BBA - Molecular Basis of Disease 1866 (2020) 165769

Contents lists available at ScienceDirect

BBA - Molecular Basis of Disease

journal homepage: www.elsevier.com/locate/bbadis

Cysteine becomes conditionally essential during hypobaric hypoxia and T

regulates adaptive neuro-physiological responses through CBS/H2S pathway
Shalini Mishraa, Gaurav Kumara, Aastha Chhabraa, Niroj Kumar Sethya, Neha Jaina,
⁎
Ram Niwas Meenaa, Rajkumar Tulsawanib, Dipti N. Prasadc, Bhuvnesh Kumara, Manish Sharmaa,
a
Peptide and Proteomics Division, Defence Institute of Physiology and Allied Sciences (DIPAS), DRDO, Delhi 110054, India
b
Chemistry Division, Defence Institute of Physiology and Allied Sciences (DIPAS), DRDO, Delhi 110054, India
c
Neurobiology Division, Defence Institute of Physiology and Allied Sciences (DIPAS), DRDO, Delhi 110054, India

A R T I C LE I N FO A B S T R A C T

Keywords: Brain is well known for its disproportionate oxygen consumption and high energy-budget for optimal func-
Hypobaric hypoxia tioning. The decrease in oxygen supply to brain, thus, necessitates rapid activation of adaptive pathways – the
Brain absence of which manifest into vivid pathological conditions. Amongst these, oxygen sensing in glio-vascular
Glio-vascular unit milieu and H2S-dependent compensatory increase in cerebral blood flow (CBF) is a major adaptive response. We
Cysteine
had recently demonstrated that the levels of H2S were significantly decreased during chronic hypobaric hypoxia
Cystathionine beta synthase
(HH)-induced neuro-pathological effects. The mechanistic basis of this phenomenon, however, remained to be
Hydrogen sulfide
deciphered. We, here, describe experimental evidence for marked limitation of cysteine during HH – both in
animal model as well as human volunteers ascending to high altitude. We show that the preservation of brain
cysteine level, employing cysteine pro-drug (N-acetyl-L-cysteine, NAC), markedly curtailed effects of HH – not
only on endogenous H2S levels but also, impairment of spatial reference memory in our animal model. We,
further, present multiple lines of experimental evidence that the limitation of cysteine was causally governed by
physiological propensity of brain to utilize cysteine, in cystathionine beta synthase (CBS)-dependent manner,
past its endogenous replenishment potential. Notably, decrease in the levels of brain cysteine manifested despite
positive effect (up-regulation) of HH on endogenous cysteine maintenance pathways and thus, qualifying cy-
steine as a conditionally essential nutrient (CEN) during HH. In brief, our data supports an adaptive, physio-
logical role of CBS-mediated cysteine-utilization pathway – activated to increase endogenous levels of H2S – for
optimal responses of brain to hypobaric hypoxia.

1. Introduction Taking cognizance of said bioenergetics considerations, it is not

Brain is established to have a huge energy budget and a distinctively
high rate of basal metabolism even in its resting state [1]. Intriguingly,
despite 2% of the body weight, a healthy brain receives nearly 15% of
total cardiac output [2] and accounts for disproportionate fraction
(~20%) of whole body oxygen consumption [1]. The oxygen delivered
to brain cells is utilized in complete glucose oxidation pathway (oxi-
dative phosphorylation) and consequent generation of ATP – whose
continuous generation and optimal availability in brain is critical for
sustaining energy-intensive electro-physiological processes [3,4]. Re-
cent studies have shown that such high rate of oxidative basal meta-
bolism is a dire necessity for maintaining the very state of consciousness
(responsiveness) in an organism [5].
difficult to envisage an inherent vulnerability of brain to systemic or
local hypoxia (the decrease in levels of oxygen). Besides numerous
pathological conditions (including stroke, asphyxia, traumatic brain
injury), the unique environmental conditions prevailing at high altitude
(hypobaria or reduced atmospheric pressure) naturally predispose brain
to hypoxic challenge and possible pathological effects (such as acute
mountain sickness, high altitude cerebral edema and cognitive im-
pairment amongst others) in the event of inadequate adaptation [6–8].
At optimal steady state (homeostasis), however, hypoxia induces the
activation of highly evolved physiological mechanisms (endogenous to
brain), which function to restore optimal supply of oxygen to brain
cells. Amongst these, hypoxic cerebral autoregulation – functionally
manifesting in cell types (astrocytes and endothelial cells) composing

⁎
Corresponding author at: Peptide & Proteomics Division, Defence Institute of Physiology and Allied Sciences (DIPAS), DRDO, Lucknow Road, Timarpur, Delhi
110054, India.
E-mail address: manishks77@gmail.com (M. Sharma).

https://doi.org/10.1016/j.bbadis.2020.165769
Received 6 September 2019; Received in revised form 2 February 2020; Accepted 12 March 2020
Available online 14 March 2020
0925-4439/ © 2020 Elsevier B.V. All rights reserved.
S. Mishra, et al.
glio-vascular units – is one of the earliest to be activated with decrease
in cerebral oxygen content. This phenomenon – intricately involved in
hypoxia sensing in brain – ensures rapid vasodilation and increased
cerebral blood flow (CBF), in response to decreased levels of oxygen,
through elevated production of H2S [9]. This process, in effect, aug-
ments oxygen delivery to brain cells by elevating RBC flux across spe-
cific regions of brain. Consistent with this natural propensity, inter-
estingly, several reports, published during past five decades, have
described strong evidence for early elevation of CBF and thus, increase
in oxygen delivery to brain in human subjects ascending to high alti-
tude [10,11].
A significant fraction of endogenous H2S in brain is produced by the
transsulfuration pathway enzyme cystathionine β-synthase, CBS [12].
CBS utilizes cysteine and/or homocysteine as its substrate and is prin-
cipally expressed in astrocytes [12] – the major cell type involved in
H2S production during hypoxia sensing in brain. Interestingly, utilizing
an animal model of hypobaric hypoxia, we had recently shown that the
exposure of animals to hypobaric hypoxia (HH, 7620 m) led to a sig-
nificant decrease in the levels of H2S in brain. Under such conditions,
we observed conspicuous glio-vascular dysfunction – cumulatively
evident with activated astrocytes (increased GFAP expression), brain
endothelial cells (increased vWF and sICAM-1 expression) and marked
loss of astrocyte end feet-basal lamina connection (loss of aquaporin 4-
laminin co-localization). Notably, further, we observed significantly
diminished neuro-vascular coupling, as evident from the loss of func-
tional hyperemia responses (studied by whisker-stimulation model) and
also, neuro-pathological responses (evident from neuronal apoptosis in
hippocampal region, concomitant with significant decline in spatial
reference memory) during such hypobaric hypoxia conditions [13].
Interestingly, the augmentation of endogenous H2S levels (utilizing an
exogenous donor) markedly prevented adverse effects of hypobaric
hypoxia on said glio-vascular and neuro-vascular dysfunctions, sug-
gesting functional role of H2S under these conditions [13].
While we could implicate functional involvement of H2S (as de-
scribed above) during pathological responses of brain under hypobaric
hypoxia challenge, the mechanistic basis for decrease in brain H2S le-
vels (in these settings) remained to be established. In the current study,
we sought to investigate the basis of this phenomenon and present
experimental evidence that the exposure to hypobaric hypoxia culmi-
nates in marked decrease in the levels of cysteine – both in rat brain as
well as blood samples from human volunteers ascending to high alti-
tude. This cysteine deficit functionally regulated lowered levels of en-
dogenous H2S as well as hypobaric hypoxia-induced neuro-pathological
responses in our animal model. Interestingly, we further demonstrate
that the decrease in brain cysteine levels was not due to inhibitory ef-
fects of HH on endogenous cysteine maintenance pathways but instead,
due to intrinsic physiological propensity for over-utilization of cysteine
by CBS-mediated biological processes during hypobaric hypoxia con-
ditions. We utilize CBS-inhibitor, AOAA, to experimentally support this
proposition in the animal model. We, further, show that the cysteine
pro-drug (NAC) – which effectively maintained endogenous levels of
cysteine despite its elevated utilization during HH – markedly pre-
vented HH-induced decrease in brain H2S levels as well as neuro-pa-
thological effects observed under such conditions. In view of these
observations, we propose cysteine as a conditionally essential factor,
necessitating supplementation for optimal brain responses to hypobaric
hypoxia.
2. Methods
2.1. Animal model and ethics statement
Male Sprague Dawley rats weighing 220–250 g were obtained from
Animal House Facility, Defence Institute of Physiology and Allied
Sciences (DIPAS, DRDO), Delhi. The study design was approved by the
‘Animal Ethical Committee’ of the Institute (DIPAS, DRDO). All the
2
BBA - Molecular Basis of Disease 1866 (2020) 165769
guidelines from ‘Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA)’ were strictly followed during
execution of experiments. The animals (4 rats per cage) were main-
tained under optimum conditions of temperature (25 ± 2 °C), relative
humidity (55 ± 2%), food and water (ad libitum). They were exposed
to 12 hr light and dark cycle in the animal house.
2.2. Human studies and ethics statement
Human studies were conducted in strict compliance with the ethical
standards of Indian Council of Medical Research (ICMR). An informed
consent was obtained from the subjects as per ‘Declaration of Helsinki’.
The fasting blood samples were collected, in EDTA vacutainers (Becton
Dickinson), from healthy (without any pre-existing clinical condition,
non-smokers) male volunteers (n = 12), 22–30 years of age. These
samples were collected after 7 days of ascent to Loma (4358 m above
sea level), Ladakh, India on the morning of 8th day. For normoxic
controls, equal number of healthy, age-matched, male subjects were
included and blood samples collected (following similar protocol) at
Pathankot, Punjab, India (332 m above sea level).
2.3. Hypobaric hypoxia exposure
The animals were exposed to simulated hypobaric hypoxia at a
constant pressure of 282 Torr (equivalent to an altitude of 7620 m, 8%
O2) in the decompression chamber for 1, 3 and 7 days. The rate of
ascent and descent to altitude was kept constant at 300 m/min. Fresh
air was continuously flushed at a fixed rate of 8 L/min to prevent CO2
build-up inside the chamber. The temperature and relative humidity of
the chamber were maintained, respectively, at 25 ± 2 °C and
55 ± 2%. The animals were exposed to 12 h of light and dark cycle in
the hypobaric hypoxia chamber to maintain circadian cycle (matching
normoxic controls). The chamber was opened once daily at a fixed time
for 15–20 min to monitor health of animals, administer drug and refill
food/water. The animals from the normoxic groups (with or without
drug treatment) were kept at normal atmospheric pressure under si-
milar conditions.
2.4. Drug administration
The animals, as per their assigned group, received a daily dose of
Sodium hydrogen sulfide, NaHS (Cayman chemical), N-Acetyl L-
Cysteine (NAC, Sigma) and/or Aminooxyacetic acid hemihydrochloride
(AOAA, Sigma) during complete hypobaric hypoxia exposure. NaHS
was dissolved fresh in 1.5 M PBS (pH 7.4) and administered (in-
traperitoneal) at a dose of 4 mg/kg of body weight per day, as described
previously [13]. NAC, dissolved in saline (0.9% NaCl, pH adjusted to
7.4 utilizing 2 N NaOH), was gavaged (orally) at a dose of 300 mg per
kg of body weight per day [14–16], beginning 3 days prior to HH ex-
posure. AOAA was prepared in saline (pH adjusted to 7.4 utilizing 1 N
NaOH) and administered intraperitoneal (starting from the day of HH
exposure) at a dose of 5 mg per kg of body weight per day [17]. The
non-drug groups received parallel treatment with the vehicle (matched)
through similar route.
2.5. Morris water maze (MWM) test
MWM test was performed to assess spatial reference memory of
animals as described previously [18]. Briefly, the animals were trained
to learn spatial location of a hidden platform in a circular water tank.
During the 5 day-training period, each rat was subjected to 4 sessions of
learning per day with an interval of 5 min between two trials. The rats
were gently placed in a different quadrant of water tank during each
trial with face towards the wall. The starting point in each quadrant was
kept fixed for all the animals. The animals were trained to search for a
submerged platform permanently located in the center of first quadrant.
S. Mishra, et al.
On reaching the platform, rats were allowed to sit for 30 s, dried
completely and then, placed back in their cage. In the event of failure to
reach the platform within 60 s, the animals were manually guided to
the platform. An overhead camera coupled to a computer-assisted
tracking system, ANY-Maze (Stoelting, USA) was used to record the
position of animal in the water tank. Latency (time taken by animal to
reach the hidden platform) and path length (distance travelled by an-
imal to reach the platform) were recorded during the experiments.
Probe trial (PT) was conducted after 5 days of training (on the 6th day,
just before initiation of HH exposure). For this test, the platform from
tank was removed and parameters, including latency (time taken by the
animal to first arrive to the coordinates where platform was placed
during training) and path length (distance travelled by rat to reach
these coordinates) were recorded. After HH exposure, animals from all
the groups were subjected to MWM test, conducted with the platform in
place (at the center of first quadrant). Latency and path length were
recorded and analyzed.
2.6. Cysteine and methionine estimation by RP-HPLC analysis
The brain tissue (cortex) was lysed in RIPA buffer (Sigma) and de-
proteinized utilizing 10% perchloric acid. The supernatant was passed
through 0.2 μm filter before utilizing for RP-HPLC analysis. The in-
strument was equipped with 515 HPLC pump, 717 plus auto-sampler
and 2487 PDA detector (Waters Corporation, USA). The separation was
achieved utilizing PicoTag 3.9 mm × 300 mm column by maintaining
isocratic flow rate of 1 ml per min for the mobile phase (40 mM am-
monium phosphate, 8 mM heptane sulphonic acid and 3.6% acetoni-
trile). The concentration of cysteine and methionine in the tissue
samples were estimated by comparing to respective standards and the
data presented as the fraction of amino acid amount observed in nor-
moxic groups.
2.7. Biochemical estimation of cysteine
The levels of cysteine in samples obtained from animals (plasma,
brain tissues) and human volunteers (plasma) were estimated utilizing
a commercially available fluorometric kit (ab211099, Abcam) fol-
lowing manufacturer's protocol.
2.8. Homocysteine estimation
The levels of homocysteine in the brain tissues (cortex, PFC, hip-
pocampus) and plasma samples were measured utilizing commercially
available kits, namely homocysteine ELISA kit (STA-670, Cell Biolabs
Inc.) and fluorometric assay kit (K531, BioVision Incorporated) re-
spectively, following manufacturer's protocol.
2.9. H2S estimation
The levels of H2S were measured employing amperometric H2S-
specific probe [19,20]. The H2S sensor (ISO-H2S-100-CXX) was con-
nected to free radical analyzer (TBR1025, World Precision Instruments,
USA) and polarized in 0.01 M PBS to obtain a stable background current
within the range of 0.1 to 2 nA. The calibration curve was, subse-
quently, prepared utilizing sodium sulfide (Na2S) at varying con-
centrations (100, 200, 400, 800 and 1600 nM). For H2S estimation in
the brain tissues, rats were anesthetized (ketamine, 50 mg per kg body
weight and xylazine, 10 mg per kg body weight, i.p.) and trans-cardially
perfused using 0.01 M ice-cold PBS to completely remove blood from
the brain tissues. The tissue samples were isolated, snap-frozen in liquid
nitrogen and homogenized at a concentration of 40 mg/ml in de-gassed
0.01 M PBS containing 50 μM diethylenetriamine pentaacetic acid
(DTPA). The supernatant was collected and H2S estimation performed
utilizing the polarized sensor. A minimum of three dilutions per sample
was utilized for estimation. For each group, H2S levels from a minimum
3
BBA - Molecular Basis of Disease 1866 (2020) 165769
of 5 animals were recorded and depicted as bar graphs shown in the
figure.
2.10. Cerebral blood flow measurement and functional hyperemia analysis
Cerebral blood flow (CBF) was measured employing laser Doppler
flowmetry as per established protocol [21]. Briefly, animals were an-
esthetized with ketamine (50 mg per kg body weight, i.p.) and their
skull was exposed. To measure CBF, the skull was thinned to translu-
cency at Bregma coordinates AP: 3.0 mm; L: 7.0 mm using stereotaxic
micro motor high-speed drill (Stoelting, USA). A needle probe (TSD144,
BIOPAC Inc., USA) was stably held at this region to monitor the total
blood cell flux (the red blood cells travelling through this region be-
tween particular time duration). LDF100C (Laser Doppler blood per-
fusion monitor, BIOPAC Inc., USA) was used to measure CBF with stable
readings for 5 min. The mean blood perfusion unit (BPU) was calculated
utilizing AcqKnowledge software (BIOPAC) where BPU is defined as an
arbitrary unit used to express the total blood cell flux. It is proportional
to the product of mean velocity and number of blood cells travelling
through this specific region. For functional hyperemia studies, the
whisker stimulation method was utilized and adapted from previously
published protocols [13,22]. Briefly, the whiskers of animals were sti-
mulated for 10–15 min using a fine brush and the mean BPU was es-
timated employing laser Doppler flowmetry.
2.11. Western blotting
30 μg of protein extracted from brain tissues or 10 μg of plasma
protein samples were separated on SDS-PAGE and transferred onto
PVDF membrane. The membrane was blocked with 5% BSA overnight
at 4 °C and incubated with specific primary antibodies at specified di-
lutions, namely CBS (ab96252, Abcam) at 1:1000 (tissue samples) or
1:2000 (plasma samples) working dilution; EAAT1 (ab416, Abcam) at
1:1500 working dilution; EAAT2 (ab41621, Abcam) at 1:1500 working
dilution; xCT (PA-1-16893, ThermoFisher) at 1:1500 working dilution;
β-Actin (SAB2100037, Sigma) at 1:5000 working dilution and Tubulin
(STJ96142, St. John's Laboratory) at 1:3000 working dilution for 2 h at
25 °C followed by incubation with suitable HRP-conjugated secondary
antibody (A0545, Sigma) at 1:15000 or 1:20000 working dilutions. The
blots were, subsequently, developed using SuperSignal™ West Femto
Maximum Sensitivity Substrate (Thermo Scientific, 34095). The re-
lative expression between various groups was calculated utilizing nor-
malized mean intensities (specific target/internal control) – estimated
utilizing widely used software, Image J (https://imagej.nih.gov/ij/) –
and represented as bar graphs (summarizing data from 3 independent
experiments).
2.12. Immunofluorescence assays
Rats were trans-cardially perfused with 0.01 M ice-cold PBS under
anesthesia (ketamine, 50 mg per kg body weight and xylazine, 10 mg
per kg body weight, i.p.) and fixed using 4% paraformaldehyde (PFA),
prepared in 0.01 M PBS. The whole brain was isolated and stored in 4%
PFA. For immunofluorescence studies, the intact brain tissue was
treated with increasing sequential concentration of sucrose (10%, 20%
for 24 h and 30% for 48–72 h). The tissue samples were, subsequently,
cut at 30-μm thickness in the hippocampal region using a cryostat. The
brain sections were washed with PBST (PBS with 0.1% Triton X-100)
and heat-induced antigen retrieval was done using sodium citrate
containing 0.05% Tween 20 (pH 6.0). Sections were blocked in 3% BSA
for 1 h at room temperature and incubated overnight (at 4 °C) with
desired primary antibody – GFAP (ab7260, Abcam) at 1:1000 working
dilution; vWF (ab6994, Abcam) at 1:500 working dilution; Aquaporin 4
(ab9512, Abcam) at 1:500 working dilution and Laminin (STJ93894,
St. John's laboratory) at 1:500 working dilution. Following this, sec-
tions were incubated with appropriate Alexa Fluor labeled secondary
S. Mishra, et al.
antibody (A11034, A11037, A11029, ThermoFisher), at working dilu-
tion of 1:1000, for 2 h at room temperature and counterstained with
4′,6-diamidino-2-phenylindole (DAPI, Sigma) utilized at a concentra-
tion of 2 μg/ml. The sections were then mounted using gelatin-based
mounting media and imaged under fluorescence microscope (Olympus
BX61, Japan) with attached digital camera. Confocal microscope
(Andor Technology, USA) was utilized to image co-labeled immuno-
fluorescence sections. A minimum of 3 sections from each animal of a
group (containing 3 animals) was acquired to obtain at least 2–4 dis-
tinct, random fields per section (preferably from both hemispheres).
Intensity thresholding method was used to quantitate mean intensities
utilizing Image J, NIH, USA (https://imagej.nih.gov/ij/).
2.13. Statistical analysis
Acquisitions of various experimental data were performed in an
unbiased manner. Statistical analyses were done using Prism 6 software
(GraphPad, San Diego, CA). Data in bar graphs are expressed as
Mean ± standard deviation (SD). Mean values between two groups
were compared employing two-tailed, unpaired Student's t-test.
Multiple group comparisons were done utilizing one-way analysis of
variance (ANOVA) with Tukey's post-hoc test. For Morris water maze
assay, the post hoc analysis, to compare the differences between groups,
wherever found significant, was performed using Bonferroni multiple
comparison test. p-value < 0.05 was considered as statistically sig-
nificant.
3. Results
3.1. Chronic hypobaric hypoxia culminates in marked lowering of cysteine
levels in brain
As described above, we previously observed that the exposure of
animals (rat) to hypobaric hypoxia lowered the levels of H2S in brain
and this phenomenon causally regulated early gliovascular dysfunction
and impending neuro-pathological effects [13]. We, therefore, sought
to understand the mechanistic basis of this critical phenomenon, uti-
lizing our animal (rat) model. The scheme of experiments is depicted in
Fig. 1A. We began by investigating the levels of expression of CBS – the
key enzyme responsible for H2S generation in brain [23] – under hy-
poxic conditions. As evident from western blotting data depicted in
Fig. 1B & C, we observed that the expression of CBS increased sig-
nificantly in response to HH, at all time points studied (1, 3 and 7 days
post HH exposure). This observation is consistent with a previous report
[24] demonstrating increased expression of CBS (transcript and pro-
tein) in neuronal cell lines during hypoxia and its regulation by HIF-1α
– the master regulator of hypoxic gene expression. Intrigued by such
apparently conflicting observations (increased CBS expression but de-
creased H2S levels in brain), we next estimated the endogenous levels of
cysteine – substrate utilized by CBS for generation of H2S – in brain,
employing RP-HPLC (Fig. 1D & E). As shown in Fig. 1E, we re-
producibly observed that the levels of endogenous cysteine decreased
significantly in the cortical region of brain under HH conditions (post 1,
3 and 7 days of HH exposure), raising a possibility that substrate lim-
itation during HH could be the likely reason for decreased levels of H2S
in brain.
Since CBS also utilizes homocysteine as its substrate, we ad-
ditionally measured the levels of homocysteine in brain (cortex) under
these conditions. As shown in Fig. 1F, we observed that the levels of
homocysteine were modestly reduced during HH, as compared to nor-
moxic animals. This observation appears consistent with the phenom-
enon of increased expression (and in every likelihood activity) of CBS
(homocysteine utilizing enzyme).
We additionally validated this important observation (pertaining to
levels of CBS substrate) in independent experiments, employing a dif-
ferent technical approach (biochemical estimation). In these
4
BBA - Molecular Basis of Disease 1866 (2020) 165769
experiments as well, we observed that hypobaric hypoxia culminated in
significant decrease in the levels of cysteine (Fig. 1G) and homocysteine
(Fig. 1H) in the brain (cortex).
Since we had observed effects of hypobaric hypoxia on memory, we
also estimated the levels of cysteine and homocysteine in the major
regions (hippocampus and prefrontal cortex, PFC) of brain regulating
this phenomenon (Fig. 1I–L). Consistent with our observations made in
Fig. 1E–H, we observed a significant reduction in the levels of cysteine
(Fig. 1I & K) and homocysteine (Fig. 1J & L), respectively in hippo-
campus and PFC as well.
We further sought to determine if the phenomenon observed in
brain could also be recapitulated utilizing plasma samples obtained
from animals exposed to hypobaric hypoxia. Intriguingly, as shown in
Fig. 1M–P, the increase in levels of expression of CBS (Fig. 1M & N) and
the decrease in levels of cysteine (Fig. 1O) and homocysteine (Fig. 1P)
could also be observed in plasma samples of the animals challenged
with hypobaric hypoxia.
3.2. Evidence for reduction in the levels of free cysteine in human volunteers
ascending to high altitude
In view of encouraging observations made with plasma samples of
animals exposed to hypobaric hypoxia, we sought to test the general
significance of phenomenon pertaining to levels of CBS, cysteine and
homocysteine (as described in Fig. 1) in plasma samples from human
volunteers ascending to high altitude (hypobaric hypoxia). We, there-
fore, estimated the levels of CBS, free cysteine and homocysteine in
plasma samples collected from human individuals, after 7 days of as-
cent to high altitude (4358 m). As evident from Fig. 2A and B, the
expression of CBS was significantly elevated in individuals ascending to
high altitude (HA), as compared to samples obtained from individuals
at sea level (SL). We further observed that the levels of free cysteine and
homocysteine were markedly reduced in these individuals (HA) in
comparison to normoxic (SL) controls (Fig. 2C & D). Taken together,
our observations (Figs. 1 & 2) suggested a conserved biological response
(of increase in the expression of CBS and decrease in the levels of free
cysteine) during exposure to hypobaric hypoxia.
3.3. Increased expression of cystine-glutamate anti-porter (xCT) and
sodium-dependent glutamate transporters (EAAT1 & 2) in hypoxic brain
We, next, sought to investigate the possible reason for decrease in
the levels of brain cysteine, during hypobaric hypoxia, utilizing our
animal model. Two critical biological processes continuously function
to maintain cellular cysteine pools (diagrammatically depicted in
Fig. 3A). These include methionine (an essential amino acid)-dependent
de novo biosynthesis and xCT (cystine-glutamate anti-porter)-mediated
in-flux of cystine into cytoplasm from extracellular regions [25]. We,
therefore, studied the possible effects of HH on both these processes in
brain. The scheme of experiment is diagrammatically depicted in
Fig. 3B. As shown in Fig. 3C, we did not observe significant change in
the endogenous levels of methionine after 1 day of exposure to hypo-
baric hypoxia (HH) – a time point at which the levels of cysteine were
significantly lowered in brain (Fig. 1E). We inferred these datasets
(Figs. 1E and 3C) as suggesting that HH-induced lowering of en-
dogenous levels of cysteine was unlikely due to limitation of methio-
nine, at least at this stage, in brain.
We next studied effect of HH on the expression of xCT (protein),
employing western blotting assays, in brain samples. As shown in
Fig. 3D, we reproducibly observed increase in the expression of this
anti-porter protein in response to hypobaric hypoxia. In this regard, it is
interesting to note that HIF-1α – the master regulator of hypoxia re-
sponses [26] – has been shown to regulate the expression of this
transporter [27,28].
Being a cationic amino-acid anti-porter, import of each cystine by
xCT comes at the cost of one glutamate molecule exported out of cells
S. Mishra, et al. BBA - Molecular Basis of Disease 1866 (2020) 165769

A. G. I. K.
Acclimatization HH
Blood collection
Days -1 0 1 2 3 4 5 6 7 Brain tissue isolation

CBS, CYS and

Sacrifice (S) S S S HCYS estimation
H. J. L.
B. 91 kDa C.
71 kDa
CBS
55 kDa
36 kDa

55 kDa
-Actin
36 kDa
M. O.

D.

N.
95 kDa
72 kDa
CBS
(Plasma) 55 kDa
P.
35 kDa
E. F.
72 kDa
55 kDa
Tubulin
35 kDa

Fig. 1. Hypobaric hypoxia modulates H2S generation pathway in brain. A) Diagrammatic representation of experimental scheme. B) Representative western im-
munoblot showing expression of CBS in brain of animals exposed to hypobaric hypoxia for 1, 3 and 7 days. β-Actin was employed as internal control for sample
loading. C) Bar graph representing normalized mean intensities (CBS expression normalized to β-Actin signal) at indicated time points (n = 3). The signal intensities
were calculated utilizing Image J, NIH. D) Representative RP-HPLC chromatogram showing retention time (in min) of amino acids, cysteine and methionine. AU:
Arbitrary units. E) Bar graph depicting relative changes (fraction of normoxic controls) in the levels of cysteine, in rat brain, after 1, 3 and 7 days of hypobaric
hypoxia exposure, n = 3. F) Bar graph representing relative changes (fraction of normoxic controls) in the levels of homocysteine in brain tissues of animals exposed
to normoxia or hypobaric hypoxia (1 Day), n = 3. G–H) Bar graphs representing the levels of cysteine, cys (G) and homocysteine, hcys (H) in cortex of animals
exposed for 1 day to HH, n = 3. I–J) Bar graphs representing the levels of cysteine (I) and homocysteine (J) in hippocampus of animals exposed for 1 day to HH,
n = 3. K–L) Bar graphs representing the levels of cysteine (K) and homocysteine (L) in prefrontal cortex of animals exposed for 1 day to HH, n = 3. M) Bar graph
representing the normalized mean intensities (CBS expression normalized to tubulin signal) in plasma samples of animals exposed to 1 and 7 days of HH exposure,
n = 3. N) Representative western immunoblot showing expression of CBS and tubulin (internal control for sample loading) in plasma of animals exposed for 1 and
7 days to HH. O–P) Bar graphs representing the levels of cysteine (O) and homocysteine (P) in plasma of animals exposed for 1 day to HH (n = 3). Mean ± SD values
are shown. *p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001. Abbreviations – Normoxic control (N); Hypobaric Hypoxia exposure for 1 day (HH_1D), 3 days (HH_3D) and 7 days
(HH_7D).

and thus, increasing the possibility of glutamate build-up in extra-
cellular regions, if not removed promptly (by astrocytes). The elevated
concentration of glutamate, a critical excitatory amino acid, under such
settings has been shown to culminate in excito-toxic neuronal damage
[28]. In astrocytes, EAATs are important class of sodium-dependent
glutamate transporters – which can potentially offset building gluta-
mate levels in wake of increased xCT activity. We, therefore, examined
the levels of expression of EAAT1 & 2 in response to HH exposure. As
shown in Fig. 3E & F, the expression of EAAT1 & 2 were also increased
in the brain, under hypobaric hypoxia conditions. Taken together, our
experiments (Fig. 3C–F) clearly suggested that the pathways involved in
maintenance of cellular cysteine levels were not inhibited (and rather
positively modulated) under hypobaric hypoxia conditions.
3.4. Excessive endogenous utilization of cysteine diminishes its levels in the
hypoxic brain
Our observations till this stage presented an intriguing paradigm
involving decreased levels of endogenous cysteine in hypoxic brain
despite increased expression of proteins (CBS, xCT, EAAT1 & 2) in-
volved in maintenance of endogenous cysteine levels under these con-
ditions. We, therefore, sought to experimentally test a hypothesis that
cellular cysteine was being excessively utilized – beyond the homeo-
static replenishment capacity of brain cells – in wake of elevated phy-
siological requirement of H2S during hypobaric hypoxia. If valid, three
independent approaches, in principle, would be expected to augment
steady state levels of the substrate (cysteine) – being utilized by the

5
S. Mishra, et al.
A. SL HA_7D B.
1 2 3 1 2 3
95 kDa
72 kDa
CBS
55 kDa
35 kDa
Tubulin 55 kDa
35 kDa
C. D.
enzyme (CBS) to generate H2S. These included: 1) direct augmentation
of substrate, 2) inhibition of enzyme utilizing the substrate and 3)
supplementation of product to shift the equilibrium towards substrate
side. As diagrammatically depicted in Fig. 4A, we utilized a cysteine
pro-drug, NAC (to directly increase cellular cysteine pool), a pharma-
cological inhibitor of CBS, AOAA, and a fast-acting H2S donor, NaHS, in
our animal model, followed by careful measurements of levels of cy-
steine and methionine in the brain (Fig. 4B–D) under normoxic or HH
conditions. As evident from Fig. 4B, the oral administration of NAC
(prior to hypoxic challenge) markedly curtailed HH-induced lowering
of endogenous brain cysteine levels. Notably, further, this treatment did
not produce any significant change in the endogenous methionine level
(Fig. 4B). Interestingly, the pre-treatment of animals with both AOAA
(Fig. 4C) and NaHS (Fig. 4D) also resulted in marked increase in cy-
steine levels in the brain, under HH conditions, with no significant
change in endogenous methionine levels. Taken together (Fig. 4B–D),
thus, we inferred these results as supporting two important phenomena
manifesting in brain: 1) a somewhat disproportionate utilization of
cysteine – far exceeding the endogenous replenishment potential –
during exposure to hypobaric hypoxia and 2) the involvement of H2S-
generating enzyme (CBS) in consuming cysteine under these conditions.
3.5. Cysteine supplementation preserves the levels of endogenous H2S and
cerebral blood flow dynamics during hypobaric hypoxia
Since the prior administration of cysteine pro-drug, NAC, effectively
maintained brain cysteine levels during hypobaric hypoxia, we next
sought to establish its effects on the levels of H2S in brain as well as
cerebral blood flow (CBF) dynamics – a process that we previously
demonstrated to be dependent on endogenous H2S levels during hy-
pobaric hypoxia [13]. The experimental scheme is diagrammatically
presented in Fig. 5A. As shown in Fig. 5B, augmentation of cysteine (by
NAC) markedly prevented HH-induced decrease in brain H2S levels.
6
BBA - Molecular Basis of Disease 1866 (2020) 165769
Fig. 2. Ascent to high altitude modulates levels of
CBS, free cysteine and homocysteine in human vo-
lunteers. A) Representative western immunoblot
showing expression of CBS in plasma samples from
human volunteers at sea level and after 7 days of
stay at high altitude (4358 m). Tubulin was em-
ployed as internal control for sample loading. B) Box
plot representing normalized mean intensities (CBS
expression normalized to Tubulin) in indicated
groups, n = 10. C–D) Box plots representing the
levels of cysteine, n = 12 (C) and homocysteine,
n = 10 (D) as estimated in plasma samples of human
volunteers from indicated groups. **p ˂ 0.01, ***p ˂
0.001. Abbreviations – Sea Level (SL), High Altitude
exposure for 7 Days (HA_7D).
This observation underscores the natural propensity of brain to increase
H2S for optimal acclimatization to HH and also, the dependence of this
phenomenon on endogenous cysteine levels (understandably, thus, the
potential to become limiting factor during HH).
We had previously shown that H2S is critical for CBF dynamics and
functional hyperemia responses during hypobaric hypoxia conditions
[13]. We, therefore, next sought to investigate the cerebral blood flow,
employing laser Doppler flowmetry and functional hyperemia re-
sponses, employing whisker stimulation model, under these conditions
(HH exposure with or without NAC pre-treatment). As shown in Fig. 5C,
we observed marked increase in the CBF in animals supplemented with
NAC in response to HH challenge. Notably, CBF in the NAC-treated
normoxic animals was only modestly elevated. Cumulatively, we in-
ferred these results as suggesting that the propensity for increasing CBF
is an inherent, physiological brain-response to HH and critically gov-
erned by endogenous levels of H2S and cysteine.
We, additionally, studied the functional hyperemia responses –
critical for multiple higher order neurological functions – utilizing
whisker stimulation model [13,22]. For the same objective, we re-
corded the relative changes in cerebral blood flow in contralateral
cortex after rhythmic whisker stimulation (functional hyperemia). We
observed that such functional hyperemia responses were significantly
better in the animals pre-treated with NAC, in both normoxic and HH
environment, as compared to untreated (Fig. 5D & E). These observa-
tions further supported the efficacy of maintaining cysteine levels
during exposure to hypobaric hypoxia.
3.6. NAC preserves glio-vascular homeostasis during hypobaric hypoxia in a
CBS-dependent manner
NAC is reported to produce beneficial effects in brain under multiple
pathological conditions – including hypobaric hypoxia [29]. In view of
our observations that NAC augmented cysteine and H2S during HH, we
S. Mishra, et al. BBA - Molecular Basis of Disease 1866 (2020) 165769

A. B. Acclimatization HH
Methionine estimation,
Days -1 0 1 Brain Tissue isolation
xCT, EAAT1 and EAAT2
(Cortex)
expression study

Sacrifice (S) S

D. E. F.

95 kDa
72 kDa 95 kDa
72 kDa
55 kDa EAAT1 72 kDa
55 kDa EAAT2
xCT 55 kDa
C. 36 kDa
36 kDa
28 kDa 36 kDa

55 kDa 55 kDa
55 kDa -Actin
-Actin
-Actin
36 kDa 36 kDa
36 kDa

Fig. 3. Evidence for increase in the expression of major cationic transporters (involved in cysteine dynamics) in brain during hypobaric hypoxia. A) Diagrammatic
representation of transsulfuration and cystine transport pathways. B) Diagrammatic representation of experimental scheme. C) Bar graph representing relative
changes (fraction of normoxic controls) in the levels of methionine (as estimated through RP-HPLC) in brain tissues of animals exposed to normoxia or hypobaric
hypoxia for 1 day (n = 3). D–F) Representative western immunoblots and corresponding bar graphs showing change in mean intensity (expression) of xCT (D),
EAAT1 (E) and EAAT2 (F) in brain tissues of animals exposed to hypobaric hypoxia for 1 day (n = 3). β-Actin was used as loading control. Mean ± SD values are
shown. *p ˂ 0.05, **p ˂ 0.01 and NS - not significant. Abbreviations – CBS: cystathionine β-synthase, CSE: cystathionine-γ-lyase, CR: cystine reductase, xCT: cystine-
glutamate antiporter, EAAT: excitatory amino acid transporter, N: Normoxic control, HH_1D: Hypobaric Hypoxia exposure for 1 day.

sought to determine the CBS-dependence of effects produced by NAC glio-vascular perturbation, the treatment of animals with AOAA under
(cysteine supplementation) during chronic hypobaric hypoxia en- these conditions (NAC supplementation during HH) markedly reversed
vironment. For this objective, we inhibited this enzyme (CBS), em- these effects. Cumulatively, the observations from experiments depicted
ploying AOAA, alongside NAC administration and subsequently, chal- in Fig. 6B–F clearly suggested that CBS was critical for effects produced
lenged these animals with chronic HH stress (7 days, Fig. 6A). Since we by NAC under HH conditions and thus, corroborating our proposition
had previously shown that the pathological effects of HH involve early about role of endogenous H2S during hypobaric hypoxia.
glio-vascular perturbation [13]; we recorded parameters indicating
endothelial dysfunction (vWF expression levels), astrocyte activation
3.7. CBS activity is critical for prevention of HH-induced impairment of
(GFAP expression levels) and structural integrity of glio-vascular units
spatial reference memory by NAC
(Aqp4-laminin dual staining) (Fig. 6B–F) utilizing the brain sections
from animals in various groups (experimental scheme depicted in
We next sought to investigate the CBS-dependence of effects pro-
Fig. 6A and specific groups indicated in individual panels).
duced by cysteine (NAC) supplementation on chronic hypobaric hy-
We observed that the supplementation of NAC to the animals sig-
poxia-induced impairment of spatial reference memory. For the same
nificantly reduced HH-induced endothelial and astrocyte activation – as
objective, we utilized groups as described in Fig. 6 and studied effects
estimated by vWF (Fig. 6B & C) and GFAP (Fig. 6D & E) staining, re-
on spatial reference memory, in these animals, as estimated by Morris
spectively, in brain sections. Interestingly, the inhibition of CBS (em-
water-maze test. The experimental strategy employed in these assays is
ploying AOAA) under these conditions (NAC supplementation) mark-
shown in Fig. 7A. As evident from Fig. 7B & C; by 5th day of training,
edly diminished the efficacy of NAC in preventing endothelial (Fig. 6B
the animals learned to reach the submerged platform utilizing the
& C) and astrocyte activation (Fig. 6D & E), suggesting that CBS activity
shortest possible path (indicated as path length) in minimum time
(and thus, endogenous H2S levels) mediated these effects.
duration (described by latency). The exposure to HH, however, culmi-
We, next, utilized confocal microscopy to study glio-vascular
nated in marked perturbation of this spatial reference memory, as
structural integrity, under these conditions. In a normal physiological
evident from significant increase in the path length and latency
state, the glio-vascular unit comprises of an astrocyte end feet (in-
(Fig. 7D–F). The animals pre-treated with NAC were markedly less af-
dicated by Aqp 4) tightly connected to basal membrane/lamina (in-
fected by exposure to hypobaric hypoxia and navigated significantly
dicated by laminin) through its interaction with Agrin. During specific
faster to the platform in these assays (Fig. 7D–F). Similar to results
neuro-pathophysiological events culminating in glio-vascular pertur-
described in Fig. 6, the treatment of animals with AOAA (CBS in-
bation, a mark disruption of this interaction is observed.
hibitor), however, curtailed the effects of NAC on preservation of higher
Experimentally, such a phenomenon can be observed as markedly
order neurological function (spatial reference memory) during hypoxia.
segregated Laminin and Aqp 4 signals in confocal microscopy. As
Taken together, we inferred that CBS activity was critical for protective
shown in Fig. 6F, while NAC supplementation prevented HH-induced
effects of NAC during chronic hypobaric hypoxia.

7
S. Mishra, et al. BBA - Molecular Basis of Disease 1866 (2020) 165769

A. B.
NAC Cysteine H2S NaHS
CBS

AOAA

1. Brain Cysteine Estimation

2. Brain Methionine Estimation

Drug Dosage & HH-exposure Schedule

Acclimatization HH
Days
-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1
AOAA
NaHS NAC

C. D.

Fig. 4. Evidence for excessive utilization of cysteine in CBS-mediated H2S generation pathway during hypobaric hypoxia. A) Diagrammatic representation of the
experimental scheme. B–D) Levels of cysteine and methionine were measured in brain tissue extracts of animals exposed to hypobaric hypoxia for 1 day, in presence
or absence of treatment with NAC (B), AOAA (C) and NaHS (D), employing RP-HPLC. Bar graphs (Mean ± SD) depict changes in the cysteine and methionine levels
(represented as fraction of normoxic control, n = 3). *p ˂ 0.05, ***p ˂ 0.001 and NS - not significant. Abbreviations – CBS: cystathionine β-synthase, NAC: N-
acetylcysteine, AOAA: Aminooxyacetic acid, NaHS: Sodium hydrogen sulfide, N: Normoxic control; HH_1D: 1 day of HH exposure; HH_1D_Cys: NAC pre-treated with
1 day of HH exposure; N_Cys: NAC pre-treated normoxic control, HH_1D_AOAA: AOAA pre-treatment with 1 day of HH exposure; N_AOAA: AOAA pre-treated
normoxic control; HH_1D_NaHS: NaHS pre-treatment with 1 day of HH exposure; N_NaHS: NaHS pre-treated normoxic control.

The findings from current study, as integrating in broad cysteine
homeostasis pathway, are diagrammatically represented in Fig. 8 and
discussed in section below.
4. Discussion
Our data, presented here, revealed three important phenomena: 1)
The exposure to hypobaric hypoxia culminated in marked decrease in
the endogenous levels of cysteine despite positive regulation of cysteine
maintenance pathways under these conditions, 2) An adaptive pro-
pensity to utilize cysteine – in a CBS-dependent manner – causally
regulated its decrease during chronic hypobaric hypoxia exposure and
3) The exogenous supplementation of cysteine (employing cysteine pro-
drug, NAC) markedly ameliorated this deficiency during HH and also,
prevented neuro-pathological effects, in a CBS/H2S dependent manner,
under these conditions. A factor endogenously produced in sufficient
amounts to meet requisite physiological needs during normal condi-
tions but requiring supplementation under stressful environmental cues
is classified as ‘conditionally essential nutrient (CEN)’ [30,31]. Such
supplementation strategy is termed as ‘orthomolecular therapy’ [31]. In
view of our observations (described above) and specific considerations
for CEN, it is reasonable to suggest cysteine as a conditionally essential
factor during chronic hypobaric hypoxia and propose cysteine supple-
mentation strategy as a potential orthomolecular therapy for high al-
titude acclimatization. Notably, similar phenomenon (of L-cysteine be-
coming conditionally essential) is also known to manifest during other
pathological/stressful conditions, including tumor proliferation/sur-
vival [32] and low-birth-weight preterm infants [33]. It is intriguing
that both these conditions (solid tumors and gestation) involve hypoxic
microenvironments and thus, raise an interesting possibility for general
association between hypoxia and ensuing cysteine limitation.
Cysteine serves a substrate for CBS (the enzyme involved in en-
dogenous H2S production) in brain and a rate-limiting precursor of GSH
[25,34,35]. Both H2S and GSH are of unparalleled significance for cyto-
protection and redox homeostasis in the nervous system [36–38]. De-
spite such critical niche, the endogenous levels of free cysteine in brain
are, intriguingly, known to be significantly lower than other organ
systems [34,39]. In normal physiological settings, most of the free cy-
steine appears to be mobilized to GSH synthesis pathway and thus, the
levels of GSH, at steady state, is an order of magnitude higher than free
cysteine in brain [34]. During hypoxia, the demand for free cysteine
increases further due to elevated production (cysteine-dependent) of
H2S – which not only regulates hypoxic cerebral autoregulation [9] but
also GSH synthesis per se by multiple mechanisms [37]. In view of these
facts, the observation that chronic hypobaric hypoxia strained the in-
tracellular cysteine pool appears reasonably plausible. In this regard
(lowering endogenous cysteine pool in hypoxic environment), an in-
dependent support emerges from a recent study [40], which reported
that the levels of free cysteine – from amongst 17 proteogenic amino
acids (studied) – decreased significantly in skeletal muscles of human
volunteers, 16 days after ascent to high altitude (5260 m). Since we too
observed, in the present study, a decrease in the levels of cysteine in

8
S. Mishra, et al. BBA - Molecular Basis of Disease 1866 (2020) 165769

A. B.
Acclimatization HH

Days -3 -2 -1 0 1

NAC
1) CBF Measurement
2) Brain Tissue Isolation
& H2S Estimation

C. E.

Blood Perfusion Units

N N_Cys

Blood Perfusion Units

Stimulated 1500
1500 Unstimulated

BPU
Stimulated
1000 1000

BPU
Unstimulated
500 500
0 0
Time (sec) Time (sec)

D.
Blood Perfusion Units

Blood Perfusion Units

HH_1D HH_1D_Cys
Stimulated 1500
1500

BPU
Unstimulated Stimulated
BPU 1000 Unstimulated
1000
500 500
0 0
Time (sec) Time (sec)

Fig. 5. Restoration of cysteine levels in brain maintains endogenous H2S levels and cerebral blood flow. A) Diagrammatic representation of the experimental scheme.
B) H2S levels in brain were estimated utilizing amperometric probe. Bar graph (Mean ± SD) representing levels of H2S (in μM) in brain tissue extracts of animals
exposed to HH for 1 day, with or without NAC pre-treatment (n = 4). C) Scatter plot representing the changes in cerebral blood flow (CBF) of animals exposed to
hypobaric hypoxia for 1 day, with or without NAC pre-treatment. The cerebral blood flow was measured using laser Doppler flowmetry and data represented as blood
perfusion units (BPU, arbitrary unit). D) Bar graph (Mean ± SD) depicting percentage (%) change in BPU after whisker stimulation (represented as % change over
un-stimulated values) of animals in indicated groups (a measure of functional hyperemia responses) (n = 4–6). **p ˂ 0.01, ***p ˂ 0.001 and NS - not significant. E)
Representative overlays of signals recorded before (blue) and after (red) whisker stimulation of animals in indicated groups. Abbreviations – N: Normoxic control;
HH_1D: 1 day HH exposed; HH_1D_Cys: 1 day HH exposed along with NAC pre-treatment; N_Cys: NAC pre-treated normoxic control. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)

plasma samples of human volunteers ascending to high altitude
(Fig. 2C), it appears likely that the phenomenon of cysteine limitation
manifests beyond brain, in other organ systems as well, and has, per-
haps, a unifying physiological niche during chronic hypobaric hypoxia
responses.
In wake of elevated cysteine demand, a homeostatic system would
be expected to mount a befitting feedback response and activate cy-
steine-maintaining pathways in a bid to preserve its endogenous levels.
Brain is, however, known to have a low basal cystathionine activity
(involved in de novo cysteine production), compared to other organ
systems [41]. Under normal physiological conditions, the trans-sul-
furation pathway (de novo cysteine generation) accounts for one third of
total cysteine requirement in astrocytes [34]. The other major cysteine
maintenance pathway involves xCT (anti-porter system engaged in
importing cystine from extracellular pools in exchange for stoichio-
metric amount of glutamate from intracellular compartments) and ac-
counts for the other major fraction of cysteine required in brain under
normal physiological conditions. It is noteworthy that the latter
pathway has evolved to respond to multiple stressors including oxida-
tive stress [42], hypoxia [27], amino acid (cysteine)- and glucose-
starvation amongst others [32]. Reasonably, therefore, the expression
of xCT is regulated by NRF2 [43,44] – the master regulator of anti-
oxidant defense response [45] and ATF4 – the central regulator of ER-
and integrated-stress response [46]. In keeping with such established
phenomena, we too observed significant increase in the expression of
cystine-glutamate transporter (xCT) during hypobaric hypoxia exposure
in our animal model. Additionally, we observed an increase in the ex-
pression of EAAT 1 & 2 in our repeated experiments. These transporters
are involved in the up-take of glutamate from extracellular milieu into
the astrocytes and thereby, serving two important functions during the
conditions of elevated xCT activity: 1) Sustained cystine import into the
cells by maintaining xCT activity – which is prone to inhibition by ever-
elevating concentration of extracellular glutamate due to its own (xCT)
activity [47] and 2) Preventing possible glutamate excitotoxicity in
neurons due to higher concentration of extracellular glutamate (owing
to elevated xCT activity) [48]. It is important to note in this context that

9
S. Mishra, et al. BBA - Molecular Basis of Disease 1866 (2020) 165769

Brain Isolation
A. Acclimatization Hypobaric Hypoxia

Hippocampal
Endothelial dysfunction (vWF)

Sections
Days -3 -2 -1 0 1 2 3 4 5 6 7 Astrocyte activation (GFAP)

Glio-vascular dysfunction
NAC
AOAA (Laminin/Aquaporin 4)

N_Cys HH_7D_
B. N N_Cys _AOAA HH_7D HH_7D_Cys Cys_AOAA C.
vWF
10 X

100 µm 100 µm 100 µm 100 µm 100 µm 100 µm

20 X

50 µm 50 µm 50 µm 50 µm 50 µm 50 µm

D. N_Cys HH_7D_ E.
N N_Cys _AOAA HH_7D HH_7D_Cys Cys_AOAA
GFAP
10 X

100 µm 100 µm 100 µm 100 µm 100 µm 100 µm

20 X

50 µm 50 µm 50 µm 50 µm 50 µm 50 µm

N_Cys HH_7D_
F. N N_Cys _AOAA HH_7D HH_7D_Cys Cys_AOAA

Laminin
20 µm 20 µm 20 µm 20 µm 20 µm 20 µm

AQUP 4
20 µm 20 µm 20 µm 20 µm 20 µm 20 µm

Merge
20 µm 20 µm 20 µm 20 µm 20 µm 20 µm

Magnified

Fig. 6. Cysteine supplementation curtails hypobaric hypoxia-induced neuro-pathological responses in a CBS/H2S-dependent manner. A) Diagrammatic re-
presentation of the experimental scheme. B–E) Representative immunofluorescence micrographs showing vWF (B) and GFAP (D) expression acquired at 10× and
20× magnification in the hippocampal sections of the indicated groups. The fluorescent signal was estimated employing intensity thresholding method in Image J,
NIH software. Corresponding bar graphs (Mean ± SD values) are shown in C and E, respectively (n = 3). *p ˂ 0.05, ***p ˂ 0.001 and NS - not significant. F)
Representative co-immunofluorescence micrographs showing expression of laminin (in red), AQP 4 (in green) and their corresponding overlays (merge) in the
hippocampal region of indicated groups. The images were acquired at 40× magnification employing confocal microscopy. Abbreviations – N: Normoxic control;
N_Cys: NAC pre-treated normoxia exposed; N_Cys_AOAA: NAC and AOAA pre-treated normoxia exposed; HH_7D: 7 days HH exposed; HH_7D_Cys: 7 days HH exposed
along with NAC pre-treatment; HH_7D_Cys_AOAA: 7 days HH exposed along with NAC and AOAA pre-treatment.

10
S. Mishra, et al. BBA - Molecular Basis of Disease 1866 (2020) 165769

A.
STAGE I II III IV V

WATER PROBE TRIAL &

TRAINING HYPOBARIC HYPOXIA
HABITUATION ACCLIMATIZATION

DAYS -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 TEST

NAC
AOAA

B. C. D. E.

F.

N N_Cys N_Cys_AOAA HH_7D HH_7D_Cys HH_7D_Cys_AOAA

Fig. 7. Cysteine supplementation ameliorates hypobaric hypoxia-induced deficit in spatial reference memory. A) Schematic representation of experiments for Morris
water maze test for spatial reference memory assay. The animals were habituated to water for 2 days and subsequently, trained in the Morris water maze for 5 days.
Probe trial was conducted prior to hypobaric hypoxia exposure. The animals were then exposed to HH for 7 days and subjected to Morris water maze test. B–C) Line
graphs representing day-wise changes in latency (B) and path length (C) over the 5-day training period of animals in Morris water maze. D–E) Bar graphs representing
the latency (D) and path length (E) during the Morris water maze test for the animals from the indicated groups (n = 5). Mean ± SD is shown. *p ˂ 0.05, **p ˂ 0.01,
***p ˂ 0.001 and NS - not significant. F) Representative track plots from Morris water maze test for the indicated groups are shown. Abbreviations – N: Normoxic
control; N_Cys: NAC pre-treated normoxic group; N_Cys_AOAA: NAC and AOAA pre-treated normoxic group; HH_7D: HH exposed for 7 days; HH_7D_Cys: 7 days HH
exposure along with NAC pre-treatment; HH_7D_Cys_AOAA: 7 days HH exposure along with NAC and AOAA pre-treatment.

some previous studies had shown inhibitory effects of hypoxia on
EAATs (thus, potential inhibitory effect on cystine transport by xCT)
[49]. In repeat experiments, however, we did not observe similar
phenomenon in our model system. There could be two possibilities
leading to observed differences in such responses: 1) Hypoxia-induced
effects on EAATs could be brain cell-type specific – information not
collected in the present study. 2) The magnitude and/or duration of
hypoxia could govern the expression of EAATs in brain cells. Not-
withstanding possible limitations, however, our observations reason-
ably support positive modulation of cysteine-maintenance pathways, in
brain, during hypobaric hypoxia but physiological inadequacy of this
response, in sustaining endogenous cysteine levels, under these condi-
tions.
Despite positive effects of hypobaric hypoxia on xCT and EAATs, the
levels of endogenous cysteine in brain were, intriguingly, diminished.
To address this paradox, we presented two lines of evidence to support
the possibility of elevated consumption past endogenous replenishment
potential of brain cells during hypoxia. First, the inhibition of CBS per se
(employing AOAA) increased the endogenous levels of cysteine during
hypoxic exposure. Second, the exogenous supplementation of the
cysteine pro-drug, NAC, effectively prevented cysteine decrease during
hypobaric hypoxia. Besides this, NAC also maintained the levels of H2S
– requiring cysteine as substrate – under these conditions. Taken to-
gether, these observations yielded compelling evidence that the CBS-
dependent H2S-generating pathway is a major consumer of cysteine
during hypobaric hypoxia conditions. Notably, further, consistent with
our previous observations (pertaining to exogenous supplementation of
H2S donor, NaHS) [13], the augmentation of cysteine (employing NAC)
ameliorated the effects of hypobaric hypoxia on glio-vascular dys-
function and higher order neurophysiological functions (impairment of
spatial reference memory) in a CBS-dependent manner. In this regard, it
is interesting to note that CBS-H2S pathway has also been implicated for
protective effects of L-Cysteine supplementation in newborn mice sub-
jected to ischemic brain injury [17]. In summary, therefore, these ob-
servations cumulatively underscore the physiological significance of
this axis (cysteine-CBS-H2S) during brain responses to chronic hypo-
baric hypoxia.
There exist contrasting views, in literature, about the mechanistic
basis of cellular effects produced by NAC (cysteine supplementation). It
was generally believed to function by augmenting endogenous levels of

11
S. Mishra, et al. BBA - Molecular Basis of Disease 1866 (2020) 165769

NO INTERVENTION CYSTEINE SUPPLIMENTATION

Hypoxia xCT Hypoxia xCT

Methionine Methionine
HIF-1 HIF-1
CBS CBS

Cysteine Cysteine NAC

CBS CBS
GCLC CDO GCLC CDO

H2 S GSH Taurine H2 S GSH Taurine

CBF CBF
ROS ROS

Neuronal Dysfunction Neuronal Dysfunction

Fig. 8. Proposed scheme of events integrating phenomena deciphered from this study with known biological processes. Hypobaric hypoxia leads to decrease in the
levels of cysteine despite elevated expression of cystine-glutamate antiporter, xCT, and CBS (involved in de novo cysteine synthesis as well as H2S generation). This
decrease in cysteine levels results in lowered levels of H2S and thus, reduced CBF in brain. Further, since cysteine is also required for GSH synthesis, it could
potentially regulate ROS levels (known to elevate during hypobaric hypoxia). The decrease in CBF (and thus, brain oxygen levels) along with increased ROS
potentially cumulate to deteriorate neuronal function. Restoration of cysteine levels via exogenous supplementation (employing cysteine pro-drug, NAC) restores
endogenous H2S levels and curtails neuronal dysfunction in a CBS-dependent manner. Abbreviations – HIF-1α: hypoxia inducible factor 1α; xCT: cystine/glutamate
transporter; CBS: cystathionine β-synthase; GCLC: glutamate-cysteine ligase catalytic subunit; GSH: reduced glutathione; CBF: cerebral blood flow; ROS: reactive
oxygen species; NAC: N-acetylcysteine.

GSH [50]. A number of observations have, however, argued for po-
tential involvement of other pathways independent of GSH levels
[51–53]. Even in the settings of high altitude, a randomized trial for
NAC (in human volunteers) demonstrated decrease in oxidative stress
in NAC groups without significant change in GSH levels in NAC-sup-
plemented individuals ascending to 3600 m and only modest (< 10%)
change at 4580 m [54]. The most noteworthy insight pertaining to the
effects of NAC has emerged from a recent study demonstrating that
NAC treatment increased endogenous levels of H2S (in a CBS-dependent
manner) in the cells and produced anti-oxidant effects through mi-
tochondrial sulfane sulfur pool [53]. Our observations pertaining to
connection between levels of free cysteine and H2S besides CBS-de-
pendence of effects produced by NAC supplementation strongly lend
support to this mechanism.
It is also important to mention here that decrease in cellular cysteine
levels have been associated with the inhibition of HIF-prolyl hydro-
xylase 2 (EGLN1) and consequent stabilization of HIF-1α [47] – the
master regulator of hypoxia responses [26]. The question, whether in-
creased endogenous levels of cysteine – due to supplementation of NAC
during hypobaric hypoxia – would hamper HIF-1α stabilization, under
these conditions, merits a careful consideration. While we did not in-
vestigate this aspect per se in our present work, we expect that the
elevated levels of H2S – due to higher availability of cysteine (CBS
substrate) – would cater for parallel route of HIF-1α stabilization under
these conditions. The elevated levels of H2S per se have been shown to
stabilize HIF-1α and its target gene VEGF [55]. Interestingly, further,
the effects of H2S on transcriptional activity of HIF appear to be in-
dependent of von Hippel-Lindau Tumor Suppressor-1 protein [56]. It is,
thus, highly plausible that the maintenance of endogenous cysteine
levels (with NAC supplementation) along with optimal CBS enzyme
activity would ‘positively’ modulate HIF-pathway and facilitate brain
adaptation to chronic hypobaric hypoxia.
Another important facet of free cysteine dynamics merits careful
consideration. In the neurovascular milieu, GSH can also serve as a
cysteine store and can be potentially utilized as a salvage pathway for
cysteine generation [57]. The enzyme ‘gamma-glutamyl transpeptidase’
regulates this biological process and responds to multiple biological
cues [58], including adaptive increase during oxidative stress – a phe-
nomenon well known to manifest during hypobaric hypoxia [59]. In-
terestingly, the levels of GSH decline during chronic hypobaric hypoxia
– both in the animal model [29,59] as well as human volunteers as-
cending to high altitude [54]. While on the prima facie (considering
GSSG levels), this observation appears consistent with the phenomenon
of increased oxidative stress during hypoxia; there is no evidence per se,
to the best of our knowledge, which can experimentally rule out the
activation of cysteine salvage pathway (similar to xCT) under these
conditions and thus, possible lowering of GSH levels, at least in part, as
a feedback response attempting to elevate levels of free cysteine in the
brain. It will, therefore, be interesting to determine the relative ex-
pression/activity of gamma-glutamyl transpeptidase during chronic
hypobaric hypoxia and render mechanistic clarity related to the niche
of GSH – as only an antioxidant peptide or equally important cysteine
store – in brain during hypoxic conditions.
Notwithstanding the above said caveat in our knowledge, however,
the phenomena of HH-induced decrease in levels of free cysteine and a
physiological propensity for its routing to CBS-dependent H2S-genera-
tion pathway (as deciphered from cumulative observations described
here) will be critical for adaptive responses of brain to hypobaric hy-
poxia.
Author contributions
MS conceived and coordinated the study. SM, GK, AC, NJ, NKS,
RNM, RT, MS performed the experiments. SM, GK, AC, NKS, RT, DNP,
BK, MS analyzed the experiments. MS, SM wrote the manuscript. All
authors reviewed the results, proofread and approved final version of
the manuscript.

12
S. Mishra, et al.
Transparency document
The Transparency document associated with this article can be
found, in online version.
Declaration of competing interest
The authors have declared that no conflict of interest exists.
Acknowledgements
The authors are thankful to Indian army and SD branch (Army HQ)
for logistic support. We sincerely acknowledge all the volunteers in this
study. We are thankful to Dr. SN Singh for kind support during various
stages of this work and Dr. R. J. Tirpude for support with experimental
animals. The work was supported by grant [No: DIP-265 (MS)] from
DRDO, India. SM & AC received fellowship from UGC, GK from CSIR
and NJ from ICMR, India.
References
[1] M.E. Raichle, D.A. Gusnard, Appraising the brain’s energy budget, Proc. Natl. Acad.
Sci. U. S. A. 99 (2002) 10237–10239.
[2] C.Y. Xing, T. Tarumi, J. Liu, Y. Zhang, M. Turner, J. Riley, C.D. Tinajero, L.J. Yuan,
R. Zhang, Distribution of cardiac output to the brain across the adult lifespan, J.
Cereb. Blood Flow Metab. 37 (2017) 2848–2856.
[3] D. Attwell, S.B. Laughlin, An energy budget for signaling in the grey matter of the
brain, J. Cereb. Blood Flow Metab. 21 (2001) 1133–1145.
[4] F. Hyder, R.K. Fulbright, R.G. Shulman, D.L. Rothman, Glutamatergic function in
the resting awake human brain is supported by uniformly high oxidative energy, J.
Cereb. Blood Flow Metab. 33 (2013) 339–347.
[5] R.G. Shulman, F. Hyder, D.L. Rothman, Baseline brain energy supports the state of
consciousness, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 11096–11101.
[6] M.H. Wilson, S. Newman, C.H. Imray, The cerebral effects of ascent to high alti-
tudes, Lancet Neurol. 8 (2009) 175–191.
[7] X. Yan, Cognitive impairments at high altitudes and adaptation, High Alt. Med. Biol.
15 (2014) 141–145.
[8] G. Willmann, F. Gekeler, K. Schommer, P. Bartsch, Update on high altitude cerebral
edema including recent work on the eye, High Alt. Med. Biol. 15 (2014) 112–122.
[9] T. Morikawa, M. Kajimura, T. Nakamura, T. Hishiki, T. Nakanishi, Y. Yukutake,
Y. Nagahata, M. Ishikawa, K. Hattori, T. Takenouchi, T. Takahashi, I. Ishii,
K. Matsubara, Y. Kabe, S. Uchiyama, E. Nagata, M.M. Gadalla, S.H. Snyder,
M. Suematsu, Hypoxic regulation of the cerebral microcirculation is mediated by a
carbon monoxide-sensitive hydrogen sulfide pathway, Proc. Natl. Acad. Sci. U. S. A.
109 (2012) 1293–1298.
[10] R.L. Hoiland, A.R. Bain, M.G. Rieger, D.M. Bailey, P.N. Ainslie, Hypoxemia, oxygen
content, and the regulation of cerebral blood flow, Am. J. Physiol. Regul. Integr.
Comp. Physiol. 310 (2016) R398–R413.
[11] P.N. Ainslie, A.W. Subudhi, Cerebral blood flow at high altitude, High Alt. Med.
Biol. 15 (2014) 133–140.
[12] H. Kimura, Hydrogen sulfide: its production and functions, Exp. Physiol. 96 (2011)
833–835.
[13] G. Kumar, A. Chhabra, S. Mishra, H. Kalam, D. Kumar, R. Meena, Y. Ahmad,
K. Bhargava, D.N. Prasad, M. Sharma, H2S regulates hypobaric hypoxia-induced
early glio-vascular dysfunction and neuro-pathophysiological effects, EBioMedicine
6 (2016) 171–189.
[14] R. Demiralay, N. Gursan, G. Ozbilim, G. Erdogan, E. Demirci, Comparison of the
effects of erdosteine and N-acetylcysteine on apoptosis regulation in endotoxin-
induced acute lung injury, J. Appl. Toxicol. 26 (2006) 301–308.
[15] C.B. Pocernich, M. La Fontaine, D.A. Butterfield, In-vivo glutathione elevation
protects against hydroxyl free radical-induced protein oxidation in rat brain,
Neurochem. Int. 36 (2000) 185–191.
[16] A. Serrano-Mollar, D. Closa, N. Prats, S. Blesa, M. Martinez-Losa, J. Cortijo,
J.M. Estrela, E.J. Morcillo, O. Bulbena, In vivo antioxidant treatment protects
against bleomycin-induced lung damage in rats, Br. J. Pharmacol. 138 (2003)
1037–1048.
[17] S. Liu, D. Xin, L. Wang, T. Zhang, X. Bai, T. Li, Y. Xie, H. Xue, S. Bo, D. Liu, Z. Wang,
Therapeutic effects of L-cysteine in newborn mice subjected to hypoxia-ischemia
brain injury via the CBS/H2S system: role of oxidative stress and endoplasmic re-
ticulum stress, Redox Biol. 13 (2017) 528–540.
[18] C.V. Vorhees, M.T. Williams, Morris water maze: procedures for assessing spatial
and related forms of learning and memory, Nat. Protoc. 1 (2006) 848–858.
[19] Y. Sun, Y. Huang, R. Zhang, Q. Chen, J. Chen, Y. Zong, J. Liu, S. Feng, A.D. Liu,
L. Holmberg, D. Liu, C. Tang, J. Du, H. Jin, Hydrogen sulfide upregulates KATP
channel expression in vascular smooth muscle cells of spontaneously hypertensive
rats, J. Mol. Med. (Berl) 93 (2015) 439–455.
[20] T. Xu, N. Scafa, L.P. Xu, S. Zhou, K. Abdullah Al-Ghanem, S. Mahboob, B. Fugetsu,
X. Zhang, Electrochemical hydrogen sulfide biosensors, Analyst 141 (2016)
1185–1195.
13
BBA - Molecular Basis of Disease 1866 (2020) 165769
[21] B.A. Sutherland, T. Rabie, A.M. Buchan, Laser Doppler flowmetry to measure
changes in cerebral blood flow, Methods Mol. Biol. 1135 (2014) 237–248.
[22] C. Lecrux, X. Toussay, A. Kocharyan, P. Fernandes, S. Neupane, M. Levesque,
F. Plaisier, A. Shmuel, B. Cauli, E. Hamel, Pyramidal neurons are “neurogenic hubs”
in the neurovascular coupling response to whisker stimulation, J. Neurosci. 31
(2011) 9836–9847.
[23] K. Abe, H. Kimura, The possible role of hydrogen sulfide as an endogenous neu-
romodulator, J. Neurosci. Off. J. Soc. Neurosci. 16 (1996) 1066–1071.
[24] N. Takano, Y.J. Peng, G.K. Kumar, W. Luo, H. Hu, L.A. Shimoda, M. Suematsu,
N.R. Prabhakar, G.L. Semenza, Hypoxia-inducible factors regulate human and rat
cystathionine beta-synthase gene expression, Biochem. J. 458 (2014) 203–211.
[25] B.D. Paul, J.I. Sbodio, S.H. Snyder, Cysteine metabolism in neuronal redox home-
ostasis, Trends Pharmacol. Sci. 39 (2018) 513–524.
[26] G.L. Semenza, Life with oxygen, Science 318 (2007) 62–64.
[27] B. Sims, M. Clarke, L. Francillion, E. Kindred, E.S. Hopkins, H. Sontheimer, Hypoxic
preconditioning involves system Xc− regulation in mouse neural stem cells, Stem
Cell Res. 8 (2012) 285–291.
[28] C.H. Hsieh, Y.J. Lin, W.L. Chen, Y.C. Huang, C.W. Chang, F.C. Cheng, R.S. Liu,
W.C. Shyu, HIF-1alpha triggers long-lasting glutamate excitotoxicity via system xc
(−) in cerebral ischaemia-reperfusion, J. Pathol. 241 (2017) 337–349.
[29] K. Jayalakshmi, S.B. Singh, B. Kalpana, M. Sairam, S. Muthuraju, G. Ilavazhagan, N-
acetyl cysteine supplementation prevents impairment of spatial working memory
functions in rats following exposure to hypobaric hypoxia, Physiol. Behav. 92
(2007) 643–650.
[30] B.S. Kendler, Supplemental conditionally essential nutrients in cardiovascular dis-
ease therapy, J. Cardiovasc. Nurs. 21 (2006) 9–16.
[31] L.K. Mischley, Conditionally essential nutrients: the state of the science, Journal of
Food and Nutrition 1 (2014) e204.
[32] J.A. Combs, G.M. DeNicola, The non-essential amino acid cysteine becomes es-
sential for tumor proliferation and survival, Cancers (Basel), 11, (2019).
[33] M.A. Riedijk, R.H. van Beek, G. Voortman, H.M. de Bie, A.C. Dassel, J.B. van
Goudoever, Cysteine: a conditionally essential amino acid in low-birth-weight
preterm infants? Am. J. Clin. Nutr. 86 (2007) 1120–1125.
[34] G.J. McBean, Cysteine, glutathione, and thiol redox balance in astrocytes,
Antioxidants (Basel), 6, (2017).
[35] O. Kranich, R. Dringen, M. Sandberg, B. Hamprecht, Utilization of cysteine and
cysteine precursors for the synthesis of glutathione in astroglial cultures: preference
for cystine, Glia 22 (1998) 11–18.
[36] R. Dringen, Metabolism and functions of glutathione in brain, Prog. Neurobiol. 62
(2000) 649–671.
[37] Y. Kimura, H. Kimura, Hydrogen sulfide protects neurons from oxidative stress,
FASEB J. 18 (2004) 1165–1167.
[38] H. Kimura, Physiological role of hydrogen sulfide and polysulfide in the central
nervous system, Neurochem. Int. 63 (2013) 492–497.
[39] R.K. Shaw, J.D. Heine, Ninhydrin positive substances present in different areas of
normal rat brain, J. Neurochem. 12 (1965) 151–155.
[40] A.J. Chicco, C.H. Le, E. Gnaiger, H.C. Dreyer, J.B. Muyskens, A. D’Alessandro,
T. Nemkov, A.D. Hocker, J.E. Prenni, L.M. Wolfe, N.M. Sindt, A.T. Lovering,
A.W. Subudhi, R.C. Roach, Adaptive remodeling of skeletal muscle energy meta-
bolism in high-altitude hypoxia: lessons from AltitudeOmics, J. Biol. Chem. 293
(2018) 6659–6671.
[41] J.D. Finkelstein, Methionine metabolism in mammals, J. Nutr. Biochem. 1 (1990)
228–237.
[42] B. Mysona, Y. Dun, J. Duplantier, V. Ganapathy, S.B. Smith, Effects of hypergly-
cemia and oxidative stress on the glutamate transporters GLAST and system xc− in
mouse retinal Muller glial cells, Cell Tissue Res. 335 (2009) 477–488.
[43] A.Y. Shih, H. Erb, X. Sun, S. Toda, P.W. Kalivas, T.H. Murphy, Cystine/glutamate
exchange modulates glutathione supply for neuroprotection from oxidative stress
and cell proliferation, J. Neurosci. Off. J. Soc. Neurosci. 26 (2006) 10514–10523.
[44] E. Habib, K. Linher-Melville, H.X. Lin, G. Singh, Expression of xCT and activity of
system xc(−) are regulated by NRF2 in human breast cancer cells in response to
oxidative stress, Redox Biol. 5 (2015) 33–42.
[45] Q. Ma, Role of nrf2 in oxidative stress and toxicity, Annu. Rev. Pharmacol. Toxicol.
53 (2013) 401–426.
[46] J. Lewerenz, P. Maher, Basal levels of eIF2alpha phosphorylation determine cellular
antioxidant status by regulating ATF4 and xCT expression, J. Biol. Chem. 284
(2009) 1106–1115.
[47] K.J. Briggs, P. Koivunen, S. Cao, K.M. Backus, B.A. Olenchock, H. Patel, Q. Zhang,
S. Signoretti, G.J. Gerfen, A.L. Richardson, A.K. Witkiewicz, B.F. Cravatt, J. Clardy,
W.G. Kaelin Jr., Paracrine induction of HIF by glutamate in breast cancer: EglN1
senses cysteine, Cell 166 (2016) 126–139.
[48] Z.C. Ye, J.D. Rothstein, H. Sontheimer, Compromised glutamate transport in human
glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters
and enhanced activity of cystine-glutamate exchange, J. Neurosci. Off. J. Soc.
Neurosci. 19 (1999) 10767–10777.
[49] M. Dallas, H.E. Boycott, L. Atkinson, A. Miller, J.P. Boyle, H.A. Pearson, C. Peers,
Hypoxia suppresses glutamate transport in astrocytes, J. Neurosci. Off. J. Soc.
Neurosci. 27 (2007) 3946–3955.
[50] S.C. Lu, Glutathione synthesis, Biochim. Biophys. Acta 1830 (2013) 3143–3153.
[51] S. Patriarca, A.L. Furfaro, C. Domenicotti, P. Odetti, D. Cottalasso, U.M. Marinari,
M.A. Pronzato, N. Traverso, Supplementation with N-acetylcysteine and taurine
failed to restore glutathione content in liver of streptozotocin-induced diabetics rats
but protected from oxidative stress, Biochim. Biophys. Acta 1741 (2005) 48–54.
[52] J. Zhou, L.D. Coles, R.V. Kartha, N. Nash, U. Mishra, T.C. Lund, J.C. Cloyd,
Intravenous administration of stable-labeled N-acetylcysteine demonstrates an in-
direct mechanism for boosting glutathione and improving redox status, J. Pharm.
S. Mishra, et al.
Sci. 104 (2015) 2619–2626.
[53] D. Ezerina, Y. Takano, K. Hanaoka, Y. Urano, T.P. Dick, N-acetyl cysteine functions
as a fast-acting antioxidant by triggering intracellular H2S and sulfane sulfur pro-
duction, Cell Chem. Biol. 25 (2018) 447–459 (e444).
[54] P. Vats, V.K. Singh, S.N. Singh, S.B. Singh, Glutathione metabolism under high-
altitude stress and effect of antioxidant supplementation, Aviat. Space Environ.
Med. 79 (2008) 1106–1111.
[55] X. Liu, L. Pan, Y. Zhuo, Q. Gong, P. Rose, Y. Zhu, Hypoxia-inducible factor-1alpha is
involved in the pro-angiogenic effect of hydrogen sulfide under hypoxic stress, Biol.
Pharm. Bull. 33 (2010) 1550–1554.
[56] M.W. Budde, M.B. Roth, Hydrogen sulfide increases hypoxia-inducible factor-1
14
BBA - Molecular Basis of Disease 1866 (2020) 165769
activity independently of von Hippel-Lindau tumor suppressor-1 in C. elegans, Mol.
Biol. Cell 21 (2010) 212–217.
[57] M.H. Hanigan, W.A. Ricketts, Extracellular glutathione is a source of cysteine for
cells that express gamma-glutamyl transpeptidase, Biochemistry 32 (1993)
6302–6306.
[58] H. Zhang, H.J. Forman, Redox regulation of gamma-glutamyl transpeptidase, Am. J.
Respir. Cell Mol. Biol. 41 (2009) 509–515.
[59] P. Maiti, S.B. Singh, A.K. Sharma, S. Muthuraju, P.K. Banerjee, G. Ilavazhagan,
Hypobaric hypoxia induces oxidative stress in rat brain, Neurochem. Int. 49 (2006)
709–716.