Adult-onset dominant muscular dystrophy in Greek families caused by Annexin A11

Objective: Mutations in the prion-like domain of RNA binding proteins cause dysfunctional stress responses and associated aggregate pathology in patients with neurogenic and myopathic phenotypes. Recently, mutations in ANXA11 have been reported in patients with amyotrophic lateral sclerosis and multisystem proteinopathy. Here we studied families with an autosomal dominant muscle disease caused by ANXA11:c.118G > T;p.D40Y. Methods: We performed deep phenotyping and exome sequencing of patients from four large Greek families, including seven affected individuals with progressive muscle disease but no family history of multi-organ involvement or ALS. Results: In our study, all patients presented with an autosomal dominant muscular dystrophy without any Paget disease of bone nor signs of frontotemporal dementia or Parkinson's disease. Histopathological analysis showed rimmed vacuoles with annexin All accumulations. Electron microscopy analysis showed myofibrillar abnormalities with disorganization of the sarcomeric structure and Z-disc dissolution, and subsarcolemmal autophagic material with myeloid formations. Molecular genetic analysis revealed ANXA11:c.118G > T;p.D4OY segregating with the phenotype. Interpretation: Although the pathogenic mechanisms associated with p.D4OY mutation in the prion-like domain of Annexin All need to be further clarified, our study provides robust and clear genetic evidence to support the expansion of the phenotypic spectrum of ANXA11.Peer reviewe

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

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

Deep Characterization of a Greek Patient with Desmin-Related Myofibrillar Myopathy and Cardiomyopathy

Desmin is a class III intermediate filament protein highly expressed in cardiac, smooth and striated muscle. Autosomal dominant or recessive mutations in the desmin gene (DES) result in a variety of diseases, including cardiomyopathies and myofibrillar myopathy, collectively called desminopathies. Here we describe the clinical, histological and radiological features of a Greek patient with a myofibrillar myopathy and cardiomyopathy linked to the c.734A>G,p.(Glu245Gly) heterozygous variant in the DES gene. Moreover, through ribonucleic acid sequencing analysis in skeletal muscle we show that this variant provokes a defect in exon 3 splicing and thus should be considered clearly pathogenic

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

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

<div><p>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 <i>Slc25a46</i> 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.</p></div

Complete rescue of the <i>ataxic</i> phenotype through complementation with the human <i>SLC25A46</i> gene.

<p><b>(A)</b> Schematic representation of the NotI digested 202.7Kb genomic fragment used for the generation of <i>TghuSLC25A46</i> mice. <b>(B)</b> Transgene copy number determination by real-time PCR in four transgenic lines, Tg1255, Tg1332, Tg1351 and Tg1358 (n = 6 per group), compared to the two <i>Slc25a46</i> copies of the WT mice, using a primer pair common for both mouse and human <i>Slc25a46</i> genes. <b>(C)</b> Expression pattern of human SLC25A46 on mitochondrial extracts from cerebrum (B), cerebellum (C), spinal cord (SC), muscle (M), heart (H), thymus (T), spleen (S) and liver (L) tissues of <i>TghuSLC25A46/Slc25a46</i>^{<i>atc/atc</i>} mice (Tg1351-<i>atc/atc</i>) through immunoblotting with antibodies against SLC25A46 and the mitochondrial protein GRP75. Complete rescue of <b>(D)</b> premature lethality (n = 8 per group), <b>(E)</b> body weight gain (n = 6 per group) and <b>(F)</b> muscle weakness (n = 6 per group) in <i>atc/atc</i> mice expressing human SLC25A46 (Tg1351-<i>atc/atc</i>). All mice used were sex matched littermates. <b>(G)</b> Confocal images of cerebellar sagittal sections immunostained for calbindin illustrate the restored PC phenotype and ML thickness in transgenic “rescue” Tg1351-<i>atc/atc</i> one-year old mice, as compared to WT and <i>atc/atc</i> mice. <b>(H)</b> Immunofluorescence of vGlut1 and vGlut2 shows normal distribution, in the ML and IGL of the cerebellum of Tg1351-<i>atc/atc</i> mice. Scale bar 40 μm.</p

Cellular alterations in the retina and optic nerve of <i>atc/atc</i> mice.

<p><b>(A)</b> and at higher magnification <b>(A’)</b> Confocal images of 4-week-old mouse retinas showing reduced MAP2 immunostaining in the internal plexiform layer (IPL) of <i>atc/atc</i> mice where the RGC dendrites lie. <b>(B)</b> Amacrine cells immunoreactive for Pax6 and GAD65 are significantly reduced in the <i>atc/atc</i> retina. Quantification of Pax6⁺ cells in the ganglion cell layer (GCL) <b>(C)</b> and the inner nuclear layer (INL) <b>(D)</b> (n = 3 mice per genotype, p = <0.0001 for the GCL and p = 0.03 for the INL). Confocal images of 4-week-old mouse retina <b>(E)</b> and optic nerve head <b>(F)</b>, showing reduced NF immunoreactivity in the innermost part of the retina where RGC axons lie and disorganization of RGC axons in the optic nerve of <i>atc/atc</i> mice. <b>(G)</b> Toluidine blue stained semi-thin optic nerve sections of 4-week-old WT and <i>atc/atc</i> mice. <b>(H)</b> Quantification of axonal numbers per optic nerve section (n = 4 nerves per genotype, p = 0.007). <b>(I)</b> Double immunofluorescence of a myelinated fiber in the optic nerve labeled for CASPR and Na_v. Scale bars (A), 10 μm; (B, E, F) 20 μm; (G) 5μm; (I) 1 μm. <b>(J)</b> Quantification of Na_v+ nodal length (n = 206 WT and 212 <i>atc/atc</i> nodes pooled from 3 mice/genotype, p<0.00001; two-tailed unpaired Student’s <i>t</i>-test; mean values ± SEM).</p

Label free proteomic analysis.

<p>Label free proteomic analysis.</p