Daniela Ferrari

The molecular mechanisms by which mutant huntingtin (mHTT) drives pathogenesis in Huntington’s disease (HD) remain incompletely defined. Here we show that neurogenesis is disrupted at multiple stages of lineage progression in both rodent and human neural stem cell (NSC) models of HD. We identify a previously unrecognized phenotype characterized by aberrant expansion of early multipotent progenitors coupled to a profound defect in astrogliogenesis, whereby HD astrocytes fail to express glial fibrillary acidic protein (GFAP). Mechanistically, this defect arises from dysregulation of an epigenetic regulatory axis involving EZH2 and LIN28 upregulation together with reduced expression of the mature let-7g microRNA. Epigenetic pharmacological interventions, targeting this pathway at distinct nodes—through EZH2 modulation, let-7g restoration, or LIN28 inhibition—rescued astroglial differentiation in human HD cells and improved motor function in a Drosophila HD model. Our findings suggest that mHTT might trigger a dual-phase astroglial failure: an early developmental impairment followed by a collapse of regenerative gliogenesis. This bimodal mechanism proposes astrocytic dysfunction as a central driver of HD pathogenesis. Finally, we identify a panel of clinically relevant epigenetic compounds that, by converging on distinct targets within this axis, hold promise for stage-spanning therapeutic strategies capable of modifying disease trajectory.

Background: Neural stem cell (NSC) transplantation holds promising therapeutic potential for neurodegenerative disorders like amyotrophic lateral sclerosis (ALS). However, pre-clinical studies and early-phase clinical trials have faced challenges hindering the effective clinical translation of this approach. Crucial hurdles include the side-effects of prolonged immunosuppression, concerns regarding cell origin and transplantation dosage, identification of the most appropriate therapeutic window, and invasiveness of surgical procedures. Here, we assessed the safety and efficacy of intracerebroventricular (ICV) hNSC transplantation as a novel and possibly more effective experimental approach for ALS. Methods: We evaluated the safety of administering up to 1 × 10⁶ hNSCs in immunodeficient mice and assessed their potential efficacy in reducing ALS hallmarks employing the SOD1^G93A mouse model. Both transient (15 days) and prolonged immunosuppression regimens, at low (15 mg/kg) and high (30 mg/kg) doses, were tested along with two different cell dosages (3 × 10⁵ and 1 × 10⁶). Results: Our study suggests that: (i) a bilateral ICV transplantation of 1 × 10⁶ hNSCs is safe and non-tumorigenic in immunodeficient hosts; (ii) sustained and high-dose immunosuppression is essential for ensuring cell survival in immunocompetent SOD1^G93A mice; and (iii) hNSCs may delay motor symptom progression and reduce spinal cord microgliosis in SOD1^G93A mice when administered in the lateral ventricles under prolonged high-dose (30 mg/kg) immunosuppression. Conclusions: ICV transplantation of hNSCs emerges as a safe and promising strategy for ALS, demonstrating potential to delay motor decline and reduce spinal cord microgliosis. However, sustained high-dose immunosuppression is crucial for therapeutic efficacy, emphasizing the need for further optimization to overcome translational challenges and achieve durable clinical benefits.

Amyotrophic Lateral Sclerosis (ALS) is an incurable neurodegenerative disease, causing degeneration of motor neurons, paralysis, and death. About 5–10% of cases are associated with gene mutations inherited from a family member (fALS). Among them, mutations in the transactive-response (TAR)-DNA-binding protein (TARDBP), which encodes for the TAR DNA binding protein 43 (TDP-43) are responsible for 4–5% of fALS but the molecular mechanisms that initiate and sustain the neurodegenerative process are largely unknown. Metabolic impairments might be involved in the pathogenesis of ALS and are currently under investigation. In order to correlate biochemical and metabolic alterations with disease progression, here, we established the metabolic fingerprint of dermal fibroblasts derived from symptomatic and asymptomatic members of a family with fALS cases carrying to the p.G376D mutation in TDP-43. We found that increased proliferation, unbalanced oxidative homeostasis and higher ATP production rate coupled with enhanced metabolic activity are underlying traits of this family. Fibroblasts from carrier individuals deploy several mechanisms to increase mitochondrial respiration to meet increasing energy demands. This is accompanied by an upregulation of glycolysis corresponding to a metabolic reprograming towards a glycolytic phenotype for ATP production during ALS progression, particularly in late disease stages. In summary, we uncover alterations in energy metabolism in TDP43^G376D patient-derived primary fibroblasts that may be used as risk biomarkers and/or to monitor ALS progression.

Background and aims: Neural stem cell (NSC) transplantation holds promising therapeutic potential for neurodegenerative disorders like amyotrophic lateral sclerosis (ALS). However, pre-clinical studies and early-phase clinical trials have faced challenges hindering the effective clinical translation of this approach. Crucial hurdles include the side-effects of prolonged immunosuppression, concerns regarding cell origin and transplantation dosage, identification of the most appropriate therapeutic window, and invasiveness of surgical procedures. Here, we show challenges and promises in optimizing a non-clinical protocol to assess safety and efficacy of human NSC (hNSC) intracerebroventricular (ICV) transplantation for ALS.Methods: We evaluated the safety of administering up to 1×10⁶hNSCs in immunodeficient mice and assessed their potential efficacy in reducing ALS hallmarks employing the SOD1^G93Amouse model. Both, transient (15 days) and prolonged immunosuppression regimens, at low (15 mg/kg) and high (30 mg/kg) doses, were tested along with two different cell dosages (3×10⁵and 1×10⁶).Results: Bilateral ICV injection of up to 1×10⁶hNSCs proved to be safe, with no evidence of tumor formation. At 40 days post-transplantation, hNSCs induced a trend toward delaying motor decline and reducing spinal cord (SC) microgliosis when transplanted under prolonged high-dose (30 mg/kg) immunosuppression.Conclusions: Our study suggests that: (i) a bilateral ICV transplantation of 1×10⁶hNSCs is safe and non-tumorigenic in immunodeficient hosts; (ii) sustained high-dose immunosuppression is essential for ensuring cell survival in immunocompetent mice; and (iii) hNSC transplantation may provide therapeutic benefits in ALS by delaying motor decline and reducing microgliosis. This study also highlights persisting hurdles that need to be further addressed, such as the aggressive murine immune response to exogenous cells.

Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by cognitive and behavioral symptoms, obesity, and sleep disturbance, and no therapy has been developed to alleviate its symptoms or delay disease onset. SMS occurs due to haploinsufficiency of the retinoic acid-induced-1 (RAI1) gene caused by either chromosomal deletion (SMS-del) or RAI1 missense/nonsense mutation. The molecular mechanisms underlying SMS are unknown. Here, we generated and characterized primary cells derived from four SMS patients (two with SMS-del and two carrying RAI1 point mutations) and four control subjects to investigate the pathogenetic processes underlying SMS. By combining transcriptomic and lipidomic analyses, we found altered expression of lipid and lysosomal genes, deregulation of lipid metabolism, accumulation of lipid droplets, and blocked autophagic flux. We also found that SMS cells exhibited increased cell death associated with the mitochondrial pathology and the production of reactive oxygen species. Treatment with N-acetylcysteine reduced cell death and lipid accumulation, which suggests a causative link between metabolic dyshomeostasis and cell viability. Our results highlight the pathological processes in human SMS cells involving lipid metabolism, autophagy defects and mitochondrial dysfunction and suggest new potential therapeutic targets for patient treatment.

Animal models currently used to test the efficacy and safety of cell therapies, mainly murine models, have limitations as molecular, cellular, and physiological mechanisms are often inherently different between species, especially in the brain. Therefore, for clinical translation of cell-based medicinal products, the development of alternative models based on human neural cells may be crucial. We have developed an in vitro model of transplantation into human brain organoids to study the potential of neural stem cells as cell therapeutics and compared these data with standard xenograft studies in the brain of immunodeficient NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice. Neural stem cells showed similar differentiation and proliferation potentials in both human brain organoids and mouse brains. Our results suggest that brain organoids can be informative in the evaluation of cell therapies, helping to reduce the number of animals used for regulatory studies.

Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by cognitive and behavioral symptoms, obesity, and sleep disturbance. There is no therapy to alleviate its symptoms or delay disease onset. SMS occurs due to haploinsufficiency of the retinoic acid-induced-1 (RAI1) gene caused by either chromosomal deletion (SMS-del) or RAI1 missense/nonsense mutation. The molecular mechanisms underlying SMS are not known. Here, we generated and characterized primary cells derived from four SMS patients, two carrying SMS-del and two carrying RAI1 point mutations, and four control subjects to investigate the pathogenetic processes underlying SMS. By combining transcriptomic and lipidomic analyses, we show altered expression of lipid and lysosomal genes, deregulation of lipid metabolism, accumulation of lipid droplets, and a block of autophagic flux. SMS cells show increased cell death associated with mitochondrial pathology and reactive oxygen species production. Treatment with N-acetylcysteine reduces cell death and lipid accumulation, suggesting a causative link between metabolic dyshomeostasis and cell viability. Our results highlight the pathological processes in human SMS cells involving lipid metabolism, autophagy defects and mitochondrial dysfunction and suggest new potential therapeutic targets for patient treatment.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of the upper and lower motor neurons (MNs). About 10% of patients have a family history (familial, fALS); however, most patients seem to develop the sporadic form of the disease (sALS). SOD1 (Cu/Zn superoxide dismutase-1) is the first studied gene among the ones related to ALS. Mutant SOD1 can adopt multiple misfolded conformation, lose the correct coordination of metal binding, decrease structural stability, and form aggregates. For all these reasons, it is complicated to characterize the conformational alterations of the ALS-associated mutant SOD1, and how they relate to toxicity. In this work, we performed a multilayered study on fibroblasts derived from two ALS patients, namely SOD1^L145F and SOD1^S135N, carrying the p.L145F and the p.S135N missense variants, respectively. The patients showed diverse symptoms and disease progression in accordance with our bioinformatic analysis, which predicted the different effects of the two mutations in terms of protein structure. Interestingly, both mutations had an effect on the fibroblast energy metabolisms. However, while the SOD1^L145F fibroblasts still relied more on oxidative phosphorylation, the SOD1^S135N fibroblasts showed a metabolic shift toward glycolysis. Our study suggests that SOD1 mutations might lead to alterations in the energy metabolism.

Recent cutting-edge human genetics technology has allowed us to identify copy number variations (CNVs) and has provided new insights for understanding causative mechanisms of human diseases. A growing number of studies show that CNVs could be associated with physiological mechanisms linked to evolutionary trigger, as well as to the pathogenesis of various diseases, including cancer, autoimmune disease and mental disorders such as autism spectrum disorders, schizophrenia, intellectual disabilities or attention-deficit/hyperactivity disorder. Their incomplete penetrance and variable expressivity make diagnosis difficult and hinder comprehension of the mechanistic bases of these disorders. Additional elements such as co-presence of other CNVs, genomic background and environmental factors are involved in determining the final phenotype associated with a CNV. Genetically engineered animal models are helpful tools for understanding the behavioral consequences of CNVs. However, the genetic background and the biology of these animal model systems have sometimes led to confusing results. New cellular models obtained through somatic cellular reprogramming technology that produce induced pluripotent stem cells (iPSCs) from human subjects are being used to explore the mechanisms involved in the pathogenic consequences of CNVs. Considering the vast quantity of CNVs found in the human genome, we intend to focus on reviewing the current literature on the use of iPSCs carrying CNVs on chromosome 15, highlighting advantages and limits of this system with respect to mouse model systems.

Stem cells are emerging as a therapeutic option for incurable diseases, such as Amyotrophic Lateral Sclerosis (ALS). However, critical issues are related to their origin as well as to the need to deepen our knowledge of the therapeutic actions exerted by these cells. Here, we investigate the therapeutic potential of clinical-grade human neural stem cells (hNSCs) that have been successfully used in a recently concluded phase I clinical trial for ALS patients (NCT01640067). The hNSCs were transplanted bilaterally into the anterior horns of the lumbar spinal cord (four grafts each, segments L3–L4) of superoxide dismutase 1 G93A transgenic rats (SOD1 rats) at the symptomatic stage. Controls included untreated SOD1 rats (CTRL) and those treated with HBSS (HBSS). Motor symptoms and histological hallmarks of the disease were evaluated at three progressive time points: 15 and 40 days after transplant (DAT), and end stage. Animals were treated by transient immunosuppression (for 15 days, starting at time of transplantation). Under these conditions, hNSCs integrated extensively within the cord, differentiated into neural phenotypes and migrated rostro-caudally, up to 3.77 ± 0.63 cm from the injection site. The transplanted cells delayed decreases in body weight and deterioration of motor performance in the SOD1 rats. At 40DAT, the anterior horns at L3–L4 revealed a higher density of motoneurons and fewer activated astroglial and microglial cells. Accordingly, the overall survival of transplanted rats was significantly enhanced with no rejection of hNSCs observed. We demonstrated that the beneficial effects observed after stem cell transplantation arises from multiple events that counteract several aspects of the disease, a crucial feature for multifactorial diseases, such as ALS. The combination of therapeutic approaches that target different pathogenic mechanisms of the disorder, including pharmacology, molecular therapy and cell transplantation, will increase the chances of a clinically successful therapy for ALS.