Aralar mRNA and protein levels in neurons and astrocytes freshly isolated from young and adult mouse brain and in maturing cultured astrocytes

Neurochemistry International 61 (2012) 1325–1332

Contents lists available at SciVerse ScienceDirect

Neurochemistry International journal homepage: www.elsevier.com/locate/nci

Aralar mRNA and protein levels in neurons and astrocytes freshly isolated from young and adult mouse brain and in maturing cultured astrocytes Baoman Li, Leif Hertz, Liang Peng ⇑ Department of Clinical Pharmacology, China Medical University, Shenyang, PR China

a r t i c l e

i n f o

Article history: Received 31 July 2012 Received in revised form 4 September 2012 Accepted 13 September 2012 Available online 24 September 2012 Keywords: Aralar Astrocyte Glutamate Neuron Protein expression Astrocyte culture

a b s t r a c t Intense glucose-based energy metabolism and glutamate synthesis by astrocytes require malate– aspartate-shuttle (MAS) activity to regenerate NAD+ from NADH formed during glycolysis, since brain lacks significant glycerophosphate shuttle activity. Aralar is a necessary aspartate/glutamate exchanger for MAS function in brain. Based on cytochemical immunoassays the absence of aralar in adult astrocytes was repeatedly reported. This would mean that adult astrocytes must regenerate NAD+ by producing lactate from pyruvate, eliminating its use by oxidative and biosynthetic pathways. We alternatively used astrocytes and neurons from adult brain, freshly isolated by fluorescence-activated cell sorting, to determine aralar protein by a specific antibody and its mRNA by real-time PCR. Both protein and mRNA expressions were identical in adult neurons and astrocytes and similar to whole brain levels. The same level of aralar expression was reached in well-differentiated astrocyte cultures, but not until late development, coinciding with the late-maturing brain capability for glutamate formation and degradation. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction During the last 10 years in vivo magnetic resonance spectroscopic (13C-NMR) assays of metabolism of 13C-labeled glucose or acetate have demonstrated that astrocytes in adult brain have a rate of oxidative metabolism of glucose in gray matter corresponding to 20–30 percent of the total, i.e., at least similar to neurons calculated per volume (reviewed by Hertz, 2011b). Even experiments using incorporation of radioactive or stable isotopes of the astrocyte-specific substrate acetate into neuronal glutamate show this high percentage (Cruz and Cerdán, 1999; Blüml et al., 2002; Lebon et al., 2002; Deelchand et al., 2009; Boumezbeur et al., 2010; Patel et al., 2010; Lanz et al. 2012), with any differences in absolute rates due to species differences and/or different use of anesthetics. Glucose-derived pyruvate is needed by astrocytes for two major purposes, (i) to supply ATP from oxidative pathways for energyconsuming processes, such as uptake of potassium ions (K+) (Somjen et al., 2008; MacAulay and Zeuthen, 2012; Wang et al., 2012) and glutamate (Danbolt, 2001) from extracellular fluid, and (ii) to produce glutamate from glucose via the anaplerotic pathway using pyruvate carboxylase, which is absent in neurons (reviewed by Hertz et al., 2007; Hertz, 2011b). This pathway is needed for de novo synthesis of glutamine, which in the brain ⇑ Corresponding author. Address: Department of Clinical Pharmacology, China Medical University, No. 92 Beier Road, Heping District, Shenyang, PR China. Tel.: +86 24 23256666x5130; fax: +86 24 23251769. E-mail address: [email protected] (L. Peng). 0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.09.009

serves as an essential precursor for the neurotransmitters glutamate and GABA. Pyruvate is generated by the glycolytic pathway in the cytosol, and its production involves one oxidative process, formation of diphosphoglycerate from glyceraldehyde 3-phosphate. In this reaction, NAD+ is reduced to H+ + NADH, which is unable to cross the mitochondrial membrane for its re-oxidation. NAD+ must be regenerated for glycolysis to continue, and this can be accomplished by a redox shuttle system that transfers ‘reducing equivalents’ to the mitochondria or by the cytoplasmic lactate dehydrogenase (LDH) reaction. Because conversion of pyruvate to lactate by LDH eliminates pyruvate as an oxidative-biosynthetic substrate, astrocytic redox shuttling is required to generate pyruvate for ATP and glutamate production, and probably also during glutamate degradation (Hertz, 2011a; Bauer et al., 2012). There are two major intracellular redox shuttle systems, the glycerol-phosphate shuttle and malate–aspartate shuttle (MAS). Both cytosolic and mitochondrial glycerol-3-phosphate dehydrogenases are present in brain, but the importance of this shuttle in brain is probably negligible because these two enzymes are expressed in different cell types (Nguyen et al., 2003; LaNoue et al., 2007). The main pathway for re-synthesis of cytosolic NAD+ in brain is the MAS. As illustrated in Fig. 1, the MAS transfers reducing equivalents from cytoplasm to mitochondria by means of coupled reactions that carry out oxidation–reduction and transamination reactions, utilizing oxaloacetate (OAA), aspartate, malate, aketoglutarate (a-KG), and glutamate as participants in the shuttle (for details, see Fig. 1 and its legend). Cycling of these compounds

1326

B. Li et al. / Neurochemistry International 61 (2012) 1325–1332

The Malate-Aspartate Shuttle Transfers Reducing Equivalents from Cytoplasm to Mitochondria NAD+

Aspartate

NADH

MDHc

OAA

AATc

Malate α-KG

Glutamate

Cytosol Mitochondrion

α-KG

Glutamate

AATm

OAA

Aspartate NAD+

MDHm

Malate

NADH 3 ATP via electron transport chain

Fig. 1. In the malate–aspartate shuttle (MAS) cytosolic malate dehydrogenase (MDHc) oxidizes NADH and converts oxaloacetate (OAA) to malate (top right of figure), which enters the mitochondria in exchange with a-ketoglutarate (a-KG). The mitochondrial malate dehydrogenase (MDHm) re-oxidizes malate to OAA, which is transaminated to aspartate by the mitochondrial aspartate aminotransferase (AATm). Aspartate leaves the mitochondria in exchange with glutamate, requiring ACG (aralar or citrin). In the mitochondria glutamate conversion to a-KG is essential for AATm activity forming aspartate from OAA and delivering a-KG for mitochondrial export. The glutamate imported into the mitochondria had been formed by cytosolic aspartate aminotransferase (AATc) from a-KG after its entry into the cytosol. Without MAS activity NADH formed in the cytosol during glycolysis would have been unable to enter the mitochondria for oxidation. Reprinted from Hertz and Dienel (2002), with permission.

between the cytoplasm and mitochondria also requires two carrier proteins, a malate/a-ketoglutarate exchanger (OGC – Slc25a10) and a glutamate/aspartate exchanger (AGC – Slc25a12 [aralar] or Slc25a13 [citrin]). There are two AGC forms in adult brain, predominantly AGC1 or aralar, with small clusters of citrin only in a few neurons (Contreras et al., 2010). In contrast, hepatocytes only express citrin (Del Arco et al., 2002). Adult brain astrocytes therefore need coordinated activities of OGC, aralar, mitochondrial and cytosolic aspartate aminotransferases, and malate dehydrogenases for MAS function (Fig. 1), and thus for both energy metabolism and transmitter synthesis. Expression of OGC and both the mitochondrial and cytosolic aspartate aminotransferases in brain is well established (Fonnum, 1968; Horio et al., 1988; McKenna et al., 2000), and aspartate aminotransferase activity is high in cultured astrocytes (Schousboe et al., 1977; Erecin´ska et al., 1993). However, the operation of MAS in astrocytes in adult brain in situ has been questioned because aralar (or citrin) was barely detectable in immunocytochemical assays (Ramos et al., 2003; Berkich et al., 2007). The highest level of cytochemically determined aralar expression was obtained when an antigen retrieval technique was used in a study by Pardo et al. (2011). However, electron microscopic analysis of aralar localization determined by immunogold-particle labeling of neuronal and astrocytic mitochondria indicated that astrocytic mitochondria contained only about 7% of the total number of labeled particles. Astrocytic processes were identified by their irregular shape and filamentous membranes that often surround both axons and dendrites and/or by the formation of gap junctions, but this determination may not include the abundant astrocytic mitochondria in fine peripheral processes of astrocytes (Lovatt et al., 2007). Complete lack of astrocytic aralar would make oxidative metabolism of glucose and glutamate biosynthesis in astrocytes impossible. However, based on the MAS turnover rates determined by Berkich et al. (2007) and the contribution of astrocytes to cerebrocortical volume, Hertz (2011a) concluded that the total amount of aralar in the experiments by Pardo et al. (2011) was enough to sustain known rates of astrocytic oxidative metabolism. Nevertheless,

the low mitochondrial expression is worrisome, and higher MAS activity may be needed for glutamate and GABA turnover. At least the immunocytochemical assays that detected even lower levels of aralar, if any, in adult astrocytes (Ramos et al., 2003; Berkich et al., 2007) accordingly appear to be discordant with the readilydetectable, high rates of glucose oxidation and anaplerotic activity conclusively determined by in vivo MRS studies (see Section 1). Together with the positive cytochemical study by Pardo et al. (2011) this raises the possibility that the other immunocytochemical assays may have failed to detect aralar antigen in astrocytes. Failure to detect astrocytic aralar in the studies that did not use an antigen-retrieval procedure (Ramos et al., 2003; Berkich et al., 2007) could have arisen from incomplete antigen exposure to the antibody. In fact, Nishino and Nowak (2004) showed that antigen retrieval substantially enhanced signals from heat shock protein (HSP) 72 and glial fibrillary acidic protein (GFAP), attenuated signals from HSP27, and did not alter the strong MAP2 signal. Alternatively, a false-negative result could arise from lack of antigenicity upon tissue fixation or tissue processing (Fritschy, 2008). At least as high mRNA expression of aralar in adult astrocytes as in neurons has been shown in cells separated by fluorescenceactivated cell sorting (FACS) from brains of transgenic mice coexpressing one fluorescent signal with an astrocytic marker and a different fluorescent signal with a neuronal marker (Lovatt et al., 2007). Although these findings open the possibility that astrocytes might also translate aralar mRNA to protein, they do not necessarily prove it. Protein expression was not studied, because rather small amounts of cells are obtained by the cell-sorting techniques, and many genes were investigated. Similar to a subsequent study by Cahoy et al. (2008) mRNA was therefore determined by microarray analysis. In the present study, the cell separation technique employed by Lovatt et al. (2007) was scaled up by using several transgenic animals, so that enough material was obtained to determine aralar protein levels by Western blotting and mRNA by real-time polymerase chain reaction (PCR). Cellular extracts were prepared from neurons and astrocytes isolated from transgenic mice at 14 and 35 days of age, and the results were compared to whole brain extracts from 70-day-old adult CD-1 mice. Similar comparisons were made in developing cultured cerebral cortical astrocytes but not in neurons, which cannot be maintained for a sufficiently long time in culture (Peng et al., 1991).

2. Material and methods 2.1. Animals and cell preparation Male and female CD-1 or FVB/NTg(GFAP GFP)14Mes/J or B6.CgTg(Thy1-YFPH)2Jrs/J mice (from The Jackson Laboratory, Bar Harbor, ME) were housed as previously detailed (Fu et al., 2012). The transgenic mice combine expression of Thy1, a marker of large projection neurons (see Lovatt et al., 2007), with a specific fluorescent signal, and the astrocyte marker GFAP, with a fluorescent signal at a different wavelength. All experiments were carried out in accordance with the USA National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised 1978, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University. After decapitation cerebral hemispheres minus olfactory bulbs, hippocampi, and basal ganglia were immediately removed and used either for cell culture and whole-brain studies (CD-1 mice) or cell sorting (transgenic mice). For the latter, cerebral hemispheres were placed in cold Hanks’ buffer containing the glutamate receptor antagonists DNQX (3 lM) and APV (100 lM). A cell

B. Li et al. / Neurochemistry International 61 (2012) 1325–1332

suspension was prepared as previously described (Lovatt et al., 2007). The cerebral hemispheres were cut into small pieces, and digested with 8 U/ml papain in Ca2+/Mg2+-free PIPES/cysteine buffer, pH 7.4, for 1 h at 37 °C/5%CO2. After washing, the tissue was further digested with 40 U/ml DNase I in Mg2+-containing minimum essential medium (MEM) with 1% bovine serum albumin (BSA) for 15 min at 37 °C/5% CO2, carefully triturated in cold MEM with 1% BSA, centrifuged over a 90% Percoll gradient to collect all cells at and above the lipid layer. This solution was further diluted five times and centrifuged to collect the pellet. The cells were resuspended in cold MEM with 1% BSA and 4 mg ml1 propidium iodide (PI). Immediately thereafter the cells were sorted into cold MEM with 1% BSA, using the BD FACSAria Cell Sorting System (35 psi sheath pressure, FACSDiva software S/W 2.2.1; BD Biosciences, San José, CA) as described by Lovatt et al. (2007). GFP and YFP were excited by a 488 nm laser, and emissions were collected by 530 nm discrimination filters. mRNA expression of cell markers of astrocytes (Connexin 30, GFAP, Glt-1 and Fgfr3), neurons (Gabra-1, KCC2, Snap25 and synaptotagmin) and oligodendrocytes (Connexin 47, Mag, Mog and Mbp) were determined (Supplementary Fig. 1 [from Fu et al., 2012], with further details in legend). No contamination with neurons and oligodendrocytes was found in the astrocyte samples or of astrocytes and oligodendrocytes in the neuronal samples (Fu et al., 2012). 2.2. Cultures Primary cultures of astrocytes were prepared from newborn male or female mice as previously described (Hertz et al., 1978, 1998; Hertz 2012) with minor modifications. The neopallia of the cerebral hemispheres were aseptically isolated as described above, freed of meninges, dissociated by vortexing, filtered through nylon meshes, diluted in culture medium, and planted in Falcon Primaria culture dishes. The culture medium was a Dulbecco’s Medium with 7.5 mM glucose, 20% horse serum, and the cultures were incubated at 37oC in a humidified atmosphere of CO2/air (5:95%). The medium was exchanged with fresh medium of similar composition on day 3, and subsequently every 3–4 days. At day 3, the serum concentration was reduced to 10%, and after the age of 2 weeks, 0.25 mM dibutyryl cyclic AMP (dBcAMP) was included in the medium. This compound increases intracellular cyclic AMP and promotes differentiation in astrocyte cultures derived from newborn brain (Hertz, 1990, 1993; Meier et al., 1991; Schubert et al., 2000). The age of 2 weeks for its addition has been determined experimentally, and is consistent with the finding by Moonen and Sensenbrenner (1976) that astrocytes need a certain stage of development in order to respond to dBcAMP, and that by Lodin et al. (1979) that astrocytes de-differntiate in vitro, unless treated with this compound. The statement by Fedoroff et al. (1984) that the dBcAMP-treated cells correspond to reactive astrocytes has proven incorrect (Wandosell et al., 1993), However, unfortunately it may have influenced most researchers using cultured astrocytes for almost 30 years, and in the process damaged the reputation of cultured astrocytes (Kimelberg, 2010). The similarities between not only levels but also development of aralar protein and mRNA expression in the cultured astrocytes and in freshly isolated astrocytes, which will be shown in ‘Results’, support the validity of cultured astrocytes obtained using these procedures as valid models of their in vivo counterparts. 2.3. Real-time PCR Although results for reverse transription PCR were already available, mRNA expression was re-determined by real time polymerase chain reaction (RT-PCR or qPCR). A cell suspension was

1327

prepared by collecting cells in Trizol. The RNA pellet was precipitated with isopropyl alcohol, washed with 70% ethyl alcohol, and dissolved in distilled water. Primers for aralar (fwd: 50 CCTCACCTCAGTTTGGTGTCACTC-30 ; rev: 50 -GTGGCCGTGGCAAG TCTGTA-30 ) and TATA-binding protein (TBP), used as a reference gene (fwd: 50 -GCCTTCCTTCTTGGGTATG-30 ; rev: 50 -GAGGTCTTTACGGATGTCAAC-30 ) were generated by TaKaRa Biotechnology (Dalian, China) and optimized to an equal annealing temperature of 60 °C. The 179 bp product has no similarity with any citrin sequence, as shown by checking the Fwd, Rev primer and the whole 175 bp sequence on line (blast.ncbi.nlm.nih.gov). It is also different from a primer previously used for reverse transcription PCR and that used by Lovatt et al., 2007, both of which were also tested and provided comparable results. Only those observed by real-time PCR will be presented in Section 3. However, the classical PCR amplification, which was performed in a Robocycler thermocycler: 3 min at 95 °C, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s, then 95 °C for 3 min, 55 °C for 1 min, followed by PCR product separation by 1% agarose gel electrophoresis resulted in a single band with desired length for both aralar and TBP. SYBR Greenbased real-time PCR with an Mx 3000P instrument and the Green Quantitative RT-PCR Kit from Agilent Technologies (Cary, NC, USA) was performed using the optimized protocol. The final PCR mixture contained 2 ll each of forward and reverse primers (1 lM), 2 ll of Fast Start DNA Master SYBR Green I (2), 0.3 ll of Ret Dye (1 lM), 2 ll of cDNA template, and it was made up to 20 ll with nuclease free water (Pérez et al., 2012). Reactions were performed in duplicate. Real-time PCR efficiency (E) for each pair of primers and target gene was determined using 5-fold serial dilutions of RT product (1 lg, 200, 40, 8 and 1.6 ng). The number of cycles (Ct) necessary to obtain a threshold fluorescent signal of target genes and reference gene, TBP was determined with 1 lg cDNA. E and Ct were calculated from MxPro QPCR Software (Agilent Technologies, Cary, NC, USA). The relative expression ratio (ratio) of a target gene was calculated based on E and Ct as follows (Pfaffl, 2001):

Ratio ¼ ð1 þ Etarget Þ½DCttarget ðcontrol  sampleÞ=ð1 þ Eref Þ  ½DCtref ðcontrol  sampleÞ

2.4. Statistical analysis The differences between multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by Fisher’s LSD multiple comparison test for unequal replications. The level of significance was set at P < 0.05.

3. Results 3.1. Selectivity and linear range of anti-aralar Western blots The anti-aralar antibody recognized a single band with the same molecular weight (70 kDa) as aralar in extracts of adult mouse brain and 3-week-old cultured astrocytes, but the protein was not detected in liver, which is known to only express citrin (entire blot with some debris shown in Fig. 2A). Scanning of 3 individual experiments showed that the expression of aralar relative to b-actin was similar in whole brain and in the cultured astrocytes (Fig. 2B). When the amounts of protein loaded on the gel were varied over the range 10–100 lg (Fig. 2C), the signal intensities for both aralar and b-actin were nearly linear up to about 80 mg (Fig. 2D). The routinely-loaded protein amount (50 lg) is thus well within the rectilinear range (Fig. 2B), indicating that increases or decreases in the amount of aralar will be reliably quantified.

1328

B. Li et al. / Neurochemistry International 61 (2012) 1325–1332

3.2. Aralar protein and mRNA levels in adult astrocytes and neurons obtained by FACS

A MW Brain

Liver

Astrocytes

Neurons

150 kDa 100 kDa 80 kDa

aralar

60 kDa 50 kDa

β-actin

40 kDa 30 kDa

B Ratio of arlar/β-actin

0.5 0.4 0.3 0.2 0.1

Fig. 3A shows an individual Western blot demonstrating selective labeling of aralar and distinct increases in protein expression of aralar between the ages of 14 and 35 days in dissociated astrocytes, neurons, and whole brain when assayed in the same gels and blots using the same amounts of protein per lane (50 lg). The amount of aralar relative to b-actin increased with age from 14 to 35 days. At each age, aralar levels were similar in astrocytes and neurons freshly-isolated from whole brain and equivalent to that in whole brain (Fig. 3B). Expression of aralar mRNA in freshly-dissociated astrocytes and neurons was similar at 14 days, but by 35 days its level was much higher in astrocytes compared with neurons and similar to that in whole brain (Fig. 3C and D). Thus, in the brain in vivo ontogenetic development of aralar is similar in both cell types (protein) or becomes eventually expressed to a greater extent in astrocytes (mRNA). 3.3. Aralar protein and mRNA levels in cultured astrocytes

0 Brain

Liver

C

Astrocytes

Neurons

Brain Tissue (μg) 10

25

50

75

100

aralar

β-actin

Early developmental increases in aralar protein with time in cultured astrocytes were modest. Two-week-old astrocyte cultures as well as 3-week-old cultures which had not received the routine differentiating treatment with dBcAMP from the age of 2 weeks had similar levels (Fig. 4A). However, dBcAMP treatment more than doubled astrocytic aralar expression, raising it to the level of that in intact brain. Thus, differentiation is a critical aspect of aralar expression in cultured astrocytes similar to what has been shown for many other astrocyte characteristics (Hertz, 1990; Meier et al., 1991). Aralar mRNA expression in the cultured astrocytes was lowest at 1 week, intermediate at 2 weeks, and highest at 3 weeks after dBcAMP-treatment (Fig. 4B). In this context it should be reemphasized that astrocytes need to reach a certain developmental stage, before they can respond to dBcAMP (Mooonen and Sensenbrenner, 1976). At this point in time aralar mRNA expression reached a similarly high level as in brain, i.e., a higher level than in freshly isolated neurons. This slow development is remarkable, as will be discussed below.

D

Intensity

4. Discussion

aralar β-actin

Brain Tissue (μg)

Fig. 2. Protein expression of aralar in brain, liver and cultured astroccytes determined with the antibody sc-271056, specific to aralar, and showing the entire gel. (A) A representative immunoblot showing protein expression for aralar and b-actin, used as a house-keeping protein. The staining of HRP-labeled 2nd antibody was photographed by fluorescent imaging system, but the molecular weight (MW) was photographed by neutral light, and is thus not visible in the Fig. at the same time. The size of aralar is 70 kDa, and that of b-actin 46 kDa. Similar results were obtained from three independent experiments. (B) Means ± SEM (n = 3) of scanned ratios between aralar and b-actin. (C) A representative immunoblot showing protein expression for aralar, determined with the antibody sc-271056, and b-actin, used as a house-keeping protein, in intact CD-1 mouse brain with applied of protein amounts between 10 and 100 lg. (D) Intensity of the expressions of aralar and b-actin at different amounts of applied protein, measured by scanning.

The question whether mature astrocytes express an astrocyteglutamate carrier, an essential component of the malate–aspartate redox shuttle system, was first raised by Ramos et al. (2003) and followed up in subsequent studies by Berkich et al. (2007) and Pardo et al. (2011). This is a tremendously important issue because it brings attention to the question how astrocytes obtain the pyruvate they require for oxidative metabolism and glutamate synthesis. In vivo NMR studies in many laboratories have established very significant rates of oxidation of [13C]glucose in astrocytes, which appears to be at odds with at least some immunocytochemical data. Although the amount of protein reflects capacity, not biological activity, extremely low aralar protein levels in astrocytes or even absence of aralar (Ramos et al., 2003; Berkich et al. 2007) are not compatible with a functioning MAS. Moreover, two studies in cultured astrocytes correlate MAS activity with function in astrocytes: Fitzpatrick et al. (1988) showing inhibited glucose metabolism, but uninhibited pyruvate metabolism after MAS inhibition, and Amaral et al. (2011) demonstrating direct relationship between pyruvate oxidation rate and MAS flux. The low and declining activity in cultured astrocytes reported by Ramos et al. (2003) can probably be explained by differences in culturing technique.

B. Li et al. / Neurochemistry International 61 (2012) 1325–1332

A

Astrocytes (GFAP) 14 days

35 days

Neuron (YFP) 14 days

1329

Brain (Control) 14 days

35 days

35 days

aralar

β-actin

B

1.5

Ratio of aralar/β-actin

14 days 1

*

35 days * *

0.5

0 Astrocytes (GFAP)

Brain

Neuron (YFP)

C

Threshold

D Relative Expression Ratio

1.5 14 days 1.0

35 days

*

0.5

*

*

0.0 Astrocytes (GFAP)

Neurons (YFP)

Brain

Fig. 3. Protein and mRNA expression of aralar in astrocytes and neurons isolated by FACS from cerebral hemispheres of 14- and 35-day old astrocyte-labeled (FVB/NTg(GFAP GFP)14Mes/J or neuron-labeled B6.Cg-Tg(Thy1-YFPH)2Jrs/J) mice and in intact brain of adult CD-1 mice. (A) A representative immunoblot showing protein expression for aralar and b-actin, used as a house-keeping protein. The size of aralar is 70 kDa, and of b-actin 46 kDa. Similar results were obtained from three independent experiments. (B) Means ± SEM of scanned ratios between aralar and b-actin. ⁄Statistically significant (P < 0.05) difference from the same preparation from 14-day old animals. (C) A representative amplification plot of aralar mRNA expression, determined by real-time PCR in astrocytes and neurons isolated by FACS from cerebral hemispheres of 14- and 35-day old astrocyte-labeled (FVB/NTg(GFAP GFP)14Mes/J or neuron-labeled B6.Cg-Tg(Thy1-YFPH)2Jrs/J) mice and in intact brain of adult CD-1 mice. Similar results were obtained from three independent experiments. (D) Means ± SEM (n = 3) of the relative expression ratio (ratio) of aralar. ⁄Statistically significant (P < 0.05) difference from the same preparation from 14-day old animals.

The present study circumvented the possibility that the reported absence or reduced expression of aralar protein in adult astrocytes compared to neurons found with immunocytochemical assays might underestimate aralar expression in astrocytic

mitochondria. It used a different approach that avoided the complexities of immunoassays and of recognition of all astrocytic mitochondria in intact tissue. Extracts of freshly-isolated astrocytes and neurons obtained from brain and separated by FACS were assayed

1330

B. Li et al. / Neurochemistry International 61 (2012) 1325–1332 Astrocytes

A

2 week

1 week

3 week

+ dBcAMP

**

**

Brain

Aralar

TBP

Ratio of aralar/β -actin

B

2.5 2 1.5 1 0.5

*

0 1 week

2 week

3 week

+ dBcAMP Brain

Astrocytes

C

Threshold

Relative Expression Ratio

D

1.5

** ** 1.0

0.5

* 0.0

+ 1 week

2 week

3 week

dBcAMP

Brain

Astrocytes

Fig. 4. Protein and mRNA expression of aralar in primary cultures of astrocytes and in intact brain of adult CD-1 mice. Astrocytes were cultured for 1 or 2 weeks, and for 3 weeks with (dBcAMP) or without (3 week) addition to the medium of 0.25 mM dibutyryl cAMP from the age of 2 weeks. (A). A representative immunoblot showing protein expression for aralar and b-actin, used as a house-keeping protein. Similar results were obtained from three independent experiments. (B) Means ± SEM (n = 3) of scanned ratios between aralar and b-actin. ⁄Statistically significant (P < 0.05) difference from 2 and 3 weeks groups in astrocytes. ⁄⁄Statistically significant (P < 0.05) difference from all other groups, but not from each other. (C) A representative amplification plot of aralar. mRNA expression of aralar determined by real-time PCR in primary cultures of astrocytes and in intact brain of adult CD-1 mice. Similar results were obtained from three independent experiments. (D) Means ± SEM (n = 3) of the relative expression ratio (ratio) of aralar. ⁄Statistically significant (P < 0.05) difference from 2 and 3 weeks groups in astrocytes. ⁄⁄Statistically significant (P < 0.05) difference from all other groups, but not from each other.

by Western blotting and qRT-PCR. Aralar protein expression levels were similar in these two cell types in both suckling and young adult mice, whereas mRNA levels were higher in astrocytes, con-

firming previous findings by Lovatt et al. (2007) of at least as high mRNA levels in astrocytes as in neurons. Moreover, the possibility of deficient translation in astrocytes was discounted by showing

B. Li et al. / Neurochemistry International 61 (2012) 1325–1332

that aralar protein levels were as high in astrocytes as in neurons. The remarkably high mRNA levels in astrocytes may indicate a readiness to respond to potential metabolic stimuli. Similar assays in cultured astrocytes showed that aralar protein and mRNA levels in astrocytes doubled between 1 and 2 weeks to eventually reach or bypass the level in neurons. The doubling of aralar protein after astrocytic differentation by dBcAMP, brought the levels up to those observed in astrocytes obtained by FACS in adult brain. These Western blot immunoassays were obtained within the linear ranges for signal intensity as a function of aralar and b-actin protein amount. They detected aralar, not citrin, since no antibody response was found in liver cells. Moreover, the aralarspecific primer for mRNA would not recognize citrin. Equivalent expression of aralar in astrocytes and neurons isolated from brain should remove a significant hurdle for acceptance of high rates of oxidative metabolism in brain astrocytes and in at least some types of cultured cells (reviewed by Hertz et al., 2007; Hertz 2011b). The developmental increases of brain aralar protein and mRNA levels between 2 and 4 weeks of age are consistent with a comparable increase in fluxes in glutamatergic and GABAergic neuronal TCA cycles. Tricarboxylic acid cycle function matures early, as shown by maximum ability of stimuli of energy production to enhance brain slice oxidation around postnatal day 15 (Holtzman et al., 1982). Increases in the activity of the glutamate (GABA)glutamine cycle between postnatal days 10 and 30 (Chowdhury et al., 2007) occur in parallel with the rise in aralar protein and mRNA levels between postnatal days 14 and 35 (Fig. 3). Other glucose-metabolizing enzymes (e.g., hexokinase, aldolase, LDH, and pyruvate dehydrogenase) also exhibit large increases in their activity between the ages of 15 and 30 days (Leong and Clark, 1984; Land et al., 1977). Synaptic mitochondria mature earlier (Almeida et al., 1995) than non-synaptic mitochondria (Bates et al., 1994), the fraction that would include astrocytic mitochondria. The cytosolic malate dehydrogenase (MDHc) which operates in the MAS, but not in the TCA cycle, has a slow developmental increase in non-synaptic mitochondria, whereas the mitochondrial malate dehydrogenase (MDHm), which functions both in the MAS (Fig. 1) and in the TCA cycle, matures much faster (Malik et al., 1993). Additional strong evidence that the late development is related to the maturation of glutamatergic and GABAergic signaling is the demonstration by Patel and Balázs (1970) that incorporation of 14C from glucose into amino acids in the rat brain in vivo increases sharply between postnatal days 10 and 20, reaching its maximum around day 25, and that the maximum increase in glutamine/glutamate specific activities (a sign of metabolic compartmentation) may even occur a few days later. The activity of glutamine synthetase, an astrocytic enzyme required to provide the precursor for neurotransmitter glutamate and GABA for neurons, also increases steeply during all of the first 3 weeks of development in cultured astrocytes and in brain in vivo (Hertz et al., 1978; Patel et al., 1982). Together these observations suggest that a considerable part of the late increase in aralar expression in both neurons and astrocytes reflects the increase of glutamate production, astrocyte-to-neuron transfer of glutamate via glutamine, GABA synthesis, and astrocytic glutamate and GABA degradation that are essential for glutamatergic and GABAergic transmitter activity. In conclusion, using the fluorescence-based technique for brain cell separation developed by Lovatt et al. (2007), we confirmed their observation that freshly-isolated astrocytes and neurons have at least similar levels of aralar mRNA expression. These findings were extended to demonstrate large increases in expression of not only aralar mRNA but also its protein in both cell types between postnatal days 14 and 35 and similar protein levels in the mature cells. The findings suggest a developmental correlation not only with TCA cycle activity, but also with formation and,

1331

perhaps, degradation of glutamate and GABA (Hertz, 2011a, b). This is consistent with the need for astrocytic MAS activity not only for energy metabolism but also for these processes. The newest and best performed immunocytochemical study (Pardo et al., 2011) is qualitatively, although not quantitatively in agreement with the observations made. Acknowledgements This study was supported by Grants 31171036 to L.P. and No. 31000479 to B.L. from the National Natural Science Foundation of China. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neuint.2012. 09.009. References Almeida, A., Brooks, K.J., Sammut, I., Keelan, J., Davey, G.P., Clark, J.B., Bates, T.E., 1995. Postnatal development of the complexes of the electron transport chain in synaptic mitochondria from rat brain. Dev. Neurosci. 17, 212–218. Amaral, A.I., Teixeira, A.P., Håkonsen, B.I., Sonnewald, U., Alves, P.M., 2011. A comprehensive metabolic profile of cultured astrocytes using isotopic transient metabolic flux analysis and C-labeled glucose. Front Neuroenerg. 3, 5. Bates, T.E., Heales, S.J., Davies, S.E., Boakye, P., Clark, J.B., 1994. Effects of 1-methyl4-phenylpyridinium on isolated rat brain mitochondria: evidence for a primary involvement of energy depletion. J. Neurochem. 63, 640–648. Bauer, D.E., Jackson, J.G., Genda, E.N., Montoya, M.M., Yudkoff, M., Robinson, M.B., 2012. The glutamate transporter, GLAST, participates in a macromolecular complex that supports glutamate metabolism. Neurochem. Int. 61, 566–574. Berkich, D.A., Ola, M.S., Cole, J., Sweatt, A.J., Hutson, S.M., LaNoue, K.F., 2007. Mitochondrial transport proteins of the brain. J. Neurosci. Res. 85, 3367–3377. Blüml, S., Moreno-Torres, A., Shic, F., Nguy, C.H., Ross, B.D., 2002. Tricarboxylic acid cycle of glia in the in vivo human brain. NMR Biomed. 15, 1–5. Boumezbeur, F., Petersen, K.F., Cline, G.W., Mason, G.F., Behar, K.L., Shulman, G.I., Rothman, D.L., 2010. The contribution of blood lactate to brain energy metabolism in humans measured by dynamic 13C nuclear magnetic resonance spectroscopy. J. Neurosci. 30, 13983–13991. Cahoy, J.D., Emery, B., Kaushal, A., Foo, L.C., Zamanian, J.L., Christopherson, K.S., Xing, Y., Lubischer, J.L., Krieg, P.A., Krupenko, S.A., Thompson, W.J., Barres, B.A., 2008. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–278. Chowdhury, G.M., Patel, A.B., Mason, G.F., Rothman, D.L., Behar, K.L., 2007. Glutamatergic and GABAergic neurotransmitter cycling and energy metabolism in rat cerebral cortex during postnatal development. J. Cereb. Blood Flow Metab. 27, 1895–1907. Contreras, L., Urbieta, A., Kobayashi, K., Saheki, T., Satrústegui, J., 2010. Low levels of citrin (SLC25A13) expression in adult mouse brain restricted to neuronal clusters. J. Neurosci. Res. 88, 1009–1016. Cruz, F., Cerdán, S., 1999. Quantitative 13C NMR studies of metabolic compartmentation in the adult mammalian brain. NMR Biomed. 12, 451–462. Danbolt, N.C., 2001. Glutamate uptake. Prog. Neurobiol. 65, 1–105. Deelchand, D.K., Nelson, C., Shestov, A.A., Ug˘urbil, K., Henry, P.G., 2009. Simultaneous measurement of neuronal and glial metabolism in rat brain in vivo using co-infusion of [1,6–13C2]glucose and [1,2–13C2]acetate. J. Magn. Reson. 196, 157–163. del Arco, A., Morcillo, J., Martínez-Morales, J.R., Galián, C., Martos, V., Bovolenta, P., Satrústegui, J., 2002. Expression of the aspartate/glutamate mitochondrial carriers aralar1 and citrin during development and in adult rat tissues. Eur. J. Biochem. 269, 3313–3320. Erecin´ska, M., Pleasure, D., Nelson, D., Nissim, I., Yudkoff, M., 1993. Cerebral aspartate utilization: near-equilibrium relationships in aspartate aminotransferase reaction. J Neurochem. 60, 1696–1706. Fedoroff, S., McAuley, W.A., Houle, J.D., Devon, R.M., 1984. Astrocyte cell lineage. V. Similarity of astrocytes that form in the presence of dBcAMP in cultures to reactive astrocytes in vivo. J. Neurosci. Res. 12, 14–27. Fitzpatrick, S.M., Cooper, A.J., Hertz, L., 1988. Effects of ammonia and betamethylene-DL-aspartate on the oxidation of glucose and pyruvate by neurons and astrocytes in primary culture. J Neurochem. 51, 1197–1203. Fonnum, F., 1968. The distribution of glutamate decarboxylase and aspartate transaminase in subcellular fractions of rat and guinea-pig brain. Biochem. J. 106, 401–412. Fritschy, J.M., 2008. Is my antibody-staining specific? How to deal with pitfalls of immunohistochemistry. Eur. J. Neurosci. 28, 2365–2370.

1332

B. Li et al. / Neurochemistry International 61 (2012) 1325–1332

Fu, H., Li, B., Hertz, L., Peng, L., 2012. Contributions in astrocytes of SMIT1/2 and HMIT to myo-inositol uptake at different concentrations and pH. Neurochem. Int. 61, 187–194. Hertz, L., 1990. Dibutyryl cyclic AMP treatment of astrocytes in primary cultures as a substitute for normal morphogenic and ‘functiogenic’ transmitter signals. In: Privat, A., Giacobini, E., Timiras, P., Vernadakis, A. (Eds.), Molecular Aspects of Development and Aging in the Nervous System. Plenum, N.Y., pp. 227–243. Hertz, L., 1993. Metabolic interactions between neurons and astrocytes. In: Fedoroff, S., Doucette, R., Juurlink, B.H.J. (Eds.), Biology and Pathology of Astrocyte–Neuron Interactions. Plenum. New. Y., pp. 1–13. Hertz, L., 2011a. Brain glutamine synthesis requires neuronal aspartate: a commentary. J. Cereb. Blood Flow Metab. 31, 384–387. Hertz, L., 2011b. Astrocytic energy metabolism and glutamate formation–relevance for 13C-NMR spectroscopy and importance of cytosolic/mitochondrial trafficking. Magn. Reson. Imaging 29, 1319–1329. Hertz, L., 2012. Isotope-based quantitation of uptake, release, and metabolism of glutamate and glucose in cultured astrocytes. Methods Mol. Biol. 814, 305–323. Hertz, L., Bock, E., Schousboe, A., 1978. GFA content, glutamate uptake and activity of glutamate metabolizing enzymes in differentiating mouse astrocytes in primary cultures. Dev. Neurosci. 1, 226–238. Hertz, L., Dienel, G.A., 2002. Energy metabolism in the brain. Int. Rev. Neurobiol. 51, 1–102. Hertz, L., Peng, L., Dienel, G.A., 2007. Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/ glycogenolysis. J. Cereb. Blood Flow Metab. 27, 219–249. Hertz, L., Peng, L., Lai, J.C., 1998. Functional studies in cultured astrocytes. Methods 16, 293–310. Holtzman, D., Olson, J., Zamvil, S., Nguyen, H., 1982. J. Neurochem. 39, 274–276. Horio, Y., Tanaka, T., Taketoshi, M., Uno, T., Wada, H., 1988. Rat cytosolic aspartate aminotransferase: regulation of its mRNA and contribution to gluconeogenesis. J. Biochem. 103, 805–808. Kimelberg, H.K., 2010. Functions of mature mammalian astrocytes: a current view. Neuroscientist 16, 79–106. Land, J.M., Booth, R.F., Berger, R., Clark, J.B., 1977. Development of mitochondrial energy metabolism in rat brain. Biochem. J. 164, 339–348. LaNoue, K.F., Carson, V., Berkich, D.A., Hutson, S.M., 2007. Mitochondrial/Cytosolic Interactions via Metabolite Shuttles and Transporters, in Handbook of Neurochemistry and Molecular Neurobiology. In: Lajtha, A., Gibson, G.E., Dienel, G.A. (Eds.). Springer-Verlag, Berlin, pp. 5616–5689, Vol. 2. Lanz, B., Uffmann, K., Wyss, T., Weber, M., Buck, B., Buck, A., Gruetter, R., 2012. A two-compartment mathematical model of neuroglial metabolism using [1-11C] acetate. J. Cereb. Blood Flow Metab. 32, 548–559. Lebon, V., Petersen, K.F., Cline, G.W., Shen, J., Mason, G.F., Dufour, S., Behar, K.L., Shulman, G.I., Rothman, D.L., 2002. Astroglial contribution to brain energy metabolism in humans revealed by 13C nuclear magnetic resonance spectroscopy: elucidation of the dominant pathway for neurotransmitter glutamate repletion and measurement of astrocytic oxidative metabolism. J. Neurosci. 22, 1523–1531. Leong, S.F., Clark, J.B., 1984. Regional enzyme development in rat brain. Enzymes associated with glucose utilization. Biochem. J. 218, 131–138. Lodin, Z., Faltin, J., Korínková, P., 1979. The effect of dibutyryl cyclic AMP on cultivated glial cells from corpus callosum of 30-day-old rats. Physiol. Bohemoslov. 28, 105–111. Lovatt, D., Sonnewald, U., Waagepetersen, H.S., Schousboe, A., He, W., Lin, J.H., Han, X., Takano, T., Wang, S., Sim, F.J., Goldman, S.A., Nedergaard, M., 2007. The transcriptome and metabolic gene signature of protoplasmic astrocytes in the adult murine cortex. J. Neurosci. 27, 12255–12266. MacAulay, N., Zeuthen, T., 2012. Glial K+ clearance and cell swelling: key roles for cotransporters and pumps. Neurochem. Res. Feb 26. [Epub ahead of print].

Malik, P., McKenna, M.C., Tildon, J.T., 1993. Regulation of malate dehydrogenases from neonatal, adolescent, and mature rat brain. Neurochem. Res. 18, 247–257. McKenna, M.C., Stevenson, J.H., Huang, X., Hopkins, I.B., 2000. Differential distribution of the enzymes glutamate dehydrogenase and aspartate aminotransferase in cortical synaptic mitochondria contributes to metabolic compartmentation in cortical synaptic terminals. Neurochem. Int. 37, 229–241. Meier, E., Hertz, L., Schousboe, A., 1991. Neurotransmitters as developmental signals. Neurochem. Int. 19, 1–15. Moonen, G., Sensenbrenner, M., 1976. Effects of dibutyryl cyclic AMP on cultured brain cells from chick embryos of different ages. Experientia 32, 40–42. Nguyen, N.H., Bråthe, A., Hassel, B., 2003. Neuronal uptake and metabolism of glycerol and the neuronal expression of mitochondrial glycerol-3dehydrogenase. J. Neurochem. 85, 831–842. Nishino, K., Nowak Jr., T.S., 2004. Time course and cellular distribution of hsp27 and hsp72 stress protein expression in a quantitative gerbil model of ischemic injury and tolerance. thresholds for hsp72 induction and hilar lesioning in the context of ischemic preconditioning. J. Cereb. Blood Flow Metab. 24, 167–178. Pardo, B., Rodrigues, T.B., Contreras, L., Garzón, M., Llorente-Folch, I., Kobayashi, K., Saheki, T., Cerdan, S., Satrústegui, J., 2011. Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation. J. Cereb. Blood Flow Metab. 31, 90–101. Patel, A.B., de Graaf, R.A., Rothman, D.L., Behar, K.L., Mason, G.F., 2010. Evaluation of cerebral acetate transport and metabolic rates in the rat brain in vivo using 1H[13C]-NMR. J. Cereb. Blood Flow Metab. 30, 1200–1213. Patel, A.J., Balázs, R., 1970. Manifestation of metabolic compartmentation during the maturation of the rat brain. J. Neurochem. 17, 955–971. Patel, A.J., Hunt, A., Gordon, R.D., Balázs, R., 1982. The activities in different neural cell types of certain enzymes associated with the metabolic compartmentation glutamate. Brain Res. 256, 3–11. Peng, L.A., Juurlink, B.H., Hertz, L., 1991. Differences in transmitter release, morphology, and ischemia-induced cell injury between cerebellar granule cell cultures developing in the presence and in the absence of a depolarizing potassium concentration. Dev. Brain Res. 63, 1–12. Pérez, L.J., Díaz de Arce, H., Cilloni, F., Salviato, A., Marciano, S., Perera, C.L., Salomoni, A., Beato, M.S., Romero, A., Capua, I., Cattoli, G., 2012. An SYBR Green-based realtime RT-PCR assay for the detection of H5 hemagglutinin subtype avian influenza virus. Mol. Cell Probes. 26, 137–145. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in realtime RT-PCR. Nucleic Acids Res. 29, e45. Ramos, M., del Arco, A., Pardo, B., Martínez-Serrano, A., Martínez-Morales, J.R., Kobayashi, K., Yasuda, T., Bogónez, E., Bovolenta, P., Saheki, T., Satrústegui, J., 2003. Developmental changes in the Ca2+-regulated mitochondrial aspartateglutamate carrier aralar1 in brain and prominent expression in the spinal cord. Brain Res. Dev. Brain Res. 143, 33–46. Schousboe, A., Svenneby, G., Hertz, L., 1977. Uptake and metabolism of glutamate in astrocytes cultured from dissociated mouse brain hemispheres. J. Neurochem. 29, 999–1005. Schubert, P., Morino, T., Miyazaki, H., Ogata, T., Nakamura, Y., Marchini, C., Ferroni, S., 2000. Cascading glia reactions: a common pathomechanism and its differentiated control by cyclic nucleotide signaling. Ann. N. Y. Acad. Sci. 903, 24–33. Somjen, G.G., Kager, H., Wadman, W.J., 2008. Computer simulations of neuron-glia interactions mediated by ion flux. J. Comput. Neurosci. 25, 349–365. Wandosell, F., Bovolenta, P., Nieto-Sampedro, M., 1993. Differences between reactive astrocytes and cultured astrocytes treated with di-butyryl-cyclic AMP. J. Neuropathol. Exp. Neurol. 52, 205–215. Wang, F., Smith, N.A., Xu, Q., Fujita, T., Baba, A., Matsuda, T., Takano, T., Bekar, L., Nedergaard, M., 2012. Astrocytes modulate neural network activity by Ca2+dependent uptake of extracellular K+. Sci. Signal 5, ra26.