Parkinson’s disease (PD) is a movement disorder characterized by a massive loss of dopaminergic neurons of nigrostriatal origins. Several genes associated with familial cases of PD encode proteins that are direct components of the 26S Ubiquitin Proteasome System (UPS) or interact with enzymes involved in the UPS. Of these genes, parkin, and its product Parkin, an ubiquitin E3 ligase, is the most well characterized. Loss-of-function mutations in parkin result in the “early onset” PD known as Autosomal Recessive Juvenile Parkinsonism (AR-JP). Most research has focused on studying in what ways do nigrostriatal dopaminergic neurons differ from other neurons in the brain and how and why do these cells die in PD. In the following report I describe studies addressing the equally important alternative question: How do other neurons of the brain differ from nigrostriatal dopaminergic neurons that allow them to survive in AR-JP? I hypothesize that another E3 ligase provides redundant functions to Parkin in surviving neurons but that this redundant UPS enzyme is absent from dopaminergic neurons of the SNC. One protein that could possibly provide such a redundant function is the Parkin-like E3 ligase, Ariadne-1. Ariadne-1 and Parkin share significant sequence identity and similarity; they share the RING-IBR-RING signature domain; they share some UPS E2 enzymes; and they bind some of the substrates. In this dissertation I show Ariadne-1 to be a component of LB in post-mortem human tissue of various neurodegenerative disease. Then, in rats, I determine that Ariadne-1 is present as both mRNA and protein in cells of the SNC. Furthermore, Ariadne-1 is globally expressed throughout the mammalian brain and this expression is restricted to neurons and absent from glial cells and white matter tracts. I also find that only a subset of nigrostriatal dopaminergic neurons express Ariadne-1. Then, using the PD model of unilateral striatal lesioning of mice, I determine that Ariadne-1 expression actually correlates more closely with an increased susceptibility to oxidative stress-induced cell death. Lastly, using two different parkin-/- mice, I determine that, in the absence of Parkin, Ariadne-1 expression correlates with a measurable advantage to dopaminergic neurons of the SNC.

Purpose. Sensory feedback from the vestibular system and neck muscle stretch receptors is critical for the regulation of posture. The relationship of the head to the trunk is a major factor determining the availability and integration of sensory feedback and can be interfered with by varying head orientation. The goal of this research was to assess (1) how adopting different head-on-trunk orientations would impact postural stability, particularly in relation to the stability boundary, during static balance tasks and (2) how adopting different head-on-trunk and head-in-space orientations would impact postural stability, movement characteristics, and multi-segmental torso coordination during a dynamic postural transition task in healthy, young participants. Methods. Healthy, young participants were asked to maintain 30 seconds of upright stance and forward lean or to move from sitting to standing with extended, flexed, and neutral head orientations. Dual force plates were used to assess postural stability from center of pressure variability, range, velocity, or time-to-contact. Six motion capture cameras were used to assess kinematics. During the sit-to-stand task, head velocities, trunk flexion, and movement phase durations were calculated. Segment cross-correlation and joint range of motion were calculated for six torso segments. Results. Extended head-on-trunk orientations decreased postural stability during upright stance, forward lean, and the sit-to-stand movement compared to flexed or neutral orientations. During the sit-to-stand task, head-on-trunk extension, with or without head-in-space extension, led to reduced head velocities, trunk flexion, movement duration, and transition phase duration. Head extension led to increased inter-segmental torso motion, and decreased temporal coordination of torso segments. Conclusions. This study demonstrated that interfering with head-trunk posture by adopting head extended orientations impairs balance and leads to sit-to-stand strategy changes that may interfere with movement and coordination. Results show that head-on-trunk extension is more critical than head-in-space extension for determining postural and movement changes. The findings suggest that vestibular system interference may not be the main route through which head extension impacts postural control, but that extensor muscle stretch receptors may be a factor in the posture and movement changes associated with head-on-trunk extension. It is possible tonic neck muscle activity is a critical factor for regulating balance and movement.

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Autism is a neurodevelopmental disorder characterized by core impairments in social reciprocity and communication, together with a repetitive/restricted pattern of behavioral interests. Up to a third of individuals with autism also suffer from epilepsy, and human cross-sectional studies have demonstrated that the co-existence of epileptic seizures tends to result in more severe autistic phenotypes. It remains unknown as to whether this phenomenon is a result of anticonvulsant medications, the underlying autism promoting insult or the effect of recurrent seizures themselves. In an attempt to establish a connection between recurrent seizures and their impact on social behavior, we designed a simple and reliable mouse model of recurrent seizures by employing daily intraperitoneal injections of the chemoconvulsant PTZ (pentylenetetrazole, a GABA antagonist). Social motivation was assayed on the three-chamber social interaction test. We observed that 24 hours following 10 daily injections of a subconvulsant dose of PTZ (30mg/kg), both male and female mice display a reduction in social interaction. Mice exposed to seizures also vocalized significantly less than control mice. These social impairments were not related to a gross impairment in locomotion, olfaction, or exploration and were found to persist up to 30 days following their last seizure. These results suggest that recurrent seizures themselves may be facilitating some of the heightened social deficits frequently seen with autism and epilepsy. These results also lend naturally to i) studies examining molecular and structural neuroplastic changes occurring in brain circuits that are important for social behavior, and ii) data that may highlight the contributions of specific genes that may accelerate or protect against the development of seizure-induced impairments in social behavior.

Neuroinflammation has been linked to the pathogenesis of many diseases, including chronic traumatic encephalopathy (CTE). CTE is a progressive neurodegenerative disease caused by exposure to repeated head impacts (RHI) from a variety of sources, including contact sports and military injury. CTE is characterized neuropathologically by the deposition of hyperphosphorylated tau (p-tau) in neurons as neurofibrillary tangles (NFT) and neurites at the depths of the cortical sulci in an irregular pattern. In addition to p-tau accumulation, there is also an accumulation of pigment-containing macrophages around small blood vessels in the white matter and widespread microglial inflammation in CTE. Macrophage and microglial inflammation can be beneficial to tissue repair, but if persistent, can precipitate neurodegeneration. This study quantified the density of perivascular CD68+ macrophages in the dorsolateral prefrontal (DLF) white matter, a brain region known to be affected early in CTE in post-mortem brain tissue from 46 individuals, 7 controls (mean age: 46.14, SD: 11.39, range: 22-55), 20 individuals exposed to RHI without CTE (mean age: 22.75, SD: 3.65, range: 17- 29), and 19 individuals, all of them American football players, with pathologically verified CTE (mean age: 25.11, SD: 2.92, range: 18-29). Brain tissue was provided by the Injury and Traumatic Encephalopathy (UNITE) brain bank and the post-traumatic stress disorder (PTSD) brain bank. Comparisons were made between controls, individuals exposed to RHI without CTE, and individuals with CTE. Fixed tissue samples of the DLF cortex and white matter were cut at 10μm and stained with CD68 to mark perivascular macrophages. Slides were imaged with a brightfield microscope at 40x magnification and analyzed using the HALO image software analysis platform. In the total population, a one-way test of variance (ANOVA) revealed a statistically significant increase in perivascular macrophages, indicated by CD68 positive pixels, in Stage III CTE compared to controls (p<0.05), a significant increase in Stage II compared to Stage I CTE (p<0.05), and a statistically significant increase in Stage III compared to Stage I CTE (p<0.01). The analysis also revealed a trend toward more CD68 pixels in Stage II CTE compared to controls (p=0.0883) and a trend toward more pixels in Stage III CTE compared to RHI no CTE (p=0.0705). Among the American football players, analyses revealed that Stage II CTE had significantly more perivascular macrophages than Stage I CTE (p<0.01), Stage II CTE had significantly more than controls (p<0.01), Stage III CTE had significantly more than Stage I CTE (p<0.05), and Stage III CTE had significantly more than controls (p<0.05). In summary, this study demonstrates that there is an increase in perivascular CD68 positive macrophages in individuals exposed to RHI with and without CTE. Perivascular macrophages and other neuroinflammatory molecules may play a critical role in the pathogenesis of CTE.

Alternative splicing is a key mechanism by which eukaryotes generate phenotypic complexity without increasing genomic load. In vertebrate evolution, cassette exon alternative splicing is prominent with increasing phenotypic complexity and is specifically enriched in the brain. Apolipoprotein receptor 2 (Apoer2) is a neuronal alternatively spliced transmembrane receptor that binds critical extracellular ligands such as neuroprotective Reelin and Alzheimer’s disease (AD) related risk factor APOE4. Inclusion and exclusion of single exons in Apoer2 regulates isoform specific roles in neuronal processes, such as long-term potentiation (LTP) and neuronal survival. Alternative splicing of APOER2 exon 18, which encodes a functional domain critical for LTP, has been reported as dysregulated in AD. However, the full repertoire and function of APOER2 isoforms in physiological and AD conditions is not well understood. We hypothesize that combinatorial APOER2 alternative splicing events generate a diverse pool of isoforms in the human brain that can become dysregulated in AD and alter receptor function in neurons. Our overall goal is to define the APOER2 transcript pool and understand whether isoform proportions and functions are altered in AD, potentially contributing to synaptic dysfunction. In this work, we observed that Apoer2 has evolved over the course of vertebrate evolution, gaining new exons that alter function at the protein level and increasing the complexity of its alternative splicing events from zebrafish to humans. We generated the first APOER2 specific long-read RNA sequencing dataset in the human cerebral cortex, which identified 48 full-length APOER2 isoforms, some of which are unique compared to full-length murine Apoer2 isoforms and indicate that Apoer2 is spliced in a species specific manner. To determine whether splicing of APOER2 is dysregulated in AD, we generated full-length APOER2 isoform maps in Control and AD parietal cortex and hippocampus. We identified over 200 unique APOER2 isoforms in each brain region with 151 isoforms common between the two brain regions. We also identified region and disease specific APOER2 isoforms suggesting APOER2 splicing is spatially regulated and altered in AD. We found AD and Control-specific APOER2 isoforms exhibited alterations in receptor processing and cleavage patterns, indicating combinatorial splicing across APOER2 dictates protein function and is changed in AD. Sequential cleavage of Apoer2 in response to Reelin generates an intracellular domain (ICD) that translocates to the nucleus and affects transcription; however, whether APOE influences Apoer2 cleavage is unclear. We found Apoer2-ICD is generated in an APOE isoform specific manner and is generated regardless of exon 19 inclusion, which encodes part of the ICD. We generated four novel mouse lines to examine the effects of Apoer2 exon 19 inclusion and APOE isoforms (APOE3 and APOE4) on hippocampal gene expression. We found Apoer2 exon 19 inclusion modulates upregulation of genes such as Serpina3n known to be induced by APOE4 expression, which has strong implications for understanding molecular mechanisms underlying APOE4 as a risk factor in AD. Lastly, since Apoer2 exon 19 confers critical functions at the protein level, including adaptor protein binding and association with the NMDA receptor, as well as potentially modulating APOE4’s transcriptional effects, we were interested in how an RNA binding protein, Srsf1, may influence Apoer2 exon 19 splicing. We and others have found SRSF1 partially represses exon 19 inclusion in primary murine neurons. Because splicing is often modulated by neuronal activity, we examined whether Apoer2 exon 19 and Srsf1 are altered in response to activity stimulation. We found upregulation of exon 19 exclusion and no strong changes in SRSF1 expression or phosphorylation, suggesting modulation of SRSF1 is not a potent regulatory mechanism of activity induced changes in Apoer2 exon 19 splicing. Overall, we have examined the Apoer2 splicing landscape in the brain across multiple vertebrate species. We identified a rich diversity of alternatively spliced APOER2 isoforms in Control and AD brains providing novel APOER2 variants that are significantly changed in AD. These AD related APOER2 isoforms have differential functional impacts on APOER2 biology that may contribute to AD pathogenesis.

When fractionated using an HPLC ion exchange column, three distinct peaks (peak I, II, and III) of phosphatase activity were observed in the supernatant of homogenized bovine adrenal medulla cells, suggesting the presence of at least three different phosphatases. These phosphatases showed different activities toward phosphocasein in the presence of Mg$\sp{2+}$ and Mn$\sp{2+}$. Peak III, which represents about 50% of the active enzyme activity when phosphocasein is used as substrate, showed a molecular weight of 140 KDa as determined by HPLC gel filtration and has been identified as a type 2A protein phosphatase. Okadaic acid, a phosphatase inhibitor (specific for type 2A and type 1) and tumor promoter, was employed to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. After 3 days cultured in the presence of 50 ng/ml NGF, 20-25% of the PC12 cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC$\sb{50}$ of about 7 nM. This inhibition was rapidly reversed after wash-out of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells indicating that continual phosphatase activity is required to maintain neurites. A 27-mer oligonucleotide was synthesized as a hybridization probe to isolate clones encoding the sequence of protein phosphatases from a bovine adrenal medulla cDNA library. A cDNA clone encoding the full length of the catalytic subunit of protein phosphatase type 2A has been isolated. The deduced protein sequence (309 residues, 35.63 KDa) is 99.7% identical to that of phosphatase 2A$\alpha$ form from rabbit skeletal muscle, human liver and porcine kidney and differs by only one amino acid (Arg-55 vs. Cys-55). At the nucleotide level, the clone showed 97% identity with that of the catalytic subunit of protein phosphatase type 2A$\alpha$ from human liver. Sequence comparison of bovine adrenal medulla clone with phosphatase type 1 from rabbit skeletal muscle and type 2B from mouse brain identifies six highly conserved domains in the three enzymes that are expected to be crucial for the catalytic activity of protein phosphatase.

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