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ORIGINAL ARTICLE
Cellular and Molecular Life Sciences (2025) 82:160
https://doi.org/10.1007/s00018-025-05689-7
Introduction
Cardiac arrest (CA), which aects nearly 500,000 people
annually, is a leading cause of mortality and a major pub-
lic health challenge worldwide [1]. Brain injury is a com-
mon sequela following CA, with up to 70% of hospitalized
patients dying from brain injury even when return of spon-
taneous circulation (ROSC) is achieved [1, 2]. In recent
years, thanks to the continuous optimization of the emer-
gency network and the popularization of CPR and debril-
lation technology, the survival probability of CA patients
has increased signicantly, but brain resuscitation after CA
remains a largely unsolved clinical problem [3].
The pathophysiology of post-cardiac arrest brain injury
(PCABI) involves both primary (ischemic) and secondary
(reperfusion) injury, which occur sequentially during car-
diac arrest, resuscitation, and the acute post-resuscitation
Wenbin Zhang, Xin Yu and Yao Lin contributed equally to this work.
Mao Zhang: Lead contact.
Jiefeng Xu
z2jexu@zju.edu.cn
Mao Zhang
z2jzk@zju.edu.cn
1 Department of Emergency Medicine, Second Aliated
Hospital, Zhejiang University School of Medicine,
Hangzhou, China
2 Zhejiang Key Laboratory of Trauma, Burn, and Medical
Rescue, Hangzhou, China
3 Zhejiang Province Clinical Research Center for Emergency
and Critical Care Medicine, Hangzhou, China
Abstract
Introduction Brain injury is a common sequela following cardiac arrest (CA), with up to 70% of hospitalized patients dying
from it. Brain microvascular endothelial cells (BMVECs) play a crucial role in post-cardiac arrest brain injury (PCABI).
However, the eects and mechanisms of targeting BMVEC energy metabolism to mitigate brain injury remain unclear.
Methods We established a mouse model of cardiac arrest by injecting potassium chloride into the right internal jugular vein.
Mass spectrometry detected targeted changes in short-chain fatty acids and energy metabolism metabolites in the CA/CPR
group compared to the sham group. Mice with overexpressed ACSS2 in BMVECs were created using an AAV-BR1 vector,
and ACSS2 knockout mice were generated using the CRE-LOXP system. The oxygen glucose deprivation/re-oxygenation
(OGD/R) model was established to investigate the role and mechanisms of ACSS2 in endothelial cells in vitro.
Results Metabolomics analysis revealed disrupted cerebral energy metabolism post-CA/CPR, with decreased acetyl-CoA
and amino acids. Overexpression of ACSS2 in BMVECs increased acetyl-CoA levels and improved neurological function.
Vascular endothelial cell-specic ACSS2 knockout mice exhibited reduced aortic sprouting in vitro. Overexpression of
ACSS2 improved endothelial dysfunction following oxygen glucose deprivation/re-oxygenation (OGD/R) and inuenced
autophagy by interacting with transcription factor EB (TFEB) and modulating the AMP-activated protein kinase α (AMPKα)
pathway.
Conclusion Our study shows that ACSS2 modulates the biological functions of BMVECs by promoting autophagy. Enhanc-
ing energy metabolism via ACSS2 may target PCABI treatment development.
Keywords ACSS2 · Cardiopulmonary resuscitation · Autophagy · Ischemia-reperfusion injury · Endothelial dysfunction
Received: 10 January 2025 / Revised: 4 March 2025 / Accepted: 30 March 2025
© The Author(s) 2025
Acetyl-CoA synthetase 2 alleviates brain injury following cardiac arrest
by promoting autophagy in brain microvascular endothelial cells
WenbinZhang1,2,3· XinYu1,2,3· YaoLin1,2,3· ChenghaoWu1,2,3· RuojieZhu1,2,3· XiangkangJiang1,2,3· JiaweiTao1,2,3·
ZiweiChen1,2,3· JiantaoHe1,2,3· XiaodanZhang1,2,3· JiefengXu1,2,3· MaoZhang1,2,3
1 3
Cellular andMolecular Life Sciences
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W. Zhang et al.
phase [4]. Brain microvascular endothelial cells (BMVECs)
constitute part of the blood-brain barrier (BBB), preventing
harmful substances from entering the brain from the blood,
and play an important role in the pathogenesis of PCABI
[5–7]. During ischemia, the endothelium undergoes rapid
activation and becomes increasingly permeable to blood
constituents [8–10]. In addition, endothelial cells upregu-
late pro-inammatory and cell adhesion molecules, leading
to intravascular cell adhesion after focal ischemia, which
further aects reperfusion post-ischemia.
Changes in endothelial cell function and integrity may be
accompanied by alterations in cellular energy demand and
metabolism. Under ischemic or inammatory conditions,
endothelial cells can rapidly switch to an angiogenic mode
to form new blood vessels [11]. Recent studies indicate
that following ischemia-reperfusion injury (IRI), endothe-
lial cells undergo metabolic reprogramming, characterized
by increased levels of intracellular reactive oxygen species
(ROS) and glutathione, reduced glucose consumption, and
elevated lactate production [12]. These changes help main-
tain redox homeostasis, suggesting that targeting BMVECs
metabolism could improve microvascular function and alle-
viate brain injury following CA/CPR.
Acetyl-CoA synthetase 2 (ACSS2), a key metabolic
enzyme converting extracellular acetate into intracellu-
lar acetyl-CoA, by participating in acetyl-CoA synthesis,
ACSS2 broadly inuences cellular energy metabolism [13],
gene expression regulation [14], signal transduction [15],
demonstrating unique adaptability and functionality, espe-
cially under metabolic stress [16, 17]. A telling example
lies in the positive correlation between ACSS2 activity and
macropinocytosis in pancreatic cancer cells - a mechanism
critical for nutrient acquisition under conditions of nutrient
scarcity [18]. While the physiological function of ACSS2
is somewhat understood, the specic workings and mecha-
nisms by which it operates in models of CA remain a strik-
ingly unexplored.
The primary objective of this study was to explore the
role of ACSS2 in BMVECs and its impact on brain injury
following CPR. Here, we employed AAV-BR1 to overex-
press ACSS2 eciently in BMVECs of mice subjected
to CPR, thereby alleviating neurological dysfunction. We
further discovered that ACSS2, as a positive regulator of
angiogenesis, modulates endothelial cell migration, tubule
formation, and enhances blood ow recovery post-isch-
emia. Mechanistically, ACSS2 activates the TFEB signaling
pathway and upregulates autophagy. Our ndings unravel
an interacting network among ACSS2, TFEB, and AMPKα,
providing a deeper molecular understanding of the regula-
tion of angiogenesis and autophagy.
Materials and methods
Experimental animals
Adult male C57BL/6 mice (6 weeks old; Vital River Labo-
ratory) were injected with a viral vector encoding ACSS2
encapsulated in the AAV-BR1 capsid (1.8 × 1011vg/animal,
Hanbio, China) via the tail vein [19]. The control group
received an equivalent dose of an empty AAV-BR1 vec-
tor. ACSS2Tek -CKO mice were bred by crossing ACSS2f/f
(oxed ACSS2 allele) with Tek-Cre transgenic mice (Gem-
Pharmatech, China). Both strains were maintained on a
C57BL/6 background and identied through genotyping.
Primer sequences are listed in Table 1. Genotyping and
experimental conditions were conducted blinded. Mice
were kept at 22 °C with 12-hour light/dark cycles and pro-
vided food and water ad libitum.
Animal model
Cardiac arrest was induced in 10-12-week-old male C57/
BL6 mice following a one-week acclimatization period.
Anesthesia was induced with 1% pentobarbital sodium
(45–50 mg/kg, i.p.), maintaining core body temperature at
36 °C using a rectal probe. The neck area was prepared for
surgery, and endotracheal intubation was performed using
a 20G venous catheter. A heparinized PE-10 catheter was
inserted into the right jugular vein for drug administra-
tion. Ventilation was supported using a ventilator (Orkort
Biotechnology, China) set at 105 breaths/min, a 1:1.2 I: E
ratio, and a tidal volume of 1.0 ml. ECG monitoring was
achieved using a BL420 system (TECHMAN, China). After
stabilizing the body temperature between 36 and 37 °C,
cardiac arrest was induced by injecting 50 µl of pre-cooled
0.5 M potassium chloride solution. After a 5-minute stand-
still, mechanical ventilation was restarted alongside chest
compressions (> 300/min) and adrenaline (0.1 ml of 32 µg/
ml) injections repeated every minute. Failure to revive the
mouse after 10 min of chest compressions indicated unsuc-
cessful CPR. Post-resuscitation, ventilation was maintained
for 1 h before reducing the tidal volume to 0.8 ml for another
hour. Two hours post-resuscitation, the PE-10 catheter and
20G stylet were removed, and the mouse was placed in a
warming chamber until fully conscious. It was then returned
to its home cage for further observation and care.
Cell culture and establishment of oxygen-glucose
deprivation/reoxygenation (OGD/R) model
Brain-derived Endothelial cells (bEnd.3) were acquired
from Procell (Wuhan, China) and cultured in high-glucose
Dulbecco’s Modied Eagle Medium (DMEM; Gibco, USA)
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160 Page 2 of 22
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Acetyl-CoA synthetase 2 alleviates brain injury following cardiac arrest by promoting autophagy in brain…
supplemented with 10% fetal bovine serum (FBS; Procell,
China). Cells were maintained at 37 °C with 5% CO2 in a
humidied incubator. For the OGD/R protocol, the culture
medium was replaced with glucose-free DMEM (Procell,
China), and cells were exposed to 0% O2, 5% CO2, and 95%
N2 for 6 h in a sealed container [20]. Following this, cells
were returned to normal culture conditions with standard
medium. A control group remained under standard condi-
tions throughout the experiment.
Compounds
TFEB activator 1 (HY-135825), VEGF164 Protein (HY-
P7312) and Dorsomorphin (Synonyms: Compound C;
BML-275) were purchased from MedChem Express. For in
vitro studies, TFEB activator 1 and Compound C were dis-
solved in DMSO, aliquoted and stored at − 20 °C.
Acetyl-CoA content measurement
Overdose anesthesia was administered to euthanize the
mice, followed immediately by rinsing the brain with
ice-cold Hank’s balanced salt solution (Procell, China) to
eliminate blood contamination. The mouse brains were then
rapidly excised, weighed, and ash-frozen in liquid nitrogen
for preservation. Frozen brain tissues were homogenized in
pre-chilled 1 N perchloric acid (PCA) at a ratio of 2 µL/mg
tissue, and the homogenate was promptly neutralized with
3 M potassium bicarbonate (KHCO3) until no more bubbles
were produced, ensuring a pH range of 6–8. After centrif-
ugation to separate the supernatant, the assay was carried
out according to the manufacturer’s protocol (Abcam, UK).
The reaction mixture was added, and the uorescence inten-
sity (SpectraMax, USA) was measured using a uoromet-
ric plate reader with excitation at 535 nm and emission at
587 nm.
Adenosine triphosphate (ATP) content
measurement
Samples of brain tissue are rapidly thawed after removal
from − 80 °C storage, weighed promptly, and then pro-
cessed according to the manufacturer’s instructions (Beyo-
time, China) by adding an appropriate ratio of ATP detection
lysis buer. The tissue is thoroughly homogenized using a
glass homogenizer (Thermo Fisher Scientic Inc., USA) to
ensure complete lysis. Following homogenization, the sus-
pension is centrifuged at 12,000 g for 5 min at 4 °C, and
the supernatant is carefully collected. This supernatant is
then mixed with the ATP detection working solution, and
its luminescence is read using a luminometer (SpectraMax,
Table 1 All primer sequences
Primer name Forward primer sequence Reverse primer sequence
ACSS2-shRNA#1 C C A T G A A A C C T G G T T C T G C T T
ACSS2-shRNA#2 C G G T T T G A G A C C A C C T A C T T T
ACSS2-shRNA#3 G A T C G A A A T G T C C A T G A G A A A
TFEB-siRNA#1 G C A G G C U G U C A U G C A U U A U T T A U A A U G C A U G A C A G C C U G C T T
TFEB-siRNA#2 C C A A G A A G G A U C U G G A C U U T T A A G U C C A G A U C C U U C U U G G T T
TFEB-siRNA#3 C C A U G G C C A U G C U A C A U A U T T A U A U G U A G C A U G G C C A U G G T T
siRNA NC U U C U C C G A A C G U G U C A C G U TT A C G U G A C A C G U U C G G A G A A T T
AMPKα-siRNA#1 C A C G A G U U G A C C G G A C A U A A A T T U U U A U G U C C G G U C A A C U C G U G T T
AMPKα-siRNA#2 G C A A U C A A G C A G U U G G A U U A U T T A U A A U C C A A C U G C U U G A U U G C T T
AMPKα-siRNA#3 G G A A G U C A U A C A A U A G A A U T T A U U C U A U U G U A U G A C U U C C T T
Rab33b A G A C G T G C C T G A C T T A C C G G T G T C C C A C A A C T G G A T C T T G
Dapk2 C T C G A T G A G G A G C C C A A A T A T G C C C G G C A C T T C T T C A C G A T
Atg4b T A T G A T A C T C T C C G G T T T G C T G A G T T C C C C C A A T A G C T G G A A A G
Atg101 A T G A A C T G T C G A T C A G A A G T G C C C T A T G G A G T A C G T G C C C T
Map1lc3a G A C C G C T G T A A G G A G G T G C C T T G A C C A A C T C G C T C A T G T T A
Bnip1 A G G C T A T G C A G A C T C T A G T C A G C A G T T C T C G G C G G T T G T A C T
Atg7 G T T C G C C C C C T T T A A T A G T G C T G A A C T C C A A C G T C A A G C G G
Atg5 T G T G C T T C G A G A T G T G T G G T T G T C A A A T A G C T G A C T C T T G G C A A
Prkn T C T T C C A G T G T A A C C A C C G T C G G C A G G G A G T A G C C A A G T T
Tfeb T C C T C T G G C G G C A G T A C T A T G T G C C A T C T A G T C C C A G G A A
Acss2 G T G A A A G G A T C T T G G A T T C C A G T C A G A T G T T T G A C C A C A A T G C A G
Actb A A G T C C C T C A C C C T C C C A A A A G A A G C A A T G C T G T C A C C T T C C C
Acss2-ox T C A C T T G A G A A C T T C C T A C C T T A G C C A G A C A C T G T G C C C G C T C A A C A T A T
Tek-iCre-Ki G G G C A G T C T G G T A C T T C C A A G C T C T T G A T T C A C C A G A T G C T G A G G T T A
Tek-iCre-Wt G G G C A G T C T G G T A C T T C C A A G C T A T A T C C C C T T G T T C C C T T T C T G C
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W. Zhang et al.
ultramicrotome and observed with a transmission electron
microscope (Hitachi, Japan).
Targeted metabolomics
Fresh whole brain tissue specimens from mice were rapidly
frozen in liquid nitrogen. After thawing, 50 mg of tissue was
weighed and placed in a 2 mL EP tube with a steel ball.
Short-chain fatty acids (SCFAs) were detected using Met-
Ware’s Agilent 7890B-7000D GC-MS/MS platform, while
targeted energy metabolites were analyzed using the AB
Sciex QTRAP 6500 LC-MS/MS platform. Pareto-scaled
principal component analysis (PCA) and orthogonal partial
least-squares discriminant analysis (OPLS-DA) were per-
formed using R. Metabolites with a Variable Importance in
the Projection (VIP) score > 1 and a p-value < 0.05 were con-
sidered signicantly regulated.
Tube formation assay
Before the experiment, cells were pre-treated with OGD/R.
Materials including growth factor-reduced Matrigel (BD
Biosciences, USA), pipette tips, 1.5 mL centrifuge tubes,
and 96-well plates were pre-cooled. The matrix gel was pre-
pared by mixing stock gel with pre-cooled DMEM at a 2:1
volume ratio. On ice, 50 µL of the mixed gel was dispensed
into each well of a 96-well plate and incubated at 37 °C
for 1 h to solidify. Cells were then seeded at 30,000 cells
per well. After a 6-hour incubation at 37 °C, tubular struc-
tures were observed under an inverted microscope (Leica,
Germany). The total number of branching points and tube
lengths were quantied using Image-Pro Plus 6 software.
Migration assay
Before the experiment, cells were pre-treated with OGD/R
prior to the experiment. For the scratch wound assay, 5 × 105
cells/well (three replicates per group) were plated into a
6-well plate and incubated to reach conuence. The mono-
layer was scratched using a tip and washed with serum-free
medium to remove detached cells. Cells were photographed
at 0 h and 24 h post-wounding. The closure area of wound
was calculated as follows: migration area (%) = (A0 h–
A24 h)/A0 h× 100, where A0 h represents the area of initial
wound area, A24 h represents the remaining area of wound at
the metering point.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cultured cells using Trizol
reagent (Invitrogen, USA). For mRNA detection, cDNA was
synthesized from 1 µg of total RNA utilizing the HiScript II
USA). The ATP concentration is determined by referencing
the values against a standard curve.
Neurological scoring
The neurological status of mice was evaluated one day
before and the rst day after surgery. To assess spontane-
ous motor ability, the open eld test was used. Mice were
placed in an unobstructed plastic container to observe their
free exploration patterns.
Neurological function was quantied using a standardized
grading scale for mice, ensuring objectivity and accuracy.
Two independent researchers conducted the assessments,
with disagreements resolved by a third expert, adopting the
majority opinion as the ocial record [21]. Additionally, the
escape circle test was performed to further evaluate mobil-
ity. Mice were placed at the center of a 12-centimeter-diam-
eter circle on a smooth surface. The latency to escape the
circle and reach the periphery was recorded at specied time
points before and after surgery, aiding in the assessment of
motor function recovery [22].
Cerebral blood ow (CBF) monitoring
CBF was monitored using the Laser Speckle Imaging Sys-
tem (RWD, China) at two critical time points: before sur-
gery and 24 h post-surgery. To enhance data reliability,
high-intensity signals from sinuses were excluded during
analysis. Changes in CBF were quantied by calculating
the mean signal intensity, serving as an indicator of cere-
bral perfusion. Researchers performing surgeries and CBF
assessments were blinded to the group allocations, ensuring
objectivity in the experiment.
Transmission electron microscope
To minimize mechanical damage, hippocampal tissue sam-
ples were handled carefully without pulling, crushing, or
excessive squeezing. Prior to the procedure, culture dishes
containing electron microscope xative (Beyotime, China)
were prepared. Tissue was cut into 1 mm³ pieces directly in
the xative-lled dish to preserve structural integrity. These
pieces were then transferred to EP tubes with fresh xative
and xed at 4 °C.
For cell processing, tissue was digested with pancreatic
enzyme (Gibco, USA), neutralized with culture medium
or serum, and transferred to EP tubes. The supernatant
was removed, and xative was added. Cell clusters were
divided into 2–3 mm pieces and washed three times with
0.1 M PBS for 10 min each. Samples were dehydrated at
room temperature, inltrated with resin, and polymerized
at 60 °C for 48 h. Ultrathin sections were prepared using an
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Acetyl-CoA synthetase 2 alleviates brain injury following cardiac arrest by promoting autophagy in brain…
Western blotting (WB)
Total protein was meticulously extracted using the RIPA
lysis buer (Beyotime, China), which included 0.1mM
phenylmethylsulfonyl uoride (PMSF) (Beyotime, China).
Equal amounts of protein were separated by SDS-polyacryl-
amide gel electrophoresis (SDS-PAGE) using an 4-20%
acrylamide gel and transferred to polyvinylidene diuoride
(PVDF) membranes (MERCK, German). Signal intensities
were quantied using ImageJ software. Antibodies used in
this study include anti-β-actin (Catalog #4970, CST), anti-
GAPDH (Catalog # 2118, CST), anti-phospho-AMPKα
(Thr172) (Catalog #2535, CST), anti- AMPKα (Catalog
#5831, CST), anti-ACSS2 (Catalog No. 16087-1-AP, Pro-
teintech), anti-p62(Catalog #23214, CST), anti-TFEB (Cat-
alog No. 13372-1-AP, Proteintech), anti-LC3A/B (Catalog
#12741, CST), anti-ATG7 (Catalog No. 10088-2-AP, Pro-
teintech), and anti-Flag (Catalog No. 66008-4-Ig, Protein-
tech). The detailed procedures were performed as previously
described [23]. The bands representing the targeted proteins
were subsequently exposed using the ChemiDoc imager
(Bio-Rad, USA). The gray value of the protein bands was
analyzed by Image J software (NIH, USA).
Co-immunoprecipitation (CO-IP)
Initially, cells were transfected with an ACSS2 overexpres-
sion vector carrying a Flag tag to ensure strong expression
of the target protein. Subsequently, the overexpressing
cells were harvested following strict protocols and lysed
thoroughly using an appropriate lysis buer (Beyotime,
China) to maintain the integrity and activity of the Flag-
tagged protein. An immunoprecipitation kit utilizing Protein
A + G magnetic beads (Beyotime, China) was employed, co-
immunoprecipitation was performed according to the manu-
facturer’s instructions. Antibodies used in this study include
anti-TFEB (Catalog No. 13372-1-AP, Proteintech) and anti-
Flag (Catalog No. 66008-4-Ig, Proteintech).
Aortic ring assay
The aortic ring assay is a method for studying vascular
development. In this experiment, we utilized aortas from
ACSS2Tek -CKO and ACSS2f/f mice as research specimens,
cleaned the surrounding tissues, and sectioned them into
1 mm long rings. Subsequently, the aortic rings were cul-
tured in growth factor-reduced matrigel (BD Biosciences,
USA) at 37 °C. On the 7th day, vascular development was
assessed using an inverted microscope platform (Leica,
China).
One Step RT-PCR Kit (Vazyme, China). Sequentially, qRT-
PCR analysis was carried out employing ChamQ SYBR
qPCR Master Mix (Vazyme, Canada). Relative mRNA
expression levels were determined using the comparative
threshold cycle (2^−ΔΔCT) method, referencing to ACTIN.
Primer sequences applied in this research are provided in
the supplementary information Table 1.
Construction of stable bEnd.3 cell lines with ACSS2
overexpression and ACSS2 knockdown
The overexpression plasmid of ACSS2 was provided by
Genray Biotech (WB9113Gn, China), while the knock-
down plasmid was provided by Rongdibio. Constructng
an overexpression plasmid using the pCDH-CMV-MCS-
EF1-copGFP-T2A-Puro cloning vector with ACSS2 as
the target gene. Transfection was performed when the cell
density reached 50-60%, with an empty vector used as the
control group. The medium was replaced with fresh culture
medium after 4 hours of transfection, followed by the addi-
tion of selective medium containing 2.5 µg/mL puromycin
(Beyotime, China) for stable cell screening after 48 h. Puro-
mycin selection pressure was maintained by changing the
medium every 2 days until the 6th day. The cells were then
adjusted to a concentration of 2 × 104 cells/mL and seeded
into 24-well plates at a volume of 1 mL per well. For clone
purication, single-cell isolation was performed using a
96-well plate, with each well pre-lled with 20 µL PBS.
Cells were digested with 0.05% trypsin-PBS solution and
carefully transferred to PBS-containing wells in the 96-well
plate. Ultimately, selected monoclonal cell lines were vali-
dated. Refer to Table 1 for specic sequence details.
RNA interference
TFEB siRNA (siTFEB #1, #2, #3) and AMPKα siRNA
(siAMPKα #1, #2, #3) were obtained from Hanbio Co.,
Ltd. Briey, the cell transfection protocol adhered to the
manufacturer’s recommendations for Lipofectamine 3000
(Invitrogen, USA), employing siTFEB, siAMPKα, and
a universal negative control siRNA (abbreviated as Con
siRNA). 24 h post-transfection, quantitative real-time PCR
was employed to systematically evaluate the inhibitory
ecacy of each siRNA sequence on target gene expres-
sion. Based on the validation results from qRT-PCR, the
most ecacious siRNA sequences in terms of inhibition
were selected for subsequent functional experiments, ensur-
ing the reliability and depth of the study ndings. Detailed
information regarding the siRNA sequences was provided
in the supplementary materials of the paper for readers’
reference.
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W. Zhang et al.
between two groups. A P-value of less than 0.05 was consid-
ered to indicate statistical signicance.
Results
Energy metabolism disruption and reduced ACSS2
expression in mouse brain following CA/CPR
As previously described, we rst induced CA by intrajugu-
lar injection of potassium chloride, followed by initiation of
CPR after a 5-minute arrest period. Representative electro-
cardiograms (ECGs) of this procedure are shown in Supple-
mentary Fig. 1A-F. We monitored potassium concentrations
in the blood prior to the procedure, 1 h post-procedure,
and 24 h post-procedure. Although there was a transient
increase in serum potassium levels observed 1 h after the
intervention, this elevation did not reach statistical signi-
cance (Figure S1G). Then, we examined changes in energy
metabolism of brain tissue 24 h after CA/CPR in mice,
compared to sham controls (Fig. 1A-C). Through metabo-
lomics sequencing analysis, we observed elevated levels
of uracil in CA/CPR group mouse brains, accompanied by
signicant reductions in L-Alanine, Tyrosine, L-citrulline,
L-Asparagine, Serine, Lysine, and Inosine, indicative of dis-
rupted cerebral energy metabolism (Fig. 1C). Of particular
importance, a marked decrease in ATP levels (Fig. 1D) and
acetyl-CoA (Fig. 1E), a core component of energy metabo-
lism, was detected in the brain tissue of mice post CA/CPR.
Against this backdrop, the signicance of short-chain fatty
acids (SCFAs) as alternative energy sources came to the fore.
Hematoxylin-eosin (HE) and Nissl staining
Mice were euthanized with an intraperitoneal injection of
3% pentobarbital sodium, followed by perfusion with PBS
and 4% paraformaldehyde to retrieve and x organs. These
organs were xed in 4% paraformaldehyde, processed for
histology, embedded in paran, and sectioned at 8 μm.
Hematoxylin and eosin (HE) staining was performed,
and cellular morphology was examined microscopically
(Hamamatsu Photonics, Japan) to assess damage. For Nissl
staining, paran-embedded brain sections were stained
according to the manufacturer’s instructions, incubated in
0.1% methylene blue for 3 min, rinsed with double-distilled
water, dehydrated, cleared, and sealed with neutral resin.
Neuronal structure and distribution in the hippocampus
were observed under a microscope (Hamamatsu Photonics,
Japan).
Immunouorescence staining
Freshly collected tissue samples were promptly frozen in
liquid nitrogen and sectioned into 8 μm-thick slices. These
slices were subjected to a series of preprocessing steps [24],
sealed, and analyzed for morphological and uorescence
signals using an advanced microscope system. Primary
antibodies used included anti-ACSS2 (Catalog No. 16087-
1-AP, Proteintech), anti-GFAP (Catalog #12389, CST), anti-
Iba1 (Catalog #17198, CST), anti-NeuN (Catalog #24307,
CST), anti-CD31 (AF3628-SP, Novus Biologicals), anti-
TFEB (Catalog No. 13372-1-AP, Proteintech), and anti-
LC3A/B (Catalog #12741, CST).
In cell culture experiments, cells were seeded at 5 × 104
cells per well in a 12-well plate. Once adhered and at the
desired growth state, cells were stimulated with experimen-
tal drugs. They were then xed, permeabilized, blocked, and
incubated with primary antibodies overnight at 4°C. The
next day, uorescently labeled secondary antibodies were
added, and slides were sealed. Target protein localization
and expression intensity were observed using a uorescence
microscope (3DHISTECH, Hungary). Primary antibodies
used included anti-ACSS2 (MA5-25697, Invitrogen) and
anti-CD31 (AF3628-SP, Novus Biologicals).
Statistical analysis
All statistical analyses were conducted using GraphPad
Prism 8.0 software. Data with a normal distribution are
presented as mean ± SD. One-way analysis of variance
(ANOVA) followed by Tukey’s post hoc test for multiple
comparisons or two-way ANOVA with Bonferroni pair-
wise comparisons was used for comparisons among mul-
tiple groups. Student’s t-test was employed for comparisons
Fig. 1 Energy metabolism disorders in mouse brain tissue induced by
CA/CPR and the role of ACSS2 in BMVECs during metabolic stress.
(A) Clustering analysis of all targeted energy metabolism metabolo-
mics. n = 6. (B) Clustering analysis of dierential metabolites in tar-
geted energy metabolism metabolomics studies. n = 6 . (C) Statistical
analysis of dierential metabolites in B. (D-E) The levels of ATP (D)
and acetyl-CoA (E) in mouse brain tissue. n = 6. (F) The content of
short-chain fatty acids in mouse brain tissue. n = 6. (G) Representative
western blot showing cortex and hippocampus expression of ACSS2 in
dierent groups. (H) Quantitative expressional analysis of WB bands
of ACSS2. n = 5. (I) The mRNA levels of ACSS2 in cortex and hip-
pocampus of dierent groups. n = 4. (J) Representative immunouo-
rescence micrographs showing the expression of ACSS2 and CD31
in the mouse hippocampus and cortex. Scale bar, 20 μm. (K-L) Co-
localization analysis of ACSS2 and CD31 in the mouse cortex (K) and
hippocampus (L), as indicated by white arrows. (M) Representative
micrographs showing nuclear localization of ACSS2 (red) in bEnd.3
cells. Scale bar, 20 μm. (N) Quantitative analysis of nuclear transloca-
tion-positive cell proportion in M. n = 3. (O-P) Co-localization analy-
sis of ACSS2 and DAPI in bEnd.3 cells in control (O) and OGD/R (P)
groups, as indicated by white arrows. Data are presented as mean ± SD.
Statistical analyses were performed using one-way ANOVA for mul-
tiple group comparisons. For comparisons between two groups, Stu-
dent’s t-test was employed. *P < 0.05, **P < 0.01, ***P < 0.001versus
the control or sham group
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Acetyl-CoA synthetase 2 alleviates brain injury following cardiac arrest by promoting autophagy in brain…
conventional glycolysis and fatty acid oxidation pathways,
hindering energy production.
Under high-energy demand or metabolic stress condi-
tions, ACSS2 plays a crucial role by utilizing non-carbo-
hydrate substrates like acetate to maintain cellular energy
homeostasis [17]. Our subsequent investigation of ACSS2
Targeted SCFA metabolomics analyses revealed that acetate
was most abundant in mouse brain tissue and showed a pro-
nounced increase following CA/CPR intervention (Fig. 1F).
Decreased levels of ATP and acetyl-CoA, along with the
accumulation of acetate, suggested impairment of both
1 3
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W. Zhang et al.
of their physiological reactions. To assess long-term health
eects, we recorded body weight changes from the injection
onset to 10 weeks of age (over a period of four weeks). This
showed no signicant dierence between the AAV-BR1
treated group and controls, attesting to the vector’s safety
concerning mouse growth and development. (Figure S4A).
Proving eective ACSS2 upregulation, Western Blots
of cortical and hippocampal tissues displayed heightened
ACSS2 and Flag-tagged proteins in mice injected with
HBAAV2/BR1-m-ACSS2, arming targeted overexpres-
sion in BMVECs (Figure S4B-C). Histological inspections
of key organs, including liver, lungs, heart, kidneys, and
spleen via H&E staining, despite EGFP presence in some,
demonstrated normal structures, suggesting AAV-BR1’s
relative safety in o-target organs (Figure S4D). NeuN
staining of hippocampal regions CA1-CA3 armed no neu-
ronal loss or abnormalities due to ACSS2 overexpression,
underlining the intervention’s precision and safety (Figure
S4E-F).
Before inducing cardiac arrest, we evaluated the basic
physiological indicators of ACSS2-OE mice and control
mice, comprising body weight, heart rate, and body tem-
perature, to guarantee uniformity between the experimental
and control groups at the beginning of the study (Table 2).
Subsequently, we systematically recorded the heart rate
at ROSC, the heart rate 60 min post-ROSC, and the tem-
perature at the end of the resuscitation process, with no
signicant dierences observed (Table 2). Throughout the
course, all mice in the CA/CPR group successfully achieved
resuscitation, with consistent key resuscitation parameters,
including CPR time to ROSC and appropriate use of adrena-
line (Table 2). Progressing, in our ACSS2-OE mouse model,
CA/CPR induction allowed for systematic neurological
outcome assessments. Post-CA/CPR, mice presented sig-
nicantly lower neurological function scores than controls,
paralleled by raised neural injury markers, NSE and S100β
(Fig. 2A-D). Notably, ACSS2-OE mice demonstrated supe-
rior neurological scores, faster motor test performances, and
milder serum marker elevations, implicating ACSS2’s neu-
roprotection (Fig. 2A-D). Increased IFN-β post-CA/CPR in
levels in brain tissue demonstrated a substantial decrease in
both cortical and hippocampal regions of mice post CA/CPR
(Fig. 1G). This diminution in ACSS2, instrumental in ace-
tate’s conversion to acetyl-CoA, may directly limit acetate
utilization, exacerbating the energy crisis. Immunouores-
cence staining conrmed ACSS2’s predominant localiza-
tion in neurons, astrocytes, and vascular endothelial cells
within the CA1 region of the hippocampus in mice (Figure
S2A-E, Fig. 1J-L), with relatively limited expression in the
latter cell type yet playing a pivotal role in acetate metab-
olism regulation [25]. Intriguingly, in an in vitro OGD/R
model, ACSS2 underwent noticeable nuclear translocation
(Fig. 1M-P), highlighting its potential importance in vas-
cular endothelial cells, especially in coping with metabolic
stress induced by CA/CPR.
Based on these observations, we hypothesized that
enhancing ACSS2 activity in BMVECs might be a novel
strategy for improving brain function post CA/CPR.
Overexpression of ACSS2 in BMVECs enhances brain
function recovery in mice following CA/CPR
To investigate directly the impact of the ACSS2 gene on
brain injury in mice following CA/CPR, particularly within
BMVECs, we established a mouse model with BMVEC-
targeted ACSS2 overexpression. Utilizing the AAV-BR1
serotype vector from Hanbio, China, known for its specic-
ity in transducing brain endothelial cells, we initiated our
study. Firstly, 6-week-old mice received AAV-BR1-EGFP
empty vectors through tail vein injections. Over the subse-
quent two weeks, live imaging tracked EGFP uorescence
intensity across multiple organs, revealing the brain as the
primary site of intense uorescence, though the liver, kid-
neys, and lungs also exhibited variable expression (Figure
S3A-B). Immunouorescence analysis further validated
ecient transduction of brain microvascular endothelium
by AAV-BR1 (Figure S3C-D).
Building on this foundation, we engineered an AAV-
BR1 vector carrying the ACSS2 gene (HBAAV2/BR1-m-
ACSS2) for administration to mice, with close monitoring
Control-
Sham
(n = 1 0)
ACSS2-OE-
Sham
(n = 1 0)
Control-CA
(n = 1 0)
ACSS2-OE-
CA/
CPR(n = 1 0)
P value
Weight, g 23.6 ± 1.5 24.7 ± 1.4 23.4 ± 2.3 24.5 ± 1.5 0.2533
Heart rate before arrest, bpm 407.4 ± 27.8 387.1 ± 20.3 409.6 ± 39.1 390.7 ± 78.4 0.6246
Temperature before arrest, o C 36.5 ± 0.5 36.2 ± 0.6 36.4 ± 0.6 36.0 ± 0.6 0.2367
CPR time to ROSC, s / / 318.0 ± 151.8 288.0 ± 144.6 0.6563
Heart rate at ROSC, bpm / / 329.2 ± 85.5 285.5 ± 164.7 0.4661
Heart rate 60 min after ROSC,
bpm
/ / 556.7 ± 50.8 525.0 ± 92.4 0.3544
Temperature at end, o C 36.2 ± 0.6 36.3 ± 0.4 36.2 ± 0.8 36.5 ± 0.4 0.6154
Epinephrine dosage (32ug/ml), ml / / 0.5 ± 0.2 0.5 ± 0.2 > 0.9999
Table 2 Baseline characteristics
of the four mouse groups
Data with a normal distribution
are presented as mean ± SD.
Student’s t-test was employed
for comparisons between two
groups. One-way ANOVA was
employed for comparisons
among four groups. P-value of
less than 0.05 was considered to
indicate statistical signicance
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Acetyl-CoA synthetase 2 alleviates brain injury following cardiac arrest by promoting autophagy in brain…
assays showing negligible variations (Fig. 3M). In harmony
with gain-of-function outcomes, ACSS2 depletion dra-
matically hindered EC migration (Fig. 3N-O) and curtailed
nodes by 34% (Fig. 3P-Q) and total length by 27% (Fig. 3P,
R), reinforcing ACSS2’s pivotal role in these dynamics.
ACSS2-mediated autophagy regulation in BMVECs
following CA/CPR
In the following research segment, we meticulously explored
the molecular mechanisms by which ACSS2 improves
endothelial dysfunction in BMVECs. Autophagy is a cel-
lular process in which cells self-digest and recycle their own
components, essential for cellular survival and maintaining
energy homeostasis, especially under conditions of nutrient
deprivation [26, 27]. We uncovered that ACSS2 potently
enhances the expression of numerous autophagy-linked
genes (Fig. 4A), implicating ACSS2 in EC regulation via
the autophagy pathway following OGD/R treatment. Nota-
bly, ACSS2 overexpression not only boosts the mRNA
transcription of pivotal autophagy genes—TFEB, and
autophagy related 7(ATG7)—but also elevates their protein
abundance, paralleled by an augmented microtubule-asso-
ciated protein 1 light chain 3 (LC3) II /LC3-I ratio (Fig. 4E
and G-H), eects starkly reversed upon ACSS2 depletion
in BMVECs following OGD/R treatment (Fig. 4F and
I-J). Concurrently, ACSS2 overexpression notably reduced
sequestosome 1 (p62/ SQSTM1) level in brain microvas-
cular endothelial cells, whereas ACSS2 knockout exhibited
an opposing outcome (Fig. 4E-F). Furthermore, heightened
ACSS2 levels resulted in a remarkable 228% increase, on
average, in autophagic lysosome counts within BMVECs
(Fig. 4B-C), a change unaccompanied by alterations in
mitochondrial counts (Fig. 4D).
To corroborate the physiological and pathophysiological
signicance of ACSS2-induced autophagy activation, we
turned to a CA/CPR mouse model. Initially, we examined
autophagic activity in the hippocampal BMVECs of CPR
group and control group mice using electron microscopy. As
illustrated in Fig. 4K-L, autophagosome formation was evi-
dent in the CPR group. There was a trend toward an increase
in the number of autolysosomes, although this increase was
not statistically signicant (Fig. 4M). Furthermore, we
observe an aberrant amplication of autophagic activity
after CPR, accompanied by unusually elevated p62 protein
levels– a hallmark indicative of impaired autophagic ux
(Fig. 4N, P), suggesting that cellular capacity for clearing
damaged materials is compromised amidst revival stress.
The highlight of our ndings is the discovery that tar-
geted overexpression of ACSS2 in BMVECs could poten-
tially reverse this detrimental trend. Relative to control
groups, genetically modied mice subjected to CPR and
controls was attenuated in the ACSS2-OE group, whereas
IL-6 levels remained unchanged (Fig. 2E-F).
Delving into ACSS2’s energy metabolic regulation,
ACSS2 overexpression signicantly boosted brain acetyl-
CoA levels and ATP levels at 24 h after CPR (Fig. 2G-H).
Cerebral blood ow assessment post-CA/CPR highlighted
ACSS2-OE’s ecacy in enhancing ow without disrupt-
ing baseline levels (Fig. 2I-J). The immunouorescence
results further demonstrated that ACSS2 overexpression
attenuated the reduction of CD31-positive vascular area in
the hippocampus, indicating its vascular protective eects
(Fig. 2K-L). Although ACSS2 overexpression exhibited
improvements in average vessel length, total number of end
points and junctions within the hippocampal region, these
changes failed to reach statistical signicance (Fig. 2M-O).
Additionally, cortical analysis revealed that ACSS2 overex-
pression signicantly increased the number of end points
(Figure S5A, D), while other parameters including total
vessels area, average vessel length, and junction numbers
showed no statistically signicant alterations (Figure S5B-
C, E).
Overexpression of ACSS2 in BMVECs improves
endothelial dysfunction following OGD/R treatment
Dysfunctional endothelial cells exhibit impaired migra-
tion, tube formation, and growth [12]. Through meticulous
application of gain- and loss-of-function methodologies, we
examined the ACSS2 gene’s impact on BMVEC functional
biology.
Initiating with ACSS2 overexpression, BMVECs under-
went lentivira. l transfection with ACSS2 expression con-
structs, followed by puromycin selection (1.5 µg/ml) to
secure stable clones (Figure S5F). Western blot and q-PCR
validation armed successful ACSS2 overexpression
(Fig. 3A-C). Prior to vascular function examination, cells
were pre-treated with OGD/R. Of particular note, while
ACSS2 exerted a profound regulatory impact on EC migra-
tion and tube formation, its direct eect on EC proliferation
was minimal, ascertained by CCK8-based cell metabolic
activity assays showing negligible variations (Fig. 3D).
Strikingly, ACSS2 surplus signicantly accelerated EC
migration (Fig. 3E-F) and, in tube formation assays, trig-
gered a profound increase in nodes count (up by 314%) and
total tube length (increased by 187%), thereby underscoring
a potent pro-angiogenic inuence (Fig. 3H and I).
On the contrary, to suppress ACSS2, BMVECs were
transfected with either control-shRNA or ACSS2-shRNA
deploying Lipo3000, leading to stable clone isolation
and verication of ACSS2 knockdown eciency through
western blot and q-PCR (Fig. 3J-L). Notably, ACSS2 had
minimal eects on EC proliferation, as evidenced by CCK8
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W. Zhang et al.
autophagic activity. We observed an increase in TFEB pro-
tein levels, indicating an upward trend, but unlike in vitro
experiments, this elevation failed to exhibit a statistically
signicant dierence (Fig. 4N, P).
exhibiting ACSS2 overexpression demonstrated not only a
substantial reduction in p62 levels (Fig. 4N, P), signifying a
signicantly enhanced clearance eciency in the autopha-
gic process, but also a marked increase in the LC3II/LC3I
ratio (Fig. 4N-O), which directly attests to heightened
autophagosome formation, further corroborating augmented
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Acetyl-CoA synthetase 2 alleviates brain injury following cardiac arrest by promoting autophagy in brain…
ecacy of our genetic manipulation (Figure S6B-C). The
aortic ring assay solidies TFEB’s centrality in ACSS2-
driven angiogenesis, with EC-specic ACSS2 deletion
mice manifesting diminished aortic outgrowth (Fig. 5J-K).
Employing TFEB activator 1 to restore TFEB nuclear local-
ization rescues angiogenic impairment [30], underscoring
TFEB’s therapeutic potential (Fig. 5J-K). AMP-activated
protein kinase-α (AMPKα) is a well-recognized key regu-
lator of autophagy [31], energy homeostasis [32] and
angiogenesis [33]. By inhibiting AMPKα activation using
compound C, we observed a reduction in the pro-angiogenic
eects of TFEB agonists (Fig. 5J-K). These ndings posi-
tion AMPKα as a critical regulator within the ACSS2-medi-
ated angiogenic cascade. Meanwhile, AMPKα silencing via
siRNA and veried the eciency of the knockdown through
western blotting and qPCR (Fig. 5L-N). AMPKα depletion
signicantly hindered the migration of ACSS2-overex-
pressing BMVEC following OGD/R treatment (Fig. 5O-
P), reducing the number of nodes by 67% (Fig. 5F-G) and
the total length by 51% (Fig. 5F, H). These ndings further
solidify that TFEB and AMPKα are required for ACSS2 to
improve endothelial dysfunction, and this eect is threat-
ened in the absence of functional AMPKα.
ACSS2 activates the AMPKα signaling pathway
Next, we proceed to demonstrate that the AMPKα signaling
pathway was enhanced by ACSS2 in BMVECs. Initially,
we employed the CCK8 assay to ascertain the eective
concentration of VEGF (Figure S5G). In the presence of
VEGF (2.5ng/ml), ACSS2 overexpression facilitated phos-
phorylation of AMPKα at the Thr172 residue (Fig. 6A-B),
echoing previous reports where phosphorylation at this site
was markedly elevated within 5 min of VEGF stimulation
(Fig. 6A-B). Consistently, under both basal conditions and
upon VEGF stimulation, knockdown of ACSS2 led to a
reduction in AMPKα phosphorylation (Fig. 6C-D).
Moving forward, we aimed to elucidate whether TFEB
activator1 modulates AMPKα phosphorylation to facilitate
ACSS2-mediated angiogenesis. Our ndings revealed that
pretreatment with TFEB activator1 in ACSS2-knockdown
BMVECs successfully enhanced AMPKα phosphorylation
levels, an eect not notably observed in non-modied con-
trol cells (Fig. 6E-F). Strikingly, in these ACSS2-decient
BMVECs, TFEB activator1 signicantly promoted cell
migration following OGD/R treatment (Fig. 6G-H) and,
in tube formation assays, it dramatically increased node
numbers by 107% (Fig. 6I-J) and the total length by 35%
(Fig. 6I, K), underscoring a substantial boost in angiogenic
capacity.
These ndings not only conrm the pivotal role of
ACSS2 in augmenting AMPKα signaling within BMVECs
TFEB and AMPKα are required for ACSS2 to improve
endothelial dysfunction
TFEB (transcription factor EB) is a transcription factor
that regulates autophagy and lysosomal biogenesis within
cells, and its function depends on translocation from the
cytoplasm to the nucleus [28]. In our study, immunouo-
rescence analysis following OGD/R treatment showed that
overexpression of ACSS2 promoted nuclear translocation
of TFEB (Figure S5H-I). Next, we investigated the inter-
action between ACSS2 and TFEB, focusing on their roles
in mitigating dysfunction in BMVECs following OGD/R
treatment. First, we systematically performed TFEB knock-
down in BMVEC with ACSS2 overexpression and veried
the eciency of the knockdown through western blotting
and qPCR (Fig. 5A-C). Remarkably, TFEB depletion sig-
nicantly hindered the migration of ACSS2-overexpressing
in BMVECs (Fig. 5D-E), reducing the number of nodes by
82% (Fig. 5F-G) and the total length by 53% (Fig. 5F, H),
emphasizing TFEB’s impact on angiogenesis. To ascertain
whether an interaction exists between ACSS2 and TFEB, we
conducted co-immunoprecipitation experiments using ag-
tagged ACSS2 protein. Consistent with previous reports,
our study results revealed a physical interaction between
ACSS2 and TFEB. (Fig. 5I).
Proceeding forward, we engineered transgenic mice
with endothelial cell-specic ACSS2 knockout utilizing the
CRE-LOXP system (Figure S6A). The aortic ring assay,
a well-established in vivo technique for evaluating angio-
genic potential, was subsequently employed [29]. Aortae
harvested from these transgenic mice, alongside those from
control mice, were subjected to western blot analysis, which
revealed a substantial reduction in ACSS2 protein levels
within the aortic tissue of transgenic mice, arming the
Fig. 2 Overexpression of ACSS2 in BMVECs enhances brain function
recovery in mice following CA/CPR. (A) Neurological function scores
of ACSS2-OE mice and control mice before CPR and 24 h after CPR.
n = 10. (B) Time to cross the circle for ACSS2-OE mice and control
mice before CPR and 24 h after CPR. n = 10. (C-F) Serum levels of
S100β (C), NSE (D), IFN-β (E), and IL-6 (F) in ACSS2-OE mice and
control mice, in both the sham and CPR-24 h groups. n = 10. (G-H)
Brain tissue levels of ATP (G) and acetyl-CoA (H) in ACSS2-OE mice
and control mice, in both the sham and CPR-24 h groups. n = 6. (I)
Representative images showing cerebral blood ow (CBF) reperfu-
sion monitored by Doppler laser ultrasound at the indicated time. (J)
Quantitative analysis of CBF relative signal intensity at specied time
points. n = 10. (K) Representative immunouorescence micrographs
showing the expression of CD31 in the mouse hippocampus. Scale bar,
100 μm. (L-O) Quantitative analysis of vessel percentage area (L),
average vessels length (M), total number of end points (N), and total
number of junctions (O) in ACSS2-OE mice and control mice, in both
the sham and CPR-24 h groups. n = 4. Data are presented as mean ± SD.
Statistical analyses were performed using one-way ANOVA for mul-
tiple group comparisons. *P < 0.05, **P < 0.01, ***P < 0.001versus the
preoperative control or sham group. #P < 0.05, ##P < 0.01, ###P < 0.001
versus the control group at 24 h following CPR
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W. Zhang et al.
Discussion
CA is a leading cause of death worldwide, imposing sub-
stantial social and economic burdens on public health [34].
This study aimed to elucidate the role and mechanisms of
but also uncovers a novel regulatory axis involving TFEB
activator 1, which particularly inuences the angiogenic
potential under conditions of reduced ACSS2 activity.
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Acetyl-CoA synthetase 2 alleviates brain injury following cardiac arrest by promoting autophagy in brain…
post-CPR, marking a signicant advancement in the eld.
This research underscores the immense potential of modu-
lating microvascular endothelial metabolic pathways via
ACSS2 as an innovative, clinically relevant strategy to sig-
nicantly enhance recovery outcomes in patients suering
from brain injuries associated with CPR procedures.
Our initial hypothesis posited that the neuroprotec-
tive eect of BMVECs ACSS2 following CPR was indi-
rect, potentially through attenuation of neuroinammation.
Analysis revealed that in ACSS2-OE mice post-CA/CPR,
the increase in IFN-β levels observed in control mice was
mitigated (Fig. 2E), suggesting ACSS2 can alleviate inam-
mation induced by CA/CPR. Despite IL-6 levels remaining
unchanged between groups (Fig. 2F), this still underscores a
selective regulatory role of ACSS2 in inammatory media-
tors. Nonetheless, the striking observation of enhanced
cerebral perfusion in ACSS2- OE mice juxtaposed against
controls in the aftermath of CA/CPR suggests an immediate
and pivotal role for ACSS2 in BMVECs (Fig. 2I-J). This
role involves expediting metabolic reprogramming, thereby
augmenting cerebral blood ow—an eect that diverges
from our preliminary conjectures. This nding prompts us
to reconsider the direct impact of endothelial ACSS2 on
regulating endothelial function.
The mammalian genome encodes three ACSS genes,
with ACSS1 and ACSS3 localized in mitochondria, while
ACSS2 resides in the cytoplasm and nuclear compartments
of mammalian cells [14, 35]. In response to adverse envi-
ronmental cues, however, ACSS2 can be phosphorylated
by AMPK and translocate to the nucleus to transcription-
ally regulate its targets [36]. Consistent with these data, we
found ACSS2 accumulation in the nucleus under oxygen
and glucose deprivation conditions (Fig. 1M-P). Nuclear
translocation of ACSS2 under hypoxia or nutrient depriva-
tion implies an adaptive and potentially protective function
for ACSS2 in BMVECs (Fig. 1M-P). Thus, AMPKα may
mediate ACSS2-dependent signaling under starvation con-
ditions. On another front, ACSS2 enhances the activity of
TFEB in BMVECs (Figure S4D-E), revealing an interac-
tion between the ACSS2 and TFEB signaling pathways. As
a novel nding, we observed ACSS2 sensitivity to nutri-
ent deprivation in BMVECs, broadening our understand-
ing of ACSS2’s molecular regulation, encompassing gene
expression and nuclear translocation. Inhibition of TFEB
or AMPKα suppresses the enhanced migratory and tubulo-
genic capacity of BMVECs induced by ACSS2 upregula-
tion (Fig. 5D-H and O-S).
CBF is meticulously regulated through interconnected
surface arteries [37], penetrating arterioles, and an extensive
capillary meshwork [38], all coordinated by neurovascular
coupling mechanisms that align blood ow with metabolic
demands of neurons [39]. Capillaries constitute the majority
BMVECs in the pathology of brain injury following CPR,
with a particular focus on how ACSS2 facilitates metabolic
reprogramming in BMVECs to enhance cerebral blood ow
and thereby alleviate injury. Our ndings revealed a dys-
regulated energy metabolism in the murine brain post-CPR,
characterized by decreased levels of various amino acids
and acetyl-CoA, alongside impaired utilization of acetate,
an alternative energy substrate (Fig. 1A-E). This metabolic
disturbance appears to stem from a reduction in ACSS2 lev-
els within brain tissue (Fig. 1G-I).
To address this, we employed AAV-BR1 to selectively
overexpress ACSS2 in brain microvascular endothelium,
the cellular layer directly interfacing with the bloodstream
(Figure S2A-D). This intervention eectively mitigated
neurological dysfunction in CPR mice without eliciting dis-
cernible adverse eects (Fig. 2A-B). Our investigation thus
pioneers the recognition of ACSS2 as a central mediator of
BMVEC functionality, casting new light on the intricate
molecular mechanisms underlying endothelial dysfunc-
tion (Fig. 7). By spotlighting ACSS2, we have uncovered a
highly promising therapeutic target for interventions target-
ing ischemia-reperfusion injuries akin to those encountered
Fig. 3 Overexpression of ACSS2 in BMVECs improves endothelial
dysfunction following OGD/R treatment. (A) Representative west-
ern blot showing expression of ACSS2 in bEnd.3 stable cell lines
transfected with GFP control or ACSS2 overexpression plasmid. (B)
Quantitative expressional analysis of WB bands of ACSS2 shown in
A. n = 3. (C) The mRNA levels of ACSS2 in bEnd.3 stable cell lines
transfected with GFP control or ACSS2 overexpression plasmid. n = 3.
(D) Cell viability at various time points in bEnd.3 stable cell lines
transfected GFP control or ACSS2 overexpression plasmid following
OGD/R treatment. n = 3. (E) Representative images of wound healing
assay for the GFP and ACSS2-OE groups following OGD/R treatment.
Scale bar, 500 μm. (F) Statistical analysis of relative migration areas at
24 h for the GFP and ACSS2-OE groups in the wound healing assay.
n = 8 . (G) Representative images of tube formation for the GFP and
ACSS2-OE groups following OGD/R treatment. Scale bar, 50 μm. (H-
I) Number of nodes (H) and total length (I) per eld were measured
by NIH imageJ and statistically analyzed. n = 10. (J) Representative
western blot showing expression of ACSS2 in bEnd.3 stable cell lines
transfected with either control-shRNA or ACSS2-shRNA plasmid. (K)
Quantitative expressional analysis of WB bands of ACSS2 shown in J.
n = 3 . (L) The mRNA levels of ACSS2 in bEnd.3 stable cell lines trans-
fected with either control-shRNA or ACSS2-shRNA plasmid. n = 3 .
(M) Cell viability at various time points in bEND.3 stable cell lines
transfected with either control-shRNA or ACSS2-shRNA plasmid fol-
lowing OGD/R treatment. n = 3. (N) Representative images of wound
healing assay for the control-shRNA and ACSS2-shRNA groups fol-
lowing OGD/R treatment. Scale bar, 500 μm. (O) Statistical analysis
of relative migration areas at 24 h for the control-shRNA and ACSS2-
shRNA groups in the wound healing assay. n = 8. (P) Representative
images of tube formation for the control-shRNA and ACSS2-shRNA
groups groups following OGD/R treatment. Scale bar, 50 μm. (Q-R)
Number of nodes (Q) and total length (R) per eld were measured by
NIH imageJ and statistically analyzed. n = 10. Data are presented as
mean ± SD. Statistical analyses were performed using Student’s t-test.
*P < 0.05, **P < 0.01, ***P < 0.001versus the GFP or control-shRNA
group
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W. Zhang et al.
role through stabilizing residual vascular structures. In
contrast, in the cortex, ACSS2 overexpression selectively
increased the number of end points (Figure S5A, D), a
phenomenon consistent with the LSCI-detected enhance-
ment of cortical blood ow (Fig. 2I). Notably, the increased
number of end points may have reected ACSS2-mediated
of this network, extensively covering all brain regions. Our
data demonstrated that ACSS2 exerted region-specic vas-
cular regulatory eects: in the ischemia-vulnerable hippo-
campus, ACSS2 signicantly preserved the CD31⁺ vascular
area (Fig. 2K-L) without improving angiogenic parameters
(average length, junctions, etc.), suggesting its protective
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Acetyl-CoA synthetase 2 alleviates brain injury following cardiac arrest by promoting autophagy in brain…
ACSS2 activity abrogates the capacity of acetate to sustain
cancer cell viability under acidic environments. Mice lack-
ing ACSS2 exhibit reduced acetate uptake in liver tumors,
accompanied by a marked decrease in tumor burden [44].
This implies that ACSS2 is a pivotal player in adapting to
metabolic stress.
Endothelial cells exhibit metabolic features akin to many
cancer cells, prominently featuring a high dependency on
glycolysis even in the absence of complete glucose oxidation
[45, 46]. Endothelial cells primarily generate ATP through
the glycolytic pathway and convert the major carbon source
of glucose into lactate for export [47]. Notably, Endothelial
cells also possess the unique ability to convert glucose into
acetate, which can then be transformed into cytoplasmic
acetyl-CoA by the ACSS2 enzyme [25]. Our study high-
lights that elevating ACSS2 expression in BMVECs of mice
subjected to cardiac arrest followed by CPR signicantly
increases acetyl-CoA content in brain tissue (Fig. 1E).
Moreover, considering that under ample glucose supply and
the citric acid cycle shuttle, cells or tissues can eciently
convert glucose into acetyl-CoA and potentially act as net
producers rather than mere consumers of acetate, this phe-
nomenon is also evident in non-genetically modied normal
mice, where ACSS2 overexpression in BMVECs is simi-
larly associated with higher acetyl-CoA levels (Fig. 1E).
Hence, endothelial cells’ capability to exploit acetate as a
metabolic resource might constitute a vital adaptive strat-
egy supporting cell proliferation and survival under nutrient
restriction or hypoxic microenvironments.
Previous studies have highlighted the pivotal role of
ACSS2 in the nervous system, particularly in mouse hip-
pocampal neurons [14], where it acts as a coactivator in
regulating histone acetylation processes, thereby promot-
ing the expression of genes associated with spatial mem-
ory formation. Of note, reduced ACSS2 levels have been
observed in postmortem temporal cortex tissues from
Alzheimer’s disease (AD) patients and in the hippocampus
and prefrontal cortex of late-stage 5×FAD mice [48]. This
nding is corroborated by observations of restored histone
acetylation levels, increased glutamate receptor expression,
and improved cognitive function following supplementa-
tion with the acetate precursor glycerol triacetate (GTA),
underscoring the importance of ACSS2 and its metabolic
pathways in maintaining synaptic plasticity and cognitive
function [48]. Similarly, in our investigation, we observed
that ACSS2 expression extends beyond neurons to include
glial cells and vascular endothelial cells (Figure S1A-E). By
specically enhancing ACSS2 activity in brain microvascu-
lar endothelial cells, we successfully alleviated neurological
decits in mice following cardiopulmonary resuscitation.
Importantly, our study transcends the conventional perspec-
tive, which primarily focuses on the neuronal functions
promotion of acute-phase vascular compensatory responses.
While current data did not denitively establish their direct
involvement in functional collateral circulation formation,
this nding highlighted the greater plasticity of the cortical
vascular network in responding to ACSS2-driven metabolic
regulation.
Notably, our quantitative analysis demonstrated that
ACSS2 overexpression signicantly enhanced the ZO-1/
CD31 area ratio during the post-resuscitation phase (Fig-
ure S7A-B). This structural alteration at neurovascular
interfaces suggests ACSS2 may confer protective eects on
BBB stability, potentially through modulation of tight junc-
tion-endothelial cell interactions. We proceeded to investi-
gate fundamental biological characteristics of EC function,
such as proliferation [40], migration [41], and tube forma-
tion [42], pathways potentially involved in the proangio-
genic phenotype induced by increased ACSS2 expression
in vitro. Our data suggest that tube formation and migra-
tion are key processes in angiogenesis critically regulated
by ACSS2 (Fig. 3E-I and O-S). However, in vitro modula-
tion of ACSS2 expression, either up or down, did not aect
EC proliferation (Fig. 3D, M). Cells might compensate for
ACSS2-mediated metabolic alterations by activating alter-
native pathways to maintain energy homeostasis and bio-
synthetic needs, preserving proliferative capacity. Indeed,
growing evidence suggests that ACSS2 promotes the use
of exogenous acetate to epigenetically and transcriptionally
regulate cell survival under acidic, hypoxic, or metabolic
stress conditions [17, 25, 43]. Extending this understand-
ing to a specic pathological context, suppression of
Fig. 4 ACSS2-mediated autophagy regulation in BMVECs following
CA/CPR. (A) The expression of autophagy-and lysosome biogene-
sis-related genes were quantied using real-time PCR. (B) Electron
micrograph of a typical stable transfectant of bEnd.3 cells with GFP
control or ACSS2 overexpression plasmid, where the white arrows
indicate autophagic lysosomes. Scale bar, 500 nm. (C-D) The aver-
age number of autolysosome (C) and mitochondria (D) per cell in B.
n = 12. (E) Representative western blot showing expression of ATG7,
TFEB, PRKN, LC3II/I in bEnd.3 stable cell lines transfected with GFP
control plasmid and ACSS2 overexpression plasmid following OGD/R
treatment. (F) Representative western blot showing expression of
ATG7, TFEB, PRKN, LC3II/I in bEnd.3 stable cell lines transfected
with control-shRNA plasmid and ACSS2-shRNA plasmid following
OGD/R treatment. (G-H) Quantitative expressional analysis of WB
bands in E. n = 5. (I-J) Quantitative expressional analysis of WB bands
of in F. n = 5. (K) Representative electron micrograph of BMVECs in
mouse hippocampal tissue, autophagosome formation is visibly indi-
cated by white arrows. Scale bar, 200 nm. (L-M) The average number
of autophagosome (L) and autolysosome (M) per eld of view. n = 5.
(N) Representative western blot showing expression of TFEB, p62 and
LC3II/I in mouse hippocampal tissue. (O-P) Quantitative expressional
analysis of WB bands in N. n = 5. Data are presented as mean ± SD.
Statistical analyses were performed using one-way ANOVA for mul-
tiple group comparisons. For comparisons between two groups, Stu-
dent’s t-test was employed. *P < 0.05, **P < 0.01, ***P < 0.001versus
the GFP, control-shRNA or sham group; ##P < 0.01 versus the control-
CPR group
1 3
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W. Zhang et al.
has far-reaching implications for maintaining brain health
and combating neurodegenerative diseases.
At the mechanistic level, ACSS2 activates AMPKα and
TFEB in BMVECs. Studies have demonstrated that ACSS2
forms complexes with TFEB in the nucleus, which are
then guided by TFEB to promoter regions of lysosome and
of ACSS2, revealing a broader protective role for ACSS2
within vascular endothelial cells. This, in turn, paints a more
comprehensive picture of ACSS2’s functional landscape,
suggesting that its sphere of inuence may be more exten-
sive than previously appreciated. It intimates that ACSS2
1 3
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Acetyl-CoA synthetase 2 alleviates brain injury following cardiac arrest by promoting autophagy in brain…
autophagy promotes in vitro tube formation, migration of
endothelial cells, and angiogenesis in vivo, depending on
cellular and molecular microenvironments [33]. In the pres-
ent study, we systematically investigated the pivotal role
of ACSS2 in the metabolic reprogramming of BMVECs
following CPR and uncovered the signicant involvement
of TFEB-mediated autophagy in ACSS2-induced tube
formation, aortic sprouting, and angiogenesis. While our
data implicate AMPKα- and TFEB-mediated autophagy in
ACSS2’s impact on angiogenesis, we cannot discount the
potential intermediary roles of other proangiogenic factors
in ACSS2-regulated angiogenesis.
AMPKα is a key regulator of autophagy [53] and angio-
genesis [54] under hypoxic conditions. Through inhibition
of AMPKα in in vitro tube formation assays (Fig. 5Q-S)
and ex vivo aortic ring sprouting assays (Fig. 5J-K), we
identied AMPKα as a novel signaling pathway mediat-
ing the proangiogenic eect of ACSS2 in BMVECs. Prior
studies have proven that AMPKα activation can enhance
phosphorylation of ACSS2 at S659, promoting its nuclear
translocation [36]. Conversely, our current research shows
that Acss2 knockout reciprocally inhibits AMPKα activa-
tion following VEGF stimulation (Fig. 6C-D), implying
that ACSS2 is not merely a downstream eector of AMPKα
but also feeds back upstream to modulate AMPKα activa-
tion status through some mechanism. Our study demon-
strates that TFEB activators can reverse the decrease in
AMPKα activation caused by Acss2 knockout, suggesting
TFEB might be a critical node linking ACSS2 and AMPKα
in this reciprocal regulation (Fig. 6E-F). Indeed, apart from
regulating autophagy, AMPKα is a multifunctional kinase
aecting metabolism [55] and cytoskeleton signaling [56],
potentially mediating angiogenesis-promoting eects inde-
pendently of autophagy [33]. Whether AMPKα can further
facilitate angiogenesis in an autophagy-independent manner
under conditions of ACSS2 upregulation remains unclear
and merits future investigation.
In our experiments, we demonstrated that AMPKα is
essential for ACSS2-dependent TFEB regulation. CAMKKβ
has been reported as vital for VEGF-induced AMPKα acti-
vation [57, 58]. Recent molecular studies on endolysosomal
Ca2+ physiology highlight the critical role of Ca2+ in con-
trolling various cellular processes and diseases [59]. It has
been discovered that TFEB upregulates MCOLN1, a protein
crucial for calcium homeostasis, which regulates intracel-
lular Ca2+ concentrations and contributes to the increase of
intracellular Ca2 + 33. Lysosomal Ca2+ activates calmodu-
lin, which induces TFEB nuclear translocation via bind-
ing and dephosphorylation [59]. The orchestrated nuclear
translocation of TFEB increased the interaction between
nuclear TFEB with AMPK-mediated ACSS2 [60]. In con-
cordance with these insights, our experimental observations
autophagosome genes it regulates [36]. Subsequently, the
acetyl-CoA produced locally from nuclear protein deacety-
lation by ACSS2 is utilized for histone H3 acetylation at
these promoter sites, leading to the expression of lysosomal
and autophagosomal genes, thereby enhancing lysosomal
biogenesis and autophagy [13]. Recent research has indi-
cated impaired autophagic ux in cortical neurons follow-
ing cardiac arrest and return of spontaneous circulation
(CA-ROSC) [49]. LC3-II protein levels peak between 6 and
12 h post-CA-ROSC, and although they decline after 24 h,
Beclin-1 and p62 proteins remain elevated [49], consistent
with our observations, autophagic ux is impeded 24 h fol-
lowing CA-ROSC in mice (Fig. 4N-P), further validating
our ndings. Autophagy is crucial for maintaining vascular
wall homeostasis [50], and its dysregulation can induce vas-
cular aging and related pathologies [51]. Autophagy protects
endothelial cells against endothelial dysfunction induced
by glucose, angiotensin II, and oxygen-glucose depriva-
tion [52]. Accumulating evidence suggests that appropriate
Fig. 5 TFEB and AMPKα are required for ACSS2 to improve endo-
thelial dysfunction. (A) Representative western blot images showing
the knockdown of TFEB in the bEnd.3 cell line with ACSS2 over-
expression. (B) Quantitative expressional analysis of WB bands of
TFEB shown in A. n = 3. (C) The mRNA levels of TFEB after its
knockdown in the bEnd.3 cell line with ACSS2 overexpression. n = 4 .
(D) Representative images of wound healing assay for control-siRNA
and TFEB-siRNA groups in the stable bEnd.3 cell line with ACSS2
overexpression following OGD/R treatment. Scale bar, 500 μm. (E)
Statistical analysis of relative migration areas at 24 h in D. n = 8. (F)
Representative images of tube formation for control-siRNA and TFEB-
siRNA groups in the stable bEnd.3 cell line with ACSS2 overexpres-
sion following OGD/R treatment. Scale bar, 50 μm. (G-H) Number of
nodes(G) and total length (H) per eld were measured by NIH imageJ
and statistically analyzed. n = 10. (I) Representative CO-IP image
using a TFEB antibody in the stable bEnd.3 cell line with ACSS2 over-
expression following OGD/R treatment. (J) Representative images of
aortic rings isolated from control or EC-ACSS2 knockout (KO) mice,
embedded in Matrigel and cultured for 7 days in the presence of TFEB
activator 1 (2 µM) or compound C (10 µM). (K) Seven days later,
the sprouts grown from the corresponding aortic rings were measured
and quantitatively analyzed. (n = 4 for each group). (L) Representative
western blot images showing the knockdown of AMPKα in the bEnd.3
cell line with ACSS2 overexpression. (M) Quantitative expressional
analysis of WB bands of AMPKα in L. n = 3. (N) The mRNA levels
of AMPKα after its knockdown in the bEnd.3 cell line with ACSS2
overexpression. n = 4. (O) Representative images of wound healing
assay for control-siRNA and AMPKα-siRNA groups in the stable
bEnd.3 cell line with ACSS2 overexpression following OGD/R treat-
ment. Scale bar, 500 μm. (P) Statistical analysis of relative migration
areas at 24 h in O. n = 8. (Q) Representative images of tube formation
for control-siRNA and AMPKα-siRNA groups in the stable bEnd.3
cell line with ACSS2 overexpression following OGD/R treatment.
Scale bar, 50 μm. (R-S) Number of nodes (R) and total length (S) per
eld were measured by NIH imageJ and statistically analyzed. n = 10.
Data are presented as mean ± SD. Statistical analyses were performed
using one-way ANOVA for multiple group comparisons. For compari-
sons between two groups, Student’s t-test was employed. *P < 0.05,
**P < 0.01, ***P < 0.001versus the control-siRNA group or ACSS2f/f
group; #P < 0.05 versus the ACSS2Tek -cko + DMSO group
1 3
Page 17 of 22 160
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W. Zhang et al.
thereby potentiating and enhancing the functional capacity
of lysosomes. However, upon the inhibition of AMPKα via
Compound C, the benecial eects of TFEB activator1 on
rescuing the profound decrement in aortic sprouting func-
tionality, precipitated by ACSS2 deciency, were intrigu-
ingly mitigated. Our data suggest that TFEB and AMPKα
underscored a parallel phenomenon: employing the TFEB
activator1 to orchestrate the nuclear relocation of TFEB sub-
stantially alleviated the compromised aortic sprouting func-
tionality that ensued from ACSS2 insuciency (Fig. 5J-K).
This interaction may subsequently amplify the transcrip-
tional activity of genes pivotal to lysosomal biogenesis,
1 3
160 Page 18 of 22
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Acetyl-CoA synthetase 2 alleviates brain injury following cardiac arrest by promoting autophagy in brain…
Evan’s Blue or sodium uorescein) with real-time imaging
modalities to comprehensively evaluate the spatiotemporal
regulation of BBB function by ACSS2.
Collectively, our study is the rst to directly demonstrate
the role of ACSS2 in brain microvascular endothelial cells
as a protector against brain injury following CPR. This not
only broadens our perspective on brain repair mechanisms
post-injury but also reveals that ACSS2 directly governs
endothelial cell function, augmenting cerebral perfusion,
modulating inammation, and fostering angiogenesis, thus
expanding our understanding of ACSS2’s functional rep-
ertoire. Additionally, by elucidating how ACSS2 interacts
with AMPKα and TFEB to regulate autophagy and angio-
genesis, we have not only rened our knowledge of ACSS2
signal transduction but also identied potential targets for
intervening in vascular diseases and neurodegenerative dis-
orders. These ndings enhance our grasp of the intricacies
in angiogenesis control and furnish a theoretical foundation
for devising therapeutic strategies tailored to diseases char-
acterized by endothelial dysfunction. Future investigations
could delve further into the therapeutic potential of ACSS2,
particularly in devising intervention strategies targeting
brain injury subsequent to CA.
activation are at least necessary for ACSS2-regulated tube
formation; however, we acknowledge that ACSS2 may also
engage in crosstalk with other angiogenic signaling path-
ways and exert specic regulatory eects on angiogenesis
under dierent contexts.
Nevertheless, our study still presents several limitations.
Although AAV-BR1 is tailored to target cerebral vascular
endothelial cells, its transduction eciency and cell-type
specicity require further validation across all experimen-
tal animals or humans. Moreover, we observed o-target
transfection in liver, lung, and kidney cells, leaving open
the possibility that overexpression of ACSS2 in these cells
may aect brain injury following CPR. Furthermore, our
study primarily focused on the protective eect of ACSS2
overexpression against brain injury post-CPR without thor-
oughly exploring the specic functions of ACSS2 down-
regulation or under natural states, potentially overlooking
ACSS2’s natural regulatory mechanisms under physiologi-
cal conditions. Another limitation lies in the broad depic-
tion of ACSS2’s protective mechanisms in brain tissue
through BMVEC metabolic reprogramming, inammatory
responses, and angiogenesis, with more in-depth investi-
gation needed to elucidate the precise molecular mecha-
nisms underlying these processes, especially the intricate
interactions with signaling pathways such as AMPKα and
TFEB. While the observed enhancement in ZO-1/CD31 co-
localization patterns suggests structural preservation of the
BBB, future investigations should integrate dynamic per-
meability assessments using tracer-based techniques (e.g.,
Fig. 6 ACSS2 activates the AMPKα signaling pathway. (A) Represen-
tative western blot images depict p-AMPKα and total AMPKα levels
in ACSS2 overexpression group and control group, following stimula-
tion with VEGF (2.5 ng/ml) over a time course of 0 to 120 min. (B)
Quantitative expressional analysis of WB bands shown in A. n = 3.
(C) Representative western blot images depict p-AMPKα and total
AMPKα levels in ACSS2 knockdown group and control group, fol-
lowing stimulation with VEGF (2.5 ng/ml) over a time course of 0 to
120 min. (D) Quantitative expressional analysis of WB bands in C.
n = 3 . (E) Representative Western blot images illustrate the proles of
p-AMPKα and total AMPKα in ACSS2 knockdown group and their
respective controls, following pre-treatment with TFEB activator 1 (2
µM) for 12 h and subsequent stimulation with VEGF (2.5 ng/ml) over
a 0–15 min period. (F) Quantitative expressional analysis of WB bands
in E. n = 3. (G) Representative images from wound healing assays are
depicted, showcasing the cellular migration dynamics in stable clones
with ACSS2 knockdown upon treatment with DMSO as a vehicle con-
trol and TFEB activator 1(2 µM). Scale bar, 500 μm. (H) Statistical
analysis of relative migration areas at 24 h in G. n = 8. (I) Representa-
tive images of tube formation in stable clones with ACSS2 knockdown
upon treatment with DMSO as a vehicle control and TFEB activator
1(2 µM). Scale bar, 50 μm. (J-K) Number of nodes (J) and total length
(K) per eld were measured by NIH imageJ and statistically analyzed.
n = 10. Data are presented as mean ± SD. Two-way ANOVA with Bon-
ferroni pairwise comparisons were used for comparisons among mul-
tiple groups. Student’s t-test was employed for comparisons between
two groups. *P < 0.05, **P < 0.01, ***P < 0.001versus respective con-
trol groups
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W. Zhang et al.
Data availability The research data generated during this study are
accessible upon a reasonable request made to the corresponding author.
Declarations
Ethical approval All experiments received approval from the ethics
committee of the Second Aliated Hospital, College of Medicine,
Zhejiang University, adhering to NIH guidelines (No. 2022 − 176).
Consent for publication Not applicable.
Conict of interest Authors declared that there were no known con-
icts of interest associated with this publication.
Open Access This article is licensed under a Creative Commons
Attribution-NonCommercial-NoDerivatives 4.0 International License,
which permits any non-commercial use, sharing, distribution and
reproduction in any medium or format, as long as you give appropri-
ate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if you modied the licensed
Supplementary Information The online version contains
supplementary material available at h t t p s : / / d o i . o r g / 1 0 . 1 0 0 7 / s 0 0 0 1 8 - 0
2 5 - 0 5 6 8 9 - 7 .
Acknowledgements We express our gratitude to the Clinical Research
Center of the Second Aliated Hospital of Zhejiang University School
of Medicine for the platform support provided for our experiment.
Author contributions Mao Zhang, Jiefeng Xu: Conceptualization, Su-
pervision, Funding acquisition, Writing - review & editing. Wenbin
Zhang: Conceptualization, Methodology, Software, Writing– original
draft and editing, Project administration, Formal analysis. Xin Yu, Yao
lin: Methodology, Project administration, Investigation, Writing– orig-
inal draft. Xiangkang Jiang, Chenghao Wu, Jiawei Tao, Ziwei Chen,
Jiantao He, Ruojie Zhu, Xiaodan Zhang: Validation, Investigation,
Project administration.
Funding This study was supported by the National Natural Science
Foundation of China (No. 82072126, No. 82372204), the Zhejiang
Provincial Key Research and Development Program of China (No.
2021C03073, No.2024C04045).
Fig. 7 Schematic illustration of ACSS2 in BMVECs alleviating
brain injury in a mouse model of CA/CPR. Via tail vein injection of
HBAAV2/br1-m-Acss2-3xag-null, we selectively augmented ACSS2
expression in brain microvascular endothelial cells (BMVECs), cata-
lyzing acetyl-CoA synthesis. Moreover, this study elucidates a dual-
pronged mechanism by which ACSS2-mediated autophagic enhance-
ment operates: On one front, activated AMPKα directly initiates the
autophagic process, accelerating organelle turnover and clearance of
cellular damage. Simultaneously, ACSS2 overexpression facilitates
nuclear translocation of TFEB, eectively unlocking a transcrip-
tional program that broadly upregulates genes associated with the
autophagy-lysosome pathway, thereby reinforcing the cell’s intrinsic
repair capabilities. This intervention strategy has proven eective in
enhancing the autophagic function of BMVECs, which leads to sig-
nicant improvements in endothelial function and cerebral blood ow
perfusion
1 3
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Acetyl-CoA synthetase 2 alleviates brain injury following cardiac arrest by promoting autophagy in brain…
18. Zhou Z et al (2022) Acetyl-Coenzyme A synthetase 2 poten-
tiates macropinocytosis and muscle wasting through meta-
bolic reprogramming in pancreatic cancer. Gastroenterology
163(5):1281–1293e1
19. Korbelin J et al (2016) A brain microvasculature endothelial cell-
specic viral vector with the potential to treat neurovascular and
neurological diseases. EMBO Mol Med 8(6):609–625
20. Li X et al (2022) Cerium oxide nanoparticles with antioxidative
neurorestoration for ischemic stroke. Biomaterials 291:121904
21. Abella BS et al (2004) Intra-arrest cooling improves outcomes in
a murine cardiac arrest model. Circulation 109(22):2786–2791
22. Allen D et al (2011) SK2 channels are neuroprotective for isch-
emia-induced neuronal cell death. J Cereb Blood Flow Metab
31(12):2302–2312
23. Xie Y et al (2024) Interleukin-21 receptor signaling promotes
metabolic dysfunction-associated steatohepatitis-driven hepa-
tocellular carcinoma by inducing immunosuppressive IgA(+) B
cells. Mol Cancer 23(1):95
24. Zhang W et al (2023) Inhibition of TANK-binding kinase1
attenuates the astrocyte-mediated neuroinammatory response
through YAP signaling after spinal cord injury. CNS Neurosci
Ther 29(8):2206–2222
25. Zhu X et al (2023) Acetate controls endothelial-to-mesenchymal
transition. Cell Metab 35(7):1163–1178e10
26. Levine B, Kroemer G (2019) Biological functions of autophagy
genes: A disease perspective. Cell 176(1–2):11–42
27. Byun S et al (2020) Fasting-induced FGF21 signaling activates
hepatic autophagy and lipid degradation via JMJD3 histone
demethylase. Nat Commun 11(1):807
28. Lu Y et al (2023) Endothelial TFEB signaling-mediated autopha-
gic disturbance initiates microglial activation and cognitive dys-
function. Autophagy 19(6):1803–1820
29. Baker M et al (2011) Use of the mouse aortic ring assay to study
angiogenesis. Nat Protoc 7(1):89–104
30. Song JX et al (2016) A novel Curcumin analog binds to and acti-
vates TFEB in vitro and in vivo independent of MTOR Inhibition.
Autophagy 12(8):1372–1389
31. Meng Y et al (2022) Targeting CRL4 suppresses chemoresistant
ovarian cancer growth by inducing mitophagy. Signal Transduct
Target Ther 7(1):388
32. Li Y et al (2023) Rapid metabolic reprogramming mediated by
the AMP-activated protein kinase during the lytic cycle of Toxo-
plasma gondii. Nat Commun 14(1):422
33. Fan Y et al (2018) Endothelial TFEB (Transcription factor
EB) positively regulates postischemic angiogenesis. Circ Res
122(7):945–957
34. Inoue A et al (2022) Extracorporeal cardiopulmonary resuscita-
tion in adult patients with out-of-hospital cardiac arrest: a ret-
rospective large cohort multicenter study in Japan. Crit Care
26(1):129
35. Garcia JA et al (2023) Acss2/HIF-2 signaling facilitates colon
cancer growth and metastasis. PLoS ONE 18(3):e0282223
36. Li X et al (2017) Nucleus-Translocated ACSS2 promotes gene
transcription for lysosomal biogenesis and autophagy. Mol Cell
66(5):684–697e9
37. Hales PW, Clark CA (2013) Combined arterial spin labeling and
diusion-weighted imaging for noninvasive Estimation of cap-
illary volume fraction and permeability-surface product in the
human brain. J Cereb Blood Flow Metab 33(1):67–75
38. Claassen J et al (2021) Regulation of cerebral blood ow in
humans: physiology and clinical implications of autoregulation.
Physiol Rev 101(4):1487–1559
39. Kisler K et al (2017) Cerebral blood ow regulation and neu-
rovascular dysfunction in alzheimer disease. Nat Rev Neurosci
18(7):419–434
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h t t p : / / c r e a t i v e c o m m o n s . o r g / l i c e n s e s / b y - n c - n d / 4 . 0 / .
References
1. Perkins GD et al (2021) Brain injury after cardiac arrest. Lancet
398(10307):1269–1278
2. Topjian AA et al (2018) Association of early postresuscitation
hypotension with survival to discharge after targeted tempera-
ture management for pediatric Out-of-Hospital cardiac arrest:
secondary analysis of a randomized clinical trial. JAMA Pediatr
172(2):143–153
3. Tsao CW et al (2023) Heart disease and stroke Statistics-2023
update: A report from the American heart association. Circulation
147(8):e93–e621
4. Sekhon MS, Ainslie PN, Griesdale DE (2017) Clinical patho-
physiology of hypoxic ischemic brain injury after cardiac arrest:
a two-hit model. Crit Care 21(1):90
5. Kondo D et al (2022) Eects of adrenaline and vasopressin on
cerebral microcirculation at baseline and during global brain isch-
emia and reperfusion in rabbits. BMC Anesthesiol 22(1):380
6. Hoiland RL et al (2023) Clinical targeting of the cerebral oxygen
cascade to improve brain oxygenation in patients with hypoxic-
ischaemic brain injury after cardiac arrest. Intensive Care Med
49(9):1062–1078
7. Sun D et al (2024) Fluid shear stress induced-endothelial phe-
notypic transition contributes to cerebral ischemia-reperfusion
injury and repair. APL Bioeng 8(1):016110
8. Abdullahi W, Tripathi D, Ronaldson PT (2018) Blood-brain bar-
rier dysfunction in ischemic stroke: targeting tight junctions and
transporters for vascular protection. Am J Physiol Cell Physiol
315(3):C343–c356
9. Wang Y et al (2021) Medioresinol as a novel PGC-1α activa-
tor prevents pyroptosis of endothelial cells in ischemic stroke
through PPARα-GOT1 axis. Pharmacol Res 169:105640
10. Reiterer M et al (2022) Hyperoxia reprogrammes microvascular
endothelial cell response to hypoxia in an Organ-Specic manner.
Cells 11(16)
11. Li X, Kumar A, Carmeliet P (2019) Metabolic pathways fueling
the endothelial cell drive. Annu Rev Physiol 81:483–503
12. Zodda E et al (2023) Autonomous metabolic reprogramming and
oxidative stress characterize endothelial dysfunction in acute
myocardial infarction. Elife 12
13. Li X, Qian X, Lu Z (2017) Local histone acetylation by ACSS2
promotes gene transcription for lysosomal biogenesis and autoph-
agy. Autophagy 13(10):1790–1791
14. Mews P et al (2017) Acetyl-CoA synthetase regulates histone acet-
ylation and hippocampal memory. Nature 546(7658):381–386
15. Xu M et al (2014) An acetate switch regulates stress erythropoi-
esis. Nat Med 20(9):1018–1026
16. Bradshaw PC (2021) Acetyl-CoA metabolism and histone acety-
lation in the regulation of aging and lifespan. Antioxid (Basel)
10(4)
17. Schug ZT et al (2015) Acetyl-CoA synthetase 2 promotes acetate
utilization and maintains cancer cell growth under metabolic
stress. Cancer Cell 27(1):57–71
1 3
Page 21 of 22 160
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
W. Zhang et al.
52. Nussenzweig SC, Verma S, Finkel T (2015) The role of autoph-
agy in vascular biology. Circ Res 116(3):480–488
53. Zhang X et al (2024) Hypoxia-mediated programmed cell death
is involved in the formation of wooden breast in broilers. J Anim
Sci Biotechnol 15(1):77
54. Fisslthaler B, Fleming I (2009) Activation and signaling by the
AMP-activated protein kinase in endothelial cells. Circ Res
105(2):114–127
55. Xiao J et al (2024) 25-Hydroxycholesterol regulates lysosome
AMP kinase activation and metabolic reprogramming to educate
immunosuppressive macrophages. Immunity 57(5):1087–1104.
e7
56. Hansen D et al (2015) Altered signaling for mitochondrial and
myobrillar biogenesis in skeletal muscles of patients with mul-
tiple sclerosis. Transl Res 166(1):70–79
57. Kim BW et al (2008) Vascular endothelial growth factor (VEGF)
signaling regulates hippocampal neurons by elevation of intra-
cellular calcium and activation of calcium/calmodulin protein
kinase II and mammalian target of Rapamycin. Cell Signal
20(4):714–725
58. Stahmann N et al (2010) Activation of AMP-activated protein
kinase by vascular endothelial growth factor mediates endothelial
angiogenesis independently of nitric-oxide synthase. J Biol Chem
285(14):10638–10652
59. Morgan AJ et al (2011) Molecular mechanisms of endolysosomal
Ca2 + signalling in health and disease. Biochem J 439(3):349–374
60. Wang X et al (2022) Long-term running exercise alleviates cogni-
tive dysfunction in APP/PSEN1 Transgenic mice via enhancing
brain lysosomal function. Acta Pharmacol Sin 43(4):850–861
Publisher’s note Springer Nature remains neutral with regard to juris-
dictional claims in published maps and institutional aliations.
40. Wu X et al (2021) Angiogenesis after acute myocardial infarction.
Cardiovasc Res 117(5):1257–1273
41. Gregorius J et al (2021) Small extracellular vesicles obtained
from hypoxic mesenchymal stromal cells have unique character-
istics that promote cerebral angiogenesis, brain remodeling and
neurological recovery after focal cerebral ischemia in mice. Basic
Res Cardiol 116(1):40
42. Kalkan BM et al (2020) CASIN and AMD3100 enhance endothe-
lial cell proliferation, tube formation and sprouting. Microvasc
Res 130:104001
43. Comerford SA et al (2014) Acetate dependence of tumors. Cell
159(7):1591–1602
44. Murthy D et al (2024) Cancer-associated broblast-derived ace-
tate promotes pancreatic cancer development by altering poly-
amine metabolism via the ACSS2-SP1-SAT1 axis. Nat Cell Biol
26(4):613–627
45. De Bock K et al (2013) Role of PFKFB3-driven Glycolysis in
vessel sprouting. Cell 154(3):651–663
46. Eelen G et al (2018) Endothelial Cell Metabolism Physiol Rev
98(1):3–58
47. Veys K et al (2020) Role of the GLUT1 glucose transporter in
postnatal CNS angiogenesis and Blood-Brain barrier integrity.
Circ Res 127(4):466–482
48. Lin Y et al (2023) ACSS2-dependent histone acetylation improves
cognition in mouse model of Alzheimer’s disease. Mol Neurode-
gener 18(1):47
49. Shao R et al (2021) The balance between AIM2-associated
inammation and autophagy: the role of CHMP2A in brain injury
after cardiac arrest. J Neuroinammation 18(1):257
50. Torisu K et al (2016) Intact endothelial autophagy is required to
maintain vascular lipid homeostasis. Aging Cell 15(1):187–191
51. Salazar G et al (2020) SQSTM1/p62 and PPARGC1A/PGC-
1alpha at the interface of autophagy and vascular senescence.
Autophagy 16(6):1092–1110
1 3
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