Targeting GPER1 to suppress autophagy as a male-specific therapeutic strategy for iron-induced striatal injury
Targeting GPER1 to suppress autophagy as a male-specific therapeutic strategy for iron- induced striatal injury
Tzu-Yun Chen 0
Chih-Lung Lin 1
Li-Fang Wang 2
Ke-Li Tsai 0
Jun-Yu Lin 0
0 Department of Physiology, faculty of Medicine, college of Medicine, Kaohsiung Medical University , Kaohsiung, t aiwan
1 Department of neurosurgery, f aculty of Medicine, c ollege of Medicine, Kaohsiung Medical University , Kaohsiung, t aiwan
2 Department of Medicinal and Applied c hemistry, c ollege of Life Science, Kaohsiung Medical University , Kaohsiung, t aiwan
3 Graduate institute of Medicine, Kaohsiung Medical University , Kaohsiung, t aiwan
4 Graduate institute of clinical Medicine, college of Medicine, Kaohsiung Medical University , Kaohsiung, t aiwan
Published: xx xx xxxx The functional outcome of intracerebral hemorrhage (ICH) in young male patients are poor than in premenopausal women. After ICH, ferrous iron accumulation causes a higher level of oxidative injury associated with autophagic cell death in striatum of male mice than in females. In rodent model of ferrous citrate (FC)-infusion that simulates iron accumulation after ICH, female endogenous estradiol (E2) suppresses autophagy via estrogen receptor ? (ER?) and contributes to less injury severity. Moreover, E2 implantation diminished the FC-induced autophagic cell death and injury in males, whose eR? in the striatum is less than females. Since, no sex difference of ER? was observed in striatum, we delineated whether ER? and G-protein-coupled estrogen receptor 1 (GPER1) mediate the suppressions of FC-induced autophagy and oxidative injury by E2 in a sex-dimorphic manner. The results showed that the ratio of constitutive GPER1 to ER? in striatum is higher in males than in females. The GPER1 and eR? predominantly mediated suppressive effects of E2 on FC-induced autophagy in males and antioxidant effect of E2 in females, respectively. This finding opens the prospect of a male-specific therapeutic strategy targeting GPER1 for autophagy suppression in patients suffering from iron overload after hemorrhage.
Intracerebral hemorrhage (ICH) is a devastating disorder associated with dismal outcome. Men have poor
survival than premenopausal women after ICH1,2, which is associated with iron accumulation and autophagy
induction. Previous reports showed that males exhibited worse free radical homeostasis and a lesser defense capacity
against oxidative brain damage than females did3. However, no effective sex-based therapy has been used in
patients suffering from long-term neurodegeneration after ICH. The prevalence of ICH is expected to increase
because the elderly population continues to grow, and the most serious complication of oral anticoagulation,
which is given for ischemic stroke prevention, is hemorrhagic stroke4. Therefore, gaining insight into sex-related
differences in endogenous protective mechanisms against hemorrhagic stroke will provide better strategies for
optimization of sex-specific treatment.
Recently, a male-specific therapeutic strategy targeting autophagic inhibition for patients suffering from
intracerebral iron overload has been suggested5. Although E2 implantation decreases FC-induced striatal injury
and autophagy in both sexes6, how to prevent the feminizing effects of exogenous E2 in males is an important
issue. Because the constitutive mRNA and protein levels of ER?, but not ER?, in the striatum are higher in female
than in male rats7, we prospect high level of endogenous E2 may protect against hemorrhagic stroke
predominantly via ER? in females, while, in males, other ERs may mediate the neuroprotection conferred by E2 against
FC-induced striatal injury. GPER1 has a high affinity for E2 and mediates both rapid signaling and transcriptional
events in response to E28. A previous report demonstrated that G1, a GPER1 agonist, had a protective effect on
cognitive function via a ctivation of PI3K/Akt and downstream mTORC19 that inhibited autophagy, while a
GPER1 antagonist, G15, increased the level of LC3B-II and the accumulation of autophagosomes10. These results
Sex differences in constitutive and inducible levels of GPER1 and ER? in the striatum. To
examine the sexual dimorphism of GPER1 and ER? in the neuroprotective mechanism of E2 against FC-induced
striatal injury, we compared the constitutive level of striatal GPER1 or ER? in intact male with that in intact
female mice. Interestingly, we found that the striatal protein level of ER? was significantly lower in intact male
mice than in intact females (Fig.?1a). No significant sex difference in striatal GPER1 was observed between intact
mice (Fig.?1b), but the ratio of GPER1 to ER? was higher in males than in females.
To know whether FC infusion induces the expression of GPER1 or ER? in the striatum in a sex-dimorphic
manner, we compared the levels of FC-induced expression of GPER1 or ER? in males with that in females. The
results showed that FC infusion significantly increased the striatal levels of both GPER1 and ER? in male mice
but not in female mice (Fig.?1a,b).
In order to exclude the influence of endogenous sex hormones, castrated male and female mice were used in
the following studies. The protein levels of ER? and GPER1 between castrated and intact mice were compared to
exclude any possible effect of castration for 2 weeks on the expression of those proteins. The results showed that
there was no significant difference in the level of GPER1 or ER? between sex-matched intact mice and castrated
mice (Supplement?S1). To ensure that the serum level of E2 after implantation was within the physiological range,
we measured the E2 concentration using an ELISA kit. As shown in Supplement?S2, the serum levels of E2 after
implantation were within the physiological range. Therefore, the following experiments were performed by using
castrated male and female mice to study the effect and mechanism of exogenous E2 on striatal injury.
Effects of E2 implantation on FC-induced behavioral deficits, striatal injury, autophagy and lipid
peroxidation in castrated male and female mice. To examine whether autophagy and lipid
peroxidation are involved in the mechanism underlying E2-based protection against iron neurotoxicity in both castrated
males and females, forelimb-use asymmetry scores and the levels of SBDP 145/150 were examined as indexes of
behavioral deficits and the severity of striatal injury, respectively. Moreover, the ratio of LC3-II to LC3-I, which
indicates LC3 lipidation and is regard as a marker for autophagy, and the level of 4-HNE, a biomarker of oxidative
stress, were examined. The results showed that FC infusion increased the levels of FC-induced behavioral deficit
(Fig.?2a), cleavage of ?-II spectrin (Fig.?2b), LC3 lipidation (Fig.?2c) and lipid peroxidation (Fig.?2d) in both
castrated male and female mice. No sex differences in FC-induced behavioral deficits (Fig.?2a) or spectrin cleavage
(Fig.?2b) between castrated male and female mice were observed. Implantation of E2 significantly decreased the
FC-induced behavioral deficits, severity of striatal injury, LC3 lipidation and lipid peroxidation in both castrated
male and female mice (Fig.?2).
Effects of E2, G1 (GPER1 agonist) and propyl pyrazole triol (PPT; ER? agonist) on FC-induced
behavioral deficits, striatal injury, autophagy and lipid peroxidation in castrated male and
female mice. G1 and PPT were used to examine the mediating roles of GPER1 and ER? in the effect of E2
on FC-induced behavior deficits, injury severity, autophagy and lipid peroxidation. The results showed that E2
decreased the levels of FC-induced behavioral deficits, spectrin cleavage, LC3 lipidation and lipid peroxidation
Effects of silencing GPER1 or ER? on FC-induced behavioral deficits, striatal injury, autophagy,
and lipid peroxidation in castrated male and female mice. To elucidate whether there is a sexually
dimorphic role of GPER1 in mediating the effects of E2 on FC-induced injury and autophagy, we used GPER1
siRNA to silence GPER1. The efficiency of GPER1 siRNA silencing was verified by Western blot analysis (as
shown in Supplement?S3a). The results showed that E2 decreased the levels of FC-induced behavior deficits
(Fig.?4a), spectrin cleavage (Fig.?4b), LC3 lipidation (Fig.?4c), and lipid peroxidation (4-HNE) (Fig.?4d) in the
striatums of castrated male and female mice treated with non-target siRNA. GPER1 siRNA significantly diminished
the suppressive effects of E2 on FC-induced behavior deficits (Fig.?4a) and spectrin cleavage (Fig.?4b) in castrated
males but not in castrated females. GPER1 siRNA also significantly diminished the suppression of FC-induced
LC3 lipidation and lipid peroxidation conferred by E2 in castrated males but not in castrated females (Fig.?4c,d).
Additionally, to further understand whether ER? has a sexually dimorphic mediating role in the mechanism
underlying neuroprotection by E2 against iron toxicity, we used ER? siRNA to silence ER?, and the knockdown
efficiency of ER? siRNA was checked by Western blot analysis (as shown in Supplement?S3b). The results showed
that E2 decreased FC-induced behavior deficits (Fig.?5a), spectrin cleavage (Fig.?5b), LC3 lipidation (Fig.?5c), and
lipid peroxidation (4-HNE) (Fig.?5d) in the striatums of castrated male and female mice treated with non-target
siRNA. ER? siRNA significantly diminished the protective effect of E2 on FC-induced behavior deficits (Fig.?5a),
spectrin cleavage (Fig.?5b), LC3 lipidation (Fig.?5c) and lipid peroxidation (Fig.?5d) in castrated females but not
in castrated males.
The present study demonstrated that FC infusion triggers higher protein expression of GPER1 in the striatum of
intact male mice than in females. A GPER1 agonist (G1) significantly decreased FC-induced LC3 lipidation only
in castrated males. And GPER1 siRNA diminished the suppressive effect of E2 on FC-induced LC3 lipidation
and lipid peroxidation in castrated males but not in castrated females. On the other hand, an ER? agonist (PPT)
significantly decreased the level of FC-induced behavior deficits only in castrated females. While ER? siRNA
diminished the suppressive effect of E2 on FC-induced behavior deficits, spectrin cleavage, LC3 lipidation and
lipid peroxidation in a female-specific manner. These results suggest that GPER1 predominantly mediates the
suppressive effect of E2 on FC-induced autophagy and injury in a male-specific manner, while ER? plays a
dominant role in anti-oxidation and neuroprotection among females.
After ICH, men have higher mortality and worse survival than premenopausal women1. Although a
sexually dimorphic therapeutic effect of tirilazad mesylate on the mortality of hemorrhagic patients was reported
in 200711, no effect on clinical outcome was observed12. The present management of ICH is mainly supportive,
including maintenance of homeostasis and treatment of brain edema13. Estrogens contribute to the sex
difference in ICH response14 due to their protective actions such as blocking lipid peroxidation reactions15; donating
hydrogen atoms16; scavenging free radicals; attenuating NADPH oxidase activation; decreasing superoxide and
reactive oxygen species generation; and reducing oxidative stress17. However, chronic administration of estrogens
may elicit unwanted side effects including increased risk of breast/endometrial cancer, and feminization in males.
Present results show that the protein level of ER? was significantly higher in intact female mice than in males and
Non-target GPER1 siRNA
Non-target GPER1 siRNA
Non-target GPER1 siRNA
Non-target GPER1 siRNA
Non-target GPER1 siRNA
Non-target GPER1 siRNA
Non-target GPER1 siRNA
Non-target GPER1 siRNA
(b) SBDP ?
Materials and Methods
Animals. A total of 384 C57BL/6 mice (192 males and 192 females) purchased from National Laboratory
Animal Center, Taipei, Taiwan, was used and no animal was excluded. To study the sex differences in constitutive
and inducible estrogen receptors, we used intact C57BL/6 mice at 12 weeks of age. To study the contribution of
exogenous E2 to the sex difference in FC-induced striatal injury, we castrated both male and female C57BL/6 mice
for two weeks to exclude the influence from endogenous sex hormones, and then implanted them with estradiol
(E2). According to our previous study, implantation of an E2 Silastic tube released E2 in serum at physiological
levels (56?92 pg/ml) and were maintained for at least 7 days38. A Silastic tube (2 mm outer diameter, 1 mm inner
diameter; 20 mm in length) containing 0.8 mmol of E2 (Sigma-Aldrich, E8515) was implanted subcutaneously
24 h before FC infusion6. Alternatively, a Silastic tube filled with 1.6 mmol of GPER1 agonist G1 (Cayman; CAS
Registry No. 881639-98-1) or with 1.6 mmol of ER? agonist PPT (propyl pyrazole triol; Cayman, CAS Registry
No. 263717-53-9) was implanted subcutaneously at 24 h before FC infusion to simulate the specific activation of
GPER1 or ER?, respectively. Three microliters of fresh prepared FC (1 nmol/?l) (Ammonium iron sulfate: Sigma,
Cat. No. 21540-6; Citric acid: Amresco, Cat. No. 0101) was infused into the right striatum (coordinates: 0.2 mm
anterior, 2.5 mm lateral, and 3.5 mm ventral to the bregma) using a microinfusion pump (CMA Microdialysis,
Sweden) at a rate of 1 ?l/min. All animals were randomly assigned to control or experiment groups. The iron
deposition was confirmed by Prussian blue assay on brain sections from mice infused with FC as shown in
Supplement?S4. Mice of normal saline-infusion were used as the control group. Two days after FC infusion, the
forelimb-use asymmetry test was performed before sacrifice. Then, the samples for Western blot were dissected
at 2 mm anterior and 2 mm posterior of the injection site, then separated the outer cortex and isolated the
striatum. All operations were performed under anesthesia with Zoletil 50 (1 ml/kg body weight intraperitoneally).
All experiments were approved by the Kaohsiung Medical University Committee for the Use of Experimental
Animals (IACUC approved No: 102117).
Administration of siRNA. GPER1 siRNA (1000 nM) or ER? siRNA (1000 nM) was mixed with an
equal volume of Invivofectamine 3.0 (Invitrogen, Cat. No. IVF3001) and was injected twice (5 ?l each time)
into the right striatum: once at 24 hours before and once at 5 hours after FC infusion. Two days later, tissue
samples containing the striatum were sampled for the detection of silencing efficiency of GPER1 or ER? by
Western blot analysis. The mouse GPER1 siRNA mixture contains three Stealth siRNA sequences (MSS233774,
MSS233775, MSS233776) (Invitrogen, Cat. No. 1320001); Stealth RNAi negative control duplexes (Invitrogen,
Cat. No. 12935-300) were used as a non-target siRNA for GPER1. The sequences of ER? siRNA were
5?-CUGGUUCAUAUGAUCAACUGG-3? and 3?-AGUUGAUCAUAUGAACCAGCU-5?. Silencer negative
control siRNA (Invitrogen, Ambion, Cat. No. AM4611) was used as a non-target siRNA for ER?.
Forelimb-use asymmetry test. To assess behavior deficits due to striatal injury, we evaluated forelimb-use
asymmetry scores two days after FC infusion. Each individual mouse was placed in a transparent cylinder (25 cm
in diameter and 30 cm in height) in the dark, and the use of ipsilateral limbs (I), contralateral limbs (C), or
simultaneous use of both forelimbs (B) was observed for a 5-minute period. The test was randomized, blind, and
repeated twice in each mouse. The forelimb-use asymmetry score was calculated using the following equation:
[I/(I + C + B)] ? [C/(I + C + B)].
Western blot analysis. Tissue samples were homogenized in 5 volumes of lysis buffer containing 50 mmol/L
Tris base, 150 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L NaF, 0.1 mmol/L Na3VO4, 1% Triton X-100, pH 7.4,
and protease inhibitor. Homogenates were clarified by centrifugation at 13000 rpm for 30 min, and the
concentration of total soluble protein was measured by using a commercially available dye reagent (Protein Assay Kit
II, Bio-Rad, Hercules) with bovine serum albumin as a standard. Protein samples were loaded on an 8 or 12%
polyacrylamide gel for electrophoresis (SDS-PAGE; Bio-Rad, Alcobendas, Spain) and then transferred to a PVDF
membrane. After being blocked with TBS-TWEEN 20 (0.05%) containing 5 or 10% nonfat milk for 1 h, the
membranes were incubated overnight at 4 ?C with the following primary antibodies: (i) rabbit polyclonal
antibodies against LC3B (Sigma, Cat. No. L7543) at 1 mg/mL; (ii) mouse monoclonal antibody raised against amino
acids 2368?2472 of human ?II-spectrin (Santa Cruz, Cat. No. sc-48382) at 1/1000 dilution; (iii) rabbit
polyclonal antibodies against GPER1 (Abcam, Cat. No. ab39742) at 1/250 dilution; (iv) rabbit monoclonal anti-ER?
antibody, clone 60 C (Millipore, Cat. No. 04?820); and (v) primary antibody against 4-HNE (Abcam, Cat. No.
ab48506). Then, the membranes were incubated in TBS-TWEEN 20 (0.05%) containing 10% nonfat milk for
1 h. The primary antibodies were revealed with horseradish-peroxidase-labeled goat anti-rabbit IgG secondary
antibodies (Thermo, Cat. No. 31460) or peroxidase-conjugated AffiniPure goat anti-mouse IgG secondary
antibodies (Jackson, Cat. No. 115-035-003) and were detected with enhanced chemiluminescence reagent. Each blots
showed in figures are derived from the same gel with margin-cropping.
Statistics. FC-induced injury and E2 neuroprotection were compared between brains from males and females
using a two-way ANOVA followed by a post hoc Scheffe test. Data on the effects of E2, G1 or PPT, GPER1 siRNA,
and ER? siRNA on FC-cytotoxicity, autophagy or lipid peroxidation were analyzed using a multiway ANOVA to
determine the effect of each factor. Significance was accepted at p< 0.05.
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This work was supported by the ?KMU-KMUH Co-Project of Key Research?, grant No. KMU-DK107007 (Dr . Hsu/Dr. Lin), and by a research grant from the Ministry of Science and Technology , R.O.C. , Grant No.
103- 2320 -B- 037 -028- MY3 (Dr. Hsu/Dr. Wang). We appreciate the help from Springer Nature English Language Editing Services for correcting this manuscript .
Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-43244-0.