A new Caenorhabditis elegans model to study copper toxicity in Wilson disease.

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.

αS1-Casein-Loaded Proteo-liposomes as Potential Inhibitors in Amyloid Fibrillogenesis: In Vivo Effects on a C. elegans Model of Alzheimer's Disease.

According to the amyloid hypothesis, in the early phases of Alzheimer's disease (AD), small soluble prefibrillar aggregates of the amyloid ß-peptide (Aß) interact with neuronal membranes, causing neural impairment. Such highly reactive and toxic species form spontaneously and transiently in the amyloid building pathway. A therapeutic strategy consists of the recruitment of these intermediates, thus preventing aberrant interaction with membrane components (lipids and receptors), which in turn may trigger a cascade of cellular disequilibria. Milk αs1-Casein is an intrinsically disordered protein that is able to inhibit Aß amyloid aggregation in vitro, by sequestering transient species. In order to test αs1-Casein as an inhibitor for the treatment of AD, it needs to be delivered in the place of action. Here, we demonstrate the use of large unilamellar vesicles (LUVs) as suitable nanocarriers for αs1-Casein. Proteo-LUVs were prepared and characterized by different biophysical techniques, such as multiangle light scattering, atomic force imaging, and small-angle X-ray scattering; αs1-Casein loading was quantified by a fluorescence assay. We demonstrated on a C. elegans AD model the effectiveness of the proposed delivery strategy in vivo. Proteo-LUVs allow efficient administration of the protein, exerting a positive functional readout at very low doses while avoiding the intrinsic toxicity of αs1-Casein. Proteo-LUVs of αs1-Casein represent an effective proof of concept for the exploitation of partially disordered proteins as a therapeutic strategy in mild AD conditions.

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

De novo CLTC mutations underlie a spectrum of early-onset neurodevelopmental phenotypes having developmental delay/intellectual disability (ID), epilepsy, and movement disorders (MD) as major clinical features. CLTC 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. Here, we assessed the functional impact of the recurrent c.2669C > T (p.P890L) substitution, which is associated with a relatively mild ID/MD phenotype. Primary fibroblasts endogenously expressing the mutated protein show reduced transferrin uptake compared to fibroblast lines obtained from three unrelated healthy donors, suggesting defective clathrin-mediated endocytosis. In vitro studies also reveal a block in cell cycle transition from G0/G1 to the S phase in patient's cells compared to control cells. To demonstrate the causative role of the p.P890L substitution, the pathogenic missense change was introduced at the orthologous position of the Caenorhabditis elegans gene, chc-1 (p.P892L), via CRISPR/Cas9. The resulting homozygous gene-edited strain displays resistance to aldicarb and hypersensitivity to PTZ, indicating defective release of acetylcholine and GABA by ventral cord motor neurons. Consistently, mutant animals show synaptic vesicle depletion at the sublateral nerve cords, and slightly defective dopamine signaling, highlighting a generalized deficit in synaptic transmission. This defective release of neurotransmitters is associated with their secondary accumulation at the presynaptic membrane. Automated analysis of C. elegans locomotion indicates that chc-1 mutants move slower than their isogenic controls and display defective synaptic plasticity. Phenotypic profiling of chc-1 (+/P892L) heterozygous animals and transgenic overexpression experiments document a mild dominant-negative behavior for the mutant allele. Finally, a more severe phenotype resembling that of chc-1 null mutants is observed in animals harboring the c.3146 T > C substitution (p.L1049P), homologs of the pathogenic c.3140 T > C (p.L1047P) change associated with a severe epileptic phenotype. Overall, our findings provide novel insights into disease mechanisms and genotype-phenotype correlations of CLTC-related disorders.

Synthesis and Characterization of a Biocompatible Nanoplatform Based on Silica-Embedded SPIONs Functionalized with Polydopamine.

Superparamagnetic iron oxide nanoparticles (SPIONs) have gained increasing interest in nanomedicine, but most of those that have entered the clinical trials have been withdrawn due to toxicity concerns. Therefore, there is an urgent need to design low-risk and biocompatible SPION formulations. In this work, we present an original safe-by-design nanoplatform made of silica nanoparticles loaded with SPIONs and decorated with polydopamine (SPIONs@SiO2-PDA) and the study of its biocompatibility performance by an ad hoc thorough in vitro to in vivo nanotoxicological methodology. The results indicate that the SPIONs@SiO2-PDA have excellent colloidal stability in serum-supplemented culture media, even after long-term (24 h) exposure, showing no cytotoxic or genotoxic effects in vitro and ex vivo. Physiological responses, evaluated in vivo using Caenorhabditis elegans as the animal model, showed no impact on fertility and embryonic viability, induction of an oxidative stress response, and a mild impact on animal locomotion. These tests indicate that the synergistic combination of the silica matrix and PDA coating we developed effectively protects the SPIONs, providing enhanced colloidal stability and excellent biocompatibility.

Nusinersen Induces Disease-Severity-Specific Neurometabolic Effects in Spinal Muscular Atrophy.

Intrathecal delivery of Nusinersen-an antisense oligonucleotide that promotes survival motor neuron (SMN) protein induction-is an approved therapy for spinal muscular atrophy (SMA). Here, we employed nuclear magnetic resonance (NMR) spectroscopy to longitudinally characterize the unknown metabolic effects of Nusinersen in the cerebrospinal fluid (CSF) of SMA patients across disease severity. Modulation of amino acid metabolism is a common denominator of biochemical changes induced by Nusinersen, with distinct downstream metabolic effects according to disease severity. In severe SMA1 patients, Nusinersen stimulates energy-related glucose metabolism. In intermediate SMA2 patients, Nusinersen effects are also related to energy homeostasis but involve ketone body and fatty acid biosynthesis. In milder SMA3 patients, Nusinersen mainly modulates amino acid metabolism. Moreover, Nusinersen modifies the CSF metabolome of a more severe clinical group towards the profile of untreated SMA patients with milder disease. These findings reveal disease severity-specific neurometabolic signatures of Nusinersen treatment, suggesting a selective modulation of peripheral organ metabolism by this CNS-directed therapy in severe SMA patients.

TGS1 impacts snRNA 3'-end processing, ameliorates survival motor neuron-dependent neurological phenotypes in vivo and prevents neurodegeneration.

Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5'-monomethylguanosine cap of small nuclear RNAs (snRNAs) to a trimethylguanosine cap. Here, we show that loss of TGS1 in Caenorhabditis elegans, Drosophila melanogaster and Danio rerio results in neurological phenotypes similar to those caused by survival motor neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3' tails that are often uridylated. snRNAs with defective 3' terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.

Extracellular Vesicles From Microalgae: Uptake Studies in Human Cells and Caenorhabditis elegans.

Extracellular vesicles (EVs) are lipid membrane nano-sized vesicles secreted by various cell types for intercellular communication, found in all kingdoms of life. Nanoalgosomes are a subtype of EVs derived from microalgae with a sustainable biotechnological potential. To explore the uptake, distribution and persistence of nanoalgosomes in cells and living organisms, we separated them from a culture of the chlorophyte Tetraselmis chuii cells by tangential flow filtration (TFF), labelled them with different lipophilic dyes and characterized their biophysical attributes. Then we studied the cellular uptake of labelled nanoalgosomes in human cells and in C. elegans, demonstrating that they enter the cells through an energy dependent mechanism and are localized in the cytoplasm of specific cells, where they persist for days. Our data confirm that nanoalgosomes are actively uptaken in vitro by human cells and in vivo by C. elegans cells, supporting their exploitation as potential nanocarriers of bioactive compounds for theranostic applications.

Anosmin-1-Like Effect of UMODL1/Olfactorin on the Chemomigration of Mouse GnRH Neurons and Zebrafish Olfactory Axons Development.

The impairment of development/migration of hypothalamic gonadotropin-releasing hormone (GnRH) neurons is the main cause of Kallmann's syndrome (KS), an inherited disorder characterized by hypogonadism, anosmia, and other developmental defects. Olfactorin is an extracellular matrix protein encoded by the UMODL1 (uromodulin-like 1) gene expressed in the mouse olfactory region along the migratory route of GnRH neurons. It shares a combination of WAP and FNIII repeats, expressed in complementary domains, with anosmin-1, the product of the ANOS1 gene, identified as the causative of KS. In the present study, we have investigated the effects of olfactorin in vitro and in vivo models. The results show that olfactorin exerts an anosmin-1-like strong chemoattractant effect on mouse-immortalized GnRH neurons (GN11 cells) through the activation of the FGFR and MAPK pathways. In silico analysis of olfactorin and anosmin-1 reveals a satisfactory similarity at the N-terminal region for the overall arrangement of corresponding WAP and FNIII domains and marked similarities between WAP domains' binding modes of interaction with the resolved FGFR1-FGF2 complex. Finally, in vivo experiments show that the down-modulation of the zebrafish z-umodl1 gene (orthologous of UMODL1) in both GnRH3:GFP and omp 2k :gap-CFP rw034 transgenic zebrafish strains leads to a clear disorganization and altered fasciculation of the neurites of GnRH3:GFP neurons crossing at the anterior commissure and a significant increase in olfactory CFP + fibers with altered trajectory. Thus, our study shows olfactorin as an additional factor involved in the development of olfactory and GnRH systems and proposes UMODL1 as a gene worthy of diagnostic investigation in KS.

Mitochondrial ribosomal protein genes connected with Alzheimer's and tellurite toxicity.

Mitochondrial diseases are a group of genetic disorders characterized by dysfunctional mitochondria. Within eukaryotic cells, mitochondria contain their own ribosomes, which synthesize small amounts of proteins, all of which are essential for the biogenesis of the oxidative phosphorylation system. The ribosome is an evolutionarily conserved macromolecular machine in nature both from a structural and functional point of view, universally responsible for the synthesis of proteins. Among the diseases afflicting humans, those of ribosomal origin - either cytoplasmic ribosomes (80S) or mitochondrial ribosomes (70S) - are relevant. These are inherited or acquired diseases most commonly caused by either ribosomal protein haploinsufficiency or defects in ribosome biogenesis. Here we review the scientific literature about the recent advances on changes in mitochondrial ribosomal structural and assembly proteins that are implicated in primary mitochondrial diseases and neurodegenerative disorders, and their possible connection with metalloid pollution and toxicity, with a focus on MRPL44, NAM9 (MNA6) and GEP3 (MTG3), whose lack or defect was associated with resistance to tellurite. Finally, we illustrate the suitability of yeast Saccharomyces cerevisiae (S. cerevisiae) and the nematode Caenorhabditis elegans (C. elegans) as model organisms for studying mitochondrial ribosome dysfunctions including those involved in human diseases.

TFEB Regulates ATP7B Expression to Promote Platinum Chemoresistance in Human Ovarian Cancer Cells.

ATP7B is a hepato-specific Golgi-located ATPase, which plays a key role in the regulation of copper (Cu) homeostasis and signaling. In response to elevated Cu levels, ATP7B traffics from the Golgi to endo-lysosomal structures, where it sequesters excess copper and further promotes its excretion to the bile at the apical surface of hepatocytes. In addition to liver, high ATP7B expression has been reported in tumors with elevated resistance to platinum (Pt)-based chemotherapy. Chemoresistance to Pt drugs represents the current major obstacle for the treatment of large cohorts of cancer patients. Although the mechanisms underlying Pt-tolerance are still ambiguous, accumulating evidence suggests that lysosomal sequestration of Pt drugs by ion transporters (including ATP7B) might significantly contribute to drug resistance development. In this context, signaling mechanisms regulating the expression of transporters such as ATP7B are of great importance. Considering this notion, we investigated whether ATP7B expression in Pt-resistant cells might be driven by transcription factor EB (TFEB), a master regulator of lysosomal gene transcription. Using resistant ovarian cancer IGROV-CP20 cells, we found that TFEB directly binds to the predicted coordinated lysosomal expression and regulation (CLEAR) sites in the proximal promoter and first intron region of ATP7B upon Pt exposure. This binding accelerates transcription of luciferase reporters containing ATP7B CLEAR regions, while suppression of TFEB inhibits ATP7B expression and stimulates cisplatin toxicity in resistant cells. Thus, these data have uncovered a Pt-dependent transcriptional mechanism that contributes to cancer chemoresistance and might be further explored for therapeutic purposes.

Deregulation of microtubule organization and RNA metabolism in Arx models for lissencephaly and developmental epileptic encephalopathy.

X-linked lissencephaly with abnormal genitalia (XLAG) and developmental epileptic encephalopathy-1 (DEE1) are caused by mutations in the Aristaless-related homeobox (ARX) gene, which encodes a transcription factor responsible for brain development. It has been unknown whether the phenotypically diverse XLAG and DEE1 phenotypes may converge on shared pathways. To address this question, a label-free quantitative proteomic approach was applied to the neonatal brain of Arx knockout (ArxKO/Y) and knock-in polyalanine (Arx(GCG)7/Y) mice that are respectively models for XLAG and DEE1. Gene ontology and protein-protein interaction analysis revealed that cytoskeleton, protein synthesis and splicing control are deregulated in an allelic-dependent manner. Decreased α-tubulin content was observed both in Arx mice and Arx/alr-1(KO) Caenorhabditis elegans ,and a disorganized neurite network in murine primary neurons was consistent with an allelic-dependent secondary tubulinopathy. As distinct features of Arx(GCG)7/Y mice, we detected eIF4A2 overexpression and translational suppression in cortex and primary neurons. Allelic-dependent differences were also established in alternative splicing (AS) regulated by PUF60 and SAM68. Abnormal AS repertoires in Neurexin-1, a gene encoding multiple pre-synaptic organizers implicated in synaptic remodelling, were detected in Arx/alr-1(KO) animals and in Arx(GCG)7/Y epileptogenic brain areas and depolarized cortical neurons. Consistent with a conserved role of ARX in modulating AS, we propose that the allelic-dependent secondary synaptopathy results from an aberrant Neurexin-1 repertoire. Overall, our data reveal alterations mirroring the overlapping and variant effects caused by null and polyalanine expanded mutations in ARX. The identification of these effects can aid in the design of pathway-guided therapy for ARX endophenotypes and NDDs with overlapping comorbidities.

Mimicking human riboflavin responsive neuromuscular disorders by silencing flad-1 gene in C. elegans: Alteration of vitamin transport and cholinergic transmission.

Riboflavin (Rf), or vitamin B2, is the precursor of FMN and FAD, redox cofactors of several dehydrogenases involved in energy metabolism, redox balance and other cell regulatory processes. FAD synthase, coded by FLAD1 gene in humans, is the last enzyme in the pathway converting Rf into FAD. Mutations in FLAD1 gene are responsible for neuromuscular disorders, in some cases treatable with Rf. In order to mimic these disorders, the Caenorhabditis elegans (C. elegans) gene orthologue of FLAD1 (flad-1) was silenced in a model strain hypersensitive to RNA interference in nervous system. Silencing flad-1 resulted in a significant decrease in total flavin content, paralleled by a decrease in the level of the FAD-dependent ETFDH protein and by a secondary transcriptional down-regulation of the Rf transporter 1 (rft-1) possibly responsible for the total flavin content decrease. Conversely an increased ETFDH mRNA content was found. These biochemical changes were accompanied by significant phenotypical changes, including impairments of fertility and locomotion due to altered cholinergic transmission, as indicated by the increased sensitivity to aldicarb. A proposal is made that neuronal acetylcholine production/release is affected by alteration of Rf homeostasis. Rf supplementation restored flavin content, increased rft-1 transcript levels and eliminated locomotion defects. In this aspect, C. elegans could provide a low-cost animal model to elucidate the molecular rationale for Rf therapy in human Rf responsive neuromuscular disorders and to screen other molecules with therapeutic potential.

Caenorhabditis elegans provides an efficient drug screening platform for GNAO1-related disorders and highlights the potential role of caffeine in controlling dyskinesia.

Dominant GNAO1 mutations cause an emerging group of childhood-onset neurological disorders characterized by developmental delay, intellectual disability, movement disorders, drug-resistant seizures and neurological deterioration. GNAO1 encodes the α-subunit of an inhibitory GTP/GDP-binding protein regulating ion channel activity and neurotransmitter release. The pathogenic mechanisms underlying GNAO1-related disorders remain largely elusive and there are no effective therapies. Here, we assessed the functional impact of two disease-causing variants associated with distinct clinical features, c.139A > G (p.S47G) and c.662C > A (p.A221D), using Caenorhabditis elegans as a model organism. The c.139A > G change was introduced into the orthologous position of the C. elegans gene via CRISPR/Cas9, whereas a knock-in strain carrying the p.A221D variant was already available. Like null mutants, homozygous knock-in animals showed increased egg laying and were hypersensitive to aldicarb, an inhibitor of acetylcholinesterase, suggesting excessive neurotransmitter release by different classes of motor neurons. Automated analysis of C. elegans locomotion indicated that goa-1 mutants move faster than control animals, with more frequent body bends and a higher reversal rate and display uncoordinated locomotion. Phenotypic profiling of heterozygous animals revealed a strong hypomorphic effect of both variants, with a partial dominant-negative activity for the p.A221D allele. Finally, caffeine was shown to rescue aberrant motor function in C. elegans harboring the goa-1 variants; this effect is mainly exerted through adenosine receptor antagonism. Overall, our findings establish a suitable platform for drug discovery, which may assist in accelerating the development of new therapies for this devastating condition, and highlight the potential role of caffeine in controlling GNAO1-related dyskinesia.

Nanoalgosomes: Introducing extracellular vesicles produced by microalgae.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

Impairment of the neurotrophic signaling hub B-Raf contributes to motoneuron degeneration in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a motoneuron disease caused by deletions of the Survival of Motoneuron 1 gene (SMN1) and low SMN protein levels. SMN restoration is the concept behind a number of recently approved drugs which result in impressive yet limited effects. Since SMN has already been enhanced in treated patients, complementary SMN-independent approaches are needed. Previously, a number of altered signaling pathways which regulate motoneuron degeneration have been identified as candidate targets. However, signaling pathways form networks, and their connectivity is still unknown in SMA. Here, we used presymptomatic SMA mice to elucidate the network of altered signaling in SMA. The SMA network is structured in two clusters with AKT and 14-3-3 ζ/δ in their centers. Both clusters are connected by B-Raf as a major signaling hub. The direct interaction of B-Raf with 14-3-3 ζ/δ is important for an efficient neurotrophic activation of the MEK/ERK pathway and crucial for motoneuron survival. Further analyses in SMA mice revealed that both proteins were down-regulated in motoneurons and the spinal cord with B-Raf being reduced at presymptomatic stages. Primary fibroblasts and iPSC-derived motoneurons from SMA patients both showed the same pattern of down-regulation. This mechanism is conserved across species since a Caenorhabditis elegans SMA model showed less expression of the B-Raf homolog lin-45 Accordingly, motoneuron survival was rescued by a cell autonomous lin-45 expression in a C. elegans SMA model resulting in improved motor functions. This rescue was effective even after the onset of motoneuron degeneration and mediated by the MEK/ERK pathway.

Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds.

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 µg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and ß-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

A Single Amino Acid Residue Regulates PTEN-Binding and Stability of the Spinal Muscular Atrophy Protein SMN.

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by decreased levels of the survival of motoneuron (SMN) protein. Post-translational mechanisms for regulation of its stability are still elusive. Thus, we aimed to identify regulatory phosphorylation sites that modulate function and stability. Our results show that SMN residues S290 and S292 are phosphorylated, of which SMN pS290 has a detrimental effect on protein stability and nuclear localization. Furthermore, we propose that phosphatase and tensin homolog (PTEN), a novel phosphatase for SMN, counteracts this effect. In light of recent advancements in SMA therapies, a significant need for additional approaches has become apparent. Our study demonstrates S290 as a novel molecular target site to increase the stability of SMN. Characterization of relevant kinases and phosphatases provides not only a new understanding of SMN function, but also constitutes a novel strategy for combinatorial therapeutic approaches to increase the level of SMN in SMA.

Protective effect of Vigna unguiculata extract against aging and neurodegeneration.

Aging and age-related neurodegeneration are among the major challenges in modern medicine because of the progressive increase in the number of elderly in the world population. Nutrition, which has important long-term consequences for health, is an important way to prevent diseases and achieve healthy aging. The beneficial effects of Vigna unguiculata on metabolic disorders have been widely documented. Here, we show that an aqueous extract of V. unguiculata beans delays senescence both in Saccharomyces cerevisiae and Drosophila melanogaster, in a Snf1/AMPK-dependent manner. Consistently, an increased expression of FOXO, SIRT1, NOTCH and heme oxygenase (HO) genes, already known to be required for the longevity extension in D. melanogaster, is also shown. Preventing α-synuclein self-assembly is one of the most promising approaches for the treatment of Parkinson's disease (PD), for which aging is a risk factor. In vitro aggregation of α-synuclein, its toxicity and membrane localization in yeast and neuroblastoma cells are strongly decreased in the presence of bean extract. In a Caenorhabditis elegans model of PD, V. unguiculata extract substantially reduces the number of the age-dependent degeneration of the cephalic dopaminergic neurons. Our findings support the role of V. unguiculata beans as a functional food in age-related disorders.

Novel Curcumin-Diethyl Fumarate Hybrid as a Dualistic GSK-3ß Inhibitor/Nrf2 Inducer for the Treatment of Parkinson's Disease.

Common copathogenic factors, including oxidative stress and neuroinflammation, are found to play a vital role in the development of neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD). Nowadays, owing to the multifactorial character of the diseases, no effective therapies are available, thus underlying the need for new strategies. Overexpression of the enzyme GSK-3ß and downregulation of the Nrf2/ARE pathway are responsible for a decrease in antioxidant defense effects. These pieces of evidence underline the usefulness of dual GSK-3ß inhibitors/Nrf2 inducers. In this regard, to design a dual modulator, the structures of a curcumin-based analogue, as GSK-3ß inhibitor, and a diethyl fumarate fragment, as Nrf2 inducer, were combined. Among the hybrids, 5 and 6 proved to effectively inhibit GSK-3ß, while 4 and 5 showed a marked ability to activate Nrf2 together to increase the neuronal resistance to oxidative stress. These last pieces of evidence translated into specific neuroprotective effects of 4 and 5 against PD pathological events including neurotoxicity elicited by α-synuclein aggregates and 6-hydroxydopamine. Hybrid 5 also showed neuroprotective effects in a C. elegans model of PD where the activation of GSK-3ß is intimately involved in Nrf2 regulation. In summary, 5 emerged as an interesting multitarget derivative, valuable to be exploited in a multitarget PD perspective.

Co-occurring WARS2 and CHRNA6 mutations in a child with a severe form of infantile parkinsonism.

OBJECTIVE: To investigate the molecular cause(s) underlying a severe form of infantile-onset parkinsonism and characterize functionally the identified variants. METHODS: A trio-based whole exome sequencing (WES) approach was used to identify the candidate variants underlying the disorder. In silico modeling, and in vitro and in vivo studies were performed to explore the impact of these variants on protein function and relevant cellular processes. RESULTS: WES analysis identified biallelic variants in WARS2, encoding the mitochondrial tryptophanyl tRNA synthetase (mtTrpRS), a gene whose mutations have recently been associated with multiple neurological phenotypes, including childhood-onset, levodopa-responsive or unresponsive parkinsonism in a few patients. A substantial reduction of mtTrpRS levels in mitochondria and reduced OXPHOS function was demonstrated, supporting their pathogenicity. Based on the infantile-onset and severity of the phenotype, additional variants were considered as possible genetic modifiers. Functional assessment of a selected panel of candidates pointed to a de novo missense mutation in CHRNA6, encoding the α6 subunit of neuronal nicotinic receptors, which are involved in the cholinergic modulation of dopamine release in the striatum, as a second event likely contributing to the phenotype. In silico, in vitro (Xenopus oocytes and GH4C1 cells) and in vivo (C. elegans) analyses demonstrated the disruptive effects of the mutation on acetylcholine receptor structure and function. CONCLUSION: Our findings consolidate the association between biallelic WARS2 mutations and movement disorders, and suggest CHRNA6 as a genetic modifier of the phenotype.