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Browsing by Subject "neuron"

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  • Karmila, Nelli (2022)
    Schizophrenia is a debilitating psychiatric disorder associated with reduced life expectancy. The biological mechanism of schizophrenia is nebulous; however, many findings point to the central nervous system and neurons, where a reduction in dendritic spines has been indicated by previous research. The genetic findings support the involvement of synapses in the pathogenesis of schizophrenia. To study the biological properties stemming from genetics, relevant model systems and efficient methods are needed. Induced pluripotent stem cell (iPSC) technology offers a robust method for modeling the biological processes underlying schizophrenia. Somatic cells, e.g. fibroblasts, can be reprogrammed back to a pluripotent state resembling embryonic stem cells, and further differentiated into any cell type of the body, which might not be otherwise accessible. This allows establishing and characterizing neuronal cultures from patient and control cell lines, potentially revealing biological differences associated to the disease phenotype. The field of schizophrenia research has adopted iPSC technology and multiple studies have been conducted. These include assessments of synaptic density in the produced neuronal cultures, many of which reported decreased density associated with schizophrenia. In this thesis, a modified version of Nehme et al. (2018) protocol was used to differentiate iPSCs into neurons in co-cultures with human iPSC-derived astrocytes. The overarching aim was to construct an immunocytochemistry (ICC) -based assay to measure synaptic density in the produced co-cultures. First, suitable markers for characterization by ICC were tested and selected. The markers were selected to inform about neuronal identity, maturity, and synapses of the differentiated neurons. Next, the culturing conditions were optimized regarding the cell density and coating of the culturing wells. Finally, to estimate the utility of the assay, a pilot study was performed with three cell lines derived from a healthy control and a monozygotic twin pair discordant for schizophrenia. iPSCs from these cell lines were differentiated into neurons in co-cultures with astrocytes, and then characterized with ICC using selected markers and image analysis software. The synaptic density was quantified for each cell line. The performance of the assay was evaluated with analysis of variance (ANOVA) and restricted maximum likelihood model (RELM). An assay to quantify synaptic structures in mature neurons was established. The average synaptic density for all cell lines was approximately 1 synapse per 100μm of neurite. Analysis of the data produced with the assay revealed a notable batch effect and technical variation. This suggests that further optimization is needed to reduce variance from undesired sources. The pilot data suggests that the differences in synaptic density between cases and controls may be modest, further highlighting the need for minimizing noise in the assay to improve signal to noise ratio. However, indicated by power analysis, large sample sizes are needed to identify meaningful differences between cases and controls. In light of these results, more attention should be drawn to the methodology in the field of iPSC-based studies, as the principals of the assay constructed here were similar to other synaptic assays used in previous publications.
  • Jasikova, Sara (2024)
    Schizophrenia (SCZ) is a chronic neuropsychiatric disorder believed to arise from the intricate interplay between genetic predisposition and environmental factors. Though the aetiology of SCZ is unknown many findings support an excessive synaptic pruning hypothesis. Maternal immune activation (MIA), encompassing prenatal infection and systemic inflammation, constitutes a significant environmental risk factor implicated in SCZ onset (Patterson, 2009; Brown, 2012). MIA induces persistent alterations in the microglia of offspring termed microglial priming, characterized by heightened reactivity to inflammatory stimuli (Choudhury and Lennox, 2021). Notably, studies have reported increased sensitivity to activation, elevated expression of inflammatory markers, and an increase in the total number of microglia (Perry and Holmes, 2014; Choudhury and Lennox, 2021). Primed microglia may contribute to excessive synaptic pruning, thereby compromising neuronal connectivity and potentially leading to the onset of SCZ. This thesis investigated the impact of microglia on neurons and explored the microglial tendency for hyperactivation in the context of SCZ predisposition. It utilized induced pluripotent stem cell (iPSC) technology to create a rat astrocyte/unaffected control human iPSC-derived neuron/induced microglia-like cell (iMGL) tri-culture model. Uniquely, iMGLs were differentiated from a library of monozygotic twin lines discordant for SCZ, and unaffected controls. This allows for exploration of the differences between iMGLs from unaffected twins with a genetic predisposition for SCZ, affected twins with clinical manifestation of SCZ, and unaffected controls without a known genetic predisposition for SCZ. The tri-culture system was subjected to lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly(I:C)) treatments to activate iMGLs, and differences in cytokine release, synapse pruning, and neuronal activity were assessed. The principal outcomes of our investigation revealed enhanced cytokine release from SCZ-derived iMGLs when exposed to inflammatory stimuli, alongside increased network connectivity among samples containing genetically predisposed iMGLs. While most of the results did not reach significance, they suggest a potential link between SCZ pathophysiology and hyperactive microglia. Future research will focus on enlarging the study cohort, establishing tri-culture models featuring neurons and iMGLs derived from the iPSCs of the same patient, conducting CBA analysis to confirm the elevated cytokines finding, and scrutinizing iMGL morphology.