease Signatures in the Spinocerebellar Ataxia Type 1 Mouse Cortex

The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) is known to lead to the progressive degeneration of specific neuronal populations, including cerebellar Purkinje cells (PCs), brainstem cranial nerve nuclei and inferior olive nuclei, and spinocerebellar tracts. The disease-causing protein ataxin-1 is fairly ubiquitously expressed throughout the brain and spinal cord, but most studies have primarily focused on the role of ataxin-1 in the cerebellum and brainstem. Therefore, the functions of ataxin-1 and the effects of SCA1 mutations in other brain regions including the cortex are not well-known. Here, we characterized pathology in the motor cortex of a SCA1 mouse model and performed RNA sequencing in this brain region to investigate the impact of mutant ataxin-1 towards transcriptomic alterations. We identified progressive cortical pathology and significant transcriptomic changes in the motor cortex of a SCA1 mouse model. We also identified progressive, region-specific, colocalization of p62 protein with mutant ataxin-1 aggregates in broad brain regions, but not the cerebellum or brainstem. A cross-regional comparison of the SCA1 cortical and cerebellar transcriptomic changes identified both common and unique gene expression changes between the two regions, including shared synaptic dysfunction and region-specific kinase regulation. These findings suggest that the cortex is progressively impacted via both shared and region-specific mechanisms in SCA1

<i>dpp</i> overexpression results in excess IOM cells.

<p>(A-B) Pupal retinas at 24 h APF stained with anti-Dlg (red; cell boundary marker) and anti-Cut (green; cone cell marker). (A) Pupal retina from <i>w¹¹¹⁸</i> shows a normal ommatidium structure. Normal eye with GFP expression by <i>lz-Gal4</i> was shown. Scale bar = 10 µm. (B) <i>lz>dpp</i> (<i>lz-Gal4/+;UAS-dpp/+</i>) eye showed an increased number of IOM cells (arrowheads). Note that the number of cone cells was not changed. (C) Statistical analysis of total number of IOM cells. Error bars are standard error of the mean; **P<0.01, <i>t-test</i>. (D) Normal eye phenotype of <i>lz>gfp</i> (<i>lz-Gal4/+; UAS-gfp/+</i>). (E) Dpp overexpression by <i>lz-Gal4</i> caused roughened and bulged eye. Scale bar = 200 µm. (F) Co-overexpression of wild-type <i>BarH1</i> suppressed bulged eye phenotypes of the <i>lz>dpp</i>. (G) Comparison of dorsal eye views of (D) and (E). (H) Comparison of dorsal views of (E) and (F). (I) Relative eye size measured from the dorsal view (see Materials and Methods). The bulged eye phenotype of <i>lz>dpp</i> was rescued by co-overexpressing <i>BarH1</i>. Error bars are standard error of the mean; *P<0.05, <i>t-test</i>. N.S. (Not Significant). (J) Proposed model for the role of Bar in the regulation of cell fate and morphogenetic cell death (see Discussion). Note that Bar is required for the repression of the indicated genes in the undifferentiated basal cells, and it is unknown whether the repression is direct.</p

Bar suppresses programmed cell death induced by <i>rpr</i>.

<p>(A) <i>GMR-rpr</i> resulted in almost complete elimination of all retinal cells and showed small adult eye. (B) The reduction of <i>Bar</i> gene dosage (50% Bar reduction) in the <i>GMR-rpr</i> background partially restored ommatidia and eye size. (C) <i>Bar</i> LOF clones with EGUF system suppressed the effect of <i>GRM-rpr</i>. (D) Statistical analysis of eye size for (A-C). More than 15 eyes were scored for each genotype. (E) Overexpression of <i>rpr</i> gene by <i>GMR-Gal4</i> showed more severe phenotypes than <i>GMR-rpr</i>. (F) Knock-down of Bar expression by overexpressing double strand RNAi against <i>BarH1</i> (<i>GMR-Gal4/UAS-rpr</i>, <i>UAS-BarH1</i> RNAi) dramatically restored the eye size. (G) <i>GMR-Gal4/UAS-BarH1</i> RNAi showed normal eye phenotype. (H) Statistical analysis of eye size for (E-G). For G and H, error bars are standard error of the mean; ** P<0.01, Student's <i>t</i>-test. Scale Bar = 400 µm.</p

Bar regulates cone cell differentiation.

<p>Several cone cell makers were examined in <i>Bar</i> mutant clones generated from the following strain; <i>yw, Df(1)B^263-20, frt19a/frt19a, ubi-mRFP, hs-flp</i>. The mosaic clones were marked by loss of RFP in eye discs. (A) Pros (green) was ectopically expressed in the absence of <i>Bar</i>. (B) dPax2 (green) or (C) <i>dPax2-lacZ</i> expression was ectopically induced within <i>Bar</i> LOF mutant clones. (D) Cut (green) expression was reduced in the <i>Bar</i> LOF mutant clone. Scale bar = 20 µm.</p

Bar is required for <i>dpp</i> repression posterior to the furrow.

<p>(A) <i>dpp-lacZ</i> (red) was ectopically induced behind the MF (arrow) within <i>Bar LOF</i> mutant clone (arrowheads) identified by the absence of GFP clone marker (green). (B) Ectopic expression of <i>dpp-lacZ</i> (red) was observed in <i>Bar</i> LOF clones near the posterior margin (arrowheads). (C) Schematic of <i>dpp-lacZ</i> expression in <i>Bar</i> LOF clones at different positions (A, B & D). (D) pMad (grey) was ectopically induced (arrows in D') behind the furrow within <i>Bar</i> LOF mutant clone identified by the absence of RFP clone marker in the larval eye discs (<i>Df(1)B^263-20, frt19a/frt19a, ubi-mRFP, hs-flp; BS3.0-dpp-lacZ/+</i>). (E) Over-expression of wild-type <i>BarH1</i> in the basal undifferentiated cells by <i>lz-Gal4</i> strongly suppressed the ectopic expression of <i>dpp-lacZ</i>, especially in the posterior region of the <i>Bar</i> LOF clones marked by arrows, where BarH1 expression was ectopically induced. Genotype is <i>lz-Gal4, Df(1)B^263-20, frt19a/frt19a, ubi-GFP, hs-flp;BS3.0-dpp-lacZ/+;UAS-dBarH1/+</i>. <i>Bar</i> LOF clones were marked by the loss of GFP staining. <i>dpp-lacZ</i> and BarH1 were marked by anti-β-gal (grey) and anti-BarH1 (BH1, red), respectively. Scale bars = 20 µm.</p