Hair follicle stem cells (HFSCs) utilize glycolytic metabolism during their activation and anagen induction. However, the role of pyruvate kinase M2 (PKM2), which catalyzes the final step of glycolysis, in hair regeneration has not been elucidated. In this study, we investigated the expression pattern and activity of PKM2 during the depilation-induced anagen progression in mice. We found that TEPP-46, a selective activator of PKM2, enhanced hair re-growth and proliferation of HFSCs. PKM2 expression was increased via up-regulation of Wnt/β-catenin signaling, which is involved in hair regeneration. Moreover, a combined treatment with KY19382, a small molecule that activates Wnt/β-catenin signaling, and TEPP-46 significantly enhanced hair re-growth and wound-induced hair follicle neogenesis (WIHN). These results indicate that simultaneous activation of the PKM2 and Wnt/β-catenin signaling could be a potential strategy for treating alopecia patients. A hair follicle is a mini-organ that regenerates itself through the hair cycle comprising three phases: growth phase (anagen), regression phase (catagen), and resting phase (telogen) . Among vThe pyruvate kinase (PK) is an enzyme that transfers phosphate from phosphoenolpyruvate to ADP and produces pyruvate and ATP in the last step of glycolysis . PK actsThe Wnt/β-catenin signaling pathway is a major pathway that regulates the proliferation and differentiation in many tissues ,16,17. IThe major available hair loss treatments, which have been approved by the FDA, are minoxidil and finasteride . These dIn this study, we identified that PKM2 plays a role in the activation of HFSCs and hair growth promotion. Pharmacological control of PKM2 activity modulated the proliferation of HFSCs and anagen induction. The expression level and localization of PKM2 correlated with those of β-catenin in the bulge region, where the stem cells reside in hair follicles. Moreover, simultaneous treatment with TEPP-46, a PKM2 activator , and KY1All animal experiments had approval from the Institutional Animal Care and Use Committee of Yonsei University . Animal studies were designated according to the ARRIVE guidelines .2 gas.Mice were housed in a computerized environmental managing system of micro-ventilation cages . Mice had free approach to food and water. Room temperature was managed at 24 °C, with a relative humidity of 40–70% and 12 light/12 dark cycles. The mice were raised with a normal diet and bedding from Central Lab Animal Inc. . Mice were housed with no more than 5 animals per cage. The mice were randomly separated into each group. After the experiments were completed, the mice were euthanized using COConsidering the 3Rs  of animal use, we first performed preliminary experiments to determine the effects of TEPP-46 and shikonin on hair growth through ex vivo vibrissa culture, and then we performed minimal animal experiments under optimal conditions.2 gas.Eight-week-old male C57BL/6N mice  were anesthetized with 2,2,2-tribromoethanol  through 400 mg/kg IP injections. The mouse backs were plucked to induce a synchronized hair cycle . To moniTissues were fixed overnight in 10% formalin, and paraffin was embedded. The paraffin-embedded skin tissues of 4 μm sections were attached to the slides. The slides were deparaffinized in three changes of xylene and rehydrated through a graded ethanol series. The sections were stained with hematoxylin for 5 min, followed by eosin for 1 min. The H&E-stained slides were photographed using a stereomicroscope . The number of follicles was quantified by counting the hair follicles in H&E staining images. Dermal thickness was calculated using Image J software V1.48.The slides with skin tissues attached were deparaffinized and rehydrated. Antigens were retrieved by microwaving them for 15 min in 10 mM sodium citrate buffer. Antigens were blocked with 5% BSA in PBS and placed with the primary antibodies: rabbit anti-PKM2 , rabbit anti-β-catenin  or mouse anti-β-catenin , rabbit anti-Ki67 , mouse anti-keratin 15 , mouse anti-PCNA , and rabbit anti-cytokeratin 17  or rabbit anti-fibroblast growth factor (fgf) 9 . After washing with PBS, antigens were placed with secondary antibodies: anti-mouse Alexa Fluor 488  or anti-rabbit Alexa Fluor 555 . After washing with PBS, slides were counterstained with 4′,6-diamidino-2-phenylindole . Fluorescent images were taken using a confocal microscope .PKM2, forward 5′-TGG CGC CCA TTA CCA GCG ACC-3′ and reverse 5′-TCC CTT CTT GAA GAA GCC TCG GGC CTT-3′ and GAPDH, forward 5′-ACC CAG AAG ACT GTG GAT GG-3′ and reverse 5′-GGA TGC AGG GAT GAT GTT CT-3′.The total RNA was separated using Trizol reagent  according to the manufacturer’s descriptions. RNA (2 μg) was reverse transcribed using reverse transcriptases (Invitrogen) at 42 °C for 1 h. For real-time PCR, the resulting cDNA (1 μL) was amplified in a reaction mixture including 10 pmol of the primer set  and iQ SYBR Green Supermix . The primer sequences were: g at 4°C for 30 min. Equal amounts of protein (1 μg) were incubated with 100 mM KCl , 50 mM Tris  pH 7.5, 5 mM, 0.6 mM ADP, 0.5 mM phosphoenolpyruvate (Sigma-Aldrich), 10 μM fructose-1,6-bisphosphate (Sigma-Aldrich), 240 μM NADH (Sigma-Aldrich), and 8 units of lactate dehydrogenase (Sigma-Aldrich). The absorbance change of NADH’s oxidation was measured at 320 nm using a FLUOstar OPTIMA luminometer .Pyruvate kinase activity assay was previously described ,31. The v/v) water, 20% propylene glycol , and 50% ethanol ; KY19382 was dissolved in polyethylene glycol 400 (PEG 400) (Sigma-Aldrich). To evaluate re-grown hair, re-grown hairs were collected using a clipper and were measured using a precision balance. After the experiments were completed, the mice were euthanized using CO2 gas.Six-week-old male C57BL/6N mice were adapted to their new environment for 1 week. After 1 week of acclimation, seven-week-old mice, whose hair follicles entered the telogen phase , were an2 full-thickness wounds were made on the backs of the mice under aseptic conditions. Twenty μL of each drug used in the hair regrowth tests was treated to the wound regions daily for up to 14 or 25 days. After the experiments were completed, the mice were euthanized using CO2 gas.Three-week-old male C57BL/6N mice were acclimated to their new environment for 3 days. After anesthesia with 2,2,2-tribromoethanol , 1 cmThe tissues were put into 5 mM EDTA in PBS at 37 °C for 6 h. The dermis layer was isolated under the microscope (Nikon) and incubated in 4% paraformaldehyde for 10 min and NBT/BCIP solution  for 10 min. The ALP staining images were obtained by using a stereomicroscope (Nikon). The number of neogenic follicles was quantified by counting dark blue dots.http://www.ncbi.nlm.nih.gov/geo/ (accessed on 1 December 2021)) using GEO accession number GSE45512.The gene expression profile results were obtained in NCBI’s GEO database (v/v) dimethyl sulfoxide , shikonin, or TEPP-46 for each well. All drugs were dissolved in DMSO. The culture medium was replaced every 2 days, and the hair shaft length of vibrissa follicles was measured at 10 days. The follicle was visualized with a stereomicroscope (Nikon). The length was measured using Image J software V1.48.Mouse vibrissa follicles were extracted from 6-week-old C57BL/6N female mice under a stereomicroscope (Nikon). The separated follicles were cultured with 500 μL DMEM supplemented with 12.5 μg/mL gentamicin  and 1% penicillin/streptomycin (Gibco) within a 24-well plate. The follicles were incubated with 0.1% . Statistical significance is indicated in the figures as follows: * We analyzed the expression profiles of PKM2 to confirm any differences in its expression in alopecia patients, compared to healthy controls (GEO: GSE45512). PKM2 expression levels were decreased in the alopecia patients, compared to the healthy controls . This reTo investigate the effect of TEPP-46, a selective PKM2 activator , on hairTo further characterize the effect of PKM2 activity on hair growth, we tested the effects of shikonin, a selective PKM2 inhibitor . The vibTo determine the correlation between PKM2 expression and Wnt/β-catenin signaling, we analyzed the PKM2 and β-catenin during the depilation-induced anagen progression. In the bulge region, the expression pattern of PKM2 was similar to that of β-catenin and PCNA . QuantitIn a recent study, we noted that PKM2 expression was induced by activating the Wnt/β-catenin signaling in colorectal cancer . To inveTo investigate the combined effect of the activators of PKM2 and Wnt/β-catenin signaling on hair re-growth, TEPP-46 was co-treated with KY19382 on dorsal skins of mice. Hair re-growth was significantly enhanced by combined treatment with TEPP-46 and KY19382, as shown by the measurement of hair weight a,b. MorePKM2 mRNA level, as well as PK activity, were increased at PWDs 17 and 25 (The WIHN assay was used to assess hair regeneration  and was 7 and 25 . OverallTo determine the combined effect of KY19382 and TEPP-46 on WIHN, KY19382 was co-treated with TEPP-46 on mice wounds. To confirm the fully regenerative follicles, we conducted whole-mount ALP staining using mouse wounds at 25 days after drug application. Whole-mount ALP staining showed that TEPP-46 induced WIHN. The combined treatment with KY19382 and TEPP-46 further increased the number of neogenic hair follicles a,b. To oAlopecia is characterized by the shortening of the anagen phase and miniaturization of hair follicles . The numPKM2 plays a crucial role in aerobic glycolysis by catalyzing the last rate-limiting step of glycolysis ,45. The In this study, we investigated the roles of activity and expression levels of PKM2 in hair regeneration. The control of PKM2 activity by the treatment of TEPP-46 or shikonin affects hair growth both ex vivo and in vivo. The role of PKM2 level in hair promotion is especially related to Wnt/β-catenin signaling, as evidenced by the effect of KY19382 in hair growth and the correlation between expression levels of β-catenin and PKM2. The pathophysiological role of PKM2 level in hair growth is supported by reduced PKM2 levels in alopecia patients. Furthermore, we found that combined treatment with Wnt/β-catenin signaling and PKM2 activators further enhanced hair re-growth and WIHN, compared to single treatment. This further hair growth is attributed to the fact that the combined treatment activates and increases PKM2 through Wnt/β-catenin signaling. These data indicate that both the level and activity of PKM2 play a role in hair growth. Moreover, an approach using both PKM2 and Wnt/β-catenin signaling activators could be an effective strategy for hair regeneration.


Aberrant Wnt/β-catenin signaling has been reported to play crucial role in pathogenesis of hepatocellular carcinoma (HCC). In this review, we focus on the regulatory role of Wnt/β-catenin signaling in cancer stemness and metabolic reprogramming, which are two emerging hallmarks of cancer. Understanding the role of Wnt/β-catenin signaling in regulation of the above processes reveals novel therapeutic strategy against this deadly disease. Hepatocellular carcinoma (HCC) is a major cause of cancer death worldwide due to its high rates of tumor recurrence and metastasis. Aberrant Wnt/β-catenin signaling has been shown to play a significant role in HCC development, progression and clinical impact on tumor behavior. Accumulating evidence has revealed the critical involvement of Wnt/β-catenin signaling in driving cancer stemness and metabolic reprogramming, which are regarded as emerging cancer hallmarks. In this review, we summarize the regulatory mechanism of Wnt/β-catenin signaling and its role in HCC. Furthermore, we provide an update on the regulatory roles of Wnt/β-catenin signaling in metabolic reprogramming, cancer stemness and drug resistance in HCC. We also provide an update on preclinical and clinical studies targeting Wnt/β-catenin signaling alone or in combination with current therapies for effective cancer therapy. This review provides insights into the current opportunities and challenges of targeting this signaling pathway in HCC. Liver cancer was the third leading cause of death from cancer worldwide in 2020 according to the World Health Organization, where hepatocellular carcinoma (HCC) is the dominant primary liver cancer, accounting for over 90% of cases . HCC is With the continuously increasing trend of HCC cases, there are several therapeutic strategies ranging from curative therapy and targeted drug therapy to immunotherapy. Liver transplantation, resection or ablation are suggested to be the suitable therapeutic modalities for HCC patients at an early stage without intrahepatic or extrahepatic metastases . There iThe Wnt signaling pathway partakes in various essential biological processes, including embryonic development, maintaining adult stem cells, and regulating proliferation and angiogenesis. It is classified into canonical and noncanonical pathways, where the former is β-catenin dependent, whereas the latter is β-catenin independent . In the The noncanonical signaling pathway is classified into the Wnt-planar cell polarity pathway (PCP) and the Wnt/calcium pathway. Both are also activated by the Wnt ligand attached to the FZD receptor but play different roles in regulating the cytoskeletons and calcium ions within the cells . In the CTNNB1, AXIN, and APC [CTNNB1 is the gene that specifically encodes β-catenin. Mutation of β-catenin is positively related to HCC progression due to its oncogenic role [Wnt/β-catenin signaling is crucial in contributing to HCC pathogenesis, where genetic mutations and epigenetic alterations are primarily revealed . Activat and APC ,17,18. Inic role . To datenic role ,21. In anic role . Previounic role , which inic role ,23. It inic role .Apart from β-catenin, deregulation of the Wnt/β-catenin signaling pathway is also caused by mutations in protein degradation complexes . These mC-Myc and cyclin D, as key Wnt-target genes, not only perform their roles as proto-oncogenes for tumor formation but also regulate liver cancer stem cell (CSC) properties by mediating various signaling pathways involved in cellular differentiation and survival [CTNNB1 mutations in HCV-related HCC rather than HBV-related HCC or nonviral HCC [Axin1 is more often found in HBV-related HCC tumors [Axin1 mutation also plays a role in exerting oncogenic effects manifested by overexpressing glutamine synthase (GS), leading to β-catenin activation that correlates to the non-canonical pathway [In addition, high levels of E-cadherin have been reported to be correlated with the accumulation of β-catenin in both the cytosol and nucleus, that drives the transcription of Wnt target genes . C-Myc asurvival ,20. As psurvival ,30. It iiral HCC . HoweverC tumors . Interes pathway . Several epigenetic dysregulations contribute to Wnt/β-catenin activation in HCC. DNA methylation is crucial in maintaining CSC properties, in which its inhibition can influence the fate of cells and gene expressions . For insFurthermore, several alterations through histone modification have been reported in HCC. Enhancer of zeste homologous 2 (EZH2) is a histone methyltransferase that plays a role in catalyzing methylation of histone H3 to achieve repression of Wnt antagonists, promoting Wnt/β-catenin signaling and hepatocarcinogenesis ,42. HistWNT3A secretion with miR-15a/16/107 binding, resulting in the activation of Wnt/β-catenin and polarizes M2 macrophage [Wnt1, suppressing the level of β-catenin and inhibiting Wnt-target gene transcription [It has been suggested that microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are critical regulators associated with various tumors, in which they are negatively regulated . Dysregucrophage . Similarcrophage . Furthercription ,51. Furtcription . All thecription . In addicription . To sum ZBTB20 has been reported in liver tumorigenesis with its role in suppressing PPARG expression and inhibiting proteasomal degradation of the β-catenin destruction complex [C-Myc is the most critical gene induced by activated Wnt/β-catenin signaling, which enhances the mechanisms of glycolysis and glutaminolysis [Apart from genetic mutations and epigenetic dysregulation, other molecules/pathways were identified to regulate Wnt/β-catenin signaling. In normoric environment, ROS is maintained at a low level; whereas a steady increase of ROS level promotes cancer development and progression . A recen complex . Overall complex . C-Myc iinolysis . This isinolysis ,77. Specinolysis . It is ainolysis . Additioinolysis , as the inolysis . Apart finolysis . Moreoveinolysis . Notablyinolysis .Altered metabolism, one of the hallmarks of cancer, was recently reported to be linked with Wnt signaling and cancer immunology. When compared with normal cells, cancer cells require more energy and glycolytic intermediates, such as glucose-6-phosphate (G6P) and NADPH, and TCA intermediates, including citrate, acetyl-CoA and lactate, for the biosynthesis of macromolecules to sustain cell proliferation . These iHCC cells are different from normal hepatocytes, as they express various metabolic enzymes . For insmTORC1 activation is caused by GS-mediated glutamine synthesis, correlated with upregulated β-catenin signaling and phosphorylation of mTOR-S2448 in human HCC, thus revealing the relationship in the GS/glutamine/p-mTOR-S2448 axis [ALDH3A1 are positively correlated, in which Wnt/β-catenin signaling is consistently upregulated in HCC [c-myc, CCND1 and MMP7, leading to HCC progression [The demand for glutamine, a free amino acid abundantly present intracellularly, is increased in cancer cells in order to satisfy their metabolic needs compared to normal cells. Glutamine is also involved in redox homeostasis mediated by glutamine transporters, mainly the SoLuteCarrier (SLC) families, which are highly expressed in the liver and serve as metabolic gateways to modulate the transport of both nutrients and metabolites, such as glucose, amino acids, vitamins and certain ions ,104. Thi448 axis . Anotherd in HCC . Branched in HCC . Anothergression . Furthergression . Additiogression ,115. SCD1 is associated with the modulation of p53, Wnt/β-catenin and autophagy [MYC has also been shown to be a transcriptional amplifier for certain PPARα target genes, which consequently potentiate to HCC progression [MYC triggers phospholipid conversion and that UFAs are increased, which is beneficial to cancer cells in terms of cell structure maintenance and energy storage [Several components of the fatty acid transportation machinery, such as CD36, are upregulated in malignant tissues compared with normoxic cells and tissues. They are associated with the enhancement of EMT through the activation of WNT/TGF-β signaling . Therefoutophagy ,124,125.gression . DNL is gression . Supportgression . Yao et  storage ,128. Int storage .GLUT1, HK, PKM2 and SLC1A5, [mTORC1 repressed GLS expression by enhancing the translation of c-myc through S6K, which led to an increase in glutaminolysis [Wnt/β-catenin signaling regulates cellular metabolism through modifying the activities of several oncogenic factors. Activated c-myc regulates glycolysis, glutaminolysis and lipid synthesis within the TME via enhancing gene expression of  SLC1A5, . In addi SLC1A5, . Further SLC1A5, . mTORC1 inolysis . Apart finolysis . Anotherinolysis ,135. Insulin receptor substrates (IRS) were induced by WNT3a stimulation which led to Akt activation in HCC . During The Wnt/β-catenin signaling pathway plays an important role in stem cell biology, by regulating stem cell proliferation and oncogenesis, in which cancer stem cells are responsible for driving tumor heterogeneity . Cancer While it is known that drug resistance has been a persistent issue in treating cancers, it is possible to overcome drug resistance by targeting CSCs. Among a number of stem cell related pathways, alternation of Wnt/β-catenin signaling in HCC was shown to play a critical role in drug resistance to sorafenib, lenvatinib, cabozantinib and regorafenib, etc. . A recenAs mentioned in the cancer metabolism section, there are several key enzymes and corresponding metabolites involved in glycolysis that play roles in the regulation of drug resistance. Enzymes such as hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK), or their metabolites such as pyruvate and lactate, play roles in glycolytic-induced drug resistance through their antiapoptotic effects . UpregulWnt/β-catenin signaling plays multiple roles, which enables multifarious therapeutic strategies to target it. Recently, several drugs and molecules have been designed to target the Wnt/β-catenin cascade in HCC . SeveralThere are several monoclonal antibodies that neutralize extracellular targets, including Wnt/FZD ligands and receptors, which play therapeutic roles. Antibodies against Wnt-1 and Wnt-2 have been raised to inhibit Wnt signaling and induce programmed cell death in HCC cell lines . Apart fThe presence of B-catenin destruction complex in the cytosol may serve as a therapeutic target for various cancers. Although it has not yet been further assessed in HCC, there are still some potentially significant reported conclusions. CGK062 is an antagonist that enhances proteasomal degradation of β-catenin by inducing β-catenin phosphorylation . WhereasVarious studies have shown the importance of forming β-catenin/LEF-TCF complexes in the nucleus, as this leads to downstream gene activation in promoting cancers. Therefore, the use of therapeutics targeting this binding could be a possible approach to treat cancers caused by the aberrant Wnt/β-catenin signaling pathway. Sorafenib is a multikinase inhibitor approved by the FDA that can suppress canonical Wnt signaling and can be used to target advanced-stage HCC. It can suppress TCF/LEF and the levels of β-catenin protein and Wnt-target gene mRNA . HoweverThe Dishevelled protein was reported to be an upstream component in the Wnt/β-catenin signaling, which was found to play an important role in inducing Wnt/Fzd signaling. Targeting DVL by inhibiting DVL/FZD interaction via a KTXXXW-containing peptide was reported to inactivate the canonical Wnt/β-catenin pathway, leading to cell death of HCC cells. This result shows the blockade of DVL to FZD as a possible way to hamper the Wnt/β-catenin signaling . InteresSince the Wnt/β-catenin signaling pathway was identified decades ago, substantial attention has been drawn to the field of HCC research due to its critical involvement in tumor initiation, progression and therapeutic resistance. The present review highlighted the role of Wnt/β-catenin in the regulation of cancer stemness and metabolic reprogramming, which are regarded as therapeutic vulnerabilities against HCC. However, our understanding of how the Wnt/β-catenin pathway contributes to these cancer hallmarks remains insufficient. Therefore, additional molecular and animal studies are required urgently to identify novel therapeutic targets against the Wnt/β-catenin pathway for HCC treatment. Recent pre-clinical study showed that Wnt/β-catenin is one of the major pathways hindering the efficiency of current immune checkpoint therapy . Echo to


After spinal cord transection (SCT) the interaction between motoneurons (MNs) and muscle is impaired, due to reorganization of the spinal network after a loss of supraspinal inputs. Rats subjected to SCT, treated with intraspinal injection of a AAV-BDNF (brain-derived neurotrophic factor) construct, partially regained the ability to walk. The central effects of this treatment have been identified, but its impact at the neuromuscular junction (NMJ) has not been characterized. Here, we compared the ability of NMJ pre- and postsynaptic machinery in the ankle extensor (Sol) and flexor (TA) muscles to respond to intraspinal AAV-BDNF after SCT. The gene expression of cholinergic molecules  was investigated in tracer-identified, microdissected MN perikarya, and in muscle fibers with the use of qPCR. In the NMJs, a distribution of VAChT, nAChR and Schwann cells was studied by immunofluorescence, and of synaptic vesicles and membrane active zones by electron microscopy. We showed partial protection of the Sol NMJs from disintegration, and upregulation of the VAChT and AChE transcripts in the Sol, but not the TA MNs after spinal enrichment with BDNF. We propose that the observed discrepancy in response to BDNF treatment is an effect of difference in the TrkB expression setting BDNF responsiveness, and of BDNF demands in Sol and TA muscles. Acetylcholine (ACh) is a key molecule to maintain locomotion at each level of the motor unit. In the spinal cord, the cholinergic propriospinal network modulates central pattern generator neurons and motoneurons (MNs), acting on the latter through the M2 muscarinic receptors ,2,3,4. IC-interneuron-derived cholinergic innervation of the lumbar MNs. That was accompanied by changes in the density of perineuronal nets in the vicinity of these MNs [Complete spinal cord transection (SCT) leads to a loss of motor ability, due to the disruption of supraspinal tracts and altered functioning of the preserved spinal network ,7. Due these MNs ,9,14. Suhese MNs ,15.Brain-derived neurotrophic factor (BDNF), expressed in the spinal interneuronal network and in MNs, maintains their morphology and function, prevents atrophy, and modulates axonal regeneration and plasticity, acting via full length tropomyosin receptor kinase B (TrkB.FL). It is abundant in multiple cell populations in the spinal cord and supraspinal motor nuclei ,18,19,20The BDNF/TrkB signaling pathway is instrumental for the stability of NMJ and proper neurotransmission ,27,28; iChanges that occur in the spinal cord after injury also lead to a strong impairment of the interplay between the MNs and all the muscle fibers that are innervated in the respective muscle ,38,39. TThese findings have set the main aim of our work, which was to determine whether intraspinal AAV-BDNF treatment of rats with SCT could counteract the post-lesion changes in cholinergic signaling in MNs and at the peripheral synapses. For that purpose, we investigated the gene expression and protein distribution of pre- and postsynaptic markers associated with vesicle packaging, ACh release, degradation, and receptivity to ACh at the second post-surgery week. We also aimed to answer the question of whether the flexor and extensor muscles responded differentially to spinal injury and treatment, and to identify structural correlates of the potential therapeutic action of BDNF. The latter question was raised in light of data showing that BDNF transported anterogradely in motor axons affected skeletal muscle, perisynaptic Schwann cells, and played a role in the activity-dependent modifications of neuromuscular transmission ,43,44,45This study shows that Sol MNs and Sol muscle, in contrast to TA muscle, were highly susceptible to intraspinal BDNF treatment after SCT. Preliminary accounts of this work have been presented elsewhere ,46.Experiments were carried out on 53 adult male Wistar rats (between 10–12 weeks of age), weighing 290–350 g prior to surgeries. The rats were obtained from the Animal Facility of the Medical University of Białystok  and housed at the Animal House of the Nencki Institute of Experimental Biology in Warsaw. They were housed in a 12 h light-dark cycle, with free access to water and pellet food, under standard humidity and temperature conditions, in groups of 4–6. After surgery, the rats were placed in individual cages. Experimental protocols involving animals, their surgery, and care were approved by the First Local Ethics Committee in Warsaw , in compliance with the guidelines of the Directive of 24 November 1986 (86/609/EEC), 22 September 2010 (2010/63/EU) and the Animal Protection Act of Poland (2017) on the protection of animals used for scientific purposes.The rats were subdivided into three groups : intact In 36 rats, a complete spinal cord transection (SCT) was performed at the Th10 level under aseptic conditions, as previously described ,48. Rats8 AAV1/2-SYN-BDNF particles or 3 × 108 AAV-1/2-SYN-EGFP particles) or sterile PBS was injected at a speed of 0.1 µL per minute with the SP101i syringe pump . AAV1/2-BDNF construct coding for BDNF under control of the hSyn promoter was obtained as described earlier [Immediately after SCT, at the site of the Th11/12 laminectomy corresponding to the spinal level L1, the dura was opened and lignocainum hydrochloricum  was applied to the surface of the spinal cord. With the support of a surgical microscope, AAV-BDNF, AAV-EGFP, or PBS was administered into the spinal cord through a fine glass capillary, approximately 0.7 mm bilaterally from the midline, and 1 mm deep. One µL of viral particles solution  of MNs received intramuscular injections of fluorescence tracers: cholera toxin conjugated with Alexa Fluor 488  was injected into the TA, and Fast Blue  was injected into the Sol muscle, to label MNs that innervated the muscles of interest. The rats were anesthetized as described above. The area of skin that covered selected muscles of the hindlimb was shaved, disinfected, and cut. The biceps femoris muscle was incised and the underlying TA and Sol muscles were exposed. Fluorescence tracers were injected into the TA (20 µL) and Sol (5 µL). Injections were performed with a Hamilton microsyringe with a 22-gauge needle attached. After 5 min of tracer delivery, the needle was kept in the muscle for at least 3 min to avoid dye leakage and then slowly removed. The injection site was cleaned with a warm 0.9% NaCl solution, and the biceps femoris muscle and skin were sutured. After surgery, the rats were placed in warm cages, covered with blankets, and inspected until they recovered from the effects of anesthesia. Subsequently, the rats were placed in individual home cages with easy access to pellets and water. The antibiotic Baytril  and the analgesic Tolfedine  were administered daily for the first three (after tracer injection) and five (after SCT) postoperative days. The rats were inspected twice daily and their bladders were manually voided. No significant health problems were observed after spinalization, except occasional bladder bleeding during the initial days after surgery.The group prepared for LMD received Baytril and Tolfedine after the first surgery (tracers’ injections), the antibiotic Sultridin  and the analgesic Vetaflunix  after SCT in the paradigm described above. If necessary, plastic collars  were placed around the necks of the animals to protect their wounds from being licked. The collars were usually removed after the first postoperative night.Two weeks after the SCT, rats were anesthetized with isoflurane and then received a lethal dose of Morbital . Then they were transcardially perfused with ice-cold saline  or 4% paraformaldehyde (TEM groups). Their vertebral columns were then excised, placed on ice, and the spinal cords were dissected. Spinal cords and selected hindlimb muscles  intended for qPCR analysis were cut with a McIlwain tissue chopper  to 0.8mm transverse sections, frozen on dry ice, and stored at −80 °C. In the LMD group, spinal cord L3-6 segments (where TA and Sol MNs are located) were surrounded by Jung tissue freezing medium  and frozen at −80 °C until they were cut into longitudinal sections, as described . The musSingle muscle fibers  were isolated under a magnifying glass. Free-floating fibers were washed in PBS for 1 h and incubated with a solution of 5% normal goat serum (NGS) in PBST (PBS with 0.05% Triton-X) for the next one hour at RT to block nonspecific labeling. To detect the presynaptic components of the NMJ and Schwann cells (SC), fibers were incubated overnight at 4 °C with anti-VAChT antibody that identified synaptic vesicles containing acetylcholine vesicular transporter , combined with anti-S100 antibody that identified SC . Both primary antibodies were diluted in PBST. For confirmation of the distribution of motoneuronal fibers and terminal Schwann cells (tSC), antibodies recognizing neurofilament  and periaxin  were used. Subsequently, the fibers were washed three times, for 5 min each, in PBS, and incubated for 1 h (at RT) with secondary antibodies. Alexa Fluor 647 goat anti-rabbit, Alexa Fluor 488 goat anti-guinea pig , and Bungarotoxin conjugated to Alexa Fluor 555  were used to detect postsynaptic nicotinic acetylcholine receptors (nAChR). After three washes, the fibers were labeled for the nuclei with Hoechst dye (1:1000). After another three washes, fibers were mounted on glass slides  and air-dried, then coverslipped  with Mowiol 4–88 medium and kept in the dark at 4 °C until analysis. Prior to regular staining, a single versus a triple labeling was verified to test whether the simultaneous triple reaction did not affect antibody binding. Negative controls (primary antibody omitted) showed the absence of immunoreactivity (data not shown).The images of the NMJ were captured with a Zeiss LSM 780 confocal microscope  using PL APO 40× (1.4 NA) DIC oil-immersion objective. The z-stacks of the 16 bit images consisted of digital slices collected at 0.21 μm intervals with a pixel size of 0.069. Scanning was sequential with a 1.6 μs dwell time. Images were collected at constant exposure parameters for each of four channels detecting fluorescence labeling for VAChT, S-100, Bungarotoxin, and Hoechst with the use of the 561 nm diode-pumped solid-state laser, the 633 nm helium neon laser, and the 488 nm argon laser.The z-stacks of the images were subjected to a deconvolution procedure using Huygens Professional software  to reduce the image distortion arising from light scattering. Based on a random set of images, an experimentally blind rater determined the parameters of the deconvolution algorithm to obtain maximum resolution for each antibody. The level of background and signal to noise ratio was selected for each channel independently; the number of iterations for the deconvolution algorithm was 40.3) and NMJ analysis was done using Imaris 7.6.1 software . The area and volume of the presynaptic and postsynaptic parts of the NMJ  and 20× (0.50 N.A. DIC M/N2) objectives, and the part of the fibers containing NMJs was cut out with a scalpel under a magnifying glass to obtain ca. 1 mm × 1 mm samples for electron microscopy (TEM).For TEM purposes, fiber samples were postfixed in 2.5% glutaraldehyde (EM grade) in a 0.1 M cacodylate buffer (CB) for 1 h at RT, washed 3 × 10 min in CB, then postfixed in 1% osmium tetroxide for 1 h in CB at RT and washed in water (3 × 10 min). Next, the samples were dehydrated by incubation in increasing ethanol concentrations  and cleared in a 1:1 mixture of ethanol + propylene oxide (1 × 10 min) and in 100% propylene oxide (2 × 15 min). During dehydration step with 70% ethanol, the samples were stained with 1% uranyl acetate (for 40 min). Finally, tissue was embedded in the Epon epoxy embedding medium (Serva). Polymerization was done at 60 °C for two days. Ultrathin sections (60 nm) were obtained using Leica Ultracut R ultramicrotome, collected on TEM slot grids of 1 mm × 2 mm . If preliminary examination did not reveal the presence of the NMJ, the sample was cut deeper until the desired structures were obtained. Sections were then post-stained with uranyl acetate and Reynold’s lead citrate. Electron micrographs were obtained with a Morada camera and a JEM 1400 transmission electron microscope at 80 kV  in the Laboratory of Electron Microscopy, at the Nencki Institute of Experimental Biology, in Warsaw, Poland.2 was used for calculation of the density parameter of the SVs. The density of the SVs was measured in the field of at least 500 nm2 remaining after exclusion of the area occupied by the axonal mitochondria. The number and size of synaptic vesicles (SVs) were measured following the protocol of Kim et al., using the cell counter function of ImageJ software . The numFor measurements of the width of synaptic clefts and for the counting of active zones (AZ) only synapses with preserved pre- and postsynaptic membranes were taken into account. The selection of images was based on the criterion of the smallest visible length of the presynaptic membrane equal to 1 um/image. AZs were identified as electron-dense material aligned with postsynaptic junctional folds, as described in the literature ,52. The ® Whole Transcriptome Kit . The concentration of cDNA was determined using a Quant-iT™ PicoGreen™ dsDNA Assay Kit .Microdissection procedures were the same as those previously described in detail . BrieflyRNA was isolated from whole spinal cord segments, microdissected MNs, and muscle samples, using the ZR RNA MiniPrepTM kit  according to the manufacturer’s protocol. An optional DNAse I digestion step was performed . The Transcriptor First Strand cDNA Synthesis Kit with random hexamer primers (Roche Applied Science) was used to convert the total RNA (0.5–1 µg) into coding DNA (cDNA). Reverse transcription was performed at 50 °C for 60 min followed by enzyme denaturation at 85 °C for 5 min. ®96 sequence detection system (Roche Applied Science). Target-specific probes and forward and reverse primers were used as designed by the Universal ProbeLibrary Assay Design Center  value, as well as the cycle number at which the amplification curve reaches the threshold line.Quantification of the Bdnf, Ntrk2, ChAT, VAChT, nAChR, Chrm2 & Chrm4, Ache, S100 beta and MBP gene transcripts was performed using TaqMan hydrolysis probes and the LightCyclern Center . Duplex https://www.graphpad.com/quickcalcs/Grubbs1.cfm, accessed on 20 June 2022). Statistical significance was accepted at p ≤ 0.05. The Shapiro–Wilk test was used to verify the normality of data distribution, and the Levene’s test was used to verify the homogeneity of variance in the groups. One-way ANOVA, followed by the generalization of HSD Tukey (Spjotvoll & Stoline) were applied for unequal sample sizes. Because the assumption of the normality of distribution or homogeneity of variances was violated in some experimental groups, the non-parametric Mann–Whitney U-test was used for the comparison of independent samples (between groups), and the t test and Wilcoxon test were used for the comparison of dependent samples within groups (TA vs. Sol MNs and TA vs. Sol muscle specimens). The STATISTICA 13.1 software  was used to analyze the data.The mean and standard deviation (SD) or standard error of the mean (SEM) were calculated for each data set. The Grubbs test of outliers was carried out for all data, and outliers were discarded (BDNF transcript levels were examined by a qPCR assay in samples of mRNA extracts from the (1) lumbar spinal segments; (2) tibialis anterior (TA) and soleus (Sol) motoneurons; and (3) muscles isolated from the Control, SCT-PBS, and SCT-BDNF rats.−3 BDNF/ref gene), which was in agreement with our previous results [−9–5 × 10−8 , and in a greater than 200-fold increase in the transgene-injected segment (not shown), confirming the effectiveness of the model. AAV-BDNF administration also resulted in high levels of BDNF mRNA in the TA MNs of four rats and in the Sol MNs of two rats; in the remaining rats, no overexpression of BDNF was found  assayed in the L3-6 segments B; in theWe also asked whether BDNF overexpression affected the levels of p75 neurotrophin receptor, which binds all neurotrophins with equal affinity. A remarkable increase in the intensity of p75 labeling was found in the SCT-PBS group, and a weaker signal was observed in the SCT-BDNF group B. In the isolated pools of the TA and Sol MNs, spinalization caused differential effects. In the TA MNs, the levels of TrkB mRNA decreased three times, and was further reduced by AAV-BDNF treatment, while in the Sol MNs, the levels of TrkB mRNA did not decrease in spinal groups (−4) and in the TA (mean = 7.1 × 10−6) muscle , suggesting that, under physiological conditions, the TA muscle was a less abundant source of neurotrophic support and a less obvious target of autocrine regulation by BDNF than the Sol muscle. In both muscles in the Control group, BDNF mRNA levels were lower than those in the spinal cords A. Howevep = 0.0002, Mann–Whitney U-test) but not in the TA muscle. Surprisingly, spinal BDNF overexpression partially counteracted that deficit . Furthermore, compared to the SCT-PBS rats, all SCT-BDNF rats revealed a higher expression of BDNF mRNA in the Sol muscle, while only two out of five SCT-BDNF rats revealed a higher expression of BDNF mRNA in the TA muscle  in the levels of BDNF mRNA in the Sol . The analysis also revealed the disparity in the abundance of NT-3 mRNA and TrkC mRNA in the control muscles, pointing to higher neurotrophin dependence of the Sol circuit. Because muscle receptors and the nerve fibers that innervate them require NT-3 neurotrophin for recovery after damage ,56, and n = 79 NMJ) and Sol (n = 98 NMJ) muscles  with perfectly labeled structures selected from the triple-labeled TA . The infolded postsynaptic membrane of the NMJs, visualized by means of fluorescent-tagged bungarotoxin (BTx) bound to nAChRs, showed a similar feature: Sol NMJs had a greater area (1.3×) of the junctional postsynaptic membrane occupied by nAChRs than TA NMJs . Endplates of predominantly slow twitch muscles of the rats were shown to be larger than in the fast twitch muscles [The image analysis of the presynaptic part of the NMJs visualized by VAChT immunofluorescence revealed that a size of the axon terminal bouton area occupied by VAChT was different between the TA and Sol NMJs in the Control animals A. Sol MN muscles ,61.p = 0.0002, Sol p = 0.0023; volume: TA p = 0.0002, Sol p = 0.0002; ANOVA and Tukey’s post hoc test). A presynaptic part of the TA NMJ was more affected than that of the Sol NMJ; in each spinal group, a complete loss of the VAChT IF signal at terminals was more frequent in the TA MNs . Both the VAChT IF signal area and volume decreased significantly in the NMJs in both muscles in response to SCT  and postsynaptic (nAChR) cholinergic components of the NMJs from the side view D. The exp = 0.07, Mann–Whitney U-test), but did not prevent this process in the TA NMJs. Additional measurements of the VAChT/BTx-nAChR labeling overlap in the en-face images, with the use of maximal intensity projection analysis, confirmed a profound decrease inthe overlapping in spinal groups, both in the TA and Sol NMJs . The decrease resulted primarily from the marked loss of the VAChT signal, and was deeper in the TA muscle . Spinal Sol NMJs E. There Next, to further examine the effect of SCT and BDNF overexpression on the presynaptic and postsynaptic parts of the NMJ, we investigated its ultrastructure. For the measurements of the SVs, density, synaptic clefts, and counting of active zones in the synapses with preserved pre-and postsynaptic membranes were selected. The number, size, and frequency size distributions of synaptic vesicles (SVs), synaptic cleft distance, and the number of active zones per 1 µm of membrane were determined in the NMJs of the Control, SCT-PBS, and SCT-BDNF rats .SCT did not change the mean density of the SVs in the populations of NMJs with maintained synaptic contacts in either the TA or Sol MN terminals, albeit in some cases in the Sol MNs, the density of the SVs was decreased, as exemplified in The average synaptic cleft distance in the Controls was 44.83 nm (SD = 5.52) in TA junctions and 52.8 (SD = 5.73) in Sol junctions. In the spinal groups, the distance was comparable and amounted to 51–52 in both NMJ types. The number of active zones was comparable between groups. We concluded that in the subpopulation of junctions with preserved morphology in all morphological parameters under study were maintained within the control range.p = 0.012, Wilcoxon test), suggesting that the ACh turnover was higher in the TA than in the Sol MN pool. The same relation was observed for choline acetyltransferase (ChAT) mRNA levels, but the result did not reach a statistical significance . Acetylcholinesterase (AChE) mRNA levels were comparable in both MNs types  in the TA MNs were significantly higher than in the Sol MNs  and a 4-fold increase in the Sol muscle (p = 0.000). After AAV-BDNF treatment, the levels of AChE transcripts did not differ from the controls in both muscles.The spinalization effect on the AChE mRNA differed between muscles. As shown in the p = 0.005, Wilcoxon test). We did not observe significant changes in the nAChR mRNA after SCT, but BDNF treatment resulted, to our surprise, in markedly lower expression of the nAChR in the Sol muscles.In the control TA and Sol muscles, the expression of postsynaptic nicotinic receptor nAChR alpha 1 subunit, which plays a role in ACh binding and channel gating, was differentiated, with much higher expression in the Sol muscles . In the SCT-PBS group, no significant change in the transcript levels of either receptor was found in the muscles, but their correlation was markedly reduced. The AAV-BDNF treatment reduced the expression of M2 in the Sol muscles, but recovered to some extent a quantitative relationship between receptors   .In this study, we showed a set of changes that pointed to a strong modulation of cholinergic signaling in the motor circuit of the Soleus muscle in response to spinal injury and BDNF treatment. We investigated the levels of expression of enzymes that catalyze acetylcholine synthesis and hydrolysis, and of the vesicular transporter of ACh responsible for packaging ACh into synaptic vesicles. A clear pattern of changes in the presynaptic component included a decrease in the AChE transcript after transection, and an opposite change in that transcript after AAV-BDNF treatment, which led to the normalization of AChE expression in the Sol MNs. BDNF treatment also led to the marked elevation of VAChT expression in the Sol MNs. Our study also pointed to a new possible mechanism contributing to the regulation of ACh release by BDNF. In both the TA and Sol muscles, AAV-BDNF treatment led to a decreased expression of M2 mAChR receptors. Because presynaptic M2 mAChRs have been reported to suppress ACh release ,67, theiWe also broadened the scope of evidence for different vulnerabilities of the TA and Sol circuits to spinal cord injury, as shown in our previous reports ,9. BasedA significantly lower level of VAChT transcripts in the Sol MNs, compared to the TA MNs detected in our study, was in line with the results by Woods and Slater, who reported lower ACh release in the Sol muscle, as compared to ACh release in another ankle joint flexor, the EDL . This chThe level of expression of AChE in the TA and Sol MNs was similar. In the muscles, the AChE mRNA level in the TA was higher than in the Sol, which was in line with studies by Sketelj and coworkers who reported several-fold higher levels of AChE in fast compared to slow muscles .in situ hybridization in multiple pools of MNs located in the L3–L4 spinal segments [This study is the first to show a very low abundance of BDNF transcripts in the TA and Sol MNs in rats. These results added to our early observation of differential BDNF mRNA expression detected by means of segments . Importasegments ; thus, tsegments ,54,72 wein situ hybridization experiment [An interesting aspect that emerged from our analysis of the TrkB mRNA expression was a significantly higher level of TrkB mRNA in the TA than in the Sol MNs. It stood in opposition to the early study by Copray and Kernell, where TrkB and BDNF mRNA expression in the Sol and EDL MNs did not show differences in a non-radioactive periment . The difDespite the low level of BDNF expression in both types of muscles, there was a difference between them, with a much higher expression of BDNF in the Sol than in the TA muscle. Similarly, NT-3 and TrkC expression were also significantly higher in the Sol than in the TA muscle. That characteristics may reflect higher neurotrophic demands of the Sol circuit and may functionally relate to the daily duration of the Sol muscle activity, which in intact animals is approximately seven times longer than that of the TA muscle . Our datThe analysis of gene expression did not show significant differences in the levels of motoneuronal BDNF mRNA in the SCT-group compared to Control group for both types of MNs. This was in line with studies reporting that a low level of BDNF mRNA observed in control MNs was not affected by SCT ,77. In cIn the muscles, SCT only had an effect on the level of Sol BDNF expression, and significantly decreased it. Considering the activity-dependent expression of BDNF, it was in line with the different states of those two muscles in paraplegic rats, as reported by us previously. In the Soleus muscle, which is constantly relaxed when the animal drags the paws on the dorsal aspect, the effect of disuse is more pronounced .The intraspinal injection of AAV-BDNF led to a high overexpression of BDNF in the L3–L6 segments of the spinal cord. The general capacity of the spinal system to respond to the treatment was confirmed by the analysis of TrkB gene expression, which showed preserved receptivity to the BDNF. No observed increase in lumbar expression of the TrkB at 2 weeks after SCT was contrary to what has been documented by Keeler and coworkers ,77. We dThe treatment led to an increase in BDNF mRNA in the MNs innervating the TA. Only one rat from the SCT-BDNF group overexpressed BDNF in the Sol MNs. This was an expected result, considering that the TA MNs were located more proximal to the site of injection as compared to the Sol MNs, thus increasing the probability of neuronal transfection by the AAV-BDNF . Few studies have explored the motoneuronal expression of BDNF after the intraspinal injection of AAV-BDNF. According to the previous result of our group , six weeIn a current study, after the intraspinal administration of AAV-BDNF, the level of BDNF mRNA in the Sol muscle increased, and tended to normalize. In the TA, no changes were observed. The source of that increased production in the Sol needs further investigation. We assumed it was not an effect of the axonal transport of transgene mRNA, as we didn’t observe any increase in the motoneuronal expression of BDNF in the Sol muscle. We hypothesize that the BDNF is expressed in muscle fibers during functional improvement, which was observed in this study, since the BDNF is target-derived and expressed in an activity-dependent manner ,31,32,79A decrease in VAChT mRNA was detected in the TA, but not in the Sol MNs at 2 weeks after SCT, and may have referred to data described by Magalhães-Gomes, who examined skeletal muscles in mice with reduced expression of VAChT in MNs, in which fast- and slow-type muscles responded differentially to cholinergic deficits. In the TA circuit, a deficiency led to muscle fiber atrophy, while in the Sol circuit, it led to muscle fiber hypertrophy . A rich In previous sections, we discussed a clear difference between the TA and Sol motor circuits in several aspects of BDNF signaling in the control and SCT conditions. Therefore, post-lesion maintenance of the NMJs and of the cholinergic activity, which are regulated by BDNF-TrkB pathway, may have occurred to varying degrees in both muscles. In general, the TA NMJs were more vulnerable to SCT than the Sol NMJs, as shown by examination of their morphology and synaptic connectivity; our observation was consistent with the result of Burns et al., who also reported destabilized synapses in the TA, but not in the Sol . PostsynThe TEM analysis revealed no significant differences between the TA and Sol NMJs, and no clear effect of SCT or BDNF-treatment on the SVs pool, synaptic cleft distance, and number of active zones. However, it should be noted that this set of data reflects the state of the SVs in the NMJ subpopulation with maintained integrity, because NMJ sections showing dispersed presynaptic structure and membrane fragmentation in the TEM were eliminated from counting and measurements. Thus, we discuss the SVs pools in these NMJs, which were preserved. Non-compliance between changes in the VAChT protein level and the quantity of SVs may also suggest the presence of non-functional or empty vesicles in rats after transection. That is in line with studies by Rodrigues and coworkers on mice with a 70% reduction in VAChT expression, who showed that the number of vesicles in the NMJs were the same compared to WT mice, and only a shape and distribution of the SVs in response to electrical stimulation differed . InteresTaking into account different effects that the SCT and BDNF treatment had on cholinergic signaling in slow and fast muscles, the search for a possible mechanism of action of BDNF should start with the characteristics of processes that differentiate the functioning of the slow and fast motor unit. It was shown that, at 1–2 months after SCT, the slow to fast fiber transition of the paralyzed muscles began ,90, and We propose that after SCT, the intraspinal injection of AAV-BDNF acted on the cholinergic system at two levels. At first, transfected interneurons, enriched in BDNF, released neurotrophin that stimulated MNs in the lumbar spinal segments. We expected this mechanism to operate mostly in Soleus MNs, due to their high receptivity to BDNF, in contrast to TA MNs, which are almost devoid of receptors. As a result, an increase in ChAT expression was induced, which was shown to be strongly correlated with locomotor performance after spinal injury , with a in vitro experiments [Terminal Schwann cells (TSC) could have also contribute to the BDNF-related maintenance of cholinergic transmission. TSCs, which surround the peripheral synapse, were shown to be regulated by BDNF , to expreriments . Taking eriments ,104, we The limitation of this study lies in the fact that the types of muscle fibers were not distinguished in the analysis; therefore, general conclusions are drawn with the assumption that the TA was fast and the Sol was slow muscle. The response of NMJs on slow-oxidative, fast-oxidative, and fast-glycolytic fibers is a key area that needs to be explored further, as well as spatial gene expression in the tripartite peripheral synapse. Enriching the TA MNs with TrkB receptors in this model could lead to beneficial changes, as BDNF improves axonal transport in fast MNs , and reg


The Wnt signaling pathway plays an important role in the normal development and regulation of ovarian follicles. Genetic or epigenetic activation of β-catenin has been implicated in tumorigenesis of a spectrum of ovarian neoplasms. This review summarizes the recent findings of the β-catenin mutations in a family of low-grade ovarian stromal tumors.The Wnt signaling pathway is important in the normal development and regulation of ovarian follicles throughout the lifecycle of females. Dysregulation of the Wnt signaling pathway, genetically or epigenetically, with subsequent activation of β-catenin has been implicated in tumorigenesis of a spectrum of ovarian neoplasms, from benign to malignant. We review the recent findings of the Wnt signaling pathway involved in regulating normal physiologic processes of the ovarian follicle cycle. We also review the β-catenin mutations in a family of low-grade ovarian stromal tumors, focusing on characterizing their shared morphological features and the utility of immunohistochemistry of β-catenin in facilitating the accurate diagnosis of these ovarian stromal tumors. The Wnt signaling pathway is one of the most critical mechanisms in regulating cell proliferation, differentiation, and morphogenesis. The Wnt signaling pathway comprises a diverse group of glycoproteins that serve as ligands and bind to transmembrane Frizzled family receptors. The ligand-receptor interactions activate the pathway and govern the downstream signaling cascades, ultimately affecting the transcriptional control of the cellular cytoskeleton, organelle dynamics, epithelial-mesenchymal interaction, and tissue remodeling in the ovary. Wnt signaling consists of two major pathways: a canonical pathway that is β-catenin-dependent and a non-canonical Wnt pathway that is β-catenin-independent. Canonical Wnt signaling is governed by the interaction of β-catenin with other molecules to regulate cellular decisions related to proliferation and differentiation. Recent studies have demonstrated that the Wnt signaling pathway plays important roles in the development and regulation of ovarian folliculogenesis and oogenesis. The Wnt pathway is critical to the development and function of the ovary in three aspects: (1) it is essential during the embryonic development of the ovary; (2) it is crucial for the development and regulation of ovarian follicles from birth to maturity; and (3) it plays an essential role in steroid hormone production which in turn regulates the development of ovarian follicles and fertility in the adult ovary.During embryonic development, the Wnt pathway plays an essential role in early ovarian development and suppression of the male reproductive tract. In the mammalian embryo, both sexes are initially indistinguishable morphologically. Specific hormones secreted by the differentiating embryonic testes, such as Mullerian inhibiting substance and testosterone, are required for sex-specific development. These hormones suppress females (Mullerian) but promote male (Wolffian) reproductive duct development. The signaling molecule Wnt-4 is essential for ovarian differentiation and oogenesis by stimulating granulosa cell differentiation and promoting female sexual development. At birth, sexual development in males with Wnt-4 mutation appears normal; however, Wnt-4-mutant females are masculinized with the absence of the Mullerian duct while developing the Wolffian duct. Wnt-4 null females have sex-reversed ovaries but with a significantly reduced number of oocytes and the expression of the genes associated with testicular development at birth. It has been shown that Wnt-4 suppresses the growth of Leydig cells in the developing ovary . The adult ovary undergoes constant dynamic changes throughout the estrous cycle as follicles progress from immature preantral follicles to more developed preovulatory follicles and eventually corpus luteum formation following ovulation. The complex process of folliculogenesis relies on synchronized efforts of hormones from the hypothalamus, pituitary, and gonads. While the initial stage of the follicles develops primarily in the absence of gonadotropin input, the transition from preantral to a preovulatory follicle occurs as a result of increased follicle-stimulating hormone (FSH) and luteinizing hormone (LH) responsiveness as well as numerous other local hormones and growth factors . It has NR5A1 and Lhcgr [Recent studies show that β-catenin is required to produce and regulate estrogen levels stimulated by FSH. The accumulation of β-catenin protein has been identified in high estrogen-producing follicles compared to those with low intrafollicular estradiol concentrations . Evidencnd Lhcgr . Togethend Lhcgr .The ovarian cortical and medulla stroma is similar in appearance and continuous with ill-defined boundaries. Ovarian stroma in reproductive-age women is highly cellular and vascular with inconspicuous supporting fibers such as reticular fiber and collagen. It comprises a mixed population of incompletely characterized stromal cells or interstitial cells typically arranged in a dense storiform pattern . StromalDuring follicle maturation, the granulosa cells proliferate to form many layers around the oocyte. A boundary layer, the zona pellucida, appears between the oocyte and the granulosa cells. Stromal cells surrounding the follicle differentiate into the theca interna and the theca externa. Cells of the theca interna become cuboidal steroid-producing cells which often contain cytoplasmic lipid droplets. Androgens from thecal cells is converted to estrogen in granulosa cells and then released into circulation. The theca externa, a mixture of fibroblasts and smooth muscle cells lacking endocrine function, is contiguous with the surrounding ovarian stroma ,21. ReceAPC and Axin genes is far more common than activating mutations of the β-catenin gene CTNNB1. Only approximately 3% of malignant neoplasms harbor CTNNB1 mutations [APC mutations are found in more than 80% of colorectal cancers, but only approximately 5% of colorectal cancers harbor CTNNB1 mutation [CTNNB1 mutations [CTNNB1 mutations have been identified in 16–54% of endometrioid adenocarcinoma, which accounts for less than 10% of all ovarian carcinomas. However, CTNNB1 mutations are rarely seen in other histologic types, such as serous, clear cell, and mucinous carcinomas [The β-catenin protein is stabilized and anchored to the cell membrane by the APC/Axin/GSK-3β complex, the so-called β-catenin destruction complex (1). When the Wnt pathway is activated during tumorigenesis, β-catenin is released and redistributed from the cell membrane to the nuclei and cytoplasm of tumor cells . Excessiutations . Moreovemutation ,25. The mutation ,27. Howeutations ,28. Geneutations . CTNNB1 rcinomas ,30.CTNNB1 gene, which has 16 exons and maps to 3p21 [CTNNB1 mutations are relatively low-grade with relatively favorable prognosis, such as desmoid tumor, renal angiomyolipoma, pulmonary lymphangioleiomyomatosis, and pancreatic solid pseudopapillary tumor [The β-catenin protein is composed of a 23.2 kb polypeptide encoded by the  to 3p21 . The pri to 3p21 . General to 3p21 ,31. Thusry tumor .CNNB1 gene have been detected in a family of low-grade stromal tumors of the ovary, which have overlapping morphological features, similar clinical presentation, and favorable prognosis. This family includes primary ovarian solid pseudopapillary neoplasm (SPPN), microcystic stromal tumor (MCST), and signet-ring stromal cell tumor (SRSCT). CTNNB1 and APC genes in ovarian SPPN, MCST and SRSCT.Recently mutations in the CTNNB1 mutation at exon 3 in ovarian SPPN [Dr. Young’s group first described SPPN in 2010 . SPPN isian SPPN . Since tCTNNB1 mutations in exon 3, identical to that of SPPN, indicating dysregulation of the Wnt/β-catenin pathway in the pathogenesis of this neoplasm. Several other studies subsequently confirmed the above findings [APC germline mutations [CTNNB1 mutation identified besides APC mutations. This has prompted a comprehensive molecular study by McCluggage’s group with the largest case series of MCST thus far [CTNNB1 mutations and 2 cases having APC mutations including one with germline APC mutation. CTNNB1 and APC mutations are mutually exclusive with no neoplasm exhibiting a mutation in both [In 2009, Irving J.A. and Young R.H. described a novel and rare ovarian stromal neoplasm and named it the microcystic stromal tumor . MCST prfindings ,39,40,41utations ,43. In butations  there isthus far . Among 1 in both . CTNNB1 gene at exon 3, along with a germline missense mutation in Janus kinase 3 (JAK3) gene [SRSCT is a rare ovarian stromal tumor that Ramzy first described in 1976 . Thus faK3) gene ,56. JAK3APC gene, but not the CTNNB1 gene, has also been reported for SRSCT. Yoshikawa et al. recently described a variant of SRSCT with the nuclear accumulation of β-catenin in an adult Asian woman harboring germline APC mutation, but lack of CTNNB1 mutations. Another unusual case of SRSCT was reported recently by Chen et al. [CTNNB1 gene was identified in Chen’s case using next-generation sequencing with a panel of 54 genes. Arguably the tumor in the latter was less typical for SRSCT, given its bilateral presentation and different immunophenotype compared to the previous reports. Immunohistochemical study in Chen’s case showed that tumor cells are diffusely positive for SF1, calretinin and SMA, and focally positive for cytokeratin AE1/3 and ER. Tumor cells are also negative for cyclin D1 [Similar to MCST, mutations of n et al. . In contyclin D1 . The repCTNNB1 and APC, can result in the same aberrant expression pattern of β-catenin protein [As mentioned above, the aberrant nuclear and cytoplasmic immunostaining pattern of β-catenin is the most striking immunohistochemical phenotype found in ovarian SPPN, MCST, and SRSCT. Immunohistochemical detection of nuclear and cytoplasmic accumulation of β-catenin has much higher sensitivity compared to molecular testing because mutations in several Wnt signaling genes, including  protein ,44,51. Additionally, these three tumors also share similar immunohistochemical profiles of other markers/antibodies . ImmunorLEF1 belongs to the T cell Factor (TCF)/LEF family of transcription factors and contains a highly conserved DNA-binding domain associated with the C terminal of β-catenin. LEF1 is one of the critical nuclear effectors downstream of the Wnt/β-catenin signaling pathway. When the Wnt signaling is inactivated, LEF1 is bound to Groucho-related co-repressors, negatively regulating the expression of Wnt signaling genes. Upon activation of the Wnt pathway via β-catenin mutation, mutant β-catenin protein displaces the Groucho-related co-repressors and promotes LEF1 transcription factor activity . CompareCTNNB1 or APC genes have been identified in a family of low-grade ovarian stromal tumors, resulting in nuclear and cytoplasmic accumulation of β-catenin. Identification of the hotspot mutations in exon 3 of the CTNNB1 gene indicates that the activation of the Wnt signaling pathway plays a crucial role in the tumorigenesis of these low-grade ovarian stromal tumors. The overlapping morphological features and shared immunophenotype seen in these low-grade ovarian stromal tumors suggest a likely common origin from the same type of ovarian stromal cells. In summary, the Wnt/β-catenin pathway plays an important role in ovarian follicular and stromal physiology. However, this pathway can become dysregulated, leading to a variety of ovarian neoplasms. Besides ovarian endometrioid adenocarcinoma, mutations in


Protein arginine methyltransferase 5 (PRMT5) is a popular anticancer target that regulates histone or nonhistone methylation and is linked to the development and poor prognosis of non-small cell lung cancer. PRMT5 inhibitors have shown great promise in clinical trials as a cancer therapy. However, most inhibitors reported recently act in a SAM-competitive mode and lack structural diversity. In this paper, a novel non-SAM inhibitor, 3039-0164, was discovered by the structure-based virtual screening method. The binding mechanism of 3039-0164 to PRMT5 was revealed via molecular docking and molecular dynamics simulations. 3039-0164 inhibited PRMT5 enzymatic activity, downregulated the expression of PRMT5 downstream target genes (FGFR3 and eIF4E), and blocked the activation of the PI3K/AKT/mTOR and ERK signaling pathways. The discovery of 3039-0164 provides precise and creative hit compounds for the design optimization of PRMT5 lead compounds in non-small cell lung cancer. Non-small cell lung cancer (NSCLC) is the most common and lethal primary lung cancer, which is difficult to detect and requires systemic treatment . Despite50 value of 22 nM, and its THIQ moiety forms the cation–π interaction with the positively charged methyl group in SAM, which makes a fundamental linkage, reduces SmD3 methylation and cell death, and shows a dose-dependent anticancer effect [Epigenetics is closely linked to cancer development, affecting gene expression and cellular function by regulating DNA or histone methylation, acetylation, phosphorylation, ubiquitination modifications, and chromatin remodeling . It is sr effect ,22. Addir effect , lung car effect ,22, and r effect ,25, and In this paper, we used the virtual screening method based on the receptor structure to choose 158 candidates acting at the PRMT5 substrate binding site (at the location of EPZ015666). Through molecular docking and molecular dynamics simulations, it could be found that the identified 3039-0164 bound to PRMT5 in a stable manner mainly by hydrogen bonding and π–π interactions. The MTT experiment showed that 3039-0164 could inhibit cell viability of the A549 non-small cell lung cancer cell line. Furthermore, protein function assays showed that 3039-0164 downregulated the expression of two PRMT5 target genes, FGFR3 and eIF4E, and impeded the activation of the PI3K/AKT/mTOR and ERK signaling pathways. The discovery of 3039-0164 opens up new avenues for research and design optimization of PRMT5 inhibitors, which can help improve the prognosis of non-small cell lung cancer and other cancers in the clinic.https://www.rcsb.org/, accessed: 25 December 2021). The complex was first processed in the Protein Preparation Wizard module. All water molecules were removed, and the protonation state of all charged residues was adjusted to neutral pH. Then the docking grid was generated with EPZ015666 as the center. Since SAM has a cation–π interaction with EPZ015666 and affects PRMT5 inhibitor binding [The Schrödinger Maestro package   was used binding  were gen binding  in a 1:5To eliminate the compounds with false-positive chemical and non-drug-like functional groups, before the virtual screening, the 1,671,908 compounds from the ChemDiv, Specs, and TargetMol databases were preliminarily screened based on the three pan-assay interference structures (PAINS) and one rapid elimination of swill (REOS) rules in Schrödinger Suite’s Canvas software. Meanwhile, the compounds meeting Lipinski’s rule of five were retained. The obtained compounds were then used for the virtual screening based on molecular docking. The top 10% (1706) of small molecules were clustered based on the Glide docking score, and the group size was set to 200. Finally, 158 compounds were selected for subsequent experimental examination by observing the binding pose of each ligand. All compounds were purchased from TopScience .https://www.rcsb.org/, accessed: 25 December 2021) and prepared using the Protein Preparation Wizard module, for example, hydrogenation and refinement of the structure, with the OPLS3 force field. Then, with EPZ015666 as the docking center, a docking grid box was generated. Next, the 3039-0164 small molecule structure file was downloaded from pubchem.ncbi.nlm.nih.gov/ and prepared in the Schrödinger software. Finally, molecular docking analysis was performed by the Glide module.Molecular docking was performed using the Schrödinger software package . PRMT5 was selected from the Protein Data Bank  restrain on protein backbone atoms. The restrain was gradually decreased within 0.9 ns from 10 to 0.01 kcal/(mol Å2). Finally, without applying any constraints, three 200 ns MD simulations were run at 310 K and 1 atm. The Cpptraj [The Amber20  software Cpptraj  module o2 incubator at 37 °C containing 5% CO2. A total of 158 compounds obtained from the virtual screening were dissolved in DMSO and placed in a −40 °C environment. To rapidly identify compounds with strong inhibitory activity, the A549 cell line was first treated with 20 μM of each compound for 72 h. For the MTT assay, cells were first inoculated in 96-well 3000 cells/well microplates and incubated overnight to allow cell adhesion, and DMSO (10.0 µM) and different concentrations  were compared with compounds, which were studied at 24, 48, and 72 h. Then, 10 µL of MTT (5 mg/mL) was added to each for 4 h incubation at 3 °C, followed by 100 µL of acid isopropanol (10% SDS and 0.01 mol/L hydrochloric acid). Finally, the absorbance of the samples at 570 nm was measured with a microplate reader . Cell viability was calculated relative to untreated controls, and data conclusions were based on at least three independent experiments.The non-small cell lung cancer cell line A549 (purchased from ATCC) was selected and cultured in RPMI 1640 medium containing 10% FBS  and 1% penicillin–streptomycin solution in a CO50 values. PRMT5 and SAM/SAH were derived from BPS Bioscience (Cat. No. 51045) and Sigma . 3039-0164 was prepared as 10 mM of backup material in DMSO and diluted in DMSO to the final concentration. PRMT5 and substrate were incubated at the indicated concentrations of 3039-0164 in 384-well plates for 60 min at room temperature. Then, the remaining substrate of PRMT5 was labeled by adding the receptor and ligand solutions. The labeling procedure was reacted at room temperature for 60 min, and then the endpoint values were read using EnSpire Alpha mode. In the in vitro enzymatic assay, 1% DMSO was used as the control.PRMT5 enzymatic assay was performed as previously done by Ji et al.  at ChemPg, 4 °C) was centrifuged for 5 min, and the supernatant fraction was collected. The protein concentration was determined using the Bio-Rad protein assay kit . The extracted proteins were separated in 10% SDS-PAGE gels (50 μg) and transferred to nitrocellulose (NC) membranes at 300 mA for 1 h at 4 °C. The proteins were then incubated with a primary antibody (1:1000) and a fluorescence-coupled secondary antibody . Additionally, eIF4E antibodies were obtained from Santa Cruz Biotechnology ; antibodies against total/phospho-AKT, total/phospho-ERK, and total/phospho-mTOR were obtained from Cell Signaling Technology .Proteins were extracted from A549 cells using RIPA lysis buffer containing protease and phosphatase inhibitors. After two washes with cold PBS, the cell lysate  was used for multiple comparisons. Structure-based virtual screening (SBVS) plays a key role in drug discovery research ,35. The MMGBSA values were preferred, when structurally similar candidates were discovered. Based on these conditions, 158 small-molecule candidates were reserved for the further biological test. We performed MTT assays on these 158 candidate compounds. Results showed that in the A549 non-small cell lung cancer cell line, 3039-0164 showed a strong inhibitory effect. Meanwhile, its ∆GMMGBSA value of 3039-0164 with PRMT5 was lower than the others . Therefore, 3039-0164 was selected for the following studies.Virtual screening was carried out for 1,671,908 chemical ligands with the previously established grid boxes. Following that, 1706 candidate compounds in the top 10% of the Glide score were submitted to 200-group cluster analysis. Compounds with more diversity, more interactions, and plausible binding patterns were chosen. Candidates with smaller molecular weights or lower ∆GMolecular docking  is a matTo obtain the stable complex, three parallel 200 ns molecular dynamics simulations were performed for the docked PRMT5-SAM-3039-0164 complex. By monitoring the RMSD values of 3039-0164 and PRMT5 active site along the simulation, we could see that the system fluctuated stably after 140 ns a. Based 50 value of 7.8 ± 1.7 μM and concentration dependence, 3039-0164 demonstrated the most significant antiproliferative effect on A549 cells in the 72 h continuous effect group. In summary, A549 was discovered to be extremely hazardous to 3039-0164.PRMT5 was shown to be highly elevated in human lung cancer cells and tissues, and inhibiting its expression decreased the proliferation of tissue-cultured lung adenocarcinoma A549 cells significantly ,41. As a50 of 63 μM and a concentration-dependent impact  as a new potent antitumor chemotype, showing the promising anticancer effects of anthraquinone compounds. Despite this, the toxic assessment of this compound or the identification of toxic moieties, such as anthraquinone and the bis (2-hydroxyethyl) amino group, will be meaningful for drug safety. Simultaneously, in the next plan, further structural optimization is considered to improve its efficiency and selectivity for PRMT5. In the meanwhile, X-ray crystallography and biophysical techniques, such as microscale thermophoresis, isothermal titration calorimetry, or surface plasmon resonance, are expected to be used for verifying the direct targeted effects of promising inhibitors against PRMT5.In in vitro studies, three features of 3039-0164’s anticancer effect against NSCLC were investigated: PRMT5 enzyme activity; expression of two target genes, FGFR3 and elF4E; and FGFR3-mediated downstream signaling pathways. The kinase experiment revealed that 3039-0164 inhibited PRMT5, but its IC studies ,53. Some studies  reportedIn conclusion, based on the virtual screening of a PRMT5-EPZ015666 substrate-binding pocket structure, this work finds a novel PRMT5 inhibitor, 3039-0164, with an anthraquinone moiety and demonstrates the influence of this inhibitor on cellular function through a series of biological tests. We hope that our research will spark some new ideas for improving the prognosis regimen of non-small cell lung cancer patients by optimizing the PRMT5 inhibitor design and developing drug-lead compounds.