Neurodegeneration and Epilepsy in a Zebrafish Model of CLN3 Disease (Batten Disease)

The neuronal ceroid lipofuscinoses are a group of lysosomal storage disorders that comprise the most common, genetically heterogeneous, fatal neurodegenerative disorders of children. They are characterised by childhood onset, visual failure, epileptic seizures, psychomotor retardation and dementia. CLN3 disease, also known as Batten disease, is caused by autosomal recessive mutations in the CLN3 gene, 80–85% of which are a ~1 kb deletion. Currently no treatments exist, and after much suffering, the disease inevitably results in premature death. The aim of this study was to generate a zebrafish model of CLN3 disease using antisense morpholino injection, and characterise the pathological and functional consequences of Cln3 deficiency, thereby providing a tool for future drug discovery. The model was shown to faithfully recapitulate the pathological signs of CLN3 disease, including reduced survival, neuronal loss, retinopathy, axonopathy, loss of motor function, lysosomal storage of subunit c of mitochondrial ATP synthase, and epileptic seizures, albeit with an earlier onset and faster progression than the human disease. Our study provides proof of principle that the advantages of the zebrafish over other model systems can be utilised to further our understanding of the pathogenesis of CLN3 disease and accelerate drug discovery

Impaired β-Cell Neogenesis in a Mouse Model of Metabolic Syndrome

Pancreatic islet β-cell plays an essential role in insulin release and hence glucose homeostasis. The maintenance of glucose homeostasis depends on β-cell ability to cope with enough insulin to fulfill metabolic and physiological demands. Adequate insulin release is the result of highly complex and dynamic interplays between acute changes in β- cell electrical activity, exocytosis and chronic adaptations in cellular function, volume, mass and proliferation. All of this appears modulated, to some extent, by the functional presence of Slc12a2 symporters, also known as Na+K+2Cl– cotransporter-1 (Nkcc1), which accumulates intracellular Cl– above its electrochemical equilibrium. Recent studies from our laboratory showed that mice lacking Nkcc1 in β-cell (Nkcc1βKO) develop a cluster of metabolic phenotypes reminiscent of the metabolic syndrome i.e., hyperglycemia, hyperinsulinemia, glucose intolerance, insulin resistance, steatohepatitis and overweight. The present study is aimed at determining the potential relationship between those phenotypes and pancreas morphometric parameters including islet size, β-cell number, volume, mass and neogenesis. The results presented here indicate that elimination of Nkcc1 from β-cell negatively impact those parameters, even before the onset of overweight and its metabolic complications. Therefore, we propose that Nkcc1 plays a potential causative role in the development of metabolic syndrome

マイクロトム変異体コレクションから単離したトマトcurl変異体の遺伝学的及び形態学的解析

筑波大学 (University of Tsukuba)201

NLRP3 inflammasome activation in primary murine microglia and monocyte-derived human microglia

openParkinson’s Disease (PD) is a neurodegenerative disorder with unclear etiology. Neuroinflammation pathways, especially linked to NLRP3 inflammasome and Interferon-γ/Interferon-β signaling, is emerging to be an important factor in the upset and progression of the disease. Microglia are the resident immune cells in the brain, triggering neuroinflammatory response upon any detection of signs for brain lesions or nervous system dysfunction. A misregulated microglia response could lead to chronic activation and cause the loss of dopaminergic neurons which is typical of PD. From an evolutionary perspective, microglia appear as a novelty, lacking in Invertebrates, and show interspecific variability in their physiology. In the present study, we aim at studying neuroinflammatory response in both mouse and human microglia, even in association with the PD-linked mutation G2019S on LRRK2 gene.Parkinson’s Disease (PD) is a neurodegenerative disorder with unclear etiology. Neuroinflammation pathways, especially linked to NLRP3 inflammasome and Interferon-γ/Interferon-β signaling, is emerging to be an important factor in the upset and progression of the disease. Microglia are the resident immune cells in the brain, triggering neuroinflammatory response upon any detection of signs for brain lesions or nervous system dysfunction. A misregulated microglia response could lead to chronic activation and cause the loss of dopaminergic neurons which is typical of PD. From an evolutionary perspective, microglia appear as a novelty, lacking in Invertebrates, and show interspecific variability in their physiology. In the present study, we aim at studying neuroinflammatory response in both mouse and human microglia, even in association with the PD-linked mutation G2019S on LRRK2 gene

Pathological Notch3 aggregation

Cerebral small vessel disease (SVD), characterized by pathological processes that affect structure and function of the brain microvasculature and result in subsequent damage of the cerebral white and deep grey matter, is the main cause for long-term disability and vascular dementia. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of monogenic SVD leading to early-onset stroke and vascular dementia, is caused by mutations in the Notch3 transmembrane receptor. Accumulation and deposition of the extracellular domain of Notch3 (Notch3-ECD) in blood vessel walls are the earliest disease manifestations. However, the underlying molecular pathomechanism is incompletely understood and specific treatment options are not available. To study the aggregation behavior of Notch3 mutants we have recently developed an in vitro aggregation assay based on recombinant Notch3-ECD fragments and their detection by the single-molecule spectroscopy technique called scanning for intensely fluorescent targets (SIFT). While the vast majority of CADASIL mutations alter the number of cysteine residues within the Notch3-ECD, over the last years several mutations not involving a cysteine have been reported to be associated with a CADASIL-like phenotype provoking a debate about their clinical significance. In the first part of this work, the in vitro assay was applied to study the pathogenic potential of five of these mutations. Three of them showed an aggregation behavior similar to cysteine-affecting mutations, a finding supported by the typical CADASIL-like clinical appearance of the mutation carriers and we thus classified them as pathogenic mutations. For the two other mutants the available clinical data had been fragmentary and in agreement with that, no significant aggregation potential was observed, strongly suggesting that they represent apathogenic polymorphisms. Thus, our in vitro assay offers new insights into the Notch3 aggregation mechanism and may serve as diagnostic tool determining the clinical relevance of cysteine-sparing mutations. In the second part of this thesis, the in vitro assay was used to search for small-molecule aggregation inhibitors. Several synthetic compounds of the diphenylpyrazole (DPP) class as well as natural polyphenolic substances with antiaggregatory properties were identified. They not only inhibited the de novo aggregation of mutant Notch3 but were also capable of dissolving pre-formed aggregates. Some of the compounds were subsequently shown to be also effective in a novel cellular assay based on the accumulation of mutant Notch3 in the extracellular matrix (ECM) of mouse embryonic fibroblasts (MEF). With this study on drug-mediated inhibition of Notch3 accumulation we have laid a basis for an antiaggregation therapy.Zerebrale Mikroangiopathien sind die häufigste Ursache für Langzeitbehinderung und vaskuläre Demenz. Sie sind durch pathologische Prozesse charakterisiert, die die Struktur und Funktion der Mikrovaskulatur im Gehirn beeinträchtigen und dadurch zu Schädigungen der weißen und tiefen grauen Substanz führen. CADASIL (zerebrale autosomaldominante Arteriopathie mit subkortikalen Infarkten und Leukoenzephalopathie), die häufigste monogene Form zerebraler Mikroangiopathien, führt zu juvenilen Schlaganfällen und vaskulärer Demenz und wird durch Mutationen im Notch3 Rezeptor verursacht. Frühestes pathologisches Merkmal ist die Akkumulation und Ablagerung der extrazellulären Domäne von Notch3 (Notch3-ECD) in den Wänden kleiner Blutgefäße. Allerdings ist bisher wenig über den zu Grunde liegenden molekularen Pathomechanismus bekannt und spezielle Behandlungsmöglichkeiten sind nicht verfügbar. Um das Aggregationsverhalten von Notch3 Mutanten zu studieren, haben wir einen in vitro Aggregationsassay entwickelt, der auf rekombinanten Notch3-ECD Fragmenten und ihrer Detektion mittels SIFT (scanning for intensely fluorescent targets) beruht, einer speziellen Einzelmolekülspektroskopietechnik. Während die Mehrheit der CADASIL Mutationen die Anzahl der Cysteinreste in der Notch3-ECD verändert, ist in den letzten Jahren mehrfach über Mutationen ohne Cysteinbeteiligung berichtet worden, die mit einem CADASIL-ähnlichen Phänotyp assoziiert sind und über deren klinische Relevanz derzeit diskutiert wird. Im ersten Teil dieser Arbeit wurde der in vitro Assay zur Untersuchung des pathogenen Potentials von fünf dieser Mutationen verwendet. Drei von ihnen zeigten ein ähnliches Aggregationspotential wie klassische Cystein-Mutationen, ein Befund, der durch die typischen CADASIL Symptome der Mutationsträger gestützt wird. Wir klassifizieren sie daher als pathogene Mutationen. Für die beiden anderen Mutanten, deren klinische Beschreibung lückenhaft war, konnte dementsprechend kein signifikantes Aggregationspotential beobachtet werden, so dass sie wahrscheinlich apathogene Polymorphismen darstellen. Somit eröffnet unser in vitro Assay neue Einblicke in den Notch3-Aggregationsmechanismus und könnte als diagnostische Methode zur Bestimmung der klinischen Relevanz von Nicht-Cystein Mutationen eingesetzt werden. Im zweiten Teil dieser Arbeit wurde der in vitro Assay für die Suche nach kleinen Molekülen mit aggregationsinhibierender Aktivität eingesetzt. Mehrere synthetische Substanzen aus der Klasse der Diphenylpyrazole (DPP), sowie natürliche Polyphenole mit antiaggregatorischen Eigenschaften wurden identifiziert. Sie hemmten nicht nur die de novo Aggregation von einer Notch3 Mutante, sie waren auch in der Lage, präformierte Aggregate wieder aufzulösen. Für einige der Substanzen konnte anschließend auch eine Wirkung in einem zellulären Assay gezeigt werden, der auf der Akkumulation einer Notch3 Mutante in der extrazellulären Matrix (ECM) muriner embryonaler Fibroblasten (MEF) beruht. Mit dieser Studie zur substanzvermittelten Hemmung der Notch3 Akkumulation haben wir den Grundstein für eine Antiaggregationstherapie gelegt

Development of in vitro Drug Screening Platforms Using Human Induced Pluripotent Stem Cell-Derived Cardiovascular Cells

Drug-induced cardiotoxicity is a critical challenge in the development of new drugs. Since the advent of human pluripotent stem cell-derived cardiomyocytes (CMs), researchers have explored ways to utilize these cells for in vitro preclinical drug screening applications. One area of interest is microphysiological systems (i.e. organ-on-a-chip), which aims to create more complex in vitro models of human organ systems, thus improving drug response predictions. In this dissertation, we investigated novel analysis methods and model platforms for detecting drug-induced cardiotoxicity using human induced pluripotent stem cell (iPSC)-derived cardiovascular cells. First, we utilized human iPSC-derived CMs (iPS-CMs) to establish optical methods of detecting cardioactive compounds. We utilized optical flow to assess the iPS-CM contractions captured using brightfield microscopy. The parameters were then analyzed using a machine learning algorithm to determine the accuracy of detection that can be obtained by the model for a given drug concentration. This result was compared to the analysis of the calcium transients measured using a genetically encoded calcium indicator (GCaMP6). The brightfield contraction analysis matched the detection sensitivity of fluorescent calcium transient analysis, while also being able to detect the effects of excitation-contraction decoupler (blebbistatin), which was not detected using calcium transient analysis. Second, we utilize iPS-CMs to model trastuzumab-related cardiotoxicity. Trastuzumab, a monoclonal antibody against ErbB2 (Her2), is used to treat Her2+ breast cancer and has known clinical cardiotoxicity. We demonstrated that an active ErbB2 signaling via binding of neuregulin-1 (NRG-1) to ErbB4 is necessary to detect the cardiotoxic effects of trastuzumab. Activation of ErbB2/4 pathway via NRG-1 is cardioprotective, and we also demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) similarly activates the ErbB2/4 pathway. Finally, we established a co-culture platform of iPS-CMs and endothelial cells (ECs), which recapitulated the physiological phenomenon of EC-secreted NRG-1 activating the ErbB2/4 pathway on the CMs. Third, we demonstrated the use of human iPSC-derived ECs (iPS-ECs) for creating 3-dimensionial vascular networks inside microfluidic devices. The iPS-ECs were characterized for EC markers and physiological functions. We utilized a CDH5-mCherry iPSC line to create iPS-ECs that expressed VE-cadherin fused to mCherry. The vascular networks formed by the iPS-ECs were patent and perfusable, retaining 70 kDa dextran within the lumen of the vessels. The vasculature responded to small molecule inhibitors, showing increased vessel formation in response to TGF-β inhibitor SB431542 and decreased vessel formation in response to multi-targeted receptor tyrosine kinase inhibitor sunitinib. Taken together, our findings advance the current understanding and utility of iPS-CMs for drug screening applications, while establishing platforms for creating microphysiological systems that incorporate iPS-EC co-culture. The use of iPSC-derived cells opens possibilities for disease-specific and patient-specific drug screening applications in the future