Desmin is essential for the structure and function of the sinoatrial node:implications for increased arrhythmogenesis

Our objective was to investigate the effect of desmin depletion on the structure and function of the sinoatrial pacemaker complex (SANcl) and its implication in arrhythmogenesis. Analysis of mice and humans (SANcl) indicated that the sinoatrial node exhibits high amounts of desmin, desmoplakin, N-cadherin, and β-catenin in structures we call “lateral intercalated disks” connecting myocytes side by side. Examination of the SANcl from an arrhythmogenic cardiomyopathy model, desmin-deficient (Des-/-) mouse, by immunofluorescence, ultrastructural, and Western blot analysis showed that the number of these lateral intercalated disks was diminished. Also, electrophysiological recordings of the isolated compact sinoatrial node revealed increased pacemaker systolic potential and higher diastolic depolarization rate compared with wild-type mice. Prolonged interatrial conduction expressed as a longer P wave duration was also observed in Des-/mice. Upregulation of mRNA levels of both T-type Ca2+ current channels, Cav3.1 and Cav3.2, in the Des-/- myocardium (1.8- and 2.3-fold, respectively) and a 1.9-fold reduction of funny hyperpolarization-activated cyclic nucleotide-gated K+ channel 1 could underlie these functional differences. To investigate arrhythmogenicity, electrocardiographic analysis of Des-deficient mice revealed a major increase in supraventricular and ventricular ectopic beats compared with wild-type mice. Heart rate variability analysis indicated a sympathetic predominance in Des-/- mice, which may further contribute to arrhythmogenicity. In conclusion, our results indicate that desmin elimination leads to structural and functional abnormalities of the SANcl. These alterations may be enhanced by the sympathetic component of the cardiac autonomic nervous system, which is predominant in the desmin-deficient heart, thus leading to increased arrhythmogenesis

Amine-storing Organelles in Soma and Dendrites of Human Locus Coeruleus Neurons

Previous studies have identified in human catecholamine neurons abundant spherical acidophilic protein bodies (PB), which originate from mitochondria retaining the double membrane (Issidorides et al., 1996). In locus coeruleus (LC), PB have somatodendritic distribution and are unequivocal storage vesicles for noradrenaline, as demonstrated by immunolocalization of Dopamine-β-Hydroxylase (Issidorides et al., 2004). This species-specific phenotype in man is the result of important physiological functions, because depletion or missing of PB is accompanied with Parkinson’s disease. The aim of this study was to investigate the composition of PB and their role in normal and pathological conditions. Post mortem brain specimens of LC were collected from 13 control subjects and 12 cases of Parkinson’s disease patients. Human adrenal medulla was used as a model tissue and histochemical and immunohistochemical correlation between PB and chromaffin granules was made. At the ultrastructural level, colloidal gold method was used for the accurate localization of macromolecules, at high resolution. The mitochondrial origin of PB was sealed with their positive immunoreactivity for mitochondrial porin. The next purpose was to reinforce the identity of PB as monoamine storage sites and to assess their potential of somatodendritic release. For this reason we studied the subcellular immunolocalization of Chromogranin A (CgA) and Vesicular Monoamine Transporter 2 (VMAT2), given the fact that their localization defines the vesicles capacity of filling with monoamine and hence exocytotic release (Schafer et al., 2010; Li et al., 2005). The data provided, demonstrate the novel ultrastructural immunolocalization of both CgA and VMAT2 in PB, supporting their involvement in somatodendritic storage and release of noradrenaline in human LC. In Parkinson’s disease, immunolocalization of VMAT2 in the LC revealed the reduction of protein compared to normal controls. Reduced expression of VMAT2 leads to defective sequestration of monoamines into vesicles, their accumulation in the cytoplasm and eventually the emergence of Parkinson’s disease phenotype. Parkinson’s disease is characterized by a progressive cellular deposition of the synaptic protein a-synuclein in diverse brain regions (Schulz, 2007). Along with the impairment of mitochondrial respiration, both mitochondrial fission/fusion have been shown to be altered (Cardoso, 2011). In view of the above, we investigated the mitochondrial ultrastructure in LC from Parkinson’s disease patients along with a-synuclein immunolocalization. The morphological study revealed disrupted mitochondrial ultrastructure indicating dysfunction in normal neurotransmitter-storing organelle production, leading to defective sequestration of monoamine into vesicles. Immunolocalization of a-synuclein in Parkinson’s disease brains revealed the accumulation of this protein in different stages in physiologically appearing neurons, as well as, in mature brainstem Lewy bodies. At the electron microscope the subcellular localization of this protein in PB, as well as, in neuromelanin of LC neurons was revealed. The study of PB, which are responsible for the somatodentritic storage and possible release of noradrenaline in human LC neurons, and their contribution in the formation of Lewy bodies, as indicated by the localization of common components among these two structures may be helpful towards the understanding of Parkinson’s disease

Histological and ultrastructural immunolocalization of enzymes and proteins of noradrenaline-storing organelles in human catecholamine Locus coeruleus neurons

Human monoamine neurons are characterized by abundant spherical acidophilic protein-bodies, which originate from mitochondria, retaining the double membrane. In Locus Coeruleus (LC), they have somatodendritic distribution and are unequivocal storage vesicles for noradrenaline. Their depletion is accompanied by Parkinson’s disease (PD) pathology. The aim of this study was to investigate their composition and role in normal and pathological conditions. Post-mortem brain specimens were collected from control subjects and PD patients. Human adrenal medulla was used as a model-tissue and histochemical and immunohistochemical correlation between protein-bodies and chromaffin granules was made. At the ultrastructural level, the colloidal immunogold method was used. The mitochondrial origin of protein-bodies was sealed with their positive immunoreactivity for mitochondrial porin. The data provided demonstrate the ultrastructural immunolocalization of both Chromogranin-A and Vesicular Monoamine Transporter-2 in protein-bodies. The study of pathological brains also revealed the localization of a-synuclein in protein-bodies, as well as, in neuromelanin of LC neurons. The study of protein-bodies, which are responsible for somatodentritic storage and possible release of noradrenaline in human LC neurons, and their contribution in the formation of Lewy bodies, as indicated by the localization of common components among these two structures may be helpful, towards the understanding of PD.Οι ανθρώπινοι μονοαμινικοί νευρώνες χαρακτηρίζονται από πολυάριθμα σφαιρικά οξεόφιλα πρωτεϊνικά σωμάτια, που προέρχονται από φυσιολογικά μιτοχόνδρια διατηρώντας τη διπλή μεμβράνη. Στον Υπομέλανα Τόπο (LC) εμφανίζουν σωματοδενδριτική κατανομή και αποτελούν αποθηκευτικά κυστίδια νοραδρεναλίνης. Η ελάττωσή τους συνδυάζεται με εκδήλωση νόσου Πάρκινσον (NΠ). Σκοπός της διατριβής ήταν η διερεύνηση της σύστασης και του ρόλου τους σε φυσιολογικές και παθολογικές καταστάσεις. Η μελέτη πραγματοποιήθηκε σε νεκροτομικό υλικό εγκεφάλων φυσιολογικών ατόμων και ασθενών με NΠ. Ως ιστός-αναφοράς χρησιμοποιήθηκε μυελώδης μοίρα ανθρώπινων επινεφριδίων και πραγματοποιήθηκε ιστοχημική και ανοσοϊστοχημική σύγκριση των πρωτεϊνικών σωματίων με τα χρωμιόφιλα κοκκία. Σε υπερμικροσκοπικό επίπεδο εφαρμόστηκε ανοσοϊστοχημεία με κολλοειδή χρυσό. Η μιτοχονδριακή προέλευση των πρωτεϊνικών σωματίων επισφραγίστηκε με ανοσοεντόπιση σε αυτά της μιτοχονδριακής πορίνης. Παράλληλα, πραγματοποιήθηκε υποκυτταρική εντόπιση των πρωτεϊνών χρωμογρανίνη Α και του κυστιδιακού μεταφορέα μονοαμινών-2, στα πρωτεϊνικά σωμάτια. Η μελέτη των παθολογικών εγκεφάλων ανέδειξε επιπλέον, την εντόπιση της α-συνουκλεΐνης στα πρωτεϊνικά σωμάτια και στη νευρομελανίνη των νευρώνων του LC. Η μελέτη των πρωτεϊνικών σωματίων που είναι υπεύθυνα για σωματοδενδριτική αποθήκευση και πιθανή απελευθέρωση της νοραδρεναλίνης στους ανθρώπινους νευρώνες του LC και η συμμετοχή τους στη δημιουργία των σωματίων Lewy, όπως υποδεικνύει η εντόπιση κοινών συστατικών ανάμεσα στις δύο αυτές δομές, θα βοηθήσει στη διερεύνηση της ΝΠ

Amelioration of desmin network defects by αB-crystallin overexpression confers cardioprotection in a mouse model of dilated cardiomyopathy caused by LMNA gene mutation

International audienceThe link between the cytoplasmic desmin intermediate filaments and those of nuclear lamins serves as a major integrator point for the intracellular communication between the nucleus and the cytoplasm in cardiac muscle. We investigated the involvement of desmin in the cardiomyopathy caused by the lamin A/C gene mutation using the LmnaH222P/H222P mouse model of the disease. We demonstrate that in these mouse hearts desmin loses its normal Z disk and intercalated disc localization and presents aggregate formation along with mislocalization of basic intercalated disc protein components, as well as severe structural abnormalities of the intercalated discs and mitochondria. To address the extent by which the observed desmin network defects contribute to the progression of LmnaH222P/H222P cardiomyopathy, we investigated the consequences of desmin-targeted approaches for the disease treatment. We showed that cardiac-specific overexpression of the small heat shock protein αΒ-Crystallin confers cardioprotection in LmnaH222P/H222P mice by ameliorating desmin network defects and by attenuating the desmin-dependent mislocalization of basic intercalated disc protein components. In addition, αΒ-Crystallin overexpression rescues the intercalated disc, mitochondrial and nuclear defects of LmnaH222P/H222P hearts, as well as the abnormal activation of ERK1/2. Consistent with that, by generating the LmnaH222P/H222PDes+/- mice, we showed that the genetically decreased endogenous desmin levels have cardioprotective effects in LmnaH222P/H222P hearts since less desmin is available to form dysfunctional aggregates. In conclusion, our results demonstrate that desmin network disruption, disorganization of intercalated discs and mitochondrial defects are a major mechanism contributing to the progression of this LMNA cardiomyopathy and can be ameliorated by αΒ-Crystallin overexpression

Impairment of chaperone-mediated autophagy induces dopaminergic neurodegeneration in rats

<p>Chaperone-mediated autophagy (CMA) involves the selective lysosomal degradation of cytosolic proteins such as SNCA (synuclein α), a protein strongly implicated in Parkinson disease (PD) pathogenesis. However, the physiological role of CMA and the consequences of CMA failure in the living brain remain elusive. Here we show that CMA inhibition in the adult rat substantia nigra via adeno-associated virus-mediated delivery of short hairpin RNAs targeting the LAMP2A receptor, involved in CMA's rate limiting step, was accompanied by intracellular accumulation of SNCA-positive puncta, which were also positive for UBIQUITIN, and in accumulation of autophagic vacuoles within LAMP2A-deficient nigral neurons. Strikingly, LAMP2A downregulation resulted in progressive loss of nigral dopaminergic neurons, severe reduction in striatal dopamine levels/terminals, increased astro- and microgliosis and relevant motor deficits. Thus, this study highlights for the first time the importance of the CMA pathway in the dopaminergic system and suggests that CMA impairment may underlie PD pathogenesis.</p

Cutaneous Vasculopathy in a COVID-19 Critically Ill Patient: A Histologic, Immunohistochemical, and Electron Microscopy Study

We describe a critically ill, SARS-CoV-2 positive patient with respiratory failure and thrombotic/livedoid skin lesions, appearing during the course of the disease. The biopsy of the lesions revealed an occlusive, pauci-inflammatory vasculopathy of the cutaneous small vessels characterized by complement and fibrinogen deposition on vascular walls, pointing to a thrombotic vasculopathy. Transmission electron microscopy of the affected skin failed to reveal any viral inclusions. Clinical evaluation and laboratory findings ruled out systemic coagulopathies and disseminated intravascular coagulation, drug-induced skin reaction, and common viral rashes. Our hypothesis is that the, herein evidenced, microvascular occlusive injury might constitute a significant pathologic mechanism in COVID-19, being a common denominator between cutaneous and pulmonary manifestations

Alterations in the levels of an H1 DNA linker histone subtype in peripheral blood leukocytes from schizophrenia patients are linked with this disorder

AbstractSchizophrenia is an illness with severe social and familial impact. However, biomarkers related to chromatin that could help prognosis so as to prevent or attenuate the symptoms of first episodes and relapses of this malady  are either few or obscurely related. To this end, we decided to  analyze the chromatin constitution of certain  H1 DNA linker histone subtypes of schizophrenia patients, since this particular epigenetic chromatin parameter has not been previously studied with respect to this disorder. We examined the abundance of three histone H1 subtypes (also called variants), i.e., H1.0, H1.3, and H1.5, as well as the total histone H1 fraction in peripheral blood lymphocytes and neutrophils of hospitalized relapsed schizophrenic patients (inpatients), chronic outpatients on medication, first-episode patients, and normal control subjects. Of the three H1 subtypes  analyzed, H1.0 protein levels were found to be significantly lower in both lymphocytes and neutrophils of all patients that participated in the study. Total histone H1 levels were also found to be decreased in all patient cases. The fact that the changes in H1.0 levels and the total H1 fraction were observed regardless of state or phase of the disorder or the administered medication implies that these epigenetic changes are most probably intrinsically associated with the etiology of the illness. Due to the importance of the histone epigenetic profile in chromatin remodeling and gene expression, the observed non physiologiocal alterations of H1.0 levels and total H1 may contribute to the psychopathology of schizophrenia  by affecting the normal expression levels of certain genes. As this is the first time that a specific histone subtype is linked to schizophrenia, our work may serve as the basis for studying this disorder from a novel perspective, encompassing chromatin epigenetic research related to the histone protein subtypes/variants and as a novel source of biomarkers for this disorder

Novel insights into SLC25A46-related pathologies in a genetic mouse model

The mitochondrial protein SLC25A46 has been recently identified as a novel pathogenic cause in a wide spectrum of neurological diseases, including inherited optic atrophy, Charcot-Marie-Tooth type 2, Leigh syndrome, progressive myoclonic ataxia and lethal congenital pontocerebellar hypoplasia. SLC25A46 is an outer membrane protein, member of the Solute Carrier 25 ( SLC25) family of nuclear genes encoding mitochondrial carriers, with a role in mitochondrial dynamics and cristae maintenance. Here we identified a loss-of-function mutation in the Slc25a46 gene that causes lethal neuropathology in mice. Mutant mice manifest the main clinical features identified in patients, including ataxia, optic atrophy and cerebellar hypoplasia, which were completely rescued by expression of the human ortholog. Histopathological analysis revealed previously unseen lesions, most notably disrupted cytoarchitecture in the cerebellum and retina and prominent abnormalities in the neuromuscular junction. A distinct lymphoid phenotype was also evident. Our mutant mice provide a valid model for understanding the mechanistic basis of the complex SLC25A46-mediated pathologies, as well as for screening potential therapeutic interventions