The Accordion Zebrafish tq206 Mutant in the Assessment of a Novel Pharmaceutical Approach to Brody Myopathy.

Brody disease (BD) is an "ultra-rare" human genetic disorder of skeletal muscle function due to defects in the atp2a1 gene causing deficiency of the SERCA protein, isoform1. The main clinical signs are exercise-induced stiffness and delayed muscular relaxation after physical exercises, even mild ones. No mouse model nor specific therapies exist for Brody myopathy, which is therefore considered an orphan disease. Bovine congenital pseudomyotonia (PMT) is a muscular disorder characterized by an impairment of muscle relaxation and is the only mammalian model of human BD. The pathogenetic mechanism underlying bovine PMT has been recently clarified. These findings prompted us to purpose a potential pharmacological approach addressing a specific population of BD patients who exhibit reduced expression but still exhibit activity of the SERCA1 pump. Preclinical research involving in vivo studies is essential and necessary before clinical trials can be pursued and SERCA protein shows a high degree of conservation among species. So far, the only animal models available to study BD in vivo are a group of zebrafish mutant lines known as accordion zebrafish (acc). In this paper, we focused on a comprehensive characterization of the "acctq206" zebrafish variant. Our aim was to use this mutant line as an experimental animal model for testing the novel therapeutic approach for BD.

The stem-like Stat3-responsive cells of zebrafish intestine are Wnt/ß-catenin dependent.

The transcription factor Stat3 is required for proliferation and pluripotency of embryonic stem cells; we have prepared and characterized fluorescent Stat3-reporter zebrafish based on repeats of minimal responsive elements. These transgenic lines mimic in vivo Stat3 expression patterns and are responsive to exogenous Stat3; notably, fluorescence is inhibited by both stat3 knockout and IL6/Jak/STAT inhibitors. At larval stages, Stat3 reporter activity correlates with proliferating regions of the brain, haematopoietic tissue and intestine. In the adult gut, the reporter is active in sparse proliferating cells, located at the base of intestinal folds, expressing the stemness marker sox9b and having the morphology of mammalian crypt base columnar cells; noteworthy, zebrafish stat3 mutants show defects in intestinal folding. Stat3 reporter activity in the gut is abolished with mutation of T cell factor 4 (Tcf7l2), the intestinal mediator of Wnt/ß-catenin-dependent transcription. The Wnt/ß-catenin dependence of Stat3 activity in the gut is confirmed by abrupt expansion of Stat3-positive cells in intestinal adenomas of apc heterozygotes. Our findings indicate that Jak/Stat3 signalling is needed for intestinal stem cell maintenance and possibly crucial in controlling Wnt/ß-catenin-dependent colorectal cancer cell proliferation.

A novel in-frame deletion in MYOT causes an early adult onset distal myopathy.

Missense mutations in MYOT encoding the sarcomeric Z-disk protein myotilin cause three main myopathic phenotypes including proximal limb-girdle muscular dystrophy, spheroid body myopathy, and late-onset distal myopathy. We describe a family carrying a heterozygous MYOT deletion (Tyr4_His9del) that clinically was characterized by an early-adult onset distal muscle weakness and pathologically by a myofibrillar myopathy (MFM). Molecular modeling of the full-length myotilin protein revealed that the 4-YERPKH-9 amino acids are involved in local interactions within the N-terminal portion of myotilin. Injection of in vitro synthetized mutated human MYOT RNA or of plasmid carrying its cDNA sequence in zebrafish embryos led to muscle defects characterized by sarcomeric disorganization of muscle fibers and widening of the I-band, and severe motor impairments. We identify MYOT novel Tyr4_His9 deletion as the cause of an early-onset MFM with a distal myopathy phenotype and provide data supporting the importance of the amino acid sequence for the structural role of myotilin in the sarcomeric organization of myofibers.

Modeling Human Muscular Dystrophies in Zebrafish: Mutant Lines, Transgenic Fluorescent Biosensors, and Phenotyping Assays.

Muscular dystrophies (MDs) are a heterogeneous group of myopathies characterized by progressive muscle weakness leading to death from heart or respiratory failure. MDs are caused by mutations in genes involved in both the development and organization of muscle fibers. Several animal models harboring mutations in MD-associated genes have been developed so far. Together with rodents, the zebrafish is one of the most popular animal models used to reproduce MDs because of the high level of sequence homology with the human genome and its genetic manipulability. This review describes the most important zebrafish mutant models of MD and the most advanced tools used to generate and characterize all these valuable transgenic lines. Zebrafish models of MDs have been generated by introducing mutations to muscle-specific genes with different genetic techniques, such as (i) N-ethyl-N-nitrosourea (ENU) treatment, (ii) the injection of specific morpholino, (iii) tol2-based transgenesis, (iv) TALEN, (v) and CRISPR/Cas9 technology. All these models are extensively used either to study muscle development and function or understand the pathogenetic mechanisms of MDs. Several tools have also been developed to characterize these zebrafish models by checking (i) motor behavior, (ii) muscle fiber structure, (iii) oxidative stress, and (iv) mitochondrial function and dynamics. Further, living biosensor models, based on the expression of fluorescent reporter proteins under the control of muscle-specific promoters or responsive elements, have been revealed to be powerful tools to follow molecular dynamics at the level of a single muscle fiber. Thus, zebrafish models of MDs can also be a powerful tool to search for new drugs or gene therapies able to block or slow down disease progression.

Glucocorticoids promote Von Hippel Lindau degradation and Hif-1α stabilization.

Glucocorticoid (GC) and hypoxic transcriptional responses play a central role in tissue homeostasis and regulate the cellular response to stress and inflammation, highlighting the potential for cross-talk between these two signaling pathways. We present results from an unbiased in vivo chemical screen in zebrafish that identifies GCs as activators of hypoxia-inducible factors (HIFs) in the liver. GCs activated consensus hypoxia response element (HRE) reporters in a glucocorticoid receptor (GR)-dependent manner. Importantly, GCs activated HIF transcriptional responses in a zebrafish mutant line harboring a point mutation in the GR DNA-binding domain, suggesting a nontranscriptional route for GR to activate HIF signaling. We noted that GCs increase the transcription of several key regulators of glucose metabolism that contain HREs, suggesting a role for GC/HIF cross-talk in regulating glucose homeostasis. Importantly, we show that GCs stabilize HIF protein in intact human liver tissue and isolated hepatocytes. We find that GCs limit the expression of Von Hippel Lindau protein (pVHL), a negative regulator of HIF, and that treatment with the c-src inhibitor PP2 rescued this effect, suggesting a role for GCs in promoting c-src-mediated proteosomal degradation of pVHL. Our data support a model for GCs to stabilize HIF through activation of c-src and subsequent destabilization of pVHL.

Mutant MYO1F alters the mitochondrial network and induces tumor proliferation in thyroid cancer.

Familial aggregation is a significant risk factor for the development of thyroid cancer and familial non-medullary thyroid cancer (FNMTC) accounts for 5-7% of all NMTC. Whole exome sequencing analysis in the family affected by FNMTC with oncocytic features where our group previously identified a predisposing locus on chromosome 19p13.2, revealed a novel heterozygous mutation (c.400G > A, NM_012335; p.Gly134Ser) in exon 5 of MYO1F, mapping to the linkage locus. In the thyroid FRTL-5 cell model stably expressing the mutant MYO1F p.Gly134Ser protein, we observed an altered mitochondrial network, with increased mitochondrial mass and a significant increase in both intracellular and extracellular reactive oxygen species, compared to cells expressing the wild-type (wt) protein or carrying the empty vector. The mutation conferred a significant advantage in colony formation, invasion and anchorage-independent growth. These data were corroborated by in vivo studies in zebrafish, since we demonstrated that the mutant MYO1F p.Gly134Ser, when overexpressed, can induce proliferation in whole vertebrate embryos, compared to the wt one. MYO1F screening in additional 192 FNMTC families identified another variant in exon 7, which leads to exon skipping, and is predicted to alter the ATP-binding domain in MYO1F. Our study identified for the first time a role for MYO1F in NMTC.

Loss-of-function mutations in the SIGMAR1 gene cause distal hereditary motor neuropathy by impairing ER-mitochondria tethering and Ca2+ signalling.

Distal hereditary motor neuropathies (dHMNs) are clinically and genetically heterogeneous neurological conditions characterized by degeneration of the lower motor neurons. So far, 18 dHMN genes have been identified, however, about 80% of dHMN cases remain without a molecular diagnosis. By a combination of autozygosity mapping, identity-by-descent segment detection and whole-exome sequencing approaches, we identified two novel homozygous mutations in the SIGMAR1 gene (p.E138Q and p.E150K) in two distinct Italian families affected by an autosomal recessive form of HMN. Functional analyses in several neuronal cell lines strongly support the pathogenicity of the mutations and provide insights into the underlying pathomechanisms involving the regulation of ER-mitochondria tethering, Ca2+ homeostasis and autophagy. Indeed, in vitro, both mutations reduce cell viability, the formation of abnormal protein aggregates preventing the correct targeting of sigma-1R protein to the mitochondria-associated ER membrane (MAM) and thus impinging on the global Ca2+ signalling. Our data definitively demonstrate the involvement of SIGMAR1 in motor neuron maintenance and survival by correlating, for the first time in the Caucasian population, mutations in this gene to distal motor dysfunction and highlight the chaperone activity of sigma-1R at the MAM as a critical aspect in dHMN pathology.

A Smad3 transgenic reporter reveals TGF-beta control of zebrafish spinal cord development.

TGF-beta (TGFß) family mediated Smad signaling is involved in mesoderm and endoderm specifications, left-right asymmetry formation and neural tube development. The TGFß1/2/3 and Activin/Nodal signal transduction cascades culminate with activation of SMAD2 and/or SMAD3 transcription factors and their overactivation are involved in different pathologies with an inflammatory and/or uncontrolled cell proliferation basis, such as cancer and fibrosis. We have developed a transgenic zebrafish reporter line responsive to Smad3 activity. Through chemical, genetic and molecular approaches we have seen that this transgenic line consistently reproduces in vivo Smad3-mediated TGFß signaling. Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. In the CNS, Smad3 reporter activity is particularly high in the subpallium, tegumentum, cerebellar plate, medulla oblongata and the retina proliferative zone. In the spinal cord, the reporter is activated at the ventricular zone, where neuronal progenitor cells are located. Colocalization methods show in vivo that TGFß signaling is particularly active in neuroD+ precursors. Using neuronal transgenic lines, we observed that TGFß chemical inhibition leads to a decrease of differentiating cells and an increase of proliferation. Similarly, smad3a and 3b knock-down alter neural differentiation showing that both paralogues play a positive role in neural differentiation. EdU proliferation assay and pH3 staining confirmed that Smad3 is mainly active in post-mitotic, non-proliferating cells. In summary, we demonstrate that the Smad3 reporter line allows us to follow in vivo Smad3 transcriptional activity and that Smad3, by controlling neural differentiation, promotes the progenitor to precursor switch allowing neural progenitors to exit cell cycle and differentiate.

Generation and application of signaling pathway reporter lines in zebrafish.

In the last years, we have seen the emergence of different tools that have changed the face of biology from a simple modeling level to a more systematic science. The transparent zebrafish embryo is one of the living models in which, after germline transformation with reporter protein-coding genes, specific fluorescent cell populations can be followed at single-cell resolution. The genetically modified embryos, larvae and adults, resulting from the transformation, are individuals in which time lapse analysis, digital imaging quantification, FACS sorting and next-generation sequencing can be performed in specific times and tissues. These multifaceted genetic and cellular approaches have permitted to dissect molecular interactions at the subcellular, intercellular, tissue and whole-animal level, thus allowing integration of cellular and developmental genetics with molecular imaging in the resulting frame of modern biology. In this review, we describe a new step in the zebrafish road to system biology, based on the use of transgenic biosensor animals expressing fluorescent proteins under the control of signaling pathway-responsive cis-elements. In particular, we provide here the rationale and details of this powerful tool, trying to focus on its huge potentialities in basic and applied research, while also discussing limits and potential technological evolutions of this approach.

Study of metabolism and potential toxicity of nine synthetic opioid analogs using the zebrafish larvae model.

The use of novel psychoactive substances (NPSs) has dramatically increased worldwide, and among them, synthetic opioids are one of the fastest growing groups, where cinnamylpiperazines and 2-benzylbenzimidazoles represent two of the most relevant subclasses. However, the data on their toxicity and metabolism are still limited. The aim of the present study was to evaluate the toxicity and metabolic pathways of some compounds belonging to these families, namely, AP-237, 2-methyl AP-237, isotonitazene, flunitazene, etodesnitazene, metonitazene, metodesnitazene, N-pyrrolidino etonitazene, and butonitazene. The study was performed using a zebrafish early life stages model. In fact, zebrafish (Danio rerio) embryos and larvae have recently been recognized as a suitable animal model in alternative to mammals, because they require less time and resources and do not need complex procedures for ethics approval. The cellular toxicity after a single administration was assessed at the fourth day post-fertilization with acridine orange staining. Possible morphological defects were evaluated with a light microscope after 24 h of exposure to 1 µmol/L concentration of each drug. Subsequently, the larvae were euthanized and underwent analysis of drug metabolites using UPLC coupled to an Orbitrap high-resolution mass spectrometer. High rates of morphological defects, as well as of cellular death, were detected, but no significant difference in mortality between treatment and control groups was observed. In addition, several metabolites, mainly produced through monohydroxylation, N-dealkylation, and O-dealkylation, were identified in the larvae extracts.

Antitumor Effect of Berberine Analogs in a Canine Mammary Tumor Cell Line and in Zebrafish Reporters via Wnt/ß-Catenin and Hippo Pathways.

The heterogeneous nature of human breast cancer (HBC) can still lead to therapy inefficacy and high lethality, and new therapeutics as well as new spontaneous animal models are needed to benefit translational HBC research. Dogs are primarily investigated since they spontaneously develop tumors that share many features with human cancers. In recent years, different natural phytochemicals including berberine, a plant alkaloid, have been reported to have antiproliferative activity in vitro in human cancers and rodent animal models. In this study, we report the antiproliferative activity and mechanism of action of berberine, its active metabolite berberrubine, and eight analogs, on a canine mammary carcinoma cell line and in transgenic zebrafish models. We demonstrate both in vitro and in vivo the significant effects of specific analogs on cell viability via the induction of apoptosis, also identifying their role in inhibiting the Wnt/ß-catenin pathway and activating the Hippo signals with a downstream reduction in CTGF expression. In particular, the berberine analogs NAX035 and NAX057 show the highest therapeutic efficacy, deserving further analyses to elucidate their mechanism of action more in detail, and in vivo studies on spontaneous neoplastic diseases are needed, aiming at improving veterinary treatments of cancer as well as translational cancer research.

A novel DSP zebrafish model reveals training- and drug-induced modulation of arrhythmogenic cardiomyopathy phenotypes.

Arrhythmogenic cardiomyopathy (AC) is an inherited disorder characterized by progressive loss of the ventricular myocardium causing life-threatening ventricular arrhythmias, syncope and sudden cardiac death in young and athletes. About 40% of AC cases carry one or more mutations in genes encoding for desmosomal proteins, including Desmoplakin (Dsp). We present here the first stable Dsp knock-out (KO) zebrafish line able to model cardiac alterations and cell signalling dysregulation, characteristic of the AC disease, on which environmental factors and candidate drugs can be tested. Our stable Dsp knock-out (KO) zebrafish line was characterized by cardiac alterations, oedema and bradycardia at larval stages. Histological analysis of mutated adult hearts showed reduced contractile structures and abnormal shape of the ventricle, with thinning of the myocardial layer, vessels dilation and presence of adipocytes within the myocardium. Moreover, TEM analysis revealed "pale", disorganized and delocalized desmosomes. Intensive physical training protocol caused a global worsening of the cardiac phenotype, accelerating the progression of the disease. Of note, we detected a decrease of Wnt/ß-catenin signalling, recently associated with AC pathogenesis, as well as Hippo/YAP-TAZ and TGF-ß pathway dysregulation. Pharmacological treatment of mutated larvae with SB216763, a Wnt/ß-catenin agonist, rescued pathway expression and cardiac abnormalities, stabilizing the heart rhythm. Overall, our Dsp KO zebrafish line recapitulates many AC features observed in human patients, pointing at zebrafish as a suitable system for in vivo analysis of environmental modulators, such as the physical exercise, and the screening of pathway-targeted drugs, especially related to the Wnt/ß-catenin signalling cascade.

Hyperactivation of Wnt/ß-catenin and Jak/Stat3 pathways in human and zebrafish foetal growth restriction models: Implications for pharmacological rescue.

Foetal Growth Restriction (FGR), previously known as Intrauterine Growth Restriction (IUGR), is an obstetrical condition due to placental insufficiency, affecting yearly about 30 million newborns worldwide. In this work, we aimed to identify and pharmacologically target signalling pathways specifically involved in the FGR condition, focusing on FGR-related cardiovascular phenotypes. The transcriptional profile of human umbilical cords from FGR and control cases was compared with the response to hypoxia of zebrafish (Danio rerio) transgenic lines reporting in vivo the activity of twelve signalling pathways involved in embryonic development. Wnt/ß-catenin and Jak/Stat3 were found as key pathways significantly dysregulated in both human and zebrafish samples. This information was used in a chemical-genetic analysis to test drugs targeting Wnt/ß-catenin and Jak/Stat3 pathways to rescue a set of FGR phenotypes, including growth restriction and cardiovascular modifications. Treatments with the Wnt/ß-catenin agonist SB216763 successfully rescued body dimensions, cardiac shape, and vessel organization in zebrafish FGR models. Our data support the Wnt/ß-catenin pathway as a key FGR marker and a promising target for pharmacological intervention in the FGR condition.

Analysis of complete mitochondrial genomes of patients with schizophrenia and bipolar disorder.

The present study aims at investigating the association between common and rare variants of mitochondrial DNA (mtDNA), and increased risk of schizophrenia (SZ) and bipolar disorder (BPD) in a cohort of patients originating from the same Italian population. The distribution of the major European mtDNA haplogroups was determined in 89 patients and their frequencies did not significantly differ from those observed in the Italian population. Moreover, 27 patients with high probability of having inherited the disease from the maternal side were selected for whole mitochondrial genome sequencing to investigate the possible presence of causative point mutations. Overall, 213 known variants and 2 novel changes were identified, but none of them was predicted to have functional effects. Hence, none of the sequence changes we found in our sample could explain the maternal component of SZ and BPD predisposition.

Genetic Determinants of the Effects of Training on Muscle and Adipose Tissue Homeostasis in Obesity Associated with Lymphedema.

It is widely accepted that metabolic changes associated with training are influenced by a person's genetic background. In this review, we explore the polymorphisms underlying interindividual variability in response to training of weight loss and muscle mass increase in obese individuals, with or without lymphedema, and in normal-weight subjects. We searched PubMed for articles in English published up to May 2019 using the following keywords: (((physical training[Title/Abstract] OR sport activity[Title/Abstract]) AND predisposition[Title/Abstract]) AND polymorphism [Title/Abstract]). We identified 38 single-nucleotide polymorphisms that may modulate the genetic adaptive response to training. The identification of genetic marker(s) that improve the beneficial effects of training may in perspective make it possible to assess training programs, which in combination with dietary intervention can optimize body weight reduction in obese subjects, with or without lymphedema. This is particularly important for patients with lymphedema because obesity can worsen the clinical status, and therefore, a personalized approach that could reduce obesity would be fundamental in the clinical management of lymphedema.

Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish.

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders.

Ubiquitylation of the ER-Shaping Protein Lunapark via the CRL3KLHL12 Ubiquitin Ligase Complex.

Cullin-RING ligases (CRLs) control key cellular processes by promoting ubiquitylation of a multitude of soluble cytosolic and nuclear proteins. Subsets of CRL complexes are recruited and activated locally at cellular membranes; however, few CRL functions and substrates at these distinct cellular compartments are known. Here, we use a proteomic screen to identify proteins that are ubiquitylated at cellular membranes and found that Lunapark, an endoplasmic reticulum (ER)-shaping protein localized to ER three-way junctions, is ubiquitylated by the CRL3KLHL12 ubiquitin ligase. We demonstrate that Lunapark interacts with mechanistic target of rapamycin complex-1 (mTORC1), a central cellular regulator that coordinates growth and metabolism with environmental conditions. We show that mTORC1 binds Lunapark specifically at three-way junctions, and lysosomes, where mTORC1 is activated, make contact with three-way junctions where Lunapark resides. Inhibition of Lunapark ubiquitylation results in neurodevelopmental defects indicating that KLHL12-dependent ubiquitylation of Lunapark is required for normal growth and development.

Progressively De-Differentiated Pancreatic Cancer Cells Shift from Glycolysis to Oxidative Metabolism and Gain a Quiescent Stem State.

Pancreatic ductal adenocarcinoma (PDAC) is typically characterized by high chemoresistance and metastatic spread, features mainly attributable to cancer stem cells (CSCs). It is of central interest the characterization of CSCs and, in particular, the study of their metabolic features in order to selectively identify their peculiarities for an efficient therapeutic approach. In this study, CSCs have been obtained by culturing different PDAC cell lines with a specific growth medium. Cells were characterized for the typical stem/mesenchymal properties at short-, medium-, and long-term culture. Metabolomics, proteomics, analysis of oxygen consumption rate in live cells, and the effect of the inhibition of lactate transporter on cell proliferation have been performed to delineate the metabolism of CSCs. We show that gradually de-differentiated pancreatic cancer cells progressively increase the expression of both stem and epithelial-to-mesenchymal transition markers, shift their metabolism from a glycolytic to an oxidative one, and lastly gain a quiescent state. These quiescent stem cells are characterized by high chemo-resistance, clonogenic ability, and metastatic potential. Re-differentiation reverts these features, re-activating their proliferative capacity and glycolytic metabolism, which generally correlates with high aggressiveness. These observations add an important piece of knowledge to the comprehension of the biology of CSCs, whose metabolic plasticity could be exploited for the generation of promising and selective therapeutic approaches for PDAC patients.

Different atrophy-hypertrophy transcription pathways in muscles affected by severe and mild spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is a neurodegenerative disorder associated with mutations of the survival motor neuron gene SMN and is characterized by muscle weakness and atrophy caused by degeneration of spinal motor neurons. SMN has a role in neurons but its deficiency may have a direct effect on muscle tissue. METHODS: We applied microarray and quantitative real-time PCR to study at transcriptional level the effects of a defective SMN gene in skeletal muscles affected by the two forms of SMA: the most severe type I and the mild type III. RESULTS: The two forms of SMA generated distinct expression signatures: the SMA III muscle transcriptome is close to that found under normal conditions, whereas in SMA I there is strong alteration of gene expression. Genes implicated in signal transduction were up-regulated in SMA III whereas those of energy metabolism and muscle contraction were consistently down-regulated in SMA I. The expression pattern of gene networks involved in atrophy signaling was completed by qRT-PCR, showing that specific pathways are involved, namely IGF/PI3K/Akt, TNF-alpha/p38 MAPK and Ras/ERK pathways. CONCLUSION: Our study suggests a different picture of atrophy pathways in each of the two forms of SMA. In particular, p38 may be the regulator of protein synthesis in SMA I. The SMA III profile appears as the result of the concurrent presence of atrophic and hypertrophic fibers. This more favorable condition might be due to the over-expression of MTOR that, given its role in the activation of protein synthesis, could lead to compensatory hypertrophy in SMA III muscle fibers.

Autosomal Dominant Tubulointerstitial Kidney Disease with Adult Onset due to a Novel Renin Mutation Mapping in the Mature Protein.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a genetically heterogeneous renal disorder leading to progressive loss of renal function. ADTKD-REN is due to rare mutations in renin, all localized in the protein leader peptide and affecting its co-translational insertion in the endoplasmic reticulum (ER). Through exome sequencing in an adult-onset ADTKD family we identified a new renin variant, p.L381P, mapping in the mature protein. To assess its pathogenicity, we combined genetic data, computational and predictive analysis and functional studies. The L381P substitution affects an evolutionary conserved residue, co-segregates with renal disease, is not found in population databases and is predicted to be deleterious by in silico tools and by structural modelling. Expression of the L381P variant leads to its ER retention and induction of the Unfolded Protein Response in cell models and to defective pronephros development in zebrafish. Our work shows that REN mutations outside of renin leader peptide can cause ADTKD and delineates an adult form of ADTKD-REN, a condition which has usually its onset in childhood. This has implications for the molecular diagnosis and the estimated prevalence of the disease and points at ER homeostasis as a common pathway affected in ADTKD-REN, and possibly more generally in ADTKD.