Proteome analysis of mouse primary astrocytes

Astrocytes play a role in energy metabolism, neuronal homeostasis and release of neuronal growth factors and several neurotransmitters. They also relate to a variety of brain diseases and contribute to restore brain dysfunction. Although current research has revealed several roles for astrocytes, knowledge on astrocytic protein expression is limited and a systematic and comprehensive proteome study of astrocytes has not been reported so far. We applied a proteomics technique based on two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF) and unambiguously identified 301 spots corresponding to 191 individual proteins in primary mouse astrocytes. The identified proteins were from antioxidant, chaperone, cytoskeleton, nucleic acid binding, signaling, proteasomal, hypothetical and miscellaneous proteins. A reference database is provided and proteins were identified in astrocytes specifically and unambiguously for the first time. A reliable analytical tool independent of antibody availability and specificity along with tentative astrocytic marker proteins is described.     

Metabolic differences between primary cultures of astrocytes and neurons from cerebellum and cerebral cortex. Effects of fluorocitrate

Astrocytes and neurons cultured from mouse cerebellum and cerebral cortex were analyzed with respect to content and synthesis of amino acids as well as export of metabolites to the culture medium and the response to fluorocitrate, an, inhibitor of aconitase. The intracellular levels of amino acids were similar in the two astrocytic populations. The release of citrate, lactate and glutamine, however, was markedly higher from cerebellar than from cortical astrocytes. Neurons contained higher levels of glutamate, aspartate and GABA than astrocytic cultures. Cortical neurons were especially high in GABA and aspartate, and the level of aspartate increased specifically when the extracellular level of glutamine was elevated. Fluorocitrate inhibited the TCA cycle in the astrocytes, but was less effective in cerebellar neurons. Whereas neurons responded to fluorocitrate with an increase in the formation of lactate, reflecting, glycolysis, astrocytes decreased the formation of lactate in the presence of fluorocitrate, indicating that astrocytes to a high degree synthesize pyruvate and hence lactate from TCA cycle intermediates.     

Proteome profile of whole cerebellum of the mature rat

Cerebellum is an important brain region involved in motor, cognition, learning and memory functions. Proteome mapping of the 21 days old rat cerebellum identified total 285 proteins, out of which 76 proteins were not reported earlier from rat brain. This includes 49 neuronal activity-specific proteins, 7 of which are reported for the first time from the cerebellum in this study. The protein sequence data for 31 proteins reported here have been integrated in the UniProt Knowledgebase.     

Gaba-activated conductance of neurons isolated from rat cerebellum and sensory ganglia

Currents activated by gamma-aminobutyric acid (GABA) were recorded in single Purkinje cells isolated from the rat cerebellum and trigeminal ganglia. All neurons tested were GABA-sensitive. Reversal potential of GABA-activated current matched equilibrium potential for chloride ions as determined by Nernst's equation. The dose-response curve was described by Langmuir's isotherm with a dissociation constant (K_d) of 1.4·10^–4 M. Nembutal did not just increase the amplitude of GABA-activated current but also activated matching ionic conductance in the absence of GABA. Ionic currents activated by GABA were reversibly blocked by bicuculline methiodide and isocoryne.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 645–652, September–October, 1988.     

Implication of apoE isoforms in cholesterol metabolism by primary rat hippocampal neurons and astrocytes

Apolipoprotein E (apoE) has been genetically linked to late-onset Alzheimer's disease. From the three common alleles (epsilon2, epsilon3 and epsilon4), epsilon4 has been suggested to promote amyloid beta (Ass) plaque fibrillation, one hallmark of Alzheimer's disease. It has been demonstrated that altered lipid content of hippocampal plasma membrane coincides with the disease. In this study, we show for the first time that the apoE dependent cholesterol metabolism in hippocampal neurons is higher than that of hippocampal astrocytes. Further, apoE-bound cholesterol is highly incorporated in membranous compartments in hippocampal neurons, whereas hippocampal astrocytes show higher intracellular distribution. This is an effect that coincides with cell-type dependent difference of low density lipoprotein receptor (LDLR) family member expression. Hippocampal neurons express high levels of the LDLR related protein (LRP), whereas hippocampal astrocytes are highly positive for LDLR. We could also demonstrate an apoE isoform (apoE2, apoE3 and apoE4) dependent cholesterol uptake in both cells types. In hippocampal neurons, we could find a decreased apoE4-bound cholesterol uptake. In contrast, hippocampal astrocytes show decreased internalization of apoE2-bound cholesterol. In addition, lipidated apoE4 is little associated with neurites in hippocampal neurons in comparison to the other two isoforms. In contrary, hippocampal astrocytes show faint apoE2 immunocytostaining intensity. Data presented indicate that the role of apoE4 in cholesterol homeostasis and apolipoprotein cell association is more pronounced in hippocampal neurons, showing significant alterations compared to the other two isoforms, suggesting that hippocampal neurons are affected by apoE4 associated altered cholesterol metabolism compared to hippocampal astrocytes.     

Synthesis and release of GABA in cerebral cortical neurons co-cultured with astrocytes from cerebral cortex or cerebellum

Cerebral cortical neurons were co-cultured for up to 7 days with astrocytes after plating on top of a confluent layer of astrocytes cultured from either cerebral cortex or cerebellum (sandwich co-cultures). Neurons co-cultured with either cortical or cerebellar astrocytes showed a high stimulus coupled release of gamma-aminobutyric acid (GABA), which is the neurotransmitter of these neurons. When the astrocyte selective GABA uptake inhibitor 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol was added during the release experiments, an increase in the stimulus coupled GABA release was seen, indicating that the astrocytes take up a large fraction of GABA released from the neurons. The activity of the GABA synthesizing enzyme glutamate decarboxylase, which is a specific marker of GABAergic neurons, was markedly increased in sandwich co-cultures of cortical neurons and cerebellar astrocytes compared to neurons cultured in the absence of astrocytes whereas in co-cultures with cortical astrocytes this increase was less pronounced. Pure astrocyte cultures did not show any detectable glutamate decarboxylase activity. The astrocyte specific marker enzyme glutamine synthetase (GS) was present at high activity in a glucocorticoid-inducible form in pure astrocytes as well as in co-cultures regardless of the regional origin of the astrocytes. When neurons were cultured on top of the astrocytes, the specific activity of GS was lower compared to astrocytes cultured alone, a result compatible with the notion that neurons are devoid of this enzyme. The results show that cortical neurons develop and differentiate when seeded on top of both homotypic and heterotypic astrocytes. Moreover, it could be demonstrated that the two cell types in the culture system communicate with each other with regard to GABA homeostasis during transmitter release.     

Functional and molecular identification of sodium-coupled dicarboxylate transporters in rat primary cultured cerebrocortical astrocytes and neurons

Na+-coupled carboxylate transporters (NaCs) mediate the uptake of tricarboxylic acid cycle intermediates in mammalian tissues. Of these transporters, NaC3 (formerly known as Na+-coupled dicarboxylate transporter 3, NaDC3/SDCT2) and NaC2 (formerly known as Na+-coupled citrate transporter, NaCT) have been shown to be expressed in brain. There is, however, little information available on the precise distribution and function of both transporters in the CNS. In the present study, we investigated the functional characteristics of Na+-dependent succinate and citrate transport in primary cultures of astrocytes and neurons from rat cerebral cortex. Uptake of succinate was Na+ dependent, Li+ sensitive and saturable with a Michaelis constant (Kt) value of 28.4 microM in rat astrocytes. Na+ activation kinetics revealed that the Na+ to succinate stoichiometry was 3:1 and the concentration of Na+ necessary for half-maximal transport was 53 mM. Although uptake of citrate in astrocytes was also Na+ dependent and saturable, its Kt value was significantly higher (approximately 1.2 mM) than that of succinate. Unlabeled succinate (2 mM) inhibited Na+-dependent [14C]succinate (18 microM) and [14C]citrate (4.5 microM) transport completely, whereas unlabeled citrate inhibited Na+-dependent [14C]succinate uptake more weakly. Interestingly, N-acetyl-L-aspartate, which is the second most abundant amino acid in the nervous system, also completely inhibited Na+-dependent succinate transport in rat astrocytes. The inhibition constant (Ki) for the inhibition of [14C]succinate uptake by unlabeled succinate, N-acetyl-L-aspartate and citrate was 15.9, 155 and 764 microM respectively. In primary cultures of neurons, uptake of citrate was also Na+ dependent and saturable with a Kt value of 16.2 microM, which was different from that observed in astrocytes, suggesting that different Na+-dependent citrate transport systems are expressed in neurons and astrocytes. RT-PCR and immunocytochemistry revealed that NaC3 and NaC2 are expressed in cerebrocortical astrocytes and neurons respectively. These results are in good agreement with our previous reports on the brain distribution pattern of NaC2 and NaC3 mRNA using in situ hybridization. This is the first report of the differential expression of different NaCs in astrocytes and neurons. These transporters might play important roles in the trafficking of tricarboxylic acid cycle intermediates and related metabolites between glia and neurons.     

Energy metabolism in glutamatergic neurons,GABAergic neurons and astrocytes in primary cultures

Several aspects of energy metabolism (glucose utilization, lactate production,¹⁴CO₂ production from labeled glucose, glutamate or pyruvate, oxygen consumption and contents of ATP and phosphocreatine) were measured in cerebellar granule cells (glutamatergic) in primary cultures and compared with corresponding data for cerebral cortical neurons (mainly GABA-ergic) and astrocytes. Cerebellar granule cells and astrocytes were metabolically more active than cerebral cortical neurons. Glutamate which is utilized as a major metabolic fuel as astrocytes and, to a lesser extent, in cerebral cortical neurons, was virtually not oxidized in cerebellar granule cells.Special Issue dedicated to Prof. Holger Hydén.     

Potassium currents in primary cultured astrocytes from the rat corpus callosum

The corpus callosum (CC) is the main white matter tract in the brain and is involved in interhemispheric communication. Using the whole-cell voltage-clamp technique, a study was made of K⁺-currents in primary cultured astrocytes from the CC of newborn rats. These cells were positive to glial fibrillary acidic protein after culturing in Dulbecco’s Modified Eagle Medium (> 95% of cells) or in serum-free neurobasal medium with G5 supplement (> 99% of cells). Astrocytes cultured in either medium displayed similar voltage-activated ion currents. In 81% of astrocytes, the current had a transient component and a sustained component, which were blocked by 4-aminopyridine and tetraethylammonium, respectively; and both had a reversal potential of ?66 mV, indicating that they were carried by K⁺ ions. Based on the Ba²⁺-sensitivity and activation kinetics of the K⁺-current, two groups of astrocytes were discerned. One group (55% of cells) displayed a strong Ba²⁺ blockade of the K⁺-current whose activation kinetics, time course of decay, and the current-voltage relationship were modified by Ba²⁺. This current was greatly blocked (52%) by Ba²⁺ in a voltage-dependent way. Another group (45% of cells) presented weak Ba²⁺-blockade, which was only blocked 24% by Ba²⁺. The activation kinetics and time course of decay of this current component were unaffected by Ba²⁺. These results may help to understand better the roles of voltage-activated K⁺-currents in astrocytes from the rat CC in particular and glial cells in general.     

Taurine-activated currents in isolated neurons of the rat cerebellum

Taurine-activated currents were investigated in rat cerebellar neurons using techniques of voltage clamping at the membrane and intracellular perfusion. Activation of both chloride and calcium conductance at the membrane were produced by applying taurine to the membrane surface. The dose-response curve for taurine-activated current is in the 1×10^–4–1×10^–1 M concentration region. The dissociation constant of the taurine-receptor complex equals 2×10^–3 M. Activation of taurine-induced currents is a cooperative process: Hill's coefficient –2. It was found that bicuculline and strychnine exert a blocking action on taurine-activated currents, while pentobarbital and oxazepam potentiate taurine action.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 6, November–December, 1990, pp. 780–786.     

Atrial natriuretic peptide-like immunoreactivity in neurons and astrocytes of human cerebellum and inferior olivary complex.

Atrial natriuretic peptide (ANP) has previously been localized in areas of mammalian brain associated with olfaction, cardiovascular function, and fluid/electrolyte homeostasis. Despite the presence of several types of natriuretic peptide receptors in mammalian cerebellum, neither intrinsic nor extrinsic sources of the natriuretic peptides have been described. In this report we describe the immunohistochemical localization of both intrinsic and extrinsic sources for ANP in human cerebellum. ANP-like immunoreactivity (ANP-LIR) was observed in climbing fibers in the cerebellar molecular layer that probably originated from isolated immunopositive neurons of the inferior olivary complex. Intrinsic sources of ANP-LIR included small subpopulations of protoplasmic and fibrous astrocytes and Bergmann glia, as well as Golgi and Lugaro neurons of the granule cell layer. These results suggest that, in addition to its presumptive roles in local vasoregulation, ANP may serve as a modulator of the activity of Purkinje neurons.     

Comparative analysis of the membrane proteins and their specificities in neurons,protoplasmic astrocytes,and oligodendrocytes from rat brain

Plasma membranes from neuronal perikarya (N), protoplasmic astrocytes (A) and oligodendrocyies (O) of rat brains were analysed with respect to their protein and glycoprotein contents and specificities. SDS-polyacrylamide gel electrophoresis revealed a total number of 23, 17, and 17 major proteins in N, A, and O respectively. Periodate-Schiff's staining showed that approximately 40–60% of these proteins were glycoproteins. The reactivity of these glycoproteins to Con A and WGA was also studied. Selective iodination of whole cells followed by electrophoresis and autoradiography indicated that of the major proteins, only 25% of neuronal and 60% of astroglial and oligodendroglial membrane proteins were exposed outside the cell surface. The overall results suggest that membrane proteins of each of the three cell types studied here have characteristically different internal and external markers differing in size, glycoprotein content, and reactivity of the glycoproteins to lectins.     

High-throughput transfection of differentiated primary neurons from rat forebrain

Primary neurons in culture are considered to be a highly relevant model in the study of neuronal development and activity. They can be cultivated and differentiated in vitro but are difficult to transfect using conventional methods. To address this problem, a capillary electroporation system called Cellaxess Elektra was developed for efficient and reproducible transfection of primary cortical and hippocampal neurons without significant impact on cell morphology and viability. The cells are transfected in any stage of differentiation and development, directly in cell culture plates. Genetic material is delivered in situ to as many as 384 samples at a time, which enables both high-throughput and high-quality screening for hard-to-transfect primary cells, meaning that gene function can be studied on a genome-wide scale in cells previously inaccessible to genetic manipulation.     

A neuropeptide precursor in cerebellum: proenkephalin exists in subpopulations of both neurons and astrocytes.

The adult rat cerebellum has minimal enkephalin immunoreactivity and is devoid of opiate-binding activity. Using novel monoclonal antibodies to the mammalian enkephalin precursor, we describe the immunofluorescent detection of proenkephalin, in the absence of mature enkephalin peptides, in subpopulations of rat cerebellar neurons and astrocytes. In cryostat sections, neurons that express proenkephalin include Golgi cells, macroneurons within deep cerebellar nuclei and a subpopulation of Purkinje cells. Proenkephalin messenger RNA and protein are present in subpopulations of both grey and white matter astrocytes, but not Bergmann glia. In dissociated glial culture, proenkephalin is expressed in process-bearing astrocytes, apparently in association with a subset of intermediate filaments. Proenkephalin within astrocytes is not seen until the second postnatal week and increases through to adulthood. Neuropeptide gene expression adds to the growing range of neuronal-type properties glial cells can display.     

Large-conductance cationic channels in the nuclear envelope of Purkinje neurons from the rat cerebellum

Using a patch-clamp technique, we studied the biophysical properties of large-conductance channels in the nuclear envelope of rat cerebellar Purkinje neurons. Our experiments showed that channels with identical conductance, selectivity, and kinetics are expressed in the external and internal nuclear membranes of these cells. These channels connect the perinuclear space with the cyto-and nucleoplasm; they are not channels of the complex of the nuclear pores for passive diffusion of ions and small molecules, as was believed earlier [17]. We hypothesize that large-conductance cationic channels in the membranes of the nuclear envelope are identical to ion channels of the endoplasmic reticulum and are necessary for functioning of the intermembrane space of the envelope as a calcium store. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 113–118, March–April, 2007.     

Similarities of adenosine uptake systems in astrocytes and neurons in primary cultures

Uptake of extracellular adenosine was studied in primary cultures of astrocytes or neurons. Both cell types showed a high affinity uptake. TheK _m values were not significantly different (6.5±3.75 M in astrocytes and 6.1±1.86 M in neurons), but the intensity of the uptake was higher in astrocytes than in neurons (V _max values of 0.16±0.030 and 0.105±0.010 nmol×min^–1×mg^–1 protein, respectively). The temperature sensitivity was similar in the two cell types. Adenosine uptake inhibitors and benzodiazepines inhibited the adenosine uptake systems in both astrocytes and neurons with IC₅₀ values in the high nanomolar or the micromolar range and the rank order of potency was similar in the two cell types. In both cell types the (–) isomers of two sets of benzodiazepine stereoisomers were more potent than the (+) isomers. Dixon analysis showed that dipyridamole, papaverine, hexobendine and chlordiazepoxide inhibited the adenosine uptake competitively and clonazepam noncompetitively in both cell types.     

Uptakes of iodide and chloride by primary cultures of mouse astrocytes and neurons

Primary cultures of both mouse astrocytes and neurons accumulate more¹²⁵I^– than³⁶Cl^– from the medium. The average cell/medium ratio of¹²⁵I^– of astrocytes (1.01) is greater than that of neurons (0.74), whereas the ratio of³⁶Cl^– of neurons (0.47) is greater than that of astrocytes (0.25). The equilibrium potentials of both¹²⁵I^– and³⁶Cl^– calculated from the cell/medium ratios in astrocytes and neurons are significantly lower than their corresponding resting transmembrane potentials which suggest that both iodide and chloride are actively transported into both cell types. With respect to different transport inhibitors, thiocyanate is more effective in inhibiting¹²⁵I^– uptake whereas furosemide is more effective in inhibiting³⁶Cl^– uptake. Radioiodide uptake by mouse astrocytes was directly proportional to the [Na⁺]_o but was not significantly affected by changes of [Cl^–]_o or [HCO ₃ ^– ]_o, except that it is low in bicarbonate-free medium. Radiochloride uptake by astrocytes was inversely related to [Cl^–]_o and [HCO ₃ ^– ]_o and was not affected [Na⁺]_o, except that it was low in sodium-free medium. Radioiodide uptake by neurons was directly related to [Na⁺]_o between 60 and 140 mM and inversely related to [HCO ₃ ^– ]_o between 10 and 40 mM, but it was not affected by [Cl^–]_o. Radiochloride uptake by neurons was directly related to [Cl^–]_o and to [Na⁺]_o between 60 and 140 mM and was not affected by [HCO ₃ ^– ]_o. However, in sodium-free medium both¹²⁵I^– and³⁶Cl^– uptakes into neurons were higher than those in [Na⁺]_o between 5 and 60 mM. These results indicate that uptake of¹²⁵I^– and³⁶Cl^– into astrocytes and neurons are different in their ion dependence and that they are under separate regulation.Special issue dedicated to Dr. Paola S. Timiras     

Induced neurotransmitter release from primary cultures of rat brain neurons

Neural cells from fetal rat brain were grown in tissue culture in the absence of serum and maintained for 4–5 weeks without medium renewal. Over 80% of the embryonic cells in the culture had a neuronal appearance and formed intercellular synaptic connections. When mature, a definite population of the neuronal cells accumulated ³H-dopamine in a sodium-dependent, benztropine inhibited process. The mature cells were also able to release ³H-dopamine in a potassium evoked, calcium-dependent process, with half maximal dopamine release achieved at a Ca²⁺ concentration of 120μM. In the maturing cells the capacity for potassium evoked, calcium-dependent dopamine release increased from an undetectable level in the first three days to a plateau level after 10–11 days $\text{in vitro}$. The fully expressed release capacity (20–30% of the neurotransmitter retained in the cells) was maintained thereafter. These results demonstrate that primary brain neurons develop a functional neurosecretion apparatus in a chemically defined medium in the absence of animal serum. This extends the utility of primary cultures of brain neurons for developmental structural and biochemical studies of neurotransmission.