Thesis advisor: Kenneth C. Williams / Individuals infected with Human Immunodeficiency Virus (HIV) are susceptible to pathological abnormalities due to the infiltration of virus into different anatomical compartments. Monocytes are a heterogeneous population that undergoes changes in phenotype with HIV infection. It is hypothesized that changes in monocyte subsets observed through the course of infection will correlate with the development of SIV-Encephalitis (SIVE). 14 CD8+ T cell depleted rhesus macaques were infected with SIVmac251 and changes in 3 monocyte subsets, defined by their CD14 and CD16 surface expression as CD14+CD16-, CD14+CD16+, and CD14-CD16+, were tracked through the course of disease. The CD14+CD16- subset increased in the absolute number of cells and decreased in percentage of the total monocyte population. The CD14+CD16+ and CD14-CD16+ subsets increased in both absolute number and percentage. These changes have a biphasic dynamic that occurs during early infection and is pronounced in encephalitic animals. Several markers showed differential expression with infection and between subsets. Mac387, an early monocyte-macrophage marker, demonstrated a considerable decrease in expression. Concomitant with this change, CD68, CD163, CD44v6, CCR2, and CD64 increased expression in the total monocyte population, with the magnitude of these changes occurring in a subset-specific manner. In conclusion, monocyte subsets undergo changes with SIV infection that correspond to the development of encephalitis, highlighting the contribution of monocytes in neuroAIDS. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Kenneth C. Williams / Elucidating the mechanisms through which viral infection and persistence in CNS occurs is critical to understanding the development and progression of neurological disease. To date, no study has demonstrated that monocyte traffic in HIV and SIV infection directly results in neuronal injury. The central hypothesis in this thesis is that continuous trafficking of monocytes into tissues is essential for pathogenesis with viral infection. In the dissertation work presented here, two studies addressed this hypothesis. In Chapter 2, experiments examining the role of peripheral monocyte activation in the development of neuroAIDS using the tetracycline antibiotic minocycline will be described. We hypothesized that decreased monocyte activation with minocycline treatment would play a neuroprotective role in the context of rapid SIV infection with a high incidence of SIV encephalitis (SIVE). We observed a reversal of neuronal injury within days of minocycline treatment that correlated with loss of monocyte activation. From these findings we concluded that decreased activation of monocytes results in lower CNS traffic. However this effect may have occurred due to lower plasma virus, decreased SIV infection of monocytes, or the ability of minocycline to cross the BBB and modulate changes within the CNS directly. In Chapter 3 of this thesis, we hypothesized that continuous traffic of activated monocytes from the periphery into the CNS is required for neuronal injury with AIDS, and that by effectively stopping monocyte accumulation, CNS pathology can be blocked or reversed. We also hypothesized that monocyte trafficking is necessary for the seeding of brain and small intestine with cell-associated virus. In order to test these hypotheses, we utilized the anti-α4 blocking antibody natalizumab (Tysabri; Biogen Idec), which selectively binds to the α4 subunit of α4β1 (VLA-4) and α4β7 integrins, preventing the interaction between α4 and its various ligands. To address the first hypothesis, natalizumab was administered after four weeks of infection once significant neuronal damage had already occurred. We found that preventing cell traffic with natalizumab is sufficient to stabilize neuronal injury and loss, demonstrating conclusively that stopping monocyte traffic stabilizes CNS disease. To address the second hypothesis, rhesus macaques were treated with natalizumab on the day of SIV infection. Natalizumab treatment completely blocked SIV infection in the brain, and virus traffic to the small intestine was significantly suppressed. Overall, these studies demonstrate that continuous traffic of monocytes is required for neuronal injury and the formation of CNS lesions, and that early trafficking of leukocytes is critical for seeding of the CNS and contributes to seeding of the small intestine with virus. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Emerging evidence suggests that opioid drugs can exacerbate neuroAIDS. Microglia are the principal neuroimmune effectors thought to be responsible for neuron damage in HIV-infected individuals, and evidence suggests that drugs acting via opioid receptors in microglia aggravate the neuropathophysiological effects of HIV. The P2X family of ATP activated ligand-gated ion channels regulates key aspects of microglial function. In addition, opioid-dependent microglial activation has been reported to be mediated through P2X4 signaling, prompting us to investigate P2X receptors contribution to the neurotoxic effects of HIV and morphine. In vitro experiments showed treatment with TNP-ATP prevented the neurotoxic effects of morphine and/or HIV Tat, or ATP alone in a concentration dependent manner. This evidence suggests P2X receptors mediate the neurotoxic effects of these insults in striatal neurons. P2X1, P2X3, and P2X7 selective receptor antagonists did not prevent Tat- and/or morphine-induced neurotoxicity, implying cellular pathways activated may not involve these subtypes. Cells from P2X4KO mice show that activation of the P2X4 receptor on glia are necessary to cause Tat and/or morphine toxicity. However, data implied that baseline neuronal function may be altered due to lack of P2X4 receptor expression, and also gave evidence for altered Tat and morphine cellular signaling when the two are given in combination versus alone. Surgeries were performed on P2X4 KO and WT mice, which received intrastriatal Tat injections and morphine and/or naltrexone pellets. WT mice showed significant increases in inflammatory markers when treated with Tat and/or morphine. Increases in inflammatory markers were not seen in P2X4 KO mice, implying P2X4 receptors play a role in neuroinflammation resulting from Tat and/or morphine. Finally, human tissue samples from the National NeuroAIDS Tissue Consortium were analyzed. Changes in P2X5 and P2X7 mRNA were found in microarray data, but only changes in P2X7 mRNA levels were confirmed by RT-PCR. No changes in P2X4 mRNA levels were detected. Our experiments indicate the P2X receptor family contributes to Tat- and morphine- related neuronal injury, and reveal that members of the P2X receptor family, especially P2X4, may be novel therapeutic targets for restricting the synaptodendritic injury and neurodegeneration that accompany neuroAIDS and opiate abuse.

The chemokine receptor CCR5 (CCR5) plays an integral role within the inflammatory network of cells. Importantly, CCR5 is a mediator in several disease states and can be targeted using small molecule antagonists. Within this work, CCR5’s role in prostate cancer and HIV/AIDS has been exploited in order to develop potential therapeutics and probes. First, a series of novel compounds was designed by using pharmacophore-based drug design based upon known CCR5 antagonists and molecular modeling studies of the CCR5 receptor’s three-dimensional conformation. Once synthesized, these compounds were tested for their CCR5 antagonism and their anti-proliferative effects in several prostate cancer cell lines. The data from both the calcium mobilization studies and the anti-proliferation studies suggests that the compounds synthesized have activity as CCR5 antagonists and as anti-proliferative agents in certain prostate cancer cell lines. In addition, a bivalent ligand containing both a mu opioid receptor (MOR) and a CCR5 antagonist pharmacophore was designed and synthesized in order to study the pharmacological profile of the putative CCR5-MOR heterodimer and its relation with NeuroAIDS. The structural-activity relationship between the bivalent ligand and the heterodimer was studied with radio-ligand binding assays, functional assays, HIV-1 fusion assays, cell fusion assays, and in silico molecular dynamics. The subsequent bivalent ligand was proven to be a potent inhibitor in both an artificial cell fusion assay mimicking HIV invasion and a native HIV-1 invasion assay using live virus. In all, two novel sets of compounds were synthesized that targeted either CCR5 or the CCR5-MOR heterodimer. For the CCR5 antagonists, as leads for prostate cancer therapeutics, further work needs to be done to ascertain and develop their structure-activity-relationship. This library of novel compounds was shown as promising leads as CCR5 and anti-prostate cancer agents. The bivalent ligand targeting the CCR5-MOR heterodimer proved to be a potent and tissue-specific inhibitor for neuroAIDS where the known treatment, maraviroc, is less efficacious and fails to inhibit virus entry in the presence of morphine. Both projects illustrate the roles that CCR5 plays in these two unique diseases.

INTRODUCTION: HIV- associated neurocognitive disorder (HAND) and pathology are common manifestations of HIV-infection, and often persist in spite of controlled peripheral viremia. Severity of HAND can range from loss of concentration and psychological changes to frank dementia. Inflammatory host-immune responses and chemotaxis of immune cells into the CNS are thought to be integral to development of NeuroAIDS and HAND. OBJECTIVES: This studies' primary aim was to determine if significant differences existed between Substance P and NK1R expression in brain tissue samples of HIV-infected individuals with neurocognitive disorder or pathology. The secondary aim was to determine whether expression of HIV viral entry receptors CCR5 and CXCR4 correlate with expression of Substance P or NK1R. The tertiary aim of this study was to determine if age at death, CNS penetration-effectiveness of antiretroviral therapy, diagnosis before HAART, average plasma CD4, or abnormal alcohol or drug use increased prevalence of neurocognitive disease. STUDY DESIGN: Cross-sectional study of HIV-infected individuals (n=60) from the larger National NeuroAIDS Tissue Consortium Cohort. Pre-death demographic data, neurocognitive assessment, alcohol and drug use, ART regimens, date of diagnosis and death, and plasma CD4 levels, as well as pathology findings at autopsy and brain tissue samples were provided by the NNTC; expression levels of Substance P, NK1R, CCR5, and CXCR4 from brain samples were provided by Dr. Steven Douglas of The Children's Hospital of Philadelphia. RESULTS: In this sample of HIV-infected individuals, Substance P expression was significantly less in the cingulate cortex of individuals with (p=0.003). Within-subject expression patterns of CCR5 and truncated-NK1R in the cingulate cortex and cerebellum were both significantly altered by neuropathology and cannabis use; CCR5 expression was also significantly affected by opiate use. CCR5 and CXCR4 expression correlated strongly with truncated-NK1R expression. No between-subject factors significantly altered prevalence of neurocognitive impairment in this HIV-infected population. CONCLUSIONS: The study found significant changes in Substance P, NK1R, and CCR5 expression associated with neuropathology. Furthermore, in heterogeneous populations, expression patterns may be more important than overall level of expression in identifying risk factors for NeuroAIDS and other chronic diseases. / Epidemiology

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Neurotoxlns represent unique chemical tools, providing a means to 1) gain insight into cellular mechanisms of apopotosis and necrosis, 2) achieve a morphological template for studies otherwise unattainable, 3) specifically produce a singular phenotype of denervation, and 4) provide the starting point to delve into processes and mechanisms of nerve regeneration and sprouting. There are many other notable uses of neurotoxins in neuroscience research, and ever more being discovered each year. The objective of this review paper is to highlight the broad areas of neuroscience in which neurotoxins and neurotoxicity mechanism come into play. This shifts the focus away from neurotoxins per se, and onto the major problems under study today. Neurotoxins broadly defined are used to explore neurodegenerative disorders, psychiatric disorders and substance use disorders. Neurotoxic mechanisms relating to protein aggregates are indigenous to Alzheimer disease, Parkinson's disease. NeuroAIDS is a disorder in which microglia and macrophages have enormous import. The gap between the immune system and nervous system has been bridged, as neuroinflammation is now considered to be part of the neurodegenerative process. Related mechanisms now arise in the process of neurogenesis. Accordingly, the entire spectrum of neuroscience is within the purview of neurotoxins and neurotoxicity mechanisms. Highlights on discoveries in the areas noted, and on selective neurotoxins, are included, mainly from the past 2 to 3 years.