In this thesis, I will discuss three studies in relation to the recovery of function after nervous system injury in hopes of answering a vital question. How do manipulations of specific inhibitors of regeneration affect the functional recovery of rodents after SCI, and what circuit-level change(s) might underlie these functional changes?

Our first study investigates the mechanism of peripheral conditioning by using a demyelinating reagent, ethidium bromide (EtBr), to test whether demyelination, rather than mechanical nerve crush of axons is sufficient to induce the regeneration-promoting, conditioning effect. After assessing regeneration, as well as functional recovery through behavioral studies, we found that demyelination is a likely contributor to the conditioning effect. These studies provide the framework for further investigation into the mechanisms of the conditioning effect.

The second study combines our novel EtBr-mediated conditioning paradigm with inhibition of the repulsive axon guidance molecule Wnt. In these studies, bone marrow stromal cells (BMSCs) secreting the Wnt inhibitor frizzled-related peptide 2 (SFRP2), were grafted at a cervical level 1 (C1) lesion site caudal to main body of dorsal funiculi, second order, sensory neurons. In our studies, better sensory functional recovery was observed in animals with SFRP2 treatment compared to naive animals. Circuit level analysis demonstrated that axons of conditioned sensory neurons sprouted and made new connections with a small subset of dorsal column neurons, caudal to the main body of dorsal column nuclei. Transient silencing or re-transection of this remodeled circuit at thoracic level 10 (T10) resulted in the loss of recovered sensory function, indicating that this remodeled circuit is sufficient to confer the observed recovery of sensory function.

Our third study aims to minimize the effect of Wnt signaling in a newly generated line of related to receptor tyrosine kinase (Ryk) conditional knockout (cKO) mice. Ryk is a repulsive receptor of Wnt, and we studied corticospinal axon plasticity in this mouse model. After C5 dorsal column lesion, better motor-related function recovery was observed in Ryk cKO mice. Circuit level analysis showed increased collateral sprouting with Ryk deletion in motor cortex. Secondary lesion at C3 resulted in a loss of both the enhanced recovery mediated by Ryk deletion, as well as a loss of corticospinal axon collaterals between levels C3 and C5. Whereas after lesioning of the corticospinal motor axons at the level of the pyramid (pyramidotomy), functional recovery was lost completely, implicating a role for the remodeled corticospinal tract in the recovery of motor function.

These studies indicate that, while regeneration is not easily achieved, local sprouting and axon plasticity might offer more attainable circuit changes after spinal injury. We have found that this plasticity leads to significant motor and sensory functional recovery in rodents, and may be a potential avenue for mediating recovery for approximately 280,000 patients affected by SCI in the United States.

The Wnt signaling pathways play important roles in many developmental processes from invertebrate to vertebrate. The Wnt/Planar cell polarity (PCP) pathway is one of the non- canonical Wnt signaling pathways that mainly regulate cytoskeleton. Without the control of PCP components, Drosophila wing epithelia would grow in random directions. Our lab previous work has shown that PCP components Celsr3, Vangl2 and Frizzled3 are responsible for neuronal migration, axon guidance and synapse formation. Prickle is one of the core components of PCP signaling pathway. Studies have reported that several neurological abnormalities were identified with Prickle mutation in human, mice, zebrafish and Drosophila. Prickle1 R104Q missense mutation was reported in three consanguineous families with Progressive Myoclonus Epilepsy-Ataxia Syndrome. Here, we used CRISPR-Cas 9 gene editing to generate this human point mutation in mice. In this thesis, we performed examination of behavioral deficits in Prickle1 R104Q mutant mice. We found that Prickle1 R104Q mutant mice have normal locomotor activity in open field assay and light-dark transition assay, while they exhibited deficit in novel object recognition test. Then we used three-chamber social interaction test and found that these Prickle1 mutant mice have abnormal sociability and normal social novelty. Using Barnes maze, we showed Prickle1 mutant mice have normal learning ability and spatial memory. In conclusion, our work has identified behavioral deficits in Prickle1 R104Q mutant mice.

The signaling mechanisms of regulating excitatory synapses are the foundation for understanding neural circuit functions and disorders but have not been fully understood. Previously our lab has found an opposing role of core planar cell polarity (PCP) components, Celsr3 and Vangl2, in regulating excitatory synapse formation. Celsr3 is required for excitatory synapse formation, while Vangl2 inhibits excitatory synapse formation. Here, we took a further step to focus on another PCP component called Prickle. We found that the deletion of Prickle homolog 2 (Prickle2/Pk2) in developing and adult mice’s dorsal hippocampus and medial prefrontal cortex (mPFC) reduced the density of excitatory synapses. Moreover, Prickle2 p.E8Q mutation, which was found in human patients diagnosed with Autism Spectrum Disorder (ASD), reduced the density of excitatory synapses in the stratum radiatum of mice CA1 region. The reduced synapse density may be due to the decreased level of NMDA receptors, AMPA receptors, and MAGUK family proteins that are essential for synapse development. We also found that the Prickle2 p.E8Q protein was less stable than WT Prickle2, which may be due to the increased interaction between Prickle2 and Vangl2 that enhanced the proteasomal degradation of Prickle2. In addition, in HEK293T cells, Prickle2 interacted with Prickle homolog 1 (Prickle1/Pk1), which contributes to ASD and epilepsy. In mice’s stratum radiatum of CA1 region and layer I-III of mPFC, Prickle2 and Prickle1 colocalized with each other while both colocalized with postsynaptic density 95 (PSD-95). These data together indicated Prickle2 plays an essential role in regulating synapse formation and function.

Depression is characterized by a loss of synaptic connections in regions of the brain involved in emotional regulation and cognitive function, and antidepressant treatments generally result in a compensatory increase in the number of these connections. Previous studies have shown that a single sub-anesthetic dose of ketamine, a rapid-acting antidepressant, increases synaptic connectivity in excitatory neurons projecting from the prefrontal cortex to other brain regions involved in cognitive regulation and function, countering the destabilization and loss of synapses observed in depression. However, there is little understanding of the molecular pathways and mechanisms involved in synapse restoration at the synapse structure level. Due to the role of proteins involved in synapse formation and maintenance, we sought to determine whether ketamine’s antidepressant mechanism is dependent on these proteins in projecting neurons of excitatory circuits that contribute to emotional regulation. Mice were divided into groups with or without chronic stress and with conditional knockouts for the proteins of interest. They were then subjected to a series of behavioral tests and their brain tissue was analyzed for changes in excitatory synapse number. We found that ketamine’s promotion of synapse formation and sustained antidepressant-like effect on behavior was hindered in knockout mice. These findings revealed that ketamine is dependent on proteins involved in synapse formation and maintenance for its rescue of depressive-like behavior and restoration of excitatory synaptic connectivity.

Glutamatergic synapses are the main excitatory synapses in the mammalian central nervous system. The assembly and regulation of these highly organized and asymmetric junctions requires extensive molecular signaling mechanisms. Although thousands of components and interactions within the synapse have been identified, the signaling logic that governs their assembly has remained elusive. We show that a conserved cell polarity signaling protein, Lgl1, is localized in the postsynaptic density. Conditional knockout of Lgl1 in hippocampal pyramidal neurons at postnatal day 7

resulted in increased synapse numbers, altered synaptic ultrastructure, altered compositions of synaptic proteins, and reduced AMPA/NMDA ratio. Conditional Lgl1 deletion from adult mice also produces synaptic phenotypes similar to what is observed in the deletion at postnatal day 7. Lgl1 and atypical Protein Kinase C mutually inhibit each other in apical-basal polarity signaling. We found that a double conditional knockout of both isoforms of atypical PKCs in hippocampal neurons at postnatal day 7 lead to changes in synaptic ultrastructure at postnatal day 14, but not synapse number. Reduction of synapse number was observed in adulthood, suggesting a role for aPKC in the maintenance of synapses rather than their initial formation. Conditional deletions of Lgl1 or atypical PKCs lead to deficits in novel object recognition and social interaction. Triple cKO of Lgl1 and atyptical PKCs restores synapse number and some of the behavioral functions, such as open field and novel object recognition, but not social behavior. The phenotypes observed following conditional deletion of Lgl1 or aPKC are consistent with their opposing roles in apical-basal polarity and their influence on other pathways such as the planar cell polarity pathway and cellular adhesion. Lgl1 is one of the genes commonly deleted in a severe but poorly known behavioral disorder, Smith-Magenis Syndrome (SMS), which is often diagnosed with autism. Lgl1+/- animals show increased synapse numbers, deficits in social interaction, and stereotyped repetitive behavior. Furthermore, three previously characterized NMDA antagonists can rescue social interaction in Lgl1+/- animals. Therefore, deletion of Lgl1 may contribute to neuropsychiatric symptoms in SMS and provides insight into cellular pathways that may underlie the behavioral phenotypes that are observed in the syndrome.

Glucose metabolism starts with the activity of the first rate-limiting step enzyme Hexokinase (HK). In the brain, which the major energy source is glucose, HK-1 is the dominant isoform, and mostly associated with the mitochondrial outer membrane. This positioning of HK1 is critical, because it couples glucose metabolism to the energy production pathways of mitochondria. Here, we report a new molecular mechanism that regulates the mitochondrial positioning and activity of HK-1 via the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes a reversible posttranslational modification of proteins by adding a GlcNAc sugar moiety to serine and threonine residues. The catalytic activity of OGT is regulated by intracellular UDP-GlcNAc concentrations, which fluctuate proportionally in response to nutrient flux through the hexosamine pathway. In this study, We show that HK-1 is dynamically modified with O-GlcNAc at its regulatory domain. O-GlcNAcylation of HK-1 is elevated when cells were transfected with OGT or treated with TMG. We further characterized O-GlcNAc modification of HK1 recruited it on mitochondria and increases its enzymatic activity. Our findings may reveal key molecular pathways that couple neuronal metabolism to mitochondrial function via OGT, and how their dysregulation leads to neurological disorders.

The segment polarity protein Dishevelled-2 (Dvl2) is a component of the Dishevelled protein family that plays an important role in the Wnt developmental signal transduction pathways expressed within the central nervous system. It has an antagonistic function to other Dishevelled proteins in facilitating endocytosis of Frizzled3 Wnt receptors, though the specific behavioral effects have yet to be properly elucidated. A recently described genomic disorder characterized by a microdeletion of the 17p13.1 region, which contains an overlapping region with Dvl2, correlated to a recognizable phenotype in human patients consisting of intellectual disability and delay in social interaction. We sought to utilize various behavioral assays to test several behaviors associated with these phenotypes in adult mice lacking Dvl2 to demonstrate if the disruptions of neural development can be replicated in rodents and to potentially implicate Dvl2 knockout with these developmental delays. The behavioral paradigms used include open field, novel object recognition, and three-chamber social interaction tests to assess locomotor activity, anxiety-like behavior, learning and memory, sociability, and social novelty. An exploratory drive towards the center of a new environment as well as less preference for novel objects in contrast to familiar ones was observed in Dvl2-deficient mice, revealing a noticeable impairment in anxiety-like behavior and in context-independent memory. Impairments in sociability and social novelty were also observed, though without completely removing functionality. Therefore, evidence is provided that a lack of Dvl2 is associated with anxiety, memory, and social behavior deficits.

Chronic stress not only decreases the quality of life, but also increases the risk of developing a stress-related neuropsychiatric disorder, cardiovascular diseases, metabolic dysfunction, and/or Alzheimer’s disease (AD). Through animal studies, a key stress signaling factor in the hypothalamic-pituitary-adrenal (HPA) axis, corticotropin-releasing factor (CRF), was found to contribute to the development of AD through binding to its cognate type-1 receptor (CRFR1). The two defining cellular pathological phenotypes in AD, amyloid-β (Aβ) plaques and hyperphosphorylated tau (p-tau) tangles are both potentiated by CRFR1 activation. Current treatments for AD offer only a temporary reprieve from symptoms and do not provide ample relief. Unfortunately, the recent experimental therapies targeting amyloidogenic pathways have been unsuccessful in the treatment of human AD. Delineating the therapeutic potential of drugs targeting the CRF system could provide a novel treatment approach to combat AD pathogenesis.To assess how CRFR1-antagonism (vehicle/R121919) and/or stress alters AD pathology, early and mature (6- and 12- month) Wt/PSAPP cohorts were subjected to a series of behavioral assays (measuring learning, memory, and cognitive rigidity), immunohistochemical imaging and analysis of Aβ plaques in cortical/hippocampal sections, and biochemically probed for soluble/insoluble Aβ species in cortical fractions. Early R121919 treatment reduces AD pathology: improving behavioral task ability, reducing plaques throughout the hippocampus and cortex, and lowering cortical highly toxic soluble Aβ oligomers while increasing the amount of in-soluble monomeric Aβ species. Interestingly, stress improves memory, increases hippocampal plaques, lowers cortical plaques, and increases the amount of cortical insoluble monomeric species of Aβ, in early vehicle groups. While early treatment with R121919 ameliorates AD pathogenesis, mature AD pathology was not affected by R121919 (treatment did lower mature cortical soluble monomeric Aβ species). Parallel to these studies, stress did not alter mature behavioral deficits, but did lower hippocampal and cortical plaques, while increasing levels of mature cortical highly neurotoxic soluble oligomeric Aβ species (vehicle-cohorts) and mature cortical soluble monomeric Aβ (R121919-cohorts). Overall, CRFR1-antagonism may provide novel therapies for AD pathogenesis.