Browsing by Author "Fan, Yan"
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Item Astrocyte-mediated HIV-1 Tat neurotoxicity and its molecular mechanisms: astrocyte activation and impaired neurogenesis(2016-08-01) Fan, Yan; He, Johnny J.; Ghorpade, Anuja; Barber, Robert C.Human immunodeficiency virus type 1 (HIV-1) invasion of the central nervous system (CNS) often causes motor and cognitive dysfunction, which is termed HIV-associated neurocognitive disorders (HAND). Although the introduction of combination antiretroviral therapy (cART) has effectively suppressed viral replication, improved immune function and increased life expectancy among HIV-infected individuals, it has failed to provide complete protection from HAND or to reverse the disease. HIV-1 Tat protein is a major pathogenic factor in HAND. Studies including ours have demonstrated that Tat is taken up by HIV-1 uninfected brain cells and alters the function of these cells, especially astrocytes, neurons and neural progenitor cells. However, the underlying mechanisms are still unclear. There are two parts to my dissertation research. In the first part, we determined the roles of signal transducer and activator of transcription 3 (STAT3) in Tat-induced glial fibrillary acidic protein (GFAP) transactivation. Astrocytes, the most abundant cells in the brain, not only provide the scaffold support in the brain, but also are essential for maintaining the homeostasis of the brain. GFAP is the specific molecular marker for astrocytes, but it also modulates astrocyte function both physiologically and pathologically. In this study, we first showed that STAT3 expression and phosphorylation led to significant increases in GFAP transcription and protein expression. Then we determined that Tat expression was associated with increased STAT3 expression and phosphorylation in Tat-expressing astrocytes and HIV-infected astrocytes. In addition, we showed that GFAP, Egr-1 and p300 transcription all showed positive response to STAT3 and its phosphorylation. Moreover, knockdown of STAT3 resulted in significant decreases in Tat-induced GFAP and Egr-1 transcription and protein expression. Taken together, these findings show that STAT3 is involved in and acts upstream of Egr-1 and p300 in the Tat-induced GFAP transactivation cascade and suggest important roles of STAT3 in controlling astrocyte proliferation and activation in the HIV-infected CNS. In the second part of the dissertation research, we took advantage of the doxycyclineinducible and astrocyte-specific HIV-1 Tat transgenic mice (iTat) and determined the relationship between Tat expression and neurogenesis. Tat expression in astrocytes was associated with detection of fewer neuron progenitor cells (NPC), fewer immature neurons and fewer mature neurons in the dentate gyrus of the hippocampus of the mouse brain. In vitro NPC-derived neurosphere assays showed that Tat-containing conditioned media from astrocytes and recombinant Tat protein inhibited NPC proliferation and migration and altered NPC differentiation, while immunodepletion of Tat from Tat-containing conditioned media or heat inactivation of recombinant Tat abrogated those effects. Notch signaling downstream gene Hes1 promoter-driven luciferase reporter gene assay and Western blotting showed that recombinant Tat and Tat-containing conditioned media activated Hes1 transcription and protein expression, which were abrogated by Tat heat inactivation, immunodepletion, and cysteine mutation at position 30. Moreover, Notch signaling inhibitor DAPT significantly rescued Tat-impaired NPC differentiation in vitro and neurogenesis in vivo. Taken together, these results show that Tat adversely affects NPC and neurogenesis through Notch signaling and point to the potential of developing Notch signaling inhibitors as HAND therapeutics.Item Characterization of a doxycycline inducible and astrocyte-specific HIV-1 Nef transgenic mouse model (iNef)(2018-03-14) He, Johnny; Luo, Xiaoyu; Fan, Yan; Tesselaar, Kiki; Wilson, KellyPurpose: Over 37 million people worldwide are currently infected with human immunodeficiency virus type 1 (HIV-1). Introduction of combination antiretroviral therapy has improved the quality and length of life, leading to increased incidents of minor cognitive and motor disorder. HIV-1 infects astrocytes, the most abundant cells of the central nervous system (CNS). Once infected, astrocytes become reactive, characterized by increased expression of astrocyte-specific protein glial fibrillary acidic protein (GFAP). However, those cells do not support productive HIV replication, primarily expressing non-structural viral proteins, such as Tat and Nef. Although the role of Tat in HIV/neuroAIDS has been extensively studied, little is known about the roles of Nef in HIV/NeuroAIDS. Nef is known to play important roles in immune evasion, T-cell depletion, and disease progression. The current study is to characterize roles of Nef in HIV/neuroAIDS. Method: A doxycycline inducible astrocyte-specific HIV-1 Nef transgenic mouse model (iNef) was created. In this model, Nef expression is under the control of both GFAP promoter and doxycycline responsive elements, which allows characterization of the effects of Nef expression on the CNS, independent of HIV-1 infection. Neuropathological outcomes (astrocytosis, neuroinflammation, and neuronal integrity) and neurobehavioral effects (motor and memory) were determined using immunofluorescence staining and neurobehavioral batteries, respectively. Results: Nef expression was confirmed in the brain of the iNef mice with doxycycline induction. Meanwhile, Increased GFAP expression and cytokine expression, loss of neuronal dendrites and decreased speed and latency to fall were found in these Nef-expressing iNef mice. Conclusion: These data show that Nef expression led to astrocytosis, neuroinflammation, compromised neuronal integrity, and impaired coordination and motor function in Nef-expressing iNef mice and suggest that Nef could be a major contributing factor to HIV/neuroAIDS. Further investigation is under way to determine the underlying molecular mechanisms.Item Chemical Reprogramming of Mouse and Human Müller glia into Retinal Ganglion-Like Cells (RGCs)(2019-03-05) Zhang, Wei; Chavala, Sai; Fan, YanChemical Reprogramming of Mouse and Human Müller glia into Retinal Ganglion-Like Cells (RGCs) Yan Fan, Wei Zhang, Sai Chavala Department of Pharmacology and Neuroscience, Graduate School of Biomedical Sciences, UNT Health Science Center, Fort Worth, TX 76107 Purpose: Glaucoma is a leading cause of irreversible blindness with increasing prevalence as the population ages. Glaucoma is characterized by loss of retinal ganglion cells (RGCs), and current therapies, whether surgical, pharmacological, or neuroprotective, do not reverse the degeneration. Stem cell approaches to replace lost RGCs are a viable option. However, the use of stem cells for RGC replacement currently faces several barriers: 1) absence of a safe, non-immunogenic, and ethical stem cell source of RGCs, 2) inefficient differentiation protocols that can take more than 40 days, and 3) RGC donor integration into the degenerate host retina are major issues that need to be overcome to be considered for clinical use. A radically new approach to restoring vision for glaucoma patients that can overcome these limitations would be to reengineer a resident cell in the retina, such as Müller glia, that could serve as a reservoir for new RGCs, and avoid the need for cell transplantation. In this study, we take the first step in realizing our long-term goal of using a chemically reprogramming strategy to replace lost RGCs and restore vision. Materials and Methods: Primary Müller glia were isolated from mouse or human retina. Small molecules were purchased from Sigma or Cayman. For the in vitro studies, Müller glia were fixed after small molecule conversion with 4% PFA. Immunofluorescence staining was performed to detect RGC specific markers: Brn3a, Brn3b, ISL-1, RBPMS, and Tuj1. Total RNA was isolated and subjected for real-time PCR for detection of neuronal marker NeuN and RGC markers: Brn3a, Brn3b, ISL-1, NefH, and Nefl. For the in vivo studies, intravitreal injection was performed to deliver small molecules and other growth factors. BrdU was injected intravitreally or intraperitoneally to label proliferating and regenerating cells. 24 hours after the final injection, eyes were enucleated, fixed, and embedded for frozen sections. Immunofluorescence staining was performed to detect proliferation with BrdU and Ki67. After 7, 21, and 35 days of final injection, BrdU and RGC markers were co-stained to detect newly generated RGC-like cells. Results: Both mouse and human Müller glia can be converted to RGC-like cells within 24 hours in vitro. In vivo injection of small molecule and growth factor cocktail into the vitreous stimulated cell proliferation, which was located between the outer nuclear layer and the inner nuclear layer where the cells stained positive with GFAP. After 7 days, BrdU positive cells migrated to the inner edge of INL. After 21 days, BrdU positive cells reached the ganglion cell layer (GCL) but no co-staining with a RGC marker was observed at this time point. Strikingly after 35 days, we detected co-stained BrdU and ISL-1 or RBPMS in the GCL. There was no retinal toxicity observed from the small molecule injection. Conclusion: Our small molecule cocktail is highly efficient in converting mouse and human Müller glia to RGC-like cells in vitro, enabling us to generate RGC-like cells in approximately one day with more than a 90% conversion efficiency. The small molecule cocktail can be injected intravitreally to stimulate cell proliferation and RGC regeneration in vivo. Additional retinal toxicity testing will be needed to evaluate the safety profile of the intravitreal small molecule cocktail.Item HIV-1 Tat adversely affects neurogenesis through Notch signaling(2016-03-23) Liu, Ying; He, Johnny PhD; Fan, YanAlterations in adult neurogenesis appear to be a common hallmark in several neurodegenerative diseases including human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorder (HAND). HIV-1 Tat is a major pathogenic factor in HIV-associated neuropathogenesis. In vitro study have revealed that Tat severely reduces the proliferation of NPCs and impacts neurogenesis. But adult neurogenesis is limited to specific brain regions in the mammalian brain, so further investigations on the in vivo effect of Tat on NPCs proliferation and differentiation, and the molecular mechanisms are still urgent and necessary. In this study, we took advantage of the Doxycycline (Dox)-inducible brain-specific HIV-1 Tat transgenic mouse model (iTat) to investigate how Tat affected neural progenitor cell (NPC) proliferation and differentiation in the dentate gyrus of hippocampus of the mouse brain. We found that Tat decreased NPC proliferation and impacted NPC differentiation by leading dynamic imbalance of neurogenesis and astrogliogenesis. When investigated the underlying mechanism, we demonstrated that HIV-1 Tat was sufficient for Hes1 activation, which is the critical molecular downstream pathway of Notch signaling. In this process, the cysteine-rich domain of Tat plays an essential role in Hes1 activation and modulating neurogenesis and astrogliogenesis. Lastly, we determined that Notch signaling was directly involved in HIV-1 Tat induced dynamic imbalance of neurogenesis and astrogliogenesis.Item HIV-1 TAT INDUCED LYSOSOMAL EXOCYTOSIS IN ASTROCYTES AND ITS CONTRIBUTIONS TO TAT NEUROTOXICITY(2014-03) Fan, Yan; He, JohnnyPurpose (a): HIV-1 Tat protein is considered to be the critical reason in the processing of HIV-associated neuropathogenesis. Our previous studies demonstrates that HIV-1 Tat expression leads to ER stress in astrocytes through GFAP aggregation and suggest that disruption of ER homeostasis, i.e., ER stress may be involved in HIV-associated neuropathogenesis. But what the neurotoxic factor is in this indirect astrocyte-mediated Tat neurotoxicity system is still unknown. In this study, we take advantage of our Tat-inducible transgenic mice, and proteomic analysis was performed to explore the neurotoxic factor in the astrocyte-mediated Tat neurotoxicity system. Methods (b): Brain-targeted inducible Tat transgenic and GFAP knockout mice were used in the study. Primary astrocytes and neurons cultures were prepared from mouse embryos. U373 cells and primary astrocytes and were either transfected with pTat.Myc or treated with doxycycline to induce Tat expression. Cells were prepared and analyzed for β–hexosaminidase activity by NAG assay after ionomycin mediated Ca2+ influx. Culture supernatants were collected for immunodepletion and analyzed for their neurotoxicity toward primary mouse or human neurons using MTT assay. TIRF microscopy is utilized to visualize lysosome exocytosis events. Results (c): Base on the protein sequencing and pathway analysis, we proposed that HIV-1 Tat induces lysosomal exocytosis in astrocytes. Then, NAG assay and TIRF microscopy provide consistent evidences that verified our hypothesis. Moreover, we attested that inhibition of Tat induced lysosomal exocytosis in astrocytes by vaculin-1 can abolish Tat induced neuron death. More interestingly, we observed that Tat induced lysosomal exocytosis and neuron death only appears in astrocytes but not in other cell types, such as 293T and Huh 7.5.1 cells. Further more, we proved that GFAP plays a critical role in Tat induced lysosomal exocytosis and neuron death. Conclusions (d): Taken together, these results demonstrate that HIV-1 Tat induces lysosomal exocytosis and hydrolytic enzymes contained within lysosome are suggested to contribute to neuronal death, which is a novel insight into astrocytes-mediated Tat neurotoxicity.Item HIV-1 TAT INDUCES ER STRESS IN ASTROCYTES AND CAUSES NEUROTOXICITY THROUGH GFAP ACTIVATION AND AGGREGATION(2013-04-12) Fan, YanPurpose: HIV-1 Tat is a major pathogenic factor for HIV-associated neurodegenerative diseases. One of the consistent hallmarks of HIV-1 infection of the central nervous system (CNS) is astrocytosis, which is characterized by increased cytoplasmic accumulation of intermediate filament glial fibrillary acidic protein (GFAP). Our previous studies show that Tat induces GFAP expression in astrocytes and GFAP is a critical regulator of Tat neurotoxicity. However, the molecular mechanisms responsible for GFAP activation-mediated Tat neurotoxicity is not known. Thus, we attempted in this study to determine the underlying unknown molecular mechanisms. Methods: Brain-targeted inducible Tat transgenic and GFAP knockout mice were used in the study. Primary astrocytes were either transfected with pTat.cMyc or treated with doxycycline to induce Tat expression. Cells were harvested to detect GFAP, eIF2a, ATF6, Oasis and Bip expression by Western blotting to , or to detect Tat, GFAP and XBP-1 by RT-PCR. Culture supernatants were collected and analyzed for neurotoxicity toward primary mouse or human neurons using MTT assay. GFAP, MAP-2 and Bip expression in astrocytes, Tat-expressing mouse brain and the brains of HIV-infected individuals were also determined by IF and IHC staining. Flow cytometry was performed to determine proteasomal activity by monitor expression intensity of proteasomal activity reporter pZsProSensor-1. Results: We showed that HIV-1 Tat-induced GFAP up-regulation and aggregation in astrocytes activated endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways, such as PERK, IRE1, ATF6 and OASIS, We further showed that supernatants from Tat-expressing astrocytes were neurotoxic. Importantly, we showed that the neurotoxicity in these culture supernatants were significantly diminished when GFAP was null or when the cultures were treated with 4-sodium phenyl butyrate (4-PBA), a chemical chaperon capable of blocking the ER stress, suggesting that GFAP activation and aggregation-induced ER stress in the presence of Tat expression is at least in part responsible for Tat neurotoxicity. Lastly, we showed that GFAP activation and aggregation resulted in lower proteasome activity and induction of autophagy in astrocytes. Conclusions: Taking together, this study demonstrates that HIV-1 Tat expression leads to ER stress in astrocytes through GFAP aggregation and suggest that disruption of ER homeostasis, i.e., ER stress may be involved in HIV-associated neuropathogenesis.Item Ketogenic Diet Increases Mitophagy in a Mouse Model of Glaucoma(2023) Morgan, Autumn; Fan, Yan; Inman, DenisePurpose: We have previously shown that limiting dietary intake to high fat, low protein, and negligible carbohydrate results in mitochondrial biogenesis, and in the case of glaucoma, a reduction in neurodegeneration of retinal ganglion cells (RGCs). In this experimental follow-up study, we wanted to examine the effect of the ketogenic diet on mitophagy, or mitochondrial recycling, within the glaucomatous retina. Methods: MitoQC mice were placed on a ketogenic diet or standard rodent chow for 5 weeks and ocular hypertension (OHT) was induced via microbead injection. The MitoQC reporter mice have a pH-sensitive mCherry-GFP tag on the outer mitochondrial membrane that results in retention of red fluorescence when mitochondria bound for recycling are engulfed by lysosomes. The FIJI (ImageJ) macro MitoQC counter was used to quantify red puncta (mitolysosomes) in sectioned retina as a measure of mitophagy within the RGCs and Müller glia. Results: Mitophagy in RGCs, as measured by red puncta, was significantly decreased by ocular hypertension in the control retina (Control + OHT) in comparison to naïve control retina (Ctrl; p<0.0001). The ketogenic diet (KD) resulted in a significant increase in mitolysosomes in RGCs when compared to Ctrl (p<0.0001), Control + OHT (p<0.0001) and KD + OHT (p=0.0089). The ketogenic mice with OHT showed a significantly higher RGC-associated mitolysosome number than Control + OHT mice (p<0.0001). In contrast, mitolysosomes quantified in the Müller glia of Control + OHT mice were significantly higher than the naïve control mice (p=0.0127). Mice in the KD (p=0.0001) and KD + OHT(p=0.0005) groups had significantly greater mitolysosomes than the control Müller glia, however there was no difference in mitophagy between the Control + OHT, KD, and KD + OHT Müller glia groups. Conclusion: Our data demonstrates that mitophagy is managed differently within RGCs and Müller glia of mouse retinas. The KD promoted mitophagy within the RGCs to a degree that overcame the decline of mitophagy after OHT in the control group. Within the Müller glia, the KD was redundant because OHT alone increased mitophagy to similar levels as the KD. These findings suggest a divergence of mitochondrial homeostasis in RGCs and Müller glia that may reflect the different metabolic needs of these cell types.Item Long-term HIV-1 Tat Expression in the Brain Led to Neurobehavioral, Pathological, and Epigenetic Changes Reminiscent of Accelerated Aging(International Society on Aging and Disease, 2020-02-01) Zhao, Xiaojie; Fan, Yan; Vann, Philip H.; Wong, Jessica M.; Sumien, Nathalie; He, Johnny J.HIV infects the central nervous system and causes HIV/neuroAIDS, which is predominantly manifested in the form of mild cognitive and motor disorder in the era of combination antiretroviral therapy. HIV Tat protein is known to be a major pathogenic factor for HIV/neuroAIDS through a myriad of direct and indirect mechanisms. However, most, if not all of studies involve short-time exposure of recombinant Tat protein in vitro or short-term Tat expression in vivo. In this study, we took advantage of the doxycycline-inducible brain-specific HIV-1 Tat transgenic mouse model, fed the animals for 12 months, and assessed behavioral, pathological, and epigenetic changes in these mice. Long-term Tat expression led to poorer short-and long-term memory, lower locomotor activity and impaired coordination and balance ability, increased astrocyte activation and compromised neuronal integrity, and decreased global genomic DNA methylation. There were sex- and brain region-dependent differences in behaviors, pathologies, and epigenetic changes resulting from long-term Tat expression. All these changes are reminiscent of accelerated aging, raising the possibility that HIV Tat contributes, at least in part, to HIV infection-associated accelerated aging in HIV-infected individuals. These findings also suggest another utility of this model for HIV infection-associated accelerated aging studies.Item Restoration of vision by chemically reprogrammed photoreceptors(2019-03-05) Kaya, Koray; Fan, Yan; Sumien, Nathalie; Shetty, Ritu; wei, Zhang; Davis, Delaney; Thomas Mock, Thomas; Batabyal, Subrata; Ni, Aiguo; Mohanty, Samarendra; Han, Zongchao; Farjo, Rafal; Forster, Michael; Swaroop, Anand; Chavala, Sai; Mahato, BirajPurpose: Many retinopathies such as Retinitis Pigmentosa, Stargardt disease, Cone-rod dystrophy, Achromatopsia, Chroideremia and Labor congenital Amaurosis (LCA) comprise a wide range of genetically and phenotypically heterogeneous conditions that share common progressive loss of photoreceptor function accompanied by irreversible vision loss. Majority of the patients affected by these diseases present with uncorrectable decreased visual acuity during their childhood years, which most often progress to legal blindness. Strategy to restore vision with photoreceptor like replacement cell has the advantage of being applied to these patients, regardless of their genetic dysfunction or stage of disease. Currently no FDA approved treatments are available to treat these disorders. We have discovered a chemical engineering method that can convert fibroblasts to chemically induced photoreceptors (CiPCs) with their ability to restore vision in retinal degeneration mouse model. Methods: A combination of small molecule (5C) was used to convert fibroblasts to CiPCs. Gene expression of CiPCs was analyzed by RNA sequencing, RT-PCR and immunofluorescence. Light responsiveness of CiPCs was tested by single cell patch clamp recording upon stimulation with light. In vivo CiPC function was examined by pupil analysis, light aversion test, visual acuity and contrast sensitivity measurement after injecting them into retinal degeneration mouse model. Results: We have identified a set of five small molecules (5C) that induces mouse embryonic fibroblasts (MEFs) and human adult dermal fibroblasts (HADF) into CiPCs both rods and cones, without the use of pluripotent cells or viral transcription factors in less than two weeks time. Detailed analyses have been performed in mouse cells, but in brief, these cells express transcript and proteins consistent with youthful photoreceptors (postnatal day 5). In vitro functional analysis indicated that CiPCs are light responsive. Moreover, when mouse ciPCs are injected into the subretinal space of retinal degeneration mutant mice (rd1), in some mice (approximately 50%) we observed cell survival for several months (more than 120 days), restored light-dark preference/discrimination, improved scotopic-Optomotry testing, fleeting but partial ERG recovery, and restoration of pupillary reflexes. Conclusions: Based upon these observations we demonstrate restoration of visual functions by CiPCs that carry extraordinary translational potential for millions of visually impaired patients worldwide.Item The ketogenic diet and hypoxia promote mitophagy in the context of glaucoma(Frontiers Media S.A., 2024-05-22) Morgan, Autumn B.; Fan, Yan; Inman, Denise M.Mitochondrial homeostasis includes balancing organelle biogenesis with recycling (mitophagy). The ketogenic diet protects retinal ganglion cells (RGCs) from glaucoma-associated neurodegeneration, with a concomitant increase in mitochondrial biogenesis. This study aimed to determine if the ketogenic diet also promoted mitophagy. MitoQC mice that carry a pH-sensitive mCherry-GFP tag on the outer mitochondrial membrane were placed on a ketogenic diet or standard rodent chow for 5 weeks; ocular hypertension (OHT) was induced via magnetic microbead injection in a subset of control or ketogenic diet animals 1 week after the diet began. As a measure of mitophagy, mitolysosomes were quantified in sectioned retina immunolabeled with RBPMS for RGCs or vimentin for Muller glia. Mitolysosomes were significantly increased as a result of OHT and the ketogenic diet (KD) in RGCs. Interestingly, the ketogenic diet increased mitolysosome number significantly higher than OHT alone. In contrast, OHT and the ketogenic diet both increased mitolysosome number in Muller glia to a similar degree. To understand if hypoxia could be a stimulus for mitophagy, we quantified mitolysosomes after acute OHT, finding significantly greater mitolysosome number in cells positive for pimonidazole, an adduct formed in cells exposed to hypoxia. Retinal protein analysis for BNIP3 and NIX showed no differences across groups, suggesting that these receptors were equivocal for mitophagy in this model of OHT. Our data indicate that OHT and hypoxia stimulate mitophagy and that the ketogenic diet is an additive for mitophagy in RGCs. The different response across RGCs and Muller glia to the ketogenic diet may reflect the different metabolic needs of these cell types.