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Identifying the mechanisms that regulate neural precursor cell (NPC) proliferation and differentiation is important for understanding CNS development among different vertebrates. My work has focused specifically on understanding how mitogenic factors, like basic fibroblast growth factor (FGF2), regulate the NPC cell cycle. Mitogenic factors and serum are thought to drive cell cycle and therefore proliferation mainly by activating G1-type cyclin-dependent kinases (CDKs). The general hypothesis being addressed here is that FGF2 also promotes cell cycle progression of NPCs through the degradation of the cell cycle inhibitor p27Kip1. I show that, in the presence of FGF2 in vitro, embryonic rat cortical NPCs express high protein levels of the CDC28 protein kinase regulatory subunit 1b (Cks1b), a component of the SCFSkp2 E3 ubiquitin ligase complex that targets p27Kip1 for proteasomal degradation. I also show that NPCs maintained in FGF2 express undetectable levels of p27KIP1, while removal of FGF2 results in increased p27Kip1 protein expression and decreased protein expression of Cks1b. RNA expression data shows that Cks1b mRNA is reduced in non-dividing NPCs but is present in dividing NPCs, suggesting that Cks1b is being regulated at the transcriptional level. Analysis of the putative promoter of Cks1b reveals numerous conserved transcription factor consensus sites that could potentially play a role in regulation of Cks1b transcription, including consensus sites for E2F and the cell cycle-dependent element (CDE) cell cycle genes homology region (CHR) tandem repressor element. I use chromatin immunoprecipitation and luciferase assays to identify which E2Fs occupy and regulate the transcription of Cks1b under different conditions of mitogen stimulation. The data show that E2F4 occupies the promoter of Cks1b in non-dividing NPCs while E2F1 binds exclusively in proliferating NPCs. Mutation of either the E2F or CDE/CHR consensus sites independently de-represses the activity of a Cks1b promoter reporter in NPCs in G0/G1, while mutation of both sites delays induction of promoter activity. Finally, I use in ovo electroporation to determine if p27Kip1 has an additional role in neuronal differentiation during early spinal cord development. I show that ectopic expression of p27Kip1 is insufficient to induce neuronal differentiation in spinal cord progenitors.

Homocysteine, an intermediate metabolite biosynthesized from the methionine cycle, is a homologue of cysteine. Homocysteine differs from cysteine by an additional methylene group, which makes it more reactive. Elevated homocysteine level is a risk factor for cardiovascular disease and cerebrovascular disease, brain atrophy, neurodegenerative diseases and cognitive dysfunctions. Recent studies suggest a bi-directional relationship between homocysteine levels and immune-inflammatory activation. Our studies sought to determine if hyperhomocysteinemia affects cell infiltrates in the Central Nervous System (CNS). Inflammatory monocytes recruitment into the CNS and microglia proliferation have been shown in several inflammatory models, and Ly-6Chi CCR2+ monocytes have been shown to be the precursor for microglia. Based on these findings, we hypothesized that hyperhomocysteinemia (HHcy) would alter CNS infiltrate composition. We investigated whether HHcy affected the total mononuclear cells composition in the CNS. We also determined whether HHcy altered the inflammatory monocyte subsets composition in the CNS. In order to determine the effects of HHcy in the CNS mononuclear cells composition, we genotyped the mice, and isolated mononuclear cells from the CNS using percoll gradient method. Then we simultaneously stained the cells with three antibodies, PE-labeled anti-mouse CD11b, PE-Cy5-labeled anti-mouse CD45, and FITC-labeled anti-mouse Ly-6C and analyzed the samples by flow cytometry method. HHcy made no difference in the percentage of lymphocytes, infiltrating monocytes and microglia in the total CNS mononuclear cells, but within infiltrating monocytes, HHcy decreased Ly-6Clo and increased Ly-6Chi subsets. These findings demonstrate that HHcy has effects on the CNS mononuclear cell composition. In summary, HHcy decreased Ly-6Clo and increased Ly-6Chi subsets of infiltrating monocytes in the CNS. There is a potential role of HHcy in increasing inflammatory monocytes infiltration. / Pharmacology

Aged HIV-positive (HIV+) individuals represent a large proportion of the HIV population as life spans are extended significantly by successful antiretroviral therapy. Increased age with HIV infection brings a unique set of central nervous system (CNS) complications including more rapid onset and progression of age-related diseases, loss of protein quality control and accumulation of aberrant proteins, such as hyperphosphorylated Tau (hpTau). In this context, we have discovered a new signaling connection between age-related neurodegeneration and HIV involving the PINCH protein. Particularly interesting new cysteine histidine-rich protein (PINCH), an adaptor protein in neuronal cells is involved in cytoskeletal organization, cell migration and cell survival. While some of the pathophysiological aspects of the PINCH-signaling cascade in tauopathy are largely conserved among neurodegenerative diseases such as Alzheimer’s disease, HIV and others, the presence of the HIV protein Tat impacts specific key points in the PINCH pathway that exacerbate CNS cell dysfunction. In virus-infected cells, Tat regulates viral replication. Even though neurons are not permissive to viral infection, the Tat protein can enter all cell types. Our studies show that Tat interferes with key PINCH signaling partners in neurons. PINCH is robustly expressed by neurons and to a lesser degree by astrocytes in HIV infection of the CNS; whereas, in the healthy CNS, PINCH is nearly undetectable. Similarly, protein phosphatase 1α (PP1α), one of the binding partners of PINCH, is increased in the neurons of HIV encephalitis patients and co-localizes with PINCH in neurons. PINCH is a non-catalytic scaffolding protein that binds integrin-linked kinase (ILK), and PP1α and mediates Akt and GSK3β kinase activities, all of which when disrupted contribute to aberrant Tau phosphorylation. In Tat transgenic mice, increased expression levels of PINCH, PP1α and hpTau were observed. Also, in vivo manipulations of expression levels of PINCH altered the levels of hpTau, where overexpression of PINCH increased hpTau levels in Tat transgenic mouse brains and PINCH knockdown decreased hpTau formation. Furthermore, our studies show that Tat increases levels of hpTau and PINCH, interacts with PP1α and changes the subcellular distribution of PINCH and PP1α in vitro. Tat alters the levels of ILK, Akt and GSK3β, which are key kinases associated with hpTau formation. Furthermore, our preliminary data using expression plasmid for PP1α also shows that overexpression of PP1α decreases Tat-induced aberrant hpTau formation. Our studies address HIV replication-independent functions of Tat in neurons linked to PINCH signaling. These studies address a novel pathway through which Tau may be aberrantly phosphorylated. Thus, understanding new pathways of communication among Tat, PINCH, PP1α, ILK and Tau will open new directions for the study of HIV-associated tauopathy and will provide opportunities for therapeutic interventions in age- and disease-related pathologies. Several studies report associations between the PINCH protein and HIV-associated CNS disease. PINCH is detected in the cerebrospinal fluid (CSF) of HIV patients and changes in levels during disease may be indicative of changes in disease status over time. PINCH binds hpTau in the brain and CSF, but little is known about the relevance of these interactions to HIV CNS disease. In this study, PINCH and hpTau levels were assessed in three separate CSF samples collected longitudinally from 20 HIV+ participants before and after initiating antiretroviral therapy, or before and after a change in the current regimen. Correlational analyses were conducted for CSF levels of PINCH and hpTau and other variables including plasma CD4+ T-cell count, plasma and CSF viral burden, CSF Neopterin, white blood cell (WBC) count, and antiretroviral CNS penetration-effectiveness (CPE). Results suggest that in these HIV participants, changes in CSF levels of PINCH appear to correlate with changes in plasma CD4+ count and with changes in CSF hpTau levels, but not with plasma or CSF viral burden, Neopterin, or WBC, or with anti-retroviral regimen CPE. Furthermore, results from our case matched HIV brain-CSF study confirms that higher levels of PINCH and hpTau are detected in HIV encephalitis brains. Additionally, correlation between PINCH and hpTau levels in brain and other clinical parameters such as age at death, date of death suggesting the era of antiretroviral therapy, CPE score and cognition in HIV patients yielded interesting results that are currently being expanded upon by investigators in the Langford laboratory. Thus, these results suggest that PINCH may be involved in Tauopathy associated neurodegeneration in the HIV CNS disease. Therefore, understanding the contribution of PINCH to HIV-associated Tauopathy may provide a new therapeutic avenue for regulating synaptodendritic dysfunction associated with Tau. Moreover, characterizing the clinical significance of PINCH in the CSF may warrant including PINCH as a member of biomarker panel to assess severity or progression of HIV-associated neurocognitive alterations. / Biomedical Neuroscience

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Thesis advisor: Kenneth C. Williams / Factors that impact the development of neuroAIDS include monocyte expansion and activation, viral neuroinvasion and replication, and accumulation of activated and infected macrophages in the CNS. To better understand changes in monocyte/macrophage biology associated with the development of SIV encephalitis (SIVE) and neuroAIDS, we: 1) performed gene expression analyses using high density microarrays to characterize the response of monocyte subsets to SIV infection, 2) serially labeled CNS macrophages with fluorescent dextrans by intracranial injection and labeled myeloid progenitors in the bone marrow with BrdU to determine the timing of SIV neuroinvasion and macrophage recruitment/turnover in the CNS, and 3) performed in vitro studies to determine the role of PCNA expression in macrophages with SIV infection. We found the majority of macrophages in SIVE lesions were present in the CNS early in infection and productively infected macrophages were recruited to the CNS terminally with AIDS. We observed differences in the timing of recruitment, rate of turnover, PCNA expression, and productive infection between CD163+ and MAC387+ macrophages in the CNS. SIV infection was associated with induction of interferon stimulated genes in all monocytes, maturation of the intermediate monocyte subset, and increased rate of monocyte/macrophage recruitment to the CNS. Greater ratios of CD163+ to MAC387+ macrophages in the CNS were associated with SIVE. We also found that PCNA expression decreased macrophage apoptosis with SIV infection. Together, these studies suggest that the development of SIVE is a dynamic process and that continuous recruitment of activated monocyte/macrophage and reintroduction of virus from the periphery is required to drive CNS disease. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Huntington's Disease (HD) is an inherited neurodegenerative disorders caused by CAG repeat expansions in exon 1 of the huntingtin gene (htt). Patients with HD experience profound region specific neural degeneration for reasons that remain incompletely understood. Early studies in HD brain suggest that transcriptional misregulation occurs early in disease, before significant tissue loss and degeneration has occurred. However, a comprehensive understanding of the events that contribute to this remain poorly understood. In this study, we investigate a functional role for small RNA or miRNAs in the central nervous system (CNS) of patients with HD. Our work identifies subsets of miRNAs misregulated in HD. A functional role for these miRNAs was investigated by identifying their predicted targets. We identify a subset of differentially detected miRNAs which are inversely correlated with predicted downstream predicted 3'UTR target genes. We also identify targets of these differentially detected miRNAs including transcriptional regulators REST, CoREST and cFOS. The transcription factor REST silences neuronal gene expression in non-neuronal cells. Polyglutamine expansions in Huntingtin, which cause HD, abrogate REST-mediated Huntingtin binding, and as a result REST translocates to the nucleus, occupies RE1 consensus sites and represses the expression of both coding and non-coding RNAs. In this work, we identify miRNAs (miRNAs) with upstream REST consensus sites that are decreased in HD patient primary motor cortices (BA4). One of these miRNAs, miRNA-9/miRNA-9* is capable of regulating the expression of two components of the REST complex: miRNA-9 targets REST and miRNA-9* targets CoREST. These data provide evidence for a double negative feedback loop between the REST silencing complex and the miRNAs it regulates. In addition to these studies, we identify CNS enriched miRNAs which may differentially regulate human versus non-human primate gene expression. We computationally identified a single nucleotide change from G to A in the 3'UTR of human cFOS 3'UTR which is predicted to be regulated by the brain enriched miRNA-7. A regulatory role for the single nucleotide change in humans (G->A) was assessed by mutating the single nucleotide in the human cFOS 3'UTR (from A->G), as well as by introducing the corresponding human mutation (G->A) into the rhesus and chimpanzee cFOS 3'UTRs. The presence of the A nucleotide in the predicted MRE for miRNA-7 was sufficient to partially abrogate miRNA-7 activity in reporter plasmids. Finally, overexpression of artificial precursor miRNAs in human HEK293 and mouse N2A cell lines confirmed differential targeting of cFOS in human versus mouse cell lines. These data provide evidence for the potential contribution of a single nucleotide change in humans as regards changes in cFOS regulated gene expression. Since cFOS is a transcription factor, downstream affects from altered expression could be significant. Together, this work provides new support for the role of brain enriched miRNAs in the CNS and identifies functional support that their misregulation or altered expression can impact expression of protein coding transcripts in disease brain, and may be relevant to primate brain evolution.