HAART Effects on Mitochondrial Function in Human Brain Cells - PowerPoint PPT Presentation

1 / 16
About This Presentation
Title:

HAART Effects on Mitochondrial Function in Human Brain Cells

Description:

Impaired short-term memory coupled with reduced ability of mental concentration ... Dayna Lucuab, Alycia Yee, Kristen Ewell, Chloe Sivigney, and the HACRP Family ... – PowerPoint PPT presentation

Number of Views:175
Avg rating:3.0/5.0
Slides: 17
Provided by: courtn4
Category:

less

Transcript and Presenter's Notes

Title: HAART Effects on Mitochondrial Function in Human Brain Cells


1
HAART Effects on Mitochondrial Function in Human
Brain Cells
Courtney Kim University of Hawaii at Manoa Cell
and Molecular Biology Monday August 4, 2008
2
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
3
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
4
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
5
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.
6
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)
7
NEURON MITOCHONDRIAL DNA
(P0.005)
(P0.009)
(P0.012)
(P0.004)
CONTROL (n4)
AZT/3TC/LPV/RTV (n3)
8
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
9
NEURON ATP
(P0.036)
CONTROL (n4)
AZT/3TC/LPV/RTV (n3)
10
Mitochondrial Oxidative Damage in Neurons by
8-oxo-dG Base Specific Repair Assay
MtDNA 16 Kb
Break Frequency - ln hOGG1 repair NO
hOGG1 repair
11
ASTROCYTE MITOCHONDRIAL DNA
CONTROL (n4)
AZT/3TC/LPV/RTV (n3)
d4T/3TC/LPV/RTV (n3)
12
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)
13
ASTROCYTE ATP
CONTROL (n4)
AZT/3TC/LPV/RTV (n3)
d4T/3TC/LPV/RTV (n3)
14
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

15
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.

16
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
Write a Comment
User Comments (0)
About PowerShow.com