Dietary cholesterol promotes repair of demyelinated lesions in the adult brain.

Multiple Sclerosis (MS) is an inflammatory demyelinating disorder in which remyelination failure contributes to persistent disability. Cholesterol is rate-limiting for myelin biogenesis in the developing CNS; however, whether cholesterol insufficiency contributes to remyelination failure in MS, is unclear. Here, we show the relationship between cholesterol, myelination and neurological parameters in mouse models of demyelination and remyelination. In the cuprizone model, acute disease reduces serum cholesterol levels that can be restored by dietary cholesterol. Concomitant with blood-brain barrier impairment, supplemented cholesterol directly supports oligodendrocyte precursor proliferation and differentiation, and restores the balance of growth factors, creating a permissive environment for repair. This leads to attenuated axon damage, enhanced remyelination and improved motor learning. Remarkably, in experimental autoimmune encephalomyelitis, cholesterol supplementation does not exacerbate disease expression. These findings emphasize the safety of dietary cholesterol in inflammatory diseases and point to a previously unrecognized role of cholesterol in promoting repair after demyelinating episodes.

Panax notoginseng saponins promotes stroke recovery by influencing expression of Nogo-A, NgR and p75NGF, in vitro and in vivo.

The spontaneous recovery of function after injury in the adult central nervous system is limited due to the several proteins, such as Nogo-A that have repulsive or inhibitory effects on growing neuritis. The Chinese herbal medicine extraction Panax notoginseng saponins (PNS) injection has been widely used and effective in repairing the function of impaired nerves, but the mechanism of this herbal medicine is still poorly understood. This project evaluated the effect of Panax notoginseng saponins on neurological functional recovery and on the expression of Nogo-A, NgR and p75 at 7, 14 and 28 d after middle cerebral artery occlusion (MCAO) in rats and also oxygen-glucose deprivation/reperfusion (OGD/R) model on SH-SY5Y cells. We found that the expression of Nogo-A, NgR and p75 of rats receiving MCAO surgery increased on the 7th day, reached a peak on the 14th or 28th day and maintained high levels and Panax notoginseng saponins significantly decreased these expressions. This may be the mechanism of Panax notoginseng saponins that contributes to the recovery of nerve function, which plays an important role in brain protection after cerebral infarction.

Multimodal analysis in acute and chronic experimental autoimmune encephalomyelitis.

Different experimental autoimmune encephalomyelitis models (EAE) have been developed. However, due to the different experimental conditions applied, observations simultaneously considering different pathological targets are still scarce. Using EAE induced in Dark Agouti rats with syngenic whole spinal cord homogenate suspended in incomplete Freund's adjuvant, we here analyze neurosteroidogenic machinery, cytokine levels, microglial cells, infiltration of inflammatory cells, myelin proteins and Na(+), K(+)-ATPase pump activity in the spinal cord. Data obtained in the acute phase of the disease confirmed that neurological signs were accompanied by the presence of perivascular infiltrating T cells (CD3(+) cells) and activated monocytic/microglial cells (ED1(+) and MHC-II(+)) in the spinal cord. In particular, the number of MHC-II(+) cells was significantly increased in association with increased expression of pro- (i.e., TNF-α, IL-1ß) and anti-inflammatory (i.e., TGF-ß) cytokines as well as with decreased expression of proteolipid protein and myelin basic protein. During the chronic phase of the disease, the number of MHC-II(+) cells was still increased, although less than in the acute phase. Changes in the number of MHC-II(+) cells were associated with decreased Na(+),K(+)-ATPase enzymatic activity. A general decrease in the levels of neuroactive steroids, with the exception of an increase in tetrahydroprogesterone and 17ß-estradiol, was detected in the acute phase. These changes were maintained or reverted in the chronic phase of EAE. In conclusion, we report that modifications in the neuroimmune response in the acute and chronic phases of EAE are associated with specific changes in myelin proteins, Na(+),K(+)-ATPase pump and in the levels of neuroactive steroids.

Identification of drug modulators targeting gene-dosage disease CMT1A.

The structural integrity of myelin formed by Schwann cells in the peripheral nervous system (PNS) is required for proper nerve conduction and is dependent on adequate expression of myelin genes including peripheral myelin protein 22 (PMP22). Consequently, excess PMP22 resulting from its genetic duplication and overexpression has been directly associated with the peripheral neuropathy called Charcot-Marie-Tooth disease type 1A (CMT1A), the most prevalent type of CMT. Here, in an attempt to identify transcriptional inhibitors with therapeutic value toward CMT1A, we developed a cross-validating pair of orthogonal reporter assays, firefly luciferase (FLuc) and ß-lactamase (ßLac), capable of recapitulating PMP22 expression, utilizing the intronic regulatory element of the human PMP22 gene. Each compound from a collection of approximately 3,000 approved drugs was tested at multiple titration points to achieve a pharmacological end point in a 1536-well plate quantitative high-throughput screen (qHTS) format. In conjunction with an independent counter-screen for cytotoxicity, the design of our orthogonal screen platform effectively contributed to selection and prioritization of active compounds, among which three drugs (fenretinide, olvanil, and bortezomib) exhibited marked reduction of endogenous Pmp22 mRNA and protein. Overall, the findings of this study provide a strategic approach to assay development for gene-dosage diseases such as CMT1A.

Paclitaxel (Taxol) attenuates clinical disease in a spontaneously demyelinating transgenic mouse and induces remyelination.

Treatment with paclitaxel by four intraperitoneal injections (20 mg/kg) 1 week apart attenuated clinical signs in a spontaneously demyelinating model, if given with onset of clinical signs. If given at 2 months of age (1 month prior to clinical signs), disease was almost completely prevented The astrogliosis, prominent in our model, was reversed by paditaxel as determined by astrocyte counts and quantitation of GFAP. Electron microscopic examination of affected regions at 2.5 months demonstrated that the myelin was generally normal. By 4 months of age, demyelination was common in the superior cerebellar peduncle, maximal at 6 months, but continued to 8 months. In addition to myelin vacuolation and nude axons, the presence of many thin myelin sheaths suggested remyelination or partial demyelination. Although no evidence of oligodendrocyte loss was seen, nuclear changes were observed. To substantiate that remyelination was occurring, we measured MBP (18.5 kDa), MBP-exon II, Golli-MBP, TP8, Golli-MBP-J37, platelet-derived growth factor alpha (PDGFR alpha) and sonic hedgehog (SHH). Of these TP8, PDGFR alpha and SHH were up-regulated in the untreated transgenic. After paditaxel treatment, MBP-Exon II, TP8, PDGFR alpha and SHH were further up-regulated. We concluded that some of the effects of paditaxel were to stimulate proteins involved in early myelinating events possibly via a signal transduction mechanism.

Selenium is required for normal upregulation of myelin genes in differentiating oligodendrocytes.

The purpose of this study was to characterize the selenium requirement for the normal differentiation of oligodendrocyte lineage cells. In primary mixed glial cultures prepared from newborn rat brains, the overall growth of cultures, as seen from the total RNA yield, was not significantly affected by selenium. However, 30 nM selenium was required for the normal upregulation the proteolipid protein, basic protein, and myelin-associated glycoprotein gene expression assessed by Northern blot analysis. Selenium deprivation during initial, rapid phase of the gene upregulation irreversibly suppressed the genes, indicating the existence of a critical period in oligodendrocyte differentiation. In purified oligodendrocyte cultures prepared by mechanical dislodging of progenitor (O-2A) cells from mixed glial cultures, total cell number and total RNA yield were virtually unaffected by selenium deprivation; however, the developmental upregulation of the myelin genes was profoundly attenuated. Immunocytochemical analysis confirmed the suppressive effect of selenium deficiency on the differentiation of oligodendrocyte lineage cells, as seen from a significant decrease in the population of GalC+ and O4+ cells. Because the number of GC+ cells was more reduced than the number of O4+ cells, the results indicate that selenium deficiency may specifically inhibit the progression from immature to mature oligodendrocytes.

Transcriptional regulation of myelin associated glycoprotein gene expression by cyclic AMP.

The treatment of rat glioma C6 cells with 10 microM isoproterenol (Ipt) for 4 days upregulated the expression of the myelin-associated glycoprotein (MAG) gene by approximately 55-fold over the control value. The constant presence of Ipt in the medium was required for the maximal upregulation, as time-restricted exposures to the drug produced only partial, or no upregulation of the gene. No difference in the MAG mRNA stability could be detected in Ipt-treated vs untreated cells indicating that the drug upregulates the MAG gene at transcriptional level. Serum (FCS) strongly attenuated the response of the MAG gene to Ipt. The stimulatory effect of Ipt was profoundly reduced by spermine and H-89, indicating that protein kinase A-dependent protein phosphorylation is involved in the MAG gene activation. Within 30 min after Ipt administration, the c-fos gene was upregulated by 10-fold, and thereafter, its message level decreased and stabilized at approximately 3-fold over control. In contrast, the c-jun gene was downregulated to approximately 20% of control within 30 min after Ipt administration. Subsequently, its message level rose and fell once again within 12 h to approximately half of control, and returned to control level within 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal hypothyroidism affects the timely expression of myelin-associated glycoprotein in the rat brain.

Congenital hypothyroidism strongly affects myelination. To assess the role of thyroid hormone on myelin gene expression, we have studied the effect of hypothyroidism on the steady state levels of myelin-associated glycoprotein (MAG) and its mRNA in rat brain during the first postnatal month. As studied by immunoblot analysis of several brain regions, MAG increased from days 10-15 onwards, reaching constant levels by days 20-25. Hypothyroid samples showed a delay in the accumulation of MAG that was more severe in rostral regions, such as cortex and hippocampus. The effect of hypothyroidism on the accumulation of the protein correlated with mRNA levels. MAG mRNA started to accumulate in the cerebrum of normal animals by postnatal day 7, reaching maximal levels by day 20. Hypothyroid rats showed a delay of several days in the onset of mRNA expression, increasing thereafter at the same rate as in normal animals, and eventually reaching similar values. When individual brain regions were analyzed, we found strong regional differences in the effect of hypothyroidism. The cerebral cortex was most affected, with messenger levels lower than in normal animals at all ages. In more caudal regions differences between control and hypothyroid rats were evident only at the earlier stages of myelination, with spontaneous recovery at later ages. By run on analysis, we found no differences in transcriptional activities of the MAG gene in normal, hypothyroid, or T4-treated rats. Therefore, the effects of hypothyroidism on MAG mRNA and protein levels were most likely caused by decreased mRNA stability. We propose that thyroid hormone contributes to enhanced myelin gene expression by affecting the stability of newly transcribed mRNA in the early phases of myelination.

Relative synthesis of myelin in different brain regions of postnatally undernourished rats.

We used a double isotope procedure and starved and normal littermate rats to compare relative protein synthesis in the cerebellar nuclear, myelin, synaptosomal, mitochondrial, and microsomal subfractions of postnatally starved animals. The remaining brain tissue was dissected into 6 additional regions (cerebral cortex, medulla oblongata, midbrain, hippocampus, striatum, and hypothalamus) and these were frozen for similar subcellular fractionation and analysis at a later date. The microsomal fraction derived from frozen tissues was discarded. The results show that early postnatal starvation specifically depresses myelin synthesis to about the same extent in all major brain regions at 18 and 21 days of age.