Recruitment and the Role of Nuclear Localization in Polyglutamine-mediated Aggregation

The inherited neurodegenerative diseases caused by an expanded glutamine repeat share the pathologic feature of intranuclear aggregates or inclusions (NI). Here in cell-based studies of the spinocerebellar ataxia type-3 disease protein, ataxin-3, we address two issues central to aggregation: the role of polyglutamine in recruiting proteins into NI and the role of nuclear localization in promoting aggregation. We demonstrate that full-length ataxin-3 is readily recruited from the cytoplasm into NI seeded either by a pathologic ataxin-3 fragment or by a second unrelated glutamine-repeat disease protein, ataxin-1. Experiments with green fluorescence protein/polyglutamine fusion proteins show that a glutamine repeat is sufficient to recruit an otherwise irrelevant protein into NI, and studies of human disease tissue and a Drosophila transgenic model provide evidence that specific glutamine-repeat–containing proteins, including TATA-binding protein and Eyes Absent protein, are recruited into NI in vivo. Finally, we show that nuclear localization promotes aggregation: an ataxin-3 fragment containing a nonpathologic repeat of 27 glutamines forms inclusions only when targeted to the nucleus. Our findings establish the importance of the polyglutamine domain in mediating recruitment and suggest that pathogenesis may be linked in part to the sequestering of glutamine-containing cellular proteins. In addition, we demonstrate that the nuclear environment may be critical for seeding polyglutamine aggregates.

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Ubiquitin-interacting motifs of ataxin-3 regulate its polyglutamine toxicity through Hsc70-4-dependent aggregation

eLife ◽

10.7554/elife.60742 ◽

2020 ◽

Vol 9 ◽

Author(s):

Sean L Johnson ◽

Bedri Ranxhi ◽

Kozeta Libohova ◽

Wei-Ling Tsou ◽

Sokol V Todi

Keyword(s):

Spinocerebellar Ataxia Type ◽

Dependent Manner ◽

Allelic Series ◽

Spinocerebellar Ataxia Type 3 ◽

Polyglutamine Expansion ◽

Polyglutamine Disorders ◽

Glutamine Repeat ◽

Type 3 ◽

The Impact

Spinocerebellar ataxia type 3 (SCA3) belongs to the family of polyglutamine neurodegenerations. Each disorder stems from the abnormal lengthening of a glutamine repeat in a different protein. Although caused by a similar mutation, polyglutamine disorders are distinct, implicating non-polyglutamine regions of disease proteins as regulators of pathogenesis. SCA3 is caused by polyglutamine expansion in ataxin-3. To determine the role of ataxin-3’s non-polyglutamine domains in disease, we utilized a new, allelic series of Drosophila melanogaster. We found that ataxin-3 pathogenicity is saliently controlled by polyglutamine-adjacent ubiquitin-interacting motifs (UIMs) that enhance aggregation and toxicity. UIMs function by interacting with the heat shock protein, Hsc70-4, whose reduction diminishes ataxin-3 toxicity in a UIM-dependent manner. Hsc70-4 also enhances pathogenicity of other polyglutamine proteins. Our studies provide a unique insight into the impact of ataxin-3 domains in SCA3, identify Hsc70-4 as a SCA3 enhancer, and indicate pleiotropic effects from HSP70 chaperones, which are generally thought to suppress polyglutamine degeneration.

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Role of Adenosine Receptors in Rare Neurodegenerative Diseases with Motor Symptoms

Current Protein and Peptide Science ◽

10.2174/1389203722666210910110126 ◽

2021 ◽

Vol 22 ◽

Author(s):

Vicent Beltran-Beltran ◽

Noelia Benetó ◽

Tamara Lapeña-Luzón ◽

Laura R. Rodríguez ◽

Federico V. Pallardó ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Adenosine Receptors ◽

Spinocerebellar Ataxia Type ◽

European Medicines Agency ◽

Receptor Subtypes ◽

Motor Symptoms ◽

Spinocerebellar Ataxia Type 3 ◽

Adenosine 2A Receptor ◽

Type 3

: The approval of istradefylline, an adenosine 2A receptor (A2AR) antagonist, as an add-on treatment in adult patients with Parkinson’s disease by the Food and Drug Administration (FDA) and European Medicines Agency (EMA), is the latest proof of the importance of the adenosinergic system in the nervous system. Adenosine is an endogenous purine nucleoside with a role as a modulator of both neurotransmission and the inflammatory response. As such, the expression pattern of the 4 adenosine receptors (A1R, A2AR, A2BR and A3R) and the extracellular adenosine levels have attracted great interest in the pathogenesis and possible treatment of rare neurodegenerative diseases with motor symptoms. These include Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), restless legs syndrome (RLS) and Machado-Joseph disease (MJD, also known as spinocerebellar ataxia type 3, SCA3). In this review, we shall focus on the role of the different adenosine receptor subtypes in the development and possible treatment of the aforementioned rare neurodegenerative diseases with motor symptoms using the currently available data. The last section discusses the possibility of a role for the adenosine receptors in the treatment of other rare diseases based on the available molecular pathology knowledge.

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Role of PICK1 in aggresome formation and spinocerebellar ataxia type 3 pathogenesis

10.14711/thesis-991012753766103412 ◽

2019 ◽

Author(s):

Ho Chun Lai

Keyword(s):

Spinocerebellar Ataxia ◽

Spinocerebellar Ataxia Type ◽

Spinocerebellar Ataxia Type 3 ◽

Aggresome Formation ◽

Type 3

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Druggable genome screen identifies new regulators of the abundance and toxicity of ATXN3, the Spinocerebellar Ataxia Type 3 disease protein

10.1101/690818 ◽

2019 ◽

Author(s):

Naila S. Ashraf ◽

Joanna R. Sutton ◽

Yemen Yang ◽

Bedri Ranxhi ◽

Kozeta Libohova ◽

...

Keyword(s):

Progenitor Cells ◽

Spinocerebellar Ataxia ◽

Mammalian Cells ◽

Spinocerebellar Ataxia Type ◽

Dependent Manner ◽

Spinocerebellar Ataxia Type 3 ◽

Neuronal Progenitor Cells ◽

Neuronal Progenitor ◽

Disease Protein ◽

Type 3

AbstractBackgroundSpinocerebellar Ataxia type 3 (SCA3, also known as Machado-Joseph disease) is a neurodegenerative disorder caused by a CAG repeat expansion encoding an abnormally long polyglutamine (polyQ) tract in the disease protein, ataxin-3 (ATXN3). No preventive treatment is yet available for SCA3. Because SCA3 is likely caused by a toxic gain of ATXN3 function, a rational therapeutic strategy is to reduce mutant ATXN3 levels by targeting pathways that control its production or stability. Here, we sought to identify genes that modulate ATXN3 levels as potential therapeutic targets in this fatal disorder.MethodsWe screened a collection of siRNAs targeting 2742 druggable human genes using a cell-based assay based on luminescence readout of polyQ-expanded ATXN3. From 317 candidate genes identified in the primary screen, 100 genes were selected for validation. Among the 33 genes confirmed in secondary assays, 15 were validated in an independent cell model as modulators of pathogenic ATXN3 protein levels. Ten of these genes were then assessed in a Drosophila model of SCA3, and one was confirmed as a key modulator of physiological ATXN3 abundance in SCA3 neuronal progenitor cells.ResultsAmong the 15 genes shown to modulate ATXN3 in mammalian cells, orthologs of CHD4, FBXL3, HR and MC3R regulate mutant ATXN3-mediated toxicity in fly eyes. Further mechanistic studies of one of these genes, FBXL3, encoding a F-box protein that is a component of the SKP1-Cullin-F-box (SCF) ubiquitin ligase complex, showed that it reduces levels of normal and pathogenic ATXN3 in SCA3 neuronal progenitor cells, primarily via a SCF complex-dependent manner. Bioinformatic analysis of the 15 genes revealed a potential molecular network with connections to tumor necrosis factor-α/nuclear factor-kappa B (TNF/NF-kB) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways.ConclusionsWe identified 15 druggable genes with diverse functions to be suppressors or enhancers of pathogenic ATXN3 abundance. Among identified pathways highlighted by this screen, the FBXL3/SCF axis represents a novel molecular pathway that regulates physiological levels of ATXN3 protein.

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In vivo molecular signatures of cerebellar pathology in spinocerebellar ataxia type 3

10.1101/2020.01.03.894337 ◽

2020 ◽

Author(s):

Maria do Carmo Costa ◽

Maria Radzwion ◽

Hayley S. McLoughlin ◽

Naila S. Ashraf ◽

Svetlana Fischer ◽

...

Keyword(s):

Spinocerebellar Ataxia ◽

Spinocerebellar Ataxia Type ◽

Resonance Spectroscopy ◽

Phospholipid Membrane ◽

Spinocerebellar Ataxia Type 3 ◽

Noninvasive Biomarkers ◽

Type 3 ◽

Mediated Reduction

AbstractBackgroundNo treatment exists for the most common dominantly inherited ataxia Machado-Joseph disease, or spinocerebellar ataxia type 3 (SCA3). Successful evaluation of candidate therapeutics will be facilitated by validated noninvasive biomarkers of aspects of disease pathology recapitulated by animal models.ObjectiveWe sought to identify shared neurochemical signatures in two mouse models of SCA3 that reflect aspects of the human disease pathology.MethodsCerebellar neurochemical concentrations in homozygous YACMJD84.2 (Q84/Q84) and hemizygous CMVMJD135 (Q135) mice were measured by magnetic resonance spectroscopy at 9.4 tesla. Motivated by the shared neurochemical abnormalities in the two models, we determined the levels of neurofilament medium (NFL, indicator of neuroaxonal integrity) and myelin basic protein (MBP, indicator of myelination) in cerebellar lysates from a subset of mice and from patients with SCA3. Finally, NFL and MBP levels were measured in cerebellar extracts of Q84/Q84 mice upon sustained silencing of the mutant ATXN3 gene from 6-8 weeks-of-age until death.ResultsBoth Q84/Q84 and Q135 mice displayed lower N-acetylaspartate than wild-type littermates, indicating neuroaxonal loss/dysfunction, and lower myo-inositol and total choline, indicating disturbances in phospholipid membrane metabolism and demyelination. Cerebellar NFL and MBP levels were accordingly lower in both models as well as in the cerebellar cortex of patients with SCA3 than controls. Furthermore, long-term sustained RNAi-mediated reduction of ATXN3 levels increased NFL and MBP in Q84/Q84 cerebella.ConclusionsN-acetylaspartate, myo-inositol and total choline levels in the cerebellum are candidate biomarkers of neuroaxonal and oligodendrocyte pathology in SCA3, which are reversible by reduction of mutant ATXN3 levels.

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A genetic model for human polyglutamine-repeat disease in Drosophila melanogaster

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1999.0458 ◽

1999 ◽

Vol 354 (1386) ◽

pp. 1057-1060 ◽

Cited By ~ 15

Author(s):

Nancy M. Bonini

Keyword(s):

Drosophila Melanogaster ◽

Genetic Model ◽

Late Onset ◽

Spinocerebellar Ataxia Type ◽

Gene Encoding ◽

Spinocerebellar Ataxia Type 3 ◽

Neural Degeneration ◽

Polyglutamine Repeat ◽

Disease Protein ◽

Type 3

To apply genetics to the problem of human polyglutamine–repeat disease, we recreated polyglutamine–repeat disease in Drosophila melanogaster . To do this, we expressed forms of the human gene encoding spinocerebellar ataxia type 3, also called Machado–Joseph disease (SCA–3/MJD). This gene is responsible for the most common form of human ataxia worldwide. Expression of a normal form of the MJD protein with 27 polyglutamines (MJDtr–Q27) had no phenotype. However, expression of a form of the protein with an expanded run of 78 glutamines (MJDtr–Q78) caused late onset progressive degeneration. In addition, the MJDtr–Q78 formed abnormal protein aggregates, or nuclear inclusions (NIs), whereas the control protein was cytoplasmic. These data indicate that the mechanisms of human polyglutamine–repeat disease are conserved to Drosophila . We are currently using this model to address potential mechanisms by which the mutant disease protein causes neural degeneration, as well as to define genes that can prevent polyglutamine–induced degeneration. By applying the power of Drosophila genetics to the problem of human polyglutamine–induced neural degeneration, we hope to identify ways to prevent and treat these diseases in humans.

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Karyopherin α-3 is a key protein in the pathogenesis of spinocerebellar ataxia type 3 controlling the nuclear localization of ataxin-3

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716071115 ◽

2018 ◽

Vol 115 (11) ◽

pp. E2624-E2633 ◽

Cited By ~ 19

Author(s):

Anna Sergeevna Sowa ◽

Elodie Martin ◽

Inês Morgado Martins ◽

Jana Schmidt ◽

Reinhard Depping ◽

...

Keyword(s):

Spinocerebellar Ataxia ◽

Nuclear Localization ◽

Neurodegenerative Disorder ◽

Nucleocytoplasmic Transport ◽

Spinocerebellar Ataxia Type ◽

Nuclear Pore ◽

Spinocerebellar Ataxia Type 3 ◽

Nuclear Localization Signals ◽

Positive Effects ◽

Type 3

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene leading to a polyglutamine expansion in the ataxin-3 protein. The nuclear presence and aggregation of expanded ataxin-3 are critical steps in disease pathogenesis. To identify novel therapeutic targets, we investigated the nucleocytoplasmic transport system by screening a collection of importins and exportins that potentially modulate this nuclear localization. Using cell, Drosophila, and mouse models, we focused on three transport proteins, namely, CRM1, IPO13, KPNA3, and their respective Drosophila orthologs Emb, Cdm, and Kap-α3. While overexpression of CRM1/Emb demonstrated positive effects in Drosophila, KPNA3/Kap-α3 emerged as the most promising target, as knockdown via multiple RNAi lines demonstrated its ability to shuttle both truncated and full-length expanded ataxin-3, rescue neurodegeneration, restore photoreceptor formation, and reduce aggregation. Furthermore, KPNA3 knockout in SCA3 mice resulted in an amelioration of molecular and behavioral disturbances such as total activity, anxiety, and gait. Since KPNA3 is known to function as an import protein and recognize nuclear localization signals (NLSs), this work unites ataxin-3 structure to the nuclear pore machinery and provides a link between karyopherins, NLS signals, and polyglutamine disease, as well as demonstrates that KPNA3 is a key player in the pathogenesis of SCA3.

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