Super-resolution microscopy reveals glioma cell footprints and exosome deposits.

Gliomas are aggressive brain tumors whose infiltrative growth is mediated by intercellular crosstalk. Exosomes, small extracellular vesicles, play a key role in cell-cell communication but are difficult to visualize using conventional microscopy. Performing immunostaining for CD63, a known exosome marker, and using STED microscopy, we demonstrate exosome secretion in primary glioma cells.

Optimization of Lipoplexes Functionalized with a Sialic Acid Mimetic (F9-PEG) to Target the C1858T Variant for Preclinical Assessment of a Novel Immunotherapy in Endocrine Autoimmunity.

The C1858T variant is strongly associated with type 1 diabetes and autoimmune thyroid disease. Current treatment is substitutive hormonal administration, which does not target the disease pathogenetic mechanism. We previously implemented a novel immunotherapy, employing siRNA directed against the C1858T variant of delivered via functionalized lipoplexes, in order to halt autoimmune disease progression.

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence.

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo.

Altered cytoskeleton dynamics in patient-derived iPSC-based model of PCDH19 clustering epilepsy.

Protocadherin 19 (PCDH19) is an adhesion molecule involved in cell-cell interaction whose mutations cause a drug-resistant form of epilepsy, named PCDH19-Clustering Epilepsy (PCDH19-CE, MIM 300088). The mechanism by which altered PCDH19 function drive pathogenesis is not yet fully understood. Our previous work showed that PCDH19 dysfunction is associated with altered orientation of the mitotic spindle and accelerated neurogenesis, suggesting a contribution of altered cytoskeleton organization in PCDH19-CE pathogenesis in the control of cell division and differentiation.

Imaging-Based Molecular Interaction Between Src and Lamin A/C Mechanosensitive Proteins in the Nucleus of Laminopathic Cells.

Laminopathies represent a wide range of genetic disorders caused by mutations in gene-encoding proteins of the nuclear lamina. Altered nuclear mechanics have been associated with laminopathies, given the key role of nuclear lamins as mechanosensitive proteins involved in the mechanotransduction process. To shed light on the nuclear partners cooperating with altered lamins, we focused on Src tyrosine kinase, known to phosphorylate proteins of the nuclear lamina.

Ketogenic Diet and Progression of Kidney Disease in Animal Models of Nephropathic Cystinosis.

Key Points: Ketogenic diet can change the metabolism in the body and helped restore the function of altered pathways in nephropathic cystinosis. Ketogenic diet had significant benefits for preventing kidney damage, even when initiated after the onset of kidney impairment. Ketogenic diet may provide a partial therapeutic alternative in countries where cysteamine therapy is too expensive.

Assessment of the FRET-based Teen sensor to monitor ERK activation changes preceding morphological defects in a RASopathy zebrafish model and phenotypic rescue by MEK inhibitor.

Background: RASopathies are genetic syndromes affecting development and having variable cancer predisposition. These disorders are clinically related and are caused by germline mutations affecting key players and regulators of the RAS-MAPK signaling pathway generally leading to an upregulated ERK activity. Gain-of-function (GOF) mutations in PTPN11, encoding SHP2, a cytosolic protein tyrosine phosphatase positively controlling RAS function, underlie approximately 50% of Noonan syndromes (NS), the most common RASopathy.

Caspase-dependent apoptosis in Riboflavin Transporter Deficiency iPSCs and derived motor neurons.

Riboflavin Transporter Deficiency (RTD) is a rare genetic, childhood-onset disease. This pathology has a relevant neurological involvement, being characterized by motor symptoms, ponto-bulbar paralysis and sensorineural deafness. Such clinical presentation is associated with muscle weakness and motor neuron (MN) degeneration, so that RTD is considered part of the MN disease spectrum.

Novel loss of function mutation in gene compromises tubulin stability and proteostasis causing spastic paraplegia and ataxia.

Microtubules are dynamic cytoskeletal structures involved in several cellular functions, such as intracellular trafficking, cell division and motility. More than other cell types, neurons rely on the proper functioning of microtubules to conduct their activities and achieve complex morphologies. Pathogenic variants in genes encoding for α and β-tubulins, the structural subunits of microtubules, give rise to a wide class of neurological disorders collectively known as "tubulinopathies" and mainly involving a wide and overlapping range of brain malformations resulting from defective neuronal proliferation, migration, differentiation and axon guidance.

The recurrent pathogenic Pro890Leu substitution in CLTC causes a generalized defect in synaptic transmission in .

mutations underlie a spectrum of early-onset neurodevelopmental phenotypes having developmental delay/intellectual disability (ID), epilepsy, and movement disorders (MD) as major clinical features. encodes the widely expressed heavy polypeptide of clathrin, a major component of the coated vesicles mediating endocytosis, intracellular trafficking, and synaptic vesicle recycling. The underlying pathogenic mechanism is largely unknown.

SARS-CoV-2 infection of thymus induces loss of function that correlates with disease severity.

Background: Lymphopenia, particularly when restricted to the T-cell compartment, has been described as one of the major clinical hallmarks in patients with coronavirus disease 2019 (COVID-19) and proposed as an indicator of disease severity. Although several mechanisms fostering COVID-19-related lymphopenia have been described, including cell apoptosis and tissue homing, the underlying causes of the decline in T-cell count and function are still not completely understood.

Objective: Given that viral infections can directly target thymic microenvironment and impair the process of T-cell generation, we sought to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on thymic function.

Dominant ARF3 variants disrupt Golgi integrity and cause a neurodevelopmental disorder recapitulated in zebrafish.

Vesicle biogenesis, trafficking and signaling via Endoplasmic reticulum-Golgi network support essential developmental processes and their disruption lead to neurodevelopmental disorders and neurodegeneration. We report that de novo missense variants in ARF3, encoding a small GTPase regulating Golgi dynamics, cause a developmental disease in humans impairing nervous system and skeletal formation. Microcephaly-associated ARF3 variants affect residues within the guanine nucleotide binding pocket and variably perturb protein stability and GTP/GDP binding.

MET Inhibition Sensitizes Rhabdomyosarcoma Cells to NOTCH Signaling Suppression.

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma. The Fusion-Positive (FP) subtype expresses the chimeric protein PAX3-FOXO1 (P3F) while the Fusion-Negative (FN) is devoid of any gene translocation. FP-RMS and metastatic FN-RMS are often unresponsive to conventional therapy.

Lamin A and the LINC complex act as potential tumor suppressors in Ewing Sarcoma.

Lamin A, a main constituent of the nuclear lamina, is involved in mechanosignaling and cell migration through dynamic interactions with the LINC complex, formed by the nuclear envelope proteins SUN1, SUN2 and the nesprins. Here, we investigated lamin A role in Ewing Sarcoma (EWS), an aggressive bone tumor affecting children and young adults. In patients affected by EWS, we found a significant inverse correlation between LMNA gene expression and tumor aggressiveness.

CAPE and its synthetic derivative VP961 restore BACH1/NRF2 axis in Down Syndrome.

The cells possess several mechanisms to counteract the over-production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), including enzymes such as superoxide dismutase, catalase and glutathione peroxidase. Moreover, an important sensor involved in the anti-oxidant response is KEAP1-NRF2-ARE signaling complex. Under oxidative stress (OS), the transcription factor NRF2 can dissociate from the KEAP1-complex in the cytosol and translocate into the nucleus to promote the transcriptional activation of anti-oxidant genes, such as heme oxygenase 1 and NADPH quinone oxidoreductase.

Preparation and In Vitro Evaluation of RITUXfab-Decorated Lipoplexes to Improve Delivery of siRNA Targeting C1858T PTPN22 Variant in B Lymphocytes.

Autoimmune endocrine disorders, such as type 1 diabetes (T1D) and thyroiditis, at present are treated with only hormone replacement therapy. This emphasizes the need to identify personalized effective immunotherapeutic strategies targeting T and B lymphocytes. Among the genetic variants associated with several autoimmune disorders, the C1858T polymorphism of the protein tyrosine phosphatase non-receptor type 22 () gene, encoding for Lyp variant R620W, affects the innate and adaptive immunity.

SPRED2 loss-of-function causes a recessive Noonan syndrome-like phenotype.

Upregulated signal flow through RAS and the mitogen-associated protein kinase (MAPK) cascade is the unifying mechanistic theme of the RASopathies, a family of disorders affecting development and growth. Pathogenic variants in more than 20 genes have been causally linked to RASopathies, the majority having a dominant role in promoting enhanced signaling. Here, we report that SPRED2 loss of function is causally linked to a recessive phenotype evocative of Noonan syndrome.

Lamin A/C Mechanosensor Drives Tumor Cell Aggressiveness and Adhesion on Substrates With Tissue-Specific Elasticity.

Besides its structural properties in the nucleoskeleton, Lamin A/C is a mechanosensor protein involved in perceiving the elasticity of the extracellular matrix. In this study we provide evidence about Lamin A/C-mediated regulation of osteosarcoma cell adhesion and spreading on substrates with tissue-specific elasticities. Our working hypothesis is based on the observation that low-aggressive and bone-resident SaOS-2 osteosarcoma cells express high level of Lamin A/C in comparison to highly metastatic, preferentially to the lung, osteosarcoma 143B cells, thereby suggesting a role for Lamin A/C in tumor cell tropism.

Dissecting the Role of PCDH19 in Clustering Epilepsy by Exploiting Patient-Specific Models of Neurogenesis.

PCDH19-related epilepsy is a rare genetic disease caused by defective function of PCDH19, a calcium-dependent cell-cell adhesion protein of the cadherin superfamily. This disorder is characterized by a heterogeneous phenotypic spectrum, with partial and generalized febrile convulsions that are gradually increasing in frequency. Developmental regression may occur during disease progression.

Biallelic mutations in RNF220 cause laminopathies featuring leukodystrophy, ataxia and deafness.

Leukodystrophies are a heterogeneous group of rare inherited disorders that mostly involve the white matter of the CNS. These conditions are characterized by primary glial cell and myelin sheath pathology of variable aetiology, which causes secondary axonal degeneration, generally emerging with disease progression. Whole exome sequencing performed in five large consanguineous nuclear families allowed us to identify homozygosity for two recurrent missense variants affecting highly conserved residues of RNF220 as the causative event underlying a novel form of leukodystrophy with ataxia and sensorineural deafness.

TFG binds LC3C to regulate ULK1 localization and autophagosome formation.

The early secretory pathway and autophagy are two essential and evolutionarily conserved endomembrane processes that are finely interlinked. Although growing evidence suggests that intracellular trafficking is important for autophagosome biogenesis, the molecular regulatory network involved is still not fully defined. In this study, we demonstrate a crucial effect of the COPII vesicle-related protein TFG (Trk-fused gene) on ULK1 puncta number and localization during autophagy induction.

Altered cytoskeletal arrangement in induced pluripotent stem cells (iPSCs) and motor neurons from patients with riboflavin transporter deficiency.

The cytoskeletal network plays a crucial role in differentiation, morphogenesis, function and homeostasis of the nervous tissue, so that alterations in any of its components may lead to neurodegenerative diseases. Riboflavin transporter deficiency (RTD), a childhood-onset disorder characterized by degeneration of motor neurons (MNs), is caused by biallelic mutations in genes encoding the human riboflavin (RF) transporters. In a patient- specific induced Pluripotent Stem Cells (iPSCs) model of RTD, we recently demonstrated altered cell-cell contacts, energy dysmetabolism and redox imbalance.