Psychiatry - Theses
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ItemAstrocyte neuropathology in autism: role of neuroinflammation & glutamatergic signalling( 2017)Alterations in excitatory glutamatergic signalling together with increased astrocytic activation and neuroinflammation have been observed in the brain of individuals with autism. Astrocytes play important roles in developmental corticogenesis and neurogenesis, as well as regulating glutamate receptor signalling and intercellular glutamate levels at the glutamatergic tripartite synapse. The overall aim of my PhD was to investigate astrocytic neuropathology in autism and its potential interaction with disruptions in mGluR5 signalling. I conducted a post-mortem stereological investigation within the white matter of the dorsolateral prefrontal cortex (DLPFC) to assess density of astrocyte and other glia utilising the optical fractionator. In addition, astrocytic somal size was assessed via the nucleator and total astrocyte process length estimated utilising the spaceballs probe. Using an in-vitro approach I then explored the effect of Poly I:C mediated astrocyte activation on mGluR5 glutamatergic signalling. This included assessing levels of the pro-inflammatory markers IL-6 and Rantes using ELISA, gene expression of astrocytic and glutamatergic genes via qPCR, as well as mGluR5 activity using a radioligand binding assay. Finally, I characterised the gene and protein expression of astrocyte markers of human pluriporent stem cells (hPSC) derived astrocytes using qPCR and immunohistochemistry, as well as cytokines levels using ELISA. The current study revealed no change in astrocyte density or activation morphology within the white matter of the DLPFC in autism versus age matched controls. There was also no alteration in astrocyte cell somal size and total process length. In-vitro Poly I:C induced astrocyte activation demonstrated reduced mGluR5 binding and mRNA expression, with disruption to other astrocytic glutamatergic elements. A novel protocol for differentiating. human pluripotent stem cells into astrocytes was developed, with hPSC-derived astrocytes displaying morphology similar to that of primary human foetal astrocytes and expressing mature astrocyte markers at the gene and protein level, as well as having the ability to be activated upon exposure to Poly I:C. My findings suggest that astrocyte activation within the brain in autism may be less severe than previously appreciated, with the absence of severe astrocytic hypertrophy and increased proliferation not observed. Results from the mechanistic in-vitro studies suggest that mGluR5 and glutamatergic signalling dysregulation can occur as a result of astrocyte activation, and that modulation of mGluR5 through positive allosteric modulation may have potential benefits in reversing some of these astrocyte activation mediated glutamatergic disruptions. Finally, our improved protocol for hPSC astrocyte differentiation provides a simple and efficient method to derive mature and functional astrocytes as an in-vitro model for autism and other neurological disorders.
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ItemFunctional in vitro modelling of the nervous system using human pluripotent stem cells; a platform to study brain disorders( 2016)Human pluripotent stem cells (hPSC) constitute a valuable resource for establishing in vitro models of nervous system development, function and dysfunction. This is particularly needed for complex developmental brain disorders whereby animal models and access to postmortem human brain tissue may be limited. A major prerequisite step to developing suitable in vitro models of the human nervous system is to establish hPSC induction protocols to derive regionally specific neuronal populations. The first major aim of this thesis was to characterize the phenotype and maturation stage of neural progenitor cell types derived from the following induction protocols: the dual SMAD inhibition protocol (DS), sonic hedgehog pathway agonist protocol (SAG), and SMAD/GSK3b inhibition protocol (CHIR). Our studies show that the dual SMAD inhibition protocol results predominantly in cortical progenitors representative of deep and intermediate cortical layer neurons, while combined early treatment of sonic hedgehog agonist during neural induction give rise to progenitors of ventral cortical identity. Using an induction protocol involving inhibition of the SMAD and GSK3b pathways, followed by later exposure to BMP2/4, hPSC are directed towards neural crest lineages, which upon further differentiation give rise to peripheral sensory neurons. Another essential component for in vitro modelling of the human nervous system using hPSC is to demonstrate neuronal activity and connectivity as measures of functionality. Whilst intercellular activity and characteristics of hPSC-derived neurons are well documented, reports on their potential to form functional network is scarce. This feature is critical for using hPSC to model psychiatric disorders such as Autism spectrum disorder (ASD) in which abnormal connectivity is one of the major characteristics of the disease. Using microelectrode arrays (MEA), the second major aim of this thesis was to assess the functional maturation rate and neuronal network activities of hPSC-derived cortical and peripheral sensory neuronal populations. Our data demonstrate that functional maturation of hPSC-derived cortical neurons occurs at a slower rate relative to hPSC-derived sensory neurons with no evidence of network activities detected over 8 weeks of differentiation. In contrast, hPSC-derived sensory neurons show faster maturation and form functional networks in vitro by week 6-7 post differentiation. Further functional characterization on MEA reveals the capability of sensory neuronal subtypes to respond to appropriate stimuli including heat, capsaicin and hypoosmotic induced stretch. The final major objective of the thesis was to employ our in vitro model of corticogenesis to interrogate neurodevelopmental disorders, specifically microcephaly and ASD. Accordingly, one aim was to investigate the function of WD Repeat Domain 62 (WDR62) in neurogenesis, a major candidate gene of autosomal recessive primary microcephaly. Studies of WDR62 utilizing human based in vitro models are lacking. Our studies show that WDR62 expression coincides with SOX2 and PAX6 expression during hPSC neural induction, the prime period of neurogenesis. Furthermore, knockdown of WDR62 expression in hPSC impacted on neurogenic and gliogenic differentiation, as shown by a reduction in TBR2 (EOMES) and S100b+ progenitor populations, respectively. The final aim was to characterize idiopathic ASD induced pluripotent stem cell (iPSC)-derived cortical neurons, focusing on the expression of genes associated with corticogenesis, glia specification, synaptic function, cell proliferation and cell death. Our data suggests higher expression of TBR1 and lower expression of MAP2AB, GFAP and GRM5 in ASD neurons compared to controls. These results implicate processes related to deep cortical layer neuronal differentiation, dendritic formation, glial differentiation and synaptic function in ASD core pathophysiology, paving the way for future studies utilizing these ASD iPSC lines. In summary, these studies provide key information for utilizing hPSC to model neural circuitry systems and demonstrate hPSC robustness as a complementary in vitro model system to investigate developmental brain disorders.
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ItemRedox biology and autism( 2016)Background: Evidence suggests that oxidative stress may be related to the aetiology of autism. This is supported by studies showing deficiencies in glutathione and other antioxidants, mitochondrial dysfunction and genetic links between autism and abnormalities in redox biology. Glutathione, an important cellular antioxidant, is therefore proposed as a potential treatment target in autism. N-acetyl cysteine (NAC), a glutathione precursor, may be an effective method of supplementing glutathione levels, and thus improving behavioural symptoms and functioning, in children with autism. Method: This study was a mixed-methods, double-blind, randomised, placebo-controlled clinical trial of 500 mg daily NAC, in addition to treatment as usual, for 6 months in children with autistic disorder, as defined by the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, revised. The primary outcomes were the core symptoms of autistic disorder: social interaction, as measured by the Social Responsiveness Scale; communication, as measured by the Children’s Communication Checklist – Second Edition; and restricted and repetitive behaviours and interests, as measured by the Repetitive Behavior Scale – Revised. Secondary outcomes were problem behaviours, as measured by the Developmental Behaviour Checklist – Primary Carer Version; adaptive functioning, as measured by the Vineland Adaptive Behavior Scales – Second Edition; and parent and clinician global impression scales. In addition, qualitative analysis of parent/guardian reports and clinicians’ observations was carried out to supplement the main efficacy study. Results: A total of 98 children (79 male, 19 female; age range = 3.1-10.1 years) were enrolled into the study, of whom 48 were randomised to receive NAC and 50 were randomised to receive placebo. The NAC and placebo groups did not differ on any demographic or baseline symptom severity measure. Seventy-one participants (34 from NAC group, 37 from placebo group) completed the 6-month trial. NAC did not differ from placebo on safety and tolerability. There were no differences between the NAC and placebo groups on any primary or secondary outcome measures. In contrast, the qualitative analysis found that NAC was associated with more frequent reports of improved calmness and verbal communication than placebo. Conclusions: This study found that NAC was not effective in improving core symptoms or functioning in children with autism, as assessed by a range of comprehensive quantitative measures. However, this study did demonstrate the potential utility of mixed-methods approaches in autism treatment trials. Overall, this study does not support the widespread use of NAC for autism, although questions remain regarding dosage, and effects on specific symptoms within the broader clinical picture.