Novel neuronal proteolipid protein isoforms encoded by the human myelin proteolipid protein 1 gene

The human myelin proteolipid protein 1 gene (hPLP1), which encodes the major structural myelin proteins of the central nervous system (CNS), is classically described as expressed in the oligodendrocytes, the CNS myelinating cells. We identified two new exons in the intron 1 of the hPLP1 gene that lead to the expression of additional mRNA and protein isoforms mainly expressed in neurons instead of oligodendrocytes. Those novel neuronal PLP isoforms are detected as soon as human fetal development and their concomitant expression is specific of the human species. As classical PLP proteins, the novel protein isoforms seem to be addressed to the plasma membrane. These results suggest for the first time that PLP may have functions in humans not only in oligodendrocytes but also in neurons and could be implicated in axono-glial communication. Moreover, this neuronal expression of the hPLP1 gene might explain the neuronal dysfunctions in patients carrying hPLP1 gene mutations.     

Selective expression of LDLR and VLDLR in myelinating oligodendrocytes.

Oligodendrocytes form myelin sheaths around axons in the central nervous system. Although cholesterol is one of the major lipid components in myelin sheath, the source of cholesterol for myelination remains to be defined. In this study, we report that low-density lipoprotein receptor (LDLR) and very low-density lipoprotein receptor (VLDLR) are selectively expressed in mature myelinating oligodendrocytes in the postnatal CNS. Both receptors are specifically expressed in differentiated oligodendrocytes in P7 spinal cord, but progressively down-regulated after P15. In adult animals, only LDLR expression can be detected in a small number of oligodendrocytes throughout the entire spinal cord. In the brain region, LDLR is expressed by the white matter oligodendrocytes of both cerebellum and cerebral cortex, whereas VLDLR has a weak expression in cerebellar oligodendrocytes. Together, our expression studies suggest that cholesterol uptake by LDLR and VLDLR may play an important role in the formation of myelin sheath.     

N-acetylaspartate as a reservoir for glutamate

N-acetylaspartate (NAA) is an intermediary metabolite that is found in relatively high concentrations in the human brain. More specifically, NAA is so concentrated in the neurons that it generates one of the most visible peaks in nuclear magnetic resonance (NMR) spectra, thus allowing NAA to serve as "a neuronal marker". However, to date there is no generally accepted physiological (primary) role for NAA. Another molecule that is found at similar concentrations in the brain is glutamate. Glutamate is an amino acid and neurotransmitter with numerous functions in the brain. We propose that NAA, a six-carbon amino acid derivative, is converted to glutamate (five carbons) in an energetically favorable set of reactions. This set of reactions starts when aspartoacylase converts the six carbons of NAA to aspartate and acetate, which are subsequently converted to oxaloacetate and acetyl CoA, respectively. Aspartylacylase is found in astrocytes and oligodendrocytes. In the mitochondria, oxaloacetate and acetyl CoA are combined to form citrate. Requiring two steps, the citrate is oxidized in the Kreb's cycle to alpha-ketoglutarate, producing NADH. Finally, alpha-ketoglutarate is readily converted to glutamate by transaminating the alpha-keto to an amine. The resulting glutamate can be used by multiple cells types to provide optimal brain functional and structural needs. Thus, the abundant NAA in neuronal tissue can serve as a large reservoir for replenishing glutamate in times of rapid or dynamic signaling demands and stress. This is beneficial in that proper levels of glutamate serve critical functions for neurons, astrocytes, and oligodendrocytes including their survival. In conclusion, we hypothesize that NAA conversion to glutamate is a logical and favorable use of this highly concentrated metabolite. It is important for normal brain function because of the brain's relatively unique metabolic demands and metabolite fluxes. Knowing that NAA is converted to glutamate will be important for better understanding myriad neurodegenerative diseases such as Canavan's Disease and Multiple Sclerosis, to name a few. Future studies to demonstrate the chemical, metabolic and pathological links between NAA and glutamate will support this hypothesis.     

Reduction of oligodendrocyte myelin glycoprotein expression following facial nerve transection

Oligodendrocyte myelin glycoprotein (OMgp) has been thought to be expressed in the oligodendrocytes and inhibit the regeneration of the nerves by binding to the Nogo receptor expressed in neurons in the central nervous system (CNS). However, OMgp is expressed in the CNS in the neurons as well as oligodendrocytes. In order to help understanding the physiological role of neuronal OMgp, we examined the change of OMgp expression in the facial nucleus after the facial nerve transection. Real-time RT-PCR and Western blot analysis showed a down-regulation of OMgp expression in the facial nucleus 5-7 (mRNA) or 5-14 (protein) days after transection. Thereafter, expression of OMgp returned to the control level at 28 days after axotomy. Subsequent analysis using in situ hybridization histochemistry and immunohistochemistry established that the decrease of OMgp expression was attributable to the expression in facial motoneurons, but not in oligodendrocytes. These findings suggest a possibility that the change of neuronal OMgp expression might be involved in reconnection of neural circuit between axotomized facial neuron and upper motor neuron after transection.     

Edaravone inhibits lipid peroxidation in neonatal hypoxic-ischemic rats: an in vivo microdialysis study

The occurrence of hypoxia-ischemia (HI) during early fetal or neonatal stages of an individual leads to the damaging of immature neurons resulting in behavioral and psychological dysfunctions. Free radical-mediated lipid peroxidation is the main cause of neurotoxicity including neonatal brain damage. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a novel anti-oxidant agent and the drug of choice in the treatment of acute ischemic brain disorders in adult patient. The purpose of this study is to determine the direct effects of edaravone in inhibiting the lipid peroxidation production in the neonatal rat brains during hypoxic-ischemic insult by electron paramagnetic resonance (EPR) spectoroscopy and in vivo brain microdialysis. Seven-day-old Wistar rats were subjected to left common carotid artery ligation and a probe was inserted in the rat hippocampus. Edaravone (5, 50, or 100 microM) or saline was perfused with a spin trap agent (alpha-(4-pyridyl-N-oxide)-N-tert-butylnitrone; POBN) before, during and after hypoxia (1h of 8% O2 exposure) and then analyzed by EPR. Edaravone (100 microM) did not show any EPR evidence of POBN adduct formation during and after hypoxic-ischemic insult. However, the EPR signal increased, but not significantly during the hypoxic period in the hypoxic and edaravone 50 microM-treated groups compared to control. Edaravone at 5 microM significantly increased the EPR signals compared to control. This study shows that edaravone directly and dose-dependently inhibited the formation of lipid free radicals produced during hypoxic-ischemic insult in the neonatal rat brain. These results suggest that edaravone is able to attenuate neuronal damage in the rat neonatal brain by inhibiting the formation of lipid radicals.     

Oligodendrocyte‐microglia cross‐talk in the central nervous system

Communication between the immune system and the central nervous system (CNS) is exemplified by cross‐talk between glia and neurons shown to be essential for maintaining homeostasis. While microglia are actively modulated by neurons in the healthy brain, little is known about the cross‐talk between oligodendrocytes and microglia. Oligodendrocytes, the myelin‐forming cells in the CNS, are essential for the propagation of action potentials along axons, and additionally serve to support neurons by producing neurotrophic factors. In demyelinating diseases such as multiple sclerosis, oligodendrocytes are thought to be the victims. Here, we review evidence that oligodendrocytes also have strong immune functions, express a wide variety of innate immune receptors, and produce and respond to chemokines and cytokines that modulate immune responses in the CNS. We also review evidence that during stress events in the brain, oligodendrocytes can trigger a cascade of protective and regenerative responses, in addition to responses that elicit progressive neurodegeneration. Knowledge of the cross‐talk between microglia and oligodendrocytes may continue to uncover novel pathways of immune regulation in the brain that could be further exploited to control neuroinflammation and degeneration.     

体内和体外条件下Nogo-A在大鼠小脑颗粒神经元上表达差异的研究

Nogo-A是网膜家族蛋白的成员之一,在抑制成年哺乳动物中枢神经系统损伤后轴突再生的过程中发挥着重要作用。Nogo-A表达于寡突胶质细胞和多种神经元,但在成年动物的小脑颗粒神经元中却未检测到。为探讨Nogo-A在小脑颗粒神经元上的表达情况及其影响因素,本实验应用免疫荧光组织化学染色法研究了Nogo-A蛋白在新生大鼠脑切片上和不同体外培养条件下小脑颗粒神经元中的表达。结果显示:在体条件下Nogo-A蛋白在新生大鼠的小脑颗粒神经元上的表达逐渐减少,至新生14d时检测不到;而在体外培养的来源于新生7d大鼠的小脑颗粒神经元中Nogo-A蛋白持续表达,可维持到14d;与胶质细胞共培养,或加入胶质细胞培养上清的小脑颗粒神经元仍然表达Nogo-A蛋白。本研究结果表明,体内和体外两种条件下Nogo-A蛋白在小脑颗粒神经元上的表达存在差异,新生期Nogo-A在小脑颗粒神经元上的表达下调可能与Purkinje细胞有关,提示Nogo-A在生后发育过程中可能与神经元的迁移或突触的形成密切相关。     

Oxidation induces autonomous activation of protein kinase C Apl I,but not protein kinase C Apl II in homogenates of Aplysia neurons

The longest central nervous system (CNS) specific isoform of the major myelin-associated inhibitor of neurite growth, Nogo-A, has previously been shown to be expressed largely in oligodendrocytes. Using an antibody raised against a recombinant fusion protein comprising amino acids 223-399 of Nogo-A, we show in this report that Nogo-A is also expressed in the cell body of a distinct set of CNS neurons. The antibody detects a single protein band of 220 kDa in rat brain lysate and neuroblastoma cell lysates. Immunofluorescent analyses reveal that Nogo-A is found largely in the endoplasmic reticulum of neuroblastoma cell lines SH-SY5Y and NIE-115. In the mouse CNS, Nogo-A can be found in specific subsets of neuronal cell bodies in addition to oligodendrocytes, but not glial fibrillary associated protein positive astrocytes or MAC-1 positive microglia. Our results provide a conclusive demonstration of the expression of Nogo-A in CNS neurons, which suggests that Nogo-A may have distinct endogenous roles in neurons other than its known ability to inhibit neurite growth.     

Myelin basic protein co-distributes with other PI(4,5)P2-sequestering proteins in Triton X-100 detergent-resistant membrane microdomains

The 18.5 kDa isoform of myelin basic protein (MBP) has recently been shown to sequester phosphatidylinositol-(4,5)-bis-phosphate (PI(4,5)P₂) in vesicular membranes in vitro, as do domains of other membrane- and cytoskeleton-associated proteins such as MARCKS (myristoylated alanine-rich C kinase substrate) and GAP-43 (growth-associated protein of 43 kDa), known collectively as “PI(4,5)P₂-modulins” [Musse et al., Biochemistry, 47 (2008) 10372–10382 (doi:10.1021/bi801302b)]. Here, we demonstrate co-localisation of MBP and MARCKS in primary rat oligodendrocytes, and co-distribution of MBP, MARCKS, and GAP-43 in lipid raft fractions recovered from Triton X-100 detergent-extracted isolated myelin and brain homogenates. The results lend further support to MBP's multifunctionality, particularly as an additional modulator of PI(4,5)P₂ availability in myelin.     

A safety trial of high dose glyceryl triacetate for Canavan disease

Canavan disease (CD MIM#271900) is a rare autosomal recessive neurodegenerative disorder presenting in early infancy. The course of the disease is variable, but it is always fatal. CD is caused by mutations in the ASPA gene, which codes for the enzyme aspartoacylase (ASPA), which breaks down N-acetylaspartate (NAA) to acetate and aspartic acid. The lack of NAA-degrading enzyme activity leads to excess accumulation of NAA in the brain and deficiency of acetate, which is necessary for myelin lipid synthesis. Glyceryltriacetate (GTA) is a short-chain triglyceride with three acetate moieties on a glycerol backbone and has proven an effective acetate precursor. Intragastric administration of GTA to tremor mice results in greatly increased brain acetate levels, and improved motor functions. GTA given to infants with CD at a low dose (up to 0.25 g/kg/d) resulted in no improvement in their clinical status, but also no detectable toxicity. We present for the first time the safety profile of high dose GTA (4.5 g/kg/d) in 2 patients with CD. We treated 2 infants with CD at ages 8 months and 1 year with high dose GTA, for 4.5 and 6 months respectively. No significant side effects and no toxicity were observed. Although the treatment resulted in no motor improvement, it was well tolerated. The lack of clinical improvement might be explained mainly by the late onset of treatment, when significant brain damage was already present. Further larger studies of CD patients below age 3 months are required in order to test the long-term efficacy of this drug.     

Prenatal expression of cholecystokinin (CCK) in the central nervous system (CNS) of mouse

Cholecystokinin (CCK) is a peptide found in both gut and brain. Although numerous studies address the role of brain CCK postnatally, relatively little is known about the ontogeny of CCK expression in the central nervous system (CNS). Recent work revealed that CCK modulates olfactory axon outgrowth and gonadotropin-releasing hormone-1 (GnRH-1) neuronal migration, suggesting that CCK may be an important factor during CNS development. To further characterize the developmental expression of CCK in the nervous system, in situ hybridization experiments were performed. CCK mRNA expression was widely distributed in the developing mouse brain. As early as E12.5, robust CCK expression is detected in the thalamus and spinal cord. By E17.5, cells in the cortex, hippocampus, thalamus and hypothalamus express CCK. In addition, CCK mRNA was also detected in the external zone of the median eminence where axons of the neuroendocrine hypophysiotropic systems terminate. Our study demonstrates that CCK mRNA is expressed prenatally in multiple areas of the CNS, many of which maintain CCK mRNA expression postnatally into adult life. In addition, we provide evidence that regions of the CNS known to integrate hormonal and sensory information associated with reproduction and the GnRH-1 system, expressed CCK already during prenatal development.     

High cholesterol level is essential for myelin membrane growth

Cholesterol in the mammalian brain is a risk factor for certain neurodegenerative diseases, raising the question of its normal function. In the mature brain, the highest cholesterol content is found in myelin. We therefore created mice that lack the ability to synthesize cholesterol in myelin-forming oligodendrocytes. Mutant oligodendrocytes survived, but CNS myelination was severely perturbed, and mutant mice showed ataxia and tremor. CNS myelination continued at a reduced rate for many months, and during this period, the cholesterol-deficient oligodendrocytes actively enriched cholesterol and assembled myelin with >70% of the cholesterol content of wild-type myelin. This shows that cholesterol is an indispensable component of myelin membranes and that cholesterol availability in oligodendrocytes is a rate-limiting factor for brain maturation.     

Blaschko line analogies in the central nervous system: A hypothesis

In X-chromosome-linked skin disorders the pattern of involvement follows Blaschko lines. Patterns of changes analogous to cutaneous Blaschko lines in different X-linked diseases existed in other organs. There is no commonly accepted analogy to Blaschko lines in the central nervous system (CNS). The objective of this study was to consider a hypothesis of the existence of Blaschko lines in the CNS in the example of incontinentia pigmenti (IP). Articles were analyzed in which brain imaging methods were used in IP patients with CNS anomalies. In IP patients with neurological signs brain lesions usually were localized and extended radially. Affected areas did not correspond to territories vascularized by any determined artery. Radially distributed brain lesions morphologically match the radial unit model of cortical development. It can be proposed that in IP in CNS Blaschko line analogies, similar to those in the skin, represent the trace of development of the clone of neurons arising from the cell marked with IKBKG mutation. The hypothesis of the existence of Blaschko line analogies in CNS is supported by radially distributed CNS image findings in IP, the radial unit model of CNS development, and the common embryonic origin of skin, CNS, and eyes.     

Minisegments of newborn rat optic nerves in vitro: gliogenesis and myelination

Summary The question of whether the development of CNS glial cells requires the presence of axons or not can be studied with in vitro systems. In order to compare the differentiation of glial cells during development in vitro with that in situ, we have selected the optic nerve, which is anatomically as well as histotypically a well defined structure. For the in vitro investigations, small explants, called minisegments, of newborn rat optic nerves were cultivated taking four major conditions into account: (1) the regular size of the minisegments should guarantee a permanent exchange of the culture medium in order to avoid cell death, (2) neither mechanical nor enzymatic dissociation of the tissue were applied, (3) the minisegments were explanted into flasks without substrate for cell adhesion and (4) the minisegments were under constant gyratory agitation. The following in situ results were obtained: optic nerves of newborn rats are morphologically characterized by the presence of naked axons, astrocytes, glial precursors, and the absence of both differentiated oligodendrocytes and myelin. At postnatal day 5 myelin sheaths are still absent. Two weeks after birth, differentiated oligodendrocytes and microglial cells are present and numerous axons are surrounded by compact myelin. The in vitro experiments show the following main results, which were obtained after 14 h, 2 d, 5 d and 14 d in culture: during time in culture, the shape of minisegment of newborn rat optic nerves undergoes drastic changes, which indicate high cellular dynamics. After 14 h in vitro, axonal profiles, cells with pyknotic nuclei as well as clusters of astrocytes and glial precursors are present. After 2 days in culture the axonal profiles disappeared and the number of degenerating cells decreased drastically. Many large cells, probably phagocytes containing inclusions and more cells are differentiated. At the stage of 5 d in vitro 4 major types of cells can be distinguished: differentiated oligodendrocytes, which form compact and loose myelin, astrocytes, large and small glioblasts and phagocytes. Immunoprecipitates for myelin basic protein and/or myelin associated glycoprotein were found in oligodendrocytes, in their processes and associated to the myelin. Processes of some astrocytes showed immunoreactive products of glial fibrillary acidic protein. After two weeks in culture, the minisegments were mostly composed of astrocytes, whereas oligodendrocytes became rare and phagocytes disappeared. It can be concluded that CNS glial cells can attain their structural and immunocytochemical characteristics in the total absence of neuronal cell bodies and axons. However, it can be speculated that neurons (or neuronal factors) could regulate the number of astrocytes and oligodendrocytes and keep these glial cells in a physiological equilibrium.     

Regional distribution of importin subtype mRNA expression in the nervous system: study of early postnatal and adult mouse

Importin-alpha and beta1 mediate the translocation of macromolecules bearing nuclear localization signals across the nuclear pore complex. Five importin-alpha isoforms have been identified in mice and six in human. Some of these importins play an important role in neural activity such as long term potentiation, but the functional differences of each isoform in the CNS are still unclear. We performed in situ hybridization (ISH) using non-isotopic probes to clarify the expression patterns of importin-alpha subtypes (alpha5, alpha7, alpha1, alpha4, alpha3) and importin-beta1 in the mouse CNS of adult and early postnatal stages. The mRNAs of the importin-alpha subtypes and importin beta1 were expressed throughout the CNS with specific patterns; importin-alpha5, alpha7, alpha3, and beta1 showed moderate to high expression levels throughout the brain and spinal cord; importin-alpha4 showed a lack of expression in limited regions; and importin-alpha1 showed a low expression level throughout the brain and spinal cord but with a moderate expression level in the olfactory bulb and reticular system. We also demonstrated that importin-alphas and beta1 mRNAs were predominantly expressed in neurons in the adult mouse brain by using double-labeling fluorescence ISH and immunohistochemistry. Moreover, importin-alphas and beta1 mRNAs were detected throughout the CNS of postnatal mice and were highly expressed in the external granule layer of the cerebellar cortex on postnatal days 0, 4, and 10. This is the first report of importin-alphas and beta1 expression throughout the CNS of adult mice, as well as in the developing brain, including cell type specific localization.     

Reduction of oligodendrocyte myelin glycoprotein expression following facial nerve transection

Oligodendrocyte myelin glycoprotein (OMgp) has been thought to be expressed in the oligodendrocytes and inhibit the regeneration of the nerves by binding to the Nogo receptor expressed in neurons in the central nervous system (CNS). However, OMgp is expressed in the CNS in the neurons as well as oligodendrocytes. In order to help understanding the physiological role of neuronal OMgp, we examined the change of OMgp expression in the facial nucleus after the facial nerve transection. Real-time RT-PCR and Western blot analysis showed a down-regulation of OMgp expression in the facial nucleus 5–7 (mRNA) or 5–14 (protein) days after transection. Thereafter, expression of OMgp returned to the control level at 28 days after axotomy. Subsequent analysis using in situ hybridization histochemistry and immunohistochemistry established that the decrease of OMgp expression was attributable to the expression in facial motoneurons, but not in oligodendrocytes. These findings suggest a possibility that the change of neuronal OMgp expression might be involved in reconnection of neural circuit between axotomized facial neuron and upper motor neuron after transection.     

The multi-faceted basis of vitamin B12 (cobalamin) neurotrophism in adult central nervous system: Lessons learned from its deficiency

Glial cells, myelin and the interstitium are the structures of the mammalian central nervous system (CNS) mainly affected by vitamin B₁₂ (cobalamin, Cbl) deficiency. Most of the response to the damage caused by Cbl deficiency seems to come from astrocytes and microglia, and is manifested as an increase in the number of cells positive for glial fibrillary acidic protein, the presence of ultrastructural signs of activation, and changes in cytokine and growth factor production and secretion. Myelin damage particularly affects the lamellae, which are disorganized by edema, as is the interstitium. Surprisingly, rat Schwann cells (myelin-forming cells of the peripheral nervous system) are fully activated but the few oligodendrocytes (myelin-forming cells of the CNS) are scarcely activated. The presence of intramyelin and interstitial edema raises questions about the integrity of the blood–brain barrier and blood–cerebrospinal fluid (CSF) barrier.     

CNS myelination requires cytoplasmic dynein function

Background: Cytoplasmic dynein provides the main motor force for minus‐end‐directed transport of cargo on microtubules. Within the vertebrate central nervous system (CNS), proliferation, neuronal migration, and retrograde axon transport are among the cellular functions known to require dynein. Accordingly, mutations of DYNC1H1, which encodes the heavy chain subunit of cytoplasmic dynein, have been linked to developmental brain malformations and axonal pathologies. Oligodendrocytes, the myelinating glial cell type of the CNS, migrate from their origins to their target axons and subsequently extend multiple long processes that ensheath axons with specialized insulating membrane. These processes are filled with microtubules, which facilitate molecular transport of myelin components. However, whether oligodendrocytes require cytoplasmic dynein to ensheath axons with myelin is not known. Results: We identified a mutation of zebrafish dync1h1 in a forward genetic screen that caused a deficit of oligodendrocytes. Using in vivo imaging and gene expression analyses, we additionally found evidence that dync1h1 promotes axon ensheathment and myelin gene expression. Conclusions: In addition to its well known roles in axon transport and neuronal migration, cytoplasmic dynein contributes to neural development by promoting myelination. Developmental Dynamics 244:134–145, 2015. © 2014 Wiley Periodicals, Inc.     

An hypothesis of function for the avian glycogen body: A novel role for glycogen in the central nervous system

Our own and other recent data have led us to hypothesize that the glycogen body, heretofore generally considered to be metabolically inert, may be functionally geared to support the process of myelin formation in the avian central nervous system (CNS). We envision that the abundant glycogen stores in this tissue, unlike those in the liver or in skeletal muscle, can serve as a recyclable substrate for the ultimate production of reducing equivalents that would be available for the synthesis of myelin lipid cholesterol. In addition, it is suggested that such glycogen also may serve as a source of organic acids which might provide alternate substrates to the CNS under conditions of metabolic stress.     

Development- and activity-dependent regulation of SNAP-25 phosphorylation in rat brain

Synaptosomal-associated protein of 25kDa (SNAP-25), a member of the SNARE proteins essential for neurotransmitter release, is phosphorylated at Ser(187) in PC12 cells and in the rat brain in a protein kinase C-dependent manner. It remains unclear how the phosphorylation of SNAP-25 is regulated during development and by neuronal activity. We studied the mode of SNAP-25 phosphorylation at Ser(187) in the rat brain using an anti-phosphorylated SNAP-25 antibody. Both the expression and phosphorylation of SNAP-25 increased remarkably during the early postnatal period, but their onsets were quite different. SNAP-25 expression was detected as early as embryonic Day 18, whereas the phosphorylation of SNAP-25 could not be detected until postnatal Day 4. A delay in the onset of phosphorylation was also observed in cultured rat hippocampal neurons. The phosphorylation of SNAP-25 was regulated in a neuronal activity-dependent manner and, in the rat hippocampus, decreased by introducing seizures with kainic acid. These results clearly indicated that the phosphorylation of SNAP-25 at Ser(187) is regulated in development- and neuronal activity-dependent manners, and is likely to play important roles in higher brain functions.