The adult mammalian brain possesses the remarkable capacity for generating new neurons throughout life. This process of adult neurogenesis is restricted to a few privileged areas of the brain such as dentate gyrus (DG) of the hippocampus. This form of extreme plasticity generates new neurons and glial cells from the resident population of neural progenitor cells (NPCs) that are important for learning and memory while disruption in this process is implicated in many disorders including depression and age-related dementia. The generation of new cells from NPCs can be affected by various intrinsic and extrinsic factors, which range from the local microenvironment of the neurogenic niche to voluntary exercise and diet. The thesis research presented here examines how the process of neurogenesis is regulated by factors such as aging and investigates role of a novel recently identified regulator Brain-specific angiogenesis inhibitor 2 (BAI2).The first part of my thesis examines the age-dependent changes in cell intrinsic properties of NPCs. Application of a modified method allowing to isolate and culture NPCs later in life when neurogenesis is decreased uncovered that while hippocampal precursors from the aged brain display same NPC characteristics as cells from the younger brain, their cell proliferation ability is diminished. Detailed cellular-level analysis demonstrated that neuronal output and NPC fate choice is driven by changes in cell-autonomous properties that contribute to the reduced number of newborn neurons in the aging brain. This study shows that in vitro models of neurogenesis in the adulthood can serve as a platform for investigating important biological questions regarding the development and differentiation of hippocampal neurons generated throughout adult life. iii The second part of my thesis examines the role of BAI2, the adhesion G-protein coupled receptor (aGPCR) family member, in regulation of adult neurogenesis. Constitutive knock out of BAI2 shows a possible cell extrinsic mechanism for regulation NPCs proliferation via VEGF growth factor. At the same time conditional deletion of BAI2 in mice resulted in the decreased cell proliferation in the neural precursor cells suggesting additional cell-autonomous regulation of cell proliferation by BAI2. Applying methodology developed in the first part of this thesis showed allowed me to disentangle extrinsic and cell autonomous role of BAI2 in cell proliferation. Altogether, this work defines BAI2 as a novel regulator of adult neurogenesis and proposes a mechanism of action that could affect future neuronal proliferation studies. Further investigation of BAI2 cellular mechanism in NPCs may inform future human disease studies and treatments associated with neurogenesis pathologies. In summary, my thesis work presented here has contributed significant novel discoveries in neurodevelopment in the adult mouse hippocampus. I have found novel cell extrinsic and cell intrinsic mechanisms for regulating hippocampal neural precursor cell proliferation, differentiation and cell fate decisions.

Learning and memory are crucial cognitive processes that promote an organism’s survival, allowing it to rapidly modify its behavior in response to changes in the environment and repeat patterns of behavior that enhance survivability and reproduction. At a cellular level, the phenomena of learning and memory are facilitated by the restructuring of plastic neural circuits that encode behaviors. The dynamic nature of dendritic spines, small, actin-rich, membranous protrusions on the dendrites of neurons, underlies this plasticity and the neural capacity for modification. Dendritic spine are the sites where neurons receive most of their excitatory inputs and several studies have linked the dynamic changes dendritic spines can undergo to learning and lasting memory. Amongst these changes are the spontaneous outgrowth and subsequent persistence of new dendritic spines. New spine outgrowth increases during learning paradigms, and the fraction of these new spines that are retained correlates with improved memory of a learned experience. Little is known about the mechanisms that determine whether a new spine stabilizes and persists, but recent work has demonstrated that strong synaptic stimulation, particularly that which induces long-lasting potentiation (LTP) of spine volume and synaptic strength at mature spines, enhances the survivorship of nascent dendritic spines. This phenomenon can be induced absent of presynaptic input using two-photon (2p) glutamate uncaging of 4-methoxy-7-nitroindolinyl (MNI)-caged glutamate at a high-frequency (HFU) that allows for the spatially restricted release of glutamate at a single dendritic spine. LTP-inducing glutamatergic stimulation initiates several molecular signaling cascades that occur within theiii spine; which of these mechanisms exist in new spines and play a role in enhancing new spine stability remains a mystery. The interaction between the GluN2B subunit of the NMDA-type glutamate receptor (NMDAR) and CaMKII that is activated downstream of NMDAR-mediated Ca2+ influx is one molecular mechanism induced during LTP that has been implicated in new spine survivorship. This interaction was found to be necessary for activity-dependent new spine stabilization and genetically disrupting GluN2B-CaMKII binding blocks the stabilizing effects of glutamatergic stimulation. The work described in this dissertation follows up on these results by further defining the role that CaMKII plays in activity-dependent new spine stabilization. Chapter 1 of this dissertation is a review of the current understanding of new spines, including their role in the dynamic restructuring of neural circuits to facilitate learning and memory, the mechanisms that govern their formation, and the current understanding of the mechanisms that promote their stabilization. Chapter 1 also reviews what is known about the signaling cascades that are involved in nascent dendritic spine stability and the role that CaMKII and its myriad functions and interactions may play in enhancing new spine stabilization. Chapter 2 of this dissertation describes experiments which further define the roles that CaMKII structural and enzymatic activity play in activity-dependent new spine stabilization. 2p imaging and HFU of MNI-caged glutamate was used to stimulate spontaneously formed nascent dendritic spines shortly after outgrowth on CA1 neurons in cultured organotypic hippocampal slices where CaMKIIα enzymatic and structural activities were altered. These experiments provided direct evidence that CaMKIIα structural and scaffolding interactions, but not its kinase activity, are required for the previously reported enhancement of new spine stability following 2p glutamate uncaging. Chapter 3 of this dissertation contains additional analysis performed on the data described in Chapter 2. These analyses corroborate previous results that HFU at a new spine, which enhances survivorship, often results in new spine enlargement, but new evidence described in this chapter suggests that this enlargement is not necessary to stabilize new spines. Also shown in Chapter 3 is evidence that CaMKIIα kinase activity inhibits spine enlargement at new spines, iv despite it being necessary for spine enlargement at mature spines. Chapter 4 describes preliminary experiments that further explore the non-enzymatic role that CaMKIIα performs at new spines by targeting two binding partners that do not require CaMKIIα to function: the 26S proteasome and synGAP-1α. The first section of Chapter 4 describes experiments that aim to determine the role of the 26S proteasome in activity-dependent new spine stabilization. Pharmacological inhibition of the 26S proteasome was utilized to determine if the proteasome had a role in enhancing new spine survivorship rates following HFU. The preliminary results of these experiments show that inhibition of the 26S proteasome increased basal rates of new spine survivorship such that any changes or deficits in activity-induced stabilization could not be determined. The second section of Chapter 4 focuses on the role of synGAP-1α, a negative regulator of synaptic stability, plasticity, and maturation, in activity-dependent new spine stabilization. Green fluorescent protein (GFP)-tagged synGAP-1α was overexpressed in CA1 neurons in cultured organotypic hippocampal slices and HFU of MNI-caged glutamate was used to stimulate individual new spines that had formed on these cells. The preliminary findings of these experiments show that synGAP-1α, which is enriched at lower levels in new spines compared to their mature counterparts, undergoes activity-dependent evacuation from new spines following HFU. This evacuation does not require CaMKIIα kinase activity, as preliminary results show that synGAP-1α dispersion from new spines still occurs during pharmacological inhibition of CaMKIIα. Together, my results demonstrate that non-enzymatic scaffolding activities of CaMKIIα, such as the interactions between CaMKII and the 26S proteasome or synGAP-1α, regulate new spine survivorship and volume changes, providing further insight into the molecular mechanisms that govern new spine stability and maturation.

Trafficking, localization, and biophysical properties of AMPA-type glutamate receptors (AMPARs) at synapses are predominant mechanisms for regulating synaptic strength underlying learning and memory. AMPARs are highly regulated by a number of auxiliary proteins such as TARPs, cornichons, and CKAMPs, as well as the members of the SynDIG/Prrt family, which will be the focus of this study. Previous studies of the transmembrane protein Synapse Differentiation Induced Gene 1 (SD1; SD1) as an AMPAR interacting protein that regulates excitatory synaptic strength and AMPAR number both in vitro and in vivo. The SynDIG family is defined by a family of four genes, SD1-4, which are expressed in distinct locations within the brain. SynDIG4 (SD4/Prrt1) is primarily expressed within the hippocampus, the main site of memory formation in both mice and humans. SD4 was identified in several independent proteomic screens in complex with AMPARs, suggesting it may also function as an AMPAR auxiliary factor. We previously observed that long-term potentiation (LTP) is abolished by single tetanus stimulation of hippocampal slices from SD4 knockout (KO) mice. In this study we demonstrate that only specific protein domains are necessary for clustering of AMPARs and SD4. Furthermore, we observed that some SD4 co-localizes with extra-synaptic GluA1-containing AMPARs in primary neurons, while loss of SD4 results in reduced extra-synaptic AMPARs, implying a role of SD4 outside the synapse. We hypothesize that SD4 regulates AMPARs through maintenance of an extra-synaptic pool of GluA1-containing AMPARs required for trafficking and LTP.

During development, Sonic hedgehog (Shh) regulates the proliferation of cerebellar granule neuron precursors (GNPs) in part via expression of Nmyc. We present evidence supporting a novel role for the Mad family member Mad3 in the Shh pathway to regulate Nmyc expression and GNP proliferation. Mad3 mRNA is transiently expressed in GNPs during proliferation. Cultured GNPs express Mad3 in response to Shh stimulation in a cyclopamine-dependent manner. Mad3 is necessary for Shh-dependent GNP proliferation as measured by bromodeoxyuridine incorporation and Nmyc expression. Furthermore, Mad3 overexpression, but not that of other Mad proteins, is sufficient to induce GNP proliferation in the absence of Shh. Structure-function analysis revealed that Max dimerization and recruitment of the mSin3 corepressor are required for Mad3-mediated GNP proliferation. Surprisingly, basic-domain-dependent DNA binding of Mad3 is not required, suggesting that Mad3 interacts with other DNA binding proteins to repress transcription. Interestingly, cerebellar tumors and pretumor cells derived from patched heterozygous mice express high levels of Mad3 compared with adjacent normal cerebellar tissue. Our studies support a novel role for Mad3 in cerebellar GNP proliferation and possibly tumorigenesis, and they challenge the current paradigm that Mad3 should antagonize Nmyc by competition for direct DNA binding via Max dimerization.