Mitochondria play important roles in neuronal function and survival, including ATP production, Ca2+ buffering, and apoptosis. Mitochondrial dysfunction is a common event in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD); however, what causes the mitochondrial dysfunction remains unclear. Mitochondrial fission is mediated by dynamin-related protein 1 (DRP1) and fusion by mitofusin 1/2 (MFN1/2) and optic atrophy 1 (OPA1), which are essential for mitochondrial function. Mutations in the mitochondrial fission and fusion machinery lead to neurodegeneration. Thus, whether defective mitochondrial dynamics participates in ALS and HD requires further investigation. ALS is a fatal neurodegenerative disease characterized by upper and lower motor neuron loss. Mutations in Cu/Zn superoxide dismutase (SOD1) cause the most common familiar form of ALS by mechanisms not fully understood. Here, a new motor neuron-astrocyte coculture system was created and live-cell imaging was used to evaluate mitochondrial dynamics. Excessive mitochondrial fission was observed in mutant SOD1G93A motor neurons, correlating with impaired axonal transport and neuronal cell death. Inhibition of mitochondrial fission restored mitochondrial dynamics and protected neurons against SOD1G93A -induced mitochondrial fragmentation and neuronal cell death, implicating defects in mitochondrial dynamics in ALS pathogenesis. iv HD is an inherited neurodegenerative disorder caused by glutamine (Q) expansion in the polyQ region of the huntingtin (HTT) protein. In the current work, mutant HTT caused mitochondrial fragmentation in a polyQ-dependent manner in both primary cortical neurons and fibroblasts from human patients. An abnormal interaction between DRP1 and HTT was observed in mutant HTT mice and inhibition of mitochondrial fission or promotion of mitochondrial fusion restored mitochondrial dynamics and protected neurons against mutant HTT-induced cell death. Thus, mutant HTT may increase mitochondrial fission by elevating DRP1 GTPase activity, suggesting that mitochondrial dynamics plays a causal role in HD. In summary, rebalanced mitochondrial fission and fusion rescues neuronal cell death in ALS and HD, suggesting that mitochondrial dynamics could be the molecular mechanism underlying these diseases. Furthermore, DRP1 might be a therapeutic target to delay or prevent neurodegeneration.

The 43 kDa TAR DNA binding protein (TDP-43) has been identified as one of the major proteins that accumulates in the cytoplasm of brain and spinal cord from the patients affected with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Under basal conditions, TDP-43 localizes in nucleus functioning as an RNA binding protein to regulate different aspects of RNA metabolism, such as alternative splicing of messenger RNA. In ALS/FTLD brains and spinal cords, TDP-43 forms well-defined cytoplasmic granules, the behavior very similar to stress granule (SG) proteins, but the mechanisms are poorly understood. To investigate the mechanism of TDP-43 granule formation and to identify potential therapeutic targets by inhibiting the granule formation, our laboratory screened a chemical library of 75,000 compounds using the inducible PC12 cells that express EGFP-tagged wild-type human TDP-43. We used the biological effect of cycloheximide on SGs as a basis for the screen, since it is known to prevent the formation of SGs and TDP-43 granules, pointing to a novel biological pathway that regulates TDP-43 granule formation. One of the candidate compounds, Compound 8 (C8) and its analog C8j dose- dependently decreased the arsenite-induced TDP-43 granules without cytotoxicity, and reduced the protein levels of full-length, truncated, high molecular weight and phosphorylated TDP-43. In addition, we found C8j reduced the phosphorylation at novel, previously unknown Thr103-Ser104 amino acid residues of human TDP-43 under arsenite stress. The phospho-mimetic mutations at these sites induced spontaneous intracellular TDP-43 granules, indicating their regulatory role in TDP-43 granule formation. We also performed a series of gene expression analysis combined with the systems biology algorithm, mode of action by network identification (MNI), to identify the mode of action of C8, and found C8 potentially targets protein metabolism and modification processes to reduce the TDP-43 granules. Our study identified a family of non-cytotoxic chemical compounds that reduces the formation of arsenite-induced TDP-43 granules and their potential mode of action. Furthermore, we identified previously unknown TDP-43 phosphorylation sites Thr103- Ser104 that are involved in the TDP-43 granule formation. We anticipate this study will elucidate the biological pathways regulating TDP-43 aggregation and potential therapeutics for ALS/FTLD-U.

The mechanisms by which mitochondria convert nutrients into cellular energy have been described in intricate detail, and yet, the regulation and compartmentalization of such metabolic pathways are poorly understood. As a result, the underlying causes of mitochondrial dysfunction and bioenergetic deficiency in diseases such as amyotrophic lateral sclerosis (ALS) remain elusive. To address this longstanding gap in the field, we first sought to understand how the metabolism of glucose and glucose-derived pyruvate are regulated in the cell. Previous research has suggested that this metabolic regulation is mediated by specialized lipid raft domains of the endoplasmic reticulum (ER) in close contact with mitochondria, referred to as mitochondria-associated ER membranes (MAM). Using density gradient ultracentrifugation and immunoblotting techniques, we found that MAM domains play a role in the compartmentalization of glycolysis by recruiting and promoting the interaction of specific glycolytic enzymes. We then performed a series of bioenergetic, proteomic, and lipidomic analyses to determine how the establishment of ER-mitochondria contact sites at MAM affects the biology of mitochondria attached at these domains. We observed a novel distinction between mitochondria in contact with ER-MAM domains (MER) and those that are free from the ER (FM), with MER displaying a higher capacity for pyruvate-driven respiration and FM being specialized for fatty acid-driven energy production. Finally, using cell and mouse models of ALS with mutations in superoxide dismutase 1 (SOD1), we found that the glycolytic deficiency in ALS is a direct consequence of the progressive disruption of MAM structure and function, which thereby hinders the use of glucose-derived pyruvate as a mitochondrial fuel. This triggers a shift in mitochondrial substrate from pyruvate to fatty acids that, when sustained over time, contributes to the death of motor neurons and the progression of this fatal disease. Overall, this work aims to advance our understanding of metabolic compartmentalization, mitochondrial substrate specificity, and the relevance of both to ALS etiology.

Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) represents a spectrum of neurodegenerative disease with clinical presentations ranging from progressive paralysis to cognitive impairment. Approximately 15% of ALS-FTD patients initially presenting with motor symptoms also receive a diagnosis of dementia, but a majority of these patients demonstrate some level of cognitive impairment over the course of disease. Identifying molecular pathways that contribute to the development of cognitive deficits in ALS-FTD has thus far been limited by the quality of clinical information and postmortem tissue preservation as well as available technologies. This thesis aims to investigate early stages of cognitive involvement in ALS-FTD using postmortem tissues from a cohort of non-demented ALS patients who have had cognitive and pathological phenotyping. Spatially resolved transcriptome profiling of prefrontal cortex tissues from this cohort identifies dysregulated pathways in non-motor regions, contributing to our understanding of molecular perturbations underlying cognitive impairment in ALS-FTD.

Bakgrund: ALS är en neurodegenerativ sjukdom som drabbar drygt 300 personer årligen i Sverige. Ofta är det anhöriga till personerna med ALS som sköter omvårdnaden i hemmet. Det har visat sig medföra stora utmaningar och anhöriga som vårdar är därmed en utsatt grupp som kan behöva stöd. Syfte: Syftet var att beskriva anhörigas upplevelser av stöd vid vård av en närstående med Amyotrofisk lateralskleros i hemmet. Metod: Resultatet baserades på 10 empiriska, kvalitativa studier som återfanns i databaserna PubMed och CINAHL. Analys av studiernas resultat inspirerades av Graneheims och Lundmans innehållsanalys för kvalitativa studier. Resultat: Analysen resulterade i två teman - Praktiskt stöd är avgörande för att hantera vardagen och Emotionellt stöd bidrar till trygghet med 8 subteman. Teman och subteman beskriver anhörigas upplevelser av stöd och inom vilka områden de upplevde sig ha behov av stöd, både under sjukdomsförloppet och efter att personen med ALS avlidit. Konklusion: Anhöriga som vårdar en närstående med ALS i hemmet ställs inför komplexa vårdsituationer som medför varierande praktiska och emotionella stödbehov. Det är viktigt att sjukvårdspersonal med ett personcentrerat förhållningssätt regelbundet bedömer anhörigas stödbehov för att säkerställa att anhöriga som vårdar inte drabbas av ohälsa. / Background: ALS is a neurodegenerative disease that annually affects around 300 people in Sweden. Relatives of the people with ALS are often those who provide care at home. This involves major challenges and relatives who provide care are therefore a vulnerable group which may need support. Aim: The aim was to describe relatives’ experiences of support when providing care for a person with Amyotrophic lateral sclerosis at home. Methods: The result was based on 10 empirical, qualitative studies found on the databases PubMed and CINAHL. The analysis of the studies results were inspired by Graneheim’s and Lundman’s content analysis for qualitative studies. Results: The analysis resulted in two themes – Practical support is vital for managing daily life and Emotional support contributes to safety with 8 subthemes. Themes and subthemes describes relatives’ experiences of support and within which aspects they needed support, both during the course of the disease and after the death of the person with ALS. Conclusion: Relatives who provide care for a person with ALS at home faces complex care situations which entails diverse practical and emotional support needs. It is important that healthcare professionals with a person-centered approach regularly assesses relatives' support needs to ensure that relatives who provide care do not suffer from ill health.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24839号 / 医博第5007号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上, 治久, 教授 寺田, 智祐, 教授 林, 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

As the most common motor neuron disease, Amyotrophic lateral sclerosis (ALS) affects around 4 in every 100,000 people worldwide with reports of increasing prevalence over the years. Characterized by progressive degeneration of motor neurons, ALS patients suffer impairments of movement and typically die from respiratory failure 2-5 years after diagnosis. Curiously, ALS exists on a disease continuum with Frontotemporal Dementia (FTD) where 30-50% of patients will be diagnosed with both diseases. In FTD, degeneration of cortical neurons results in diverse behavioural changes including deficits in executive and social skills as well as language and memory. A central connection between ALS and FTD is TDP-43 (encoded by TARDBP), an essential DNA/RNA binding protein known to serve critical functions in numerous cellular processes. Despite mutations in TARDBP constituting a small percentage of familial cases, TDP 43 nuclear-to-cytoplasmic mislocalization is a pathological hallmark of most ALS-FTD cases. Therefore, therapeutic targets to rectify pathology and disease may be uncovered by identifying factors that regulate TDP-43. While it is currently established TDP-43 is ubiquitinated and phosphorylated in diseased states, our lab recently found TDP-43 is SUMOylated in response to stress. Of note, perturbations in the stress response are becoming increasingly implicated in neurodegenerations. Furthermore, TDP-43 plays critical roles in the stress response which become perturbed in ALS/FTD. We developed a TDP-43 "SUMO dead" mouse allele to gain an understanding of how disrupting this may contribute to the pathogenesis of ALS-FTD. Longitudinal characterization of the model explored behavioural and histological in vivo consequences following loss of TDP-43 SUMOylation. However, the phenotypes observed in the mutant mice were less robust in comparison to established ALS/FTD mouse models. Mutant mice did not have consistent differences in tests for similar outcomes, trials of the same test, or across age. Female mutant mice presented with early hyperactivity and disinhibition along with altered social grooming behaviour. At later age, these female mice developed impairments in spatial working memory. Male mice developed apathetic behaviour and motor deficits at the middle age timepoint. Histologically, various forms of pathological TDP-43 were observed in the absence of neurodegeneration. These data reveal that TDP-43 SUMOylation may play an important role in ALS/FTD pathogenesis.

Human microglia play a pivotal role in neurological diseases, but few targeted therapies that directly modulate microglial state or function exist due to an incomplete understanding of microglial heterogeneity. This thesis aims to advance our understanding of microglial heterogeneity by using single-cell RNA sequencing to profile live human microglia from autopsies or surgical resections across diverse neurological diseases and using computational tools to infer chemical and genetic regulators of specific microglial substates. Chapter 1 provides an overview of microglial ontogeny, function, and known heterogeneity, especially in disease contexts. It also describes the steadily increasing disease burden seen in neurological disease as well as the lack of efficacious treatments and future directions for microglia-targeted therapies. Chapter 2 focuses on microglial heterogeneity in an understudied disease, ALS, describing population structure shifts seen in ALS across cortex and spinal cord. Chapter 3 instead focuses on exploring underlying cross-disease microglial population structure, identifying subsets with metabolic and functional properties, as well as subsets enriched in susceptibility genes for neurodegenerative disease. We then demonstrate applications of this type of data by using our resource to annotate other datasets. Chapter 4 leverages this data in another way, by identifying and validating candidates for chemically and genetically inducing subtype-specific states in vitro. Notably, we show that Camptothecin downregulates the transcriptional signature of disease-enriched subsets and upregulates a signature previously shown to be depleted in Alzheimer’s. Finally, I review our findings and discuss future directions for the field.

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