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Abstract Copper (Cu) is a vitally important micronutrient, whose balance between essential and toxic levels requires a tightly regulated network of proteins. Dysfunction in key components of this network leads to the disruption of Cu homeostasis, resulting in fatal disorders such as Wilson disease, which is caused by mutations in the hepatic Cu efflux transporter ATP7B. Unfortunately, the molecular targets for normalizing Cu homeostasis in Wilson disease remain poorly understood. Here, using genome-wide screening, we identified the cellular prion protein (PrP) as an important mediator of Cu toxicity in WD. Loss of ATP7B stimulates hepatic expression of PrP, which promotes endocytic Cu uptake, leading to toxic Cu overload. Suppression of PrP significantly reduces Cu toxicity in cell and animal models of Wilson disease. These findings highlight the critical regulatory role of PrP in copper metabolism and open new avenues for exploring the therapeutic potential of PrP suppression in Wilson disease.

Abstract Inherited retinal diseases (IRDs) are a group of diseases whose common landmark is progressive photoreceptor loss. The development of gene‐specific therapies for IRDs is hampered by their wide genetic heterogeneity. Mitochondrial dysfunction is proving to constitute one of the key pathogenic events in IRDs; hence, approaches that enhance mitochondrial activities have a promising therapeutic potential for these conditions. We previously reported that miR‐181a/b downregulation boosts mitochondrial turnover in models of primary retinal mitochondrial diseases. Here, we show that miR‐181a/b silencing has a beneficial effect also in IRDs. In particular, the injection in the subretinal space of an adeno‐associated viral vector (AAV) that harbors a miR‐181a/b inhibitor (sponge) sequence (AAV2/8‐GFP‐Sponge‐miR‐181a/b) improves retinal morphology and visual function both in models of autosomal dominant (RHO‐P347S) and of autosomal recessive (rd10) retinitis pigmentosa. Moreover, we demonstrate that miR‐181a/b downregulation modulates the level of the mitochondrial fission‐related protein Drp1 and rescues the mitochondrial fragmentation in RHO‐P347S photoreceptors. Overall, these data support the potential use of miR‐181a/b downregulation as an innovative mutation‐independent therapeutic strategy for IRDs, which can be effective both to delay disease progression and to aid gene‐specific therapeutic approaches.

Abstract Pompe disease is a metabolic myopathy due to acid alpha‐glucosidase deficiency. In addition to glycogen storage, secondary dysregulation of cellular functions, such as autophagy and oxidative stress, contributes to the disease pathophysiology. We have tested whether oxidative stress impacts on enzyme replacement therapy with recombinant human alpha‐glucosidase (rhGAA), currently the standard of care for Pompe disease patients, and whether correction of oxidative stress may be beneficial for rhGAA therapy. We found elevated oxidative stress levels in tissues from the Pompe disease murine model and in patients’ cells. In cells, stress levels inversely correlated with the ability of rhGAA to correct the enzymatic deficiency. Antioxidants (N‐acetylcysteine, idebenone, resveratrol, edaravone) improved alpha‐glucosidase activity in rhGAA‐treated cells, enhanced enzyme processing, and improved mannose‐6‐phosphate receptor localization. When co‐administered with rhGAA, antioxidants improved alpha‐glucosidase activity in tissues from the Pompe disease mouse model. These results indicate that oxidative stress impacts on the efficacy of enzyme replacement therapy in Pompe disease and that manipulation of secondary abnormalities may represent a strategy to improve the efficacy of therapies for this disorder.

Abstract Mitochondrial diseases (MDs) are a heterogeneous group of devastating and often fatal disorders due to defective oxidative phosphorylation. Despite the recent advances in mitochondrial medicine, effective therapies are still not available for these conditions. Here, we demonstrate that the microRNAs miR‐181a and miR‐181b (miR‐181a/b) regulate key genes involved in mitochondrial biogenesis and function and that downregulation of these miRNAs enhances mitochondrial turnover in the retina through the coordinated activation of mitochondrial biogenesis and mitophagy. We thus tested the effect of miR‐181a/b inactivation in different animal models of MDs, such as microphthalmia with linear skin lesions and Leber's hereditary optic neuropathy. We found that miR‐181a/b downregulation strongly protects retinal neurons from cell death and significantly ameliorates the disease phenotype in all tested models. Altogether, our results demonstrate that miR‐181a/b regulate mitochondrial homeostasis and that these miRNAs may be effective gene‐independent therapeutic targets for MDs characterized by neuronal degeneration.

The retina is among the most metabolically active tissues with high-energy demands. The peculiar distribution of mitochondria in cells of retinal layers is necessary to assure the appropriate energy supply for the transmission of the light signal. Photoreceptor cells (PRs), retinal pigment epithelium (RPE), and retinal ganglion cells (RGCs) present a great concentration of mitochondria, which makes them particularly sensitive to mitochondrial dysfunction. To date, visual loss has been extensively correlated to defective mitochondrial functions. Many mitochondrial diseases (MDs) show indeed neuro-ophthalmic manifestations, including retinal and optic nerve phenotypes. Moreover, abnormal mitochondrial functions are frequently found in the most common retinal pathologies, i.e., glaucoma, age-related macular degeneration (AMD), and diabetic retinopathy (DR), that share clinical similarities with the hereditary primary MDs. MicroRNAs (miRNAs) are established as key regulators of several developmental, physiological, and pathological processes. Dysregulated miRNA expression profiles in retinal degeneration models and in patients underline the potentiality of miRNA modulation as a possible gene/mutation-independent strategy in retinal diseases and highlight their promising role as disease predictive or prognostic biomarkers. In this review, we will summarize the current knowledge about the participation of miRNAs in both rare and common mitochondria-mediated eye diseases. Definitely, given the involvement of miRNAs in retina pathologies and therapy as well as their use as molecular biomarkers, they represent a determining target for clinical applications.

Since SARS-CoV-2 outbreak in December 2019, world health-system has been severely impacted with increased hospitalization, Intensive-Care-Unit (ICU) access and high mortality rates, mostly due to severe acute respiratory failure and multi-organ failure. Excessive and uncontrolled release of proinflammatory cytokines (cytokine release/storm syndrome, CRS) have been linked to the development of these events. The recent advancements of immunotherapy for the treatment of hematologic and solid tumors shed light on many of the molecular mechanisms underlying this phenomenon, thus rendering desirable a multidisciplinary approach to improve COVID-19 patients’ outcome. Indeed, currently available therapeutic-strategies to overcome CRS, should be urgently evaluated for their capability of reducing COVID-19 mortality. Notably, COVID-19 shares different pathogenic aspects with acute graft-versus-host-disease (aGVHD), hemophagocytic-lymphohistiocytosis (HLH), myelofibrosis, and CAR-T-associated CRS. Specifically, similarly to aGVHD, an induced tissue damage (caused by the virus) leads to increased cytokine release (TNFα and IL-6) which in turn leads to exaggerated dendritic cells, macrophages (like in HLH) and lymphocytes (as in CAR-T) activation, immune-cells migration, and tissue-damage (including late-stage fibrosis, similar to myelofibrosis). Janus Kinase (JAK) signaling represents a molecular hub linking all these events, rendering JAK-inhibitors suitable to limit deleterious effects of an overwhelming inflammatory-response. Accordingly, ruxolitinib is the only selective JAK1 and JAK2-inhibitor approved for the treatment of myelofibrosis and aGVHD. Here, we discuss, from a molecular and hematological point of view, the rationale for targeting JAK signaling in the management of COVID-19 patients and report the clinical results of a patient admitted to ICU among the firsts to be treated with ruxolitinib in Italy.

Parkinson’s disease (PD) is characterized by motor dysfunctions including bradykinesia, tremor at rest and motor instability. These symptoms are associated with the progressive degeneration of dopaminergic neurons originating in the substantia nigra pars compacta and projecting to the corpus striatum, and by accumulation of cytoplasmic inclusions mainly consisting of aggregated alpha-synuclein, called Lewy bodies. PD is a complex, multifactorial disorder and its pathogenesis involves multiple pathways and mechanisms such as α-synuclein proteostasis, mitochondrial function, oxidative stress, calcium homeostasis, axonal transport, and neuroinflammation. Motor symptoms manifest when there is already an extensive dopamine denervation. There is therefore an urgent need for early biomarkers to apply disease-modifying therapeutic strategies. Visual defects and retinal abnormalities, including decreased visual acuity, abnormal spatial contrast sensitivity, color vision defects, or deficits in more complex visual tasks are present in the majority of PD patients. They are being considered for early diagnosis together with retinal imaging techniques are being considered as non-invasive biomarkers for PD. Dopaminergic cells can be found in the retina in a subpopulation of amacrine cells; however, the molecular mechanisms leading to visual deficits observed in PD patients are still largely unknown. This review provides a comprehensive analysis of the retinal abnormalities observed in PD patients and animal models and of the molecular mechanisms underlying neurodegeneration in parkinsonian eyes. We will review the role of α-synuclein aggregates in the retina pathology and/or in the onset of visual symptoms in PD suggesting that α-synuclein aggregates are harmful for the retina as well as for the brain. Moreover, we will summarize experimental evidence suggesting that the optic nerve pathology observed in PD resembles that seen in mitochondrial optic neuropathies highlighting the possible involvement of mitochondrial abnormalities in the development of PD visual defects. We finally propose that the eye may be considered as a complementary experimental model to identify possible novel disease’ pathways or to test novel therapeutic approaches for PD.

Gene therapy is proving to be an effective approach to treat or prevent ocular diseases ensuring a targeted, stable, and regulated introduction of exogenous genetic material with therapeutic action. Retinal diseases can be broadly categorized into two groups, namely monogenic and complex (multifactorial) forms. The high genetic heterogeneity of monogenic forms represents a significant limitation to the application of gene-specific therapeutic strategies for a significant fraction of patients. Therefore, mutation-independent therapeutic strategies, acting on common pathways that underly retinal damage, are gaining interest as complementary/alternative approaches for retinal diseases. This review will provide an overview of mutation-independent strategies that rely on the modulation in the retina of key genes regulating such crucial degenerative pathways. In particular, we will describe how gene-based approaches explore the use of neurotrophic factors, microRNAs (miRNAs), genome editing and optogenetics in order to restore/prolong visual function in both outer and inner retinal diseases. We predict that the exploitation of gene delivery procedures applied to mutation/gene independent approaches may provide the answer to the unmet therapeutic need of a large fraction of patients with genetically heterogeneous and complex retinal diseases.

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo‐lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua‐1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua‐1 mutant animals exhibited very poor resistance to Cu compared to the wild‐type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua‐1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA‐1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B‐H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B‐H1069Q reduced Cu toxicity in cua‐1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA‐1 function. Taken together, our findings suggest that the newly generated cua‐1 mutant strain represents an excellent model for Cu toxicity studies in WD. [ABSTRACT FROM AUTHOR]

Abstract Mitochondrial‐dependent (intrinsic) programmed cell death (PCD) is an essential homoeostatic mechanism that selects bioenergetically proficient cells suitable for tissue/organ development. However, the link between mitochondrial dysfunction, intrinsic apoptosis and developmental anomalies has not been demonstrated to date. Now we provide the evidence that non‐canonical mitochondrial‐dependent apoptosis explains the phenotype of microphthalmia with linear skin lesions (MLS), an X‐linked developmental disorder caused by mutations in the holo‐cytochrome c‐type synthase (HCCS) gene. By taking advantage of a medaka model that recapitulates the MLS phenotype we demonstrate that downregulation of hccs, an essential player of the mitochondrial respiratory chain (MRC), causes increased cell death via an apoptosome‐independent caspase‐9 activation in brain and eyes. We also show that the unconventional activation of caspase‐9 occurs in the mitochondria and is triggered by MRC impairment and overproduction of reactive oxygen species (ROS). We thus propose that HCCS plays a key role in central nervous system (CNS) development by modulating a novel non‐canonical start‐up of cell death and provide the first experimental evidence for a mechanistic link between mitochondrial dysfunction, intrinsic apoptosis and developmental disorders.

BackgroundProtein adsorption is the first of a complex series of events that regulates many phenomena at the nano-bio interface, e.g. cell adhesion and differentiation, in vivo inflammatory responses and protein crystallization. A quantitative understanding of how nanoscale morphology influences protein adsorption is strategic for providing insight into all of these processes, however this understanding has been lacking until now.Methodology/principal findingsHere we introduce novel methods for quantitative high-throughput characterization of protein-surface interaction and we apply them in an integrated experimental strategy, to study the adsorption of a panel of proteins on nanostructured surfaces. We show that the increase of nanoscale roughness (from 15 nm to 30 nm) induces a decrease of protein binding affinity (Conclusions/significanceThese results show that the adsorption of proteins depends significantly on surface nanostructure and that the relevant morphological parameter regulating the protein adsorption process is the nanometric pore shape. These new findings improve our understanding of the role of nanostructures as a biomaterial design parameter and they have important implications for the general understanding of cell behavior on nanostructured surfaces.