A key prerequisite for cellular and organismal health is a functional proteome. A variety of human protein misfolding diseases are associated with the occurrence of amyloid protein aggregates, such as amyotrophic lateral sclerosis (ALS) or Huntington’s disease. The proteins involved in disease manifestation all contain aggregation-prone sequences of low compositional complexity. Such sequences are also known as prion-like, because of their sequence similarity to yeast prions. Yeast prion proteins are a specific subset of amyloid forming proteins with distinct physio-chemical and functional features, which give them transmissible properties. The aggregation properties of yeast prions and disease-related prion-like proteins reside in structurally independent, prion-forming domains (PrDs). These domains are highly enriched for uncharged polar amino acids, such as glutamine (Q) and asparagine (N). These compositional features can be used to predict prion-like proteins bioinformatically. To investigate the prevalence of prion-like proteins across different organisms, we analyzed a range of eukaryotic proteomes. Our analysis revealed that the slime mold D. discoideum contains the highest number of prion-like N/Q-rich proteins of all organisms. Based on this finding, we hypothesized that D. discoideum could be a valuable model system to study protein homeostasis (proteostasis) and the molecular basis of protein misfolding diseases. To explore how D. discoideum manages its highly aggregation-prone proteome, we analyzed the behavior of several well-characterized misfolding-prone marker proteins (variants of the disease-causing exon 1 of the huntingtin protein as well as wildtype and variant versions of the Q/N-rich yeast prion Sup35NM). Intriguingly, these proteins did not form cytosolic aggregates in D. discoideum, as they do in other organisms. Aggregates, however, formed as a result of heat stress, which indicates that the tested proteins have the capacity to aggregate, but are kept under tight control under normal conditions. Furthermore, when the stress level was reduced, the stress-induced aggregates dissolved, suggesting that D. discoideum has evolved mechanisms to reverse aggregation after a period of acute stress. Together, these findings reveal an unusual resilience of D. discoideum to aggregation-prone proteins, which very likely results from specific adaptations in its proteostasis network. By studying these specific adaptations, we could get important insight into the strategies that nature employs to control and maintain a highly aggregation-prone proteome. So far, our experimental investigations have revealed evidence for three specific adaptations. First, we identified the disaggregase Hsp101 as a key player in the acute stress response of D. discoideum. A functional analysis of Hsp101 in yeast and D. discoideum revealed that it supports thermotolerance. Second, we found evidence for an important role of the nucleus and nucleolus in proteostasis. We discovered that a small fraction of highly aggregation-prone proteins accumulated in the nucleus or nucleolus of D. discoideum cells. The magnitude of this nuclear accumulation could be increased by proteasome impairment, which suggests that the ubiquitin-proteasome system (UPS) is involved. This finding is consistent with previous studies in other organisms and hints at the possibility that D. discoideum disposes of aggregation-prone proteins by degrading them in the nucleus/nucleolus. Third and finally, we found that cells containing nuclear accumulations are asymmetrically distributed in the multicellular developmental stage (slug), suggesting that D. discoideum employs cell-sorting mechanisms to dispose of cells with accumulated protein damage. Although our current understanding of proteostasis in D. discoideum is preliminary, we have gained important insight into the molecular mechanisms and cellular pathways that D. discoideum uses to counteract protein aggregation. Findings from this work will inform similar comparative studies in other organisms and will impact our molecular understanding of protein misfolding diseases and aging. / Eine wesentliche Voraussetzung für die Gesundheit von Zellen und Organismen ist ein funktionales Proteom. Eine Reihe von humanen Protein- Missfaltungs-Erkrankungen, wie Chorea Huntington und Amyotrophe Lateralsklerose (ALS) werden mit dem Auftreten von amyloiden Protein- Aggregaten in Verbindung gebracht. Sämtliche Proteine, die in der Pathogenese dieser Krankheiten eine Rolle spielen, enthalten aggregations-anfällige Sequenzen mit geringer Sequenzkomplexität. Solche Sequenzen werden als Prion-ähnlich bezeichnet, da sie in ihrer Zusammensetzung den Prionen aus der Hefe S. cerevisiae gleichen. Die Prion-Proteine der Hefe gehören zu einer Unterart von amyloid-aggregierenden Proteinen, die durch bestimmte physikochemische und funktionelle Eigenschaften einen infektiösen Charakter erhalten. Die Aggregations-Eigenschaften von Hefeprionen und aggregationsanfällige Proteinen, die mit Erkrankungen in Verbindung gebracht werden, basieren auf strukturell unabhängigen, Prion-bildenden Domänen (prion domain, PrD). Diese Domänen sind angereichert mit polaren Aminosäuren wie Glutamin und Asparagin. Diese Zusammensetzung kann dazu verwendet werden prion-ähnliche Proteine bioinformatisch vorherzusagen. Um die Verbreitung von Prion-ähnlichen Proteinen in verschiedenen Organismen zu untersuchen, analysierten wir eine Reihe von eukaryotischen Proteomen. Unsere Analyse zeigte, dass der Schleimpilz D. discoideum die höchste Anzahl von Prion-ähnlichen N/Q-reichen Proteinen aufzeigt. Aufgrund dieser Erkenntnisse erstellten wir die Hypothese, dass D. discoideum ein nützlicher Modellorganismus sein könnte, um Protein Homöostase (Proteostase) sowie die molekulare Basis von Proteins-Missfaltungs-Erkrankungen zu ergründen. Um zu analysieren, wie D. discoideum mit seinem höchst aggregations-anfälligen Proteom umgehen kann, untersuchten wir das Verhalten mehrerer bereits charakterisierter aggregations-anfälliger Marker-Proteine in D. discoideum. Hierbei verwendeten wir Varianten des krankheits-erzeugenden Exon 1 des humanen Huntingtin Protein sowie den wild-typ und Varianten des N/Q-reichen Hefe Prions Sup35. Interessanterweise bildeten diese Proteine, anders als in anderen Organismen, keine zytosolischen Aggregate in D. discoideum aus. Aggregate wurden jedoch unter Hitzestress-Bedingungen gebildet. Dies deutet darauf hin, dass die getesteten Proteine durchaus das Vermögen zu aggregieren besitzen, jedoch unter normalen Wachstumsbedingungen streng kontrolliert werden. Wenn, darüberhinaus das Stress- Level gesenkt wurde, kam es zur Auflösung der stress-induzierten Aggregate. Dies deutet darauf hin, dass D. discoideum Mechanismen entwickelt hat, um Aggregate nach Perioden von akutem Stress wieder aufzulösen. Zusammengenommen enthüllen diese Erkenntnisse eine ungewöhnliche Widerstandsfähigkeit gegenüber aggregations-anfälligen Proteinen. Diese beruht höchstwahrscheinlich auf spezifischen Modifikationen im Proteostase Netzwerk. Durch die Analyse dieser spezifischen Anpassungen könnten wichtige Einblicke in die Strategien gewährt werden, welche die Natur benutzt, um ein höchst aggregations-anfälliges Proteom zu erhalten und zu kontrollieren. Bisher erbrachten unsere Experimente Anhaltspunkte für drei spezifische Anpassungen. Erstens zeigten wir, dass die Disaggregase Hsp101 eine Schlüsselrolle in der akuten Stressantwort in D. discoideum einnimmt. Eine funktionale Analyse von Hsp101 in D. discoideum und Hefe zeigte, dass die Disaggregase Thermotoleranz fördert. Zweitens haben wir Anhaltspunkte, dass der Nukleus und der Nukleolus eine wichtige Rolle in der Proteostase einnehmen. Eine geringe Fraktion der überaus aggregations-anfälligen Proteine akkumuliert im Nukleus oder Nukleolus von D. discoideum. Das Ausmaß der nuklearen Akkumulation konnte erhöht werden, wenn das Proteasom beeinträchtigt wird. Dies deutet darauf hin, dass das Ubiquitin-Proteasom-System involviert sein könnte. Diese Beobachtung ist im Einklang mit jüngsten Berichten aus anderen Organismen und daraus folgt, dass D. discoideum möglicherweise aggregations-anfällige Proteine durch Abbau im Nukleus entsorgt. Drittens konnten wir feststellen, dass Zellen, die nukleare Akkumulationen enthalten, asymmetrisch in der multizellulären Entwicklungs-Struktur des Pseudoplasmodiums verteilt sind. Dies deutet darauf hin, dass D. discoideum möglicherweise den Zellsortierungsmechanismus während der Entwicklung nutzen kann, um Zellen mit angereicherten Protein-Schäden zu beseitigen. Auch wenn das gegenwärtige Verständnis der Proteostase in D. discoideum nur vorläufig ist, haben wir wichtige Einblicke in die molekularen Mechanismen und zellulären Prozesse erhalten, die D. discoideum verwendet, um Protein-Aggregation zu verhindern. Die Ergebnisse dieser Arbeit werden ähnliche vergleichende Studien in anderen Organismen beeinflussen und Auswirkungen auf unser molekulares Verständnis über Protein-Missfaltungs-Erkrankungen und das Altern haben.

The hallmark feature of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), is the accumulation of cytoplasmic inclusions of key disease-linked proteins. Two of these proteins, TDP-43 and SOD1, represent a significant proportion of sporadic and familial ALS cases, respectively. The population of potentially aggregation-prone partially-folded states on the folding free-energy landscape may serve as a common mechanism for ALS pathogenesis. A detailed biophysical understanding of the folding and misfolding energy landscapes of TDP-43 and SOD1 can provide critical insights into the design of novel therapeutics to delay onset and progression in ALS. Equilibrium unfolding studies on the RNA recognition motif (RRM) domains of TDP-43 revealed the population of a stable RRM intermediate in RRM2, with residual structure localized to the N-terminal half of the domain. Other RRM domains from FUS/TLS and hnRNP A1 similarly populate RRM intermediates, suggesting a possible connection with disease. Mutations, which enhance the population of the RRM2 intermediate, could serve as tools for deciphering the functional and misfolding roles of this partially-folded state in disease models, leading to the development of new biomarkers to track ALS progression. ALS mutations in SOD1 have been shown to destabilize the stable homodimer to result in increased populations of the monomeric and unfolded forms of SOD1. Mechanistic insights into the misfolding of SOD1 demonstrated that the unfolded state is a key species in the initiation and propagation of aggregation, suggesting that limiting these populations may provide therapeutic benefit to ALS patients. An in vitro time-resolved Förster Resonance Energy Transfer assay to screen small molecules that stabilize the native state of SOD1 has identified several lead compounds, providing a pathway to new therapeutics to treat ALS.

La protéostasie désigne l’ensemble de stratégies développées par la cellule pour assurer la préservation de son protéome. Parmi celles-ci on peut citer le contrôle du repliement, de la concentration, et de la distribution des protéines. Les neurones en raison de leur importante activité métabolique représentent une population cellulaire particulièrement vulnérable à l’altération de la protéostasie, auquel cas on parle de protéinopathie. C’est notamment le cas des tauopathies et β-amyloidopathies, deux troubles neurodégénératifs, respectivement caractérisés par le dysfonctionnement de la protéine tau et du peptide amyloïde-β (Aβ). La protéine tau par le biais de son état de phosphorylation contrôle la stabilisation des microtubules, tandis que l’Aβ issu du clivage de l’APP (Amyloid Precursor Protein) serait impliqué dans la plasticité synaptique ; de telle sorte que l’altération du fonctionnement ou de la protéostasie de ces deux molécules engendre de graves troubles neuronaux. Le glaucome, principale cause de cécité irréversible au monde, est une neuropathie dégénérative caractérisée par la perte spécifique des somas des cellules ganglionnaires de la rétine (CGR) et de leurs axones dans le nerf optique. Bien que l’hypertension oculaire (HTO) soit le principal facteur de risque, on ignore la cause du glaucome raison pour laquelle il n’existe aucun remède contre la maladie. La maladie d’Alzheimer (MA), principale cause de démence, est caractérisée par la présence d’enchevêtrement neurofibrillaires formés de la protéine tau dans les neurones et de plaques séniles constitué d’agrégats d’Aβ dans le parenchyme cérébral. De manière surprenante, de nombreuses études révèlent que le glaucome et la MA présentent de nombreux points communs. C’est ainsi que des agrégats d’Aβ et de tau ont été trouvés dans les CGR de sujets atteints du glaucome. De même les sujets victimes de la MA présentent des déficits visuels et une dégénérescence des CGR. Vu l’importance de tau pour la physiologie neuronale et son rôle de médiateur de la toxicité d’Aβ, nous proposons l’hypothèse selon laquelle le dysfonctionnement de la protéine tau résulte en la perte des CGR. Les résultats présentés dans cette thèse reposent sur deux modèles expérimentaux de neurodégénérescence : un modèle de glaucome dépendant de HTO chez les rats (modèle de Morrison) et le modèle 3xTg de la MA chez lequel les souris expriment des mutations dans la protéine tau et la voie Aβ (PS1M146V, APPSWE, TauP301L). Chez ces animaux nous avons prélevé la rétine, le nerf optique et le cerveau, sur lesquels nous avons étudié l’expression, la distribution, et la neurotoxicité de tau par western blot, immunohistochimie et PCR quantitative. Nos résultats révèlent que comparativement aux contrôles sains, les rétines malades (glaucome et MA) présentent une accumulation de tau anormalement phosphorylée, tandis que son expression génique reste inchangée. Cette hausse de tau est la conséquence de sa relocalisation vers le compartiment somatodendritique et le segment axonal intrarétinien des CGR, ceci au détriment des axones myélinisés inclus dans le nerf optique. Nos données montrent que les CGR 3xTg présentent une baisse drastique du transport axonal antérograde, indiquant que l’altération de la distribution de tau pourrait être à la base de cette perte de fonction axonale. Finalement, nous démontrons que l’accumulation de tau dans la rétine malade provoque éventuellement la mort des CGR. Au total, cette thèse démontre que les rétines atteintes du glaucome et de la MA présentent les manifestations cardinales des tauopathies à savoir l’accumulation, l’altération de la phosphorylation, et une distribution anormale de tau le tout culminant en la perte de fonction et la dégénérescence des CGR. / Proteostasis refers to a set of strategies developed by the cell to ensure the maintenance of its proteome. These strategies include the control of protein folding, the amount, and the distribution of the proteins. Neurons are endowed with a high metabolic rate and, as such, are highly vulnerable to alterations in proteostasis, a situation referred to as proteinopathy. Tauopathies and β-amyloidopathies are two such instances wherein tau and amyloid-β, respectively, undergo dysfunction. Tau protein is a microtubule stabilising protein which function is regulated by its phosphorylation state, while Aβ a product of the cleavage of APP (Amyloid Precursor Protein) which is thought to be involved in the regulation of synaptic plasticity. Therefore, functional or proteostatic alterations of these proteins result in harmful consequences for neurons. Glaucoma, the main cause of irreversible blindness, is a degenerative optic neuropathy characterised by the selective loss of retinal ganglion cells (RGC) and their axons in the optic nerve. Although ocular hypertension (OHT) is the main risk factor for the development of glaucoma, the cause of the disease is still unknown. There is currently no cure for glaucoma and the only available treatment is to reduce OHT pharmacologically or surgically. Alzheimer’s disease, the main cause of dementia, is characterized by the presence of neurofibrillary tangles made of tau protein in neurons and senile plaques made of Aβ in the cerebral parenchyma. Intriguingly, several studies have shown that glaucoma and AD share several common features. For instance, aggregates of tau and Aβ have been described in the retina of glaucoma subjects. Likewise, AD patients show visual defects associated with RGC degeneration. Mindful of the importance of tau for neuronal physiology, and of its role as mediator of Aβ toxicity, we put forward the hypothesis that tau protein alterations leads to RGC dysfunction and death. vii The results presented in this thesis were based on two experimental models of neurodegeneration: a model of OHT-dependent glaucoma in rats leading to RGC death (Morrison model), and the 3xTg model of AD wherein mice overexpress mutant forms of tau and Aβ (PS1M146V, APPSWE, TauP301L). Using these animals, we collected retina, optic nerve, and brains which we used to study tau expression, distribution and neurotoxicity by western blot, immunohistochemistry and real-time PCR. Our results show that, when compared to healthy controls, the diseased retina (glaucoma or AD) display accumulation of abnormally phosphorylated tau while its gene expression remains unchanged. The increase of retinal tau protein might result from the redistribution of the protein in the somatodendritic compartment and intraretinal axonal segment of RGCs at the expense of the extraocular axonal segment enclosed within the optic nerve. Our data also demonstrate that RGCs from 3xTg mice show a drastic reduction of anterograde axonal transport suggesting that missorted tau might underlie these functional deficits. Lastly, we demonstrate that tau accumulation in the diseased retina eventually promotes RGC death. Altogether, this thesis demonstrates that the glaucomatous and AD retinas present the cardinal features of tauopathies including tau accumulation, altered phosphorylation, and mislocalization which contribute to RGC dysfunction and subsequent death.

Les protéines sont responsables de pratiquement toutes les fonctions performées au sein du corps cellulaire et de ses alentours. Le contrôle de l’expression génique détermine l’abondance, la localisation et le moment de la production de protéines dans la cellule. Il s’agit de l’un des processus centraux à la régulation de la physiologie et du fonctionnement cellulaire. La moindre perte de balance dans ce complexe système engendre des conséquences majeures sur l’intégrité cellulaire, menant au développement de plusieurs maladies parfois incurables. La traduction de l’ARN messager en produit protéique constitue la dernière étape de l’expression génique. Elle est régulée de plusieurs façons, intrinsèques et extrinsèques à la séquence. Il s’agit également du processus cellulaire le plus coûteux en termes d’énergie. Le profilage des ribosomes (Ribo-Seq) figure parmi les récentes et prometteuses technologies ayant permis une meilleure étude des mécanismes de régulation de la traduction. Ces résultats contiennent toutefois la présence de variabilité et de bruits de nature infondée. Ce travail présente la mise en place d’une stratégie permettant la dissociation de signaux d’origine biologique de ceux ayant une origine technique. Ceci est effectué au travers de la mise en place de profiles consensus de densité ribosomale extrait d’une analyse comparative de plusieurs expériences de Ribo-Seq chez la levure (Saccharomyces cerevisiae). Les signaux biologiques dérivés par les profils consensus correspondent avec les signatures de pauses ribosomales connues, telles que les scores de repliements de l’ARNm et la charge des acides aminés. Épatamment, notre stratégie a également permis l’identification de séquences différentiellement transcrites (DT). Ces dernières jouent un rôle sur la cinétique de la phase d’élongation de la traduction, elles comportent notamment une surreprésentation de codons associés aux modifications des ARNs de transfert (tRNAs). Elles se retrouvent d’ailleurs impliquées dans le maintien de l’homéostase cellulaire, ayant une présence marquée chez des gènes prenants part aux mécanismes de biosynthèse de la macromolécule ribosomale ainsi que chez les ARNms aux sublocalisations cellulaires précises, notamment chez les mitochondries et le réticulum endoplasmique (ER). En plus de démontrer les possibilités de découvertes offertes par la technique du Ribo-Seq, cette étude présente une évidence de la nature dynamique et hétérogène du processus de traduction chez la cellule eucaryote. Elle démontre également le rôle de l’information directement encodée dans la séquence dans l’optimisation générale de l’homéostasie cellulaire. / Proteins are responsible for virtually all functions performed within and in the surroundings of a cell. The control of gene expression, which determines the amount, localisation and timing of protein production in the cell, is the central processes in the regulation of cellular physiology and function. Any disturbance in this complex system can generate important consequences on cellular integrity, sometimes leading to incurable diseases. The translation of messenger RNA into a protein product is the last step of the gene expression mechanism. It can be regulated in manifold ways, both intrinsically and extrinsically to the transcript sequence. It is also the costliest cellular process in terms of energy. Ribosome profiling (Ribo-Seq) is one of the recent and promising technologies making it possible to better study the mechanisms of translation regulation. Its results have however been shown to display variability in reproducibility and to contain noise of uncharted sources. This work presents the implementation of a strategy for dissociating signals of biological origin from those of technical origin. This is performed by the computation of a consensus profile of ribosomal density derived from a comparative analysis of several Ribo-Seq experiments in yeast (Saccharomyces cerevisiae). The biological signals derived by the consensus profiles correspond with signatures of known ribosomal pauses, such as mRNA folding strength and amino acid charges. Amazingly, our strategy also enabled the identification of differentially transcribed (DT) sequences. The latter have shown an over-representation of codons associated with modifications of transfer RNAs (tRNAs). They are also involved in the control of cellular homeostasis, exhibiting a marked presence in genes involved in ribosome biosynthesis as well as in mRNAs with precise translation sub-localization, particularly in mitochondria and the endoplasmic reticulum (ER). In addition to demonstrating the possibilities of discovery offered by the Ribo-Seq technique, this study also presents evidence of the dynamic and heterogeneous nature of the translation process in the eukaryotic cell. It also showcases its diverse regulatory mechanisms and the role of information directly encoded in the sequence in the general optimization of cellular homeostasis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a loss of voluntary movement over time, leading to paralysis and death. While 10% of ALS cases are inherited or familial (FALS), the majority of cases (90%) are sporadic (SALS) with unknown etiology. Approximately 20% of FALS cases are genetically linked to a mutation in the anti-oxidizing enzyme, superoxide dismutase (SOD1). SALS and FALS are clinically indistinguishable, suggesting a common pathogenic mechanism exists for both types. Since such a large number of genetic mutations in SOD1 result in FALS (>170), it is reasonable to suspect that non-genetic modifications to SOD1 induce structural perturbations that result in ALS pathology as well. In fact, misfolded SOD1 lacking any genetic mutation was identified in end stage spinal cord tissues of SALS patients using misfolded SOD1-specific antibodies. In addition, this misfolded WT SOD1 found in SALS tissue inhibits axonal transport in vitro, supporting the notion that misfolded WT SOD1 exhibits toxic properties like that of FALS-linked SOD1. Indeed, aberrant post-translational modifications, such as oxidation, cause WT SOD1 to mimic the toxic properties of FALS-linked mutant SOD1. Based on these data, I hypothesize that modified, misfolded forms of WT SOD1 contribute to SALS disease progression in a manner similar to FALS linked mutant SOD1 in FALS. The work presented in this dissertation supports this hypothesis. Specifically, one common misfolded form of SOD1 is defined and exposure of this toxic region is shown to enhance SOD1 toxicity. Preventing exposure, or perhaps stabilization, of this “toxic” region is a potential therapeutic target for a subset of both familial and sporadic ALS patients. Further, the possibility of exploiting this misfolded SOD1 species as a biomarker is explored. For example, an over-oxidized SOD1 species was identified in peripheral blood mononuclear cells (PBMCs) from SALS patients that is reduced in controls. Moreover, 2-dimensional gel electrophoresis revealed a more negatively charged species of SOD1 in PBMCs of healthy controls greatly reduced in SALS patients. This species is hypothesized to be involved in the degradation of SOD1, further implicating both misfolded SOD1 and altered protein homeostasis in ALS pathogenesis.

Cu, Zn superoxide dismutase (SOD1) is a dimeric, β-sandwich, metalloenzyme responsible for the dismutation of superoxide. Mutations covering nearly 50% of the amino acid sequence of SOD1 have been found to acquire a toxic gain-of-function leading to amyotrophic lateral sclerosis. A hallmark of this disease is the presence of insoluble aggregates containing SOD1 found in the brain and spinal cord. While it is unclear how these aggregates or smaller, precursor oligomeric species may be the source of the toxicity, mutations leading to increased populations of unstable, partially folded species along the folding pathway of SOD1 may be responsible for seeding and propagating aggregation. In an effort to determine the responsible species, we have systematically characterized the stability and folding kinetics of five well studied ALS variants: A4V, L38V, G93A, L106V and S134N. The effect of the amino acid substitutions was determined on a variety of different constructs characterizing the various post-translational maturation steps of SOD1: folding, disulfide bond formation and Zn binding. Zn was found to bind progressively tighter along the folding pathway of SOD1, minimizing populations of monomeric species. In contrast, ALS variants were found to have the greatest perturbation in the equilibrium populations of the folded and unfolded state for the most immature, disulfide-reduced metal-free SOD1. In this species, at physiological temperature, four out of five ALS variants were >50% unfolded. Finally the energetic barriers in the folding and unfolding reaction were studied to investigate the unusually slow folding of SOD1. These results reveal that both unfolding and refolding are dominated by enthalpic barriers which may be explained by the desolvation of the chain and provide insights into the role of sequence in governing the folding pathway and rate.

No description available.

<p dir="ltr">Skeletal muscle accounts for 40% of total body weight and the homeostasis of muscle tissue is critical in maintaining proper body function. Skeletal muscle develops during the embryonic stages from the muscle progenitor cells derived from the dermomyotome structure. The myogenic progenitor cells contribute to the primary myogenesis by forming the primary myotubes which are the founding structures that the secondary myogenesis continues to build on. A portion of the myogenic progenitor cells makes up the adult muscle stem cells residing in homeostatic muscle tissue. The adult muscle stem cells contribute substantially for the adult muscle regeneration. Due to the significance of the muscle tissue and the importance of muscle stem cells, dysregulation of the muscle homeostasis or the muscle stem cell homeostasis will result in severe pathological conditions such as myopathy.</p><p dir="ltr">Mitochondria are cellular organelles that are responsible for generating energy needed for cellular processes, especially for muscle tissue where muscle contraction requires the presence of ATP. On the other hand, mitochondria also serve as signaling molecules and provide macromolecules for the biosynthesis. FAM210A (Family With Sequence Similarity 210 Member A) protein was shown to impact the lean mass of human subjects yet a detailed study on the effect of FAM210A in skeletal muscle was not performed, nor has the molecular mechanisms through which FAM210A function been elucidated. Therefore, I take on the task to unveil the function of FAM210A in muscle development, muscle homeostasis and muscle stem cell behavior by using a combination of mouse models with different myogenic promoters to target <i>Fam210a</i> at different developmental stages.</p><p dir="ltr">In the first part of the thesis, I investigated the role of FAM210A in post differentiation myofibers. Using the <i>Myl1</i>^<em>Cre</em> driven deletion of <i>Fam210a</i>, I found that <i>Fam210a</i>^<em>MKO</em> had normal development before 3 weeks of age, but the growth was stagnant from 4 weeks on, and the mice did not survive past 8 weeks of age. I found that the assembly of the ribosomes in the <i>Fam210a</i>^<em>MKO</em> was defective, leading to impaired translation which attenuated the muscle atrophy phenotype. I identified through proteomics that the mitochondrial autophagy and proteostatic control pathways were significantly induced yet mitochondrial organization and energetic proteins were downregulated. Metabolomics analysis showed that the signaling metabolite acetyl-CoA was increased in the <i>Fam210a</i>^<em>MKO</em> which led to increased protein acetylation, specifically, we showed that the ribosomal proteins were hyperacetylated, and that the acetylation increase was elicited by the <i>Fam210a</i>-null mitochondria.</p><p dir="ltr">In the second part of the thesis, I investigated the function of FAM210A in muscle progenitor cells. In the <i>FamMKO</i> mice, I found that deletion of <i>Fam210a</i> from embryonic myogenic progenitor cells led to developmental arrest and postnatal death at day 6. In the <i>FamPKO</i> mice, I found that <i>Fam210a</i> is needed for adult muscle stem cell to contribute to regeneration. Loss of <i>Fam210a</i> leads to the regenerative defects when the muscle was exposed to injury cues. We further showed that <i>Fam210a</i> deletion in muscle stem cells resulted in disruption of the proteostatic control over muscle stem cell activation, thereby forbidding the translational increase necessary to facilitate activation and proliferation. Furthermore, I showed that <i>Fam210a</i> deletion leads to excessive OPA1 cleavage, which contributes to the regenerative failure of muscle stem cells as fusion is required for the mitochondrial network remodeling during regeneration. Therefore, <i>Fam210a</i> safeguards the mitochondrial network and proteostasis during regeneration.</p><p dir="ltr">In summary, my studies characterized the functional contribution of FAM210A during embryonic muscle development, muscle mass maintenance and adult muscle stem cell homeostasis. The regulation of FAM210A in these three processes impinge on the translational regulation. My studies further demonstrated the importance of mitochondrial regulated protein translation in skeletal muscle and muscle stem cells.</p>

[ES] El equilibrio de la homeostasis de proteínas es esencial para asegurar la funcionalidad celular. La expresión de proteínas propensas a plegarse mal induce la formación de agregados tóxicos que alteran el correcto funcionamiento de estos sistemas de control, lo que conduce a un desequilibrio de la homeostasis de proteínas, y por consiguiente a una afectación patológica. Varias enfermedades neurodegenerativas, como la enfermedad de Huntington (EH), Parkinson, Alzheimer, entre otras, tienen en común esta marca patológica molecular. Especialmente, la EH pertenece a un grupo de patologías producidas por proteínas que contienen expansiones de poliQs (varias ataxias espinocerebelosas, la atrofia muscular bulbar y espinal y la atrofia dentatorubro-pálidoluisiana), que hacen que estos péptidos sean propensos a la agregación, y causen los problemas que hemos descrito. A pesar de que estas enfermedades son genéticas, y se conoce su causa molecular, se cree que la presencia de variantes alélicas en otros genes puede exacerbar o ralentizar la agregación de proteínas con poliQs. Por tanto, la identificación de estos genes permitirá profundizar en los mecanismos moduladores de la dinámica de agregación de poliQs e identificar dianas terapéuticas frente a la toxicidad de poliQs. También es ampliamente conocido que el ARN que porta tripletes CAG tiene carácter patogénico. En este trabajo hemos desarrollado modelos, empleando C. elegans, que muestran signos indirectos de que están expresando transcritos CAG patogénicos, puesto que los tejidos diana están alterados. Usamos uno de estos modelos, que perturba la función de las neuronas GABAérgicas del gusano, para realizar un cribado de 85 compuestos farmacológicos, que nos llevó a identificar cuatro compuestos que reducían los defectos motores en estos animales. Además de poliQs y transcritos CAG, también se producen péptidos derivados de una traducción no canónica, conocida como traducción RAN. En relación a esto, hemos profundizado en la identificación y modelización de estos péptidos en C. elegans mediante la expresión de expansiones CAG fusionadas a proteínas fluorescentes para su detección in vivo. Además hemos empleado otros modelos ya existentes, para caracterizar potenciales dianas terapéuticas, como AMPK, la cual puede ser activada por diferentes sustancias. Sin embargo, algunas sustancias, como la metformina o el salicilato, son pleiotrópicas. Por tanto, hemos caracterizado la activación sinérgica de AMPK, mediante metformina y salicilato, para reducir el estrés por agregados de poliQs, con lo que podríamos evitar que otras dianas resulten activadas. Por último, hemos demostrado que este tratamiento sinérgico reduce la toxicidad inducida por la α -sinucleína implicada en la enfermedad de Parkinson. Por otro lado, hemos caracterizado en C. elegans un nuevo alelo que potencia la la agregación de poliQs, vlt10, en el gen unc-1. Nuestros resultados sugieren que la mutación unc-1(vlt10) perturba la sinapsis eléctrica entre las neuronas sensoriales IL2 y ASJ, induciendo un exceso de señalización hormonal que requiere la función de la sulfotransferasa SSU-1. También hemos demostrado que la diana de esta señal es un receptor nuclear llamado NHR-1. En este mismo capítulo, hemos identificado otra ruta de señalización hormonal, mediada por otro receptor nuclear (DAF-12), que tiene un papel protector. Se desconoce qué hormona modula la actividad de NHR-1. Sin embargo, hemos identificado que algunos de los genes regulados por NHR-1 están relacionados con el metabolismo lipídico. Además, hemos observado que la disrupción de unc-1 induce la acumulación de lípidos en los gusanos, pero a la vez estos animales contienen menos ácidos grasos totales. Esto sugiere que las grasas juegan un papel fundamental en la modulación de la agregación de poliQs, y quizás señala posibles dianas terapéuticas contra enfermedades causadas / [CA] L'equilibri de l'homeòstasi de proteïnes és essencial per a assegurar la funcionalitat cel·lular. L'expressió de proteïnes propenses a plegar-se malament indueix la formació d'agregats tòxics i altera el correcte funcionament dels sistemes de control, cosa que condueix a un desequilibri de l'homeòstasi de proteïnes, i per consegüent a una afectació patològica. Diverses malalties neurodegeneratives, com la malaltia de Huntington (MH), Parkinson i Alzheimer, entre altres, tenen en comú aquesta signatura patològica. Especialment, la MH pertany a un grup de patologies produïdes per proteïnes que contenen expansions de poliQs (s'inclouen diverses atàxies espinocerebel·loses 1, 2, 3, 6, 7 i 17, l'atròfia muscular bulbar i espinal, i l'atròfia dentatorúbrica-pàl·lidaluisiana), que fan que aquests pèptids siguen propensos a l'agregació, i causen els problemes que hem descrit. Malgrat que aquestes malalties són genètiques i es coneix la seva causa molecular, es creu que la presència de variants al·lèliques en gens pot incrementar o alentir l'agregació de proteïnes amb poliQs. Per tant, la identificació de gens modificadors permet aprofundir en els mecanismes moduladors de la dinàmica d'agregació de poliQs ens permet trobar dianes terapèutiques enfront de la toxicitat de poliQs. També és àmpliament conegut que l'ARN que contenen expansions CAG té caràcter patogènic. Per tant, hem desenvolupat models, utilitzant C. elegans, en els quals es mostren signes indirectes que expressen transcrits patogènics, ja que els teixits diana revelen una funcionalitat alterada. Utilitzant aquests models, que alteren la funció de les neurones GABAèrgiques del cuc, hem realitzat un cribratge de 85 compostos farmacològics, que ens va portar a identificar quatre compostos que reduïen els defectes motors en aquests animals. A més de poliQs i l'ARN que conté expansions de CAG, es produeixen pèptids derivats d'una traducció no canònica, coneguda com a traducció RAN. En relació amb això, hem aprofundit en la identificació i modelització d'aquests pèptids en C. elegans mitjançant l'expressió d'expansions CAG fusionades a proteïnes fluorescents per a la seva detecció in vivo. A més hem emprat models ja generats, per a caracteritzar potencials dianes terapèutiques, com AMPK puga ser activat per diversos fàrmacs. No obstant això, alguns activadors, com la metformina o el salicilat, són substàncies pleiotròpiques. Per tant, hem caracteritzat l'activació sinèrgic de AMPK, mitjançant metformina i salicilat, per a reduir l'estrés induït per agregats de poliQs i s'evita l'activació de dianes no desitjades. I finalment, hem demostrat que aquest tractament sinèrgic podria ser utilitzat per a reduir proteïnes tòxiques com la α-sinucleína, causant de la malaltia de Parkinson. D'altra banda, hem caracteritzat un nou modulador de l'agregació de poliQs, vlt10, en el gen unc-1, que produeix un augment d'agregació de poliQs. Els nostres resultats suggereixen que la mutació unc-1(vlt10) pertorba la sinapsi elèctrica entre les neurones sensorials IL2 i ASJ, induint un excés de senyalització hormonal que requereix la funció de la sulfotransferasa SSU-1. També hem demostrat que la diana d'aquest senyal és un receptor nuclear anomenat NHR-1. En el mateix capítol, hem identificat una altra ruta de senyalització hormonal, mitjançada per un altre receptor nuclear (DAF-12), que té un paper protector. Es desconeix l'hormona que modula l'activitat de NHR-1. No obstant això, hem identificat que alguns del genes regulats per NHR-1 estan relacionats amb el metabolisme lipídic. els resultats han sigut confirmats. També hem observat que la disrupció de unc-1 indueix l'acumulació de lípids en els cucs, però simultàniament aquests animals contenen menys àcids grassos totals. Això suggereix que els greixos juguen un paper fonamental en la modulació de l'agregació de poliQs, i potser assenyala / [EN] The balance of protein homeostasis is essential to ensure cellular functionality. The expression of proteins that are prone to misfolding induces the formation of toxic aggregates, which leads to an imbalance in protein homeostasis and pathological consequences. Several neurodegenerative diseases, such as Huntington disease (HD), Parkinson, Alzheimer, among others, have this molecular pathological mark in common. HD belongs to a group of pathologies produced by proteins that contain expansions of polyQs (several spinocerebellar ataxias 1, 2, 3, 6, 7 and 17, bulbar and spinal muscular atrophy and the dentatorubral-pallidoluysian atrophy), which make these peptides prone to aggregation, and cause the problems described above. Although these diseases are genetic, and their molecular cause is known, it is believed that the presence of allelic variants in other genes can exacerbate or slow the polyQ aggregation. Therefore, the identification of these genes will allow delving into the modulating mechanisms of the polyQ aggregation dynamics and find therapeutic targets against the polyQ toxicity. In addition, RNA that contains CAG triplets is pathogenic. In this work we have developed models using C. elegans that show indirect signs of pathogenic transcript expression since altered functionality was observed in target tissues. We use a model, which disturbs the function of the worm's GABAergic neurons, to screen for 85 pharmacological compounds, which led us to identify four compounds that reduced motor defects in these animals. In addition to polyQs and CAG transcripts, also are produced peptides derived from a non-canonical translation, known as RAN translation. In this regard, we have delved into the identification and modelling of these peptides in C. elegans through the expression of CAG expansions fused to fluorescent proteins to investigate them in vivo. Furthermore, we have used models previously generated to characterize potential therapeutic targets, such as AMPK. This enzyme can be activated using different compounds. However, some activators, such as metformin or salicylate, are pleiotropic substances. Therefore, in the second chapter of this thesis, we have characterised the synergistic activation of AMPK, using metformin and salicylate, to reduce the stress induced by polyQ aggregates and prevent possible unwanted targets from being activated. Finally, we have shown that this synergistic treatment could be used to reduce α-synuclein toxicity, which is involved in Parkinson disease. On the other hand, we have characterised a new allele that enhances polyQ aggregation, vlt10, in the unc-1 gene. Our results suggest that the unc-1(vlt10) mutation disturbs the electrical synapse between sensory neurons IL2 and ASJ, inducing an excess of hormonal signalling that requires the function of the sulfotransferase SSU-1. We have also shown that the target of this signal is the nuclear receptor NHR-1. In the same chapter, we have identified another hormonal signalling pathway, mediated by another nuclear receptor (DAF-12), which has a protective role. It is unknown which hormone modulates the activity of NHR-1. However, we have identified several genes that regulate lipid metabolism and whose expression could be modulated by NHR-1. In addition, we have observed that the disruption of unc-1 induces the accumulation of lipids in worms, but at the same time these animals contain less total fatty acids. Thus, our results suggest that the metabolism of fats play a key role in modulating polyQs aggregation, highlighting potential therapeutic targets against diseases caused by aggregation-prone proteins. / Gómez Escribano, AP. (2021). Modelos de toxicidad inducidos por microsatélites CAG y caracterización de dianas terapéuticas en C. elegans [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/166783