OLIG2 is an in vivo bookmarking transcription factor in the developing neural tube in mouse.

Cells possess intrinsic features that are inheritable via epigenetic regulation, such as DNA methylation and histone modification. These inheritable features maintain a unique gene expression pattern, underlying cellular memory. Because of the degradation or displacement of mitotic chromosomes, most transcription factors do not contribute to cellular memory. However, accumulating in vitro evidence indicates that some transcription factors can be retained in mitotic chromosomes called as bookmarking. Such transcription factors may contribute to a novel third mechanism of cellular memory. Since most findings of transcription factor bookmarking have been reported in vitro, little is currently known in vivo. In the neural tube of mouse embryos, we discovered that OLIG2, a basic helix loop helix (bHLH) transcription factor that regulates proliferation of neural progenitors and the cell fate of motoneurons and oligodendrocytes, binds to chromatin through every cell cycle including M-phase. OLIG2 chromosomal localization coincides with mitotic cell features such as the phosphorylation of histone H3, KI67, and nuclear membrane breakdown. Chromosomal localization of OLIG2 is regulated by an N-terminus triple serine motif. Photobleaching analysis revealed slow OLIG2 mobility, suggesting a high affinity of OLIG2 to DNA. In Olig2 N-terminal deletion mutant mice, motoneurons and oligodendrocyte progenitor numbers are reduced in the neural tube, suggesting that the bookmarking regulatory domain is important for OLIG2 function. We conclude that OLIG2 is a de novo in vivo bookmarking transcription factor. Our results demonstrate the presence of in vivo bookmarking in a living organism and illustrate a novel function of transcription factors.

Sulfatide with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs in renal intercalated cells.

Diverse molecular species of sulfatide with differences in FA lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. Immunohistochemical analysis revealed that vacuolar H+-ATPase-positive vesicles were accumulated in intercalated cells in sulfatide-deficient kidneys. Seventeen sulfatide species were detected in the murine kidney by iMScope MALDI-MS analysis. The distribution of the specific sulfatide species was classified into four patterns. Although most sulfatide species were highly expressed in the outer medullary layer, two unique sulfatide species of m/z 896.6 (predicted ceramide structure: t18:0-C22:0h) and m/z 924.6 (predicted ceramide structure: t18:0-C24:0h) were dispersed along the collecting duct, implying expression in intercalated cells. In addition, the intercalated cell-enriched fraction was purified by fluorescence-activated cell sorting using the anti-vacuolar H+-ATPase subunit 6V0A4, which predominantly contained sulfatide species (m/z 896.6 and 924.6). The Degs2 and Fa2h genes, which are responsible for ceramide hydroxylation, were expressed in the purified intercalated cells. These results suggested that sulfatide molecular species with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs, which were characteristically expressed in intercalated cells, were involved in the excretion of NH3 and protons into the urine.

Establishment of neural stem cell culture from the central nervous system of the Iberian ribbed newt Pleurodeles waltl.

Urodele amphibians have exceptional regeneration ability in various organs. Among these, the Iberian ribbed newt (Pleurodeles waltl) has emerged as a useful model organism for investigating the mechanisms underlying regeneration. Neural stem cells (NSCs) are an important source of regeneration in the central nervous system (CNS) and their culture method in vitro has been well established. NSCs form spherical cell aggregates called neurospheres and their formation has been demonstrated in various vertebrates, including some urodele species, but not in P. waltl. In this study, we reported neurosphere formation in brain- and spinal cord-derived cells of post-metamorphic P. waltl. These neurospheres showed proliferative activity and similar expression of marker proteins. However, the surface morphology was found to vary according to their origin, implying that the characteristics of the neurospheres generated from the brain and spinal cord could be similar but not identical. Subsequent in vitro differentiation analysis demonstrated that spinal cord-derived neurospheres gave rise to neurons and glial cells. We also found that cells in neurospheres from P. waltl differentiated to oligodendrocytes, whereas those from axolotls were reported not to differentiate to this cell type under standard culture conditions. Based on our findings, implantation of genetically modified neurospheres together with associated technical advantages in P. waltl could reveal pivotal gene(s) and/or signaling pathway(s) essential for the complete spinal cord regeneration ability in the future.

AUF1, an mRNA decay factor, has a discordant role in Cpeb1 expression.

Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates polyadenylation and subsequent translation of CPE-containing mRNAs involved in various physiological and pathological phenomena. Although the significance of CPEB1-mediated translational regulation has recently been reported, the detailed regulatory mechanism of Cpeb1 expression remains unclear. To elucidate the post-transcriptional regulatory mechanisms of Cpeb1 expression, we constructed reporter plasmids containing various deletions or mutations in the Cpeb1 mRNA 3' untranslated region (3'UTR). We investigated their expression levels in Neuro2a neuroblastoma cells. We found that Cpeb1 expression is regulated through an AU-rich element in its 3'UTR. Furthermore, the mRNA decay factor AU-rich binding factor 1 (AUF1) regulates Cpeb1 expression, and knockdown of AUF1 upregulates Cpeb1 mRNA expression but results in a decrease in CPEB1 protein levels. These findings indicate that AUF1 has a discordant role in the expression of Cpeb1.

Distribution, fine structure, and three-dimensional innervation of lamellar corpuscles in rat plantar skin.

Lamellar corpuscles function as mechanoreceptors in the skin, composed of axon terminals and lamellae constructed by terminal Schwann cells. They are classified into Pacinian, Meissner, and simple corpuscles based on histological criteria. Lamellar corpuscles in rat dermal papilla cells have been reported; however, the morphological aspects have yet to be thoroughly investigated. In the present study, we analyzed the enzyme activity, distribution, fine structure, and three-dimensional innervation of lamellar corpuscles in rat plantar skin. The lamellar corpuscles exhibiting non-specific cholinesterase were densely distributed in rat footpads, evident as notable skin elevations, especially at the apex, the highest portion of the ridges in each footpad. In contrast, only a few lamellar corpuscles were found in other plantar skin areas. Lamellar corpuscle was considered composed of a flat axon terminal Schwann cell lamellae, which were roughly concentrically arranged in the dermal papilla. These histological characteristics correspond to those of the simple corpuscle. Moreover, the axon tracing method revealed that one trunk axon innervated several simple corpuscles. The territory of the trunk axons overlapped with each other. Finally, the animals' footprints were analyzed. During the pausing and walking phases, footpads are often in contact with the floor. These results demonstrate that the type of lamellar corpuscles in the dermal papillae of rat plantar skin is a simple corpuscle and implies that their distribution pattern in the plantar skin is convenient for efficient sensing and transmission of mechanical stimuli from the ground.

Change in phospholipid species of retinal layer in traumatic optic neuropathy model.

Injured optic nerves induce death in almost all retinal ganglion cells (RGC) and cause a loss of axons. To date, we have studied injured RGC axon regeneration by using a traumatic optic nerve injury (TONI) rodent model, and we revealed that axonal regeneration is induced by the graft of an autologous peripheral nerve. The efficient approach to the regeneration of axons thus needs an environmental adjustment of RGC. However, the RGC environment induced by TONI remains unknown. Here, we analyzed female and male C57BL/6 mouse retinal tissue alterations in detail after TONI and focused on the major phospholipid species that are enriched in the whole retina. Reactive astrocyte accumulation, glia scar formation, and demyelination were observed in the injured optic nerve area, while RGC cell death, astrocyte accumulation, and Glial fibrillary acidic protein (GFAP) positive Müller cell increases were detected in the retinal layer. Furthermore, phosphatidylinositol (PI) 18:0/20:4 was localized to three nuclear layer structures: the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer (ONL) in control retina; however, the localization of 18:0/20:4 PI in TONI was disturbed. Meanwhile, phosphatidylserine (PS) 18:0/22:6 showed that the expression was specifically in the inner plexiform layer (IPL) with similar signal intensity in both cases. Other PS species and phosphatidylethanolamine (PE) were differentially localized in the retinal layer; however, the expressions of PE including docosahexaenoic acid (DHA) were affected by TONI. These results suggest that not only GCL but also other retinal layers were influenced by TONI.

Scaffold attachment factor B: distribution and interaction with ERα in the rat brain.

Scaffold attachment factor (SAFB) 1 and its homologue SAFB2 are multifunctional proteins that are involved in various cellular mechanisms, including chromatin organization and transcriptional regulation, and are also corepressors of estrogen receptor alpha (ERα). Both SAFBs are expressed at high levels in the brain. However, the distributions of SAFB1 and SAFB2 have yet to be characterized in detail and it is unclear whether both proteins interact with ERα in the brain. In this study, we investigated the expression and distribution of both SAFBs and their interaction with ERα in adult male rat brain. Immunohistochemical staining showed that SAFB1 and SAFB2 have a similar distribution pattern and are widely expressed throughout the brain. Double-fluorescence immunohistochemical and immunocytochemical analyses in primary cultures showed that the two SAFB proteins are localized in nuclei of neurons, astrocytes, and oligodendrocytes. Of note, SAFB2 was also found in cytoplasmic regions in these cell lineages. Both SAFB proteins were also expressed in ERα-positive cells in the medial preoptic area (MPOA) and arcuate and ventromedial hypothalamic nuclei. Co-immunoprecipitation experiments revealed that both SAFB proteins from the MPOA reciprocally interact with endogenous ERα. These results indicate that, in addition to a role in basal cellular function in the brain, the SAFB proteins may serve as ERα corepressors in hormone-sensitive regions.

Origin of Oligodendrocytes in the Vertebrate Optic Nerve: A Review.

One of the unsolved problems in the research field of oligodendrocyte (OL) development has been the site(s) of origin of optic nerve OLs and its precursor cells (OPCs). It is generally accepted that OLs in the optic nerve are derived from the brain, and thus optic nerve OLs are immigrant cells. We previously demonstrated the brain origin of optic nerve OPCs in chick embryos. However, the site of optic nerve OPC origin has not been examined experimentally in developing rodents for the past two decades. We have recently reported that optic nerve OPCs in mice arise in the preoptic area by E12.5 and gradually migrate caudally and enter the optic nerve. These OPCs give rise to myelinating OLs in the optic nerve in the postnatal or adult stages. Surprisingly, there are species differences with respect to the origin of optic nerve OPCs between chicks and mice. Here, we summarize the site of OPC origin in the optic nerve based on our own previous and recent results, and discuss possible mechanisms underlying these species differences.

Sulfatide species with various fatty acid chains in oligodendrocytes at different developmental stages determined by imaging mass spectrometry.

HSO3-3-galactosylceramide (Sulfatide) species comprise the major glycosphingolipid components of oligodendrocytes and myelin and play functional roles in the regulation of oligodendrocyte maturation and myelin formation. Although various sulfatide species contain different fatty acids, it is unclear how these sulfatide species affect oligodendrogenesis and myelination. The O4 monoclonal antibody reaction with sulfatide has been widely used as a useful marker for oligodendrocytes and myelin. However, sulfatide synthesis during the pro-oligodendroblast stage, where differentiation into the oligodendrocyte lineage has already occurred, has not been examined. Notably, this stage comprises O4-positive cells. In this study, we identified a sulfatide species from the pro-oligodendroblast-to-myelination stage by imaging mass spectrometry. The results demonstrated that short-chain sulfatides with 16 carbon non-hydroxylated fatty acids (C16) and 18 carbon non-hydroxylated fatty acids (C18) or 18 carbon hydroxylated fatty acids (C18-OH) existed in restricted regions of the early embryonic spinal cord, where pro-oligodendroblasts initially appear, and co-localized with Olig2-positive pro-oligodendroblasts. C18 and C18-OH sulfatides also existed in isolated pro-oligodendroblasts. C22-OH sulfatide became predominant later in oligodendrocyte development and the longer C24 sulfatide was predominant in the adult brain. Additionally, the presence of each sulfatide species in a different area of the adult brain was demonstrated by imaging mass spectrometry at an increased lateral resolution. These findings indicated that O4 recognized sulfatides with short-chain fatty acids in pro-oligodendroblasts. Moreover, the fatty acid chain of the sulfatide became longer as the oligodendrocyte matured. Therefore, individual sulfatide species may have unique roles in oligodendrocyte maturation and myelination. Read the Editorial Highlight for this article on page 356.

Sox2 promotes survival of satellite glial cells in vitro.

Sox2 is a transcriptional factor expressed in neural stem cells. It is known that Sox2 regulates cell differentiation, proliferation and survival of the neural stem cells. Our previous study showed that Sox2 is expressed in all satellite glial cells of the adult rat dorsal root ganglion. In this study, to examine the role of Sox2 in satellite glial cells, we establish a satellite glial cell-enriched culture system. Our culture method succeeded in harvesting satellite glial cells with the somata of neurons in the dorsal root ganglion. Using this culture system, Sox2 was downregulated by siRNA against Sox2. The knockdown of Sox2 downregulated ErbB2 and ErbB3 mRNA at 2 and 4 days after siRNA treatment. MAPK phosphorylation, downstream of ErbB, was also inhibited by Sox2 knockdown. Because ErbB2 and ErbB3 are receptors that support the survival of glial cells in the peripheral nervous system, apoptotic cells were also counted. TUNEL-positive cells increased at 5 days after siRNA treatment. These results suggest that Sox2 promotes satellite glial cell survival through the MAPK pathway via ErbB receptors.

Sox2 in the adult rat sensory nervous system.

Sex-determining region Y (SRY)-box 2 (Sox2) is a member of the Sox family transcription factors. In the central nervous system, Sox2 is expressed in neural stem cells from neurogenic regions, and regulates stem cell proliferation and differentiation. In the peripheral nervous system, Sox2 is found only in the immature and dedifferentiated Schwann cells, and is involved in myelination inhibition or N-cadherin redistribution. In the present immunohistochemical study, we found that Sox2 is also expressed in other cells of the adult rat peripheral nervous system. Nuclear Sox2 was observed in all satellite glial cells, non-myelinating Schwann cells, and the majority of terminal Schwann cells that form lamellar corpuscles and longitudinal lanceolate endings. Sox2 was not found in myelinating Schwann cells and terminal Schwann cells of subepidermal free nerve endings. Satellite glial cells exhibit strong Sox2 immunoreactivity, whereas non-myelinating Schwann cells show weak immunoreactivity. RT-PCR confirmed the presence of Sox2 mRNA, indicating that the cells are likely Sox2 expressors. Our findings suggest that the role of Sox2 in the peripheral nervous system may be cell-type-dependent.

G protein-coupled receptor 30 contributes to improved remyelination after cuprizone-induced demyelination.

Estrogen exerts neuroprotective and promyelinating actions. The therapeutic effect has been shown in animal models of multiple sclerosis, in which the myelin sheath is specifically destroyed in the central nervous system. However, it remains unproven whether estrogen is directly involved in remyelination via the myelin producing cells, oligodendrocytes, or which estrogen receptors are involved. In this study, we found that the membrane-associated estrogen receptor, the G protein-coupled receptor 30 (GPR30), also known as GPER, was expressed in oligodendrocytes in rat spinal cord and corpus callosum. Moreover, GPR30 was expressed throughout oligodendrocyte differentiation and promyelinating stages in primary oligodendrocyte cultures derived from rat spinal cords and brains. To evaluate the role of signaling via GPR30 in promyelination, a specific agonist for GPR30, G1, was administered to a rat model of demyelination induced by cuprizone treatment. Histological examination of the corpus callosum with oligodendrocyte differentiation stage-specific markers showed that G1 enhanced oligodendrocyte maturation in corpus callosum of cuprizone-treated animals. It also enhanced oligodendrocyte ensheathment of dorsal root ganglion (DRG) neurons in co-culture and myelination in cuprizone-treated animals. This study is the first evidence that GPR30 signaling promotes remyelination by oligodendrocytes after demyelination. GPR30 ligands may provide a novel therapy for the treatment of multiple sclerosis.

Visualization of Amyloid Oligomers in the Brain of Patients with Alzheimer's Disease.

In the pathogenesis of Alzheimer's disease (AD), highly neurotoxic amyloid-ß (Aß) oligomers appear early, they are thus considered to be deeply involved in the onset of Alzheimer's disease. However, Aß oligomer visualization is challenging in human tissues due to their multiple forms (e.g., low- and high-molecular-weight oligomers, including protofibrils) as well as their tendency to rapidly change forms and aggregate. In this review, we present two visualization approaches for Aß oligomers in tissues: an immunohistochemical (using the monoclonal antibody TxCo1 against toxic Aß oligomer conformers) and imaging mass spectrometry using the small chemical Shiga-Y51 that specifically binds Aß oligomers. TxCo1 immunohistochemistry revealed Aß oligomer distributions in postmortem human brains with AD. Using Shiga-Y51, imaging mass spectrometry revealed Aß oligomer distributions in the brain of a transgenic mouse model for AD. These two methods would potentially contribute to elucidating the pathological mechanisms underlying AD.

Differential responses of endogenous adult mouse neural precursors to excess neuronal excitation.

Adult neurogenesis in the subgranular zone of the hippocampus (SGZ) is enhanced by excess as well as mild neuronal excitation, such as chemoconvulsant-induced brief seizures. Because most studies of neurogenesis after seizures have focused on the SGZ, the threshold of neuronal excitation required to enhance neurogenesis in the subventricular zone (SVZ) is not clear. Therefore, we examined the responses of SVZ precursors to brief generalized clonic seizures induced by a single administration of the chemoconvulsant pentylenetetrazole (PTZ). Cell cycle progression of precursors was analysed by systemic administration of thymidine analogues. We found that brief seizures immediately resulted in cell cycle retardation in the SVZ. However, the same effect was not seen in the SGZ. This initial cell cycle retardation in the SVZ was followed by enhanced cell cycle re-entry after the first round of mitosis, leading to precursor pool expansion, but the cell cycle retardation and expansion of the precursor pool were transient. Cell cycle progression in the PTZ-treated group returned to normal after one cell cycle. The numbers of precursors in the SVZ and new neurons in the olfactory bulb, which are descendants of SVZ precursors, were not significantly different from those in control mice more than 2 days after seizures. Because similar effects were observed following electroconvulsive seizures, these responses are likely to be general effects of brief seizures. These results suggest that neurogenesis in the SVZ is more tightly regulated and requires stronger stimuli to be modified than that in the SGZ.

Cpeb1 expression is post-transcriptionally regulated by AUF1, CPEB1, and microRNAs.

Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates the translation of numerous mRNAs. We previously showed that AU-rich binding factor 1 (AUF1) regulates Cpeb1 expression through the 3' untranslated region (3'UTR). To investigate the molecular basis of the regulatory potential of the Cpeb1 3'UTR, here we performed reporter analyses that examined expression levels of Gfp reporter mRNA containing the Cpeb1 3'UTR. Our findings indicate that CPEB1 represses the translation of Cpeb1 mRNA and that miR-145a-5p and let-7b-5p are involved in the reduction in Cpeb1 expression in the absence of AUF1. These results suggest that Cpeb1 expression is post-transcriptionally regulated by AUF1, CPEB1, and microRNAs.

Cytoplasmic Polyadenylation Element-Binding Protein 1 Post-transcriptionally Regulates Fragile X Mental Retardation 1 Expression Through 3' Untranslated Region in Central Nervous System Neurons.

Fragile X syndrome (FXS) is an inherited intellectual disability caused by a deficiency in Fragile X mental retardation 1 (Fmr1) gene expression. Recent studies have proposed the importance of cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in FXS pathology; however, the molecular interaction between Fmr1 mRNA and CPEB1 has not been fully investigated. Here, we revealed that CPEB1 co-localized and interacted with Fmr1 mRNA in hippocampal and cerebellar neurons and culture cells. Furthermore, CPEB1 knockdown upregulated Fmr1 mRNA and protein levels and caused aberrant localization of Fragile X mental retardation protein in neurons. In an FXS cell model, CPEB1 knockdown upregulated the mRNA levels of several mitochondria-related genes and rescued the intracellular heat shock protein family A member 9 distribution. These findings suggest that CPEB1 post-transcriptionally regulated Fmr1 expression through the 3' untranslated region, and that CPEB1 knockdown might affect mitochondrial function.

Nuclear lamins are differentially expressed in retinal neurons of the adult rat retina.

Lamins are type V intermediate filament proteins that support nuclear membranes. They are divided into A-type lamins, which include lamin A and C, and B-type lamins, which include lamin B1 and B2. In the rat brain, lamin A and C are expressed in relatively equal amounts, while the expressions of lamin B1 and B2 vary depending on the cell type. Lamins play important roles in normal morphogenesis and function. In the nervous system, their abnormal expression causes several neurodegenerative diseases such as peripheral neuropathy, leukodystrophy and lissencephaly. The retina belongs to the central nervous system (CNS) and has widely been used as a source of CNS neurons. We investigated the expression patterns of lamin subtypes in the adult rat retina by immunohistochemistry and found that the staining patterns differed when compared with the brain. All retinal neurons expressed lamin B1 and B2 in relatively equal amounts. In addition, horizontal cells and a subpopulation of retinal ganglion cells expressed lamin A and C, while photoreceptor cells expressed neither lamin A nor C, and all other retinal neurons expressed lamin C only. This differential expression pattern of lamins in retinal neurons suggests that they may be involved in cellular differentiation and expression of cell-specific genes in individual retinal neurons.

Keto form of curcumin derivatives strongly binds to Aß oligomers but not fibrils.

The accumulation of ß-amyloid (Aß) aggregates in the brain occurs early in the progression of Alzheimer's disease (AD), and non-fibrillar soluble Aß oligomers are particularly neurotoxic. During binding to Aß fibrils, curcumin, which can exist in an equilibrium state between its keto and enol tautomers, exists predominantly in the enol form, and binding activity of the keto form to Aß fibrils is much weaker. Here we described the strong binding activity the keto form of curcumin derivative Shiga-Y51 shows for Aß oligomers and its scant affinity for Aß fibrils. Furthermore, with imaging mass spectrometry we revealed the blood-brain barrier permeability of Shiga-Y51 and its accumulation in the cerebral cortex and the hippocampus, where Aß oligomers were mainly localized, in a mouse model of AD. The keto form of curcumin derivatives like Shiga-Y51 could be promising seed compounds to develop imaging probes and therapeutic agents targeting Aß oligomers in the brain.

Myelin glycosphingolipids, galactosylceramide and sulfatide, participate in carbohydrate-carbohydrate interactions between apposed membranes and may form glycosynapses between oligodendrocyte and/or myelin membranes.

Glycosphingolipids (GSLs) can interact with each other by homotypic or heterotypic trans carbohydrate-carbohydrate interactions across apposed membranes, resulting in cell-cell adhesion. This interaction can also provide an extracellular signal which is transmitted to the cytosolic side, thus forming a glycosynapse between two cells. The two major GSLs of myelin, galactosylceramide (GalC) and its sulfated form, galactosylceramide I(3)-sulfate (SGC), are an example of a pair of GSLs which can participate in these trans carbohydrate-carbohydrate interactions and trigger transmembrane signaling. These GSLs could interact across apposed oligodendrocyte membranes at high cell density or when a membranous process of a cell contacts itself as it wraps around the axon. GalC and SGC also face each other in the apposed extracellular surfaces of the multilayered myelin sheath. Communication between the myelin sheath and the axon regulates both axonal and myelin function and is necessary to prevent neurodegeneration. Participation of transient GalC and SGC interactions in glycosynapses between the apposed extracellular surfaces of mature myelin might allow transmission of signals throughout the myelin sheath and thus facilitate myelin-axonal communication.

The localization and non-genomic function of the membrane-associated estrogen receptor in oligodendrocytes.

There is general acceptance that the estrogen receptor can act as a transcription factor. However, estrogens can also bind to receptors that are located at the plasma membrane and stimulate rapid intracellular signaling processes. We recently showed that a membrane-associated estrogen receptor (mER) is present within myelin and at the oligodendrocyte (OL) plasma membrane. To understand the physiological function of mER in OLs, we investigated its cellular localization and involvement in rapid signaling in CG4 cells and OL primary cultures. An ERalpha was expressed along the lacy network of veins in the membrane sheets and in the perikaryon and nucleus in OLs. ERbeta was located in the nucleus, and to a lesser extent along the veins. The expression of ERalpha and ERbeta in OL membranes was confirmed by Western analysis of isolated membranes. OL membranes mainly had truncated forms of ERalpha, 53 and 50 kDa, in addition to some 65 kDa form, whereas ERbeta was a 54 kDa form. CG4 cells and OLs were pulsed with 17alpha- and 17beta-estradiol for various times and the total lysates were analyzed for phosphorylated kinases. Both 17alpha- and 17beta-estradiol elicited rapid phosphorylation of p42/44MAPK, Akt, and GSK-3beta within 8 min. This rapid signaling is consistent with estradiol ligation of a membrane form of ER. Since 17alpha-estradiol is produced at higher concentrations than 17beta-estradiol in the brain of both sexes, signaling via 17alpha-estradiol-liganded mER may have an important function in OLs.