BackgroundThe key pathological signature of ALS/ FTLD is the mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm. However, TDP-43 gain of function in the cytoplasm is still... Show moreBackgroundThe key pathological signature of ALS/ FTLD is the mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm. However, TDP-43 gain of function in the cytoplasm is still poorly understood since TDP-43 animal models recapitulating mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm are missing.MethodsCRISPR/Cas9 technology was used to generate a zebrafish line (called CytoTDP), that mis-locates endogenous TDP-43 from the nucleus to the cytoplasm. Phenotypic characterization of motor neurons and the neuromuscular junction was performed by immunostaining, microglia were immunohistochemically localized by whole-mount tissue clearing and muscle ultrastructure was analyzed by scanning electron microscopy. Behavior was investigated by video tracking and quantitative analysis of swimming parameters. RNA sequencing was used to identify mis-regulated pathways with validation by molecular analysis.ResultsCytoTDP fish have early larval phenotypes resembling clinical features of ALS such as progressive motor defects, neurodegeneration and muscle atrophy. Taking advantage of zebrafish’s embryonic development that solely relys on yolk usage until 5 days post fertilization, we demonstrated that microglia proliferation and activation in the hypothalamus is independent from food intake. By comparing CytoTDP to a previously generated TDP-43 knockout line, transcriptomic analyses revealed that mis-localization of endogenous TDP-43, rather than TDP-43 nuclear loss of function, leads to early onset metabolic dysfunction.ConclusionsThe new TDP-43 model mimics the ALS/FTLD hallmark of progressive motor dysfunction. Our results suggest that functional deficits of the hypothalamus, the metabolic regulatory center, might be the primary cause of weight loss in ALS patients. Cytoplasmic gain of function of endogenous TDP-43 leads to metabolic dysfunction in vivo that are reminiscent of early ALS clinical non-motor metabolic alterations. Thus, the CytoTDP zebrafish model offers a unique opportunity to identify mis-regulated targets for therapeutic intervention early in disease progression. Show less
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterised by a progressive loss of motor function. Despite the strives into growing knowledge on ALS, fundamental... Show moreAmyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterised by a progressive loss of motor function. Despite the strives into growing knowledge on ALS, fundamental obstacles remain into understanding disease causation and nominating drug targets for a cure. With this work, we widen our understanding of ALS through the analysis of single-nuclei RNA-sequencing dataset of patients’ and control motorcortices. We show intrinsically higher expression of ALS-related genes in Extratelencephalic Neurons accompanied by selective vulnerability of subsets of cortical motor neurons. These changes are found with alterations in oligodendrocytes and microglia that widen our knowledge of cell-to-cell interactions in ALS. We use hiPSC-derived in vitro systems to model the molecular changes identified. Secondly, we offer a wide view on models of human brain cells in a dish to encompass protocols useful for modelling complex cell-to-cell interactions in ALS. Moreover, we describe a human in vitro system for the study of motor neuron that is highly reproducible, scalable and high-throughput. This new method allows the assessment of multiple cell lines in the same dish and could provide insights into heterogeneity in human populations and mechanisms disrupted in disease. We then use these models to further dissect molecular mechanisms disrupted in ALS. We undertake a discussion onto the future of the field and hopefully the opening to a more holistic approach to the understanding of ALS, where multidisciplinary techniques and the use of different models might expand our perspectives on the disease. Show less
Degenerative diseases of the nervous system, such as Alzheimer's, Parkinson's and ALS, are severe, progressive and ultimately fatal. Most existing drugs for these neurodegenerative diseases only... Show moreDegenerative diseases of the nervous system, such as Alzheimer's, Parkinson's and ALS, are severe, progressive and ultimately fatal. Most existing drugs for these neurodegenerative diseases only temporarily relieve symptoms, increase mobility or relieve pain, but do not slow disease progression.This dissertation describes a method to efficiently carry out the development of new drugs that could inhibit disease progression in neurodegenerative diseases. Namely, by using pharmacodynamic biomarkers. These are signaling substances to measure the magnitude of a drug response.These biomarkers can be used in early clinical-pharmacological studies in healthy volunteers or small groups of patients to select the best drug candidates and their expected therapeutic doses as early as possible in the development stage. This helps to make informed choices to advance a potential new drug into large and expensive phase 2 and 3 (registration) studies, or conversely to discontinue development of a non-potential drug as early as possible. This biomarker method was applied in this dissertation to investigate 2 new drugs that could potentially slow disease progression in Alzheimer's and ALS (a RIPK1 inhibitor) or Parkinson's disease (a LRRK2 inhibitor). The research results from multiple early clinical-pharmacological studies in healthy volunteers and patients described in this thesis form the basis for larger phase 2 and 3 follow-up studies that have now been initiated with ALS patients and Parkinson's disease patients. Both with the goal of confirming whether these agents can indeed slow disease progression, which would represent a major breakthrough in the treatment of these conditions. Show less
Cappelli et al. reported that Nitric Oxide Synthase 1 Adaptor Protein is a co-regulated transcript of the TAR DNA-binding protein 43 kDa, reduced in amyotrophic lateral sclerosis and frontotemporal... Show moreCappelli et al. reported that Nitric Oxide Synthase 1 Adaptor Protein is a co-regulated transcript of the TAR DNA-binding protein 43 kDa, reduced in amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients with TAR DNA-binding protein 43 kDa pathology. Overall, their results highlight Nitric Oxide Synthase 1 Adaptor Protein as a novel druggable disease-relevant gene in TAR DNA-binding protein 43 kDa-related proteinopathies.Many lines of evidence have highlighted the role played by heterogeneous nuclear ribonucleoproteins in amyotrophic lateral sclerosis. In this study, we have aimed to identify transcripts co-regulated by TAR DNA-binding protein 43 kDa and highly conserved heterogeneous nuclear ribonucleoproteins which have been previously shown to regulate TAR DNA-binding protein 43 kDa toxicity (deleted in azoospermia-associated protein 1, heterogeneous nuclear ribonucleoprotein -Q, -D, -K and -U). Using the transcriptome analyses, we have uncovered that Nitric Oxide Synthase 1 Adaptor Protein mRNA is a direct TAR DNA-binding protein 43 kDa target, and in flies, its modulation alone can rescue TAR DNA-binding protein 43 kDa pathology. In primary mouse cortical neurons, we show that TAR DNA-binding protein 43 kDa mediated downregulation of Nitric Oxide Synthase 1 Adaptor Protein expression strongly affects the NMDA-receptor signalling pathway. In human patients, the downregulation of Nitric Oxide Synthase 1 Adaptor Protein mRNA strongly correlates with TAR DNA-binding protein 43 kDa proteinopathy as measured by cryptic Stathmin-2 and Unc-13 homolog A cryptic exon inclusion. Overall, our results demonstrate that Nitric Oxide Synthase 1 Adaptor Protein may represent a novel disease-relevant gene, potentially suitable for the development of new therapeutic strategies. Show less
This thesis describes the development of microRNA-based gene therapies for ALS andSCA3. Other aspects of a successful gene therapy including the administration routes toreach the target organs and... Show moreThis thesis describes the development of microRNA-based gene therapies for ALS andSCA3. Other aspects of a successful gene therapy including the administration routes toreach the target organs and regulation of the transgene expression were also investigated.Due to the permanent nature of gene therapy, a method to modulate the transgeneexpression is desirable. Show less
The discovery of human pluripotent stem cells has enabled the development of assays to model human disease in vitro. The research described in this thesis has focused on modeling Amyotrophic... Show moreThe discovery of human pluripotent stem cells has enabled the development of assays to model human disease in vitro. The research described in this thesis has focused on modeling Amyotrophic Lateral Sclerosis and Lesch Nyhan Syndrome using pluripotent stem cells. Chapter 1 contains the aim and outline of this thesis, Chapter 2 is introduction to the field and provides an overview of recent advances in stem cell modeling of Amyotrophic Lateral Sclerosis (ALS). Chapter 3 contains an introduction to the field of X-chromosome inactivation. In Chapter 4 we identify the Prostaglandin D2 DP1 receptor (DP1) as a therapeutic target for ALS and more generally validate that insights found in stem cell models of disease can be validated in vivo. In Chapter 5, we address whether non-steroidal anti-inflammatory drugs (NSAIDs) affect survival of motor neurons in ALS. In Chapter 6, we develop a stem cell assay to model Lesch Nyhan Syndrome (LNS). Finally, Chapter 7 is the general discussion of the work and contains a short perspective of the future. Show less
