Cytoplasm Of Oligodendrocytes Research Articles

Noggin-mediated effects on metabolite profiles of microglia and oligodendrocytes after ischemic insult

Recent studies show that shifts in energy metabolism in activated microglia are linked to their functions and immune responses in the ischemic brain. We previously reported that an antagonist of the bone morphogenetic protein, noggin, enhanced myelination in the ischemic brain during the chronic phase, and conditioned media (CM) from activated BV2 microglia treated with noggin after ischemia/reperfusion (I/R) increased the expression of myelin basic protein (MBP) in oligodendrocytes (MO3.13). To determine whether noggin induced changes in cell metabolism, metabolite profiles in BV2 and MO3.13 cells were analyzed by untargeted metabolomics using 1H nuclear magnetic resonance spectroscopy. Compared to vehicle-treated BV2 cells, noggin treatment (100 ng/mL for 3 h after I/R) suppressed the I/R-induced increase in intracellular glucose and lactate levels but increased extracellular levels of glucose and several amino acids. When MO3.13 cells were exposed to noggin CM from BV2 cells, most of the vehicle CM-induced changes in the levels of metabolites such as choline, formate, and intermediates of oxidative phosphorylation were reversed, while the glycerol level was markedly increased. An increase in glycerol level was also observed in the noggin-treated ischemic brain and was further supported by the expression of glycerol-3-phosphate dehydrogenase 1 (required for glycerol synthesis) in the cytoplasm of MBP-positive oligodendrocytes in the ischemic brains treated with noggin. These results suggest that noggin-induced changes in the metabolism of microglia provide a favorable environment for myelin synthesis in oligodendrocytes during the recovery phase after ischemic stroke.

Early and extensive alterations of glial connexins, distal oligodendrogliopathy type demyelination, and nodal/paranodal pathology are characteristic of multiple system atrophy.

The pathological hallmark of multiple system atrophy (MSA) is aberrant accumulation of phosphorylated α-synuclein in oligodendrocytes, forming glial cytoplasmic inclusions (GCIs). Extensive demyelination occurs particularly in the olivopontocerebellar and striatonigral pathways, but its precise mechanism remains elusive. Glial connexins (Cxs), which form gap junction channels between astrocytes and oligodendrocytes, play critical roles in myelin maintenance, and have not been studied in MSA. Therefore, we immunohistochemically investigated glial Cx changes in the cerebellar afferent fibers in 15 autopsied patients with MSA. We classified demyelinating lesions into three stages based on Klüver-Barrera staining: early (Stage I), intermediate (Stage II), and late (Stage III) stages showing subtle, moderate, and severe myelin reduction, respectively. Myelin-associated glycoprotein, but not myelin oligodendrocyte glycoprotein, was preferentially decreased in Stage I, suggesting distal oligodendrogliopathy type demyelination. Accumulation of phosphorylated α-synuclein in oligodendrocytes was frequently seen in Stage I but less frequently observed in Stages II and III. Tubulin polymerization-promoting protein (TPPP/p25α)-positive oligodendrocytes were preserved in Stage I but successively decreased in Stages II and III. Even at Stage I, Cx32 was nearly absent from myelin, despite the relative preservation of other nodal proteins, such as neurofascin, claudin-11/oligodendrocyte-specific protein, and contactin-associated protein 1, which successively decreased in the later stages. Cx32 was re-distributed in the oligodendrocyte cytoplasm and co-localized with GCIs. Cx47 gradually decreased at the oligodendrocyte surface in a stage-dependent manner but was not co-localized with GCIs. Astrocytic Cx43 was down-regulated in Stage I but up-regulated in Stages II and III, reflecting astrogliosis. Cx43/Cx47 gap junctions significantly decreased from Stage I to III. Activated microglia/macrophages and T cells infiltrated in Stage I rather than Stages II and III. Therefore, early and extensive alterations of glial Cxs, particularly Cx32 loss, occur in MSA and may accelerate distal oligodendrogliopathy type demyelination and nodal/paranodal dysfunction through disruption of inter-glial communication.

NEP1‑40 promotes myelin regeneration via upregulation of GAP‑43 and MAP‑2 expression after focal cerebral ischemia in rats.

Axon regeneration after lesions to the central nervous system (CNS) is largely limited by the presence of growth inhibitory molecules expressed in myelin. Nogo-A is a principal inhibitor of neurite outgrowth, and blocking the activity of Nogo-A can induce axonal sprouting and functional recovery. However, there are limited data on the expression of Nogo-A after CNS lesions, and the mechanism underlying its influences on myelin growth remains unknown. The aim of the present study was to observe the time course of Nogo-A after cerebral ischemia/reperfusion in rats using immunohistochemistry and western blot techniques, and to test the effect of its inhibitor Nogo extracellular peptide 1–40 (NEP1-40) on neural plasticity proteins, growth-associated binding protein 43 (GAP-43) and microtubule associated protein 2 (MAP-2), as a possible mechanism underlying myelin suppression. A classic model of middle cerebral artery occlusion (MCAO) was established in Sprague-Dawley rats, which were divided into three groups: i) MCAO model group; ii) MCAO + saline group; and iii) MCAO + NEP1-40 group. Rats of each group were divided into five subgroups by time points as follows: days 1, 3, 7, 14 and 28. Animals that only received sham operation were used as controls. The Nogo-A immunoreactivity was located primarily in the cytoplasm of oligodendrocytes. The number of Nogo-A immunoreactive cells significantly increased from day 1 to day 3 after MCAO, nearly returning to the control level at day 7, increased again at day 14 and decreased at day 28. Myelin basic protein (MBP) immunoreactivity in the ipsilateral striatum gradually decreased from day 1 to day 28 after ischemia, indicating myelin loss appeared at early time points and continuously advanced during ischemia. Then, intracerebroventricular infusion of NEP1-40, which is a Nogo-66 receptor antagonist peptide, was administered at days 1, 3 and 14 after MCAO. It was observed that GAP-43 considerably increased from day 1 to day 7 and then decreased to a baseline level at day 28 compared with the control. MAP-2 expression across days 1–28 significantly decreased after MCAO. Administration of NEP1-40 attenuated the reduction of MBP, and upregulated GAP-43 and MAP-2 expression at the corresponding time points after MCAO compared with the MCAO + saline group. The present results indicated that NEP1-40 ameliorated myelin damage and promoted regeneration by upregulating the expression of GAP-43 and MAP-2 related to neuronal and axonal plasticity, which may aid with the identification of a novel molecular mechanism of restriction in CNS regeneration mediated by Nogo-A after ischemia in rats.

Role of VAPB and vesicular profiles in α-synuclein aggregates in multiple system atrophy.

The pathological hallmark of multiple system atrophy (MSA) is fibrillary aggregates of α‐synuclein (α‐Syn) in the cytoplasm and nucleus of both oligodendrocytes and neurons. In neurons, α‐Syn localizes to the cytosolic and membrane compartments, including the synaptic vesicles, mitochondria, and endoplasmic reticulum (ER). α‐Syn binds to vesicle‐associated membrane protein‐binding protein B (VAPB) in the ER membrane. Overexpression of wild‐type and familial Parkinson's disease mutant α‐Syn perturbs the association between the ER and mitochondria, leading to ER stress and ultimately neurodegeneration. We examined brains from MSA patients (n = 7) and control subjects (n = 5) using immunohistochemistry and immunoelectron microscopy with antibodies against VAPB and phosphorylated α‐Syn. In controls, the cytoplasm of neurons and glial cells was positive for VAPB, whereas in MSA lesions VAPB immunoreactivity was decreased. The proportion of VAPB‐negative neurons in the pontine nucleus was significantly higher in MSA (13.6%) than in controls (0.6%). The incidence of cytoplasmic inclusions in VAPB‐negative neurons was significantly higher (42.2%) than that in VAPB‐positive neurons (3.6%); 67.2% of inclusion‐bearing oligodendrocytes and 51.1% of inclusion‐containing neurons were negative for VAPB. Immunoelectron microscopy revealed that α‐Syn and VAPB were localized to granulofilamentous structures in the cytoplasm of oligodendrocytes and neurons. Many vesicular structures labeled with anti‐α‐Syn were also observed within the granulofilamentous structures in the cytoplasm and nucleus of both oligodendrocytes and neurons. These findings suggest that, in MSA, reduction of VAPB is involved in the disease process and that vesicular structures are associated with inclusion formation.

Alpha-Synuclein Inclusion Formation in Human Oligodendrocytes.

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by presence of α-synuclein-positive inclusions in the cytoplasm of oligodendrocytes. These glial cytoplasmic inclusions (GCIs) are considered an integral part of the pathogenesis of MSA, leading to demyelination and neuronal demise. What is most puzzling in the research fields of GCIs is the origin of α-synuclein aggregates in GCIs, since adult oligodendrocytes do not express high levels of α-synuclein. The most recent leading hypothesis is that GCIs form via transfer and accumulation of α-synuclein from neurons to oligodendrocytes. However, studies regarding this subject are limited due to the absence of proper human cell models, to demonstrate the entry and accumulation of neuronal α-synuclein in human oligodendrocytes. Here, we generated mature human oligodendrocytes that can take up neuron-derived α-synuclein and form GCI-like inclusions. Mature human oligodendrocytes are derived from neural stem cells via “oligosphere” formation and then into oligodendrocytes, treating the cells with the proper differentiation factors at each step. In the final cell preparations, oligodendrocytes consist of the majority population, while some astrocytes and unidentified stem cell-like cells were present as well. When these cells were exposed to α-synuclein proteins secreted from neuron-like human neuroblastoma cells, oligodendrocytes developed perinuclear inclusion bodies with α-synuclein immunoreactivity, resembling GCIs, while the stem cell-like cells showed α-synuclein-positive, scattered puncta in the cytoplasm. In conclusion, we have established a human oligodendrocyte model for the study of GCI formation, and the characterization and use of this model might pave the way for understanding the pathogenesis of MSA.

MSA: From basic mechanisms to experimental therapeutics

Multiple system atrophy (MSA) is a rare and fatal neurodegenerative disorder characterized by rapidly progressive autonomic and motor dysfunction. Pathologically, MSA is mainly characterized by the abnormal accumulation of misfolded α-synuclein in the cytoplasm of oligodendrocytes, which plays a major role in the pathogenesis of the disease. Striatonigral degeneration and olivopontecerebellar atrophy underlie the motor syndrome, while degeneration of autonomic centers defines the autonomic failure in MSA. At present, there is no treatment that can halt or reverse its progression. However, over the last decade several studies in preclinical models and patients have helped to better understand the pathophysiological events underlying MSA. The etiology of this fatal disorder remains unclear and may be multifactorial, caused by a combination of factors which may serve as targets for novel therapeutic approaches. In this review, we summarize the current knowledge about the etiopathogenesis and neuropathology of MSA, its different preclinical models, and the main disease modifying therapies that have been used so far or that are planned for future clinical trials.

TDP-43 proteinopathy in Theiler's murine encephalomyelitis virus infection.

TDP-43, an RNA-binding protein that is primarily nuclear and important in splicing and RNA metabolism, is mislocalized from the nucleus to the cytoplasm of neural cells in amyotrophic lateral sclerosis (ALS), and contributes to disease. We sought to investigate whether TDP-43 is mislocalized in infections with the acute neuronal GDVII strain and the persistent demyelinating DA strain of Theiler’s virus murine encephalomyelitis virus (TMEV), a member of the Cardiovirus genus of Picornaviridae because: i) L protein of both strains is known to disrupt nucleocytoplasmic transport, including transport of polypyrimidine tract binding protein, an RNA-binding protein, ii) motor neurons and oligodendrocytes are targeted in both TMEV infection and ALS. TDP-43 phosphorylation, cleavage, and cytoplasmic mislocalization to an aggresome were observed in wild type TMEV-infected cultured cells, with predicted splicing abnormalities. In contrast, cells infected with DA and GDVII strains that have L deletion had rare TDP-43 mislocalization and no aggresome formation. TDP-43 mislocalization was also present in neural cells of TMEV acutely-infected mice. Of note, TDP-43 was mislocalized six weeks after DA infection to the cytoplasm of oligodendrocytes and other glial cells in demyelinating lesions of spinal white matter. A recent study showed that TDP-43 knock down in oligodendrocytes in mice led to demyelination and death of this neural cell [1], suggesting that TMEV infection mislocalization of TDP-43 and other RNA-binding proteins is predicted to disrupt key cellular processes and contribute to the pathogenesis of TMEV-induced diseases. Drugs that inhibit nuclear export may have a role in antiviral therapy.

THE ULTRASTRUCTURAL STUDY OF THE SPINAL CORD AND THE DELTOID MUSCLE OF THE CLINICAL CASE OF THE VERDNIG-HOFFMANN SYNDROME

The Werdnig-Hoffmann syndrome is an infantile form of hereditary spinal progressive muscle atrophy with an autosomal recessive mode of inheritance with a frequency of 1 per 10,000 population. The data on the complex ultramicroscopic study of the spinal cord and deltoid muscle of a clinical case of Verdnig-Hoffmann syndrome in a newborn male child who died at the age of 1 month are given. Rubella virus patterns of the RNA were isolated in serum and spinal cord tissue samples. Changes in the structure of the spinal cord and deltoid muscle are described. An ultrastructural study showed the presence of a virus in the tissue of the spinal cord and vascular endothelial cells of the muscle. In the tissue of the spinal cord against the destruction of neurons was detected rubella virus. Viral particles were localized both in the cytoplasm of preserved oligodendrocytes and diffusely in destructively altered tissue of the spinal cord. The number of cells in the spinal cord tissue was reduced, the structure of their mitochondria was disturbed, and vacuolization of the cytoplasm was detected. For myelin fibers there is inherent heterogeneity of the thickness of the membranes, uneven distribution, deformation of the contours, and variability of diameters. Myelin fibers stratified, fragmented, osmiophilia lost. In the study of muscle samples prevailed destructively modified muscle fibers. At the same time, against the background of an unevenly pronounced atrophy of muscle fibers, the reproduction of the rubella virus in the cytoplasm of capillary endotheliocytes and pericytes of muscle vessels was detected. The etiological role of the rubella virus in the onset of the clinical symptoms constituting spinal muscular atrophy syndrome as a variant of congenital rubella has been suggested. .

Ultrastructural pathology of oligodendrocytes in white matter in continuous attack-like schizophrenia and a role for microglia

Previously the authors have reported the ultrastructural pathology and deficits of oligodendrocytes in gray and white matter of the prefrontal cortex in continuous paranoid schizophrenia. The aim of the present work was to study the effects of microglia on the ultrastructure of oligodendrocytes in white matter underlying the prefrontal cortex (BA10) in attack-like schizophrenia. Postmortem morphometric electron microscopic study of oligodendrocytes in close apposition to microglia was performed in white matter underlying the prefrontal cortex (BA10). Nine cases of chronic attack-like schizophrenia and 20 normal controls were studied. Areas of oligodendrocytes, volume density (Vv) and the number of mitochondria, vacuoles of endoplasmic reticulum and lipofuscin granules were estimated. Group comparison was performed using ANCOVA. The schizophrenia group differed from the control group by paucity of ribosomes in cytoplasm of oligodendrocytes, cytoplasm swelling, a significant increase in Vv and number of vacuoles and lipofuscin granules. Significant correlations between the parameters of vacuoles and lipofuscin granules and mitochondria were found only in the schizophrenia group. Dystrophic alterations of oligodendrocytes apposed microglial cells were found in the white matter of the prefrontal cortex in chronic schizophrenia as compared to controls. Microglia might contribute to abnormalities of lipid and protein metabolism of oligodendrocytes.

Ultrastructural pathology of oligodendrocytes in the white matter in continuous paranoid schizophrenia: a role for microglia

Previously the authors have reported the ultrastructural pathology and deficit of oligodendrocytes in gray and white matter of the prefrontal cortex in schizophrenia. The aim of the study was to determine of the effects of microglia on the ultrastructure of oligodendrocytes in the white matter underlying the prefrontal cortex in continuous schizophrenia. Postmortem morphometric electron microscopic study of oligodendrocytes in close apposition to microglia was performed in white matter underlying the prefrontal cortex (BA10). Eleven cases of chronic continuous schizophrenia and 11 normal controls were studied. Areas of oligodendrocytes, of their nuclei and cytoplasm, volume density (Vv) and the number of mitochondria, vacuoles of endoplasmic reticulum and lipofuscin granules were estimated. Group comparison was performed using ANCOVA. The schizophrenia group differed from the control group by paucity of ribosomes in the cytoplasm of oligodendrocytes, a significant decrease in Vv and the number of mitochondria and increase in the number of lipofuscin granules. Significant correlations between the parameters of lipofuscin granules, mitochondria and vacuoles were found only in the schizophrenia group. The number of lipofuscin granules were correlated positively with the illness duration. Dystrophic alterations of oligodendrocytes attached to microglial cells were found in the white matter of the prefrontal cortex in chronic paranoid schizophrenia as compared to controls. The data obtained suggest that microglia might contribute to abnormalities of energy, lipid and protein metabolism of oligodendrocytes in schizophrenia.

Olig1 Acetylation and Nuclear Export Mediate Oligodendrocyte Development.

The oligodendrocyte transcription factor Olig1 is critical for both oligodendrocyte development and remyelination in mice. Nuclear to cytoplasmic translocation of Olig1 protein occurs during brain development and in multiple sclerosis, but the detailed molecular mechanism of this translocation remains elusive. Here, we report that Olig1 acetylation and deacetylation drive its active translocation between the nucleus and the cytoplasm in both mouse and rat oligodendrocytes. We identified three functional nuclear export sequences (NES) localized in the basic helix-loop-helix domain and one specific acetylation site at Lys 150 (human Olig1) in NES1. Olig1 acetylation and deacetylation are regulated by the acetyltransferase CREB-binding protein and the histone deacetylases HDAC1, HDAC3, and HDAC10. Acetylation of Olig1 decreased its chromatin association, increased its interaction with inhibitor of DNA binding 2 and facilitated its retention in the cytoplasm of mature oligodendrocytes. These studies establish that acetylation of Olig1 regulates its chromatin dissociation and subsequent translocation to the cytoplasm and is required for its function in oligodendrocyte maturation.

Region-Specific Alterations of Matrix Metalloproteinase Activity in Multiple System Atrophy.

MSA is a sporadic progressive neurodegenerative disorder characterized by a variable combination of parkinsonism, cerebellar ataxia, and autonomic dysfunction. The pathological hallmark of MSA is the accumulation of alpha-synuclein aggregates in the cytoplasm of oligodendrocytes along with neuronal loss and neuroinflammation, as well as blood-brain barrier dysfunction and myelin deterioration. Matrix metalloproteinases are zinc-dependent endopeptidases involved in the remodeling of the extracellular matrix, demyelination, and blood-brain barrier permeability. Several lines of evidence indicate a role for these enzymes in various pathological processes, including stroke, multiple sclerosis, Parkinson's, and Alzheimer's disease. This study aimed to assess potential alterations of matrix metalloproteinase-1, -2, -3, and -9 expression or activity in MSA postmortem brain tissue. Gelatin zymography revealed increased matrix metalloproteinase-2 activity in the putamen, but not in the frontal cortex, of MSA patients relative to controls. Immunohistochemistry revealed increased number of glial cells positive for matrix metalloproteinase-1, -2, and -3 in the putamen and frontal cortex of MSA patients. Double immunofluorescence revealed that matrix metalloproteinase-2 and -3 were expressed in astrocytes and microglia. Only matrix metalloproteinase-2 colocalized with alpha-synuclein in oligodendroglial cytoplasmic inclusions. These results demonstrate widespread alterations of matrix metalloproteinase expression in MSA and a pattern of increased matrix metalloproteinase-2 expression and activity affecting preferentially a brain region severely affected (putamen) over a relatively spared region (frontal cortex). Elevated matrix metalloproteinase expression may thus contribute to the disease process in MSA by promoting blood-brain barrier dysfunction and/or myelin degradation.

Role of synucleins in traumatic brain injury — an experimental in vitro and in vivo study in mice.

Synucleins are small prone to aggregate proteins associated with several neurodegenerative diseases (NDDs), however their role in traumatic brain injury (TBI) is an emerging area of investigation. Using in vitro scratch injury model and in vivo mouse weight-drop model we have found that the injury causes alterations in the expression and localization of synucleins near the damaged area. Before injury, α-synuclein is diffused in the cytoplasm of neurons and γ-synuclein is both in the cytoplasm and nucleus of oligodendrocytes. After the scratch injury of the mixed neuronal and glial culture, α-synuclein forms punctate structures in the cytoplasm of neurons and γ-synuclein is almost completely localized to the nucleus of the oligodendrocytes. Furthermore, the amount of post-translationally modified Met38-oxidized γ-synuclein is increased 3.8 fold 24 h after the scratch. α- and γ-synuclein containing cells increased in the initially cell free scratch zone up to 24 h after the scratch.Intracellular expression and localization of synucleins are also changed in a mouse model of focal closed head injury, using a standardized weight drop device. γ-Synuclein goes from diffuse to punctate staining in a piriform cortex near the amygdala, which may reflect the first steps in the formation of deposits/inclusions. Surprisingly, oxidized γ-synuclein co-localizes with cofilin-actin rods in the thalamus, which are absent in all other regions of the brain. These structures reach their peak amounts 7 days after injury. The changes in γ-synuclein localization are accompanied by injury-induced alterations in the morphology of both astrocytes and neurons.

Novel therapeutic approaches in multiple system atrophy.

Multiple system atrophy (MSA) is a sporadic, adult onset, relentlessly progressive neurodegenerative disease characterized by autonomic abnormalities associated with parkinsonism, cerebellar dysfunction, pyramidal signs, or combinations thereof. Treatments that can halt or reverse the progression of MSA have not yet been identified. MSA is neuropathologically defined by the presence of α-synuclein-containing inclusions, particularly in the cytoplasm of oligodendrocytes (glial cytoplasmic inclusions, GCIs), which are associated with neurodegeneration. The mechanisms by which oligodendrocytic α-synuclein inclusions cause neuronal death in MSA are not completely understood. The MSA neurodegenerative process likely comprises cell-to-cell transmission of α-synuclein in a prion-like manner, α-synuclein aggregation, increased oxidative stress, abnormal expression of tubulin proteins, decreased expression of neurotrophic factors, excitotoxicity and microglial activation, and neuroinflammation. In an attempt to block each of these pathogenic mechanisms, several pharmacologic approaches have been tried and shown to exert neuroprotective effects in transgenic mouse or cellular models of MSA. These include sertraline, paroxetine, and lithium, which hamper arrival of α-synuclein to oligodendroglia; rifampicin, lithium, and non-steroidal anti-inflammatory drugs, which inhibit α-synuclein aggregation in oligodendrocytes; riluzole, rasagiline, fluoxetine and mesenchymal stem cells, which exert neuroprotective actions; and minocycline and intravenous immunoglobulins, which reduce neuroinflammation and microglial activation. These and other potential therapeutic strategies for MSA are summarized in this review.

Lipid dysfunction and pathogenesis of multiple system atrophy

Multiple system atrophy (MSA) is a progressive neurodegenerative disease characterized by the accumulation of α-synuclein protein in the cytoplasm of oligodendrocytes, the myelin-producing support cells of the central nervous system (CNS). The brain is the most lipid-rich organ in the body and disordered metabolism of various lipid constituents is increasingly recognized as an important factor in the pathogenesis of several neurodegenerative diseases. α-Synuclein is a 17 kDa protein with a close association to lipid membranes and biosynthetic processes in the CNS, yet its precise function is a matter of speculation, particularly in oligodendrocytes. α-Synuclein aggregation in neurons is a well-characterized feature of Parkinson’s disease and dementia with Lewy bodies. Epidemiological evidence and in vitro studies of α-synuclein molecular dynamics suggest that disordered lipid homeostasis may play a role in the pathogenesis of α-synuclein aggregation. However, MSA is distinct from other α-synucleinopathies in a number of respects, not least the disparate cellular focus of α-synuclein pathology. The recent identification of causal mutations and polymorphisms in COQ2, a gene encoding a biosynthetic enzyme for the production of the lipid-soluble electron carrier coenzyme Q10 (ubiquinone), puts membrane transporters as central to MSA pathogenesis, although how such transporters are involved in the early myelin degeneration observed in MSA remains unclear. The purpose of this review is to bring together available evidence to explore the potential role of membrane transporters and lipid dyshomeostasis in the pathogenesis of α-synuclein aggregation in MSA. We hypothesize that dysregulation of the specialized lipid metabolism involved in myelin synthesis and maintenance by oligodendrocytes underlies the unique neuropathology of MSA.

Increased Expression of ABCA8 in Multiple System Atrophy Brain is Associated with Changes in Pathogenic Proteins

Multiple system atrophy (MSA) is a fatal neurodegenerative disease of unknown aetiology characterised by the accumulation of insoluble α-synuclein (α-syn) aggregates in the cytoplasm of myelin-producing oligodendrocytes. Dysfunction of the lipid-rich myelin membrane may precede α-syn pathology in MSA pathogenesis. ATP-binding cassette transporter A8 (ABCA8) is a little-studied lipid transporter, which has recently been found to be highly expressed in oligodendrocyte-rich white matter regions of the human brain. ABCA8 expression promotes sphingomyelin production in oligodendrocytes in vitro, suggesting a role in myelin formation and maintenance. We hypothesise that aberrant ABCA8 expression in oligodendrocytes plays a role in the early pathogenesis of MSA by impacting myelin stability and regulation of α-syn and p25α. We measured the expression of ABCA8, α-syn and p25α in MSA brains in disease-affected grey matter (GM, putamen and cerebellum), disease-affected white matter (WM, under the motor cortex) and an unaffected brain region (visual cortex). We transfected human oligodendrocytes with ABCA8 and assessed its impact on α-syn and p25α expression. ABCA8 expression was significantly increased in the disease-affected GM and WM with no significant change in the unaffected brain region. α-syn and p25α expression were significantly increased in the disease-affected WM and GM respectively. Overexpression of ABCA8 in oligodendrocytes caused significant increases in both α-syn and p25α expression. These data suggest a direct relationship between the levels of ABCA8 and the ectopic expression of α-syn and increased expression of p25α. As these data reflect results found in MSA, we hypothesise that increased ABCA8 may precipitate MSA pathology.

Identification and Quantification of Oligodendrocyte Precursor Cells in Multiple System Atrophy, Progressive Supranuclear Palsy and Parkinson's Disease

Multiple system atrophy is a neurodegenerative disorder characterized pathologically by abnormal accumulations of α-synuclein in the cytoplasm of oligodendrocytes, which are termed glial cytoplasmic inclusions (GCIs). Oligodendrocytes are responsible for myelinating axons and providing neurotrophic support, but in MSA, myelin loss, axonal loss and gliosis are consistent features suggesting that GCIs play a central role in disease pathogenesis. Oligodendroglial, myelin and axonal degeneration are also features of multiple sclerosis (MS) in which recent studies have highlighted the robust remyelination capacity of the central nervous system (CNS). The cells responsible for remyelination are called oligodendroglial precursor cells (OPCs). In this study, we investigated the role of OPCs in the pathogenesis of MSA and progressive supranuclear palsy (PSP), a neurodegenerative disease in which neuropathological changes include oligodendroglial inclusions composed of microtubule-associated protein tau. Despite the lability of OPC-specific antigens, we successfully identified OPCs and demonstrated that tau and α-synuclein do not accumulate in OPCs. We also showed that the density of OPCs was increased in a white matter region of the MSA brain, which is also severely affected by GCIs and myelin degeneration. These findings raise the possibility that OPCs could be available to repair disease-associated damage in MSA, consistent with their biological function.

Quantitative Analysis of the Detergent-Insoluble Brain Proteome in Frontotemporal Lobar Degeneration Using SILAC Internal Standards

A hallmark of neurodegeneration is the aggregation of disease related proteins that are resistant to detergent extraction. In the major pathological subtype of frontotemporal lobar degeneration (FTLD), modified TAR-DNA binding protein 43 (TDP-43), including phosphorylated, ubiquitinated, and proteolytically cleaved forms, is enriched in detergent-insoluble fractions from post-mortem brain tissue. Additional proteins that accumulate in the detergent-insoluble FTLD brain proteome remain largely unknown. In this study, we used proteins from stable isotope-labeled (SILAC) human embryonic kidney 293 cells (HEK293) as internal standards for peptide quantitation across control and FTLD insoluble brain proteomes. Proteins were identified and quantified by liquid-chromatography coupled with tandem mass spectrometry (LC-MS/MS) and 21 proteins were determined to be enriched in FTLD using SILAC internal standards. In parallel, label-free quantification of only the unlabeled brain derived peptides by spectral counts (SC) and G-test analysis identified additional brain-specific proteins significantly enriched in disease. Several proteins determined to be enriched in FTLD using SILAC internal standards were not considered significant by G-test due to their low total number of SC. However, immunoblotting of FTLD and control samples confirmed enrichment of these proteins, highlighting the utility of SILAC internal standard to quantify low-abundance proteins in brain. Of these, the RNA binding protein PTB-associated splicing factor (PSF) was further characterized because of structural and functional similarities to TDP-43. Full-length PSF and shorter molecular weight fragments, likely resulting from proteolytic cleavage, were enriched in FTLD cases. Immunohistochemical analysis of PSF revealed predominately nuclear localization in control and FTLD brain tissue and was not associated with phosphorylated pathologic TDP-43 neuronal inclusions. However, in a subset of FTLD cases, PSF was aberrantly localized to the cytoplasm of oligodendrocytes. These data raise the possibility that PSF directed RNA processes in oligodendrocytes are altered in neurodegenerative disease.

A Missense Mutation in Myelin Oligodendrocyte Glycoprotein as a Cause of Familial Narcolepsy with Cataplexy

Narcolepsy is a rare sleep disorder characterized by excessive daytime sleepiness and cataplexy. Familial narcolepsy accounts for less than 10% of all narcolepsy cases. However, documented multiplex families are very rare and causative mutations have not been identified to date. To identify a causative mutation in familial narcolepsy, we performed linkage analysis in the largest ever reported family, which has 12 affected members, and sequenced coding regions of the genome (exome sequencing) of three affected members with narcolepsy and cataplexy. We successfully mapped a candidate locus on chromosomal region 6p22.1 (LOD score ¼ 3.85) by linkage analysis. Exome sequencing identified a missense mutation in the second exon of MOG within the linkage region. A c.398C>G mutation was present in all affected family members but absent in unaffected members and 775 unrelated control subjects. Transient expression of mutant myelin oligodendrocyte glycoprotein (MOG) in mouse oligodendrocytes showed abnormal subcellular localization, suggesting an altered function of the mutant MOG. MOG has recently been linked to various neuropsychiatric disorders and is considered as a key autoantigen in multiple sclerosis and in its animal model, experimental autoimmune encephalitis. Our finding of a pathogenic MOG mutation highlights a major role for myelin and oligodendrocytes in narcolepsy and further emphasizes glial involvement in neurodegeneration and neurobehavioral disorders. [corrected].

Oxidative damage in multiple sclerosis lesions

Multiple sclerosis is a chronic inflammatory disease of the central nervous system, associated with demyelination and neurodegeneration. The mechanisms of tissue injury are currently poorly understood, but recent data suggest that mitochondrial injury may play an important role in this process. Since mitochondrial injury can be triggered by reactive oxygen and nitric oxide species, we analysed by immunocytochemistry the presence and cellular location of oxidized lipids and oxidized DNA in lesions and in normal-appearing white matter of 30 patients with multiple sclerosis and 24 control patients without neurological disease or brain lesions. As reported before in biochemical studies, oxidized lipids and DNA were highly enriched in active multiple sclerosis plaques, predominantly in areas that are defined as initial or ‘prephagocytic’ lesions. Oxidized DNA was mainly seen in oligodendrocyte nuclei, which in part showed signs of apoptosis. In addition, a small number of reactive astrocytes revealed nuclear expression of 8-hydroxy-d-guanosine. Similarly, lipid peroxidation-derived structures (malondialdehyde and oxidized phospholipid epitopes) were seen in the cytoplasm of oligodendrocytes and some astrocytes. In addition, oxidized phospholipids were massively accumulated in a fraction of axonal spheroids with disturbed fast axonal transport as well as in neurons within grey matter lesions. Neurons stained for oxidized phospholipids frequently revealed signs of degeneration with fragmentation of their dendritic processes. The extent of lipid and DNA oxidation correlated significantly with inflammation, determined by the number of CD3 positive T cells and human leucocyte antigen-D expressing macrophages and microglia in the lesions. Our data suggest profound oxidative injury of oligodendrocytes and neurons to be associated with active demyelination and axonal or neuronal injury in multiple sclerosis.