\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[greek,ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{tDCS confers neuroprotection by regulating LAT1-isoleucine-CBFB-PTEN
signaling after rat cerebral ischemia-reperfusion injury}



\author[1]{Xujin  Yao}%
\author[1]{Jinyang  Ren}%
\author[1]{Yu Cui}%
\author[2]{Songfeng Chen}%
\author[3]{Jing Cheng}%
\author[1]{Jingchen  Gao}%
\author[1]{Xiaohong Huang}%
\author[1]{Jiangdong  Sun}%
\author[1]{Xiangyu Xu}%
\author[1]{Xiangyi Kong}%
\author[1]{Wenjie Hu}%
\author[1]{Tao Lin}%
\author[4]{Fengyuan Che}%
\author[1]{Qi Wan}%
\affil[1]{Qingdao University}%
\affil[2]{Tsinghua University}%
\affil[3]{Wuhan University}%
\affil[4]{Linyi People's Hospital}%


\vspace{-1em}



  
  \date{June 10, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Background and Purpose: Isoleucine is a branched-chain amino acid
serving as an essential nutrient resource and metabolic. However, its
role in cerebral ischemic stroke remains unknown. Experimental Approach:
Middle cerebral artery occlusion (MCAO) was used to mimic in vivo model
of stroke. Oxygen-glucose deprivation insult (OGD) was used to injure
cultured cortical neurons. High-Performance Liquid Chromatography (HPLC)
was used to measure the level of isoleucine. A western blot assay and
immunofluorescent staining were used to measure the level of CBFB and
PTEN. TTC staining was used to measure the infarct size. Cell death and
viability were assessed by LDH and CCK8 assays. DCS was used to
stimulate cortical neurons. tDCS was used to stimulate the cortex. Key
Results: Extraneuronal isoleucine is decreased and intraneuronal
isoleucine is increased after rat cerebral I/R injury. Reducing
intraneuronal isoleucine via inhibition of its transporter, LAT1
promotes neuronal survival whereas supplementing isoleucine aggravates
neuronal damage. Isoleucine downregulates the expression of CBFB, and
that acts upstream of PTEN to mediate isoleucine-induced neuronal damage
after OGD insult. To identify the therapeutic approach that suppresses
the ischemia-induced increase of intraneuronal isoleucine, we tested the
effect of tDCS on isoleucine. Our data suggest that Cathodal tDCS can
reduce cerebral infarct size. And such neuroprotection is mediated
through reducing LAT1-dependent increase of intraneuronal isoleucine.
Conclusions and Implications: This study identifies LAT1- dependent
increase of intraneuronal isoleucine promotes neuronal death after rat
cerebral I/R injury. Our results indicate that tDCS protects against rat
cerebral I/R injury through regulating LAT1-isoleucine-CBFB-PTEN
signaling.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Isoleucine is one of the three branched chain amino acids (BCAAs).
Ingestion of BCAAs is effective for promoting rehabilitation of
post-stroke patients and decreasing inflammation (1, 2). In the brain,
isoleucine readily crosses the BBB and contributes to the production of
neurotransmitters as nitrogen donors (3). Metabolite profiling has
identified a decreased level of isoleucine in the plasma of acute stroke
patients (4). Recent studies suggest that high concentration of
isoleucine is associated with increased risk of cardiovascular diseases
(5). However, the role of isoleucine in cerebral ischemic stroke is not
clear.

CBFB (core binding factor \selectlanguage{greek}β\selectlanguage{english}) plays a critical role in enhancing the
DNA-binding activity and the stability of Runx protein (6). CBFB
deficiency has been shown to result in embryo death (7). CBFB is also
found to involve in hematopoiesis and bone development (6, 7). However,
little is known about CBFB in cerebral ischemic stroke.

Transcranial direct-current stimulation (tDCS) is a promising
non-invasive method to induce neuroplasticity within the cerebral cortex
and augment stroke recovery (8, 9). However, the molecular mechanisms
mediating the neuroprotective role of tDCS remains largely unknown.
Direct-current electrical fields (DCSs) promote nerve growth and axon
regeneration (10). Our previous study provides the first evidence that
DCSs at physiological strengths guide the migration of neuronal
stem/progenitor cells (NSPCs)(10), suggesting DCS as a potential
approach in the treatment of neurological disorders.

In this study, we set out to elucidate the role of isoleucine in the
injured neurons following cerebral ischemia-reperfusion injury. We found
unexpectedly that intraneuronal isoleucine is aggregated after cerebral
ischemia-reperfusion injury. Increased activity of LAT1 (an L-type amino
acid transporter) leads to the elevation of intraneuronal isoleucine in
cortical cultures subjected to oxygen-glucose deprivation (OGD).
Reducing the level of intraneuronal isoleucine promotes cell survival
after cerebral ischemia-reperfusion injury, and supplementing isoleucine
aggravates the neuronal damage. We reveal that isoleucine acts upstream
to reduce the expression of CBFB, and that the phosphatase PTEN acts
downstream of CBFB to mediate isoleucine-induced neuronal damage after
OGD insult. Interestingly, we demonstrate that DCS reduces the level of
intraneuronal isoleucine in cortical cultures subjected to OGD, and that
tDCS decreases the cerebral infarct volume of rat following middle
cerebral artery occlusion (MCAO) through reducing LAT1-depencent
increase of intraneuronal isoleucine. Together, these results lead us to
conclude that LAT1 overactivation-dependent isoleucine-CBFB-PTEN signal
transduction pathway may mediate ischemic neuronal injury, and that tDCS
exerts its neuroprotective effect by suppressing LAT1
overactivation-dependent signaling after cerebral ischemia-reperfusion
injury.

\textbf{METHODS}

\textbf{Animals}

All animal experiments were approved and carried out in compliance with
the IACUC guidelines of Qingdao University School of Medicine. All
animal use and experimental protocols were approved and carried out in
compliance with the IACUC guidelines and Animal Care and Ethics
Committee of Qingdao University. All studies involving animals are
reported in accordance with the ARRIVE guidelines for reporting
experiments involving animals(11). Male Sprague-Dawley rats (250 - 300g,
Qingdao Daren Fucheng Animal Husbandry Co. Ltd, China) were housed with
2--3 rats per cage on a 12 h light/dark cycle in a
temperature-controlled room (23 \selectlanguage{ngerman}°C--25 °C), with free access to food and
water. Animals were allowed at least 3 days to acclimatize before
experimentation. Samples were assigned to the experimental groups by
random, for data collection and procession. Rats were placed in a
plastic restraining holder, CSF (\selectlanguage{english}[?]50 \selectlanguage{greek}μ\selectlanguage{english}L) was collected from the cisterna
magna at 0 h, 3 h and 6 h after reperfusion, using a 27-gauge winged
needle set attached to a 1 cm syringe. The experiments were performed by
investigators blinded to the groups for which each animal was assigned.

\textbf{PTEN knockout mice}

Tamoxifen was used to induce the development of conditionally
PTEN-knockout mice. First, we performed an indoor hybridization of the
Ubc-CreER transgenic line of the Shanghai Model Biological Research
Center with the PTEN LoxP/LoxP mice of Qingdao University. Genomic DNA
was extracted from the tail tissues of F1 and F2 progeny, and then the
genotype of each mouse (CreER transgene, focusing on PTEN) was studied.
Finally, the genotypes were evaluated by PCR using PTEN LoxP and Cre as
primers. PTEN LoxP forward CTCCTCTACTCCATTCTTCCC, and reverse,
ACTCCCACCAATGAACAAAC; Cre forward, GCGGTCTGG CAGTAAAAACTATC, and
reverse, GTGAAACAGCATTGCTGTCACTT. At the age of 4--6 weeks, the
Pre-mutant mice (PTENLoxP/LoxP Ubc- CreER +) and control mice (PTEN
LoxP/LoxP Ubc-CreER-) were given intraperitoneal infusion. Investigation
of PTEN mouse phenotype was at the earliest 3 days after the ultimate
infusion. The mice (tamoxifen-induced recombination) usually co-exist
with different proportions of PTEN-wild-type cells and PTEN-knockout
cells. PTEN was not removed from all cells.

\textbf{Transient focal cerebral ischemia}

Transient focal cerebral ischemia was operated by suture occlusion
technique as described in our previous study (12). The whole process is
applied as our laboratory described. Male SD rats weighing 250-- 300 g
were anesthetized under gas condition of 4\% isoflurane in 70\%
N\textsubscript{2}O and 30\% O\textsubscript{2} and body temperature was
remained during and after surgery. The middle incision was made on the
neck, the right external carotid artery (ECA) was carefully exposed and
dissected. A 3--0 monofilament nylon suture was inserted from the ECA
into the right internal carotid artery to occlude the origin of the
right middle cerebral artery (MCA) (approximately 22 mm). After 60 min
of occlusion, the suture was removed to allow reperfusion, the ECA was
ligated, and the wound was closed. Sham-operated rats underwent
identical surgery except that the suture was inserted and withdrawn
immediately. Rectal temperature was maintained at 37.0 \selectlanguage{ngerman}± 0.5°C using a
heating pad and heating lamp. Brain samples were acquired after 24 h
after middle cerebral artery occlusion (MCAO), before reperfusion with
ice-cold 0.9\% saline and decapitated, rats were anesthetized with 4\%
isoflurane in 70\% N\textsubscript{2} and 30\% O\textsubscript{2}. The
peri-infarct tissues of ipsilateral hemispheres of the brains were
rapidly removed for Western blotting, frozen sectioning and the sliced
brain for TTC (2,3,5-triphenyltetrazolium chloride) staining. JPH203(10
\selectlanguage{greek}μ\selectlanguage{english}M, 1\selectlanguage{greek}μ\selectlanguage{english}L, S8667, Selleckchem, USA) were i. c. v. injected prior to MCAO
surgery. At 1h after MCAO insult, isoleucine (10 mg/kg) (I2752, Sigma,
USA) was intraperitoneally administrated.

\textbf{Infarct measurement}

TTC assay and infarct volume measurement were performed as described
previously by us(12). Infarct volumes (in mm\textsuperscript{\textbf{3}}
) were derived after the animals were sacrificed 24 h after MCAO onset
and the brains harvested and frozen to facilitate sectioning into 5\selectlanguage{ngerman}×2mm
coronal sections. Brain slices were stained for 30 min at 37\selectlanguage{english} with 2\%
TTC (2,3,5-triphenyltetrazolium chloride: Sigma Aldrich, St. Louis, MD,
USA) in phosphate-buffered saline (PBS) and the slices were fixed in 4\%
paraformaldehyde at 4 degC. The normal area of brain was stained red,
whereas infarct area remained unstained. All images were collected and
photographed with a scanner. The areas of the infarct, ipsilateral
hemisphere and contralateral hemisphere were measured using NIH Image
(Software version 1.52a) and volumes were derived by integrating the
edema-corrected infarct areas (area of contralateral hemisphere minus
area of healthy tissue in ipsilateral hemisphere).

\textbf{Primary cortical neuron culture}

The cortical neuronal cultures were prepared from rat at gestation day
17 as described by us previously (13). Briefly, dissociated neurons were
suspended in plating medium (Neurobasal medium, 2\% B-27 supplement,
0.5\% FBS, 0.5 \selectlanguage{greek}μ\selectlanguage{english}M L-glutamine, and 25 \selectlanguage{greek}μ\selectlanguage{english}M glutamic acid) and plated on
poly-D-lysine coated Petri dishes. After 1.0 day in culture, half of the
plating medium was removed and replaced with maintenance medium
(Neurobasal medium, 2\% B-27 supplement, and 0.5 mM L-glutamine).
Thereafter, maintenance medium was changed in the same manner every 3.0
days. The cultured neurons were used for experiments at 12 days after
plating.

\textbf{Oxygen-glucose deprivation insult}

The method of oxygen-glucose deprivation insult (OGD) was described in
our previous study (13). To initiate the OGD challenge, cells were
transferred to deoxygenated glucose-free extracellular solution (ECS)
(in mM: 116 NaCl, 5.4 KCl, 0.8 MgSO\textsubscript{4}, 1.0
NaH\textsubscript{2}PO\textsubscript{4}, 1.8 CaCl\textsubscript{2}, and
26 NaHCO\textsubscript{3}), introduced into a specialized chamber, and
maintained at 37\selectlanguage{ngerman}°C in 95\% N\textsubscript{2} and 5\%
CO\textsubscript{2} for 2 h. Neurons were removed from the chamber,
transferred to maintenance medium, and returned to the incubator. For
sham group treatment, cultures were transferred to the standard ECS (in
mM: 116 NaCl, 5.4 KCl, 0.8 MgSO\textsubscript{4}, 1.0
NaH\textsubscript{2}PO\textsubscript{4}, 1.8 CaCl\textsubscript{2}, 26
NaHCO\textsubscript{3}, and 33 glucose), introduced into the chamber
maintained at 37°C in 95\% air and 5\% CO\textsubscript{2}. After 1 h
incubation, the neurons were transferred to maintenance medium and
returned to the original incubator. JPH203(5 \selectlanguage{greek}μ\selectlanguage{english}M in 0.05\% DMSO, S8667,
Selleckchem, USA) were applied prior to the OGD insult. At 1h after
reoxygenation and glucose deprivation, isoleucine (50 \selectlanguage{greek}μ\selectlanguage{english}M) was
administrated. Bisperoxovanadium (pyridine-2-carboxyl) {[}BpV(pic){]}
(Santa Cruz, CA) was applied to primary neuron 30min before OGD insult
(14).

\textbf{Western blotting analysis}

Western blotting was performed as previously described by us (15). A
membrane was blocked with 5\% (wt/vol) BSA or 5\% (wt/vol) nonfat dry
milk in TBST (TBS containing 0.1\% Tween 20) at room temperature for 60
min, incubated with appropriate primary antibodies at 4 \selectlanguage{ngerman}°C overnight and
then with HRP-conjugated secondary antibodies at room temperature for 60
min. Antigen--antibody complexes were detected using chemiluminescence
reagents (Millipore). Primary antibody CBFB (rabbit, 1:1000, DF3273,
Affinity, China), PTEN (rabbit, 1:100, \#9188, Cell Signaling
Technology, USA) and \selectlanguage{greek}β-\selectlanguage{english}actin (rabbit, 1:2000, bs-0061R, Bioss, China).

\textbf{Immunofluorescent staining and confocal microscopy}

Immunofluorescence staining was performed as described previously by us
(16). For double-immunofluorescence staining, primary cultured neurons
cells were seeded in 24-well culture plates plated with cell-climbing
slices. Neurons were fixed in 4 \% paraformaldehyde (PFA) for 20 min.
Fixed cells were then incubated in blocking buffer (5 \% donkey serum,
0.3 \% Triton X-100 in PBS) for 2 hours. Incubation with primary
antibodies were carried out in 4 , overnight. Phosphate Buffered Saline
(PBS) instead of primary antibody for negative control overnight at 4 .
Primary antibodies used are CBFB (anti-rabbit, 1:500, DF3273, Affinity,
China), MAP2 (anti-mouse, 1:1000, Sigma-Aldrich, M4403) and NeuN
(anti-mouse, 1:500, MAB377, Millipore Sigma, USA). After washing with
PBS for three times, Primary neuron or sections were washed 3 times with
1X PBS, then incubated with the appropriate secondary antibodies in 1X
Phosphate buffer TBS (1:500, anti-mouse 4408S; anti-rabbit 4412S Cell
Signaling Technology, USA) for 2 h at room temperature and then were
rinsed with PBS for 3 times. Finally, glass coverslips or sections were
kept in an oven at 30--32*C for 10--15min to dry and covered using Cover
Glass (Fisher Scientific) with Gold antifade (Life Technologies, catalog
number P36941). Confocal images were acquired using a fluorescence
confocal microscope (C2 Si, Nikon, Tokyo, Japan). All digital images
were processed using the same settings to improve the contrast.

\textbf{High performance liquid chromatography}

CSF sample (50 \selectlanguage{greek}μ\selectlanguage{english}L) was pipetted into 2.0 mL micro-centrifuge tube and 5
\selectlanguage{greek}μ\selectlanguage{english}L of DTT (for reduction of disulphide bonds and to release bound
cysteine and homocysteine from proteins) was added to it (17). Then the
solution was vortexed for 30 s. After that an equal volume (50 \selectlanguage{greek}μ\selectlanguage{english}L) of 10
\% sulfosalicylic acid was added to precipitate the proteins, followed
by vortex mixing for 2 min. Then the samples were centrifuged at 25,000
rpm for 10 min at 4 \selectlanguage{ngerman}°C yielding a clear and kept at
-80\textsuperscript{o}C. Sample was derivatized as described. The
chromatographic system (1290 Infinity II, Agilent Technologies,
Germany). Amino acids were detected based on the retention time
established for the individual amino acid under defined experimental
conditions. Calculation was based on the area under peak established for
a given amino acid of known concentration.

\textbf{Lentivirus transfection}

The lentiviral-based PLKO.1 plasmid vector encoding two shRNA specific
for CBFB shRNA were purchased from Genchem Biotechnology Company
(Shanghai, China). The sequences of the two CBFB-specific shRNAs, CBFB-
1: TGAGATTAAGTACACGGGCTT, CBFB- 2: CCGCGAGTGTGAGATTAAGTA. The
CBFB-recombinant lentivirus and PTEN-recombinant lentivirus were
constructed by Genchem Biotechnology Company (Shanghai, China). The
transfection was performed 3 days prior to OGD insult as described (18).

\textbf{DCS and tDCS application}

In OGD model, steady DCSs were applied to cultured neurons in the
culture chambers using methods described previously by us (10). For the
DCS stimulation, agar-salt bridges were used to connect silver/silver
chloride electrodes in beakers of Steinberg's solution, to pools of
excess culture medium at either side of the chamber. Field strengths
were measured directly at the beginning and end of the observation
period. Culture conditions in control were identical except no DCSs were
applied. HEPES acid (20 mM) was added to the culture medium, with pH
adjusted to 7.4. The cells were stimulated by DCS at the current
strength of 250 mV/mm for 20 min at 3 h after reoxygenation following
OGD.

In MCAO model, tDCS was applied in rat without re-anesthesia by a
constant current stimulator (Schneider Electronics, Gleichen, Germany)
that was specifically designed for application of low-intensity currents
in small mammals (19). Epicranial electrode implant was carried out in
rat 7 days before the MCAO operation. One electrode was positioned on
each side of the cranium in a symmetrical way and fixed with nontoxic
glass ionomer cement. The electrode over the ischemic cortex was
connected to the cathodal terminal and the other electrode was connected
to anodal terminal. Prior to stimulation, epicranial implanted electrode
was filled with saline solution. The contact area of the electrode
toward the skull was 3.5 mm\textsuperscript{2}. The tDCS was applied in
rat at current intensity of 100 \selectlanguage{greek}μ\selectlanguage{english}A with a current density of 2.86
mA/cm\textsuperscript{2}. Rat underwent tDCS at 3 h after I/R for 10
min, followed by 3 min rest and then 10 min stimulation, for a total 8
times of 10 min stimulation. To avoid a stimulation break effect, the
current strength was ramped for 10 s. The sham rat underwent the same
procedure of stimulated groups, but no current was applied.

\textbf{Analysis of lactate dehydrogenase release and cell viability}

Neuronal viability or injury was evaluated using Cell Counting Kit 8
(CCK8; Dojindo, CK04) or LDH Release Assay Kit (Biovision, K311-400).
The two methods were performed under the manufacturer's instructions.
For the CCK8 assay, 10 \selectlanguage{greek}μ\selectlanguage{english}L CCK8 reagent was added to the culture medium 4
h before analysis and Optical density (OD)\textsubscript{450} values
were measured using a microplate reader (Molecular Devices, SpectraMax
ABS). For the LDH assay, 100\% cell death was induced with 0.5\% Triton
X-100 in sister culture and OD\textsubscript{490} values were measured.
The relative assessments of neuronal injury or viability were normalized
by comparison with 100\% cell death (LDH release) or with control cell
as 100\% cell survival (CCK8 assay).

\textbf{Statistics}

Student's \emph{t} test or ANOVA test was used where appropriate to
examine the statistical significance of the differences between groups
of data. All results are presented as mean \selectlanguage{ngerman}± SEM. Significance was
placed at \emph{p} \textless{} 0.05.

\textbf{RESULTS}

\textbf{Isoleucine is decreased in cerebral spinal fluid after rat
cerebral ischemia-reperfusion injury}

To study the role of isoleucine in cerebral ischemia-reperfusion injury,
we first measured the level of isoleucine in rat cerebral spinal fluid
(CSF) after middle cerebral artery occlusion (MCAO, an in vivo rat model
of cerebral ischemia-reperfusion injury) at 0, 3 and 6 h after
ischemia-reperfusion. The samples were analyzed by High Performance
Liquid Chromatography (HPLC). Our results show that the concentration of
isoleucine in CSF is significantly decreased after cerebral
ischemia-reperfusion injury (Fig. 1a). These data suggest that the level
of extracellular isoleucine in the damaged brain is decreased after rat
cerebral ischemic-reperfusion injury.

\textbf{The level of intraneuronal isoleucine is increased after
oxygen-glucose deprivation}

To determine whether the level of intraneuronal isoleucine is also
decreased after rat cerebral ischemic-reperfusion injury, we performed
HPLC to measure intracellular isoleucine in rat neuronal culture after
oxygen-glucose deprivation (OGD). Surprisingly, we found that the level
of intraneuronal isoleucine is increased after OGD insult (Fig. 1b).

\textbf{Increased activity of LAT1 leads to the elevation of
intraneuronal isoleucine after OGD insult}

To understand how OGD induces the elevation of intraneuronal isoleucine,
we examined the effect of L-type amino acid transporter 1 (LAT1) on the
level of intraneuronal isoleucine since isoleucine is known to enter
into cell via LAT1 (20, 21). HPLC analyses show that of JPH203, a potent
LAT1 inhibitor (22-24), blocks OGD-induced increase of intraneuronal
isoleucine (Fig. 1b), implying that an increased activity of LAT1 leads
to the increase of intraneuronal isoleucine after OGD insult.

\textbf{Reducing the level of intraneuronal isoleucine promotes cell
survival after cerebral ischemia-reperfusion injury}

To determine whether the increased intraneuronal isoleucine has impact
on the neuronal survival after cerebral ischemia-reperfusion injury, we
first test the effect of isoleucine treatment in both MCAO and OGD
models. We show that supplementing isoleucine increases the infarct
volume in MCAO rats and also causes more severe damage in cortical
neuronal cultures after OGD (Fig. 2a-c). We then tested the effect of
LAT1 inhibitor JPH203 in rat MCAO model. We found that injection of
JPH203 decreases the infarct volume in MCAO rats (Fig. 2d). In cultured
cortical neurons, JPH203 treatment reduces the neuronal death after OGD
insult (Fig. 2e-f). Together, these results indicate that the elevation
of intraneuronal isoleucine by the increased activity of LAT1 promotes
neuronal death and that reducing the level of intraneuronal isoleucine
by LAT1 inhibitor protects against neuronal damage after cerebral
ischemia-reperfusion injury.

\textbf{Increase of intraneuronal isoleucine promotes neuronal death
through CBFB after OGD insult}

To understand the molecular mechanisms by which the increase of
intraneuronal isoleucine promotes ischemic neuronal death, we examined
the role of CBFB, a co-binding factor of Runx protein in ischemia
injury. Our results show that CBFB is expressed in rat brain, and that
the expression of CBFB is decreased after ischemic stroke in the
penumbra brain tissue of cerebral cortex and in primary neuron culture
after OGD insult (Supplementary Fig. 1a; Fig. 3a-c; Supplementary Fig.
1b-c). We further show that CBFB knockdown by lentiviral CBFB shRNA
treatment reduces neuronal viability in OGD-insulted cortical cultures,
and that CBFB overexpression promotes neuronal survival (Supplementary
Fig. 2a-d).

Based on the TRANSFAC Predicted Transcription Factor Targets dataset
(https://genexplain.com/transfac/), we predicted that there might be
interaction between Mammalian Target of Rapamycin pathway (mTOR) and
CBFB (25). Since isoleucine is a regulator of mTOR signaling (26-28), we
reason that isoleucine may be the upstream signaling of CBFB. As
expected, we found that intraperitoneal injection of isoleucine in rat
leads to a decreased level of CBFB in brain cortex tissue (Fig. 4a,
Supplementary Fig. 3 a). In consistent with this observation, isoleucine
treatment reduces the level of CBFB in neuronal culture (Fig. 4b,
Supplementary Fig. 3b). Thus, intraneuronal isoleucine acts an upstream
signal to downregulate CBFB in neurons.

The observed downregulation of CBFB by isoleucine leads us to propose
that intraneuronal isoleucine may acts through CBFB to promote neuronal
death after OGD insult. CBFB was overexpressed in cultured neurons
before subjected to OGD insult. We found that overexpressing CBFB blocks
isoleucine-induced enhancement of neuronal damage after OGD insult while
knockdown of CBFB further increases isoleucine-induced neuronal damage
(Fig. 4c-f). These data suggest that CBFB mediates isoleucine-induced
neuronal damage.

\textbf{PTEN acts downstream of CBFB to mediate isoleucine
increase-induced neuronal death after OGD insult}

PTEN is a key regulator in cell survival (29-31). PTEN-PI3K-AKT
signaling is mediated by Runx (32, 33). Our data show that PTEN is
decreased after overexpression of CBFB (Fig. 5a). On the contrast,
knockdown of CBFB leads to the upregulation of PTEN (Fig. 5b). However,
overexpression of PTEN does not alter the level of CBFB (Fig. 5c).
Furthermore, there is no detected change of CBFB expression in cerebral
cortex of \emph{Pten} \textsuperscript{-/-} mouse (Fig. 5d,
Supplementary Fig. 1d). These data suggest that PTEN is the downstream
signal of CBFB in neurons.

We further show that there is an upregulation of PTEN in primary
cortical neuron after OGD insult (Fig. 5e). Overexpression of CBFB
reduces PTEN expression, whereas knockdown of CBFB upregulates PTEN
expression in OGD-insulted neurons (Fig. 5f). These results support the
notion that downregulation of CBFB leads to the upregulation of PTEN
after OGD insult.

To determine whether PTEN acts downstream of CBFB to mediate isoleucine
increase-induced neuronal death after OGD insult, we first show that
isoleucine causes downregulation of CBFB but upregulation of PTEN (Fig.
6a). Then, by performing cell viability assay and LDH release assay we
demonstrate that treatment of PTEN inhibitor bpV(pic) blocks isoleucine
supplement-induced neuronal death after OGD insult (Fig. 6b-c). Taken
together, we conclude that CBFB-PTEN signal pathway may mediate
isoleucine increase-induced neuronal death after OGD insult.

\textbf{Application of tDCS alleviates ischemic neuronal injury through
the reduction of intraneuronal isoleucine via inhibiting LAT1}

Our data thus far indicate that overactivation of LAT1 promotes ischemic
neuronal damage through isoleucine-CBFB-PTEN signaling pathway after rat
cerebral ischemia-reperfusion injury. As cathodal tDCS is shown to
protect against cerebral ischemia injury(8, 9), we set up to determine
whether LAT1-dependent signaling mediates cathodal tDCS-induced
neuroprotective effect. We first confirmed that cathodal tDCS exerts
neuroprotective effect in rat MCAO model (Fig. 7a) We then show that DCS
results in a decreased level of intraneuronal isoleucine in the neuronal
cultures (Fig. 7b). LAT1 inhibitor decreases intraneuronal isoleucine
but does not further decrease the level of intraneuronal isoleucine
(Fig. 7b), suggesting that DCS decreases intraneuronal isoleucine via
inhibition of LAT1 in ischemic neurons. Finally, in OGD and MCAO models,
we demonstrate that isoleucine supplement blocks DCS- and tDCS-induced
neuroprotective effects, whereas the treatment of LAT1 inhibitor JPH203
does not further decrease ischemic neuronal injury after DCS or tDCS
application (Fig. 7). Collectively, we conclude that tDCS decreases
ischemic neuronal damage through the reduction of intraneuronal
isoleucine via inhibiting LAT1.

\textbf{DISCUSSION}

Isoleucine is used as nutrition source in clinical vignettes (34-36). In
the brain, isoleucine serves as important metabolic precursors, which
are required for the synthesis of proteins and neurotransmitters,
including dopamine, serotonin and norepinephrine (37). Studies have
shown that acute supplementation of isoleucine reduces seizure
propagation for patients with epilepsy (38). However, the role of
isoleucine in cerebral ischemic stroke is not clear. The present study
reveals a non-canonical role of isoleucine in rat model of cerebral
ischemia-reperfusion injury.

We found that isoleucine is decreased in CSF in rats after cerebral
ischemic reperfusion injury, which is consistent with the recent
metabolomics profiling on plasma of the acute stroke patients (39-42).
Unexpectedly, we further show that intraneuronal isoleucine is increased
after OGD insult. Reducing intraneuronal isoleucine ameliorates the
neuronal death, whereas injection of isoleucine aggravates the neuronal
damage. Thus, aggregation of intraneuronal isoleucine is a potential
target for the development of new ischemic stroke therapy.

CBFB is a non-DNA-binding partner of Runt-related transcription
factors1-3 (Runx1-3)(43). CBFB forms a heterodimer with Runx, which
enhances the transcription ability of Runx protein(44, 45). Although
CBFB is expressed in the brain, little is known about the functional
role of CBFB in the central nervous system. We found for the first time
that CBFB is decreased in ischemic neurons, which contributes to the
neuronal death after ischemia-reperfusion injury. Moreover, we
demonstrate that the increase of isoleucine leads to the reduced level
of CBFB in ischemic neurons, whereas PTEN acts downstream of CBFB to
mediate isoleucine increase-induced neuronal death after OGD insult.

As a noninvasive brain stimulation, tDCS has been shown to exert
neuroprotective effect in cerebral ischemia-reperfusion injury by
inhibition of neuroinflammation. DCS protects against ischemic neuronal
death by reducing the activity of LAT1 and subsequent reduction of
intraneuronal isoleucine. Thus, this study reveals a molecular mechanism
by which tDCS confers neuroprotection through regulating
LAT1-isoleucine-dependent signaling after cerebral ischemia-reperfusion
injury. These findings support tDCS as a potential neuroprotection
therapy for stroke patients.

Our data indicate that isoleucine supplement promotes neuronal damage
after cerebral ischemia-reperfusion injury, which does not support the
application that isoleucine supplement in ischemic stroke therapy.
Recent studies seem to suggest the non-beneficial roles of isoleucine in
several pathological conditions. Ingestion of BCAAs significantly
promotes human platelet activity and arterial thrombosis formation in
mice (46). Long-term application of BCAAs could lead to decreased
tryptophan and reduced neuronal serotonin (47). Chronic high level of
plasma isoleucine is shown to be positively associated with AD (48, 49).
Thus, in-depth study is required to further uncover the role of
isoleucine in pathological conditions.

This study has several limitations. First, we only show the increased
import of isoleucine, which results in the aggregation of intraneuronal
isoleucine. Further study needs to be done to determine how LAT1 is
activated in ischemic conditions. Second, the dysfunction of isoleucine
catabolism is likely to contribute to ischemia-induced reduction of
isoleucine (50). Future studies will be performed to test this
possibility. Third, although this study indicates that isoleucine
results in downregulation of CBFB, more detailed studies are needed to
establish how isoleucine downregulates CBFB. In addition, due to the
unavailability of anti-isoleucine antibody, we were unable to label and
measure the reduction of intraneuronal isoleucine in the ischemic brain
tissue in the rat MCAO model.

In conclusion, our study demonstrates that ischemia-induced increase of
intraneuronal isoleucine promotes ischemic neuronal death. We provide
evidence that tDCS treatment protects against ischemic neuronal death
through regulating LAT1-isoleucine-CBFB-PTEN signaling after rat
cerebral ischemia-reperfusion injury.

\textbf{DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR}

This Declaration acknowledges that this paper adheres to the principles
for transparent reporting and scientific rigour of preclinical research
as stated in the BJP guidelines for Design \& Analysis, Immunoblotting
and Immunochemistry, and Animal Experimentation, and as recommended by
funding agencies, publishers and other organizations engaged with
supporting research.

\textbf{REFERENCE}

1. Cole JT, Mitala CM, Kundu S, Verma A, Elkind JA, Nissim I, et al.
Dietary branched chain amino acids ameliorate injury-induced cognitive
impairment. Proc Natl Acad Sci U S A. 2010;107(1):366-71.

2. Cao Y, Liu K, Liu S, Guo L, Cai C, Yao JJBri. Isoleucine Regulates
the Synthesis of Pancreatic Enzymes via the Activation of mRNA
Expression and Phosphorylation in the Mammalian Target of Rapamycin
Signalling Pathways in Pancreatic Tissues. Br J Pharmacol.
2019;2019:6302950.

3. Hutson S, Sweatt A, Lanoue KJTJon. Branched-chain {[}corrected{]}
amino acid metabolism: implications for establishing safe intakes. J
Nutr. 2005;135:1557S-64S.

4. Wang D, Kong J, Wu J, Wang X, Lai MJNl. GC-MS-based metabolomics
identifies an amino acid signature of acute ischemic stroke. Neurosci
Letters. 2017;642:7-13.

5. Ruiz-Canela M, Toledo E, Clish CB, Hruby A, Liang L, Salas-Salvado J,
et al. Plasma Branched-Chain Amino Acids and Incident Cardiovascular
Disease in the PREDIMED Trial. Clin Chem. 2016;62(4):582-92.

6. Qin X, Jiang Q, Matsuo Y, Kawane T, Komori H, Moriishi T, et al. Cbfb
regulates bone development by stabilizing Runx family proteins. J Bone
Miner Res. 2015;30(4):706-14.

7. Wang CQ, Chin DW, Chooi JY, Chng WJ, Taniuchi I, Tergaonkar V, et al.
Cbfb deficiency results in differentiation blocks and stem/progenitor
cell expansion in hematopoiesis. Leukemia. 2015;29(3):753-7.

8. Zhang K, Rui G, Zhang J, Guo L, An G, Lin J, et al. Cathodal tDCS
exerts neuroprotective effect in rat brain after acute ischemic stroke.
BMC Neuroscience . 2020;21(1):21.

9. Peruzzotti-Jametti L, Cambiaghi M, Bacigaluppi M, Gallizioli M, Gaude
E, Mari S, et al. Safety and efficacy of transcranial direct current
stimulation in acute experimental ischemic stroke. Stroke.
2013;44(11):3166-74.

10. Li L, El-Hayek Y, Liu B, Chen Y, Gomez E, Wu X, et al.
Direct-current electrical field guides neuronal stem/progenitor cell
migration. Stem Cells. 2008;26(8):2193-200.

11. Curtis M, Alexander S, Cirino G, Docherty J, George C, Giembycz M,
et al. Experimental design and analysis and their reporting II: updated
and simplified guidance for authors and peer reviewers. British Journal
of Pharmacology. 2018;175(7):987-93.

12. Zhang Z, Chen J, Han X, Zhang Y, Liao H, Lei R, et al.
Bisperoxovandium (pyridin-2-squaramide) targets both PTEN and ERK1/2 to
confer neuroprotection. Bisperoxovandium (pyridin-2-squaramide) targets
both PTEN and ERK1/2 to confer neuroprotection. British Journal of
Pharmacology. 2017;174(8):641-56.

13. Liu B, Liao M, Mielke J, Ning K, Chen Y, Li L, et al. Ischemic
insults direct glutamate receptor subunit 2-lacking AMPA receptors to
synaptic sites. 2006;26(20):5309-19.

14. Liu R, Tang J, Pan M, Zhuang Y, Zhang Y, Liao H, et al. ERK 1/2
Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal
Cerebral Ischemia-Reperfusion Injury. 2018;43(7):1424-38.

15. Ning K, Pei L, Liao M, Liu B, Zhang Y, Jiang W, et al. Dual
neuroprotective signaling mediated by downregulating two distinct
phosphatase activities of PTEN. The Journal of Neuroscience.
2004;24(16):4052-60.

16. Ning K, Li L, Liao M, Liu B, Mielke J, Chen Y, et al. Circadian
regulation of GABAA receptor function by CKI epsilon-CKI delta in the
rat suprachiasmatic nuclei. Nature Neuroscience. 2004;7(5):489-90.

17. Sharma G, Attri SV, Behra B, Bhisikar S, Kumar P, Tageja M, et al.
Analysis of 26 amino acids in human plasma by HPLC using AQC as
derivatizing agent and its application in metabolic laboratory. Amino
Acids. 2014;46(5):1253-63.

18. Gao C, Wang X, Lu J, Li Z, Jia H, Chen M, et al. Mesenchymal stem
cells transfected with sFgl2 inhibit the acute rejection of heart
transplantation in mice by regulating macrophage activation. Stem Cell
Res Ther. 2020;11(1):241.

19. Liebetanz D, Koch R, Mayenfels S, König F, Paulus W, Nitsche
MJCnojotIFoCN. Safety limits of cathodal transcranial direct current
stimulation in rats. Clinical Neuroscience. 2009;120(6):1161-7.

20. Li Y, Gao L, Luo X, Wang L, Gao X, Wang W, et al. Epigenetic
silencing of microRNA-193a contributes to leukemogenesis in t(8;21)
acute myeloid leukemia by activating the PTEN/PI3K signal pathway.
Blood. 2013;121(3):499-509.

21. Sano A, Shi H, Suzuki R, Shirataki Y, Sakagami H. Change in Amino
Acid Pools During Neuronal Differentiation of PC12 Cells. In Vivo.
2018;32(6):1403-8.

22. Johansen ML, Bak LK, Schousboe A, Iversen P, Sorensen M, Keiding S,
et al. The metabolic role of isoleucine in detoxification of ammonia in
cultured mouse neurons and astrocytes. Neurochem Int.
2007;50(7-8):1042-51.

23. Okunushi K, Furihata T, Morio H, Muto Y, Higuchi K, Kaneko M, et al.
JPH203, a newly developed anti-cancer drug, shows a preincubation
inhibitory effect on L-type amino acid transporter 1 function. J
Pharmacol Sci. 2020;144(1):16-22.

24. Patel M, Dalvi P, Gokulgandhi M, Kesh S, Kohli T, Pal D, et al.
Functional characterization and molecular expression of large neutral
amino acid transporter (LAT1) in human prostate cancer cells. Int J
Pharm. 2013;443(1-2):245-53.

25. Tandon M, Chen Z, Pratap JJBcrB. Runx2 activates PI3K/Akt signaling
via mTORC2 regulation in invasive breast cancer cells. Breast Cancer
Research. 2014;16(1):R16.

26. Ye J, Palm W, Peng M, King B, Lindsten T, Li MO, et al. GCN2
sustains mTORC1 suppression upon amino acid deprivation by inducing
Sestrin2. Genes Dev. 2015;29(22):2331-6.

27. Li H, Ye D, Xie W, Hua F, Yang Y, Wu J, et al. Defect of
branched-chain amino acid metabolism promotes the development of
Alzheimer's disease by targeting the mTOR signaling. Bioscience Reports.
2018;38(4).

28. Tenno M, Shiroguchi K, Muroi S, Kawakami E, Koseki K, Kryukov K, et
al. Cbf\selectlanguage{greek}β\selectlanguage{english}2 deficiency preserves Langerhans cell precursors by lack of
selective TGF\selectlanguage{greek}β \selectlanguage{english}receptor signaling. The Journal of Experimental Medicine.
2017;214(10):2933-46.

29. Zhao D, Qin XP, Chen SF, Liao XY, Cheng J, Liu R, et al. PTEN
Inhibition Protects Against Experimental Intracerebral
Hemorrhage-Induced Brain Injury Through PTEN/E2F1/beta-Catenin Pathway.
Front Mol Neurosci. 2019;12:281.

30. Kath C, Goni-Oliver P, M\selectlanguage{ngerman}üller R, Schultz C, Haucke V, Eickholt B, et
al. PTEN suppresses axon outgrowth by down-regulating the level of
detyrosinated microtubules. PLoS One. 2018;13(4):e0193257.

31. Sarn N, Jaini R, Thacker S, Lee H, Dutta R, Eng CJMp.
Cytoplasmic-predominant Pten increases microglial activation and
synaptic pruning in a murine model with autism-like phenotype. Molecular
Psychiatry. 2020.

32. Lim M, Zhong C, Yang S, Bell AM, Cohen MB, Roy-Burman P. Runx2
regulates survivin expression in prostate cancer cells. Lab Invest.
2010;90(2):222-33.

33. Farina N, Zingiryan A, Akech J, Callahan C, Lu H, Stein J, et al. A
microRNA/Runx1/Runx2 network regulates prostate tumor progression from
onset to adenocarcinoma in TRAMP mice. Oncotarget . 2016;7(43):70462-74.

34. Mori T, Yoshioka K. Quick and effective improvement of leucine
enriched dietary supplement on malnutrition in acute stroke patients
receiving enteral tube feeding. BMC Emerg Med. 2020;20(1):56.

35. De Simone R, Vissicchio F, Mingarelli C, De Nuccio C, Visentin S,
Ajmone-Cat MA, et al. Branched-chain amino acids influence the immune
properties of microglial cells and their responsiveness to
pro-inflammatory signals. Biochim Biophys Acta. 2013;1832(5):650-9.

36. D'Antona G, Ragni M, Cardile A, Tedesco L, Dossena M, Bruttini F, et
al. Branched-chain amino acid supplementation promotes survival and
supports cardiac and skeletal muscle mitochondrial biogenesis in
middle-aged mice. Cell Metab. 2010;12(4):362-72.

37. Holecek M. Branched-chain amino acids in health and disease:
metabolism, alterations in blood plasma, and as supplements. Nutr Metab
(Lond). 2018;15:33.

38. Gruenbaum SE, Dhaher R, Rapuano A, Zaveri HP, Tang A, de Lanerolle
N, et al. Effects of Branched-Chain Amino Acid Supplementation on
Spontaneous Seizures and Neuronal Viability in a Model of Mesial
Temporal Lobe Epilepsy. J Neurosurg Anesthesiol. 2019;31(2):247-56.

39. Szpetnar M, Matras P, Boguszewska-Czubara A, Kielczykowska M, Rudzki
S, Musik I. Is additional enrichment of diet in branched-chain amino
acids or glutamine beneficial for patients receiving total parenteral
nutrition after gastrointestinal cancer surgery? Adv Clin Exp Med.
2014;23(3):423-31.

40. Holecek M. Branched-Chain Amino Acids and Branched-Chain Keto Acids
in Hyperammonemic States: Metabolism and as Supplements. Metabolites.
2020;10(8).

41. Wang D, Kong J, Wu J, Wang X, Lai M. GC-MS-based metabolomics
identifies an amino acid signature of acute ischemic stroke. Neurosci
Lett. 2017;642:7-13.

42. Ruiz-Canela M, Guasch-Ferré M, Toledo E, Clish C, Razquin C, Liang
L, et al. Plasma branched chain/aromatic amino acids, enriched
Mediterranean diet and risk of type 2 diabetes: case-cohort study within
the PREDIMED Trial. Diabetologia. 2018;61(7):1560-71.

43. Qin X, Jiang Q, Matsuo Y, Kawane T, Komori H, Moriishi T, et al.
Cbfb regulates bone development by stabilizing Runx family proteins. J
Bone Miner Res. 2015;30(4):706-14.

44. Wang C, Chin D, Chooi J, Chng W, Taniuchi I, Tergaonkar V, et al.
Cbfb deficiency results in differentiation blocks and stem/progenitor
cell expansion in hematopoiesis. Leukemia. 2015;29(3):753-7.

45. Davis J, Rogers D, Adams L, Yong T, Jung J, Cheng B, et al.
Association of core-binding factor \selectlanguage{greek}β \selectlanguage{english}with the malignant phenotype of
prostate and ovarian cancer cells. Journal of Cellular Physiology.
2010;225(3):875-87.

46. Xu Y, Jiang H, Li L, Chen F, Liu Y, Zhou M, et al. Branched-Chain
Amino Acid Catabolism Promotes Thrombosis Risk by Enhancing
Tropomodulin-3 Propionylation in Platelets. Circulation.
2020;142(1):49-64.

47. Choi S, Disilvio B, Fernstrom MH, Fernstrom JD. Oral branched-chain
amino acid supplements that reduce brain serotonin during exercise in
rats also lower brain catecholamines. Amino Acids. 2013;45(5):1133-42.

48. Li H, Ye D, Xie W, Hua F, Yang Y, Wu J, et al. Defect of
branched-chain amino acid metabolism promotes the development of
Alzheimer's disease by targeting the mTOR signaling. Biosci Rep.
2018;38(4).

49. Griffin JW, Bradshaw PC. Amino Acid Catabolism in Alzheimer's
Disease Brain: Friend or Foe? Oxid Med Cell Longev. 2017;2017:5472792.

50. Cole JT, Sweatt AJ, Hutson SM. Expression of mitochondrial
branched-chain aminotransferase and alpha-keto-acid dehydrogenase in rat
brain: implications for neurotransmitter metabolism. Front Neuroanat.
2012;6:18.

\textbf{tDCS confers neuroprotection by regulating
LAT1-isoleucine-CBFB-PTEN signaling after rat cerebral
ischemia-reperfusion injury}

\textbf{Running title:} Regulation of LAT1-isoleucine-CBFB-PTEN
signaling by tDCS

Xujin Yao\textsuperscript{a}, Jinyang Ren\textsuperscript{a}, Yu
Cui\textsuperscript{a}, Songfeng Chen\textsuperscript{b}, Jing
Cheng\textsuperscript{b}, Xiaohong Huang\textsuperscript{a}, Jingchen
Gao\textsuperscript{a}, Jiangdong Sun\textsuperscript{a}, Xiangyu
Xu\textsuperscript{c}, Xiangyi Kong\textsuperscript{a}, Wenjie
Hu\textsuperscript{a}, Tao Lin\textsuperscript{a}, Fengyuan
Che\textsuperscript{d}, Qi Wan\textsuperscript{a,e}

\emph{\textsuperscript{a}Institute of Neuroregeneration \&
Neurorehabilitation, Department of Pathophysiology, Qingdao University,
308 Ningxia Street, Qingdao 266071, China}

\emph{\textsuperscript{b}Department of Physiology, School of Medicine,
Wuhan University, 185 Donghu Street, Wuhan 430071, China}

\emph{\textsuperscript{c}Department of Rehabilitation, Affiliated
Hospital of Qingdao University, Qingdao, 266000, China.}

\emph{\textsuperscript{d}Central Laboratory, Department of Neurology,
Linyi People's Hospital, Qingdao University, 27 East Jiefang Road,
Linyi, Shandong, China}

\emph{\textsuperscript{e}Qingdao Gui-Hong Intelligent Medical Technology
Co. Ltd, 7 Fenglong Road, Qingdao High-tech Industrial Development
District, Qingdao, China}

Correspondence:

Qi Wan, Institute of Neuroregeneration \& Neurorehabilitation,
Department of Pathophysiology, Qingdao University, 308 Ningxia Street,
Qingdao 266071, China. Email: qiwan1@hotmail.com

Fengyuan Che, Central Laboratory, Department of Neurology, Linyi
People's Hospital, Qingdao University, 27 East Jiefang Road, Linyi,
Shandong, China

Email: che1971@126.com\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Supplementary Fig. 1 CBFB is decreased in neuron after ischemic
injury}

\begin{enumerate}
\tightlist
\item
  Immunofluorescence of CBFB in cerebral cortex. Neuron (NeuN, green),
  CBFB (red)
\item
  Western blotting analysis of CBFB in cortex tissue after I/R injury.
  Peri-infarct cortex tissue was prepared for western blotting analysis
  at 3 h, 6 h after 60min occlusion (n=6 in each group, *p \textless{}
  0.05 versus 3 h sham, \textsuperscript{**}p \textless{} 0.05 versus 6
  h sham, one-way ANOVA test).
\item
  Western blotting analysis of CBFB in primary neuron after OGD insult.
  Primary cortical neuron was prepared for western blotting analysis at
  3 h, 6 h after 60min OGD insult (n=6 in each
  group,\textsuperscript{\#}p \textless{} 0.05 versus 3 h
  sham,\textsuperscript{\#\#}p \textless{} 0.05 versus 6 h sham, one-way
  ANOVA test)
\item
  Genotyping of \emph{Pten} conditional\emph{-} knockout mouse compared
  to wild-type, \emph{Pten} -/-(a), \emph{Pten} +/- (b), WT(c)
\end{enumerate}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Supplementary Fig. 2 Downregulation of CBFB mediates neuronal
death after OGD insult}

\begin{enumerate}
\tightlist
\item
  Cell viability test shows overexpression of CBFB reduces neuronal
  death after OGD insult. CBFB cDNA is transfected into neuron 72h prior
  to OGD insult (n=6 in each group, \textsuperscript{*}p \textless{}
  0.05 versus OGD + Vehicle, one-way ANOVA test).
\item
  LDH release test shows overexpression of CBFB reduces neuronal death
  after OGD insult. CBFB cDNA was transfected into neuron 72h prior to
  OGD insult (n=6 in each group, \textsuperscript{\#}p \textless{} 0.05
  versus OGD + Vehicle, one-way ANOVA test).
\item
  Cell viability test shows knockdown of CBFB promotes neuronal death
  after OGD insult. CBFB shRNA was transfected into neuron 72h prior to
  OGD insult (n=6 in each group, \textsuperscript{**}p \textless{} 0.05
  versus OGD + Vehicle, one-way ANOVA test).
\item
  LDH release test shows knockdown of CBFB promotes neuronal death after
  OGD insult. CBFB shRNA was transfected into neuron 72h prior to OGD
  insult (n=6 in each group, \textsuperscript{\#\#}p \textless{} 0.05
  versus OGD + Vehicle, one-way ANOVA test).
\end{enumerate}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Supplementary Fig. 3 Isoleucine decreases the expression of CBFB
in rat neurons}

Isoleucine decreases the expression of CBFB in cortex. Isoleucine (10
mg/kg) is intraperitoneally injected 6h before the rat cortex is
prepared for western blotting analysis (n=6 in each
group,\textsuperscript{*}p \textless{} 0.05 versus control, one-way
ANOVA test).

Isoleucine decreases the expression of CBFB in primary neuron.
Isoleucine is supplemented to neuron 6h before primary neuron culture is
prepared for western blotting analysis (n=6 in each
group,\textsuperscript{**}p \textless{} 0.05 versus control, one-way
ANOVA test).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig/Fig}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fih/Fih}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fii/Fii}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fij/Fij}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fik/Fik}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fil/Fil}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fim/Fim}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

