Hippocampal demyelination and memory dysfunction are associated with increased levels of the neuronal microRNA miR-124 and reduced AMPA receptors.

OBJECTIVE: 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 >40% of individuals with MS. How demyelination alters neuronal gene expression is unknown. METHODS: To explore whether loss of hippocampal myelin alters expression of neuronal microRNAs (miRNAs), we compared miRNA profiles from myelinated and demyelinated hippocampi from postmortem MS brains and performed validation studies. RESULTS: 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 AMPA2 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. INTERPRETATION: 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.

Cortical remyelination: a new target for repair therapies in multiple sclerosis.

OBJECTIVE: 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). Because 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. METHODS: Postmortem brain tissue from 22 patients with MS (age 27-77 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. RESULTS: New oligodendrocytes that were actively forming myelin sheaths were identified in 30 of 42 remyelinated subpial cortical lesions, including lesions from 3 patients in their 70s. 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 upregulate CD44, hyaluronan, and versican, molecules that form complexes that inhibit oligodendrocyte maturation and remyelination. INTERPRETATION: 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.

Multiple sclerosis normal-appearing white matter: pathology-imaging correlations.

OBJECTIVE: The study was undertaken to determine the pathologic basis of subtle abnormalities in magnetization transfer ratio (MTR) and diffusion tensor imaging (DTI) parameters observed in normal-appearing white matter (NAWM) in multiple sclerosis brains. METHODS: Brain tissues were obtained through a rapid postmortem protocol that included in situ magnetic resonance imaging (MRI). Four types of MRI-defined regions of interest (ROIs) were analyzed: (1) regions that were abnormal on all images (T2T1MTR lesions); (2) NAWM regions with slightly abnormal MTR located close to white matter lesions (sa-WM Close); (3) NAWM regions with slightly abnormal MTR located far from lesions (sa-WM Far); and (4) NAWM regions with normal MTR (NAWM). Immunohistochemical analysis for each ROI comprised immunostaining for myelin, axonal markers, activated microglia/macrophages, astrocytes, plasma proteins, and blood vessels. RESULTS: Forty-eight ROIs from 4 secondary progressive MS brains were analyzed. sa-WM Close ROIs were associated with significantly more axonal swellings. There were more enlarged major histocompatibility complex II(+) microglia and macrophages detected in sa-WM Far, sa-WM Close, and T2T1MTR lesions than in NAWM. Across all ROIs, MTR and DTI measures were moderately correlated with myelin density, axonal area, and axonal counts. Excluding T2T1MTR lesions from analysis revealed that MTR and DTI measures in nonlesional white matter (WM) were correlated with activated microglia, but not with axonal or myelin integrity. INTERPRETATION: The pathologic substrates for MRI abnormalities in NAWM vary based on distance from focal WM lesions. Close to WM lesions, axonal pathology and microglial activation may explain subtle MRI changes. Distant from lesions, microglial activation associated with proximity to cortical lesions might underlie MRI abnormalities.

Demyelination causes synaptic alterations in hippocampi from multiple sclerosis patients.

OBJECTIVE: Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the human central nervous system. Although the clinical impact of gray matter pathology in MS brains is unknown, 30 to 40% of MS patients demonstrate memory impairment. The molecular basis of this memory dysfunction has not yet been investigated in MS patients. METHODS: To investigate possible mechanisms of memory impairment in MS patients, we compared morphological and molecular changes in myelinated and demyelinated hippocampi from postmortem MS brains. RESULTS: Demyelinated hippocampi had minimal neuronal loss but significant decreases in synaptic density. Neuronal proteins essential for axonal transport, synaptic plasticity, glutamate neurotransmission, glutamate homeostasis, and memory/learning were significantly decreased in demyelinated hippocampi, but not in demyelinated motor cortices from MS brains. INTERPRETATION: Collectively, these data support hippocampal demyelination as a cause of synaptic alterations in MS patients and establish that the neuronal genes regulated by myelination reflect specific functions of neuronal subpopulations.

Hyaluronan accumulates in demyelinated lesions and inhibits oligodendrocyte progenitor maturation.

Demyelination is the hallmark of numerous neurodegenerative conditions, including multiple sclerosis. Oligodendrocyte progenitors (OPCs), which normally mature into myelin-forming oligodendrocytes, are typically present around demyelinated lesions but do not remyelinate affected axons. Here, we find that the glycosaminoglycan hyaluronan accumulates in demyelinated lesions from individuals with multiple sclerosis and in mice with experimental autoimmune encephalomyelitis. A high molecular weight (HMW) form of hyaluronan synthesized by astrocytes accumulates in chronic demyelinated lesions. This form of hyaluronan inhibits remyelination after lysolecithin-induced white matter demyelination. OPCs accrue and do not mature into myelin-forming cells in demyelinating lesions where HMW hyaluronan is present. Furthermore, the addition of HMW hyaluronan to OPC cultures reversibly inhibits progenitor-cell maturation, whereas degrading hyaluronan in astrocyte-OPC cocultures promotes oligodendrocyte maturation. HMW hyaluronan may therefore contribute substantially to remyelination failure by preventing the maturation of OPCs that are recruited to demyelinating lesions.

Reticulon family members modulate BACE1 activity and amyloid-beta peptide generation.

Inhibiting the activity of the beta-amyloid converting enzyme 1 (BACE1) or reducing levels of BACE1 in vivo decreases the production of amyloid-beta. The reticulon family of proteins has four members, RTN1, RTN2, RTN3 and RTN4 (also known as Nogo), the last of which is well known for its role in inhibiting neuritic outgrowth after injury. Here we show that reticulon family members are binding partners of BACE1. In brain, BACE1 mainly colocalizes with RTN3 in neurons, whereas RTN4 is more enriched in oligodendrocytes. An increase in the expression of any reticulon protein substantially reduces the production of Abeta. Conversely, lowering the expression of RTN3 by RNA interference increases the secretion of Abeta, suggesting that reticulon proteins are negative modulators of BACE1 in cells. Our data support a mechanism by which reticulon proteins block access of BACE1 to amyloid precursor protein and reduce the cleavage of this protein. Thus, changes in the expression of reticulon proteins in the human brain are likely to affect cellular amyloid-beta and the formation of amyloid plaques.

Beta4 tubulin identifies a primitive cell source for oligodendrocytes in the mammalian brain.

We have identified a novel population of cells in the subventricular zone (SVZ) of the mammalian brain that expresses beta4 tubulin (betaT4) and has properties of primitive neuroectodermal cells. betaT4 cells are scattered throughout the SVZ of the lateral ventricles in adult human brain and are significantly increased in the SVZs bordering demyelinated white matter in multiple sclerosis brains. In human fetal brain, betaT4 cell densities peak during the latter stages of gliogenesis, which occurs in the SVZ of the lateral ventricles. betaT4 cells represent <2% of the cells present in neurospheres generated from postnatal rat brain but >95% of cells in neurospheres treated with the anti-mitotic agent Ara C. betaT4 cells produce oligodendrocytes, neurons, and astrocytes in vitro. We compared the myelinating potential of betaT4-positive cells with A2B5-positive oligodendrocyte progenitor cells after transplantation (25,000 cells) into postnatal day 3 (P3) myelin-deficient rat brains. At P20, the progeny of betaT4 cells myelinated up to 4 mm of the external capsule, which significantly exceeded that of transplanted A2B5-positive progenitor cells. Such extensive and rapid mature CNS cell generation by a relatively small number of transplanted cells provides in vivo support for the therapeutic potential of betaT4 cells. We propose that betaT4 cells are an endogenous cell source that can be recruited to promote neural repair in the adult telencephalon.

Imaging correlates of decreased axonal Na+/K+ ATPase in chronic multiple sclerosis lesions.

OBJECTIVE: Degeneration of chronically demyelinated axons is a major cause of irreversible neurological decline in the human central nervous system disease, multiple sclerosis (MS). Although the molecular mechanisms responsible for this axonal degeneration remain to be elucidated, dysfunction of axonal Na+/K+ ATPase is thought to be central. To date, however, the distribution of Na+/K+ ATPase has not been studied in MS lesions. METHODS: The percentage of axons with detectable Na+/K+ ATPase was determined in 3 acute and 36 chronically demyelinated lesions from 13 MS brains. In addition, we investigated whether postmortem magnetic resonance imaging profiles could predict Na+/K+ ATPase immunostaining in a subset (20) of the chronic lesions. RESULTS: Na+/K+ ATPase subunits alpha1, alpha3, and beta1 were detected in the internodal axolemma of myelinated fibers in both control and MS brains. In acutely demyelinated lesions, Na+/K+ ATPase was detectable on demyelinated axolemma. In contrast, 21 of the 36 chronic lesions (58%) contained less than 50% Na+/K+ ATPase-positive demyelinated axons. In addition, magnetic resonance imaging-pathology correlations of 20 chronic lesions identified a linear decrease in the percentage of Na+/K+ ATPase-positive axons and magnetization transfer ratios (p < 0.0001) and T1 contrast ratios (p < 0.0006). INTERPRETATION: Chronically demyelinated axons that lack Na+/K+ ATPase cannot exchange axoplasmic Na+ for K+ and are incapable of nerve transmission. Loss of axonal Na+/K+ ATPase is likely to be a major contributor to continuous neurological decline in chronic stages of MS, and quantitative magnetization transfer ratios and T1 contrast ratios may provide a noninvasive surrogate marker for monitoring this loss in MS patients.

Neurogenesis in the chronic lesions of multiple sclerosis.

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.

Activation of the ciliary neurotrophic factor (CNTF) signalling pathway in cortical neurons of multiple sclerosis patients.

Neuronal and axonal degeneration results in irreversible neurological disability in multiple sclerosis (MS) patients. A number of adaptive or neuroprotective mechanisms are thought to repress neurodegeneration and neurological disability in MS patients. To investigate possible neuroprotective pathways in the cerebral cortex of MS patients, we compared gene transcripts in cortices of six control and six MS patients. Out of 67 transcripts increased in MS cortex nine were related to the signalling mediated by the neurotrophin ciliary neurotrophic factor (CNTF). Therefore, we quantified and localized transcriptional (RT-PCR, in situ hybridization) and translational (western, immunohistochemistry) products of CNTF-related genes. CNTF-receptor complex members, CNTFRalpha, LIFRbeta and GP130, were increased in MS cortical neurons. CNTF was increased and also expressed by neurons. Phosphorylated STAT3 and the anti-apoptotic molecule, Bcl2, known down stream products of CNTF signalling were also increased in MS cortical neurons. We hypothesize that in response to the chronic insults or stress of the pathogenesis of multiple sclerosis, cortical neurons up regulate a CNTF-mediated neuroprotective signalling pathway. Induction of CNTF signalling and the anti-apoptotic molecule, Bcl2, thus represents a compensatory response to disease pathogenesis and a potential therapeutic target in MS patients.