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Funding information Research and Innovation Foundation of Cyprus, Grant/Award Number: SOUNDPET (INTEGRATED/0918/0008); Piccolo Grande Guerriero ‐ Associazione Malattia PMLD, Grant/Award Number: 2020‐22 Grant Abstract Background: Focussed Ultrasound (FUS) combined with microbubbles (MBs) was proven a promising modality for non‐invasive blood brain barrier disruption (BBBD). Herein, two devices for FUS‐mediated BBBD in rodents are presented. Methods: A two‐axes robotic device was manufactured for navigating a single element FUS transducer of 1 MHz relative to the brain of rodents. A second more compact device featuring a single motorized vertical axis was also developed. Their performance was assessed in terms of motion accuracy, MRI compatibility and trans‐skull BBBD in wild type mice using MBs in synergy with pulsed FUS. Results: Successful BBBD was evidenced by the Evans Blue dye method, as well as by Fibronectin and Fibrinogen immunostaining. BBB permeability was enhanced when the applied acoustic intensity was increased. Conclusions: The proposed devices constitute a cost‐effective and ergonomic solution for FUS‐mediated BBBD in small animal models. Further experimentation is needed to examine the repeatability of results and optimise the therapeutic protocol.

Induction of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), in connexin 32 (Cx32) or Cx47 knockout (KO) mice with deficiency in oligodendrocyte gap junctions (GJs) results in a more severe disease course. In particular, Cx47 KO EAE mice experience an earlier EAE onset and more pronounced disease severity, accompanied by dysregulated pro-inflammatory responses preceding the disease manifestations. In this study, analysis of relevant pro-inflammatory cytokines in wild type EAE, Cx32 KO EAE, and Cx47 KO EAE mice revealed altered expression of Vcam-1 preceding EAE [7 days post injection (dpi)], of Ccl2 at the onset of EAE (12 dpi), and of Gm-csf at the peak of EAE (24 dpi) in Cx47 KO EAE mice. Moreover, Cx47 KO EAE mice exhibited more severe blood-spinal cord barrier (BSCB) disruption, enhanced astrogliosis with defects in tight junction formation at the glia limitans, and increased T-cell infiltration prior to disease onset. Thus, Cx47 deficiency appears to cause dysregulation of the inflammatory profile and BSCB integrity, promoting early astrocyte responses in Cx47 KO EAE mice that lead to a more severe EAE outcome. Further investigation into the role of oligodendrocytic Cx47 in EAE and multiple sclerosis pathology is warranted.

Charcot-Marie-Tooth disease is one of the most common inherited neurological disorders affecting the peripheral nervous system. The common clinical manifestations of the disease are distal muscle weakness and atrophy, often associated with a characteristic steppage gait and foot deformities. Transient acute and recurrent or chronic central nervous system manifestations, predominantly, dysarthria, dysphagia, motor weakness, and ataxia, have been recognized as a feature of the X-linked type 1 of CMT (CMTX1). The CNS symptoms occur typically in young age and often precede the clinical manifestation of the polyneuropathy. Several predisposing factors such as exercise, fever, and returning from areas of high altitude have been described as triggers of the CNS symptoms; however, in many cases, a substantial cause remains undetermined. In this report, we describe a patient with three attacks of transient CNS deficits at the ages of 11, 21, and 38 years, respectively, which were also accompanied by transient white matter abnormalities on MRI. Two of the attacks occurred after prolonged exposure to sunlight. In our knowledge, this is the first documented case with such long latency periods between CNS attacks as well as the only report describing intense sun exposure as a possible provoking factor.

Background and Purpose. Progressive multifocal leukoencephalopathy (PML) is a debilitating disease of the central nervous system caused by the ubiquitous polyomavirus JC (JCV) in immunocompromised hosts. In recent years, a new subpopulation of patients at risk for PML has emerged, due to the growing use of immunomodulatory or immunosuppressive therapies in autoimmune diseases such as multiple sclerosis (MS). The anti-JCV antibody index is used as a stratification tool in assessing the risk of developing PML. The objective of this study was to retrospectively describe the prevalence of anti-JCV antibodies in the MS population in Cyprus. Methods. We retrospectively collected the demographics of 214 MS patients in Cyprus who were screened for anti-JCV antibodies using the STRATIFY JCV™ assay between September 2011 and June 2018. Logistic regression analysis was used to examine the effect of demographic variables on seropositivity, and bivariate tests were used to assess the association between demographic characteristics and JCV AI index. Results. A total of 214 MS patients in Cyprus were tested. Overall anti-JCV antibody prevalence was 45.8% (95% confidence interval 37.2%–55.8%). We could not establish a significant association between seropositivity and increasing age or sex. In the subgroup analysis of natalizumab-treated patients, the annual seroconversion rate was 4.5%. Conclusions. Overall seroprevalence of anti-JCV antibodies in MS patients in Cyprus using the STRATIFY JCV assay was lower than the worldwide reported mean. Although previously reported, in our study, the anti-JCV antibody seropositivity was not associated with increasing age or sex.

Seronegativemyastheniagravis(MG) presents a serious gap in MG diagnosis and understanding. We applied acellbasedassay(CBA) for the detection of muscle specific kinase (MuSK) antibodies undetectable by radioimmunoassay. We tested 633 triple-seronegative MG patients' sera from 13 countries, detecting 13% as positive.MuSKantibodies were found, at significantly lower frequencies, in 1.9% of healthy controls and 5.1% of other neuroimmune disease patients, including multiple sclerosis and neuromyelitis optica. The clinical data of the newly diagnosedMuSK-MG patients are presented. 27% of ocular seronegative patients wereMuSKantibody positive. Moreover, 23% had thymic hyperplasia suggesting that thymic abnormalities are more common than believed.

CMT1X, an X-linked inherited neuropathy, is caused by mutations in GJB1, which codes for Cx32, a gap junction protein expressed by Schwann cells and oligodendrocytes. Many GJB1 mutations cause central nervous system (CNS) abnormality in males, including stable subclinical signs and, less often, short-duration episodes characterized by motor difficulties and altered consciousness. However, some mutations have no apparent CNS effects. What distinguishes mutations with and without CNS manifestations has been unclear. Here we studied a total of 14 Cx32 mutations, 10 of which are associated with florid episodic CNS clinical syndromes in addition to peripheral neuropathy. The other 4 mutations exhibit neuropathy without clinical or subclinical CNS abnormalities. These “PNS-only” mutations (Y151C, V181M, R183C and L239I) form gap junction plaques and produce levels of junctional coupling similar to those for wild-type Cx32. In contrast, mutants with CNS manifestations (F51L, E102del, V139M, R142Q, R142W, R164W T55I, R164Q and C168Y) either form no morphological gap junction plaques or, if they do, produce little or no detectable junctional coupling. Thus, PNS and CNS abnormalities may involve different aspects of connexin function.

Objective. Pantothenate kinase-associated neurodegeneration (PKAN) is an autosomal recessive disorder with variable onset, rate of progression, and phenotypic expression. Later-onset, more slowly progressive PKAN often presents with neuropsychiatric as well as motor manifestations that include speech difficulties, progressive dystonia, rigidity, and parkinsonism. PKAN is caused by biallelic PANK2 mutations, a gene that encodes pantothenate kinase 2, a regulatory enzyme in coenzyme A biosynthesis. Current therapeutic strategies rely on symptomatic relief. We describe the treatment of the first, later-onset PKAN patient with oral fosmetpantotenate (previously known as RE-024), a novel replacement therapy developed to bypass the enzymatic defect. Methods. This was an open-label, uncontrolled, 12-month treatment with fosmetpantotenate of a single patient with a later-onset, moderately severe, and slowly progressive form of PKAN. Results. The patient showed improvement in all clinical parameters including the Unified Parkinson’s Disease Rating Scale (UPDRS), Barry-Albright Dystonia Scale, the EuroQol five-dimensional three-level (EQ-5D-3L) scale, timed 25-foot walk test, and electroglottographic speech analysis. Fosmetpantotenate was well-tolerated with only transient liver enzyme elevation which normalized after dose reduction and did not recur after subsequent dose increases. Conclusions. Fosmetpantotenate showed promising results in a single PKAN patient and should be further studied in controlled trials.

Double-seronegative myasthenia gravis (dSN-MG, without detectable AChR and MuSK antibodies) presents a serious gap in MG diagnosis and understanding. Recently, autoantibodies against the low-density lipoprotein receptor-related protein 4 (LRP4) have been identified in several dSN-MG sera, but with dramatic frequency variation (similar to 2-50%). We have developed a cell based assay (CBA) based on human LRP4 expressing HEK293 cells, for the reliable and efficient detection of LRP4 antibodies. We have screened about 800 MG patient sera from 10 countries for LRP4 antibodies. The overall frequency of LRP4-MG in the dSN-MG group (635 patients) was 18.7% but with variations among different populations (range 7-32.7%). Interestingly, we also identified double positive sera: 8/107 anti-AChR positive and 10/ 67 anti-MuSK positive sera also had detectable LRP4 antibodies, predominantly originating from only two of the participating groups. No LRP4 antibodies were identified in sera from 56 healthy controls tested, while 4/110 from patients with other neuroimmune diseases were positive. The clinical data, when available, for the LRP4-MG patients were then studied. At disease onset symptoms were mild (81% had MGFA grade I or II), with some identified thymic changes (32% hyperplasia, none with thymoma). On the other hand, double positive patients (AChR/LRP4-MG and MuSK/LRP4-MG) had more severe symptoms at onset compared with any single positive MG subgroup. Contrary to MuSK-MG, 27% of ocular dSN-MG patients were LRP4 antibody positive. Similarly, contrary to MuSK antibodies, which are predominantly of the IgG4 subtype, LRP4 antibodies were predominantly of the IgG1 and IgG2 subtypes. The prevalence was higher in women than in men (female/male ratio 2.5/1), with an average disease onset at ages 33.4 for females and 41.9 for males. Overall, the response of LRP4-MG patients to treatment was similar to published responses of AChR-MG rather than to MuSK-MG patients.

Objective: A study was undertaken to describe the clinical spectrum, voltage-gated potassium channel (VGKC) complex antibody specificities, and central nervous system localization of antibody binding in 29 patients diagnosed with Morvan syndrome (MoS). Methods: Clinical data were collected using questionnaires. Radioimmunoassay, cell-based assays, and mouse brain immunohistochemistry were used to characterize the serum antibodies. Results: Neuromyotonia (100%), neuropsychiatric features (insomnia 89.7%, confusion 65.5%, amnesia 55.6%, hallucinations 51.9%), dysautonomia (hyperhidrosis 86.2%, cardiovascular 48.3%), and neuropathic pain (62.1%) were the most common manifestations. A total of 93.1% of MoS patients were male. VGKC-complex antibodies were present in 23 of 29 (79%) MoS patients at referral; 24 of 27 available sera had CASPR2, LGI1, or both CASPR2 and LGI1 antibodies (3 also with contactin-2 antibodies). CASPR2 antibodies were generally higher titer than LGI1 antibodies. Tumors (41.4%), mainly thymomas, were associated with CASPR2 antibodies and a poor prognosis, whereas LGI1 antibodies were associated with serum hyponatremia. In brain tissue regions including the hypothalamus, raphe, and locus coeruleus, commercial antibodies to LGI1 bound to neuronal cell bodies including the antidiuretic hormone-secreting and orexin-secreting hypothalamic neurons, whereas CASPR2 commercial antibodies bound more often to the neuropil. MoS antibodies bound similarly, but there was evidence of additional antibodies in some sera that were not adsorbed by LGI1- or CASPR2-expressing cells and bound to mouse Caspr2−/− tissue. Interpretation: MoS is clinically distinct from other VGKC-complex antibody-associated conditions, and usually is associated with high-titer CASPR2 antibodies, often accompanied by lower-titer LGI1 antibodies. CASPR2 and LGI1 antibodies bind to multiple brain regions, which helps to explain the multifocal clinical features of this disease, but other antibodies are likely to play a role in some patients and need to be characterized in future studies. ANN NEUROL 2012

Ion channels are complex transmembrane proteins that orchestrate the electrical signals necessary for normal function of excitable tissues, including the central nervous system, peripheral nerve, and both skeletal and cardiac muscle. Progress in molecular biology has allowed cloning and expression of genes that encode channel proteins, while comparable advances in biophysics, including patch-clamp electrophysiology and related techniques, have made the functional assessment of expressed proteins at the level of single channel molecules possible. The role of ion channel defects in the pathogenesis of numerous disorders has become increasingly apparent over the last two decades. Neurological channelopathies are frequently genetically determined but may also be acquired through autoimmune mechanisms. All of these autoimmune conditions can arise as paraneoplastic syndromes or independent from malignancies. The pathogenicity of autoantibodies to ion channels has been demonstrated in most of these conditions, and patients may respond well to immunotherapies that reduce the levels of the pathogenic autoantibodies. Autoimmune channelopathies may have a good prognosis, especially if diagnosed and treated early, and if they are non-paraneoplastic. This review focuses on clinical, pathophysiologic and therapeutic aspects of autoimmune ion channel disorders of the nervous system.

Antibodies that immunoprecipitate (125)I-alpha-dendrotoxin-labelled voltage-gated potassium channels extracted from mammalian brain tissue have been identified in patients with neuromyotonia, Morvan's syndrome, limbic encephalitis and a few cases of adult-onset epilepsy. These conditions often improve following immunomodulatory therapies. However, the proportions of the different syndromes, the numbers with associated tumours and the relationships with potassium channel subunit antibody specificities have been unclear. We documented the clinical phenotype and tumour associations in 96 potassium channel antibody positive patients (titres >400 pM). Five had thymomas and one had an endometrial adenocarcinoma. To define the antibody specificities, we looked for binding of serum antibodies and their effects on potassium channel currents using human embryonic kidney cells expressing the potassium channel subunits. Surprisingly, only three of the patients had antibodies directed against the potassium channel subunits. By contrast, we found antibodies to three proteins that are complexed with (125)I-alpha-dendrotoxin-labelled potassium channels in brain extracts: (i) contactin-associated protein-2 that is localized at the juxtaparanodes in myelinated axons; (ii) leucine-rich, glioma inactivated 1 protein that is most strongly expressed in the hippocampus; and (iii) Tag-1/contactin-2 that associates with contactin-associated protein-2. Antibodies to Kv1 subunits were found in three sera, to contactin-associated protein-2 in 19 sera, to leucine-rich, glioma inactivated 1 protein in 55 sera and to contactin-2 in five sera, four of which were also positive for the other antibodies. The remaining 18 sera were negative for potassium channel subunits and associated proteins by the methods employed. Of the 19 patients with contactin-associated protein-antibody-2, 10 had neuromyotonia or Morvan's syndrome, compared with only 3 of the 55 leucine-rich, glioma inactivated 1 protein-antibody positive patients (P < 0.0001), who predominantly had limbic encephalitis. The responses to immunomodulatory therapies, defined by changes in modified Rankin scores, were good except in the patients with tumours, who all had contactin-associated-2 protein antibodies. This study confirms that the majority of patients with high potassium channel antibodies have limbic encephalitis without tumours. The identification of leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 as the major targets of potassium channel antibodies, and their associations with different clinical features, begins to explain the diversity of these syndromes; furthermore, detection of contactin-associated protein-2 antibodies should help identify the risk of an underlying tumour and a poor prognosis in future patients.

The gap junction (GJ) protein connexin32 (Cx32) is expressed by myelinating Schwann cells and oligodendrocytes and is mutated in X-linked Charcot-Marie-Tooth disease. In addition to a demyelinating peripheral neuropathy, some Cx32 mutants are associated with transient or chronic CNS phenotypes. To investigate the molecular basis of these phenotypes, we generated transgenic mice expressing the T55I or the R75W mutation and an IRES-EGFP, driven by the mouseCnppromoter. The transgene was expressed in oligodendrocytes throughout the CNS and in Schwann cells. Both the T55I and the R75W mutants were localized in the perinuclear cytoplasm, did not form GJ plaques, and did not alter the expression or localization of two other oligodendrocytic GJ proteins, Cx47 and Cx29, or the expression of Cx29 in Schwann cells. On wild type background, the expression of endogenous mCx32 was unaffected by the T55I mutant, but was partly impaired by R75W. Transgenic mice with the R75W mutation and all mutant animals withGjb1-null background developed a progressive demyelinating peripheral neuropathy along with CNS myelination defects. These findings suggest that Cx32 mutations result in loss of function in myelinated cells withouttrans-dominant effects on other GJ proteins. Loss of Cx32 function alone in the CNS causes myelination defects.

Mutations in the gene encoding the K+channel KCNQ2 cause neonatal epilepsy and myokymia, indicating that KCNQ2 regulates the excitability of CNS neurons and motor axons, respectively. We show here that KCNQ2 channels are functional components of axon initial segments and nodes of Ranvier, colocalizing with ankyrin-G and voltage-dependent Na+channels throughout the CNS and PNS. Retigabine, which opens KCNQ channels, diminishes axonal excitability. Linopirdine, which blocks KCNQ channels, prolongs the repolarization of the action potential in neonatal nerves. The clustering of KCNQ2 at nodes and initial segments lags that of ankyrin-G during development, and both ankyrin-G and KCNQ2 can be coimmunoprecipitated in the brain. KCNQ3 is also a component of some initial segments and nodes in the brain. The diminished activity of mutant KCNQ2 channels accounts for neonatal epilepsy and myokymia; the cellular locus of these effects may be axonal initial segments and nodes.

Gap junctions (GJs) are vital for oligodendrocyte survival and myelination. In order to examine how different stages of inflammatory demyelination affect oligodendrocyte GJs, we studied the expression of oligodendrocytic connexin32 (Cx32) and Cx47 and astrocytic Cx43 in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS) induced by recombinant myelin oligodendrocyte glycoprotein. EAE was characterized by remissions and relapses with demyelination and axonal loss. Formation of GJ plaques was quantified in relation to the lesions and in normal appearing white matter (NAWM). During acute EAE at 14 days postimmunization (dpi) both Cx47 and Cx32 GJs were severely reduced within and around lesions but also in the NAWM. Cx47 was localized intracellularly in oligodendrocytes while protein levels remained unchanged, and this redistribution coincided with the loss of Cx43 GJs in astrocytes. Cx47 and Cx32 expression increased during remyelination at 28 dpi but decreased again at 50 dpi in the relapsing phase. Oligodendrocyte GJs remained reduced even in NAWM, despite increased formation of Cx43 GJs toward lesions indicating astrogliosis. EAE induced in Cx32 knockout mice resulted in an exacerbated clinical course with more demyelination and axonal loss compared with wild-type EAE mice of the same backcross, despite similar degree of inflammation, and an overall milder loss of Cx47 and Cx43 GJs. Thus, EAE causes persistent impairment of both intra- and intercellular oligodendrocyte GJs even in the NAWM, which may be an important mechanism of MS progression. Furthermore, GJ deficient myelinated fibers appear more vulnerable to CNS inflammatory demyelination. © 2012 Wiley Periodicals, Inc.

Gap junctions (GJs) provide channels for direct cell-to-cell connectivity serving the homeostasis in several organs of vertebrates including the central (CNS) and peripheral (PNS) nervous systems. GJs are composed of connexins (Cx), which show a highly distinct cellular and subcellular expression pattern. Oligodendrocytes, the myelinating cells of the CNS, are characterized by extensive GJ connectivity with each other as well as with astrocytes. The main oligodendrocyte connexins forming these GJ channels are Cx47 and Cx32. The importance of these channels has been highlighted by the discovery of human diseases caused by mutations in oligodendrocyte connexins, manifesting with leukodystrophy or transient encephalopathy. Experimental models have provided further evidence that oligodendrocyte GJs are essential for CNS myelination and homeostasis, while a strong inflammatory component has been recognized in the absence of oligodendrocyte connexins. Further studies revealed that connexins are also disrupted in multiple sclerosis (MS) brain, and in experimental models of induced inflammatory demyelination. Moreover, induced demyelination was more severe and associated with higher degree of CNS inflammation in models with oligodendrocyte GJ deficiency, suggesting that disrupted connexin expression in oligodendrocytes is not only a consequence but can also drive a pro-inflammatory environment in acquired demyelinating disorders such as MS. In this review, we summarize the current insights from human disorders as well as from genetic and acquired models of demyelination related to oligodendrocyte connexins, with the remaining challenges and perspectives.

Human hGDH2 arose via duplication in the apes and driven by positive selection acquired enhanced catalytic ability under conditions inhibitory to its precursor hGDH1 (common to all mammals). To explore the biological advantage provided by the novel enzyme, we studied, by immunohistochemistry (IHC) and immunofluorescence (IF), hGDH1 and hGDH2 expression in the human brain. Studies on human cortical tissue using anti-hGDH1-specific antibody revealed that hGDH1 was expressed in glial cells (astrocytes, oligodendrocytes, and oligodendrocyte precursors) with neurons being devoid of hGDH1 staining. In contrast, an hGDH2-specific antiserum labeled both astrocytes and neurons. Specifically, hGDH2 immunoreactivity was found in the cytoplasm of large neuronal cells within coarse structures resembling mitochondria. These were distributed either in the perikaryon or in the cell periphery. Double immunofluorescence (IF) suggested that the latter represented hGDH2-labeled mitochondria of presynaptic nerve terminals. Hence, hGDH2 evolution bestowed large human neurons with enhanced glutamate metabolizing capacity, thus strengthening cortical excitatory transmission.

The X-linked form of Charcot-Marie-Tooth disease (CMT1X) is the second most common form of hereditary motor and sensory neuropathy. The clinical phenotype is characterized by progressive muscle atrophy and weakness, areflexia, and variable sensory abnormalities; central nervous system manifestations occur, too. Affected males have moderate to severe symptoms, whereas heterozygous females are usually less affected. Neurophysiology shows intermediate slowing of conduction and distal axonal loss. Nerve biopsies show more prominent axonal degeneration than de/remyelination. More than 400 different mutations in GJB1, the gene that encodes the gap junction (GJ) protein connexin32 (Cx32), cause CMT1X. Many Cx32 mutants fail to form functional GJs, or form GJs with abnormal biophysical properties. Schwann cells and oligodendrocytes express Cx32, and the GJs formed by Cx32 play an important role in the homeostasis of myelinated axons. Animal models of CMT1X demonstrate that loss of Cx32 in myelinating Schwann cells causes a demyelinating neuropathy. An effective therapy remains to be developed.

Greig cephalopolysyndactyly syndrome (GCPS) is typically characterized by preaxial or mixed preaxial and postaxial polydactyly with or without syndactyly and craniofacial features including hypertelorism and macrocephaly. Although GLI3 shows considerable pleiotropy, it is the only gene known to cause this particular phenotype. We report on a patient with GCPS caused by a novel GLI3 mutation. In addition, the patient had asymmetry of the calf muscles, most likely secondary to chronic hypertrophic radiculopathy. The GLI3 mutation identified by targeted Sanger sequencing analysis in our patient is predicted to lead to premature termination of translation. This is the first report of a Cypriot patient with a GCPS because of a novel GLI3 mutation. The report provides additional evidence in support of the rich variability in phenotypic expression, the mutational heterogeneity and ethnic diversity associated with this rare condition.

The segregation of myelinated fibers into distinct domains around the node of Ranvier--the perinodal areas--is crucial for nervous system homeostasis and efficient nerve conduction. Perinodal areas are formed by axo-glial interactions, namely the interaction of molecules between the axon and the myelinating glia. In a variety of demyelinating pathologies including multiple sclerosis, the molecular architecture of the myelinated fiber is disrupted, leading to axonal degeneration. In this study we have analyzed the alterations of TAG-1, Caspr2, and voltage-gated potassium channels (VGKCs), forming the juxtaparanodal tripartite complex, in relation to adjacent paranodal and nodal molecules, in two different models of CNS demyelination, the experimental autoimmune encephalomyelitis (EAE) and the cuprizone model of toxic demyelination. We found extensive alterations of the juxtaparanodal molecular architecture under de- and remyelinating conditions. Inflammation alone was sufficient to disrupt the borders between the domains leading to the diffusion of juxtaparanodal components to the adjacent paranodal area. EAE induction and cuprizone-induced demyelination resulted initially in paranodal domain elongation with subsequent diffusion of the juxtaparanodal components and the reduction of their expression levels. At later stages, with decreasing inflammation and spontaneous remyelination there was a partial restoration of the paranodal domain but not sufficient re-organization of the juxtaparanodes. The latter were re-formed only when complete remyelination was allowed in the cuprizone model, indicating that juxtaparanodal domain reorganization is a later event that may remain incomplete in a hostile inflammatory milieu.

X-linked Charcot-Marie-Tooth disease (CMT1X) is a common form of inherited neuropathy resulting from different mutations affecting the gap junction (GJ) protein connexin32 (Cx32). A subset of CMT1X patients may additionally present with acute fulminant CNS dysfunction, typically triggered by conditions of systemic inflammation and metabolic stress. To clarify the underlying mechanisms of CNS phenotypes in CMT1X we studied a mouse model of systemic inflammation induced by lipopolysaccharide (LPS) injection to compare wild type (WT), connexin32 (Cx32) knockout (KO), and KO T55I mice expressing the T55I Cx32 mutation associated with CNS phenotypes. Following a single intraperitoneal LPS or saline (controls) injection at the age of 40–60 days systemic inflammatory response was documented by elevated TNF-α and IL-6 levels in peripheral blood and mice were evaluated 1 week after injection. Behavioral analysis showed graded impairment of motor performance in LPS treated mice, worse in KO T55I than in Cx32 KO and in Cx32 KO worse than WT. Iba1 immunostaining revealed widespread inflammation in LPS treated mice with diffusely activated microglia throughout the CNS. Immunostaining for the remaining major oligodendrocyte connexin Cx47 and for its astrocytic partner Cx43 revealed widely reduced expression of Cx43 and loss of Cx47 GJs in oligodendrocytes. Real-time PCR and immunoblot analysis indicated primarily a down regulation of Cx43 expression with secondary loss of Cx47 membrane localization. Inflammatory changes and connexin alterations were most severe in the KO T55I group. To examine why the presence of the T55I mutant exacerbates pathology even more than in Cx32 KO mice, we analyzed the expression of ER-stress markers BiP, Fas and CHOP by immunostaining, immunoblot and Real-time PCR. All markers were increased in LPS treated KO T55I mice more than in other genotypes. In conclusion, LPS induced neuroinflammation causes disruption of the main astrocyte-oligodendrocyte GJs, which may contribute to the increased sensitivity of Cx32 KO mice to LPS and of patients with CMT1X to various stressors. Moreover the presence of an intracellularly retained, misfolded CMT1X mutant such as T55I induces ER stress under inflammatory conditions, further exacerbating oligodendrocyte dysfunction and pathological changes in the CNS. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0369-5) contains supplementary material, which is available to authorized users.

Introduction: Parkinson's disease (PD) is a neurodegenerative disorder affecting a substantial proportion of the elderly Cypriot population. The objective of this study was to evaluate PD risk variants that have been identified previously in Genome Wide Association Studies (GWAS) and to find environmental factors that are predictors for PD onset in the Cypriot population. Methods: A case-control study was conducted with a total of 235 PD patients and 464 healthy controls of Greek-Cypriot ethnicity. Demographic and lifestyle characteristics, exposure to PD risk factors and clinical data were collected. Moreover, 13 previously GWAS-identified PD risk variants were genotyped. Univariate and multivariate regression analyses examined the association between a number of environmental and genetic factors and PD. Results: Multivariable regression analysis revealed that exposure to both pesticides and other toxic substances (P = 0.03), severe head injury accompanied with fainting (P = 0.001), nuts consumption (P = 0.004), red meat consumption (P = 0.02), and soft drinks consumption (P = 0.008) were increasing the risk for PD, whereas cumulative smoking (P = 0.02), and fish consumption (P = 0.02) were decreasing the risk for PD. Five out of the 13 tested SNPs (rs12185268, rs6599389, rs356220, rs13312, and rs17649553) were confirmed to be nominally significantly associated (P < 0.05) with PD risk in the Cypriot population. Conclusions: Collectively, this case-control study has shed some light on the nature of PD epidemiology in Cyprus, by demonstrating a number of genetic and environmental determinants of PD in the Cypriot population.

Introduction Amyotrophic lateral sclerosis (ALS) is a rare, progressive neurodegenerative disease. There is heterogeneity of clinical phenotypes while a clinical characterization of ALS in Cyprus is still lacking. The aim of this 30-year retrospective study of ALS in Cyprus is to determine the demographic characteristics of patients, the clinical features of the disease, the uptake of supportive therapies and factors influencing survival. Methods All ALS patients seen at the Cyprus Institute of Neurology and Genetics from January 1985 until July 2015 were included. Medical records of eligible patients were used for data extraction and compilation of an ALS database. Clinical features were compared between gender categories using univariate tests, while survival was assessed using Kaplan-Meier curves. Cox proportional hazards models were used to identify prognostic factors for survival. Results One hundred and seventy-nine ALS patients were included in the study, of whom 7 had a positive family history. Most clinical characteristics of ALS did not differ from what is observed in other European countries. However, some clinical characteristics were unique to our population, such as an increased acceptability and utilisation of supportive treatments such as gastrostomy. Conclusions Overall, clinical characteristics of patients with ALS in the Republic of Cyprus do not differ from other European counties. Our study demonstrates a high acceptance and utilisation of supportive interventions enhancing survival, in the context of a multidisciplinary approach offered in the single tertiary centre that services the whole Cypriot ALS population. The findings of this paper are of value to the health professionals treating ALS in Cyprus.

Pelizaeus-Merzbacher-like disease or hypomyelinating leukodystrophy-2 is an autosomal recessively inherited leukodystrophy with childhood onset resulting from mutations in the gene encoding the gap junction protein connexin 47 (Cx47, encoded by GJC2). Cx47 is expressed specifically in oligodendrocytes and is crucial for gap junctional communication throughout the central nervous system. Previous studies confirmed that a cell autonomous loss-of-function mechanism underlies hypomyelinating leukodystrophy-2 and that transgenic oligodendrocyte-specific expression of another connexin, Cx32 (GJB1), can restore gap junctions in oligodendrocytes to achieve correction of the pathology in a disease model. To develop an oligodendrocyte-targeted gene therapy, we cloned the GJC2/Cx47 gene under the myelin basic protein promoter and used an adeno-associated viral vector (AAV.MBP.Cx47myc) to deliver the gene to postnatal Day 10 mice via a single intracerebral injection in the internal capsule area. Lasting Cx47 expression specifically in oligodendrocytes was detected in Cx47 single knockout and Cx32/Cx47 double knockout mice up to 12 weeks post-injection, including the corpus callosum and the internal capsule but also in more distant areas of the cerebrum and in the spinal cord. Application of this oligodendrocyte-targeted somatic gene therapy at postnatal Day 10 in groups of double knockout mice, a well characterized model of hypomyelinating leukodystrophy-2, resulted in significant improvement in motor performance and coordination at 1 month of age in treated compared to mock-treated mice, as well as prolonged survival. Furthermore, immunofluorescence and morphological analysis revealed improvement in demyelination, oligodendrocyte apoptosis, inflammation, and astrogliosis, all typical features of this leukodystrophy model in both brain and spinal cord. Functional dye transfer analysis confirmed the re-establishment of oligodendrocyte gap junctional connectivity in treated as opposed to untreated mice. These results provide a significant advance in the development of oligodendrocyte-cell specific gene therapy. Adeno-associated viral vectors can be used to target therapeutic expression of a myelin gene to oligodendrocytes. We show evidence for the first somatic gene therapy approach to treat hypomyelinating leukodystrophy-2 preclinically, providing a potential treatment for this and similar forms of leukodystrophies.