Age-related changes in glial fibrillary acidic protein mRNA in the mouse brain

Neurobiologyof Aging, Vol. 12, pp. 165-170. ~ Pergamon Press plc. 1991. Printed in the U.S.A.

0197-4580/91 $3.00 + .00

Age-Related Changes in Glial Fibrillary Acidic Protein mRNA in the Mouse Brain J A M E S R. G O S S , C A L E B E. F I N C H A N D D A V I D G. M O R G A N

Andrus Gerontology Center and Department of Biological Sciences University of Southern California, Los Angeles, CA 90089--0191 R e c e i v e d 19 M a r c h 1990; A c c e p t e d 13 July 1990

GOSS, 1. R., C. E. FINCH AND D. G. MORGAN. Age-relatedchanges in glialfibrillary acidic protein mRNA in the mouse brain. NEUROBIOL AGING 12(2) 165-170, 1991.--Several RNA sequences were tested for age-related changes in prevalence levels in the mouse cerebral cortex, hippocampus, and cerebellum. In all three regions, there were increased levels of RNA for glial fibrillary acidic protein, an astrocyte-specific protein, by RNA gel-blot analysis and by a solution hybridization assay. There was no change in glutamine synthetase mRNA level, another glial protein. The only other mRNA sequence which changed was Thy-1 antigen, a neuronal protein, which decreased slightly in the hippocampus. We conclude that with age there is an age-related increase in glial fibrillary acidic protein RNA prevalence potentially reflecting an increase in the size, number, and/or fibrous character of astrocytes. Glial fibrillary acidic protein

Thy-1 antigen

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A number of studies have sought changes in neuronal chemical markers with aging (32). However, to fully understand the aging process in the brain, it is necessary to consider not only neurons but the nonneuronal cells as well. The studies described here have concentrated on age-related changes which occur in astrocytes; specifically, changes in selected RNA transcript levels. Age-related changes in RNA prevalence would imply that genomic changes in gene expression underlie functional changes with age. Astrocytes were originally thought to play only supportive roles within the brain (43). However, recent evidence suggests new roles for astrocytes, including neurotransmitter uptake (4,20), synthesis and secretion of trophic factors (13, 14, 19, 38), targets for hormones and immune system cytokines (10, 27, 48), aid in repair and regeneration of wounds (19, 29, 40), blocking of axonal regeneration (28), regulation of synaptic density (30), and regulation of cerebral blood flow (37). Obviously, astrocytes serve important functions; the study of these cells, therefore, is critical when assessing age-related changes in the brain. The vast majority of data regarding age-related changes in astrocytes are histological and deal with astrocyte numbers and/or size. Some studies have reported an actual change in the number of astrocytes (17, 41, 44, 46), while others have found an increase in the size and fibrous character of astrocytes with age without a concomitant increase in number (3, 15, 18, 21, 22, 40). One problem with these histological approaches is the difficulty in quantifying the results. The polymorphic forms of neurons and glia, the difficulty in distinguishing between the many different types of glia, problems with tissue shrinkage, the use of different stereological correction formulas, and the labor inten-

Solution hybridization assay

Aging

sive methods needed to perform accurate cell counts within large areas of the brain all combine to make interlaboratory comparisons difficult. Two approaches which can help alleviate this problem are chemical and molecular analysis. There are surprisingly very few studies which use either of these approaches to study aging astrocytes (7,47). We have used a molecular approach employing RNA gel-blot analysis (Northern blots) and solution hybridization (RNase protection assay) to quantify changes in selected RNA levels within three different brain regions and four different age groups. The major significant findings were an increase for the astrocyte-specific RNA for glial fibrillary acidic protein (GFAP), within all brain regions studied, and a small decrease in Thy-1 antigen RNA, a predominantly neuronal protein (33), in the hippocampus. METHOD

Animals Male C57BL/6J mice were used throughout. Mice were obtained from Jackson Laboratories (Bar Harbor, ME). Animals were housed three to five per cage in an aging colony within the double-barrier vivarium of the Andrus Gerontology Center. They were maintained on a 12-hour light/dark cycle with food and water available. Animals were killed by cervical dislocation followed by decapitation and necropsy. Mice with observable tumors or other gross pathological lesions were culled from the study. Whole brains were removed and dissected on ice. Cerebral cortex, cere-

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bellum, and hippocampus were taken and immediately placed on dry ice and stored at - 7 0 ° C until needed.

[32p]Labeled cRNA Probes RNA transcripts were synthesized from cDNA's inserted into the transcription vector Bluescribe (Stratagene, San Diego, CA) and labeled with [32p]UTP to a specific activity of 109 CPM/Ixg. Clones used were as follows. Chicken [3-tubulin: 1.2 kb in length, corresponding to base pairs 475-1817 (45); mouse GFAP: 1.1 kb in length, corresponding to the entire coding region (26); feline glutamic acid decarboxylase (GAD): 1.25 kb in length, corresponding to amino terminal coding region; rat glutamine synthetase (GS): 1.6 kb in length, corresponding to 3' untranslated region; human somatostatin (SS): 0.45 kb in length, corresponding to part of the preprohormone coding region (42); mouse Thy-1 antigen: 0.76 kb in length, corresponding to the entire coding region (5).

Northern-Blot Analysis Total RNA was extracted from pooled tissue samples using the guanidine thiocyanate and cesium chloride centrifugation method (pools consisted of 5 cerebral cortices, 8 cerebella, and 16 agematched hippocampi per RNA sample). RNA yields were estimated by A26o (1.0 O.D. =43 ~g RNA/ml). Eight txg of each RNA sample were electrophoresed through a 1% agarose gel containing 6% formaldehyde, and transferred to nylon membranes. Membranes were hybridized to one of five RNA transcripts ([3tubulin, GFAP, GAD, SS, or Thy-1). Blots were hybridized overnight at 77°C in 5 x SSC containing 0.5% nonfat dry milk, 1% SDS, 10% dextran sulfate, 25 I~g/ml poly(A)RNA, 25 Ixg/ml poly(C)RNA, and 100 txg/ml sheared salmon sperm DNA, Blots were washed a total of five times with decreasing concentrations of SSC to a final criterion of 0.5 x SSC at 77°C. Kodak XOMAT film was used for autoradiographs. Autoradiographs were analyzed using a computer-aided densitometry program (see legend, Fig. 2).

Solution Hybridization Analysis Total RNA was isolated as per the Northern-blot analysis except that four cerebella and seven hippocampi were pooled per sample, cRNA probes were transcribed as described above. Prevalence of selected messages was measured using a solution hybridization/RNase protection assay (24). This assay measures hybrid formation between an excess amount of [32p]labeled antisense cRNA and increasing amounts of sample RNA. For the analysis of selected RNA sequences measured in the cerebral cortex, six amounts of total RNA were used (0-5 izg, with 1 tzg increments). Each of these were hybridized with 1 ng of a [32p]labeled antisense RNA probe. Hybridizations were performed in 5 ml conical glass tubes which had been treated with dimethyldichlorosilane. Hybridization conditions consisted of a 20-~1 reaction containing 0.4 M NaC1, 50% formamide, sample RNA and yeast tRNA to a total of 10 txg of RNA per tube. Incubation at 50°C for 4 hours gave >99% hybridization. After hybridization, remaining single-stranded [32p]cRNA was digested by adding 100 Ixl of a solution containing 0.4 M NaC1; 50 txg of sheared salmon sperm DNA; 10 Ixg of RNase A; and 300 units of RNase T1, and incubating for 1 hour in a 37°C shaking water bath. The protected duplex RNA was precipitated by adding 1 ml of cold 1 M HC1 with 0.1 M Na4P207 and placing the tubes in an ice bath for 15 minutes. The contents of each tube were filtered through Whatman GF/C filters using a Brandel cell harvester and rinsed four times with 5 ml of 1 M HC1, 0.1 M

GOSS, FINCH AND MORGAN

Na4P207. Filters were air-dried and counted using an LKB scintillation counter. Each assay was performed in triplicate. RNA sequences in the cerebellum and hippocampus were measured as above except that the six amounts of total RNA used were from 0--4 ixg with 0.8 Ixg increments for GFAP and 0-2 txg with 0.4 txg increments for Thy-1 antigen. Also hybridizations were carried out for 18 hours using 0.1 ng of [32P]labeled probe ( S A = 10 9 CPM/Ixg) in 4.5 ml conical plastic tubes (Stockwell Scientific). The decrease in the amount of probe used and the use of the plastic tubes greatly reduced the background counts found without added sample RNA. Linear regression analyses were performed for each titration and the resultant slopes were calculated. RNA prevalence levels were calculated in pg of message per txg of total RNA by dividing the slope by the specific activity of the probe, and correcting for radioactive decay and for the ratio of probe length to actual message length for each RNA sequence.

Statistical Analysis RNA prevalence levels for each probe were compared between age groups using a one-way analysis of variance (ANOVA). All differences discussed are significant to the p<0.005 level unless otherwise stated. All averages are expressed as plus and minus one standard error of the mean. Age group means were subsequently compared using Newman-Keul's range test. RESULTS

In a preliminary study we used three age groups of mice; 6 months, 12 months, and 29 months. RNA was extracted from the cortex, cerebellum, and hippocampus. There was one RNA sample per age group for the cortex and the hippocampus, and two RNA samples per age group for the cerebellum. The average yield for all the samples was 670 -+ 20 txg RNA/g tissue. There was no effect of age or region on the yields. RNA gel-blot (Northern) hybridizations were performed on all the samples using the five [32p]labeled cRNA probes discussed above. Figure 1 shows representative blots for each of the five sequences examined. The only age-related change detected was an increase in the signal for GFAP in the 29-month samples from each brain region (Fig. 2). Computer-aided densitometry indicated a three-fold increase in signal intensity between 6 and 29 months in the cortex and the cerebellum. The difference found in the hippocampus was a 2.5fold increase between 6 and 29 months (Fig. 2). Based on these preliminary findings, we extended our analysis by examining 4 age groups of mice and using the more quantifiable solution hybridization/RNase protection assay. RNA was isolated from cortex, cerebellum, and hippocampus as above. There was no effect of age on RNA yields; however, the yields were different for the three regions (850_+40, 4 3 0 ± 4 0 , and 720_+70 txg RNA/g tissue respectively). Sequence prevalence levels were measured as described in the Method section. Figure 3 shows a representative plot for the hybridization of a cRNA probe to GFAP using hippocampal total RNA taken from each age group. The four RNA sequences measured in cortex were: GFAP, GS, Thy-1 antigen, and [3-tubulin. The only significant change was an 80% increase in GFAP message in the old samples as compared to the other three age groups (Fig. 4a). Limiting amounts of RNA permitted assays of only two RNA sequences, GFAP and Thy-1 antigen, in both cerebellum and hippocampus. In the hippocampus there was both a 40% increase in GFAP RNA level with age and a 13% decrease in Thy-1 RNA level (p<0.02) (Fig. 5). In the cerebellum the only significant change was a 75% increase in GFAP RNA between young and

INCREASED GFAP RNA WITH AGE IN MOUSE BRAIN

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old ( p < 0 . 0 5 ) . Thy-1 R N A level did not c h a n g e (Fig. 6). DISCUSSION

This study shows age-related increases of GFAP RNA in the mouse cortex, cerebellum, and hippocampus. Except for the slight decrease in Thy-1 in the hippocampus, there were no changes in the other RNA sequences measured by either Northern-blot anal-

ysis or solution hybridization. This age-related increase in GFAP RNA is predicted to increase the concentration of this intermediate filament at the protein level. A preliminary report (31) found a roughly 2-fold age-related increase in striatal GFAP by immunoassay in female C57BL/6J mice. While striatum was not examined in the present study, our results suggest these increases in GFAP RNA occur globally with brain aging. In rat brain, others have found age-related increases in both GFAP RNA (N. Holbrook, NIA, personal communication) and GFAP protein levels

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Age (months) FIG. 4. RNA prevalence levels in cortex with aging. RNA was extracted from pooled mouse cortex and prevalence levels of the RNA were measured using a solution hybridization assay. Bars represent the average amount of target RNA per total RNA of four samples _+S.E.M. for each age group. GFAP = glial fibrillary acidic protein, GS = glutamine synthetase, THY-l = Thy-I antigen, BETA-TUP = [3-tubulin.

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(J. O'Callaghan, EPA, personal communication). Our research group has recently obtained preliminary data using semiquantitative immunocytochemistry that staining for GFAP is increased in these regions by 50-100% (16). Additional evidence consistent with these results is derived from the histological data summarized in the introduction. Since GFAP is the intermediate filament associated with astrocytes, any increase in their fibrous character or increase in their size would be expected to be paralleled by an increase in the amount of RNA for GFAP. Glutamine synthetase distribution in the adult CNS is localized primarily, if not exclusively, in astrocytes (36). The failure of GS RNA to increase in parallel with GFAP RNA implies that the primary astrocytic change with age is an increase in fibrous character, rather than increases in size or number. However, independent down-regulation of GS RNA (per cell), or small increases in GS RNA cannot be ruled out. Importantly, GS enzymatic activity does appear to increase by around 25% with age in a variety of brain regions (7). In future experiments we plan to measure GS RNA level in the hippocampus and cerebellum as well as cortex. An increase in the fibrous nature of the astrocytes may reflect astrocytes undergoing reactive gliosis. Reactive gliosis is a process by which astrocytes increase in size, and perhaps number; the processes become larger and more numerous, and there is an increase in glial filaments including the intermediate filaments formed by GFAP (8,9). This hypertrophy may be a reaction to the degeneration of neighboring synapses, neurites or entire neurons (1, 15, 21). GFAP RNA was increased in all the brain regions studied; but Thy-1 antigen, the neuronal marker examined by solution hybridization, decreased only in the hippocampus. One interpretation is that neuronal degeneration or loss is not the stimulus for astrocytic hypertrophy in normally aging brain, and that some other factor is responsible. Because synapse loss appears more extensive than neuron loss in rodent brain (1, 12, 15, 25) this may be the stimulus for increasing reactive astrogliosis over the life span. However, in hippocampus, neuron loss may also be a contributing factor (23), consistent with the loss of Thy1 antigen RNA in this structure. Almost all age-related studies involving specific molecules deal with proteins or enzyme activities. Typically age-related changes in these chemical markers are less than 30% (32,39).

INCREASED GFAP RNA WITH AGE IN MOUSE BRAIN

Few studies have dealt with age-related changes in brain mRNA. Studies from Finch's group (6) and others (2) have found no detectable change of poly(A)RNA yield or sequence complexity with age, while another study has found a selective decrease in POMC mRNA in the hypothalamus but not in the pituitary (35). In comparison our increases in GFAP mRNA (80% in cortex, 75% in cerebellum, and 40% in hippocampus) are very large. These results are supportive of a general hypothesis concerning aging and brain chemistry, recently elaborated by one of us [D.G.M.; (11)]. This hypothesis purports that many aging changes in synaptic neurochemistry reflect an underlying change in the cellular composition of the brain with age, with an increase in astrocyte size or number, and a decrease in neuron size or number (see Introduction). Hence, homogenates of aged brain should contain more astrocytic protein and less neuronal protein. Recently, a number of purportedly synaptic proteins, such as neurotransmitter receptors, have been found in astrocyte cultures (34). The hypothesis predicts that astrocyte proteins should increase with age, neuronal proteins should decrease with age, and proteins shared by neurons and glia should remain stable. The in-

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crease in GFAP with age is consistent with this proposal. In summary, we have found a substantial increase in GFAP mRNA in the cortex, cerebellum, and hippocampus of aged mice. This increase was shown by both RNA gel-blot hybridization and by direct measurement of prevalence levels by solution hybridization. We conclude that this reflects an increase in the size and/or number of astrocytes possibly combined with an increase in their fibrous character. Future studies will use in situ hybridization and semiquantitative immunochemistry to resolve these issues. ACKNOWLEDGEMENTS The authors wish to thank Drs. Jeffrey Masters and Nancy Nichols for their generous gift of the GS cDNA. These studies were supported by grants from the National Institute on Aging (AG-07892 to D.G.M. and AG-07909 to C.E.F.), The Anna Greenwall Award from the American Federation for Aging Research, The American Heart Association (891079) to D.G.M., and The John D. and Katherine T. Mac Arthur Foundation to C.E.F.D.G.M is an Established Investigator of the American Heart Association. J.R.G. was supported by a predoctoral fellowship from the National Institute on Aging (AG-00093).

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