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1.  Hippocampal demyelination and memory dysfunction are associated with increased levels of the neuronal microRNA miR-124 and reduced AMPA receptors 
Annals of neurology  2013;73(5):637-645.
Hippocampal demyelination, a common feature of postmortem multiple sclerosis (MS) brains, reduces neuronal gene expression and is a likely contributor to the memory impairment that is found in greater than 40% of individuals with (MS). How demyelination alters neuronal gene expression is unknown.
To explore if loss of hippocampal myelin alters expression of neuronal microRNAs (miRNA), we compared miRNA profiles from myelinated and demyelinated hippocampi from postmortem MS brains and performed validation studies.
A network-based interaction analysis depicts a correlation between increased neuronal miRNAs and decreased neuronal genes identified in our previous study. The neuronal miRNA miR-124, was increased in demyelinated MS hippocampi and targets mRNAs encoding 26 neuronal proteins that were decreased in demyelinated hippocampus, including the ionotrophic glutamate receptors, AMPA 2 and AMPA3. Hippocampal demyelination in mice also increased miR-124, reduced expression of AMPA receptors and decreased memory performance in water maze tests. Remyelination of the mouse hippocampus reversed these changes.
We establish here that myelin alters neuronal gene expression and function by modulating the levels of the neuronal miRNA miR-124. Inhibition of miR-124 in hippocampal neurons may provide a therapeutic approach to improve memory performance in MS patients.
PMCID: PMC3679350  PMID: 23595422
Multiple sclerosis; myelin; microRNA
2.  Mechanisms of Primary Axonal Damage in a Viral Model of Multiple Sclerosis 
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Recent studies have demonstrated that significant axonal injury also occurs in MS patients and correlates with neurological dysfunction, but it is not known whether this neuronal damage is a primary disease process, or occurs only secondary to demyelination. In the current studies, neurotropic strains of mouse hepatitis virus (MHV) that induce meningitis, encephalitis, and demyelination in the CNS, an animal model of MS, were used to evaluate mechanisms of axonal injury. The pathogenic properties of genetically engineered isogenic spike protein recombinant demyelinating and non-demyelinating strains of MHV were compared. Studies demonstrate that a demyelinating strain of MHV causes concomitant axonal loss and macrophage-mediated demyelination. The mechanism of axonal loss and demyelination in MHV infection is dependent on successful transport of virus from gray matter to white matter using the MHV host attachment spike glycoprotein. Our data show that axonal loss and demyelination can be independent direct viral cytopathic events, and suggest similar direct axonal damage may occur in MS. These results have important implications for the design of neuroprotective strategies for CNS demyelinating disease, and our model identifies the spike protein as a therapeutic target to prevent axonal transport of neurotropic viruses.
PMCID: PMC2747667  PMID: 19692601
Multiple Sclerosis; Axonal loss; demyelination; axonal transport; macrophages; neurotropic viruses
3.  Neurogenesis in the chronic lesions of multiple sclerosis 
Brain  2008;131(9):2366-2375.
Subcortical white matter in the adult human brain contains a population of interneurons that helps regulate cerebral blood flow. We investigated the fate of these neurons following subcortical white matter demyelination. Immunohistochemistry was used to examine neurons in normal-appearing subcortical white matter and seven acute and 59 chronic demyelinated lesions in brains from nine patients with multiple sclerosis and four controls. Seven acute and 44 of 59 chronic multiple sclerosis lesions had marked neuronal loss. Compared to surrounding normal-appearing white matter, the remaining 15 chronic multiple sclerosis lesions contained a 72% increase in mature interneuron density, increased synaptic densities and cells with phenotypic characteristics of immature neurons. Lesion areas with increased neuron densities contained a morphologically distinct population of activated microglia. Subventricular zones contiguous with demyelinated lesions also contained an increase in cells with phenotypes of neuronal precursors. These results support neurogenesis in a subpopulation of demyelinated subcortical white matter lesions in multiple sclerosis brains.
PMCID: PMC2525445  PMID: 18669500
multiple sclerosis; white matter neurons; neurogenesis
4.  Perivenous demyelination: association with clinically defined acute disseminated encephalomyelitis and comparison with pathologically confirmed multiple sclerosis 
Brain  2010;133(2):333-348.
Distinction between acute disseminated encephalomyelitis and acute multiple sclerosis is often clinically difficult. Perivenous demyelination is the pathological hallmark of acute disseminated encephalomyelitis, whereas confluent demyelination is the hallmark of acute multiple sclerosis. We investigated whether perivenous demyelination versus confluent demyelination distinguishes acute disseminated encephalomyelitis from multiple sclerosis. Patients with perivenous demyelination (n = 13; median age 43 years, range 5–67) on brain biopsy and/or autopsy, ascertained retrospectively, were compared with a cohort with confluent demyelination only (n = 91; 84% multiple sclerosis, 16% isolated syndrome at follow-up; median age 39 years, range 10–69). Clinical presentation, course and the International Paediatric Multiple Sclerosis Study Group clinical criteria for acute disseminated encephalomyelitis were assessed in both cohorts. Among the perivenous demyelination cohort, 10 patients had only perivenous demyelination and three also had confluent demyelination. All but one patient with perivenous demyelination only had a monophasic course, whereas two of three with both types had a relapsing course. The perivenous demyelination cohort was more likely than the confluent demyelination cohort to present with encephalopathy (P < 0.001), depressed level of consciousness (P < 0.001), headache (P < 0.001), meningismus (P = 0.04), cerebrospinal fluid pleocytosis (P = 0.04) or multifocal enhancing magnetic resonance imaging lesions (P < 0.001). A distinct pattern of cortical microglial activation and aggregation without associated cortical demyelination was found among six perivenous demyelination patients, all of whom had encephalopathy and four of whom had depressed level of consciousness. This pattern of cortical pathology was not observed in the confluent demyelination cohort, even in one patient with depressed level of consciousness. Clinical criteria were 80% sensitive and 91% specific for pathologically defined acute disseminated encephalomyelitis (perivenous demyelination), but misdiagnosed acute disseminated encephalomyelitis among 9% of patients with confluent demyelination and multiple sclerosis diagnosis at last follow-up. Perivenous demyelination is associated with meningoencephalopathic presentations and a monophasic course. Depressed level of consciousness is a more specific clinical criterion for pathologically confirmed acute disseminated encephalomyelitis than encephalopathy, which over-diagnosed acute disseminated encephalomyelitis among multiple sclerosis patients. A distinct pattern of cortical microglial activation without cortical demyelination may be the pathological correlate of depressed level of consciousness in acute disseminated encephalomyelitis. Although pathological evidence of perivenous demyelination may be superior to clinical criteria for diagnosing acute disseminated encephalomyelitis, the co-occurrence of perivenous and confluent demyelination in some individuals suggests pathogenic overlap between acute disseminated encephalomyelitis and multiple sclerosis and misclassification even with biopsy.
PMCID: PMC2822631  PMID: 20129932
multiple sclerosis; magnetic resonance imaging; neuropathology; immune-mediated demyelination; demyelinating disease
5.  Possible Effects of Synaptic Imbalances on Oligodendrocyte–Axonic Interactions in Schizophrenia: A Hypothetical Model 
A model of glial–neuronal interactions is proposed that could be explanatory for the demyelination identified in brains with schizophrenia. It is based on two hypotheses: (1) that glia–neuron systems are functionally viable and important for normal brain function, and (2) that disruption of this postulated function disturbs the glial categorization function, as shown by formal analysis. According to this model, in schizophrenia receptors on astrocytes in glial–neuronal synaptic units are not functional, loosing their modulatory influence on synaptic neurotransmission. Hence, an unconstrained neurotransmission flux occurs that hyperactivates the axon and floods the cognate receptors of neurotransmitters on oligodendrocytes. The excess of neurotransmitters may have a toxic effect on oligodendrocytes and myelin, causing demyelination. In parallel, an increasing impairment of axons may disconnect neuronal networks. It is formally shown how oligodendrocytes normally categorize axonic information processing via their processes. Demyelination decomposes the oligodendrocyte–axonic system making it incapable to generate categories of information. This incoherence may be responsible for symptoms of disorganization in schizophrenia, such as thought disorder, inappropriate affect and incommunicable motor behavior. In parallel, the loss of oligodendrocytes affects gap junctions in the panglial syncytium, presumably responsible for memory impairment in schizophrenia.
PMCID: PMC3102422  PMID: 21647404
glial–neuronal interactions; demyelination; schizophrenia; synaptic imbalance
Annals of neurology  2012;72(6):918-926.
Generation and differentiation of new oligodendrocytes in demyelinated white matter is the best described repair process in the adult human brain. However, remyelinating capacity falters with age in patients with multiple sclerosis. (MS). Since demyelination of cerebral cortex is extensive in brains from MS patients, we investigated the capacity of cortical lesions to remyelinate and directly compared the extent of remyelination in lesions that involve cerebral cortex and adjacent subcortical white matter.
Postmortem brain tissue from 22 patients with MS (age 27 to 77 years) and 6 subjects without brain disease were analyzed. Regions of cerebral cortex with reduced myelin were examined for remyelination, oligodendrocyte progenitor cells, reactive astrocytes, and molecules that inhibit remyelination.
“New” oligodendrocytes that were actively forming myelin sheaths were identified in 30/42 remyelinated subpial cortical lesions, including lesions from three patients in their 70's. Oligodendrocyte progenitor cells were not decreased in demyelinated or remyelinated cortices when compared to adjacent normal-appearing cortex or controls. In demyelinated lesions involving cortex and adjacent white matter, the cortex showed greater remyelination, more actively remyelinating oligodendrocytes and fewer reactive astrocytes. Astrocytes in the white-matter, but not in cortical portions of these lesions, significantly up-regulate CD44, hyaluronan, and versican, molecules that form complexes that inhibit oligodendrocyte maturation and remyelination.
Endogenous remyelination of the cerebral cortex occurs in individuals with MS regardless of disease duration or chronological age of the patient. Cortical remyelination should be considered as a primary outcome measure in future clinical trials testing remyelination therapies.
PMCID: PMC3535551  PMID: 23076662
multiple sclerosis; remyelination
7.  Deep Gray Matter Demyelination Detected by Magnetization Transfer Ratio in the Cuprizone Model 
PLoS ONE  2013;8(12):e84162.
In multiple sclerosis (MS), the correlation between lesion load on conventional magnetic resonance imaging (MRI) and clinical disability is weak. This clinico-radiological paradox might partly be due to the low sensitivity of conventional MRI to detect gray matter demyelination. Magnetization transfer ratio (MTR) has previously been shown to detect white matter demyelination in mice. In this study, we investigated whether MTR can detect gray matter demyelination in cuprizone exposed mice. A total of 54 female C57BL/6 mice were split into one control group () and eight cuprizone exposed groups (). The mice were exposed to (w/w) cuprizone for up to six weeks. MTR images were obtained at a 7 Tesla Bruker MR-scanner before cuprizone exposure, weekly for six weeks during cuprizone exposure, and once two weeks after termination of cuprizone exposure. Immunohistochemistry staining for myelin (anti-Proteolopid Protein) and oligodendrocytes (anti-Neurite Outgrowth Inhibitor Protein A) was obtained after each weekly scanning. Rates of MTR change and correlations between MTR values and histological findings were calculated in five brain regions. In the corpus callosum and the deep gray matter a significant rate of MTR value decrease was found, per week () and per week () respectively. The MTR values correlated to myelin loss as evaluated by immunohistochemistry (Corpus callosum: . Deep gray matter: ), but did not correlate to oligodendrocyte density. Significant results were not found in the cerebellum, the olfactory bulb or the cerebral cortex. This study shows that MTR can be used to detect demyelination in the deep gray matter, which is of particular interest for imaging of patients with MS, as deep gray matter demyelination is common in MS, and is not easily detected on conventional clinical MRI.
PMCID: PMC3875491  PMID: 24386344
8.  Docosahexaenoic acid promotes hippocampal neuronal development and synaptic function 
Journal of neurochemistry  2009;111(2):510-521.
Docosahexaenoic acid (DHA, 22:6n-3), the major polyunsaturated fatty acid accumulated in the brain during development, has been implicated in learning and memory, but underlying cellular mechanisms are not clearly understood. Here, we demonstrate that DHA significantly affects hippocampal neuronal development and synaptic function in developing hippocampi. In embryonic neuronal cultures, DHA supplementation uniquely promoted neurite growth, synapsin puncta formation and synaptic protein expression, particularly synapsins and glutamate receptors. In DHA-supplemented neurons, spontaneous synaptic activity was significantly increased, mostly because of enhanced glutamatergic synaptic activity. Conversely, hippocampal neurons from DHA-depleted fetuses showed inhibited neurite growth and synaptogenesis. Furthermore, n-3 fatty acid deprivation during development resulted in marked decreases of synapsins and glutamate receptor subunits in the hippocampi of 18-day-old pups with concomitant impairment of long-term potentiation, a cellular mechanism underlying learning and memory. While levels of synapsins and NMDA receptor subunit NR2A were decreased in most hippocampal regions, NR2A expression was particularly reduced in CA3, suggesting possible role of DHA in CA3-NMDA receptor-dependent learning and memory processes. The DHA-induced neurite growth, synaptogenesis, synapsin, and glutamate receptor expression, and glutamatergic synaptic function may represent important cellular aspects supporting the hippocampus-related cognitive function improved by DHA.
PMCID: PMC2773444  PMID: 19682204
docosahexaenoic acid; hippocampal development; long-term potentiation; neurite growth; synaptic function; synaptogenesis
9.  Increased mitochondrial content in remyelinated axons: implications for multiple sclerosis 
Brain  2011;134(7):1901-1913.
Mitochondrial content within axons increases following demyelination in the central nervous system, presumably as a response to the changes in energy needs of axons imposed by redistribution of sodium channels. Myelin sheaths can be restored in demyelinated axons and remyelination in some multiple sclerosis lesions is extensive, while in others it is incomplete or absent. The effects of remyelination on axonal mitochondrial content in multiple sclerosis, particularly whether remyelination completely reverses the mitochondrial changes that follow demyelination, are currently unknown. In this study, we analysed axonal mitochondria within demyelinated, remyelinated and myelinated axons in post-mortem tissue from patients with multiple sclerosis and controls, as well as in experimental models of demyelination and remyelination, in vivo and in vitro. Immunofluorescent labelling of mitochondria (porin, a voltage-dependent anion channel expressed on all mitochondria) and axons (neurofilament), and ultrastructural imaging showed that in both multiple sclerosis and experimental demyelination, mitochondrial content within remyelinated axons was significantly less than in acutely and chronically demyelinated axons but more numerous than in myelinated axons. The greater mitochondrial content within remyelinated, compared with myelinated, axons was due to an increase in density of porin elements whereas increase in size accounted for the change observed in demyelinated axons. The increase in mitochondrial content in remyelinated axons was associated with an increase in mitochondrial respiratory chain complex IV activity. In vitro studies showed a significant increase in the number of stationary mitochondria in remyelinated compared with myelinated and demyelinated axons. The number of mobile mitochondria in remyelinated axons did not significantly differ from myelinated axons, although significantly greater than in demyelinated axons. Our neuropathological data and findings in experimental demyelination and remyelination in vivo and in vitro are consistent with a partial amelioration of the supposed increase in energy demand of demyelinated axons by remyelination.
PMCID: PMC3122369  PMID: 21705418
multiple sclerosis; axon; demyelination; mitochondria; remyelination
10.  A Mutation in the Gene Encoding Mitochondrial Mg2+ Channel MRS2 Results in Demyelination in the Rat 
PLoS Genetics  2011;7(1):e1001262.
The rat demyelination (dmy) mutation serves as a unique model system to investigate the maintenance of myelin, because it provokes severe myelin breakdown in the central nervous system (CNS) after normal postnatal completion of myelination. Here, we report the molecular characterization of this mutation and discuss the possible pathomechanisms underlying demyelination. By positional cloning, we found that a G-to-A transition, 177 bp downstream of exon 3 of the Mrs2 (MRS2 magnesium homeostasis factor (Saccharomyces cerevisiae)) gene, generated a novel splice acceptor site which resulted in functional inactivation of the mutant allele. Transgenic rescue with wild-type Mrs2-cDNA validated our findings. Mrs2 encodes an essential component of the major Mg2+ influx system in mitochondria of yeast as well as human cells. We showed that the dmy/dmy rats have major mitochondrial deficits with a markedly elevated lactic acid concentration in the cerebrospinal fluid, a 60% reduction in ATP, and increased numbers of mitochondria in the swollen cytoplasm of oligodendrocytes. MRS2-GFP recombinant BAC transgenic rats showed that MRS2 was dominantly expressed in neurons rather than oligodendrocytes and was ultrastructurally observed in the inner membrane of mitochondria. Our observations led to the conclusion that dmy/dmy rats suffer from a mitochondrial disease and that the maintenance of myelin has a different mechanism from its initial production. They also established that Mg2+ homeostasis in CNS mitochondria is essential for the maintenance of myelin.
Author Summary
The myelin sheath that surrounds the axon of a neuron acts as a biological insulator. Its major function is to increase the speed at which impulses propagate along myelinated fibers in the central nervous system, as well as the peripheral nervous system. Alterations or damage affecting this structure (demyelination) result in the disruption of signals between the brain and other parts of the body. In the rat, mutations producing demyelination have been frequently identified and characterized and have contributed to a better understanding of the genetics of myelin development, physiology, and pathology. This paper reports the molecular characterization of a recessive allele responsible for the progressive disruption of myelin that was initially observed in mutant rats, previously named demyelination (dmy). This mutation generates an additional splicing acceptor site in an intron of the mitochondrial Mg2+ transporter gene (Mrs2), resulting in the insertion of a 83-bp genomic DNA segment into the Mrs2 transcript and complete functional inactivation of the mutant allele. We firstly defined the biological function of MRS2 in mammals and demonstrated the crucial and unexpected role of MRS2 in myelin physiology. Our findings might be helpful in the development of new therapeutic strategies for demyelinating syndromes.
PMCID: PMC3017111  PMID: 21253565
11.  Hippocampal CA1 atrophy and synaptic loss during experimental autoimmune encephalomyelitis, EAE 
Over half of multiple sclerosis (MS) patients experience cognitive deficits, including learning and memory dysfunction, and the mechanisms underlying these deficits remain poorly understood. Neuronal injury and synaptic loss have been shown to occur within the hippocampus in other neurodegenerative disease models, and these pathologies have been correlated with cognitive impairment. Whether hippocampal abnormalities occur in MS models is unknown. Using experimental autoimmune encephalomyelitis (EAE), we evaluated hippocampal neurodegeneration and inflammation during disease. Hippocampal pathology began early in EAE disease course, and included decreases in CA1 pyramidal layer volume, loss of inhibitory interneurons and increased cell death of neurons and glia. It is interesting to note that these effects occurred in the presence of chronic microglial activation, with a relative paucity of infiltrating blood-borne immune cells. Widespread diffuse demyelination occurred in the hippocampus, but there was no significant decrease in axonal density. Furthermore, there was a significant reduction in pre-synaptic puncta and synaptic protein expression within the hippocampus, as well as impaired performance on a hippocampal-dependent spatial learning task. Our results demonstrate that neurodegenerative changes occur in the hippocampus during autoimmune-mediated demyelinating disease. This work establishes a preclinical model for assessing treatments targeted toward preventing hippocampal neuropathology and dysfunction in MS.
PMCID: PMC3033772  PMID: 20157291
EAE; hippocampus; MS; pathology
12.  Mesenchymal stromal-cell transplants induce oligodendrocyte progenitor migration and remyelination in a chronic demyelination model 
Cell Death & Disease  2013;4(8):e779-.
Demyelinating disorders such as leukodystrophies and multiple sclerosis are neurodegenerative diseases characterized by the progressive loss of myelin that may lead toward a chronic demyelination of the brain's white matter, impairing normal axonal conduction velocity and ultimately causing neurodegeneration. Current treatments modifying the pathological mechanisms are capable of ameliorating the disease; however, frequently, these therapies are not sufficient to repress the progressive demyelination into a chronic condition and permanent loss of function. To this end, we analyzed the effect that bone marrow-derived mesenchymal stromal cell (BM-MSC) grafts exert in a chronically demyelinated mouse brain. As a result, oligodendrocyte progenitors were recruited surrounding the graft due to the expression of various trophic signals by the grafted MSCs. Although there was no significant reaction in the non-grafted side, in the grafted regions oligodendrocyte progenitors were detected. These progenitors were derived from the nearby tissue as well as from the neurogenic niches, including the subependymal zone and dentate gyrus. Once near the graft site, the cells matured to myelinating oligodendrocytes. Finally, electrophysiological studies demonstrated that axonal conduction velocity was significantly increased in the grafted side of the fimbria. In conclusion, we demonstrate here that in chronic demyelinated white matter, BM-MSC transplantation activates oligodendrocyte progenitors and induces remyelination in the tissue surrounding the stem cell graft.
PMCID: PMC3763464  PMID: 23990019
mesenchymal stromal cells; demyelinating mouse model; trophic release; oligodendrocyte activation; remyelination
13.  The Role of Myelin in Theiler's Virus Persistence in the Central Nervous System 
PLoS Pathogens  2007;3(2):e23.
Theiler's virus, a picornavirus, persists for life in the central nervous system of mouse and causes a demyelinating disease that is a model for multiple sclerosis. The virus infects neurons first but persists in white matter glial cells, mainly oligodendrocytes and macrophages. The mechanism, by which the virus traffics from neurons to glial cells, and the respective roles of oligodendrocytes and macrophages in persistence are poorly understood. We took advantage of our previous finding that the shiverer mouse, a mutant with a deletion in the myelin basic protein gene (Mbp), is resistant to persistent infection to examine the role of myelin in persistence. Using immune chimeras, we show that resistance is not mediated by immune responses or by an efficient recruitment of inflammatory cells into the central nervous system. With both in vivo and in vitro experiments, we show that the mutation does not impair the permissiveness of neurons, oligodendrocytes, and macrophages to the virus. We demonstrate that viral antigens are present in cytoplasmic channels of myelin during persistent infection of wild-type mice. Using the optic nerve as a model, we show that the virus traffics from the axons of retinal ganglion cells to the cytoplasmic channels of myelin, and that this traffic is impaired by the shiverer mutation. These results uncover an unsuspected axon to myelin traffic of Theiler's virus and the essential role played by the infection of myelin/oligodendrocyte in persistence.
Author Summary
Theiler's virus persists in the central nervous system of mice and causes a chronic disease that resembles multiple sclerosis, a common demyelinating disease of humans. The virus infects neurons for one to two weeks, but later on it persists in the white matter, in oligodendrocytes and also in macrophages. Oligodendrocytes are the myelin-making cells of the central nervous system. Strikingly, in mice with a genetic defect of myelin, the virus infects neurons normally but is unable to persist. Understanding the reason for the lack of persistence in this mutant mouse should pinpoint an essential step in the complex process resulting in persistence. In this article, we show that resistance to persistent infection is not mediated by the immune system and is not due to inefficient viral replication in oligodendrocytes or macrophages. Instead, we show that virus transported in axons traffics into the myelin, and that this traffic is interrupted by the myelin mutation. This unsuspected axon to myelin traffic of Theiler's virus is necessary for viral persistence. Our results warrant looking for a similar phenomenon in other persistent infections of the nervous system, including in humans.
PMCID: PMC1797621  PMID: 17305428
14.  Antibody to Aquaporin 4 in the Diagnosis of Neuromyelitis Optica 
PLoS Medicine  2007;4(4):e133.
Neuromyelitis optica (NMO) is a demyelinating disease of the central nervous system (CNS) of putative autoimmune aetiology. Early discrimination between multiple sclerosis (MS) and NMO is important, as optimum treatment for both diseases may differ considerably. Recently, using indirect immunofluorescence analysis, a new serum autoantibody (NMO-IgG) has been detected in NMO patients. The binding sites of this autoantibody were reported to colocalize with aquaporin 4 (AQP4) water channels. Thus we hypothesized that AQP4 antibodies in fact characterize NMO patients.
Methods and Findings
Based on these observations we cloned human water channel AQP4, expressed the protein in a eukaryotic transcription/translation system, and employed the recombinant AQP4 to establish a new radioimmunoprecipitation assay (RIPA). Indeed, application of this RIPA showed that antibodies against AQP4 exist in the majority of patients with NMO (n = 37; 21 positive) as well as in patients with isolated longitudinally extensive transverse myelitis (n = 6; six positive), corresponding to a sensitivity of 62.8% and a specificity of 98.3%. By contrast, AQP4 antibodies were virtually absent in 291 other participants, which included patients with MS (n = 144; four positive), patients with other inflammatory and noninflammatory neurological diseases (n = 73; one positive), patients with systemic autoimmune diseases (n = 45; 0 positive), and healthy participants (n = 29; 0 positive).
In the largest series reported so far to our knowledge, we quantified AQP4 antibodies in patients with NMO versus various other diseases, and showed that the aquaporin 4 water channel is a target antigen in a majority of patients with NMO. The newly developed assay represents a highly specific, observer-independent, and easily reproducible detection method facilitating clinically relevant discrimination between NMO, MS, and other inflammatory diseases.
A newly developed method to detect antibodies to the aquaporin 4 water channel can help discriminate between neuromyelitis optica, multiple sclerosis, and other inflammatory diseases.
Editors' Summary
Neuromyelitis optica (NMO or Devic syndrome) is a rare disease in which the immune system destroys the myelin (fatty material that insulates nerve fibers so that the body and the brain can communicate using electrical messages) in the optic nerve and spinal cord. Myelin destruction (demyelination) in these parts of the central nervous system (CNS) causes pain and swelling (inflammation) of the optic nerve (optic neuritis) and spinal cord (myelitis). The resultant disruption of communication along these nerves means that patients with NMO experience temporary or permanent blindness in one or both eyes that is preceded or followed by limb weakness or paralysis and loss of bladder and bowel control. These two sets of symptoms can occur many months apart and may happen once during a person's lifetime or recur at intervals. There is no cure for NMO, but corticosteroids or plasmapheresis reduce inflammation during acute attacks and, because NMO is an autoimmune disease (one in which the immune system attacks the body's own tissues instead of foreign organisms), long-term immunosuppression may prevent further attacks.
Why Was This Study Done?
There are many inflammatory/demyelinating diseases of the CNS with clinical symptoms similar to those of NMO. It is particularly hard to distinguish between NMO and multiple sclerosis, an autoimmune disease that involves widespread demyelination. Neurologists need to make a correct diagnosis before starting any treatment and usually use clinical examination and magnetic resonance imaging (to detect sites of inflammation) to help them in this task. Recently, however, a biomarker for NMO was identified. Many patients with NMO make autoantibodies (proteins that recognize a component of a person's own tissues) called NMO-IgGs. These recognize aquaporin 4 (AQP4), a protein that allows water to move through cell membranes. It is not known how often patients with NMO or other demyelinating diseases make antibodies to AQP4, so it is unclear whether testing for these antibodies would help in the diagnosis of NMO. In this study, the researchers have developed a new assay for antibodies to AQP4 and then quantified the antibodies in patients with NMO and other demyelinating diseases.
What Did the Researchers Do and Find?
The researchers made radioactively labeled AQP4 in a test tube, then incubated samples of this with serum (the liquid portion of blood), added small beads coated with protein A (a bacterial protein that binds to antibodies) and allowed the beads to settle. The amount of radioactivity attached to the beads indicates the amount of antibody to AQP4 in the original serum. The researchers used this radioimmunoprecipitation assay to measure antibodies to AQP4 in sera from 37 patients with NMO and from six with another neurological condition, longitudinally extensive transverse myelitis (LETM), which is characterized by large demyelinated lesions across the width of the spinal cord but no optic neuritis; these patients often develop NMO. They also measured antibodies to AQP4 in the sera of nearly 300 other people including patients with multiple sclerosis, other neurological conditions, various autoimmune diseases, and healthy individuals. Nearly two-thirds of the patients with NMO and all those with LETM made antibodies against AQP4; very few of the other study participants made these antibodies. In particular, only four of the 144 patients with multiple sclerosis made AQP4 antibodies.
What Do These Findings Mean?
These findings indicate that testing for antibodies to AQP4 could help neurologists distinguish between NMO and multiple sclerosis and between NMO and other demyelinating diseases of the CNS. In addition, the new radioimmunoprecipitation assay provides a standardized, high-throughput way to quantitatively test for these antibodies, whereas the indirect immune fluorescence assay for measurement of unspecific NMO-IgG is observer-dependent and nonquantitative. Although these findings need to be confirmed in more patients and the assay's reliability demonstrated in different settings, the measurement of antibodies to AQP4 by radioimmunoprecipitation may become a standard part of the differential diagnosis of NMO. Additional research will determine whether AQP4 is the only protein targeted by autoantibodies in NMO and whether this targeting is a critical part of the disease process.
Additional Information.
Please access these Web sites via the online version of this summary at
US National Institute of Neurological Disorders and Stroke has information for patients who have neuromyelitis optica, transverse myelitis, and multiple sclerosis
The Transverse Myelitis Association offers information and useful links for patients and their carers about transverse myelitis and neuromyelitis optica (in several languages, including English and Spanish)
Mayo Clinic information for patients on Devic's syndrome
Medline Plus encyclopedia pages discuss autoimmune disorders (in English and Spanish)
A brief overview of aquaporins is available from the University of Miami
The American MS Society has information on MS
PMCID: PMC1852124  PMID: 17439296
15.  Antineuroinflammatory and neurotrophic effects of CNTF and C16 peptide in an acute experimental autoimmune encephalomyelitis rat model 
Experimentalallergic encephalomyelitis (EAE) is an animal model for inflammatory demyelinating autoimmune disease, i.e., multiple sclerosis (MS). In the present study, we investigated the antineuroinflammatory/neuroprotective effects of C16, an ανβ3 integrin-binding peptide, and recombinant rat ciliary neurotrophic factor (CNTF), a cytokine that was originally identified as a survival factor for neurons, in an acute rodent EAE model. In this model, C16 peptide was injected intravenously every day for 2 weeks, and CNTF was delivered into the cerebral ventricles with Alzet miniosmotic pumps. Disease severity was assessed weekly using a scale ranging from 0 to 5. Multiple histological and molecular biological assays were employed to assess inflammation, axonal loss, neuronal apoptosis, white matter demyelination, and gliosis in the brain and spinal cord of different groups. Our results showed that the EAE induced rats revealed a significant increase in inflammatory cells infiltration, while C16 treatment could inhibit the infiltration of leukocytes and macrophages down to 2/3–1/3 of vehicle treated EAE control (P < 0.05). The delayed onset of disease, reduced clinical score (P < 0.01) in peak stage and more rapid recovery also were achieved in C16 treated group. Besides impairing inflammation, CNTF treatment also exerted direct neuroprotective effects, decreasing demyelination and axon loss score (P < 0.05 versus vehicle treated EAE control), and reducing the neuronal death from 40 to 50% to 10 to 20% (P < 0.05). Both treatments suppressed the expression of cytokine tumor necrosis factor-α and interferon-γ when compared with the vehicle control (P < 0.05). Combined treatment with C16 and CNTF produced more obvious functional recovery and neuroprotective effects than individually treatment (P < 0.05). These results suggested that combination treatment with C16 and CNTF, which target different neuroprotection pathways, may be an effective therapeutic alternative to traditional therapy.
PMCID: PMC3874474  PMID: 24416000
multiple sclerosis; anti-inflammatory; demyelination; neuroprotective effects
16.  Theiler's Virus Infection: a Model for Multiple Sclerosis 
Clinical Microbiology Reviews  2004;17(1):174-207.
Both genetic background and environmental factors, very probably viruses, appear to play a role in the etiology of multiple sclerosis (MS). Lessons from viral experimental models suggest that many different viruses may trigger inflammatory demyelinating diseases resembling MS. Theiler's virus, a picornavirus, induces in susceptible strains of mice early acute disease resembling encephalomyelitis followed by late chronic demyelinating disease, which is one of the best, if not the best, animal model for MS. During early acute disease the virus replicates in gray matter of the central nervous system but is eliminated to very low titers 2 weeks postinfection. Late chronic demyelinating disease becomes clinically apparent approximately 2 weeks later and is characterized by extensive demyelinating lesions and mononuclear cell infiltrates, progressive spinal cord atrophy, and axonal loss. Myelin damage is immunologically mediated, but it is not clear whether it is due to molecular mimicry or epitope spreading. Cytokines, nitric oxide/reactive nitrogen species, and costimulatory molecules are involved in the pathogenesis of both diseases. Close similarities between Theiler's virus-induced demyelinating disease in mice and MS in humans, include the following: major histocompatibility complex-dependent susceptibility; substantial similarities in neuropathology, including axonal damage and remyelination; and paucity of T-cell apoptosis in demyelinating disease. Both diseases are immunologically mediated. These common features emphasize the close similarities of Theiler's virus-induced demyelinating disease in mice and MS in humans.
PMCID: PMC321460  PMID: 14726460
17.  Demyelinating and Nondemyelinating Strains of Mouse Hepatitis Virus Differ in Their Neural Cell Tropism▿  
Journal of Virology  2008;82(11):5519-5526.
Some strains of mouse hepatitis virus (MHV) can induce chronic inflammatory demyelination in mice that mimics certain pathological features of multiple sclerosis. We have examined neural cell tropism of demyelinating and nondemyelinating strains of MHV in order to determine whether central nervous system (CNS) cell tropism plays a role in demyelination. Previous studies demonstrated that recombinant MHV strains, isogenic other than for the spike gene, differ in the extent of neurovirulence and the ability to induce demyelination. Here we demonstrate that these strains also differ in their abilities to infect a particular cell type(s) in the brain. Furthermore, there is a correlation between the differential localization of viral antigen in spinal cord gray matter and that in white matter during acute infection and the ability to induce demyelination later on. Viral antigen from demyelinating strains is detected initially in both gray and white matter, with subsequent localization to white matter of the spinal cord, whereas viral antigen localization of nondemyelinating strains is restricted mainly to gray matter. This observation suggests that the localization of viral antigen to white matter during the acute stage of infection is essential for the induction of chronic demyelination. Overall, these observations suggest that isogenic demyelinating and nondemyelinating strains of MHV, differing in the spike protein expressed, infect neurons and glial cells in different proportions and that differential tropism to a particular CNS cell type may play a significant role in mediating the onset and mechanisms of demyelination.
PMCID: PMC2395180  PMID: 18385249
18.  A 17 year-old girl with a demyelinating disease requiring mechanical ventilation: a case report 
BMC Research Notes  2013;6:22.
Demyelinating diseases cause destruction of the myelin sheath, while axons are relatively spared. Pathologically, demyelination can be the result of an inflammatory process, viral infection, acquired metabolic derangement and ischemic insult. Three diseases that can cause inflammatory demyelination of the CNS are: Multiple sclerosis (MS), Acute disseminated encephalomyelitis (ADEM) and Acute hemorrhagic leucoencephalitis. Differentiation is not always easy and there is considerable overlaping. Data about adults with acute demyelination requiring ICU admission is limited.
Case presentation
A 17 year old Greek female was hospitalised in the ICU because of acute respiratory failure requiring mechanical ventilation. She had a history of febrile disease one month before, acute onset of paraplegia, diplopia, progressive arm weakness and dyspnea. Her consciousness was not impaired. A demyelinating central nervous system (CNS) disease, possibly post infectious encephalomyelitis (ADEM) was the underlying condition. The MRI of the brain disclosed diffused expanded cerebral lesions involving the optic nerve, basal ganglia cerebellum, pons and medulla oblongata. There was also extended involvement of the cervical and thoracic part of the spinal cord. CSF leukocyte count was elevated with lymphocyte predominance. The patient required mechanical ventilation for two months. Then she was transferred to a rehabilitation centre. Three years later she remains paraplegic. Since then she has not suffered any other demyelination attack.
Demyelinating diseases can cause acute respiratory failure when the spinal cord is affected. Severe forms of these diseases, making necessary ICU admission, is less frequently reported. Intensivists should be aware of the features of these rare diseases.
PMCID: PMC3579686  PMID: 23331922
19.  K+ Channel Alterations in the Progression of Experimental Autoimmune Encephalomyelitis 
Neurobiology of Disease  2012;47(2):280-293.
Voltage-gated K+ (Kv) channels play critical roles not only in regulating synaptic transmission and intrinsic excitability of neurons, but also in controlling the function and proliferation of other cells in the central nervous system (CNS). The non-specific Kv channel blocker, 4-AminoPyridine (4-AP) (Dalfampridine, Ampyra®), is currently used to treat multiple sclerosis (MS), an inflammatory demyelinating disease. However, little is known how various types of Kv channels are altered in any inflammatory demyelinating diseases. By using established animal models for MS, Experimental Autoimmune Encephalomyelitis (EAE), we report that expression and distribution patterns of Kv channels are altered in the CNS correlating with EAE severity. The juxtaparanodal (JXP) targeting of Kv1.2/Kvβ2 along myelinated axons is disrupted within demyelinated lesions in the white matter of spinal cord in EAE. Moreover, somatodendritic Kv2.1 channels in the motor neurons of lower spinal cord significantly decrease correlating with EAE severity. Interestingly, Kv1.4 expression surrounding lesions is markedly up-regulated in the initial acute phase of both EAE models. Its expression in glial fibrillary acidic protein (GFAP)-positive astrocytes further increases in the remitting phase of remitting-relapsing EAE (rrEAE), but decreases in late chronic EAE (chEAE) and the relapse of rrEAE, suggesting that Kv1.4-positive astrocytes may be neuroprotective. Taken together, our studies reveal myelin-dependent and -independent alterations of Kv channels in the progression of EAE and lay a solid foundation for future study in search of a better treatment for MS.
PMCID: PMC3367054  PMID: 22560931
voltage-gated potassium (Kv) channel; experimental autoimmune encephalomyelitis (EAE); 4-AminoPyridine (4-AP); myelin; astrocyte; spinal cord
20.  Autotaxin and lysophosphatidic acid1 receptor-mediated demyelination of dorsal root fibers by sciatic nerve injury and intrathecal lysophosphatidylcholine 
Molecular Pain  2010;6:78.
Although neuropathic pain is frequently observed in demyelinating diseases such as Guillain-Barré syndrome and multiple sclerosis, the molecular basis for the relationship between demyelination and neuropathic pain behaviors is poorly understood. Previously, we found that lysophosphatidic acid receptor (LPA1) signaling initiates sciatic nerve injury-induced neuropathic pain and demyelination.
In the present study, we have demonstrated that sciatic nerve injury induces marked demyelination accompanied by myelin-associated glycoprotein (MAG) down-regulation and damage of Schwann cell partitioning of C-fiber-containing Remak bundles in the sciatic nerve and dorsal root, but not in the spinal nerve. Demyelination, MAG down-regulation and Remak bundle damage in the dorsal root were abolished in LPA1 receptor-deficient (Lpar1-/-) mice, but these alterations were not observed in sciatic nerve. However, LPA-induced demyelination in ex vivo experiments was observed in the sciatic nerve, spinal nerve and dorsal root, all which express LPA1 transcript and protein. Nerve injury-induced dorsal root demyelination was markedly attenuated in mice heterozygous for autotaxin (atx+/-), which converts lysophosphatidylcholine (LPC) to LPA. Although the addition of LPC to ex vivo cultures of dorsal root fibers in the presence of recombinant ATX caused potent demyelination, it had no significant effect in the absence of ATX. On the other hand, intrathecal injection of LPC caused potent dorsal root demyelination, which was markedly attenuated or abolished in atx+/- or Lpar1-/- mice.
These results suggest that LPA, which is converted from LPC by ATX, activates LPA1 receptors and induces dorsal root demyelination following nerve injury, which causes neuropathic pain.
PMCID: PMC2989310  PMID: 21062487
21.  Mitochondrial changes within axons in multiple sclerosis 
Brain : a journal of neurology  2009;132(Pt 5):1161-1174.
Multiple sclerosis is the most common cause of non-traumatic neurological impairment in young adults. An energy deficient state has been implicated in the degeneration of axons, the pathological correlate of disease progression, in multiple sclerosis. Mitochondria are the most efficient producers of energy and play an important role in calcium homeostasis. We analysed the density and function of mitochondria using immunohistochemistry and histochemistry, respectively, in chronic active and inactive lesions in progressive multiple sclerosis. As shown before in acute pattern III and Balo’s lesions, the mitochondrial respiratory chain complex IV activity is reduced despite the presence of mitochondria in demyelinated axons with amyloid precursor protein accumulation, which are predominantly located at the active edge of chronic active lesions. Furthermore, the strong non-phosphorylated neurofilament (SMI32) reactivity was associated with a significant reduction in complex IV activity and mitochondria within demyelinated axons. The complex IV defect associated with axonal injury may be mediated by soluble products of innate immunity, as suggested by an inverse correlation between complex IV activity and macrophage/microglial density in chronic lesions. However, in inactive areas of chronic multiple sclerosis lesions the mitochondrial respiratory chain complex IV activity and mitochondrial mass, judged by porin immunoreactivity, are increased within approximately half of large (>2.5 μm diameter) chronically demyelinated axons compared with large myelinated axons in the brain and spinal cord. The axon-specific mitochondrial docking protein (syntaphilin) and phosphorylated neurofilament-H were increased in chronic lesions. The lack of complex IV activity in a proportion of Na+/K+ ATPase α-1 positive demyelinated axons supports axonal dysfunction as a contributor to neurological impairment and disease progression. Furthermore, in vitro studies show that inhibition of complex IV augments glutamate-mediated axonal injury (amyloid precursor protein and SMI32 reactivity). Our findings have important implications for both axonal degeneration and dysfunction during the progressive stage of multiple sclerosis.
PMCID: PMC3605917  PMID: 19293237
Mitochondria; axonal degeneration; multiple sclerosis
22.  Gene Expression in Temporal Lobe Epilepsy is Consistent with Increased Release of Glutamate by Astrocytes 
Molecular Medicine  2007;13(1-2):1-13.
Patients with temporal lobe epilepsy (TLE) often have a shrunken hippocampus that is known to be the location in which seizures originate. The role of the sclerotic hippocampus in the causation and maintenance of seizures in temporal lobe epilepsy (TLE) has remained incompletely understood despite extensive neuropathological investigations of this substrate. To gain new insights and develop new testable hypotheses on the role of sclerosis in the pathophysiology of TLE, the differential gene expression profile was studied. To this end, DNA microarray analysis was used to compare gene expression profiles in sclerotic and non-sclerotic hippocampi surgically removed from TLE patients. Sclerotic hippocampi had transcriptional signatures that were different from non-sclerotic hippocampi. The differentially expressed gene set in sclerotic hippocampi revealed changes in several molecular signaling pathways, which included the increased expression of genes associated with astrocyte structure (glial fibrillary acidic protein, ezrin-moesin-radixin, palladin), calcium regulation (S100 calcium binding protein beta, chemokine (C-X-C motif) receptor 4) and blood-brain barrier function (Aquaaporin 4, Chemokine (C-C- motif) ligand 2, Chemokine (C-C- motif) ligand 3, Plectin 1, intermediate filament binding protein 55kDa) and inflammatory responses. Immunohistochemical localization studies show that there is altered distribution of the gene-associated proteins in astrocytes from sclerotic foci compared with non-sclerotic foci. It is hypothesized that the astrocytes in sclerotic tissue have activated molecular pathways that could lead to enhanced release of glutamate by these cells. Such glutamate release may excite surrounding neurons and elicit seizure activity.
PMCID: PMC1869627  PMID: 17515952
23.  Clinically feasible MTR is sensitive to cortical demyelination in MS 
Neurology  2013;80(3):246-252.
Presently there is no clinically feasible imaging modality that can effectively detect cortical demyelination in patients with multiple sclerosis (MS). The objective of this study is to determine if clinically feasible magnetization transfer ratio (MTR) imaging is sensitive to cortical demyelination in MS.
MRI were acquired in situ on 7 recently deceased patients with MS using clinically feasible sequences at 3 T, including relatively high-resolution T1-weighted and proton density–weighted images with/without a magnetization transfer pulse for calculation of MTR. The brains were rapidly removed and placed in fixative. Multiple cortical regions from each brain were immunostained for myelin proteolipid protein and classified as mostly myelinated (MMctx), mostly demyelinated (MDctx), or intermediately demyelinated (IDctx). MRIs were registered with the cortical sections so that the cortex corresponding to each cortical section could be identified, along with adjacent subcortical white matter (WM). Mean cortical MTR normalized to mean WM MTR was calculated for each cortical region. Linear mixed-effects models were used to test if mean normalized cortical MTR was significantly lower in demyelinated cortex.
We found that mean normalized cortical MTR was significantly lower in cortical tissue with any demyelination (IDctx or MDctx) compared to MMctx (demyelinated cortex: least-squares mean [LSM] = 0.797, SE = 0.007; MMctx: LSM = 0.837, SE = 0.006; p = 0.01, n = 89).
This result demonstrates that clinically feasible MTR imaging is sensitive to cortical demyelination and suggests that MTR will be a useful tool to help detect MS cortical lesions in living patients with MS.
PMCID: PMC3589181  PMID: 23269598
24.  Molecular reorganization of endocannabinoid signalling in Alzheimer’s disease 
Brain  2011;134(4):1041-1060.
Retrograde messengers adjust the precise timing of neurotransmitter release from the presynapse, thus modulating synaptic efficacy and neuronal activity. 2-Arachidonoyl glycerol, an endocannabinoid, is one such messenger produced in the postsynapse that inhibits neurotransmitter release upon activating presynaptic CB1 cannabinoid receptors. Cognitive decline in Alzheimer’s disease is due to synaptic failure in hippocampal neuronal networks. We hypothesized that errant retrograde 2-arachidonoyl glycerol signalling impairs synaptic neurotransmission in Alzheimer’s disease. Comparative protein profiling and quantitative morphometry showed that overall CB1 cannabinoid receptor protein levels in the hippocampi of patients with Alzheimer’s disease remain unchanged relative to age-matched controls, and CB1 cannabinoid receptor-positive presynapses engulf amyloid-β-containing senile plaques. Hippocampal protein concentrations for the sn-1-diacylglycerol lipase α and β isoforms, synthesizing 2-arachidonoyl glycerol, significantly increased in definite Alzheimer’s (Braak stage VI), with ectopic sn-1-diacylglycerol lipase β expression found in microglia accumulating near senile plaques and apposing CB1 cannabinoid receptor-positive presynapses. We found that microglia, expressing two 2-arachidonoyl glycerol-degrading enzymes, serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase, begin to surround senile plaques in probable Alzheimer’s disease (Braak stage III). However, Alzheimer’s pathology differentially impacts serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase in hippocampal neurons: serine hydrolase α/β-hydrolase domain-containing 6 expression ceases in neurofibrillary tangle-bearing pyramidal cells. In contrast, pyramidal cells containing hyperphosphorylated tau retain monoacylglycerol lipase expression, although at levels significantly lower than in neurons lacking neurofibrillary pathology. Here, monoacylglycerol lipase accumulates in CB1 cannabinoid receptor-positive presynapses. Subcellular fractionation revealed impaired monoacylglycerol lipase recruitment to biological membranes in post-mortem Alzheimer’s tissues, suggesting that disease progression slows the termination of 2-arachidonoyl glycerol signalling. We have experimentally confirmed that altered 2-arachidonoyl glycerol signalling could contribute to synapse silencing in Alzheimer’s disease by demonstrating significantly prolonged depolarization-induced suppression of inhibition when superfusing mouse hippocampi with amyloid-β. We propose that the temporal dynamics and cellular specificity of molecular rearrangements impairing 2-arachidonoyl glycerol availability and actions may differ from those of anandamide. Thus, enhanced endocannabinoid signalling, particularly around senile plaques, can exacerbate synaptic failure in Alzheimer’s disease.
PMCID: PMC3069704  PMID: 21459826
glia; human; neurodegeneration; retrograde signalling; synapse
25.  Myelination of rodent hippocampal neurons in culture 
Nature protocols  2012;7(10):1774-1782.
Axons of various hippocampal neurons are myelinated mainly postnatally, which is important for the proper function of neural circuits. Demyelination in the hippocampus has been observed in patients with multiple sclerosis, Alzheimer’s disease or temporal lobe epilepsy. However, very little is known about the mechanisms and exact functions of the interaction between the myelin-making oligodendrocytes and the axons within the hippocampus. This is mainly attributable to the lack of a system suitable for molecular studies. We recently established a new myelin coculture from embryonic day (E) 18 rat embryos consisting of hippocampal neurons and oligodendrocytes, with which we identified a novel intra-axonal signaling pathway regulating the juxtaparanodal clustering of Kv1.2 channels. Here we describe the detailed protocol for this new coculture. It takes about 5 weeks to set up and use the system. This coculture is particularly useful for studying myelin-mediated regulation of ion channel trafficking and for understanding how neuronal excitability and synaptic transmission are regulated by myelination.
PMCID: PMC3536533  PMID: 22955693

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