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.

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.

GABA transmission via ATP-dependent K+ channels regulates α-synuclein secretion in mouse striatum.

α-Synuclein is readily released in human and mouse brain parenchyma, even though the normal function of the secreted protein has not been yet elucidated. Under pathological conditions, such as in Parkinson's disease, pathologically relevant species of α-synuclein have been shown to propagate between neurons in a prion-like manner, although the mechanism by which α-synuclein transfer induces degeneration remains to be identified. Due to this evidence extracellular α-synuclein is now considered a critical target to hinder disease progression in Parkinson's disease. Given the importance of extracellular α-synuclein levels, we have now investigated the molecular pathway of α-synuclein secretion in mouse brain. To this end, we have identified a novel synaptic network that regulates α-synuclein release in mouse striatum. In this brain area, the majority of α-synuclein is localized in corticostriatal glutamatergic terminals. Absence of α-synuclein from the lumen of brain-isolated synaptic vesicles suggested that they are unlikely to mediate its release. To dissect the mechanism of α-synuclein release, we have used reverse microdialysis to locally administer reagents that locally target specific cellular pathways. Using this approach, we show that α-synuclein secretion in vivo is a calcium-regulated process that depends on the activation of sulfonylurea receptor 1-sensitive ATP-regulated potassium channels. Sulfonylurea receptor 1 is distributed in the cytoplasm of GABAergic neurons from where the ATP-dependent channel regulates GABA release. Using a combination of specific agonists and antagonists, we were able to show that, in the striatum, modulation of GABA release through the sulfonylurea receptor 1-regulated ATP-dependent potassium channels located on GABAergic neurons controls α-synuclein release from the glutamatergic terminals through activation of the presynaptic GABAB receptors. Considering that sulfonylurea receptors can be selectively targeted, our study highlights the potential use of the key molecules in the α-synuclein secretory pathway to aid the discovery of novel therapeutic interventions for Parkinson's disease.

Chromogranin A and vesicular monoamine transporter 2 immunolocalization in protein bodies of human locus coeruleus neurons.

Our previous histochemical and ultrastructural studies have identified, in human catecholamine neurons, abundant spherical acidophilic protein bodies (pb), which originate from regular mitochondria, retaining their double membrane. In locus coeruleus (LC) neurons, pb have somatodendritic distribution and are unequivocal storage vesicles for noradrenaline, as demonstrated by immunolocalization of Dopamine-ß-Hydroxylase. In the present study, in order to reinforce the identity of pb as monoamine storage sites in human LC, and to assess their potential of somatodendritic release, 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. The data provided in the present study, demonstrate the novel ultrastructural immunolocalization of both CgA and VMAT2 in protein bodies, supporting their involvement in somatodendritic storage and release of noradrenaline in human LC. Since the molecular mechanism of LC somatodendritic exocytosis remains largely elusive, the present study may shed light to a better understanding of this mechanism.

Microglia Activation in the Midbrain of the Human Neonate: The Effect of Perinatal Hypoxic-Ischemic Injury.

Perinatal hypoxia-ischemia (PHI) is a major risk factor for the development of neuropsychiatric deficits later in life. We previously reported that after prolonged PHI, the dopaminergic neurons of the human neonate showed a dramatic reduction of tyrosine hydroxylase (TH) in the substantia nigra, without important signs of neuronal degeneration despite the significant reduction in their cell size. Since microglia activation could precede neuronal death, we now investigated 2 microglia activation markers, ionized calcium-binding adapter molecule 1 (Iba1), and the phagocytosis marker Cd68. The highest Iba1 immunoreactivity was found in neonates with neuropathological lesions of severe/abrupt PHI, while the lowest in subjects with moderate/prolonged or older PHI. Subjects with very severe/prolonged or chronic PHI showed an increased Iba1 expression and very activated microglial morphology. Heavy attachment of microglia on TH neurons and remarkable expression of Cd68 were also observed indicating phagocytosis in this group. Females appear to express more Iba1 than males, suggesting a gender difference in microglia maturation and immune reactivity after PHI insult. PHI-induced microglial "priming" during the sensitive for brain development perinatal/neonatal period, in combination with genetic or other epigenetic factors, could predispose the survivors to neuropsychiatric disorders later in life, possibly through a sexually dimorphic way.

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 A11 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.D40Y segregating with the phenotype. INTERPRETATION: Although the pathogenic mechanisms associated with p.D40Y mutation in the prion-like domain of Annexin A11 need to be further clarified, our study provides robust and clear genetic evidence to support the expansion of the phenotypic spectrum of ANXA11.

Chromatin alterations in leukocytes of first-episode schizophrenic patients.

Studies of peripheral blood leukocytes of schizophrenic patients have shown in electron microscopy (EM) that decondensation of the chromatin constitutes a biological marker indicating increased genomic expression. Since this increase depends on chromatin relaxation by dissociation of lysine-rich histone H1 from nucleosomes, with exposure of arginine residues of core histones, the ratio of arginine to lysine residues in each nucleus represents a reliable measure of activation. Lysine- and arginine-rich proteins are demonstrable in light microscopy (LM), differentially, as yellow and black, respectively, with the ammoniacal silver reaction (ASR). Application of ASR on leukocyte pellets before they are dehydrated and embedded in epoxy resins gives reliable results in semithin sections. In thin sections the ASR method localizes only the amino acid arginine by forming deposits of electron-opaque particles, visualized in the EM. Leukocytes of 12 first-episode schizophrenic patients and 5 controls were used. Light micrographs of the semithin sections were inserted in a personal computer. The percentage of lysine and arginine was measured in 300 nuclei per subject. Morphometry showed that lymphocytes of schizophrenic patients have increased ratios of arginine to lysine, compared to controls, indicating activation; neutrophils of the patients have even a higher ratio, indicating an abnormal condition of the genome. Chromatin conformational changes are also evident by phosphotungstic acid hematoxylin (PTAH) block staining, which reveals condensed chromatin as an electron-lucent area in the nuclei, and decondensed chromatin as an electron-dense area. Because decondensed chromatin is a biological marker of schizophrenia, the efficacy of these methods to demonstrate this particular state offers a tool for early diagnosis, since first-episode schizophrenic patients have a better prognosis when treatment is started promptly, at the beginning of the disease.

Apoptotic Markers in the Midbrain of the Human Neonate After Perinatal Hypoxic/Ischemic Injury.

Our previous postmortem studies on neonates with neuropathological injury of perinatal hypoxia/ischemia (PHI) showed a dramatic reduction of tyrosine hydroxylase expression (dopamine synthesis enzyme) in substantia nigra (SN) neurons, with reduction of their cellular size. In order to investigate if the above observations represent an early stage of SN degeneration, we immunohistochemically studied the expression of cleaved caspase-3 (CCP3), apoptosis inducing factor (AIF), and DNA fragmentation by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin 3'-end-labeling (TUNEL) technique in the SN of 22 autopsied neonates (corrected age ranging from 34 to 46.5 gestational weeks), in relation to the severity/duration of PHI injury, as estimated by neuropathological criteria. No CCP3-immunoreactive neurons and a limited number of apoptotic TUNEL-positive neurons with pyknotic characteristics were found in the SN. Nuclear AIF staining was revealed only in few SN neurons, indicating the presence of early signs of AIF-mediated degeneration. By contrast, motor neurons of the oculomotor nucleus showed higher cytoplasmic AIF expression and nuclear translocation, possibly attributed to the combined effect of developmental processes and increased oxidative stress induced by antemortem and postmortem factors. Our study indicates the activation of AIF, but not CCP3, in the SN and oculomotor nucleus of the human neonate in the developmentally critical perinatal period.

Chromatin ultrastructural abnormalities in leukocytes, as peripheral markers of bipolar patients.

This study investigated the ultrastructural conformation changes of the chromatin in blood leukocytes of bipolar patients, versus normal controls, by using the phosphotungstic acid-hematoxylin (PTAH) block-staining method, modified for electron microscopy, and the immunohistochemical localization of the histone H1, by the immunogold method. These two methods are basically complementary. If histone H1 immunolabeling is used, it shows that the immunogold labeling on chromatin is different in the three phases of the illness, i.e., high in normothymia and low in depression as well as in mania. However, in this particular tissue fixation (4% paraformaldehyde-1% glutaraldehyde in 0,1 M phosphate buffer), the heterochromatin in the nuclei remains identical in the three phases of the illness. On the other hand, the PTAH method shows exactly the area of electron-lucent condensed chromatin, separate from the area of electron-dense, decondensed, chromatin. The present data confirmed that both the clinical state of depression as well as that of mania display activated lymphocytes and neutrophils with their characteristic relaxed de-condensed chromatin. On the contrary, the state of normothymia shows a reversion to the condensed state of the chromatin, as it is observed in the leukocytes of the normal controls. The ultrastructural conformations of the chromatin, revealed by the PTAH method, in combination with the histone H1 immunogold labeling, applied in blood leukocytes, supports the use of these two methods, as screening methods of choice in investigating blood biological markers in mental illness.

Impairment of chaperone-mediated autophagy induces dopaminergic neurodegeneration in rats.

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.

Noradrenaline storage function of species-specific protein bodies, markers of monoamine neurons in human locus coeruleus demonstrated by dopamine-beta-hydroxylase immunogold localization.

Our histochemical and ultrastructural studies have identified, in human catecholamine locus coeruleus (LC) neurons, abundant and large spherical protein bodies (PB), containing histone-like, arginine-rich proteins, which originate as dense bodies in mitochondria. This species-specific phenotype in the neurons of man is highly intriguing. In the electron microscope PB are disrupted in LC neurons in depressed individuals, where noradrenaline is known to be reduced. This coincidence of ultrastructure and neurochemistry raises the question whether these bodies could qualify as noradrenaline-storing organelles in the human LC. Our rationale was to examine, in known model tissues that contain catecholamines--sympathetic ganglia and tumors of the autonomic nervous system--if vesicles show the same fine structure and histochemistry as the PB of the human LC. Hence, we selected biopsy tissues of five ganglioneuromas and postmortem tissues of LC from 25 control subjects. Since dopamine-beta-hydroxylase (DBH) is a hallmark of noradrenaline identity and present in dense core vesicles, the investigation of DBH localization with the immunogold method constituted the experiment of choice for this study. Histochemical determinations of arginine with Carmoisine L, and of lipids with Rhodamine B complemented the study of similarities between the PB of the human LC and ganglioneuromas. Our results showed, with the colloidal gold method, that DBH immunogold labeling was localized in the core and in the double membranes of the PB, and also in the adjacent mitochondria. These results indicate that protein bodies (a) are unequivocal storage vesicles of noradrenaline, and (b) derive from regular mitochondria and represent a new phenotype in man, which is probably an evolutionary adaptation of amine-storing organelles.

Lymphocytes from bipolar and schizophrenic patients share common biochemical markers related to histone synthesis and histone cell membrane localization characteristic of an activated state.

In a previous communication, based on the total histone and histone variants' synthesis rates, biochemical parameters used for the characterization of the activation state of lymphocytes, we showed that a portion of the lymphocyte population obtained from peripheral blood of patients with bipolar disorder in the manic and/or depressed phases of the illness were in an activated state as opposed to normothymic patients and control subjects whose lymphocytes are in a resting, Go, state. In light of these previous findings, in the present investigation, we have analyzed total histone synthesis rates and the H2A and H3 histone variants' synthesis pattern of acid-extracted histones from the lymphocytes' nuclear fraction obtained from control subjects, patients with bipolar disorder in all phases of the illness, and patients with schizophrenia. Additional biochemical parameters, such as total cellular protein and DNA synthesis rates, were also studied. Moreover, recent findings from other investigators showed the association of histones on the plasma membrane fraction of PHA-activated, but not Go resting lymphocytes. Based on these results, acid-extracted proteins from the plasma membrane fraction obtained from control, bipolar patients in all phases of the illness, and schizophrenic patients were analyzed by immunoblotting using a polyclonal histone antibody, anti-H2B. All biochemical parameters tested show that a portion of the lymphocyte population from bipolar, i.e. manic and depressive, as well as schizophrenic patients are in an activated state and clearly indicate that the unusual for lymphocytes cell cycle-related histone biochemical properties are common to both disorders.