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NIBP is a prototype member of a novel protein family. It forms a novel subcomplex of NIK-NIBP-IKKB and enhances cytokine-induced IKKB-mediated NFKB activation. It is also named TRAPPC9 as a key member of trafficking particle protein (TRAPP) complex II, which is essential in trans-Golgi networking (TGN). The signaling pathways and molecular mechanisms for NIBP actions remain largely unknown. The aim of this research is to identify potential proteins interacting with NIBP, resulting in the regulation of NFKB signaling pathways and other unknown signaling pathways. At the laboratory of Dr. Wenhui Hu in the Department of Neuroscience, Temple University, sixteen partner proteins were experimentally identified that potentially bind to NIBP. NIBP is a novel protein with no entry in the Protein Data Bank. From a computational and bioinformatics standpoint, we use prediction of secondary structure and protein disorder as well as homology-based structural modeling approaches to create a hypothesis on protein-protein interaction between NIBP and the partner proteins. Structurally, NIBP contains three distinct regions. The first region, consisting of 200 amino acids, forms a hybrid helix and beta sheet-based domain possibly similar to Sybindin domain. The second region comprised of approximately 310 residues, forms a tetratrico peptide repeat (TPR) zone. The third region is a 675 residue long all beta sheet and loops zone with as many as 35 strands and only 2 helices, shared by Gryzun-domain containing proteins. It is likely to form two or three beta sheet sandwiches. The TPR regions of many proteins tend to bind to the peptides from disordered regions of other proteins. Many of the 16 potential binding proteins have high levels of disorder. These data suggest that the TPR region in NIBP most likely binds with many of these 16 proteins through peptides and other domains. It is also possible that the Sybindin-like domain and the Gryzun-like domain containing beta sheet sandwiches bind to some of these proteins. / Bioengineering

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. / Biomedical Sciences

Yes / Animals have evolved diverse seasonal variations in physiology and reproduction to accommodate yearly changes in environmental and climatic conditions. These changes in physiology are initiated by changes in photoperiod (daylength) and are mediated through melatonin, which relays photoperiodic information to the pars tuberalis of the pituitary gland. Melatonin drives thyroid‐stimulating hormone transcription and synthesis in the pars tuberalis, which, in turn, regulates thyroid hormone and retinoic acid synthesis in the tanycytes lining the third ventricle of the hypothalamus. Seasonal variation in central thyroid hormone signalling is conserved among photoperiodic animals. Despite this, different species adopt divergent phenotypes to cope with the same seasonal changes. A common response amongst different species is increased hypothalamic cell proliferation/neurogenesis in short photoperiod. That cell proliferation/neurogenesis may be important for seasonal timing is based on (i) the neurogenic potential of tanycytes; (ii) the fact that they are the locus of striking seasonal morphological changes; and (iii) the similarities to mechanisms involved in de novo neurogenesis of energy balance neurones. We propose that a decrease in hypothalamic thyroid hormone and retinoic acid signalling initiates localised neurodegeneration and apoptosis, which leads to a reduction in appetite and body weight. Neurodegeneration induces compensatory cell proliferation from the neurogenic niche in tanycytes and new cells are born under short photoperiod. Because these cells have the potential to differentiate into a number of different neuronal phenotypes, this could provide a mechanistic basis to explain the seasonal regulation of energy balance, as well as reproduction. This cycle can be achieved without changes in thyroid hormone/retinoic acid and explains recent data obtained from seasonal animals held in natural conditions. However, thyroid/retinoic acid signalling is required to synchronise the cycles of apoptosis, proliferation and differentiation. Thus, hypothalamic neurogenesis provides a framework to explain diverse photoperiodic responses. / MRC. Grant Number: MR/P012205/1 - Scottish Government - BBSRC. Grant Number: BB/K001043/1 - Physiological Society