Hu, Wenhui; Qin, Xuebin; Barbe, Mary F.; Kim, Seonhee; Ramirez, Servio H.; Bethea, John R. (Temple University. Libraries, 2018)
      While antiretroviral therapy (ART) regimens have significantly decreased the mortality rate in patients with HIV-1 infection and subsequent opportunistic infections, the co-morbidities continue to rise. Some of these co-morbidities include cardiomyopathies, metabolic dysfunction, accelerated aging, and most notably, neurocognitive deficits. HIV-1 associated neurocognitive disorders (HAND) denote a spectrum of neurocognitive deficits that are either asymptomatic in nature (asymptomatic neurocognitive impairments, ANI), mild to moderate in intensity (mild neurocognitive disorders, MND), or robust in nature (HIV-associated dementia, HAD). Thanks to the development of ART regimens, the incidence of HAD dramatically decreased. However, the emergence of ANI and MND continues to increase in the HIV-1 patient population. While the multifaceted nature behind the central nervous system (CNS) neuropathology of HIV-1 infection is not completely understood, dysregulated blood-brain barrier (BBB) integrity and the “Trojan-Horse” type mechanism of HIV-1 infection have been proposed as the cellular mechanisms underlying HAND. HIV-1 infects CD4+ T-lymphocytes and monocytes in the peripheral circulatory system. After these infected cells cross the BBB into the CNS, they release toxic viral proteins and viral particles onto microglia and astrocytes. These glial cells become activated, and release a plethora of inflammatory cytokines that further damage neurons via dysregulated neurotransmitter homeostasis, synaptodendritic damage, and calcium-mediated apoptotic pathways. At the same time, the virus may establish a state of latency in these microglia, perivascular macrophages, and astrocytes, which would allow for the long-term persistence of HIV-1 in the CNS. Recently, several studies have demonstrated that neural stem cells (NSCs) are capable of being productively and latently infected with HIV-1. This may be due to the fact that the hippocampal subgranular zone (SGZ), the subventricular zone (SVZ), and the circumventricular organs are highly vascularized, allowing potential direct contact of HIV-1 with NSCs. Additionally, the “Trojan” T-cells and macrophages could possibly release viral particles directly onto NSCs, and also transmit the virus through the formation of immunological synapses with NSCs. Therefore, the central hypothesis in this dissertation is that NSCs may serve as a novel CNS reservoir through which HIV-1 infection persists, and subsequently lead to neurocognitive impairments through dysregulating adult neurogenesis. Adult neurogenesis is a dynamic process that describes the generation of new neurons and glial cells from NSCs and neural progenitor cells (NPCs). This process mainly takes place in two areas of the brain: the SVZ around the lateral ventricles, and the SGZ within the dentate gyrus of the hippocampus. New neurons generated in these two neurogenic niches integrate into their respective circuitries to modulate olfactory stimuli and aid in memory acquisition/consolidation processes. Most of previous studies on the role of HIV-1 in neurogenesis focused on single viral proteins rather than the entire integrated proviral genome, and did not correlate these neurogenic deficits to neurobehavioral outcomes. Therefore, the overall objective of the studies proposed in this dissertation is to further validate the feasibility and efficiency of HIV-1 infection in NSCs at both the in vitro and in vivo levels, and explore the correlation of HIV-induced adult neurogenic deficits with neurocognitive dysfunction. The first set of studies utilized an EcoHIV reporter virus to infect mouse NSCs both in vitro and in vivo. This was done because the native HIV-1 virus is incapable of infecting non-human cells, while EcoHIV has been engineered to infect murine cells using the gp80 envelope protein. Our initial studies revealed that EcoHIV preferentially infected NSCs rather than NPCs. Additionally, a 3-day live imaging study revealed that some NSCs were infected at different time points when compared to other cells. This raised credence to the possibility that these infected NSCs/NPCs were generating new viruses which were seeding new infection. NSCs were also capable of propagating higher levels of EcoHIV transcription after treatment with latency reversing agents. Furthermore, EcoHIV infection persisted in a small number of astrocytes during the differentiation process. Subsequent studies assessed whether differentiated neurons and glial cells were vulnerable to EcoHIV infection. Our studies showed that only a small percentage of astrocytes and oligodendrocytes were infected by EcoHIV. Throughout these studies, differentiated neurons were shown to be resistant to HIV-1 infection. These in vitro findings were further validated in vivo. Histological analysis revealed that NSCs were more vulnerable to EcoHIV infection than NPCs. Notably, a small percentage of neuroblasts harbored EcoHIV, though microglia cells were infected at a significantly higher number. Altogether, these findings further solidify NSCs as a novel reservoir through which HIV-1 infection can persist in the CNS. Such findings raised the possibility that HIV-1 in NSCs may dysregulate neurogenesis. The next set of studies in this dissertation elucidated the possible role of HIV-1 infection or viral protein productions in NSCs in regulating adult neurogenesis. Specific parameters analyzed included NSC quiescence, early-stage and middle-stage lineage differentiation, and late-stage neuronal maturation. We performed a series of in vitro and in vivo studies using the HIV-1 Tg26 transgenic mouse model, which mimics HIV-1 patients suffering from low-level and chronic stress from HIV-1 viral proteins in the ART era. NSC culture studies from HIV-1 Tg26 transgenic mice and their wild-type (WT) littermates revealed that Tg26 mouse NSCs were unable to form as many primary neurospheres as WT NSCs. Additionally, when the NSCs were stratified by size, Tg26 NSCs formed lower numbers of smaller-sized primary neurospheres and more larger-sized primary neurospheres. These findings demonstrated that low-level chronic HIV-1 infection robustly reduces the NSC pool, and hampers the initial differentiation process from NSCs to NPCs. In vitro differentiation analyses revealed that compared to WT NSCs, Tg26 NSCs had a lower propensity to differentiate towards a neuronal phenotype, and instead generated more astrocytes. These findings were further confirmed through in vivo hippocampal neural lineage analysis in the SGZs of both WT and Tg26 mice. Subsequent retroviral labeling studies in the SGZ revealed that newborn dentate granule neurons in Tg26 mice had lower dendritic complexity and decreased apical dendritic spine density, when compared to dentate granule neurons from WT mice. These studies further demonstrated that adult neurogenesis is dysregulated upon persistent HIV-1 challenge or infection in NSCs. Further studies sought to examine if HIV-1 Tg26 transgenic mice had any cognitive deficits. We specifically focused on middle-aged WT and Tg26 mice, since the HIV-1 patient population is increasing in age thanks to ART regimens, and thus are more susceptible to cognitive decline than younger HIV-1 patients. We also took into account the factor of biological sex into the behavioral studies. Five types of behavioral assessments revealed sex-specific deficits in Tg26 mice. Specifically, male Tg26 mice exhibited social novelty deficits, and short and long-term spatial memory impairments. On the other hand, female Tg26 mice only manifested spatial learning deficits and short-term spatial memory impairments. Both male and female Tg26 mice had preserved physiological and reflexive functioning, in addition to intact contextual and cued fear conditioning responses. We speculated that these sex-specific differences were due to defects in adult neurogenesis during aging. Through hippocampal neurogenic analysis, we showed that middle-aged male Tg26 mice had an accelerated depletion of the NSC pool and decreased number of neuroblasts. Middle-aged female Tg26 mice have decreased pools of NSCs and NPCs, as well as decreased number of neuroblasts. In conclusion, we have effectively demonstrated that HIV-1 is capable of infecting NSCs at relatively low efficiencies. While differentiated neurons were incapable of sustaining HIV-1 infection, a small percentage of differentiated astrocytes, oligodendrocytes, neuroblasts, and microglia were susceptible to infection. These results led us to investigate the role of dysregulated adult neurogenesis in HIV-1 Tg26 mice, and if this process led to the progression of HAND. Our comprehensive in vitro and in vivo studies demonstrated that HIV-1 induced NSC quiescence, inhibited neuronal differentiation, and promoted astroglial lineage differentiation. Additionally, newborn dentate granule neurons in Tg26 mice had lower dendritic complexity and dendritic spine densities. Finally, both male and female Tg26 mice had varying degrees of cognitive deficits, which was attributed to differing hippocampal neurogenic dynamics during the aging process. Further studies should explore how to restore the neurogenic process during aging in these Tg26 mice. Transcriptomic analysis, such as single cell RNA-sequencing studies, could also possibly assist in further understanding HIV-1 proviral expression changes in differing cellular types along the NSC lineage progression.
    • Regulation and Functional Impact of Opioid Receptor Splicing in Response to Morphine

      Khalili, Kamel, 1951-; Gordon, Jennifer; Langford, Dianne; Hu, Wenhui; Tuszynski, George P.; Nonnemacher, Michael R. (Temple University. Libraries, 2015)
      Multiple classes of pharmaceuticals, including acetaminophen, aspirin, and other nonsteroidal anti-inflammatory drugs (NSAIDs), are used to relieve mild to moderate pain; however, one of the oldest classes of pharmaceuticals, opioids, remains the primary class of drugs used in the management of severe pain. For decades, the unique pharmacological profiles of opioid compounds have suggested the existence of multiple opioid receptor subtypes and, accordingly, four opioid receptors have been cloned to date; the mu (μ)-opioid receptor, the kappa (κ)-opioid receptor, the delta (δ)-opioid receptor, and the nociceptin/orphanin FQ receptor. Additionally, each receptor is encoded by its own distinct gene; the OPRM1, OPRK1, OPRD1, and OPRL1, respectively. Despite the identification and characterization of these four opioid receptor subtypes, pharmacological data, particularly from opioid receptor knockout mice, does not conform to the predications of a four opioid receptor model and instead suggests the existence of additional receptor subtypes. Additional opioid receptors have since been proposed but corresponding genes have either been unidentified or found to be genetically unrelated. Interestingly, this problem is not unique to opioid receptors, as there is a large discrepancy between the number of protein encoding genes and the repertoire of mRNA transcripts and encoded proteins they produce, with gene products far more numerous than estimates would predict. It is now understood that this discrepancy is due to the generation of multiple RNA transcripts from a single gene. Several mechanisms are utilized in order to generate mRNA transcript variants, or isoforms, from a single gene; however, the primary mechanism, known as alternative splicing, involves a complex macromolecular machine, referred to as the spliceosome, through which specific portions of the precursor mRNA (pre-mRNA) sequence are selectively removed and the remaining nucleotide sequences are ligated to form a unique mRNA transcript. Recently, multiple opioid receptor isoforms, particularly for the μ-opioid receptor, have been identified; however, both their regulation and their functional significance are poorly characterized. As such, multiple studies are needed to more precisely describe alternatively spliced μ-opioid receptor isoforms, particularly the regulation of spliceosome components that determine the splicing specificity of particular isoforms as well as the distinct signaling pathways utilized by particular isoforms both constitutively and following agonist binding. Using a model of dopaminergic neurons, this study sought to examine these questions and found that expression of a particular splice variant, MOR-1X, was up-regulated by morphine through a mechanism involving the essential splicing factor ASF/SF2. Structural comparison of this isoform to the prototypical variant MOR-1 found that the unique distal portion of C-terminal domain contains two additional PKA phosphorylation sites as well as a second agonist-induced phosphorylation motif highly conserved among opioid receptors. Functional comparison of MOR-1 and MOR-1X found distinct signaling differences, both constitutively and following morphine treatment, in MAPK signaling cascades, particularly ERK1/2. While the pharmacological significance of MOR-1X expression and signaling remains unclear, the clinical importance of this finding extends beyond a mechanism of opioid analgesic variability, as the physiological roles of opioids also include immunomodulation and have been implicated specifically in the exacerbation of HIV viral replication and pathology, particularly neurocognitive dysfunction. Accordingly, the HIV viral protein Tat was found to block morphine-mediated increases in MOR-1X expression by similarly blocking morphine-mediated increases in ASF/SF2 expression. Consequently, MOR-1X and HIV viral proteins were found to have a unique and synergistic role in the regulation of intrinsic apoptotic signaling cascades, specifically Bax expression, and in cell proliferation. Therefore, the regulation of alternative splicing events by both opioids and HIV viral proteins involves, in part, the inverse regulation of ASF/SF2 protein expression, through which the expression of the MOR-1X isoform is subsequently and significantly altered. This, in turn, may lead to functional consequences in opioid pharmacokinetics as well as in opioid-related pathology, such as the exacerbation of HIV associated neurocognitive dysfunction, as MOR-1X contains unique functional regions which may be responsible for the observed differences in MAPK and intrinsic apoptotic signaling and cellular proliferation. Collectively, these findings support previous studies that suggest alternative splicing of the MOR is altered by exogenous factors, such as morphine and HIV, identify unique signaling pathways for various opioid receptor isoforms, and are the first to suggest a potential mechanism through which pharmacological interventions could be utilized to alter opioid receptor isoform expression, thereby altering the pharmacological and physiological effects of opioids.

      Amini, Shohreh; Khalili, Kamel, 1951-; Waring, Richard B.; Yang, Weidong, Dr.; Nonnemacher, Michael R. (Temple University. Libraries, 2020)
      Neuronal homeostasis is an essential process to protect neurons from over/under-stimulation driven from systematic changes such as synapsis plasticity or tissue damage. Functional stability in neurons relays on the homeostatic plasticity that its disturbance causes irreversible injuries. Hence, a large body of studies elaborated to investigate the underlying mechanism for changes in synaptic connectivity and neuronal function. HIV-1 Tat (Transactivation of transcription), is a well-established neurotoxic protein, released by HIV-1 infected cells in the brain and disturbs neuronal homeostasis. The effects of Tat have been addressed in numerous studies investigating the molecular events associated with neuronal cell survival and death. The emergence of lncRNAs as critical players in disease etiology placed them in the spotlight to study pathogenesis of human diseases. Due to its capacity to modulate host transcriptome, HIV-1 Tat protein has been subjected to increasing genome-wide examinations. This study showed that exposing primary rat neurons to Tat resulted in the up-regulation of an uncharacterized long-non-coding RNA (lncRNA), LOC102549805 (lncRNA-U1). Evidence exists that increased expression of lncRNA-U1 in neurons disrupts bioenergetic pathways by dysregulating homeostasis of Ca2+, mitigating mitochondrial oxygen reduction, and decreasing ATP production leading to mitochondrial impairment in neurons. These changes were associated with imbalances in autophagy and apoptosis pathways via the Tat-mediated lncRNA-U1 induction. Additionally, this study showed the ability of Tat to modulate the expression of the neuropeptide B/W receptor 1 (NPBWR1) gene via the up-regulation of lncRNA-U1. Collectively, my results identified the Tat-mediated lncRNA-U1 elevation disturbs neuronal homeostasis. Our observations of lncRNA-U1 knock-down experiments indicated the novel lncRNA LOC102549805 (U1) as a viable therapeutic target to prevent HIV-1 Tat neurotoxicity.