Identificación de CHIP y Ataxina-2 como modificadores de la toxicidad inducida por la Ataxina-1 utilizando un modelo de ataxia espinocerebelosa tipo 1 en "Drosophila melanogaster"

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 22-11-200

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

Huntingtin proteolysis releases non-polyQ fragments that cause toxicity through dynamin 1 dysregulation

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N-ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full-length HTT remain elusive. Moreover, the contribution of non-polyQ C-terminal fragments is unknown. Using time- and site-specific control of full-length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N-ter fragments, the C-ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C-ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis-induced alteration of this function may be relevant to disease. Synopsis The development of a time and site-specifically controlled cleavage of the mutant huntingtin protein reveals a pathogenic mechanism induced by the non-polyQ-containing fragments that are generated upon proteolysis during disease progression. Huntingtin proteolysis generates N-ter fragments that contain the toxic polyQ stretch but also the corresponding C-ter fragments. N-ter to C-ter intramolecular interactions present in full-length huntingtin are abrogated by sequential cleavages. Whereas the N-ter polyQ fragments translocate into the nucleus, the non-polyQ C-ter huntingtin fragments remain in the cytoplasm and cause ER dilation, stress and cell death. C-ter huntingtin fragments bind and inactivate dynamin 1 at the ER thus causing ER dilation and toxicity. Site-specifically controlled cleavage of the mutant huntingtin protein reveals a pathogenic mechanism induced by non-polyQ-containing fragments that are generated upon proteolysis during disease progression.</p

Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice.

To gain insight into how mutant huntingtin (mHtt) CAG repeat length modifies Huntington's disease (HD) pathogenesis, we profiled mRNA in over 600 brain and peripheral tissue samples from HD knock-in mice with increasing CAG repeat lengths. We found repeat length-dependent transcriptional signatures to be prominent in the striatum, less so in cortex, and minimal in the liver. Coexpression network analyses revealed 13 striatal and 5 cortical modules that correlated highly with CAG length and age, and that were preserved in HD models and sometimes in patients. Top striatal modules implicated mHtt CAG length and age in graded impairment in the expression of identity genes for striatal medium spiny neurons and in dysregulation of cyclic AMP signaling, cell death and protocadherin genes. We used proteomics to confirm 790 genes and 5 striatal modules with CAG length-dependent dysregulation at the protein level, and validated 22 striatal module genes as modifiers of mHtt toxicities in vivo

Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein.

The discovery of the causative gene for Huntington's disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons. In two Drosophila models of Huntington's disease, genetic knockdown of PIP4K ameliorated neuronal dysfunction and degeneration as assessed using motor performance and retinal degeneration assays respectively. Together, these results suggest that PIP4Kγ is a druggable target whose inhibition enhances productive autophagy and mHtt proteolysis, revealing a useful pharmacological point of intervention for the treatment of Huntington's disease, and potentially for other neurodegenerative disorders

Metal homeostasis regulators suppress FRDA phenotypes in a drosophila model of the disease

Friedreich's ataxia (FRDA), the most commonly inherited ataxia in populations of European origin, is a neurodegenerative disorder caused by a decrease in frataxin levels. One of the hallmarks of the disease is the accumulation of iron in several tissues including the brain, and frataxin has been proposed to play a key role in iron homeostasis. We found that the levels of zinc, copper, manganese and aluminum were also increased in a Drosophila model of FRDA, and that copper and zinc chelation improve their impaired motor performance. By means of a candidate genetic screen, we identified that genes implicated in iron, zinc and copper transport and metal detoxification can restore frataxin deficiency-induced phenotypes. Taken together, these results demonstrate that the metal dysregulation in FRDA includes other metals besides iron, therefore providing a new set of potential therapeutic targets.This work was supported by grants from the European Community’s Seventh Framework Program FP7/2007-2013 [grant agreement no. 242193 EFACTS], the Fundació la Marató TV3 of Spain [exp 101932] and the Prometeo Program from Generalitat Valenciana [PROMETEOII/2014/067]. Work in the Botas laboratory was supported by the R01-NS42179 NIH grant. S.S. was a recipient of a fellowship from Ministerio de Ciencia e Innovación of Spain; P.C.-Q. is a recipient of a fellowship from Generalitat Valenciana of Spain, J.V.L. was supported by a research contract from the European Community’s Seventh Framework Program FP7/2007-2013 [grant agreement no. 242193 EFACTS] and L.G. is the beneficiary of a postdoctoral grant from the AXA Research Fund.Peer reviewe

Metal Homeostasis Regulators Suppress FRDA Phenotypes in a Drosophila Model of the Disease

Friedreich's ataxia (FRDA), the most commonly inherited ataxia in populations of European origin, is a neurodegenerative disorder caused by a decrease in frataxin levels. One of the hallmarks of the disease is the accumulation of iron in several tissues including the brain, and frataxin has been proposed to play a key role in iron homeostasis. We found that the levels of zinc, copper, manganese and aluminum were also increased in a Drosophila model of FRDA, and that copper and zinc chelation improve their impaired motor performance. By means of a candidate genetic screen, we identified that genes implicated in iron, zinc and copper transport and metal detoxification can restore frataxin deficiency-induced phenotypes. Taken together, these results demonstrate that the metal dysregulation in FRDA includes other metals besides iron, therefore providing a new set of potential therapeutic targets

Smaug/SAMD4A Restores Translational Activity of CUGBP1 and Suppresses CUG-Induced Myopathy

<div><p>We report the identification and characterization of a previously unknown suppressor of myopathy caused by expansion of CUG repeats, the mutation that triggers Myotonic Dystrophy Type 1 (DM1). We screened a collection of genes encoding RNA–binding proteins as candidates to modify DM1 pathogenesis using a well established <i>Drosophila</i> model of the disease. The screen revealed <i>smaug</i> as a powerful modulator of CUG-induced toxicity. Increasing <i>smaug</i> levels prevents muscle wasting and restores muscle function, while reducing its function exacerbates CUG-induced phenotypes. Using human myoblasts, we show physical interactions between human Smaug (SMAUG1/SMAD4A) and CUGBP1. Increased levels of SMAUG1 correct the abnormally high nuclear accumulation of CUGBP1 in myoblasts from DM1 patients. In addition, augmenting SMAUG1 levels leads to a reduction of inactive CUGBP1-eIF2α translational complexes and to a correction of translation of MRG15, a downstream target of CUGBP1. Therefore, Smaug suppresses CUG-mediated muscle wasting at least in part via restoration of translational activity of CUGBP1.</p></div

SMAUG1 and CUGBP1 co-localize and physically interact in DM1 myoblasts.

<p>A. Immunofluorescense and in situ images of DM1 myoblasts transfected with GFP. In control DM1 myoblasts transfected with GFP (green) CUGBP1 is predominantly in the nucleus (red); CUG foci are detected in the nuclei with a Cy3-labelled 5′-CAG-3′ LNA probe (CAG probe, white). B–C. Immunofluorescense and in situ images of DM1 myoblasts transfected with SMAUG1-ECFP. SMAUG1 is detected in the cytoplasm (SMAUG1-ECFP, green). Note that nuclear CUGBP1 signal (α-CUGBP1, red) is clearly diminished in DM1 myoblasts transfected with SMAUG1 (B, arrowhead). Longer exposure of CUGBP1 signal shows cytoplasmic CUGBP1 (C) and its co-localization with SMAUG1 in granules (arrows). D. Bar graph representing the intensity of CUGBP1 nuclear signal in DM1 myoblasts transfected with GFP (DM1-GFP, green bar), versus DM1 myoblasts transfected with SMAUG1 (DM1-SMAUG1, blue bar). Data was analyzed with ANOVA followed by Student's t test, p<0.0001. Black dots represent individual observations, red lines are the standard error of the mean. E. Western blot revealing co-immunoprecipitation between CUGBP1 and human SMAUG1 in extracts from SMAUG1-V5-transfected normal and DM1 human myoblasts. Pull down was carried out using anti-CUGBP1 antibody. SMAUG1 was visualized with anti-V5-HRP antibody. White lines in A–C delineate the nuclei. Scale bar: A–C: 10 µm.</p

Expression of SMAUG1 in control human myoblasts does not affect CUGBP1 nuclear localization.

<p>A–B. Nuclear localization of CUGBP1 (α-CUGBP1, red) is not altered by expression of SMAUG1 (SMAUG1-ECFP, green) in control unaffected primary human myoblasts. CUGBP1 co-localizes with SMAUG1 cytoplasmic granules in control myoblasts (arrow in B). Scale bar: 10 µm.</p