DIAPH3 is a master regulator of the cytoskeleton with key roles in cell division. In the mouse brain, DIAPH3-deficient neural stem cells exhibit abnormalities in karyokinesis and cytokinesis, leading to cell cycle arrest, aneuploidy, and mitotic catastrophe. Here, we investigated the role of DIAPH3 in glioma genesis in mouse models. We selectively deleted the Diaph3 and Trp53 genes in the mouse cerebral cortex and thoroughly analyzed single (Diaph3 cKO and Trp53 cKO) and double (dcKO) conditional knockout mice. The tumors appeared earlier in dcKO than in Trp53 cKO mice, and this was associated with increased whole chromosome copy number alterations, endogenous DNA damage, and shorter survival of dcKO mice. We performed a comparative transcriptomic analysis prior to the onset of tumors and identified changes in cancer gene signatures specifically in dcKO, suggesting that the loss of DIAPH3 hastens the tumorigenic process. We isolated cancer stem-like cells and assessed their sensitivity to ionizing radiation and found that DIAPH3 regulates the resistance of glioma stem-like cells to irradiation. Our data suggest that DIAPH3 has a tumor-suppressor function and that its deficiency promotes aneuploidy and genome instability, accelerating tumorigenesis and leading to early onset of high-grade diffuse glioma with DNA damage, and resistance to ionizing radiation.

Human brain development is highly regulated by several spatiotemporal processes, which disruption can result in severe neurological disorders. Emerging evidence highlights the pivotal role of mitochondrial function as one of these fundamental pathways involved in neurodevelopment. Our study investigates the role of 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) protein in cortical neurogenesis and mitochondrial activity, since mutations in the HPDL gene are associated with a childhood-onset form of hereditary spastic paraplegia characterized by corticospinal tract degeneration and cortical abnormalities. Starting from mutant neuroblastoma cells, we demonstrated that HPDL is important to respiratory chain supercomplex assembly and cellular redox balance. Moreover, RNA-seq studies revealed dysregulated pathways related to brain development. Generation of cortical neurons and organoids from HPDL patient-derived induced pluripotent stem cells exhibited premature neurogenesis at early differentiation stages, likely leading to depletion of cortical progenitors, as evidenced by decreased proliferation, slight increase of apoptosis, and unbalanced cortical type composition at later stages. Cortical organoids showed failure to grow at a normal rate, a feature highly reminiscent of the “microcephaly” observed in severe HPDL children. Mitochondrial morpho-functional characterization in mutant neurons confirmed disruption of OxPhos chain functionality in neuroblastoma knock-out model cells and HPDL mutant cortical progenitors also displayed defects in respirasome assembly and increased ROS generation rate. Treatment of mutant cortical cells with antioxidants and CoQ10 intermediates partially rescued premature neurogenesis depending on the mutational context, suggesting potential future personalized therapeutic strategies. Our findings reveal a critical role for HPDL in coordinating cortical progenitor proliferation, neurogenesis, and mitochondrial function, shedding light on a better understanding of the related clinical presentations.

Hereditary cerebellar ataxias are progressive neurodegenerative disorders for which disease-modifying treatments remain lacking. Although these conditions have traditionally been investigated from a neuron-centered perspective, evidence from several ataxia models indicates that changes in the cerebellar immune microenvironment can arise before overt neuronal loss and may contribute to early circuit dysfunction. This review examines hereditary cerebellar ataxias through the lens of early neuroimmune regulation, with particular attention to the region-specific properties of cerebellar microglia and their roles in synaptic refinement, inflammatory tone modulation and circuit homeostasis. We further discuss zebrafish as a useful experimental system for this question, because they combine in vivo imaging, genetic manipulation, and scalable functional assays in an intact vertebrate model. In this context, flavonoids—and especially naringenin—are not considered as immediate therapeutic candidates, but as mechanistically informative experimental probes to investigate how modulation of neuroimmune signaling affects disease-relevant phenotypes in vivo. By integrating genetic ataxia models with dynamic neuroimmune readouts, functional behavioral assays, and circuit-level analyses, zebrafish-based approaches can help identify early windows during which neuroimmune signaling influences cerebellar resilience and disease progression and can guide subsequent validation in mammalian systems.

Biallelic pathological variants in CYP2U1 are associated with SPG56, a complex ultra-rare form of hereditary spastic paraplegia. CYP2U1 encodes a member of the cytochrome P450 family 2, which is highly expressed in the brain and involved in arachidonic acid metabolism and lipid hydroxylation. Little is known about the early stages of neurodevelopment in SPG56. To gain insight into the molecular mechanisms of the disease, we generated and characterized both a zebrafish knock-out model (cyp2u1-/-) and cortical neurons derived from induced pluripotent stem cells (iPSCs) of two patients affected by SPG56. cyp2u1-/- zebrafish showed increased mortality and locomotor impairment, and transcriptome analysis revealed defects in the cell cycle, neural development, and oxidative phosphorylation pathways, consistent with observed decreased cell proliferation, increased apoptosis, and bioenergetic impairment. To assess whether these molecular alterations impact network function, we performed local field potential recordings and whole-brain calcium imaging in mutant larvae. Local field potential analyses revealed prolonged high-frequency events despite reduced overall power, and calcium imaging showed increased skewness of neuronal activity distributions, findings indicating altered brain network dynamics in mutants. Transcriptional profiling of cortical cultures performed at neural progenitor, early-born, and mature neuron stages pointed to altered neural network development as one of the main pathogenic mechanisms of the disease and highlighted several similarities with the zebrafish model. Multiple features of the cyp2u1-/- zebrafish recall the human SPG56 phenotype, and combined transcriptomics profiling in cyp2u1-/- zebrafish and SPG56 patient iPSC-derived cortical neurons supports impaired neural network development as a key disease mechanism.

Autosomal Dominant Optic Atrophy plus syndrome (ADOA, OMIM #125250) is a mitochondrial optic neuropathy characterized by progressive degeneration of retinal ganglion cells (RGCs), leading to worsening visual impairment. The disease is caused by pathogenic variants in the Optic Atrophy 1 (OPA1) gene, a member of the guanosine triphosphatase (GTPase) family that plays a central role in mitochondrial fusion and fission, mitophagy regulation, and mitochondrial DNA (mtDNA) maintenance. To model this disorder, we generated and characterized a human induced pluripotent stem cell (hiPSC) line from primary fibroblasts obtained from a patient affected by ADOA syndrome.

Abstract Biallelic variants in HPDL cause a severe neurological disorder in childhood, but the mechanisms linking early developmental abnormalities to later cortical degeneration remain unclear. Here, using long-term iPSC-derived human cortical cultures derived from four patients, we investigated the late consequences of HPDL deficiency through quantitative immunofluorescence and bulk RNA-seq. We found that HPDL-deficient cortical cultures undergo progressive synaptic impairment, with marked loss of PSD95-positive postsynaptic puncta despite largely preserved general neuronal maturation markers. This phenotype is accompanied by a profound reduction in astrocytes and oligodendrocytes, together with increased neuronal apoptosis and transcriptional dysregulation of genes linked to extracellular matrix organization, cellular stress, and neurodegeneration. Unexpectedly, late-stage mutant cultures also show reactivation of early developmental programs, including aberrant expression of NEUROD4 and other proneural regulators, suggesting instability of cell identity during cortical maturation. Together, these findings support a model in which HPDL deficiency first perturbs cortical developmental timing and later drives collapse of the neuro-glial unit, linking premature neurogenesis to synaptic failure, glial loss, and progressive neurodegeneration.

Charcot-Marie-Tooth disease type 2A (CMT2A; OMIM 609260) is a rare sensorimotor neuropathy caused by mutations in the MFN2 gene (1p36.22). We successfully reprogrammed fibroblasts from an 8-year-old girl carrying a de novo MFN2 mutation into induced pluripotent stem cells using non-integrative vectors. The line shows normal karyotype, pluripotency, and trilineage differentiation, providing a valuable in vitro model to study disease mechanisms.

Early diagnosis is critical for the effective management of neurodegenerative disorders, and retinal alterations have emerged as promising early biomarkers due to the retina’s close developmental and functional link to the brain. The zebrafish (Danio rerio), with its rapid development, transparent embryos, and evolutionarily conserved visual system, represents a powerful and versatile model for studying retinal degeneration. This review discusses a range of behavioral assays—including visual adaptation, motion detection, and color discrimination—that are employed to evaluate retinal function in zebrafish. These methods enable the detection of subtle visual deficits that may precede overt anatomical damage, providing a non-invasive, efficient strategy for early diagnosis and high-throughput drug screening. Importantly, these behavioral tests also serve as sensitive functional readouts to evaluate the efficacy of pharmacological treatments over time. Compared to traditional murine models, zebrafish offer advantages such as lower maintenance costs, faster development, optical transparency for live imaging, and ethical benefits due to reduced use of higher vertebrates. However, variability in experimental protocols highlights the need for standardization to ensure reliability and reproducibility.

Lafora disease (LD) is an ultra-rare and still incurable neurodegenerative condition. Although several therapeutic strategies are being explored, including gene therapy, there are currently no treatments that can alleviate the course of the disease and slow its progression. Recently, gliflozins, a series of SGLT2 transporter inhibitors approved for use in type 2 diabetes mellitus, heart failure and chronic kidney disease, have been proposed as possible repositioning drugs for the treatment of LD. With this in mind, we tested dapagliflozin (50 µM), canagliflozin (2.5 µM) and empagliflozin (200 µM) in our epm2a−/− zebrafish model, investigating their effects on pathological behaviour. In the case of dapagliflozin, we also investigated the possible mechanisms of action. Overall, the gliflozins reduced or rescued neuronal hyperexcitability and locomotor impairment. Dapagliflozin also reduced spontaneous seizure-like events in epm2a−/− larvae. At the biochemical and molecular level, dapagliflozin was found to slightly reduce glycogen content, and suppress inflammation and oxidative stress. It also ameliorates autophagic homeostasis and improves lysosomal markers. In conclusion, our preclinical study showed that dapagliflozin was able to ameliorate part of the pathological phenotype of epm2a-/- zebrafish larvae and could potentially be a suitable drug for repurposing in LD. However, since our model does not present Lafora bodies (LBs), at this early disease stage at least, it would be important to use mouse models in order to ascertain whether it is able to prevent or reduce LB formation.

The inaccessibility of human cerebellar tissue and the complexity of its development have historically hindered the study of cerebellar ataxias, a genetically diverse group of neurodegenerative disorders. Induced pluripotent stem cell (iPSC) technology offers a powerful solution, enabling the generation of patient-specific cerebellar models that retain individual genetic backgrounds. This review examines recent progress in iPSC-derived cerebellar models and their application in relation to major hereditary ataxias, including Friedreich’s ataxia, ataxia–telangiectasia, and spinocerebellar ataxias (SCAs). These models have provided valuable insights into disease mechanisms and supported the development of therapeutic strategies, such as gene therapy and high-throughput drug screening. However, challenges remain, particularly in achieving the full maturation of cerebellar cell types and incorporating microglial interactions. Moreover, emerging evidence suggests that neurodevelopmental alterations may act as early contributors to degeneration. Despite the current limitations, the advancement of patient-derived iPSC cerebellar models holds great promise for uncovering novel disease pathways and for driving precision medicine approaches in cerebellar ataxia research.

Sequences in the 5'-untranslated regions of cellular and viral mRNAs can function as internal ribosome entry sites (IRESs), driving cis-acting translation of the downstream protein-coding open reading frame. Here we demonstrate that RNA sequences with either newly identified or well-characterized IRES activity can also induce trans-acting translation of an independent mRNA species through an antisense sequence. SINEUPs are antisense long non-coding RNAs that enhance the translation of overlapping sense mRNAs in trans by employing two critical domains: the invSINEB2 sequence, which up-regulates translation (effector domain), and an antisense region providing target specificity (binding domain). First, we show that the invSINEB2 from the natural SINEUP AS Uchl1 RNA acts as an IRES when functioning in cis. Next, we establish that known viral and cellular sequences with IRES activity can operate in trans as an effector domain in synthetic SINEUPs. To identify natural IRES-containing non-coding RNAs with transactivity, we found that the non-coding hsa_circ_0 085 533 (circMyc), transcribed from the c-myc locus, enhances protein expression of PX Domain Containing Serine/Threonine Kinase Like (PXK) by promoting mRNA association with polysomes through antisense sequences. These results suggest that SINEUPs and some circular RNAs are trans-acting IRESs, expanding the repertoire of molecular mechanisms to regulate translation.

Abstract Ageing is the major risk factor for Alzheimer’s disease (AD), the most common neurodegenerative disorder. DNA damage is a hallmark of ageing, particularly when occurring at telomeres, genomic regions vulnerable to oxidative damage and often challenging for the cell to repair. Here, we show that brains of 3xTg-AD mice, an established AD model characterized by amyloid-β (Aβ)-induced pathology, exhibit increased activation of DNA damage response (DDR) pathways at telomeres. Exposure of mouse primary hippocampal neurons to 42-residue Aβ (Aβ42) oligomers, a significant pathogenetic contributor to AD, triggers telomeric DDR by increasing the levels of reactive oxygen species caused by calcium imbalance. Antisense oligonucleotides targeting non-coding RNAs generated at damaged telomeres in vivo (in 3xTg-AD mice) and in vitro reduce neurotoxicity in iPSC-derived human cortical neurons and mouse primary neurons while inhibiting Aβ42-induced telomeric DDR, and restore transcriptional pathways altered by Aβ and found dysregulated in AD patients. These results unveil an unexpected role of telomeric DNA damage responses in Alzheimer’s disease pathogenesis, and suggest a novel target for the development of RNA-based therapies.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare inherited condition described worldwide and characterized by a wide spectrum of heterogeneity in terms of genotype and phenotype. How sacsin loss leads to neurodegeneration is still unclear, and current knowledge indicates that sacsin is involved in multiple functional mechanisms. We hence hypothesized the existence of epigenetic factors, in particular alterations in methylation patterns, that could contribute to ARSACS pathogenesis and explain the pleiotropic effects of SACS further than pathogenic mutations. To investigate this issue, we recruited eight patients affected by ARSACS, four characterized by early onset of the disease and four with late onset. We performed Whole Genome Bisulfite Sequencing using DNA from peripheral blood to define the methylome of patients and compared them with a control group. Our analysis showed that patients with ARSACS exhibit an altered methylation pattern and that the observed differences exist also among affected individuals with different age of onset. Our study provides valuable insights for employing epigenetic biomarkers to assess the severity and progression of this disorder and propels further investigations into the role of epigenetic processes in ARSACS pathogenesis.

Biallelic mutations in the SACS gene, encoding sacsin, cause early-onset autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), a neurodegenerative disease also characterized by unique and poorly understood retinal abnormalities. While two murine models replicate the phenotypic and neuronal features observed in patients, no retinal phenotype has been described so far. In a zebrafish knock-out strain that faithfully mirrors the main aspects of ARSACS, we observed impaired visual function due to photoreceptor degeneration, likely caused by cell cycle defects in progenitor cells. RNA-seq analysis in embryos revealed dysfunction in proteins related to fat-soluble vitamins (e.g., TTPA, RDH5, VKORC) and suggested a key role of neuroinflammation in driving the retinal defects. Our findings indicate that studying retinal pathology in ARSACS could be crucial for understanding the impact of sacsin depletion and may offer insights into halting disease progression.

Human brain development is highly regulated by several spatiotemporal processes, which disruption can result in severe neurological disorders. Emerging evidence highlights the pivotal role of mitochondrial function as one of these fundamental pathways involved in neurodevelopment. Our study investigates the role of 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) protein in cortical neurogenesis and mitochondrial activity, since mutations in the HPDL gene are associated with SPG83, a childhood-onset form of hereditary spastic paraplegia characterized by corticospinal tract degeneration and cortical abnormalities. Starting from mutant neuroblastoma cells, we demonstrated that HPDL is essential to mitochondrial respiratory chain supercomplex assembly and cellular redox balance. Moreover, transcriptomic analyses revealed dysregulated pathways related to neurogenesis, implicating HPDL role in early cortical development. To further elucidate the role of HPDL, we generated cortical neurons and organoids from SPG83 patient-derived induced pluripotent stem cells. Mutant cells exhibited premature neurogenesis at early differentiation stages, likely leading to depletion of cortical progenitors, as evidenced by decreased proliferation, slight increase of apoptosis, and unbalanced cortical type composition at later stages. Furthermore, cortical organoids derived from SPG83 patients showed impaired growth, reminding microcephaly observed in severe cases. In addition, mitochondrial morpho-functional characterization in mutant neurons confirmed disruption of OxPhos chain functionality and increased ROS generation rate. Treatment of cortical cells with two antioxidant compounds, could partially revert premature neurogenesis. In conclusion, our findings reveal a critical role for HPDL in coordinating cortical progenitor proliferation, neurogenesis, and mitochondrial function. These insights shed light on a mechanistical understanding of SPG83 pathology and underscore the therapeutic potential of targeting oxidative stress in this and related neurological disorders. ### Competing Interest Statement The authors have declared no competing interest. Ministero della SaluteMinistero della Salute, , RC2024 Telethon FoundationTelethon Foundation, , Telethon Grant GJC21131

Hereditary spastic paraplegias (HSPs) comprise a family of degenerative diseases mostly hitting descending axons of corticospinal neurons. Depending on the gene and mutation involved, the disease could present as a pure form with limb spasticity, or a complex form associated with cerebellar and/or cortical signs such as ataxia, dysarthria, epilepsy, and intellectual disability. The progressive nature of HSPs invariably leads patients to require walking canes or wheelchairs over time. Despite several attempts to ameliorate the life quality of patients that have been tested, current therapeutical approaches are just symptomatic, as no cure is available. Progress in research in the last two decades has identified a vast number of genes involved in HSP etiology, using cellular and animal models generated on purpose. Although unanimously considered invaluable tools for basic research, those systems are rarely predictive for the establishment of a therapeutic approach. The advent of induced pluripotent stem (iPS) cells allowed instead the direct study of morphological and molecular properties of the patient’s affected neurons generated upon in vitro differentiation. In this review, we revisited all the present literature recently published regarding the use of iPS cells to differentiate HSP patient-specific neurons. Most studies have defined patient-derived neurons as a reliable model to faithfully mimic HSP in vitro, discovering original findings through immunological and –omics approaches, and providing a platform to screen novel or repurposed drugs. Thereby, one of the biggest hopes of current HSP research regards the use of patient-derived iPS cells to expand basic knowledge on the disease, while simultaneously establishing new therapeutic treatments for both generalized and personalized approaches in daily medical practice.

Hereditary spastic paraplegias (HSPs) a group of rare, clinically, and genetically heterogeneous disorders characterized by progressive degeneration of the corticospinal tract. Among these HSPs, SPG31 is due to autosomal dominant mutations in the receptor expression-enhancing protein 1 (REEP1) gene. Over 80 genes have been associated with HSPs, and the list is constantly growing as research progresses. This study is aimed to create a patient-derived human induced pluripotent stem cell (hiPSC) line with a specific nonsense mutation to better characterize the etiopathogenesis of the disease.

Hereditary spastic paraplegias are rare genetic disorders characterized by corticospinal tract impairment. Spastic paraplegia 83 (SPG83) is associated with biallelic mutations in the HPDL gene, leading to varied severities from neonatal to juvenile onset. The function of HPDL is unclear, though it is speculated to play a role in alternative coenzyme Q10 biosynthesis. Here, we report the generation of hiPS lines from primary skin fibroblasts derived from three SPG83 patients with different HPDL mutations, using episomal reprogramming. The patients’ clinical characteristics are carefully listed. The hiPS lines were meticulously characterized, demonstrating typical pluripotent characteristics through immunofluorescence assays for stemness markers (OCT4, TRA1-60, NANOG, and SSEA4) and RT-PCR for endogenous gene expression. Genetic integrity and identity were confirmed via Sanger sequencing and short tandem repeat analysis. These hiPS cells displayed typical pluripotent characteristics and were able to differentiate into neocortical neurons via a dual SMAD inhibition protocol. In addition, HPDL mutant neurons assessed via long-term culturing were able to achieve effective maturation, similarly to their wild-type counterparts. The HPDL hiPS lines we generated will provide a valuable model for studying SPG83, offering insights into its molecular mechanisms and potential for developing targeted therapies.

Summary SINEUPs are antisense long non-coding RNAs that enhance translation of overlapping sense mRNAs through the activity of two domains: a SINE B2 sequence UP- regulating translation (Effector Domain, ED) and an antisense region providing target specificity (Binding Domain, BD). In this study, we demonstrate that the invSINEB2 sequence from the natural SINEUP AS Uchl1 RNA is an Internal Ribosomal Entry Site (IRES) when acting in cis and that known viral and cellular IRES sequences can act as Effector Domain in synthetic SINEUPs. To identify natural IRES-containing, non-coding RNAs with SINEUP-like activity, we focused on circular RNAs showing that the non-coding circ5533 , transcribed from the c-myc locus , enhances endogenous protein expression of its target PX Domain Containing Serine/Threonine Kinase Like ( Pxk) by increasing mRNA association to polysomes. In summary, this study shows that natural and synthetic SINEUPs include linear and circular transcripts with an embedded IRES sequence as ED.

Background: Valproic acid (VPA), an anticonvulsant used in epilepsy, has deleterious effects on embryonic development and is considered an environmental risk factor for autism spectrum disorders. There is a growing need for easy and rapid ways to study its effects on embryonic development. The zebrafish model is a cost-and time-effective tool able to facilitate mechanistic studies and high-throughput drug screening. Epileptic patients are increasingly looking to natural compounds to avoid the strong side effects of synthetic drugs, and cannabinoids appear promising. We evaluated the potential of cannabidiol to mitigate the negative effects of VPA on developing zebrafish embryos.Methods: Wild-type AB embryos, untreated or exposed to VPA (5, 10 or 20 mu M) and/or cannabidiol (1, 2 or 3 mu M), were evaluated at up to 120 hours post-fertilization. Developmental endpoints: survival, hatching, heart rate, morphology, and locomotor behavior (tail coiling and visual motor response test). Three replicates were evaluated per group, for a total of 120 larvae per treatment.Results: Although VPA-treated groups showed significantly reduced survival rates compared to control group fish (p <= 0.01), zebrafish simultaneously treated with VPA (5 and 10 mu M) and cannabidiol (3 mu M) displayed survival rates similar to those of untreated controls. Hatching rate, body length and eye area were not influenced by any treatment, but the highest VPA dose and all cannabidiol doses caused a significant increase in burst activity (p <= 0.0001). Compared with controls, the pericardial area was larger only in larvae treated with VPA at the highest concentration (p <= 0.01). Each VPA treatment caused tachycardia (p <= 0.0001), while cannabidiol 3 mu M induced bradycardia (p <= 0.01). Finally, simultaneous treatment with VPA 5 mu M and cannabidiol 3 mu M avoided almost all the adverse effects of the two compounds administered individually, stabilizing heart rate and locomotor behavior at control levels.Conclusions: This study adds further information on the embryotoxic effect of VPA in the zebrafish model and offers new insights into the use of cannabidiol as an alternative natural drug able to mitigate the deleterious effects of VPA. Multi-laboratory large-scale validation and new genomic and molecular analyses are required to clarify the mechanism of action of VPA on developing embryos and the role of cannabidiol as a potential natural protective agent against its toxic effects.