Adenylyl cyclase activity in Alzheimer's disease brain: stimulatory and inhibitory signal transduction pathways are differently affected

BRAIN RESEARCH ELSEVIER

Brain Research (,44 (1994) 291-296

Research Report

Adenylyl cyclase activity in Alzheimer's disease brain: stimulatory and inhibitory signal transduction pathways are differently affected Anke Schnecko a, Klaus Witte ~, Jiirgen Bohl c, Thomas O h m b, Bj6rn Lemmer "'* " Zentrum der Pharmakologie, h Zentrum der Morphologie, Johann Wolfgang Goethe-Unit~ersith't, Theodor-Stern-Kai 7, D-60590 Frankfitrt, Germany c Abteilung fiir Neuropathologie, Johannes Gutenberg- Unit,ersitdt, Langenbeckstrafle, D-55131 Mainz, German) (Accepted 18 January 1994)

Abstract

Adenylyl cyclase (AC) activity was studied in post mortem hippocampus and cerebellum from eight patients with Alzheimer's disease/senile dementia of the Alzheimer type (AD/SDAT) and seven non-demented control patients. AC was stimulated via stimulatory guanine nucleotide binding proteins (Gs) using guanosine triphosphate (GTP) and GppNHp (both 1 0 - 4 M ) or directly with either forskolin (10-aM) or Mn 2÷ (10-2M). Inhibition of AC via Al-receptors was performed with N6-cyclohe xyladenosine (CHA) under basal conditions and in the presence of forskolin (10-SM). In both brain regions AC activity was significantly reduced in A D / S D A T when compared to controls. Under basal conditions and after stimulation via Gs mean reduction in hippocampus and cerebellum was 47,7% and 58.2%, respectively. The reduction was less pronounced after direct activation of the AC, amounting to 21.8% in hippocampus and 28.1% in cerebellum. CHA inhibited basal and forskolin-stimulated AC concentration-dependently by about 20% (basal) and 30% (forskolin). Inhibition by CHA was similar in hippocampus and cerebellum and tended to be more pronounced in A D / S D A T than in controls. Since the reduction of AC activity in A D / S D A T is greater after stimulation via Gs than after direct activation of the catalytic subunit, we suggest that both Gs and the catalytic subunit seem to be impaired. The fact that CHA-mediated inhibition of AC is not significantly different in A D / S D A T and controls, indicates that in contrast to Gs-, inhibitory G-proteins (Gi)-coupling to AC remains intact in Alzheimer's disease. Key words: Alzheimer's disease; Adenylyl cyclase; Hippocampus; Cerebellum; A l-receptor; G-protein; Coupling; Signal transduction

I. Introduction

A l z h e i m e r ' s d i s e a s e / s e n i l e d e m e n t i a of the Alzheimer type ( A D / S D A T ) is an irreversible neurodegenerative disorder which is characterized clinically by a progressive dementia. Since Davies' observation of a marked decrease in choline acetyltransferase in the cerebral cortex nearly 20 years ago [12] numerous neurochemical alterations have been documented in A D / S D A T . Most research has been focussed on in-vitro analysis of transmitter levels, enzyme activities and receptor densities in brain tissue (for a recent

* Corresponding author. Fax: (49) (69) 6301-7531. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 1 3 5 - Y

review, see [17]). Comparably few studies have examined signal transduction pathways beyond receptor levels in A D / S D A T . Investigation of the functional integrity of receptor mediated signal transduction is, however, essential for evaluating the potential significance of neurotransmitter replacement therapies in this disorder. Cyclic adenosine monophosphate (cAMP) is one of the major second messengers involved ifi neuronal signalling. It is generated from A T P by the enzyme adenylyl cyclase (AC). The A C is coupled to numerous neurotransmitter receptors which either stimulate or inhibit the enzyme. A C activation is mediated via stimulatory guanine nucleotide binding proteins (Gs) whereas inhibitory G-proteins (Gi) lead to enzyme inhibition.

292

A. Schnecko et al. / Brain Research 644 (1994) 291-296

It has recently b e e n r e p o r t e d by our group [23,31] a n d two o t h e r groups [9,34] that A C activity is r e d u c e d in different b r a i n regions of p a t i e n t s with A D / S D A T . Several a t t e m p t s have b e e n m a d e to elucidate at what level the signal t r a n s d u c t i o n pathway is disturbed. C o w b u r n and colleagues f o u n d an u n c o u p l i n g of Gs from the A C in several b r a i n regions of p a t i e n t s with A D / S D A T w h e n s t i m u l a t i n g the enzyme with the stable g u a n o s i n e t r i p h o s p h a t e ( G T P ) a n a l o g u e g u a n o s i n e 5 ' - O - ( 3 - t h i o t r i p h o s p h a t e ) ( G T P y S ) [9]. By modifying the e x p e r i m e n t a l c o n d i t i o n s they m a n a g e d to inhibit A C activity with G p p N H p . T h e results of this investigation indicate that the c o u p l i n g of Gi to the A C is preserved in A l z h e i m e r ' s disease b r a i n [10]. I n a very recent p u b l i c a t i o n Ross et al. r e p o r t e d on a r e d u c e d activity of the G - p r o t e i n associated enzyme, high-affinity G T P a s e , in different b r a i n regions of A D / S D A T p a t i e n t s [34]. T h e aim of the p r e s e n t study was to f u r t h e r characterize the m e c h a n i s m s which lead to the r e d u c t i o n of A C activity in A l z h e i m e r ' s disease. For the first time A C s t i m u l a t i o n as well as i n h i b i t i o n via different levels of the signal t r a n s d u c t i o n cascade were p e r f o r m e d within the same m e m b r a n e h o m o g e n a t e . T h e A C was s t i m u l a t e d via Gs with G T P or its n o n - h y d r o l y z a b l e a n a l o g u e G p p N H p . Direct activation of the A C was achieved with forskolin or Mn2+-ions. W h e r e a s forskolin also acts via a G - p r o t e i n m e d i a t e d m e c h a nism [37], M n 2+ solely stimulates the catalytic s u b u n i t [38]. U n f o r t u n a t e l y , no r e c e p t o r m e d i a t e d s t i m u l a t i o n of the A C could be studied. F r o m our experience [35] and from several indications in [32,33,41] it a p p e a r s that n e u r o n a l receptors which stimulate the A C u n c o u ple from the signal t r a n s d u c t i o n complex in the course of the m e m b r a n e p r e p a r a t i o n . In contrast, inhibitory receptors r e m a i n c o u p l e d [32,39] so that we were able to e x a m i n e a d e n o s i n e A ~-receptor m e d i a t e d i n h i b i t i o n of the A C in A D / S D A T . It is well established that a d e n o s i n e is a p o t e n t n e u r o m o d u l a t o r in the central n e r v o u s system. A d e n o s i n e inhibits n e u r o n a l firing a n d n e u r o t r a n s m i t t e r release (e.g., acetylcholine, G A B A , d o p a m i n e , n o r e p i n e p h r i n e , glutamate). Both effects are m e d i a t e d via A~-receptors that have b e e n r e p o r t e d to be c o u p l e d in an inhibitory fashion to adenylyl cyclase whereas A 2 - r e c e p t o r s which are also p r e s e n t in the central nervous system stimulate A C [14]. W e used N('-cyclohexyladenosine ( C H A ) as selective A~-receptot agonist. T h e A~-receptor m e d i a t e d i n h i b i t i o n of A C in p o s t - m o r t e m h u m a n b r a i n tissue has not b e e n described in the literature so far. As b r a i n regions which would be of interest for such an investigation we decided u p o n h i p p o c a m p u s a n d c e r e b e l l u m . T h e f o r m e r region was chosen as typically showing severe histopathological changes in A l z h e i m e r ' s disease a n d the latter b e c a u s e it is free of A l z h e i m e r - t y p i c n e u r o f i b r i l l a r y changes [19].

2. M a t e r i a l s

and methods

2.1. Materials

Rolipram was a gift from Schering AG (Berlin, Germany). We used a water-soluble forskolin analogue (forskolin-7/3-deacetyl-7/3-(~N-methylpiperazino)-butyryl,dihydrochloride, Calbiochem)which has been shown to stimulate the AC in a concentration-dependent manner [24]. The cAMP radioassay kit (TRK 432) was purchased from Amersham Buchler (Braunschweig, Germany). All other chemicals were of the highest purity commercially available. 2.2. Brain tissue

Brain tissue was obtained at autopsy from eight patients who suffered from AD/SDAT and from seven control subjects without neurological or psychiatric disorders (see Table I for details). The two groups were matched for post-mortem delay but could not strictly be regarded as age-matched. This was not considered a problem, since there was no dose correlation between AC activity and age in post-mortem human brain tissue [9,31]. Brains were bisected sagitally and the left hemisphere was used for biochemical studies. Tissue blocks were prepared from the hippocampus (at the level of the geniculate body) and cerebellum. The tissue samples were immediately frozen in liquid nitrogen and were stored at 70°C until determination of AC activity. The right hemisphere was fixed in 10% formalin for histological investigations. 10(I-/xm-tbick PEG-embedded sections were stained selectively with specific silverimpregnation-techniques in order to demonstrate Alzheimer's histopathological changes [7,16]. This staining procedure had been shown to be as sensitive and selective as immunocytochemical techniques for abnormally phosphorylated ~-protein and synthetic A4//3-amyloid [6,30]. Diagnoses were established according to the neuropathological stageing scheme by Braak and Braak [5]. The patients' tissues were included in the study if they were either classified stage 0-1 (controls) or stage V-VI (AD/SDAT). Biochemical experiments were performed with blinded tissues. 2.3. Tissue preparation

Frozen tissue samples were homogenized in 20 ml ice-cold buffer (Tris-HCI 50 mM, MgCI2 10 mM, pH 7.4) using an Ultra Turrax Table 1 Patients' characteristics Sex (m/f) Controls: 1 f 2 m 3 m 4 f 5 m 6 f 7 m AD/SDAT: 1 f 2 m 3 f 4 m 5 f 6 f 7 f 8 f

Age (years)

P o s t - m o r t e m Causeof delay (h) death

80 75 70 81 62 83 63

14 19 37 41 22 18 32

? acute heart failure bronchopneumonia lung embolia hepatocell, carc. myocard infarction acute heart failure

86 86 84 86 91 68 89 79

56 23 3 14 '? 39 16 27

myocard infarction lung embolia lung embolia bronchopneumonia acute heart failure lung embolia acute heart failure bronchopneumonia

A. Schnecko et al. / Brain Research 044 (1994) 291 296 homogenizer. The homogenate was divided into subsets. After centrifugation (25,000 g, 4°C, 10 min) the pellets were resuspended in 20 ml buffer (Tris-HCI 50 mM, MgCI z 10 raM, pH 7.4 for stimulatory experiments or Tris-HCl 50 mM, MgCI 2 2 raM, NaCI 100 mM, pH 7.4 for inhibitory experiments). In order to stay as close as possible to the "physiological situation' the m e m b r a n e s were not processed any further after this initial centrifugation step. The crude m e m b r a n e suspension was immediately used in the incubation. Protein content was determined according to Lowry et al. [25]. 2.4. Adenylyl cTclase assay Aliquots of the crude m e m b r a n e preparation were added to the pre-warmed incubation mixture containing an ATP-regenerating system (ATP 0.5 raM, creatine phosphate 10 mM, creatine phosphokinase 0.1 m g / m l ) , a phosphodiesterase inhibitor (3-isobutyl-l-methylxanthine, IBMX, 1 m M for stimulatory experiments or rolipram 0.3 m M for inhibitory experiments) and GTP, G p p N H p , forskolin, Mn 2 ÷ or C H A at the concentrations indicated. W h e n measuring Mn 2+mediated stimulation of the AC, no Mg 2+ was present in the incubation buffer. T h e inhibition of A C by C H A required the presence of G T P 10 5 M and adenosine deaminase 5 U / m l . The incubation was carried out at 37°C for 8 min. The reaction was stopped by heating the samples at 120°C in a thermoblock. After centrifugation, the cAMP-content in the supernatant was determined using a commercial radioassay kit. 2.5. Data analysis and statistics The cAMP concentrations measured with the radioassay as well as the c o n c e n t r a t i o n - r e s p o n s e curves were calculated with the nonlinear fitting program P H A R M F I T [26]. The subprogram for analyzing c o n c e n t r a t i o n - r e s p o n s e curves uses the Hill-equation for calculating maximum enzyme stimulation (E~ax), half-maximum stimulatory concentration (ECs0) and the Hill-coefficient. A C activity is expressed as 'generated c A M P / m g p r o t e i n / m i n ' . For statistical analysis the program BIAS was used [1]. Significance of the C H A mediated inhibition of A C activity was tested by analysis of variance using the Friedman test. To test for significant differences of Emaxvalues between controls and A D / S D A T the two-tailed t-test was applied. P < 0.05 was regarded as significant.

3. Results

3.1. Stimulation of the adenylyl cyclase The concentrations of the stimulating agents (10 -4 M for GTP, GppNHp and forskolin; 10 -2 M for Mn 2÷) had been shown in preliminary concentration-response studies to yield maximum stimulation of the enzyme (data not shown). The ratios of GTP-, GppNHp-, forskolin-, Mn 2+- and Mn2÷+ forskolin-stimulated to basal activity (-fold stimulation) in control tissue were 1.2, 2, 10, 7 and 11.5, respectively. The AC activities in hippocampi and cerebella of A D / S D A T - and control patients obtained in the stimulatory experiments are summarized in Table 2. No significant differences were observed between the two brain regions investigated. The data show a consistent reduction of AC activity in brains of A D / S D A T - p a tients compared to non-demented control subjects. The

2~

Table 2 AC activities in brain tissue from A D / S D A T patients and controls

Hippocampus: Basal GTP GppNHp Forskolin Mn 2+ Mn 2. + Forskolin Cerebellum: Basal GTP GppNHp Forskolin Mn 2+ Mn 2+ + Forskolin

Controls

AI)/SDAT

(n = 7)

(it = 8 )

J

133± 14 159_+ 20 259_+ 42 1375_+173 959_+ 76 1523_+130

71± 11 84+ 12 131_+ 21 966+-211 768_+ 69 1283+200

--46.6 ÷: 47.2 * -49.4" 2%L7 - 19.9" 15.8

158_+ 16 192_+ 25 322_+ 57 1 601 +_ 168 1105+-126 1 813 _+ 151

68+_ t4 85+_ 18 123_+ 30 1045 _+295 877_+ 67 1 287_+ 188

-57.0* -55.7 * -61.8 * - 34.7 -20.6* - 29.0 *

A C activity is expressed as pmol c A M P / m g / m i n . Values represent means-+ S.E.M. The concentrations of the stimulating agents were 10 4 M for GTP, G p p N H p and forskolin and 10 -2 M for Mn 2+. The right column shows the amount of disease related reduction of AC activity (A %). * = P < 0.05 ( A D / S D A T vs. controls).

reduction was more pronounced under basal conditions and when stimulating the AC via Gs (mean values in hippocamus and cerebellum being 47.7 and 58.2%, respectively) than after direct activation of the catalytic subunit with forskolin or Mn 2÷ (mean values in hippocamus and cerebellum being 21.8 and 28.1%, respectively); see Table 1.

3. 2. Inhibition of the adenylyl cyclase When using a slightly modified incubation buffer for the inhibitory experiments [20], we again found the marked reduction of AC activity in hippocampus and cerebellum of patients with A D / S D A T compared to non-demented control subjects. Mean values of the Al-mediated inhibition of AC activity under 'basal' conditions (i.e., in the presence GTP 10 -5 M) as well as under addition of forskolin 10 -5 M are given in Table 3. The CHA-mediated inhibition varied between approx. 20% of the basal activity and approx. 30% in the presence of forskotin (the exact % inhibition values are included in Table 3). There were neither significant differences in the amount of CHA-mediated enzyme inhibition between the two brain regions nor between the A D / S D A T and the control group. Although the CHA mediated inhibition of AC activity tended to be more pronounced in brain tissue from A D / S D A T patients than in control subjects this tendency did not reach statistical significance. C o n c e n t r a t i o n - r e s p o n s e curves for C H A w e r e p e r f o r m e d in t h e p r e s e n c e o f forskolin 10 -5 M. T h e m e a n

cAMP values with the corresponding fitted curves are shown in Fig. 1 (hippocampi) and Fig. 2 (cerebella).

A. Schneeko et al./Brain Research 644 (19941 291-296

294

Table 3 CHA-mediated inhibition of AC in brain tissue from A D / S D A T patients and controls Controls (n = 6) Hippocampus: Basal + ( ' H A ll) ~ M Forskolin + C H A 10 " M Cerebellum: Basal + C t t A 10 ~M Forskolin + C H A II) 4 M

E .... (%)

AD/SDAT (n = 7)

90157_+ 137+ 703 + 527+

18 16 115 76

185+ 149+858 + 664+

27 27 85 63

88± 68± 448 + 315+

-12.7 -25.11 #

v

17 13 85 56

-22.7 #

-22.6 #

100

-311.3 #

0

controls

•

AD/SDA]

l

CL

3s 80

70

' '"'"'I

10 9

' ''""I

10 ~

''

''"1

10 '

Cyclohexylodenosine

' ''""'1

1 0 -6

70-

-31.5 #

90-

'/'/'"'"i

<(

80-

109+ 18 76+ 11 650 +- 119 445+- 67

-19.5 #

n

-29.7 #

The curves demonstrate that C H A inhibited AC activity in a concentration dependent manner. The inhibitory effect of C H A was significant in the two brain regions of both controls and A D / S D A T (Friedman, P < 0.05). The ECs~-values in hippocampal membranes for the control group and for the A D / S D A T group were 5.7. 10 s M and 2.1.10 7 M, respectively. The ECs~rvalues in cerebellar membranes were 1.7. 10 -6 M for controls and 1.6' 10 6 M for A D / S D A T . The ECs~rvalues for C H A in the hippocampus are lower by one order of potency when compared to cerebellar tissue. Hill-coefficients were less than unity (except for A D / S D A T cerebella) indicating an inhomogenous receptor population (0.56 and 0.60 for control hippocampi and cerebella; 0.69 and 0.99 for A D / S D A T

0

AD/SDAT

E ..... (%)

AC activity is expressed as pmol c A M P / m g / m i n . Values represent means ± S.E.M. CHA mediated inhibition of AC was studied under 'basal" conditions and after stimulation with 10 5 M forskolin. 111-5 M GTP was present in all assays. # = P < 0.05 (CHA mediated effect).

''"'"1

controls

I00

........

1 0 -5

I

10 •

' ''"'"'

1 0 -s

(tool/I)

Fig. 1. CtlA-mediated inhibition of AC in hippocampi from A D / S D A T (e) and non-demented control patients (o). AC activity was determined in the presence of 10 s M GTP and 10 5 M forskolin. Data points represent mean values±S.E.M, of seven A D / S D A T and six control hippocampi.

t

0

1 0 .9

1 0 -~

10 7

Cyclohexylodenosine

10-6

10-~

10-'

1 0 -~

(tool/' 0

Fig. 2. CHA-mediated inhibition of AC in cerebella from A D / S D A T (o) and non-demented control patients (o). AC activity was determined in the presence of 10 5 M GTP and 10 5 M forskolin. Data points represent mean values+S.E.M, of six A D / S D A T and six control cerebella.

hippocampi and cerebella). The maximum C H A mediated inhibition of AC was 22.0% in hippocampi and 21.9% in cerebella of the control group and 28.3% and 28.1% in the respective brain regions of the A D / S D A T group. These Emax-values differ slightly from the corresponding data in Table 3, since they were calculated from the fitted curves.

4. Discussion

In the present study we developed an experimental procedure with which we could stimulate as well as inhibit the AC in the same m e m b r a n e homogenate of hippocampal and cerebellar tissue samples of A D / SDAT- and control patients. We found a consistent reduction of AC activity under basal conditions as well as after stimulation with GTP, G p p N H p , forskolin and Mn 2+ in hippocampus and cerebellum of A D / S D A T patients when compared to non-demented control patients. The reduced AC activity in A D / S D A T after stimulation via Gs as well as after direct activation of the enzyme could be due to a loss of either Gs-proteins a n d / o r catalytic subunits in the A D / S D A T brain. Experimental data on quantitative changes of G-proteins seem to rule out a loss of Gs-proteins. McLaughlin et al. [27,34] using immunoblotting techniques reported on unchanged levels of several different G a subunits, whereas Harrison et al. [ 18] by applying in-situ hybridisation techniques even found slightly elevated levels of G s a m R N A in brain tissue of A D / S D A T patients. Quantitative changes of the AC itself have not yet been reported. Therefore, the reduction of AC activity in A D / S D A T brains after direct activation of

A. Schnecko el at. /Brain Research 044 (1994) 291-29t}

the enzyme could be explained not only by a functional impairment of the AC but also by a quantitative loss of catalytic subunits. Since, however, the reduction of AC activity in A D / S D A T was more pronounced after stimulation via G-proteins than after direct activation of the catalytic subunit with Mn 2+ (and forskolin) the coupling of Gs to AC must be disturbed by the disease. These data are in good agreement with results reported by Cowburn et al. who - - by applying different experimental conditions - - also found an impaired coupling of Gs-proteins to the AC [9]. It could be speculated that the increase in G s a m R N A reported by Harrison et al. [18] represents a response by A D / S D A T neurons to an abnormality in Gs protein coupling. In contrast to other groups our results demonstrate a consistent reduction of basal AC activity, the extent of the reduction being similar to that after G-protein mediated stimulation of the AC. Since we use a very crude membrane preparation one could imagine that under these conditions the G-proteins remain closely coupled to the AC. 'Basal' AC activity would - - at least to a certain extent - - also reflect the influence of G-proteins. To our surprise basal and stimulated AC activity was reduced to a similar extent in hippocampus and cerebellum of A D / S D A T patients. Until recently it has been generally accepted that the limbic system is histopathologically severely affected in A D / S D A T whereas the cerebellum was thought to be relatively free of abnormal protein deposits. However, there is now growing evidence that the cerebellum is not completely free of histopathological changes [8,21,22]. Several groups have been able to demonstrate amyloid deposits in cerebellar tissue of A D / S D A T patients whereas no neurofibrillary changes seem to occur in this brain region [4]. Since it has been reported that AC activity in hippocampi of A D / S D A T patients is negatively correlated with the amount of amyloid found in this region but not with neuritic plaques, neurofibrillary tangles or neuropil threads [31] the reduction of AC activity in cerebella of A D / S D A T patients is not so surprising after all. Thus, there is now good evidence that the cerebellum cannot be regarded as an internal control tissue for biochemical investigations in A D / S D A T and that the cerebellum seems to be more severely affected by the disease than may have been assumed so far. Since the coupling of neuronal inhibitory receptors appears to be more stable during membrane preparation than the coupling of stimulatory receptors [39] we could investigate adenosine Al-receptor mediated inhibition of AC activity. In control tissue CHA inhibited basal AC activity by about 20% and pre-stimulated AC activity by about 30%. These data are in good agreement with the

ua

results obtained in rat brain tissue [3,15,35]. The effect was concentration dependent (see Figs. I and 2) and could also be demonstrated in brain tissue from A D / S D A T patients. Hill-coefficients of the concentration-response curves were below 1. This fact is not particularly surprising as we expected both tissues (hippocampus, cerebellum) to contain A2-receptors which are positively coupled to the AC [15]. The maximum inhibition (Em~) was identical in hippocampus and cerebellum within the control as welt as the A D / S D A T group. There was a tendency, however, towards a greater CHA mediated inhibition in both brain regions from A D / S D A T patients when compared to control subjects. The results of these inhibitory experiments indicate that in contrast to Gs- the Gi-coupling to the AC is preserved in brain tissue of A D / S D A T patients. Interestingly, a similar pattern of disease related changes seems to occur at the level of receptor G-protein interactions. Binding studies have revealed that in A D / S D A T receptors which stimulate the AC uncouple from Gs [11,13] whereas the coupling of inhibitory receptors to Gi remains intact [2,28,29,40]. Thus, there is now growing evidence that Atzheimer's disease leads to an overall impairment of the stimulatory cAMP dependent signal transduction pathway whereas the entire inhibitory pathway seems to be preserved.

Acknowledgements This work was supported by grants of the Deutsche Forschungsgemeinschaft. Part of this work was presented at the joint meeting of the 'Deutsche Gesellschaft ffir Pharmakologie und Toxikologie' and the 'Association Fran~aise des Pharmacologistes' in Lille, 1993 [36].

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