abstract: The Erk/MAPK pathway plays a major role in cell growth, differentiation, and survival. Genetic mutations that cause dysregulation in this pathway can result in the development of Rasopathies, a group of several different syndromes including Noonan Syndrome, Costello Syndrome, and Neurofibromatosis Type-1. Since these mutations are germline and affect all cell types it is hard to differentiate the role that Erk/MAPK plays in each cell type. Previous research has shown that individual cell types utilize the Erk/MAPK pathway in different ways. For example, the morphological development of lower motor neuron axonal projections is Erk/MAPK-independent during embryogenesis, while nociceptive neuron projections require Erk/MAPK to innervate epidermal targets. Here, we tested whether Erk/MAPK played a role in the postnatal development of lower motor neurons during crucial periods of activity-dependent circuit modifications. We have generated Cre-dependent conditional Erk/MAPK mutant mice that exhibit either loss or gain of Erk/MAPK signaling specifically in ChAT:Cre expressing lower motor neurons. Importantly, we found that Erk/MAPK is necessary for the development of neuromuscular junction morphology by the second postnatal week. In contrast, we were unable to detect a significant difference in lower motor neuron development in Erk/MAPK gain-of-function mice. The data suggests that Erk/MAPK plays an important role in postnatal lower motor neuron development by regulating the morphological maturation of the neuromuscular junction. / Dissertation/Thesis / Masters Thesis Biology 2017

abstract: Development of the cerebral cortex requires the complex integration of extracellular stimuli to affect changes in gene expression. Trophic stimulation activates specialized intracellular signaling cascades to instruct processes necessary for the elaborate cellular diversity, architecture, and function of the cortex. The canonical RAS/RAF/MEK/ERK (ERK/MAPK) cascade is a ubiquitously expressed kinase pathway that regulates crucial aspects of neurodevelopment. Mutations in the ERK/MAPK pathway or its regulators give rise to neurodevelopmental syndromes termed the “RASopathies.” RASopathy individuals present with neurological symptoms that include intellectual disability, ADHD, and seizures. The precise cellular mechanisms that drive neurological impairments in RASopathy individuals remain unclear. In this thesis, I aimed to 1) address how RASopathy mutations affect neurodevelopment, 2) elucidate fundamental requirements of ERK/MAPK in GABAergic circuits, and 3) determine how aberrant ERK/MAPK signaling disrupts GABAergic development. Here, I show that a Noonan Syndrome-linked gain-of-function mutation Raf1L613V, drives modest changes in astrocyte and oligodendrocyte progenitor cell (OPC) density in the mouse cortex and hippocampus. Raf1L613V mutant mice exhibited enhanced performance in hippocampal-dependent spatial reference and working memory and amygdala-dependent fear learning tasks. However, we observed normal perineuronal net (PNN) accumulation around mutant parvalbumin-expressing (PV) interneurons. Though PV-interneurons were minimally affected by the Raf1L613V mutation, other RASopathy mutations converge on aberrant GABAergic development as a mediator of neurological dysfunction. I therefore hypothesized interneuron expression of the constitutively active Mek1S217/221E (caMek1) mutation would be sufficient to perturb GABAergic circuit development. Interestingly, the caMek1 mutation selectively disrupted crucial PV-interneuron developmental processes. During embryogenesis, I detected expression of cleaved-caspase 3 (CC3) in the medial ganglionic eminence (MGE). Interestingly, adult mutant cortices displayed a selective 50% reduction in PV-expressing interneurons, but not other interneuron subtypes. PV-interneuron loss was associated with seizure-like activity in mutants and coincided with reduced perisomatic synapses. Mature mutant PV-interneurons exhibited somal hypertrophy and a substantial increase in PNN accumulation. Aberrant GABAergic development culminated in reduced behavioral response inhibition, a process linked to ADHD-like behaviors. Collectively, these data provide insight into the mechanistic underpinnings of RASopathy neuropathology and suggest that modulation of GABAergic circuits may be an effective therapeutic option for RASopathy individuals. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2019

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Signaling scaffolds are critical for the correct spatial organization of enzymes within the ERK1/2 signaling pathway and proper transmission of intracellular information. However, mechanisms that control molecular dynamics within scaffolding complexes, as well as biological activities regulated by the specific assemblies, remain unclear. The scaffold protein Shoc2 is critical for transmission of the ERK1/2 pathway signals. Shoc2 accelerates ERK1/2 signaling by integrating Ras and RAF-1 enzymes into a multi-protein complex. Germ-line mutations in shoc2 cause Noonan-like RASopathy, a disorder with a wide spectrum of developmental deficiencies. However, the physiological role of Shoc2, the nature of ERK1/2 signals transduced through this complex or mechanisms regulating the function of Shoc2 remain largely unknown. My dissertation addresses the mechanisms by which Shoc2 accelerates ERK1/2 signal transmission and the biological outputs of the Shoc2-guided signals. To delineate Shoc2-mediated ERK1/2 signals, I have utilized a vertebrate zebrafish model. I demonstrated that loss of Shoc2 protein expression leads to early embryonic lethality resulting from a significant reduction in the number of circulating erythropoietic and myelopoietic blood cells, underdeveloped neurocranial and pharyngeal cartilages, and a profound delay in calcification of bone structures. Together, this data demonstrates that the Shoc2 scaffolding module transmits ERK1/2 signals in neural crest development and blood cell differentiation. This dissertation also addresses the mechanistic basis of how allosteric ubiquitination of Shoc2 and RAF-1 is controlled. I have characterized a molecular interaction of Shoc2 with its previously unknown binding partner Valosin-Containing Protein (VCP/p97). These studies demonstrated that hexametric ATPase VCP modulates ubiquitination of Shoc2 and RAF-1 through the remodeling of the scaffolding complex in a spatial-restricted manner. Experiments utilizing fluorescence microscopy and biochemical methods show that VCP/p97 sequesters the E3 ligase HUWE1 from the Shoc2 module, thereby altering the ubiquitination of Shoc2 and RAF-1 as well as the amplitude of ERK1/2 signals. These studies also show that the levels of Shoc2 ubiquitination and ERK1/2 phosphorylation are imbalanced in fibroblasts isolated from Inclusion Body Myopathy with Paget’s disease of bone and Frontotemporal Dementia (IBMPFD) patients harboring VCP germline mutations. This data also suggests that ERK1/2 pathway deregulation is part of IBMPFD pathogenesis. In summary, these studies make a significant advance in our understanding of the mechanisms by which the Shoc2 scaffold regulates specificity and the dynamics of the ERK1/2 signaling networks. They also make important insights into our understanding of biological activities and targets of Shoc2-mediated ERK1/2 signals at the early stages of embryonic development and disease.