Glia are the understudied brain cells that perform many functions essential to maintain nervous system homeostasis and protect the brain from injury. If brain damage occurs, glia rapidly adopt the reactive state and elicit a series of cellular and molecular events known as reactive gliosis, the hallmark of many neurodegenerative diseases. However, the molecular pathways that trigger and regulate this process remain poorly defined. The fruit fly Drosophila melanogaster has glial cells that are strikingly similar to mammalian glia, and which also exhibit reactive responses after neuronal injury. By exploiting its powerful genetic toolbox, we are uniquely positioned to identify the genes that activate and execute glial responses to neuronal injury in vivo. In this dissertation, I use Wallerian degeneration in Drosophila as a model to characterize molecular pathways responsible for glia to recognize neural injury, become activated, and ultimately engulf and degrade axonal debris. I demonstrate a novel role for the GEF (guanine nucleotide exchange factors) complex DRK/DOS/SOS upstream of small GTPase Rac1 in glial engulfment activity and show that it acts redundantly with previously discovered Crk/Mbc/dCed-12 to execute glial activation after axotomy. In addition, I discovered an exciting new role for the TNF receptor associated factor 4 (TRAF4) in glial response to axon injury. I find that interfering with TRAF4 and the downstream kinase misshapen (msn) function results in impaired glial activation and engulfment of axonal debris. Unexpectedly, I find that TRAF4 physically associates with engulfment receptor Draper – making TRAF4 only second factor to bind directly to Draper – and show it is essential for Draper-dependent activation of downstream engulfment signaling, including transcriptional activation of engulfment genes via the JNK and STAT transcriptional cascades. All of these pathways are highly conserved from Drosophila to mammals and most are known to be expressed in mouse brain glia, suggesting functional conservation. My work should therefore serve as an excellent starting point for future investigations regarding their roles in glial activation/reactive gliosis in various pathological conditions of the mammalian central nervous system.

Neurons comprise the main information processing cells of the nervous system. To integrate and transmit information, neurons elaborate dendritic structures to receive input and axons to relay that information to other cells. Due to their intricate structures, dendrites and axons are susceptible to damage whether by physical means or via disease mechanisms. Studying responses to axon injury, called Wallerian degeneration, in the neuronal processes of Drosophila melanogaster has allowed the identification of genes that are required for injury responses. Screens in Drosophila have identified dsarm and highwire as two genes required for axon degeneration; when these genes are mutated axons fail to degenerate after injury, even when completely cut off from the neuronal cell body. We found that these genes are also required for dendrite degeneration after injury in vivo. Further, we reveal differences between axon and dendrite injury responses using in vivo timelapse recordings and GCaMP indicators of intracellular and mitochondrial calcium transients. These data provide insights into the neuronal responses to injury, and better define novel targets for the treatment of neurodegenerative diseases.

L'α-Synuclein (αSyn) fibrillaire, impliqué dans la maladie de Parkinson et d’autres synucleinopathies, peut se propager entre cellules de manière « prion-like » et cette propagation est liée à la progression de la maladie. Durant cette étude, nous nous sommes tournés vers les chaperons moléculaires impliqués dans l’agrégation de l’αSyn ou bien dans sa toxicité afin de trouver des candidats capables d’interférer avec la propagation. Nous avons ensuite testé l’effet des chaperons capables de se lier aux fibres d’αSyn sur l’internalisation des fibres d’αSyn par les cellules Neuro-2a. Nous démontrons que l’interaction avec l’αSyn agrégeant avec αB-crystallin (αBc) ou Carboxyl terminus of Hsc70-interacting protein (CHIP) a mené à la formation de fibres qui sont moins internalisées par les cellules. Enfin, en passant par une stratégie de pontage chimique optimisé couplé à la spectrométrie de masse, nous avons identifié les zones d’interaction entre l’αSyn fibrillaire et soit αBc, soit CHIP. Ces résidus issus des chaperons, se trouvant à proximité des fibres d’αSyn dans les complexes, pourraient être développés dans des mini-chaperons peptidiques, capables d’enrober la surface des fibres et ainsi de bloquer la liaison à la membrane et l’internalisation des fibres. De surcroît, des polypeptides issus des partenaires précédemment identifiés d’αSyn ont été testé pour leur liaison aux fibres et leur effet sur la propagation des fibres. / Fibrillar α-Synuclein (αSyn) is the molecular hallmark of Parkinson’s Disease and other synucleinopathies. Its prion-like propagation between cells is linked to disease progression. In this study, we looked to molecular chaperones previously implicated in αSyn fibrillation and/or toxicity to identify proteins capable of binding αSyn fibrillar aggregates in order to target their propagation. We further assessed the effect of the fibril-binding chaperones on internalization of αSyn fibrils by Neuro-2a cells. We demonstrate that the interaction of aggregating αSyn with αB-crystallin (αBc) or Carboxyl terminus of Hsc70-interacting protein (CHIP) led to the formation of fibrils that are less internalized by cells. Finally, using an optimized chemical cross-linking and mass spectrometry strategy, we identified the interaction areas between fibrillar αSyn and either αBc or CHIP. These chaperone residues, located proximally to αSyn fibrils, could be subsequently developed into peptidic mini chaperones, capable of coating the fibril surface and thereby blocking fibrillar cell binding and internalization. Furthermore, polypeptides derived from previously identified αSyn binding partners were tested for their binding to αSyn fibrils and subsequent effect on fibril propagation.

No description available.

Aggregation of microtubule-associated protein tau is involved in the pathology of several neurodegenerative diseases, including Alzheimer’s disease. The affibody TP4 has been shown to inhibit this aggregation process, and its target-binding positions were previously grafted onto a dimeric affibody scaffold, creating the sequestrin seqTP4. This project constitutes a part of the affinity maturation process of seqTP4, using two different bacterial display methods. An error-prone PCR library was first expressed on Staphylococcus carnosus cells for selection of variants with improved tau-binding properties, resulting in a library of 1.4×107 transformants. Flow cytometric tests indicated difficulties in the setup due to nonspecific interactions, and whereas several different approaches to alleviate the problems were investigated, two cell sorting attempts were ultimately unsuccessful. Subcloning of seqTP4 and the library to an Escherichia coli surface display vector resulted in functional surface expression of seqTP4 on E. coli JK321 and BL21 cells, and a BL21 library size of 1.6×109 transformants. An initial flow cytometric test of this library indicates the presence of improved tau-binding variants, paving the way for future cell sorting. / Aggregering av mikrotubuli-associerat protein tau är involverad i patologin av flera neurodegenerativa sjukdomar, däribland Alzheimers sjukdom. Affibodymolekylen TP4 har visat sig inhibera denna aggregeringsprocess, och överföring av dess målbindande positioner till ett dimeriskt affibodyprotein har tidigare gett upphov till seqTP4, en så kallad sequestrin. Detta projekt utgör ett led i processen att affinitetsmaturera seqTP4, med hjälp av två olika metoder för presentation av proteiner på ytan av bakterieceller. Ett error-prone PCR-bibliotek uttrycktes först på ytan av Staphylococcus carnosus-celler för selektion av varianter med ökad affinitet för tau, vilket resulterade i ett bibliotek av 1.4×107 transformanter. Flödescytometriska tester tydde på svårigheter i detta upplägg på grund av ospecifika interaktioner, och emedan flera olika angreppssätt för att förmildra dessa problem undersöktes, misslyckades slutligen två cellsorteringsförsök. Omkloning av seqTP4 och biblioteket till en vektor för ytpresentation på Escherichia coli resulterade i funktionellt ytuttryck av seqTP4 på E. coli JK321- och BL21-celler, och ett BL21-bibliotek bestående av 1.6×109 transformanter. Ett första flödescytometriskt test av detta bibliotek tyder på närvaron av varianter med förbättrad förmåga att binda tau, och vägen ligger nu relativt öppen för cellsortering.

As the aging population is increasing worldwide, so is the prevalence of neurodegenerativediseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal dementia(FTD) and amyotrophic lateral sclerosis (ALS). Reliable biomarkers able to aid the diagnosis anddifferentiation of these diseases are needed in order to start the right treatment as early as possible.Due to its representative state of the central nervous system, cerebrospinal fluid (CSF) is afavorable sample material for biomarker discovery within neurodegenerative diseases. Alteredprotein levels of this body fluid might serve as a biomarker, but further validation of earlierfindings is needed. The aim of this project was to validate earlier studies suggesting potentialprotein biomarkers in CSF. From a list of 80 potential biomarkers in the CSF of patient samples,eight were chosen to be included in this validation effort. By utilizing a suspension bead array ina sandwich assay setup, 21 antibodies were tested in an initial screening. Antibody pairs that couldmeasure the protein levels in a dilution dependent manner was further optimized before individualpatient samples were analyzed. Sandwich assays targeting the three proteins Amphiphysin(AMPH), Chitotriosidase-1 (CHIT1) and Beta-synuclein (SNCB) were successfully developed andcorrelated to earlier generated data using a suspension bead array with a single binder setup.Therefore, the earlier findings of elevated levels of AMPH and SNCB in AD patients and CHIT1in ALS patients were successfully validated. / Prevalensen av neurodegenerativa sjukdomar såsom Alzheimers sjukdom (AD), Parkinsonssjukdom (PD), frontallobsdemens (FTD) och amyotrofisk lateralskleros (ALS) ökar i takt med denåldrande populationen. Pålitliga biomarkörer som kan hjälpa till vid diagnostiseringen av dessasjukdomar behövs för att starta rätt behandling så tidigt som möjligt. Ryggmärgsvätska, enkroppsvätska tillhörande det centrala nervsystemet, kan ge en inblick i det centrala nervsystemetstillstånd. Förändrade proteinnivåer i denna kroppsvätska skulle därför kunna fungera sombiomarkörer. Målet i detta projekt var att validera tidigare föreslagna proteinbiomarkörer iryggmärgsvätska. Utifrån en lista av 80 tidigare analyserade proteiner i ryggmärgsvätska hospatienter, inkluderades åtta proteiner i detta valideringsförsök. En antikroppsbaserad så kalladsandwich assay användes i en suspension bead array för att testa 21 stycken antikroppar i ett initialtscreeningsförsök. Antikroppspar som kunde mäta proteinnivåer på ett spädningsberoende vis i detinitiala screeningsförsöket optimerades vidare innan den utvecklade sandwich assayn användes föratt analysera proteinnivåer i individuella prover. Sandwich assays gentemot Amphiphysin(AMPH), Chitotriosidase-1 (CHIT1) och Beta-synuclein (SNCB) kunde bli framtagna ochkorrelerade gentemot tidigare genererat data från en single binder assay på ett framgångsrikt sätt.Projektet kunde därmed validera tidigare fynd som indikerat förhöjda nivåer av AMPH och SNCBi AD patienter, samt förhöjda nivåer av CHIT1 i ALS patienter.

The second identified substrate of PAS kinase discussed is Pbp1. The human homolog of Pbp1 is ataxin-2, mutations in which are a known risk factor for amyotrophic lateral sclerosis (ALS). As diet and sex have been shown to be important factors regarding PAS kinase function, they also are strong contributing factors to ALS and are extensively reviewed herein. Pbp1 is known to be sequestered by PAS kinase under glucose depravation, and it can sequester additional proteins along with it to regulate different cellular pathways. To shed light on the pathways affected by Pbp1, we performed a yeast two-hybrid assay and mass spectrometry, identifying 32 novel interacting partners of Pbp1 (ataxin-2). We provide further analysis of the direct binding partner Ptc6, measuring mitophagy, mitochondrial content, colocalization, and respiration. This work elucidates novel molecular mechanisms behind the function of PAS kinase and yields valuable insights into the role of PAS kinase in disease.

La sclérose latérale amyotrophique (SLA) et la démence fronto-temporale (DFT) sont des maladies neurodégénératives fatales, actuellement sans traitement. Ces maladies entrainent la dégénérescence des neurones moteurs et corticaux, engendrant des troubles moteurs et cognitifs et ultimement menant à la mort des patients souvent par détresse respiratoire trois à cinq ans après l’apparition des premiers symptômes. À l’échelle d’une vie, le risque de développer ces pathologies est de 1 pour 300-400 pour la SLA et 1 pour 742 pour la DFT, faisant de ces pathologies un risque majeur. Avec le vieillissement de la population que nous connaissons actuellement, il est évident que l’incidence de ces maladies deviendra de plus en plus élevée. Ainsi il est essentiel de comprendre les mécanismes moléculaires sous-jacents à ces pathologies dans le but de développer des thérapies effectives et prévenir l’impact de ces pathologies dans notre société. À ce jour, l’étiologie de la SLA-DFT est encore débattue, cependant la communauté scientifique s’accorde sur le fait que l’interaction entre la génétique et l’environnement joue un rôle essentiel dans le développement de ces maladies. La caractéristique moléculaire principale de ces pathologies est la localisation cytoplasmique de la protéine, normalement, nucléaire TDP-43. TDP-43 est un régulateur clef de l’homéostasie des ARNs. Parmi ces nombreuses fonctions, TDP-43 régule la formation des granules de stress, en régulant leur protéine régulatrice G3BP1. Ces granules formés d’ARN et de protéines se forment pour protéger les cellules durant une période de stress. Récemment, ces granules ont fait l’objet de nombreuses études et leurs dysfonctions ont été associées à la SLA-DFT. Dans cette thèse, nous avons approfondi l’étude de la régulation de TDP-43 sur G3BP1. Nous avons défini que TDP-43 stabilise les transcrits de G3BP1 de par une liaison forte à une séquence conservée à travers l’évolution se situant dans le 3’UTR de G3BP1. La perte de localisation nucléaire, la présence de mutations ou de TDP-35, une isoforme pathologique de TDP-43, sont associées à une diminution des niveaux de G3BP1. Également, d’un point de vue histopathologique, dans le cortex orbitofrontal des patients atteints de SLA-DFT, les neurones présentant une localisation cytoplasmique de TDP-43 ont une perte des niveaux transcriptionnels de G3BP1, associant alors directement G3BP1 à la maladie. Par la suite, nous avons défini que la perte de fonction en tant que stabilisateur, permet la liaison de microARNs sur les transcrits de G3BP1, engendrant leur dégradation. Le blocage de la liaison de microARNs sur G3BP1 empêche la dégradation des transcrits et restaure les fonctions de la protéine. Ainsi, nous avons déterminé un moyen de contrer la perte de fonction de TDP-43 sur G3BP1. De façon intéressante, en plus de la formation des granules de stress, G3BP1 est essentielle pour l’homéostasie neuronale et la survie neuronale post-stress. Par conséquent, la restauration de la protéine est potentiellement une avenue thérapeutique multi-approche pour le traitement de ces maladies. / Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two fatal neurodegenerative diseases, currently without cure. These diseases lead to the degeneration of motor and cortical neurons, causing motor and cognitive disorders and ultimately leading to death, often from respiratory distress three to five years after the onset. Over a lifetime, the risk of developing these conditions is 1 in 300-400 for ALS and 1 in 742 for FTD, making these conditions a major risk. With the current aging of the population, it is evident that the incidence of these diseases will become increasingly high. It is therefore essential to understand the molecular mechanisms underlying these pathologies in order to develop effective therapies. To this day, the etiology of ALS-FTD is still debated. However, the scientific community agrees that the interaction between genetics and the environment play an essential role in the development of these diseases. The main molecular characteristic of these pathologies is the cytoplasmic localization of the normally nuclear protein TDP-43. TDP-43 is a key regulator of RNA homoeostasis. Among these many functions, TDP-43 regulates the formation of stress granules, by regulating their nucleator protein G3BP1. These granules of RNA and protein form to protect cells during times of stress. Recently these granules have been the subject of several studies and their dysfunction has been associated with ALS-FTD. In this thesis, we have deepened the study of the regulation of TDP-43 on G3BP1. We have defined that TDP-43 stabilizes G3BP1 transcripts by strong binding to a sequence conserved through evolution located in the 3'UTR of G3BP1. Loss of nuclear localization, the presence of mutations or of TDP-35, a pathological isoform of TDP-43, are associated with decreased levels of G3BP1. Also, histopathologically, in the orbitofrontal cortex of patients with ALS-DFT, neurons with cytoplasmic localization of TDP-43 have a loss of transcriptional levels of G3BP1, directly associating G3BP1 with the disease. Subsequently, we defined that TDP-43 loss of function as a stabilizer allows the binding of two microRNAs on the G3BP1 transcripts, causing their degradation. Blocking the binding of these microRNAs to G3BP1 prevents the degradation of the transcripts and restores the functions of the protein. Thus, we have determined a way to counter the loss of function of TDP-43 on G3BP1. Interestingly, in addition to the formation of stress granules, G3BP1 is essential for neuronal homoeostasis and post-stress neuronal survival. Therefore, the restoration of the protein is potentially a multi-approach therapeutic avenue for the treatment of these diseases.

<p dir="ltr">Neurotrauma encompasses a broad category of injuries affecting the central nervous system (CNS), which includes both the traumatic brain injury (TBI) and spinal cord injury (SCI). These injuries can result from various causes, including accidents, falls, sports-related incidents, and other traumatic events, affecting millions of individuals annually. Traumatic injuries are the leading cause of disability, and moreover are associated with elevated risk of developing cognitive impairments and neurodegenerative diseases (ND) such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD). The elevated ND risk arising from neurotrauma poses significant burdens on healthcare systems and affect life quality of affected individuals, emphasizing the critical need for research aimed at understanding the underlying mechanisms conferring ND risk from the lesion center to CNS. The goal of my thesis is to understand persistent molecular changes post SCI associated with ND using a combination of a rat animal model and neuronal cultures derived from human induced pluripotent stem cells.</p><p dir="ltr">I started with Sprague-Dawley rats with T10 spinal cord contusive injury; and assessed immediate and persistent changes in transcriptomic and epigenetic markers via next generation sequencing (NGS) at primary lesion site and distal spinal cord tissue. Along with global changes in chromatin arrangements and DNA methylation, we observed significant transcriptomic changes enriched for pathways of inflammatory responses, and synaptogenesis. These changes were further verified using immunohistochemistry and super resolution microscopy. To further understand the long-term brain abnormality linked to SCI, we investigated persistent alterations in the composition and molecular profiles of both the male and female motor cortex 30 days after injury. Immunohistochemistry revealed that SCI leads to neuronal loss and changes in synaptic density and morphology; and significant alterations in the neuron-astrocyte ratio and astrocyte morphology, in male motor cortex supporting our hypothesis that SCI may increase the risk of neurodegeneration by affecting the motor cortex. Comparison of transcriptomic data collected at a sub-acute stage in male rats, namely 7 days post injury, with 30 days post injury, identified persistent and de novo changes that occur primarily after recovery of spinal cord injury, which are enriched for neuronal and synaptic function related pathways. Interestingly, neuroendocrine-related pathways were prominently implicated at the chronic stage of SCI, with Esr1 identified as a major upstream regulator offering protective effects in females that did not exhibit significant alterations in cellular composition or morphology after SCI. Collectively, our study paved the way towards understanding sexual dimorphism in brains after spinal cord injury and provides a plausible connection between spinal cord injury and neurodegeneration later in life that were further investigated using a humanized culture model.</p><p dir="ltr">We established the feasibility of using hiPSC derived neurons to examine long term neurotoxic mechanism using lead (Pb) as a model chemical with strong associations with elevated AD risks later in life. A similar culture system was then used to assess persistent neurotoxicity of acrolein, a chemical that is known to emerge in brains post traumatic injury. We found that acrolein induced alterations in neuronal network morphology, synaptic density, and excitability. Furthermore, acrolein exposure negatively impacted mitochondrial function and persistently altered neuronal resilience towards a secondary stressor of mitochondria, namely MPP+. Acrolein exposure also alters the expression of tau and tau phosphorylation which collectively result in increased cellular vulnerability toward paired helical filament (PHF-tau) seeding, a known neurotoxin associated with ND. These findings collectively provide molecular insights as to how acrolein can partake alterations in neural function and resilience to stressors; and relay ND risks in neurotrauma patients later in life.</p><p dir="ltr">In conclusion, our comprehensive investigation employing both rat and hiPSC models uncovers plausible molecular pathways connecting SCI to neurodegenerative diseases, providing insights into the enduring consequences of these injuries on affected patients.</p>

The protein Ataxin-2 is a known positive regulator of stress granules in humans, mice and yeast (known as yeast PBP1). Due to the role that stress granules play in diseases including Amyotrophic Lateral Sclerosis (ALS) and cancer, this thesis investigates the role of Ataxin-2 and its protein binding partners in stress granule development and its effects on various metabolic phenotypes of the cell. PAS kinase is a sensory protein kinase, conserved from yeast to man, which regulates respiration and lipid biosynthesis. Our lab discovered that PAS kinase phosphorylates and activates Ataxin-2 in yeast, and that PAS kinase overexpression enhances localization of Ataxin-2 to stress granules. Our preliminary results from yeast show that PAS kinase positively regulates stress granule formation in response to metabolic stress. Ataxin-2 normally functions to promote stress granule formation and it has been specifically shown to sequester and inhibit mammalian target of rapamycin complex I (mTORC1), a major player in the regulation of cell growth, to stress granules in both yeast and mammalian cells. To build upon this knowledge we performed a large-scale yeast interactome to identify Pbp1 binding partners through yeast-two hybrid and mass spectrometry. We identified 32 novel putative binding partners. A protein of note was Ptc6, a known regulator of mitophagy with human homolog PPM1K, which is not known to be involved in stress granules. Through colocalization with Ppb1 we determined that Ptc6 is sequestered to stress granules under glucose depravation. Under Pbp1 overexpression, Ptc6 was shown to increase localization to a stress granule marker, Pab1, showing that Pbp1 may be actively promoting Ptc6 to stress granules. We investigated the effects of eliminating Pbp1 and Ptc6 in yeast cells, including on mitophagy, mitochondrial quantification, whole cell respiration and mitochondrial reactive oxidative species. In a separate project, due to the outbreak of a worldwide pandemic and early concerns that currency could be a potential SARS-CoV-2 fomite, we investigated whether the virus could survive on varying types of currency. We conducted environmental studies and found no viable virus on bank notes or money cards. In vitro studies with live virus suggested SARS-CoV-2 was highly unstable on banknotes, however SARS-CoV-2 displayed increased stability on money cards with live virus detected after 48 hours.