John M. Candy

Molecular Forms of Acetylcholinesterase and Butyrylcholinesterase in the Aged Human Central Nervous System

The distributions of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) molecular forms and their solubility characteristics were examined, using density gradient centrifugation, in various regions of the postmortem human CNS. Total AChE activity varied extensively (50‐fold) among the regions investigated, being highest in the telencephalic subcortical structures (caudate nucleus and nucleus of Meynert); intermediate in the substantia nigra, cerebellum, and spinal cord; and least in the fornix and cortical regions (hippocampus and temporal and parietal cortex). Total BChE activity was, in contrast, much more evenly distributed, with only a threefold variation between the regions studied. Although the patterns of molecular forms of each enzyme were broadly similar among the different areas, regional variations in the distribution and abundance of the various forms of AChE were much greater than those of BChE. Thus, although the tetrameric G4 form of AChE constituted the majority of the total AChE activity in all regions examined, the ratio of the G4 form to the monomeric Gl form, the latter of which constituted the majority of the remaining activity, varied markedly, ranging from 21 in the caudate nucleus to 1.7 in the tem poral cortex. In addition to the G4 and Gl forms of AChE, the dimeric G2 form was observed in the nucleus of Meynert and a fast‐sedimenting (16S) species was found in samples of both the parietal cortex and spinal cord. In contrast, the G4 and Gl forms of BChE were the only molecular species observed in the different areas and the G4.G1 ratio varied from 3.3 in the substantia nigra to 0.9 in the temporal cortex. Regarding the solubility characteristics of the individual AChE and BChE molecular forms, the majority of the G4 form of AChE was extractable only in the presence of detergent, indicating a predominantly membrane‐bound localization of this species. The smaller AChE forms (Gl and G2) and both the Gl and G4 forms of BChE were all relatively evenly distributed between soluble and membrane‐bound species. These findings are discussed in relation to neurochemical and neuroanatomical, particularly cholinergic, features of the regions examined.

Alzheimer's-disease-like changes in tau protein processing: association with aluminium accumulation in brains of renal dialysis patients

Tau protein is a major structural protein of the paired helical filaments (PHFs) found in both neuritic senile plaques and neurofibrillary tangles in Alzheimers disease (AD). Senile plaques also contain amyloid beta protein (A beta). We did an immunochemical analysis of frontal cortex from 15 dialysis cases, 5 Alzheimers disease patients, and 6 control cases to see whether AD-like changes in A beta deposition and tau protein were linked to aluminium accumulation. Dialysis patients were used because they are frequently exposed to increased levels of aluminium. 8 of the 15 dialysis cases had insoluble A beta, but there was no association between its presence and the accumulation of aluminium. However, we found AD-like changes in the processing of tau protein. In white matter, truncated tau protein in the PHF-core fraction and endogenously truncated tau in the supernatant fraction were both increased in association with aluminium accumulation in the brain. In grey matter, normal tau protein was depleted and insoluble hyperphosphorylated tau increased in association with aluminium concentration. Protease-resistant PHFs were present in grey matter in 2 dialysis cases, a frequency above that expected for AD in this age group. PHF-core tau in both grey and white matter correlated with decreased levels of normal tau protein in white matter. These findings are consistent with a role for aluminium in the development of AD-like pathology in patients subjected to prolonged aluminium exposure.

Iron, selenium and glutathione peroxidase activity are elevated in sporadic motor neuron disease.

Oxygen free radical damage is strongly implicated in the pathogenesis of familial motor neuron disease (MND) associated with mutation of the Cu/Zn superoxide dismutase gene, and may be relevant in sporadic MND. Selenium (Se) and iron (Fe) have important roles in free radical metabolism. Using neutron activation analysis we have demonstrated significant elevation of Se and Fe in lumbar spinal cord in MND cases (38) compared to controls (22). Analysis of enzymes involved in free radical scavenging showed a significant and specific increase in the activity of the selenoprotein enzyme glutathione peroxidase in MND spinal cord.

Brain iron homeostasis

The anatomical and cellular distribution of non-haem iron, ferritin, transferrin, and the transferrin receptor have been studied in postmortem human brain and these studies, together with data on the uptake and transport of labeled iron, by the rat brain, have been used to elucidate the role of iron and other metal ions in certain neurological disorders. High levels of non-haem iron, mainly in the form of ferritin, are found in the extrapyramidal system, associated predominantly with glial cells. In contrast to non-haem iron, the density of transferrin receptors is highest in cortical and brainstem structures and appears to relate to the iron requirement of neurones for mitochondrial respiratory activity. Transferrin is synthesized within the brain by oligodendrocytes and the choroid plexus, and is present in neurones, consistent with receptor mediated uptake. The uptake of iron into the brain appears to be by a two-stage process involving initial deposition of iron in the brain capillary endothelium by serum transferrin, and subsequent transfer of iron to brain-derived transferrin and transport within the brain to sites with a high transferrin receptor density. A second, as yet unidentified mechanism, may be involved in the transfer of iron from neurones possessing transferrin receptors to sites of storage in glial cells in the extrapyramidal system. The distribution of iron and the transferrin receptor may be of relevance to iron-induced free radical formation and selective neuronal vulnerability in neurodegenerative disorders.

The quantitative autoradiographic distribution of [3H]MK-801 binding sites in the normal human spinal cord

The distribution of NMDA receptors in the normal human spinal cord has been investigated using the non-competitive channel blocking agent MK-801. Specific [3H]MK-801 binding was present throughout the spinal grey matter at all segmental levels, the greatest density of binding being found in the substantia gelatinosa. Focal areas of high binding were also found in a distribution corresponding to lower motor neurones in the ventral horns. This study provides anatomical evidence that NMDA receptors are likely to be important in motor as well as sensory spinal synaptic transmission. The anatomical distribution of NMDA receptors in relation to motor neurone somata may have important implications in selective vulnerability to excitotoxic injury.

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: Alzheimers disease, Parkinsons disease, Downs syndrome, alcoholic dementia, Huntingtons chorea, and motor‐neurone disease. A moderate decrease in the density of both high‐affinity pirenzepine and high‐affinity agonist subtypes was found in Alzheimers 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 Alzheimers disease and nondemented parkinsonian cases were highly significant. As previously reported, the levels of both choline acetyltransferase and acetylcholinesterase were markedly reduced in both Alzheimers disease and Parkinsons 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 Downs 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 Parkinsons and Alzheimers diseases may be related to the greater severity of dementia in the latter disease. Key Words: Muscarinic receptor subtypes—Human hippocampus—Alzheimers disease—Parkinsons disease —Downs syndrome. Smith C. J. et al. Muscarinic cholinergic receptor subtypes in hippocampus in human cognitive disorders.

Neuropathological diagnoses in elderly patients in Oslo: Alzheimer's disease, lewy body disease, vascular lesions

Neuropathological changes in elderly residents of Oslo, Norway were characterised with respect to the cerebral substrates of dementia. Ninety-two brains were examined, representing 41% of all deaths occurring in 10 nursing homes during a 9-month period. The autopsy cohort showed a similar mean age (85 years) and sex ratio (73% female) and proportion of demented patients (75%) compared to all the patients resident in these homes who died during the same period. Clinical data was compiled retrospectively. Diagnosis was made using the CERAD protocol, and criteria for the diagnosis of Lewy body dementia. Lewy body formation was present in 20% and cerebral infarction in 21% of patients. In the demented group (69 patients) 90% fulfilled CERAD criteria for definite or probable Alzheimers disease. Eight demented cases had absent neocortical neurofibrillary tangles and 6 other cases showed Lewy body dementia (9% of demented patients). A further 8 of these demented cases had brain stem Lewy bodies with only minimal cortical involvement. Thirteen cases (19% of the sample) had cerebral infarcts but these were considered to be clinically significant in only 4 (6%). In the non-demented patients (23) 4 patients had brain stem Lewy bodies and 6 had cerebral infarcts. Despite inclusion criteria biased towards the collection of Alzheimers disease and normal patients, both Lewy body dementia (7%) and cerebral infarcts contributing to dementia (6%) were frequent.

A neuronal basis for the alerting action of (+)‐amphetamine

1 (+)‐Amphetamine mimicked the excitatory and inhibitory actions of (—)‐noradrenaline on single neurones in the brain stem of acute halothane‐anaesthetized rats when these compounds were applied by iontophoresis. (+)‐Amphetamine had no actions on neurones unaffected by (—)‐noradrenaline. 2 These mimicking actions of (+)‐amphetamine could not be observed 20 h after treatment of the animals with reserpine 5 mg/kg. 3 The enzyme inhibitors α‐methyl‐p‐tyrosine and FLA 63 also greatly reduced the number of (—)‐noradrenaline‐mimicking responses to (+)‐amphetamine. 4 In animals pretreated with α‐methyl‐p‐tyrosine, but not in those pretreated with FLA 63, excitatory actions of (+)‐amphetamine on neurones excited by (—)‐noradrenaline could be elicited 45–90 min after systemic injection of l‐DOPA. 5 These results indicate that (+)‐amphetamine can release noradrenaline from presynaptic sites in the brain stem, which may be a basis for its alerting actions.

Actions of noradrenaline, other sympathomimetic amines and antagonists on neurones in the brain stem of the cat

1 The effects of (–)‐noradrenaline ((–)‐NA) and related compounds on brain stem neurones in decerebrate unanaesthetized cats have been investigated using the technique of iontophoretic application from micropipettes. 2 Four types of response to (–)‐NA have been described. These were short lasting inhibition, long lasting inhibition, excitation, and a biphasic response consisting of short lasting inhibition followed by excitation. A variable amount of desensitization of the excitatory response, but not of inhibitory responses, was observed. 3 Experiments in which small currents were used to pass (–)‐NA from pipettes with smaller tips did not lead to any appreciable change in the proportions of neurones excited or inhibited. 4 A variety of sympathomimetic agonists was tested. Short lasting inhibition was less sensitive than excitation to changes in molecular structure. Long lasting inhibition was more sensitive to molecular change and was not mimicked by some of the agonists which mimicked short lasting inhibition. 5 Although agonists without one ring hydroxyl had weaker effects than those with both, compounds in which both ring hydroxyl groups were absent (β‐hydroxyphenylethylamine, ephedrine and amphetamine) mimicked excitation strongly. It is possible that the compounds without both ring hydroxyl groups had some effect other than simple agonistic activity. 6 A dissociation was observed between responses to dopamine and (–)‐NA. p‐Tyramine mimicked dopamine, rather than (–)‐NA. 7 Neither the α‐agonist, phenylephrine nor the β‐agonist, isoprenaline mimicked neuronal responses to (–)‐NA. The α‐antagonists phentolamine and phenoxybenzamine and the β‐antagonists dichloroisoprenaline, propranolol and d(–)‐INPEA and combinations of propranolol with phentolamine or phenoxybenzamine were ineffective in blocking either excitation or inhibition. Thus, the central receptors appear to be different from peripheral α‐ and β‐receptors. 8 The most effective antagonist of excitation was (–)‐α‐methylnoradrenaline. Metaraminol and dihydroergotamine also had some antagonistic activity. None of the compounds tested blocked inhibition. The effects of (–)‐α‐methylnoradrenaline have been discussed in relation to the hypotensive action of α‐methyldopa.

Aluminium accumulation in relation to senile plaque and neurofibrillary tangle formation in the brains of patients with renal failure

The effects of long-term exposure to aluminium on the development of Alzheimer-type neuropathological changes have been studied post-mortem in patients with chronic renal failure who did not have dialysis encephalopathy. Administration of aluminium-containing phosphate binding compounds appears to be a major factor in the accumulation of aluminium in the brain of dialysis patients. The mean serum aluminium concentrations determined during life and brain aluminium concentrations determined post-mortem correlated with both the duration and total amount of aluminium hydroxide administered to these patients. No correlation was found between the presence of bone aluminium and either the mean serum or brain aluminium concentration. Longitudinal monitoring of serum aluminium concentrations may provide a more reliable index than bone biopsy of brain aluminium concentrations in dialysis patients. Dynamic secondary ion mass spectrometry revealed focal accumulations of aluminium associated with cortical pyramidal neurones. The majority of patients also showed immunostaining in pyramidal neurones with an antibody to the N-terminal region of the beta/A4 amyloid precursor protein, while staining was absent in age-matched control cases. One-third of the patients exhibited beta/A4-positive amorphous senile plaques in the cerebral cortex. However, there was no clear correlation between either the presence and intensity of beta/A4 amyloid precursor immunostaining or the presence of senile plaques and the concentration of aluminium in the cerebral cortex. Cortical neurofibrillary tangles were not observed in any of the dialysis patients. These data suggest that it is unlikely that aluminium plays any major role in neurofibrillary tangle formation and that its putative role in senile plaque formation is likely to be only part of a complex cascade of changes.