FTY720-Mitoxy reduces toxicity associated with MSA-like α-synuclein and oxidative stress by increasing trophic factor expression and myelin protein in OLN-93 oligodendroglia cell cultures.

Multiple system atrophy (MSA) is a fatal demyelinating disorder lacking any disease-modifying therapies. MSA pathology stems from aggregated α-synuclein (aSyn) accumulation in glial cytosolic inclusions of oligodendroglial cell (OLGs), the myelinating cells of brain. In MSA brains and in MSA animal models with aSyn accumulation in OLGs, aberrant expression of brain-derived neurotrophic factor (BDNF) and glial-cell-line-derived neurotrophic factor (GDNF) occur. Nerve growth factor (NGF) expression can also be altered in neurodegenerative diseases. It is unclear if oxidative stress impacts the viability of aSyn-accumulating OLG cells. Here, we show that OLN-93 cells stably expressing human wild type aSyn or the MSA-associated-aSyn-mutants G51D or A53E, are more vulnerable to oxidative stress. In dose response studies we found that OLN-93 cells treated 48 h with 160 nM FTY720 or our new non-immunosuppressive FTY720-C2 or FTY720-Mitoxy derivatives sustained normal viability. Also, FTY720, FTY720-C2, and FTY720-Mitoxy all stimulated NGF expression at 24 h. However only FTY720-Mitoxy also increased BDNF and GDNF mRNA at 24 h, an effect paralleled by increases in histone 3 acetylation and ERK1/2 phosphorylation. Myelin associated glycoprotein (MAG) levels were also increased in OLN-93 cells after 48 h treatment with FTY720-Mitoxy. FTY720, FTY720-C2, and FTY720-Mitoxy all prevented oxidative-stress-associated-cell-death of OLN-93 cells that lack any aSyn expression. However, only FTY720-Mitoxy protected MSA-like aSyn-expressing-OLN-93-cells against oxidative-cell-death. These data identify potent protective effects for FTY720-Mitoxy with regard to trophic factors as well as MAG expression by OLG cells. Testing of FTY720-Mitoxy in mice is thus a judicious next step for neuropharmacological preclinical development.

Effects of dietary folic acid on the expression of myelin basic protein in the brain and spinal cord of pregnant and lactating rats.

BACKGROUND/AIMS: This study investigated the effects of dietary folic acid on the expression of myelin basic protein (MBP) in the maternal brain and spinal cord during pregnancy and lactation. METHODS: Female Sprague-Dawley rats were fed either a folic-acid-supplemented diet (FS, 8 mg/kg diet) or a folic-acid-deficient diet (FD, 0 mg/kg diet) from 2 weeks prior to mating until the end of lactation. The expressions of MBP were analyzed using Western blot analysis and immunohistochemistry, and myelin oligodendrocyte glycoprotein (MOG), and neuronal nuclear antigen by immunohistochemistry. RESULTS: The cerebrocortical expression of MBP was 87% higher at day 20 of pregnancy than before pregnancy in FS animals (p < 0.05) but did not change significantly in FD animals. No significant change was observed in the hippocampus or spinal cord in either dietary treatment group. The cerebrocortical MOG and NeuN expressions were significantly lower in FD animals than in FS animals before pregnancy and increased at day 20 of pregnancy but did not differ with the dietary folic acid level. CONCLUSIONS: Folic acid deficiency did not increase the expression level of MBP in the cerebral cortex during pregnancy, suggesting that folate intake during pregnancy plays an important role in the maintenance of myelin.

Myelin glycoproteins targeted by lead in the rodent model of prolonged exposure.

The aim of these experiments was to discern whether prolonged exposure of rats to lead (Pb) in water, as occurs in typical environmental exposure, affects the structure of myelin in the brain, the protein levels of the myelin glycoproteins myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG). During chronic lead intoxication, Pb levels in myelin fractions significantly increases. Prolonged lead exposure was found to decrease the expression of both MAG and MOG to a similar degree (80-85% of control values). Additionally, the electron microscopic studies have indicated the existence of structural changes in the axonal myelin sheath by revealing disintegration of its multilamellar structure. These morphological disturbances in Pb-intoxicated rats were paralleled by changes in myelin membrane fluidity as measured by spectrofluorometry and electron paramagnetic resonance (EPR) techniques employing a fatty acid spin label. In Pb-treated rats, enhanced membrane fluidity was observed, as indicated both by decreased anisotropy of the membrane and decreased order parameter. These results suggest that Pb influences the integrity of the myelin sheath in brain of adult rats and decreased level of both glycoproteins may significantly contribute to that effect.

Mimetics of the tri- and tetrasaccharide epitope of GQ1balpha as myelin-associated glycoprotein (MAG) ligands.

The synthesis of phenoxyphenyl, phenoxybenzyl, biphenyl, and phenyltriazole substituted sialic acid derivatives as mimics of the tri- and tetrasaccharide epitopes of GQ1balpha is described. These synthetically easily available sialosides show comparable or even enhanced affinity to MAG compared with the natural tri- and tetrasaccharide epitopes and form a new class of potential MAG antagonists.

Polyunsaturated fatty acid supplementation stimulates differentiation of oligodendroglia cells.

Dietary polyunsaturated fatty acids (PUFAs) have been postulated as alternative supportive treatment for multiple sclerosis, since they may promote myelin repair. We set out to study the effect of supplementation with n-3 and n-6 PUFAs on OLN-93 oligodendroglia and rat primary oligodendrocyte differentiation in vitro. It appeared that OLN-93 cells actively incorporate and metabolise the supplemented PUFAs in their cell membrane. The effect of PUFAs on OLN-93 differentiation was further assessed by morphological and Western blot evaluation of markers of oligodendroglia differentiation: 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), zonula occludens-1 (ZO-1) and myelin-associated glycoprotein (MAG). Supplementation of the OLN-93 cells with n-3 and n-6 PUFAs increased the degree of differentiation determined by morphological analysis. Moreover, CNP protein expression was significantly increased by gamma-linolenic acid (GLA, 18:3n-6) supplementation. In accordance with the OLN-93 results, studies with rat primary oligodendrocytes, a more advanced model of cell differentiation, showed GLA supplementation to promote oligodendrocyte differentiation. Following GLA supplementation, increased numbers of proteolipid protein (PLP)-positive oligodendrocytes and increased myelin sheet formation was observed during differentiation of primary oligodendrocytes. Moreover, increased CNP, and enhanced PLP and myelin basic protein expression were found after GLA administration. These studies provide support for the dietary supplementation of specific PUFAs to support oligodendrocyte differentiation and function.

Polychlorinated biphenyls exert selective effects on cellular composition of white matter in a manner inconsistent with thyroid hormone insufficiency.

Developmental exposure to polychlorinated biphenyls (PCBs) is associated with a variety of cognitive deficits in humans, and recent evidence implicates white matter development as a potential target of PCBs. Because PCBs are suspected of interfering with thyroid hormone (TH) signaling in the developing brain, and because TH is important in oligodendrocyte development, we tested the hypothesis that PCB exposure affects the development of white matter tracts by disrupting TH signaling. Pregnant Sprague Dawley rats were exposed to the PCB mixture Aroclor 1254 (5 mg/kg), with or without cotreatment of goitrogens from gestational d 7 until postnatal d 15. Treatment effects on white matter development were determined by separately measuring the cellular density and proportion of myelin-associated glycoprotein (MAG)-positive, O4-positive, and glial fibrillary acidic protein (GFAP)-positive cells in the genu of the corpus callosum (CC) and in the anterior commissure (AC). Hypothyroidism decreased the total cell density of the CC and AC as measured by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and produced a disproportionate decrease in MAG-positive oligodendrocyte density with a simultaneous increase in GFAP-positive astrocyte density. These data indicate that hypothyroidism reduces cellular density of CC and AC and fosters astrocyte development at the expense of oligodendrocyte density. In contrast, PCB exposure significantly reduced total cell density but did not disproportionately alter MAG-positive oligodendrocyte density or change the ratio of MAG-positive oligodendrocytes to GFAP-positive astrocytes. Thus, PCB exposure mimicked some, but not all, of the effects of hypothyroidism on white matter composition.

Gene expression profiling in the brains of human cocaine abusers.

Chronic cocaine abuse induces long-term neurochemical, structural and behavioural changes thought to result from altered gene expression within the nucleus accumbens and other brain regions playing a critical role in addiction. Recent methodological advances now allow the profiling of gene expression in human postmortem brain. In this article, we review studies in which we have used Affymetrix oligonucleotide microarrays to identify transcripts that are differentially expressed in the nucleus accumbens of cocaine abusers in comparison to well-matched control subjects. Of the approximately 39,000 gene transcripts interrogated, the expression of only a fraction of 1% is significantly modified in cocaine abusers. Found within this list are equivalent incidences of increased and decreased transcript abundance, including known gene transcripts clustered into several functional categories. A striking exception is a group of myelin-related genes, consisting of multiple transcripts representing myelin basic protein (MBP), proteolipid protein (PLP) and myelin-associated oligodendrocyte basic protein (MOBP), which as a group are substantially decreased in cocaine abusers compared to controls. These data, suggesting a possible dysregulation of myelin in cocaine abusers, are discussed in the context of myelin-related changes in other human brain disorders. Finally, the effects of cocaine abuse on the profile of gene expression in some other brain regions critical for addiction (the prefrontal cortex and ventral midbrain) are briefly reviewed.

Temporal expression pattern of peripheral myelin protein 22 during in vivo and in vitro myelination.

Peripheral myelin protein 22 (PMP22) was initially described as a minor component of peripheral myelin. Mutations affecting the PMP22 gene cause demyelinating neuropathies, supporting a role for the protein in PNS myelination. Furthermore, PMP22 carries the L2/HNK-1 carbohydrate epitope suggesting an adhesion/recognition function. Despite advances in characterizing the PMP22 gene, the specific role(s) of the protein in myelin remains unknown. In this study we determined the temporal expression pattern of PMP22 in comparison to galactocerebroside (GalC) and myelin associated glycoprotein (MAG), early constituents of PNS myelin, and to protein zero (P0) and myelin basic protein (MBP), late components of myelin. In sciatic nerve lysates, PMP22 was detected at postnatal day 3, after MAG, but before MBP expression. The same results were obtained in cocultures of dorsal root ganglion neurons and Schwann cells (SCs). Low levels of PMP22 were found in early, anti-MAG and anti-GalC immunoreactive, myelinating cocultures. However, PMP22 could only be detected in the SC plasma membrane after basal lamina formation. In long-term myelinating cocultures PMP22 levels continued to increase and the protein was found in anti-P0 and anti-MBP immunoreactive myelin segments. Furthermore, PMP22, MBP, and P0 protein levels were greatly enhanced by progesterone treatment of the cocultures. The highest levels of PMP22 expression were associated with late stages of myelination; however the presence of the protein in nonmyelinating SCs and in SCs commencing myelination supports multiple roles for PMP22 in peripheral nerve biology.

Expression, purification, and encephalitogenicity of recombinant human myelin oligodendrocyte glycoprotein.

Myelin oligodendrocyte glycoprotein (MOG), a putative autoantigen in multiple sclerosis (MS), is a quantitatively minor component of the CNS. In view of the difficulties associated with the purification of MOG from brain tissues, the extracellular domain of human MOG corresponding to the N-terminal 121 amino acids was expressed in Escherichia coli as a glutathione sulfotransferase fusion protein. The expressed protein was localized to inclusion bodies, and varying the growth parameters resulted in the solubilization of small amounts of GST-MOG that could be affinity purified on glutathione agarose columns. The fusion protein found in the inclusion bodies could be solubilized with urea. The solubilized fusion protein was cleaved with thrombin, and the extracellular domain was purified by CM Sephadex 50 chromatography to homogeneity. Injection of recombinant human MOG into different strains of mice resulted in the induction of an MS-like disease, characterized by severe neurological impairment and extensive CNS demyelinated lesions. Recombinant MOG produced in E. coli should prove to be useful as a highly purified biological reagent for immunological, pathological, functional, and structural studies.