Gain-of-function defects of astrocytic Kir4.1 channels in children with autism spectrum disorders and epilepsy

Dysfunction of the inwardly-rectifying potassium channels Kir4.1 (KCNJ10) represents a pathogenic mechanism contributing to Autism-Epilepsy comorbidity. To define the role of Kir4.1 variants in the disorder, we sequenced KCNJ10 in a sample of affected individuals, and performed genotype-phenotype correlations. The effects of mutations on channel activity, protein trafficking, and astrocyte function were investigated in Xenopus laevis oocytes, and in human astrocytoma cell lines. An in vivo model of the disorder was also explored through generation of kcnj10a morphant zebrafish overexpressing the mutated human KCNJ10. We detected germline heterozygous KCNJ10 variants in 19/175 affected children. Epileptic spasms with dysregulated sensory processing represented the main disease phenotype. When investigated on astrocyte-like cells, the p.R18Q mutation exerted a gain-of-function effect by enhancing Kir4.1 membrane expression and current density. Similarly, the p.R348H variant led to gain of channel function through hindrance of pH-dependent current inhibition. The frequent polymorphism p.R271C seemed, instead, to have no obvious functional effects. Our results confirm that variants in KCNJ10 deserve attention in autism-epilepsy, and provide insight into the molecular mechanisms of autism and seizures. Similar to neurons, astrocyte dysfunction may result in abnormal synaptic transmission and electrical discharge, and should be regarded as a possible pharmacological target in autism-epilepsy. Supplementary information accompanies this paper in the files section.peer-reviewe

Pilot Scale Evaluation of Wild Saccharomyces cerevisiae Strains in Aglianico

In winemaking, the influence of Saccharomyces cerevisiae strains on the aromatic components of wine is well recognized on a laboratory scale, but few studies deal with the comparison of numerous strains on a pilot scale fermentation. In this scenario, the present work aimed to validate the fermentative behavior of seven wild S. cerevisiae strains on pilot-scale fermentations to evaluate their impact on the aromatic profiles of the resulting wines. The strains, isolated from grapes of different Italian regional varieties, were tested in pilot-scale fermentation trials performed in the cellar in 1 hL of Aglianico grape must. Then, wines were analyzed for their microbiological cell loads, main chemical parameters of enological interest (ethanol, total sugars, fructose, glucose, total and volatile acidity, malic and lactic acids) and volatile aroma profiles by GC/MS/SPME. Seventy-six volatile compounds belonging to six different classes (esters, alcohols, terpenes, aldehydes, acids, and ketones) were identified. The seven strains showed different trends and significant differences, and for each class of compounds, high-producing and low-producing strains were found. Since the present work was performed at a pilot-scale level, mimicking as much as possible real working conditions, the results obtained can be considered as a validation of the screened S. cerevisiae strains and a strategy to discriminate in real closed conditions strains able to impart desired wine sensory features

Modeling riboflavin transporter deficiency type 2: from iPSC-derived motoneurons to iPSC-derived astrocytes

Introduction: Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters, RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide), which are involved in different redox reactions, including the energetic metabolism processes occurring in mitochondria. To date, human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons, which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far, no attention has been dedicated to astrocytes. Results and discussion: Here, we demonstrate that in vitro differentiation of astrocytes, which guarantee trophic and metabolic support to neurons, from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons, RTD2 does not compromise the morpho-functional features of astrocytes

Stereocontrolled synthesis of new iminosugar lipophilic derivatives and evaluation of biological activities

Iminosugars' similarity to carbohydrates determines the exceptional potential for this class of polyhydroxylated alkaloids to serve as potential drug candidates for a wide variety of diseases such as diabetes, lysosomal storage diseases, cancer, bacterial and viral infections. The presence of lipophilic substituents has a significant impact on their biological activities. This work reports the synthesis of three new pyrrolidine lipophilic derivatives O-alkylated in C-6 position. The biological activities of our iminosugars' collection were tested in two cancer cell lines and, due to the pharmaceutical potential, in the model yeast system Saccharomyces cerevisiae to assess their toxicity

Precision mouse models with expanded tropism for human pathogens

A major limitation of current humanized mouse models is that they primarily enable the analysis of human-specific pathogens that infect hematopoietic cells. However, most human pathogens target other cell types, including epithelial, endothelial and mesenchymal cells. Here, we show that implantation of human lung tissue, which contains up to 40 cell types, including nonhematopoietic cells, into immunodeficient mice (lung-only mice) resulted in the development of a highly vascularized lung implant. We demonstrate that emerging and clinically relevant human pathogens such as Middle East respiratory syndrome coronavirus, Zika virus, respiratory syncytial virus and cytomegalovirus replicate in vivo in these lung implants. When incorporated into bone marrow/liver/thymus humanized mice, lung implants are repopulated with autologous human hematopoietic cells. We show robust antigen-specific humoral and T-cell responses following cytomegalovirus infection that control virus replication. Lung-only mice and bone marrow/liver/thymus-lung humanized mice substantially increase the number of human pathogens that can be studied in vivo, facilitating the in vivo testing of therapeutics

Human iPSC-Derived Astrocytes: A Powerful Tool to Study Primary Astrocyte Dysfunction in the Pathogenesis of Rare Leukodystrophies

Astrocytes are very versatile cells, endowed with multitasking capacities to ensure brain homeostasis maintenance from brain development to adult life. It has become increasingly evident that astrocytes play a central role in many central nervous system pathologies, not only as regulators of defensive responses against brain insults but also as primary culprits of the disease onset and progression. This is particularly evident in some rare leukodystrophies (LDs) where white matter/myelin deterioration is due to primary astrocyte dysfunctions. Understanding the molecular defects causing these LDs may help clarify astrocyte contribution to myelin formation/maintenance and favor the identification of possible therapeutic targets for LDs and other CNS demyelinating diseases. To date, the pathogenic mechanisms of these LDs are poorly known due to the rarity of the pathological tissue and the failure of the animal models to fully recapitulate the human diseases. Thus, the development of human induced pluripotent stem cells (hiPSC) from patient fibroblasts and their differentiation into astrocytes is a promising approach to overcome these issues. In this review, we discuss the primary role of astrocytes in LD pathogenesis, the experimental models currently available and the advantages, future evolutions, perspectives, and limitations of hiPSC to study pathologies implying astrocyte dysfunctions.</jats:p

Human iPSC-Derived Astrocytes: A Powerful Tool to Study Primary Astrocyte Dysfunction in the Pathogenesis of Rare Leukodystrophies

Astrocytes are very versatile cells, endowed with multitasking capacities to ensure brain homeostasis maintenance from brain development to adult life. It has become increasingly evident that astrocytes play a central role in many central nervous system pathologies, not only as regulators of defensive responses against brain insults but also as primary culprits of the disease onset and progression. This is particularly evident in some rare leukodystrophies (LDs) where white matter/myelin deterioration is due to primary astrocyte dysfunctions. Understanding the molecular defects causing these LDs may help clarify astrocyte contribution to myelin formation/maintenance and favor the identification of possible therapeutic targets for LDs and other CNS demyelinating diseases. To date, the pathogenic mechanisms of these LDs are poorly known due to the rarity of the pathological tissue and the failure of the animal models to fully recapitulate the human diseases. Thus, the development of human induced pluripotent stem cells (hiPSC) from patient fibroblasts and their differentiation into astrocytes is a promising approach to overcome these issues. In this review, we discuss the primary role of astrocytes in LD pathogenesis, the experimental models currently available and the advantages, future evolutions, perspectives, and limitations of hiPSC to study pathologies implying astrocyte dysfunctions