Autophagy

ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/kaup20

BNIP3L/NIX degradation leads to mitophagy

deficiency in ischemic brains

Xiaoli Wu, Yanrong Zheng, Mengru Liu, Yue Li, Shijia Ma, Weidong Tang,
Wenping Yan, Ming Cao, Wanqing Zheng, Lei Jiang, Jiaying Wu, Feng Han,
Zhenghong Qin, Liang Fang, Weiwei Hu, Zhong Chen & Xiangnan Zhang

To cite this article: Xiaoli Wu, Yanrong Zheng, Mengru Liu, Yue Li, Shijia Ma, Weidong Tang,
Wenping Yan, Ming Cao, Wanqing Zheng, Lei Jiang, Jiaying Wu, Feng Han, Zhenghong Qin,
Liang Fang, Weiwei Hu, Zhong Chen & Xiangnan Zhang (2021) BNIP3L/NIX degradation
leads to mitophagy deficiency in ischemic brains, Autophagy, 17:8, 1934-1946, DOI:
10.1080/15548627.2020.1802089

To link to this article: https://doi.org/10.1080/15548627.2020.1802089

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AUTOPHAGY
2021, VOL. 17, NO. 8, 1934–1946
https://doi.org/10.1080/15548627.2020.1802089

RESEARCH PAPER

BNIP3L/NIX degradation leads to mitophagy deficiency in ischemic brains

Xiaoli Wua*, Yanrong Zhenga*, Mengru Liua, Yue Lia, Shijia Ma a, Weidong Tanga, Wenping Yanb, Ming Caoa,
Wanqing Zhenga, Lei Jianga, Jiaying Wub, Feng Hanc, Zhenghong Qind, Liang Fang e, Weiwei Hua, Zhong Chen a
,
and Xiangnan Zhang a
a
Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Key Laboratory of Medical Neurobiology of the Ministry of Health of
China, Zhejiang University, Hangzhou, China; bThe First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China; cKey
Laboratory of Cardiovascular & Cerebrovascular Medicine, School of Pharmacy, Nanjing Medical University, Nanjing, China; dDepartment of
Pharmacology and Laboratory of Aging and Nervous Diseases, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-
Diseases, Soochow University School of Pharmaceutical Sciences, Suzhou, China; eAcademy for Advanced Interdisciplinary Studies and Department
of Biology, Southern University of Science and Technology, Shenzhen, China

ABSTRACT ARTICLE HISTORY

Mitophagy, the elimination of damaged mitochondria through autophagy, promotes neuronal survival Received 5 September 2019
in cerebral ischemia. Previous studies found deficient mitophagy in ischemic neurons, but the mechan­ Revised 17 July 2020
isms are still largely unknown. We determined that BNIP3L/NIX, a mitophagy receptor, was degraded by Accepted 22 July 2020
proteasomes, which led to mitophagy deficiency in both ischemic neurons and brains. BNIP3L exists as KEYWORDS
a monomer and homodimer in mammalian cells, but the effects of homodimer and monomer on BNIP3L/NIX; carfilzomib;
mitophagy are unclear. Site-specific mutations in the transmembrane domain of BNIP3L (S195A and cerebral ischemia;
G203A) only formed the BNIP3L monomer and failed to induce mitophagy. Moreover, overexpression of mitophagy; ubiquitin-
wild-type BNIP3L, in contrast to the monomeric BNIP3L, rescued the mitophagy deficiency and pro­ proteasome pathway
tected against cerebral ischemic injury. The macroautophagy/autophagy inhibitor 3-MA and the protea­
some inhibitor MG132 were used in cerebral ischemic brains to identify how BNIP3L was reduced. We
found that MG132 blocked the loss of BNIP3L and subsequently promoted mitophagy in ischemic
brains. In addition, the dimeric form of BNIP3L was more prone to be degraded than its monomeric
form. Carfilzomib, a drug for multiple myeloma therapy that inhibits proteasomes, reversed the BNIP3L
degradation and restored mitophagy in ischemic brains. This treatment protected against either acute or
chronic ischemic brain injury. Remarkably, these effects of carfilzomib were abolished in bnip3l-/- mice.
Taken together, the present study linked BNIP3L degradation by proteasomes with mitophagy defi­
ciency in cerebral ischemia. We propose carfilzomib as a novel therapy to rescue ischemic brain injury by
preventing BNIP3L degradation.
Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; ATG7: autophagy related 7;
BCL2L13: BCL2-like 13 (apoptosis facilitator); BNIP3L/NIX: BCL2/adenovirus E1B interacting protein
3-like; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CFZ: carfilzomib; COX4I1: cytochrome
c oxidase subunit 4I1; CQ: chloroquine; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP:
green fluorescent protein; I-R: ischemia-reperfusion; MAP1LC3A/LC3A: microtube-associated protein 1
light chain 3 alpha; MAP1LC3B/LC3B: microtube-associated protein 1 light chain 3 beta; O-R: oxygen and
glucose deprivation-reperfusion; OGD: oxygen and glucose deprivation; PHB2: prohibitin 2; pMCAO:
permanent middle cerebral artery occlusion; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; PT:
photothrombosis; SQSTM1: sequestosome 1; tMCAO: transient middle cerebral artery occlusion;
TOMM20: translocase of outer mitochondrial membrane 20; TTC: 2,3,5-triphenyltetrazolium
hydrochloride.

Introduction mitophagy, is essential in controlling mitochondrial quality

[5]. Autophagic machinery eliminates damaged mitochon­
Cerebral ischemia results in high mortality and disability
dria and prevents mitochondrial-dependent apoptosis in
and remains one of the most refractory human diseases
ischemic neurons [6]. Enhanced mitophagy has thus been
[1,2]. Besides being the source of bioenergy, mitochondria
proposed as a therapeutic strategy to protect neurons from
function directly in regulating programmed cell death [3]
ischemic insults [7–9]. Unfortunately, few drugs can mod­
and mitochondrial damage has been well-documented as
ulate mitophagy in ischemic brains [10]. Mitophagy defi­
a cause of neuronal death in ischemic neurons [4].
ciency leads to accumulation of damaged mitochondria in
Mitochondrial elimination by autophagy, termed
brains, causing bioenergetic dysfunction and neuronal

CONTACT Xiangnan Zhang xiangnan_zhang@zju.edu.cn; Zhong Chen chenzhong@zju.edu.cn Institute of Pharmacology & Toxicology, College of
Pharmaceutical Sciences, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang University, Hangzhou 310058, China
*These authors contributed equally to this work
Supplemental data for this article can be accessed here.
© 2020 Informa UK Limited, trading as Taylor & Francis Group

inflammation [11]. Notably, mitochondria accumulate in
a variety of hypoxic and/or ischemic neuronal injury mod­
els [12,13], suggesting the insufficiency of mitophagy in
ischemia [7]. In particular, our previous studies showed
that mitophagy can be activated by ischemia-reperfusion,
while ischemia alone did not trigger mitophagy in spite of
autophagy activation [7,14]. These results implied that
ischemic brains without reperfusion might be rescued by
restoring mitophagy. However, the molecular mechanisms
underlying neuronal mitophagy deficiency in ischemic neu­
rons remain largely unclear.
BNIP3L/NIX is located on the mitochondrial outer
membrane, where it serves as a mitophagy receptor in
either cell development or pathological conditions [15,16].
We previously found that Bnip3l gene deletion significantly
blocked ischemia-reperfusion-induced neuronal mitophagy
and consequently exacerbated ischemic brain injury. These
results highlighted BNIP3L as a potential target of mito­
phagy for stroke intervention [6,8,17]. Nevertheless, it
remained unclear how to manipulate mitophagy in
ischemic neurons by regulating BNIP3L. Intriguingly,
BNIP3L was reduced along with the differentiation of either
erythroblast [16] or cardiac progenitor cells [18]. Likewise,
reduced BNIP3L was found in several disorders with insuf­
ficient mitophagy, e.g., traumatic brain injury [19], chronic
obstructive pulmonary disease [20] and acute myeloid leu­
kemia [21]. These findings implied the potential association
of reduced BNIP3L with lower mitophagy activity.
However, whether the abundance of BNIP3L controls the
duration of mitophagy remains unclear, and how BNIP3L
was reduced under pathological conditions is also ambig­
uous. BNIP3L forms either a monomer or homodimer in
mammalian cells [22,23] and it seems that a homodimer of
BNIP3L is required for its proapoptotic effects in tumor
cells [24,25]. However, the contributions of homodimer and
monomer of BNIP3L to mitophagy have not been clarified
[26,27]. Therefore, further characterization of the molecular
regulation of BNIP3L in ischemic brains may provide novel
therapies for cerebral ischemia.
In the present study, we aimed to explore how neuronal
BNIP3L was regulated in ischemic neurons. We unexpectedly
found that BNIP3L was degraded by proteasomes, which led
to mitophagy deficiency. Carfilzomib, a proteasome inhibitor
for multiple myeloma therapy, protected brains from ischemic
injury by rescuing BNIP3L degradation.
Results
Ischemia alone leads to neuronal mitophagy deficiency
To identify mitophagy induction in ischemic brains, we
determined the expression of mitophagy-related proteins
in mice subjected to either transient middle cerebral artery
occlusion (tMCAO) or permanent MCAO (pMCAO). Both
tMCAO and pMCAO led to increased LC3B-II and reduced
SQSTM1, indicating autophagy activation. The mitochon­
drial proteins COX4I1 and TOMM20, which reflect mito­
chondrial content, decreased in tMCAO- but not in
pMCAO-treated mice brains (Figure 1A,B). These data
AUTOPHAGY 1935
were in line with previous observations [4,7,12,14] and
implied deficiency in neuronal mitophagy with ischemia
alone. To confirm this phenotype, primary cultured cortical
neurons were subjected to oxygen and glucose deprivation
(OGD) or OGD-reperfusion (O-R) treatment. Similarly,
both O-R and OGD alone caused LC3B lipidation and
reduced SQSTM1. However, OGD alone failed to cause
mitochondrial loss as in O-R treated neurons. We con­
firmed the autophagic mitochondrial loss because of chlor­
oquine treatment resulted in further accumulation of LC3B-
II and reversed the degradation of SQSTM1, TOMM20, and
COX4I1 [28] (Figure 1C,D).
To further identify mitophagy deficiency, atg7 (autophagy
related 7) knockout primary cortical neurons (Atg7fl/fl; hSyn-
Cre) [29] were used. atg7 knockout blocked the mitophagy in
O-R neurons but not neurons after OGD alone (Figure 1E,F).
We transfected neurons with mCherry-LC3B and Mito-GFP,
which labeled autophagosomes and mitochondria, respec­
tively. Both O-R and OGD treatment increased the number
of autophagosomes. OGD alone did not promote the overlap
of autophagosomes with mitochondria (Fig. S1A and S1B). As
an alternative approach to identify mitophagy, primary cul­
tured neurons were transfected with Mito-QC, which is
widely used to monitor mitophagy [30]. Consistent with the
western blot data, O-R treatment enhanced the mitochondria
ratio in mCherry-alone. In contrast, OGD alone reduced
mitochondria fusion with lysosomes (Fig. S1C and S1D).
Together, these data revealed that ischemia alone compro­
mised neuronal mitophagy despite the activation of
autophagy.
BNIP3L relates to mitophagy deficiency in cerebral
ischemia
Since ischemia did not compromise autophagy activation either
in vivo or in vitro, we thus assumed a mitophagy-specific
mechanism deficiency. We examined the protein levels of
PRKN/PARK2, BNIP3L, FUNDC1, BCL2L13, PHB2 (prohibi­
tin 2) and BNIP3 in pMCAO brains [6,31–34]. The results
showed significant BNIP3L reduction with pMCAO treatment
while the expression of PRKN, FUNDC1, BCL2L13, PHB2 and
BNIP3 remained intact. These results suggested that these
mitophagy receptors, excepting BNIP3L, may have little impact
on mitophagy deficiency in ischemic brains (Fig. S2). To con­
firm the involvement of BNIP3L loss in mitophagy deficiency,
we infected the mice brain cortex and striatum with adeno-
associated viruses carrying the cDNA of GFP-BNIP3L (AAV-
GFP-Bnip3l) two weeks prior to pMCAO. The mitophagy
deficiency was rescued by ectopic expression of BNIP3L as
revealed by loss of mitochondrial markers COX4I1 and
TOMM20 (Figure 2A,B).
In line with these findings, we found that endogenous
BNIP3L decreased after OGD treatment in primary cul­
tured neurons, while the mitochondrial content was not
significantly affected (Figure 2C). These phenotypes were
rescued by overexpression of Flag-BNIP3L (Figure 2D). The
immunostaining showed the same results (Fig. S3). Because
the abundance of mitophagy receptors cannot reflect the
mitochondrial mass [35–37], we determined the COX4I1,
1936 X. WU ET AL.

Figure 1. Ischemia alone leads to neuronal mitophagy deficiency. (A) Mice were subjected to 1 h of occlusion followed by 3 h of transient middle cerebral artery
occlusion (tMCAO) or 4 h of permanent middle cerebral artery occlusion (pMCAO). The expression of SQSTM1, LC3B, COX4I1, and TOMM20 in ischemic penumbra was
detected by western blot. Duplicate lanes are shown for each grou(B) Semi-quantitative analyses are shown (n = 5 mice for each group). (C) Primary cultured mice
cortical neurons were subjected to 1 h of oxygen and glucose deprivation (OGD) followed by 1 h of reperfusion (O-R) or 2 h of OGD. The neurons were incubated
with 20 μM chloroquine for 4 h prior to the OGD procedure. The protein expression of SQSTM1, LC3B, COX4I1, and TOMM20 was detected by western blot, and (D)
relative protein markers were analyzed (n = 3 from independent experiments). Ctrl, control; Veh, vehicle; CQ, chloroquine. (E) Primary cultured cortical neurons of WT
and Atg7fl/fl; hSyn-Cre were subjected to 1 h of OGD followed by 1 h of reperfusion (O-R) or 2 h of OGD. The protein levels of SQSTM1, LC3B, COX4I1, TOMM20, and
ATG7 were detected by western blot, and (F) relative protein markers were analyzed (n = 3 from independent experiments). Data are expressed as mean ± SEM.
Statistical comparisons were performed as follows: one-way ANOVA for (B, D, and F). *P < 0.05; **P < 0.01; n.s. vs. the indicated group.

TOMM20, and expression of BNIP3L along with the dura­ BNIP3L rescues mitophagy deficiency and protects
tion of pMCAO (Figure 2E). Results showed mitochondrial against cerebral ischemia
accumulation (mitophagy deficiency) and BNIP3L loss
The aforementioned results raised questions about the
along with pMCAO duration. These data indicated an
requirement of BNIP3L dimer and monomer in mitophagy
increasing duration of mitophagy deficiency. The expres­
activation. To investigate this, we constructed the BNIP3L
sions of PRKN, FUNDC1, BCL2L13 and PHB2 were not
monomer by mutating the transmembrane (TM) domain
significantly altered after pMCAO. Abundance analysis of
of BNIP3L (S195A, G203A) [39] (Figure 3A). Constructs
the correlations of PRKN, FUNDC1, BCL2L13 and PHB2
of the single site-specific BNIP3L mutants (S195A, G203A)
with mitochondrial marker COX4I1 showed no significant
showed only a monomer form in the immunoblot (Figure
changes (Figure 2F).
3B). Moreover, these mutants remained localized to the
BNIP3L can be detected at 40 and 80 kDa, represent­
mitochondria in COS7 cells and primary neurons while
ing the monomer and homodimer of BNIP3L, respec­
the TM domain deletion mutant (ΔTM) lost mitochondrial
tively [38]. The quantification of opti-density of
distribution [25] (Fig. S4A and S4B). We then transfected
immunoblots showed that the BNIP3L dimer decreased
HeLa cells with 3× Flag-fused BNIP3L or its mutants
significantly and the monomeric form decreased at
(S195A, G203A and ΔTM). Results showed that the mito­
a slower rate along with the duration of pMCAO
chondrial contents (COX4I1 and TOMM20) were signifi­
(Figure 2G). We then determined the correlation of
cantly reduced after wild-type BNIP3L overexpression,
BNIP3L dimer/monomer with mitochondrial content
while treatment of S195A and G203A mutants had
in each individual sample with distinct pMCAO dura­
a minor effect in inducing mitochondrial loss in normal
tion. The results clearly indicated a higher correlation
conditions (Figure 3C,D). The wild-type BNIP3L still
of BNIP3L dimer loss than monomer loss with the
induced mitochondrial reduction under CCCP treatment.
increased mitochondrial content (Figure 2H,I). Overall,
In contrast, both S195A and G203A mutation failed to
we found that BNIP3L dimer loss was related to mito­
reinforce CCCP-induced mitochondrial loss (Figure 3E,F).
phagy deficiency in cerebral ischemia.
 AUTOPHAGY 1937

Figure 2. BNIP3L relates to mitophagy deficiency in cerebral ischemia. (A) Mice were infected with AAV-GFP-Bnip3l or AAV-GFThe mice were subjected to pMCAO for
4 h, then the protein levels of GFP, SQSTM1, LC3B, COX4I1 and TOMM20 in brain tissues were determined by western blot. Empty arrows indicate exogenous BNIP3L.
Duplicate lanes are shown for each grou(B) Semi-quantitative analyses of SQSTM1, LC3B, COX4I1 and TOMM20 protein levels are shown (n = 3 mice for each group).
(C) Primary neurons were subjected to OGD for 1 h. The expression of BNIP3L and TOMM20 were detected by western blot. Semi-quantitative analyses are shown
(n = 3 from independent experiments). (D) The neurons overexpression Flag-BNIP3L were subjected to OGD for 1 h, and the expression of Flag-BNIP3L and TOMM20
were detected by western blot (n = 3 from independent experiments). (E) Mice were subjected to 1, 3 or 6 h of pMCAO. The BNIP3L, PRKN, FUNDC1, BCL2L13, PHB2,
SQSTM1, LC3B, COX4I1 and TOMM20 protein levels were determined by western blot. Black arrows indicate endogenous BNIP3L. Duplicate lanes are shown for each
grou(F-I) Analyses of the correlations between BNIP3L, PRKN, FUNDC1, BLC2L13, PHB2, and mitochondrial proteins are shown (n = 3 mice for each group). Data are
expressed as mean ± SEM. Statistical comparisons were performed as follows: one-way ANOVA for (B and F-I) and t-test for (C and D). *P < 0.05; **P < 0.01;
***P < 0.001; n.s. vs. the indicated group.

Furthermore, exogenous BNIP3L reduced COX4I1 and
TOMM20 levels, which was reversed by chloroquine, indicat­
ing mitophagic degradation. However, the BNIP3L monomer
mutants were not able to reduce the mitochondrial contents
(Figure 3C-F). MitoTracker Red was used to visualize the
mitochondrial mass after BNIP3L and mutant transfection
in HeLa cells (Fig. S4 C). Compared with BNIP3L, the mono­
mer mutants failed to decrease the mitochondrial area (Fig.
S4D). BNIP3L interacts with ATG8-family proteins (LC3 and
GABARAP subfamilies) and thus induces mitophagy [15]. By
carrying out immunoprecipitation in HeLa cells with BNIP3L
gene deletion by CRISPR-Cas9, we recapitulated the interac­
tion of wild-type BNIP3L with either lipidated forms of LC3A
or LC3B. However, the interaction of mutant BNIP3Ls with
unlipidated LC3s was weaker (Figure 3G,H). Taken together,
these data indicated the incompetency of BNIP3 L monomer
for inducing mitophagy and suggested the requirement of
BNIP3L dimer to activate mitophagy.
We next asked whether wild-type BNIP3L may rescue
neuronal mitophagy deficiency in cerebral ischemia. After
being subjected to OGD, the primary neurons transfected
with Flag-BNIP3L significantly reduced mitochondrial con­
tent by measuring COX4I1 and TOMM20 immunoblots.
The BNIP3LG203A mutant construct, however, failed to
reduce mitochondrial mass in OGD-treated neurons
(Figure 4A,B). The neuron immunostaining showed the
same results (Fig. S5). These data provided evidence sup­
porting the involvement of wild-type BNIP3L in mitophagy
activation in ischemia. This notion was further verified in
ischemic mice brains by determining mitophagy following
virus-mediated expression. Results showed that GFP-
BNIP3L transduction to brains reversed the accumulation
of mitochondrial markers COX4I1 and TOMM20 after
pMCAO treatment. In contrast, GFP-BNIP3LG203A did not
alter the mitochondrial mass in ischemic brains (Figure 4C,
D). These data from in vitro and in vivo ischemic models
indicated that wild-type BNIP3L rescued mitophagy defi­
ciency in ischemic brains.
Our previous studies indicated the neuroprotective effects
of mitophagy against ischemic insults [6,31]. To further test
whether mitophagy rescued by wild-type BNIP3L may also
produce neuroprotective effects, AAV-GFP-Bnip3l and AAV-
1938 X. WU ET AL.

Figure 3. BNIP3L monomeric mutants failed to induce mitophagy in HeLa cells. (A) Structural diagram of mouse BNIP3L and mutants of the transmembrane (TM)
domain were shown. (B) HeLa cells were transfected with Flag, Flag-Bnip3l, Flag-Bnip3lS195A, Flag-Bnip3lG203A, or Flag-Bnip3lΔTM for 24 h. The relative protein levels in
HeLa cells were determined by western blot. Empty arrows indicate exogenous BNIP3L. (C) Hela cells were transfected with Flag, Flag-Bnip3l, Flag-Bnip3lS195A, Flag-
Bnip3lG203A, or Flag-Bnip3lΔTM for 24 h. Cells were treated with PBS as a control or 20 μM chloroquine for 4 h. The expression of Flag, COX4I1, TOMM20 and GAPDH
were determined by western blot. (D) Semi-quantitative analyses of COX4I1 and TOMM20 protein levels are shown (n = 3 from independent experiments). (E) HeLa
cells were transfected with Flag, Flag-Bnip3l, Flag-Bnip3lS195A, or Flag-Bnip3lG203A, Flag-Bnip3lΔTM for 24 h. Cells were treated with PBS as a control or 20 μM
chloroquine for 4 h and then incubated with CCCP (10 μM) for 6 h. The exogenous expression of BNIP3L and mitochondrial markers in HeLa cells were assessed by
western blot. (F) Semi-quantitative analyses of COX4I1 and TOMM20 protein levels are shown (n = 3 from independent experiments). (G-H) Protein-protein
interactions of MYC-LC3 with Flag, Flag-BNIP3L, Flag-BNIP3LS195A, Flag-BNIP3LG203A, Flag-BNIP3LΔLIR were confirmed by immunoprecipitation in BNIP3L KO HeLa
cells. Plasmids were co-transfected for 24 h. After 6 h of 10 μM CCCP incubation, the expression of LC3s and BNIP3L were assessed by western blot. Empty arrows
indicate exogenous BNIP3L. Semi-quantitative analyses of IP: MYC-LC3-I/IP: Flag are shown (n = 3 from independent experiments). Data are expressed as mean ±
SEM. Statistical comparisons were performed as follows: one-way ANOVA for (D, F, G and H). *P < 0.05; **P < 0.01; ***P < 0.001; n.s. vs. the indicated group.

GFP-Bnip3lG203A were separately injected into wild-type mice
brains. The ectopic BNIP3L expression produced significant
protection by reducing the brain infarct volumes from 51.9%
± 2.2% to 35.1% ± 2.5% (Figure 4E,F). This neuroprotection
was also reflected by improved neurological deficit score
(NDS) (3.67 ± 0.21 vs. 2.57 ± 0.20, P < 0.01, Figure 4G)
24 h after ischemia onset. As a comparison, the
BNIP3LG203A failed to rescue the infarct volumes (51.9% ±
2.2% vs. 47.8% ± 2.5%, n.s., Figure 4F) as well as NDS
(3.67 ± 0.21 vs. 3.57 ± 0.20, n.s., Figure 4G). We thus
concluded that wild-type BNIP3L was required for its neuro­
protective effect against ischemic injury.
BNIP3L is degraded by ubiquitin-proteasome pathway
Our data supported an intimate link of BNIP3L loss with
mitophagy deficiency in cerebral ischemia. Multiple lines of
evidence indicated that both autophagy-lysosome pathway
and ubiquitin-proteasome pathway were activated in
 AUTOPHAGY 1939

Figure 4. BNIP3L rescues mitophagy deficiency and protects against cerebral ischemia. (A) Primary cultured cortical neurons transfected with Flag, Flag-Bnip3l, or
Flag-Bnip3lG203A were subjected to 1 h of OGD. The exogenous expression of Flag, COX4I1, TOMM20 and GAPDH were determined by western blot. (B) Semi-
quantitative analyses of COX4I1 and TOMM20 are shown (n = 3 from independent experiments). (C) Mice were infected with AAV-GFP, AAV-GFP-Bnip3l or AAV-GFP-
Bnip3lG203A for at least two weeks and then subjected to 6 h of pMCAO. The AAVs, COX4I1 and TOMM20 protein levels in brain tissues were determined by western
blot. Empty arrows indicate exogenous BNIP3L. Duplicate lanes are shown for each grou(D) Semi-quantitative analyses of COX4I1 and TOMM20 are shown (n = 3
mice for each group). (E) Mice brains were infected with AAVs expressing GFP, GFP-Bnip3l, or GFP-Bnip3lG203A and then subjected to 24 h of pMCAO. The
representative TTC-stained brain slices from each group are shown. (F) The brain infarct volumes and (G) neurological deficit scores of each group were determined
(n = 6–7 mice for each group). Data are expressed as mean ± SEM. Statistical comparisons were performed as follows: one-way ANOVA for (B, D, F and G). **P < 0.01;
***P < 0.001; n.s. vs. the indicated group.

ischemic brains. We thus blocked these two degradation
pathways by 3-MA and MG132, respectively. The LC3B-II
protein levels were inhibited by 3-MA. MG132 treatment led
to an increase in level of TRP53/p53 [40], suggesting protea­
some inhibition (Figure 5A). The proteasome inhibitor
MG132, rather than autophagy inhibitor 3-MA, rescued
wild-type BNIP3L degradation. This result suggested that
that proteasomal activation was involved in wild-type
BNIP3L loss. To investigate this, we determined the ubiqui­
tination of wild-type BNIP3L and its mutants by immuno­
precipitation in BNIP3L-/- HeLa cells. Wild-type BNIP3L
was more ubiquitylated compared with its monomer
mutants (Figure 5B,C). We cannot exclude the possibility
that the smear of ubiquitin chains on wild-type BNIP3L
comes from other proteins interacting with BNIP3L and
being ubiquitinated. Further studies are needed to address
this issue.
We next determined whether wild-type BNIP3L was ubi­
quitylated in ischemic brains. To this end, mice brains were
injected with viruses to express the wild-type or monomer
BNIP3L in bnip3l-/- mice. After being subjected to pMCAO,
ubiquitination of BNIP3Ls was assayed using immunopreci­
pitation. Both BNIP3L dimer and monomer were ubiquiti­
nated, and the ubiquitylation was further increased with
MG132 treatment (Figure 5D). Additionally, MG132 pre­
vented BNIP3L loss in ischemic mice brains when adminis­
tered systemically. Rescue of BNIP3L loss was accompanied
by enhanced mitophagy as revealed by decreased levels of
COX4I1 and TOMM20 (Figure 5E). Taken together, these
data indicated that BNIP3L was degraded by the ubiquitin-
1940 X. WU ET AL.

Figure 5. BNIP3L is degraded by the ubiquitin-proteasome pathway. (A) The mice were injected (i.c.v.) with 3-MA (7.5 μg) or MG132 (4 mg/kg) at the onset of
occlusion and then subjected to 6 h of pMCAO. The BNIP3L, LC3 and TP53 protein levels were determined by western blot. (B-C) Protein-protein interactions of MYC-
Ub with Flag-BNIP3L, Flag-BNIP3LS195A, Flag-BNIP3LG203A, or Flag-BNIP3LΔTM were confirmed by immunoprecipitation in BNIP3L KO HeLa cells. Plasmids were co-
transfected for 24 h. CCCP (10 μM) and MG132 (10 μM) were incubated for 6 h before harvesting. The protein levels of ubiquitin and exogenous expression of BNIP3L
were detected by using anti-Flag and anti-MYC antibodies, respectively. Empty arrows indicate exogenous BNIP3L. (D) The bnip3l-/- mice were infected with AAVs
expressing GFP, GFP-Bnip3l, or GFP-Bnip3lG203A for two weeks and then subjected to 6 h of pMCAO. The protein-protein interactions of ubiquitin and overexpression
of BNIP3L in vivo were determined by immunoprecipitation. The ubiquitylation levels of exogenous BNIP3L were detected by using anti-ubiquitin and anti-BNIP3L
antibodies. A ratio t-test was applied (n = 3 from independent experiments). (E) The wild-type mice treated with MG132 (4 mg/kg) at the onset of occlusion were
subjected to 6 h of pMCAO. Duplicate lanes are shown for each group. The BNIP3L, LC3B, COX4I1 and TOMM20 protein levels were assessed by western blot. Black
arrows indicate endogenous BNIP3L. Data are expressed as mean ± SEM. Statistical comparisons were performed as follows: t-test for (D). **P < 0.01 vs. the indicated
group.

proteasomal pathway and thus led to mitophagy deficiency in
ischemic brains.
Carfilzomib rescues BNIP3L degradation and protects
against ischemic injury
The fact that BNIP3L degradation by proteasomes contributed
to mitophagy deficiency in ischemic brains led us to hypothe­
size that proteasome inhibitors may serve to prevent BNIP3L
loss and thus alleviate ischemic brain injury. Some protea­
some inhibitors are used in multiple myeloma therapy [41].
Among these drugs, carfilzomib (CFZ) showed advantages in
pharmacokinetics and safety [42]. We confirmed that BNIP3L
degradation caused by pMCAO was blocked by CFZ treat­
ment. As a consequence, the administration of CFZ reduced
the COX4l1 and TOMM20 protein levels, indicating the
restoration of mitophagy deficiency (Figure 6A).
CFZ also led to accumulation of total ubiquitin in ischemic
brains. In particular, after pull-down of BNIP3L in ischemic
brains, we found that CFZ treatment increased the level of
ubiquitylated BNIP3L (Figure 6B). These data strengthened
the notion that BNIP3L was ubiquitylated in ischemic brains.
We next determined the neuroprotective effect of CFZ in the
pMCAO model. CFZ (2 mg/kg) was injected intraperitoneally
at the onset of ischemia. The infarct volumes and NDS were
determined 24 h after injury. Treatment with CFZ signifi­
cantly reduced the brain infarct volumes (50.6% ± 1.6% to
31.2% ± 3.9%, P < 0.001) and NDS (3.62 ± 0.18 vs. 2.50 ± 0.19,
P < 0.05, Figure 6C,D). Importantly, neuroprotection from
CFZ treatment was almost abolished in bnip3l-/- mice, sug­
gesting that BNIP3 L was required for the therapeutic effects
of CFZ.
Short therapeutic time-window is a major limitation of
anti-stroke drugs. To address the time-window of CFZ for
acute stroke therapy, CFZ was administered to mice at 3 or
6 h after ischemia onset. Our results showed that administra­
tion of CFZ at 3 h after pMCAO was still competent to
alleviate brain infarct volumes (49.3% ± 1.1% to 28.0% ±
3.4%, P < 0.001) as well as NDS (3.56 ± 0.20 vs. 2.71 ± 0.28,
P < 0.05, Figure 6E,F), but delayed administration at 6 h
abolished the benefits of CFZ treatment. These results
 AUTOPHAGY 1941

Figure 6. Carfilzomib (CFZ) rescues BNIP3L degradation and protects against ischemic injury. (A) Mice treated with 2 mg/kg carfilzomib at the onset of occlusion were
subjected to 6 h of pMCAO. The protein levels of BNIP3L, SQSTM1, LC3B, COX4I1 and TOMM20 were detected by western blot. Duplicate lanes are shown for each
group. (B) Wild-type or bnip3l-/- mice were injected with CFZ at the onset of occlusion and then subjected to 6 h of pMCAO. The protein-protein interactions of
BNIP3L and ubiquitin after CFZ treatment were detected by immunoprecipitation in vivo. The ubiquitylation levels of endogenous BNIP3L were detected using anti-
ubiquitin and anti-BNIP3L antibodies. A ratio t-test was applied (n = 3 from independent experiments). (C) Wild-type or bnip3l-/- mice were given CFZ at the onset of
occlusion and subjected to pMCAO for 24 h. The brain slices were stained by TTC and representative slices from each group are shown. (D) The brain infarct volumes
and neurological deficit scores of each group were determined (n = 6–8 mice for each group). (E) The mice were treated with CFZ at 3 or 6 h after the onset of
occlusion. Representative brain slices are shown. (F) After 24 h of occlusion, the infarct volumes were determined by TTC staining and neurological deficit scores were
evaluated (n = 6–7 mice for each group). (G) The protein levels of BNIP3L, SQSTM1, LC3B, COX4I1 and TOMM20 were determined by western blot. Duplicate lanes are
shown for each grouBlack arrows indicate endogenous BNIP3L. Data are expressed as mean ± SEM. Statistical comparisons were performed as follows: t-test for (B);
one-way ANOVA for (D and F). *P < 0.05; **P < 0.01; ***P < 0.001; n.s. vs. the indicated group.

suggested that the effective time-window of CFZ for stroke
therapy may extend to at least 3 h post-ischemia. We identi­
fied the BNIP3L in ischemic brains and found that delayed
administration of CFZ at 3 h, but not 6 h, rescued BNIP3L
loss as well as mitophagy deficiency (Figure 6G). This obser­
vation further emphasized that BNIP3L is associated with
effective mitophagy in ischemic brains.
We further determined whether CFZ treatment may also
attenuate chronic ischemic brain injury. To do this, mice were
subjected to photothrombosis (PT) [43] and their motor func­
tion was measured every other day based on a grid-walking
task and forelimb symmetry in the cylinder task over the next
13 d. We found that CFZ treatment (2 mg/kg, i.p.) every
other day significantly reduced the number of foot faults
and the forelimb asymmetry during the 13 d after PT onset
(Fig. S6A and S6B). The brain infarct volumes were detected
at 13 d after PT using toluidine blue stain. The brain infarct
volumes were significantly decreased (saline 0.85 ± 0.07 mm3
vs. CFZ 0.59 ± 0.04 mm3, P < 0.05, Fig. S6 C and S6D),
indicating that CFZ treatment could reduce infarct volumes
after stroke. Taken together, these data suggested that CFZ
could be a promising drug for stroke.
Discussion
In the present study, we identified a previously undetermined
link between BNIP3L degradation and mitophagy deficiency
in ischemic brains. BNIP3L dimer underwent a time-
dependent decrease along with accumulation in ischemic
brains (Figure 2E). Moreover, ectopic expression of wild-
type BNIP3L, but not its monomer, reversed mitophagy defi­
ciency (Figures 3 and 4). Of note, neuronal autophagy was not
impaired by ischemia (Figure 1), further suggesting that mito­
phagy deficiency was not caused by defective autophagy
machinery. We previously found that mitophagy was acti­
vated in brains that underwent ischemia-reperfusion (I-R) in
which BNIP3L dimer was not reduced [14]. Conversely,
bnip3l-/- caused mitophagy deficiency in I-R [6]. Prior to
this study, little was known about the mechanisms of neuro­
nal mitophagy deficiency under ischemic conditions [44,45].
1942 X. WU ET AL.
Based on our results, we concluded that BNIP3L degradation
played a causative role in mitophagy deficiency of ischemic
brains.
Another finding of the present study is that BNIP3L mono­
mer mutants failed to induce mitophagy. Although BNIP3L is
present as both a dimer and monomer in cells, their respective
contributions to mitophagy remained largely unexplored. As
a result, BNIP3L was unequivocally determined to be
a mitophagy receptor in most studies [46,47]. Previous
research indicated that BNIP3, the homolog of BNIP3L [48],
formed a homodimer to promote autophagic flux and cell
death [49]. Paradoxically, it remained undetermined whether
BNIP3L dimer would be indispensable for its biological func­
tions [24]. BNIP3L dimer loss showed a much stronger cor­
relation with mitophagy deficiency than its monomeric form
in ischemic brains (Figure 2H,I). Furthermore, the single site-
specific mutations (S195A, G203A) of the TM domain in
BNIP3L formed only monomer and were sufficient to abolish
the mitophagy activation (Figure 3C-F). BNIP3L monomer
failed to rescue mitophagy deficiency in ischemic neurons and
brains (Figure 4A-D). Our results highlighted that the mono­
mer mutants (S195A and G203A) were not sufficient to
induce mitophagy. Neurons prohibit mitophagy by degrading
BNIP3L and may restore mitophagy by allowing homodimer­
ization of the remaining BNIP3L monomers. This mechanism
may provide a way for ischemic neurons to promptly control
mitophagy. In addition, although mutant BNIP3L (S195A and
G203A) showed only the monomeric form, we cannot exclude
the possibility that the mutants may have effects besides pre­
venting dimerization. Moreover, a dramatic drop in intracel­
lular ATP or glycolysis perturbation, as a stress caused by
ischemia, may also block mitophagy [50–52]. Although we
found BNIP3L loss was attributed to proteasomal degrada­
tion, it was not clear whether this degradation was triggered
by energy crisis. It remains unclear whether and how glucose
metabolism alterations and BNIP3L degradation work
together to regulate neuronal mitophagy in ischemia-related
scenarios.
Detailed mechanisms of BNIP3L homodimer in triggering
mitophagy were not fully understood. An emerging study
added a mechanistic perspective to our findings that
BNIP3L dimerization may associate with its phosphorylation
in TM domain [53]. Mutations in the TM domain did not
alter their mitochondrial location (Fig. S4A and S4B). We
confirmed that the mitochondrial distribution of BNIP3L
was required (compared with the TM domain deletion muta­
tion) for mitophagy. However, here we showed that anchoring
to mitochondria was not sufficient for BNIP3L to serve as
a mitophagy receptor. Remarkably, BNIP3L homodimer was
prone to interact with LC3A/B-II and the mutants also had
a weaker interaction with LC3A/B-I (Figure 3E,F). It is likely
that the homodimer of BNIP3L might alter the conformation
of its LIR motif, which faces LC3A/B-II proteins and thus
facilitates recognition of mitochondria by autophagosomes. In
addition to its function in inducing mitophagy, we revealed
the requirement of BNIP3L homodimer for its neuroprotec­
tive effect in ischemic brains (Figure 4E-G). The neuropro­
tective feature of BNIP3L was distinct from that of BNIP3,
which promotes cell death in its monomeric form [54]. The
present study emphasized the previously underestimated roles
of BNIP3L dimer in mitophagy activation and advanced our
understanding of how BNIP3L serves to protect ischemic
brain injury [6]. Therefore, promoting BNIP3L dimer forma­
tion in ischemic brains could be a promising strategy in
rescuing ischemic brain injury, which should be addressed
in future studies.
Ubiquitination has been shown to engage in mitophagy
regulation in ischemic neurons [55,56] but the detailed
mechanisms have not been fully illustrated. Here we showed,
for the first time, that BNIP3L underwent proteasome-
dependent rather than autophagy-dependent degradation.
Ubiquitylated BNIP3L, particularly in its dimeric form, accu­
mulated after ischemia (Figure 5), but the ubiquitination sites
and corresponding E3 ligases for BNIP3L degradation should
be further addressed. Importantly, proteasomal inhibitors,
MG132 and carfilzomib prevented BNIP3L degradation as
well as mitophagy deficiency (Figures 5E and 6A). The
BNIP3L loss-induced mitophagy deficiency was significant
for ischemic neuronal injury, since restored BNIP3L attenu­
ated brain infarct volumes and NDS (Figure 4E-G). We thus
hypothesized that preventing BNIP3L degradation could be
a novel therapy for cerebral ischemia. As expected, a single
dosage of carfilzomib at the onset of ischemia showed signifi­
cant neuroprotection in the MCAO model.
Carfilzomib is a selective proteasomal inhibitor approved
by the FDA for multiple myeloma [57]. Carfilzomib showed
improved safety profiles over bortezomib, another proteaso­
mal inhibitor with profound neurotoxicity [58]. Some other
studies indicated the neuroprotection of proteasome inhibi­
tors [59,60], but it was not fully understood how proteasome
inhibition attenuated ischemic injury [61,62]. We found that
BNIP3L deletion abolished the neuroprotection of carfilzomib
(Figure 6C,D), indicating that BNIP3L-mediated mitophagy
was the prominent mechanism, if not the only mechanism,
underlying the benefits of proteasomal inhibitors. We cannot
exclude that other pathways induced by proteasomal blockage
may also contribute to neuronal survival besides mitophagy
induction. For example, proteasomal inhibition altered neu­
ronal glucose metabolism, which may also promote neuronal
survival [63]. Surprisingly, we also found that the administra­
tion of carfilzomib as late as 3 h after ischemia onset was still
effective in rescuing brain injury. Prolonged administration of
carfilzomib to 6 h failed to alleviate brain injury (Figure 6E,F).
Coincidently, BNIP3L cannot be restored by carfilzomib at
6 h after ischemia, which further strengthened the significance
of BNIP3L-mediated mitophagy in ischemic brains. Finally,
we identified the efficacy of carfilzomib in the chronic ische­
mia model (Fig. S6). Although more complicated pathophy­
siological mechanisms may be involved, a variety of studies
indicated the importance of neuronal mitochondrial quality in
the late phases after stroke [64]. Overall, this is the first
demonstration of carfilzomib as a promising therapy for
diverse states of stroke.
Taken together, our work has unraveled critical contribu­
tions of BNIP3L degradation to compromised mitophagy in
ischemic brains. Preventing BNIP3L degradation by inhibiting
the ubiquitin-proteasome pathway rescued mitophagy defi­
ciency and attenuated cerebral ischemic injury. We identified

carfilzomib, a proteasomal inhibitor, as a promising strategy
for stroke therapy.
Materials and methods
Animals
Male C57BL/6 mice and male BALB/c mice weighing 22–25 g
(8–10 weeks old) were used. Mice were raised on a 12 h light/
dark cycle with free access to water and food. The bnip3l-/-
mice (BALB/c strain background) were provided by Prof. Paul
Ney (St. Jude Children’s Research Hospital). The Atg7fl/fl mice
were kindly provided by Masaaki Komatsu (Tokyo
Metropolitan Institute of Medical Science, Tokyo, Japan). All
experiments were approved by and conducted in accordance
with the ethical guidelines of the Zhejiang University Animal
Experimentation Committee and were in complete compli­
ance with the National Institutes of Health Guide for the Care
and Use of Laboratory Animals. Efforts were made to reduce
any pain and the number of animals required.
MCAO mouse models and drug administration
Mice were anesthetized with isoflurane during surgery.
Cerebral blood flow (CBF) was detected in the middle cerebral
artery (MCA) by laser Doppler flowmetry (Model Moor
VMS-LDF2, Moor Instruments Ltd, UK). A fiber-optic
probe was fixed to the skull from 2-mm caudal to bregma
and 6-mm lateral to midline over the cortex supplied by the
proximal part of the right MCA. A 6–0 nylon monofilament
suture, blunted at the tip and coated with 1% poly-l-lysine,
was inserted 10 mm into the internal carotid to occlude the
origin of the MCA. Animals were excluded from the study if
the reduction in CBF was less than 80%. Body temperature
was maintained at 37°C by a heat lamp (FHC, Bowdoinham,
ME, USA) until the mice woke uFor transient MCAO, reper­
fusion was allowed 1 h after ischemia by gently removing the
monofilament suture. Focal cerebral ischemia was induced by
MCAO without reperfusion.
Mice were given an intracerebroventricular (i.c.v.) injection
of 7.5 μg 3-MA (Sigma, M9281) at the onset of ischemia in
pMCAO as we described previously [7]. MG132 (Selleckchem,
S2619) was injected (4 mg/kg, i.c.v.) at the onset of ischemia.
CFZ (Selleckchem, S2853) was also injected intraperitoneally
(2 mg/kg, i.p.) at ischemia onset and at 3 h or 6 h after
ischemia. Control mice were injected with the same volume
of saline.
Neurological deficit scores were evaluated at 24 h after
MCAO as follows: 0, no deficit; 1, flexion of contralateral
forelimb upon lifting the whole animal by the tail; 2, circling
to the contralateral side; 3, falling to the contralateral side; and
4, no spontaneous motor activity.
Mice were sacrificed using a lethal dose of isoflurane at
24 h after surgery and brain sections were stained with 2,3,5, -
triphenyltetrazolium chloride (TTC; 0.25%; Sigma, T8877).
Brain infarct volumes were quantified with ImageJ software
and determined by an indirect method that corrects for
edema [6].
AUTOPHAGY 1943
Cell culture, OGD/O-R procedures
For culturing primary cortical neurons, pregnant mice with
embryonic (E17) fetuses were used. The primary cortical
neuronal culture experiments were performed as described
previously [31]. Briefly, the cortical neurons of fetal mice
were digested with 0.25% trypsin (Invitrogen, 25,200–056).
Approximately 2 × 105 cells/cm2 were seeded onto poly-
L-lysine (10 μg/ml)-coated plates and dishes. The neurons
were cultured in Neurobasal medium (Invitrogen, 21,103–­
049) containing 2% B27 (Invitrogen,17,504–044), 10 U/ml
penicillin-streptomycin (Gibco, 15,140–122) and 0.5 mmol/L
glutamine (Gibco, 25,030,081) at 37°C in a humidified atmo­
sphere with 5% CO2 + 95% N2. Cultures were maintained for
7 d before further treatment and were routinely observed
under a phase-contrast inverted microscope. To produce
atg7 knockout neurons, pregnant Atg7fl/fl mice with embryo­
nic (E17) fetuses were used. At the fifth day, Atg7fl/fl cortical
neurons (2 × 106) were transfected with hSyn-Cre virus (2 μl;
Obio Technology Crop., H4942). Neurons were maintained
for 2 d before further treatment.
For OGD treatment, cells were washed twice with glucose-
free DMEM (Invitrogen, 12,800–017), and then refreshed with
O2- and glucose-free DMEM (pre-balanced in an O2-free
chamber at 37°C). Then, we put the neurons in a sealed
chamber (Billups Rothenburg, MIC-101) ventilated with
mixed gas (5% CO2 + 95% N2) for 7 min at 25 L/min. The
chambers were sealed and incubated for 2 h at 37°C. OGD-
reperfusion (O-R) was performed by adding oxygen and glu­
cose to the culture medium for 1 h at 37°C.
BNIP3L gene knockout using CRISPR-Cas9 system in HeLa
cells
To knockout BNIP3L, two gRNAs (5ʹ-
CGGCGGCGGCTCGACTAGGT-3ʹ, 5ʹ-GCGGCGGCGGCTC
GACTAGG-3ʹ) targeting the adjacent downstream sequence
of its start codon were designed using http://crispr.mit.edu/
and inserted into a pSpCas9(BB)-2A-GFP plasmid (Addgene,
48,138; deposited by Liang Fang) according to a previously
described protocol [65]. HeLa cells (ATCC, CCL-2) were co-
transfected with two plasmids using Lipofectamine 2000
(Thermo Fisher Scientific, 11,668,027), and 48 h after trans­
fection, GFP+ cells were FAC-sorted into 96-well plates as
single cells to generate mutant cell clones. The successful
knockout was validated using western blot.
Transfection
p3× Flag-Bnip3l, and p3× Flag-Bnip3lG203A were transfected
into primary cultured neurons using Amaxa electroporation
(Lonza, VPG-1001) as described previously [14]. Briefly, the
primary neurons (5 × 106) were electroporated with 3 μg
plasmids. The cuvette with cell/DNA suspension was inserted
into the Nucleofector Cuvette Holder. After electroporation,
the cells were resuspended in the medium mentioned above
and then transferred into the prepared culture dishes. The
dishes were maintained in an incubator for 7 d at 37°C. The
plasmids were transfected into wild-type HeLa and BNIP3L
1944 X. WU ET AL.
KO HeLa according to the jet PRIME (Polyplus, nl14-15)
protocol.
Lentivirus injection in vivo
Mice were anesthetized with isoflurane and mounted in
a stereotaxic apparatus (Stoelting, 512,600) and adeno-
associated virus (AAV, designed, produced and identified by
Obio Technology Crop., Ltd., Shanghai, China) including
AAV-GFP, AAV-GFP-Bnip3l, and AAV-GFP-Bnip3lG203A
(1 μl) were injected into the cortex (AP + 0.02 mm;
L − 3.2 mm; V − 1.5 mm) and corpus striatum (AP +
0.5 mm; L − 2.0 mm; V − 3.0 mm) with a 5-μl syringe and
a 34 gauge needle at 100 nl/min using an injection pump
(Micro 4, WPI, Sarasota, Fl, USA). After each injection, the
needle was left in place for an additional 10 min and then
slowly withdrawn. The virus was allowed to express target
proteins for a minimum of 2 weeks.
Immunoblotting and immunoprecipitation
Brain tissues and cells were homogenized in RIPA buffer
(50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100
[Diamond, A110694], 1% sodium deoxycholate [Sigma-
Aldrich, 30,970], 0.1% SDS [Biofroxx, 3250GR500]) with
protease inhibitor cocktail (Roche, 04693132001). The sam­
ples come from three independent experiments. A 40 μg ali­
quot of protein from each sample was separated using SDS-
PAGE. The following primary antibodies were used: ATG7
(1:1,000; Cell Signaling Technology, 8558 T), BCL2L13
(1:1000; Proteintech; 16,612-1-AP), BNIP3L (1:1,000; Cell
Signaling Technology, 12396s), COX4I1 (1:1,000; Cell
Signaling Technology, 4850), Flag (1:1,000; Cell Signaling
Technology, 14793s), FUNDC1 (1:1,000; Abcam, ab74834),
GAPDH (1:3,000; KangChen, KC-5G4), GFP (1:1,000; MBL;
598), LC3B (1:1,000; Sigma-Aldrich, L7543), MYC (1:1,000;
MBL; M192-3), PHB2/prohibitin 2 (1:1000; Proteintech;
12,295-1-AP); PRKN/PARK2 (1:1,000; Cell Signaling
Technology, 2132s), SQSTM1 (1:1,000; Cell Signaling
Technology, 5114), TOMM20 (1:1,000; Ambo
Biotechnology, c16678), TP53 (1:1000; Cell Signaling
Technology, 2524 T) and ubiquitin (1:1000, Cell Signaling
Technology, 3936s). Secondary antibodies conjugated with
HRP against either rabbit or mouse IgG (1:3,000; Cell
Signaling Technology, 7071 and 7072) were applied. Digital
images were quantified using densitometric measurement
with Quantity-One software (Bio-Rad).
For immunoprecipitation, BNIP3L KO HeLa cells were
transiently transfected. At 24 h post-transfection, the cells
were suspended with the immunoprecipitation (IP) lysis
buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40
(Sangon Biotech, A100109), 0.25% sodium deoxycholate)
with protease inhibitor cocktail and immunoprecipitated
using EZview Red ANTI-Flag M2 Affinity Gel beads
(Sigma-Aldrich, F2426). Then the immunoprecipitates
were eluted with 100 μl 3× Flag peptide solution (Sigma-
Aldrich, F4799). For MYC IP, the supernatant was incu­
bated with 4 μl anti-MYC (MBL; M192-3) and mixed
overnight with gentle rotation at 4°C. Then 50 μl protein
A Sepharose (GE Healthcare, 17,078,001) was added for
4 h at 4°C.
For endogenous BNIP3 L IP, bnip3l-/- mice were injected
with AAVs expressing GFP, GFP-Bnip3l, or GFP-Bnip3lG203A
for at least two weeks and then were subjected to 6 h of
pMCAO. The brain tissues were homogenized in RIPA buffer
with protease inhibitor cocktail. The suspensions were cen­
trifuged at 12,000 g for 20 min. Part of the supernatant
(300 μg) was transferred to a new tube as input. In total,
20 μl Protein A Sepharose was added to the supernatant for
the preclear process for 4 h and was subsequently discarded.
The supernatant (1.5–3 μg) was incubated with 4–8 μl anti-
BNIP3L (Cell Signaling Technology, 12396s) overnight at 4°
C. Then 50 μl of Protein A Sepharose was added for 4 h at 4°
C. Subsequently, the immunoprecipitates were washed with
lysis buffer 3 times. The immunoprecipitates were then eluted
by boiling for 5 min in 2 × Laemmli sample buffer (Sigma-
Aldrich, S3401) and subjected to western blot analysis.
Statistical analysis
All data were collected and analyzed in a blind manner. Data
are presented as mean ± SEM. The single comparisons were
determined by a two-tailed Student’s t-test. One-way ANOVA
(analysis of variance) with Dunnett’s T3 post-hoc test applied
for multiple comparisons. Sample sizes were based on our
previous study and prior literature to achieve reliable mea­
surements. Statistical analyses were conducted using
GraphPad Prism 5.0 (GraphPad Software, San Diego, CA,
USA). P < 0.05 was considered statistically significant.
Acknowledgments
We are grateful to Prof. Paul Ney from St. Jude Children’s Research
Hospital for offering the bnip3l-/- mice and Professor Masaaki Komatsu
of Tokyo Metropolitan Institute of Medical Science for offering the
Atg7fl/fl mice. We are grateful to the Core Facilities of Zhejiang
University Institute of Neuroscience and Imaging Facilities.
Disclosure statement
No potential conflicts of interest are disclosed.
Funding
This work was funded by the National Natural Science Foundation of
China (81822044, 81773703 and 81872844) and the Fundamental
Research Funds for the Central Universities (2019XZZX004-17)
ORCID
Shijia Ma http://orcid.org/0000-0001-9409-9272
Liang Fang http://orcid.org/0000-0003-4502-1756
Zhong Chen http://orcid.org/0000-0003-4755-9357
Xiangnan Zhang http://orcid.org/0000-0002-7603-7403
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