Epidemiological data from assisted reproductive technologies (ART) link vitamin B12 (VitB12) deficiency to shorter gestation, low birth weight, and reduced live birth rates. VitB12 also plays a critical role in fertility for both sexes by supporting gamete quality. Despite some contradictions, emerging evidence suggests early-life VitB12 deficiency may affect learning and cognitive development. Collectively, these findings suggest that VitB12 is a key determinant of both reproductive and developmental health. Mechanistically, VitB12 acts as a coenzyme in one-carbon metabolism, and its deficiency leads to elevated homocysteine (Hcy) and methylmalonic acid (MMA) levels, promoting oxidative stress and thereby impairing fertility. Although this remains the most established mechanism, other yet unidentified pathways may also mediate the effects of VitB12 on reproductive function. While the effects of severe VitB12 deficiency are well established, mild deficiencies may develop silently in individuals following plant-based diets or those with gastrointestinal disorders. Plant-based sources of VitB12 remain controversial, as many provide only trace amounts or biologically inactive analogs such as pseudovitamin B12, which may interfere with absorption. Further research is needed to evaluate their bioavailability and clinical effectiveness. This review synthesizes clinical evidence, mechanistic insights, and dietary considerations to highlight how VitB12 status shapes reproductive health.

IntroductionPeroxisomes are highly dynamic organelles that contribute to cellular homeostasis by coordinating lipid metabolism, reactive oxygen species handling, and adaptive responses to metabolic stress. Their plasticity is particularly relevant in the nervous system, where peroxisomes cooperate with mitochondria to maintain redox and metabolic balance in neuronal and glial cells. Peroxisome proliferator-activated receptor alpha (PPARα), together with its coactivator PGC-1α, represents a major transcriptional regulator of peroxisomal and mitochondrial metabolic programs.MethodsBV2 microglial cells were exposed to rotenone to investigate how mitochondrial oxidative stress influences the peroxisomal compartment. Mitochondrial and peroxisomal morphology, intracellular distribution, oxidative damage, and the expression of proteins involved in peroxisomal metabolism and PPARα signaling were evaluated.ResultsRotenone induced a stress-associated microglial phenotype characterized by cytoskeletal remodeling, mitochondrial network disruption, and oxidative damage. These alterations were accompanied by a coordinated reorganization of the peroxisomal compartment, including increased abundance of peroxisomal membrane markers, redistribution of peroxisomes toward the perinuclear region, and enhanced expression of enzymes associated with peroxisomal fatty acid oxidation. An increased spatial association between peroxisomes and mitochondria was also observed, suggesting that peroxisomal remodeling forms part of a broader inter-organelle adaptation to mitochondrial dysfunction. These changes occurred together with increased nuclear phosphorylated PPARα and enhanced PGC-1α immunoreactivity, supporting the engagement of a PPARα–PGC-1α-related transcriptional response.DiscussionOverall, our findings suggest that mitochondrial dysfunction in microglial cells activates a PPARα-associated adaptive program that promotes peroxisomal remodeling and strengthens mitochondria–peroxisome coordination. This response may represent an attempt to preserve lipid and redox homeostasis during cellular stress and identifies PPARα-regulated peroxisomal pathways as potentially relevant components of microglial metabolic adaptation in neurodegenerative conditions.

Background/Objectives: Parkinson’s disease (PD) is one of the most prevalent neurodegenerative disorders. Despite its multifactorial etiology, PD pathophysiology shared specific features such as cytoplasmic α-synuclein inclusions, oxidative stress, mitochondrial dysfunction, and impaired autophagy. Bromodomain and Extra-Terminal domain (BET) proteins, functioning as epigenetic readers, have recently emerged as promising therapeutic targets due to their regulatory role in redox homeostasis, neuroinflammation, and autophagy. However, their potential involvement in PD pathophysiology remains largely unexplored. Therefore, we aimed at evaluating whether BET modulation could ameliorate the parkinsonian phenotype in two cellular models. Methods: Differentiated SH-SY5Y and N1E-115 neuronal cells were exposed to rotenone toxin to mimic PD phenotype and co-treated with the small BET inhibitor JQ1. Results: BET inhibition significantly counteracted rotenone-induced cell death, neuromorphological alterations, and α-synuclein accumulation. These protective effects were accompanied by restoration of redox balance, as indicated by enhanced activation of the antioxidant system and suppression of the pro-oxidant NADPH oxidase complex. Moreover, JQ1 treatment alleviated mitochondrial dysfunction and corrected autophagy impairments triggered by rotenone. Conclusions: These data highlight a novel role for BET proteins in neurodegeneration, suggesting that their modulation may represent a promising approach to counteract PD neuropathology.

Introduction:Parkinson's disease (PD) is a progressive neurodegenerative disorder, characterized by dopaminergic neuronal loss, mitochondrial dysfunction, oxidative stress, and chronic neuroinflammation. Microglial activation plays a primary role in disease progression, by amplifying inflammatory response and redox imbalance. The low-affinity neurotrophin receptor p75NTR has recently emerged as a potential therapeutic target in neurodegenerative disorders, due to its involvement in cell survival, apoptosis, and inflammatory signaling. In the present study, we investigated whether pharmacological modulation of p75NTR by LM11A-31 could protect microglial cells against Rotenone (Rot)-induced toxicity, a widely used tool to mimic PD. Methods:BV2 microglial cells were exposed to 50 nM Rot in the presence or absence of 0.5 μM LM11A-31. Cell viability, apoptotic signaling, oxidative stress, inflammatory response, and cytoskeletal organization were evaluated using immunofluorescence, Western blotting, TUNEL assay, and Scanning Electron Microscopy (SEM). Results:Rot exposure significantly increased p75NTR expression and induced marked microglial dysfunction, characterized by activation of apoptosis, oxidative stress, and inflammatory phenotype. LM11A-31 treatment improved cell survival and reduced apoptotic features, as shown by decreased TUNEL+ cells and cleaved caspase-3 immunoreactivity. In parallel, LM11A-31 restored microglial morphology and cytoskeletal integrity, also reducing ultrastructural alterations induced by Rot. Moreover, p75NTR modulation significantly blunted microglial activation markers, including Iba1 and CD68. LM11A-31 also reduced oxidative stress by limiting NOX-related signaling and lipid peroxidation, while partially restoring antioxidant defenses through modulation of Nrf2, PPARα, and glutathione-associated pathways. Conclusion:Our findings demonstrate that pharmacological modulation of p75NTR by LM11A-31 protects microglial cells against Rot-induced cytotoxicity and inflammatory activation. These protective effects involve the rescue of redox balance, suppression of pro-inflammatory signaling, preservation of cytoarchitecture, also resulting in increased cell survival. Overall, targeting p75NTR may represent a promising therapeutic strategy to counteract microglial dysfunction and neuroinflammatory processes associated with Parkinson's disease.

Protocadherin 19 (PCDH19) is an adhesion molecule involved in cell-cell interaction whose mutations cause a drug-resistant form of epilepsy, named PCDH19-Clustering Epilepsy (PCDH19-CE, MIM 300088). The mechanism by which altered PCDH19 function drive pathogenesis is not yet fully understood. Our previous work showed that PCDH19 dysfunction is associated with altered orientation of the mitotic spindle and accelerated neurogenesis, suggesting a contribution of altered cytoskeleton organization in PCDH19-CE pathogenesis in the control of cell division and differentiation. Here, we evaluate the consequences of altered PCDH19 function on microfilaments and microtubules organization, using a disease model obtained from patient-derived induced pluripotent stem cells. We show that iPSC-derived cortical neurons are characterized by altered cytoskeletal dynamics, suggesting that this protocadherin has a role in modulating stability of MFs and MTs. Consistently, the levels of acetylated-tubulin, which is related with stable MTs, are significantly increased in cortical neurons derived from the patient’s iPSCs compared to control cells, supporting the idea that the altered dynamics of the MTs depends on their increased stability. Finally, performing live-imaging experiments using fluorescence recovery after photobleaching and by monitoring GFP-tagged end binding protein 3 (EB3) “comets,” we observe an impairment of the plus-end polymerization speed in PCDH19-mutated cortical neurons, therefore confirming the impaired MT dynamics. In addition to altering the mitotic spindle formation, the present data unveil that PCDH19 dysfunction leads to altered cytoskeletal rearrangement, providing therapeutic targets and pharmacological options to treat this disorder.

The p75 neurotrophin receptor (p75NTR) plays a dual role in regulating both pro-survival and pro-apoptotic cascades in various physiological and pathological conditions, including within dopaminergic neuronal population. Notably, its overexpression has been documented in post-mortem Parkinson's disease (PD) brains, where it correlates with a significant downregulation in neuroprotective intracellular mediators. In this study, we aimed at investigating the neuroprotective effects of p75NTR modulation by the small molecule LM11A-31 in a rotenone-induced neuronal model of PD. Differentiated SH-SY5Y cells were treated with 100 nM rotenone, with or without 500 nM LM11A-31. Our results show that LM11A-31 effectively mitigates PD phenotype by enhancing cell viability, reducing apoptosis, mitigating α-synuclein aggregation, and partially restoring neuromorphological features. Mitochondrial integrity was preserved, likely through the upregulation of transcription factors involved in mitochondrial biogenesis, namely PGC-1α and PPARs. LM11A-31 treatment also reduced oxidative damage to macromolecules, normalizing Nrf2 expression and enhancing protein S-glutathionylation. The antioxidant effect of p75NTR modulation may be partially attributed to the suppression of the NADPH oxidase regulatory subunits p22PHOX and p47PHOX. Additionally, LM11A-31 restored cholesterol homeostasis disrupted by rotenone, as evidenced by the increased NPC1 expression and lysosomal localization, normalized HMGCR levels, and reduced intracellular cholesterol accumulation. Collectively, these findings demonstrate that p75NTR modulation via LM11A-31 exerts neuroprotective effects by targeting key pathological features of PD, including oxidative damage, mitochondrial derangements, and cholesterol dysmetabolism, supporting its potential as a promising therapeutic tool in PD treatment.

Pathological tau aggregates cause cognitive decline in neurodegenerative tauopathies, including Alzheimer’s disease (AD). These aggregates are prevalent within intracellular compartments. Current tau immunotherapies have shown limited efficacy in clearing intracellular tau aggregates and improving cognition in clinical trials. In this study, we developed toxic tau conformation–specific monoclonal antibody-2 (TTCM2), which selectively recognized pathological tau aggregates in brain tissues from patients with AD, dementia with Lewy bodies (DLB), and progressive supranuclear palsy (PSP). TTCM2 potently inhibited tau-seeding activity, an essential mechanism underlying tauopathy progression. To effectively target intracellular tau aggregates and ensure rapid delivery to the brain, TTCM2 was loaded in micelles (TTCM2-ms) and administered through the intranasal route. We found that intranasally administered TTCM2-ms efficiently entered the brain in hTau-tauopathy mice, targeting pathological tau in intracellular compartments. Moreover, a single intranasal dose of TTCM2-ms effectively cleared pathological tau, elevated synaptic proteins, and improved cognitive functions in aged tauopathy mice. Mechanistic studies revealed that TTCM2-ms cleared intracellular, synaptic, and seed-competent tau aggregates through tripartite motif-containing 21 (TRIM21), an intracellular antibody receptor and E3 ubiquitin ligase known to facilitate proteasomal degradation of cytosolic antibody-bound proteins. TRIM21 was found to be essential for TTCM2-ms–mediated clearance of tau pathology. Our study collectively provides evidence of the effectiveness of nasal tau immunotherapy in targeting and clearing intracellular tau pathology through TRIM21 and enhancing cognition in aged tauopathy mice. This study could be valuable in designing effective tau immunotherapies for AD and other tauopathies.

Amyotrophic Lateral Sclerosis (ALS) is considered the prototype of motor neuron disease, characterized by motor neuron loss and muscle waste. A well-established pathogenic hallmark of ALS is mitochondrial failure, leading to bioenergetic deficits. So far, pharmacological interventions for the disease have proven ineffective. Trimetazidine (TMZ) is described as a metabolic modulator acting on different cellular pathways. Its efficacy in enhancing muscular and cardiovascular performance has been widely described, although its molecular target remains elusive. We addressed the molecular mechanisms underlying TMZ action on neuronal experimental paradigms. To this aim, we treated murine SOD1G93A-model-derived primary cultures of cortical and spinal enriched motor neurons, as well as a murine motor-neuron-like cell line overexpressing SOD1G93A, with TMZ. We first characterized the bioenergetic profile of the cell cultures, demonstrating significant mitochondrial dysfunction that is reversed by acute TMZ treatments. We then investigated the effect of TMZ in promoting autophagy processes and its impact on mitochondrial morphology. Finally, we demonstrated the effectiveness of TMZ in terms of the mitochondrial functionality of ALS-rpatient-derived peripheral blood mononuclear cells (PBMCs). In summary, our results emphasize the concept that targeting mitochondrial dysfunction may represent an effective therapeutic strategy for ALS. The findings demonstrate that TMZ enhances mitochondrial performance in motor neuron cells by activating autophagy processes, particularly mitophagy. Although further investigations are needed to elucidate the precise molecular pathways involved, these results hold critical implications for the development of more effective and specific derivatives of TMZ for ALS treatment.

Riboflavin Transporter Deficiency (RTD) is a rare genetic, childhood-onset disease. This pathology has a relevant neurological involvement, being characterized by motor symptoms, ponto-bulbar paralysis and sensorineural deafness. Such clinical presentation is associated with muscle weakness and motor neuron (MN) degeneration, so that RTD is considered part of the MN disease spectrum. Based on previous findings demonstrating energy dysmetabolism and mitochondrial impairment in RTD induced Pluripotent Stem cells (iPSCs) and iPSC-derived MNs, here we address the involvement of intrinsic apoptotic pathways in disease pathogenesis using these patient-specific in vitro models by combined ultrastructural and confocal analyses. We show impaired neuronal survival of RTD iPSCs and MNs. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) documents severe alterations in patients’ cells, including deranged mitochondrial ultrastructure, and altered plasma membrane and nuclear organization. Occurrence of aberrantly activated apoptosis is confirmed by immunofluorescence and TUNEL assays. Overall, our work provides evidence of a role played by mitochondrial dysfunction in RTD, and identifies neuronal apoptosis as a contributing event in disease pathogenesis, indicating intrinsic apoptosis pathways as possible relevant targets for more effective therapeutical approaches.

Vitamin B12 (VitB12) is a micronutrient and acts as a cofactor for fundamental biochemical reactions: the synthesis of succinyl-CoA from methylmalonyl-CoA and biotin, and the synthesis of methionine from folic acid and homocysteine. VitB12 deficiency can determine a wide range of diseases, including nervous system impairments. Although clinical evidence shows a direct role of VitB12 in neuronal homeostasis, the molecular mechanisms are yet to be characterized in depth. Earlier investigations focused on exploring the biochemical shifts resulting from a deficiency in the function of VitB12 as a coenzyme, while more recent studies propose a broader mechanism, encompassing changes at the molecular/cellular levels. Here, we explore existing study models employed to investigate the role of VitB12 in the nervous system, including the challenges inherent in replicating deficiency/supplementation in experimental settings. Moreover, we discuss the potential biochemical alterations and ensuing mechanisms that might be modified at the molecular/cellular level (such as epigenetic modifications or changes in lysosomal activity). We also address the role of VitB12 deficiency in initiating processes that contribute to nervous system deterioration, including ROS accumulation, inflammation, and demyelination. Consequently, a complex biological landscape emerges, requiring further investigative efforts to grasp the intricacies involved and identify potential therapeutic targets.

Neurotrophins play pivotal roles in the development and proper functioning of the nervous system. The effects of these growth factors are mediated through the binding of high-affinity receptors, known as Trks, as well as the low-affinity receptor p75NTR. The latter has the capacity to induce complex signal pathways, as it favors both pro-survival and pro-apoptotic cascades depending on the physiopathological condition. Recent findings have indicated that p75NTR expression is increased in post-mortem Parkinson's disease (PD) brains, and this upregulation is associated with a significant reduction in neuroprotection. Given its double-edged sword nature, p75NTR has recently been identified as a promising therapeutic target to counteract neurodegenerative events. The present study aims to assess the neuroprotective effects of p75NTR modulation in a rotenone-induced neuronal model of PD. To this end, differentiated SH-SY5Y cells were exposed to rotenone to mimic the PD phenotype, and the small molecule LM11A-31 was used to modulate p75NTR activity. The main results revealed that LM11A-31 significantly mitigated the hallmarks of PD, including cell death, neuromorphological aberrations, and α-synuclein accumulation. Pharmacological manipulation of p75NTR also reduced oxidative damage by increasing the expression of transcriptional factors that regulate the antioxidant response and by decreasing the expression of the pro-oxidant NADPH-oxidase modulatory subunits. Furthermore, LM11A-31 hampered cholesterol buildup induced by rotenone, normalizing the expression of proteins involved in cholesterol biosynthesis, uptake and intracellular trafficking. Taken together, these findings suggest that p75NTR modulation may represent a novel approach to counteracting PD abnormalities of redox and cholesterol metabolism. ### Competing Interest Statement The authors have declared no competing interest.

Aberrant response to physiological cell stress is part of the mechanisms underlying the development of diverse human diseases, including neuropathologies. Neuroglobin (NGB), an intracellular monomeric globin, has gained attention for its role in endogenous stress response pathways in neuroprotection. To date, evidence supports the concept of NGB as an inducible protein, triggered by physiological and pathological stimuli via transcriptional and/or post-transcriptional mechanisms, offering cell-autonomous neuroprotective functions under various cellular stresses. Notably, recent evidence suggests the extracellular occurrence of NGB. We aimed to explore whether NGB redistribution in the cell microenvironment may serve in transmitting resilience capability in a model with neuronal characteristics. Results obtained in SH-SY5Y demonstrated that intracellular NGB upregulation is associated with the promotion of the extracellular release of the globin. Additionally, cell secretome from NGB-overexpressing cells, characterized by globin accumulation, exhibits protective effects against oxidative stress and mitochondrial toxicity, as evidenced by reduced apoptosis and preserved mitochondrial structure. These findings shed light on the potential significance of extracellular NGB as part of a common cell response to physiological and stress conditions and as a factor promoting cell resilience. Furthermore, the potential for neuroprotection of extracellular NGB paves the way for future therapeutic opportunities.

BET proteins function as histone code readers of acetylated lysins that determine the positive regulation in transcription of genes involved in cell cycle progression, differentiation, inflammation, and many other pathways. In recent years, thanks to the development of BET inhibitors, interest in this protein family has risen for its relevance in brain development and function. For example, experimental evidence has shown that BET modulation affects neuronal activity and the expression of genes involved in learning and memory. In addition, BET inhibition strongly suppresses molecular pathways related to neuroinflammation. These observations suggest that BET modulation may play a critical role in the onset and during the development of diverse neurodegenerative and neuropsychiatric disorders, such as Alzheimer's disease, fragile X syndrome, and Rett syndrome. In this review article, we summarize the most recent evidence regarding the involvement of BET proteins in brain physiology and pathology, as well as their pharmacological potential as targets for therapeutic purposes.

The relationships between motor neurons and the skeletal muscle during development and in pathologic contexts are addressed in this Chapter.We discuss the developmental interplay of muscle and nervous tissue, through neurotrophins and the activation of differentiation and survival pathways. After a brief overview on muscular regulatory factors, we focus on the contribution of muscle to early and late neurodevelopment. Such a role seems especially intriguing in relation to the epigenetic shaping of developing motor neuron fate choices. In this context, emphasis is attributed to factors regulating energy metabolism, which may concomitantly act in muscle and neural cells, being involved in common pathways.We then review the main features of motor neuron diseases, addressing the cellular processes underlying clinical symptoms. The involvement of different muscle-associated neurotrophic factors for survival of lateral motor column neurons, innervating MyoD-dependent limb muscles, and of medial motor column neurons, innervating Myf5-dependent back musculature is discussed. Among the pathogenic mechanisms, we focus on oxidative stress, that represents a common and early trait in several neurodegenerative disorders. The role of organelles primarily involved in reactive oxygen species scavenging and, more generally, in energy metabolism-namely mitochondria and peroxisomes-is discussed in the frame of motor neuron degeneration.We finally address muscular involvement in amyotrophic lateral sclerosis (ALS), a multifactorial degenerative disorder, hallmarked by severe weight loss, caused by imbalanced lipid metabolism. Even though multiple mechanisms have been recognized to play a role in the disease, current literature generally assumes that the primum movens is neuronal degeneration and that muscle atrophy is only a consequence of such pathogenic event. However, several lines of evidence point to the muscle as primarily involved in the disease, mainly through its role in energy homeostasis. Data from different ALS mouse models strongly argue for an early mitochondrial dysfunction in muscle tissue, possibly leading to motor neuron disturbances. Detailed understanding of skeletal muscle contribution to ALS pathogenesis will likely lead to the identification of novel therapeutic strategies.

Breast cancer is the first leading tumor in women in terms of incidence worldwide. Seventy percent of cases are estrogen receptor (ER) α-positive. In these malignancies, 17β-estradiol (E2) via ERα increases the levels of neuroglobin (NGB), a compensatory protein that protects cancer cells from stress-induced apoptosis, including chemotherapeutic drug treatment. Our previous data indicate that resveratrol (RSV), a plant-derived polyphenol, prevents E2/ERα-induced NGB accumulation in this cellular context, making E2-dependent breast cancer cells more prone to apoptosis. Unfortunately, RSV is readily metabolized, thus preventing its effectiveness. Here, four different RSV analogs have been developed, and their effect on the ERα/NGB pathway has been compared with RSV conjugated with highly hydrophilic gold nanoparticles as prodrug to evaluate if RSV derivatives maintain the breast cancer cells’ susceptibility to the chemotherapeutic drug paclitaxel as the original compound. Results demonstrate that RSV conjugation with gold nanoparticles increases RSV efficacy, with respect to RSV analogues, reducing NGB levels and enhancing the pro-apoptotic action of paclitaxel, even preventing the anti-apoptotic action exerted by E2 treatment on these cells. Overall, RSV conjugation with gold nanoparticles makes this complex a promising agent for medical application in breast cancer treatment.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase holoenzyme, which adds telomeric DNA repeats on chromosome ends to counteract telomere shortening. In addition, there is evidence of TERT non-canonical functions, among which is an antioxidant role. In order to better investigate this role, we tested the response to X-rays and H2O2 treatment in hTERT-overexpressing human fibroblasts (HF-TERT). We observed in HF-TERT a reduced induction of reactive oxygen species and an increased expression of the proteins involved in the antioxidant defense. Therefore, we also tested a possible role of TERT inside mitochondria. We confirmed TERT mitochondrial localization, which increases after oxidative stress (OS) induced by H2O2 treatment. We next evaluated some mitochondrial markers. The basal mitochondria quantity appeared reduced in HF-TERT compared to normal fibroblasts and an additional reduction was observed after OS; nevertheless, the mitochondrial membrane potential and morphology were better conserved in HF-TERT. Our results suggest a protective function of TERT against OS, also preserving mitochondrial functionality.

The existence of individuals who remain cognitively intact despite presenting histopathological signs of Alzheimer's disease (AD), here referred to as “Nondemented with AD neuropathology” (NDAN), suggests that some mechanisms are triggered to resist cognitive impairment. Exposed phosphatidylserine (ePS) represents a neuronal “eat‐me” signal involved in microglial‐mediated phagocytosis of damaged synapses. A possible mediator of this process is TREM2, a microglial surface receptor activated by ligands including PS. Based on TREM2 role in the scavenging function of microglia, we hypothesize that an efficient microglial phagocytosis of damaged synapses underlies synaptic resilience in NDAN, thus protecting from memory deficits. Using immunofluorescence microscopy, we performed a comparative study of human post‐mortem frontal cortices of aged‐matched, AD and NDAN individuals. We studied the distribution of activated microglia (IBA1, IBA1+/CD68+ cells) and phagocytic microglia‐related proteins (TREM2, DAP12), demonstrating higher microglial activation and TREM2 expression in NDAN versus AD. A study of the preservation of synapses around plaques, assessed using MAP2 and βIII tubulin as dendritic and axonal markers, respectively, and PSD95 as a postsynaptic marker, revealed preserved axonal/dendritic structure around plaques in NDAN versus AD. Moreover, high levels of PSD95 around NDAN plaques and the colocalization of PSD95 with CD68 indicated a prompt removal of damaged synapses by phagocytic microglia. Furthermore, Annexin V assay on aged‐matched, AD and NDAN individuals synaptosomes revealed increased levels of ePS in NDAN, confirming damaged synapses engulfment. Our results suggest a higher efficiency of TREM2‐induced phagocytic microglia in removing damaged synapses, underlying synaptic resilience in NDAN individuals.

Riboflavin transporter deficiency (RTD) is a rare genetic disorder characterized by motor, sensory and cranial neuropathy. This childhood-onset neurodegenerative disease is caused by biallelic pathogenic variants in either SLC52A2 or SLC52A3 genes, resulting in insufficient supply of riboflavin (vitamin B2) and consequent impairment of flavoprotein-dependent metabolic pathways. Current therapy, empirically based high-dose riboflavin supplementation, ameliorates the progression of the disease, even though response to treatment is variable and partial. Recent studies have highlighted concurrent pathogenic contribution of cellular energy dysmetabolism and cytoskeletal derangement. In this context, patient specific RTD models, based on induced pluripotent stem cell (iPSC) technology, have provided evidence of redox imbalance, involving mitochondrial and peroxisomal dysfunction. Such oxidative stress condition likely causes cytoskeletal perturbation, associated with impaired differentiation of RTD motor neurons. In this review, we discuss the most recent findings obtained using different RTD models. Relevantly, the integration of data from innovative iPSC-derived in vitro models and invertebrate in vivo models may provide essential information on RTD pathophysiology. Such novel insights are expected to suggest custom therapeutic strategies, especially for those patients unresponsive to high-dose riboflavin treatments.

Mandibular hypoplasia, Deafness and Progeroid features with concomitant Lipodystrophy is a rare, genetic, premature aging disease named MDPL Syndrome, due to almost always a de novo variant in POLD1 gene, encoding the DNA polymerase δ. In previous in vitro studies, we have already described several hallmarks of aging, including genetic damage, telomere shortening, cell senescence and proliferation defects. Since a clear connection has been reported between telomere shortening and mitochondria malfunction to initiate the aging process, we explored the role that mitochondrial metabolism and activity play in pathogenesis of MDPL Syndrome, an aspect that has not been addressed yet. We thus evaluated mtDNA copy number, assessing a significant decrease in mutated cells. The expression level of genes related to mitochondrial biogenesis and activity also revealed a significant reduction, highlighting a mitochondrial dysfunction in MDPL cells. Even the expression levels of mitochondrial marker SOD2, as assessed by immunofluorescence, were reduced. The decrease in this antioxidant enzyme correlated with increased production of mitochondrial ROS in MDPL cells, compared to WT. Consistent with these data, Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) analysis revealed in MDPL cells fewer mitochondria, which also displayed morphological abnormalities. Accordingly, we detected autophagic vacuoles containing partially digested mitochondria. Overall, our results demonstrate a dramatic impairment of mitochondrial biogenesis and activity in MDPL Syndrome. Administration of Metformin, though unable to restore mitochondrial impairment, proved efficient in rescuing nuclear abnormalities, suggesting its use to specifically ameliorate the premature aging phenotype.

ABSTRACT The cytoskeletal network plays a crucial role in the differentiation, morphogenesis, function and homeostasis of the nervous tissue, so that alterations in any of its components may lead to neurodegenerative diseases. Riboflavin transporter deficiency (RTD), a childhood-onset disorder characterized by degeneration of motor neurons (MNs), is caused by biallelic mutations in genes encoding the human riboflavin (RF) transporters. In a patient-specific induced pluripotent stem cells (iPSCs) model of RTD, we recently demonstrated altered cell-cell contacts, energy dysmetabolism and redox imbalance. The present study focuses on cytoskeletal composition and dynamics associated to RTD, utilizing patients' iPSCs and derived MNs. Abnormal expression and distribution of α- and β-tubulin (α- and β-TUB), as well as imbalanced tyrosination of α-TUB, accompanied by an impaired ability to re-polymerize after nocodazole treatment, were found in RTD patient-derived iPSCs. Following differentiation, MNs showed consistent changes in TUB content, which was associated with abnormal morphofunctional features, such as neurite length and Ca2+ homeostasis, suggesting impaired differentiation. Beneficial effects of RF supplementation, alone or in combination with the antioxidant molecule N-acetyl cystine (NAC), were assessed. RF administration resulted in partially improved cytoskeletal features in patients' iPSCs and MNs, suggesting that redundancy of transporters may rescue cell functionality in the presence of adequate concentrations of the vitamin. Moreover, supplementation with NAC was demonstrated to be effective in restoring all the considered parameters, when used in combination with RF, thus supporting the therapeutic use of both compounds.