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The retromer complex and associated sorting nexins (SNXs) comprise a critical trafficking machinery which mediates endosomal protein sorting. Retromer and/or SNX dysfunction has been linked to several neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Down’s syndrome (DS). In AD, deficiency of the retromer complex or its cargo proteins impairs endosomal trafficking of amyloid precursor protein (APP), resulting in the overproduction of β-amyloid (Aβ). Several SNX components directly interact with APP or APP-cleaving enzymes (β- and γ-secretases) to regulate amyloidogenic APP processing and Aβ generation. In addition, PD-linked mutations in retromer components cause mistrafficking of retromer cargo proteins and mitochondrial dysfunction, and dysregulation retromer-mediated trafficking has been considered as an important cause of hereditary spastic paraplegia (HSP) and neuronal ceroid lipofuscinoses (NCLs). Moreover, SNX27 deficiency is an important contributor for synaptic and cognitive impairment in DS. Here we review recent findings describing the retromer complex and/or SNXs-mediated endosomal sorting in neurodegenerative disorders.

Abstract STUDY QUESTION Does metformin inhibit excessive androgen-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) in vivo and in vitro? SUMMARY ANSWER Metformin inhibits testosterone-induced ER stress and unfolded protein response (UPR) activation by suppressing p38 MAPK phosphorylation in ovarian GCs. WHAT IS KNOWN ALREADY Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism. Excessive testosterone induces ER stress and UPR activation in human cumulus cells, leading to cell apoptosis. Metformin has potential inhibitory effects on ER stress and UPR activation, as demonstrated in human pancreatic beta cells and obese mice. STUDY DESIGN, SIZE, DURATION Cumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our IVF centre. A dihydrotestosterone (DHT)-induced PCOS mouse model was constructed and treated with or without metformin. Primary mouse GCs and cumulus-oocyte complexes (COCs) were cultured with testosterone, metformin, a p38 MAPK inhibitor, or p38 MAPK small interfering RNA. PARTICIPANTS/MATERIALS, SETTING, METHODS The levels of UPR sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients by real-time quantitative PCR (qPCR) and western blot. The ovaries, oocytes, GCs and COCs were collected from PCOS mice treated with metformin and controls. The expressions of ER stress markers and p38 MAPK phosphorylation were assessed by qPCR, western blot and immunofluorescence. A subsequent in vitro analysis with primary cultured GCs and COCs was used to confirm the influence of metformin on ER stress activation by qPCR and western blot. Finally, the effects of ER stress activation on GCs and COCs in relation to LH responsiveness were examined by qPCR and COC expansion. MAIN RESULTS AND THE ROLE OF CHANCE The expression of the ER stress markers GRP78, CHOP and XBP1s in the cumulus cells was higher in PCOS patients than in control patients, as were the levels of the UPR sensor proteins p-IRE1α, p-EIF2α and GRP78. Compared to those of control mice, the ovaries, GCs and COCs of DHT-treated PCOS mice showed increased levels of ER stress marker genes and proteins. Hyperandrogenism in PCOS mouse ovaries also induced p38 MAPK phosphorylation in COCs and GCs. Metformin inhibited ER stress activation was associated with decreased p-p38 MAPK levels. In vitro experiments, testosterone-induced ER stress was mitigated by metformin or p38 MAPK inhibition in primary cultured GCs and COCs. COCs expanded rapidly in the presence of testosterone during LH administration, and ovulation-related genes, namely, Areg, Ereg, Ptgs2, Sult1e1, Ptx3 and Tnfaip6, were strongly expressed in the COCs and GCs. These effects were reversed by treatment with metformin, an ER stress inhibitor or by knockdown of p38 MAPK. LIMITATIONS, REASONS FOR CAUTION The number of PCOS patients in this study was small. WIDER IMPLICATIONS OF THE FINDINGS This study provides further evidence for metformin as a PCOS treatment. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800), the Key Research and Development Program of Zhejiang Province (2017C03022), the Zhejiang Province Medical Science and Technology Plan Project (2017KY085, 2018KY457), the National Natural Science Foundation of China (31701260, 81401264, 81701514), and the Special Funds for Clinical Medical Research of the Chinese Medical Association (16020320648). The authors report no conflict of interest in this work and have nothing to disclose. TRIAL REGISTRATION NUMBER N/A.

Autophagy can be dynamically induced in response to stresses and is an essential, ubiquitous intracellular recycling system that impacts the fate of damaged resident cells, thereby influencing wound healing. Endometrial fibrosis is a form of abnormal wound healing that causes intrauterine adhesion (IUA) and infertility. We previously demonstrated that overactivated sonic hedgehog (SHH) signaling exacerbated endometrial fibrosis, but the role of autophagy in this process is still unknown. Here, we report that impaired autophagy participates in SHH pathway-induced endometrial fibrosis. Endometrial stroma-myofibroblast transition accompanied by autophagy dysfunction was present in both endometrial biopsies of IUA patients and Amhr2cre/+R26-SmoM2+/− (AM2) transgenic mouse. Mechanistically, SHH pathway negatively regulated autophagy through pAKT-mTORC1 in a human endometrial stromal cell line (T-HESCs). Furthermore, SHH pathway-mediated fibrosis was partly counteracted by autophagy modulation in both T-HESCs and the murine IUA model. Specifically, the impact of SHH pathway inhibition (GANT61) was reversed by the pharmacological autophagy inhibitor chloroquine (CQ) or RNA interference of autophagy-related gene ATG5 or ATG7. Similar results were obtained from the murine IUA model treated with GANT61 and CQ. Moreover, promoting autophagy with rapamycin reduced fibrosis in the AM2 IUA model to baseline levels. In summary, defective autophagy is involved in SHH pathway-driven endometrial fibrosis, suggesting a potential novel molecular target for IUA treatment.

Early embryonic arrest and fragmentation (EEAF) is a common phenomenon leading to female infertility, but the genetic determinants remain largely unknown. The Moloney sarcoma oncogene (MOS) encodes a serine/threonine kinase that activates the ERK signaling cascade during oocyte maturation in vertebrates. Here, we identified four rare variants of MOS in three infertile female individuals with EEAF that followed a recessive inheritance pattern. These MOS variants encoded proteins that resulted in decreased phosphorylated ERK1/2 level in cells and oocytes, and displayed attenuated rescuing effects on cortical F‐actin assembly. Using oocyte‐specific Erk1/2 knockout mice, we verified that MOS‐ERK signal pathway inactivation in oocytes caused EEAF as human. The RNA sequencing data revealed that maternal mRNA clearance was disrupted in human mature oocytes either with MOS homozygous variant or with U0126 treatment, especially genes relative to mitochondrial function. Mitochondrial dysfunction was observed in oocytes with ERK1/2 deficiency or inactivation. In conclusion, this study not only uncovers biallelic MOS variants causes EEAF but also demonstrates that MOS‐ERK signaling pathway drives human oocyte cytoplasmic maturation to prevent EEAF.

In mammalian ovaries, primordial follicles remain in a quiescent state until activation by the surrounding microenvironment. Ovarian intervention, for example, ovarian cystectomy, ovarian wedge resection or laser drilling therapies for polycystic ovarian syndrome, has long been reported to change follicular development by an unknown mechanism(s). Herein, we established a murine model with partial ovarian resection of one ovary unilaterally, with the contralateral ovary undamaged. We found the injury accelerated follicular activation and development through the mTORC1 signaling pathway. Moreover, the stimulation of primordial follicles was restricted near the incision site where the mTORC1 pathway showed sequential activation beginning at the interstitial cells and proceeding to the primordial follicles. Total and polysome-associated RNA-seq revealed the increase of the nerve growth factor (NGF) family member, in both two fractions and immunostaining showed the restricted induction of NGF near the incision site. In cultured newborn ovaries, NGF demonstrated increase of follicular activation, and moreover, the NGF inhibitor K252a effectively blocked activation of primordial follicles stimulated by the surgery. We liken ovulation in mammals to minor tissue trauma, which happens naturally and cyclically in the body. As the increase in NGF accompanied the accumulation of activated primordial follicles after ovulation, our study may represent a common mechanism for selective follicular activation induced by a localized increase in NGF in interstitial cells and mediated via the mTOR signaling pathway. In addition, the NGF inhibitor K252a and the mTOR inhibitor rapamycin constitute good candidates for protecting follicular reserve against over exhaustion after ovarian surgery.

miR-129-5p is implicated in many diseases, such as laryngeal cancer and breast cancer. In this study, we studied the mechanism underlying the role of BMP2 in intervertebral disc degeneration (IDD). We used a luciferase assay system to determine the relationship between BMP2 and miR-129-5 expression. In addition, Western blot and real-time PCR were used to confirm the regulatory relationship between miR-129-5p and its targets, while flow cytometry was used to evaluate the effect of miR-129-5p on the apoptosis of neural progenitor cells (NPCs). BMP2 was confirmed as a direct target of miR-129-5p. Furthermore, the expression of miR-129 was downregulated along with upregulated BMP2 expression in IDD patients. Meanwhile, BMP2 was validated as the target of miR-129-5p in cells transfected with miR-129-5p and BMP2 siRNA. Also, compared with NPCs transfected with blank/scramble controls or miR-129-5p inhibitors, the NPCs treated with miR-129-5p mimics or BMP2 siRNA exhibited evidently elevated viability and inhibited apoptosis. The data demonstrated that miR-129-5p was poorly expressed in IDD patients, and the dysregulation of miR-129-5p might contribute to the development of IDD by targeting BMP2 expression. K E Y W O R D Sapoptosis, BMP2, intervertebral disc degeneration, microRNA-129-5p, nucleus pulposus cell

Cyclophosphamide (CTX) is widely used in various cancer therapies and in immunosuppression, and patients can still have babies after CTX chemotherapy. CTX directly causes primordial follicle loss with overactivation and DNA damage-induced apoptosis. Previous studies have shown that maternal exposure to CTX before conception increases the incidence of birth abnormalities and alters the methylation of genes in the oocytes of offspring. Mice were treated with a single dose of CTX (100 mg/kg) at post-natal day 21 and sacrificed 47 days later when primordial follicles surviving chemotherapy developed to the antral stage. Acute DNA damage and acceleration of the activation of primordial follicles after CTX treatment were repaired within several days, but the remaining follicle numbers remarkably decrease. Although partial surviving primordial follicle were developed to mature oocyte, oocyte quality hemostasis was impaired exhibiting aberrant meiosis progression, abnormal spindle and aneuploidy, mitochondrial dysfunction and increased endoplasmic reticulum stress. Thereafter, embryo development competency significantly decreased with fewer blastocyst formation after CTX exposure. CTX treatment resulted in alteration of DNA methylations and histone modifications in fully grown GV oocytes. Single-cell RNA-seq revealed CTX treatment suppressed multiple maternal genes’ transcription including many methyltransferases and maternal factor YAP1, which probably accounts for low quality of CTX-repaired oocyte. In vitro addition of lysophosphatidic acid (LPA) to embryo culture media to promote YAP1 nuclear localization improved CTX-repaired embryo developmental competence. This study provides evidence for the consistent toxic effect of CTX exposure during follicle development, and provide a new mechanism and new insights into future clinical interventions for fertility preservation.