Aging is associated with divergent effects on Nf-L and GFAP transcription in rat brain

Neurobiologyof Aging, Vol. 17, No. 6, pp. 833-841, 1996 Copyright © 1996 ElsevierScienceInc. Printed in the USA. All rights reserved 0197-4580/96 $15.00 + .00 ELSEVIER

S0197-4580(96)00078-4

Aging Is Associated With Divergent Effects on Nf-L and GFAP Transcription in Rat Brain C R A I G A. K R E K O S K I , * I R M A M. P A R H A D , * ' ~ 1 T A K S. FUNG:~ A N D A R T H U R W. C L A R K * t 2

Neuroscience Research Group, * Departments o f Pathology, * Clinical Neurosciences, I" and Academic Computing Services,~: University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1 Canada R e c e i v e d 1 June 1995; R e v i s e d 31 O c t o b e r 1995; A c c e p t e d 19 January 1996 KREKOSKI, C. A., I. M. PARHAD, T. S, FUNG AND A. W. CLARK. Aging is associated with divergent effects on Nf-L and GFAP transcription in rat brain. NEUROBIOL AGING 17(6) 833-841, 1996.--We studied the effects of advancing age on the expression of several proteins irrLportant in the structure and function of the nervous system. Brains of young (3 month), middle-aged (13 month), and old (29 month) male Fischer 344 rats were examined. Run-on transcription and Northern blot hybridizations were used to determine gene-specific transcription rates and mRNA levels, respectively. With advancing age, there was a decrement in the transcription rate and mRNA levels for neurofilament-light subunit (Nf-L), but an increment in the transcription rate and mRNA levels for glial fibrillary acidic protein (GFAP). Proteolipid protein (PLP) mRNA levels were attenuated between 3 and 13 months of age, whereas amyloid precursor protein (APP) mRNA levels were attenuated in the middle-aged but not the old animals. Transcription rates for a-actin and fos, and mRNA level s for a-actin, were unaffected. These observations indicate divergent transcriptional regulation of several genes, notably Nf-L and GFAP, in the aging mammalian forebrain. Copyright © 1996 Elsevier Science Inc. Aging Rat forebrain Neurofilament protein Transcription rates mRNA levels

Glial fibrillary acidic protein

THE rodent nervous system undergoes a number of morphological changes with advancing age including atrophy and loss of neurons (25), hypertrophy of glia (1,28,53), and defects of myelination (40,42). A decline in aggregate synthesis of protein (22,70) and of RNA (16,51,85) might well influence the structure of the aging nervous system. But it is difficult to correlate such aggregate effects with specific morphological changes. Gene-specific information is needed to understand their molecular basis. Our laboratory has recently identified a gene-specific molecular correlate of neuronal atrophy in the peripheral nervous system of aging Fischer 344 rats. Age-related atrophy of dorsal root ganglion (DRG) neurons is a,;sociated with a decrease in neurofilament mRNA and protein expression (65). In contrast, glial fibrillary acidic protein (GFAP), the major intermediate filament of astrocytes, increases in tl~e aging rodent brain. This change is regulated largely at the pretranslational level and is manifest morphologically by an increase in the size and fibrous character of astrocytes (29,45,63,64). Thus, there is evidence that aging is associated with decreased expression of intermediate filament proteins in the neuron and increased expression of intermediate filament proteins in the astrocyte. These changes could be due to altered stability of the transcripts or to altered tr~.nscription rates of the gene. The possibility that in the aging nervous system the transcription of certain structural genes increases, while that of others decreases, is par-

Proteolipid protein

Actin

fos

ticularly intriguing; but there has been little direct evidence to support this. In the present study, we evaluated the effects of aging on the expression of specific structural genes in Fischer 344 rat forebrain. In addition to Northern blot hybridizations to measure mRNA levels, we applied a nuclear run-on transcription assay to measure gene-specific transcription rates in young, middle-aged, and old rats. The genes selected for study encode a major intermediate filament protein of neurons (neurofilament light subunit, Nf-L) (79), the major intermediate filament protein of astrocytes (GFAP) (9), a major myelin protein of oligodendrocytes (proteolipid protein, PLP) (55), a microfilament protein common to most cell types (a-actin) (4), a protein important in Alzheimer's disease (AD) pathogenesis (amyloid precursor protein, APP) (77), and the protooncogene fos (80). METHOD

Animals and Tissue Collection Male Fischer 344 rats 3 to 32 months of age were obtained from National Institute of Aging colonies maintained by Harlan Sprague-Dawley (Indianapolis, IN). Upon delivery the rats were kept on a 12 L:I2 D cycle in plastic-bottomed cages and fed a standard diet and water ad lib. The rats were anaesthetized with chloral hydrate (800 mg/kg, intraperitoneally) and decapitated.

1 Deceased. 2 To whom requests for reprints should be addressed. Arthur W. Clark, M.D., Department of Pathology, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1. 833

834 The brains were removed and the forebrain dissected and stored at -80°C. A 50-100 mg sample from the frontal cortex was used for Northern blot analysis and the remainder of the forebrain was used for run-on transcription assays.

Chemicals and Reagents The chemicals used in this study were molecular biology grade and from GIBCO/BRL Life Technologies Inc. (Burlington, Ont, Can) and Fisher Scientific Inc. (Edmonton, Alta, Can). Formamide for hybridizations was deionized with AG 501-X8 mixed bed resin (Bio-Rad, Mississanga, Ont, Can) according to the manufacturer's instructions. Restriction endonucleases and proteinase K were from Boehringer-Mannheim (Laval, Que, Can), RQI DNase and RNasin ribonuclease inhibitor from Promega Corporation (Madison, WI), and radionucleotides from Du Pont Canada Inc. (Mississauga, Ont, Can).

DNA Clones The DNA clones used were as follows: a 2000 basepair (bp) mouse Nf-L complementary DNA (cDNA) [gift of J-P Julien] (38), a 3000 bp human GFAP cDNA [gift of SA Johnson], a 650 bp rat PLP cDNA [gift of SA Johnson], a 2400 bp human APP cDNA [gift of D Liston] (14), an 1150 bp mouse a-actin cDNA [gift of GA Schultz] (59), an 1100 bp genomic fragment of the human fos gene encompassing exons 3 and 4 [gift of R Johnston] (18) and a 1400 bp genomic fragment from the middle of the human 28S ribosomal RNA (rRNA) gene [gift of JE Sylvestor] (83). Clones were amplified and gene inserts isolated using standard recombinant techniques (73).

Nuclear Runon Transcription Assay Nuclei were isolated essentially as previously described (23). All steps were performed at 4°C. Forebrains from individual animals (1 g samples) were minced, suspended in 25 ml of lysis buffer [0.3 M sucrose, 10 mM Tris (pH 8.0), 2.5 mM MgC1z, 0.25% Triton X-100, 1.0 mM dithiothreitol (DTT), 0.5 mM phenylmethyl sulfonyl fluoride (PMSF)], and homogenized in a glass Dounce homogenizer. The homogenate was filtered, mixed with an equal volume of sucrose buffer [2.0 M sucrose, 10 mM Tris (pH 8.0), 2.5 mM MgC12, 0.1% Triton X-100, 1.0 mM DTT, 0.5 rnM PMSF], layered onto a 10 ml cushion of sucrose buffer and centrifuged (60,000 × g, 1 h). The pelleted nuclei were resuspended in 2 ml of 10% glycerol, 50 mM Tris (pH 8.0), 5 mM MgClz, 1 raM DTT, 0.5 mM PMSF, transferred to 2.0 ml microtubes (Sigma Chemical Company, St. Louis, MO) and repelleted (2000 × g, 5 min). The buffer was removed and 40% glycerol, 50 mM Tris (pH 8.0), 5 mM MgC12, 1.0 mM DTT, 0.5 mM PMSF added to 275 Ixl. Nuclei were resuspended by trituration, split into two (125 ill) aliquots, and immediately stored at -80°C. Yields ranged from 2-3 × 107 nuclei/g forebrain. Nuclei (1-2 × 107) were thawed and an equal volume (125 txl) of 2 x reaction buffer containing 250 txCi of both 32p-UTP and 32p-CTP (800 Ci/mmol) added to give final concentrations of 20% glycerol, 50 mM "Iris (pH 8.0), 5 mM MgC12, 150 mM KC1, 10 mM creatine phosphate, 1.0 mM DTT, 0.5 mM ATP, 0.5 mM GTP, 1.25 p,M 32p-labeled UTP and CTP, and 1 unit/Ia.1ribonuclease inhibitor. The nuclei were incubated at 30°C for 30 min. DNase (I0 units) was added and after incubating at 37°C for 10 rain, proteinase K (10 mg/ml), SDS (20%) and EDTA (0.4 M) were added to final concentrations of 200 ixg/ml, 1% and 20 mM, respectively, and the incubation continued at 55°C for 1 h. After adding 0.3 ml of 2 m sodium acetate and 0.3 ml of a solution of guanidinium thiocyanate (4m), sodium citrate (25 raM, pH 7.0),

KREKOSKI ET AL. and 13-mercaptoethanol (100 mM), the run-on transcripts were purified by phenol:chloroform extraction. Yeast transfer RNA (tRNA; 100 txg) was added as carrier to the final aqueous phase and RNA precipitated with an equal volume of isopropanol. The RNA precipitate was collected by centrifugation, dissolved in diethylpyrocarbonate treated (DEPC)-H20 and to remove unincorporated ribonucleotides, reprecipitated three times with 1/2 volume 7.5 M ammonium acetate and 2.5 vol ethanol. The final RNA precipitate was redissolved in DEPC-H20 and the incorporation of labeled ribonucleotides into trichloroacetic acid (TCA)precipitable material determined. Samples were spotted onto Whatman GFC glass fiber discs, washed with ice-cold 5% TCA in 20 mM sodium pyrophosphate using a vacuum manifold, added to Optiphase Hisafe III scintillation cocktail (Fisher Scientific Inc.) and radioactivity measured in a LKB-Wallac scintillation counter. Gene-specific transcripts were detected by hybridizing the labeled RNA to panels of membrane-bound DNA clones. DNA clones in 0.4 N NaOH, 2 x standard saline citrate (SSC) (lx SSC = 0.15 M sodium chloride, 0.015 M sodium citrate) were slotblotted onto Zetaprobe nylon membrane (Bio-rad) prewetted with 2x SSC. The membranes were neutralized by immersion in 0.2 M Tris (pH 7.5), 2× SSC, 4 mM EDTA, blot-dried, and baked at 80°C for 30 min; prehybridized at 55°C for 8-10 h in 30% formamide, 0.25 M Na2HPO4, 1 mM EDTA, 5% SDS, 1% bovine serum albumin (BSA), 0.5 mg/ml yeast tRNA; hybridized at 55°C for 36-40 h in prehybridization buffer with 0.1 mg/ml yeast tRNA plus 5% dextran sulfate and 3-6 x 107 TCA-precipitable counts of run-on transcripts; washed for 20 min once with 2x SSC, 0.1% SDS, once with 0.5x SSC, 0.1% SDS, and twice with 0.2x SSC 0.1% SDS; and exposed to x-ray film (Kodak XAR, Eastman Kodak, Rochester, NY) with intensifying screens at -80°C for 1-7 days.

Northern Blot Analysis Total RNA was isolated using the acid-quanidinium-phenolchloroform extraction technique (13) and quantitated by absorbance at 260 nm (1.0 o.d. = 45 mg RNA/ml) using a Beckman DU65 spectrophotometer and software (Beckman Instruments Inc, Fnllerton, CA). 260 nm:280 nm ratios were consistently greater than 1.9. Total RNA samples (10 p.g) were fractionated by 1.2% agarose/2.2 M formaldehyde gel electrophoresis (73), and transferred onto Zetaprobe membrane by downward capillary blot using 3x SSC in 10 mM NaOH (12). The uniformity and quality of the RNA transfer was monitored by ethidium bromide staining of the RNA and visualization under UV light. DNA clones were labeled with 32p-dCTP (3000 Ci/mmol) by random priming to specific activities > 5 x 108 cprn/ixg DNA (21). The RNA transfers were prehybridized at 55°C for 6-8 h in 33% formamide, 0.25 M Na2HPO4, 1 mM Na2EDTA, 5% SDS, 1% BSA, 0.25 mg/ml salmon sperm DNA; hybridized at 55-60°C for 16-20 h in prehybridization buffer with 0.1 mg/ml salmon sperm DNA plus 5% dextran sulfate, and 1-2 x 107 cpm]ml SzP-labeled DNA probe; washed to final stringencies of 0.2x SSC, at 60--65°C; and exposed to Kodak XAR x-ray film with intensifying screens at -80°C for 1--4 days. RNA transfers were then stripped of old probe by washing with 0.1x SSC, 0.5% SDS at 85°C, checked for completeness of stripping by autoradiography and reprobed with a different probe up to three times.

Experimental Design and Data Analysis All averages in the text and figures are expressed as mean _+one standard error of the mean (SEM). The rats were divided into three age groups: young (3.4 _+0.3 months, range 2-6 months, n = 11),

GENE EXPRESSION IN AGING RAT FOREBRAIN

middle-aged (13.0 _+ 0.5 mc,nths, range 12-17 months, n = 10) and old (29.0 -+ 1.0 months, range 23-32 months, n = 11). In a separate analysis the old aniraals were subdivided into two groups, an old group (23-29 months, n = 6) and a very old group (32 months, n --- 5). There were no differences between these two groups (data not shown). Each transcription assay included a young, middle-aged, and old animal, and five independent trials were conducted for a total of five animals/age group. An equivalent amount of labeled RNA from each animal was hybridized to two membranes, one containing 2 p~g of unlabeled Nf-L, GFAP, PLP, fos, APP, and ct-actin DNA, along with plasmid DNA as a control for nonspecific hybridization, and the other containing 2-8 ng of rDNA. The density and area of the rRNA signals were quantitated by computerized videodensitometry (Bioquant Systems IV, R & M Biometrics, Nashville, TN) and shown to be linear over the fourfold range of rDNA used. To correct for differences in specific activity and exposure times, densitometric signals for the other transcripts were normalized to the rRNA signal using the total rRNA signal as the denominator. The mean _+ SEM signal was then determined for each transcript and age group. Nf-L, GFAP, PLP, APP, and et-actin mRNA levels were evaluated in 8-11 animals/age group, fos mRNA levels were not evaluated as they were generally beyond the sensitivity of the Northern blot technique used. The lq2qA transfers contained samples from each age group or from all of the young animals. Multiple autoradiograms were obtained after each probing and the area and density of specific mRNA bands quantitated using the Bioquant system. Autoradiograms within the linear range of the densitometer were used. Densitometric signals were expressed as a percentage of the mean value of the young animals and the mean _+ SEM signal determined for each mRNA and age group. Statistics were performed using SPSS software (SPSS Inc., Chicago, IL). Age-group means were compared using a one-way analysis of variance (ANOVA). Post hoc linear analysis of contrasts were used to compare individual age group means. All of the data were normally distributed as evaluated by KolmogorovSmirnov goodness of fit tests, and age-group variances were homogeneous as evaluated using Cochran's C and Barlett-Box Ftests. RESULTS

Changes in Gene-Specific Transcription Rates There was no difference among the age groups in the recovery of nuclear run-on transcripts, F(2, 12) = 0.99, p = 0.40. Yields from 3-, 13-, and 29-month-old age groups were 5.9 -+ 0.79, 4.9 + 0.55, and 4.6 _+0.78 x 107 cprrdg tissue, respectively. The decrease between 3 and 13 months of age did not reach statistical significance (t --- 1.06, p = 0.357). Representative autoradiograms of the transcription assay hybridizations and quantitative evaluations of gene-specific transcription rates are illustrated in Fig. 1. There was a significant difference among the age groups in Nf-L transcription rates, F(2, 12) = 7.30, p = 0.008. Nf-L transcription rates decreased in the 29 month vs. the 3 month (t = 3.23,p = 0.007) and 13 month age groups (t = 3.38, p = 0.005), but were not significantly different in the 3 month vs. the 13 month age group (t = -0.147,p = 0.886). Based on the means, Nf-L transcription decreased by about 70% between 13 and 29 months of age. There was a significant difference among the age groups in GFAP transcription rates, F(2, 12) = 6.77, p = 0.011. GFAP transcription rates increased in the 29 month vs. the 3 month (t = -3.08, p = 0.009), and 13 month age groups (t = -3.23, p = 0.007), but were not significantly different in the 3-month vs. the

835

13-month-age group (t --- -0.197, p = 0.847). Based on the means, GFAP transcription increased by about 250% between 13 and 29 months of age. There were no significant differences among the age groups in PLP, F(2, 12) = 0.180, p = 0.838, APP, F(2, 12) = 0.242, p = 0.789, et-actin F(2, 12) 0.068, p = 0.934, orfos transcription rates, F(2, 12) = 0.019, p = 0.982. However, the results for PLP and APP were inconclusive due to low or absent densitometric signals. On the other hand, tx-actin and fos transcription rates were maintained at a relatively high and constant level (Fig. 1).

Changes in mRNA Expression Total RNA recovery from frontal cortex did not change with age, F(2, 30) = 0.05, p = 0.951. Yields from young, middle-aged, and old animals were 1263 _+ 42, 1282 _+ 25, and 1263 +_ 66 i~g/g tissue, respectively. Representative autoradiograms of the Northern blot hybridizations and quantitative evaluations of mRNA levels are shown in Fig. 2. There was a significant difference among the age groups in Nf-L mRNA levels, F(2, 28) = 9.76, p = 0.0006. Nf-L mRNA levels decreased in the 29-month vs. the 3-month (t = 4.27, p < 0.0001) and 13-month age groups (t = 3.16, p = 0.004), but were not significantly different in the 3-month vs. the 13-month age group (t = 1.03, p = 0.312). Based on the means, Nf-L mRNA levels decreased by about 40% between 13 and 29 months of age. There was a significant difference among the age groups in GFAP mRNA levels, F(2, 25) = 13.8, p = 0.0001. GFAP mRNA levels increased in the 29-month vs. the 3-month (t = -5.13, p < 0.0001) and 13-month age groups (t = -3.82, p = 0.001), but were not significantly different in the 3-month vs. the 13-month age group (t = -1.39, p = 0.178). Based on the means, GFAP mRNA levels increased by about 250% between 13 and 29 months of age. There was a significant difference among the age groups in PLP mRNA levels, F(2, 29) = 8.12, p = 0.0016. PLP mRNA levels decreased in the 13-month vs. the 3-month age group (t = 2.61, p = 0.014) and in the 29-month vs. the 3-month age group (t = 3.95, p < 0.0001), but were not significantly different in the 13-month vs. the 29-month age group (t = 1.25, p = 0.223). Based on the means, PLP mRNA levels decreased by about 25% between 3 and 13 months of age. There was a difference among the age groups in APP mRNA levels that just reached significance, F(2, 26) = 3.60, p = 0.042). APP mRNA levels decreased in the 13-month vs. the 3-month age group (t = 2.63, p = 0.014), but were not significantly different in the 3-month vs. the 29-month age group (t = 0.82, p = 0.420). In addition, APP mRNA levels showed a trend toward an increase in the 29-month vs. the 13-month age group (t = -1.74, p = 0.093). Based on the means, APP mRNA levels decreased by about 25% between 3 and 13 months of age and recovered to near 3 month levels by 29 months of age. a-actin mRNA levels were unaffected by age, F(2, 27) = 0.065, p = 0.937. DISCUSSION

The present study documents several age-related changes in gene expression in male Fischer 344 rat forebrain and provides evidence that some of these changes are regulated at the level of gene transcription. The changes occur at different stages in the aging process. PLP mRNA expression showed a significant decline between 3 and 13 months of age, whereas a decrement in Nf-L expression and an increment in GFAP expression were significant only for the 13- to 29-month interval. APP mRNA ex-

836

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FIG. 1. Runon transcription hybridization analysis of various genes in aging rat forebrain. (A) Equivalent amounts of run-on transcripts (3 x 107 TCA-precipitable cpms) from a young (3 month), middle-aged (13 month), and old (29 month) animal were hybridized to limiting amounts of rDNA (2-8 ng) and an excess of Nf-L, GFAP, PLP, fos, APP, and t~-actin DNA insert (2 ~xg) immobilized on nylon membranes, pGEM plasmid DNA (2 I~g) was included as a control for nonspecific hybridization. (B) Gene-specific hybridization signals were quantitated by videodensitometry and (to correct for differences in specific activity and exposure times) normalized to the rRNA signal using the total rRNA signal as the denominator. The horizontal bars represent the age group means and the symbols represent values from individual animals.

pression decreased between 3 and 13 months of age and tended to increase between 13 and 29 months of age. Constitutive a-actin expression and fos transcription rates were unaffected by age. We did not detect any significant effects of advancing age on the incorporation of labeled RNA precursors in isolated forebrain nuclei or on total RNA content in frontal cortex. In contrast, previous studies have shown a significant age-related decline in the incorporation of labeled RNA precursors in vivo in cortex (16,88) and in isolated cortical nuclei (85), and in total RNA content in cortex (16). The 20% decrease in incorporation between 3 and 29

months detected in the present study using whole forebrain is in the range of the decreases previously reported for cortex (2040%), but did not reach statistical significance. It is possible that there is a difference between cortex and subcortex with regard to overall RNA synthesis, the subcortical structures showing less age-related change. Total RNA yields from frontal cortex in the present study were constant with advancing age, showed minimal intragroup variability, and were about twofold greater than those previously reported for Wistar cortex (16). Cell populations of the rat brain are subject to divergent age-

GENE EXPRESSION IN AGING RAT FOREBRAIN

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FIG. 2. Northern blot hybridization analysis of various mRNAs in aging rat forebrain. (A) Representative autoradiograms following hybridization of total RNA (10 I~g) from frontal cortex of young (3 month), middle-aged (13 month), and old (29 month) rats (two different rats/age group) with Nf-L (4.0 and 2.5 kb mRNAs), GFAP (3.3 kb mRNA), F'LP (1.6 kb mRNA), APP (3.3 kb mRNA), and a-actin (2.1 kb mRNA) cDNA probes. Sizes were determined~ by comparison with a BRL RNA ladder. The ethidium bromide staining intensity of 28S and 18S ribosomal bands demonstrate equivalent loading of total RNA in each lane. (B) Autoradiographic signals of specific mRNA bands were quantitated by videodensitometry with the data expressed as a percent of the mean value of the young age group. The horizontal bars represent the age group means and the symbols represent values from individual animals (8-10 animals/age group). The scale for GFAP is twice that of the other mRNAs evaluated.

related effects on gene expression. With advanced aging, chromatin structure changes towards a more condensed and presumably less active state in neurorts and nonastrocytic glia, but not in astrocytes (7,74). Incorporation of RNA precursors reportedly decreases with advanced aging in neuron- and oligodendroglia- but not in astrocyte-enriched preparations of cortical nuclei (51,85). The increments in GFAP gene expression and the decrements in

Nf-L and PLP gene expression found in the present study further support the idea that astrocytic gene expression is generally increased, whereas neuronal and oligodendroglial gene expression are generally attenuated, with advanced aging. There is also reason to infer gene-specific regulatory influences of aging within each of the cell subpopulations. For example, among neuronal mRNAs previously studied in the rat brain, there

838 are a number that decrease (20,26,35,57,58,66.75) and others that remain unchanged or increase with advanced aging (37,44,54,75, 86). We have recently shown that in rat DRG mRNAs for neurofilament and the nerve growth factor (NGF) receptor proteins trkA and p75 decrease with advanced aging, while growth associated protein (GAP)-43 mRNA remains unchanged and may increase in some very old animals (65). Interestingly, neuronal genes whose expression tends to remain stable or increase with advanced aging, including GAP-43, neural cell adhesion molecule (NCAM) (54), dynorphin (37), brain-derived neurotrophic factor (44), [3-tubulin, and MAP1B (86), encode proteins associated with neurite outgrowth and synaptic plasticity. An age-related increase in their expression may reflect the formation of new connections by surviving neurons as they sprout into adjacent areas denervated by the aging process (86). APP may also play a role in neurite outgrowth and synapse plasticity (77). Studies of APP mRNA levels in rat brain have reported both increases (6,32) and decreases (52) in APP mRNA expression with advanced aging. There is a marked increase in APP mRNA levels in the thalamus of 17- vs. 3-year-old cynomolgus monkeys (81). APP mRNA is upregulated with senescence in human fibroblasts (2) and rat astrocytes (87) in culture. In the present study, APP mRNA levels in rat cortex decreased between 3 and 13 months and tended to increase between 13 and 29 months. We believe this increase to be real, at least in some of the old animals. On the other hand, some of the old animals had APP mRNA levels that appeared to be decreased as compared to the 13-month-old animals. Note the difference in APP mRNA levels between the two old animals in Fig. 2 a. This variability could explain why we did not detect a significant change in APP mRNA levels between 13 and 29 months. We could not draw any conclusions from our data on transcription rates, however, because the low densitometric signals precluded meaningful interpretation of the results. Our data indicate no effect of aging on constitutive transcription rates for the early response genefos. Previous studies offos gene expression in the rat brain have shown that while the ability to induce fos expression is unaffected in hippocampus (39), constitutive levels of fos mRNA decrease in cortex and cerebellum (41) with advanced aging. The transient and highly inducible nature offos expression, however, must be taken into consideration when determining constitutive levels of expression. Instead of reflecting more durable factors associated with aging, substantial interindividualor intergroup differences might be accounted for by incidental differences in the immediate antemortem setting (3). a-Actin transcription and mRNA levels in this study were constant with advancing age, and there was little variability between animals within the same age group. Brain mRNA levels for a second member of the actin multigene family, 13-actin,are likewise unaffected by advancing age (26,35,82). The constancy of actin expression may be useful as an internal control for other brainspecific genes in aging studies. However, actin is ubiquitously expressed and these data do not resolve whether there are any cell-type specific effects of aging on actin expression. Individual cell analysis by in situ hybridization or a comparison of gray matter vs. white matter could be used to address this issue. PLP is a major protein constituent of CNS myelin; its mRNA appears about postnatal day 5 and increases as myelination proceeds, reaching a peak about postnatal day 28. Thereafter, PLP mRNA levels decline; in the 60-day-old rat they are about onethird those of the 28-day-old animal (27,62). To our knowledge, there have been no previous reports of PLP mRNA expression in rat brain after 60 days of age. Our data indicate a further decline after this time point. Because of weak hybridization signals in our run-on transcription assay, we could not ascribe the decrement in

KREKOSKI ET AL. mRNA levels to changes in transcription rate. This possibility is supported, however, by previously published evidence for transcriptional regulation of PLP mRNA levels in postnatal rat brain (17). GFAP is one of the most intensively studied proteins of the CNS. Its mRNA is detectable in embryonic rat brain, but it is not until the postnatal period that GFAP replaces vimentin as the dominant intermediate filament protein of astrocytes (19). Transcriptional upregulation in postnatal mouse brain (71) leads to near adult levels by postnatal day 16 (49); rat brain GFAP mRNA follows a similar postnatal profile (84). Further increases of GFAP mRNA occur in aging rodent brain, including rat cerebral cortex, hippocampus, and striatum (45,63), and in mouse cerebral cortex, hippocampus, and striatum (64). Similar changes in GFAP mRNA have been reported in aging human brain (63,72). The present study demonstrates that the age-related increase in GFAP expression in rat brain is primarily due to an increase in GFAP transcription. In contrast, a recent report found no evidence of changes in GFAP transcription rates proportionate to the rise in GFAP mRNA levels in pooled hippocampi or individual cerebral cortices of aged rats, suggesting that GFAP mRNA stability is enhanced with advancing age (45). Regulation of GFAP mRNA levels through altered mRNA stability has been observed in some settings (78,84). On the other hand, GFAP transcription in rat brain, as judged by nuclear run-on assays, is increased by lesioning (46) and inhibited by glucocorticoids (47). Reasons for the discrepancy between our findings and Laping and colleagues (45) on the effect of aging on GFAP transcription are not obvious. There is evidence that control of GFAP transcription is different for cortical astrocytes and astrocytes in other parts of the forebrain (8,9). The design of our study included both cortical and suhcortical astroglia, and this might contribute to the difference between our results and those of Laping's group. Other known distinctions in GFAP transcriptional control, such as sex differences for the influence of gonadal steroids [see (63)], seem less likely to be relevant because the sex and strain used in our study and that of Laping and colleagues are identical. Points of concordance between the two studies should also be emphasized. In both, the late age group showed relatively large variation in GFAP transcription rates. Two of our aged rats showed GFAP transcription rates in the range of those for middle-aged animals. Transcriptional activation of GFAP may be subject to substantial individual variation in the older age group, perhaps due to individual differences in one of the factors known to influence GFAP gene expression (48) or to other factors not yet defined. An additional point of concordance is the emphasis by Laping and colleagues on the fact that GFAP expression is maintained in old age, and that transcriptional activity of many genes may continue without decrement throughout the lifespan, a view in accord with our results for GFAP, c~-actin and fos gene transcription rates. Neurofilaments consist of three protein subunits, L(ight), M(edium), and H(eavy). During embryogenesis, only Nf-L and Nf-M are expressed; Nf-H appears after postnatal day 5; thereafter, the three subunits are coregulated (76,79). In the rodent, maximal steady state mRNA levels are reached around postnatal day 28 and decline thereafter [(43), present study]. Evidence from transgenic mice (5,60) and transfected cells (36,68) indicate that neurofilamerit expression is regulated at both pre- and posttranscriptional levels. In tissue culture (PC12 cells) the administration of NGF increases neurofilament expression: Nf-L mostly through transcription; Nf-M through transcription and stabilization of mRNA; and Nf-H perhaps through posttranscriptional or translational events (50).

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839

Morphological studies of the rodent brain have reported changes in many neuronal pc,pulations with advanced aging; these changes are not uniformly present and the degree of involvement is often system specific [revi~ewed in (25)]. A common degenerative change is perikaryal atrophy, which may be a prelude to cell loss. The results of this study and previous studies showing a direct correlation between the level of neurofilament gene expression and axonal and perikaryal size (33,61) suggest that the age-related atrophy of neurons is due in part to a decline in neurofilament transcription. In a concomitant study we evaluated the effects of advancing age on neurofilarnent expression in male Fischer 344 rat DRG. A decrease of 40-50'% in mRNA levels for all three neurofilament proteins was detected which approximates the 40% decrease in Nf-L mRNA level s in forebrain detected in the present study. Furthermore, Western blot analyses showed a corresponding decrease in neurofilame:at protein, and morphometric analysis showed that the decline in neurofilament expression was associated with a decrease in neurofilament numbers and perikaryal and axonal atrophy (65). Thus, age-related decrements in neurofilament gene expression occur in both the central and peripheral nervous system in the rat ~ad may be causally linked to the agerelated atrophy of neurons. The present study documents gene-specific changes in transcription rates in aging mammalian brain. Physiologic and behavioral differences that distinguish individuals of identical chronological age (15) may derive in part from differences at the level of gene transcription. Mechanisms that might be involved include age-related changes in the level of certain hormones (30,31) or in

the phosphorylation of gene regulatory proteins such as fos (56). Oxidative or other cumulative damage to DNA could also play a role (34,69). There is accumulating evidence that the aging mechanism can be retarded by manipulating gene expression. Transgenic stains of Drosophila melanogaster that overexpress the antioxidant gene superoxide dismutase have a slight but significant increase in mean lifespan (24). Dietary restriction in rodents increases mean and maximum lifespan, and there is evidence that changes in gene transcription play an important role [reviewed in (31)]. Dietary restriction in rodents has also been shown to antagonize agerelated cognitive deterioration (67) and astrocyte hypertrophy (10, 11). As age-related changes of gene transcription rates become better characterized, it may be feasible to modify transcriptional regulators experimentally, and produce long-term changes in gene expression in the mammalian brain. It will be of interest to determine whether manipulating the expression of specific genes can retard age-related degeneration of the brain. ACKNOWLEDGEMENTS This work was supported by grants from the Alzheimer Society of Lethbridge (C.A.K.), and the Medical Research Council of Canada (I.M.P.). I.M.P. was a Medical Research Council of Canada Scientist and an Alberta Heritage Foundation for Medical Research Scholar; A.W.C. was the recipient of a Career Scientist Award from the Alzheimer Society of Canada. The authors wish to thank Drs. G. A. Schultz, J.-P. Julien, S. A. Johnson, R. Johnston, and J. E. Sylvestor for their generous gifts of DNA clones, and L. A. Cellars for excellent technical assistance.

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