SVCT2 Expression and Function in Reactive Astrocytes Is a Common Event in Different Brain Pathologies

Ascorbic acid (AA), the reduced form of vitamin C, acts as a neuroprotector by eliminating free radicals in the brain. Sodium/vitamin C co-transporter isoform 2 (SVCT2) mediates uptake of AA by neurons. It has been reported that SVCT2 mRNA is induced in astrocytes under ischemic damage, suggesting that its expression is enhanced in pathological conditions. However, it remains to be established if SVCT expression is altered in the presence of reactive astrogliosis generated by different brain pathologies. In the present work, we demonstrate that SVCT2 expression is increased in astrocytes present at sites of neuroinflammation induced by intracerebroventricular injection of a GFP-adenovirus or the microbial enzyme, neuraminidase. A similar result was observed at 5 and 10 days after damage in a model of traumatic injury and in the hippocampus and cerebral cortex in the in vivo kindling model of epilepsy. Furthermore, we defined that cortical astrocytes maintained in culture for long periods acquire markers of reactive gliosis and express SVCT2, in a similar way as previously observed in situ. Finally, by means of second harmonic generation and 2-photon fluorescence imaging, we analyzed brain necropsied material from patients with Alzheimer’s disease (AD), which presented with an accumulation of amyloid plaques. Strikingly, although AD is characterized by focalized astrogliosis surrounding amyloid plaques, SVCT2 expression at the astroglial level was not detected. We conclude that SVCT2 is heterogeneously induced in reactive astrogliosis generated in different pathologies affecting the central nervous system (CNS).     

The Na+-dependent l-ascorbic acid transporter SVCT2 expressed in brainstem cells, neurons, and neuroblastoma cells is inhibited by flavonoids

Ascorbic acid (AA) is best known for its role as an essential nutrient in humans and other species. As the brain does not synthesize AA, high levels are achieved in this organ by specific uptake mechanisms, which concentrate AA from the bloodstream to the CSF and from the CSF to the intracellular compartment. Two different isoforms of sodium–vitamin C co-transporters (SVCT1 and SVCT2) have been cloned. Both SVCT proteins mediate high affinity Na⁺-dependent l -AA transport and are necessary for the uptake of vitamin C in many tissues. In the adult brain the expression of SVCT2 was observed in the hippocampus and cortical neurons by in situ hybridization; however, there is no data regarding the expression and distribution of this transporter in the fetal brain. The expression of SVCT2 in embryonal mesencephalic neurons has been shown by RT-PCR suggesting an important role for vitamin C in dopaminergic neuronal differentiation. We analyze SVCT2 expression in human and rat developing brain by RT-PCR. Additionally, we study the normal localization of SVCT2 in rat fetal brain by immunohistochemistry and in situ hybridization demonstrating that SVCT2 is highly expressed in the ventricular and subventricular area of the rat brain. SVCT2 expression and function was also confirmed in neurons isolated from brain cortex and cerebellum. The kinetic parameters associated with the transport of AA in cultured neurons and neuroblastoma cell lines were also studied. We demonstrate two different affinity transport components for AA in these cells. Finally, we show the ability of different flavonoids to inhibit AA uptake in normal or immortalized neurons. Our data demonstrates that brain cortex and cerebellar stem cells, neurons and neuroblastoma cells express SVCT2. Dose-dependent inhibition analysis showed that quercetin inhibited AA transport in cortical neurons and Neuro2a cells.     

Spatio-temporal properties of 5-lipoxygenase expression and activation in the brain after focal cerebral ischemia in rats

The role of 5-lipoxygenase (5-LOX) in brain injury after cerebral ischemia has been reported; however, the spatio-temporal properties of 5-LOX expression and the enzymatic activation are unclear. To determine these properties, we observed post-ischemic 5-LOX changes from 3 h to 14 days after reperfusion in rats with transient focal cerebral ischemia induced by 30 min of middle cerebral artery occlusion. We found that the expression of 5-LOX, both mRNA and protein, was increased in the ischemic core 12-24 h after reperfusion, and in the boundary zone adjacent to the ischemic core 7-14 days after reperfusion. The increased 5-LOX was primarily localized in the neurons in the ischemic core at 24 h, but in the proliferated astrocytes in the boundary zone 14 days after reperfusion. As 5-LOX metabolites, the level of cysteinyl-leukotrienes in the ischemic brain was substantially increased 3 h to 24 h, near control at 3 days, and moderately increased again 7 days after reperfusion; whereas the level of LTB(4) was increased mildly 3 h but substantially 7-14 days after reperfusion. Thus, we conclude that 5-LOX expression and the enzymatic activity are increased after focal cerebral ischemia, and spatio-temporally involved in neuron injury in the acute phase and astrocyte proliferation in the late phase.     

High-affinity sodium-vitamin C co-transporters (SVCT) expression in embryonic mouse neurons

The sodium-vitamin C co-transporters SVCT1 and SVCT2 transport the reduced form of vitamin C, ascorbic acid. High expression of the SVCT2 has been demonstrated in adult neurons and choroid plexus cells by in situ hybridization. Additionally, embryonic mesencephalic dopaminergic neurons express the SVCT2 transporter. However, there have not been molecular and kinetic analyses addressing the expression of SVCTs in cortical embryonic neurons. In this work, we confirmed the expression of a SVCT2-like transporter in different regions of the fetal mouse brain and in primary cultures of neurons by RT-PCR. Kinetic analysis of the ascorbic acid uptake demonstrated the presence of two affinity constants, 103 microM and 8 microM. A K(m) of 103 microM corresponds to a similar affinity constant reported for SVCT2, while the K(m) of 8 microM might suggest the expression of a very high affinity transporter for ascorbic acid. Our uptake analyses also suggest that neurons take up dehydroascorbic acid, the oxidized form of vitamin C, through the glucose transporters. We consider that the early expression of SVCTs transporters in neurons is important in the uptake of vitamin C, an essential molecule for the fetal brain physiology. Vitamin C that is found at high concentration in fetal brain may function in preventing oxidative free radical damage, because antioxidant radical enzymes mature only late in the developing brain.     

Tricellulin expression in brain endothelial and neural cells

Tricellulin is a tight junction (TJ) protein, which is not only concentrated at tricellular contacts but also present at bicellular contacts between epithelial tissues. We scrutinized the brain for tricellulin expression in endothelial and neural cells by using real-time polymerase chain reaction, Western blot and immunohistochemical and immunocytochemical analysis of cultured brain cells and paraffin sections of brain. Tricellulin mRNA was detected in primary cultures and in a cell line of human brain microvascular endothelial cells. Protein expression was confirmed by Western blot and immunofluorescence analysis, which further highlighted the localization of tricellulin in the cell membrane at tricellular and along bicellular contacts, and in the nucleus and perinuclear region. Compared with the well-studied TJ protein, zonula occludens-1, tricellulin expression was less marked at the cell membrane but more evident in the nuclear and perinuclear regions. The presence of tricellulin in cultured endothelial cells was corroborated by immunohistochemical and immunofluorescence staining in brain blood vessels, where it was colocalized with another TJ protein, claudin-5. Tricellulin mRNA was detected in neurons and astrocytes, whereas protein expression was observed in astrocytes but not in neurons, as shown by immunofluorescence analysis. This study reveals the presence and subcellular distribution of tricellulin in brain endothelial cells, both in vitro and in situ and its colocalization with other relevant TJ proteins. Moreover, it demonstrates the expression of the protein in astrocytes opening new avenues for future research to establish the biological significance of tricellulin expression in glial cells.     

Differential regulation of the ascorbic acid transporter SVCT2 during development and in response to ascorbic acid depletion

The sodium-dependent vitamin C transporter-2 (SVCT2) is the only ascorbic acid (ASC) transporter significantly expressed in brain. It is required for life and is critical during brain development to supply adequate levels of ASC. To assess SVCT2 function in the developing brain, we studied time-dependent SVCT2 mRNA and protein expression in mouse brain, using liver as a comparison tissue because it is the site of ASC synthesis. We found that SVCT2 expression followed an inverse relationship with ASC levels in the developing brain. In cortex and cerebellum, ASC levels were high throughout late embryonic stages and early post-natal stages and decreased with age, whereas SVCT2 mRNA and protein levels were low in embryos and increased with age. A different response was observed for liver, in which ASC levels and SVCT2 expression were both low throughout embryogenesis and increased post-natally. To determine whether low intracellular ASC might be capable of driving SVCT2 expression, we depleted ASC by diet in adult mice unable to synthesize ASC. We observed that SVCT2 mRNA and protein were not affected by ASC depletion in brain cortex, but SVCT2 protein expression was increased by ASC depletion in the cerebellum and liver. The results suggest that expression of the SVCT2 is differentially regulated during embryonic development and in adulthood.     

Sodium-dependent vitamin C transporter isoforms in skin: Distribution, kinetics, and effect of UVB-induced oxidative stress

Two sodium-dependent vitamin C transporter isoforms (SVCT1 and SVCT2) were identified as ascorbic acid transporters, but their roles in skin have, as yet, not been elucidated. Here we analyze the expression and function of SVCTs in healthy human skin cells and skin tissues, and in UVB-induced cutaneous tissue injury. SVCT1 was primarily found in the epidermis expressed by keratinocytes, whereas SVCT2 expression was in the epidermis and dermis in keratinocytes, fibroblasts, and endothelial cells. Uptake experiments revealed that ascorbic acid affinity of SVCT1 was lower than SVCT2 (K(m)=75 muM and K(m)=44 muM, respectively), but maximal velocity was 9-times higher (36 nmol/min/well). In keratinocytes, SVCT1 was found to be responsible for vitamin C transport, although SVCT2 gene expression was higher. On UVB irradiation, SVCT1 mRNA expression in murine skin declined significantly in a time- and dose-dependent manner, whereas SVCT2 mRNA levels were unchanged. Furthermore, UVB irradiation of keratinocytes in vitro was accompanied by reduced ascorbic acid transport. In summary, these data indicate that the two vitamin C transporter isoforms fulfill specific functions in skin: SVCT1 is responsible for epidermal ascorbic acid supply, whereas SVCT2 mainly facilitates ascorbic acid transport in the dermal compartment. UVB-induced oxidative stress in mice resulted in depletion of SVCT1 mRNA levels and led to significantly decreased ascorbic acid uptake in keratinocytes, providing evidence on why ascorbic acid levels are decreased on UVB irradiation in vivo.     

Glial perspectives of metabolic states during cerebral hypoxia--calcium regulation and metabolic energy

Cooperation between astrocytes and neurons is a unique interaction between two highly specialized cell types of the brain. Therefore, lack of nutrient supply during ischemia requires tight coordination of metabolism between astrocytes and neurons to keep the brain functions intact. To understand the impact of energy limitation on astrocytes, the functions of astrocytes have to be considered: (i) supplementation of neuronal cells, (ii) modulation of the extracellular milieu, mainly of the glutamate level, and (iii) elimination of reactive oxygen species (ROS). In cultured astrocytes and neurons inhibition of oxidative phosphorylation, using rotenone, was tested. Interestingly, this had only a negligible effect on Ca2+ homeostasis in astrocytes, even in combination with a severe glutamate stress. In contrast, in neurons glutamate in the presence of rotenone induced Ca2+ deregulation. Ca2+ homeostasis is very critical for cell survival. A massive and prolonged Ca2+ rise will lead to deregulation of many processes in such a way that the cells affected can hardly survive. Ca2+ homeostasis depends on the energy-consuming processes, which maintain the steep gradient between intracellular and extracellular Ca2+ concentration. Deprivation of oxygen and glucose during ischemia leads to a depletion of ATP in the brain, due to inhibited glycolytic and mitochondrial activity, whereas energy-consuming processes like ion pumps drain the ATP pools. On the other hand, specific mechanisms can protect brain structures against the massive insult of ischemia. Glycogen, stored in astrocytes, can maintain both neurons and astrocytes alive during short limitation of oxygen and glucose. Moreover, astrocytes can fuel ATP generation by providing lactate for neurons.     

Ginkgolide B preconditioning on astrocytes promotes neuronal survival in ischemic injury via up-regulating erythropoietin secretion

Although ischemic preconditioning (IP) can provide powerful protection on brain against ischemic insult, it is rarely used in clinic to prevent the occurrence of ischemic stroke because of safety concerns. It is therefore necessary to seek the safer stimuli to initiate pharmacological preconditioning. Our previous work demonstrated that ginkgolide B (GB) could protect neurons against ischemia-induced apoptosis. Astrocytes are the most numerous cells in mammalian central nervous system and there is a close bi-directional communication between neurons and astrocytes in brain. Besides neurons, whether GB can exert the role of preconditioning on astrocytes through which to further improve neuronal survival under ischemic condition is not yet known. In the present study, primary cultured astrocytes were treated with GB for 24 h or short-term ischemia (ischemia for 3 h, as ischemic preconditioning/IP), and then cultured back to normoxia and normal medium for 24 h to induce the preconditioning response. Astrocyte-conditioned medium (ACM) was then collected and used to incubate the cultured neurons for 24 h before neurons were subjected to severe ischemia. Our results demonstrated that not only GB and IP increased astrocytic viability in ischemia, but also the conditioned medium from astrocytes treated with GB or IP increased cell viability and decreased the number of apoptosis of neurons in ischemia. We also found that GB and IP significantly stimulated astrocytes to express and secrete erythropoietin (EPO) into ACM, and the addition of anti-EPO antibody blocked the protective effect of GB or IP-treated astrocytes culture medium on neurons in ischemia. Further study of above protection revealed that ACM from astrocytes treated with GB or IP induced the inactivation of proapoptotic factor Bad by phosphorylation at serine 136 and 112 (¹³⁶p-Bad and ¹¹²p-Bad) in neurons. Together, our results suggest that GB is capable of preconditioning on astrocytes as IP and then protects neurons against ischemia-induced apoptosis, which is mediated by EPO.     

Neurotensin type 2 receptor is involved in fear memory in mice

Neurotensin receptor subtype 2 (Ntsr2) is a levocabastine-sensitive neurotensin receptor expressed diffusely throughout the mouse brain. Previously, we found that Ntsr2-deficient mice have an abnormality in the processing of thermal nociception. In this study, to examine the involvement of Ntsr2 in mouse behavior, we performed a fear-conditioning test in Ntsr2-deficient mice. In the contextual fear-conditioning test, the freezing response was significantly reduced in Ntsr2-deficient mice compared with that of wild-type mice. This reduction was observed from 1 h to 3 weeks after conditioning, and neither shock sensitivity nor locomotor activity was altered in Ntsr2-deficient mice. In addition, we found that Ntsr2 mRNA was predominantly expressed in cultured astrocytes and weakly expressed in cultured neurons derived from mouse brain. The combination of in situ hybridization and immunohistochemistry showed that Ntsr2 mRNA was dominantly expressed in glial fibrillary acidic protein positive cells in many brain regions including the hypothalamus, while Ntsr2 gene was co-expressed with neuron-specific microtubule associated protein-2 in limited numbers of cells. These results suggest that Ntsr2 in astrocytes and neurons may have unique function like a modulation of fear memory in the mouse brain.     

SVCT2 transporter expression is post‐natally induced in cortical neurons and its function is regulated by its short isoform

Different studies have demonstrated the importance of micronutrients, such as vitamins, for normal adult brain function and development. Vitamin C is not synthesized in the brain, but high levels are detected in this organ because of the existence of specific uptake mechanisms, which concentrate ascorbic acid from the bloodstream to the cerebrospinal fluid and then into neurons and glial cells. Two different isoforms of sodium–vitamin C cotransporters (SVCT1 and SVCT2) have been cloned. SVCT2 expression has been observed in the adult hippocampus and cortical neurons by in situ hybridization. In addition, the localization of SVCT2 in the rat fetal brain has been studied by immunohistochemistry and in situ hybridization, demonstrating that SVCT2 is highly expressed in the ventricular and subventricular areas of the brain cortex. However, there are currently no immunohistochemical data regarding SVCT2 expression and function in the post‐natal brain. Therefore, we analyzed SVCT2 expression in the developing brain cortex of mice, and demonstrated an increase in SVCT2 mRNA in mice at 1–15 days of age. The expression of a short isoform, SVCT2sh, was also detected within the same period. SVCT2 expression was concentrated in neurons within the inner layer of the brain cortex. Both SVCT2 isoforms were coexpressed in N2a cells to obtain functional data. Fluorescence resonance energy transfer analysis revealed a molecular interaction between SVCT2wt and SVCT2sh. Finally, differences in transport ratios suggested that SVCT2sh expression inhibited ascorbic acid uptake in N2a cells when both isoforms were coexpressed.

     

SVCT2 Is Expressed by Cerebellar Precursor Cells,Which Differentiate into Neurons in Response to Ascorbic Acid

Ascorbic acid (AA) is a known antioxidant that participates in a wide range of processes, including stem cell differentiation. It enters the cell through the sodium-ascorbate co-transporter SVCT2, which is mainly expressed by neurons in the adult brain. Here, we have characterized SVCT2 expression in the postnatal cerebellum in situ, a model used for studying neurogenesis, and have identified its expression in granular precursor cells and mature neurons. We have also detected SVCT2 expression in the cerebellar cell line C17.2 and in postnatal cerebellum-derived neurospheres in vitro and have identified a tight relationship between SVCT2 expression and that of the stem cell-like marker nestin. AA supplementation potentiates the neuronal phenotype in cerebellar neural stem cells by increasing the expression of the neuronal marker β III tubulin. Stable over-expression of SVCT2 in C17.2 cells enhances β III tubulin expression, but it also increases cell death, suggesting that AA transporter levels must be finely tuned during neural stem cell differentiation.     

Identification of novel mRNAs expressed in oligodendrocytes

To identify new proteins, which are expressed in oligodendrocytes and which may have a functional role in myelination, a rat oligodendrocyte cDNA library was screened using differential and subtractive screening techniques. Ten clones that have elevated levels of expression in brain were isolated. Two of these clones were characterized further and one clone, pC26.H2, was found to be closely related to mouse stearoyl-CoA desaturase 2 (SCD2), which catalyzes the synthesis of unsaturated fatty acid. From Northern blot and in situ hybridization studies, SCD2 mRNA was expressed primarily in brain with lower levels found in lung and spleen. In brain sections, SCD2 mRNA was found primarily in oligodendrocytes, although mRNA was detected at a low level in neurons, in particular in Purkinje cells in the cerebellum. Northern blot analysis of the other clone, p973.HB, indicated that it was expressed more selectively in brain. In mixed glial cultures oligodendrocytes were the only cells that expressed this mRNA, whereas in brain, neurons expressed this mRNA at a higher level than in oligodendrocytes. This clone is being actively pursued because of its unique expression exclusively in oligodendrocytes and neurons.Special issue dedicated to Dr. Marjorie B. Lees.     

Temporal differences in microRNA expression patterns in astrocytes and neurons after ischemic injury

MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules that modulate gene translation. Their expression is altered in many central nervous system (CNS) injuries suggesting a role in the cellular response to stress. Current studies in brain tissue have not yet described the cell-specific temporal miRNA expression patterns following ischemic injury. In this study, we analyzed the expression alterations of a set of miRNAs in neurons and astrocytes subjected to 60 minutes of ischemia and collected at different time-points following this injury. To mimic ischemic conditions and reperfusion in vitro, cortical primary neuronal and astrocytic cultures prepared from fetal rats were first placed in oxygen and glucose deprived (OGD) medium for 60 minutes, followed by their transfer into normoxic pre-conditioned medium. Total RNA was extracted at different time-points after the termination of the ischemic insult and the expression levels of miRNAs were measured. In neurons exposed to OGD, expression of miR-29b was upregulated 2-fold within 6 h and up to 4-fold at 24 h post-OGD, whereas induction of miR-21 was upregulated 2-fold after 24 h when compared to expression in neurons under normoxic conditions. In contrast, in astrocytes, miR-29b and miR-21 were upregulated only after 12 h. MiR-30b, 107, and 137 showed expression alteration in astrocytes, but not in neurons. Furthermore, we show that expression of miR-29b was significantly decreased in neurons exposed to Insulin-Like Growth Factor I (IGF-I), a well documented neuroprotectant in ischemic models. Our study indicates that miRNAs expression is altered in neurons and astrocytes after ischemic injury. Furthermore, we found that following OGD, specific miRNAs have unique cell-specific temporal expression patterns in CNS. Therefore the specific role of each miRNA in different intracellular processes in ischemic brain and the relevance of their temporal and spatial expression patterns warrant further investigation that may lead to novel strategies for therapeutic interventions.     

Post-ischemic transcriptional and translational responses of EC-SOD in mouse brain and serum

Extracellular superoxide dismutase (EC-SOD) is neuroprotective, but its role in cerebral ischemia remains to be determined. We herein describe the topographical localization and quantitative changes in EC-SOD and its mRNA expression following cerebral ischemia in mice. Mice were subjected to transient forebrain ischemia and varied intervals of reperfusion. The measurements of EC-SOD using ELISA showed increased brain EC-SOD after 24 h of reperfusion and an increase in EC-SOD brain/serum ratio after 3 h. The immunohistochemical examination in normal mice showed strong EC-SOD immunoreactivity in the choroid plexus, pia mater, and ventral tuberal area of the hypothalamus. EC-SOD immunoreactivity in cortical and striatal capillary wall was conspicuous after 3 h. EC-SOD immunoreactivity was also noted in cortical neurons after 24 h. Northern blot analysis showed an increased EC-SOD mRNA expression in the brain after 24 h. An in situ hybridization study in normal mice demonstrated the mRNA expression of EC-SOD in choroid plexus and neurons through the brain especially in the cortex or ventral tuberal area of the hypothalamus, but demonstrated no mRNA expression of EC-SOD in the capillary wall. These findings suggest that EC-SOD accumulates on endothelial cells in response to this injury by an unknown mechanism, while cortical neurons produce EC-SOD themselves after cerebral ischemia with reperfusion.     

Autophagy was activated in injured astrocytes and mildly decreased cell survival following glucose and oxygen deprivation and focal cerebral ischemia

The present study evaluated autophagy activation in astrocytes and its contribution to astrocyte injury induced by cerebral ischemia and hypoxia. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO) in rats. In vitro hypoxia in cultured primary astrocytes was induced by the oxygen-glucose deprivation (OGD). Alterations of astrocytes were evaluated with astroglia markers glial fibrillary acidic protein (GFAP). The formation of autophagosomes in astrocytes was examined with transmission electron microscopy (TEM). The expression of autophagy-related proteins were examined with immunoblotting. The role of autophagy in OGD or focal cerebral ischemia-induced death of astrocytes was assessed by pharmacological inhibition of autophagy with 3-methyladenine (3-MA) or bafilomycin A1 (Baf). The results showed that GFAP staining was reduced in the infarct brain areas 3-12 h following pMCAO. Cerebral ischemia or OGD induced activation of autophagy in astrocytes as evidenced by the increased formation of autophagosomes and autolysosomes and monodansylcadaverine (MDC)-labeled vesicles; the increased production of microtubule-associated protein 1 light chain 3 (LC3-II); the upregulation of Beclin 1, lysosome-associated membrane protein 2 (LAMP2) and lysosomal cathepsin B expression; and the decreased levels of cytoprotective Bcl-2 protein in primary astrocytes. 3-MA inhibited OGD-induced the increase in LC3-II and the decline in Bcl-2. Furthermore, 3-MA and Baf slightly but significantly attenuated OGD-induced death of astrocytes. 3-MA also significantly increased the number of GFAP-positive cells and the protein levels of GFAP in the ischemic cortex core 12 h following pMCAO. These results suggest that ischemia or hypoxia-induced autophagic/lysosomal pathway activation may at least partly contribute to ischemic injury of astrocytes.     

Astroglial regulation of apolipoprotein E expression in neuronal cells. Implications for Alzheimer's disease

Although apolipoprotein (apo) E is synthesized in the brain primarily by astrocytes, neurons in the central nervous system express apoE, albeit at lower levels than astrocytes, in response to various physiological and pathological conditions, including excitotoxic stress. To investigate how apoE expression is regulated in neurons, we transfected Neuro-2a cells with a 17-kilobase human apoE genomic DNA construct encoding apoE3 or apoE4 along with upstream and downstream regulatory elements. The baseline expression of apoE was low. However, conditioned medium from an astrocytic cell line (C6) or from apoE-null mouse primary astrocytes increased the expression of both isoforms by 3-4-fold at the mRNA level and by 4-10-fold at the protein level. These findings suggest that astrocytes secrete a factor or factors that regulate apoE expression in neuronal cells. The increased expression of apoE was almost completely abolished by incubating neurons with U0126, an inhibitor of extracellular signal-regulated kinase (Erk), suggesting that the Erk pathway controls astroglial regulation of apoE expression in neuronal cells. Human neuronal precursor NT2/D1 cells expressed apoE constitutively; however, after treatment of these cells with retinoic acid to induce differentiation, apoE expression diminished. Cultured mouse primary cortical and hippocampal neurons also expressed low levels of apoE. Astrocyte-conditioned medium rapidly up-regulated apoE expression in fully differentiated NT2 neurons and in cultured mouse primary cortical and hippocampal neurons. Thus, neuronal expression of apoE is regulated by a diffusible factor or factors released from astrocytes, and this regulation depends on the activity of the Erk kinase pathway in neurons.     

The magnetism responsive gene Ntan1 in mouse brain

We have previously identified Ntan1 as a magnetism response gene by differential display screening in cultured rat hippocampal neurons. Ntan1 mRNA was ubiquitously expressed in all the mouse tissues examined but relatively abundant in brain, retina and testis. Ntan1 mRNA expression was detectable in the embryonic 12-day mouse brain and gradually increased with ageing. In situ hybridization analysis showed high localization of Ntan1 mRNA in pyramidal cell layer of CA region and granular cell layer of dentate gyrus in the hippocampus, and Purkinje and granular cell layers in the cerebellum, respectively. Ntan1 mRNA expression was significantly increased about two-fold 12 h after brief exposure for 15 min to magnetism at 100 mT with a gradual decrease thereafter in cultured mouse hippocampal neurons. When embryonic 12-day-old or newborn mice were successively exposed to magnetic fields at 100 mT for 2 h, four times per day until the postnatal seventh day, Ntan1 mRNA was significantly increased about 1.5-2-fold in the hippocampus in vivo. The mice exposed to magnetic fields under the same condition showed significantly decreased locomotor activity. These results suggest that magnetic exposure affects higher order neural functions through modulation of genes expression.