Browsing by Author "O'ryan, Colleen"

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    Autism Spectrum Disorder and Mitochondrial Dysfunction: The Role of Mitochondrial Dynamics
    (2022) Buchanan, Erin; O'ryan, Colleen
    Numerous genes and biological pathways are implicated in the aetiology of the neurodevelopmental disorder, autism spectrum disorder (ASD). Our research group reported that mitochondrial dysfunction was associated with ASD in South African children diagnosed with ASD using differential methylation and metabolomics studies. Propionyl-CoA Carboxylase Subunit Beta (PCCB) was differentially methylated in our cohort ASD study, and its dysregulation can lead to the accumulation of propionic acid. This links PCCB's function to a well-established animal model that uses sodium propionate (NaP) to study ASD in rats. This thesis aimed to i) examine ASD-associated mitochondrial dysfunction in a neuronal-like cell model using NaP and to investigate its effects on mitochondrial dynamics and morphology; ii) investigate the application of this in a South African ASD cohort by measuring differential methylation of essential genes involved in mitochondrial dynamics. Undifferentiated, neuronal-like SH-SY5Y cells were treated with NaP at 1.5mM, 3mM and 5mM to induce mitochondrial stress. The effects of NaP mitochondrial-induced stress were quantified using MTT and ATP assays. Transmission electron microscopy (TEM), using cryogenic techniques, was used to examine mitochondrial morphology by measuring nine parameters: area, area2 , form factor, area-weighted form factor, aspect ratio, perimeter, circularity, Feret's diameter and roundness. TEM images were analysed using Fiji/Image J. Mitochondrial DNA (MT-DNA) copy number and STOML2 expression were measured using RTqPCR, and these data were compared to TEM data. STOML2 was the most differentially methylated gene in our ASD cohort in our previous study. Differential methylation of six essential genes (DRP1, FIS1, MFN1, MFN2, OPA1, STOML2) involved in mitochondrial fusion and fission were examined using targeted next-generation bisulfite sequencing (tNGBS) between ASD and control participants in a South African cohort. Significance for all experiments was determined using unpaired t-tests, one-way ANOVA and correlation analysis (p< 0.05). SH-SY5Y cells treated with NaP showed mitochondrial dysfunction as reflected in changes in ATP levels and no changes in cell viability were observed except at 9mM NaP. In addition, NaP treatments led to significant changes in mitochondrial morphology with significant decreases in mitochondrial area, perimeter, form factor and Feret's diameter between NaP treatments and control and a significant increase in circularity. These changes in morphological data were supported by a significant increase in MT-DNA copy number at 5mM and significant decreases in STOML2 expression at all concentrations. Together, the TEM and expression data highlight the balancing act between mitochondrial fusion and fission, with increasing levels of fission occurring with increasing NaP concentrations. In the South African cohort, there were significant differences in methylation between the ASD group compared to controls at two CpG sites in MFN2, two CpG sites in STOML2 and one CpG site in FIS1 (significant increases) and two CpG sites in OPA1 (significant decreases). The increase in FIS1 and decrease in OPA1 methylation are consistent with this balancing act between mitochondrial fusion and fission in mitochondrial dysfunction in this cohort. These results suggest a potential link between mitochondrial dysfunction and the fluctuation in mitochondrial dynamics and morphology. The inclusion of TEM demonstrates its unique ability to visualise mitochondrial ultrastructure and the effect changes in mitochondrial dynamics have on morphology as opposed to only examining these changes through gene expression and metabolic assays. Although the exact relationship between the TEM and gene expression data could not be fully explained in this study, it highlights the need to explore further the relationship between mitochondrial dynamics, biogenesis and mitophagy in the context of ASD aetiology and neuronal development.
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    In vitro investigation of the molecular relationship between mitochondrial dysfunction and neurogenesis in autism aetiology
    (2024) Van Der Watt, Mignon; O'ryan, Colleen
    Mitochondrial function is integral in the regulation of neurogenesis. Our research group previously reported differentially methylated genes and metabolomic evidence converging on mitochondrial dysfunction in a South African autism spectrum disorder (ASD) cohort. Thus, subtle mitochondrial dysfunction is hypothesized to contribute to the pathogenesis of a subset of ASD by altering early neurogenesis. Given the inaccessibility of the developing brain, cell culture model systems are needed to investigate the molecular relationship between mitochondrial dysfunction and neuronal differentiation as it pertains to ASD aetiology. A key differentially methylated gene was Propionyl-CoA Carboxylase Subunit B (PCCB), a mitochondrial enzyme involved in metabolizing short fatty acids like propionic acid (PPA). Disruption of PCCB function could result in toxic accumulation of PPA. Furthermore, PPA is used to induce mitochondrial dysfunction in in vitro models and recapitulate ASD-like behaviours in animal models. Thus, PPA treatments can be used to model the effect of PCCB disruption to investigate molecular links between ASD-associated mitochondrial dysfunction and neurogenesis in vitro. The aims of this thesis were to i) induce and characterise mitochondrial dysfunction in the SH SY5Y cell culture model, ii) investigate the temporal cellular responses to PPA-induced mitochondrial stress, iii) confirm and characterise successful neuronal differentiation of SH SY5Y cells induced by a de novo differentiation protocol, and iv) investigate the effects of PPA exposure on morphological and biochemical changes seen during neuronal differentiation of the SH-SY5Y cell line. Undifferentiated neuron-like SH-SY5Y cells were treated with PPA concentrations ranging from 0-10mM PPA to induce mitochondrial stress. Cell viability and cytotoxicity under the range of PPA concentrations were examined using MTT assays to determine dosages of PPA which induced mitochondrial stress. The expression of mitochondrial homeostasis (mitostasis) genes previously associated with ASD were measured in response to PPA exposure. Reverse transcription quantitative polymerase chain reaction (RT-qPCRs) of mitochondrial biogenesis (mitogenesis) (C-MYC, PGC-α, NRF1, NFE2L2, TFAM) and mitochondrial fusion (STOML2, OPA1) genes were performed to identify PPA-responsive genes and to get initial insights on the effect of PPA treatments on transcriptional regulation of mitostasis. Mitochondrial membrane potential (DYm) was measured in response to acute PPA stress using TMRE (Tetramethylrhodamine, ethyl ester) assays. Additionally, RT-qPCRs were used to measure the temporal expression of C-MYC and PGC-1α, two integral factors in regulating interactions between mitochondrial function and neurogenesis, in response to chronic PPA exposure. Differentiation of SH-SY5Y cells was induced using retinoic acid and brain-derived neurotrophic factor. Successful neuronal differentiation was morphologically quantified using phase-contrast microscopy and the ‘Neuron J’ plugin in Image J. Neuronal differentiation was also biochemically confirmed using immunocytochemistry for the 5 immature neuron marker, Nestin, in comparison to the mature neuron marker, Beta-III tubulin. The same phase-contrast microscopy and immunocytochemistry measurements were used to determine the effect of 5mM PPA treatments on the process of neuronal differentiation. PPA was found to induce mitochondrial stress in SH-SY5Y cells by the dose-dependent decreases in cell viability and DYm. Furthermore, PPA disrupted the transcriptional regulation of mitostasis, mitogenesis, and mitochondrial fusion. Dose-dependent decreases in mRNA expression were observed for NRF1, NFE2L2, TFAM, STOML2, and C-MYC. Additionally, PPA disrupted the dynamic regulatory crosstalk between mitochondrial function and neurogenesis as highlighted by temporal attenuation of C-MYC and the multi-directional expression of PGC-1α. The de novo neuronal differentiation protocol induced increased neurite elongation and branching, cell cycle withdrawal, and upregulated Beta-III tubulin expression which indicates successful SH-SY5Y differentiation. However, PPA exposure altered neuronal differentiation. PPA treatment resulted in decreased neurite elongation and branching along with altered expression and localization of the neuronal markers. This study establishes a reductionist ex vivo cell culture model system to study neurodevelopment in vitro. Furthermore, the application of this model provides insights into the molecular relationship between ASD-associated mitochondrial dysfunction and neurogenesis. ASD-associated mitochondrial dysfunction results in disrupted transcriptional regulation of mitostasis. This transcriptional dysregulation has implications on the rate and nature of neuronal differentiation and maturation. Although this research provides initial insights into the molecular mechanisms between ASD-associated mitochondrial dysfunction and neurogenesis, there is a need for further research to gain greater understanding of this molecular relationship and its outcomes.
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    Mitochondrial mechanisms in autism spectrum disorder: characterizing the neurotoxic effects of propionic acid in vitro
    (2025) Mahony, Caitlyn; O'ryan, Colleen
    Autism Spectrum Disorder (ASD) is a chronic neurodevelopmental condition with a complex molecular aetiology shaped by genetic, epigenetic and environmental factors. The poorly understood nature of ASD aetiology significantly impedes diagnosis and management of the condition, which substantially impairs quality of life. A role for mitochondrial dysfunction in ASD has become increasingly well-recognised, while mitochondria are also emerging as central regulators of neurodevelopment, physiology and function. Moreover, recent work in a South Africa (SA) ASD cohort reported differential DNA methylation (DNAm) signatures converging on mitochondrial metabolism and Propionyl CoA Carboxylase B (PCCB). This project aimed to better characterise the mitochondrial component of ASD aetiology using an in vitro system to mechanistically interrogate the molecular signatures found in an understudied SA population. Building on established preclinical models for both PCCB dysfunction and ASD, Propionic Acid (PPA) was used to recapitulate mitochondrial dysfunction in neuronal-like SH-SY5Y cells. The Thiazolyl Blue Tetrazolium Bromide (MTT), Luminescent ATP Detection and Tetramethylrhodamine, ethyl ester (TMRE) assays revealed a dose- and time-dependent decrease in cell viability from 4 to 72 hours, catalysed by an acute loss of mitochondrial membrane potential at 4 hours. In conjunction, reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays showed an acute and sustained suppression of PCCB expression and a systemic dysregulation of the genes that govern mitochondrial metabolism (PGC1α, c-MYC), biogenesis (TFAM, NRF1), fission (DRP1) and fusion (STOML2, MFN1/2, OPA1). Confocal microscopy demonstrated associated disruptions to mitochondrial network morphology and integrity, characterised by decreased mitochondrial volume, increased mitochondrial fragmentation and perturbations to mitophagic flux. Finally, live-cell respirometry illustrated functional impairments to metabolic state marked by significant deficits in both oxidative and glycolytic ATP production. Having effectively recapitulated mitochondrial dysfunction in vitro, a model for neurogenesis was developed to examine the interplay between neuronal metabolism and maturation. Phase contrast microscopy, immunocytochemistry and respirometry assays confirmed the efficacy of differentiation, demonstrating successful cell cycle exit, efficient neurite production, elongation and branching, an increased β-III-tubulin (β3T): Nestin (NES) ratio and enhanced mitochondrial capacity. This system was harnessed to characterise PPA-induced disruptions to neurogenesis, demonstrating gross morphological aberrations and significant changes to the composition and connectivity of neuronal networks. Complementary image analysis workflows showed that PPA reduced the length and complexity of neurite processes, disrupted the localisation and expression of cytoskeletal markers and decreased the β3T:NES ratio, indicating substantial impairments to neuronal morphology and maturation. Lastly, global RNA sequencing was used to characterise the molecular mechanisms that disrupt neuronal maturation under PPA stress, revealing transcriptional signatures of developmental delay and neurotoxicity. PPA upregulated processes involved in pluripotency, proliferation and glycolysis, impaired epigenetic and metabolic reprogramming and inhibited synaptogenesis and neurotransmission. An interplay between stress-responsive, metabolic and neurogenic signalling emerged at the centre of the PPA transcriptome, highlighting reciprocal mechanisms that modulate substrate utilisation, differentiation, synaptogenesis and survival. Moreover, PPA systemically altered convergent mechanisms that shape extracellular matrix dynamics, leading to signatures of neuronal injury, apoptosis and excitotoxicity. Together, transcriptomic profiles reflected impairments to neurogenesis under PPA stress, driven by disruptions to the intrinsic and extrinsic landscapes that shape neuronal cell fate. This project established an in vitro model for neurogenesis and developmental neurotoxicity that lays the groundwork for mechanistic studies on neurodevelopment and disease. The work presented here provides a quantitative assessment of mitochondrial morphology, dynamics and function under PPA stress and is the first study to functionally demonstrate that PPA alters neuronal metabolism and differentiation in the SH-SY5Y model. Moreover, the transcriptional signatures of PPA exposure highlight novel processes that function at the interface between metabolism, neurodevelopment and neurotoxicity. Ultimately, these insights contribute to a mechanistic understanding of the mitochondrial component of ASD aetiology, which could inform the development of novel biomarkers and therapeutic strategies to improve long-term clinical outcomes.