Brain Research, 575 (1992) 309-314 (~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

BRES 25072

309

Short Communications

Post-transcriptional regulation of loss of rat striatal D2 dopamine receptor during aging Masahiro Sakata a, Shakeel M. Farooqui a and Chandan Prasad a'b aLaboratory of Neuroscience, Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA (USA) and bSection of Endocrinology, Department of Medicine, Louisiana State University Medical Center, New Orleans, LA (USA)

(Accepted 19 November 1991) Key words: Dopamine D2 receptor; Striatum; Aging; Northern blot analysis; Western blot analysis

The mechanism(s) underlying age-associated diminutions in the rat striatal D 2 dopamine receptor (DE-receptor) number was investigated. The levels of DE-receptor mRNA in 4-, 12- and 18-month-old rat striata were found not to change. In contrast, the levels of 110 kDa protein, labeled with a DE-receptor specific antibody, decreased in parallel with [aH]YM-09151-2 binding to striatal membranes. These data suggest a role for post-transcriptional mechanism(s) in age-associated decrease in DE-receptor. A notable and consistent biochemical manifestation of aging in the central nervous system is the progressive loss of dopamine receptors from the striatum in a number of species including mice 23, rats 10'12'16'19'21, rabbits a° and humans 1s'2°'36. Such losses are not accompanied with changes in binding affinity (Kd). Both D1 and D 2 subtypes of D A receptor have been shown to change with age and such changes appear to be at least partially responsible for altered motor behavioral patterns in aging 27. The precise mechanism(s) underlying attenuation of DE-receptor in the aged is not clear. However, studies on the turnover of DE-receptor in rodents after irreversible receptor blockade with N-ethoxycarbonyl-2-ethoxy1,2-dihydroquinoline (EEDQ), a non-selective receptor blocker, suggest a decreased synthesis and increased receptor degradation during aglng 9'15. Two recent developments, the availability of a cloned DE-receptor complimentary D N A (cDNA) and a polyclonal antibody highly specific for DE-receptor, have made it possible now to examine potential changes in DE-receptor during aging at transcriptional and post-transcriptional levels 2'6. The results of this study show an age-dependent decrease in the binding of [3H](+)-cis-N-(1-benzyl-2- methylpyrrolidin-3-yl)-5-chloro-2-methoxy-4-methylaminbenzamide ([3H]YM-09151-2) (80 Ci/mmol) (NENDuPont, Boston, MA), a selective D2 antagonist, and the levels of a 110 kDa protein, recognized by DE-re-

ceptor specific antibody on Western blot, in striatal membrane with no concomitant change in DE-receptor specific m R N A level. In conclusion, it appears that the changes in DE-receptor density during aging may be at post-transcriptional levels. Male Fisher-344 rats (4, 12 and 18 months old) were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN). The animals were housed individually under controlled temperature (22-24°C) and lighting (lights on from 07.00 to 19.00 h) with free access to tap water and Purina chow. Animals were adapted to our animal quarter for 7 days before sacrifice. The striatum, liver, and cerebellum were collected immediately after decapitation, frozen in liquid nitrogen and kept at -80°C until assay. The sera were also collected and kept at -20°C until testosterone radioimmunoassay (RIA). DE-receptor binding assay was performed as described elsewhere 6'29. Briefly, tissue was homogenized in 20 vols. of ice-cold buffer containing 50 mM Tris-HC1, 8 mM MgCI2, 5 mM EDTA, pH 7.15, the homogenate centrifuged at 18,000 x g for 20 min at 4°C, the pellet resuspended in the same buffer and recentrifuged as above. The final pellet was resuspended in a buffer containing 50 mM Tris-HC1, 120 mM NaC1, 5 mM KCI, 5 mM MgCI 2, 1.5 mM CaCI 2, 1 mM EDTA, pH 7.4, 10/~M pargyline hydrochloride, and 0.1% ascorbic acid. Membranes (150/~g protein) were incubated for 1 h at 25°C under reduced light with 20 pM or 800 pM [3H]YIVI-

Reprint requests: C. Prasad, Laboratory of Neuroscience, Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Road, Baton Rouge, LA 70808, USA. Correspondence: C. Prasad, Section of Endocrinology, Department of Medicine LSU, Medical Center 1542 Tulane Ave., New Orleans, LA 70112, USA. Fax: (1) (504) 568-4159.

310 09151-2 in a total volume of 1.5 ml; the non-specific binding was defined in the presence of 100/~M S ( - ) sulpiride. The bindings were terminated by rapid filtration of the reaction mixture through Whatmann GF/B glass fiber filters (Whatman International Ltd., Maidstone, UK), followed by 4 washes with 4 mt of the incubation buffer. The filters were dried and counted for radioactivity in 10 ml of liquid scintillation fluid. The specific binding was defined as the amount [3H]YM09151-2 bound to membranes in the absence of sulpiride (total binding) minus the amount bound in the presence of sulpiride (non-specific binding). Total RNA from 6 hemistriata was prepared by the guanidium isothiocyanate-CsCl method 4. Twenty/~g of total RNA was electrophoresed on 1% agarose-formaldehyde gel and blotted into nylon filter membrane (BioRad, Richmond, CA). After vacuum drying at 80°C for 2 h, filters were prehybridized in 0.25 M NaHPO4, pH 7.2, 0.25 M NaCI, 7% SDS, 1 mM EDTA, 50% formamide at 43°C for 4 h. Fragment PD-2 derived from digestion of RGB-2 cDNA clone 2 with restriction enzymes PstI and E c o R I served as the probe which recognizes both of short and long m R N A of D2-receptor 7'17. This fragment was random prime labelled with deoxycytidine 5'-[a-32p]triphosphate (dCTP) (3000 Ci/mmol) (Amersham, Arlington Heights, IL) using multiprime D N A labeling kit (Amersham, Arlington Heights, IL). Hybridization was carried out in the same buffer containing 10 6 cpm/ml of spun columned, labeled PD-2 probe at 43°C for 18 h. Filters were washed twice in 2 x SSC and 0.1% SDS at room temperature for 15 min, once in 0.5 × SSC and 0.1% SDS at room temperature for 15 min, twice in 0.1 x SSC and 0.1% SDS at 65°C for 10 min. Filters were exposed overnight to 2 days at -80°C to Kodak X-Omat films with an intensifying screen. The filters were then stripped and rehybridized with random prime labelled fl-actin probe for normalization of signal. The autoradiograms were scanned using Soft Laser Scanning densitometer (Biomed Instruments Inc., Fullerton, CA). To control the same amount of total RNA present in each lane, the signals from PD-2 probe were normalized to those from fl-actin probe. Four hemistriata, one cerebellum and pieces of liver were homogenized separately in a buffer containing 0.32 M sucrose, 10 mM Tris-HC1, pH 7.4, 2 mM EDTA, 10 /~g/ml each of leupeptin, pepstatin A, aprotinin and antipan, 50 pg/ml each of soybean trypsin inhibitor and benzamide, and 1 mM phenylmethylsulfonyl fluoride (PMSF) as described previously22'35. The homogenate was centrifuged at 900 x g for 10 min, and the resulting supernatant was centrifuged at 100,000 × g for 1 h. The final pellet was resuspended in a buffer containing 50 mM Tris-HCl, 10 mM EDTA, 100 mM NaC1, 8 mM

TABLE I Age-dependent changes in the binding of [JH]YM-09151-2 to striatal membranes and serum testosterone levels Age (months)

[3H]YM-09151-2 binding (fmol/mg protein)

Testosterone (ng/ml)

Ligand concentration

4 12 18

20 pM

800 pM

114 + 9 (7) 122 + 12 (7) 79 _+ 15" (7)

602 _+ 45 (7) 517 __ 41 (7) 368 -- 33** (7)

3.0 +_ 0.5 (6) 2.9 + 0.6 (6) 1.2 + 0.2** (6)

[3H]YM-09151-2 binding to striatal membranes in 4-, 12- and 18month-old male Fisher-344 rats was analyzed at two ligand concentrations. The data are expressed as mean + S.E.M. with the number of observations shown in the parentheses. Both the receptor densities at 20 pM and 800 pM ligand and serum levels of testosterone in 18-month-old rats were significantly. (*P < 0.05 and **P < 0.01) lower than those in 4- or 12-month-old rats.

MgC12, pH 7.4. After denaturation of membrane at 100°C for 3 min, membranes (100/~g protein/lane) were applied onto 8% SDS- P A G E and electrophoresed 13. Electrophoretic transfer of the resolved proteins from the polyacrylamide gel onto 0,45/tin nitrocellulose filter (Bio-Rad, Richmond, CA) was performed as described previously 34. The filters were then quenched in 5% nonfat dry-milk in Tris-buffered saline (TBS; 10 mM TrisHC1, 150 mM NaCI, pH 7.4) at room temperature for 2 h or at 4°C for overnight. The nitrocellulose filters were incubated at room temperature for 1.5 h with De-receptor specific antibody (1:500 dilution) in TBS 6. After 4 washes with TBS containing 0.05% Tween-20 (v/v), the filters were incubated with goat anti-rabbit immunoglobulin conjugated to alkaline phosphatase (Bio-Rad, Richmond, CA) and subsequently developed using 5-bromo4-chloro-3-indolyl phosphate/nitoro blue tetrazolium (BCIP/NBT) as substrates. The intensities of the bands were analyzed by the densitometry. Protein concentration was measured using Bradford assay 1. Serum levels of testosterone were measured using RIA kit (Diagnostic Product Corp., Los Angeles, CA). Data were expressed as mean + S.E.M. and analyzed statistically using analysis of variance followed by t-test. A decline in circulating levels of testosterone serves as a good marker of the aging of hypothalamo-pituitarygonadal axis in the male rat. The data presented in Table I show a significant decrease in serum testosterone levels in 18-month-old rats compared to 4- or 12-monthold rats (F-ratio2,t5 = 4.6, P < 0.01). The binding of [3H]YM-09151-2 to striatal membranes from 4-, 12- and 18-month-old rats was measured at a ligand concentration close to the receptor K d (20 pM) and several-fold

311

PD-2

28s-

18s-

Actin

months

4

12

18

Fig. 1. Northern blot analysis of D2 dopamine receptor mRNA in striata of 4-, 12- and 18-month-oldmale Fisher-344 rats. Each lane contains 20/~g of total RNA. The upper panel shows a Northern blot hybridized with 32p-labeled PD-2 probe specific for D~ dopamine receptor. The positions of 28 S and 18 S RNA are indicated on the left. The lower panel shows the same Northern blot rehybridized with riP-labeled fl-actin probe.

higher than the K d (800 pM). The results of this study showing a significant decrease in the number of binding sites during aging at both ligand concentrations (20 pM: F-ratio2.1s = 3.6, P < 0.05; 800 pM: F-ratio2,1s = 8.9, P < 0.01; Table I) are consistent with numerous previous studies on aging of D2-receptor 1°'12'16'19m. It must be pointed out, however, that [3H]YM-09151-2 may recognize not only D 2 but also D 3 and D 4 dopamine receptors25'32; however, the striatal level of mRNA for D3-receptor is relatively low compared to that for D2- o r D 4r e c e p t o r s 2'25'32. Therefore, the changes observed here may reflect a sum total of changes in one or all receptor subtypes. Unfortunately, ligands specific for D2-, D 3- or D 4- receptor subtypes are not available to establish the nature of the observed changes. To determine whether the observed changes in D2-receptor binding (Table I) could be due to a decrease in the transcription of D2-receptor gene during aging, the steady-state level of D2-receptor mRNA in the striata of 4-, 12- and 18 month-old rats was determined. Typical data presented in Fig. 1 showed that the relative signal

of D2-receptor mRNA when corrected for the expression of fl-actin mRNA did not change significantly with age (4 months: 1.02; 12 months: 1.04; and 18 months: 1.00). In a separate experiment using total RNA from young rat striata, we (unpublished observation) and others 8 have established that a 10% difference in the RNA loaded on the gel, when probed with PD-2, can be measured. Therefore, we conclude that during aging the change in D2-receptor mRNA must be absent or less than 10%. While the expression of mRNA for fl-actin is known to change during ontogenyTM,its expression does not vary beyond adulthood in mice 14, rats n'24 and humans 11. For example, in developing mice, the level of fl-actin mRNA in whole brain reaches the highest level by day 1 after birth, then steadily declines to a low level by day 14 that persists for at least up to 1 year of age14; similar ontogenic changes have been seen in rats 5. In contrast, the levels of fl-actin mRNA when measured in whole brain or liver of Wistar rats 24 or brain regions (cerebellum, cerebral cortex and subcortical area) of Fisher-344 rats 11 or human striata 11 (ages 18-90 years) did not exhibit any significant age-associated changes. Therefore, we chose to use the expression of fl-actin mRNA to correct for the potential changes in the signal for striatal D2-receptor during aging. Next we therefore investigated whether decreases observed in D2-receptor binding (Table I) could be due to changes in post-transcriptional events such as receptor maturation. To this end, a specific antibody against synthetic peptide predicted from the nucleotide sequence of rat D 2 dopamine receptor was used to measure the level of D2-receptor protein 6. The antibody recognizes peptide(24-34) from the NH2-terminus of the receptor located in the first outer membrane loop of rat D2-receptor. The specificity of the antibody in Western blot analysis was performed using rat striatal and liver membranes. The data presented in Fig. 2A show that the antibody recognized a weak 50 kDa band and a strong 110 kDa band (lane 1) in striatal membrane. In liver membrane, however, only a weak 42 kDa band was seen (Fig. 2A, lane 2). When probed with preimmune serum, none of the above bands were seen (Fig. 2A, lanes 3 and 4). Having established the specificity of the antibody on Western blot analysis, the sensitivity of the assay to predict small changes in the amount of 110 kDa protein was determined. To this end, samples (total 100/zg protein/ lane) containing 25, 50, 75 and 100 pg of striatal membane of proteins mixed with 75, 50, 25 and 0/~g of liver proteins, respectively, were subjected to Western blot analysis. The results of the study showed that a 25 /~g change (Fig. 2B) in D2-receptor protein was easily discernable. In contrast, lane C (Fig. 2B) containing 100/zg cerebellar membrane protein exhibited the 50 kDa band

312

200K-

200K-

II6K-

116K-

66K-

66K~i i!~i~i~i~~i! i! ~

43K43K-

~i i iiiii!ili~i~! !ii! ii i~!!i~i~iill ~i~ '

A

1

2

3

4

B

loo

75

50

25

c

Fig. 2. A: Western blot analysis of D 2 dopamine receptor in rat striatal and liver membranes. A sample of 100 gg of protein was applied onto 8% SDS-PAGE and transferred to nitrocellulose filter for Western blot. Lane 1, striatal membrane with immune serum (antibody) (1:500 dilution); lane 2, liver membrane with antibody (1:500 dilution); lane 3, striatal membrane with preimmune serum (1:500 dilution); lane 4, liver membrane with preimmune serum (1:500 dilution). The molecular weight markers are indicated on the left. B: Western blot analysis of D z dopamine receptor in striatal, liver and cerebellar membranes. Lane 100, 75, 50 and 25 contained not only 100, 75, 50 and 25 /~g of striatal membrane protein but also 0, 25, 50 and 75 pg of liver membrane protein, respectively. Lane C contained 100/xg of cerebellar membrane. The antibody was used at a dilution of 1:500.

similar that of 100/tg striatal membrane protein with a total absence of 110 kDa band. Since the m R N A of D2receptor could not be detected in cellebellar tissues z, it is conceivable that 110 kDa but not 50 kDa protein is the true D2-receptor gene product. With this background information we proceeded to determine potential changes in the level of 110 kDa protein in the striatal membranes during aging. Results presented in Fig. 3 showed that the levels of the 110 kDa protein in the striatal membrane of 12- and 18-month-old male rats were 84% and 66% of that of 4-month-old rats, respectively, with no apparent changes in 50 kDa protein by densitometry analysis. Although the measurements of the changes in D2-re-

ceptor by [3H]YM-09151-2 binding may be confounded by the fact that the above ligand may also recognize D 3 o r D 4 subtypes of dopamine receptor e5'32, the changes in Dz-receptor protein observed by Western blot analysis are highly specific. The D2-receptor antibody 6 used in this experiment was raised against an undecapeptide (GS E G K A - D R P H Y ) z sequence from the NHz-terminus of the D2-receptor; the above peptide sequence when compared with the peptide sequence in the same region in D 1 ( G - L P A E - R D F S F R ) 37, D 3 ( G - V N - R A - - R P H - ) eS, D 4 ( G A S - - - A G L A G Q G ) 32, D5 ( G A ' P P L - G P - S - Q ) e8 receptors exhibit very low homology. We have reported that a decrease in the number of D2-receptor binding sites (Table I) during aging which is

313 above changes in the DE-receptor may be due to decreased level of a 110 k D a DE-receptor specific protein (Fig. 3) rather than the changes in the level of DE-receptor m R N A (Fig. 1). The decrease in the number of DE-receptor between 4 and 18 months of age was between 31% and 39% (Table I) which was very similar to the observed decrement (34%) in 110 kDa protein in 18-month-old striatum (Fig. 3). On the other hand, the changes in the level of D Ereceptor m R N A must be absent or less than 10%. Earlier studies designed to investigate the mechanism(s) underlying regulation of striatal DE-receptor density have failed to observe a quantitative relationship between binding sites of DE-receptor and the level of DE-receptor m R N A . For example, chronic haloperidol treatment, known to increase DE-receptor binding 3's'31, has resulted into marginal to a lack of any change in the D2-receptor m R N A levels in the striatum 8'26'31.

;tOOK-

116K-

66K-

43K-

36. However, we showed here for the first time that the

DE-receptor has been purified from many tissues to represent a range of molecular weights between 95 k D a in striata 33 and 120 k D a in anterior pituitaryEE. In our study, where absolute care was taken to minimize proteolysis, the antibody against DE-receptor recognized 110 k D a protein in rat striatal membrane. These differences in the molecular weight may represent differences due to species, tissue specificity and proteolysis during receptor solubilization and purification. The size of DE-receptor core protein from c D N A sequence 2 should be about 47 k D a protein; the 110 k D a protein, therefore, must be the product of post-translational maturation of the receptor. Therefore, it appears that some steps beyond transcription of DE-receptor gene may play an important role in the physiological and pharmacological regulation of DE-receptor including during aging. These may include changes in receptor synthesis, degradation, and/or postsynthetic maturation of the receptor.

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months

4

12

18

Fig. 3. Western blot analysis of D 2 dopamine receptor in striatal membranes (100 /~g/lane) from 4-, 12- and 18-month-old male Fisher-344 rats using the antibody at a dilution of 1:500. The molecular weight markers are indicated on the left. The relative intensities of 110 kDa band in 12- and 18-month-old rats, analyzed by densitometry, were 84% and 66% of that of the 4-month-old rats, respectively.

consistent with numerous earlier reports 1°'12'16'18-21'23'3°'

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Post-transcriptional regulation of loss of rat striatal D2 dopamine receptor during aging.

The mechanism(s) underlying age-associated diminutions in the rat striatal D2 dopamine receptor (D2-receptor) number was investigated. The levels of D...
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