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SummaryWolfram syndrome 1 (WS1) is a rare genetic disorder caused by mutations in the WFS1 gene leading to a wide spectrum of clinical dysfunctions, among which blindness, diabetes and neurological deficits are the most prominent. WFS1 encodes for the endoplasmic reticulum (ER) resident transmembrane protein Wolframin with multiple functions in ER processes. However, the WFS1-dependent etiopathology in retinal cells is unknown. Herein, we showed that Wfs1 mutant mice developed early retinal electrophysiological impairments followed by marked visual loss. Interestingly, axons and myelin disruption in the optic nerve preceded the degeneration of the retinal ganglion cell bodies in the retina. Transcriptomics at pre-degenerative stage revealed the STAT3-dependent activation of proinflammatory glial markers with reduction of the homeostatic and pro-survival factors Glutamine synthetase and BDNF. Furthermore, label-free comparative proteomics identified a significant reduction of the monocarboxylate transport isoform 1 (MCT1) and its partner Basigin that are highly enriched on retinal astrocytes and myelin-forming oligodendrocytes in optic nerve together with Wolframin. Loss of MCT1 caused a failure in lactate transfer from glial to neuronal cell bodies and axons leading to a chronic hypometabolic state. Thus, this bioenergetic impairment is occurring concurrently both in the axonal regions and cell bodies of the retinal ganglion cells, selectively endangering their survival while impacting less on other retinal cells. This metabolic dysfunction occurs months before the frank RGC degeneration suggesting an extended time window for intervening with new therapeutic strategies focused on boosting retinal and optic nerve bioenergetics in WS1.

Summary The generation of inducible pluripotent stem cells (iPSCs) is a revolutionary technique allowing production of pluripotent patient-specific cell lines used for disease modeling, drug screening, and cell therapy. Integrity of nuclear DNA (nDNA) is mandatory to allow iPSCs utilization, while quality control of mitochondrial DNA (mtDNA) is rarely included in the iPSCs validation process. In this study, we performed mtDNA deep sequencing during the transition from parental fibroblasts to reprogrammed iPSC and to differentiated neuronal precursor cells (NPCs) obtained from controls and patients affected by mitochondrial disorders. At each step, mtDNA variants, including those potentially pathogenic, fluctuate between emerging and disappearing, and some having functional implications. We strongly recommend including mtDNA analysis as an unavoidable assay to obtain fully certified usable iPSCs and NPCs., Graphical abstract, Highlights • mtDNA deep sequencing is mandatory in quality control of iPSCs • mtDNA variants fluctuate at each step from fibroblasts/PBMC, to iPSCs and NPCs • mtDNA variants greatly affect iPSC phenotype, reflecting their healthiness • Results could be misinterpreted if mtDNA variants presence has not been assessed, Tiranti and colleagues observed that quality control of iPSCs rarely includes the analysis of mitochondrial DNA (mtDNA), a small circular molecule present in the mitochondria. However, mtDNA variants greatly affect the final phenotype of iPSCs. They highlighted that iPSC-derived neuronal precursors could also present mtDNA variants and underlined the paramount importance of screening mtDNA.

Leber's Hereditary Optic Neuropathy (LHON) is a maternally inherited disorder caused by homoplasmic mutations of mitochondrial DNA (mtDNA). LHON is characterized by the selective degeneration of the retinal ganglion cells (RGC). Almost all LHON maternal lineages are homoplasmic mutant (100% mtDNA copies are mutant) for one of three frequent mtDNA mutations now found in over 90% of patients worldwide (m.11778G > A/MT-ND4, m.3460G > A/MT-ND1, m.14484 T > C/MT-ND6). Human induced pluripotent stem cells (hiPSCs) were generated from a patient carrying the homoplasmic m.3460G > A/MT-ND1 mutation using the Sendai virus non-integrating virus.

Pearson marrow pancreas syndrome (PMPS) is a sporadic mitochondrial disease, resulting from the clonal expansion of a mutated mitochondrial DNA (mtDNA) molecule bearing a macro-deletion, and therefore missing essential genetic information. PMPS is characterized by the presence of deleted (Δ) mtDNA that co-exist with the presence of a variable amount of wild-type mtDNA, a condition termed heteroplasmy. All tissues of the affected individual, including the haemopoietic system and the post-mitotic, highly specialized tissues (brain, skeletal muscle, and heart) contain the large-scale mtDNA deletion in variable amount. We generated human induced pluripotent stem cells (hiPSCs) from two PMPS patients, carrying different type of large-scale deletion.

Background Anthracyclines, alone or in combination with other drugs, are among the most effective chemotherapeutic agents to treat breast cancer both in the adjuvant and neoadjuvant settings. Unfortunately, anthracycline-associated dose-dependent cardiotoxicity is a limiting factor in clinical use. Extensive efforts have been devoted to identifying strategies to prevent anthracycline-induced cardiotoxicity. However, most cardioprotective agents have shown little efficacy in clinical trials. We hypothesized that myocardial damage by anthracyclines could be rationally prevented by using trimetazidine (TMZ), previously reported to interfere with anthracycline- and trastuzumab-induced cardiotoxicity. Therefore, we planned a randomized, controlled, open trial to determine whether TMZ may prevent the development of left ventricular (LV) dysfunction in patients receiving standard treatment for breast cancer. Methods The trial included 73 patients (41.2±8.1 years) undergoing surgery for breast cancer, who were scheduled for adjuvant epirubicin-containing chemotherapy and, if indicated, trastuzumab. Patients were randomly allocated in a 1:1 ratio to receive TMZ or baseline therapy only (control group). The main study endpoint was a reduction in the deterioration of left ventricular ejection fraction (LVEF), as evaluated by serial echocardiography performed at randomization and then every 3 months after the start of chemotherapy and for 1 year after its completion. Secondary outcome measures included echocardiographic indices of LV diastolic dysfunction, structural myocardial alterations, as assessed by speckle tracking echocardiography, and changes in cardiac biomarkers (troponin and brain natriuretic peptide). Results We found no significant differences between the two groups regarding baseline clinical and echocardiographic parameters. The two groups reached a similar cumulative dose of doxorubicin. No patient died during the study and no patients withdrew from chemotherapy. Three months after the start of chemotherapy, nonsignificant changes were observed in LVEF, shortening fraction, and LV diameters. No significant changes in cardiac biomarkers were observed in either group. Tissue Doppler imaging detected a significant decrease in myocardial velocities (P=0.001) in the control group, indicating LV diastolic dysfunction. In the same group, speckle tracking imaging revealed a statistically significant alteration in ventricular deformation (P=0.01), which means a decrease in LV systolic function. In the TMZ group, no significant alterations in LV diastolic function were observed. Conclusions Tissue Doppler imaging and speckle tracking imaging are more sensitive than conventional echocardiograms in the early diagnosis of cardiac dysfunction and TMZ seems to have an important role in the prevention of cardiotoxicity. Funding Acknowledgement Type of funding source: None

Friedreich’s ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons., Friedreich’s ataxia (FRDA) is an autosomal-recessive disorder. Here the authors describe a DRG organoid from patient derived-neurons and co-culture with muscle cells to mimic the disorder in vitro and demonstrate potential correction of the phenotype by CRISPR based editing.

Rett syndrome is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles since MECP2 is expressed throughout the brain and its duplication leads to severe neurological conditions as well. Herein, we use the AAV-PHP.eB to deliver an instability-prone Mecp2 (iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viral Mecp2 transgene, limits supraphysiological Mecp2 protein levels. Intravenous injections of the PHP.eB-iMecp2 virus in symptomatic Mecp2 mutant mice significantly improved locomotor activity, lifespan and gene expression normalization. Remarkably, PHP.eB-iMecp2 administration was well tolerated in female Mecp2 mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated male Mecp2 mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of iMecp2 provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain.

Rett syndrome (RTT) is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles sinceMECP2is expressed throughout the brain and its duplication leads to severe neurological conditions as well. However, the recent introduction of AAV-PHP.eB, an engineered capsid with an unprecedented efficiency in crossing the blood-brain barrier upon intravenous injection, has provided an invaluable vehicle for gene transfer in the mouse nervous system. Herein, we use AAV-PHP.eB to deliver an instability-proneMecp2(iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viralMecp2transgene, limits supraphysiological Mecp2 protein levels in transduced neural tissues. Intravenous injections of the PHP.eB-iMecp2virus in symptomatic male and femaleMecp2mutant mice significantly ameliorated the disease progression with improved locomotor activity, coordination, lifespan and normalization of altered gene expression and mTOR signaling. Remarkably, PHP.eB-iMecp2administration did not result in severe toxicity effects either in femaleMecp2mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated maleMecp2mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of theiMecp2cassette provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain. This combination defines a novel viral system with significant therapeutic efficacy and increased safety which can contribute to overcome the hurdles that are delaying clinical applications of gene therapy for RTT.One Sentence SummaryGlobal brain transduction of the instability-proneMecp2transgene by systemic AAV-PHP.eB administration is both safe and effective in protecting male and femaleMecp2mutant mice from the RTT disease phenotype.

Background The aim of this study was to evaluate radiological and histological characteristics of fresh-frozen homologous bone as grafting material for maxillary sinus floor augmentation. Radiological, histological and clinical evaluations were made. Methods Twenty-three patients with a 2 mm to 6 mm alveolar ridge height in the posterior maxilla have been enrolled. Unilateral or bilateral sinus floor augmentations were performed with fresh frozen morcelized homologous bone. Together with implant placement, 7 months after surgery, a bone core was harvested for histological analysis. Radiological measurements were obtained by superimposition of CT scans carried out at the surgery time and six months later. A total of 93 implants were positioned. Results A mean (±SD) increase in mineralized tissue height of 10.74±2.82 mm was noticed by comparing the CT scans. Histological analysis revealed the presence of newly formed bone in the grafted sites. The follow up period after the prosthetic load ranged from 8 to 31 months. One implant failure occurred. Conclusions Fresh frozen homologous bone seems to have a good healing pattern and to be a successful and steady grafting material for the treatment of maxillary ridge atrophy. It might be considered a valid alternative to autologous bone in sinus floor augmentation procedures.