Regional variation in the activation threshold for 1,3-DNB-induced mitochondrial permeability transition in brainstem and cortical astrocytes

1,3-Dinitrobenzene (DNB) produces edematous, glio-vascular lesions in brainstem nuclei with high energy demands. Astrocytes in vulnerable brainstem nuclei appear to be an early and selective target of DNB and other nitroaromatic compounds, though the molecular basis of this susceptibility is poorly understood. It has been postulated that mitochondria are a principal target of DNB in sensitive cell types [Neuropathol. Appl. Neurobiol. 13 (5) (1987) 371], where redox-cycling of DNB increases levels of reactive oxygen species and disrupts cellular energy metabolism. The present study investigates the role of regional differences in activation of the mitochondrial permeability transition pore (mtPTP) by DNB in brainstem and cortical astrocytes and examines the expression of Bcl-2 proteins as potential regulators of mtPTP function. Neonatal rat astrocytes were cultured from both DNB-sensitive (brainstem) and insensitive (cortex) brain regions and evaluated for DNB-induced alterations in cell morphology and mitochondrial function. Exposure to DNB resulted in rapid changes in the morphology of brainstem astrocytes consistent with loss of ion homeostasis and initiation of necrotic cell death. These changes were not observed in cortical astrocytes at corresponding concentrations of DNB and were prevented in brainstem astrocytes by the mtPTP inhibitor, bongkrekic acid, suggesting that mitochondrial dysfunction is involved in DNB-induced morphological changes in brainstem astrocytes. Mitochondrial depolarization in brainstem astrocytes was observed at DNB concentrations as low as 10 microM, whereas no loss of mitochondrial membrane potential (DeltaPsi(mt)) occurred in cortical astrocytes at less than 100 microM DNB. DNB-induced loss of DeltaPsi(mt) followed apparent first-order kinetics, with EC(50)-values for half-maximal rates of mitochondrial depolarization of approximately 23 and approximately 290 microM in brainstem cortical astrocytes, respectively. DNB-induced mitochondrial depolarization was prevented by pretreatment with bongkrekic acid, indicating that loss of DeltaPsi(mt) was mediated by activation of the mtPTP. Inhibition of succinate dehydrogenase (SDH) activity occurred in astrocytes from both brain regions exposed to DNB and was blocked in brainstem, but not cortical, astrocytes by bongkrekic acid. Constitutive expression of Bcl-X(L) was high in cortical tissue and astrocytes, whereas Bax expression was low. However, Bax was highly expressed in brainstem tissue and astrocytes and Bcl-X(L) expression was markedly lower. The expression of Bcl-2 was similar in both brain regions. These data suggest that the selective vulnerability of brainstem astrocytes to DNB is due to a lower threshold for activation of the mtPTP that is be mediated, in part, by distinct expression patterns of Bcl-2 proteins rather than by intrinsic differences in susceptibility of the electron transport chain.     

1,3-Dinitrobenzene inhibits mitochondrial complex II in rat and mouse brainstem and cortical astrocytes

1,3-Dinitrobenzene (DNB) produces edematous, glio-vascular lesions that are initially confined to brainstem nuclei with high energy requirements in rats and mice. Perturbation of energy producing processes in the cell is known to induce formation of the mitochondrial permeability transition pore (mtPTP) complex. Selective vulnerability of brainstem astrocytes to DNB is mediated by a 10-fold lower threshold for opening of the cyclosporin A-inhibitable mitochondrial permeability transition (MPT) pore than their cortical counterparts. Other nitrocompounds, such as 3-nitropropionic acid, selectively interfere with regional energy metabolism, including mitochondrial succinate dehydrogenase activity. However, the link between DNB-induced onset of the MPT and disruption of energy producing processes in the astrocyte remains unclear. The effects of DNB on succinate dehydrogenase activity were evaluated in cultured neonatal rat and mouse brainstem and cortical astrocytes. Both histochemical and spectrophotometric assays confirmed significant temporal inhibition of SDH activity in brainstem and cortical astrocytes 0.5, 2 and 5h following exposure to 100 microM DNB in vitro. Although DNB-induced inhibition of SDH was significantly decreased by CsA pretreatment in brainstem astrocytes after 0.5 and 2h and with a second pore inhibitor, bongkrekic acid (BKA) after 5h, both inhibitors failed to reduce inhibition of SDH activity in cortical astrocytes. These data suggest that DNB-induced inhibition of SDH may be independent of differential regional activation of the mtPTP complex in astrocytes and that an unidentified cyclosporin A-inhibitable factor mediates DNB-induced loss of SDH function.     

Differential cellular regulation of the mitochondrial permeability transition in an in vitro model of 1,3-dinitrobenzene-induced encephalopathy

Exposure to 1,3-dinitrobenzene (DNB) is associated with neuropathologic changes in specific brainstem nuclei, mediated by oxidative stress and mitochondrial dysfunction. The expression of Bcl-2-family proteins as a function of sensitivity to 1, 3-dinitrobenzene (DNB)-induced mitochondrial permeability transition (MPT) was examined in C6 glioma and SY5Y neuroblastoma cells. Neuroblastoma cells were 10-fold more sensitive than glioma cells to DNB-induced decreases in mitochondrial reducing potential, measured by reduction of the tetrazolium compound, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The IC(50) values for DNB-related inhibition of MTT reduction were 107+/-25 microM in SY5Y cells and 1047+/-101 microM in C6 cells. Levels of reactive oxygen species (ROS) were increased in both SY5Y and C6 cells following DNB exposure by 4.6- and 6.0-fold above control, respectively. DNB caused abrupt depolarization of mitochondria in both neuroblastoma and glioma cells that was inhibited by trifluoperazine. The first order rate constants for mitochondrial depolarization were: C6, k=0.31+/-0.02 min(-1); SY5Y, k=0.14+/-0.01 min(-1). Onset of MPT occurred at 10-fold lower concentration of DNB in SY5Y cells than in C6 cells. The antioxidants, deferoxamine and alpha-tocopherol, effectively prevented DNB-induced MPT in C6 and SY5Y cells, suggesting involvement of ROS in the initiation of MPT. Exposure to DNB resulted in decreased cellular ATP content in SY5Y cells and efflux of mitochondrial calcium in both SY5Y and C6 cells, concurrent with onset of MPT. The expression of Bcl-2, Bcl-X(L), and Bax was evaluated in both cell types by Western blot analysis. C6 glioma cells strongly expressed Bcl-X(L) and only weakly expressed Bcl-2 and Bax, whereas SY5Y neuroblastoma cells expressed lower levels of Bcl-X(L) and higher levels of both Bcl-2 and Bax. Collectively, these results suggest that higher constitutive expression of Bcl-X(L), rather than Bcl-2, correlates with resistance to DNB-induced MPT in SY5Y and C6 cells and that differential regulation of the permeability transition pore may underlie the cell-specific neurotoxicity of DNB.     

Expression of apoptosis-related proteins in model of anoxia in vitro

There has been growing evidence that different modes of cell death exist, among them the apoptosis is thought to be an important mechanism of nerve cell loss implicated in various pathological states. A number of proteins mediated with apoptotic process have been identified, including p53, BAX, BCL-2 and BCL-X. We examined the expression of proteins related to programmed cell death in hippocampal neurons in vitro, exposed to pure anoxia or pretreated with apoptosis modulating agents: zinc and zinc chelator - TPEN. The results evidenced the noticeable differences in the expression of pro- and anti-apoptotic proteins in particular experiments. In the cultures exposed to pure anoxia, a significant increase of p53 and BAX immunoreactivity, associated with the decreased level of BCL-2 and BCL-X immunopositive cells was observed, related to the activation of apoptotic process. Hippocampal cultures pretreated with ZnCl2 before anoxia showed decreased immunoreactivity for p53 and BAX, connected with BCL-2 overexpression, whereas the cultures exposed to zinc chelating agent - TPEN or TPEN connected with anoxia showed significant increase of immunorectivity for p53 and BAX. This strong immunoreactivity of proapototic proteins (p53 and BAX) in hippocampal cultures exposed to anoxia or/and TPEN correlated with previous ultrastructural evidences of anoxia- and TPEN-induced apoptosis, while the overexpression of anti-apoptotic protein (BCL-2 and BCL-X) in zinc-pretreated cultures evidenced the protective ability of this metal against apoptosis in model of anoxia in vitro.     

BCL-2 family protein expression in initial and recurrent glioblastomas: modulation by radiochemotherapy

OBJECTIVE: In vitro studies indicate a role of apoptosis regulatory proteins of the BCL-2 family in the resistance of glioblastoma multiforme to irradiation and chemotherapy. To date, no study has compared the expression of these proteins in initial and recurrent tumours. The differences of expression of BCL-2, BCL-X, BAX, and MCL-1 proteins of paired first resection and recurrence glioblastoma specimens were examined. METHODS: Immunohistochemistry was performed in 37 cases of glioblastoma multiforme with paraffin embedded tissue from first resections and their recurrences in three treatment groups (15 radiochemotherapy, 15 irradiation, seven untreated). Ten high power fields were evaluated with an arbitrary score (< 5%=1, 5-50%=2, >50%=3), and cumulative scores for each antigen calculated. RESULTS: In the whole group, we found a significant up regulation of antiapoptotic BCL-2 (median cumulative score of 15 in the primary, 19 at recurrence; p<0.0001 in the Wilcoxon test), BCLX (median scores 20 and 25, respectively, p<0.0001), and MCL-1 (median scores 11 and 14, p=0.0395), and a significant down regulation of proapoptotic BAX (median scores 14 and 11, p<0.0001). In the subgroups, these trends were also found. No association between protein expression and treatment regimen was found, although significant changes were restricted to the subgroups that received adjuvant chemotherapy. No significant correlation with clinical prognosis was detected with the Kaplan-Meier method. CONCLUSIONS: In the development from initial to recurrent glioblastoma multiforme, the BCL-2 family rheostat shifts towards antiapoptotic adjustment in vivo. Importantly, the changes in BCL-2 family protein expression characterised here were also seen in the subgroup of patients who did not receive adjuvant radiotherapy or chemotherapy, suggesting that the changes of BCL-2 family protein expression result not only from radiochemotherapy but also reflect the natural course of disease.     

Changes in the astrocytic aquaporin‐4 and inwardly rectifying potassium channel expression in the brain of the amyotrophic lateral sclerosis SOD1G93A rat model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting upper and lower motor neurons. Dysfunction and death of motor neurons are closely related to the modified astrocytic environment. Astrocytic endfeet, lining the blood–brain barrier (BBB), are enriched in two proteins, aquaporin‐4 (AQP4) and inwardly rectifying potassium channel (Kir) 4.1. Both channels are important for the maintainance of a functional BBB astrocytic lining. In this study, expression levels of AQP4 and Kir4.1 were for the first time examined in the brainstem and cortex, along with the functional properties of Kir channels in cultured cortical astrocytes of the SOD1^G93A rat model of ALS. Western blot analysis showed increased expression of AQP4 and decreased expression of Kir4.1 in the brainstem and cortex of the ALS rat. In addition, higher immunoreactivity of AQP4 and reduced immunolabeling of Kir4.1 in facial and trigeminal nuclei as well as in the motor cortex were also observed. Particularly, the observed changes in the expression of both channels were retained in cultured astrocytes. Furthermore, whole‐cell patch‐clamp recordings from cultured ALS cortical astrocytes showed a significantly lower Kir current density. Importantly, the potassium uptake current in ALS astrocytes was significantly reduced at all extracellular potassium concentrations. Consequently, the Kir‐specific Cs⁺‐ and Ba²⁺‐sensitive currents were also decreased. The changes in the studied channels, notably at the upper CNS level, could underline the hampered ability of astrocytes to maintain water and potassium homeostasis, thus affecting the BBB, disturbing the neuronal microenvironment, and causing motoneuronal dysfunction and death. © 2012 Wiley Periodicals, Inc.     

Regional heterogeneity of the astroglial immunoreactive phenotype: effect of lipopolysaccharide.

In order to find out whether the concept of regional heterogeneity in astrocytes also applies to the immunoreactive phenotype, we studied cultured primary rat astrocytes originating from five different brain regions (cortex, hippocampus, striatum, septum, and brainstem).We investigated this heterogeneity through the ability of lipopolysaccharide (LPS) to differentially induce several parameters that are known to characterize activated astroglia: major histocompatibility complex (MHC) class II and intercellular adhesion molecule (ICAM)-1 expression, nitric oxide (NO) production, synthesis of interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha). Under basal conditions, some of these parameters are already heterogeneic. The presence of LPS enhances these differences: expression of MHC class II increases after a 48-hour incubation with LPS, with brainstem and hippocampus astrocytes reaching the highest levels; NO production is induced by an LPS incubation, with the brainstem showing low NO production levels; septum and striatum instead show higher cytokine (TNF-alpha, IL-6) productions. The baseline expression of ICAM-1 also shows major regional differences, with the brainstem displaying the highest ICAM-1 expression. Our results demonstrate that the immunoreactive abilities of astrocytes show regional heterogeneities. This specialization may be implicated in the pathophysiological pathways of several neurodegenerative disorders.     

干细胞因子对糖尿病小鼠皮层神经元凋亡的保护作用

目的 探讨干细胞因子对糖尿病所致神经元凋亡的干预效应及其可能机制.方法 成年雄性C57小鼠27只,随机分为正常对照组、糖尿病组、糖尿病干细胞因子组,每组9只.用链脲佐菌素建立糖尿病小鼠模型,TUNEL染色反映大脑神经元的凋亡情况,Western blot检测BCL-2、BAX、CASPASE 3及P-ERK/ERK蛋白的表达.结果 与对照组相比,糖尿病小鼠大脑皮层神经元凋亡数量增加且有活性的CASPASE 3表达升高,应用干细胞因子干预后,上述情况可以得到有效的改善.糖尿病小鼠大脑皮层内的BCL-2、BAX均较正常组明显升高,应用于细胞因子后可抑制BAX的表达而对BCL-2无明显影响;相应的BCL-2/BAX比值在正常组和糖尿病组之间无显著性差异存在,但干细胞因子的应用能明显升高其表达.糖尿病小鼠P-ERK表达水平明显降低,应用于细胞因子后可有效增加ERK的磷酸化甚至超过正常水平.结论 干细胞因子可能通过增加ERK的磷酸化而影响BCL-2/BAX的表达发挥对糖尿病神经元的抗凋亡作用.     

Valproic acid mediates the synaptic excitatory/inhibitory balance through astrocytes - A preliminary study

Valproic acid (VPA) is one of the most widely used anticonvulsant and mood-stabilizing agents for the treatment of epilepsy and bipolar disorder. However, the underlying therapeutic mechanisms of the treatment of each disease remain unclear. Recently, the anti-epileptic effect of VPA has been found to lead to modulation of the synaptic excitatory/inhibitory balance. In addition, the therapeutic action of VPA has been linked to its effect on astrocytes by regulating gene expression at the molecular level, perhaps through an epigenetic mechanism as a histone deacetylase (HDAC) inhibitor. To provide insight into the mechanisms underlying the actions of VPA, this study investigated whether the synaptic excitatory/inhibitory (E/I) balance could be mediated by VPA through astrocytes. First, using the primary rat neuronal, astroglial, and neuro-glial mixed culture systems, we demonstrated that VPA treatment could regulate the mRNA levels of two post-synaptic cell adhesion molecules(neuroligin-1 and neuregulin-1) and two extracellular matrices (neuronal pentraxin-1and thrombospondin-3) in primary rat astrocyte cultures in a time- and concentration-dependent manner. Moreover, the up-regulation effect of VPA was noted in astrocytes, but not in neurons. In addition, these regulatory effects could be mimicked by sodium butyrate, a HDAC inhibitor, but not by lithium or two other glycogen synthase kinase-3 beta inhibitors. With the known role of these four proteins in regulating the synaptic E/I balance, we further demonstrated that VPA increased excitatory post-synaptic protein (postsynaptic density 95) and inhibitory post-synaptic protein (Gephyrin) in cortical neuro-glial mixed cultures. Our results suggested that VPA might affect the synaptic excitatory/inhibitory balance through its effect on astrocytes. This work provides the basis for future evaluation of the role of astroglial cell adhesion molecules and the extracellular matrix on the control of excitatory and inhibitory synapse formation.     

Are cerebral prostanoids of astroglial origin? Studies on the prostanoid forming system in developing rat brain and primary cultures of rat astrocytes

Prostanoid forming capacity in vitro and convulsion-induced prostanoid formation in vivo were studied in the developing rat brain. For comparison, prostanoid synthesis in homogenates of primary astrocyte cultures of different ages was also examined. There was no significant prostanoid production in homogenates from primary astrocyte cultures prepared one week after cultivation. Two-week-old astrocyte cultures possessed a prostanoid synthesizing system of high specific activity. The relative proportions of the products were similar to those obtained in brain homogenates of adult rats, prostaglandin D2 (PGD2) being the major product. Prostanoid forming capacity of brain homogenates was low at birth, increased during development and nearly reached adult values by day 21. Generalized convulsions could be evoked by pentylenetetrazol (PTZ) irrespective of age, but convulsion-induced prostanoid formation characteristic of adult rodents did not take place before the third week of postnatal life. The close similarities between the characteristic features of prostanoid synthesis in both brain and astroglial homogenates, together with the coincidence during brain development of the expression of cerebral prostanoid synthesis with the appearance of mature astrocytes suggest that astrocytes are an important source of brain prostanoids.     

Astrocytic exportin‐7 responds to ischemia through mediating LKB1 translocation from the nucleus to the cytoplasm

The superfamily of importin‐β–related proteins is the largest class of nuclear transport receptors and can be generally divided into importins and exportins according to their transport directions. Eleven importins and seven exportins have been identified, and the expression patterns of both classes are important for their functions in nucleocytoplasmic transport activities. This study demonstrates that all of the importins (importin‐β; transportin‐1, ‐2, and ‐3; and importin‐4, ‐5, ‐7, ‐8, ‐9, ‐11, and ‐13) and all the exportins (exportin‐1, ‐2, ‐4, ‐5, ‐6, ‐7, and ‐t) are differentially expressed in the cerebral cortex, cerebellum, hippocampus, and brainstem and in primary cultures of cerebral cortical astrocytes and neurons. For astrocytes, we observed that different importins and exportins displayed different expression changes during 0–6 hr of ischemia treatment, especially an increase of both the mRNA and the protein of exportin‐7. Immunostaining showed that exportin‐7 accumulated inside the nucleus and around the nuclear envelope. In addition, we noticed an increased cytoplasmic distribution of one of the cargo proteins of exportin‐7, LKB1, an important element in maintaining energy homeostasis. This increased cytoplasmic distribution was accompanied by an increased expression of exportin‐7 under ischemia in astrocytes. We demonstrate that exportin‐7 responds to ischemia in astrocytes and that this response involves translocation of LKB1, a protein that plays important roles during metabolic stress, from the nucleus to the cytoplasm. © 2014 Wiley Periodicals, Inc.     

Proteomic identification of brainstem cytosolic proteins in a neuropathic pain model

Neuropathic pain involves co-regulation of many genes and their translational products in both peripheral and central nervous system. We used proteomics approaches to investigate expressional changes in cytosolic protein levels in rat brainstem tissues following ligation of lumbar 5 and 6 (L5, L6) spinal nerves, which generates a model of peripheral neuropathic pain (NP). Proteins from brainstem tissue homogenates of NP and SHAM animals were fractionated by two-dimensional (2-DE) gel electrophoresis to produce a high-resolution map of the brainstem soluble proteins. Proteins showing altered expression levels between NP and SHAM were selected. Isolated proteins were in-gel trypsin-digested and the resulting peptides were analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Using the mass spectrometric data, we were able to identify 17 proteins of interest through searches of the Swiss-Prot and NCBi nonredundant protein sequence database. Several of the identified proteins, including fatty acid binding protein-brain (FABP-B), major histocompatibility complex (MHC) class 1, T-cell receptor (TCR) alpha chain, and interleukin-1 (IL-1), showed significantly higher levels in the NP rat brainstem. Proteomic analysis has identified several proteins with differential expression levels in NP as compared to SHAM. However, the function of the proteins identified is postulated; therefore, further experiments are required to determine the true role of each protein in NP.     

ADDLs对原代培养小鼠皮层神经元及星形胶质细胞混合培养体系Notch-1信号转导及HIF-1α表达的影响

目的探讨Aβ寡聚体(ADDLs)对原代培养小鼠皮层神经元及星形胶质细胞混合培养体系Notch-1信号转导及HIF-1α蛋白表达的影响。方法在原代培养小鼠皮层神经元及星形胶质细胞混合培养体系的基础之上应用CCK-8法观察Aβ寡聚体对混合培养体系细胞活力的影响,并应用免疫印迹的方法检测Aβ寡聚体对混合培养体系的NICD及HIF-1α水平的影响。另外,应用qPCR的方法检测凋亡相关caspase-3mRNA水平。结果 Aβ寡聚体干预浓度在高于2μM的情况下可使混合培养体系细胞活力水平明显降低,并呈现浓度依赖。10μM的ADDLs明显升高了原代混合培养体系的NICD及HIF-1α蛋白表达水平,且均呈现出相对的时间依赖性,同时这两种蛋白表达水平的变化表现出基本一致的趋势。同时,10μM的ADDLs明显上调了混合体系的caspase-3 mRNA水平。结论 Aβ寡聚对原代培养小鼠皮层神经元及星形胶质细胞混合培养体系的毒性作用与上调NICD及HIF-1α表达水平密切相关。     

Characterization of “plasma proteins” secreted by cultured rat macroglial cells

The brain is isolated behind a blood-tissue barrier that restricts the access of circulating proteins to neural cells. There is evidence that some of these proteins are synthesized within the central nervous system. The present study examines the synthesis and secretion of such proteins by cultured macroglial cells. Primary glial cultures were derived from cortical and subcortical regions of neonatal rat brains, and subsequent secondary cultures were enriched in type-1 astrocytes, type-2 astrocytes, or oligodendrocytes. Newly synthesized proteins were immunoprecipitated from the culture media using antisera directed against whole rat serum. All three types of glial cells secreted a range of plasma proteins. In general, type-1 astrocytes secreted more of these proteins than did type-2 astrocytes or oligodendrocytes, although the one-dimensional polyacrylamide gel electrophoresis (PAGE) profiles were specific for each cell type. Antisera directed against specific plasma proteins identified three of the most abundant proteins secreted by type-1 astrocytes as transferrin, α-2-macroglobulin, and ceruloplasmin. Northern blot analysis of cellular RNA confirmed that type-1 astrocytes contained transferrin mRNA, and that it was more abundant in cultures derived from subcortical regions than from cortical regions. In situ hybridization studies revealed that virtually all type-1 and type-2 astrocytes contained transferrin mRNA. Since the proteins identified in this study have been proposed to have a variety of neurotrophic roles in the central nervous system, these data further extend the range of possible functions that glial cells may serve in the CNS.     

Plasma membrane calcium ATPase isoforms in astrocytes.

The plasma membrane Ca(2+)-ATPase (PMCA) is an essential component of the machinery responsible for cellular Ca(2+) homeostasis. Together with the Na(+)/Ca(2+) exchanger, the plasma membrane Ca(2+)-ATPase (PMCA) is responsible for the extrusion of Ca(2+) from the cytosol. Although both PMCAs and Na(+)/Ca(2+) exchangers are present in high amounts in the brain, it is thought that only the latter localize to glia. This study investigates whether PMCAs are also present in astrocytes and thus are components of Ca(2+) signalling in this cell type. Membrane proteins and mRNA were isolated from primary cultures of rat cortical astrocytes and C6 glioma cells. PMCA isoforms were investigated with isoform specific antibodies and the splice variant pattern was studied in RT-PCR experiments using specific oligonucleotides. The PMCA1, 2, and 4 isoforms were detected in rat cortical astrocytes, whereas only PMCA1 and 2 were found in C6 cells. While neurons express both the CI and CII splice variants, only the splice variant CI of PMCA1, 2, and 4 was detected in astrocytes. Thus, the PMCA pump is present in mammalian glial cells. These results also show that the amounts of PMCA1 and 4 isoforms in astrocytes are comparable to those found in neurons. In contrast, astrocytes contain smaller amounts of PMCA2. Furthermore, PMCA2 and PMCA4 underwent an evident time dependent up-regulation in astrocytes cultured in vitro.     

C2H2-171 : A novel human cDNA representing a developmentally regulated poz domain/zinc finger protein preferentially expressed in brain

We describe a novel human zinc finger cNDA, C2H2-171. This cDNA represents an mRNA which encodes a protein of 484 amino acids and a calculated molecular weight of 54 kD. Four zinc finger-like domains are found in the C-terminal end of the protein. At the N-terminus, C2H2-171 contains a POZ/tramtrack-like domain similar to that found in the tumor associated zinc finger proteins LAZ-3/BCL-6 and PLZ-F, as well as in non-zinc finger proteins. C2H2-171 RNA is preferentially expressed in the brain, and increases during the course of murine development, with maximal expression in the adult. C2H2-171 RNA is differentially expressed in brain regions, with the highest level of expression in the cerebellum. C2H2-171 RNA was expressed at high levels in primary cerebellar granule cell neurons compared to astrocytes. The gene encoding C2H2-171 is highly conserved in vertebrates, and maps to the terminus of human chromosome 1 (1q44-ter). This chromosomal location is associated with a number of cytogenetic aberrations including those involving brain developmental anomalies and tumorigenesis. These data suggest that C2H2-171 may play an important role in vertebrate brain development and function.     

Developmental regulation of GAP-43, glutamine synthetase and beta-actin mRNA in rat cortical astrocytes.

Steady-state levels of mRNA encoding growth-associated protein 43 (GAP-43), glutamine synthetase (GS) and beta-actin were measured during development of neonatal rat cortical astrocytes in primary culture. GAP-43 mRNA and protein decreased rapidly during the first 2 weeks and slowly thereafter. In contrast, GS mRNA increased approximately 3-fold during the first 2 weeks and reached maximum by day 15. Actin mRNA first increased up to 8 days and decreased thereafter reaching a constant amount of 15 days, similar to the initial low value. Thus, GAP-43, GS and beta-actin mRNA levels are differentially regulated during development of astrocytes in primary culture. Because the patterns of expression of astrocytic markers GS and GFAP (shown previously) in vitro and in vivo are similar to each other, primary cultures of astrocytes may be an excellent system for investigating mechanisms of developmental regulation of these genes.