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MECP2 deficiency causes a broad spectrum of neuropsychiatric disorders that can affect both genders. Rett syndrome is the most common and is characterized by an apparently normal growth period followed by a regression phase in which patients lose most of their previously acquired skills. After this dramatic period, various symptoms progressively appear, including severe intellectual disability, epilepsy, apraxia, breathing abnormalities and motor deterioration. MECP2 encodes for an epigenetic transcription factor that is particularly abundant in the brain; consequently, several transcriptional defects characterize the Rett syndrome brain. The well-known deficiency of several neurotrophins and growth factors, together with the positive effects exerted by trofinetide, a synthetic analogue of insulin-like growth factor 1, in Rett patients and in mouse models of Mecp2 deficiency, prompted us to investigate the therapeutic potential of nerve growth factor. Initial in vitro studies demonstrated a healing effect of recombinant human GMP-grade NGF (rhNGF) on neuronal maturation and activity in cultured Mecp2-null neurons. Subsequently, we designed in vivo studies with clear translational potential using intranasally administered rhNGF already used in the clinic. The efficacy of rhNGF in vivo in Mecp2-null hemizygous male mice and heterozygous female mice was assessed. General well-being was evaluated by a conventional phenotypic score and motor performance through the Pole and Beam Walking tests, while cognitive function and interaction with the environment were measured by the Novel Object Recognition test and the Marble Burying test, respectively. At the end of the treatment, mouse cortices were dissected and bulk RNA sequencing was performed to identify the molecular pathways involved in the protective effects of rhNGF. In both male and female mouse models of Rett syndrome, rhNGF exerted positive effects on cognitive and motor functions. In male hemizygous mice, which suffer from significantly more severe and rapidly advancing symptoms, the drug's ability to slow the disease's progression was more pronounced. The unbiased research for the molecular mechanisms triggering the observed benefits revealed a strong positive effect on gene sets related to oxidative phosphorylation, mitochondrial structure and function. These results were validated by demonstrating the drug's ability to improve mitochondrial structure and respiration in Mecp2-null cerebral cortices. Furthermore, Gene Ontology analyses indicated that NGF exerted the expected improvement in neuronal maturation. We conclude that intranasal administration of rhNGF is a non-invasive and effective route of administration for the treatment of Rett syndrome and possibly for other neurometabolic disorders with overt mitochondrial dysfunction., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Objective: In autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) disease, severity and age of onset vary greatly, hindering to objectively measure and predict clinical progression. Thickening of the retinal nerve fiber layer is distinctive of ARSACS patients, as assessed by optical coherence tomography, whereas conventional brain magnetic resonance imaging findings include both supratentorial and infratentorial changes. Because longitudinal imaging studies in ARSACS patients are not available to define these changes as biomarkers of disease progression, we aimed to address this issue in the ARSACS mouse model., Methods: We performed longitudinal retinal OCT and brain MRI in the Sacs -/- ARSACS mouse model, alongside motor and coordination assessment in the beam walking test. We also investigated visual function and the molecular mechanisms underlying RNFL increased thickness by histology and immunofluorescence., Results: We demonstrated that RNFL thickening by OCT gradually increases in the early stages of pathology in the Sacs -/- mouse model, reflecting the progression of motor impairment, and later reaches a plateau when thinning of the posterior corpus callosum becomes detectable by MRI. Mechanistically, we unveiled that RNFL thickening is associated with aberrant accumulation of non-phosphorylated neurofilament H and glial fibrillary acidic protein. We also uncovered mild signs of myelin pathology coherent with increased latency of visual evoked potentials, and altered retinal activation by photopic electroretinography., Interpretation: We show that both RNFL thickening and MRI changes may represent biomarkers of disease progression in the Sacs -/- mouse model. Our data gathers knowledge instrumental to clinical studies, holding potential as readout for treatment efficacy. ANN NEUROL 2025;97:425-434., (© 2024 The Author(s). Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Autosomal recessive spastic ataxia of Charlevoix-Saguenay is a rare neurodegenerative disease caused by biallelic variants in the SACS gene encoding for sacsin. More than 200 pathogenic variants have been identified to date, most of which are missense. It is likely that the prevalence of autosomal recessive spastic ataxia of Charlevoix-Saguenay is underestimated due to the lack of an efficient diagnostic tool able to validate variants of uncertain significance. We have previously shown that sacsin is almost absent in fibroblasts of patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay regardless of the type of SACS variant, because sacsin carrying missense variants is cotranslationally degraded. In this work, we aimed to establish the pathogenicity of SACS variants by quantifying sacsin protein in blood samples, with relevant implications for autosomal recessive spastic ataxia of Charlevoix-Saguenay diagnosis. We developed a protocol to assess sacsin protein levels by western blot using small amounts of peripheral blood mononuclear cells, which can be propagated in culture and cryopreserved. The study involves eight patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay (including a novel case) carrying variants of different types and positions along the SACS gene and two parents who are carriers of heterozygous missense variants. We show that patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay (carrying either missense or truncating variants) almost completely lacked sacsin in peripheral blood mononuclear cells. Moreover, both carriers of a SACS missense variant showed 50% reduction in sacsin protein levels compared to controls. We also describe a patient with uniparental isodisomy carrying a homozygous nonsense variant near the 3' end of the SACS gene. This resulted in a stable sacsin protein lacking the last 202 amino acids, probably due to escape of nonsense-mediated decay of mRNA. In conclusion, we have optimized a minimally invasive diagnostic tool for autosomal recessive spastic ataxia of Charlevoix-Saguenay in blood samples based on sacsin protein level assessment. Indeed, our results provide definite evidence that sacsin carrying missense pathogenic variants undergoes cotranslational degradation. The quantitative reduction in sacsin levels in the case of missense variants of uncertain significance allows defining them as pathogenic variants, something which cannot be predicted bioinformatically with high certainty., Competing Interests: The authors report no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane. Heterozygous AFG3L2 mutations cause spinocerebellar ataxia type 28 (SCA28) or dominant optic atrophy type 12 (DOA12), while biallelic AFG3L2 mutations result in the rare and severe spastic ataxia type 5 (SPAX5). The clinical spectrum of SPAX5 includes childhood-onset cerebellar ataxia, spasticity, dystonia and myoclonic epilepsy. We previously reported that the absence or mutation of AFG3L2 leads to the accumulation of mitochondria-encoded proteins, causing the overactivation of the stress-sensitive protease OMA1, which over-processes OPA1, leading to mitochondrial fragmentation. Recently, OMA1 has been identified as the pivotal player communicating mitochondrial stress to the cytosol via a pathway involving the inner mitochondrial membrane protein DELE1 and the cytosolic kinase HRI, thus eliciting the integrated stress response. In general, the integrated stress response reduces global protein synthesis and drives the expression of cytoprotective genes that allow cells to endure proteotoxic stress. However, the relevance of the OMA1-DELE1-HRI axis in vivo, and especially in a human CNS disease context, has been poorly documented thus far. In this work, we demonstrated that mitochondrial proteotoxicity in the absence/mutation of AFG3L2 activates the OMA1-DELE1-HRI pathway eliciting the integrated stress response. We found enhanced OMA1-dependent processing of DELE1 upon depletion of AFG3L2. Also, in both skin fibroblasts from SPAX5 patients (including a novel case) and in the cerebellum of Afg3l2-/- mice we detected increased phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α), increased levels of ATF4 and strong upregulation of its downstream targets (Chop, Chac1, Ppp1r15a and Ffg21). Silencing of DELE1 or HRI in SPAX5 fibroblasts (where OMA1 is overactivated at basal state) reduces eIF2α phosphorylation and affects cell growth. In agreement, pharmacological potentiation of integrated stress response via Sephin-1, a drug that selectively inhibits the stress-induced eIF2alpha phosphatase GADD34 (encoded by Ppp1r15a), improved cell growth of SPAX5 fibroblasts and cell survival and dendritic arborization ex vivo in primary Afg3l2-/- Purkinje neurons. Notably, Sephin-1 treatment in vivo extended the lifespan of Afg3l2-/- mice, improved Purkinje neuron morphology, mitochondrial ultrastructure and respiratory capacity. These data indicate that activation of the OMA1-DELE1-HRI pathway is protective in the context of SPAX5. Pharmacological tuning of the integrated stress response may represent a future therapeutic strategy for SPAX5 and other cerebellar ataxias caused by impaired mitochondrial proteostasis., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS gene encoding sacsin, a huge protein highly expressed in cerebellar Purkinje cells (PCs). Patients with ARSACS, as well as mouse models, display early degeneration of PCs, but the underlying mechanisms remain unexplored, with no available treatments. In this work, we demonstrated aberrant calcium (Ca2+) homeostasis and its impact on PC degeneration in ARSACS. Mechanistically, we found pathological elevation in Ca2+-evoked responses in Sacs-/- PCs as the result of defective mitochondria and ER trafficking to distal dendrites and strong downregulation of key Ca2+ buffer proteins. Alteration of cytoskeletal linkers, which we identified as specific sacsin interactors, likely account for faulty organellar trafficking in Sacs-/- cerebellum. Based on this pathogenetic cascade, we treated Sacs-/- mice with Ceftriaxone, a repurposed drug that exerts neuroprotection by limiting neuronal glutamatergic stimulation and, thus, Ca2+ fluxes into PCs. Ceftriaxone treatment significantly improved motor performances of Sacs-/- mice, at both pre- and postsymptomatic stages. We correlated this effect to restored Ca2+ homeostasis, which arrests PC degeneration and attenuates secondary neuroinflammation. These findings disclose key steps in ARSACS pathogenesis and support further optimization of Ceftriaxone in preclinical and clinical settings for the treatment of patients with ARSACS.

Background: Neurodevelopmental disorders (NDDs) are heterogeneous conditions due to alterations of a variety of molecular mechanisms and cell dysfunctions. SETD5 haploinsufficiency leads to NDDs due to chromatin defects. Epigenetic basis of NDDs has been reported in an increasing number of cases while mitochondrial dysfunctions are more common within NDD patients than in the general population., Methods: We investigated in vitro neural stem cells as well as the brain of the Setd5 haploinsufficiency mouse model interrogating its transcriptome, analyzing mitochondrial structure, biochemical composition, and dynamics, as well as mitochondrial functionality., Results: Mitochondrial impairment is facilitated by transcriptional aberrations originated by the decrease of the SETD5 enzyme. Low levels of SETD5 resulted in fragmented mitochondria, reduced mitochondrial membrane potential, and ATP production both in neural precursors and neurons. Mitochondria were also mislocalized in mutant neurons, with reduced organelles within neurites and synapses., Limitations: We found several defects in the mitochondrial compartment; however, we can only speculate about their position in the hierarchy of the pathological mechanisms at the basis of the disease., Conclusions: Our study explores the interplay between chromatin regulation and mitochondria functions as a possible important aspect of SETD5-associated NDD pathophysiology. Our data, if confirmed in patient context, suggest that the mitochondrial activity and dynamics may represent new therapeutic targets for disorders associated with the loss of SETD5., (© 2023. The Author(s).)