Characterization of the Expression of Vacuolar Protein Sorting 11 (Vps11) in Mammalian Oligodendrocytes.

A founder mutation in human VPS11 (Vacuolar Protein Sorting 11) was recently linked to a genetic leukoencephalopathy in Ashkenazi Jews that presents with the classical features of white matter disorders of the central nervous system (CNS). The neurological deficits include hypomyelination, hypotonia, gradual loss of vision, and seizures. However, the cells expressing the mutation were not identified. Here we describe, using immunocytochemistry, the strong expression of Vps11 in mouse oligodendrocytes and, specifically, its localization with Myelin Associated Glycoprotein (MAG) in the inner tongue of myelin. In longitudinal sections of myelin, it forms a bead-like structure, alternating with Myelin Basic Protein (MBP). Immunofluorescent staining with Vps11 and neurofilament proteins indicates the absence of Vps11 in axons in vivo. Finally, changes in Vps11 expression are associated with altered proteolipid protein (PLP) levels based upon mice with duplications or deletions of the Plp1 gene. To determine potential functional contributions of Vps11, we combined Vps11 with Platelet Derived Growth Factor Receptor-α (PDGFRα) in vitro and in vivo: in both conditions, co-localization of the two proteins was frequently found in round vesicles of OPCs/oligodendrocytes, suggesting retrograde transport for degradation by the endolysosomal system. Neuron-to-glial communication has been invoked to explain degenerative changes in myelin followed by degenerative changes in axons, and vice versa; but to our knowledge, no specific proteins in retrograde transport from the myelin inner tongue to oligodendrocyte perikarya have been identified. The identification of mutations in VPS11 and its localization at the axon-myelin interface should open new avenues of research.

Insertion of proteolipid protein into mitochondria but not DM20 regulates metabolism of cells.

Proteolipid protein (PLP), besides its adhesive role in myelin, has been postulated to have multiple cellular functions. One well-documented function of PLP is regulation of oligodendrocyte (Olg) apoptosis. In contrast, DM20, an alternatively spliced product of the PLP1/Plp1 gene, has been proposed to have functions that are unique from PLP but these functions have never been elucidated. Here, we compare metabolism of PLP and DM20, and show that oxidative phosphorylation (OxPhos) was significantly decreased in Plp1 but not DM20 or EGFP expressing cells. The reserve OxPhos capacity of Plp1 expressing cells was half of control cells, suggesting that they are very vulnerable to stress. ATP in media of Plp1 expressing cells is significantly increased more than two-fold compared to controls; markers of apoptosis are increased in cells over-expressing Plp1, indicating that abnormal metabolism of PLP is most likely the direct cause leading to Olg apoptosis. We hypothesize that abnormal metabolism, mediated by increased insertion of PLP into mitochondria, underlies demyelination in Pelizaeus-Merzbacher Disease (PMD) and in models of PMD. To understand why PLP and DM20 function differently, we mutated or deleted amino acids located in the PLP-specific region. All these mutations and deletions of the PLP-specific region prevented insertion of PLP into mitochondria. These findings demonstrate that the PLP-specific region is essential for PLP's import into mitochondria, and now offer an explanation for deciphering unique functions of PLP and DM20.

Novel pathologic findings in patients with Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is an X-linked inherited hypomyelinating disorder caused by mutations in the gene encoding proteolipid protein (PLP), the major structural protein in central nervous system (CNS) myelin. Prior to our study, whether hypomyelination in PMD was caused by demyelination, abnormally thin sheaths or failure to form myelin was unknown. In this study, we compared the microscopic pathology of myelin from brain tissue of 3 PMD patients with PLP1 duplications to that of a patient with a complete PLP1 deletion. Autopsy tissue procured from PMD patients was embedded in paraffin for immunocytochemistry and plastic for electron microscopy to obtain highresolution fiber pathology of cerebrum and corpus callosum. Through histological stains, immunocytochemistry and electron microscopy, our study illustrates unique pathologic findings between the two different types of mutations. Characteristic of the patient with a PLP1 deletion, myelin sheaths showed splitting and decompaction of myelin, confirming for the first time that myelin in PLP1 deletion patients is similar to that of rodent models with gene deletions. Myelin thickness and g-ratios of some fibers, in relation to axon diameter was abnormally thin, suggesting that oligodendrocytes remain metabolically functional and/or are attempting to make myelin. Many fibers showed swollen, progressive degenerative changes to axons in addition to the dissolution of myelin. All three duplication cases shared remarkable fiber pathology including swellings, constriction and/or transection and involution of myelin. Characteristic of PLP1 duplication patients, many axons showed segmental demyelination along their length. Still other axons had abnormally thick myelin sheaths, suggestive of continued myelination. Thus, each type of mutation exhibited unique pathology even though commonality to both mutations included involution of myelin, myelin balls and degeneration of axons. This pathology study describes findings unique to each mutation that suggests the mechanism causing fiber pathology is likewise heterogeneous.

Glial cell biology in the Great Lakes region.

We report on the tenth bi-annual Great Lakes Glial meeting, held in Traverse City, Michigan, USA, September 27-29 2015. The GLG meeting is a small conference that focuses on current research in glial cell biology. The array of functions that glial cells (astrocytes, microglia, oligodendrocytes, Schwann cells) play in health and disease is constantly increasing. Despite this diversity, GLG meetings bring together scientists with common interests, leading to a better understanding of these cells. This year's meeting included two keynote speakers who presented talks on the regulation of CNS myelination and the consequences of stress on Schwann cell biology. Twenty-two other talks were presented along with two poster sessions. Sessions covered recent findings in the areas of microglial and astrocyte activation; age-dependent changes to glial cells, Schwann cell development and pathology, and the role of stem cells in glioma and neural regeneration.

Insertion of proteolipid protein into oligodendrocyte mitochondria regulates extracellular pH and adenosine triphosphate.

Proteolipid protein (PLP) and DM20, the most abundant myelin proteins, are coded by the human PLP1 and non-human Plp1 PLP gene. Mutations in the PLP1 gene cause Pelizaeus-Merzbacher disease (PMD) with duplications of the native PLP1 gene accounting for 70% of PLP1 mutations. Humans with PLP1 duplications and mice with extra Plp1 copies have extensive neuronal degeneration. The mechanism that causes neuronal degeneration is unknown. We show that native PLP traffics to mitochondria when the gene is duplicated in mice and in humans. This report is the first demonstration of a specific cellular defect in brains of PMD patients; it validates rodent models as ideal models to study PMD. Insertion of nuclear-encoded mitochondrial proteins requires specific import pathways; we show that specific cysteine motifs, part of the Mia40/Erv1 mitochondrial import pathway, are present in PLP and are required for its insertion into mitochondria. Insertion of native PLP into mitochondria of transfected cells acidifies media, partially due to increased lactate; it also increases adenosine triphosphate (ATP) in the media. The same abnormalities are found in the extracellular space of mouse brains with extra copies of Plp1. These physiological abnormalities are preventable by mutations in PLP cysteine motifs, a hallmark of the Mia40/Erv1 pathway. Increased extracellular ATP and acidosis lead to neuronal degeneration. Our findings may be the mechanism by which microglia are activated and proinflammatory molecules are upregulated in Plp1 transgenic mice (Tatar et al. (2010) ASN Neuro 2:art:e00043). Manipulation of this metabolic pathway may restore normal metabolism and provide therapy for PMD patients.

Gait abnormalities and progressive myelin degeneration in a new murine model of Pelizaeus-Merzbacher disease with tandem genomic duplication.

Pelizaeus-Merzbacher disease (PMD) is a hypomyelinating leukodystrophy caused by mutations of the proteolipid protein 1 gene (PLP1), which is located on the X chromosome and encodes the most abundant protein of myelin in the central nervous sytem. Approximately 60% of PMD cases result from genomic duplications of a region of the X chromosome that includes the entire PLP1 gene. The duplications are typically in a head-to-tail arrangement, and they vary in size and gene content. Although rodent models with extra copies of Plp1 have been developed, none contains an actual genomic rearrangement that resembles those found in PMD patients. We used mutagenic insertion chromosome engineering resources to generate the Plp1dup mouse model by introducing an X chromosome duplication in the mouse genome that contains Plp1 and five neighboring genes that are also commonly duplicated in PMD patients. The Plp1dup mice display progressive gait abnormalities compared with wild-type littermates. The single duplication leads to increased transcript levels of Plp1 and four of the five other duplicated genes over wild-type levels in the brain beginning the second postnatal week. The Plp1dup mice also display altered transcript levels of other important myelin proteins leading to a progressive degeneration of myelin. Our results show that a single duplication of the Plp1 gene leads to a phenotype similar to the pattern seen in human PMD patients with duplications.

Synaptic dysfunction in the hippocampus accompanies learning and memory deficits in human immunodeficiency virus type-1 Tat transgenic mice.

BACKGROUND: Human immunodeficiency virus (HIV) associated neurocognitive disorders (HAND), including memory dysfunction, continue to be a major clinical manifestation of HIV type-1 infection. Viral proteins released by infected glia are thought to be the principal triggers of inflammation and bystander neuronal injury and death, thereby driving key symptomatology of HAND. METHODS: We used a glial fibrillary acidic protein-driven, doxycycline-inducible HIV type-1 transactivator of transcription (Tat) transgenic mouse model and examined structure-function relationships in hippocampal pyramidal cornu ammonis 1 (CA1) neurons using morphologic, electrophysiological (long-term potentiation [LTP]), and behavioral (Morris water maze, fear-conditioning) approaches. RESULTS: Tat induction caused a variety of different inclusions in astrocytes characteristic of lysosomes, autophagic vacuoles, and lamellar bodies, which were typically present within distal cytoplasmic processes. In pyramidal CA1 neurons, Tat induction reduced the number of apical dendritic spines, while disrupting the distribution of synaptic proteins (synaptotagmin 2 and gephyrin) associated with inhibitory transmission but with minimal dendritic pathology and no evidence of pyramidal neuron death. Electrophysiological assessment of excitatory postsynaptic field potential at Schaffer collateral/commissural fiber-CA1 synapses showed near total suppression of LTP in mice expressing Tat. The loss in LTP coincided with disruptions in learning and memory. CONCLUSIONS: Tat expression in the brain results in profound functional changes in synaptic physiology and in behavior that are accompanied by only modest structural changes and minimal pathology. Tat likely contributes to HAND by causing molecular changes that disrupt synaptic organization, with inhibitory presynaptic terminals containing synaptotagmin 2 appearing especially vulnerable.

Determinants of central nervous system adult neurogenesis are sex, hormones, mouse strain, age, and brain region.

Multiple sclerosis is a sexually dimorphic (SD) disease that causes oligodendrocyte death, but SD of glial cells is poorly studied. Here, we analyze SD of neural progenitors in 6-8 weeks and 6-8 months normal C57BL/6, SJL/J, and BALB/c mice in the subventricular zone (SVZ), dorsolateral horn (DLC), corpus callosum (CC), and parenchyma. With a short 2-h bromodeoxyuridine (BrdU) pulse, no gender and strain differences are present at 6-8 weeks. At 6-8 months, the number of BrdU(+) cells decreases twofold in each sex, strain, and region, indicating that a common aging mechanism regulates BrdU incorporation. Strikingly, 2× more BrdU(+) cells are found in all brain regions in 6-8 months C57BL/6 females versus males, no gender differences in 6-8 months SJL/J, and fewer BrdU(+) cells in females versus males in BALB/cs. The number of BrdU(+) cells modestly fluctuates throughout the estrous cycle in C57BL/6 and SJLs. Castration causes a dramatic increase in BrdU(+) cells in SVZ and DLC. These findings indicate that testosterone is a major regulator of adult neural proliferation. At 6-8 months, the ratio of PDGFRα(+) cells in the CC to BrdU(+) cells in the DLC of both strains, sexes, estrous cycle, and castrated mice was essentially the same, suggesting that BrdU(+) cells in the DLC differentiate into CC oligodendrocytes. The ratio of TUNEL(+) to BrdU(+) cells does not match proliferation, indicating that these events are differentially regulated. Differential regulation of these two processes leads to the variation in glial numbers between gender and strain. Explanations of neural proliferation based upon data from one sex or strain may be very misleading.

Increased Plp1 gene expression leads to massive microglial cell activation and inflammation throughout the brain.

PMD (Pelizaeus-Merzbacher disease) is a rare neurodegenerative disorder that impairs motor and cognitive functions and is associated with a shortened lifespan. The cause of PMD is mutations of the PLP1 [proteolipid protein 1 gene (human)] gene. Transgenic mice with increased Plp1 [proteolipid protein 1 gene (non-human)] copy number model most aspects of PMD patients with duplications. Hypomyelination and demyelination are believed to cause the neurological abnormalities in mammals with PLP1 duplications. We show, for the first time, intense microglial reactivity throughout the grey and white matter of a transgenic mouse line with increased copy number of the native Plp1 gene. Activated microglia in the white and grey matter of transgenic mice are found as early as postnatal day 7, before myelin commences in normal cerebra. This finding indicates that degeneration of myelin does not cause the microglial response. Microglial numbers are doubled due to in situ proliferation. Compared with the jp (jimpy) mouse, which has much more oligodendrocyte death and hardly any myelin, microglia in the overexpressors show a more dramatic microglial reactivity than jp, especially in the grey matter. Predictably, many classical markers of an inflammatory response, including TNF-α (tumour necrosis factor-α) and IL-6, are significantly up-regulated manyfold. Because inflammation is believed to contribute to axonal degeneration in multiple sclerosis and other neurodegenerative diseases, inflammation in mammals with increased Plp1 gene dosage may also contribute to axonal degeneration described in patients and rodents with PLP1 increased gene dosage.

Different proteolipid protein mutants exhibit unique metabolic defects.

PMD (Pelizaeus-Merzbacher disease), a CNS (central nervous system) disease characterized by shortened lifespan and severe neural dysfunction, is caused by mutations of the PLP1 (X-linked myelin proteolipid protein) gene. The majority of human PLP1 mutations are caused by duplications; almost all others are caused by missense mutations. The cellular events leading to the phenotype are unknown. The same mutations in non-humans make them ideal models to study the mechanisms that cause neurological sequelae. In the present study we show that mice with Plp1 duplications (Plp1tg) have major mitochondrial deficits with a 50% reduction in ATP, a drastically reduced mitochondrial membrane potential and increased numbers of mitochondria. In contrast, the jp (jimpy) mouse with a Plp1 missense mutation exhibits normal mitochondrial function. We show that PLP in the Plp1tg mice and in Plp1-transfected cells is targeted to mitochondria. PLP has motifs permissive for insertion into mitochondria and deletions near its N-terminus prevent its co-localization to mitochondria. These novel data show that Plp1 missense mutations and duplications of the native Plp1 gene initiate uniquely different cellular responses.

Sexual dimorphism in the white matter of rodents.

Sexual dimorphism of astrocytes and neurons is well documented in many brain and spinal cord structures. Sexual dimorphism of oligodendrocytes (Olgs) and myelin has received less attention. We recently showed that density of Olgs in corpus callosum, fornix, and spinal cord of wild-type male rodents is more densely packed than in females; myelin proteins and myelin gene expression are likewise greater in males than in female rodents. However, glial cell proliferation and cell death were two times greater in female corpus callosum. Endogenous sex hormones, specifically lack of androgens, produce an Olg female phenotype in castrated male mouse. In vitro studies using Olgs culture also showed differences between males and females Olg survival and signaling pathways in response to sexual hormones. Sexual dimorphism of white matter tracts and glia in rodents indicates the necessity for controlling gender in the experimental studies of neurodegenerative disorders. Most importantly, our studies suggest that hormones may contribute to sexual dimorphism observed in certain human diseases including multiple sclerosis.

Neuronal loss in Pelizaeus-Merzbacher disease differs in various mutations of the proteolipid protein 1.

Mutations affecting proteolipid protein 1 (PLP1), the major protein in central nervous system myelin, cause the X-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD). We describe the neuropathologic findings in a series of eight male PMD subjects with confirmed PLP1 mutations, including duplications, complete gene deletion, missense and exon-skipping. While PLP1 mutations have effects on oligodendrocytes that result in mutation-specific degrees of dysmyelination, our findings indicate that there are also unexpected effects in the central nervous system resulting in neuronal loss. Although length-dependent axonal degeneration has been described in PLP1 null mutations, there have been no reports on neuronal degeneration in PMD patients. We now demonstrate widespread neuronal loss in PMD. The patterns of neuronal loss appear to be dependent on the mutation type, suggesting selective vulnerability of neuronal populations that depends on the nature of the PLP1 disturbance. Nigral neurons, which were not affected in patients with either null or severe misfolding mutations, and thalamic neurons appear particularly vulnerable in PLP1 duplication and deletion patients, while hippocampal neuronal loss was prominent in a patient with complete PLP1 gene deletion. All subjects showed cerebellar neuronal loss. The patterns of neuronal involvement may explain some clinical findings, such as ataxia, being more prominent in PMD than in other leukodystrophies. While the precise pathogenetic mechanisms are not known, these observations suggest that defective glial functions contribute to neuronal pathology.

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.

Plasticity of neurons and glia following neonatal hypoxic-ischemic brain injury in rats.

Periventricular white matter injury in premature infants is linked to chronic neurological dysfunction. Periventricular white matter injury is caused by many mechanisms including hypoxia-ischemia (HI). Animal models of HI in the neonatal rodent brain can replicate some important features of periventricular white matter injury. Most rodent studies have focused upon early cellular and tissue events following unilateral neonatal HI that is elicited by unilateral carotid artery ligation and followed by timed exposure to moderate hypoxia. Milder hypoxic-ischemic insults elicit preferential white matter injury. Little information is available about long-term cellular effects of unilateral HI. One month after unilateral neonatal hypoxia ischemia, we show that all the components for structural reorganization of the brain are present in moderately injured rats. These components in the injured side include extensive influx of neurites, axonal and dendritic growth cones, abundant immature synapses, and myelination of many small axons. Surprisingly, this neural recovery is often found in and adjacent to cysts that have the ultrastructural features of bone extracellular matrix. In contrast, brains with severe hypoxia ischemia one month after injury still undergo massive neuronal degeneration. While massive destruction of neurons and glia are striking events shortly after brain HI, neural cells re-express their intrinsic properties and attempt an anatomical recovery long after injury.

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. Mu-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.

Anti-IL-16 therapy reduces CD4+ T-cell infiltration and improves paralysis and histopathology of relapsing EAE.

Infiltration of the central nervous system (CNS) by CD4+ Th1 cells precedes onset and relapses of experimental autoimmune encephalomyelitis (EAE). We reported that (B6xSJL) F1 (H-2b/s) mice with severe relapsing-remitting disease had extensive infiltration by CD4+ T cells compared to that in C57BL/6 (B6) (H-2b) mice, which developed mild low-relapsing disease in response to myelin oligodendrocyte peptide 35-55 (MOG(35-55)). This observation led us to search for mechanisms that specifically regulate trafficking of CD4+ cells in relapsing H-2b/s mice. We show that the CD4+ cell chemoattractant cytokine interleukin (IL)-16 has an important role in regulation of relapsing EAE induced by MOG(35-55) in the (B6xSJL) F1 (H-2b/s) mice. We found production of IL-16 in the CNS of mice with EAE. IL-16 levels in the CNS correlated well with the extent of CD4+ T-cell and B-cell infiltration during acute and relapsing disease. Infiltrating CD4+ T cells, B cells, and to a lesser extent CD8+ T cells all contained IL-16 immunoreactivity. Treatment with neutralizing anti-IL-16 antibody successfully reversed paralysis and ameliorated relapsing disease. In treated mice, diminished infiltration by CD4+ T cells, less demyelination, and more sparing of axons was observed. Taken together, our results show an important role for IL-16 in regulation of relapsing EAE. We describe a novel therapeutic approach to specifically impede CD4+ T cell chemoattraction in EAE based on IL-16 neutralization. Our findings have high relevance for the development of new therapies for relapsing EAE and potentially MS.

New oligodendrocytes are generated after neonatal hypoxic-ischemic brain injury in rodents.

Neonatal hypoxic-ischemic (HI) white matter injury is a major contributor to chronic neurological dysfunction. Immature oligodendrocytes (OLGs) are highly vulnerable to HI injury. As little is known about in vivo OLG repair mechanisms in neonates, we studied whether new OLGs are generated after HI injury in P7 rats. Rats received daily BrdU injections at P12-14 or P21-22 and sacrificed at P14 to study the level of cell proliferation or at P35 to permit dividing OLG precursors to differentiate. In P14 HI-injured animals, the number of BrdU+ cells in the injured hemisphere is consistently greater than controls. At P35, sections were double-labeled for BrdU and markers for OLGs, astrocytes, and microglia. Double-labeled BrdU+/myelin basic protein+ and BrdU+/carbonic anhydrase+ OLGs are abundant in the injured striatum, corpus callosum, and the infarct core. Quantitative studies show four times as many OLGs are generated from P21-35 in HI corpora callosa than controls. Surprisingly, the infarct core contains many newly generated OLGs in addition to hypertrophied astrocytes and activated microglia. These glia and non-CNS cells may stimulate OLG progenitor proliferation or induce their migration. At P35, astrogliosis and microgliosis are dramatic ipsilaterally but only a few microglia and some astrocytes are BrdU+. This finding indicates microglial and astrocytic hyperplasia occurs shortly after HI but before the P21 BrdU injections. Although the neonatal brain undergoes massive cell death and atrophy the first week after injury, it retains the potential to generate new OLGs up to 4 weeks after injury within and surrounding the infarct.

Oligodendrocyte progenitor proliferation and maturation is differentially regulated by male and female sex steroid hormones.

Using primary cultures of oligodendrocyte progenitors isolated from male and female neonatal rodent brains, we observed more oligodendrocytes in female-derived compared to male-derived cultures. To determine whether the observed differences were due to a differential effect of sex hormones on proliferation, we treated cultures with increasing doses of 17beta-estradiol, testosterone or progesterone and labeled cells with bromodeoxyuridine to identify cells in S phase. Treatment with 17beta-estradiol, but not progesterone or testosterone, delayed the exit of oligodendrocyte progenitor cells from the cell cycle. In addition, 17beta-estradiol treatment enhanced membrane sheet formation, while progesterone increased cellular branching. Interestingly, the estrogen modulator tamoxifen mimicked the effect of 17beta-estradiol on cell cycle exit, but not on membrane formation. Immunocytochemical localization of estrogen receptors (ERs) showed ERbeta mainly localized to the cytoplasm of oligodendrocytes, suggesting that the effect of 17beta-estradiol on membrane formation could be mediated by interaction with this receptor. We conclude that sex steroids differentially regulate oligodendrocyte progenitor number and myelin formation, possibly contributing to gender-specific differences in repair.

Detection of oligodendrocytes in tissue sections using PCR synthesis of digoxigenin-labeled probes.

Oligodendrocytes, the myelin-forming cells in the central nervous system, were visualized with excellent resolution at the light microscopic level using in situ hybridization (ISH). Digoxigenin (Dig)-tagged probes were synthesized and efficiently labeled by PCR. Specific probes to myelin genes were made by RT from brain total RNAs, followed by PCR with designed specific primers in the presence of Dig-11-dUTP. Probes specific to proteolipid protein (PLP), PLP and its isoform DM20 (PLP/DM20), and myelin oligodendrocyte glycoprotein (MOG) were synthesized and labeled. ISH was then applied on vibratomed tissue sections from mouse brains. Despite a low expression of MOG-specific and PLP-specific mRNAs in adult and newborn mouse brains, an oligodendrocyte population was detected. The specificity of Dig-labeled probes was confirmed with the double labeling of carbonic anhydrase II (CA II) and glial fibrillary acidic protein (GFAP) immunocytochemistry and ISH. This versatile and easy method for synthesis and labeling of specific probes to oligodendrocytes can be also applied to detect many other mRNAs in the nervous system and in other tissues.

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 microM 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.