Methylmercury (MeHg) is a global pollutant that can elicit a range of adverse health effects in both humans and wildlife populations. Humans are often exposed to MeHg through the consumption of contaminated seafood. Developing fetuses are especially susceptible to the effects of MeHg as it can cross the blood-brain barrier and the placenta. At high doses in utero MeHg causes developmental disorders and congenital disabilities, but long-term low-dose effects are still not fully known. Using a culture model of cerebral cortex development, our lab has shown that low-dose MeHg promotes premature neuronal differentiation. Autism spectrum disorder (ASD) has been associated with prenatal MeHg exposure and is correlated with neuronal overproduction, but a cause-effect relationship has not been shown. In this thesis, I aim to test the hypothesis that prenatal exposure to low-dose MeHg can cause ASD-like symptoms in the offspring following premature neuronal differentiation. My results showed that adult mice prenatally exposed to MeHg exhibited key ASD characteristics including impaired communication, reduced sociability, and increased restrictive repetitive behaviours. Furthermore, I explored the underlying cellular and molecular mechanism that promotes premature neuronal differentiation caused by prenatal MeHg exposure. To reverse the MeHg-induced premature neuronal differentiation, I utilized metformin, an FDA-approved diabetes drug. Overall, these findings provide insights into the toxicology of MeHg and its relationship with ASD etiology, including the underlying mechanism, and a potential therapeutic strategy.

The excitatory, ionotropic glutamatergic AMPA receptor is the most common membrane-bound receptor in the central nervous system. AMPARs and the NMDA receptors are central to synaptic plasticity, memory, and mechanisms of neurodegeneration. The AMPAR is an obligate heterotetramer, composed of subunits GluA1-4. Subunit permutation determines ion conductance, trafficking and other functional characteristics. Few available antibodies are subunit-specific, disabling researchers from accurately visualizing differential AMPAR subunit distribution in the nervous system. This study sought to visualize a novel monoclonal GluA1/2/3 antibody with functional avidity for three of four receptor subunits and to characterize the ultrastructural localization of these receptors using confocal and electron microscopy.

Neuronal differentiation encompasses an elaborate developmental program which until recently was difficult to study in vitro. The advent of several cell lines able to differentiate in culture proved to be the turning point for gaining an understanding of molecular neuroscience. In particular the olfactory epithelium provides an attractive tool with which to investigate fundamental questions relating to neuronal differentiation, as it displays a unique capacity to regenerate and to retain a neurogenetic potential from its genesis and throughout adult life. The coordinated regulation of gene expression is fundamental to the control of neuronal differentiation. In order to reveal active processes at the molecular level and to dissect key components of molecular pathways, differential gene expression studies provide a foundation for the elucidation of dynamic molecular mechanisms. This thesis identified genes involved in neuronal differentiation by utilising a clonal olfactory receptor neuronal cell line (OLF442). Gene expression levels were identified using differential display and oligonucleotide array technology before and after serum deprivation. Differential display revealed two kinases whose expression levels were elevated during the differentiation of OLF442, identified as focal adhesion kinase (FAK) related non-kinase (FRNK) and mammalian ste20 like (MST)2 kinase. Furthermore, analysis of the oligonucleotide array data confirmed the expression of genes involved in altering presentation of extracellular matrix molecules, in mediating cytoskeletal rearrangements, and in ceasing the cell cycle, supporting the use of OLF442 as a model for studying differentiation. The differentiation of OLF442 results from the synchronisation of multiple transduction cascades and cellular responses as evidenced by the microarray data. A protein that can synchronise such signalling is the non-receptor protein tyrosine kinase, FAK. Thus the finding of the endogenous FAK inhibitor FRNK by differential display was intriguing as there was no difference in the expression level of FAK induced by differentiation, contrasting that of FRNK. This induced FRNK expression was derived autonomously as it was not responsive to the caspase-3 inhibitor, DEVD-CHO. This is particularly pertinent since the primary role of FRNK is to act as an inhibitor of FAK by competing with its substrates and reducing the phosphorylation of both FAK and its associated proteins. Differential display also revealed the upregulation of another kinase, which had 90% homology with rat MST2 kinase within the 3' UTR. Both mouse MST2 kinase (sequence submitted to GenBank, accession number AY058922) and the closely related family member MST1 kinase were sequenced and cloned. Moreover, evidence to support an autonomously expressed carboxyl-terminal domain of MST2 kinase is presented in Chapter 3 and provides a unique way in which MST2 may regulate its own activity. To further understand the role of MST in neuronal differentiation, a series of stable OLF442 transfections (with mutant and wild-type MST constructs) were carried out. MST was localised with cytoplasmic structures that may represent actin stress fibres, indicating a potential cytoskeletal role during neuronal differentiation. This indicated that MST1 may play a role in the morphological processes involved in neuronal differentiation. The identification of two kinases by differential display provided the motivation to understand the cellular context of OLF442 and to determine the phosphorylation status of the mitogen-activated protein kinase (MAPK) signalling cascades. Differentiation of OLF442 induced high-level phosphorylation of a putative B-Raf isoform, MEK2 and ERK1/2. Interestingly, there was a switch between preferential phosphorylation of MEK1 in undifferentiated OLF442 to preferential phosphorylation of MEK2 following differentiation. SAPK/JNK was also phosphorylated, as was the transcription factor c-Jun, which is a common substrate of both the ERK and SAPK/JNK signalling modules. The mapping of the cellular context of differentiating OLF442 has identified a promising model of a novel MAPK module. This consists of FAK signalling through Rap1 to ERK providing sustained activation, which is buffered or terminated by the expression of the endogenous FAK inhibitor FRNK. Furthermore, MST kinase could potentially play a role in regulating the cytoskeletal re-arrangements that are necessary for neuronal differentiation. MST kinase may signal transiently via the SAPK pathway to provide concomitant activation of c-Jun that is required for neuronal differentiation. An understanding of the gene expression pattern of the normal neuronal differentiation program allows a greater understanding of potential developmental aberrations. This could provide an opportunity for therapies to be conceived, while understanding the complexity of neuronal determination could also provide opportunities for stem cell transplantation.