SOMAscan Proteomics Identifies Novel Plasma Proteins in Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex disease characterized by the interplay of genetic and environmental factors for which, despite decades of intense research, diagnosis remains rather delayed, and most therapeutic options fail. Therefore, unravelling other potential pathogenetic mechanisms and searching for reliable markers are high priorities. In the present study, we employ the SOMAscan assay, an aptamer-based proteomic technology, to determine the circulating proteomic profile of ALS patients. The expression levels of ~1300 proteins were assessed in plasma, and 42 proteins with statistically significant differential expression between ALS patients and healthy controls were identified. Among these, four were upregulated proteins, Thymus- and activation-regulated chemokine, metalloproteinase inhibitor 3 and nidogen 1 and 2 were selected and validated by enzyme-linked immunosorbent assays in an overlapping cohort of patients. Following statistical analyses, different expression patterns of these proteins were observed in the familial and sporadic ALS patients. The proteins identified in this study might provide insight into ALS pathogenesis and represent potential candidates to develop novel targeted therapies.

Pro-Inflammatory and Cytotoxic Effects of Polystyrene Microplastics on Human and Murine Intestinal Cell Lines.

Plastic is a polymer extremely resistant to degradation that can remain for up to hundreds or thousands of years, leading to the accumulation of massive amounts of plastic waste throughout the planet's ecosystems. Due to exposure to various environmental factors, plastic breaks down into smaller particles named microplastics (1-5000 µm) and nanoplastics (<1 µm). Microplastics (MPs) are ubiquitous pollutants but, still, little is known about their effects on human and animal health. Herein, our aim is to investigate cytotoxicity, oxidative stress, inflammation and correlated gene modulation following exposure to polystyrene microplastics (PS-MPs) in HRT-18 and CMT-93 epithelial cell lines. After 6, 24 and 48 h PS-MPs treatment, cell viability (MTT) and oxidative stress (SOD) assays were performed; subsequently, expression changes and cytokines release were investigated by Real-Time PCR and Magnetic-beads panel Multiplex Assay, respectively. For each exposure time, a significantly increased cytotoxicity was observed in both cell lines, whereas SOD activity increased only in CMT-93 cells. Furthermore, Magnetic-beads Multiplex Assay revealed an increased release of IL-8 in HRT-18 cells' medium, also confirmed by gene expression analysis. Results obtained suggest the presence of a pro-inflammatory pattern induced by PS-MPs treatment that could be related to the observed increase in cytotoxicity.

A SOX2-engineered epigenetic silencer factor represses the glioblastoma genetic program and restrains tumor development.

Current therapies remain unsatisfactory in preventing the recurrence of glioblastoma multiforme (GBM), which leads to poor patient survival. By rational engineering of the transcription factor SOX2, a key promoter of GBM malignancy, together with the Kruppel-associated box and DNA methyltransferase3A/L catalytic domains, we generated a synthetic repressor named SOX2 epigenetic silencer (SES), which induces the transcriptional silencing of its original targets. By doing so, SES kills both glioma cell lines and patient-derived cancer stem cells in vitro and in vivo. SES expression, through local viral delivery in mouse xenografts, induces strong regression of human tumors and survival rescue. Conversely, SES is not harmful to neurons and glia, also thanks to a minimal promoter that restricts its expression in mitotically active cells, rarely present in the brain parenchyma. Collectively, SES produces a significant silencing of a large fraction of the SOX2 transcriptional network, achieving high levels of efficacy in repressing aggressive brain tumors.

Next-Generation Sequencing Advances the Genetic Diagnosis of Cerebral Cavernous Malformation (CCM).

Cerebral Cavernous Malformation (CCM) is a cerebrovascular disease of genetic origin that predisposes to seizures, focal neurological deficits and fatal intracerebral hemorrhage. It may occur sporadically or in familial forms, segregating as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. Its pathogenesis has been associated with loss-of-function mutations in three genes, namely KRIT1 (CCM1), CCM2 and PDCD10 (CCM3), which are implicated in defense mechanisms against oxidative stress and inflammation. Herein, we screened 21 Italian CCM cases using clinical exome sequencing and found six cases (~29%) with pathogenic variants in CCM genes, including a large 145−256 kb genomic deletion spanning the KRIT1 gene and flanking regions, and the KRIT1 c.1664C>T variant, which we demonstrated to activate a donor splice site in exon 16. The segregation of this cryptic splicing mutation was studied in a large Italian family (five affected and seven unaffected cases), and showed a largely heterogeneous clinical presentation, suggesting the implication of genetic modifiers. Moreover, by analyzing ad hoc gene panels, including a virtual panel of 23 cerebrovascular disease-related genes (Cerebro panel), we found two variants in NOTCH3 and PTEN genes, which could contribute to the abnormal oxidative stress and inflammatory responses to date implicated in CCM disease pathogenesis.

Towards precision nanomedicine for cerebrovascular diseases with emphasis on Cerebral Cavernous Malformation (CCM).

Introduction: Cerebrovascular diseases encompass various disorders of the brain vasculature, such as ischemic/hemorrhagic strokes, aneurysms, and vascular malformations, also affecting the central nervous system leading to a large variety of transient or permanent neurological disorders. They represent major causes of mortality and long-term disability worldwide, and some of them can be inherited, including Cerebral Cavernous Malformation (CCM), an autosomal dominant cerebrovascular disease linked to mutations in CCM1/KRIT1, CCM2, or CCM3/PDCD10 genes.Areas covered: Besides marked clinical and etiological heterogeneity, some commonalities are emerging among distinct cerebrovascular diseases, including key pathogenetic roles of oxidative stress and inflammation, which are increasingly recognized as major disease hallmarks and therapeutic targets. This review provides a comprehensive overview of the different clinical features and common pathogenetic determinants of cerebrovascular diseases, highlighting major challenges, including the pressing need for new diagnostic and therapeutic strategies, and focusing on emerging innovative features and promising benefits of nanomedicine strategies for early detection and targeted treatment of such diseases.Expert opinion: Specifically, we describe and discuss the multiple physico-chemical features and unique biological advantages of nanosystems, including nanodiagnostics, nanotherapeutics, and nanotheranostics, that may help improving diagnosis and treatment of cerebrovascular diseases and neurological comorbidities, with an emphasis on CCM disease.

A substrate-specific mTORC1 pathway underlies Birt-Hogg-Dubé syndrome.

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1-3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB-unlike other substrates of mTORC1, such as S6K and 4E-BP1- is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt-Hogg-Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.

Next Generation Sequencing (NGS) Strategies for Genetic Testing of Cerebral Cavernous Malformation (CCM) Disease.

The application of next generation sequencing (NGS) technique has a great impact on complex disease studies. Indeed, genetic heterogeneity, phenotypic variability, and disease rarity are all factors that make the traditional diagnostic approach to genetic disorders, whereby a specific gene is selected for sequencing based on the clinical phenotype, very challenging and obsolete.Exome sequencing, which sequences the protein-coding region of the genome, has been rapidly applied to variant discovery in research settings. Recent coverage and accuracy improvements have accelerated the development of clinical exome sequencing (CES) platforms targeting disease-related genes and enabling variant identification in patients with suspected genetic diseases. Nowadays, CES is rapidly becoming the diagnostic test of choice in patients with suspected Mendelian diseases, especially for those with heterogeneous etiology and clinical presentation. Reporting large CES series can improve guidelines on best practices for test utilization, and a better variant interpretation through clinically oriented data sharing.Herein, we suggest a feasible CES procedure for the genetic testing of Cerebral Cavernous Malformation (CCM) disease, including proband identification, library preparation, data analysis, and variant interpretation.

KRIT1 Loss-Of-Function Associated with Cerebral Cavernous Malformation Disease Leads to Enhanced S-Glutathionylation of Distinct Structural and Regulatory Proteins.

Loss-of-function mutations in the KRIT1 gene are associated with the pathogenesis of cerebral cavernous malformations (CCMs), a major cerebrovascular disease still awaiting therapies. Accumulating evidence demonstrates that KRIT1 plays an important role in major redox-sensitive mechanisms, including transcriptional pathways and autophagy, which play major roles in cellular homeostasis and defense against oxidative stress, raising the possibility that KRIT1 loss has pleiotropic effects on multiple redox-sensitive systems. Using previously established cellular models, we found that KRIT1 loss-of-function affects the glutathione (GSH) redox system, causing a significant decrease in total GSH levels and increase in oxidized glutathione disulfide (GSSG), with a consequent deficit in the GSH/GSSG redox ratio and GSH-mediated antioxidant capacity. Redox proteomic analyses showed that these effects are associated with increased S-glutathionylation of distinct proteins involved in adaptive responses to oxidative stress, including redox-sensitive chaperonins, metabolic enzymes, and cytoskeletal proteins, suggesting a novel molecular signature of KRIT1 loss-of-function. Besides providing further insights into the emerging pleiotropic functions of KRIT1, these findings point definitively to KRIT1 as a major player in redox biology, shedding new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell sensitivity to oxidative stress, which may eventually lead to cellular dysfunctions and CCM disease pathogenesis.

mTOR-dependent phosphorylation controls TFEB nuclear export.

During starvation the transcriptional activation of catabolic processes is induced by the nuclear translocation and consequent activation of transcription factor EB (TFEB), a master modulator of autophagy and lysosomal biogenesis. However, how TFEB is inactivated upon nutrient refeeding is currently unknown. Here we show that TFEB subcellular localization is dynamically controlled by its continuous shuttling between the cytosol and the nucleus, with the nuclear export representing a limiting step. TFEB nuclear export is mediated by CRM1 and is modulated by nutrient availability via mTOR-dependent hierarchical multisite phosphorylation of serines S142 and S138, which are localized in proximity of a nuclear export signal (NES). Our data on TFEB nucleo-cytoplasmic shuttling suggest an unpredicted role of mTOR in nuclear export.