Monoamine oxidase isoenzymes in rat brain: Differential changes during postnatal development but not aging

Neurobiology of Aging, Vol. 16, No. 5, pp. 833-836, 1995 Copyright © 1995 Elsevier Science Inc. Printed in the USA. All rights reserved 0197-4580/95 $9.50 + .00

Pergamon 0197-4580(95)00061-5

Monoamine Oxidase Isoenzymes in Rat Brain: Differential Changes During Postnatal Development But Not Aging K. M A M A T H A

R A O , S. N . N A G E N D R A

1 AND M. N. SUBHASH

D e p a r t m e n t o f Neurochemistry, National Institute o f M e n t a l Health and Neurosciences, Bangalore 560 029, India R e c e i v e d 9 A u g u s t 1993; R e v i s e d 30 D e c e m b e r 1994; A c c e p t e d 9 J a n u a r y 1995 MAMATHA, RAO, K., S. N. NAGENDRA AND M. N. SUBHASH. Monoamine oxidase isoenzymes in rat brain: Differential changes during postnatal development but not aging. NEUROBIOL AGING 16(5) 833-836, 1995. -- Differential development of monoamine oxidase (MAO) isoenzymes in rat whole brain is described in postnatally developing Sprague-Dawley rats. Total MAO and isoenzyme activity was measured using nonspecific and specific substrates. Total MAO activity measured using tyramine, increased postnataUy up to 24 weeks of age and attained a plateau afterward. The increase in total MAO activity was significant at all age groups (18 days to 36 months) investigated as compared to new born rats. MAO-A and MAO-B activities were measured using octopamine and benzylamine respectively. We also observed a marginal increase of MAO-A activity and a significant increase of MAO-B activity upon development. Furthermore, at 12 weeks of age, MAO-B activity increased by 10-fold as compared to new born and was consistent up to 36 mouths of age. The qualitative localization of the enzyme activity on non SDS-PAGE by nitroblue tetrazolium staining confirmed the increase of MAO-B during the development. It is suggested that the maturational increase of total MAO activity in brain is predominantly due to the increase of MAO-B isoenzyme. Monoamine oxidase

Isoenzymes

Rat brain

Development

Aging

nated by M A O - A , whereas M A O - B is responsible for deamination of dopamine (16). Several investigators have reported the increase of M A O - B but not M A O - A activity with age in human brain (5), though this finding is disputed (20). Major preponderance of M A O - B during development has been reported in microvessels of rat brain (10). Studies have indicated elevated brain MAO-B in certain neurodegenerative diseases like Parkinson's (15), Huntington's disease (HD), and Alzheimer's disease (AD) (4,9), without any change of M A O - A activity (3). Experiments with animals have indicated that aminergic neuronal systems on the aging brain have differential M A O isoenzyme profiles (23). Detailed studies of the developmental changes in M A O activity are difficult to perform in man due to the heterogeneity of the tissue material with regard to age, sex, cause of death, variable delay between death, and autopsy and other factors (1). In experimental animal studies, such factors can be eliminated, allowing the assay of M A O in well defined conditions where only age is the variable. We report the results of analysis of

M O N O A M I N E oxidase (MAO: E C 1.4.3.4) has an important role in the regulation of oxidative deamination of endogenous and exogenous monoamines in the central nervous system. Subcellular localization studies have shown M A O to be present in mitochondrial membranes and in brain capillary walls (2). Regional differentiation (25) and differential postnatal development (10) of M A O has also been reported earlier in rat brain. The functional development of brain M A O was poor at birth and reached adult levels after several weeks postnatally as and when functional activity of brain developed. Similar results were also reported f r o m human autopsy brain study (17). The concept of two functionally distinct forms of M A O has been generally accepted in the last decade. Using pargyline binding and specific inhibitors, M A O - A and M A O - B forms have been reported in different tissues, including brain of rat, mice, and humans (11). M A O - A is inhibited by nanomolar and MAO-B by m i c r o m o l a r concentrations of clorgyline (8) whereas both are inhibited by 10 -7 M L-deprenyl (12). In h u m a n brain, 5-hydroxytryptamine and noradrenaline is oxidatively deami-

~Requests for reprints should be addressed to S. N. Nagendra, Department of Pharmacology & Toxicology, 5040 Malott, School of Pharmacy, University of Kansas, Lawrence, KS 66045. 833

834

RAO, N A G E N D R A A N D S U B H A S H

M A O activities in rat brain at various stages o f its development. The M A O o n t o g e n y o f b r a i n is also d e m o n s t r a t e d by staining the enzyme activities o n n o n d e n a t u r i n g gel electrophoresis. METHOD

P r o t e i n c o n t e n t was d e t e r m i n e d by the m e t h o d o f Lowry et al. (13). Statistical analysis o f changes a m o n g s t groups was carried out by one-way analysis of variance (ANOVA) followed by post hoc c o m p a r i s o n o f m e a n s by B o n f e r r o n i ' s test. Values were considered significant w h e n p < 0.05.

Chemicals T y r a m i n e - H C l , benzylamine-HC1, octopamine-HC1, horse radish peroxidase, homovanillic acid, bovine serun, albumin, ~litro blue t e t r a z o l i u m , a n d p h e n a z i n e m e t h o s u l f a t e were p u r c h a s e d from Sigma C h e m . Co. (St. Louis, MO). All other chemicals used were o f analytical grade, p u r c h a s e d f r o m Glaxo (India) Ltd. Male Sprague-Dawley rats were obtained from Central Animal Research Facility ( C A R F ) o f N a t i o n a l Institute o f M e n t a l H e a l t h a n d Neurosciences ( N I M H A N S ) . Rats o f varying age groups such as new b o r n , 18 days, 4, 8, 12, 24 weeks 12, 15,18, 24, 30, a n d 36 m o n t h s were used for this study. They were housed u n d e r controlled temperature a n d humidity with n o r m a l 12L:12D schedule a n d free access to chow pellets a n d water. Rats were mildly anesthetized with ether and decapitated. Brain was removed a n d washed with ice cold 0.9°70 saline to get rid o f meninges a n d blood clots. W h o l e brain was h o m o g e n i z e d (1:10 w/v) in 0.1 M Na-K p h o s p h a t e , p H 7.8 c o n t a i n i n g 0.1% triton X-100 a n d centrifuged at x 2 0 , 0 0 0 g for 30 rain a n d the supern a t a n t was used for the M A O assay. Triton X-100 has been successfully used to solubilize the m e m b r a n e M A O elsewhere (7).

M A O Assay The M A O activity was measured by the fluorometric method o f Snyder a n d Hendley (22). Briefly, 3 ml o f final assay system consisted o f 0.1 M Na-K p h o s p h a t e , p H 7.8, horse radish peroxidase (20 U), enzyme p r e p a r a t i o n (~-100-125/~g protein) and respective substrates (0.1 mM), tyramine (for total M A O activity), o c t o p a m i n e ( M A O - A ) (26), b e n z y l a m i n e (MAO-B). The reaction was initiated by adding the substrate followed by HVA (0.25 mg) to a preincubated (10 rain at 37°C) assay system. After 1-h incubation in a shaking water bath at 37°C, the reaction was terminated by chilling the tubes at 4°C for 10 min. The reaction mixture was centrifuged for 10 min at ×2000 g to o b t a i n a clear s u p e r n a t a n t . Fluorescent m e a s u r e m e n t was m a d e with A m i n c o B o w m a n s p e c t r o p h o t o f l u o r i m e t e r with a n emission at 425 n m a n d excitation at 315 n m . Simultaneously, a tissue b l a n k was processed w i t h o u t the exogenous substrate. The enzyme activity was calculated after measuring the fluorescence intensity against a s t a n d a r d curve established with increasing concentrations (1-10 /~M) o f H202. The exact molarity of H2O 2 was d e t e r m i n e d by t i t r a t i o n with p o t a s s i u m p e r m a n g a n a t e . M A O activity is designated as u n i t / r a g protein wherein, one unit is defined as n m o l p r o d u c t f o r m e d / m i n .

M A O Localization on PAGE N o n d e n a t u r i n g electrophoresis on 8°7o polyacrylamide gel ( P A G E ) was carried out using b r a i n h o m o g e n a t e p r e p a r a t i o n s (~-100 t~g p r o t e i n ) o b t a i n e d f r o m a rat o f each group. Similar protein concentration f r o m each age group was carefully loaded o n P A G E wells. A f t e r the electrophoresis the activity staining was carried out by the m e t h o d of Shih a n d E i d u s o n (21). The gels were i n c u b a t e d at 37°C for 30 min with 3 ml o f reaction mixture c o n t a i n i n g 0.1 M Na-K p h o s p h a t e , p H 7.8, n i t r o b l u e tetrazolium (1 mM), phenazine methosulfate (1 mM), horse radish peroxidase (20 U), a n d 0.1 m M o f benzylamine. Gels were washed well with water and dark colored bands indicating enzyme activities were visualized.

RESULTS T h e enzyme activity is expressed as activity per mg protein hence, the analysis o f protein content (data not included) of rat b r a i n tissue during the d e v e l o p m e n t a n d aging was necessary.

Whole Brain M A O Activity M A O activity in homogenates of brain tissue from young and aging rats was assayed using tyramine, o c t o p a m i n e a n d benzyla m i n e as substrates. Total M A O activity increased by four fold in postnatally developing rats at 12 weeks o f age a n d r e m a i n e d u n c h a n g e d up to 36 m o n t h s o f age (Table 1). The developmental increase o f total M A O activity was significant ( p < 0.001) at all age groups (18 days to 36 m o n t h s ) studied as c o m p a r e d to new b o r n g r o u p (Table 1).

MAO-A and MAO-B Activity o f Young and Aging Rats The d e v e l o p m e n t a l increase of M A O - B activity f r o m new b o r n to aging rats was highly p r o n o u n c e d and significant. However, the d e v e l o p m e n t a l increase o f M A O - A was m a r g i n a l a n d was significant only at 24, 30, and 36 m o n t h s of age as compared to new b o r n rats. A p h e n o m e n a l 10-fold increase o f M A O - B activity was observed at 12 weeks o f age as c o m p a r e d to new b o r n . The ratio o f M A O - A to M A O - B activities in new b o r n was 3:1 which dramatically changed to 1:4 at 12 weeks of age (Table 1). M A O A to B ratio (1:4) was consistent from adult h o o d (12 weeks) to 18 m o n t h s o f age. However, at 24 m o n t h s o f age A to B ratio increased to 1:3 a n d was consistent up to 36

TABLE 1 ONTOGENY OF RAT BRAIN MONOAM1NE OX1DASE ACTIVITY Age New Born 18 days 4weeks 8weeks 12weeks 24 weeks 12 months 15months 18months 24months 30months 36months

Total MAO

MAO-A$

MAO-B$

(A/B) Ratio

0.830 _+ 0.20 1.600 + 0.20*** 2.110_+0.18"** 2.750_+0.20*** 3.300_+0.15"** 3.350 _+ 0.20*** 3.200 _+ 0.20*** 3.356_+0.11"** 3.237_+0.12"** 3.558_+0.11"** 3.438-+0.10"** 3.581+0.10"**

0.500 + 0.20 0.630 _+ 0.13 0.650+_0.11 0.680_+0.08 0.700_+0.10 0.750 _+ 0.10 0.650 _+ 0.20 0.642-+0.03 0.612_+0.05 0.880_+0.08* 0.850-+0.05* 0.910+0.06"*

0.250 + 0.05 0.720 +_+0.06*** 1.250_+0.13"** 2.120_+0.11"** 2.660_+0.10"** 2.600 -+ 0.10"** 2.610 + 0.20*** 2.660_+0.09*** 2.614_+0.12"** 2.658_+0.08*** 2.664_+0.10"** 2.718_+0.12"**

3:1 1:1 1:2 1:3 1:4 1:4 1:4 1:4 1:4 1:3 1:3 1:3

Values are mean + SD for rats (n = 6) from new born to 12 months of age and (n = 4) from 15 to 36 months of age. Sunits/mg protein. Statistical analysis of changes amongst groups was performed by ANOVA followed by post hoc multiple comparison of means by Bonferroni's test. The statistical significance between different age groups and new born is shown with: *p < 0.05; **p < 0.01 ; ***p < 0.001. The statistical significance of difference amongst groups is shown with: Total MAO: F(148.5),p < 0.001; MAO-A: F(5), p < 0.001; MAO-B: F(339),p < 0.001.

D I F F E R E N T I A L POSTNATAL C H A N G E S OF MAO months (Table 1). The developmental increase of MAO-B activity was confirmed by localizing the enzyme activity on nondenaturing PAGE by NBT staining. Figure 1 indicates the increase of MAO-B activity from new born to 12 months of age. DISCUSSION

In general, the developmental pattern of brain MAO demonstrated in this study is consistent with the previous reports (18,27). However, the attainment of peak activity was found to be at 12 weeks and not 3 weeks of age as reported in earlier studies (18,27). There was no further significant change of total MAO activity from 12 to 36 months of age. This finding is in agreement with the results of Strolin and Keane (23) who observed no significant increase of total MAO activity in aging rats. It is reported that MAO-B exists as a predominant form in animal brain including human brain (6). Although, MAO activity has been shown to be altered during the development of brain, there is controversy as to the type of MAO present in brain. The interesting finding of this study is the disproportionate appearance of MAO-A and MAO-B of brain during the development which is in agreement with earlier studies (10,14). The developmental increase of MAO-A activity from new born to 12 months was marginal whereas the increase in MAO-B activity was highly significant. The ratio of MAO-A to MAO-B activities in new born (3:1) gradually changed to 1:4 at 12 weeks of age. Thus, a significant change in the ratio of A to B activities during the development was observed with no further change from adulthood to 18 months of age. Kalaria and Harik (11) observed A / B ratio of 1:3 in adult rat cortex and 1:1 in human cerebral cortex. Our findings are in accordance with the above reports suggesting that MAO-B is largely responsible for maturational increase of M A O in cerebral cortex. It is concluded that the developmental pattern of MAO-A and MAO-B in rat brain is similar to that of human brain particularly with respect to cerebral cortex. The developmental changes of MAO-B was confirmed by localizing and staining the enzyme activity on non-

835 SDS-PAGE by NBT method. The MAO-A activity was more or less unchanged from new born to adulthood whereas, MAO-B activity recorded a significant developmental increase. This may be due to the fact that number of MAO-B molecules mainly glial increases, whereas that of MAO-A mainly, neuronal do not change with aging in rodent brain (24). However, a significant increase of MAO activity was observed with increased age, which correlates with the developmental increase of a predominant form present in brain, MAO-B (Fig. 1). An increase in enzyme activity could be due to an increase in affinity, represented by a decrease in Km. MAO-B is largely responsible for maturational changes of MAO activity of whole brain, hence the changes seen in total MAO activity in certain neurodegenerative disorders, may be due to MAO-B and not MAO-A. At birth there was meager amount of MAO-B that dramatically increased during first 12 postnatal weeks of age. However, no such dramatic change with respect to MAO-A was observed and this could be due to lack of specific function or the substrate in brain. This study indicate that MAO-B activity presumably reaches peak level by the 12th postnatal week with approximately 10-fold increase of enzyme activity as compared with the MAO-B activity at birth. In addition, the activities measured at 4 and 8 weeks showed a steady decrease of MAO-A to MAO-B ratio from 1:2 to 1:3 (Table 1). Thus, the enzyme activities were rapidly changing during the maturation phase until a peak activity was achieved at 12 weeks of age wherein the ratio of A to B was maintained at 1:4 up to 18 months of age. However, Kalaria and Harik (10) have shown a 20-fold increase in cerebral microvessels during first 2 postnatal weeks, with a slight decline at around 20 days of postnatal age. This observation was also evident in previous studies using rat brain homogenates (19) and the reason for this is not clear. The differential development suggests that MAO-A and MAO-B are regulated separately by genetic or hormonal factors. Thus, it is possible that only one form is affected in different conditions. Cross et al. (4) have observed a significant selective increase of MAO-B activity in Alzheimer's brain due

FIG. 1. Quantitative localization of MAO-B activity of rat brain during the development by non-SDS-PAGE (8°70). After the electrophoresis, the activities were stained as described in method. Lanes 1 to 5 represent activities present in protein obtained from new born, 18 days, 12, 24 weeks, and 12-month-old rat brains, respectively.

836

RAO, NAGENDRA

to r e a c t i v e g l i o s i s t h a t o c c u r s in A D w h e r e a s M A O - A a c t i v i t y w a s u n c h a n g e d . T h i s is likely in n e u r o d e g e n e r a t i v e diseases s u c h as A D , H D , a n d in o t h e r n e u r o - p s y c h i a t r i c d i s o r d e r s (4,9,15). B e c a u s e M A O - A is s h o w n to be l o c a l i z e d p r e d o m i n a n t l y in t h e n e u r o n s a n d M A O - B is localized e x t r a n e u r o n a l l y (24), t h e d a t a p r e s e n t e d in t h e p r e s e n t s t u d y s u g g e s t t h a t t h e activity o f M A O B in r a t b r a i n s h o w d e v e l o p m e n t a l i n c r e a s e . It is s u g g e s t e d t h a t

AND SUBHASH

t h e selective i n c r e a s e o f M A O - B a c t i v i t y is d u e to its p r e f e r e n tial e x t r a n e u r o n a l l o c a l i z a t i o n . ACKNOWLEDGEMENT This study was supported by N I M H A N S grant and ICMR fellowship to R. K. Mamatha. We thank B. S. S. Rao for the encouragement for this work.

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