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1.  Proteolipid protein cannot replace P0 protein as the major structural protein of peripheral nervous system myelin 
Glia  2014;63(1):66-77.
The central nervous system (CNS) of terrestrial vertebrates underwent a prominent molecular change when proteolipid protein (PLP) replaced P0 protein as the most abundant protein of CNS myelin. However, PLP did not replace P0 in peripheral nervous system (PNS) myelin. To investigate the possible consequences of a PLP to P0 shift in PNS myelin, we engineered mice to express PLP instead of P0 in PNS myelin (PLP-PNS mice). PLP-PNS mice had severe neurological disabilities and died between 3 and 6 months of age. Schwann cells in sciatic nerves from PLP-PNS mice sorted axons into one-to-one relationships but failed to form myelin internodes. Mice with equal amounts of P0 and PLP had normal PNS myelination and lifespans similar to wild-type (WT) mice. When PLP was overexpressed with one copy of the P0 gene, sciatic nerves were hypomyelinated; mice displayed motor deficits but had normal lifespans. These data support the hypothesis that while PLP can co-exist with P0 in PNS myelin, PLP cannot replace P0 as the major structural protein of PNS myelin.
PMCID: PMC4237650  PMID: 25066805
Schwann cell; hypomyelination; myelin compaction; myelin evolution; axon
2.  Selective inhibitors of nuclear export avert progression in preclinical models of inflammatory demyelination 
Nature neuroscience  2015;18(4):511-520.
Axonal damage has been associated with aberrant protein trafficking. This study characterizes a novel class of compounds targeting nucleo-cytoplasmic shuttling, by binding to the catalytic groove of the nuclear export protein XPO1/CRM1 (chromosome region maintenance protein1). Oral administration of novel reversible CRM1 inhibitors in preclinical murine models of demyelination significantly attenuated disease progression, even when started after the onset of paralysis. Clinical efficacy was associated with decreased proliferation of immune cells, characterized by nuclear accumulation of cell cycle inhibitors, and preservation of cytoskeletal integrity even in demyelinated axons. Neuroprotection was not limited to models of demyelination, but observed also in other mouse models of axonal damage (i.e. kainic acid injection) and detected in cultured neurons after knockdown of Xpo1, the gene encoding for CRM1. A proteomic screen for target molecules revealed that CRM1 inhibitors in neurons prevented nuclear export of molecules associated with axonal damage while retaining transcription factors modulating neuroprotection.
PMCID: PMC4522902  PMID: 25706475
3.  Lipopolysaccharide-Induced Microglial Activation and Neuroprotection against Experimental Brain Injury Is Independent of Hematogenous TLR4 
Intraperitoneal injection of the Gram-negative bacterial endotoxin lipopolysaccharide (LPS) elicits a rapid innate immune response. While this systemic inflammatory response can be destructive, tolerable low doses of LPS render the brain transiently resistant to subsequent injuries. However, the mechanism by which microglia respond to LPS stimulation and participate in subsequent neuroprotection has not been documented. In this study, we first established a novel LPS treatment paradigm where mice were injected intraperitoneally with 1.0 mg/kg LPS for four consecutive days to globally activate CNS microglia. By using a reciprocal bone marrow transplantation procedure between wild-type and Toll-like receptor 4 (TLR4) mutant mice, we demonstrated that the presence of LPS receptor (TLR4) is not required on hematogenous immune cells but is required on cells that are not replaced by bone marrow transplantation, such as vascular endothelia and microglia, to transduce microglial activation and neuroprotection. Furthermore, we showed that activated microglia physically ensheathe cortical projection neurons, which have reduced axosomatic inhibitory synapses from the neuronal perikarya. In line with previous reports that inhibitory synapse reduction protects neurons from degeneration and injury, we show here that neuronal cell death and lesion volumes are significantly reduced in LPS-treated animals following experimental brain injury. Together, our results suggest that activated microglia participate in neuroprotection and that this neuroprotection is likely achieved through reduction of inhibitory axosomatic synapses. The therapeutic significance of these findings rests not only in identifying neuroprotective functions of microglia, but also in establishing the CNS location of TLR4 activation.
PMCID: PMC4461442  PMID: 22915113
4.  Microglial displacement of inhibitory synapses provides neuroprotection in the adult brain 
Nature Communications  2014;5:4486.
Microglia actively survey the brain microenvironment and play essential roles in sculpting synaptic connections during brain development. While microglial functions in the adult brain are less clear, activated microglia can closely appose neuronal cell bodies and displace axosomatic presynaptic terminals. Microglia-mediated stripping of presynaptic terminals is considered neuroprotective, but the cellular and molecular mechanisms are poorly defined. Using 3D electron microscopy, we demonstrate that activated microglia displace inhibitory presynaptic terminals from cortical neurons in adult mice. Electrophysiological recordings further establish that the reduction in inhibitory GABAergic synapses increased synchronized firing of cortical neurons in γ-frequency band. Increased neuronal activity results in the calcium-mediated activation of CaM kinase IV, phosphorylation of CREB, increased expression of antiapoptotic and neurotrophic molecules and reduced apoptosis of cortical neurons following injury. These results indicate that activated microglia can protect the adult brain by migrating to inhibitory synapses and displacing them from cortical neurons.
Microglia play essential roles in sculpting synaptic connections during brain development but their role in the adult brain is less clear. Here the authors show that activated microglia can prophylactically protect the adult rodent brain from injury by migrating to and displacing inhibitory synapses from cortical neurons.
PMCID: PMC4109015  PMID: 25047355
5.  Mapping and morphometric analysis of synapses and spines on fusiform cells in the dorsal cochlear nucleus 
Fusiform cells are the main integrative units of the mammalian dorsal cochlear nucleus (DCN), collecting and processing inputs from auditory and other sources before transmitting information to higher levels of the auditory system. Despite much previous work describing these cells and the sources and pharmacological identity of their synaptic inputs, information on the three-dimensional organization and utltrastructure of synapses on these cells is currently very limited. This information is essential since an understanding of synaptic plasticity and remodeling and pathologies underlying disease states and hearing disorders must begin with knowledge of the normal characteristics of synapses on these cells, particularly those features that determine the strength of their influence on the various compartments of the cell. Here, we employed serial block face scanning electron microscopy (SBFSEM) followed by 3D reconstructions to map and quantitatively characterize synaptic features on DCN fusiform cells. Our results reveal a relative sparseness of synapses on the somata of fusiform cells but a dense distribution of synapses on apical and basal dendrites. Synapses on apical dendrites were smaller and more numerous than on basal dendrites. The vast majority of axosomatic terminals were found to be linked to other terminals connected by the same axon or different branches of the same axon, suggesting a high degree of divergent input to fusiform cells. The size of terminals was correlated with the number of mitochondria and with the number of active zones, which was highly correlated with the number of postsynaptic densities, suggesting that larger terminals exert more powerful influence on the cell than smaller terminals. These size differences suggest that the input to basal dendrites, most likely those from the auditory nerve, provide the most powerful sources of input to fusiform cells, while those to apical dendrites (e.g., parallel fiber) are weaker but more numerous.
PMCID: PMC4172007  PMID: 25294990
serial block face scanning electron microscopy; dorsal cochlear nucleus; fusiform cells; dendritic spine volumes; parallel fibers; synaptic vesicles; active zone; postsynaptic density
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.  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
8.  Demyelination Causes Synaptic Alterations in Hippocampi from Multiple Sclerosis Patients 
Annals of neurology  2011;69(3):445-454.
Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the human central nervous system. While the clinical impact of gray matter pathology in MS brains is unknown, 30–40% of MS patients demonstrate memory impairment. The molecular basis of this memory dysfunction has not yet been investigated in MS patients.
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.
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.
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.
PMCID: PMC3073544  PMID: 21446020
Multiple Sclerosis; hippocampus; demyelination; memory
9.  Myelination and axonal electrical activity modulate the distribution and motility of mitochondria at CNS nodes of Ranvier 
Energy production presents a formidable challenge to axons as their mitochondria are synthesized and degraded in neuronal cell bodies. To meet the energy demands of nerve conduction, small mitochondria are transported to and enriched at mitochondrial stationary sites located throughout the axon. In this study, we investigated whether size and motility of mitochondria in small myelinated central nervous system axons was differentially regulated at nodes, and whether mitochondrial distribution and motility are modulated by axonal electrical activity. The size/volume of mitochondrial stationary sites was significantly larger in juxtaparanodal/internodal axoplasm than in nodal/paranodal axoplasm. By 3-dimensional electron microscopy, we observed that axonal mitochondrial stationary sites were composed of multiple mitochondria of varying length, except at nodes where mitochondria were uniformly short and frequently absent altogether. Mitochondrial transport speed was significantly reduced in nodal axoplasm when compared to internodal axoplasm. Increased axonal electrical activity decreased mitochondrial transport and increased the size of mitochondrial stationary sites in nodal/paranodal axoplasm. Decreased axonal electrical activity had the opposite effects. In cerebellar axons of the myelin deficient rat, which contains voltage-gated Na+ channel clusters but lacks paranodal specializations, axonal mitochondrial motility and stationary site size were similar at Na+ channel clusters and other axonal regions. These results demonstrate juxtaparanodal/internodal enrichment of stationary mitochondria and neuronal activity-dependent dynamic modulation of mitochondrial distribution and transport in nodal axoplasm. In addition, the modulation of mitochondrial distribution and motility requires oligodendrocyte-axon interactions at paranodal specializations.
PMCID: PMC3139464  PMID: 21593309
myelination; mitochondria; axonal transport; node of Ranvier; action potential
10.  Demyelination increases axonal stationary mitochondrial size and the speed of axonal mitochondrial transport 
Axonal degeneration contributes to permanent neurological disability in inherited and acquired diseases of myelin. Mitochondrial dysfunction has been proposed as a major contributor to this axonal degeneration. It remains to be determined, however, if myelination, demyelination or remyelination alter the size and distribution of axonal mitochondrial stationary sites or the rates of axonal mitochondrial transport. Using live myelinated rat dorsal root ganglion (DRG) cultures, we investigated whether myelination and lysolecithin-induced demyelination affect axonal mitochondria. Myelination increased the size of axonal stationary mitochondrial sites by 2.3 fold. Following demyelination, the size of axonal stationary mitochondrial sites was increased by an additional 2.2 fold and the transport velocity of motile mitochondria was increased by 47%. These measures returned to the levels of myelinated axons following remyelination. Demyelination induced activating transcription factor (ATF) 3 in DRG neurons. Knockdown of neuronal ATF3 by shRNA abolished the demyelination-induced increase in axonal mitochondrial transport and increased nitrotyrosine immunoreactivity in axonal mitochondria, suggesting that neuronal ATF3 expression and increased mitochondrial transport protect demyelinated axons from oxidative damage. In response to insufficient ATP production, demyelinated axons increase the size of stationary mitochondrial sites and thereby balance ATP production with the increased energy needs of nerve conduction.
PMCID: PMC2885867  PMID: 20463228
Demyelination; mitochondria; adaptive response; axonal transport; axon; ATF3
11.  Beta-4 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 beta-4 tubulin (βT4) and has properties of primitive neuroectodermal cells. βT4 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, βT4 cell densities peak during the latter stages of gliogenesis, which occurs in the SVZ of the lateral ventricles. βT4 cells represent less than 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. βT4 cells produce oligodendrocytes, neurons, and astrocytes in vitro. We compared the myelinating potential of βT4-positive cells with A2B5-positive oligodendrocyte progenitor cells following transplantation (25,000 cells) into postnatal day 3 (P3) myelin deficient rat brains. At P20, the progeny of βT4 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 βT4 cells. We propose that βT4 cells are an endogenous cell source that can be recruited to promote neural repair in the adult telencephalon.
PMCID: PMC2742370  PMID: 19535576
multiple sclerosis; subventricular zone; neural stem cell; myelin; oligodendrocyte; transplantation
12.  P0 Protein Is Required For and Can Induce Formation of Schmidt-Lantermann Incisures in Myelin Internodes 
Axons in the peripheral (PNS) and central (CNS) nervous systems are ensheathed by multiple layers of tightly compacted myelin membranes. A series of cytoplasmic channels connect outer and inner margins of PNS, but not CNS myelin internodes. Membranes of these Schmidt-Lantermann (S-L) incisures contain the myelin-associated glycoprotein (MAG), but not P0 or proteolipid protein (PLP), the structural proteins of compact PNS (P0) and CNS (PLP) myelin. We show here that incisures are present in MAG-null and absent from P0-null PNS internodes. To test the possibility that P0 regulates incisure formation, we replaced PLP with P0 in CNS myelin. S-L incisures formed in P0-CNS myelin internodes. Furthermore, axoplasm ensheathed by 65% of the CNS incisures examined by electron microscopy had focal accumulations of organelles, indicating that these CNS incisures disrupt axonal transport. These data support the hypotheses that P0 protein is required for and can induce S-L incisures and that P0-induced CNS incisures can be detrimental to axonal function.
PMCID: PMC2682947  PMID: 18614675
myelin; P0 protein; proteolipid protein; myelin-associated glycoprotein; axon; axonal transport
13.  Axo-Glial Septate Junctions 
The Journal of Cell Biology  2000;150(3):97-100.
PMCID: PMC2175180  PMID: 10931879

Results 1-13 (13)