Galanin-Like Immunoreactivity Is Unchanged in Alzheimer''s Disease and Parkinson''s Disease Dementia Cerebral Cortex

Galanin is a recently isolated neuropeptide that is of particular interest in dementing disorders because of its known colocalization with choline acetyltransferase in magnocellular neurons of the basal nucleus of Meynert. These neurons degenerate in Alzheimer's disease, and there is a corresponding deficiency of cortical choline acetyltransferase activity. In the present study, galanin-like immunoreactivity was measured in the postmortem cerebral cortex and hippocampus of 10 controls and 14 patients who had had Alzheimer's disease. Significant reductions of choline acetyltransferase activity (50-60%) were found in all regions examined; however, there was no significant effect on concentrations of galanin-like immunoreactivity. Similar measurements were made in postmortem tissues of 12 control and 13 demented Parkinsonian patients who had had Alzheimer-type cortical pathology. Choline acetyltransferase activity was again significantly decreased in all regions examined but there were no significant reductions in galanin-like immunoreactivity. Experimental lesions of the fornix in rats produced parallel significantly correlated reductions of both choline acetyltransferase activity and galanin-like immunoreactivity in the hippocampus. Galanin-like immunoreactivity in the human hypothalamus consisted of two molecular-weight species on gel-permeation chromatography, and two forms were resolved by reverse-phase HPLC. The paradoxical preservation of galanin-like immunoreactivity, despite depletion of the activity of choline acetyltransferase, with which it is colocalized, is as yet unexplained. Recent studies have shown that galanin inhibits both acetylcholine release in the hippocampus and memory acquisition; therefore, preserved galanin may exacerbate the cholinergic and cognitive deficits that accompany dementia.     

Differential Alteration of Various Cholinergic Markers in Cortical and Subcortical Regions of Human Brain in Alzheimer''s Disease

The main objective of the present study was to determine whether cholinergic markers (choline acetyltransferase activity and nicotinic and muscarinic receptors) are altered in Alzheimer's disease. Choline acetyltransferase activity in Alzheimer's brains was markedly reduced in various cortical areas, in the hippocampus, and in the nucleus basalis of Meynert. The maximal density of nicotinic sites, measured using the novel nicotinic radioligand N-[3H]methylcarbamylcholine, was decreased in cortical areas and hippocampus but not in subcortical regions. M1 muscarinic cholinergic receptor sites were assessed using [3H]pirenzepine as a selective ligand; [3H]pirenzepine binding parameters were not altered in most cortical and subcortical structures, although the density of sites was modestly increased in the hippocampus and striatum. Finally, M2-like muscarinic sites were studied using [3H]-acetylcholine, under muscarinic conditions. In contrast to M1 muscarinic sites, the maximal density of M2-like muscarinic sites was markedly reduced in all cortical areas and hippocampus but was not altered in subcortical structures. These findings reveal an apparently selective alteration in the densities of putative nicotinic and muscarinic M2, but not M1, receptor sites in cortical areas and in the hippocampus in Alzheimer's disease.     

Presynaptic Cholinergic Dysfunction in Patients with Dementia

Abstract: Indices of presynaptic cholinergic nerve endings were assayed in neocortical biopsy samples from patients with presenile dementia. For those patients in whom Alzheimer's disease was histologically confirmed, [¹⁴C]acetylcholine synthesis, choline acetyltransferase activity and choline uptake were all found to be markedly reduced (at least 40%) below mean control values. The changes occurred in samples from both the frontal and temporal lobes and for [¹⁴C]acetylcholine synthesis the decrease was similar under conditions of high and low neuronal activity (as assessed by incubations in 31 mM and 5 mM K⁺ respectively). Samples from other demented patients, in whom the histological features of Alzheimer's disease were not detected, produced values for all three biochemical parameters which were similar to controls. For the total group of patients with presenile dementia there were correlations between values for the three markers of presynaptic cholinergic nerve endings suggestive of a loss of functional activity at these sites in Alzheimer's disease.     

Is the neuronal basis of Alzheimer's disease cholinergic or glutamatergic?

The hypothesis that the symptomatology of Alzheimer's disease is attributable to cholinergic dysfunction is supported by postmortem studies that have demonstrated reduced choline acetyltransferase (ChAT) activity across all areas of cerebral cortex and diminished numbers of perikarya in the basal forebrain nucleus basalis of Meynert. Biopsy studies of ChAT activity, choline uptake, and acetylcholine synthesis also suggest that cholinergic denervation occurs relatively early in the course of the disease, and in confirmation of postmortem data, correlates with the severity of cognitive impairment. An alternative hypothesis to explain the dementia of Alzheimer's disease is the glutamatergic hypothesis. This is based largely on postmortem evidence indicating reduced binding and uptake of D[3H]aspartate, as well as loss of a number of other putative markers, such as phosphate-activated glutaminase activity, glutamate concentration, and the number of pyramidal cell perikarya, with this latter change correlating with the severity of dementia. Short-comings of each hypothesis are discussed and the merits of single neuron hypotheses to explain the dementia of Alzheimer's disease are considered.     

Characterization of l-[3H]Nicotine Binding in Human Cerebral Cortex: Comparison Between Alzheimer''s Disease and the Normal

Putative nicotine receptors in the human cerebral cortex were characterized with L-[3H]nicotine, L-[3H]Nicotine binding was enhanced by the addition of Ca2+ and abolished in the presence of Na3EDTA. Association and dissociation of the ligand were rapid at 25 degrees C with t1/2 values of 2 and 3 min, respectively. Saturation binding analysis revealed an apparent single class of sites with a dissociation constant of 5.6 nM and a Hill coefficient of 1.05. There was no effect of postmortem interval on the density of binding sites assayed up to 24 h in rat frontoparietal cortex. Nicotine binding in human cortical samples was also unaltered by increasing sampling delay. In human cortical membranes, binding site density decreased with normal aging. Receptor affinity and concentration in samples of frontal cortex (Brodmann area 10) from patients with Alzheimer's disease were comparable to age-matched control values. Samples of infratemporal cortex (Brodmann area 38) from patients with Alzheimer's disease had a 50% reduction in the number of L-[3H]nicotine sites. Choline acetyltransferase activity was significantly decreased in both cortical areas. Enzyme activities in the temporal pole were reduced to 20% of control values. These data indicate that postsynaptic nicotine receptors are spared in the frontal cortex in Alzheimer's disease. In the infratemporal cortex, significant numbers of receptors remain despite the severe reduction in choline acetyltransferase activity. Replacement therapy directed at these sites may be warranted in Alzheimer's disease.     

Comparative Alterations of Nicotinic and Muscarinic Binding Sites in Alzheimer''s and Parkinson''s Diseases

We have recently reported on the differential alterations of various cholinergic markers in cortical and subcortical regions in Alzheimer's disease (AD). The main purpose of the present study was to determine if cholinergic deficits observed in patients with AD are unique to this disorder or can be generalized to others such as idiopathic Parkinson's disease (PD) and PD with Alzheimer-type dementia (PD/AD). Muscarinic M1, M2, and nicotinic receptor binding parameters (KD and Bmax) were determined in various cortical and subcortical areas using selective radioligands ([3H]pirenzepine, [3H]AF-DX 116, and N[3H]methylcarbamylcholine). Choline acetyltransferase activity was also determined as a marker of the integrity of cholinergic innervation. Alterations of cholinergic markers are comparable in cortical areas in AD, PD, and PD/AD brains. In frontal and temporal cortices, as well as in the hippocampus, choline acetyltransferase activity and binding capacities of M2 and nicotinic binding sites are similarly decreased in these three disorders compared with age-matched control values. M1 receptor binding parameters are not significantly modified in cortical areas in patients with these disorders. In contrast, important differences between AD and PD brain tissues are found in subcortical areas such as the striatum and the thalamus. The density of M1 sites is significantly increased in striatal areas only in patients with AD, whereas densities of nicotinic sites are decreased in thalamus and striatum in PD and PD/AD, but not AD, brain tissues. The binding capacity of M2 sites is apparently unchanged in subcortical areas in all three disorders, although tendencies toward reductions are observed in the striatum of PD and PD/AD patients. Thus, although comparable alterations of various cholinergic markers are observed in cortical areas in the three neurological disorders investigated in the present study, important differences are seen in subcortical areas. This may be relevant to the respective etiological and clinical profiles of AD and PD.     

Choline acetyltransferase activity at different ages in brain of Ts65Dn mice, an animal model for Down's syndrome and related neurodegenerative diseases

Ts65Dn mice, trisomic for a portion of chromosome 16 segmentally homologous to human chromosome 21, are an animal model for Down's syndrome and related neurodegenerative diseases, such as dementia of the Alzheimer type. In these mice, cognitive deficits and alterations in number of basal forebrain cholinergic neurons have been described. We have measured in Ts65Dn mice the catalytic activity of the cholinergic marker, choline acetyltransferase (ChAT), as well as the activity of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), in the hippocampus and in cortical targets of basal forebrain cholinergic neurons. In mice aged 10 months, ChAT activity was significantly higher in Ts65Dn mice, compared to 2N animals, in the hippocampus, olfactory bulb, olfactory cortex, pre-frontal cortex, but not in other neocortical regions. At 19 months of age, on the other hand, no differences in ChAT activity were found. Thus, alterations of ChAT activity in these forebrain areas seem to recapitulate those recently described in patients scored as cases of mild cognitive impairment or mild Alzheimer's disease. Other neurochemical markers putatively associated with the disease progression, such as those implicating astrocytic hyperactivity and overproduction of amyloid precursor protein family, were preferentially found altered in some brain regions at the oldest age examined (19 months).     

A method for parallel determination of choline acetyltransferase and muscarinic cholinergic receptors: Application in aged-rat brain

We have devised a method for the parallel determination of choline acetyltransferase (CAT) and muscarinic cholinergic receptor (mCh-R) in the same brain tissue. The method for CAT activity determination is more rapid, simplified, stable, and economical than the usual Fonnum's method. With our method, age-associated changes in CAT activity and mCh-R levels were examined. Although CAT activity hardly changed with age except in a few areas, mCh-R binding of aged-rats was markedly reduced in all areas. These results suggest that the change in mCh-R represents an age-associated biochemical change in the brain and that determination of CAT activity is not sufficient for the study of age-associated changes in the brain cholinergic system.     

Muscarinic Cholinergic Receptor Subtypes in Hippocampus in Human Cognitive Disorders

Total muscarinic receptor levels, the levels of the subtypes exhibiting high and low affinity for pirenzepine, and the high- and low-affinity agonist states of the receptor were investigated in hippocampal tissue obtained at autopsy from mentally normal individuals and the following pathological groups: Alzheimer's disease, Parkinson's disease, Down's syndrome, alcoholic dementia, Huntington's chorea, and motor-neurone disease. A moderate decrease in the density of both high-affinity pirenzepine and high-affinity agonist subtypes was found in Alzheimer's disease, whereas a trend towards an increase in the overall muscarinic receptor density was apparent in the parkinsonian patients without dementia, mainly due to an increase in the low-affinity agonist state; the differences between the Alzheimer's disease and nondemented parkinsonian cases were highly significant. As previously reported, the levels of both choline acetyltransferase and acetylcholinesterase were markedly reduced in both Alzheimer's disease and Parkinson's disease--with a greater loss of both enzymes in the demented subgroup of parkinsonian patients. Activities of the cholinergic enzymes were also extensively reduced in Down's syndrome, accompanied by a loss of high-affinity pirenzepine binding. There were no significant receptor or enzyme alterations in the other groups studied. These observations suggest that in the human brain, extensive degeneration of cholinergic axons to the hippocampus, as indicated by a loss of cholinergic enzymes, is not necessarily accompanied by extensive muscarinic receptor abnormalities (as might be expected if a major subpopulation were presynaptic). Moreover, the opposite changes in muscarinic binding in Parkinson's and Alzheimer's diseases may be related to the greater severity of dementia in the latter disease.     

Reciprocal Regulation of Ornithine Decarboxylase and Choline Kinase Activities by Their Respective Reaction Products in the Developing Rat Cerebellar Cortex

Changes in the activity of choline kinase were measured in the cerebellum during development. Early transient increase was found in the enzyme activity just prior to and during birth. This period of increase did not coincide with the periods of transient elevation in ornithine decarboxylase and choline acetyltransferase previously observed in the developing cerebellum. The effects of the naturally occurring polyamines (putrescine, spermidine, and spermine) on choline kinase and choline acetyltransferase activities, and of phosphorylcholine (the product of the reaction catalyzed by choline kinase) on ornithine decarboxylase and choline acetyltransferase activities, were also examined. Choline acetyltransferase activity was not influenced by either polyamines or phosphorylcholine. However, choline kinase activity from 7-day-old, but not from adult, cerebellum was increased 25% in the presence of 4 mM spermine. In contrast, low spermidine concentrations (less than 2 mM) inhibited choline kinase activity selectively in 7-day-old cerebellum. Ornithine decarboxylase activity from 7-day-old cerebellum was inhibited in a concentration-dependent manner by phosphorylcholine. The present data together with other previous reports suggest that: (a) polyamines may play a role in choline utilization during development via their regulation of choline kinase activity, on the one hand, and of acetylcholinesterase activity on the other; and (b) during development, a reciprocal regulation of choline kinase and ornithine decarboxylase activities by their respective reaction products may exist, whereby choline kinase activity is regulated in a complex manner by polyamines and, in turn, ornithine decarboxylase is inhibited by phosphorylcholine.     

Presynaptic Serotonergic Markers in Community-Acquired Cases of Alzheimer's Disease: Correlations with Depression and Neuroleptic Medication

Abstract: Presynaptic serotonergic markers, serotonin uptake sites, and concentrations of serotonin and 5-hydroxyindoleacetic acid were studied in the frontal and temporal cortex of 20 community-acquired cases of Alzheimer's disease and 16 controls matched for age, sex, postmortem delay, and storage. Clinical assessments, including behavioural symptoms, of the Alzheimer patients were made at 4-month intervals during life. There was a significant reduction in the number of serotonin uptake sites in Alzheimer cases in temporal but not frontal cortex. There was no significant alteration in the concentrations of serotonin or 5-hydroxyindoleacetic acid in either region. Alzheimer patients who had persistent depressive symptoms during life had significantly fewer serotonin uptake sites in both cortical areas compared with Alzheimer patients without these symptoms. In addition, Alzheimer patients who were receiving chronic neuroleptic medication had significantly lower concentrations of serotonin in frontal cortex and 5-hydroxyindoleacetic acid in temporal cortex than those patients not receiving such treatment. These data suggest previous studies that reported uniform serotonergic dysfunction may have been subject to unintentional selection of behaviourally disturbed Alzheimer cases or those receiving chronic neuroleptic medication. This study also provides a basis for the treatment of behaviourally disturbed Alzheimer patients with serotonomimetics.     

Changes of Activity and Isozyme Pattern of Phosphofructokinase in the Brains of Patients with Alzheimer's Disease

Abstract: A severe reduction of the in vivo cerebral glucose consumption rate is generally found in patients with Alzheimer's disease. In postmortem studies changes in the activities of key regulatory glycolytic enzymes, including 6-phosphofructokinase (PFK), have been reported in Alzheimer's disease brains, but the results obtained so far are inconsistent and controversial. We reevaluated the activity of PFK in brain tissue from clinically and neuropathologically confirmed cases of Alzheimer's disease using optimized tissue disintegration and assay methods and determined the PFK isozyme pattern. PFK activity in brains from patients with Alzheimer's disease was significantly increased in frontal and temporal cortex and unchanged in the other brain areas studied when compared with control brains. All three PFK isozymes were detected in each of the brain areas studied. In brains of Alzheimer's disease patients the level of the C-type PFK was slightly reduced at the expense of the M- and L-type subunits. The data presented do not support the results of other groups, which reported up to a 90% reduction of PFK activity in Alzheimer's disease. In contrast, the data presented clearly rule out the suggestion that changes of PFK activity might be one of the causes for the reduced glucose consumption in Alzheimer's disease brains.     

Evidence for the extreme overestimation of choline acetyltransferase in human sperm, human seminal plasma and rat heart: a case of mistaking carnitine acetyltransferase for choline acetyltransferase

Detection of choline acetyltransferase (ChAc) in a number of non-neuronal tissues has been extremely overestimated. There are two major types of errors encountered. Type 1 error occurs when endogenous substrates (e.g. L-carnitine) are acetylated by acetyltransferase enzymes (e.g. carnitine acetyltransferase ( CarAc ) ) yielding an acetylated product mistaken for acetylcholine (AcCh). In the past, human sperm and human seminal plasma putative ChAc activity has been extremely overestimated due to Type 1 error. This study demonstrates (1) an endogenous acetyltransferase and substrate activity in human sperm and human seminal plasma forming an acetylated product that is not AcCh but probably acetylcarnitine ( AcCar ); (2) that the addition of 5 mM choline substrate does not significantly increase acetyltransferase activity; (3) that boiled seminal plasma contains an endogenous acetyltransferase substrate which is not choline, but probably L-carnitine. Type 2 error occurs when endogenous carnitine acetyltransferase synthesizes true AcCh, resulting in mistaken evidence for ChAc. This is demonstrated by the fact that the choline substrate Km-value for the neuronal or true ChAc from mouse brain is 0.73 +/- 0.06 mM while the Km-value of choline substrate for purified CarAc from pigeon breast muscle is 108 +/- 4 mM. Type 2 error has occurred for the estimation of putative ChAc in rat heart. The rat heart ChAc was measured in previous studies utilizing a concentration of 30 mM choline substrate. While saturation of neuronal ChAc is observed at 2-5 mM choline, saturation of the rat heart CarAc enzyme is not reached until over 800 mM. Purified CarAc significantly synthesizes AcCh at 30 mM choline. Thus, putative ChAc has been greatly overestimated in the scientific literature for mammalian sperm, human seminal plasma and rat heart.     

Postnatal Development of the Acetylcholine System in Different Parts of the Rat Cerebellum

Abstract: The components of the cholinergic nervous system, i.e., choline acetyltransferase, acetylcholinesterase, sodium-dependent high-affinity choline uptake, acetylcholine, and the muscarinic acetylcholine receptors, in the developing archi- and paleocerebellum of the rat have been investigated by biochemical methods. A close correlation between the development of the different elements of the system has been demonstrated in the two areas. The cholinergic structure develops first in the archicerebellum, which displays high levels of choline acetyltransferase, acetylcholinesterase, acetylcholine, and sodium-dependent high-affinity choline uptake. The paleocerebellum receives a sparser cholinergic innervation during development. The differences in the values for these components in the cerebellum as a whole may reflect the development of cholinergic and noncholinergic neuronal structures. It is concluded that the development of the cholinergic system cannot be analyzed in the cerebellum as a whole; rather specific regions such as the archi-, paleo-, or neocerebellum must be examined.     

Projections and actions of tachykininergic, cholinergic, and serotonergic neurones in the intestine of the Atlantic cod

The native tachykinins cod neurokinin A and cod substance P, serotonin and acetylcholine have excitatory effects on the circular smooth muscle of the cod intestine. Furthermore, immunoreactivities to the cod tachykinins, serotonin and two markers for cholinergic neurones, viz. choline acetyltransferase and vesicular acetylcholine transporter, have been demonstrated in myenteric neurones of the cod intestine. In order to elucidate whether the neurones containing these substances project orally and thus might be involved in the ascending excitatory reflex of peristalsis, myotomy operations have been performed on the cod intestine. The immunoreactive areas of the myenteric plexus immediately oral and anal to the myotomy operations have been measured by using confocal laser scanning microscopy. Large accumulations of immunoreactivity to the tachykinins are found on the anal side of the myotomies, indicating oral projections of tachykininergic neurones. The areas immunoreactive to serotonin and choline acetyltransferase are of equal size on the oral and anal sides. Since the tachykinin containing neurones of the intestine project orally, and since cod neurokinin A and cod substance P have excitatory effects on circular smooth muscle, we conclude that tachykininergic neurones are involved in the ascending excitatory reflex of peristalsis in the cod intestine. Received: 6 March 1997 / Accepted: 15 September 1997     

Identification of a mutation in the CHAT gene of Old Danish Pointing Dogs affected with congenital myasthenic syndrome

The presence of a recessive inherited muscle disease in Old Danish Pointing Dogs has been well known for years. Comparisons of this disease with myasthenic diseases of other dog breeds and humans have pointed toward a defect in the synthesis of the neurotransmitter acetylcholine possibly due to decreased activity of the enzyme choline acetyltransferase. We sequenced exons 5-18 of the gene encoding choline acetyltransferase (CHAT) in 2 affected and 2 unaffected dogs and identified a G to A missense mutation in exon 6. The mutation causes a valine to methionine substitution and segregates in agreement with the inheritance of the disease. The mutation was not detected in 50 dogs representing 25 other dog breeds. A DNA test has been developed and is now available to the breeders of Old Danish Pointing Dogs.     

Cholinergic development in chick optic tectum and retina reaggregated cell cultures

The developmental profiles of acetylcholinesterase and choline acetyltransferase in chick optic tectum and retina cell aggregates, over a 30-day period, have been determined and compared with the corresponding developmental curves obtained in vivo. Both acetylcholinesterase and choline acetyltransferase activities in retina cell aggregates and the acetylcholinesterase activity in optic tectum cell aggregates usually lie between 40 and 90% of the values measured in vivo for the same cell (tissue) type and developmental age. However, the choline acetyltransferase activity in tectum aggregates increases only during the first 7 days of culture, and then decreases to reach a low value of 8% of that measured in vivo, by day 24. This fact, which is associated with widespread degeneration and cell death, could be attributed to the condition of natural deafferentiation occurring in a tectum cell aggregate. A parallel has been drawn between this behavior of a tectum cell aggregate and the effect of early embryonic eye removal on the development of the contralateral optic tectum in vivo. Thus, the tectum may have a biphasic pattern of development, with an autonomous period of growth of about 2 wk, followed by an afference-dependent phase, while the retina behaves, from a cholinergic point of view, as a relatively self-sufficient structure.Abbreviations AChE acetylcholinesterase - ChAT choline acetyltransferase - ACh acetylcholine - BW284 C51 dibromide 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide     

Choline acetyltransferase.

Acetylcholine is essential to neural function. It synthesis is catalyzed by choline acetyltransferase, the enzyme responsible for the acetylation of choline by acetyl coenzye A, a reaction favored slightly thermodymodynamically and not at all kinetically. An analytically pure enzyme still has not been obtained; however, method of purification have been greatly improved recently. Numerous inhibitors of the enzyme have been synthesized and their structure-action relationships examained. Evidence has been accumulated showing the essential involvement of an imidazole group in the active site of choline acetyltransferase. The literature regarding the controversial role to thiol groups in choline acetyltransferase is reviewed. Recently, derivatives of coenzyme A have been introduced as inhibitors of this enzyme and the specificity of coenzyme A binding has been examined. Possible mechanisms responsible for the control fo acetylcholine synthesis are discussed.     

Intralaminar Neurochemical Distributions in Human Midtemporal Cortex: Comparison Between Alzheimer''s Disease and the Normal

The intralaminar distributions of transmitter and nontransmitter enzyme activities and amino acid levels were determined in the midtemporal cortices from normal individuals and established cases of Alzheimer's disease. In the normal, choline acetyltransferase (CAT) and acetylcholinesterase (AChE) activities were relatively high in the outer cortical layers, particularly, for CAT, in the two granular layers (II and IV). Both activities were reduced in Alzheimer's disease at all, although generally most extensively in the outer and middle layers of the grey matter whereas activities were near normal in the white matter. Further, the enzyme distribution patterns of these cholinergic activities were also disrupted in Alzheimer's disease and the activity of CAT throughout the cortex was generally reduced to that found in the white matter. No such differences in distribution were found for two other enzymes, pseudocholinesterase and lactate dehydrogenase. Assessment of the gamma-aminobutyric acid (GABA) system in the normal revealed a much more extensive intralaminar variation in the enzyme, glutamate decarboxylase, compared with the level of GABA itself. In contrast with the cholinergic enzymes, neither the levels nor intralaminar patterns of GABA were altered in Alzheimer's disease. From an analysis of free amino acids at the different cortical levels, the cortical pattern of glutamic acid in the normal was different from that for GABA, aspartic acid, or nontransmitter amino acids such as alanine. Neither of the putative amino acids, glutamate or aspartate, was altered in Alzheimer's disease. These findings demonstrate the relatively selective nature of microchemical changes occurring in the cortex in Alzheimer's disease and suggest that a functional abnormality in cholinergic input to the outer neocortical layers (I-IV) with predominantly receptive and associative functions may be an important feature of the disease.