Polyglutamine Genes Interact to Modulate the Severity and Progression of Neurodegeneration in Drosophila

The expansion of polyglutamine tracts in a variety of proteins causes devastating, dominantly inherited neurodegenerative diseases, including six forms of spinal cerebellar ataxia (SCA). Although a polyglutamine expansion encoded in a single allele of each of the responsible genes is sufficient for the onset of each disease, clinical observations suggest that interactions between these genes may affect disease progression. In a screen for modifiers of neurodegeneration due to SCA3 in Drosophila, we isolated atx2, the fly ortholog of the human gene that causes a related ataxia, SCA2. We show that the normal activity of Ataxin-2 (Atx2) is critical for SCA3 degeneration and that Atx2 activity hastens the onset of nuclear inclusions associated with SCA3. These activities depend on a conserved protein interaction domain of Atx2, the PAM2 motif, which mediates binding of cytoplasmic poly(A)-binding protein (PABP). We show here that PABP also influences SCA3-associated neurodegeneration. These studies indicate that the toxicity of one polyglutamine disease protein can be dramatically modulated by the normal activity of another. We propose that functional links between these genes are critical to disease severity and progression, such that therapeutics for one disease may be applicable to others.     

Overexpression of Cystathionine γ-Lyase Suppresses Detrimental Effects of Spinocerebellar Ataxia Type 3

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin-3 (ATXN3) gene resulting in toxic protein aggregation. Inflammation and oxidative stress are considered secondary factors contributing to the progression of this neurodegenerative disease. There is no cure that halts or reverses the progressive neurodegeneration of SCA3. Here we show that overexpression of cystathionine γ-lyase, a central enzyme in cysteine metabolism, is protective in a Drosophila model for SCA3. SCA3 flies show eye degeneration, increased oxidative stress, insoluble protein aggregates, reduced levels of protein persulfidation and increased activation of the innate immune response. Overexpression of Drosophila cystathionine γ-lyase restores protein persulfidation, decreases oxidative stress, dampens the immune response and improves SCA3-associated tissue degeneration. Levels of insoluble protein aggregates are not altered; therefore, the data implicate a modifying role of cystathionine γ-lyase in ameliorating the downstream consequence of protein aggregation leading to protection against SCA3-induced tissue degeneration. The cystathionine γ-lyase expression is decreased in affected brain tissue of SCA3 patients, suggesting that enhancers of cystathionine γ-lyase expression or activity are attractive candidates for future therapies.     

Interaction of Akt-phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCA1) is one of several neurological disorders caused by a CAG repeat expansion. In SCA1, this expansion produces an abnormally long polyglutamine tract in the protein ataxin-1. Mutant polyglutamine proteins accumulate in neurons, inducing neurodegeneration, but the mechanism underlying this accumulation has been unclear. We have discovered that the 14-3-3 protein, a multifunctional regulatory molecule, mediates the neurotoxicity of ataxin-1 by binding to and stabilizing ataxin-1, thereby slowing its normal degradation. The association of ataxin-1 with 14-3-3 is regulated by Akt phosphorylation, and in a Drosophila model of SCA1, both 14-3-3 and Akt modulate neurodegeneration. Our finding that phosphatidylinositol 3-kinase/Akt signaling and 14-3-3 cooperate to modulate the neurotoxicity of ataxin-1 provides insight into SCA1 pathogenesis and identifies potential targets for therapeutic intervention.     

Genome-wide screen for modifiers of ataxin-3 neurodegeneration in Drosophila

Spinocerebellar ataxia type-3 (SCA3) is among the most common dominantly inherited ataxias, and is one of nine devastating human neurodegenerative diseases caused by the expansion of a CAG repeat encoding glutamine within the gene. The polyglutamine domain confers toxicity on the protein Ataxin-3 leading to neuronal dysfunction and loss. Although modifiers of polyglutamine toxicity have been identified, little is known concerning how the modifiers function mechanistically to affect toxicity. To reveal insight into spinocerebellar ataxia type-3, we performed a genetic screen in Drosophila with pathogenic Ataxin-3-induced neurodegeneration and identified 25 modifiers defining 18 genes. Despite a variety of predicted molecular activities, biological analysis indicated that the modifiers affected protein misfolding. Detailed mechanistic studies revealed that some modifiers affected protein accumulation in a manner dependent on the proteasome, whereas others affected autophagy. Select modifiers of Ataxin-3 also affected tau, revealing common pathways between degeneration due to distinct human neurotoxic proteins. These findings provide new insight into molecular pathways of polyQ toxicity, defining novel targets for promoting neuronal survival in human neurodegenerative disease.     

A Drosophila homolog of the polyglutamine disease gene SCA2 is a dosage-sensitive regulator of actin filament formation

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in ataxin-2, the SCA2 gene product. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration remain unknown. In this study we have used genetic and molecular approaches to investigate the function of a Drosophila homolog of the SCA2 gene (Datx2). Like human ataxin-2, Datx2 is found throughout development in a variety of tissue types and localizes to the cytoplasm. Mutations that reduce Datx2 activity or transgenic overexpression of Datx2 result in female sterility, aberrant sensory bristle morphology, loss or degeneration of tissues, and lethality. These phenotypes appear to result from actin filament formation defects occurring downstream of actin synthesis. Further studies demonstrate that Datx2 does not assemble with actin filaments, suggesting that the role of Datx2 in actin filament formation is indirect. These results indicate that Datx2 is a dosage-sensitive regulator of actin filament formation. Given that loss of cytoskeleton-dependent dendritic structure defines an early event in SCA2 pathogenesis, our findings suggest the possibility that dysregulation of actin cytoskeletal structure resulting from altered ataxin-2 activity is responsible for neurodegeneration in SCA2.     

Phosphorylation of S776 and 14-3-3 Binding Modulate Ataxin-1 Interaction with Splicing Factors

Ataxin-1 (Atx1), a member of the polyglutamine (polyQ) expanded protein family, is responsible for spinocerebellar ataxia type 1. Requirements for developing the disease are polyQ expansion, nuclear localization and phosphorylation of S776. Using a combination of bioinformatics, cell and structural biology approaches, we have identified a UHM ligand motif (ULM), present in proteins associated with splicing, in the C-terminus of Atx1 and shown that Atx1 interacts with and influences the function of the splicing factor U2AF65 via this motif. ULM comprises S776 of Atx1 and overlaps with a nuclear localization signal and a 14-3-3 binding motif. We demonstrate that phosphorylation of S776 provides the molecular switch which discriminates between 14-3-3 and components of the spliceosome. We also show that an S776D Atx1 mutant previously designed to mimic phosphorylation is unsuitable for this aim because of the different chemical properties of the two groups. Our results indicate that Atx1 is part of a complex network of interactions with splicing factors and suggest that development of the pathology is the consequence of a competition of aggregation with native interactions. Studies of the interactions formed by non-expanded Atx1 thus provide valuable hints for understanding both the function of the non-pathologic protein and the causes of the disease.     

Molecular genetics of spinocerebellar ataxia type 8 (SCA8)

We previously reported that a transcribed but untranslated CTG expansion causes a novel form of ataxia, spinocerebellar ataxia type 8 (SCA8) (Koob et al., 1999). SCA8 was the first example of a dominant spinocerebellar ataxia that is not caused by the expansion of a CAG repeat translated into a polyglutamine tract. This slowly progressive form of ataxia is characterized by dramatic repeat instability and a high degree of reduced penetrance. The clinical and genetic features of the disease are discussed below.     

Pathogenic mechanisms of a polyglutamine-mediated neurodegenerative disease, spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCA1) is one of nine inherited neurodegenerative diseases caused by the expansion of a CAG trinucleotide repeat encoding a polyglutamine tract. SCA1 patients lose motor coordination and develop slurred speech, spasticity, and cognitive impairments. Difficulty with coordinating swallowing and breathing eventually causes death. Genetic evidence indicates that the disease mutation induces a toxic gain of function in the SCA1 encoded protein ATXN1. The discovery that residues in ATXN1 outside of the polyglutamine tract are crucial for pathogenesis hinted that alterations in the normal function of this protein are linked to its toxicity. Biochemical and genetic studies provide evidence that the polyglutamine expansion enhances interactions that are normally regulated by phosphorylation at Ser(776) and a subsequent alteration in its interaction with other cellular proteins. Moreover, the finding that other ATXN1 interactions are decreased in disease suggests that the polyglutamine expansion contributes to disease by both a gain-of-function mechanism and partial loss of function.     

Spinocerebellar ataxia 7 (SCA7)

Spinocerebellar ataxia 7 (SCA7) is a progressive autosomal dominant neurodegenerative disorder characterized clinically by cerebellar ataxia associated with progressive macular dystrophy. The disease affects primarily the cerebellum and the retina, but also many other CNS structures as the disease progresses. SCA7 is caused by expansion of an unstable trinucleotide CAG repeat encoding a polyglutamine tract in the corresponding protein, ataxin-7. Normal SCA7 alleles contain 4-35 CAG repeats, whereas pathological alleles contain from 36-306 CAG repeats. SCA7 has a number of features in common with other diseases with polyglutamine expansions: (i) the appearance of clinical symptoms above a threshold number of CAG repeats (>35); (ii) a correlation between the size of the expansion and the rate of progression of the disease: the larger the repeat, the faster the progression; (iii) instability of the repeat sequence (approximately 12 CAG/transmission) that accounts for the marked anticipation of approximately 20 years/generation. The CAG repeat sequence is particularly unstable and de novo mutations can occur during paternal transmissions of intermediate size alleles (28-35 CAG repeats). This can explain the persistence of the disease in spite of the anticipation that should have resulted in its extinction.     

Silencing Mutant Ataxin-3 Rescues Motor Deficits and Neuropathology in Machado-Joseph Disease Transgenic Mice

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein – ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.     

Novel Polyglutamine Model Uncouples Proteotoxicity from Aging

Polyglutamine expansions in certain proteins are the genetic determinants for nine distinct progressive neurodegenerative disorders and resultant age-related dementia. In these cases, neurodegeneration is due to the aggregation propensity and resultant toxic properties of the polyglutamine-containing proteins. We are interested in elucidating the underlying mechanisms of toxicity of the protein ataxin-3, in which a polyglutamine expansion is the genetic determinant for Machado-Joseph Disease (MJD), also referred to as spinocerebellar ataxia 3 (SCA3). To this end, we have developed a novel model for ataxin-3 protein aggregation, by expressing a disease-related polyglutamine-containing fragment of ataxin-3 in the genetically tractable body wall muscle cells of the model system C. elegans. Here, we demonstrate that this ataxin-3 fragment aggregates in a polyQ length-dependent manner in C. elegans muscle cells and that this aggregation is associated with cellular dysfunction. However, surprisingly, this aggregation and resultant toxicity was not influenced by aging. This is in contrast to polyglutamine peptides alone whose aggregation/toxicity is highly dependent on age. Thus, the data presented here not only describe a new polyglutamine model, but also suggest that protein context likely influences the cellular interactions of the polyglutamine-containing protein and thereby modulates its toxic properties.     

Molecular clearance of ataxin-3 is regulated by a mammalian E4

Insoluble aggregates of polyglutamine-containing proteins are usually conjugated with ubiquitin in neurons of individuals with polyglutamine diseases. We now show that ataxin-3, in which the abnormal expansion of a polyglutamine tract is responsible for spinocerebellar ataxia type 3 (SCA3), undergoes ubiquitylation and degradation by the proteasome. Mammalian E4B (UFD2a), a ubiquitin chain assembly factor (E4), copurified with the polyubiquitylation activity for ataxin-3. E4B interacted with, and thereby mediated polyubiquitylation of, ataxin-3. Expression of E4B promoted degradation of a pathological form of ataxin-3. In contrast, a dominant-negative mutant of E4B inhibited degradation of this form of ataxin-3, resulting in the formation of intracellular aggregates. In a Drosophila model of SCA3, expression of E4B suppressed the neurodegeneration induced by an ataxin-3 mutant. These observations suggest that E4 is a rate-limiting factor in the degradation of pathological forms of ataxin-3, and that targeted expression of E4B is a potential gene therapy for SCA3.     

CHIP protects from the neurotoxicity of expanded and wild-type ataxin-1 and promotes their ubiquitination and degradation

CHIP (C terminus of Hsc-70 interacting protein) is an E3 ligase that links the protein folding machinery with the ubiquitin-proteasome system and has been implicated in disorders characterized by protein misfolding and aggregation. Here we investigate the role of CHIP in protecting from ataxin-1-induced neurodegeneration. Ataxin-1 is a polyglutamine protein whose expansion causes spinocerebellar ataxia type-1 (SCA1) and triggers the formation of nuclear inclusions (NIs). We find that CHIP and ataxin-1 proteins directly interact and co-localize in NIs both in cell culture and SCA1 postmortem neurons. CHIP promotes ubiquitination of expanded ataxin-1 both in vitro and in cell culture. The Hsp70 chaperone increases CHIP-mediated ubiquitination of ataxin-1 in vitro, and the tetratricopeptide repeat domain, which mediates CHIP interactions with chaperones, is required for ataxin-1 ubitiquination in cell culture. Interestingly, CHIP also interacts with and ubiquitinates unexpanded ataxin-1. Overexpression of CHIP in a Drosophila model of SCA1 decreases the protein steady-state levels of both expanded and unexpanded ataxin-1 and suppresses their toxicity. Finally we investigate the ability of CHIP to protect against toxicity caused by expanded polyglutamine tracts in different protein contexts. We find that CHIP is not effective in suppressing the toxicity caused by a bare 127Q tract with only a short hemagglutinin tag, but it is very efficient in suppressing toxicity caused by a 128Q tract in the context of an N-terminal huntingtin backbone. These data underscore the importance of the protein framework for modulating the effects of polyglutamine-induced neurodegeneration.     

The Role of Interruptions in polyQ in the Pathology of SCA1

At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission.     

Cell biology of spinocerebellar ataxia

Ataxia is a neurological disorder characterized by loss of control of body movements. Spinocerebellar ataxia (SCA), previously known as autosomal dominant cerebellar ataxia, is a biologically robust group of close to 30 progressive neurodegenerative diseases. Six SCAs, including the more prevalent SCA1, SCA2, SCA3, and SCA6 along with SCA7 and SCA17 are caused by expansion of a CAG repeat that encodes a polyglutamine tract in the affected protein. How the mutated proteins in these polyglutamine SCAs cause disease is highly debated. Recent work suggests that the mutated protein contributes to pathogenesis within the context of its "normal" cellular function. Thus, understanding the cellular function of these proteins could aid in the development of therapeutics.