Instruction manuals

Biphasic and RegionSpecific MAO-B Response to Aging in Normal Human Brain

Description
Biphasic and RegionSpecific MAO-B Response to Aging in Normal Human Brain
Published
of 11
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
Share
Transcript
  Biphasic and Region-Specific MAO-B Responseto Aging in Normal Human Brain J. SAURA,* N. ANDRE´S,* C. ANDRADE,* J. OJUEL,† K. ERIKSSON* AND N. MAHY* 1 *Biochemistry Unit and †Statistics Department, School of Medicine, University of Barcelona, Barcelona, Spain Received 22 April 1996; Revised 29 April 1997; Accepted 30 April 1997 SAURA, J., N. ANDRES, C. ANDRADE, J. OJUEL, K. ERIKSSON AND N. MAHY.  Biphasic and region-specific MAO B responseto aging in normal human brain.  NEUROBIOL AGING 18(5) 497–507, 1997.—Variations of monoamine oxidases (MAO) A and Bwere studied during aging in 27 human subjects (age range 17–93 years) in 18 brain structures of temporal cortex, frontal gyrus,hippocampal formation, striatum, cerebellum, and brainstem. [ 3 H]Ro41-1049 and [ 3 H]lazabemide were used as selective radioligandsto image and quantify MAO-A and MAO-B respectively by enzyme autoradiography. Postmortem delay or time of tissue storage didnot affect MAO-A or MAO-B levels. There was, moreover, no evidence of sexual dimorphism. A marked age-related increase inMAO-B was observed in most structures. This increase started at the age of 50–60 years. Before this age, MAO-B levels were constantin all structures studied. MAO-B-rich senile plaques were observed in some cortical areas but they did not significantly influence theage-related MAO-B increase. Surprisingly, no age-related MAO-B changes were observed in the substantia nigra. In contrast toMAO-B, no clear age-related changes in MAO-A were observed, indicating an independent regulation of the two isoenzymes, alsosuggested by the cross-correlation analysis of these data. © 1997 Elsevier Science Inc.MAO A MAO B Aging human brain MONOAMINE oxidases (MAO, EC 1.4.3.4) are key enzymes inthe deamination of monoamine neurotransmitters and neuromodu-lators. There are two isoenzymes, MAO-A and MAO-B, that differin their selectivity for substrates (62), inhibitors (9), and cellularlocalization, and that are encoded by two separate genes (2). Inthe human brain, serotonin and noradrenaline are preferred phys-iological substrates for MAO-A (29,32) and dopamine is acommon substrate for both isoenzymes (3,29,32,49). There is onlyindirect evidence, from experiments on human brain in vitro orfrom experiments on rodents in vivo, as to which isoenzymephysiologically deaminates other MAO substrates. These studiessuggest that adrenaline may be a preferred MAO-A substrate(18,33) and phenethylamine (41,45) and tele-methyl-histamine(63) may be preferred MAO-B substrates. MAO-A is found innoradrenergic and adrenergic neurons (10,24,25,54,65) andMAO-B is found in serotonergic and histaminergic neurons andastrocytes (10,24, 25,39,40,54,65). Only some dopaminergic neu-rons have MAO-B (12,35), and the presence of MAO-A iscontroversial (24,25, 35,54,65). Therefore, the cellular local-ization of the isoenzymes does not always match their presumedphysiological substrates, suggesting that in some neuronal typesthe role of MAO is the inactivation of extraneous neurotrans-mitters and also that some monoamines are partly deaminated inastrocytes.Many studies have shown increased levels of MAO-B in theaging human brain (1,16,19,20,26,48,52,53,58) and in the brains of Alzheimer’s patients (19,48,53,56,57). This increase is thought tobe caused by the presence of MAO-B-rich reactive astrocytes inresponse to neuronal degeneration. Increased MAO-B levels couldbe harmful to cells because of the increased production of oxygenfree radicals (11). The beneficial effects of the MAO-B inhibitordeprenyl (selegiline) in aging (17,22,34) and in Alzheimer’sdisease (7,31,36,46,60, see however 14) could be interpreted inthis way.Two radioligands, [ 3 H]Ro41-1049 and [ 3 H]lazabemide([ 3 H]Ro19-6327), have recently been characterized as selectivetools for imaging and quantifying MAO-A and MAO-B in tissuesections by enzyme autoradiography in vitro (55). In the presentstudy we have used this approach to analyze, for the first time,age-related changes in MAO-A and MAO-B in 18 structures of normal human brain with a high-resolution technique.This study had four aims: 1) to determine whether the increaseof MAO-B is constant throughout the lifespan as suggested in onestudy (16) or whether it begins at a certain age, as other studiesseem to indicate (26,50); 2) to compare the brain MAO-B contentbetween females and males. Many studies have shown higherMAO-B levels in females in platelets (15,21,38,44,51), whereas inthe brain the question is controversial—some authors have ob-served higher MAO-B in the female brain (52) and others havefound no differences between the sexes (16,26,66); 3) to delineatethe pattern of age-related changes in MAO-A in human cerebellumbecause MAO-A levels in the cerebellum are increased in old rats(27,59) and, to our knowledge, this structure has never beenincluded in human studies; and 4) to evaluate whether the pattern 1 To whom requests for reprints should be addressed: N. Mahy, Unitat de Bioquı´mica, Facultat de Medicina, Universitat de Barcelona, C/Casanova, 143,08036 Barcelona, Spain. Neurobiology of Aging, Vol. 18, No. 5, pp. 497–507, 1997Copyright © 1997 Elsevier Science Inc.Printed in the USA. All rights reserved0197-4580/97 $17.00  .00 PII:S0197-4580(97)00113-9 497  of variations in MAO-A and MAO-B between 14 and 93 years wassimilar in each of the brain structures studied. MATERIALS AND METHODS  Human Tissue Postmortem human brains were obtained through the localBank of Neurological Tissue (School of Medicine, University of Barcelona, Barcelona, Spain) from 27 subjects (10 female, 17male) with no known history of neurological or psychiatricdisorder, ranging in age from 14 to 93 years. Cases with a numberof senile plaques above the age threshold, assessed according toKhatchaturian’s criteria (4), were not included in the study.Postmortem delay ranged from 1.5 h to 24 h and the time of storage of tissue blocks prior to experiment ranged from 3 to 29months. Causes of death were cardiac failure (aged 35, 45, 52, 66,67, 73, 73, 75, 75, 85, 86 and 86 years), sepsis (aged 14, 22, 27, 65and 71 years), digestive hemorrhage (aged 55, 65, 79 and 87years), drowning (aged 17 years), thoracic trauma (aged 29 years),abdominal neoplasm (aged 66 years), ruptured abdominal aneu-rysm (aged 70 years), lung tromboembolism (aged 82 years), andbronchopneumonia (aged 93 years). No significant correlation wasfound between age and time postmortem ( r   0.02), age and tissuestorage time ( r     0.25), or postmortem delay and tissue storagetime ( r   0.16). No significant differences between females andmales were observed in any of these three parameters (see footnoteto Table 1).At autopsy, the brains were dissected into blocks of tissue lessthan 3 cm thick, immediately frozen with dry ice, and kept at  80°C until cryostat sectioning or homogenization (28). In thepresent study we used blocks of temporal cortex (Brodmann area22) ( n    26), frontal gyrus (Brodmann area 4) ( n    27),hippocampus ( n  20), striatum ( n  23), brainstem at the level of the substantia nigra ( n  23), and cerebellar hemispheres ( n  27).  Enzyme Autoradiography Cryostat sections (12-  m thick) were obtained from all blocks,mounted on gelatinized slides, air dried, and kept at  30°C untilthe experiment. Sections were stored for no longer than 2 months.Enzyme autoradiography was performed essentially as previouslydescribed (55).To reveal MAO-A, sections were incubated for 60 min at 37°Cwith 8 nM [ 3 H]Ro41-1049 (27.0 Ci/mmol) in a buffer solution(pH  7.4) containing 50 mM Tris, 120 mM NaCl, 1 mM MgCl 2 ,5 mM KCl and 0.5 mM EGTA. Incubation was terminated by a 1min  1 min wash in cold buffer followed by a quick dip in colddistilled water. Nonspecific binding was defined by coincubationwith 1  M clorgyline.To reveal MAO-B, adjacent sections were incubated for 90 minTABLE 1 EFFECT OF SEX ON MAO IN HUMAN BRAINMAO-A MAO-Bmale femalepercentdifference male femalepercentdifference Temporal cortexGray matter 1394  42 (17) 1486  77 (9)   6.6 2621  153 (17) 2857  252 (9)   9.0White matter 297  31 (17) 309  42 (9)   4.0 1472  191 (17) 1636  238 (9)   11.1Frontal gyrusGray matter 1268  50 (17) 1210  73 (10)   4.6 2185  142 (17) 2348  159 (9)   7.5White matter 134  13 (17) 188  24 (10)   40.3* 973  99 (17) 1234  126 (10)   26.8Hippocampal formationDentate gyrus 1475  51 (13) 1518  101 (6)   2.9 5571  300 (12) 5666  220 (5)   1.7Pyramidal layer of CA1 1652  46 (14) 1738  93 (6)   5.2 3049  254 (12) 3229  178 (5)   5.9White matter of entorhinal area194  27 (14) 211  41 (6)   8.8 2520  288 (14) 2660  373 (6)   5.6Basal gangliaPutamen 1243  133 (15) 1406  111 (8)   13.2 2051  137 (15) 2166  156 (8)   5.6Globus pallidus, lateralis 1624  277 (15) 1385  195 (8)   14.7 2123  139 (15) 1968  169 (8)   7.3Globus pallidus, medialis 1639  250 (14) 1460  269 (7)   10.9 2117  165 (14) 2039  167 (7)   3.7Internal capsule 296  36 (12) 276  37 (8)   7.1 1182  110 (14) 1387  120 (7)   17.4Cerebellar hemispheresGranular layer 1036  39 (16) 968  60 (8)   6.6 1926  162 (12) 2002  119 (9)   3.9Molecular layer 645  36 (16) 561  42 (8)   13.0 1556  145 (11) 1536  78 (8)   1.3White matter 125  21 (17) 127  31 (10)   1.6 1220  78 (17) 1334  150 (10)   9.3Dentate nucleus 2496  122 (12) 2156  129 (7)   13.6 3087  237 (12) 3344  270 (7)   8.3BrainstemSubstantia nigra 1664  150 (13) 1681  166 (9)   1.0 3215  148 (14) 3372  127 (9)   4.9Cerebellar peduncle 211  25 (14) 226  18 (9)   7.3 713  56 (14) 829  64 (9)   16.2Pontine reticularformation1334  95 (10) 1404  144 (7)   5.3 2462  120 (11) 2320  117 (7)   5.8Data shows Mean  SEM and is expressed in fmol/mg prot. Number of cases are shown in parentheses. Percent difference indicates the variationbetween male and female. No significant differences were found between males ( n  17) and female ( n  10) related to age (62.2  5.0 vs. 60.3  8.3 years), postmortem delay (11.9  1.4 vs. 11.4  1.7 h) and tissue storage time (14.4  1.7 vs. 13.9  1.8 months) (Student’s  t  -test).*  p  0.05. 498 SAURA ET AL.  at 22°C in the above buffer containing 3 nM [ 3 H]lazabemide (20.2Ci/mmol). The washing protocol was identical to that for MAO-Aand coincubation with 1  M deprenyl was used to define nonspe-cific binding.After drying under a stream of cold air, sections were apposedto [ 3 H]-sensitive film (Hyperfilm TM , Amersham, Bucks, UK) for26 days (MAO-A) or 12 days (MAO-B). Films were developedand analyzed densitometrically after calibration with plastic stan-dards ([ 3 H]-microscales, Amersham, Bucks, UK) using a comput-er-assisted Image Analysis System (Interdens, Microm, Barcelona,Spain). The average brain protein content was estimated as 8%.  Homogenate Binding Samples of gray matter of approximately 1.5 g were dissectedfrom frozen temporal cortex blocks from 12 subjects, 6 young (3female, 3 male, age range 14–35 years) and 6 old (3 female, 3male, age range 73–86 years). Tissue was homogenized in 5 mL of 50 mM Tris buffer (pH    7.7) with a Polytron (Kinematica,Kriens-Luzern, Switzerland) and centrifuged (45000  g  10 min,4°C). The pellet was homogenized and centrifuged twice more asdescribed above. The final pellet was homogenized in 5 mL of theTris incubation buffer (pH    7.4) used in autoradiography (seeabove). Protein was determined by the method of Bradford (5)using bovine serum albumin as the standard. Protein contentranged from 1.5 to 3 mg/mL. Samples of a young and old subjectof the same sex were processed in parallel in every experiment.Homogenates were incubated with gentle agitation with [ 3 H]laza-bemide (1–20 nM, 6 concentrations, triplicate samples) for 90 minat 25°C in the above Tris buffer as previously described (8).Nonspecific binding was determined in parallel samples contain-ing 10   M  L -(-)deprenyl. At the end of the incubations, sampleswere washed three times in ice-cold Tris buffer and vacuumfiltered with a Brandel cell harvester (Biomedical Research &Development Laboratories, Gaithersburg, MD) through WhatmanGF/C filters (Kent, UK). Filters were transferred to vials that werekept in agitation overnight before radioactivity counting.Binding isotherms were analyzed by nonlinear least squarecurve fitting with the program INPLOT (GraphPad, San Diego,CA) to determine the maximal number of binding sites (  B max ) andequilibrium dissociation constant ( K  D ). Statistics Values are expressed as mean    standard error of the mean(SEM). The effect of age, postmortem delay, and tissue storagetime on MAO-A and MAO-B, the relationship between MAO-Aand MAO-B, and the relationship between the monoamine oxi-dases in different brain structures were analyzed by linear regres-sion and Pearson’s linear correlation coefficient (product moment).In order to eliminate the effect of age in the study of therelationship between MAO-A or MAO-B in the same structure,partial correlations adjusted for age were performed. For compar-ison between males and females the Student’s  t   test (two-tailed)was used and an analysis of covariance (ANCOVA) controlling forage was performed if necessary. A  p  of less than 0.05 wasconsidered as significant. Data were analyzed with statisticalsoftware SPSS 6.1 (Marija Norusis/SPSS Inc., Chicago, IL) andSTATGRAPHICS 6.0 (STSC Inc., Rockville, MD).  Materials [ 3 H]Ro41-1049 and [ 3 H]lazabemide were kindly provided byDr. J.G. Richards, Hoffmann La Roche, Basel, Switzerland. L -(-)-Deprenyl and clorgyline were supplied by Research Biomed-ical Inc. (Natick, MA). All other chemicals were of analyticalgrade and were obtained from various sources. RESULTS  Distribution Binding of [ 3 H]Ro41-1049 and [ 3 H]lazabemide to human brainwas highly specific (nonspecific binding lower than 100fmol/mgprot). The pattern of distribution agreed with that previouslyreported (54) and is shown in Table 1 and Figures 1 and 2. Thehighest levels of MAO-A and MAO-B were measured in thedentate nucleus of the cerebellum and in the dentate gyrus of thehippocampus, respectively. In most individuals, the abundance of MAO-A in the globus pallidus and putamen was similar, aspreviously shown (54). However, in three cases, aged 22, 65 and87 years, the globus pallidus was particularly enriched in MAO-Awith approximately twice the MAO-A content of the respectiveputamen (Figures 3 and 4). MAO-B-rich patches like thosedescribed as formed by senile plaque-associated astrocytes (39,56)were found in four cases in the gray matter of the frontal gyrus(0.2, 0.3, 1.7, and 2.4 patches/mm 2 ) and in two cases in the graymatter of the temporal cortex (1.2 and 2.3 patches/mm 2 ). In noneof the other brain structures studied were such formations ob-served.When [ 3 H]Ro41-1049 and [ 3 H]lazabemide specific bindingwere compared, positive correlations were obtained in all struc-tures ( r   from 0.10 to 0.68) and these were statistically significant(  p    0.05) in 8 out of 18 structures. The degree of correlationbetween MAO-A and MAO-B was especially high in the whitematter of the entorhinal area ( r   0.68,  p  0.001), gray matter of the temporal cortex ( r     0.55,  p    0.01), white matter of thecerebellum (  p  0.01), and the putamen ( r   0.57,  p  0.01).  Effect of Postmortem Delay and Tissue Storage Time No significant correlation between postmortem delay andMAO-A or MAO-B was found in any of the brain structuresstudied; in addition, the time of tissue storage and MAO did notcorrelate with MAO-A or MAO-B in any structure (data notshown). These results are expected because MAO-A and MAO-Bare known to be stable proteins in the range of postmortem delay(16,19,20) and tissue storage times (26) of the present study.  Effect of Sex No significant differences between females and males werefound in any of the brain regions studied either in MAO-A or inMAO-B, except in the white matter of the frontal gyrus, where theMAO-A concentration of females was significantly higher (Table1).Differences between genders were also assessed by splitting thedata into two age groups: younger than 56 ( n  9) and older than64 ( n    18). This analysis resulted in significant differences insome cerebellar structures. In the group of younger cases, femaleshad higher MAO-A levels in the white matter of the cerebellum(  220%,  p    0.05) and had higher levels of MAO-B in thegranular layer (  35%,  p  0.05) and dentate nucleus (  22%,  p  0.05). In the group of older cases, females had lower MAO-Alevels in the granular layer (  14%,  p  0.05) and dentate nucleus(  19%,  p  0.05).  Age-Related Changes MAO-B showed a significant positive correlation with age invarious brain areas (Table 2). This correlation was especiallystrong in the putamen, globus pallidus medialis, granular layer of the cerebellum, and dentate nucleus. Exponential instead of linearregression did not yield higher correlation coefficients. From the499MAO IN THE AGING HUMAN BRAIN  observation of the MAO-B/age plots it became apparent that theage-related MAO-B increase was restricted to patients older than50–60 years. Indeed, when cases below the age of 60 wereconsidered, a negative, though not significant, correlation wasfound between MAO-B and age in 16 out of 18 structures. Incontrast, when only the cases older than 45 were considered, thestrength of the correlation between age and MAO-B was main-tained or even enhanced, especially significant in the pyramidallayer of CA1 (Table 2). This analysis was repeated for thepopulations older than 45, 50, and 60 years, and the strongestcorrelations were obtained in the first group.When the effect of age on brain MAO-B was studied in malesand females separately, similar regression coefficients were ob-tained in both groups with the exception of the hippocampalstructures, where the age-related MAO-B increase was restricted tomales ( r   in females ranged from -0.01 to -0.18). FIG. 1. Distribution of [ 3 H]Ro41-1049 binding to sections of frontal gyrus (  A, B ), hippocampal formation ( C, D ),striatum (  E, F  ), and substantia nigra ( G, H  ). Left and right column show tissues from young cases (17–35 years)and aged cases (79–86 years), respectively. Both images of the same structure were obtained from the same filmand were developed in identical conditions to reflect differences in the abundance of binding sites. Similardistribution patterns and abundance between young and aged individuals are observed in all tissues. Calibration bar,3 mm. 500 SAURA ET AL.  In order to examine whether the age-related increase in[ 3 H]lazabemide could be due to a change in affinity rather than toa change in the number of binding sites, saturation experiments intissue homogenates of temporal cortex gray matter were per-formed. No significant differences in affinity between young andold individuals were observed. Mean K  D  values were 3.8  0.6 nMand 6.6    1.9 nM for the groups of young and old cases,respectively. However,  B max  estimates were significantly higher(  p  0.05) in the group of old individuals: 8.5  0.9 pmol/mg prot(young) vs 16.3  3.1 pmol/mg prot (old) (Figure 5).No significant correlation between age and MAO-A contentwas found in any of the structures studied (Table 2). The visualobservation of the MAO-A/age plots confirmed the absence of age-related changes, but in some structures (e.g., the white matterof the entorhinal area, cerebellar peduncle) V-shape patterns weresuggested. The data were then analyzed splitting the cases intoyounger and older than 45, 50, or 60 years. No significantcorrelations between age and MAO-A were found in the youngergroups and most correlations were negative (15 out of 18 in theyounger-than-60 group). In contrast, most coefficients were posi- FIG. 2. Distribution of [ 3 H]lazabemide binding to human brain sections adjacent to those shown in Figure 1 (seelegend for details). Note the similar pattern of distribution of MAO-B in young and aged individuals but the higherabundance of MAO-B in the aged individuals in frontal gyrus, hippocampal formation and striatum. This increaseis not due to the presence of MAO-B-rich senile plaques. In the substantia nigra, no differences between youngand aged individuals were found in the abundance of MAO-B. Calibration bar, 3 mm. 501MAO IN THE AGING HUMAN BRAIN
Search
Similar documents
View more...
Tags
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks