Ubiquitin-binding site 1 of pathogenic ataxin-3 regulates its toxicity in Drosophila models of Spinocerebellar Ataxia Type 3

Spinocerebellar Ataxia Type 3 (SCA3) is a member of the family of polyglutamine (polyQ) diseases that are caused by anomalous CAG triplet repeat expansions in several genes. SCA3 results from abnormal polyQ expansion in the deubiquitinase (DUB), ataxin-3 (Atxn3). To understand the role of the different domains of mutant Atxn3 on its pathogenicity, with the hope that they can be explored for therapeutic interventions, we have systematically studied their individual and collective effects on its toxicity. One such domain is ubiquitin-binding site 1 (UbS1) on the catalytic domain of Atxn3; UbS1 is necessary for the enzymatic activity of Atxn3. Here, we investigated the importance of UbS1 on the toxicity of pathogenic Atxn3. We generated transgenic Drosophila melanogaster lines that express polyQ-expanded Atxn3 with and without a functional UbS1. We found that mutating UbS1 markedly exacerbates the toxicity of pathogenic Atxn3. Additional studies indicated that UbS1 regulates the toxicity of Atxn3 not by affecting its aggregation or sub-cellular localization, but by impacting its role in ubiquitin processing. Our findings provide additional insights into the role of Atxn3’s domains in the pathogenicity of SCA3

Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin‐3

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102210/1/emboj2008289-sup-0001.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102210/2/emboj2008289.pd

A Phenotypically Robust Model of Spinal and Bulbar Muscular Atrophy in Drosophila

Spinal and bulbar muscular atrophy (SBMA) is an X-linked disorder that affects males who inherit the androgen receptor (AR) gene with an abnormal CAG triplet repeat expansion. The resulting protein contains an elongated polyglutamine (polyQ) tract and causes motor neuron degeneration in an androgen-dependent manner. The precise molecular sequelae of SBMA are unclear. To assist with its investigation and the identification of therapeutic options, we report here a new model of SBMA in Drosophila melanogaster. We generated transgenic flies that express the full-length, human AR with a wild-type or pathogenic polyQ repeat. Each transgene is inserted into the same safe harbor site on the third chromosome of the fly as a single copy and in the same orientation. Expression of pathogenic AR, but not of its wild-type variant, in neurons or muscles leads to consistent, progressive defects in longevity and motility that are concomitant with polyQ-expanded AR protein aggregation and reduced complexity in neuromuscular junctions. Additional assays show adult fly eye abnormalities associated with the pathogenic AR species. The detrimental effects of pathogenic AR are accentuated by feeding flies the androgen, dihydrotestosterone. This new, robust SBMA model can be a valuable tool toward future investigations of this incurable disease

The Deubiquitinating Enzyme Ataxin-3 Regulates Ciliogenesis and Phagocytosis in the Retina

Expansion of a CAG repeat in ATXN3 causes the dominant polyglutamine disease spinocerebellar ataxia type 3 (SCA3), yet the physiological role of ATXN3 remains unclear. Here, we focus on unveiling the function of Ataxin-3 (ATXN3) in the retina, a neurological organ amenable to morphological and physiological studies. Depletion of Atxn3 in zebrafish and mice causes morphological and functional retinal alterations and, more precisely, photoreceptor cilium and outer segment elongation, cone opsin mislocalization, and cone hyperexcitation. ATXN3 localizes at the basal body and axoneme of the cilium, supporting its role in regulating ciliary length. Abrogation of Atxn3 expression causes decreased levels of the regulatory protein KEAP1 in the retina and delayed phagosome maturation in the retinal pigment epithelium. We propose that ATXN3 regulates two relevant biological processes in the retina, namely, ciliogenesis and phagocytosis, by modulating microtubule polymerization and microtubule-dependent retrograde transport, thus positing ATXN3 as a causative or modifier gene in retinal/macular dystrophies

Ubiquitin-Specific Protease 25 Functions in Endoplasmic Reticulum-Associated Degradation

Endoplasmic Reticulum (ER)-associated degradation (ERAD) discards abnormal proteins synthesized in the ER. Through coordinated actions of ERAD components, misfolded/anomalous proteins are recognized, ubiquitinated, extracted from the ER and ultimately delivered to the proteasome for degradation. It is not well understood how ubiquitination of ERAD substrates is regulated. Here, we present evidence that the deubiquitinating enzyme Ubiquitin-Specific Protease 25 (USP25) is involved in ERAD. Our data support a model where USP25 counteracts ubiquitination of ERAD substrates by the ubiquitin ligase HRD1, rescuing them from degradation by the proteasome

Myosin VIIA, Important for Human Auditory Function, Is Necessary for Drosophila Auditory Organ Development

BACKGROUND: Myosin VIIA (MyoVIIA) is an unconventional myosin necessary for vertebrate audition [1]-[5]. Human auditory transduction occurs in sensory hair cells with a staircase-like arrangement of apical protrusions called stereocilia. In these hair cells, MyoVIIA maintains stereocilia organization [6]. Severe mutations in the Drosophila MyoVIIA orthologue, crinkled (ck), are semi-lethal [7] and lead to deafness by disrupting antennal auditory organ (Johnston's Organ, JO) organization [8]. ck/MyoVIIA mutations result in apical detachment of auditory transduction units (scolopidia) from the cuticle that transmits antennal vibrations as mechanical stimuli to JO. PRINCIPAL FINDINGS: Using flies expressing GFP-tagged NompA, a protein required for auditory organ organization in Drosophila, we examined the role of ck/MyoVIIA in JO development and maintenance through confocal microscopy and extracellular electrophysiology. Here we show that ck/MyoVIIA is necessary early in the developing antenna for initial apical attachment of the scolopidia to the articulating joint. ck/MyoVIIA is also necessary to maintain scolopidial attachment throughout adulthood. Moreover, in the adult JO, ck/MyoVIIA genetically interacts with the non-muscle myosin II (through its regulatory light chain protein and the myosin binding subunit of myosin II phosphatase). Such genetic interactions have not previously been observed in scolopidia. These factors are therefore candidates for modulating MyoVIIA activity in vertebrates. CONCLUSIONS: Our findings indicate that MyoVIIA plays evolutionarily conserved roles in auditory organ development and maintenance in invertebrates and vertebrates, enhancing our understanding of auditory organ development and function, as well as providing significant clues for future research

Understanding the Role of the Josephin Domain in the PolyUb Binding and Cleavage Properties of Ataxin-3

Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites through which it anchors polyubiquitin chains of different linkages that are then cleaved by the N-terminal catalytic (Josephin) domain. The properties of the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well established. Very little is known, however, about how two recently identified ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain binding and cleavage. In the current study, we sought to define the specific contribution of the Josephin domain to the catalytic properties of ataxin-3 and assess how the topology and affinity of these binding sites modulate ataxin-3 activity. Using NMR we modeled the structure of diUb/Josephin complexes and showed that linkage preferences are imposed by the topology of the two binding sites. Enzymatic studies further helped us to determine a precise hierarchy between the sites. We establish that the structure of Josephin dictates specificity for K48-linked chains. Site 1, which is close to the active site, is indispensable for cleavage. Our studies open the way to understand better the cellular function of ataxin-3 and its link to pathology

Alpha- and beta-adrenergic octopamine receptors in muscle and heart are required for Drosophila exercise adaptations.

Endurance exercise has broadly protective effects across organisms, increasing metabolic fitness and reducing incidence of several age-related diseases. Drosophila has emerged as a useful model for studying changes induced by chronic endurance exercise, as exercising flies experience improvements to various aspects of fitness at the cellular, organ and organismal level. The activity of octopaminergic neurons is sufficient to induce the conserved cellular and physiological changes seen following endurance training. All 4 octopamine receptors are required in at least one target tissue, but only one, Octβ1R, is required for all of them. Here, we perform tissue- and adult-specific knockdown of alpha- and beta-adrenergic octopamine receptors in several target tissues. We find that reduced expression of Octβ1R in adult muscles abolishes exercise-induced improvements in endurance, climbing speed, flight, cardiac performance and fat-body catabolism in male Drosophila. Importantly, Octβ1R and OAMB expression in the heart is also required cell-nonautonomously for adaptations in other tissues, such as skeletal muscles in legs and adult fat body. These findings indicate that activation of distinct octopamine receptors in skeletal and cardiac muscle are required for Drosophila exercise adaptations, and suggest that cell non-autonomous factors downstream of octopaminergic activation play a key role

Polyglutamine length-dependent toxicity from α1ACT in Drosophila models of spinocerebellar ataxia type 6

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease that results from abnormal expansion of a polyglutamine (polyQ) repeat. SCA6 is caused by CAG triplet repeat expansion in the gene CACNA1A, resulting in a polyQ tract of 19-33 in patients. CACNA1A, a bicistronic gene, encodes the α1A calcium channel subunit and the transcription factor, α1ACT. PolyQ expansion in α1ACT causes degeneration in mice. We recently described the first Drosophila models of SCA6 that express α1ACT with a normal (11Q) or hyper-expanded (70Q) polyQ. Here, we report additional α1ACT transgenic flies, which express full-length α1ACT with a 33Q repeat. We show that α1ACT33Q is toxic in Drosophila, but less so than the 70Q version. When expressed everywhere, α1ACT33Q-expressing adults die earlier than flies expressing the normal allele. α1ACT33Q causes retinal degeneration and leads to aggregated species in an age-dependent manner, but at a slower pace than the 70Q counterpart. According to western blots, α1ACT33Q localizes less readily in the nucleus than α1ACT70Q, providing clues into the importance of polyQ tract length on α1ACT localization and its site of toxicity. We expect that these new lines will be highly valuable for future work on SCA6