The motivation for this PhD thesis was to understand the molecular basis of how cells regulate the formation of an organised and mechanically strong extracellular matrix (ECM). In tendon this process begins during embryogenesis with the appearance of bundles of narrow-diameter (~30 nm) collagen fibrils that are parallel to the tendon long axis. At the onset of collagen fibrillogenesis, the cells elongate, the fibrils are constrained within plasma membrane channels with their ends contained in tension-sensitive actin-stabilised plasma membrane protrusions. The mechanism by which actin is reorganised during cell elongation and the formation of tension-sensitive plasma membrane protrusions is poorly understood. The small GTPase RhoA is the major regulator of actin reorganisation into stress fibres, which have been implicated in mechanotransduction, ECM assembly and remodelling. The hypothesis tested by this PhD thesis was that the organisation and tensioning of extracellular collagen fibrils is generated on a blueprint of tensioned actin filaments within the cell. Rho activity is regulated specifically by Rho GTPase activating proteins (RhoGAPs). By comparing the global gene expression of tendon tissues at different developmental stages, Arhgap28, a novel RhoGAP, which is expressed during tendon development but not during postnatal maturation, was identified.Arhgap28 belongs to a large family of RhoGAPs containing the closely related members, Arhgap6 and Arhgap18, which have previously been shown to regulate RhoA and stress fibre formation. Arhgap28 expression was upregulated in embryonic fibroblasts cultured in a 3D, tensioned embryonic tendon-like construct compared to monolayer culture. Arhgap28 expression was further enhanced during the development of mechanical strength and stiffness of the tendon constructs, but downregulated when the tension in tendon constructs was released. Overexpression of a C-terminal V5-tagged Arhgap28 protein caused a reduction in RhoA activation and disruption of stress fibre assembly. Modulation of Rho signalling using lysophosphatidic acid and Y27632 showed that collagen remodelling by cells in collagen gels and tendon constructs is regulated by RhoA signalling. A tissue-wide qPCR analysis identified Arhgap28 in several tissues including tendon, bone, and skin. An Arhgap28 reporter mouse (Arhgap28gt) and an Arhgap28 knockout mouse (Arhgap28del) were also studied to investigate the role of Arhgap28 in tissue organisation in vivo. Arhgap28gt mice showed Arhgap28 expression in bones at E18.5. Homozygous Arhgap28del mice were viable, appeared normal but expressed a truncated Arhgap28 transcript, which if translated, would produce a protein lacking the RhoGAP domain. Therefore, it was hypothesised that knockout mice were normal due to compensation from another RhoGAP. Overexpression of Arhgap6 in Arhgap28-null bone tissues was confirmed. Upregulation in RhoA expression was also detected, further suggesting that Arhgap28 regulates RhoA. Interestingly, a microarray comparison of bone tissues from wild type and Arhgap28-null mice showed that genes linked to bone dysplasia are downregulated in Arhgap28-null bone. Together, these results suggest that formation of a strong and organised collagen ECM is mediated by RhoA-generated cellular tension and that Arhgap28 and Arhgap6 might be co-regulators of this process.

Our ability to learn relies on the potential of neuronal networks to change through experience. The primary visual cortex (V1) has become a popular system for studying how experience shapes cortical neuronal networks. Experience-dependent plasticity in V1 has been extensively studied in young animals, revealing that experiences in early postnatal life substantially shape neuronal activity in the developing cortex. In contrast, less is known about how experiences modify the representation of visual stimuli in the adult brain. In addition, adult experience-dependent plasticity remains largely unexplored in neurodevelopmental disorders. To address this issue, we established a two-photon calcium imaging set-up, suitable for chronic imaging of neuronal activity in awake-behaving mice. We implemented protocols for the reliable expression of genetically encoded calcium indicators (GCaMP6), for the implantation of a chronic cranial window and for the analysis of chronic calcium imaging data. This approach enables us to monitor the activity of hundreds of neurons across days, and up to 4-5 weeks. We used this technique to determine whether the daily exposure to high-contrast gratings would induce experience-dependent changes in V1 neuronal activity. We monitored the activity of putative excitatory neurons and of three non-overlapping populations of inhibitory interneurons in layer 2/3 of adult mice freely running on a cylindrical treadmill. We compared the results obtained from mice that were exposed daily to either a high-contrast grating or to a grey screen and characterized their neuronal response properties. Our results did not reveal significant differences in neuronal properties between these two groups, suggesting a lack of stimulus-specific plasticity in our experimental conditions. However, we did observe and characterize, in both groups, a wide range of activity changes in individual cells over time. We finally applied the same method to investigate impairments in experience-dependent plasticity in a mouse model of intellectual disability (ID), caused by synaptic GTPase-activating protein (SynGAP) haploinsufficiency. SynGAP haploinsufficiency is a common de novo genetic cause of non-syndromic ID and is considered a Type1 risk for autism spectrum disorders. While the impact of Syngap gene mutations has been thoroughly studied at the molecular and cellular levels, neuronal network deficits in vivo remain largely unexplored. In this study, we compared in vivo neuronal activity before and after monocular deprivation in adult mutant mice and littermate controls. These results revealed differences in baseline network activity between both experimental groups. These impairments in cortical neuronal network activity may underlie sensory and cognitive deficits in patients with Syngap gene mutations.

Thesis (Ph.D.)--McMaster University, 2003. / Advisor: David W. Andrews. Includes bibliographical references (leaves 122-141) Also available via World Wide Web.

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SYNGAP1 haploinsufficiency is one of the most common monogenic causes of nonsyndromic moderate to severe intellectual disability (NSID) and autism (Hamdan et al., 2009; Pinto et al., 2010). De novo truncating or frameshift mutations in the SYNGAP1 gene lead to the loss of the encoded protein Synaptic GTPase activating protein (SynGAP), one of the most abundant of postsynaptic proteins (Hamdan et al., 2011). SynGAP, present at excitatory and inhibitory synapses (Kim et al., 1998), acts as a key regulator of highly conserved signaling pathways linked to AMPA- and NMDA-receptor dependent plasticity at the post synaptic density (Krapivisky et al., 2004; Vazquez et al., 2004). The Syngap mouse model has been extensively used to understand the pathophysiology underlying abnormal SynGAP-mediated signaling. Syngap heterozygous (het) mice demonstrate a range of physiological and behavioural abnormalities from development to adulthood (Komiyama et al., 2002; Muhia et al., 2010). However, recent advances in techniques for genome manipulation have allowed for the generation of rat models of neurodevelopmental disorders, including Syngap; enabling phenotypes to be validated across species and to address cognitive and social dysfunction, using paradigms that are more difficult to assess in mice. In this study, we examined the pathophysiology associated with a heterozygous deletion of the C2 and catalytic GAP domain of the protein, in Long-Evans rats (het). In contrast with het mice, het rats do not present with hyperactivity and can be habituated to an open field environment. To examine associative recognition memory, we tested the rats in five spontaneous exploration tasks for short-term and long-term memory, object-recognition (OR), object-location (OL), object-place (OP), object-context (OC) and object-place-context (OPC). Both groups were able to perform short-term memory tasks, but only wild type rats performed above chance in OL with a 24hour delay, suggesting deficits in long- term spatial memory. We also tested if partial loss of the GAP domain in SynGAP affects social behaviour in rats and we found that het rats exhibited impaired short- term social memory, with no signs of social isolation. These findings do not fully recapitulate previous abnormalities reported in the mouse model of SYNGAP1 haploinsufficiency, suggesting that some key behavioural phenotypes may be species-specific. Furthermore, based on physiological deficits that Syngap het mice exhibit, such as alterations in mEPSC/mIPSC amplitude and frequency and evoked cortical hyperexcitability in vitro (Guo et al., 2009; Ozkan et al., 2014), we also aimed to test if in vivo neuronal activity and circuit properties are altered. Using two-photon calcium imaging in awake mice, we focused on two areas of the cortex; a primary sensory area, the binocular region of the visual cortex (V1), and an association area, the medial posterior parietal cortex (PPC). Both areas have been found to maintain activity during visual discrimination tasks but to present with divergent activity trajectories (Harvey et al., 2012; Goard et al., 2016). We found preliminary evidence that neurons in layer 2-3 of the PPC of Syngap mice are hypoactive in basal conditions when animals are still in the dark, compared to wild type controls. When we assessed whether that changes when animals are running, we found that during locomotion neurons of both genotypes increase their activity, consistent with previous findings in wild type mice (McGinley et al., 2015; Pakan et al., 2016). However, this response gain is exaggerated in Syngap het neurons of the PPC. In contrast to above findings in PPC, results in V1 show that layer 2-3 neurons are hyperactive during both behavioural states, suggesting seemingly different computations of these two cortical areas. This work provides the first evidence for a dysregulated neuronal circuit in vivo in both visual and parietal cortex of Syngap mice, two areas critical for sensory processing that has been found to be affected in individuals with NSID and autism (Joosten and Bundy, 2010). We also provide first evidence of the effect of loss of SynGAP activity in behaviour of rats, complimenting existing data in the literature in a species-specific manner and providing greater insight into sensory and cognitive dysfunction associated with dysregulation in SynGAP-mediated signaling.

ADP Ribosylation Factor 1 (ARF1) is a universal small GTP binding protein which has an important role in vesicular trafficking between endoplasmic reticulum and Golgi. ARF1 is a basic component of Coat Protein I (COPI) vesicles which have functions in both formation of coatomer complex and recruitment of cargo proteins. In this study, the expression ARF1 was analyzed in pea (P. sativum L. cv. Araka) grown at different developmental stages. Because of the differential hormonal levels at corresponding stages, the effects of hormones on ARF1 expression were also studied. The results of present research show that ARF1 expression in embryos and 2 days grown plants after germination is lower when compared to 6 days grown plants. In order to see the hormonal effect, 3 weeks old plants were supplied with 50&micro / M of each hormone for 3 times on alternate days. Protein extraction, cell fractionation,Western blot was carried out and immunoblot analysis was conducted with AtARF1 polyclonal antibodies. It was shown that, in pea shoots, abscisic acid and gibberellin increases the inactive GDP bound ARF1 by hydrolyzing ARF-GTP through activating ARFGTPase activating protein (ARF-GAP) or partially inhibiting ARF-Guanine Nucleotide Exchange Factor (ARF-GEF). In roots, ARF-GDP (cytosolic fraction), ARF-GTP (microsomal fraction) and total amount of ARF1 (13.000 x g supernatant fraction) were down regulated by ~11, ~19 and ~11 fold respectively with the application of gibberellin / and by ~11, ~7 and ~3 fold respectively with the application of abscisic acid / when compared to control plants. These results indicate the importance of plant hormones in the regulation of ARF1 in pea.

Activated receptor tyrosine kinases recruit many signalling proteins to initiate downstream cell proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K), a heterodimer consisting of a p85 regulatory protein and a p110 catalytic protein. Our laboratory has previously shown the p85α protein also has in vitro GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle trafficking events for activated receptors. Expression of a p85α protein containing an arginine to alanine substitution at position 274 (p85R274A) that affects its GAP activity, caused sustained levels of activated platelet-derived growth factor receptors (PDGFRs), enhanced downstream signalling, and resulted in cellular transformation. Together with other data, this suggested that in p85R274A-expressing cells, PDGFRs are more rapidly trafficked through the endocytic pathway, which reduces opportunities for sorting events necessary for receptor degradation. Our laboratory has observed previously that p85 was capable of binding to both Rab5-GDP, as well as Rab5-GTP, which is an atypical characteristic of GAP proteins, whereas p110β had previously been reported to bind Rab5-GTP selectively. Based on these observations, this thesis project was designed to test the hypothesis that both proteins contributed GAP activity towards Rab5, with p85 providing a catalytic arginine residue (R274) and p110β providing switch stabilization functions specific to the GTP-bound state. To accomplish the thesis objective, cells expressing individual p85 defects (lacking GAP activity, R274A; or lacking p110-binding ability through deletion of residues 478-513, Δ110) were compared to cells expressing a double mutant missing both functions. Stable clonal NIH 3T3 cell lines were generated and selected in G418 and clones expressing similar levels of FLAG-tagged p85 wild type or mutants compared to the control cell lines (NIH 3T3, FLAG-vector control, p85 wild type, and p85R274A) were chosen for analysis. A time-course of PDGF stimulation showed that cells expressing p85R274A or p85Δ110+R274A have sustained phosphorylation levels of the PDGFR, reduced rates of PDGFR degradation and sustained MAPK/Erk signalling. Contrary to the cellular transformation previously reported for p85R274A-expressing cells, expression of p85Δ110+R274A did not lead to cellular transformation. These divergent results suggest that p85-associated p110 serves two functions. As the catalytic subunit of PI3K, one function is the localized generation of PI3,4,5P3 lipids at the plasma membrane for Akt activation, and possibly during receptor endocytosis where it could impact MAPK/Erk activation/deactivation kinetics and cell transformation. These results support a second function for p110 in the regulation of PDGFR activation/deactivation kinetics and PDGFR half-life, both strongly influenced by alterations in PDGFR trafficking. This suggests that p110β may regulate PDGFR trafficking by providing Rab5-GTP switch stabilization that complements the catalytic arginine residue (R274) within p85, and that p85α and p110β work together as a Rab5 GAP. The role of PDGFR in the localization of the RabGAP function of p85 to specific subcellular compartments was also examined. It was hypothesized that PDGFR may help localize the RabGAP function of p85 to vesicles containing Rab5 or Rab4 through the binding of p85 to phosphorylated tyrosine residues on activated PDGFR. Stable cell lines expressing individual p85 defects (lacking GAP activity, R274A; or lacking PDGFR-binding ability through site-directed mutation of residues 358 and 649 from arginine to alanine, ΔR; or a double mutant missing both functions) demonstrated that p85R274A or p85ΔR+R274A expression leads to sustained PDGFR activation and signalling, and to delayed PDGFR degradation in response to PDGF stimulation. The sustained signalling observed resulted in cellular transformation in cells expressing p85R274A or p85ΔR+R274A. The data suggests that PDGFR does not play a role in the localization of the RabGAP activity of p85. The findings of this study elucidates important non-canonical functions of the PI3K heterodimer and contributes to our understanding of how specific mutations in both p85 and p110β within regions implicated in the regulation of RabGAP activity can alter signalling events and lead to enhancement of tumour-associated phenotypes.

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