Recent Findings from Analysis of HIV Clade C in India
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1 Recent Findings from Analysis of HIV Clade C in India Pankaj Seth, Ph.D Associate Professor, Molecular & Cellular Neuroscience National Brain Research Centre (NBRC) Manesar, INDIA pseth@nbrc.res.in
2 NeuroAIDS Over reaction of the immune response in the brain by triggering a range of inflammatory molecules; Neuronal damage
3 HIV Neuropathogenesis G. Jones and C. Power; Neurobiology of Disease 2006
4
5 Genetic Features of HIV-1 Tat Sequences Ranga et al J. Virology 78: 2586, 2004
6 Genetic Features of HIV-1 Tat Sequences Ranga et al J. Virology 78: 2586, 2004
7 DOMAIN STRUCTURE OF HIV TRANSACTIVATING (TAT) PROTEIN NEUROTOXIC REGION N CORE 86 C CYSTEINE RICH DOMAIN TAR BINDING DOMAIN TRANSACTIVATION CO-FACTOR BINDING From Dr. U Ranga, JNCSAR
8 The Question HIV-1 B or Viral Protein Tat B HIV-1 C or Viral Protein Tat C? Neuronal Cell Death Differences in HAD in HIV-1 B and C Endemic areas
9 Challenges in NeuroAIDS Field Understanding how HIV-1 infection of CNS causes neurological complications remains a challenging task, mainly due to - lack of good animal models for HIV-1 limited access to brain tissue with different stages of HIV infection or AIDS For improved understanding of neuropathogenesis in HAD, use of an in vitro system is mandatory
10 Human Fetal Brain cells : Culture Procedure Brain dissected Aseptically (8-16 weeks Gs) (Dr. S. Sharma & Dr. N. Thapar, Civil Hospital Gurgaon) Cultured in PDL Coated culture flasks for 5-7 days in appropriate media (Without serum) Cells Passaged Identified by Morphology and Immunostaining with cell specific markers For purity of cell population
11 Human Fetal Brain Derived Cells: Differentiated Human Fetal Brain Derived Neurosphere: Neuronal Lineage Tuj-1 Nestin Nestin / Tuj-1 Merge
12 DOMAIN STRUCTURE OF HIV TRANSACTIVATING (TAT) PROTEIN NEUROTOXIC REGION N CORE 86 C CYSTEINE RICH DOMAIN TAR BINDING DOMAIN TRANSACTIVATION CO-FACTOR BINDING From Dr. U Ranga, JNCSAR
13 Experimental Design Treated with Tat protein B or C (Direct Damage) OR Transfected with pcdna / pcdna Tat B / C or Mutant Tat C (Indirect Damage) Collection of Sups at 24 hrs Human Fetal Brain Cells (Astrocytes / Neurons) Chemokine Secretion MCP-1 / CCL2 Neuronal Death In situ TUNEL staining MTT assay Caspase-3 Activation Oxidative Stress
14 Direct Cell Death By HIV Tat -Exposure of human neurons with HIV-1 Tat Protein
15 Apoptosis in Human Primary Neurons Following Treatment With Tat Protein: TUNEL Assay 50 Con 40 Tat B Tat C ** %TUNEL+/DAPI 30 Tat B 20 * P < 0.05 ** P < * 10 Tat C ng/ml 50 ng/ml 100 ng/ml 25 ng/ml 50 ng/ml 100 ng/ml
16 Neuronal Damage Following Treatment With Tat Protein: MTT Assay Cell Viability (%) ** * P < 0.01 vs Tat B ** P < Vs Control * 0 Control Tat B Tat C
17 Reactive Oxygen Species Levels In Human Neurons Following Treatment With Tat Protein Mean Fluorescence Intensity / ug protein ** ** Control Tat B Tat C ** P < 0.001
18 Indirect Cell Death By HIV Tat - Transfection of Astrocytes with Tat Expressing Vectors and Exposed Human Neurons to Spent Astrocyte Media
19 Isogenic Variants of HIV Tat C Tat C Tat CC Mutant (Like Tat B) Tat SC Mutant Ranga et al J. Virology 78: 2586, 2004
20 HIV-1 Tat Expressing Human Astrocytes: Stained with Tat Polyclonal Ab pdesred Tat DAPI / Tat Overlay pdesred Tat / Tat Overlay
21 Apoptosis in Human Neurons Following Exposure To Supernatant From Tat and Mutant Tat Transfected Astrocytes 100 ** ** Tat C Tat CC Mutant (Like Tat B) Tat SC Mutant ** P < %TUNEL+/DAPI Vector Control pcdna Tat B 0 pcdna Tat C Mutant Tat C (CC) Vector alone pcdna Tat B pcdna+tat C Mutant Tat C (CC)
22 Neuronal Damage in Human Neurons Exposed to Tat Transfected Astrocyte Supernatants : MTT Assay 125 Tat C Tat CC Mutant (Like Tat B) Tat SC Mutant * P < 0.01 Vs Con Cell Viability (%) * * 0 pcdna Tat B Tat C mutant CC mutant SC
23 Reactive Oxygen Species Levels in Human Neurons Exposed to Tat Transfected Astrocyte Supernatants Mean Fluorescence Intensity / ug protein ** ** ** P < pcdna TatB Tat C Mut CC Mut SC
24 Live Cell Pictures of Human Neurons Exposed To Media from Tat Transfected Astrocytes pcdna Tat B Tat C Mutant CC Mutant SC
25 Mitochondrial Membrane Potential Depolarization of Human Primary Neurons Following Exposure to Astrocyte Supernatants: JC-1 Green Aggregates Tat C Mutant CC
26 Active Caspase 3 Expression in Human Neurons Exposed to Tat Transfected Astrocyte Supernatants
27 CCL2/MCP-1 Production from Human Astrocytes Exposed to Tat Protein or Tat Expressing Vectors
28 Conclusions Functional differences in HIV-1 Tat B and Tat C, in terms of damage to human neurons, oxidative stress, depolarization of mitochondrial membrane and production of CCL2 / MCP-1 chemokine. The natural variation of C to S at the amino acid at position number 31 in Tat protein is responsible for the changes seen in Tat C. Our findings correlate with clinical observations made in AIDS patients in India where there is a low prevalence of HIV-1 associated dementia.
29 Acknowledgements National A: NBRC Mamta Mishra, S. Vitrevel B: Civil Hospital Gurgaon Dr. S. Sharma & Dr. N. Thapar C: JNCASR, Bangalore Dr. Udaykumar Ranga International A: NINDS, National Institutes of Health, USA Dr. E. Major B: John Hopkins University, USA Prof. A. Nath
30
31 Future Plans 1. Co-culture studies with Tat B & Tat C expressing stable glial cell lines and neurons - Co-culture of Tat expressing astrocytes with neurons to assess modulation of neuronal growth and survival - Modulation of Neuronal growth, survival in presence of HIV-1 B and HIV-1 C virus 2. To investigate whether HIV-1 Tat modulates differentiation of human brain progenitor cell - Alterations in expression of genes important for glia-neuron interplay 3. To investigate effect of Viral protein and drugs of abuse on Human fetal brain cells - Growth, Proliferation and differentiation of Neural stem cells
32 Recent Findings from Analysis of HIV Clade C in India Pankaj Seth Mamata Mishra (Ph.D Student) S. Vitrevel (Project Assistant) Durga Lal Meena (Technician) Molecular and Cellular Neuroscience
33 Active Caspase 3 Expression in Human Neurons Exposed to Tat Transfected Astrocyte Supernatants Act. Caspase-3 Beta Tubulin C Tat B Mutant C Tat C 19 KDa 17 KDa
34
35 Human Fetal Brain Derived Neurosphere: Neuronal Lineage Tuj-1 Nestin Nestin / Tuj-1 Merge
36 Human Fetal Brain Derived Cells Developed at NBRC Differentiated
37 Research Goals of The NeuroAIDS Lab LONG TERM GOAL Delineate the Cellular & Molecular Mechanisms of HIV-1 Induced Neurodegeneration Understand Differences in Neurobiology of the HIV-1 B & HIV-1 C Strain IMMEDIATE GOAL Investigate Neurobiology of HIV-1 Transactivating Viral Protein Tat B and C in Human Fetal Brain Cells Investigate the Effect of HIV-1 Viral Protein Tat on Differentiating Human Fetal CNS Progenitor Cells
38 Future Plans 2. To investigate whether HIV-1 Tat modulates differentiation of human brain progenitor cell - Alterations in expression of genes important for glianeuron interplay (such as release of neurotrophic factor / growth factors from glia cells, AEG-1, Glutamate transporters, Calcium mobalization, TNF, IL-1b, IL-6, MCP-1/CCL2, neurotrophic factor) 3. To investigate effect of Viral protein and drugs of abuse on Human fetal brain cells - Growth, Proliferation and differentiation of Neural stem cells (SAC approval requested)
39 HIV-1 Tat B and C Induce Differential Expression of MCP-1/CCL2 Production From Human Astrocytes.
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