M. Said Ghandour

Brain dysmyelination and recovery assessment by noninvasive in vivo diffusion tensor magnetic resonance imaging.

Diffusion tensor magnetic resonance imaging (DT‐MRI) was applied for in vivo quantification of myelin loss and regeneration. A transgenic mouse line (Oligo‐TTK) expressing a truncated form of the herpes simplex virus 1 thymidine kinase gene (hsv1‐tk) in oligodendrocytes was studied along with two induced phenotypes of myelin pathology. Myelin loss and axonal abnormalities differentially affect values of DT‐MRI parameters in the brain of transgenic mice. Changes in the anisotropy of the white matter were assessed by calculating and mapping the radial (D⊥) and axial (D∥) water diffusion to axonal tracts and fractional anisotropy (FA). A significant increase in D⊥ attributed to the lack of myelin was observed in all selected brain white matter tracts in dysmyelinated mice. Lower D∥ values were consistent with the histological observation of axonal modifications, including reduced axonal caliber and overexpression of neurofilaments and III β‐tubulin. We show clearly that myelination and axonal changes play a role in the degree of diffusion anisotropy, because FA was significantly decreased in dysmyelinated brain. Importantly, myelin reparation during brain postnatal development induced a decrease in the magnitude of D⊥ and an increase in FA compared with the same brain before recovery. The progressive increase in D∥ values was attributed to the gain in normal axonal morphology. This regeneration was confirmed by the detection of enlarged oligodendrocyte population, newly formed myelin sheaths around additional axons, and a gradual increase in axonal caliber.

Proliferation and death of oligodendrocytes and myelin proteins are differentially regulated in male and female rodents.

Sexual dimorphism of neurons and astrocytes has been demonstrated in different centers of the brain, but sexual dimorphism of oligodendrocytes and myelin has not been examined. We show, using immunocytochemistry and in situ hybridization, that the density of oligodendrocytes in corpus callosum, fornix, and spinal cord is 20–40% greater in males compared with females. These differences are present in young and aged rodents and are independent of strain and species. Proteolipid protein and carbonic anhydrase-II transcripts, measured by real-time PCR, are approximately two to three times greater in males. Myelin basic protein and 2′, 3′-cyclic nucleotide 3′-phosphodiesterase, measured by Western blots, are 20–160% greater in males compared with females. Surprisingly, both generation of new glia and apoptosis of glia, including oligodendrocytes, are approximately two times greater in female corpus callosum. These results indicate that the lifespan of oligodendrocytes is shorter in females than in males. Castration of males produces a female phenotype characterized by fewer oligodendrocytes and increased generation of new glia. These findings indicate that exogenous androgens differentially affect the lifespan of male and female oligodendrocytes, and they can override the endogenous production of neurosteroids. The data imply that turnover of myelin is greater in females than in males. μ-Calpain, a protease upregulated in degeneration of myelin, is dramatically increased at both transcriptional and translational levels in females compared with males. These morphological, molecular, and biochemical data show surprisingly large differences in turnover of oligodendrocytes and myelin between sexes. We discuss the potential significance of these differences to multiple sclerosis, a sexually dimorphic disease, whose progression is altered by exogenous hormones.

Recovery from Chronic Demyelination by Thyroid Hormone Therapy: Myelinogenesis Induction and Assessment by Diffusion Tensor Magnetic Resonance Imaging

The failure of the remyelination processes in multiple sclerosis contributes to the formation of chronic demyelinated plaques that lead to severe neurological deficits. Long-term cuprizone treatment of C57BL/6 mice resulted in pronounced white matter pathology characterized by oligodendrocyte depletion, irreversible demyelination and persistent functional deficits after cuprizone withdrawal. The use of a combination of in vivo diffusion tensor magnetic resonance imaging (DT-MRI) and histological analyses allowed for an accurate longitudinal assessment of demyelination. Injection of triiodothyronine (T3) hormone over a 3 week interval after cuprizone withdrawal progressively restored the normal DT-MRI phenotype accompanied by an improvement of clinical signs and remyelination. The effects of T3 were not restricted to the later stages of remyelination but increased the expression of sonic hedgehog and the numbers of Olig2+ and PSA-NCAM+ precursors and proliferative cells. Our findings establish a role for T3 as an inducer of oligodendrocyte progenitor cells in adult mouse brain following chronic demyelination.

Astrocytic hypertrophy in dysmyelination influences the diffusion anisotropy of white matter

The effect of a proteolipid protein (PLP) mutation on the developing white matter anisotropy was examined by diffusion tensor magnetic resonance imaging (DT‐MRI) in a noninvasive study of a mouse model of Pelizaeus‐Merzbacher disease (PMD). The jimpy PLP mutation in mice produces an irreversible dysmyelination in jimpy males, whereas heterozygous females exhibit a transient hypomyelination, as assessed by a longitudinal study of the same mice during development. Modifications of the different individual DT‐MRI parameters were highlighted by specific changes in tissue structures caused by the mutation that includes the hypomyelination, axonal abnormalities, and recovery. Astrocytic hypertrophy is a striking cellular event in dysmyelinated jimpy brain, where most axons or bundles of fibers are entirely wrapped by astrocyte cytoplasmic processes, so its influences on DT‐MRI parameters in dysmyelination were examined for the first time. DT‐MRI data of the jimpy brain were compared with those obtained from dysmyelination of (oligo‐TTK) transgenic mice, induced by oligodendrocyte killing, which have a mild astrocyte hypertrophy (Jalabi et al., 2005 ), and from recovering jimpy females, which have reduced astrocyte hypertrophy. The unique morphological feature of astrocytes in jimpy males coupled with an increase in the water channel protein aquaporin 4 (AQP4) was found to facilitate the directional water diffusion in the white matter. In addition to the major changes of DT‐MRI parameters in the two dysmyelinated mice caused by the myelin loss and axonal modifications, the amplified magnitude of radial and axial diffusions in jimpy males was attributed principally to the strongly pronounced astrocyte hypertrophy.

25-hydroxycholesterol provokes oligodendrocyte cell line apoptosis and stimulates the secreted phospholipase A2 type IIA via LXR beta and PXR.

In several neurodegenerative diseases of the CNS, oligodendrocytes are implicated in an inflammatory process associated with altered levels of oxysterols and inflammatory enzymes such as secreted phospholipase A2 (sPLA2). In view of the scarce literature related to this topic, we investigated oxysterol effects on these myelinating glial cells. Natural oxysterol 25‐hydroxycholesterol (25‐OH; 1 and 10 μM) altered oligodendrocyte cell line (158N) morphology and triggered apoptosis (75% of apoptosis after 72 h). These effects were mimicked by 22(S)‐OH (1 and 10 μM) which does not activate liver X receptor (LXR) but not by a synthetic LXR ligand (T0901317). Therefore, oxysterol‐induced apoptosis appears to be independent of LXR. Interestingly, sPLA2 type IIA (sPLA2‐IIA) over‐expression partially rescued 158N cells from oxysterol‐induced apoptosis. In fact, 25‐OH, 24(S)‐OH, and T0901317 stimulated sPLA2‐IIA promoter and sPLA2 activity in oligodendrocyte cell line. Accordingly, administration of T0901317 to mice enhanced sPLA2 activity in brain extracts by twofold. Short interfering RNA strategy allowed to establish that stimulation of sPLA2‐IIA is mediated by pregnane X receptor (PXR) at high oxysterol concentration (10 μM) and by LXR β at basal oxysterol concentration. Finally, GC coupled to mass spectrometry established that oligodendrocytes contain oxysterols and express their biosynthetic enzymes, suggesting that they may act through autocrine/paracrine mechanism. Our results show the diversity of oxysterol signalling in the CNS and highlight the positive effects of the LXR/PXR pathway which may open new perspectives in the treatment of demyelinating and neurodegenerative diseases.

An immortalized jimpy oligodendrocyte cell line: Defects in cell cycle and cAMP pathway

Normal and jimpy oligodendrocytes in secondary cultures were transfected with plasmids containing the SV40 T‐antigen gene expressed under the control of the mouse metallothionein‐I promoter. Two immortalized stable cell lines, a normal (158N) and jimpy (158JP) cell line, expressed transcripts and proteins of oligodendrocyte markers, including proteolipid protein (PLP), myelin basic protein (MBP), and carbonic anhydrase II (CAII). Galactocerebroside and sulfatide were also detected with immunocytochemistry. Immunoelectron microscopy using gold particles showed that the truncated endogenous jimpy PLP was distributed throughout the cytoplasm and in association with the plasma membrane of cell bodies and processes. The length of the cell cycle in the jimpy oligodendrocytes in the absence of zinc was 31 h, about a 4‐h longer cell cycle than the normal line. In the presence of 100 μM zinc, the cell cycle became 3 h shorter for both cell lines, with the jimpy cell cycle duration remaining 4 h longer than the normal line. Interestingly, the jimpy cell line showed a significant deficiency in stimulation via the cAMP pathway. While the level of oligodendrocyte markers (PLP, MBP, and CAII) were significantly increased by dibutyryl cAMP (dbcAMP) treatment in the normal cell line, no changes were observed in the jimpy cell lines. This observation, together with previous results showing jimpy oligodendrocytes failure to respond to basic fibroblast growth factor (bFGF), suggests a role for PLP in a signal transduction pathway. Jimpy and normal oligodendrocytes transfected with the SV40T antigen gene, driven by the wild‐type promoter of mouse metallothionein‐I, continue to express properties of oligodendrocytes and therefore provide a powerful model to explore the function of myelin proteins and to dissect the complexity of the jimpy phenotype. GLIA 34:241–252, 2001.

Mitochondrial carbonic anhydrase in the nervous system: expression in neuronal and glial cells.

Abstract: Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C‐terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30‐kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell‐specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion‐induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.

Peroxisomal and mitochondrial status of two murine oligodendrocytic cell lines (158N, 158JP): potential models for the study of peroxisomal disorders associated with dysmyelination processes.

In some neurodegenerative disorders (leukodystrophies) characterized by myelin alterations, the defect of peroxisomal functions on myelin‐producing cells (oligodendrocytes) are poorly understood. The development of in vitro models is fundamental to understanding the physiopathogenesis of these diseases. We characterized two immortalized murine oligodendrocyte cell lines: a normal (158N) and a jimpy (158JP) cell line mutated for the proteolipid protein PLP/DM20. Fluorescence microscopy, flow cytometry, and western blotting analysis allow to identify major myelin proteins (PLP colocalizing with mitochondria; myelin basic protein), oligodendrocyte (CNPase and myelin oligodendrocyte glycoprotein), and peroxisomal markers [adrenoleukodystrophy protein, PMP70, acyl‐CoA oxidase 1 (ACOX1), l‐peroxisomal bifunctional enzyme, and catalase]. Using electron microscopy, peroxisomes were identified in the two cell lines. Gene expression (ATP‐binding cassette, Abcd1, Abcd2, Abcd3, and Acox1) involved in peroxisomal transport or β‐oxidation of fatty acids was evaluated using quantitative PCR. 4‐phenylbutyrate treatment increases expression of ACOX1, l‐peroxisomal bifunctional enzyme, PLP, myelin oligodendrocyte glycoprotein, and CNPase, mainly in 158N cells. In both cell lines, 4‐phenylbutyrate‐induced ACOX1 and catalase activities while only Abcd2 gene was up‐regulated in 158JP. Moreover, the higher mitochondrial activity and content observed in 158JP were associated with higher glutathione content and increased basal production of reactive oxygen species revealing different redox statuses. Altogether, 158N and 158JP cells will permit studying the relationships between peroxisomal defects, mitochondrial activity, and oligodendrocyte functions.

Trafficking of PLP/DM20 and cAMP signaling in immortalized jimpy oligodendrocytes.

The synthesis, transport, and insertion of jimpy proteolipid protein and DM20 were studied in normal (158N) and jimpy (158JP) immortalized oligodendrocyte lines. Four different expression vectors encoding fusion proteins composed of native PLP and DM20 or jimpy PLP or DM20 were linked to enhanced green fluorescent protein (EGFP). All four transfected fusion proteins had similar distributions in the cell bodies and processes of the two cell types. Both normal and jimpy PLP‐EGFP and DM20‐EGFP were detected in both cell lines as far as 200 μm from the cell body, indicating synthesis and transport of mutated PLP and DM20 toward the plasma membrane. Immunocytochemistry of fixed normal and jimpy cells with the O10 antibody, which recognizes a conformationally sensitive PLP/DM20 epitope, confirmed that normal and jimpy PLP and DM20 were transported to the plasma membrane. Live staining of normal and jimpy cells transiently transfected with the native PLP showed positive staining, indicating PLP was correctly inserted into the membrane of both normal and jimpy oligodendrocytes. However, live staining of normal and jimpy cells transiently transfected with jimpy PLP showed no positive staining, indicating the mutated protein is abnormally inserted into the plasma membrane. Electrophysiological recordings of the resting membrane potential measured in the whole cell mode of the patch‐clamp technique showed the absence of a developmentally regulated negative shift in the membrane potential in jimpy cells compared to normal native or immortalized oligodendrocytes. Treatment of 158N cells and native oligodendrocytes with dibutyryl cAMP (dbcAMP) caused morphological and biochemical differentiation, but failed to do so in 158JP cells, suggesting an abnormal signaling pathway in jimpy. The defect in cAMP signaling in jimpy oligodendrocytes was associated with the suppression of increase in mRNA level of the inducible cAMP early repressor (ICER). When the jimpy oligodendrocyte line was transfected with normal PLP or DM20 and exposed to dbcAMP, the cells failed to differentiate. This finding suggests that improper insertion of jimpy protein into the plasma membrane alters the membrane in such a way that certain signaling pathways are permanently altered. The abnormal insertion of jimpy PLP/DM20 into the plasma membrane may be the basis for the lack of cell signaling and abnormal resting potential in jimpy oligodendrocytes. GLIA 40:300–311, 2002.

Recovery of Myelin after Induction of Oligodendrocyte Cell Death in Postnatal Brain

A transgenic mouse line (Oligo-TTK) was established to monitor oligodendrocyte cell death and myelin formation in the CNS. The expression of a conditionally toxic gene, the herpes simplex virus-1 thymidine kinase (HSV1-TK), was made under control of the MBP (myelin basic protein) gene promoter. A truncated form of the HSV1-TK (TTK) gene was used to avoid both bystander effect resulting from leaking in thymidine kinase activity and sterility in transgenic males observed in previous transgenic mice. The transgene was expressed in the CNS with a restricted localization in oligodendrocytes. Oligodendrocyte proliferation and myelin formation are therefore tightly controlled experimentally by administration of ganciclovir (GCV) via the induction of oligodendrocyte cell death. The most severe and irreversible hypomyelination was obtained when GCV was given daily from postnatal day 1 (P1) to P30. Oligodendrocyte plasticity and myelin recovery were analyzed in another phenotype generated by GCV treatment from P1 to P15. In this model, after dysmyelination, an apparent normal behavior was restored with no visible pathological symptoms by P30. Proliferating cells, which may be implicated in myelin repair in this model, are detected primarily in myelin tracts expressing the oligodendrocyte phenotype. Therefore, the endogenous potential of oligodendrocytes to remyelinate was clearly demonstrated in the mice of this study.