Investigation of GLT-1d in juvenile, adult, and pathological mouse brains

This thesis describes, for the first time, the expression, localisation, and potential relevance of GLT-1d, a newly discovered splice variant of the glutamate transporter GLT-1, in mouse brain. GLT-1d was identified in both rat and human brain during preliminary investigations. Importantly, during these preliminary investigations, GLT-1d was shown to be a functional and clinically relevant excitatory amino acid transporter that warranted further investigation. Since known GLT-1 splice variants are differentially expressed in terms of development, distribution, and disease, it was anticipated that GLT-1d may likewise be differentially expressed and distributed. Accordingly, investigation of the expression and distribution of GLT-1d in juvenile, adult, and pathological mouse brains was contemplated and ultimately led to the research for this thesis. The aim of the research for this thesis was to investigate the expression and distribution of GLT-1d in juvenile, adult, and pathological mouse brains. In order to achieve this aim, the research for this thesis was carried out in four studies: First, antisera were produced using synthetic peptides that corresponded to the unique C-terminal amino acid sequences of rat and human GLT-1d. The antisera were then validated for use in the investigation of the expression and distribution of GLT-1d in mouse brain. This study established that one of the antisera produced, namely Rb 1713 antiserum, was a useful research tool to investigate GLT-1d in mouse brain. Interestingly, heating of samples prior to western blotting led to a $\sim$50 kDa downward shift in the apparent molecular weight of a higher molecular weight oligomer form of GLT-1d. It is plausible that this downward shift in apparent molecular weight of the higher molecular weight form of GLT-1d may be due to heat-induced dissociation of a protein partner, potentially PICK1. Second, the expression and regional and cellular distribution profile of GLT-1d was examined in juvenile mouse brain. This study demonstrated that GLT-1d exhibited a temporal expression and distribution profile in juvenile mouse brain that was coordinated with the expression profiles of GLT-1a, GLT-1b, GLAST, glutamine synthetase, and VGLUT1, and that GLT-1d protein was expressed in astrocytes in juvenile mouse brain, a juvenile mouse being defined as postnatal day 1 (P1)--P21. GLT-1d expression increased most noticeably from P7--P14 in juvenile mouse brain, which suggested that there was an increasing demand for GLT-1d activity during mouse brain development and maturation. The increase in expression of GLT-1d was largely coincident with gliogenesis, synaptogenesis, a transition to mature forms and expression levels of glutamate receptors, and myelination. Based on the findings of this study, it is plausible that GLT-1d may be associated with the inception of and ensuing glutamatergic neurotransmission in juvenile mouse brain. Third, the expression, and regional and cellular distribution profile of GLT-1d was examined in adult mouse brain. This study established that GLT-1d mRNA and protein expression and distribution was highest in adult mouse forebrain, with enriched GLT-1d immunoreactivity observed in neuronal somata in layers IV--VI of the neocortex, CA1 and CA3 regions of the hippocampus, and the dentate gyrus, but also observed in astrocytes. The GLT-1d expression decreased in a rostrocaudal manner with the lowest level in the cerebellum. It is plausible that GLT-1d may play an important role in glutamate homeostasis in adult brain, particularly in the forebrain. Interestingly, the optic and auditory vestibular nerves and white matter of the cerebellum exhibited high levels of GLT-1d expression. The high level of GLT-1d expression in the white matter of the cerebellum, despite this region having the lowest level in general, suggested a possible role for GLT-1d protein in white matter tracts. The elevated GLT-1d expression in white matter tracts points to GLT-1d as a potential therapeutic target for neurodegenerative disease associated with white matter tracts, e.g., spinal injury and Alzheimer's disease. Finally, the expression profile of GLT-1d in acute and chronic pathological adult mouse brains was examined in two mouse models. The mouse models were a transient focal cerebral ischaemic insult and reperfusion mouse model, i.e., the acute pathological condition, and an hSOD1G93A amyotrophic lateral sclerosis mouse model, i.e., the chronic pathological condition. The final study suggested that the modest reduction in GLT-1d expression observed in the contralateral hemisphere may have been due to dysfunction within/associated with the terminals of the intercortical glutamatergic neurons projecting into the contralateral hemisphere and/or changes in secretion by intercortical neurons projecting into the contralateral hemisphere of neurotrophic factors that are known to modulate GLT-1 expression. Intriguingly, no significant change was observed for GLT-1a mRNA in both the ipsilateral and contralateral hemispheres. One possible explanation for this finding may be the cellular distribution of GLT-1a and GLT-1d. GLT-1a, is predominantly expressed in astrocytes, whereas GLT-1d was expressed in both neurons and astrocytes in adult mouse brain. It is plausible, therefore, given the substantial differences in sensitivity of neurons and astrocytes to glutamate that the neuronal and astrocytic GLT-1d may exhibit different kinetics in response to a mild cerebral ischaemic insult--as was the case in this study--relative to the predominantly astrocytic GLT-1a. Indeed, the sensitivity of GLT-1d to a mild cerebral ischaemic insult is an important finding. In the chronic pathological condition, the elevated GLT-1a and GLT-1d mRNA expression observed in the pre-symptomatic hSOD1G93A mouse motor cortex relative to the age-matched wildtype may be a compensatory phase during which neurons were more active in a hostile environment. The marked decline in GLT-1a and GLT-1d mRNA expression as the hSOD1G93A mouse motor cortex neurons approached the symptomatic stage, i.e., onset stage, may have been due to the neurons dying, which was followed by further compensatory phase during which the remaining neurons endeavoured to restore glutamate homeostasis in their immediate environment. The characterisation of GLT-1d as set out in this thesis expanded the knowledge regarding GLT-1 and its association with the development, normal function, and pathological conditions of mouse brains. It is plausible that the knowledge derived here with respect to mouse brains may be useful in understanding the role of GLT-1d, and perhaps GLT-1 in general, in other mammalian brains.