HAART Effects on Mitochondrial Function in Human Brain Cells Courtney Kim University of Hawaii at Manoa Cell and Molecular Biology Monday August 4, 2008

Characteristics of HIV-Associated Dementia (HAD) Impaired short-term memory coupled with reduced ability of mental concentrationImpaired short-term memory coupled with reduced ability of mental concentration Motor dysfunction and speech problemsMotor dysfunction and speech problems Leg weakness, slowness of hand movement and gaitLeg weakness, slowness of hand movement and gait Depression, personality changes (apathy and social withdrawal)Depression, personality changes (apathy and social withdrawal)

Apoptotic neurons have been observed in cerebral cortex of HIV-patients (Adle-Biassette 1995) Apoptotic neurons have been observed in cerebral cortex of HIV-patients (Adle-Biassette 1995) Astrocytes exposed to HIV-1 or gp120 have impaired glutamate uptake in vitro (Wang 2003)Astrocytes exposed to HIV-1 or gp120 have impaired glutamate uptake in vitro (Wang 2003) ddC induces oxidative stress and pro-apoptotic proteins in isolated mitochondria from gerbil brain, causing apoptosis (Opii 2007)ddC induces oxidative stress and pro-apoptotic proteins in isolated mitochondria from gerbil brain, causing apoptosis (Opii 2007) NRTIs inhibit mt transmembrane potential in rat primary dorsal root ganglion neurons, leading to energy failure, axonal degeneration, and cell death (Keswani 2003)NRTIs inhibit mt transmembrane potential in rat primary dorsal root ganglion neurons, leading to energy failure, axonal degeneration, and cell death (Keswani 2003) Reduction of mtDNA was found in PC-12 cells, rat chromaffin neuroendocrine cell line, exposed to a high doses of ddI and ddC (Cui 1997)Reduction of mtDNA was found in PC-12 cells, rat chromaffin neuroendocrine cell line, exposed to a high doses of ddI and ddC (Cui 1997) OBSERVATIONS IN HIV NEUROPATHIES AND MITOCHONDRIA Photograph:

Multi-Hit Model of HIV and Mitochondrial Dysfunction DNA polymerase- DNA polymerase-  UncouplingUncoupling TransportTransport Oxidative StressOxidative Stress ApoptosisApoptosis PhosphorylationPhosphorylation Proteolytic ProcessingProteolytic Processing GlycosylationGlycosylation Day L, et al. Mitochondrion 4 (2004) 95–109. AACTG Metabolic Guides: McComsey GA, Morrow JD.. J Acquir Immune Defic Syndr 2003;34:45-9. Dagan, T., Sable, C., Bray, J. Gerschenson, M. Mitochondrion, 1: , Gerschenson, M. and Brinkman, K. Mitochondrion, NEUROPATHIESHAD Peripheral Neuropathy MCMD HIV CYTOKINES ART Mitochondria Cartoon:

Mitochondrial Dysfunction Caused From NRTIs Velsor LW. Toxicol Appl Pharmacol. 2004;99: McComsey, G. & Gerschenson, M., Antiviral Therapy, 2008, In press. (AZT or d4T) NRTI decreases mitochondrial function by inhibiting mtDNA and mtRNA synthesis and/or mtRNA

In Vitro Model Human Astrocytes Human Cortical Neurons or Medium FBS pen/strep Control (n=4) 0.2  M AZT or 0.2  M d4T / 0.75  M 3TC / 0.03  m RTV / 0.03  m LPV Experimental (n=3) HARVEST! 16,000 cells/flask ATP CONCENTRATION (nmol/mg) DNA RNA CYTOCHROME-B (CYTB) NADH DEHYDROGENASE I (ND1) NADH DEHYDROGENASE VI (ND6) Relative to ribosomal L13 MTDNA (copies/cell) MT NADH DEHYDROGENASE SUBUNIT II NUCLEAR FAS GENE Real-time PCR (Roche) Every 2-3 days BCA Protein ATP HSII Kit (Roche) CDNA Astrocytes HCN-1A Neurons

NEURON MITOCHONDRIAL DNA (P=0.004) (P=0.012) (P=0.009) (P=0.005) CONTROL (n=4) AZT/3TC/LPV/RTV (n=3)

NEURON MT TRANSCRIPTS CONTROL (n=4) AZT/3TC/LPV/RTV (n=3) * SIGNIFICANCE P < 0.05 ** SIGNIFICANCE P < * * ** * CYTOCHROME-B NADH DEHYDROGENASE SUBUNIT I NADH DEHYDROGENASE SUBUNIT VI

NEURON ATP (P=0.036) CONTROL (n=4) AZT/3TC/LPV/RTV (n=3)

Mitochondrial Oxidative Damage in Neurons by 8-oxo-dG Base Specific Repair Assay Break Frequency = - ln hOGG1 repair NO hOGG1 repair NO hOGG1 repair MtDNA 16 Kb

ASTROCYTE MITOCHONDRIAL DNA CONTROL (n=4) AZT/3TC/LPV/RTV (n=3) d4T/3TC/LPV/RTV (n=3)

ASTROCYTE MT TRANSCRIPTS CONTROL (n=4) AZT/3TC/LPV/RTV (n=3) d4T/3TC/LPV/RTV (n=3) NADH DEHYDROGENASE SUBUNIT I NADH DEHYDROGENASE SUBUNIT VI CYTOCHROME-B

ASTROCYTE ATP CONTROL (n=4) AZT/3TC/LPV/RTV (n=3) d4T/3TC/LPV/RTV (n=3)

MtDNA copies/cell were decreased significantly at day-2, and increased at day-14, day-16, and day-18 in AZT- treated neurons compared to untreated controls MtDNA copies/cell were decreased significantly at day-2, and increased at day-14, day-16, and day-18 in AZT- treated neurons compared to untreated controls CytB and ND1 mtRNA transcripts were significantly decreased at day-11 in AZT-treated neurons CytB and ND1 mtRNA transcripts were significantly decreased at day-11 in AZT-treated neurons ND1 was significantly increased at day-9 and day-16 in AZT-treated neurons ND1 was significantly increased at day-9 and day-16 in AZT-treated neurons ATP significantly increased at day-11 in AZT-treated neurons ATP significantly increased at day-11 in AZT-treated neurons 8-oxo-deoxyguanine damage was increased in mtDNA of neurons treated with D4T over time 8-oxo-deoxyguanine damage was increased in mtDNA of neurons treated with D4T over time HAART did not affect astrocytes in vitro HAART did not affect astrocytes in vitro RESULTS

CONCLUSIONS Alterations were observed in mtDNA, mtRNA, and ATP levels in HCN-1a neurons exposed to human CSF doses of AZT/3TC/LPV/RTV after long-term exposure.Alterations were observed in mtDNA, mtRNA, and ATP levels in HCN-1a neurons exposed to human CSF doses of AZT/3TC/LPV/RTV after long-term exposure. MtDNA copies/cells were significantly increased in AZT- treated neurons after day-11, suggesting that mitochondria may be compensating for decreases observed in CytB, ND1, and ND6 mt transcripts at day-11.MtDNA copies/cells were significantly increased in AZT- treated neurons after day-11, suggesting that mitochondria may be compensating for decreases observed in CytB, ND1, and ND6 mt transcripts at day-11. Astrocytes were unaffected after one week of HAART. Astrocytic changes may occur after longer exposure.Astrocytes were unaffected after one week of HAART. Astrocytic changes may occur after longer exposure. 8-oxo-dG damage was greatest in neurons treated with d4T, suggesting d4T may cause oxidative damage to mtDNA.8-oxo-dG damage was greatest in neurons treated with d4T, suggesting d4T may cause oxidative damage to mtDNA.

MAHALO!! OUR LAB: Mariana Gerschenson, Ph.D., Daniel Libutti, Julia Choi, Michelle Tsang, Dayna Lucuab, Alycia Yee, Kristen Ewell, Chloe Sivigney, and the HACRP Family This research was funded by NIH RR16467