HAART Effects on Mitochondrial Function in Human Brain Cells

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HAART Effects on Mitochondrial Function in Human
Brain Cells
Courtney Kim University of Hawaii at Manoa Cell
and Molecular Biology Monday August 4, 2008
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Characteristics ofHIV-Associated Dementia (HAD)

• Impaired short-term memory coupled with reduced
  ability of mental concentration
• Motor dysfunction and speech problems
• Leg weakness, slowness of hand movement and gait
• Depression, personality changes (apathy and
  social withdrawal)

http//africascience.blogspot.com/2007/08/hiv-laun
ches-two-pronged-attack-on.html
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OBSERVATIONS IN HIV NEUROPATHIES AND MITOCHONDRIA

• 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)
• 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)
• 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)

Photograph http//www.iscr.ed.ac.uk/news/press-re
leases-2005aug16.htm
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Multi-Hit Model of HIV and Mitochondrial
Dysfunction
HIV
CYTOKINES
ART

• DNA polymerase-?
• Uncoupling
• Transport
• Oxidative Stress
• Apoptosis
• Phosphorylation
• Proteolytic Processing
• Glycosylation

NEUROPATHIES HAD Peripheral Neuropathy MCMD
Day L, et al. Mitochondrion 4 (2004) 95109.
AACTG Metabolic Guides http//aactg.s-3.com/metab
olic/lactic.pdf. McComsey GA, Morrow JD.. J
Acquir Immune Defic Syndr 20033445-9. Dagan,
T., Sable, C., Bray, J. Gerschenson, M.
Mitochondrion, 1397-412, 2002. Gerschenson, M.
and Brinkman, K. Mitochondrion, 2004.
Mitochondria Cartoon www.nsf.gov/news/overviews/b
iology/interact08.jsp
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Mitochondrial Dysfunction Caused From NRTIs
(AZT or d4T)
NRTI decreases mitochondrial function by
inhibiting mtDNA and mtRNA synthesis
and/or mtRNA
Velsor LW. Toxicol Appl Pharmacol.
20049910-19. McComsey, G. Gerschenson, M.,
Antiviral Therapy, 2008, In press.
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In Vitro Model
Astrocytes
Control (n4)
Human Cortical Neurons or
Experimental (n3)
Human Astrocytes
0.2?M AZT or 0.2?M d4T / 0.75?M 3TC / 0.03?m RTV
/ 0.03?m LPV
HCN-1A Neurons
Medium FBS pen/strep
Every 2-3 days
DNA
HARVEST!
MTDNA (copies/cell) MT NADH DEHYDROGENASE SUBUNIT
II NUCLEAR FAS GENE
RNA
16,000 cells/flask
CDNA
BCA Protein
ATP HSII Kit (Roche)
CYTOCHROME-B (CYTB) NADH DEHYDROGENASE I
(ND1) NADH DEHYDROGENASE VI (ND6) Relative to
ribosomal L13
Real-time PCR (Roche)
ATP CONCENTRATION (nmol/mg)
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NEURON MITOCHONDRIAL DNA
(P0.005)
(P0.009)
(P0.012)
(P0.004)
CONTROL (n4)
AZT/3TC/LPV/RTV (n3)
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NEURON MT TRANSCRIPTS
NADH DEHYDROGENASE SUBUNIT I



CYTOCHROME-B

NADH DEHYDROGENASE SUBUNIT VI
CONTROL (n4)
AZT/3TC/LPV/RTV (n3)
SIGNIFICANCE P lt 0.05
SIGNIFICANCE P lt 0.001
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NEURON ATP
(P0.036)
CONTROL (n4)
AZT/3TC/LPV/RTV (n3)
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Mitochondrial Oxidative Damage in Neurons by
8-oxo-dG Base Specific Repair Assay
MtDNA 16 Kb
Break Frequency - ln hOGG1 repair NO
hOGG1 repair
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ASTROCYTE MITOCHONDRIAL DNA
CONTROL (n4)
AZT/3TC/LPV/RTV (n3)
d4T/3TC/LPV/RTV (n3)
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ASTROCYTE MT TRANSCRIPTS
NADH DEHYDROGENASE SUBUNIT I
CYTOCHROME-B
NADH DEHYDROGENASE SUBUNIT VI
CONTROL (n4)
AZT/3TC/LPV/RTV (n3)
d4T/3TC/LPV/RTV (n3)
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ASTROCYTE ATP
CONTROL (n4)
AZT/3TC/LPV/RTV (n3)
d4T/3TC/LPV/RTV (n3)
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RESULTS

• 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
• ND1 was significantly increased at day-9 and
  day-16 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
• HAART did not affect astrocytes in vitro

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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.
• 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.
• 8-oxo-dG damage was greatest in neurons treated
  with d4T, suggesting d4T may cause oxidative
  damage to mtDNA.

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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