Recent findings highlight NAD+ as a central regulator of various cellular processes, including energy metabolism, stress response, and aging. Growing evidence of the benefits associated with dietary NAD+ precursors has elevated NAD+ to a promising therapeutic target for addressing female infertility. This review aims to evaluate existing literature on the mechanisms governing the availability and utilization of NAD+ in the ovaries and its alterations in female reproductive disorders, with a particular focus on ovarian aging and dysfunction including polycystic ovary syndrome and premature ovarian insufficiency. Alongside data from in vivo and in vitro studies on various NAD+ boosters, this review incorporates findings from research on genetic mutations, polymorphisms in human and animal populations, and insights from transgenic animal models. The present work emphasizes that NAD+ deficiency is largely driven by a combination of factors, including heightened consumption, impaired utilization efficiency, and diminished biosynthesis or transport. Based on this analysis, we suggest that the ovary possesses its own unique NAD+ metabolism, but our understanding of its regulatory mechanisms is still in its infancy. Key questions remain unanswered, such as how NAD+ and its precursors are transported into oocytes and ovarian cells, their specific preferences for different NAD+ precursors, as well as the specific changes associated with different ovarian dysfunctions. Finally, we reviewed methods for studying NAD+ metabolism as essential tools for investigating the potential of NAD+ boosting therapies to counteract ovarian aging and dysfunction.

Rett syndrome (RTT), caused primarily by mutations in the X-linked MECP2 gene, is a neurodevelopmental disorder marked by systemic alterations, including mitochondrial dysfunction, chronic oxidative stress, and persistent subclinical inflammation. This OxInflammatory state suggests that disruption of redox-inflammatory regulatory pathways may contribute to disease pathophysiology. In this context, NF-κB and Nrf2 represent two important signaling regulators that operate in a coordinated crosstalk to balance inflammatory activation and antioxidant defense. In this study, we explored the functional status of NF-κB/Nrf2 crosstalk in primary dermal fibroblasts derived from RTT patients and healthy controls (CTR). Under basal conditions, RTT fibroblasts exhibited increased nuclear localization of NF-κB p65 and higher levels of acetylated NF-κB, indicating a constitutive inflammatory condition. In contrast, Nrf2 activation was not proportionally enhanced, suggesting an imbalance between inflammatory and antioxidant signaling. Following LPS stimulation, CTR fibroblasts displayed the expected coordinated activation of NF-κB and Nrf2 pathways, along with induction of downstream target genes. RTT fibroblasts, however, failed to activate either pathway and showed blunted transcriptional responses. These findings support the presence of a dysregulated signaling axis consistent with a chronic OxInflammatory state. Analysis of regulatory mechanisms revealed increased basal CBP/p300 levels in RTT cells without a compensatory increase in SIRT1, pointing toward altered acetylation dynamics that may favor persistent NF-κB activity. To mechanistically study the NF-κB/Nrf2 crosstalk, LPS-stimulated cells were treated with the Nrf2 activator sulforaphane (SFN), alone or combined with the NF-κB inhibitor BAY-117082. In RTT fibroblasts, these combined interventions significantly reduced pro-inflammatory cytokine expression and consistently enhanced HMOX1 transcription and HO-1 protein levels. Although nuclear localization changes were modest at the selected time point, downstream gene expression patterns indicated that coordinated modulation of inflammatory and antioxidant pathways can partially rebalance cellular responses. Taken together, our findings provide preliminary evidence that RTT exhibits a dysfunctional NF-κB/Nrf2 regulatory axis characterized by basal inflammatory activation and impaired antioxidant compensation. Modulation of NF-κB signaling, in combination with Nrf2 activation, may represent a promising strategy to counteract the persistent OxInflammatory milieu associated with RTT and warrants further investigation.

Rett Syndrome (RTT) is a rare neurodevelopmental disorder, primarily affecting girls (1:10,000 live births), largely caused by mutations in the X-linked gene MECP2, an epigenetic regulator encoding for the methyl-CpG binding protein 2 (MeCP2). Recent evidence links ferroptosis, an iron-dependent cell death characterized by lipid peroxide accumulation, to neurodegenerative and neurodevelopmental disorders like autism. Several RTT hallmarks, including redox imbalance, excess labile iron, increased lipid peroxidation, and impaired antioxidant enzyme activity, align with ferroptosis characteristics. Therefore, we investigated ferroptosis's role in RTT using human primary fibroblasts from healthy and RTT subjects, treating them with ferroptosis inducers: erastin and RSL3. Our findings show RTT cells are highly susceptible to ferroptosis, marked by elevated lipid peroxidation and mitochondrial reactive oxygen species (mtROS) production, crucial for ferroptotic cell death. We also observed altered iron metabolism and dysregulated ferritinophagy. RTT fibroblasts exhibited an imbalanced antioxidant defense, particularly after ferroptotic stimuli, and ferroptosis inducers worsened redox imbalance compared to controls. Importantly, a ferroptosis inhibitor (Ferrostatin-1) and a SOD mimetic (mito-TEMPO) prevented these effects and normalized the altered basal conditions of RTT cells. In conclusion, our results reveal a general dysregulation in RTT cells contributing to increased ferroptosis sensitivity. This suggests a significant role for ferroptosis in RTT pathophysiology and progression, potentially opening new therapeutic avenues for this condition.

Rett syndrome (RTT), a neurodevelopmental disorder primarily affecting females, is characterized by mutations in the MECP2 gene, leading to systemic oxidative stress and mitochondrial dysfunction. This study investigates the role of Coenzyme Q10 (CoQ10), particularly its reduced form ubiquinol, in modulating oxidative stress and mitochondrial function in primary dermal fibroblasts derived from RTT patients with distinct MeCP2 mutations. Baseline assessments revealed significant CoQ10 deficiencies and elevated reactive oxygen species (ROS) levels, notably in fibroblasts with the T158M mutation. Ubiquinol supplementation effectively restored CoQ10 levels and improved redox balance in these cells. Additionally, treatment influenced mitochondrial dynamics, as evidenced by alterations in the expression of fission and fusion proteins and modulated the activity of paraoxonase 2 (PON2), an enzyme involved in cellular antioxidant defense. In conclusion, our data suggest that CoQ10 supplementation could mitigate oxidative damage and preserve mitochondrial integrity, but we are far from being able to claim that it can represents an effective therapeutic strategy to complement current pharmacological treatments in RTT patients. Further research is warranted to explore the potential of CoQ10 as an adjunctive treatment, particularly during the early stages of RTT.

Recent studies emphasize the role of neuroendocrine dysfunctions and sirtuins in polycystic ovarian syndrome (PCOS). We investigated whether altered SIRT1 and SIRT3 levels contribute to brain changes and oxidative stress, identifying these pathways as potential therapeutic targets for PCOS-related complications. Using a DHEA-induced PCOS mouse model, we examined brain expression of pathways related to SIRT1 and SIRT3 and to oxidative/glycative stress changes. SH-SY5Y cells treated with DHEA were used to confirm direct neuronal effects. We found decreased levels of Sirt1 and Sirt3 transcripts but increased protein expression and activity of both sirtuins in brains of DHEA-treated mice. The DHEA group showed elevated oxidative and glycative stress, including an overall increased lipid peroxidation and DNA damage, as well as accumulation of advanced glycation endproducts (AGEs) in isocortices. Differences in Cpt1 isoform expressions suggested disrupted metabolic processing in the PCOS brains. Neuronal degeneration was also observed, alongside unchanged Bdnf and TrkB mRNA levels in DHEA brains. Exposure of differentiated SH-SY5Y neuron-like cells to high concentrations (≥ 100 µM) led to increased oxidative stress, altered sirtuins expression, and ultimately cell toxicity. While low concentrations of DHEA (1 µM) did not elicit such responses. These findings reveal a complex interplay between oxidative stress, metabolic dysregulation, and neuronal health in PCOS brain, underscoring the need for further investigations into the underlying mechanisms, including research in genetic components. This research provides foundational insights into how PCOS may influence neurobiological processes and helps clarify some aspects of its pathogenesis.

Abstract Study question Are nano-microplastics (NMPs) taken up into human granulosa cells causing redox alterations? Summary answer NMPs enter into human granulosa cells and influence viability, energy production and antioxidant response. What is known already In recent decades, anthropogenic activities have increased the production of global plastic with millions of tons produced every year. Under the action of different physical, chemical or biological agents, plastic waste breaks down into (NMP) particles that propagate in the environment posing threats for human health. NMPs translocate from digestive tract to circulatory systems reaching organs and cells, including female gonads. Nevertheless, there is insufficient knowledge of their effects on reproductive functions in mammals to allow for an accurate risk assessment to be conducted and any risks managed in animal and human being contexts. Study design, size, duration Human ovarian granulosa cells (KGN cell line) were exposed to NMPs of different sizes (40 nm, 70 nm, 100 nm, and 200 nm) at concentrations ranging from 5 to 1000 μg/ml for 24 h and then processed for cellular and biochemical analyses Participants/materials, setting, methods NMPs uptake by granulosa cells was tested by the employment of fluorescent NMPs and observation under confocal laser scanning microscopy. Cell viability was assed by Cell Counting Kit8. The effects of NMPs on mitochondrial bioenergetics was evaluated by MitoStress kit (Seahorse Xfe96, Agilent). ATP production was evaluated by Cell Titer-Glo ATP assay kit (Promega). Key enzymes of antioxidant response were analysed at transcript and protein level by using real-time Taqman PCR and Western blotting, respectively. Main results and the role of chance We demonstrated that NMPs are taken up by granulosa cells at all tested concentrions along with significant decrease of cell vitality with all sizes and concentrations. Seahorse analysis revealed an altered bioenergetic profiles. ATP levels increase at 70, 100 and 200 nm (one way ANOVA p = 0.001) at all tested concentrations (one way ANOVA p < 0.001). The gene and protein expression of catalase (CAT) superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) increased at 5 and 100 µg/ml with all sizes except 100 nm, whereas the level of sirtuins (SIRT1 and SIRT3) transcripts and protein decreased. Reduced levels of phosphorylated NRF2, the transcription factor that activates antioxidant responsive elements (ARE)-mediated gene expression were observed. Limitations, reasons for caution The use of human cell lines requires careful consideration when transferring to human testing. Wider implications of the findings Our results contribute to understanding the effects of NMPs on mammalian fertility in order to find possible protective approaches. Considering that the level of susceptibility of mammalian female germ cells to NMPs is still unknown, present results contribute to evidence-based strategies allowing living and working in a health promoting environment. Trial registration number Effects of combined exposure to nano/microplastics and plastic additives on mammalian female fertility (CUP E53D2301100006)

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication and the presence of restricted interests and repetitive behavior. To date, no single cause has been demonstrated but both genetic and environmental factors are believed to be involved in abnormal brain development. In recent years, immunological and mitochondrial dysfunctions acquired particular interest in the study of the molecular mechanisms underlying the pathophysiology of ASD. For this reason, our study focused on evaluating the mitochondrial component and activation of the NLRP3 inflammasome, a critical player of the innate immune system. The assembly of NLRP3 with ASC mediates activation of Caspase-1, which in turn, by proteolytic cleavage, activates Gasdermin D and the proinflammatory cytokines IL-1β/IL-18 with their subsequent secretion. Using primary fibroblasts of autistic and control patients we studied basal and stimulated conditions. Specifically, LPS and ATP were used to activate the NLRP3 inflammasome and MCC950 for its inhibition. In addition, FCCP was used as a mitochondrial stressor and MitoTEMPO as a scavenger of mitochondrial ROS. Our results showed a hyperactivation of NLRP3 inflammasome in ASDs, as evidenced by the co-localization of the two main components, NLRP3 and ASC, by the higher levels of ASC specks, oligomers and dimers and by the increased amounts of active Caspase-1 and IL-1β. In addition, increased mitochondrial superoxide anion and reduced mitochondrial membrane potential were detected in ASD cells. These data are in accordance with the abnormal mitochondrial morphology evidenced by transmission electron microscopy analysis. Interestingly, NLRP3 inflammasome inhibition with MCC950 improved mitochondrial parameters, while the use of MitoTEMPO, in addition to decrease mitochondrial ROS production, was able to prevent NLRP3 inflammasome activation suggesting for the first time an abnormal bidirectional crosstalk between mitochondria and NLRP3 inflammasome in ASD.

To date, Rett syndrome (RTT), a genetic disorder mainly caused by mutations in the X-linked MECP2 gene, is increasingly considered a broad-spectrum pathology, instead of just a neurodevelopmental disease, due to the multitude of peripheral co-morbidities and the compromised metabolic pathways, affecting the patients. The altered molecular processes include an impaired mitochondrial function, a perturbed redox homeostasis, a chronic subclinical inflammation and an improper cholesterol metabolism.The persistent subclinical inflammatory condition was first defined ten years ago, as a previously unrecognized feature of RTT, playing a role in the pathology progress and modulation of phenotypical severity. In light of this, the present work aims at reviewing the current knowledge on the chronic inflammatory status and the altered immune/inflammatory functions in RTT, as well as investigating the emerging mechanisms underlying this condition with a special focus on the latest findings about inflammasome system, autoimmunity responses and intestinal micro- and mycobiota. On these bases, although further research is needed, future therapeutic strategies able to re-establish an adequate immune/inflammatory response could represent potential approaches for RTT patients.