mTORC1/rpS6 and p-FAK-Y407 signaling regulate spermatogenesis: Insights from studies of the adjudin pharmaceutical/toxicant model.

In rodents and humans, the major cellular events at spermatogenesis include self-renewal of spermatogonial stem cells and undifferentiated spermatogonia via mitosis, commitment of spermatogonia to differentiation and transformation to spermatocytes, meiosis, spermiogenesis, and the release of spermatozoa at spermiation. While details of the morphological changes during these cellular events have been delineated, knowledge gap exists between the morphological changes in the seminiferous epithelium and the underlying molecular mechanism(s) that regulate these cellular events. Even though many of the regulatory proteins and biomolecules that modulate spermatogenesis are known based on studies using genetic models, the underlying regulatory mechanism(s), in particular signaling pathways/proteins, remain unexplored since much of the information regarding the signaling regulation is unknown. Studies in the past decade, however, have unequivocally demonstrated that the testis is using several signaling proteins and/or pathways to regulate multiple cellular events to modulate spermatogenesis. These include mTORC1/rpS6/Akt1/2 and p-FAK-Y407. While selective inhibitors and/or agonists and antagonists are available to examine some of these signaling proteins, their use have limitations due to their specificities and also potential systemic cytotoxicity. On the other hand, the use of genetic models has had profound implications for our understanding of the molecular regulation of spermatogenesis, and these knockout (null) models have also revealed the factors that are critical for spermatogenesis. Nonetheless, additional studies using in vitro and in vivo models are necessary to unravel the signaling pathways involved in regulating seminiferous epithelial cycle. Emerging data from studies, such as the use of the adjudin pharmaceutical/toxicant model, have illustrated that this non-hormonal male contraceptive drug is utilizing specific signaling pathways/proteins to induce specific defects in spermatogenesis, yielding mechanistic insights on the regulation of spermatogenesis. We sought to review these recent data in this article, highlighting an interesting approach that can be considered for future studies.

Adjudin improves beta cell maturation, hepatic glucose uptake and glucose homeostasis.

AIMS/HYPOTHESIS: Recovering functional beta cell mass is a promising approach for future diabetes therapies. The aim of the present study is to investigate the effects of adjudin, a small molecule identified in a beta cell screen using zebrafish, on pancreatic beta cells and diabetes conditions in mice and human spheroids. METHODS: In zebrafish, insulin expression was examined by bioluminescence and quantitative real-time PCR (qPCR), glucose levels were examined by direct measurements and distribution using a fluorescent glucose analogue, and calcium activity in beta cells was analysed by in vivo live imaging. Pancreatic islets of wild-type postnatal day 0 (P0) and 3-month-old (adult) mice, as well as adult db/db mice (i.e. BKS(D)-Leprdb/JOrlRj), were cultured in vitro and analysed by qPCR, glucose stimulated insulin secretion and whole mount staining. RNA-seq was performed for islets of P0 and db/db mice. For in vivo assessment, db/db mice were treated with adjudin and subjected to analysis of metabolic variables and islet cells. Glucose consumption was examined in primary human hepatocyte spheroids. RESULTS: Adjudin treatment increased insulin expression and calcium response to glucose in beta cells and decreased glucose levels after beta cell ablation in zebrafish. Adjudin led to improved beta cell function, decreased beta cell proliferation and glucose responsive insulin secretion by decreasing basal insulin secretion in in vitro cultured newborn mouse islets. RNA-seq of P0 islets indicated that adjudin treatment resulted in increased glucose metabolism and mitochondrial function, as well as downstream signalling pathways involved in insulin secretion. In islets from db/db mice cultured in vitro, adjudin treatment strengthened beta cell identity and insulin secretion. RNA-seq of db/db islets indicated adjudin-upregulated genes associated with insulin secretion, membrane ion channel activity and exocytosis. Moreover, adjudin promoted glucose uptake in the liver of zebrafish in an insulin-independent manner, and similarly promoted glucose consumption in primary human hepatocyte spheroids with insulin resistance. In vivo studies using db/db mice revealed reduced nonfasting blood glucose, improved glucose tolerance and strengthened beta cell identity after adjudin treatment. CONCLUSIONS/INTERPRETATION: Adjudin promoted functional maturation of immature islets, improved function of dysfunctional islets, stimulated glucose uptake in liver and improved glucose homeostasis in db/db mice. Thus, the multifunctional drug adjudin, previously studied in various contexts and conditions, also shows promise in the management of diabetic states. DATA AVAILABILITY: Raw and processed RNA-seq data for this study have been deposited in the Gene Expression Omnibus under accession number GSE235398 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235398 ).

Spermiation: Insights from Studies on the Adjudin Model.

Spermatogenesis is comprised of a series of cellular events that lead to the generation of haploid sperm. These events include self-renewal of spermatogonial stem cells (SSC), proliferation of spermatogonia by mitosis, differentiation of spermatogonia and spermatocytes, generation of haploid spermatids via meiosis I/II, and spermiogenesis. Spermiogenesis consists of a series of morphological events in which spermatids are being transported across the apical compartment of the seminiferous epithelium while maturing into spermatozoa, which include condensation of the genetic materials, biogenesis of acrosome, packaging of the mitocondria into the mid-piece, and elongation of the sperm tail. However, the biology of spermiation remains poorly understood. In this review, we provide in-depth analysis based on the use of bioinformatics tools and an animal model that mimics spermiation through treatment of adult rats with adjudin, a non-hormonal male contraceptive known to induce extensive germ cell exfoliation across the seminiferous epithelium, but nost notably elongating/elongated spermatids. These analyses have shed insightful information regaridng the biology of spermiation.

mTORC1/rpS6 signaling complex modifies BTB transport function: an in vivo study using the adjudin model.

Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it "tighter." However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier "leaky." Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stages VIII-IX of the epithelial cycle, and it coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating that rpS6 is a BTB-modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E, wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240, and 244 from the NH2 terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier "leaky." This thus promoted the entry of the nonhormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.

Research update and opportunity of non-hormonal male contraception: Histone demethylase KDM5B-based targeting.

With the continued increase in global human population, diverse contraception approaches have become increasingly essential, including non-hormonal male contraception. Non-hormonal approaches to contraception are very convenient; however, such options are limited because data regarding the identification and characterization of tissue/cell-specific targets and appropriate small molecule candidate contraceptives are lacking. Based on in-silico studies of genomics, transcriptomics, and proteomics, performed by mining datasets in PubMed, we first reviewed testis-, epididymis-, and germline cell-specific genes/proteins, with the aim of presenting evidence that many of these could become 'druggable' targets for the development of non-hormonal male contraceptives in the future. Although many hurdles remain before the successful therapeutic use of non-hormonal contraceptive, to facilitate this approach, we describe here the changing perspectives on several potential non-hormonal contraceptives (e.g. small molecules, plant extracts, etc.) that are under development; continued effort may yield marketable products. Further, we highlight specific enzymes within the histone lysine demethylase subfamily that play a central role in germ line regulation. In particular, we focused on several prospective candidate small-molecules suggested to interact with the catalytic domain of histone lysine demethylase KDM5B, which is ubiquitously expressed in the testis/spermatozoa of both mice and human.

A sirtuin activator and an anti-inflammatory molecule-multifaceted roles of adjudin and its potential applications for aging-related diseases.

Adjudin was originally developed as an improved analog of lonidamine to serve as a non-hormonal reversible male contraceptive that could cause exfoliation of the immature sperms from the seminiferous epithelium. Recently, the functionality spectrum of adjudin expands beyond as an anti-spermatogenic agent, namely, it could function as an anti-cancer drug potentially useful for combination chemotherapy, and as an anti-inflammatory molecule that could protect against ischemic stroke injury. Most strikingly, adjudin acts through activation of mitochondrion-located Sirt3 to safeguard hair cells of the cochlea from ototoxicant such as gentamycin. Recent studies also indicate that adjudin could attenuate oxidative stress and cellular senescence. These findings suggest wider applications of this small molecule, particularly in aging-related diseases.

Adjudin protects blood-brain barrier integrity and attenuates neuroinflammation following intracerebral hemorrhage in mice.

Secondary brain injury exacerbates neurological dysfunction and neural cell death following intracerebral hemorrhage (ICH), targeting the pathophysiological mechanism of the secondary brain injury holds promise for improving ICH outcomes. Adjudin, a potential male contraceptive, exhibits neuroprotective effects in brain injury disease models, yet its impact in the ICH model remains unknown. In this study, we investigated the effects of adjudin on brain injury in a mouse ICH model and explored its underlying mechanisms. ICH was induced in male C57BL/6 mice by injecting collagenase into the right striatum. Mice received adjudin treatment (50 mg/kg/day) for 3 days before euthanization and the perihematomal tissues were collected for further analysis. Adjudin significantly reduced hematoma volume and improved neurological function compared with the vehicle group. Western blot showed that Adjudin markedly decreased the expression of MMP-9 and increased the expression of tight junctions (TJs) proteins, Occludin and ZO-1, and adherens junctions (AJs) protein VE-cadherin. Adjudin also decreased the blood-brain barrier (BBB) permeability, as indicated by the reduced albumin and Evans Blue leakage, along with a decrease in brain water content. Immunofluorescence staining revealed that adjudin noticeably reduced the infiltration of neutrophil, activation of microglia/macrophages, and reactive astrogliosis, accompanied by an increase in CD206 positive microglia/macrophages which exhibit phagocytic characteristics. Adjudin concurrently decreased the generation of proinflammatory cytokines, such as TNF-α and IL-1ß. Additionally, adjudin increased the expression of aquaporin 4 (AQP4). Furthermore, adjudin reduced brain cell apoptosis, as evidenced by increased expression of anti-apoptotic protein Bcl-2, and decreased expression of apoptosis related proteins Bax, cleaved caspase-3 and fewer TUNEL positive cells. Our data suggest that adjudin protects against ICH-induced secondary brain injury and may serve as a potential neuroprotective agent for ICH treatment.

Adjudin prevents neuronal damage and neuroinflammation via inhibiting mTOR activation against pilocarpine-induced status epilepticus.

Inflammatory responses in the brain play an etiological role in the development of epilepsy, suggesting that finding novel molecules for controlling neuroinflammation may have clinical value in developing the disease-modifying strategies for epileptogenesis. Adjudin, a multi-functional small molecule compound, has pleiotropic effects, including anti-inflammatory properties. In the present study, we aimed to investigate the effects of adjudin on pilocarpine-induced status epilepticus (SE) and its role in the regulation of reactive gliosis and neuroinflammation. SE was induced in male C57BL/6 mice that were then treated with adjudin (50 mg/kg) for 3 days after SE onset. Immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and western blot analysis were used to evaluate the effects of adjudin treatment in the hippocampus after SE. Our results showed that adjudin treatment significantly mitigated apoptotic cell death in the hippocampus after SE onset. Moreover, adjudin treatment suppressed SE-induced glial activation and activation of mammalian target of rapamycin signaling in the hippocampus. Concomitantly, adjudin treatment significantly reduced SE-induced inflammatory processes, as confirmed by changes in the expression of inflammatory mediators such as tumor necrosis factor-α, interleukin-1ß, and arginase-1. In conclusion, these findings suggest that adjudin may serve as a potential neuroprotective agent for preventing pathological mechanisms implicated in epileptogenesis.

Cell-Cell Interaction-Mediated Signaling in the Testis Induces Reproductive Dysfunction-Lesson from the Toxicant/Pharmaceutical Models.

Emerging evidence has shown that cell-cell interactions between testicular cells, in particular at the Sertoli cell-cell and Sertoli-germ cell interface, are crucial to support spermatogenesis. The unique ultrastructures that support cell-cell interactions in the testis are the basal ES (ectoplasmic specialization) and the apical ES. The basal ES is found between adjacent Sertoli cells near the basement membrane that also constitute the blood-testis barrier (BTB). The apical ES is restrictively expressed at the Sertoli-spermatid contact site in the apical (adluminal) compartment of the seminiferous epithelium. These ultrastructures are present in both rodent and human testes, but the majority of studies found in the literature were done in rodent testes. As such, our discussion herein, unless otherwise specified, is focused on studies in testes of adult rats. Studies have shown that the testicular cell-cell interactions crucial to support spermatogenesis are mediated through distinctive signaling proteins and pathways, most notably involving FAK, Akt1/2 and Cdc42 GTPase. Thus, manipulation of some of these signaling proteins, such as FAK, through the use of phosphomimetic mutants for overexpression in Sertoli cell epithelium in vitro or in the testis in vivo, making FAK either constitutively active or inactive, we can modify the outcome of spermatogenesis. For instance, using the toxicant-induced Sertoli cell or testis injury in rats as study models, we can either block or rescue toxicant-induced infertility through overexpression of p-FAK-Y397 or p-FAK-Y407 (and their mutants), including the use of specific activator(s) of the involved signaling proteins against pAkt1/2. These findings thus illustrate that a potential therapeutic approach can be developed to manage toxicant-induced male reproductive dysfunction. In this review, we critically evaluate these recent findings, highlighting the direction for future investigations by bringing the laboratory-based research through a translation path to clinical investigations.

The Non-hormonal Male Contraceptive Adjudin Exerts its Effects via MAPs and Signaling Proteins mTORC1/rpS6 and FAK-Y407.

Adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly called AF-2364), is a nonhormonal male contraceptive, since it effectively induces reversible male infertility without perturbing the serum concentrations of follicle stimulating hormone (FSH), testosterone, and inhibin B based on studies in rats and rabbits. Adjudin was shown to exert its effects preferentially by perturbing the testis-specific actin-rich adherens junction (AJ) at the Sertoli-spermatid interface known as apical ectoplasmic specialization (apical ES), thereby effectively inducing spermatid exfoliation. Adjudin did not perturb germ cell development nor germ cell function. Also, it had no effects on Sertoli cell-cell AJ called basal ectoplasmic specialization (basal ES), which, together with tight junction constitute the blood-testis barrier (BTB), unless an acute dose of adjudin was used. Adjudin also did not perturb the population of spermatogonial stem cells nor Sertoli cells in the testis. However, the downstream signaling protein(s) utilized by adjudin to induce transient male infertility remains unexplored. Herein, using adult rats treated with adjudin and monitored changes in the phenotypes across the seminiferous epithelium between 6 and 96 h in parallel with the steady-state protein levels of an array of signaling and cytoskeletal regulatory proteins, recently shown to be involved in apical ES, basal ES and BTB function. It was shown that adjudin exerts its contraceptive effects through changes in microtubule associated proteins (MAPs) and signaling proteins mTORC1/rpS6 and p-FAK-Y407. These findings are important to not only study adjudin-mediated male infertility but also the biology of spermatogenesis.

Adjudin-loaded redox-sensitive paclitaxel-prodrug micelles for overcoming multidrug resistance with efficient targeted Colon cancer therapy.

Multidrug resistance (MDR) is the primary cause for the failure of chemotherapy in the treatment of colon cancer. Recent research has indicated that the combination of a chemotherapeutic agent and a mitochondrial inhibitor might represent a promising strategy to help overcome MDR. However, for this approach to be clinically effective, it is important that the two drugs can be actively and simultaneously delivered into tumor cells at an optimal ratio and completely released drug within cells. To address these challenges, we designed and prepared a folate receptor-targeted and redox-responsive drug delivery system (FA- ss -P/A) that was able to co-deliver paclitaxel (PTX) and adjudin (ADD) to reverse colon cancer MDR. The PTX prodrug was obtained by conjugating PTX to dextrin via a disulfide-linkage. Then, folic acid (FA) was modified on the PTX prodrug. Finally, ADD, a mitochondrial inhibitor, was encapsulated in the PTX prodrug-formed micelles. A series of in vitro and in vivo experiments subsequently demonstrated that FA- ss -P/A can effectively reverse MDR by increasing cell uptake, inhibiting PTX efflux, and improving drug release.

The novel platinum(IV) prodrug with self-assembly property and structure-transformable character against triple-negative breast cancer.

Herein, a small library of Pt(IV) prodrugs based on cisplatin and chemosensitizer adjudin (ADD) were explored for efficient cisplatin resistant triple-negative breast cancer (TNBC) treatment. We further elucidated the detail relationship of chemical structure, alkyl chain length (ethyl to dodecyl) and ADD substituted degree, with respect to the self-assembly ability and cytotoxic effect of prodrugs. It demonstrated that all prodrugs could self-assemble into nanomedicine, which was in consist with the molecule structure building and self-assembly simulation. All nanomedicines possessed small particle size, uniform morphology and ultra-high drug loading content (84.0%-86.5%). Moreover, the length of alkyl chain was of great importance for the structure-transformable character and cytotoxicity of nanomedicines. Interestingly, ADD monosubstituted with butyl or hexyl contralateral substituted prodrug (C4-Pt-ADD or C6-Pt-ADD) assembled nanomedicine could convert to wire or sheet structure. These transformable nanoparticles showed great potential in improving the sensitivity of cisplatin to TNBC with up to 266-fold lower IC50 value and significantly enhanced in vivo tumor growth inhibition. Therefore, the self-assembled nanomedicine based on Pt(IV)-ADD could be a promising strategy for TNBC therapy.

Modulating the Blood-Testis Barrier Towards Increasing Drug Delivery.

Testicular cells produce several biologically active peptides that exert their downstream effects by activating distinct signaling proteins. These biomolecules are now known to support spermatogenesis and effectively enhance paracellular and transcellular diffusion of drugs (e.g., adjudin) across the blood-testis barrier (BTB). We briefly discuss the biomolecules that maintain the BTB: these provide new insights into how the BTB can be modulated to allow therapeutic drugs, including male contraceptives, to be transported across the BTB and more generally across blood-tissue barriers. Information gleaned by studying the BTB, as well as other blood-tissue barriers, augments our understanding of blood-tissue barriers and provides new insights into how drugs can be delivered to organs that are effectively protected by tissue barriers.

Adjudin synergizes with paclitaxel and inhibits cell growth and metastasis by regulating the sirtuin 3-Forkhead box O3a axis in human small-cell lung cancer.

BACKGROUND: Small-cell lung cancer (SCLC), a malignant tumor, is usually widely metastatic when diagnosed. The lack of important therapeutic clinical advances makes it difficult to treat. Previous studies showed that Adjudin had anticancer effects in many other human cancers, and it was synergetic with cisplatin in non-small cell lung cancer. However, the mechanism on SCLC was unclear. METHODS: We investigated the potential mechanism and effect of Adjudin on SCLC both in vitro and in vivo. RESULTS: An SCLC xenograft model showed that Adjudin inhibited tumor growth and was significantly synergetic with paclitaxel (in vitro as well). Cell Counting Kit-8 assays, flow cytometric analysis and western blotting showed that Adjudin effectively suppressed SCLC cell proliferation by inducing S phase arrest and caspase-dependent apoptosis. Moreover, Transwell and scratch assays showed that Adjudin also effectively inhibited migration and invasion. Furthermore, Adjudin activated the sirtuin 3 (SIRT3)-Forkhead box O3a (FOXO3a) pathway. Downregulating SIRT3 or FOXO3a significantly attenuated Adjudin-induced anticancer effects. Furthermore, higher expression of SIRT3 and FOXO3a were positively correlated, and both were associated with longer survival in lung cancer patients. CONCLUSION: Overall, the present study is the first to show that Adjudin synergizes with paclitaxel and inhibits cell growth and metastasis by regulating the SIRT3-FOXO3a axis in SCLC; thus, Adjudin has great potential to be an anticancer agent.

mTORC1/rpS6 and spermatogenic function in the testis-insights from the adjudin model.

mTORC1/rpS6 signaling complex promoted Sertoli blood-testis barrier (BTB) remodeling by perturbing Sertoli cell-cell adhesion site known as the basal ectoplasmic specialization (ES). mTORC1/rpS6 complex also promoted disruption of spermatid adhesion at the Sertoli-spermatid interface called the apical ES. Herein, we performed analyses using the adjudin (a non-hormonal male contraceptive drug under development) model, wherein adjudin was known to perturb apical and basal ES function when used at high dose. Through direct administration of adjudin to the testis, adjudin at doses that failed to perturb BTB integrity per se, overexpression of an rpS6 phosphomimetic (i.e., constitutively active) mutant (i.e., p-rpS6-MT) that modified BTB function considerably potentiated adjudin efficacy. This led to disorderly spatial expression of proteins necessary to maintain the proper cytoskeletal organization of F-actin and microtubules (MTs) across the seminiferous epithelium, leading to germ cell exfoliation and aspermatogenesis. These findings yielded important insights regarding the role of mTORC1/rpS6 signaling complex in regulating BTB homeostasis.

Adjudin Attenuates Cerebral Edema and Improves Neurological Function in Mice with Experimental Traumatic Brain Injury.

Adjudin, a small molecular compound that is used as a male contraceptive, has been reported to play a neuroprotective role in an ischemic stroke injury model. However, its effect on traumatic brain injury (TBI) has not been assessed. Hence, we investigated the effects of adjudin on cerebral edema using a mouse model of TBI and explored the underlying mechanisms. Adult male C57BL/6 mice received controlled cortical impact (CCI) injury, then an injection of adjudin (50 mg/kg). The mice were euthanized 3 days post-CCI injury, and samples were collected for further analysis. Cultured primary mouse astrocytes were used for in vitro experiments. Adjudin treatment significantly attenuated cerebral edema on Day 3 and improved neurobehavioral outcomes on Days 3, 7, and 14 after CCI injury, compared with the vehicle group. Additionally, the evaluation of Evans blue extravasation and expression of tight junction proteins demonstrated remarkable effects of adjudin on blood-brain barrier protection. Further, adjudin treatment significantly decreased the gene and protein expression of aquaporin 4 in post-injury mice and inhibited progression of neuroinflammation in both mice and cultured astrocytes. The Western blot results of the peritraumatic protein samples demonstrated that adjudin significantly blocked the phosphorylation of IKKα, IκBα/ß, and NF-κB p65, which resulted in a reduction of NF-κB p65 nuclear translocation. In conclusion, adjudin attenuated the development of TBI-induced cerebral edema at least partly via anti-inflammatory effects and inhibition of the NF-κB pathway. These findings suggest that adjudin is a potential therapeutic intervention preventing the development of cerebral edema after TBI.

Sirt3 Mediates the Inhibitory Effect of Adjudin on Astrocyte Activation and Glial Scar Formation following Ischemic Stroke.

In response to stroke-induced injury, astrocytes can be activated and form a scar. Inflammation is an essential component for glial scar formation. Previous study has shown that adjudin, a potential Sirt3 activator, could attenuate lipopolysaccharide (LPS)- and stroke-induced neuroinflammation. To investigate the potential inhibitory effect and mechanism of adjudin on astrocyte activation, we used a transient middle cerebral artery occlusion (tMCAO) model with or without adjudin treatment in wild type (WT) and Sirt3 knockout (KO) mice and performed a wound healing experiment in vitro. Both our in vivo and in vitro results showed that adjudin reduced astrocyte activation by upregulating Sirt3 expression. In addition, adjudin treatment after stroke promoted functional and neurovascular recovery accompanied with the decreased area of glial scar in WT mice, which was blunted by Sirt3 deficiency. Furthermore, adjudin could increase Foxo3a and inhibit Notch1 signaling pathway via Sirt3. Both the suppression of Foxo3a and overexpression of N1ICD could alleviate the inhibitory effect of adjudin in vitro indicating that Sirt3-Foxo3a and Sirt3-Notch1 signaling pathways were involved in the inhibitory effect of adjudin in wound healing experiment.

Adjudin-preconditioned neural stem cells enhance neuroprotection after ischemia reperfusion in mice.

BACKGROUND: Transplantation of neural stem cells (NSCs) has been proposed as a promising therapeutic strategy for the treatment of ischemia/reperfusion (I/R)-induced brain injury. However, existing evidence has also challenged this therapy on its limitations, such as the difficulty for stem cells to survive after transplantation due to the unfavorable microenvironment in the ischemic brain. Herein, we have investigated whether preconditioning of NSCs with adjudin, a small molecule compound, could enhance their survivability and further improve the therapeutic effect for NSC-based stroke therapy. METHOD: We aimed to examine the effect of adjudin pretreatment on NSCs by measuring a panel of parameters after their transplantation into the infarct area of ipsilateral striatum 24 hours after I/R in mice. RESULTS: We found that pretreatment of NSCs with adjudin could enhance the viability of NSCs after their transplantation into the stroke-induced infarct area. Compared with the untreated NSC group, the adjudin-preconditioned group showed decreased infarct volume and neurobehavioral deficiency through ameliorating blood-brain barrier disruption and promoting the expression and secretion of brain-derived neurotrophic factor. We also employed H2O2-induced cell death model in vitro and found that adjudin preconditioning could promote NSC survival through inhibition of oxidative stress and activation of Akt signaling pathway. CONCLUSION: This study showed that adjudin could be used to precondition NSCs to enhance their survivability and improve recovery in the stroke model, unveiling the value of adjudin for stem cell-based stroke therapy.

Toxicants target cell junctions in the testis: Insights from the indazole-carboxylic acid model.

There are numerous types of junctions in the seminiferous epithelium which are integrated with, and critically dependent on the Sertoli cell cytoskeleton. These include the basal tight junctions between Sertoli cells that form the main component of the blood-testis barrier, the basal ectoplasmic specializations (basal ES) and basal tubulobulbar complexes (basal TBC) between Sertoli cells; as well as apical ES and apical TBC between Sertoli cells and the developing spermatids that orchestrate spermiogenesis and spermiation. These junctions, namely TJ, ES, and TBC interact with actin microfilament-based cytoskeleton, which together with the desmosomal junctions that interact with the intermediate filament-based cytoskeleton plus the highly polarized microtubule-based cytoskeleton are working in concert to move spermatocytes and spermatids between the basal and luminal aspect of the seminiferous epithelium. In short, these various junctions are structurally complexed with the actin- and microtubule-based cytoskeleton or intermediate filaments of the Sertoli cell. Studies have shown toxicants (e.g., cadmium, bisphenol A (BPA), perfluorooctanesulfonate (PFOS), phthalates, and glycerol), and some male contraceptives under development (e.g., adjudin, gamendazole), exert their effects, at least in part, by targeting cell junctions in the testis. The disruption of Sertoli-Sertoli cell and Sertoli-germ cell junctions, results in the loss of germ cells from the seminiferous epithelium. Adjudin, a potential male contraceptive under investigation in our laboratory, produces loss of spermatids from the seminiferous tubules through disruption of the Sertoli cell spermatid junctions and disruption of the Sertoli cell cytoskeleton. The molecular and structural changes associated with adjudin administration are described, to provide an example of the profile of changes caused by disturbance of Sertoli-germ cell and also Sertoli cell-cell junctions.

Adjudin disrupts spermatogenesis by targeting drug transporters: Lesson from the breast cancer resistance protein (BCRP).

For non-hormonal male contraceptives that exert their effects in the testis locally instead of via the hypothalamic-pituitary-testicular axis, such as adjudin that disrupts germ cell adhesion, a major hurdle in their development is to improve their bioavailability so that they can be efficiently delivered to the seminiferous epithelium by transporting across the blood-testis barrier (BTB). If this can be done, it would widen the gap between their efficacy and general toxicity. However, Sertoli cells that constitute the BTB, peritubular myoid cells in the tunica propria, germ cells at different stages of their development, as well as endothelial cells that constitute the microvessels in the interstitium are all equipped with multiple drug transporters, most notably efflux drug transporters, such as P-glycoprotein, multidrug resistance-related protein 1 (MRP1) and breast cancer resistance protein (BCRP) that can actively prevent drugs (e.g., adjudin) from entering the seminiferous epithelium to exert their effects. Recent studies have shown that BCRP is highly expressed by endothelial cells of the microvessels in the interstitium in the testis and also peritubular myoid cells in tunica propria even though it is absent from Sertoli cells at the site of the BTB. Furthermore, BCRP is also expressed spatiotemporally by Sertoli cells and step 19 spermatids in the rat testis and stage-specifically, limiting to stage VII‒VIII of the epithelial cycle, and restricted to the apical ectoplasmic specialization [apical ES, a testis-specific F-actin-rich adherens junction (AJ)]. Interestingly, adjudin was recently shown to be capable of downregulating BCRP expression at the apical ES. In this Opinion article, we critically discuss the latest findings on BCRP; in particular, we provide some findings utilizing molecular modeling to define the interacting domains of BCRP with adjudin. Based on this information, it is hoped that the next generation of adjudin analogs to be synthesized can improve their efficacy in downregulating BCRP and perhaps other drug efflux transporters in the testis to improve their efficacy to traverse the BTB by modifying their interacting domains.