Cholinergic deficit in Alzheimer's disease: A study based on CSF and autopsy data

Cholinesterase (ChE) activity was measured as a possible marker of cholinergic neurotransmission of the brain in CSF of 93 patients with probable Alzheimer's disease/senile dementia of the Alzheimer type (AD/SDAT) and of 29 control patients. ChE activity in CSF was decreased significantly in the AD/SDAT patients as compared to the controls. This reduction correlated significantly with the various measures of the severity of dementia. However, the reduction of ChE activity was only moderate (25–30%) even in patients with the most severe dementia and nonsignificant in patients with early symptoms of AD/SDAT. The significance of various confounding factors, which may interfere with CSF ChE measurements is discussed. Our findings seem to indicate that the deficiency of cholinergic neurons is not directly reflected in CSF and that the measurements of ChE activities in CSF are not helpful in diagnosing AD/SDAT. In the autopsy study the activities of cholineacetyltransferase (ChAT) and ChE were determined for ten brain areas of 20 AD/SDAT patients and of 14 controls. In AD/SDAT patients ChAT activity was profoundly decreased (50–85% decrease) in the cortical areas and hippocampus, but was unchanged or only mildly reduced in other subcortical brain areas. This study further confirms that the affection of cholinergic neurons is limited to projections from nucleus basalis to cortex and hippocampus, whereas other cholinergic neurons, like in striatum, seem to be relatively spared. In general, the activities of ChAT and ChE were lower in Alzheimer patients dying at younger age suggesting more severe disease process with these patients.     

Cortical Substance P-Like Immunoreactivity in Cases of Alzheimer's Disease and Senile Dementia of the Alzheimer Type

Abstract: The concentration of substance P-like immunoreactive material (SPLI) and somatostatin-like immunoreactive material (SLI) and the activity of acetyl-CoA: choline- O -acetyltransferase (ChAT; EC 2.3.1.6.) were measured in eight brain regions of 13 normal patients and 12 patients with Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT). SPLI was significantly lower in five of eight regions in the patients with AD/SDAT. Younger patients with AD/SDAT had significantly lower SLI in the parietal cortex than older patients. ChAT activity and SPLI in the parietal cortex of the presenile patients with ADISDAT were not significantly different from values found in older patients.     

Influence of Region-Specific Alterations of Neuropeptidase Content on the Catabolic Fates of Neuropeptides in Alzheimer's Disease

Abstract: We established the cartography of 11 exo- and endopeptidases in the frontal and parietal cortices and in the cerebellum of brains of patients diagnosed with a senile dementia of the Alzheimer's type (SDAT). Comparison with those of four subjects who had died without known neurologic or psychiatric illness indicated that there existed a region-specific alteration of the peptidase contents in the disease. In the frontal area of SDAT brains, postproline dipeptidyl aminopeptidase and aminopeptidase M activities were significantly reduced. In the parietal cortex of SDAT brain, activities of three additional endopeptidases—angiotensin-converting enzyme, proline endopeptidase, and endopeptidase 24.15—were also drastically reduced. In contrast, the cerebellum displayed a set of proteolytic activities that remained unaffected in SDAT brain. The putative influence of the disease on the catabolic fates of neurotensin, neuropeptide Y, and somatostatin(1–14) was investigated. Neurotensin was catabolized at identical rates in the frontal and parietal cortices in nondemented and SDAT brains. In contrast, neuropeptide Y metabolism was slowed down in SDAT brains in the frontal but not in the parietal cortex. Finally, the degradation velocities of somatostatin(1–14) were lowered in both cortical areas of SDAT brains. It is interesting that, by means of specific peptidase inhibitors, we demonstrated that endopeptidase 24.15 participated in somatostatin(1–14) inactivation in the parietal but not in the frontal cortex. It is suggested that the lowering of the rate of somatostatin(1–14) inactivation in the parietal cortex of SDAT brains likely results from the depletion of endopeptidase 24.15 in this brain region.     

Tyrosine Hydroxylase, Tryptophan Hydroxylase, Biopterin, and Neopterin in the Brains of Normal Controls and Patients with Senile Dementia of Alzheimer Type

The activities of tyrosine hydroxylase and tryptophan hydroxylase, and the concentrations of the biopterin cofactor and the precursor neopterin were measured in 14 regions of postmortem brains from four histologically verified patients of senile dementia of the Alzheimer type (SDAT) and eight histologically normal controls. Neopterin concentrations were measured in the human brain for the first time. The activities of tyrosine hydroxylase and tryptophan hydroxylase in the brains of patients with SDAT were significantly reduced in the substantia nigra and in the lateral segment of the globus pallidus, locus ceruleus, and substantia nigra, respectively. The concentrations of total biopterin in the brains of patients with SDAT were significantly reduced in the putamen and substantia nigra, but the total neopterin concentrations did not change significantly. These results suggest that the reduction in biogenic amines in SDAT might be related to reductions in biosynthetic enzymes associated with biogenic amines, due to destruction of monoaminergic neurons.     

In vivo studies of the effect of magnetic field exposure on ontogeny of choline acetyltransferase in the rat brain

Developmental increases of the activity of choline acetyltransferase (ChAT) were examined in the brains of fetuses and offspring from parent rats continuously exposed to a 500 mG, 60 Hz circularly polarized (CP) magnetic field (MF) prior to pregnancy, and further, during pregnancy and lactation. In developing rats between 12 days and 20 days of embryogenesis that were housed in a control unit, i.e., nonexposed to MF, the specific activity of ChAT in whole brain specimens increased from 2.4% to 6.9% of adult activity, while specific activity of ChAT in rat brain specimens between 12 days of embryogenesis and 10 days of postpartum increased from 2.4% to 21.6% of adult activity. On the other hand, the specific activity of ChAT in whole brain specimens from rats under housed MF exposure conditions was found to increase from 2.6% to 6.7% of adult activity between 12 days and 20 days of embryogenesis and from 2.6% to 21.6% of adult activity between 12 days of embryogenesis and 10 days postpartum. Furthermore, the effect of the same test magnetic environment on the specific activity of ChAT in the brains of parental rats was examined in order to determine whether magnetic field exposure of parental rats might reflect onto the development of fetal brain. It was observed that continuous exposure of parental rats to a 500 mG, 60 Hz CP MF did not show any significant changes in the specific activity of ChAT in the septodiagonal band complex, dorsal and ventral hippocampus, striatum, and frontoparietal cerebral cortex, as compared with the same brain regions of control subjects. As far as the development of cholinergic neural circuits was concerned, these test magnetic environments did not interfere in their development and it suggested that parental-fetal intercommunication might provide relatively stable states for neural development, even under these test magnetic environments. © 1993 Wiley-Liss. Inc.     

微波辐射对小鼠脑内乙酰胆碱含量及胆碱乙酸转移酶活性的影响

用频率为2450MHz功率密度为10mW/Cm~2(WBASAR约11.4W/kg)的微波(连续波)对置于微波暗室内的昆明种雄性小鼠急性全身照射1小时后,立即按常规方法断头,取脑,制成样品,然后用放射免疫测定法测量小鼠脑内乙酰胆碱(ACh)含量及胆碱乙酰转移酶(ChAT)活性。结果表明:照射组的ACh含量为11.6±1.4pmol/mg(脑鲜重),ChAT活性为45.4±8.7pmolACh/min.mg(脑鲜重);而对照组的分别为16.0±2.1pmol/mg和61.0±13.8pmolACh/min.mg。证明微波照射后可引起动物脑内ACh水平和ChAT活性下降,提示微波辐射对中枢胆碱能系统确有不利影响。     

A Search for Discrete Cholinergic Nuclei in the Human Ventral Forebrain

Abstract: Slices cut from five frozen human brains were dissected into 2-mm cubes and assayed for choline acetyltransferase (ChAT) activity and protein content. A pattern of enrichment of ChAT activity was found ventral to the anterior commissure; this finding is consistent with the location of the enzyme in the cells of the nucleus basalis of Meynert. The region beneath the anterior commissure was the only place a discrete enrichment of activity could be found, and the precise topography of the enrichment was somewhat variable from brain to brain. The results are discussed in the light of recent knowledge concerning the source of the cortical cholinergic innervation.     

A Modified Hortega-Globus Stain is Superior to Bielschowsky and Bodian Stains for Demonstrating Neuritic Plaques

The quantitative assessment of the age-dependent number of neuritic plaques is essential for the diagnosis of Alzheimer type dementia. This study reports the superiority of a modified Hortega-Globus stain compared to Bielschowsky and Bodian stains applied to samples obtained from ten brains of patients with a clinical history of progressive dementia. In two of ten cases only the modified Hortega-Globus stain allowed confirmation of the diagnosis of senile dementia of the Alzheimer type (SDAT). The counts of neuritic plaques in sections stained by other methods were not sufficient to establish the histological diagnosis of SDAT. These results indicate that the choice of the most sensitive staining method is critical for the correct histopathologic diagnosis of the Alzheimer type dementia.     

Effects of Aging and Amyloid-β Peptides on Choline Acetyltransferase Activity in Rat Brain

Choline acetyltransferase (ChAT, acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6), involved in the learning and memory processes is responsible for the synthesis of acetylcholine. There are many discrepancies in literature concerning ChAT activity during brain aging and the role of amyloid beta peptides in modulation of this enzyme. The aim of the study was to investigate the mechanism of ChAT regulation and age-related alteration of ChAT activity in different parts of the brain. Moreover the effect of A peptides on ChAT activity in adult and aged brain was investigated. The enzyme activity was determined in the brain cortex, hippocampus and striatum in adult (4-months-old), adult-aged (14-months-old) and aged (24-months-old) animals. The highest ChAT activity was observed in the striatum. We found that inhibitors of protein kinase C, A, G and phosphatase A2 have no effect on ChAT activity and that this enzyme is not dependent on calcium ions. About 70% of the total ChAT activity is present in the cytosol. Arachidonic acid significantly inhibited cytosolic form of this enzyme. In the brain cortex and striatum from aged brain ChAT activity is inhibited by 50% and 37%, respectively. The aggregated form of A 25-35 decreased significantly ChAT activity only in the aged striatum and exerted inhibitory effect on this enzyme in adult, however, statistically insignificant. ChAT activity in the striatum was diminished after exposure to 1 mM H₂O₂. The results from our study indicate that aging processes play a major role in inhibition of ChAT activity and that this enzyme in striatum is selectively sensitive for amyloid beta peptides.     

Down''s Syndrome Individuals Begin Life with Normal Levels of Brain Cholinergic Markers

We measured the activities of the cholinergic marker enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in autopsied brains of seven infants (age range 3 months to 1 year) with Down's syndrome (DS), a disorder in which virtually all individuals will develop by middle age the neuropathological changes of Alzheimer's disease accompanied by a marked brain cholinergic reduction. When compared with age-matched controls cholinergic enzyme activity was normal in all brain regions of the individuals with infant DS with the exception of above-normal activity in the putamen (ChAT) and the occipital cortex (AChE). Our neurochemical observations suggest that DS individuals begin life with a normal complement of brain cholinergic neurons. This opens the possibility of early therapeutic intervention to prevent the development of brain cholinergic changes in patients with DS.     

Effect of Unilateral Decortication on Choline Acetyltransferase Activity in the Nucleus Basalis and Other Areas of the Rat Brain

Acetyl-coenzyme A: choline O-acetyltransferase (EC 2.3.1.6) (ChAT) enzyme activity was measured in the nucleus basalis and other microscopically identified brain areas at various times after unilateral cortical lesions were made in the rat. Initially, a significant decrease in ChAT activity was detected in the nucleus basalis ipsilateral to the lesion. However, after 120 days ChAT activity had apparently recovered, as levels of the enzyme at that time were not significantly different from control values. No changes in ChAT activity could be detected in any of the other brain areas similarly studied. The significance of these findings and their relationship to the morphological changes seen in neurones of the nucleus basalis after cortical lesions are discussed.     

Distributions of choline acetyltransferase and acetylcholinesterase activities in layers of rat superior colliculus

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were assayed in samples dissected from sagittal sections through rat superior colliculus. The magnitude of ChAT activity was about half to equal that found in rat whole brain in all layers except stratum griseum intermediale, where the average activity was higher than whole brain. AChE activity was three to four times that found in rat whole brain in superficial layers and about the same as average brain in deeper layers, except in the statum griseum intermediale, where the average activity was about twice whole brain. Rostral-caudal gradients in both ChAT and AChE activities occurred in stratum griseum intermediale, with activities in the caudal region of some animals as high as four times those in the rostral. ChAT activity in samples associated with locations of patches or spots of AChE staining product in stratum griseum intermediale was significantly higher than in samples from "nonpatch" regions. Results are discussed relative to inputs into the colliculus, whose terminations may correlate in location with the distributions of the enzyme activities.     

Correlative Regulation of Nerve Growth Factor Level and Choline Acetyltransferase Activity by Thyroxine in Particular Regions of Infant Rat Brain

Abstract: Effects of thyroxine (T₄) on nerve growth factor (NGF) level and choline acetyltransferase (ChAT) activity of rat brains were investigated. Repetitive intraperitoneal administration of T₄ caused increases in both NGF level and ChAT activity in the frontal cortex, septum, hippocampus, and striatum and decreases in the cerebellum in 2-day-old rats. Only ChAT activity was elevated in the olfactory bulb, and the NGF level remained unchanged there. No changes were observed in the midbrain and pons/medulla. Furthermore, T₄ was effective on the post-natal rats only up to day 11. These results suggest that T₄ plays a role in the developmental regulation of NGF level and ChAT activity in rat brain in a region- and/or stage-specific manner. That (1) changes in NGF level and ChAT activity occurred in regions nearly identical to those that contained NGF-responding neurons, and (2) the change in NGF level in the hippocampus and frontal cortex was followed by the change of ChAT activity after a single injection of T₄ suggest that the effects of T₄ on cholinergic differentiation are, at least in part, mediated via NGF, which itself is quantitatively regulated by T₄.     

125I-Galanin Binding Sites in Alzheimer's Disease: Increases in Hippocampal Subfields and a Decrease in the Caudate Nucleus

Abstract: Using iodinated human galanin and autoradiography, galanin binding sites were studied in cortical and hippocampal areas and in some brainstem nuclei in the brains of eight patients with senile dementia of the Alzheimer type (SDAT) and of nine matched control cases. The highest density of binding sites was found in the substantia nigra with a less intense labeling in the hippocampus and cortical regions. In the SDAT cases a significant increase in number of galanin binding sites was found in some hippocampal areas, a decrease in the caudate nucleus, and no significant changes in frontal and entorhinal cortices. These findings suggest that some central galanin systems may be deranged in SDAT.     

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.     

Developmental Differences Between Soluble and Membrane-Bound Fractions of Choline-O-Acetyltransferase in Neonatal Mouse Brain

Three fractions (one soluble and two membrane-bound) of choline acetyltransferase (ChAT) isolated from a nerve ending fraction of mouse forebrain, which have previously been reported to differ in several biochemical and physical aspects, were also found to differ in their rates of postnatal development. At 2 days of age, the activity in all three fractions was very low. Sodium phosphate buffer-soluble (cytoplasmic) ChAT activity increased significantly by 8 days of age, whereas the ChAT activity of the two membrane-bound fractions (NaCl- and Triton-soluble) did not increase until 13 days of age. These results suggested that the differences observed between the three fractions of ChAT prepared from mouse brain are not solely artifacts of the isolation procedure.     

Developmental changes in choline acetyltransferase and glutamate decarboxylase activity in various regions of the brain of the male,female, and neonatally androgenized female rat

In an attempt to discern effects of sex hormones on the development of neurotransmitter systems in the rat brain, choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) have been measured at postnatal days 8, 12, 25, and 60 in five regions (amygdala, anterior hypothalamus, hippocampus, olfactory bulbs, and cerebral cortex) of the brains of normal male, normal female, and neonatally androgen-treated female rats. Essentially no associations between sex or of neonatal androgenization on either enzyme were found. The data, however, provide new information on the relative rates of development of ChAT and GAD in five regions of the rat brain which supplement the limited information already available in the literature. ChAT activity was highest in amygdala and hypothalamus, but developed most rapidly in hippocampus and cerebral cortex. The relative activities and patterns of development of GAD activity were similar to those of ChAT.     

Chromatographic separation of reaction products from the choline acetyltransferase and carnitine acetyltransferase assay: differential ChAT and CrAT activity in brain extracts from Alzheimer's disease versus controls

Choline acetyltransferase (ChAT) catalyzes the reaction between choline and acetylcoenzyme A (AcCoA) to form acetylcholine (ACh) in nerve terminals. ACh metabolism has implications in numerous aspects of physiology and varied disease states, such as Alzheimer's disease. Therefore a specific, sensitive, and reliable method for detecting ChAT enzyme activity is of great utility in a number of situations. Using an existing radionuclide-based enzyme activity assay, we have observed detectable ChAT signals from non-cholinergic cells, suggesting a contaminant in the assay producing an artifactual signal. Previous reports have suggested that L-acetylcarnitine (LAC) contaminates many assays of ChAT activity, because of difficulties in separating LAC from ACh by organic extraction. To determine the source of this hypothesized artifact and to rectify the problem, we have developed a paper chromatography-based assay for the detection of acetylcholine and other contaminating reaction products of this assay, including LAC. Our first goal was to develop a simple and economical method for resolving and verifying the identities of various reaction products or contaminants that could be performed in most laboratories without specialized equipment. Our second goal was to apply this separation method in postmortem human brain tissue samples. Our assay successfully detected several contaminants, especially in assays using brain tissue, and allowed the separation of the intended ACh product from these contaminants. We further demonstrate that this assay can be used to measure carnitine acetyltransferase (CrAT) activity in the same samples, and assays comparing ChAT and CrAT show that CrAT is highly active in neuronal tissues and in neuronal cell cultures relative to ChAT. Thus, the simple chromatography-based assay we describe allows the measurement of specific reaction products separated from contaminants using commonly available and inexpensive materials. Further, we show that ChAT activity is significantly reduced in brain extracts from Alzheimer's disease compared to controls.     

Effects of corticosterone on brain cholinergic enzymes in chick embryos

The effects of corticosterone on the cholinergic enzymes, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied in the chick embryonic brain. Chick embryos received either 0.25, 0.5, or 1.0 g of corticosterone via the air sac daily for three days during either embryonic days 6 through 8 (E6-E8), of cerebral neurogenesis, or days 10 through 12 (E10-E12), a period of cerebellar neurogenesis. Enzyme activities were determined in cerebral hemispheres, optic lobes, cerebellum and remaining brain at 10, 15, and 20 days of incubation. In embryos treated from E6 to E8, ChAT activity was generally higher at day 10 in cerebral hemispheres and optic lobes (cerebellum was not determined) while AChE activity was not affected. At day 20 ChAT activity of treated chick embryos was lower in the cerebral hemispheres and optic lobes, but not in the cerebellum; AChE activity was higher in the cerebral hemispheres, lower in the optic lobes, and not changed in the cerebellum as compared to controls. However, in embryos treated from E10 to E12 both cerebellar ChAT and AChE activities were higher at day 15 in comparison to controls. These data show that the hormonal effects were most prominent only in the brain areas undergoing neurogenesis during the period of hormonal treatment. Since AChE activity is also present in nonneuronal cells, the observed alterations caused by corticosterone may reflect glial cell responses to the hormone. Whether the hormone affects the final number and/or maturation of cholinergic neurons and/or glial cells remain to be investigated.