Myelin Synthesis Research Articles

An anti-inflammatory role for N-acetyl aspartate in stimulated human astroglial cells

Although N-acetyl-l-aspartate (NAA) has been shown to be important to myelin synthesis and osmotic regulation, the biological rationale for the high levels of NAA found in the brain remains unknown. Here, a human astroglial cell line (STTG) was treated with NAA and stimulated with ionomycin, ionomycin/PMA, or IL-1β. PGE2 levels in ionomycin-stimulated STTG cells decreased by 76% and >95% at NAA concentrations of 10 and 20mM, respectively. NAA also decreased the levels of COX-2 protein and activated NF-κB in IL-1β-stimulated STTG cells but had little effect on unstimulated cells. Also, NAA significantly decreased intracellular calcium levels in ionomycin/PMA-stimulated cells. NAA had no effect on total COX-2 activity or COX-2 mRNA. Acetylation of IκBα kinase, an acetylation target of aspirin, was not observed when NAA was present. These results demonstrate that NAA appears to be important in the modulation of inflammation in the human STTG astroglial cell line. The results of these findings are discussed in relation to neuronal pathologies that exhibit abnormal NAA levels within the brain.

Insulin-like growth factor binding protein-1-6 expression in activated microglia

In the CNS insulin-like growth factor-1 (IGF-1) enhances survival of neurons, promotes myelin synthesis and acts as a mitogen for microglia. The effects of IGF-1 are regulated by a family of 6 IGF binding proteins (IGFBPs). We investigated mRNA expression patterns of IGFBPs in primary rat microglia under basal conditions and after activation with lipopolysaccharide (LPS). Under basal conditions, microglia expressed IGFBP-2 to -6, whereas, IGFBP-1 could not be detected. Following 2 h treatment with LPS mRNA levels for IGFBP-4 and -6 displayed a down regulation, and IGFBP-5 became undetectable. Levels of IGFBP-2 and -3 remained unaltered. Expression patterns of IGFBPs might play an important role in regulating the autocrine/paracrine IGF-1 actions on microglia under inflammatory conditions.

Progesterone up-regulates neuronal brain-derived neurotrophic factor expression in the injured spinal cord

Progesterone (PROG) provides neuroprotection to the injured central and peripheral nervous system. These effects may be due to regulation of myelin synthesis in glial cells and also to direct actions on neuronal function. Recent studies point to neurotrophins as possible mediators of hormone action. Here, we show that the expression of brain-derived neurotrophic factor (BDNF) at both the mRNA and protein levels was increased by PROG treatment in ventral horn motoneurons from rats with spinal cord injury (SCI). Semiquantitative in situ hybridization revealed that SCI reduced BDNF mRNA levels by 50% in spinal motoneurons (control: 53.5±7.5 grains/mm 2 vs. SCI: 27.5±1.2, P<0.05), while PROG administration to injured rats (4 mg/kg/day during 3 days, s.c.) elicited a three-fold increase in grain density (SCI+PROG: 77.8±8.3 grains/mm 2, P<0.001 vs. SCI). In addition, PROG enhanced BDNF immunoreactivity in motoneurons of the lesioned spinal cord. Analysis of the frequency distribution of immunoreactive densities (χ 2: 812.73, P<0.0001) showed that 70% of SCI+PROG motoneurons scored as dark stained whereas only 6% of neurons in the SCI group belonged to this density score category ( P<0.001). PROG also prevented the lesion-induced chromatolytic degeneration of spinal cord motoneurons as determined by Nissl staining. In the normal intact spinal cord, PROG significantly increased BDNF inmunoreactivity in ventral horn neurons, without changes in mRNA levels. Our findings suggest that PROG enhancement of endogenous neuronal BDNF could provide a trophic environment within the lesioned spinal cord and might be part of the PROG activated-pathways to provide neuroprotection.

Steroid hormone signaling between Schwann cells and neurons regulates the rate of myelin synthesis.

Fluorescence digital imaging microscopy was used to develop a method that allows the continuous monitoring and quantitative measurement of a single myelin internode throughout its development. Using this technique, steroid hormones such as progesterone and dexamethasone were shown to reduce the time required for the initiation and to regulate the rate of myelin synthesis. Progesterone was capable of increasing the rate of myelin synthesis in Schwann cell/neuronal co-cultures in a dose-dependent manner. RT-PCR and in situ hydridization studies revealed that the mRNAs for P450scc and 3beta-hydroxysteroid dehydrogenase, the enzymes involved in progesterone biosynthesis, were induced at the onset of myelin synthesis. The progesterone receptor protein translocated into the nucleus of the neurons during myelin synthesis, suggesting that progesterone could also be affecting neuronal gene expression. Changes in gene expression caused by progesterone are being examined to identify additional factors that may control myelin formation.

Differential effects of progestins on the brain

Interactions exist between progestins and the γ-aminobutyric acid (GABA) receptor subtype A where C 21-steroids function as activators. Other interactions between progesterone and neurotransmitter systems include stimulation of dopamine release in striatal tissue, stimulation of GnRH release from hypothalamic neurons and inhibition of opioid receptor binding and activation. Cyproterone acetate increases dopaminergic responses and binds to opiate receptors independently of its classical effect on the androgen receptor. Progesterone substitution in perimenopausal women promotes length and quality of sleep. This effect seems most prominent for progesterone administered vaginally. Progestins also play a role in the pathogenesis of migraine. Migraine symptoms occur predominantly during the perimenstrual stage. Women who suffer from menstrual migraine triggered by premenstrual progesterone loss often benefit from cyclic progesterone administration. This may be because progesterone and allopregnenolone reduce meningeal release of substance P and inhibit the development of neurogenic oedema. Women whose migraine symptoms subside during pregnancy, however, benefit from intramuscular medroxyprogesterone acetate. Progesterone, generated from pregnenolone by Schwann cells, also enhances myelin synthesis. Myelination of axons is promoted when progesterone is added to cultures of rat dorsal root ganglia. No reliable data exist with respect to the effects of other progestins on demyelinating disease. Progestins promote the growth of meningioma as progesterone receptors predominate in meningioma tissue. Progesterone and synthetic progestins should therefore not be prescribed in these patients.

Effect of Iron-Deficiency Anemia on Cognitive Skills and Neuromaturation in Infancy and Childhood

Iron-deficiency anemia in infancy has been consistently shown to negatively influence performance in tests of psychomotor development. In most studies of short-term follow-up, lower scores did not improve with iron therapy, despite complete hematologic replenishment. The negative impact on psychomotor development of iron-deficiency anemia (IDA) in infancy has been well documented in more than a dozen studies during the last two decades. Two studies will be presented here to further support this assertion. Additionally, we will present some data referring to longer follow-up at 5 and 10 years as well as data concerning recent descriptions of the neurologic derangements that may underlie these behavioral effects. To evaluate whether these deficits may revert after long-term observation, a cohort of infants was re-evaluated at 5 and 10 years of age. Two studies have examined children aged 5 years who had anemia as infants using comparable tools of cognitive development showing persisting and consistent important disadvantages in those who were formerly anemic. These tests were better predictors of future achievement than psychomotor scores. These children were again examined at 10 years and showed lower school achievement and poorer fine-hand movements. Studies of neurologic maturation in a new cohort of infants aged 6 months included auditory brain stem responses and naptime 18-lead sleep studies. The central conduction time of the auditory brain stem responses was slower at 6, 12, and 18 months and at 4 years, despite iron therapy beginning at 6 months. During the sleep-wakefulness cycle, heart-rate variability--a developmental expression of the autonomic nervous system--was less mature in anemic infants. The proposed mechanisms are altered auditory-nerve and vagal-nerve myelination, respectively, as iron is required for normal myelin synthesis.

P0 mRNA expression increases during gradual nerve elongation in adult rats

Leg lengthening with nerve elongation is a common clinical treatment. We investigated morphological and molecular changes in peripheral nerves associated with femoral lengthening using animal models. Sciatic nerves of 13 week old male Wistar rats ( n = 35) were elongated indirectly by leg lengthening for 14 days at 1 mm/day. At 3, 7, 14, 21, and 35 days following initiation of elongation, sciatic nerves on the elongated side and contralateral (control) side were excised at the midpoint of the femur. Internodal length was increased by 17%. Light and electron microscopic observation of transverse sections at 14 days showed elongated nerves appearing similar to control nerves with no degenerating axons and normal myelin thickness. We next examined changes of mRNA expression of a major myelin glycoprotein, P0, in elongated nerves using a quantitative reverse transcription–polymerase chain reaction and in situ hybridization. P0 mRNA expression in elongated nerves was increased during the first 3 weeks, with expression reaching 160% of control nerve expression at 14 days. Results of in situ hybridization were confirmatory. We concluded that myelin synthesis occurred during gradual nerve elongation. In adulthood, Schwann cells retain ability to synthesize myelin in response to nerve stretching.

Autoimmunoreactivity to Schwann cells in patients with inflammatory neuropathies.

Inflammatory demyelinating neuropathies are characterized by a loss of peripheral nerve myelin. Myelin breakdown is thought to result from an autoimmune reaction towards nerve components. Schwann cells play a crucial role in the synthesis and maintenance of peripheral nerve myelin. An immune attack targeting Schwann cells could therefore affect myelin integrity, leading to disease. We studied the reactivity of sera from patients with Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) towards Schwann cells using immunofluorescence microscopy. We found 24% of the GBS (56 out of 233) and 26% of the CIDP (12 out of 46) patients to have circulating immunoglobulin G autoantibodies against proliferating, non-myelinating human Schwann cells. In contrast, healthy donors showed positive staining in only two out of 34 sera. No reaction was found with sera from patients with non-inflammatory neurological disorders. Immunofluorescence was localized at the distal tips (leading lamella) of the Schwann cell processes. Distal tips of neurites (nerve-growth-cones) of in vitro differentiated non-myelinated hNT2 neurons also stained strongly. GBS and CIDP serum immunoreactivity was also observed in teased nerve fibre preparations. These data suggest that, at least part of the immunoreactivity is not directed against myelin, but towards non-myelin proteins and epitopes possibly involved in Schwann cell-axon interaction.

Normal metabolism but different physical properties of myelin from mice deficient in proteolipid protein.

Proteolipid protein (PLP) is the primary protein component of CNS myelin, yet myelin from the PLP(null) mouse has only minor ultrastructural abnormalities. Might compensation for a potentially unstable structure involve increased myelin synthesis and turnover? This was not the case; neither accumulation nor in vivo synthesis rates for the myelin-specific lipid cerebroside was altered in PLP(null) mice relative to wild-type (wt) animals. However, the yield of myelin from PLP(null) mice, assayed as levels of cerebroside, was only about 55% of wt control levels. Loss of myelin occurred during initial centrifugation of brain homogenate at 20,000g for 20 min, which is sufficient to sediment almost all myelin from wt mice. Cerebroside-containing fragments from PLP(null) mice remaining in the supernatant could be sedimented by more stringent centrifugation, 100,000g for 60 min. Both the rapidly and the more slowly sedimenting cerebroside-containing membranes banded at the 0.85/0.32 M sucrose interface of a density gradient, as did myelin from wt mice. These results suggest at least some myelin from PLP(null) mice differs from wt myelin with respect to physical stability (fragmented into smaller particles during dispersion) and/or density. Alternatively, slowly sedimenting cerebroside-containing particles could be myelin precursor membranes that, lacking PLP, were retarded in their processing toward mature myelin and thus differ from mature myelin in physical properties. If this is so, recently synthesized cerebroside should be preferentially found in these "slower-sedimenting" myelin precursor fragments. Metabolic tracer experiments showed this was not the case. We conclude that PLP(null) myelin is physically less stable and/or less dense than wt myelin.

Effect of Iron-Deficiency Anemia on Cognitive Skills and Neuromaturation in Infancy and Childhood

Iron-deficiency anemia in infancy has been consistently shown to negatively influence performance in tests of psychomotor development. In most studies of short-term follow-up, lower scores did not improve with iron therapy, despite complete hematologic replenishment.The negative impact on psychomotor development of iron-deficiency anemia (IDA) in infancy has been well documented in more than a dozen studies during the last two decades. Two studies will be presented here to further support this assertion. Additionally, we will present some data referring to longer follow-up at 5 and 10 years as well as data concerning recent descriptions of the neurologic derangements that may underlie these behavioral effects.To evaluate whether these deficits may revert after long-term observation, a cohort of infants was re-evaluated at 5 and 10 years of age. Two studies have examined children aged 5 years who had anemia as infants using comparable tools of cognitive development showing persisting and consistent important disadvantages in those who were formerly anemic. These tests were better predictors of future achievement than psychomotor scores. These children were again examined at 10 years and showed lower school achievement and poorer fine-hand movements. Studies of neurologic maturation in a new cohort of infants aged 6 months included auditory brain stem responses and naptime 18-lead sleep studies. The central conduction time of the auditory brain stem responses was slower at 6, 12, and 18 months and at 4 years, despite iron therapy beginning at 6 months. During the sleep-wake-fulness cycle, heart-rate variability—a developmental expression of the autonomic nervous system—was less mature in anemic infants. The proposed mechanisms are altered auditory-nerve and vagal-nerve myelination, respectively, as iron is required for normal myelin synthesis.

Developmental increase of aspartoacylase in oligodendrocytes parallels CNS myelination

Canavan disease, an autosomal-recessive neurogenetic disorder, is caused by mutations in aspartoacylase, an enzyme that deacetylates N-acetylaspartate to generate free acetate in the brain. Earlier studies have shown that aspartoacylase is primarily restricted to myelin synthesizing cells (oligodendroglia) in the CNS. These findings have led us to investigate the developmental expression of aspartoacylase gene in the rat brain in an attempt to shed more light on the role of this enzyme in myelination. In situ hybridization using a 35S riboprobe based on murine aspartoacylase cDNA was used in this study. The probe hybridized mostly to the white matter tracts with different densities depending on the age of the animal and region of the brain examined. Little or no hybridization signals were detected in the 1-day-old rats, whereas the signal was clearly detectable in most of the white matter regions of the CNS in the 11-day-old rats. The signal density markedly increased at postnatal day 17, the peak of myelination. Thereafter, the hybridization signals decreased somewhat but still could be observed in the adult animals. Thus, the developmental expression pattern of aspartoacylase gene in the postnatal brain closely parallels myelination in the CNS. In the CNS, the hybridization signal of ASPA appeared to be restricted primarily to oligodendrocytes, the primary myelin synthesizing cell type in the CNS. However, the signal was not detectable in rat sciatic nerve (Schwann cells) of the peripheral nervous system. These findings indicate that the role of N-acetylaspartate in myelin synthesis is restricted to the CNS. Furthermore, they provide additional support for the acetate deficiency hypothesis of Canavan disease and also make a stronger case for acetate supplementation as an immediate and inexpensive therapy for Canavan disease.

TNFalpha downregulates PPARdelta expression in oligodendrocyte progenitor cells: implications for demyelinating diseases.

TNFalpha has been implicated in several demyelinating disorders, including multiple sclerosis (MS) and X-adrenoleukodystrophy (X-ALD). TNFalpha abundance is greatly increased in the areas surrounding damaged regions of the central nervous system of patients with MS and X-ALD, but its role in the observed demyelination remains to be elucidated. A class of nuclear receptors, the peroxisome proliferator-activated receptors (PPARs), has been implicated in several physiological and pathological processes. In particular, PPARdelta has been shown to promote oligodendrocyte (OL) survival and differentiation and PPARgamma has been implicated in inflammation. In the present study, we investigate on the effects of TNFalpha on OLs during differentiation in vitro. The results obtained show that TNFalpha treatment impairs PPARdelta expression with concomitant decrease of lignocerolyl-CoA synthase and very-long-chain fatty acid beta-oxidation as well as plasmalogen biosynthesis. We propose a hypothetical model possibly explaining the perturbation effects of proinflammatory cytokines on myelin synthesis, maturation, and turnover.

Aspartoacylase is restricted primarily to myelin synthesizing cells in the CNS: therapeutic implications for Canavan disease

Canavan disease is a devastating neurodegenerative childhood disease caused by mutations in aspartoacylase, an enzyme that deacetylates N-acetylaspartate to generate free acetate in the brain. Localization of aspartoacylase in different cell types in the rat brain was examined in an attempt to understand the pathogenesis of Canavan disease. In situ hybridization histochemistry with a riboprobe based on murine aspartoacylase cDNA was used in this study. The hybridization signal was detectable primarily in the myelin-synthesizing cells, namely oligodendroglia. These findings provide strong additional support for insufficient myelin synthesis as the pathogenic basis of Canavan disease and make a compelling case for acetate supplementation as a simple and noninvasive therapy for this fatal disease with no treatment.

Decreased GFAP-mRNA expression in spinal cord of cobalamin-deficient rats.

We have demonstrated previously that chronic vitamin B12 [cobalamin (Cbl)] deficiency preferentially affects glial cells in the rat central nervous system (CNS) and severely affects peripheral glial cells independently of and concomitantly with the central neuropathy. In this study, we determined the mRNA levels for myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in different CNS areas of rats made Cbl-deficient by total gastrectomy, as well as the mRNA levels for glycoprotein Po and peripheral myelin protein (PMP)22 in the sciatic nerve. GFAP-mRNA levels were significantly decreased in the spinal cord (SC) and hypothalamus, but not in the cortex, hippocampus, or striatum of totally gastrectomized (TGX) rats. No differences in GFAP protein levels were found in the SC and hypothalamus of the TGX rats treated or not with Cbl. MBP-mRNA levels were significantly decreased only in the hypothalamus, and the levels of mRNA for both glial markers returned to normal with Cbl replacement therapy. The levels of mRNA for the various myelin proteins in the sciatic nerve were not modified by Cbl deficiency. These results demonstrate that: a) the neurotrophic action of Cbl in rat CNS occurs in a zonal manner; and b) Cbl deficiency does not affect myelin synthesis (with the sole exception of the hypothalamus).

Deciphering peripheral nerve myelination by using Schwann cell expression profiling.

Although mutations in multiple genes are associated with inherited demyelinating neuropathies, the molecular components and pathways crucial for myelination remain largely unknown. To approach this question, we performed genome-wide expression analysis in several paradigms where the status of peripheral nerve myelination is dynamically changing. Anchor gene correlation analysis, a form of microarray analysis that integrates functional information, using correlation-based clustering, with a statistically rigorous test, the Westfall and Young step-down algorithm, was applied to this data set. Biological pathways active in myelination, genes encoding proteins involved in myelin synthesis, and genes whose mutation results in myelination defects were identified. Many known genes and previously uncharacterized ESTs not heretofore associated with myelination were also identified. One of these ESTs, MASR (myelin-associated SUR4 protein), encodes a member of the SUR4 family of fatty acid desaturases, enzymes involved in elongation of very long chain fatty acids. Its specific localization in myelinating Schwann cells indicates a crucial role for MASR in normal myelin lipid synthesis.

Iron metabolism and requirements in early childhood: do we know enough?: a commentary by the ESPGHAN Committee on Nutrition.

*University of Lancashire, Lancashire, United Kingdom; †University of Milano, Milano, Italy; ‡University of Lund, Malmo, Sweden; §Hopital des Enfants Malades, Paris, France; Hopital Necker Enfants-Malades, Paris, France, ¶Umea University, Umea, Sweden; #University of Munich, Munich, Germany; **Free University of Amsterdam, Amsterdam, The Netherlands; ††Royal Veterinary and Agricultural University, Frederiksberg, Denmark; ‡‡CHUV University Hospital, Lausanne, Switzerland; §§University of Liege, Liege, Belgium; Medical University of Warsaw, Warsaw, Poland; and ¶¶University of Glasgow, Glasgow, United Kingdom

A new algorithm for analysis of oligonucleotide arrays: application to expression profiling in mouse brain regions

Oligonucleotide arrays are a powerful technology for measuring the expression of thousands of genes simultaneously. Improvements in the sensitivity and precision of the measurements, which often pose a challenge to users, would assist the practical application of the technology. Here, we describe a new analysis algorithm for assessing changes in gene expression using oligonucleotide arrays. Changes in expression are detected in terms of the statistical significance ( S-score) of change, which combines signals detected by multiple probe pairs according to an error model characteristic of oligonucleotide arrays. We show that the S-score is sensitive and reliable, enabling us to obtain more consistent results than with existing methods. Cluster analysis of S-score data of four brain regions exhibits patterns that are more distinctive because of improved data quality. In our case study of two mouse brain regions, over 200 genes were identified to have detectable changes between the ventral tegmental area and the prefrontal cortex. The genes with the most distinctive changes are found to be related to myelin or neurofilament synthesis, calcium signaling, and transcription factors. Many of these findings are in agreement with previous studies, using other techniques, such as in situ hybridization. Overall, our findings suggest that this new algorithm may have broad applicability for improving the analysis of oligonucleotide array data.

Glial cells as targets for cytotoxic immune mediators

Oligodendrocytes and Schwann cells are the glia principally responsible for the synthesis and maintenance of myelin. Damage may occur to these cells in a number of conditions, but perhaps the most studied are the idiopathic inflammatory demyelinating diseases, multiple sclerosis in the CNS, and Guillain-Barré syndrome and its variants in the peripheral nervous system (PNS). This article explores the effects on these cells of cytotoxic immunological and inflammatory mediators: similarities are revealed, of which perhaps the most important is the sensitivity of both Schwann cells and oligodendrocytes to many such agents. This area of research is, however, characterised and complicated by numerous and often very substantial inter-observer discrepancies. Marked variability in cell culture techniques, and in assays of cell damage and death, provide artifactual explanations for some of this variability; true inter-species differences also contribute. Not the least important conclusion centres on the limited capacity of in vitro studies to reveal disease mechanisms: cell culture findings merely illustrate possibilities which must then be tested ex vivo using human tissue samples affected by the relevant disease.

Canine distemper infections, with special reference to South Africa, with a review of the literature.

Canine distemper virus is a member of the genus Morbillivirus of the family Paramyxoviridae that causes severe disease in dogs and a range of wild mammals. The clinical signs relate essentially to the respiratory, gastrointestinal and central nervous systems. In South Africa, infection with Ehrlichia canis and canine parvovirus may present similarly Many dogs will initially present with a wide range of central nervous system signs without any history of systemic disease. A recent South African study evaluating ante mortem diagnosis highlighted the importance of recognising clinical signs, cerebrospinal fluid IgG titres, serum IgM titres and immunocytochemistry of epithelial tissue. A 2-year retrospective evaluation of cerebrospinal fluid samples collected from dogs presented to the Onderstepoort Veterinary Academic Hospital indicates that distemper infection is common, and this disease should routinely be suspected in cases of diverse neurological disease in dogs. The South African dog population is specifically at high risk for the disease because of the large pool of unvaccinated, reproductively-active dogs that expose the wildlife resources of the country to risk of fatal disease. Outbreaks of disease in dogs continue to occur in developed and developing communities in both vaccinated and unvaccinated dogs worldwide, and have also been described in a wide range of free-ranging wildlife, including seals, dolphins and lions, and in endangered zoo animals. Modified live virus vaccines have contributed markedly to disease control in the dog population but have caused mortality in some wild carnivores. New recombinant vaccines are being developed that will be safe in wild animals. The pathogenesis of CNS demyelination has been compared to various important demyelinating diseases in humans and, amongst other things, relates to down-regulation of the oligodendrocyte gene coding for myelin synthesis and non-immunocyte CNS cell expression of type II major histocompatibility receptors. Early CNS lesions are characterised by demyelination and later lesions by perivascular round cell cuffing. Treatment is supportive.

Membrane traffic in myelinating oligodendrocytes

In the central nervous system (CNS), the myelin sheath is synthesised by oligodendrocytes as a specialised subdomain of an extended plasma membrane, reminiscent of the segregated membrane domains of polarised cells. Myelination takes place within a relatively short period of time and oligodendrocytes must have adapted membrane sorting and transport mechanisms to achieve such a high rate of myelin synthesis and to maintain the unique organisation of the myelin membrane. In adult life, maintenance of the functional myelin sheath requires a carefully orchestrated balance of myelin synthesis and turnover. Imbalance in these processes may cause dys- or demyelination and disease. This review summarises what is currently known about myelin protein trafficking and mistrafficking in oligodendrocytes. We also present data demonstrating distinct transport pathways for myelin structural proteins and the expression of SNARE proteins in differentiating oligodendrocytes. Myelinating glial cells may well serve as a model system for studying general aspects of membrane trafficking and organisation of membrane domains.