Nuran Ercal MD PhD
Department of Chemistry,
Missouri University of Science and Technology, Rolla, MO
&
Department of Internal Medicine, St. Louis University, St. Louis, MO

Toxic HIV Proteins
Addictive Drugs
(METH)
OXIDATIVE
STRESS
BBB
BBB integrity disrupted

 Increased generation of
ROS and/or a decrease in the antioxidant capacity of cells result in “ Oxidative
Stress”.
 Oxidative stress compromises crucial cellular functions.

Sources of Reactive Oxygen Species
1) Non mitochondrial:
NADPH Oxidases
Microsomal cytochrome P-450
Cyclooxygenases
Monoamine oxidases
Peroxisomal b oxidation of fatty acids
Phagocytes
2) >90% is mitochondrial electron transport chain contains several redox centers that may leak electrons to oxygen---- Superoxide radical!

ROS
 Superoxide (O



2
¯• ) –
No direct effects on targets
Penetrates important sites
Subsequently converted to other ROI

Hydrogen Peroxide (H

2
O
2
) –
Dismutation of superoxide radical
2H + + 2O
2
¯•
SOD
H
2
O
2
+ O
2



Reacts with thiols
Bacteriocidal only at higher concentrations
Secondary oxidants from H
2
O
2 responsible for killing


Hydroxyl Radicals (OH • ) – Fenton Reaction

Fe 2+
OH •
+H
2
O
2
Fe 3+ + OH ¯ bacteriocidal arsenal is controversial
+ OH as a major component of neutrophil
•

Limited radius of action

Consequences of oxidative stress
Oxidative Damage to Lipids
Membrane peroxidation
Decreased membrane fluidity
Marker
F
2
-Isoprostanes
8-iso-PGF
2 a
Oxidative Damage to DNA
Oxidative Damage to Proteins
Mutations
Deletions
O
H
N oxo8dG
NH
NH
N
Sugar
NH
2
Oxidation of sulfhydryl groups
Reactions with aldehydes
Protein aggregation
Protein carbonyls
PROTEIN

 Introduction (background)
 HIV Associated Dementia
 Methamphetamine
 Blood Brain Barrier (BBB)
 Proposed Research Goals
 Experimental Methods
 Previous Results: RBE4
 Recent Results: HBMVEC
 Future Studies: immortal HBMVEC and transgenic animals
 Conclusion



 HIV-associated dementia (HAD)
 HIV-related encephalitis (HIVE)
 More commonly minor neurocognitive problems
 DEMENTIA is the most challenging complications of HIV infection.
Avindra Nath et al International Review of Psychiatry, Feb 2008, 20 (1), 25-31.

Symptoms include:
 Sluggish mental capabilities and motor control
 Marked apathy
 Loss of interest in previously enjoyable activities
 Tremors and impaired balance, slow eye movement, abnormal reflexes..
 The disorder continues to escalate, eventually resulting in severe dementia
 Some experience mania or even psychosis

 One third of adults and half of children with HIV have dementia in the western countries
 HAD is the most common case of dementia among people aged 40 or less and a significant independent risk factor for death due to AIDS!
Avindra Nath et al International Review of Psychiatry, Feb 2008, 20 (1), 25-31.
 HAART ( H ighly A ctive A nti R etroviral T herapy) therapy has reduced HAD in infected population some but it does not seem to be effective.
 In the era of HAART, the course of HIV dementia appears to have changed.
Steiner J et al , Antioxidants &Redox Signaling, 2006; McArthur JC et al , Neurology, 1993;
Bouwman FH et al , Neurology, 1998

 Highly active antiretroviral therapy (HAART) is introduced in the mid-90s.
 HAART consists of combination of


 nucleoside reverse transcriptase inhibitors non-nucleoside RT inhibitors and protease inhibitors
 There are more than 20 (zidovudine, lamivudine, stavudine, emtriva, crixivan, kaletra and more) approved antiretroviral drugs.
 HAART therapy has been shown to prolong survival in
AIDS patients.
Current Pharmaceutical Design, 2006, 12,2031-2055

 Over 22 million people have died from AIDS
 Over 42 million people are living with HIV/AIDS
 Over 19 million women are living with HIV/AIDS
 There are over 14,000 new infections every day
(95% are in developing countries) http://www.until.org/statistics.shtml



The UN estimates that, currently, there are 14 million
AIDS orphans and that by 2010 there will be 25 million
By the year 2010, five countries (Ethiopia, Nigeria,
China, India, and Russia) with 40% of the world
’ s population will add 50 to 75 million infected people to the worldwide pool of HIV disease if nothing is done to help stop the spread of HIV/AIDS http://www.until.org/statistics.shtml

 One million people are currently living with HIV in the
U.S., with approximately 40,000 new infections occurring each year
 70% of these new infections occur in men and 30% occur in women
 75% of the new infections in women are heterosexually transmitted
 Half of all new infections occur in people 25 years old or younger http://www.until.org/statistics.shtml

 HIV envelope protein (gp120) and transregulatory protein
(Tat) may play a role in the development of HAD by increasing the production of reactive oxygen species
(ROS) in blood brain barrier (BBB).
 Oxidative stress may be involved in HIV neuropathogenesis.

Oxidative Stress Related Disorders










Parkinson’s Disease
Alzheimer’s Disease
Stroke
Rheumatoid arthritis
Atherosclerosis
Vascular dysfunction
Multiple sclerosis
Inflammatory bowel disease
H. pylori -associated gastritis
Systemic inflammatory response syndrome
 And the list is growing…













Autoimmune thyroid disease
Cystic fibrosis
Diabetes
Aging
Macular degeneration
HIV/AIDS
Cancer
Septic shock
Heavy metal toxicity
Nanoparticle toxicity
EtOH abuse
Meth
ALS

 Analyses of brain tissue and CSF of patients with
HIV-1 dementia shows evidence for oxidative stress correlated with disease pathogenesis and cognitive impairment.
 Evidences are:
 4-HNE is high
 Protein carbonyls are high
 Nitrated tyrosine residues are increased in HIV dementia brains

 It is widely known that many people with HIV-1 use addictive drugs. Among them:

Alcohol

Methamphetamine

Cocaine

Nitrite Inhalants

Hallucinogens
 Nearly 50% of HIV positive women in the US contract the infection via drug use!!
 Methamphetamine abuse is common


9.4 million people in the US
Midwest
– 90% of all drug cases
 Methamphetamine (METH) induces ROS.

Crystal
Powder
Pill

Picture source: US Department of Justice (www.usdoj.gov)

 Street names of methamphetamine
(METH)
 Speed


Ice
Crystal Meth
 Chalk
 Consumption
 Smoke



Snort
Inject
Oral Ingestion
 Surge in catecholamine levels:



Dopamine (DA)
Serotonin
Norepinephrine (NE)
National Drug Intelligence Center, National Drug Threat Assessment 2008, October 2007;
Picture Source: http://cerhr.niehs.nih.gov/chemicals/stimulants/amphetamines/methamphetamine.gif

 Oxidative stress has been shown to play an important role in the toxic effects of
METH.
 METH increases levels of dopamine.
 Dopamine can react to form ROS through several different pathways.
Dopamine enzymatic oxidation
DA + O
2
MAO
+ H
2
O-------------------

3,4,dihydroxyphenylacetic acid + NH
3
+
H
2
O
2
Dopamine autoxidation
DA + O
2
 SQ · + O
2
· - + H +
DA + O
2
· + 2 H+  SQ
·
+ H
2
O
2
Josephine W.S et al., Annals of the New York Academy of Sciences (2000)
Zecca, L., et al ., Nature Reviews (2004)



 The BBB has been implicated in the development of HIV Dementia.
 The BBB is a barrier between the blood and the fluid that surrounds the cells of the brain.
 The BBB is selectively permeable, allowing some substances to cross and not others. It is generally more permeable to lipophilic substances.
 The cells that line the capillaries of the rest of the body usually have small gaps between them, that allow substance exchange. The BBB lacks these small gaps, prohibiting much exchange.

What is the function of the BBB?
 The BBB maintains that delicate balance by regulating the entry and exit of substances.
 The BBB also provides protection by preventing toxic chemicals from entering the brain.
 The structural integrity of the BBB is compromised in HIV/AIDs demented patients.
 What is the cause of this destruction?

What’s causing the blood-brain barrier to fail?


Toxic HIV Proteins
Gp120 and tat
OXIDATIVE
STRESS
BBB
Addictive Drugs
(METH)
BBB integrity disrupted

 gp120 is a protein on the outer envelope of the HIV-1 virus.
 It binds to a receptor on
CD4 cells and aides in the injection of viral nucleic acid into the host cell.
 The amino acids responsible for this binding interaction are highly conserved amongst strains of the virus.

Gp120 (red) in complex with a CD4 surface protein (yellow)

More on gp120
 gp120 is composed of an outer and inner domain made of α helices and β sheets.

The negatively charged central cavity contains the binding site for a positively charged region of a surface protein on CD4 (electrostatic interactions).

 gp120 changes confirmation when bound to different proteins
Picture Source: Tongqing Zhou et al . Nature 2007

 It is a viral protein released from
HIV-1-infected T cells and monocytes/macrophages
 Transactivator of Transcription
(Tat) protein consists of between
86 and 101 amino acids depending on the subtype
Jeang, K. T. (1996) In: Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic
Acid and Amino Acid Sequences. Los Alamos National Laboratory (Ed.) pp. III-3 –III-18; Picture
Source: Grant R. Campbell et al. J. Biol. Chem. 2004

 Although the mechanism is not explicitly known, it has been shown that both gp120 and tat increase oxidative stress levels in cells.
 Tat



Decreases GSH by inhibiting GSH synthetase
Increases NO secretion which induces apoptosis
Increases calcium uptake resulting in production of ROS
 gp120





Decreases GSH by inhibiting GSH synthetase
Increases cell permeability
Promotes calcium overload
Oxyradical production
Mitochondrial dysfunction by altering ion channels
Choi J et al , J Biol Chem 2000; Toneatto S et al , AIDS 1999; Visalli, Valeria, Neuroscience 2007

METH
Dopamine
Oxidation by MAO
Autooxidation
H
2
O
2
Quinones
O
2
•¯ , OH •
ROS
RNS
Gp120
TAT
Glutamate mobilization by NMDAR activation
Microglial activation
ERK1/2
MAPK
Cytokines &
Chemokines
IL-1B,IL-8,TNF-a
CCL2, CCL4,CXCL8 iNOS eNOS
Ca 2+ overload
O
2
• ¯ nNOS
• NO
NOO ¯
DNA oxidation
Protein oxidation
Lipid Peroxidation
Oxidative
Stress
Antioxidants

 Analyses of brain tissue and CSF of patients with
HIV-1 dementia shows evidence for oxidative stress correlated with disease pathogenesis and cognitive impairment.
 Evidences are:
 4-HNE is high
 Protein carbonyls are high
 Nitrated tyrosine residues are increased in HIV dementia brains

 Overlap of pathways leading to neurodegeneration in
HAD
 METH use enhances gp120 and Tat mediated neurotoxicity
 Oxidative stress common to viral protein and METH mediated toxicity
 Therapeutic approaches
 Block rise in intracellular calcium
 Antagonize NMDA receptors
 Target oxidative damage

 Disruption of the BBB is seen more in AIDS/HIV dementia patients then in non-demented AIDS/HIV patients.
 The disruption of the BBB may be due to an increase in
ROS by viral proteins

Tat and gp120 induce oxidative stress
 Stimulation of iNOS and production of NO
 Glutamate mediated excitotoxicity
 Disruption of calcium homeostasis
 METH may potentiate Tat and gp120 induced oxidative stress on BBB .

 Highly active antiretroviral therapy (HAART) does not prevent BBB disruption nor does it decrease ROS production.
 Therefore, ANTIOXIDANTS should be included in the treatment to prevent HAD.
 Dealing with BBB

Most antioxidants (AO) unable to cross BBB

Design AO capable of crossing BBB

Low MW thiol antioxidants with ability to cross BBB

 The most important biological thiol, Glutathione (GSH), protects cells against ROS.
 GSH deficiency has been associated with various neurodegenerative diseases.
 A significant decrease in GSH levels in patients with AIDS/HIV in various biological samples (blood, liver, brain).
 Thiol antioxidants in HAD:
 NAC (decreased the mortality rate of HIV-infected patients)
 NAC analogs (N-acetyl-L-cysteinyl)-S-acetylcysteamine
(NACA, possible candidate)
Pocernich CB et al ., Brain Research Reviews , 2005 .

NAC NACA

 NACA in radiation damage
 NACA in neurological complications of
 HIV Dementia
 Lead poisoning (possibly via Glu)
 NACA in medicinal and abusive drug complications
 Acetaminophen poisoning
 METH neurotoxicity
 NACA in macular degeneration

30-day percentage survival rates of SD-rats after irradiation with pre-treatment of NAC or NACA and posttreatment of NAC or NACA.
100%
75%
50%
25%
0%
A B C D E
Animal groups
F G H
A: XRT only; B: XRT + NAC (pre-treated); C: XRT + NACA (pre-treated);D: Control
(no XRT and any treatment); E:NAC only; F: NACA only; G:XRT + NAC (posttreated); H: XRT + NACA (post-treated).

Control
NACA only (750 μM)
Glutamate (10 mM) + NACA (750 μM)
Glutamate only (10 mM)
PC12 cells were plated at a density 25 x 10 3 cells/well in a 24 well plate and grown for 24 h in culture medium; then they were treated or not (control) with 10 mM Glu with or without NACA.. Twenty four h later, cells were examined and photographed.

1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
Control
***
METH 1mM
###
METH +NACA NACA0.5mM
HCMVEC/D3 Cells were pretreated with 1mM NACA for 2 h, followed by
24 h incubation with METH 1mM. NACA protected HCMVEC/D3 cells from cytotoxicity caused by METH. Values were the means +SD of three samples.Statistically different values of ***p <0.0001 were determined, compared to control.
### p < 0.0001 compared to METH 1mM group.

 Leading cause of blindness in people over 55
 Degeneration of macular region of the eye
 Macula – oval yellow spot near the center of the retina of the eye
 Near its center is the fovea, a small pit containing the largest concentration of cone cells of the eye. Responsible for central vision
 Disease begins when transport of nutrients and waste via retinal pigment epithelial cells begins to slow and down, leading to accumulation of waste products

Image Source: www.sfn.org
(Society for Neuroscience)








Metabolically active cells
Large oxygen fluxes across its boundary
Exposure to sunlight
RPE phagocytose photoreceptor outer segments containing PUFAS which can undergo lipid peroxidation
Age decreases antioxidants in RPE, increasing effect of oxidative insults


Cell model – Adult retinal pigment epithelial cells
(ARPE-19)

Oxidative stress induction – tert butylhydroperoxide (tBHP), a cell permeant oxidant that causes lipid peroxidation and loss of
GSH

Antioxidant – Various concentrations of Nacetycysteine amide

Pretreat cells for 24 h with antioxidant, followed by treatment with tBHP (0.4 mM) for 4 h


Cell model – Adult retinal pigment epithelial cells
 Oxidative stress induction – tert butylhydroperoxide (tBHP), a cell permeant oxidant that causes lipid peroxidation and loss of
GSH

Antioxidant – Various concentrations of Nacetycysteine amide

Pretreat cells for 24 h with antioxidant, followed by treatment with tBHP (0.4 mM) for 4 h

 tBHP induced cell death and protection by NACA pretreatment
100
80
60
40
*
20
0
Co nt ro l st ar os po rin e tB
HP tB
HP
+
5 mM
N
AC
A n=3, *p<0.0001 as compared to control

 tBHP induced production of ROS (incubation with tBHP for 45 min)
400 **
**
300
*
200
100
0
0
0.
2
0.
4
Concentration of tBHP(

M )
0.
8 n=3, *p<0.00 as compared to control, **p<0.005 as compared to control

 Protection by NACA pretreatment
*
200
150
100
50
0
Co nt ro l
#
0.
4 mM
tB
HP
0.
4 mM
tB
HP
+
5 mM
N
A n=3, *p<0.0001 as compared to control, #p<0.005 as compared to tBHP treated group

 Restoration of GSH levels by NACA pretreatment
80
60
40
20
*
#
0
Co nt ro l
0.
4 m
M
tB
HP
0.
4 m
M
tB
HP
+
5 m
M
N
A n=3, * p<0.005 as compared to control, # p<0.0001 as compared to tBHP treated group.

 NACA is capable of supplying GSH to cells undergoing tBHP induced oxidative stress
 NACA has a protective effect against tBHP induced cell death and reactive oxygen species production

 XRT-induced oxidative stress


Glutamate-toxicity
 HIV-proteins (gp120and Tat)-METH-induced oxidative stress
Acetaminophen-toxicity
 Macular degeneration

 XRT-induced oxidative stress


Glutamate-toxicity
 HIV-proteins (gp120and Tat)-METH-induced oxidative stress
Acetaminophen-toxicity
 Macular degeneration

A reversed-phase HPLC method developed to separate and quantify NAC and NACA by fluorescence detection by using
N-(1-pyrenyl) maleimide (NPM) as the derivatizing agent.
Biological thiols such as glutathione (GSH), cysteine
(CYS), and homocysteine
(HCYS) can be determined simultaneously.
O
N
O
R HS thiol
H
H H
S R
O
N
O
NPM NPM-thiol derivative
Wu W et al ., Biomedical Chromatography, 20: 415-422 (2006)

Chromatogram of a P lasma Sample from an Animal Sacrificed 30 min
After Administration of 500 mg/kg Body Weight NACA.
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
5.5
5.0
4.5
4.0
3.5
1 2 3 4 5 6 7
Minutes
8 9 10 11 12 13 14
3.0
2.5
2.0
1.5
1.0
0.5
15
0.0
5.5
5.0
4.5
4.0
3.5

 Replenishes GSH through deacetylation to cysteine
 Low oral bioavailability (30%) due to negative charge
 Low solubility and tissue distribution
 May cause a severe, anaphylaxis-like allergic reaction when given intravenously http://www.medicinenet.com/acetylcysteine-injection/article.htm

 Neutral charge: Lipophilic
 Easily crosses the cell membranes
 Crosses blood brain barrier, scavenges free-radicals , chelates copper, and protects red blood cells from oxidative stress.
 Current research areas using NACA include:

Parkinson’s treatment
 cerebral ischemia
 other neurodegenerative diseases
Penugonda S et.al., Brain Research, September 21, 2005

 A few antioxidants have been used in clinical trials in
HIV dementia.
 OPC-14117 (lipophilic compound structurally similar to
Vit E), Selegiline (L-Deprenyl, MAO inhibitor), and CPI-
1189 (lipophilic antioxidant scavenging superoxide radicals).
 Unfortunately, the findings are very disappointing with side effects of cataract formation, elevation of hepatic enzymes and decrease in mean corpuscular volume.
Steiner J et al, Antioxidants & Redox Signaling, 2006

Toxic HIV Proteins
Addictive Drugs
(METH)
OXIDATIVE
STRESS
BBB
BBB integrity disrupted

Aim I: To determine whether oxidative stress induced by gp120 and Tat at the BBB is potentiated by METH.
Aim II: To determine whether the potent antioxidant
NACA protects the BBB from gp120, Tat, or
METH alone and in combination.

 RBE4-rat blood brain barrier cell model
(Banks Lab)
 HBMEC-primary human brain microvascular endothelial cells (Banks Lab)
 Immortilized HBMEC (Banks Lab)

 Reduced glutathione (GSH) and oxidized glutathione
(GSSG) assay by HPLC method
 ROS measurement by DCF fluorescence-DCFH2DA converted by intracellular ROS into fluorescent DCF
 Lipid peroxidation by-product (MDA) by HPLC
 Caspase-3 for apoptosis
 Antioxidant enzyme levels

 GSH (glutathione)


-glutamyl-cyteinyl-glycine
NH3+
HOOC CH CH2 CH2
O
C N CH
H CH2
SH
O
C N CH2
H
COOH




GSH is the most abundant non-protein thiol
Critical in maintaining the redox environment
Cellular GSH is increased in times of stress, and down-regulated after a challenge has been faced.
 GSH is crucial antioxidant in the BRAIN because:
 20% oxygen is used in the brain (2% of the BW)

Result: large quantities of ROS in the brain
 In addition,

There is high levels of iron in some parts of the brain

Brain is rich in pufas

Brain has very low amounts of SOD, CAT and GPx
So, defense against ROS by GSH becomes crucial in the brain.




RBE4 cells divided into groups:




Control
METH only (100

M)
Tat only (40nM) gp120 only (3nM)





NACA only (1mM)
Tat (40nM)+ METH (100

M) gp120 (3nM) + METH (100

M)
Tat (40nM)+ METH (100

M) + NACA (1mM) gp120 (3nM) + METH (100

M) + NACA (1mM)
Incubation times – 3 h METH + 12 h for gp120 and
Tat groups
Serine-Borate buffer used to prevent artifactual oxidation

Effect of METH on GSH Levels
Effects of increasing concentrations of METH on GSH levels in RBE4 cells.
N=4; *Significantly different when compared with control p<0.005, **p<0.0005, ***p<0.00003. (n=3/group).

Effect of NACA on GSH Levels in gp120 & METH Treated Cells
140
120
100
80
60
40
20
0
GSH Levels (% control)
**
*
#
***
**
*
Co nt ro l
N
A
CA
(1
m
M
) gp
12
0
(3
n
M
)
M
ET
H
(1
00
u
M
) H gp
12
0+
M
ET gp
12
0+
M
ET
H+
N
A
CA
***
##
#
*
Gp120+Meth+NACA treated cells have similar GSH levels as controls.
N=4; *p<0.05, **p<0.05, ***p<0.05, #p<0.05, ##p<0.003, ###p<0.001.

Effect of NACA on GSH Levels in Tat and METH Treated Cells
NACA significantly increased the GSH levels in Tat+METH-incubated RBE4 cells.
N=4; *p<0.05 compared with control, **p<0.05, ***p<0.02, #p<0.03, ##p<0.02.

 Intracellular ROS production was measured using the dye 2,7-dichlorofulrescin diacetate (DCFH2-DA) method.
 This measurement of cell oxidation is based on ROSmediated conversion of nonfluorescent compound
(DCFH2-DA) into a highly fluorescent compound
(DCF).

Effects of NACA on the Generation of ROS in gp120 Treated Cells
1 nM gp120 treated cells have significantly higher intracellular ROS as compared to controls. The increase of ROS was inhibited in the presence of ROS scavenger
NACA (1 mM)
N=4; *p<0.05, **p<0.05

Effects of NACA on the Generation of ROS in Tat Treated Cells
10nm Tat treated cells have significantly higher intracellular ROS as compared to controls. The increase of ROS was inhibited in the presence of ROS scavenger
NACA (1mM).
N=4; *p<0.05, **p<0.05

Malondialdehyde (MDA) levels:
Polyunsaturated fatty acids, exposed to free radicals, can be oxidized to hydroperoxides which decompose (in the presence of metals) to hydrocarbons and aldehydes such as malondialdehyde
(MDA).

Lipid Peroxidation Levels in gp120 Treated Cells
NACA (1mM) attenuated lipid peroxidation in gp120 treated cells.1nM gp120 induced significant increase in MDA levels
N=4; *p<0.05, **p<0.05

Lipid Peroxidation Levels in Tat Treated Cells
NACA (1mM) attenuated lipid peroxidation in Tat treated cells. 10 nM Tat induced significant increases in MDA levels.
N=4; ***p<0.05, **p<0.05, #p<0.01

 Caspase-3 activity was measured by a spectrophotometric assay kit. The protease activity was measured by the addition of a specific peptide substrate for caspsase-3. The cleavage of peptide by the caspase releases the chromophore, which can be quantitated spectrophotometrically at a wavelength of
405nm.

Effect of NACA on Caspase 3 Activity in gp120 Treated Cells
NACA (1mM) treated cells have significantly lower caspase-3 activity as compared to the gp120 alone treated cells.
N=4; *p<0.05, **p<0.05

Effect of NACA on caspase 3 activity in Tat treated cells
NACA (1mM) treated cells have significantly lower caspase-3 activity as compared to the Tat alone treated cells.
N=4; *p<0.05

Effect of NACA on Antioxidant Enzyme Levels in RBE4 Cells
Groups
Control gp120
Tat gp120+NACA
Tat+NACA
Catalase
(mU/mg protein)
6.5 ± 0.7
3.38 ± 0.14*
5.02 ± 0.16
8.45 ± 0.68**
9.2 ± 0.8
#
GPx
(mU/mg protein)
6.25 ± 0.73
3.62 ± 0.25*
3.96 ± 0.52*
8.87 ± 0.47**
10.12 ± 0.51
#
GR
(mU/mg protein)
13.94 ± 1.4
9.82 ± 0.09
*
9.73 ± 0.33
*
11.82 ± 0.23
**
11.71 ± 1.44
#
N=4
*p<0.005-0.01
, Compared with control group
**p<0.005-0.01
, Compared with gp120 group
#p<0.005-0.01
, Compared with Tat group

 Determine whether the potent antioxidant NACA protects the BBB from gp120, Tat, or METH (alone and/or in combination)
 In vitro : Use Human brain microvascular endothelial cells (HBMVECs)
 New cell line :immortilized Human brain microvascular endothelial cells (HBMVECs)
 In vivo : Use transgenic mice over expressing gp120 or Tat.
 Permeability experiments in BBB models both in
HBMVECs and cells isolated from the transgenic mice




HBMVEC cells divided into groups:




Control
METH only (100

M)
Tat only (40nM) gp120 only (3nM)





NACA only (1mM)
Tat (40nM)+ METH (100

M) gp120 (3nM) + METH (100

M)
Tat (40nM)+ METH (100

M) + NACA (1mM) gp120 (3nM) + METH (100

M) + NACA (1mM)
Incubation times – 3 h METH + 12 h for gp120 and
Tat groups
Serine-Borate buffer used to prevent artifactual oxidation

MTS tetrazolium compound
NADH or NADPH (in living cells)
NAD + or NADP +
Colored MTS Formazan Product
(The absorbance at 490nm is proportional to the number of living cells in culture.)

140
120
100
80
60
40
20
0
0.5
5
Concentration (m M)
* p<0.05 as compared to NACA
N=4
10
*
NACA
NAC
NACA treated cells have significantly higher cell viability as compared to NAC treated cells.

Effect of gp120 on cell viability
NACA + gp120 treated cells have significantly higher cell viability as compared to gp120 alone treated cells. (Cells were treated with gp120 for 12 hours, followed by NACA treatment for 12 hours)
N=4; *P<0.05
Control vs. 5 nM gp120, **P<0.05
5 nM gp120 vs. 5 nM gp120+ 1 mM NACA

Protective Effect of NACA on Tat and METH Treated cells
90
80
70
60
50
40
30
20
*
#
10
0
Ta t
M
E
TH
M
E
TH
+
T at
M
E
TH
+
N
AC
A
* p<0.005 as compared to METH only group M
E
TH
+
T at
+
N
AC
A
# p < 0.05 as compared to METH + Tat group
Tat (100nM) +METH (4nM)+NACA treated cells have significantly higher cell viability as compared to Tat+METH alone treated group. Cell viability was measured using MTS assay.
Cells were exposed to Tat and/or METH for 24 hours.

1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
Control
***
METH 1mM
###
METH +NACA NACA0.5mM
HCMVEC/D3 Cells were pretreated with 1mM NACA for 2 h, followed by
24 h incubation with METH 1mM. NACA protected HCMVEC/D3 cells from cytotoxicity caused by METH. Values were the means +SD of three samples.Statistically different values of ***p <0.0001 were determined, compared to control.
### p < 0.0001 compared to METH 1mM group.

 The BBB cells (rat and human) do show oxidative stress when exposed to HIV proteins (gp120 and tat) and Meth.
 NACA reverses all the oxidative stress parameters to their control levels.
 How does it affect the functionality of BBB?
 Meaning, what happens to its permeability?

 TEER was measured using Millicell- electric resistance system. The cells were placed in the upper chamber of 24-well tissue inserts and were cultured for 4 days before use. Change in TEER during experimental conditions was calculated as a percentage of the corresponding baseline values. Unit: ohm/cm 2
(Ohm: resistance, cm 2 : surface area of the monolayer).

N=3
NACA treated cells have higher electric resistance as compared to Tat and gp120 alone treated groups.

 TEER decreases with gp120, tat and both
 NACA again returns it to its control level.
 Meaning:
 BBB permeability increases in the presence of gp120, tat or both.
 BBB integrity DISRUPTED.
 NACA helps keep BBB integrity.

Gp120 transgenic (SJL/(C57BL/6/SV129) and nontransgenic (C57BL/6) mice were used. Genotyping was done by PCR using DNA isolated from tail cuts. Brain samples were removed and GPx activities were measured.
Groups
Non-transgenic gp120-transgenic
GPx
(mU/mg protein)
17.22 ± 1.83
20.49 ± 1.64*
*p<0.05 compared to the control group. (N = 4/group)

 HIV proteins, gp120 and tat, along with many addictive drugs, specifically methamphetamine, can induce oxidative stress in the blood-brain barrier.
 Oxidative stress disturbs the integrity of BBB.
 New thiol antioxidants may protect the BBB against oxidative stress and help reduce the risk of neurodegenerative diseases.

1.
Ates B, Abraham LS, Ercal N.
In vitro Antioxidant and Free-Radical
Scavenging Properties of N-Acetylcysteine Amide: A Novel Thiol
Antioxidant. (Accepted, Free Radical Research ).
2.
Wu W, Abraham LS, Ogony J, Matthews R, Ercal N . Effects of N-
Acetylcysteine Amide, a Novel Thiol Antioxidant on Radiation Induced
Cytotoxicity in Chinese Hamster Ovary cells. (Accepted, Life Sciences ).
3.
Yan M, Shen J, Person MD, Kuang X, Lynn WS, Atlas D, Wong PK.
Endoplasmic reticulum stress and unfolded protein response in Atmdeficient thymocytes and thymic lymphoma cells are attributable to oxidative stress.
Neoplasia.
2008;10(2):160-7.
4.
Lee KS, Kim SR, Park HS, Park SJ, Min KH, Lee KY, Choe YH,
Hong SH, Han HJ, Lee YR, Kim JS, Atlas D, Lee YC. A novel thiol compound, N-acetylcysteine amide, attenuates allergic airway disease by regulating activation of NF-kappaB and hypoxia-inducible factor-
1alpha. E xp Mol Med . 200;39(6):756-68.
5.
Amer J, Atlas D, Fibach E. N-acetylcysteine amide (AD4) attenuates oxidative stress in beta-thalassemia blood cells. Biochim Biophys Acta .
2008;1780(2):249-55.

6 .
Penugonda S, Mare S, Lutz P, Banks WA, Ercal N .
Potentiation of lead-induced cell death in PC12 cells by glutamate: protection by Nacetylcysteine amide (NACA), a novel thiol antioxidant. Toxicol Appl
Pharmacol . 2006;216(2):197-205.
7.
Price TO., Uras F, Banks WA, Ercal N . A novel antioxidant Nacetylcysteine amide prevents gp120- and Tat-induced oxidative stress in brain endothelial cells. Exp Neurol.
2006;201(1):193-202.
8.
Bartov O, Sultana R, Butterfield DA, Atlas D. Low molecular weight thiol amides attenuate MAPK activity and protect primary neurons from
Abeta(1-42) toxicity. Brain Res.
2006;1069(1):198-206.
9.
Wu W, Goldstein G, Adams C, Matthews RH, Ercal N . Separation and quantification of N-acetyl-l-cysteine and N-acetyl-cysteine-amide by HPLC with fluorescence detection. Biomed Chromatogr.
2006;
20(5):415-22.
10. Sadan O, Bahat-Stromza M, Gilgun-Sherki Y, Atlas D, Melamed E,
Offen D . A novel brain-targeted antioxidant (AD4) attenuates haloperidol-induced abnormal movement in rats: implications for tardive dyskinesia. Clin Neuropharmacol . 2005;28(6):285-8.

11. Penugonda S, Mare S, Goldstein G, Banks WA, Ercal N .
Effects of Nacetylcysteine amide (NACA), a novel thiol antioxidant against glutamate-induced cytotoxicity in neuronal cell line PC12. Brain Res.
2005; 1056(2): 132-8.
12. Gilgun-Sherki Y, Barhum Y, Atlas D, Melamed E, Offen D. Analysis of gene expression in MOG-induced experimental autoimmune encephalomyelitis after treatment with a novel brain-penetrating antioxidant.
J Mol Neurosci . 2005;27(1):125-35.
13. Grinberg L, Fibach E, Amer J, Atlas D. N-acetylcysteine amide, a novel cell-permeating thiol, restores cellular glutathione and protects human red blood cells from oxidative stress. Free Radic Biol Med .
2005;38(1):136-45.
14. Offen D, Gilgun-Sherki Y, Barhum Y, Benhar M, Grinberg L, Reich
R, Melamed E, Atlas D. A low molecular weight copper chelator crosses the blood-brain barrier and attenuates experimental autoimmune encephalomyelitis. J Neurochem . 2004;89(5):1241-51.



NIH R15
AIDS Reagent Program










Tulin Price-Otamis
William A. Banks
Glenn Goldstein
Linu Abraham
Xinsheng Zhang
Karissa Braaten
Atrayee Banerjee
Shinya Dohgu
Burhan Ates
Wei Wu