Mitofusin 1 and 2 are outer-mitochondrial membrane proteins that have been shown to be involved in fusion. Mitofusin 2 has also been associated with apoptosis and development. When Mfn1 and Mfn2 were each conditionally knocked out from the cerebellum, Purkinje cells in Mfn2 deficient cerebellum during development had undergone neurodegeneration. Mutations in Mfn2 have also been associated with the Charcot Marie Tooth Type 2A (CMT2A). We want to asses the effect Mfn2 and Mfn1 might have on the development of other regions of the brain such as the telencephalon. We generated Mfn1 and Mfn2 conditional knockouts in the telencephalon by crossing them with Foxg1 Cre - a cre expressed in the telencephalon. We found that Mfn1 deficient mice have lost their corpus callosum at the midline, but survive over 6 months with a decrease in progenitor cells postnatally. Mfn2 deficient mice die between P9 and P12 with a decrease in progenitor cells postnatally and a decrease in number of neurons in the cortex. Therefore, our results suggest that Mfn1 and Mfn2 play a significant role in the development of the telencephalon.

Mitofusin 1 and 2 are outer-mitochondrial membrane proteins that have been shown to be involved in fusion. Mitofusin 2 has also been associated with apoptosis and development. When Mfn1 and Mfn2 were each conditionally knocked out from the cerebellum, Purkinje cells in Mfn2 deficient cerebellum during development had undergone neurodegeneration. Mutations in Mfn2 have also been associated with the Charcot Marie Tooth Type 2A (CMT2A). We want to asses the effect Mfn2 and Mfn1 might have on the development of other regions of the brain such as the telencephalon. We generated Mfn1 and Mfn2 conditional knockouts in the telencephalon by crossing them with Foxg1 Cre - a cre expressed in the telencephalon. We found that Mfn1 deficient mice have lost their corpus callosum at the midline, but survive over 6 months with a decrease in progenitor cells postnatally. Mfn2 deficient mice die between P9 and P12 with a decrease in progenitor cells postnatally and a decrease in number of neurons in the cortex. Therefore, our results suggest that Mfn1 and Mfn2 play a significant role in the development of the telencephalon.

Mitofusin 1 and 2 are outer-mitochondrial membrane proteins that have been shown to be involved in fusion. Mitofusin 2 has also been associated with apoptosis and development. When Mfn1 and Mfn2 were each conditionally knocked out from the cerebellum, Purkinje cells in Mfn2 deficient cerebellum during development had undergone neurodegeneration. Mutations in Mfn2 have also been associated with the Charcot Marie Tooth Type 2A (CMT2A). We want to asses the effect Mfn2 and Mfn1 might have on the development of other regions of the brain such as the telencephalon. We generated Mfn1 and Mfn2 conditional knockouts in the telencephalon by crossing them with Foxg1 Cre - a cre expressed in the telencephalon. We found that Mfn1 deficient mice have lost their corpus callosum at the midline, but survive over 6 months with a decrease in progenitor cells postnatally. Mfn2 deficient mice die between P9 and P12 with a decrease in progenitor cells postnatally and a decrease in number of neurons in the cortex. Therefore, our results suggest that Mfn1 and Mfn2 play a significant role in the development of the telencephalon.

Master of Philosophy (Medicine) / The actin cytoskeleton is important in many cellular processes such as motility, and establishing and maintaining cell morphology. Members of the tropomyosin protein family associate with the actin cytoskeleton along the major groove of actin filaments (F-actin), stabilising them and regulating actin-filament dynamics. To date over 40 non-muscle tropomyosin isoforms have been identified, which are encoded by 4 different genes (α, β, γ, δ). Individual tropomyosin isoforms define functionally distinct F-actin populations. Previous studies have shown that tropomyosins sort to distinct subcellular compartments at different stages of development in polarised cells. Neuronal growth cones are highly dynamic polarised structures, dependent on a constant reorganisation of the actin cytoskeleton. By eliminating tropomyosins in a knockout (KO) mouse model, we investigated the role of two tropomyosin isoforms, Tm5NM1 and Tm5NM2 (γTm gene products) in growth cone dynamics and neurite outgrowth. Growth cone protrusion rates were significantly increased in one day old Tm5NM1/2 KO hippocampal neurons compared to WT controls. Neuritogenesis was significantly affected by the elimination of Tm5NM1/2, with a slight decrease in neurite length and an increase in neuronal branching in neurons cultured for four days. At the molecular level, the depletion of Tm5NM1/2 had no impact on the protein levels and activity of ADF/cofilin in hippocampal neurons while in cortical neurons a subtle but significant increase in ADF/cofilin activity was observed. The subtle phenotype in the early stages of neuritogenesis observed from eliminating Tm5NM1/2 may be explained with functional compensation by other tropomyosin isoforms. Functional compensation for the loss of Tm5NM1/2 may be provided by isoforms Tm5a/5b, TmBr2 and Tm4 as they localise to the growth cones, structures where Tm5NM1/2 are normally found. These results suggest that Tm5NM1/2 may not be required for early stages of neuritogenesis but may still play a fine-tuning role for this process.

Master of Philosophy (Medicine) / The actin cytoskeleton is important in many cellular processes such as motility, and establishing and maintaining cell morphology. Members of the tropomyosin protein family associate with the actin cytoskeleton along the major groove of actin filaments (F-actin), stabilising them and regulating actin-filament dynamics. To date over 40 non-muscle tropomyosin isoforms have been identified, which are encoded by 4 different genes (α, β, γ, δ). Individual tropomyosin isoforms define functionally distinct F-actin populations. Previous studies have shown that tropomyosins sort to distinct subcellular compartments at different stages of development in polarised cells. Neuronal growth cones are highly dynamic polarised structures, dependent on a constant reorganisation of the actin cytoskeleton. By eliminating tropomyosins in a knockout (KO) mouse model, we investigated the role of two tropomyosin isoforms, Tm5NM1 and Tm5NM2 (γTm gene products) in growth cone dynamics and neurite outgrowth. Growth cone protrusion rates were significantly increased in one day old Tm5NM1/2 KO hippocampal neurons compared to WT controls. Neuritogenesis was significantly affected by the elimination of Tm5NM1/2, with a slight decrease in neurite length and an increase in neuronal branching in neurons cultured for four days. At the molecular level, the depletion of Tm5NM1/2 had no impact on the protein levels and activity of ADF/cofilin in hippocampal neurons while in cortical neurons a subtle but significant increase in ADF/cofilin activity was observed. The subtle phenotype in the early stages of neuritogenesis observed from eliminating Tm5NM1/2 may be explained with functional compensation by other tropomyosin isoforms. Functional compensation for the loss of Tm5NM1/2 may be provided by isoforms Tm5a/5b, TmBr2 and Tm4 as they localise to the growth cones, structures where Tm5NM1/2 are normally found. These results suggest that Tm5NM1/2 may not be required for early stages of neuritogenesis but may still play a fine-tuning role for this process.

Mitofusin 1 and 2 are outer-mitochondrial membrane proteins that have been shown to be involved in fusion. Mitofusin 2 has also been associated with apoptosis and development. When Mfn1 and Mfn2 were each conditionally knocked out from the cerebellum, Purkinje cells in Mfn2 deficient cerebellum during development had undergone neurodegeneration. Mutations in Mfn2 have also been associated with the Charcot Marie Tooth Type 2A (CMT2A). We want to asses the effect Mfn2 and Mfn1 might have on the development of other regions of the brain such as the telencephalon. We generated Mfn1 and Mfn2 conditional knockouts in the telencephalon by crossing them with Foxg1 Cre - a cre expressed in the telencephalon. We found that Mfn1 deficient mice have lost their corpus callosum at the midline, but survive over 6 months with a decrease in progenitor cells postnatally. Mfn2 deficient mice die between P9 and P12 with a decrease in progenitor cells postnatally and a decrease in number of neurons in the cortex. Therefore, our results suggest that Mfn1 and Mfn2 play a significant role in the development of the telencephalon.

Lactate, a metabolic end product of glycolysis in mammals, has emerged as an important energy substrate for the brain. In addition to its energetic role, lactate was shown to modulate the excitability of neurons, to have a neuroprotective role and to participate in long-term memory formation. One previous investigation from our group reported that lactate modulates 4 synaptic plasticity-associated genes and potentiates the activity of the N-Methyl-D-aspartic acid (NMDA) receptor, a major receptor type involved in glutamatergic neurotransmission. The current thesis aimed at first to extend these findings by examining genome-wide transcriptional responses to this metabolite in cortical neurons. Using ribonucleic acid(RNA) sequencing to evaluate expression changes in protein-coding genes, we found that lactate modulates robustly after 1h, 20 genes involved in the mitogen-activated protein kinase (MAPK) signaling pathway and in synaptic plasticity in a NMDA receptor activitydependent manner and that nicotinamide adenine dinucleotide, reduced (NADH), but not pyruvate, reproduces the modulatory effects of lactate on 70% of all differentially expressed genes. In a time course experiment, genes modulated after lactate treatment for 6h and 24h were also identified; these are involved in 9 signaling pathways including circadian rhythm, drug addiction, and retrograde endocannabinoid signaling. Bioinformatics analyses indicated CREB1 and CREM as candidate master regulators of gene expression and the modulatory effect of lactate was prevented by inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, indicating a role for this kinase in mediating lactate signaling. An examination of changes in dendritic spines’ morphology and density - a morphologicalcorrelate of synaptic plasticity – has shown that lactate modulated spine density changes induced by potassium chloride (KCl) and carbachol. An additional investigation described in this thesis indicated that different gut microbiota manipulations (germ-free, prebiotics, high-fat diet) regulated mRNA expression of genes involved in the Astrocyte-Neuron Lactate Shuttle (ANLS) - a metabolic cooperation mechanism between astrocytes and glutamatergic neurons. Overall, the results of this thesis help to establish a role for lactate as a signaling molecule in the brain, highlight mechanisms implicated in its signaling, and open new avenues for investigation of links between the gut microbiota and brain energy metabolism.

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