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Cats are known to be affected by hippocampal sclerosis, potentially causing antiseizure drug(s) resistance. In order to lay the foundation for a standardized, systematic classification and diagnosis of this pathology in cats, this prospective study aimed at evaluating normal reference values of cellular densities and the cytoarchitecture of the feline hippocampus. Three transverse sections (head, body and tail) of each left hippocampus were obtained from 17 non-epileptic cats of different brachycephalic and mesocephalic breeds and age classes (range: 3–17 years). Histological (hematoxylin and eosin, Nissl) and immunohistochemical (NeuN, GFAP) staining was performed to investigate neuron and astroglial cell populations, as well as the layer thickness of the pyramidal cell layer and granule cell layer. Significant differences in neuronal density (in CA2-CA4 and the granule cell layer) and layer thickness (in CA1-CA3 and the granule cell layer) were evidenced throughout the longitudinal hippocampal axis (p<0.05); on the other hand, the astrocyte density did not differ. Moreover, reference ranges were defined for these parameters in the pyramidal cell layer and in the granule cell layer. The findings did not differ according to breed or age. In veterinary medicine these parameters have not been evaluated in cats so far. As surgical treatment may become a therapeutic option for cats with temporal lobe epilepsy, estimating normal values of the hippocampal cytoarchitecture will help in the standardized histopathological examination of resected hippocampal specimens to reach a diagnosis of hippocampal sclerosis. [ABSTRACT FROM AUTHOR]

Objective: Precursors of peptide hormones undergo posttranslational modifications within the trans-Golgi network (TGN). Dysfunction of proteins involved at different steps of this process cause several complex syndromes affecting the central nervous system (CNS). We aimed to clarify the genetic cause in a group of patients characterized by hypopituitarism in combination with brain atrophy, thin corpus callosum, severe developmental delay, visual impairment, and epilepsy.Methods: Whole exome sequencing was performed in seven individuals of six unrelated families with these features. Postmortem histopathological and HID1 expression analysis of brain tissue and pituitary gland were conducted in one patient. Functional consequences of the homozygous HID1 variant p.R433W were investigated by Seahorse XF Assay in fibroblasts of two patients.Results: Bi-allelic variants in the gene HID1 domain-containing protein 1 (HID1) were identified in all patients. Postmortem examination confirmed cerebral atrophy with enlarged lateral ventricles. Markedly reduced expression of pituitary hormones was found in pituitary gland tissue. Colocalization of HID1 protein with the TGN was not altered in fibroblasts of patients compared to controls, while the extracellular acidification rate upon stimulation with potassium chloride was significantly reduced in patient fibroblasts compared to controls.Interpretation: Our findings indicate that mutations in HID1 cause an early infantile encephalopathy with hypopituitarism as the leading presentation, and expand the list of syndromic CNS diseases caused by interference of TGN function. ANN NEUROL 2021;90:149-164. [ABSTRACT FROM AUTHOR]

• Weakness of the feet dorsiflexors can evolve in IOPD after long-term ERT. • This distal weakness is due to progression of the myopathy. • A neuropathy in IOPD constitutes a further symptom that may evolve despite ERT. Enzyme replacement therapy in infantile onset Pompe disease has led to a new phenotype with features not known in the pre-enzyme replacement therapy era. We investigated the origin of a rapidly emerging and severe weakness of the foot dorsiflexors in a 7-year-old boy after 6.5 years of enzyme replacement therapy. Electroneurography yielded normal findings except low compound muscle action potentials of the extensor digitorum brevis muscles after stimulation of the peroneal nerves. Electromyography of the tibial muscle demonstrated a myopathic pattern. Tibial muscle, sural nerve, and skin biopsy showed a myopathy with empty and glycogen containing vacuoles, a mild loss of myelinated and unmyelinated axons, and a moderately reduced intraepidermal nerve fiber density. These findings provide evidence for a severe distal muscle involvement and a mild sensory neuropathy evolving during the course of disease after long-term enzyme replacement therapy, thereby expanding the new emerging phenotype of infantile onset Pompe disease. [ABSTRACT FROM AUTHOR]

Native American myopathy (NAM) is an autosomal recessive congenital myopathy, up till now exclusively described in Lumbee Indians who harbor one single homozygous mutation (c.1046G>C, pW284S) in the STAC3 gene, encoding a protein important for proper excitation-contraction coupling in muscle. Here, we report the first non- Amerindian patient of Turkish ancestry, being compound heterozygous for the mutations c.862A>T (p.K288) and c.432þ4A>T (aberrant splicing with skipping of exon 4). Symptoms in NAM include congenital muscle weakness and contractures, progressive scoliosis, early ventilatory failure, a peculiar facial gestalt with mild ptosis and downturned corners of the mouth, short stature, and marked susceptibility to malignant hyperthermia. This case shows that NAM should also be considered in non- Indian patients with congenital myopathy, and suggests that STAC3 mutations should be taken into account as a potential cause of malignant hyperthermia. [ABSTRACT FROM AUTHOR]

Background: Human, hairy skin contains a subgroup of C-fibers, the C-low threshold mechanoreceptive afferents ((C-LTMR) C-tactile or C-touch (CT) fibers) that are linked with the signaling of affective aspects of human touch. Recent studies suggest an involvement of these afferents in the modulation of pain in healthy volunteers. Small fiber neuropathy (SFN) is associated with a damage of C-fibers. Therefore, an impairment of C-LTMRs can be assumed. We aimed to elaborate a possible role of CT-afferents in pain modulation by investigating healthy volunteers and SFN-patients.Methods: Experiment I: 20 SFN-patients (12 women, median age 52.0 years) and 20 healthy controls (14 women, median age 43.0 years) participated in this prospective fMRI and psychophysical study. Heat-pain (HP), CT-targeted touch (slow brushing) and HP combined with CT-targeted touch were applied in randomized order to the left shank in a block design. The participants rated pain intensity on a visual analogue scale. Experiment II: We investigated a possible impact of pain intensity on CT induced pain modulation (10 healthy participants). The intensity of HP stimulation was chosen to induce pain intensity 50/100 (NRS). HP stimulation was applied with and without CT-targeted touch.Results: Experiment I: CT-stimulation was sufficient to reduce heat pain in healthy participants (p = 0.016), but not in SFN-patients. HP induced pain intensity was significantly higher (32,2 vs 52,6) in SFN-patients. During HP, bold responses in pain associated areas were observed in both groups. Additional CT-stimulation elicited no significant difference of bold responses compared to HP. Experiment II: In healthy volunteers, we reproduced a significant reduction of HP intensity by CT-stimulation (p = 0.038).Conclusions: CT input seems to be sufficient to modulate pain, independent of intensity of the pain stimulus. As a prerequisite, the CT fibers have to be intact as in healthy volunteers. If CT fibers are impaired - as in SFN -, CT-targeted touch does not modulate pain intensity. The location of CT-induced pain modulation might be attributed to the level of the dorsal horn since the cortical activation pattern of heat pain with and without CT-targeted touch did not differ in healthy subjects and in SFN-patients. [ABSTRACT FROM AUTHOR]

The effects of enzyme replacement therapy (ERT) in infantile Pompe disease are variable, necessitating the identification of biomarkers to assess the severity of disease and response to ERT. The aims of this study were to investigate whether quantification of muscle pathology in infantile Pompe disease prior to and during ERT is feasible at the light microscope, and to develop a score that summarizes the degree of muscle pathology in a comprehensive manner from PAS-stained resin sections alone. We, therefore, determined glycogen load, extent of muscle fibre disruption, and amount of autophagic vacuoles in resin-embedded muscle biopsy specimens from 11 infantile Pompe patients and 2 with early childhood phenotype by quantitative methods, correlated the findings with ultrastructural analyses, compared PAS-stained resin sections with conventional PAS-stained cryosections, and related the quantified degree of muscle damage from infantile patients to the effects of ERT. Comparison of electron and light microscopic findings demonstrated that important alterations of skeletal muscle morphology can also be depicted by examining PAS stained resin sections. Infantile patients with good response to ERT had lower muscle pathology score values prior to and during ERT than those with moderate and poor response, but the number of tissue samples available for evaluation was limited. These findings suggest that quantification of muscle pathology by analysing PAS stained resin sections is in principle feasible and useful to monitor disease progression and therapy response. These results have to be validated by investigating a larger group of patients. [ABSTRACT FROM AUTHOR]

Introduction Familial amyloid polyneuropathy is a rare autosomal dominant disorder caused by mutations in the transthyretin gene, TTR. Diagnosis can be challenging, especially if other family members are not affected or an obvious systemic involvement is lacking. The patients are often misdiagnosed, leading to a delay in the initiation of therapy. Case presentation A 35-year-old woman of Turkish origin presented to our outpatient clinic with severe polyneuropathy associated with distally pronounced tetraparesis and hypesthesia of 2 to 3 years' duration. In addition, small nerve fiber involvement with impaired detection of cold temperatures and tingling pain in the lower legs was reported. She did not complain of autonomic dysfunction or visual disturbance. Her family history was empty regarding neuromuscular disorders. The routine diagnostic work-up was unremarkable. A sural nerve biopsy disclosed amyloid deposits, which led to the identification of a rare heterozygous transthyretin mutation (p.Glu74Gly; old classification: pGlu54Gly). Conclusions Few cases with this very heterozygous mutation can be found in the literature. In contrast to the case of our patient, all of the previously described patients in the literature presented with additional severe autonomic symptoms, involvement of the eyes and a positive family history. In this case report, we emphasize that, in patients with progressive neuropathy with small fiber involvement, an amyloid neuropathy should be considered in the differential diagnosis, even if the family history is empty and other organs are not affected. [ABSTRACT FROM AUTHOR]

Biotin-thiamine-responsive basal ganglia disease ( BTBGD) is a potentially treatable disorder caused by mutations in the SLC19A3 gene, encoding the human thiamine transporter 2. Manifestation of BTBGD as acute encephalopathy triggered by a febrile infection has been frequently reported, but the underlying mechanisms are not clear. We investigated a family with two brothers being compound heterozygous for the SLC19A3 mutations p.W94R and p.Q393*fs. Post-mortem analysis of the brain of one brother showed a mixture of acute, subacute and chronic changes with cystic and necrotic lesions and hemorrhage in the putamen, and hemorrhagic lesions in the caudate nucleus and cortical layers. SLC19A3 expression was substantially reduced in the cortex, basal ganglia and cerebellum compared with an age-matched control. Importantly, exposure of fibroblasts to stress factors such as acidosis or hypoxia markedly upregulated SLC19A3 in control cells, but failed to elevate SLC19A3 expression in the patient's fibroblasts. These results demonstrate ubiquitously reduced thiamine transporter function in the cerebral gray matter, and neuropathological alterations similar to Wernicke's disease in BTBGD. They also suggest that episodes of encephalopathy are caused by a substantially reduced capacity of mutant neuronal cells to increase SLC19A3 expression, necessary to adapt to stress conditions. [ABSTRACT FROM AUTHOR]

Background: In juvenile idiopathic inflammatory myopathies (IIMs), morphological characteristic features of distinct subgroups are not well defined. New treatment strategies require a precise diagnosis of the subgroups in IIM, and, therefore, knowledge about the pathomorphology of juvenile IIMs is warranted. Methods: Muscle biopsies from 15 patients (median age 8 (range 3–17) years, 73% female) with IIM and seven controls were analyzed by standard methods, immunohistochemistry, and transmission electron microscopy (TEM). Detailed clinical and laboratory data were accessed retrospectively. Results: Proximal muscle weakness and skin symptoms were the main clinical symptoms. Dermatomyositis (DM) was diagnosed in 9/15, antisynthetase syndrome (ASyS) in 4/15, and overlap myositis (OM) in 2/15. Analysis of skeletal muscle tissues showed inflammatory cells and diffuse upregulation of MHC class I in all subtypes. Morphological key findings were COX-deficient fibers as a striking pathology in DM and perimysial alkaline phosphatase positivity in anti-Jo-1-ASyS. Vascular staining of the type 1 IFN-surrogate marker, MxA, correlated with endothelial tubuloreticular inclusions in both groups. None of these specific morphological findings were present in anti-PL7-ASyS or OM patients. Conclusions: Morphological characteristics discriminate IIM subtypes in juvenile patients, emphasizing differences in aetiopathogenesis and supporting the notion of individual and targeted therapeutic strategies. [ABSTRACT FROM AUTHOR]

The generation of new neurons in the olfactory bulb (OB) persists into adulthood and is a multistep process that includes proliferation, fate choice, migration, survival, and differentiation. Neural precursor cells destined to form olfactory interneurons arise in the subventricular zone (SVZ) and migrate along the rostral migratory stream (RMS) to the OB. Recently, some factors classically known from their effects on the vascular system have been found to influence different steps of adult neurogenesis. In the present study, we report a modulatory function for the vascular endothelial growth factor receptor-1 (VEGFR-1) in adult olfactory neurogenesis. We identified expression of VEGFR-1 in GFAP-positive cells within regions involved in neurogenesis of the adult mouse brain. To determine functions for VEGFR-1 in adult neurogenesis, we compared neural progenitor cell proliferation, migration, and differentiation from wild-type and VEGFR-1 signaling-deficient mice (Flt-1TK-/-mice). Our data show that VEGFR-1 signaling is involved in the regulation of proliferation of neuronal progenitor cells within the SVZ, migration along the RMS, and in neuronal differentiation and anatomical composition of interneuron subtypes within the OB. RMS migration in Flt-1TK-/- mice was altered mainly as a result of increased levels of its ligand VEGF-A, which results in an increased phosphorylation of VEGFR-2 in neuronal progenitor cells within the SVZ and the RMS. This study reveals that proper RMS migration is dependent on endogenous VEGF-A protein. [ABSTRACT FROM AUTHOR]

Using ferric chloride (FeCl3) to induce experimental superior sagittal sinus (SSS) thrombosis might interfere with magnetic resonance imaging (MRI)-assisted visualization and evaluation of the thrombus, the brain parenchyma, and the quality of the occlusion. The aim of this study was to investigate whether aluminum chloride (AlCl3)-induced thrombosis of the SSS has comparable properties to those of FeCl3 without causing artifacts in MRI. SSS thrombosis was induced in 14 male Wistar rats by exposure of the SSS and subsequent topical application of a filter paper strip soaked in AlCl3 (n = 7) or FeCl3 (n = 7) over a period of 15 min. The animals with AlCl3-induced SSS thrombosis showed a constant and complete occlusion with in histological analysis large thrombi. Blood flow measurements indicated a significant reduction on the first and seventh postoperative day compared to preoperative measurements. MRI enabled visualization and subsequent evaluation of the thrombus and the surrounding parenchyma. In comparison, FeCl3-induced SSS thrombosis could not be evaluated by MRI due to artifacts caused by the paramagnetic properties and increased susceptibility of FeCl3. The occluded sinus and the surrounding area appeared hypointense. The quality of SSS occlusion by AlCl3 was comparable to that of FeCl3. AlCl3 therefore represents a significant alternative substance in experimental SSS thrombosis ideally suited for studies using MRI. [ABSTRACT FROM AUTHOR]

The article discusses research on the prevalence of progressive multifocal leukoencephatolopathy in a patient with non-Hodgkin lymphoma following his treatment with cladribine. Topics discussed include the case of a female patient with disoriented mental status, homonymous hemianopsia, and progressive visual disturbances. Also mentioned are the patient's medical history of follicular lymphoma, and the results of the magnetic resonance imaging (MRI) and computed tomography (CT).

Cardiomyocyte maturation during pre- and postnatal development requires multiple intertwined processes, including a switch in energy generation from glucose utilization in the embryonic heart towards fatty acid oxidation after birth. This is accompanied by a boost in mitochondrial mass to increase capacities for oxidative phosphorylation and ATP generation required for efficient contraction. Whether cardiomyocyte differentiation is paralleled by augmented capacities to deal with reactive oxygen species (ROS), physiological byproducts of the mitochondrial electron transport chain (ETC), is less clear. Here we show that expression of genes and proteins involved in redox homeostasis and protein quality control within mitochondria increases after birth in the mouse and human heart. Using primary embryonic, neonatal and adult mouse cardiomyocytes in vitro we investigated how excessive ROS production induced by mitochondrial dysfunction affects cell survival and stress response at different stages of maturation. Embryonic and neonatal cardiomyocytes largely tolerate inhibition of ETC complex III by antimycin A (AMA) as well as ATP synthase (complex V) by oligomycin but are susceptible to complex I inhibition by rotenone. All three inhibitors alter the intracellular distribution and ultrastructure of mitochondria in neonatal cardiomyocytes. In contrast, adult cardiomyocytes treated with AMA undergo rapid morphological changes and cellular disintegration. At the molecular level embryonic cardiomyocytes activate antioxidative defense mechanisms, the integrated stress response (ISR) and ER stress but not the mitochondrial unfolded protein response upon complex III inhibition. In contrast, adult cardiomyocytes fail to activate the ISR and antioxidative proteins following AMA treatment. In conclusion, our results identified fundamental differences in cell survival and stress response in differentiated compared to immature cardiomyocytes subjected to mitochondrial dysfunction. The high stress tolerance of immature cardiomyocytes might allow outlasting unfavorable intrauterine conditions thereby preventing fetal or perinatal heart disease and may contribute to the regenerative capacity of the embryonic and neonatal mammalian heart. [Display omitted] • Capacities for redox homeostasis increase parallel to cardiomyocyte maturation. • Embryonic and neonatal cardiomyocytes largely tolerate mitochondrial dysfunction. • The site of mitochondrial ROS generation determines outcome in immature cardiomyocytes. • Adult cardiomyocytes are highly susceptible to mitochondrial dysfunction. • Cardiomyocyte maturation alters the molecular response to mitochondrial impairment. [ABSTRACT FROM AUTHOR]

Background: Dilated cardiomyopathy (DCM) is a leading cause of death in children with heart failure. The outcome of pediatric heart failure treatment is inconsistent, and large cohort studies are lacking. Progress may be achieved through personalized therapy that takes age- and disease-related pathophysiology, pathology, and molecular fingerprints into account. We present single nuclei RNA sequencing from pediatric patients with DCM as the next step in identifying cellular signatures.Methods: We performed single nuclei RNA sequencing with heart tissues from 6 children with DCM with an age of 0.5, 0.75, 5, 6, 12, and 13 years. Unsupervised clustering of 18 211 nuclei led to the identification of 14 distinct clusters with 6 major cell types.Results: The number of nuclei in fibroblast clusters increased with age in patients with DCM, a finding that was confirmed by histological analysis and was consistent with an age-related increase in cardiac fibrosis quantified by cardiac magnetic resonance imaging. Fibroblasts of patients with DCM >6 years of age showed a profoundly altered gene expression pattern with enrichment of genes encoding fibrillary collagens, modulation of proteoglycans, switch in thrombospondin isoforms, and signatures of fibroblast activation. In addition, a population of cardiomyocytes with a high proregenerative profile was identified in infant patients with DCM but was absent in children >6 years of age. This cluster showed high expression of cell cycle activators such as cyclin D family members, increased glycolytic metabolism and antioxidative genes, and alterations in ß-adrenergic signaling genes.Conclusions: Novel insights into the cellular transcriptomes of hearts from pediatric patients with DCM provide remarkable age-dependent changes in the expression patterns of fibroblast and cardiomyocyte genes with less fibrotic but enriched proregenerative signatures in infants. [ABSTRACT FROM AUTHOR]

Synaptopodin-2 (SYNPO2) is a protein associated with the Z-disc in striated muscle cells. It interacts with α-actinin and filamin C, playing a role in Z-disc maintenance under stress by chaperone-assisted selective autophagy (CASA). In smooth muscle cells, SYNPO2 is a component of dense bodies. Furthermore, it has been proposed to play a role in tumor cell proliferation and metastasis in many different kinds of cancers. Alternative transcription start sites and alternative splicing predict the expression of six putative SYNPO2 isoforms differing by extended amino- and/or carboxy-termini. Our analyses at mRNA and protein levels revealed differential expression of SYNPO2 isoforms in cardiac, skeletal and smooth muscle cells. We identified synemin, an intermediate filament protein, as a novel binding partner of the PDZ-domain in the amino-terminal extension of the isoforms mainly expressed in cardiac and smooth muscle cells, and demonstrated colocalization of SYNPO2 and synemin in both cell types. A carboxy-terminal extension, mainly expressed in smooth muscle cells, is sufficient for association with dense bodies and interacts with α-actinin. SYNPO2 therefore represents an additional and novel link between intermediate filaments and the Z-discs in cardiomyocytes and dense bodies in smooth muscle cells, respectively. In pathological skeletal muscle samples, we identified SYNPO2 in the central and intermediate zones of target fibers of patients with neurogenic muscular atrophy, and in nemaline bodies. Our findings help to understand distinct functions of individual SYNPO2 isoforms in different muscle tissues, but also in tumor pathology. [ABSTRACT FROM AUTHOR]

This article explores the distribution and composition of the ferritin complex in the adult brain and its connection to neuroferritinopathy (NF), a type of neurodegeneration with brain iron accumulation (NBIA). NF is characterized by symptoms like movement disturbances and tremors. The study identifies a novel variant in the ferritin heavy chain gene (FTH1) that is associated with NF and provides insights into the role of ferritin in both healthy and diseased brains. The authors, a diverse group of researchers, present their findings in a respectful and culturally sensitive manner, making this report a valuable resource for library patrons researching this topic. [Extracted from the article]

Cats, similar to humans, are known to be affected by hippocampal sclerosis (HS), potentially causing antiepileptic drug (AED) resistance. HS can occur as a consequence of chronic seizure activity, trauma, inflammation, or even as a primary disease. In humans, temporal lobe resection is the standardized therapy in patients with refractory temporal lobe epilepsy (TLE). The majority of TLE patients are seizure free after surgery. Therefore, the purpose of this prospective cadaveric study is to establish a surgical technique for hippocampal resection in cats as a treatment for AED resistant seizures. Ten cats of different head morphology were examined. Pre-surgical magnetic resonance imaging (MRI) and computed tomography (CT) studies of the animals' head were carried out to complete 3D reconstruction of the head, brain, and hippocampus. The resected hippocampal specimens and the brains were histologically examined for tissue injury adjacent to the hippocampus. The feasibility of the procedure, as well as the usability of the removed specimen for histopathological examination, was assessed. Moreover, a micro-CT (mCT) examination of the brain of two additional cats was performed in order to assess temporal vasculature as a reason for possible intraoperative complications. In all cats but one, the resection of the temporal cortex and the hippocampus were successful without any evidence of traumatic or vascular lesions in the surrounding neurovascular structures. In one cat, the presence of mechanical damage (a fissure) of the thalamic surface was evident in the histopathologic examination of the brain post-resection. All hippocampal fields and the dentate gyrus were identified in the majority of the cats via histological examination. The study describes a new surgical approach (partial temporal cortico-hippocampectomy) offering a potential treatment for cats with clinical and diagnostic evidence of temporal epilepsy which do not respond adequately to the medical therapy. [ABSTRACT FROM AUTHOR]

Small fiber neuropathy (SFN) affects unmyelinated and thinly myelinated nerve fibers causing neuropathic pain with distal distribution and autonomic symptoms. In idiopathic SFN (iSFN), 30% of the cases, the underlying aetiology remains unknown. Gadolinium (Gd)-based contrast agents (GBCA) are widely used in magnetic resonance imaging (MRI). However, side-effects including musculoskeletal disorders and burning skin sensations were reported. We investigated if dermal Gd deposits are more prevalent in iSFN patients exposed to GBCAs, and if dermal nerve fiber density and clinical parameters are likewise affected. 28 patients (19 females) with confirmed or no GBCA exposure were recruited in three German neuromuscular centers. ISFN was confirmed by clinical, neurophysiological, laboratory and genetic investigations. Six volunteers (two females) served as controls. Distal leg skin biopsies were obtained according to European recommendations. In these samples Gd was quantified by elemental bioimaging and intraepidermal nerve fibers (IENF) density via immunofluorescence analysis. Pain phenotyping was performed in all patients, quantitative sensory testing (QST) only in a subset (15 patients; 54%). All patients reported neuropathic pain, described as burning (n = 17), jabbing (n = 16) and hot (n = 11) and five QST scores were significantly altered. Compared to an equal distribution significantly more patients reported GBCA exposures (82%), while 18% confirmed no exposures. Compared to unexposed patients/controls significantly increased Gd deposits and lower z-scores of the IENF density were confirmed in exposed patients. QST scores and pain characteristics were not affected. This study suggests that GBCA exposure might alter IENF density in iSFN patients. Our results pave the road for further studies investigating the possible role of GBCA in small fiber damage, but more investigations and larger samples are needed to draw firm conclusions. [ABSTRACT FROM AUTHOR]

Background: More than 90% of individuals with Noonan syndrome (NS) with mutations clustered in the CR2 domain of RAF1 present with severe and often lethal hypertrophic cardiomyopathy (HCM). The signaling pathways by which NS RAF1 mutations promote HCM remain elusive, and so far, there is no known treatment for NS-associated HCM.Methods: We used patient-derived RAF1S257L/+ and CRISPR-Cas9-generated isogenic control inducible pluripotent stem cell (iPSC)-derived cardiomyocytes to model NS RAF1-associated HCM and to further delineate the molecular mechanisms underlying the disease.Results: We show that mutant iPSC-derived cardiomyocytes phenocopy the pathology seen in hearts of patients with NS by exhibiting hypertrophy and structural defects. Through pharmacological and genetic targeting, we identify 2 perturbed concomitant pathways that, together, mediate HCM in RAF1 mutant iPSC-derived cardiomyocytes. Hyperactivation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), but not extracellular regulated kinase 1/2, causes myofibrillar disarray, whereas the enlarged cardiomyocyte phenotype is a direct consequence of increased extracellular regulated kinase 5 (ERK5) signaling, a pathway not previously known to be involved in NS. RNA-sequencing reveals genes with abnormal expression in RAF1 mutant iPSC-derived cardiomyocytes and identifies subsets of genes dysregulated by aberrant MEK1/2 or ERK5 pathways that could contribute to the NS-associated HCM.Conclusions: Taken together, the results of our study identify the molecular mechanisms by which NS RAF1 mutations cause HCM and reveal downstream effectors that could serve as therapeutic targets for treatment of NS and perhaps other, more common, congenital HCM disorders. [ABSTRACT FROM AUTHOR]

Noonan syndrome (NS), the most common among RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770 C > T missense change. We characterize the molecular, structural, and functional consequences of aberrant RAF1–associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes and myocardial tissue biopsies. The aforementioned changes correlated with the isoform shift of titin from a longer (N2BA) to a shorter isoform (N2B) that also affected the active force generation and contractile tensions. The genotype-phenotype correlation was confirmed using cardiomyocyte progeny of an isogenic gene-corrected RAF1S257L-iPSC line and was mainly reversed by MEK inhibition. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease. Studies on 3D bioartificial cardiac tissues reveal the impacts of hypertrophic cardiomyopathy-associated RAF1 mutations on sarcomere structure, contractile behavior, Ca2+ handling, and intracellular signaling. [ABSTRACT FROM AUTHOR]

Pompe disease is a rare genetic metabolic disorder caused by mutations in acid-alpha glucoside (GAA) leading to pathological lysosomal glycogen accumulation associated with skeletal muscle weakness, respiratory difficulties and cardiomyopathy, dependent from the GAA residual enzyme activity. This study aimed to investigate early proteomic changes in a mouse model of Pompe disease and identify potential therapeutic pathways using proteomic analysis of skeletal muscles from pre-symptomatic Pompe mice. For this purpose, quadriceps samples of Gaa6neo/6neo mutant (Pompe) and wildtype mice, at the age of six weeks, were studied with three biological replicates for each group. The data were validated with skeletal muscle morphology, immunofluorescence studies and western blot analysis. Proteomic profiling identified 538 significantly upregulated and 16 significantly downregulated proteins in quadriceps muscles derived from Pompe animals compared to wildtype mice. The majority of significantly upregulated proteins were involved in metabolism, translation, folding, degrading and vesicular transport, with some having crucial roles in the etiopathology of other neurological or neuromuscular diseases. This study highlights the importance of the early diagnosis and treatment of Pompe disease and suggests potential add-on therapeutic strategies targeting protein dysregulations. [ABSTRACT FROM AUTHOR]

Peroxisomes are eukaryotic organelles that rapidly change in number depending on the metabolic requirement of distinct cell types and tissues. In the brain, these organelles are essential for neuronal migration and myelination during development and their dysfunction is associated with age-related neurodegenerative diseases. Except for one study analysing ABCD3-positive peroxisomes in neurons of the frontal neocortex of Alzheimer disease (AD) patients, no data on other brain regions or peroxisomal proteins are available. In the present morphometric study, we quantified peroxisomes labelled with PEX14, a metabolism-independent peroxisome marker, in 13 different brain areas of 8 patients each either with low, intermediate or high AD neuropathological changes compared to 10 control patients. Classification of patient samples was based on the official ABC score. During AD-stage progression, the peroxisome density decreased in the area entorhinalis, parietal/occipital neocortex and cerebellum, it increased and in later AD-stage patients decreased in the subiculum and hippocampal CA3 region, frontal neocortex and pontine gray and it remained unchanged in the gyrus dentatus, temporal neocortex, striatum and inferior olive. Moreover, we investigated the density of catalase-positive peroxisomes in a subset of patients (> 80 years), focussing on regions with significant alterations of PEX14-positive peroxisomes. In hippocampal neurons, only one third of all peroxisomes contained detectable levels of catalase exhibiting constant density at all AD stages. Whereas the density of all peroxisomes in neocortical neurons was only half of the one of the hippocampus, two thirds of them were catalase-positive exhibiting increased levels at higher ABC scores. In conclusion, we observed spatiotemporal differences in the response of peroxisomes to different stages of AD-associated pathologies. [ABSTRACT FROM AUTHOR]

RASopathies are caused by mutations in genes that alter the MAP kinase pathway and are marked by several malformations with cardiovascular disorders as the predominant cause of mortality. Mechanistic insights in the underlying pathogenesis in affected cardiac tissue are rare. The aim of the study was to assess the impact of RASopathy causing mutations on the human heart. Using single cell approaches and histopathology we analyzed cardiac tissue from children with different RASopathy-associated mutations compared to age-matched dilated cardiomyopathy (DCM) and control hearts. The volume of cardiomyocytes was reduced in RASopathy conditions compared to controls and DCM patients, and the estimated number of cardiomyocytes per heart was ∼4–10 times higher. Single nuclei RNA sequencing of a 13-year-old RASopathy patient (carrying a PTPN11 c.1528C > G mutation) revealed that myocardial cell composition and transcriptional patterns were similar to <1 year old DCM hearts. Additionally, immaturity of cardiomyocytes is shown by an increased MYH6/MYH7 expression ratio and reduced expression of genes associated with fatty acid metabolism. In the patient with the PTPN11 mutation activation of the MAP kinase pathway was not evident in cardiomyocytes, whereas increased phosphorylation of PDK1 and its downstream kinase Akt was detected. In conclusion, an immature cardiomyocyte differentiation status appears to be preserved in juvenile RASopathy patients. The increased mass of the heart in such patients is due to an increase in cardiomyocyte number (hyperplasia) but not an enlargement of individual cardiomyocytes (hypertrophy). [Display omitted] • Pediatric patients with RASopathies exhibit an increased heart mass. • Cardiomyocyte size and volume are reduced in pediatric patients with RASopathies. • Cardiomyocyte numbers per heart are increased in pediatric RASopathy patients. • Cardiomyocytes in juvenile RASopathy have an immature state. • Elevated LV mass in RASopathy patients is mainly due to cardiomyocyte hyperplasia. [ABSTRACT FROM AUTHOR]

Background: Cardiomyopathies (CMs) are a heterogeneous and severe group of diseases that shows a highly variable cardiac phenotype and an incidence of app. 1/100.000. Genetic screening of family members is not yet performed routinely. Patients and methods: Three families with dilated cardiomyopathy (DCM) and pathogenic variants in the troponin T2, Cardiac Type (TNNT2) gene were included. Pedigrees and clinical data of the patients were collected. The reported variants in the TNNT2 gene showed a high penetrance and a poor outcome, with 8 of 16 patients dying or receiving heart transplantation. The age of onset varied from the neonatal period to the age of 52. Acute heart failure and severe decompensation developed within a short period in some patients. Conclusion: Family screening of patients with DCM improves risk assessment, especially for individuals who are currently asymptomatic. Screening contributes to improved treatment by enabling practitioners to set appropriate control intervals and quickly begin interventional measures, such as heart failure medication or, in selected cases, pulmonary artery banding. [ABSTRACT FROM AUTHOR]

The Popeye domain containing (POPDC) genes encode sarcolemma-localized cAMP effector proteins. Mutations in blood vessel epicardial substance (BVES) also known as POPDC1 and POPDC2 have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms. Biopsy material of patients carrying mutations in BVES were immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein–protein interaction. Patients carrying a novel homozygous variant, BVES (c.547G > T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. In contrast, variants such as BVES p.Q153X or POPDC2 p.W188X were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1–POPDC2 complex. Mutations in POPDC proteins that cause an impairment in membrane localization affect POPDC complex formation while mutations which leave protein–protein interaction intact likely affect some other essential function of POPDC proteins. [ABSTRACT FROM AUTHOR]

Popeye domain containing protein 1 (POPDC1) is a highly conserved transmembrane protein essential for striated muscle function and homeostasis. Pathogenic variants in the gene encoding POPDC1 (BVES, Blood vessel epicardial substance) are causative for limb-girdle muscular dystrophy (LGMDR25), associated with cardiac arrhythmia. We report on four affected children (age 7–19 years) from two consanguineous families with two novel pathogenic variants in BVES c.457C>T(p.Q153X) and c.578T>G (p.I193S). Detailed analyses were performed on muscle biopsies from an affected patient of each family including immunofluorescence, electron microscopy and proteomic profiling. Cardiac abnormalities were present in all patients and serum creatine kinase (CK) values were variably elevated despite lack of overt muscle weakness. Detailed histological analysis of skeletal muscle, however indicated a myopathy with reduced sarcolemmal expression of POPDC1 accompanied by altered sarcolemmal and sarcoplasmatic dysferlin and Xin/XIRP1 abundance. At the electron microscopic level, the muscle fiber membrane was focally disrupted. The proteomic signature showed statistically significant dysregulation of 191 proteins of which 173 were increased and 18 were decreased. Gene ontology-term analysis of affected biological processes revealed - among others - perturbation of muscle fibril assembly, myofilament sliding, and contraction as well as transition between fast and slow fibers. In conclusion, these findings demonstrate that the phenotype of LGMDR25 is highly variable and also includes younger children with conduction abnormalities, no apparent muscular problems, and only mildly elevated CK values. Biochemical studies suggest that BVES mutations causing loss of functional POPDC1 can impede striated muscle function by several mechanisms. • Two novel mutations in BVES are associated with an early onset of LGMD25. • Loss of POPDC1 is accompanied by altered sarcolemmal and sarcoplasmatic dysferlin and Xin/XIRP1. • In children with cardiac symptoms, a neuromuscular disorder should be ruled out. • Expression of POPDC1 in cardiac muscle highlights the cardio-skeletal link in LGMD. [ABSTRACT FROM AUTHOR]

Multi-centre MRI studies of the brain are essential for enrolling large and diverse patient cohorts, as required for the investigation of heterogeneous neurological and psychiatric diseases. However, the multi-site comparison of standard MRI data sets that are weighted with respect to tissue parameters such as the relaxation times (T1, T2) and proton density (PD) may be problematic, as signal intensities and image contrasts depend on site-specific details such as the sequences used, imaging parameters, and sensitivity profiles of the radiofrequency (RF) coils. Water or gel phantoms are frequently used for long-term and/or inter-site quality assessment. However, these phantoms hardly mimic the structure, shape, size or tissue distribution of the human brain. The goals of this study were: (1) to validate the long-term stability of a human post-mortem brain phantom, performing quantitative mapping of T1, T2, and PD, and the magnetization transfer ratio (MTR) over a period of 18 months; (2) to acquire and analyse data for this phantom and the brain of a healthy control (HC) in a multi-centre study for MRI protocol standardization in four centres, while conducting a voxel-wise as well as whole brain grey (GM) and white matter (WM) tissue volume comparison. MTR, T2, and the quotient of PD in WM and GM were stable in the post-mortem brain with no significant changes. T1 was found to decrease from 267/236 ms (GM/WM) to 234/216 ms between 5 and 17 weeks post embedment, stabilizing during an 18-month period following the first scan at about 215/190 ms. The volumetric measures, based on T1-weighted MP-RAGE images obtained at all participating centres, revealed inter- and intra-centre variations in the evaluated GM and WM volumes that displayed similar trends in both the post-mortem brain as well as the HC. At a confidence level of 95%, brain regions such as the brainstem, deep GM structures as well as boundaries between GM and WM tissues were found to be less reproducible than other brain regions in all participating centres. The results demonstrate that a post-mortem brain phantom may be used as a reliable tool for multi-centre MR studies. [ABSTRACT FROM AUTHOR]

The transgenic TGR(mREN-2)27 rat is not only characterized by fulminant hypertension, but also by a disturbance in circadian blood pressure regulation, resulting in inverse circadian blood pressure profiles. The reasons for these alterations are not very well understood at present. We therefore investigated the circadian rhythms in several hormones participating in blood pressure regulation. From TGR and Sprague-Dawley (SPRD) control rats synchronized to 12h light and 12h dark (LD 12:12) blood was collected at different circadian times (07, 11, 15, 19, 23, 03, and 07 again, 5 rats per strain and time). The activities of plasma renin and converting enzyme, as well as plasma concentrations of corticosterone and aldosterone, were determined by radioimmunoassay (RIA). SPRD rats showed significant circadian rhythms in all variables except plasma renin activity, with maxima occurring during the day. TGR rats showed significant circadian rhythmicity in plasma renin activity and corticosterone and daily variation in aldosterone; angiotensin-converting enzyme (ACE) activity did not reach statistical significance. In TGR rats, 24h means in plasma renin activity and aldosterone were approximately sevenfold and fourfold higher, respectively, than in SPRD rats. Peak concentrations in corticosterone around 15h were more than two times higher in TGR rats than in SPRD rats, whereas no differences were observed during the night. It is concluded that, in TGR rats, the overall increase in plasma renin activity and aldosterone may contribute to the elevated blood pressure. The comparatively high levels in corticosterone and plasma renin activity during daytime may be involved in the inverse circadian blood pressure profiles in the transgenic animals. (Chronobiology International, 17(5), 645–658, 2000) [ABSTRACT FROM AUTHOR]

Dermatomyositis (DM) is a systemic idiopathic inflammatory disease affecting skeletal muscle and skin, clinically characterized by symmetrical proximal muscle weakness and typical skin lesions. Recently, myositis‐specific autoantibodies (MSA) became of utmost importance because they strongly correlate with distinct clinical manifestations and prognosis. Antibodies against transcription intermediary factor 1γ (TIF‐1γ) are frequently associated with increased risk of malignancy, a specific cutaneous phenotype and limited response to therapy in adult DM patients. Anti‐Mi‐2 autoantibodies, in contrast, are typically associated with classic DM rashes, prominent skeletal muscle weakness, better therapeutic response and prognosis, and less frequently with cancer. Nevertheless, the sensitivity of autoantibody testing is only moderate, and alternative reliable methods for DM patient stratification and prediction of cancer risk are needed. To further investigate these clinically distinct DM subgroups, we herein analyzed 30 DM patients (n = 15 Mi‐2+ and n = 15 TIF‐1 γ+) and n = 8 non‐disease controls (NDC). We demonstrate that the NanoString technology can be used as a very sensitive method to clearly differentiate these two clinically distinct DM subgroups. Using the nCounter PanCancer Immune Profiling Panel™, we identified a set of significantly dysregulated genes in anti‐TIF‐1γ+ patient muscle biopsies including VEGFA, DDX58, IFNB1, CCL5, IL12RB2, and CD84. Investigation of type I IFN‐regulated transcripts revealed a striking type I interferon signature in anti‐Mi‐2+ patient biopsies. Our results help to stratify both subgroups and predict, which DM patients require an intensified diagnostic procedure and might have a poorer outcome. Potentially, this could also have implications for the therapeutic approach. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Scale bar: 100 m. c and d The expression levels of c POPDC1 and d POPDC2 in the sarcolemma normalized to SGCA, were quantified in individual fibers. 3The expression of POPDC1 and POPDC2 is greatly reduced at the sarcolemma of skeletal muscle fibers expressing POPDC1 p.Q153X. a and b Transverse sections of skeletal muscle biopsies from a patient (PT) carrying the POPDC1 p.Q153X variant in homozygosity and a matched control (CT) were stained for a POPDC1 or b POPDC2, along with SGCA as a sarcolemma marker. [Extracted from the article]

TRAPPC11 was identified as a component of the TRAPP III complex that functions in membrane trafficking and autophagy. Variants in TRAPPC11 have been reported to be associated with a broad spectrum of phenotypes but all affected individuals display muscular pathology. Identifying additional variants will further our understanding of the clinical spectrum of phenotypes and will reveal regions of the protein critical for its functions. Here we report three individuals from unrelated families that have bi-allellic TRAPPC11 variants. Subject 1 harbors a compound heterozygous variant (c.1287 + 5G > A and c.3379_3380insT). The former variant results in a partial deletion of the foie gras domain (p.Ala372_Ser429del), while the latter variant results in a frame-shift and extension at the carboxy terminus (p.Asp1127Valfs*47). Subjects 2 and 3 both harbour a homozygous missense variant (c.2938G > A; p.Gly980Arg). Fibroblasts from all three subjects displayed membrane trafficking defects manifested as delayed endoplasmic reticulum (ER)-to-Golgi transport and/or a delay in protein exit from the Golgi. All three individuals also show a defect in glycosylation of an ER-resident glycoprotein. However, only the compound heterozygous subject displayed an autophagic flux defect. Collectively, our characterization of these individuals with bi-allelic TRAPPC11 variants highlights the functional importance of the carboxy-terminal portion of the protein. [ABSTRACT FROM AUTHOR]

Introduction: Small fiber neuropathies (SFN) are associated with a reduction in quality of life. In adults, epidermal nerve fiber density (END) analysis is recommended for the diagnosis of SFN. In children, END assessment is not often performed. We analyzed small nerve fiber innervation to elucidate the potential diagnostic role of skin biopsies in young patients with pain.Methods: Epidermal nerve fiber density and sudomotor neurite density (SND) were assessed in skin biopsies from 26 patients aged 7 to 20 years (15 female patients) with unexplained chronic pain. The results were compared with clinical data.Results: Epidermal nerve fiber density was abnormal in 50% and borderline in 35% of patients. An underlying medical condition was found in 42% of patients, including metabolic, autoimmune, and genetic disorders.Discussion: Reduction of epidermal nerve fibers can be associated with treatable conditions. Therefore, the analysis of END in children with pain may help to uncover a possible cause and guide potential treatment options. [ABSTRACT FROM AUTHOR]

Human cold perception and nociception play an important role in persisting pain. However, species differences in the target temperature of thermosensitive ion channels expressed in peripheral nerve endings have fueled discussions about the mechanism of cold nociception in humans. Most frequently implicated thermosensors are members of the transient receptor potential (TRP) ion channel family TRPM8 and TRPA1. Regularly observed, distinct cold pain phenotype groups suggested the existence of interindividually differing molecular bases. In 28 subjects displaying either high or medium sensitivity to local cooling of the skin, the density at epidermal nerve fibers of TRPM8, but not that of TRPA1 expression, correlated significantly with the cold pain threshold. Moreover, reproducible grouping of the subjects, based on high or medium sensitivity to cooling, was reflected in an analogous grouping based on high or low TRPM8 expression at epidermal nerve fibers. The distribution of TRPM8 expression in epidermal nerve fibers provided an explanation for the previously observed (bi)modal distribution of human cold pain thresholds which was reproduced in this study. In the light of current controversies on the role of human TRPA1 ion channels in cold pain perception, the present observations demonstrating a lack of association of TRPA1 channel expression with cold sensitivity-related measures reinforce doubts about involvement of this channel in cold pain in humans. Since TRP inhibitors targeting TRPM8 and TRPA1 are currently entering clinical phases of drug development, the existence of known species differences, in particular in the function of TRPA1, emphasizes the increasing importance of new methods to directly approach the roles of TRPs in humans. [ABSTRACT FROM AUTHOR]

Mass spectrometric analysis of glioblastoma cyst fluids has disclosed a protein peak with m/z 6424–6433. Among the proteins, potentially generating this peak are ApoC1 and LuzP6. To further elucidate protein expression of glioblastoma cells, we analyzed MALDI–TOF results of cyst fluid, performed immunohistochemistry and mRNA analysis. MALDI–TOF protein extraction from 24 glioblastoma cyst fluids was performed with a weak cation exchange. 50 glioblastoma samples were stained with two custom-made antibodies against LuzP6 and commercial antibodies against ApoC1, C12orf75 and OCC-1 and analyzed. For mRNA detection, 16 tissue samples were stored in RNAlater, extracted using the miRNeasy kit and reversely transcribed. For 12 patients, synopsis of results from all three examinations was possible. MALDI–TOF confirmed the peak at 6433 Da in 75% of samples. Immunohistochemically, LuzP6 was detected in 92% (LuzP61–29) and 96% (LuzP630–58) of samples and ApoC1 in 66%. Mean mRNA levels were highest for ApoC1, followed by LuzP6. No correlation between mRNA expression, immunohistochemical staining and intensity of the MALDI–TOF peaks was found. An unequivocal identification of one protein as the source for the 6433 peak is not possible, but our results point to ApoC1 and LuzP6 as the underlying proteins. [ABSTRACT FROM AUTHOR]

Introduction: Noonan Syndrome (NS) is caused by mutations that affect the RAS/ERK1/2-MAPK pathway. More than 90% of NS individuals with mutations clustered in the CR2 domain of RAF1 present with severe and often lethal hypertrophic cardiomyopathy (HCM). The signaling pathways by which NS RAF1 mutations promote HCM remain elusive, and so far, there is no known treatment for NS-associated-HCM. Methods: Here, we used patient-derived RAF1S257L/+ and CRISPR-Cas9-generated isogenic control iPSC-derived cardiomyocytes (iCMs) to delineate the molecular mechanisms underlying HCM in NS-associated RAF1 mutations. Results: We found that mutant iCMs phenocopy the pathology seen in NS by exhibiting hypertrophy and structural defects (n=5). At the molecular level, while RAF1S257L/+ iCMs had elevated RAF1 and MEK1/2 activities, ERK1/2 itself was modestly enhanced. To test whether ERK1/2 activation was responsible for increased size of RAF1S257L/+ iCMs, we inhibited MEK1/2 activity with three different inhibitors (U0126, PD or Tram) and found that, while neither PD nor Tram reduced mutant iCMs area, both rescued myofibrillar disarray (n=3), suggesting that MEK1/2 and/or ERK1/2 regulate myofilament organization but not cell size. Since, PD and Tram block MEK1/2 activation by RAF, whereas U0126 prevents activation of ERK1/2 by active MEK1/2, we hypothesized that MEK1/2 rather than ERK1/2 regulates myofilament organization. Accordingly, we found that specific inhibition or ERK1/2 activity by ectopic expression of MKP3 did not rescue myofibrillar disarray in mutant RAF1 iCMs. Strikingly, U0126 was able to reduce iCMs surface area, suggesting that an ERK1/2-independent pathway was responsible for hypertrophy. Since U0126 also targets MEK5, we treated RAF1S257L/+ iCMs with a specific MEK5/ERK5 inhibitor and found that hypertrophy was drastically reduced (n=3), indicating that the ERK5 pathway triggered hypertrophy in RAF1S257L/+ iCMs. Finally, RNA-sequencing reveals genes with abnormal expression in RAF1 mutant iCMs and identifies subsets of genes dysregulated by aberrant MEK1/2 or ERK5 pathways and known as critical regulators of cardiac development, as well as extracellular matrix, cytoskeletal and actin assembly homeostasis that could contribute to the NS-associated HCM. Conclusion: Taken together, our study identifies the molecular mechanisms by which NS RAF1 mutations cause HCM and reveals downstream effectors that could serve as therapeutic targets for treatment of HCM in NS patients. [ABSTRACT FROM AUTHOR]

A subset of patients with polyglucosan body myopathy was found to have underlying mutations in the RBCK1 gene. Affected patients may display diverse symptoms ranging from skeletal muscular weakness, cardiomyopathy to chronic autoinflammation and immunodeficiency. It was suggested that the exact localization of the mutation within the gene might be responsible for the specific phenotype, with N-terminal mutations causing severe immunological dysfunction and mutations in the middle or C-terminal part leading to a myopathy phenotype. We report the clinical, immunological and genetic findings of two unrelated individuals suffering from a childhood-onset RBCK1-asscociated disease caused by the same homozygous truncating mutation (NM_031229.2:c.896_899del, p.Glu299Valfs*46) in the middle part of the RBCK1 gene. Our patients suffered from a myopathy with cardiac involvement, but in contrast to previous reports on mutations in this part of the gene, also displayed signs of autoinflammation and immunodeficiency. Our report suggests that RBCK1 mutations at locations that were previously thought to lack immunological features may also present with immunological dysfunction later in the disease course. This notably broadens the genotype–phenotype correlation of RBCK1-related polyglucosan body myopathy. [ABSTRACT FROM AUTHOR]

Massively parallel sequencing of whole genomes and exomes has facilitated a direct assessment of causative genetic variation, now enabling the identification of genetic factors involved in rare diseases (RD) with Mendelian inheritance patterns on an almost routine basis. Here, we describe the illustrative case of a single consanguineous family where this strategy suffered from the difficulty to distinguish between two etiologically distinct disorders, namely the co-occurrence of hereditary hypophosphatemic rickets (HRR) and congenital myopathies (CM), by their phenotypic manifestation alone. We used parametric linkage analysis, homozygosity mapping and whole exome-sequencing to identify mutations underlying HRR and CM. We also present an approximate approach for assessing the probability of co-occurrence of two unlinked recessive RD in a single family as a function of the degree of consanguinity and the frequency of the disease-causing alleles. Linkage analysis and homozygosity mapping yielded elusive results when assuming a single RD, but whole-exome sequencing helped to identify two mutations in two genes, namely SLC34A3 and SEPN1, that segregated independently in this family and that have previously been linked to two etiologically different diseases. We assess the increase in chance co-occurrence of rare diseases due to consanguinity, i.e. under circumstances that generally favor linkage mapping of recessive disease, and show that this probability can increase by several orders of magnitudes. We conclude that such potential co-occurrence represents an underestimated risk when analyzing rare or undefined diseases in consanguineous families and should be given more consideration in the clinical and genetic evaluation. [ABSTRACT FROM AUTHOR]

Extracellular RNA (eRNA) released from injured cells promotes tissue permeability, thrombosis, and inflammation in vitro and in vivo, and RNase1 pretreatment can reduce all these effects. In this study, we investigated the role of the eRNA/RNase1 system in tumor progression and metastasis. Under quiescent and stimulatory conditions, tumor cells released much higher levels of endogenous extracellular RNA (eRNA) than nontumor cells. In glioblastomas, eRNA was detected at higher levels in tumors than nontumor tissue. eRNA induced tumor cells to adhere to and migrate through human cerebral microvascular endothelial cells (HCMEC/D3), in a manner requiring activation of VEGF signaling. In addition, eRNA liberated TNF-α from macrophages in a manner requiring activation of the TNF-α-converting enzyme TACE. Accordingly, supernatants derived from eRNA-treated macrophages enhanced tumor cell adhesion to HCMEC/D3. TNF-α release evoked by eRNA relied upon signaling activation of mitogen-activated protein kinases and the NF-κB pathway. In subcutaneous xenograft models of human cancer, administration of RNase1 but not DNase decreased tumor volume and weight. Taken together, these results suggest that eRNA released from tumor cells has the capacity to promote tumor cell invasion through endothelial barriers by both direct and indirect mechanisms, including through a mechanism involving TNF-α release from tumor-infiltrating monocytes/macrophages. Our findings establish a crucial role for eRNA in driving tumor progression, and they suggest applications for extracellular RNase1 as an antiinvasive regimen for cancer treatment. [ABSTRACT FROM AUTHOR]

Glioma growth and progression depend on a specialized subpopulation of tumour cells, termed tumour stem cells. Thus, tumour stem cells represent a critical therapeutic target, but the molecular mechanisms that regulate them are poorly understood. Hypoxia plays a key role in tumour progression and in this study we provide evidence that the hypoxic tumour microenvironment also controls tumour stem cells. We define a detailed molecular signature of tumour stem cell genes, which are overexpressed by tumour cells in vascular and perinecrotic/hypoxic niches. Mechanistically, we show that hypoxia plays a key role in the regulation of the tumour stem cell phenotype through hypoxia-inducible factor 2α and subsequent induction of specific tumour stem cell signature genes, including mastermind-like protein 3 (Notch pathway), nuclear factor of activated T cells 2 (calcineurin pathway) and aspartate beta-hydroxylase domain-containing protein 2. Notably, a number of these genes belong to pathways regulating the stem cell phenotype. Consistently, tumour stem cell signature genes are overexpressed in newly formed gliomas and are associated with worse clinical prognosis. We propose that tumour stem cells are maintained within a hypoxic niche, providing a functional link between the well-established role of hypoxia in stem cell and tumour biology. The identification of molecular regulators of tumour stem cells in the hypoxic niche points to specific signalling mechanisms that may be used to target the glioblastoma stem cell population. [ABSTRACT FROM PUBLISHER]

Background: Pulmonary affection (PA) is associated with a substantial increase in morbidity and mortality in patients with idiopathic inflammatory myopathies (IIM). However, the underlying immune mechanisms of PA remain enigmatic and prompt deeper immunological analyses. Importantly, the Janus-faced role of natural killer (NK) cells, capable of pro-inflammatory as well as regulatory effects, might be of interest for the pathophysiologic understanding of PA in IIM. Methods: To extend our understanding of immunological alterations in IIM patients with PA, we compared the signatures of NK cells in peripheral blood using multi-color flow cytometry in IIM patients with (n = 12, of which anti-synthetase syndrome = 8 and dermatomyositis = 4) or without PA (n = 12). Results: We did not observe any significant differences for B cells, CD4, and CD8 T cells, while total NK cell numbers in IIM patients with PA were reduced compared to non-PA patients. NK cell alterations were driven by a particular decrease of CD56dim NK cells, while CD56bright NK cells remained unchanged. Comparisons of the cell surface expression of a large panel of NK receptors revealed an increased mean fluorescence intensity of NKG2D+ on NK cells from patients with PA compared with non-PA patients, especially on the CD56dim subset. NKG2D+ and NKp46+ cell surface levels were associated with reduced vital capacity, serving as a surrogate marker for clinical severity of PA. Conclusion: Our data illustrate that PA in IIM is associated with alterations of the NK cell repertoire, suggesting a relevant contribution of NK cells in certain IIMs, which might pave the way for NK cell-targeted therapeutic approaches. [ABSTRACT FROM AUTHOR]

Objective: With a combination of different sympathetic tests, we aimed to elucidate whether impairment of sympathetic function in Parkinson's disease (PD) is the consequence of a central or peripheral efferent dysfunction. Methods: Thirty‐five patients with early‐to‐intermediate PD (median age: 63 years; IQR: 57–67 years; disease duration 1–9 years, 15 women) and 20 age‐ and sex‐matched healthy controls (median age: 64.5 years; IQR: 58–68 years; 10 women) were recruited. Autonomic testing was performed in two subgroups and included the assessment of resting cardiovascular parameters, postprandial hypotension (PPH), orthostatic hypotension (OH), and vasoconstriction induced by intradermal microdialysis with different concentrations of norepinephrine (NE; 10–5; 10–6; 10–7; 10–8) and by cold through forehead cooling. We also used sympathetic multiunit microneurography (muscle sympathetic nerve activity; MSNA; burst frequency (BF): bursts per minute; burst incidence (BI): bursts per 100 heart beats) and evaluated the presence of phosphorylated α‐synuclein deposits in skin innervation in biopsies from the thighs by immunohistohemistry. Results: Diastolic blood pressure was higher in the PD group at rest (p <.001) and during OH (F = 6.533; p =.022). Vasoconstriction induced by NE microdialysis and cold was unchanged in PD patients. MSNA was lower in PD patients than in controls (BF: p =.001; BI: p =.025). Phosphorylated α‐synuclein deposits could be found only in PD patients. Conclusion: We did not find indications for peripheral sympathetic nerve fiber dysfunction or adrenoreceptor sensitivity changes. The decreased MSNA argues in favor of central sympathetic impairment. [ABSTRACT FROM AUTHOR]

Mitochondrial complex I deficiency is the most frequent mitochondrial disorder presenting in childhood and the mutational spectrum is highly heterogeneous. The NDUFB11 gene is one of the recently identified genes, which is located in the short arm of the X-chromosome. Here we report clinical, biochemical, functional and genetic findings of two male patients with lactic acidosis, hypertrophic cardiomyopathy and isolated complex I deficiency due to de novo hemizygous mutations (c.286C > T and c.328C > T) in the NDUFB11 gene. Neither of them had any skin manifestations. The NDUFB11 gene encodes a relatively small integral membrane protein NDUFB11, which is essential for the assembly of an active complex I. The expression levels of this protein was decreased in both patient cells and a lentiviral complementation experiment also supported the notion that the complex I deficiency in those two patients is caused by NDUFB11 genetic defects. Our findings together with a review of the thirteen previously described patients demonstrate a wide spectrum of clinical features associated with NDUFB11 -related complex I deficiency. However, histiocytoid cardiomyopathy and/or congenital sideroblastic anemia could be indicative for mutation in the NDUFB11 gene, while the clinical manifestation of the same mutation can be highly variable. [ABSTRACT FROM AUTHOR]