Estrogen Deficiency Induces Memory Loss Via Altered Hippocampal HB-EGF and Autophagy

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Journal of R Pandey et al. OVX-induced neuronal 244:1 53–70 Endocrinology autophagy and memory loss RESEARCH Estrogen deficiency induces memory loss via altered hippocampal HB-EGF and autophagy

Rukmani Pandey1,2, Pallavi Shukla1, Baby Anjum3, Himanshu Pawankumar Gupta2,4, Subhashis Pal5, Nidhi Arjaria6, Keerti Gupta1,2, Naibedya Chattopadhyay5, Rohit A Sinha3 and Sanghamitra Bandyopadhyay1,2

1Developmental Toxicology Laboratory, Systems Toxicology & Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhavan, Lucknow, Uttar Pradesh, India 2Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India 3Department of Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India 4Embryotoxicology Laboratory, Environmental Toxicology Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhavan, Lucknow, Uttar Pradesh, India 5Division of Endocrinology, CSIR-Central Drug Research Institute (CSIR-CDRI), Lucknow, Uttar Pradesh, India 6Electron Microscopy Laboratory, CSIR-Indian Institute of Toxicology Research (CSIR–IITR), Vishvigyan Bhawan, Lucknow, Uttar Pradesh, India

Correspondence should be addressed to S Bandyopadhyay or R A Sinha: [email protected], [email protected] or [email protected]

Abstract

Estrogen deficiency reduces estrogen receptor-alpha (ERα) and promotes apoptosis Key Words in the hippocampus, inducing learning-memory deficits; however, underlying ff estrogen deficiency mechanisms remain less understood. Here, we explored the molecular mechanism ff growth factor in an ovariectomized (OVX) rat model, hypothesizing participation of autophagy and ff AKT growth factor signaling that relate with apoptosis. We observed enhanced hippocampal ff autophagy autophagy in OVX rats, characterized by increased levels of autophagy proteins, ff neurodegeneration presence of autophagosomes and inhibition of AKT-mTOR signaling. Investigating upstream effectors of reduced AKT-mTOR signaling revealed a decrease in hippocampal heparin-binding epidermal growth factor (HB-EGF) and p-EGFR. Moreover, 17β-estradiol and HB-EGF treatments restored hippocampal EGFR activation and alleviated downstream autophagy process and neuronal loss in OVX rats. In vitro studies using estrogen receptor (ERα)-silenced primary hippocampal neurons further corroborated the in vivo observations. Additionally, in vivo and in vitro studies suggested the participation of an attenuated hippocampal neuronal HB-EGF and enhanced autophagy in apoptosis of hippocampal neurons in estrogen- and ERα-deficient conditions. Subsequently, we found evidence of mitochondrial loss and mitophagy in hippocampal neurons of OVX rats and ERα-silenced cells. The ERα-silenced cells also showed a reduction in ATP production and an HB-EGF-mediated restoration. Finally in concordance with molecular studies, inhibition of autophagy and treatment with HB-EGF in OVX rats restored cognitive performances, assessed through Y-Maze and passive avoidance tasks. Overall, our study, for the first time, links neuronal HB-EGF/EGFR signaling and autophagy with Journal of Endocrinology ERα and memory performance, disrupted in estrogen-deficient condition. (2019) 244, 53–70

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-19-0197 Journal of R Pandey et al. OVX-induced neuronal 244:1 54 Endocrinology autophagy and memory loss

Introduction Nonetheless, whether estrogen deficiency had any impact on hippocampal neuronal autophagy and whether the The primary female sex hormone, estrogen, activates latter related to apoptosis, cognitive impairments or had its receptors, ER and ER , and exhibits physiological α β any association with mitochondria awaits investigation. functions within the brain (Mukai et al. 2010, Hara et al. The intersection between growth factors, apoptosis 2015). Correspondingly, ovarian failure and estrogen and autophagy serves as an attractive target for disease deficiency, which characterize menopause in women, intervention within the brain (Garcia-Huerta et al. 2016). suppress cerebral ER levels (Qu et al. 2013, Fang et al. Growth factor signal transduction pathways often stimulate 2018) and alter neuronal functions, including cognition PI3K-AKT-mTOR mechanism and inhibit neuronal (Kim et al. 2016, Djiogue et al. 2018). Estrogen deficiency autophagy (Kalkman & Feuerbach 2017). Although induces hippocampal apoptosis, neuronal loss (Sales primarily studied in glial cells, epidermal growth factor et al. 2010, Yazgan & Naziroglu 2017) and cognitive receptor (EGFR) signaling constitutes one such pathway that dysfunctions (Kim et al. 2016, Djiogue et al. 2018), controls autophagy (Ghildiyal et al. 2013). Dysregulated while estradiol therapy reduces apoptosis and improves EGFR signaling inactivates Beclin-1, reduces endogenous learning-memory performances (Sales et al. 2010, Uzum LC3II and p62 levels and activates PI3K/AKT/mTOR cascade et al. 2016, Yazgan & Naziroglu 2017). However, the for suppressing glial lethality (Palumbo et al. 2014). In underlying mechanisms remain obscure. fact, combined EGFR-autophagy modulation has been The biological self-degradative phenomenon, envisaged as a therapeutic strategy for reduced glioblastoma autophagy, sustains cell survival and plays an essential cell invasiveness and improved neurological performances role in CNS homeostasis (Cheung & Ip 2011). Although (Palumbo et al. 2014, Tini et al. 2015). However, co-existence autophagy helps to remove denatured proteins and of neuronal EGFR and autophagy signaling pathways, damaged organelles, unrestrained autophagy activation particularly within the hippocampus, is unknown. prompts undue neuronal self-digestion and cognitive The key EGFR ligands include EGF, heparin-binding deficits (Wong & Cuervo 2010, Li et al. 2014). Accordingly, epidermal growth factor (HB-EGF), transforming growth deregulated neuronal expression of autophagy regulators, factor alpha (TGFα) and betacellulin, and their genetic mammalian target of rapamycin (mTOR) and Unc-51 like ablation or reduced expression within the hippocampal autophagy-activating kinase (ULK1), autophagy-related neurons caused psychomotor impairments and cognitive proteins, ATG7 and ATG5/12, microtubule-associated dysfunctions (Sasaki et al. 2015, Maurya et al. 2016). protein 1A/1B-light chain 3 (LC3) and Beclin-1 triggers EGFR regulates estradiol-mediated functions in the cognitive decline (Herrera et al. 2008, Yu et al. 2017). brain, with reports suggesting participation of TGFα and Additionally, an altered expression of mitochondrial DNA- EGF, particularly in the astroglial and neural stem cells encoded genes, cytochrome B (CytB) and cytochrome c (Brannvall et al. 2002, Lee et al. 2012). However, whether oxidase subunit II (COXII), and mitochondrial markers, any link existed between estrogen and EGFR signaling voltage-dependent anion channel 1 (VDAC1) and COXIV, within the hippocampal neurons remains un-elucidated. together with selective autophagic mitochondria removal, By using ovariectomy (OVX) model of estrogen loss, that is, mitophagy, contributed toward impaired neuronal supported by in vitro studies, we focused on the effect of functions (Lu et al. 2014, Shaerzadeh et al. 2014). A PTEN- estrogen deficiency on autophagy, mitochondrial content induced kinase 1 (PINK1)/Parkin pathway is also important and EGFR signaling and their link with hippocampal for regulating autophagy-mediated mitochondrial clearance neuronal apoptosis and cognition. Overall, our study in the neurons (Amadoro et al. 2014). Moreover, although identifies a novel estradiol-induced neuroprotective less studied in neurons, the mitochondrial BCL2/adenovirus mechanism that sustains hippocampal neuronal cell E1B 19 kDa protein-interacting protein 3 (BNIP3) and survival and cognitive functions. FUN14 domain containing 1 (FUNDC1) played a key role in regulating mitophagy (Lampert et al. 2019). A complex cross-talk between apoptosis and autophagy has also been reported, where enhanced autophagy Materials and methods ameliorated cognitive deficits through activation of Reagents hippocampal neuronal apoptosis (Li et al. 2016). Contrarily, reduced ATG5 levels via attenuated caspase-mediated Cell lytic reagents, In Situ Cell Death Detection Kit apoptosis appeared neuroprotective (Zhang et al. 2016a, b). Fluorescein (ref. no. 11684795910), Bradford reagent and

Journal of R Pandey et al. OVX-induced neuronal 244:1 55 Endocrinology autophagy and memory loss

17β-estradiol (cat no. E8875) were procured from Sigma. at 24 ± 2°C, 40–60% humidity and 12-h light–darkness Immobilon Western chemiluminescent HRP Substrate cycle with ad libitum availability of diet and water. The and 3-Methyladenine (3-MA; cat no. 189490) were from rats were subjected to sham- and OVX (bilateral)-surgery Millipore. Lipofectamine 2000 transfection reagent, non- following our previously described protocol (Sharan et al. targeting (NT)-siRNA (cat no. AM4611), Becn1-siRNA 2011). Briefly, rats were anesthetized with ketamine and (Assay ID s137745), Esr1-siRNA (Assay ID s128616) and xylazine (intraperitoneally, 60 and 20 mg/kg body weight, MitoTracker™ Red CMXRos (cat no. M7512) were from respectively). An incision was then made at the abdominal Invitrogen. Superscript™ III First-Strand Synthesis kit (cat wall, adipose tissues cleaned and ovaries removed (OVX). no. 18080051) and Maxima SYBR Green/ROX qPCR master The sham-operated rats underwent same surgical procedure mix (2×) were from Thermo Fischer Scientific. Wizard® excepting ovary removal (Sham). Rats were injected with Genomic DNA Purification Kit (A1120) was from Promega. gentamicin (50 mg/kg, intramuscular for 3 consecutive Recombinant HB-EGF was from R&D systems. Vectashield days) to minimize post-surgical infection. To determine Antifade Mounting Medium with DAPI was from Vector the effect of 17β-estradiol (E2; 100 µg/kg/day), an earlier Laboratories. Tandem-tagged mt-RFP EGFP plasmid was protocol with slight modifications was followed Yazgan( & a kind gift from Dr Andreas Till (Institute of Clinical Naziroglu 2017), where the rats were treated subcutaneously Molecular Biology, Christian-Albrechts-University of Kiel, for fourteen days from seventh day post OVX. To determine Kiel, Germany). Serum estradiol (E2) kit (cat no. E-EL-0065) the effect of HB-EGF and autophagy, HB-EGF (100 ng in 2 µL was from Elabscience (Houston, TX, USA). sterile saline) and the autophagy inhibitor, 3-MA (300 nM in DMSO) were injected into the hippocampus once on the Antibodies 14th day post-OVX surgery, following the protocol described before (Pandey et al. 2017). Tissue samples were collected Rabbit polyclonal ERα, goat polyclonal HB-EGF and on the 21st day after OVX surgery, following earlier studies horseradish peroxidase (HRP)-conjugated mouse showing hippocampal damage at this time point (Sales monoclonal β-Actin were from Santa Cruz Biotechnology. et al. 2010, Yazgan & Naziroglu 2017), and supported by Rabbit polyclonal LC3B, phospho-Akt, Akt and phospho- our selection criteria of autophagy (showing significantly ULK1 and rabbit monoclonal phospho-AMPKα, AMPKα, increased LC3-II) (Fig. 1A). For isolating samples for ULK1, phospho-EGFR, EGFR, ATG-5/12 conjugate, Western blotting, rats were killed by cervical dislocation VDAC1, COXIV, PINK1, Parkin, cleaved caspase-3 and and brain dissected out. Hippocampal tissues were isolated, PARP were from Cell Signaling Technology. Rabbit quickly snap frozen in liquid nitrogen, stored in −80°C and monoclonal phospho-mTOR and rabbit polyclonal mTOR processed for Western blotting. For immunohistochemistry were from Abcam. Mouse monoclonal, Neuronal Nuclei, (IHC), rats were anesthetized, transcardially perfused using NeuN, was from Millipore. Rabbit polyclonal Beclin-1, 4% PFA and 0.2% picric acid in phosphate-buffered saline ATG-7 and p62/SQSTM1, and HRP-conjugated secondary and the whole brains were taken, as described before anti-mouse, anti-rabbit and anti-goat antibodies were (Pandey et al. 2017). Blinding and randomization were not from Sigma. Alexa Fluor®546 goat anti-rabbit IgG, Alexa carried out for the study. No sample size calculation through Fluor®488 goat anti-mouse IgG and Alexa Flour®594 statistically methods was performed. However, there were chicken anti-goat IgG were from Invitrogen. no sample size differences between the beginning and end of the experiments. Animal ethical approval

Wistar rats were used after obtaining approval, specific Serum estradiol assay to this study, from the Institutional Animal Ethics Committee of CSIR-CDRI and CSIR-IITR. For maintenance Blood was collected from rats, centrifuged at 1500 g for and handling of the rats, guidelines and regulations of the 10 min and serum was isolated. Serum E2 was measured ethics committee were strictly followed. using an ELISA assay kit, following the manufacturer’s protocol, and expressed as pg/mL.

Animal treatment, surgery and hippocampal tissue isolation Primary neuronal culture

For OVX surgery, adult female rats (250–300 g) were Primary hippocampal neurons were cultured kept in a hygienic condition in well ventilated cages from embryonic-16 (E16) rats, as described earlier

Figure 1 OVX induces hippocampal autophagy. Hippocampal tissues were isolated at the 14th, 21st and 42nd day post sham- and -OVX surgery of rats. (A) Representative Western blot and densitometry of LC3-II normalized with β-actin. Hippocampal tissues from Sham, OVX and OVX + E2-treated rats were isolated at the 21st day post surgery. (B and D) Representative Western blot and densitometry of autophagy markers (B) and autophagy regulators (D) normalized with β-actin (B) and respective non-phospho counterparts (D). (C) qPCR analysis showing mRNA levels of P62 normalized with housekeeping gene, Gapdh. (E) Electron micrographs, and inset for OVX, showing autophagosome. Data are representatives of three rats/group and means ± s.e. ***P < 0.001, **P < 0.01 and *P < 0.05 compared to sham or as indicated.

(Pandey et al. 2017). Briefly, the hippocampus was isolated and sodium fluoride for phosphorylated proteins. The in Hank’s Balanced Salt Solution (HBSS), meninges lysates were then centrifuged (20,000 g, 30 min, 4°C). removed, followed by mechanical and enzymatic (0.05% For mitochondrial protein-based studies, we isolated trypsin-EDTA) digestion. The hippocampal suspension crude mitochondria from hippocampal tissues through was then centrifuged (2000 g, 10 min). The pellet obtained differential centrifugation following an earlier protocol was suspended in neurobasal medium and streptomycin (Dixit et al. 2013). Protein content in supernatant was (100μg/mL) and penicillin (100 units/mL), supplemented estimated using Bradford reagent, and SDS-PAGE (4–15%) with N2 (1%), B-27 (2%) and l-glutamine (2mM) performed with the samples (50 μg), as described previously (complete medium) and seeded onto Poly-l-Lysine (PLL)- (Pandey et al. 2017). Following transfer onto PVDF coated culture flasks. membrane, blots were probed with LC3B, Beclin-1, ATG-7, ATG-5/12, p62, p-Akt, Akt, p-mTOR, mTOR, p-AMPKα, AMPKα, p-ULK1, ULK1, HB-EGF, p-EGFR, EGFR, VDAC1, Protein extraction and Western blotting COXIV, PINK1, PARKIN, cleaved caspase-3, PARP (1:1000, Hippocampal tissues and neurons were homogenized in overnight) and β-actin (1:5000, 2 h) primary antibodies. lysis buffer containing dithiothreitol (1 mM) and protease Blots were then washed with TBST (Tris buffer pH 7.4 inhibitor cocktail, in addition to sodium-orthovanadate (10 mM Tris and 150 mM NaCl) with 0.01% Tween 20)) and

Journal of R Pandey et al. OVX-induced neuronal 244:1 57 Endocrinology autophagy and memory loss incubated with HRP-conjugated secondary antibodies at The hippocampal sections were probed with ERα, HB-EGF, double the primary antibody dilution. Protein bands were LC3B, Beclin-1, VDAC1, cleaved caspase-3 (1:100) and detected with Immobilon Western Chemiluminescent NeuN (1:200) antibodies overnight and re-probed with HRP Substrate in an Amersham Imager 600 system (GE Alexa Fluor secondary antibodies (double dilution relative Healthcare Life Sciences) and quantified using image to primary antibodies, 2 h). After mounting in Vectashield analyzer software Quantity One (Bio-Rad). antifade medium containing DAPI, photomicrography of hippocampus was performed under Nikon Eclipse Ni fluorescence microscope using NIS-Elements microscope Transmission electron microscopy (TEM) imaging software (Nikon). Following perfusion-fixation of the rats in PFA and glutaraldehyde, hippocampal tissues were post-fixed in TUNEL assay 1% osmium-tetroxide, dehydrated using acetone series (15–100%) and embedded in araldite-dodecenyl succinic Hippocampal sections were probed with TdT and anhydride mixture (Ladd Research Industries, Burlington, fluorescein-labeled dUTP (2 h, 37°C) and immunostained VT, USA). After baking (60°C), the sections were cut with 1:200 dilution of anti-rabbit NeuN (overnight, (60–80 nm) using Ultra-microtome (Leica) and picked 4°C°), as previously described (Pandey et al. 2017). The onto 200-mesh copper grids. Sections were double-stained sections were mounted in Vectashield mounting medium with uranyl-acetate and lead-citrate and observed under containing DAPI and fluorescence photomicrography FEI Tecnai G2 spirit twin transmission electron microscope performed. Apoptotic index (%) was determined as the equipped with Gatan digital CCD camera (Pleasanton, number of TUNEL-positive cells/100 nuclei, counted CA, USA) at 80 KV as described before (Rai et al. 2013). manually and randomly in five different areas using ImageJ 1.48v plugins.

Real-time PCR (quantitative PCR, qPCR) Learning and memory tests RNA was isolated from the hippocampal tissue using TRIzol reagent and subjected to cDNA synthesis Passive avoidance and Y-Maze tests were performed as using reverse transcription PCR (Superscript III, Life described earlier (Pandey et al. 2017). For passive avoidance Technologies), as described before (Pandey et al. 2017). test, rats were first subjected to acquisition in gated light- The synthesized cDNA was subjected to qPCR using dark chambers, involving an initial acclimatization (30s) SYBR green dye and primers (Table 1) (Integrated DNA in the light compartment following by a foot shock at Technologies, Coralville, IA, USA) over sequential reaction the dark compartment (0.5 mA, 10 s). The rats were then comprising denaturation (95°C, 15 s), annealing (60°C, made to undergo shock-free retention trials of 300 s each 30 s) and extension (72°C, 30 s) steps for 40 cycles in an at 24 h, 48 h and 72 h, and transfer latency time (TLT) Applied Biosystems™ Real-Time PCR Instruments (Life Technologies). Relative mRNA expression was calculated Table 1 List of primer sequences for qPCR. using the Relative Quantity (RQ) equation, RQ = 2−ΔΔCycle Primers Sequence threshold method. Egf F 5′-GTCAGCTAATGGATCGAGTCA-3′ R 5′-CTGCTCCCAGTTTCTACAGAAC-3′ Immunohistochemistry Hb-egf F 5′-AGCTCCGTATTCCCTCGTG-3′ R 5′-CACATATGACCACACTACCGTCTTG-3′ Rats were anesthetized and perfused with Tgfα F 5′-CGCCTATGGTACCTGAACATGA-3′ R 5 -ACGTACCCAGAGTGGCAGAC-3 paraformaldehyde (PFA 4%) and picric acid (0.2%), and ′ ′ Betacellulin F 5′-TGAAACCAATGGCTCTCTTTG-3′ IHC performed following earlier protocol (Pandey et al. R 5′-TGTCCTGGGTCTTGTGATTC-3′ 2017). Briefly, rat brain was isolated, post-fixed in PFA and P62 F 5′-CCTTTGGCCACCTCTCTG-3′ cryoprotected in sucrose-gradient (10, 20 and 30%). Ten R 5′-AGGACGTGGGCTCCAGTT-3′ Fundc1 F 5 -CCAAGACTATGAGAGCGATGAC-3 micrometer coronal sections from the brain containing ′ ′ R 5′-CCGGAACTGTGGCCAAATA-3′ the hippocampus were made using cryomicrotome Bnip3 F 5′-CAGCAATGGCAACGGTAATG-3′ (Microm HM 520, Labcon, Heppenheim, Germany), R 5′-CCAGAAGGATCTTCTCCATGTC-3′ mounted onto PLL-coated slides, antigen-retrieved with Gapdh F 5′-TGGGAAGCTGGTCATCAAC-3′ R 5′-GCATCACCCCATTTGATGTT-3′ citrate buffer (pH 6.0), followed by 5% BSA-blocking.

was calculated as the time taken to shift from light to N2 (1%), B-27 (2%) and l-glutamine (2 mM), and then dark compartment, where increased TLT indicated better incubated with NT-si RNA (100 nM), ERα-si RNA (100 nM), learning-memory performances. For Y-Maze test, rats were NT-siRNA + HB-EGF (5 ng/mL) or ERα si RNA + HB-EGF for subjected to a training period of thirty trials, where they 48 h. The cells were washed with PBS and XF Base minimal were allowed to freely move around the three arms of a DMEM (Agilent Technologies), used as mitochondrial Y-shaped apparatus, with one arm being shock-free and respiration assay medium. The mitochondrial respiration having 15-W light bulb (safe arm), while the other two was assayed by stepwise addition of A: oligomycin arms being dark and having foot-shocks (1–5 mA) (unsafe (1 μM), B: carbonyl cyanide-4-(trifluoromethoxy) arm). The Y-Maze memory test was performed at 24 h, 48 h phenylhydrazone (FCCP) (2 μM) and C: rotenone (0.5 μM). and seventh day post training, and % saving memory was Three measurement cycles of a 2-min mix, 1-min wait, calculated as (Etraining−Etest) × 100/Etraining, where denoted and 5-min measure were performed after each addition, running toward the unsafe arm. using the Sea-horse Bioscience XFe-24 analyzer (Agilent Technologies) (Pal et al. 2019).

Neuronal transfection Statistics For siRNA silencing, 70–80% primary neurons were transfected with NT-, ERα- or Beclin-1-siRNA (100nM), as For statistical analyses, GraphPad Prism (GraphPad described earlier (Pandey et al. 2017). Briefly, the siRNA and Software) was used. Unpaired Student t-test with two- Lipofectamine 2000 transfection reagent were suspended tailed statistics were conducted for comparison between in neurobasal medium (20 min, 37°C), and the complex two groups of independent samples. For comparison obtained incubated with neurons (CO2 incubator, 4–6 h). between more than two groups, one-way ANOVA was The medium was replaced with complete neurobasal performed, followed by the Tukey’s post hoc multiple medium, and study performed at 48 h post-transfection. comparisons test. Two-way ANOVA followed by Tukey’s To examine mitophagy, 70–80% primary neurons post hoc multiple comparisons test was performed for were transfected with tandem-tagged mt-RFP-EGFP comparisons between more than two groups and more plasmid, pAT016 (0.5 μg/well of four-well chamber slide), than one parameter. encoding a mitochondrial-targeting signal sequence fused in-frame with RFP and EGFP genes (Kim et al. 2013, Sinha et al. 2015). The cells were co-transfected with Results ERα- or NT-siRNA. After 48 h, the cells were mounted in Vectashield medium containing DAPI, and fluorescence Effect of OVX on hippocampal autophagy photomicrography was performed. Image analysis was We generated OVX female rats and investigated conducted with ImageJ software. hippocampal autophagy in the rats. We detected a significantly increased LC3-II level at day 21 post OVX MitoTracker Red staining surgery (Fig. 1A), and hence, detailed experiments were performed at this time point. A reduced serum level Primary neurons, plated on PLL-coated chamber slides, verified estrogen deficiency in the OVX ratsTable ( 2). were incubated with MitoTracker Red for 30–45 min at Along with LC3-II, we observed increased levels 37°C, as described earlier (Kaplan et al. 2012). The cells of autophagy markers, Beclin-1, ATG-7 and ATG5/12 were permeabilized with PBST (PBS + 0.1% Triton X 100), conjugate, in the hippocampus of OVX rats compared mounted in Vectashield medium containing DAPI, and to sham (Fig. 1B). We then examined whether the fluorescence photomicrography was performed.

Table 2 Serum estradiol levels in rats. Mitochondrial respiration and ATP production Groups Estradiol (pg/mL) measurement Sham 94.88 ± 8.24 Primary hippocampal neurons were plated as OVX 36.46 ± 5.65a b 40,000 cells/well onto the PLL-coated Sea-horse V7-PS Flux OVX+E2 84.07 ± 7.19 24-well polystyrene 24-well plates. The cells were grown Data represent means ± s.e. of three rats/group. for 5 days in neurobasal medium supplemented with aP < 0.001 and bP < 0.001 compared to Sham and OVX respectively.

Journal of R Pandey et al. OVX-induced neuronal 244:1 59 Endocrinology autophagy and memory loss serum estrogen deficiency could be responsible for this rats was linked to autophagy. We first examined the increased hippocampal autophagy and found that E2 effect of intra-hippocampal HB-EGF administration on supplementation, which restored serum E2 levels (Table AKT-mTOR pathway, and observed that hippocampal 2), reduced the OVX-induced changes in hippocampal levels of p-AKT, p-mTOR and p-ULK1 (Ser757) in OVX autophagy markers (Fig. 1B). To determine hippocampal rats were restored to that achieved by the E2 administered autophagic flux in OVX rats, we measured P62 (also group (Fig. 3A). Besides reduction in AKT-mTOR signaling, known as Sequestosome-1 (SQSTM1)), which undergoes we also observed increased AMPKα activation in OVX rat autolysosomal degradation during autophagy (Bjorkoy hippocampus, and this effect was restored via HB-EGF and et al. 2009). We observed a decrease in hippocampal p62 E2 treatment (Fig. 3A). Furthermore, the OVX-induced levels in the OVX rats and its recovery by E2 (Fig. 1B). changes in hippocampal LC3-II, Beclin-1, ATG-7 and p62 Nonetheless, measuring the hippocampal P62 mRNA levels (Fig. 3B) were reversed upon HB-EGF treatment in showed its up-regulated expression in OVX rats (Fig. 1C). the OVX rats, on a par with E2-treated group. Thus, our Thus, our results demonstrate that despite increased P62 data indicate that OVX causes an increased hippocampal transcription, its protein levels were significantly reduced, autophagy via reduced HB-EGF/EGFR signaling. indicating an OVX-induced enhanced autophagic flux. We further explored the autophagy regulators, and detected Role of ERα and effect of OVX on HB-EGF/EGFR and an OVX-mediated decrease in p-AKT/AKT, p-mTOR/mTOR autophagy in hippocampal neurons and p-ULK1 (S757)/ULK1 in the hippocampus, and their E2-mediated recovery (Fig. 1D), suggesting an induction We validated our findings through in vitro studies in and initiation of hippocampal autophagy in OVX primary hippocampal neurons (that regulates cognitive condition. Our TEM data demonstrating enhanced OVX- functions). We used ERα-silenced primary hippocampal mediated hippocampal accumulation of autophagosome neurons, following our in vivo observation showing reduced and its E2-mediated reduction (Fig. 1E) validated the ERα in the NeuN-expressing cells of the hippocampus increased OVX-induced autophagy. (ERβ levels remained unchanged, data not shown) of OVX rats and its recovery by E2 treatment (Fig. 4A). Efficacy of ERα silencing has been shown in Fig. 4B. In line with Effect of OVX on hippocampal EGFR signaling and its our in vivo findings Figs( 1, 2 and 3), we observed an link with autophagy ERα-siRNA-mediated reduction in HB-EGF (Fig. 4C), EGFR signaling often regulates the cellular AKT pathway p-EGFR, p-AKT/AKT, p-mTOR/mTOR and p-ULK1/ULK1 (Jin et al. 2005), and given our results showing OVX- levels (Fig. 4D), along with a concomitant increase in mediated reduction in hippocampal p-AKT/p-mTOR LC3-II, Beclin-1, ATG-7 and ATG5/12 and decrease in p62 levels, we asked if EGFR signaling participated in the expression levels in the primary hippocampal neurons OVX-induced effects. For this, we first checked the (Fig. 4E). The ERα-siRNA-mediated changes could be effects of OVX on hippocampal EGFR signaling. We blocked by HB-EGF (Fig. 4D and E). detected an OVX-mediated reduction in hippocampal We verified the above observations in OVX rats, p-EGFR, indicating decreased EGFR activation, which which showed decreased HB-EGF (Fig. 5A) and increased could be inhibited by E2 treatment (Fig. 2A). We next autophagy (Fig. 5B and C) in the NeuN-expressing screened the EGFR ligands through qPCR, and identified matured neurons in the hippocampus. Thus, our data an OVX-mediated decrease in hippocampal HB-EGF indicate an ERα downregulation, which in turn induces mRNA, while the other ligands were unaffected (Fig. 2B). increased autophagy in the hippocampal neurons via The Western blotting data validated the OVX-induced reduced HB-EGF/EGFR signaling. HB-EGF reduction and recovery by E2 treatment (Fig. 2C). To prove the HB-EGF and EGFR link, we administered Role of altered HB EGF and autophagy in HB-EGF in the hippocampus of OVX rats. We observed OVX and ERα-siRNA-induced hippocampal an HB-EGF-mediated recovery in p-EGFR levels of OVX neuronal apoptosis rats, comparable to that in the OVX + E2 group (Fig. 2D), overall, suggesting that hippocampal HB-EGF/EGFR We next examined whether the reduced HB-EGF and signaling was E2 dependent. augmented autophagy participated in OVX-induced We then investigated whether the attenuated hippocampal neuronal apoptosis, reported earlier hippocampal HB-EGF/EGFR signaling in the OVX (Sales et al. 2010, Yazgan & Naziroglu 2017). By treating

Figure 2 OVX downregulates hippocampal HB-EGF/EGFR signaling. Hippocampal tissues from Sham, OVX, OVX+E2− or OVX+HB-EGF-treated rats were isolated. (A, C and D) Representative Western blots and densitometry of p-EGFR (A and D) and HB-EGF (C) normalized with EGFR (A and D) and β-actin (C). (B) qPCR analysis showing mRNA levels of EGFR ligands normalized with housekeeping gene, Gapdh. Data represent means ± s.e. of three rats/group. ***P < 0.001 and **P < 0.01 compared to sham or as indicated.

HB-EGF or the autophagy inhibitor, 3-MA (of note, the and increased autophagy-mediated neuronal apoptosis in efficacy of 3-MA as an autophagy inhibitor in vivo was the hippocampus of OVX rats. A reduced hippocampal verified by analyzing LC3-II levels (Fig. 6A)), we found a NeuN level, suggesting neuronal loss, in the OVX reduced neuronal cleaved caspase-3 expression (Fig. 6B) rats, and its HB-EGF and 3-MA-mediated recovery and apoptotic index (%) (by TUNEL assay) (Fig. 6C), at (Fig. 6B, C and D) supported the apoptosis concept. par with E2-treated group, in the hippocampus of OVX Further, our in vitro data validated the in vivo findings, rats. Thus, our data indicate an attenuated HB-EGF/EGFR showing decreased cleaved caspase-3 and cleaved

Figure 3 OVX induces hippocampal autophagy via decreased HB-EGF/EGFR signaling. Hippocampal tissues from Sham, OVX, OVX+HB-EGF− and OVX+E2-treated rats were isolated. (A and B) Representative Western blots and densitometry of autophagy regulators (A) and the autophagy markers (B) normalized with respective non- phospho counterparts (A) and β-actin (B). Data represent means ± s.e. of three rats/group. ***P < 0.001, **P < 0.01 and *P < 0.05 compared to sham or as indicated.

Journal of R Pandey et al. OVX-induced neuronal 244:1 61 Endocrinology autophagy and memory loss

Figure 4 ERα depletion suppresses HB-EGF/EGFR signaling, inducing autophagy in hippocampal neurons. Hippocampal sections were made from Sham, OVX and OVX+E2-treated rats and immunofluorescence performed. (A) Representative fluorescence photomicrograph (20×) and quantification relative to sham of ERα and NeuN co-immunostaining and nuclear DAPI counter-staining. Scale bar: 100 μm. Data represent means ± s.e. of three rats/group. ***P < 0.001 and **P < 0.01 compared to sham or as indicated. Rat primary hippocampal neurons were transfected with NT-siRNA or ERα-siRNA. (B and C) Representative Western blot and densitometry of ERα (B) and HB-EGF (C) normalized with β-actin. NT-siRNA or ERα-siRNA was transfected in rat primary hippocampal neurons, with/without HB-EGF. (D and E) Representative Western blot and densitometry of p-EGFR (D), autophagy regulators (D) and autophagy markers (E) normalized with respective non-phospho counterparts (D) and β-actin (E). ***P < 0.001, **P < 0.01 and *P < 0.05 compared to NT-siRNA or as indicated. A full colour version of this figure is available athttps://doi.org/10.1530/JOE-19-0197 .

PARP/PARP levels following HB-EGF treatment or showing increased activation of AMPKα, possibly autophagy inhibition using Beclin-siRNA (of note, suggesting ATP deficit in OVX hippocampus, we the efficacy of Beclin-siRNA was verified (Fig. 6E)) in examined mitophagy, the process of excessive targeting of ERα-siRNA-treated primary hippocampal neurons (Fig. 6F). mitochondria by selective autophagy, earlier reported to induce neuronal apoptosis under different conditions (Ha et al. 2017, Feng et al. 2018). To test this, we assessed the OVX-mediated apoptosis: possible involvement of VDAC1 and COXIV protein levels in the hippocampus, and excessive mitophagy observed their reduction in OVX group compared to sham, and a 3-MA (autophagy inhibitor)-mediated recovery (Fig. We investigated the basis of hippocampal autophagy- 7A). Thus, our results indicate an autophagy-dependent mediated apoptosis in OVX rats. Based on our results mitochondrial loss in the hippocampus of OVX rats.

Figure 5 OVX reduces HB-EGF and induces autophagy in the neurons of hippocampus, which are suppressed by HB-EGF or E2 treatment. Hippocampal sections were made from Sham, OVX, OVX+HB-EGF- or OVX+E2-treated rats and immunofluorescence performed. (A, B and C) Representative fluorescence photomicrographs (20×) and quantification relative to sham of HB-EGF (A), LC3 (B) and Beclin-1 (C) co-immunolabeled with NeuN and counter- stained with nuclear DAPI. Scale bar: 100 μm. Data represent means ± s.e. of three rats/group. ***P < 0.001 and **P < 0.01 compared to sham or as indicated. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0197.

We related the OVX-induced mitochondrial loss with in the OVX rat hippocampus. Hence, mitophagy mediators, HB-EGF and E2 and observed a recovery in VDAC1 and other than PINK/Parkin, Bnip3 and Fundc1 further need COXIV protein levels (Fig. 7B) and mitochondrial CytB and to be investigated for understanding mechanisms of COXII genes (Fig. 7C) following HB-EGF and E2 treatment. OVX-induced hippocampal mitochondrial loss. We further observed an OVX-mediated reduction in VDAC1 expression in NeuN-expressing cells of the hippocampus, Effect of ERα-silencing on mitophagy and ATP and its E2, HB-EGF and 3-MA-mediated recovery (Fig. 7D). production in primary hippocampal neurons We next investigated mitophagic mediators, PINK1/Parkin, Bnip3 and Fundc1 (Amadoro et al. 2014, We further explored mitophagy in ERα-silenced primary Cummins & Gotz 2018, Lampert et al. 2019) at protein hippocampal neurons, using a tandem-tagged mtRFP- or mRNA levels in the hippocampus of OVX rats. We did EGFP chimeric plasmid that relies on different stabilities not find significant changes in mitochondrial PINK1 and of RFP and GFP in an acidic environment (Kim et al. Parkin levels in the hippocampus during OVX-mediated 2013, Sinha et al. 2015). We observed that in NT-siRNA- mitophagy from that observed under basal condition transfected cells, both EGFP and RFP signals co-localized (Sham), despite significant enrichment of LC3-II in the (yellow: red + green, indicating mitochondrial presence mitochondrial fraction (that indicates mitophagy) and its in cytosol), whereas the knockdown of ERα increased E2-mediated reduction (Fig. 7E). Similarly, we also observed RFP signal alone, indicating autolysosomal-resident an unaltered expression of Bnip3 and Fundc1 (Fig. 7F), mitochondria where EGFP signal was quenched at an known to participate in mitophagy (Liu et al. 2014), acidic pH, suggesting mitophagy (Fig. 8A). We further

Journal of R Pandey et al. OVX-induced neuronal 244:1 63 Endocrinology autophagy and memory loss

Figure 6 OVX and ERα-silencing induces hippocampal neuronal apoptosis via reduced HB-EGF and increased autophagy. Western blotting and IHC were performed on hippocampal tissues and sections of Sham, OVX, OVX+3-MA, OVX+HB-EGF− or OVX+E2-treated rats. (A and D) Representative Western blot and densitometry of LC3-II (A) and NeuN (D) normalized with β-actin. (B and C) Representative fluorescence photomicrographs (20× magnification) and quantification relative to sham of cleaved caspase-3 (c-Cas 3) and NeuN co-immunostaining (B), TUNEL and NeuN antibody co-staining (C), with nuclear DAPI counter-staining (B and C) in the rat hippocampal sections. Scale bar: 100 μm. Data represent means ± s.e. of three rats/group. ***P < 0.001 and **P < 0.01 compared to sham or as indicated. NT-siRNA or ERα-siRNA was transfected in rat primary hippocampal neurons, with/without HB-EGF or co-transfected with Beclin-siRNA. (E and F) Representative Western blot and densitometry of Beclin-1 (E), cleaved caspase-3 (c-Cas 3) and cleaved PARP/PARP (F) normalized with β-actin. Data represent means ± s.e. of three independent primary neuron culture preparations. ***P < 0.001 and **P < 0.01 compared to NT-siRNA or as indicated. A full colour version of this figure is available athttps://doi.org/10.1530/JOE-19-0197 . verified the reduction in mitochondrial content in ERα- production, using Sea-horse Bioscience XFe-24 analyzer silenced primary hippocampal neurons using MitoTracker (Fig. 8C). Red dye (Fig. 8B). An HB-EGF-mediated reduction in mitophagy and recovery in mitochondrial content in the Effect of HB-EGF, 3-MA and E2 on OVX-induced primary hippocampal neurons (Fig. 8A and B) suggested cognitive impairment a protective role of the growth factor against autophagy- induced mitochondrial loss in ERα-suppressed condition. We finally linked our observations of reduced Furthermore, we also observed an HB-EGF-mediated hippocampal neuronal HB-EGF and increased autophagy rescue from excessive ERα-siRNA-induced loss in ATP with learning-memory performances in OVX rats.

Figure 7 OVX induces mitochondrial loss in hippocampal neurons, which is restored by 3-MA, HB-EGF or E2 treatments. Hippocampal tissues were isolated for Western blotting and mitochondrial DNA content measurement, and hippocampal sections were made from Sham, OVX, OVX+3-MA, OVX+HB-EGF− or OVX+E2-treated rats. (A, B and E) Representative Western blots and densitometry of mitochondrial markers normalized with β-actin (A and B) and mitochondrial PINK1, Parkin and LC3-II levels normalized with VDAC1 (E). (C and F) qPCR analysis shows relative mtDNA (CytB and COXII) normalized with nuclear DNA (18S rRNA gene) (C) and Fundc1 and Bnip3 normalized with Gapdh (F). (D) Representative fluorescence photomicrographs (20×) and quantification relative to sham of VDAC1 co-immunolabeled with NeuN and counter-stained with nuclear DAPI. Scale bar: 100 μm. Data represent means ± s.e. of three rats/group. ***P < 0.001 and **P < 0.01 compared to sham or as indicated. A full colour version of this figure is available athttps:// doi.org/10.1530/JOE-19-0197.

We found that while OVX caused a reduction in TLT in Uzum et al. 2016, Yazgan & Naziroglu 2017), and passive avoidance test (Fig. 9A) and increased error (%) the present study establishes the contribution of a and decreased saving memory (%) in Y-Maze test (Fig. controlled autophagy mechanism in estrogen-mediated 9B), HB-EGF and 3-MA could inhibit these changes. The neuroprotection. Here, we essentially demonstrated effects of HB-EGF and 3-MA were comparable to that of that estrogen deficiency up-regulates the expression OVX+E2 (Fig. 9A and B), overall indicating an attenuated of autophagy proteins such as Beclin1, LC3 and ATG7 HB-EGF/EGFR and increased autophagy-dependent along with the levels of ATG5/ATG12 conjugate within leaning-memory dysfunction in OVX rats. the hippocampus, indicative of an estrogen-regulated phagophore elongation and association with the autophagic vesicles. We then explored the regulatory Discussion pathway of autophagy, and our results pointed to the nodal role of ULK complex in hippocampal neuronal The current study reveals a novel mechanism of survival, where estrogen-mediated responses extended to hippocampal neuronal damage and cognitive the autophagy machinery via a coordinated balance with dysfunction in adult females caused by ovariectomy. AKT/mTOR. Consistent with our observations, earlier Through the study, we propose a mechanism of OVX- reports showed an estrogen deficiency-induced autophagy induced ERα deficiency and HB-EGF/EGFR inactivation, in fibroblasts, osteocyte-like cell line, differentiating triggering a reduced p-AKT/mTOR-mediated increased osteoclasts and blastocysts in the uterus, where 17β-estradiol autophagy and mitochondrial loss, ultimately leading to treatment or inhibition of autophagy reversed skeletal, hippocampal neuronal apoptosis and learning-memory bone and uterine damage and reduced obesity via LC3-II impairment (Fig. 10). and mTOR-related signaling cascades (Choi et al. 2014, Lin Estrogen prevents aberrant hippocampal neuronal et al. 2016, Leu et al. 2017, Fu et al. 2018, Tan et al. 2018). apoptosis and cognitive deficits (Sales et al. 2010, Conversely, increased autophagy helped maintaining the

Journal of R Pandey et al. OVX-induced neuronal 244:1 65 Endocrinology autophagy and memory loss

Figure 8 ERα depletion induces mitophagy, mitochondrial loss and reduced ATP production in primary hippocampal neurons, which are reduced by HB-EGF treatment. NT-siRNA or ERα-siRNA was transfected in rat primary hippocampal neurons, with/without HB-EGF. (A) Representative fluorescence photomicrograph (60×) and quantitative analysis of cells transfected with tandem-tagged mt-RFP-EGFP plasmid. Fluorescence signals indicate the expression of mt-RFP-EGFP targeting mitochondria: yellow color, no mitophagy or cytosolic mitochondria; red color, mitophagy or mitochondria inside lysosomes. Scale bar: 20 μm. Transfected cells from three-four random fields were taken for analysis. (B) Representative fluorescence photomicrograph (60×) and quantification of Mitotracker and nuclear DAPI co-staining. Enlarged: Inset area of merged image. Scale bar: 20 μm. Cells from three-four random fields were taken for analysis. (C) Sea-horse analysis of ATP production (in fold change). Data represent means ± s.e. of three independent primary neuron culture preparations. ***P < 0.001, **P < 0.01 and *P < 0.05 compared to NT-siRNA or as indicated. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0197. normal functions of alveolar bone osteocytes (Florencio- mixture (Yao et al. 2018), and the other claiming Silva et al. 2018), osteoblasts derived from elderly females increased hippocampal autophagy following OVX (Fang (Camuzard et al. 2016), aged female heart (Garvin et al. et al. 2018), marked by altered p62, Beclin-1, ATG5 and 2017) and bone marrow mesenchymal stem cells (Qi et al. LC3-II protein levels. However, unlike ours, the studies 2017) following OVX. Likewise, autophagy contributed to did not delve into the autophagy regulation mechanism the estradiol-mediated protective effect on endotoxemia- particularly in the hippocampal neurons. The current induced multiple organ dysfunction (Chung et al. 2017), study provided a detailed understanding on ovariectomy- overall indicating a cell type specific effect of estrogen induced autophagy using suitable modulators, and also deficiency on autophagy. In terms of the brain, there offered a growth factor regulated mechanism supporting have been two in vivo reports, one demonstrating a short- the concept. Our study further extended the observations and long-term ovarian hormone-dependent differential in terms of mitophagy, hippocampal neuronal apoptosis response on autophagy in the cortex-hippocampus and neuronal loss, culminating in cognitive decline.

Figure 9 Reduced HB-EGF and increased autophagy induce cognitive deficits in Figure 10 OVX rats. Passive avoidance and Y-Maze tests for learning-memory Schematic proposing deregulated HB-EGF/EGFR-dependent autophagy, performances were carried out with Sham, OVX, OVX+HB-EGF , − mitochondrial loss, hippocampal neuronal apoptosis and cognitive OVX+3-MA− or OVX+ E2-treated rats. (A) Representative bar graphs deficits in E2/ERα deficiency. E2 deficiency attenuates αER and then depicting transfer latency time (TLT) in passive avoidance test. Data suppresses HB-EGF/-dependent EGFR activation in hippocampal neurons. represent means ± s.e. of eight rats/group. ###P < 0.001 compared to Reduced HB-EGF/EGFR signaling further down-regulates AKT/mTOR/ULK1 acquisition trial. ***P < 0.001 and $$P < 0.01 compared to Sham and OVX pathway of autophagy regulation, resulting in increased LC3/Beclin and for a particular retention trial respectively. (B) Representative bar graphs decreased p62-mediated autophagy, autophagosome formation and depicting % number of errors and % saving memory assessed at 24, 48 h mitochondrial loss. This deregulated HB-EGF/EGFR and autophagy and 7 days post-learning in Y-maze test. Data represent means ± s.e. of pathway forms a key reason for hippocampal neuronal apoptosis and seven rats/group. ***P < 0.001 and **P < 0.01 compared to sham; learning-memory impairment in OVX rats, which may be inhibited by E2 $$P < 0.01 and $P < 0.05 compared to OVX. or HB-EGF supplementation. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0197. Our data for the first time add HB-EGF/EGFR to the list of growth factor signaling mechanisms, viz. nerve growth Cross-talk between E2 and EGFR has been mainly factor/tropomyosin receptor kinase A (TrkA) (Sarvari reported in triple-negative breast cancer, and is et al. 2017, Liu et al. 2018), brain-derived neurotrophic characterized by dysregulated PI3K/AKT/mTOR signaling factor/TrkB (Murphy et al. 1998) and insulin-like growth (Araki & Miyoshi 2018). Irrespective of estrogen factor (Witty et al. 2013), controlling estrogen-induced treatment, an EGFR, PI3K/AKT/mTOR and autophagy responses to hippocampal neurons. Additionally, link is also known, primarily in cancerous cell, such as, although an altered EGFR signaling is well-reported in hepatic cancer, glioblastoma, and so forth (Palumbo et al. relation to autophagy for various cell types and conditions 2014, Dai et al. 2018). Our data reflect a combination of (Graham et al. 2016, Wang et al. 2017), our study all these pathways, demonstrating the regulatory role of emerged first in linking HB-EGF ligand with autophagy, hippocampal neuronal AKT/mTOR at the cross-road of thereby, opening a new direction to HB-EGF-induced E2, EGFR and downstream autophagy, rarely explored in signaling mechanisms within the hippocampus. Hence, the non-malignant cells. Interestingly, our study draws for both E2-dependent and -independent conditions, support from an earlier concept (Shafi 2016) hypothesizing mitochondrial dynamics presumably regulates the typical an inverse relation between cancer and Alzheimer’s functions of HB-EGF, viz., survival, mitogenesis, anti- Disease-induced hippocampal neurodegeneration, excitotoxicity, and so forth, particularly in hippocampal where the former generally involves an upregulated neurons. Likewise, for amyloid beta toxicity, aging, EGFR-PI3K/AKT/mTOR signaling and reduced autophagy learning memory impairments (Shariatpanahi et al. 2016, as opposed to the latter. However, analyzing our Huang et al. 2018), and so forth marked by hippocampal results indicate that the reduction in HB-EGF/EGFR autophagy, an intermediate involvement of HB-EGF seems activation, following estrogen deficiency, suppresses worth exploring. the membrane recruitment of cytoplasmic PI3K and

Journal of R Pandey et al. OVX-induced neuronal 244:1 67 Endocrinology autophagy and memory loss subsequent phoshatidylinositol-3,4,5-trisphosphate and Mitochondrial activity is necessary for both neuronal AKT-dependent mTOR activation. Thus, taken together, function and its survival wherein, mitochondrial pruning although estrogen receptor itself appears capable of for removing damaged mitochondria is essential to sustain activating PI3K (Toss & Cristofanilli 2015), our findings mitochondrial health in cells, including neurons (Sinha reveal the essential involvement of an activated EGFR or et al. 2015). This selective degradation of malfunctioning rather cross-talk between EGFR and estrogen receptor for mitochondria is executed by mitophagy, defined as an recruiting PI3K/AKT/mTOR and sustaining mitochondrial autophagy-dependent mitochondrial degradation (Sinha homeostasis and an ultimate hippocampal neuronal et al. 2015). Intriguingly, mitophagy, like autophagy, is not survival. Nonetheless, detailed future studies are necessary always protective, as increased mitophagy may also lead to understand the interactions of estrogen receptor and to increased neuronal loss (Chakrabarti et al. 2009, Wong HB-EGF/EGFR signaling toward regulating the complex & Cuervo 2010, Shi et al. 2014, Ha et al. 2017, Feng et al. association between autophagy and mitochondria. Our 2018). In line with these reports, our results also support study further addressed in details the relationship between the notion that increased mitochondrial loss under E2/ERα estrogen deficiency, autophagy and neuronal apoptosis deficiency may result in energy crisis, leading to enhanced within the hippocampus through in vivo experiments, apoptosis. However, since mitophagy involves membrane and then validated the process in ERα-deficient cultured depolarization and different mitophagy mediators hippocampal neurons. Secondly, through in vivo and (Grenier et al. 2013), the participation of these mitophagy in vitro methods using appropriate autophagy inhibitors, pathways in E2 deficiency-induced hippocampal our results demonstrated a direct autophagy-dependent neuronal apoptosis and cognitive impairment still need apoptosis within hippocampal neurons during estrogen to be explored. Hence, studies are currently underway or ERα-deficiency, and further associated this process toward a detailed understanding of the mechanistic basis with cognitive deficits. All these events were blocked of OVX-induced mitophagy, and especially the role of by E2, thereby defining novel mechanisms by which mitophagy proteins during mitochondrial clearance in this hormone affords protection against hippocampal the hippocampal neurons. Particularly, although OVX neurodegeneration. Moreover, an ERα antagonist or failed to impact PINK/Parkin (mitochondrial), BniP3 and EGFR inhibitor-mediated increase in LC3-II and Beclin-1 Fundc1 expression levels in hippocampus, the likelihood expression levels in the primary hippocampal neurons of their functional involvement in ERα-deficient (data not shown) supported that E2 deficiency-mediated hippocampal neurons also appears worth probing. ERα reduction and EGFR inactivation induce autophagy In conclusion, the current study showed that E2 in the hippocampal neurons. governs HB-EGF/EGFR-dependent autophagy and Although autophagy is generally neuroprotective, mitochondrial integrity in hippocampal neurons, and been reported for memory formation (Glatigny et al. 2019) this event may be disrupted in menopause triggering and as a survival mechanism in dementia (Hu et al. 2017, hippocampal neurodegeneration. Overall, our study Lee et al. 2017) its overactivation in neurons has also been reveals intersection points of HB-EGF, autophagy, known to induce neurodegeneration. In hypoxic ischemic mitochondrial loss and apoptosis as key players in brain injury, autophagy gene-deletion significantly E2-mediated hippocampal neuroprotection, which may prevented severe hippocampal damage in neonatal or be pharmacologically targeted for restricting cognitive adult mice (Koike et al. 2008). Similarly, beneficial effects dysfunction, particularly in elderly women. of autophagic inhibition against neuronal cell death have also been demonstrated in a 6-hydroxydopamine model Declaration of interest of substantia nigral injury, which exhibits increased The authors declare that there is no conflict of interest that could be autophagy (Li et al. 2011). Additionally, neuronal death perceived as prejudicing the impartiality of the research reported. elicited by dysfunctional ESCRT-III, which is associated with frontotemporal dementia linked to chromosome Funding 3, could be rescued by both pharmacological and This work was supported by Science and Engineering Research Board, genetic inhibition of autophagy, suggesting a pro-death Govt. of India [GAP340]. function of neuronal autophagy (Lee & Gao 2009). Interestingly, our results suggest that increased mitophagy Author contribution statement could be the mechanism how estrogen deficiency- R P contributed to the experimental plan, design, performance (animal induced autophagy leads to neuronal apoptosis and loss. treatment, majority Western blotting, IHC, ATP assay and neurobehavioral

assay), data analysis and paper writing. P S contributed to the animal carcinoma activity by suppressing EGFR and PI3K/Akt/mTOR treatment and Western blotting. B A contributed to Western blotting of all signaling. Oncology Reports 40 3235–3248. (https://doi.org/10.3892/ ATG5/12 and Fig. 7B and E. H P G contributed to qPCR, statistical analyses or.2018.6754) and data analyses. S P contributed to mitochondrial DNA content and Dixit A, Srivastava G, Verma D, Mishra M, Singh PK, Prakash O & ATP-based experiment. N A carried out TEM. K G contributed to animal Singh MP 2013 Minocycline, levodopa and MnTMPyP induced treatment and neurobehavioral assay. N C contributed to experimental changes in the mitochondrial proteome profile of MPTP and designing and supervision. R A S contributed to the planning, data maneb and paraquat mice models of Parkinson’s disease. Biochimica analyses and paper writing for all mitochondria-associated experiments. and Biophysica Acta 1832 1227–1240. (https://doi.org/10.1016/j. S B contributed to the overall experimental planning, designing, supervision bbadis.2013.03.019) and paper writing. Djiogue S, Djiyou Djeuda AB, Seke Etet PF, Ketcha Wanda GJM, Djikem Tadah RN & Njamen D 2018 Memory and exploratory behavior impairment in ovariectomized Wistar rats. Behavioral and Brain Functions 14 14. (https://doi.org/10.1186/s12993-018-0146-7) Fang YY, Zeng P, Qu N, Ning LN, Chu J, Zhang T, Zhou XW & Tian Q Acknowledgement 2018 Evidence of altered depression and dementia-related proteins in R P was supported by UGC fellowship, Govt. of India. Juhi Mishra (SRF, CSIR- the brains of young rats after ovariectomy. Journal of Neurochemistry IITR) and Rafat Malik (P. A., SERB) provided suggestions following thorough 146 703–721. (https://doi.org/10.1111/jnc.14537) reading of the manuscript. CSIR-IITR manuscript number is 3573. 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Received in final form 5 September 2019 Accepted 19 September 2019