Myelin protein zero mutation-related hereditary neuropathies: Neuropathological insight from a new nerve biopsy cohort.

Myelin protein zero (MPZ/P0) is a major structural protein of peripheral nerve myelin. Disease-associated variants in the MPZ gene cause a wide phenotypic spectrum of inherited peripheral neuropathies. Previous nerve biopsy studies showed evidence for subtype-specific morphological features. Here, we aimed at enhancing the understanding of these subtype-specific features and pathophysiological aspects of MPZ neuropathies. We examined archival material from two Central European centers and systematically determined genetic, clinical, and neuropathological features of 21 patients with MPZ mutations compared to 16 controls. Cases were grouped based on nerve conduction data into congenital hypomyelinating neuropathy (CHN; n = 2), demyelinating Charcot-Marie-Tooth (CMT type 1; n = 11), intermediate (CMTi; n = 3), and axonal CMT (type 2; n = 5). Six cases had combined muscle and nerve biopsies and one underwent autopsy. We detected four MPZ gene variants not previously described in patients with neuropathy. Light and electron microscopy of nerve biopsies confirmed fewer myelinated fibers, more onion bulbs and reduced regeneration in demyelinating CMT1 compared to CMT2/CMTi. In addition, we observed significantly more denervated Schwann cells, more collagen pockets, fewer unmyelinated axons per Schwann cell unit and a higher density of Schwann cell nuclei in CMT1 compared to CMT2/CMTi. CHN was characterized by basal lamina onion bulb formation, a further increase in Schwann cell density and hypomyelination. Most late onset axonal neuropathy patients showed microangiopathy. In the autopsy case, we observed prominent neuromatous hyperinnervation of the spinal meninges. In four of the six muscle biopsies, we found marked structural mitochondrial abnormalities. These results show that MPZ alterations not only affect myelinated nerve fibers, leading to either primarily demyelinating or axonal changes, but also affect non-myelinated nerve fibers. The autopsy case offers insight into spinal nerve root pathology in MPZ neuropathy. Finally, our data suggest a peculiar association of MPZ mutations with mitochondrial alterations in muscle.

Collagen VI-Related Myopathy Caused by Compound Heterozygous Mutations of COL6A3 in a Consanguineous Kurdish Family.

ABSTRACT: Collagen VI-related myopathies are caused by mutations of COL6A1, COL6A2, and COL6A3 and present with a wide phenotypic spectrum ranging from severe Ulrich congenital muscular dystrophy to mild Bethlem myopathy. Here, we report a consanguineous Kurdish family with 3 siblings affected by autosomal-recessive Bethlem myopathy caused by compound heterozygous mutations of COL6A3. We found the previously described missense mutation c.7447A > G/p.(Lys2483Glu) and a novel large deletion encompassing the exon 1-39 of the COL6A3 gene. Apart from the classical clinical symptoms, all patients had keratoconus, which expands the phenotype of the collagen VI-related myopathies.

Integrative Multi-Omics Analysis in Calcific Aortic Valve Disease Reveals a Link to the Formation of Amyloid-Like Deposits.

Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease in the developed world, yet no pharmacological therapy exists. Here, we hypothesize that the integration of multiple omic data represents an approach towards unveiling novel molecular networks in CAVD. Databases were searched for CAVD omic studies. Differentially expressed molecules from calcified and control samples were retrieved, identifying 32 micro RNAs (miRNA), 596 mRNAs and 80 proteins. Over-representation pathway analysis revealed platelet degranulation and complement/coagulation cascade as dysregulated pathways. Multi-omics integration of overlapping proteome/transcriptome molecules, with the miRNAs, identified a CAVD protein-protein interaction network containing seven seed genes (apolipoprotein A1 (APOA1), hemoglobin subunit ß (HBB), transferrin (TF), α-2-macroglobulin (A2M), transforming growth factor ß-induced protein (TGFBI), serpin family A member 1 (SERPINA1), lipopolysaccharide binding protein (LBP), inter-α-trypsin inhibitor heavy chain 3 (ITIH3) and immunoglobulin κ constant (IGKC)), four input miRNAs (miR-335-5p, miR-3663-3p, miR-21-5p, miR-93-5p) and two connector genes (amyloid beta precursor protein (APP) and transthyretin (TTR)). In a metabolite-gene-disease network, Alzheimer's disease exhibited the highest degree of betweenness. To further strengthen the associations based on the multi-omics approach, we validated the presence of APP and TTR in calcified valves from CAVD patients by immunohistochemistry. Our study suggests a novel molecular CAVD network potentially linked to the formation of amyloid-like structures. Further investigations on the associated mechanisms and therapeutic potential of targeting amyloid-like deposits in CAVD may offer significant health benefits.

A single supratentorial high-grade neuroepithelial tumor with two distinct BCOR mutations, exceptionally long complete remission and survival.

Here, we present a patient with high-grade neuroepithelial tumors with mutations in the BCL6 corepressor BCOR (HGNET-BCOR), a rare, highly malignant brain tumor with poor prognosis. The patient underwent gross total tumor resection (GTR), high-dose chemotherapy, and, after local relapse, GTR, proton radiation, and chemotherapy. After a 7.5 year-long complete remission, the tumor recurred locally, was treated by GTR, and responded to temozolomide treatment. In addition to an internal tandem duplication in BCOR common to the majority of HGNET-BCOR cases, molecular analysis revealed a second BCOR mutation in this tumor: a frame shift mutation. The combination of these mutations was associated with relatively low BCOR expression compared to other HGNET-BCOR cases.

Age-Related Gliosis Promotes Central Nervous System Lymphoma through CCL19-Mediated Tumor Cell Retention.

How lymphoma cells (LCs) invade the brain during the development of central nervous system lymphoma (CNSL) is unclear. We found that NF-κB-induced gliosis promotes CNSL in immunocompetent mice. Gliosis elevated cell-adhesion molecules, which increased LCs in the brain but was insufficient to induce CNSL. Astrocyte-derived CCL19 was required for gliosis-induced CNSL. Deleting CCL19 in mice or CCR7 from LCs abrogated CNSL development. Two-photon microscopy revealed LCs transiently entering normal brain parenchyma. Astrocytic CCL19 enhanced parenchymal CNS retention of LCs, thereby promoting CNSL formation. Aged, gliotic wild-type mice were more susceptible to forming CNSL than young wild-type mice, and astrocytic CCL19 was observed in both human gliosis and CNSL. Therefore, CCL19-CCR7 interactions may underlie an increased age-related risk for CNSL.

The ubiquitin ligase PHR promotes directional regrowth of spinal zebrafish axons.

To reconnect with their synaptic targets, severed axons need to regrow robustly and directionally along the pre-lesional trajectory. While mechanisms directing axonal regrowth are poorly understood, several proteins direct developmental axon outgrowth, including the ubiquitin ligase PHR (Mycbp2). Invertebrate PHR also limits regrowth of injured axons, whereas its role in vertebrate axonal regrowth remains elusive. Here we took advantage of the high regrowth capacity of spinal zebrafish axons and observed robust and directional regrowth following laser transection of spinal Mauthner axons. We found that PHR directs regrowing axons along the pre-lesional trajectory and across the transection site. At the transection site, initial regrowth of wild-type axons was multidirectional. Over time, misdirected sprouts were corrected in a PHR-dependent manner. Ablation of cyfip2, known to promote F-actin-polymerization and pharmacological inhibition of JNK reduced misdirected regrowth of PHR-deficient axons, suggesting that PHR controls directional Mauthner axonal regrowth through cyfip2- and JNK-dependent pathways.

Dynein promotes sustained axonal growth and Schwann cell remodeling early during peripheral nerve regeneration.

Following injury, axons of the peripheral nervous system have retained the capacity for regeneration. While it is well established that injury signals require molecular motors for their transport from the injury site to the nucleus, whether kinesin and dynein motors play additional roles in peripheral nerve regeneration is not well understood. Here we use genetic mutants of motor proteins in a zebrafish peripheral nerve regeneration model to visualize and define in vivo roles for kinesin and dynein. We find that both kinesin-1 and dynein are required for zebrafish peripheral nerve regeneration. While loss of kinesin-1 reduced the overall robustness of axonal regrowth, loss of dynein dramatically impaired axonal regeneration and also reduced injury-induced Schwann cell remodeling. Chimeras between wild type and dynein mutant embryos demonstrate that dynein function in neurons is sufficient to promote axonal regrowth. Finally, by simultaneously monitoring actin and microtubule dynamics in regenerating axons we find that dynein appears dispensable to initiate axonal regrowth, but is critical to stabilize microtubules, thereby sustaining axonal regeneration. These results reveal two previously unappreciated roles for dynein during peripheral nerve regeneration, initiating injury induced Schwann cell remodeling and stabilizing axonal microtubules to sustain axonal regrowth.

A small molecule screen identifies in vivo modulators of peripheral nerve regeneration in zebrafish.

Adult vertebrates have retained the ability to regenerate peripheral nerves after injury, although regeneration is frequently incomplete, often leading to functional impairments. Small molecule screens using whole organisms have high potential to identify biologically relevant targets, yet currently available assays for in vivo peripheral nerve regeneration are either very laborious and/or require complex technology. Here we take advantage of the optical transparency of larval zebrafish to develop a simple and fast pectoral fin removal assay that measures peripheral nerve regeneration in vivo. Twenty-four hours after fin amputation we observe robust and stereotyped nerve regrowth at the fin base. Similar to laser mediated nerve transection, nerve regrowth after fin amputation requires Schwann cells and FGF signaling, confirming that the fin amputation assay identifies pathways relevant for peripheral nerve regeneration. From a library of small molecules with known targets, we identified 21 compounds that impair peripheral nerve regeneration. Several of these compounds target known regulators of nerve regeneration, further validating the fin removal assay. Twelve of the identified compounds affect targets not previously known to control peripheral nerve regeneration. Using a laser-mediated nerve transection assay we tested ten of those compounds and confirmed six of these compounds to impair peripheral nerve regeneration: an EGFR inhibitor, a glucocorticoid, prostaglandin D2, a retinoic acid agonist, an inhibitor of calcium channels and a topoisomerase I inhibitor. Thus, we established a technically simple assay to rapidly identify valuable entry points into pathways critical for vertebrate peripheral nerve regeneration.

Myosin phosphatase Fine-tunes Zebrafish Motoneuron Position during Axonogenesis.

During embryogenesis the spinal cord shifts position along the anterior-posterior axis relative to adjacent tissues. How motor neurons whose cell bodies are located in the spinal cord while their axons reside in adjacent tissues compensate for such tissue shift is not well understood. Using live cell imaging in zebrafish, we show that as motor axons exit from the spinal cord and extend through extracellular matrix produced by adjacent notochord cells, these cells shift several cell diameters caudally. Despite this pronounced shift, individual motoneuron cell bodies stay aligned with their extending axons. We find that this alignment requires myosin phosphatase activity within motoneurons, and that mutations in the myosin phosphatase subunit mypt1 increase myosin phosphorylation causing a displacement between motoneuron cell bodies and their axons. Thus, we demonstrate that spinal motoneurons fine-tune their position during axonogenesis and we identify the myosin II regulatory network as a key regulator.

In Vivo Longitudinal (1)H MRS Study of Transgenic Mouse Models of Prion Disease in the Hippocampus and Cerebellum at 14.1 T.

In vivo (1)H MR spectroscopy allows the non invasive characterization of brain metabolites and it has been used for studying brain metabolic changes in a wide range of neurodegenerative diseases. The prion diseases form a group of fatal neurodegenerative diseases, also described as transmissible spongiform encephalopathies. The mechanism by which prions elicit brain damage remains unclear and therefore different transgenic mouse models of prion disease were created. We performed an in vivo longitudinal (1)H MR spectroscopy study at 14.1 T with the aim to measure the neurochemical profile of Prnp -/- and PrPΔ32-121 mice in the hippocampus and cerebellum. Using high-field MR spectroscopy we were able to analyze in details the in vivo brain metabolites in Prnp -/- and PrPΔ32-121 mice. An increase of myo-inositol, glutamate and lactate concentrations with a decrease of N-acetylaspartate concentrations were observed providing additional information to the previous measurements.

ZyFISH: a simple, rapid and reliable zygosity assay for transgenic mice.

Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations.

Phenotypic variation of autosomal-dominant corticobasal degeneration.

Neurodegenerative tauopathies may be inherited as autosomal-dominant disorders with variable clinicopathological phenotypes, and causative mutations in the microtubule-associated protein tau (MAPT) gene are not regularly seen. Herein, we describe a patient with clinically typical and autopsy-proven corticobasal degeneration (CBD). Her mother was diagnosed to have Parkinson's disease, but autopsy showed CBD pathology as in the index patient. The sister of the index patient had the clinical symptoms of primary progressive aphasia (PPA), but no pathology was available to date. Molecular analysis did not reveal any mutation in the MAPT or progranulin (GRN) genes. Our findings illustrate that CBD, progressive supranuclear palsy and PPA may be overlapping diseases with a common pathological basis rather than distinct entities. Clinical presentation and course might be determined by additional, yet unknown, genetic modifying factors.

Treatment of inclusion body myositis: is low-dose intravenous immunoglobulin the solution?

Inclusion body myositis (IBM), the most common inflammatory myopathy in the elderly, is often resistant to various forms of therapy. Placebo-controlled treatment trials with high dose intravenous immunoglobulins (IVIG) have shown disease amelioration in some but not all patients. Here, we present the informative case of a 70-year-old woman with diagnosed inclusion body myositis that showed progressive muscle weakness without treatment and following immuno-suppressive treatment with corticosteroids and azathioprine. A trial with low-dose intravenous immunoglobulins was started at that time. The patient responded rapidly to low dose IVIG treatment with amelioration of muscle strength and normalization of CK serum activities. Our results demonstrate that IBM patients may respond to low-dose IVIG treatment which has important clinical and economic consequences.

Quantitative and integrative proteome analysis of peripheral nerve myelin identifies novel myelin proteins and candidate neuropathy loci.

Peripheral nerve myelin facilitates rapid impulse conduction and normal motor and sensory functions. Many aspects of myelin biogenesis, glia-axonal interactions, and nerve homeostasis are poorly understood at the molecular level. We therefore hypothesized that only a fraction of all relevant myelin proteins has been identified so far. Combining gel-based and gel-free proteomic approaches, we identified 545 proteins in purified mouse sciatic nerve myelin, including 36 previously known myelin constituents. By mass spectrometric quantification, the predominant P0, periaxin, and myelin basic protein constitute 21, 16, and 8% of the total myelin protein, respectively, suggesting that their relative abundance was previously misestimated due to technical limitations regarding protein separation and visualization. Focusing on tetraspan-transmembrane proteins, we validated novel myelin constituents using immuno-based methods. Bioinformatic comparison with mRNA-abundance profiles allowed the categorization in functional groups coregulated during myelin biogenesis and maturation. By differential myelin proteome analysis, we found that the abundance of septin 9, the protein affected in hereditary neuralgic amyotrophy, is strongly increased in a novel mouse model of demyelinating neuropathy caused by the loss of prion protein. Finally, the systematic comparison of our compendium with the positions of human disease loci allowed us to identify several candidate genes for hereditary demyelinating neuropathies. These results illustrate how the integration of unbiased proteome, transcriptome, and genome data can contribute to a molecular dissection of the biogenesis, cell biology, metabolism, and pathology of myelin.

Mutation-specific IDH1 antibody differentiates oligodendrogliomas and oligoastrocytomas from other brain tumors with oligodendroglioma-like morphology.

Isocitrate dehydrogenase 1 (IDH1) mutations are frequent in astrocytomas, oligoastrocytomas and oligodendrogliomas. We previously reported the generation of a mutation-specific antibody that specifically detects R132H mutated IDH1 protein (clone H09). Here, we investigate the feasibility of H09 immunohistochemistry to differentiate between oligodendrogliomas/oligoastrocytomas and other tumors with similar morphology. A total of 274 brain tumors presenting with focal or extensive clear cell morphology were investigated. High numbers of H09-positive cases were observed in adult grade II oligodendrogliomas (67 of 74, 91%), grade III oligodendrogliomas (65 of 69, 94%), grade II oligoastrocytomas (11 of 14, 79%) and grade III oligoastrocytomas (10 of 11, 91%). All cases of pediatric oligodendrogliomas (n = 7), neurocytomas (n = 41, 35 central, 4 extraventricular, 2 cerebellar liponeurocytomas), dysembryoplastic neuroepithelial tumors (n = 21), clear cell ependymomas (n = 8), clear cell meningiomas (n = 9) as well as 12 primary glioblastomas with oligodendroglial differentiation and 5 pilocytic astrocytomas with oligodendroglial-like differentiation were negative for H09 immunohistochemistry. Three oligodendrogliomas with neurocytic differentiation had evidence of IDH1/IDH2 mutations either by H09 immunohistochemistry or direct sequencing. We conclude that in tumors with an oligodendroglioma-like morphology, binding of H09 is highly specific for oligodendrogliomas or oligoastrocytomas and substantially helps in the discrimination from other clear cell tumors. Negative H09 immunohistochemistry of an adult oligodendroglioma or oligoastrocytoma should prompt the consideration of other clear cell neoplasms. Further, our observations firmly assign oligodendrogliomas with neurocytic differentiation to the group of oligodendrogliomas and demonstrate that H09 is especially helpful for the difficult discrimination of such lesions from extraventricular neurocytomas.

Ablation of Dicer from murine Schwann cells increases their proliferation while blocking myelination.

The myelin sheaths that surround the thick axons of the peripheral nervous system are produced by the highly specialized Schwann cells. Differentiation of Schwann cells and myelination occur in discrete steps. Each of these requires coordinated expression of specific proteins in a precise sequence, yet the regulatory mechanisms controlling protein expression during these events are incompletely understood. Here we report that Schwann cell-specific ablation of the enzyme Dicer1, which is required for the production of small non-coding regulatory microRNAs, fully arrests Schwann cell differentiation, resulting in early postnatal lethality. Dicer(-/-) Schwann cells had lost their ability to myelinate, yet were still capable of sorting axons. Both cell death and, paradoxically, proliferation of immature Schwann cells was markedly enhanced, suggesting that their terminal differentiation is triggered by growth-arresting regulatory microRNAs. Using microRNA microarrays, we identified 16 microRNAs that are upregulated upon myelination and whose expression is controlled by Dicer in Schwann cells. This set of microRNAs appears to drive Schwann cell differentiation and myelination of peripheral nerves, thereby fulfilling a crucial function for survival of the organism.

Paraneoplastic Necrotizing Myopathy with a Mild Inflammatory Component: A Case Report and Review of the Literature.

Inflammatory myopathies such as dermatomyositis and polymyositis are well-recognized paraneoplastic syndromes. Little is known, however, about necrotizing myopathies in association with cancer. We here describe a case of paraneoplastic necrotizing myopathy with a mild inflammatory infiltrate in a patient with adenocarcinoma. After the rapid development of a severe, disabling muscle weakness, the patient experienced near complete recovery within 4 months under oral prednisone treatment. In the context of the presented case, we will review current knowledge about paraneoplastic necrotizing myopathies.

Axonal prion protein is required for peripheral myelin maintenance.

The integrity of peripheral nerves relies on communication between axons and Schwann cells. The axonal signals that ensure myelin maintenance are distinct from those that direct myelination and are largely unknown. Here we show that ablation of the prion protein PrP(C) triggers a chronic demyelinating polyneuropathy (CDP) in four independently targeted mouse strains. Ablation of the neighboring Prnd locus, or inbreeding to four distinct mouse strains, did not modulate the CDP. CDP was triggered by depletion of PrP(C) specifically in neurons, but not in Schwann cells, and was suppressed by PrP(C) expression restricted to neurons but not to Schwann cells. CDP was prevented by PrP(C) variants that undergo proteolytic amino-proximal cleavage, but not by variants that are nonpermissive for cleavage, including secreted PrP(C) lacking its glycolipid membrane anchor. These results indicate that neuronal expression and regulated proteolysis of PrP(C) are essential for myelin maintenance.

A lymphotoxin-driven pathway to hepatocellular carcinoma.

Hepatitis B and C viruses (HBV and HCV) cause chronic hepatitis and hepatocellular carcinoma (HCC) by poorly understood mechanisms. We show that cytokines lymphotoxin (LT) alpha and beta and their receptor (LTbetaR) are upregulated in HBV- or HCV-induced hepatitis and HCC. Liver-specific LTalphabeta expression in mice induces liver inflammation and HCC, causally linking hepatic LT overexpression to hepatitis and HCC. Development of HCC, composed in part of A6(+) oval cells, depends on lymphocytes and IKappa B kinase beta expressed by hepatocytes but is independent of TNFR1. In vivo LTbetaR stimulation implicates hepatocytes as the major LT-responsive liver cells, and LTbetaR inhibition in LTalphabeta-transgenic mice with hepatitis suppresses HCC formation. Thus, sustained LT signaling represents a pathway involved in hepatitis-induced HCC.