Andrew W. Procter

Cortical Pyramidal Neurone Loss May Cause Glutamatergic Hypoactivity and Cognitive Impairment in Alzheimer's Disease: Investigative and Therapeutic Perspectives

In the 1960s it became generally accepted that the cognitive impairment associated with old age was due to disorders with specific histological features rather than being an inevitable part of the aging process (see, e.g., Corsellis, 1962). Furthermore, two disorders appeared to account for the majority of cases of dementia amongst the elderly, one characterised by prominent disease of the cerebral vasculature and one with histological features similar to those described in a patient in her fifties by Alois Alzheimer early in the century. Alzheimer’s disease (AD) was therefore recognised as a major cause ofdementia, rather than a rare neurodegenerative disease giving rise to presenile dementia. This observation, coupled with the identification of the neurochemical pathology underlying Parkinson’s disease and the success of L-DOPA treatment following its introduction in 1968, set the scene for the systematic biochemical study ofdementia in old age with the hope of producing similarly dramatic treatments. The demonstration of substantial cholinergic abnormalities in the brains of patients with AD suggested a basis for such rational pharmacological treatments. However, cases have been reported that raise some doubts as to the validity of the view of AD as a primary disorder of the cholinergic system (Bowen et al., 1977). One subset of patients with dementia had typical neuropathological findings of AD, yet their cortical choline acetyltransferase (ChAT) activity was not selectively reduced (Palmer et al., 1986). Other demented patients with AD had normal numbers of cholinergic neurones in the nucleus basalis of Meynert (Perry et al., 1982; Pearson et al., 1983). A reduction in numbers of basal forebrain neurones and cortical ChAT specific activity of a magnitude similar to that seen in moderate to severe AD occurs in another neurodegenerative condition, olivopontocerebellar atrophy, yet cognitive impairment in this condition is not prominent (Kish et al., 1988). It appears likely that the neocortical cholinergic deficit in AD can explain only a part of the entire clinical syndrome. Since 1982 this group (Bowen, 1983) has focused much attention on the intrinsic neurones of the cerebral cortex. An extensive body of literature describes effects on learning and memory in humans exerted by lesions of the cerebral cortex and the hippocampus (Dudai, 1989). Experimental studies in animals have also sought to define the role of these structures in cognition. Lashley (as reviewed by Dudai, 1989) used conditioned rats and monkeys to perform various tasks, mechanically damaged the neocortex either before or after training, and then measured the effect of the lesions on acquisition and retention. He found that the amount of reduction in learning was dependent on the amount of neocortical tissue removed and, also, that the more complex the task, the greater the effect of the removal of neocortex. Studies have been extended to include the hippocampus and have also increased in subtlety by using excitotoxins, with analogous changes in behaviour (Francis et al., 1992~). The excitatory amino acids (EAA), glutamic (Glu) and aspartic acid, are the proposed transmitters of the cortical pyramidal cells and have been the subject of detailed studies in recent years. There is now strong evidence for an excitotoxic role of these amino acids in the pathogenesis of cerebral ischaemia (German0 et al.. 1987: Park et al., 1988: Sheardown et al., 1990).

Evidence of Glutamatergic Denervation and Possible Abnormal Metabolism in Alzheimer's Disease

Excitatory dicarboxylic amino acids previously have been ascribed several functions in the brain. Here their total concentration and proposed neurochemical markers of neurotransmitter function have been measured in brain from patients with Alzheimers disease (AD) and controls. Specimens were obtained antemortem (biopsy) approximately 3 years after emergence of symptoms and promptly (<3 h) postmortem some 10 years after onset. Early in the disease a slight elevation in aspartic acid concentration of cerebral cortex was observed in the patients with AD. A reduction in glutamic acid concentration of a similar magnitude was found. It is argued that this, together with a decrease in CSF glutamine content and lack of change in the phosphate‐activated brain glutaminase activity of tissue, reflects an early metabolic abnormality. Later in the disease evidence of glutamatergic neurone loss is provided by the finding that in many regions of the cerebral cortex the Na+‐dependent uptake of D‐[3H]aspartic acid was almost always lowest in AD subjects compared with control when assessed by a method designed to minimise artifacts and epiphenomena. Release of endogenous neuro‐transmitters from human brain tissue postmortem did not appear to have the characteristics of that from human tissue antemortem and rat brain.

Topographical distribution of neurochemical changes in Alzheimer's disease

Biochemical indices of cortical nerve cells affected in Alzheimers disease have been proposed (excitatory dicarboxylic amino acid, EDAA, sodium-dependent carrier; phosphate-activated glutaminase activity; serotonin type 2 recognition site; somatostatin-like immunoreactivity). These and the content of EDAAs and two related amino acids, and choline acetyltransferase (ChAT) activity have been measured in up to 13 areas of cerebral cortex and the cerebellar cortex from 16 patients with Alzheimers disease and 17 controls. Reduction of the index of the serotonin recognition site, somatostatin content and another biochemical index of interneurones coincide and indicate a rather unexpected focal loss of such neurones from the parietal lobe. No unequivocal measure of the integrity of pyramidal neurones could be established as the content of no amino acid was reduced, the index of the EDAA carrier showed evidence of change in few brain regions and glutaminase activity was subject to unexplained variability. ChAT activity alone closely paralleled a previous report of the distribution of morphological degeneration. The results are discussed in relation to therapy and positron emission tomography.

Excitatory amino acid-releasing and cholinergic neurones in Alzheimer's disease

Brains of normal controls and patients with primary degenerative dementia were investigated for choline acetyltransferase (ChAT) activity in the frontal, temporal and parietal cortex, hippocampus, amygdala and thalamus. A few patients with Alzheimers disease were unusual as the cholinergic marker was unaffected, except in the amygdala. Other patients with dementia and undiagnosed neurodegenerative disorder had elevated cortical ChAT activity. The interpretations offered are: (a) the syndrome of dementia and Alzheimer pathologic change precedes significant loss of cortical cholinergic innervation; (b) denervation in dementia can occur early in olfactory areas, exemplified here by the amygdala; (c) dementia is associated with the loss of non-cholinergic structure. An indication of structures involved was given by loss of a marker of excitatory amino acid-releasing neurones.

Reduced glycine stimulation of [3H]MK-801 binding in Alzheimer's disease.

The novel N‐methyl‐D‐aspartate recjeptor channel ligand (+)‐[3H]5‐methyl‐J0, l1‐dihydro‐5H‐dibenzo[a, d]‐cyclohepten‐5, 10‐imine maleate ([3H]MK‐801) has been utilized to label this receptor in human brain tissiie. Characteristics of [3H]MK‐801 binding to well‐washeq membranes from 17 control subjects and 16 patients with Alzheimers disease were determined in frontal, parietal, and temporal cerebral cortex and cerebellar cortex. In control tissue the pharmacological specificity of the binding of this substance is entirely consistent with the profile previously reported for rat brain. Binding could be stimulated by the addition of glutamic acid to the incubation medium; addition of glycine produced further enhancement which was not prevented by strychnine. The specificity of the effects of the and other amino acids on the binding was the same as in the rat. In Alzheimers disease significantly less binding Was observed in the frontal cortex under glutamate‐ and glycine‐stimulated conditions. This appears to be associated with a reduced affinity of the site whereas the pharmacological specificity of the site remained unchanged. The effect did not appear to be due to differences in mode of death between Alzheimers disease and control subjects and is unlikely to be related to factors for which the groups were matched. In contrast, binding was not altered in the absence of added amino acids and presence of glutamate alone. These results imply that in the cerebral cortex the agonist site and a site in the cation channel of the receptor are not selectively altered, but that their coupling to a strychnine‐insensitive glycine recognition site is impaired.

Brain amino acid concentrations and Ca2+-dependent release in intractable depression assessed antemortem

The concentrations of 3 putative neurotransmitters (glutamate, aspartate and gamma-aminobutyrate), 4 related amino acids and 5 non-transmitter-related amino acids have been measured in neurosurgical samples (frontal cortex) from patients with intractable depression and controls. In addition, the glutamate receptor agonist 2-amino-4-sulpho-butanoic acid (homocysteic acid) has been identified in human brain and measured in these samples. There were no changes in the concentrations of amino acids in depressed patients compared to control with the exception of aspartic and homocysteic acids which were elevated in a sub-group of patients with depression compared to control. The Ca2+-dependent release (K+-stimulated) of putative neurotransmitters has been demonstrated for the first time from brain tissue of depressed patients. Glutamate release was unaltered from the control value. Aspartate values showed unexplained variability but its release and that of gamma-aminobutyrate were elevated in some depressed subjects. These results do not support the hypothesis of reduced amino acid function in depressive illness.

D-cycloserine, a putative cognitive enhancer, facilitates activation of the N-methyl-D-aspartate receptor-ionophore complex in Alzheimer brain.

The action of D-Cycloserine (DCS) at the strychnine-insensitive glycine recognition site of the NMDA receptor-ionophore complex has been studied with membranes from inferior parietal cortex of patients with Alzheimers disease. The maximal response of the site, measured using [3H]MK-801 binding, was 64% of that observed with glycine. Stimulation of binding induced by DCS in the presence of fixed concentrations of glycine resulted in a family of dose-response curves, consistent with the antibiotic having the property of a partial agonist at this glycine site. It is proposed that because of circumscribed glutamatergic pyramidal cell pathology DCS will have benefit for Alzheimers patients over and above all other types of cognitive impairment.

Serotonergic Pathology Is Not Widespread in Alzheimer Patients Without Prominent Aggressive Symptoms

Behavioural symptoms of Alzheimers disease, such as aggression, may determine the care patients required. Most postmortem neurochemical studies have been of institutionalized patients and conclusions drawn from these may not be valid for all patients. We have shown that serotonin 2 receptors are not lost from 12 of the 13 areas of cerebral cortex examined in the patients assessed to be free of aggressive symptoms. This has been interpreted as representing the relative preservation of cortical interneurones. In contrast choline acetyltransferase activity was reduced in all areas whereas serotonin content was reduced in only 2 of the 4 areas examined.

Characterisation of the Glycine Modulatory Site of the N‐Methyl‐d‐Aspartate Receptor‐Ionophore Complex in Human Brain

Abstract: [3H]Glycine binding and glycine modulation of [3H]MK‐801 binding have been used to study the glycine allosteric site associated with the N‐methyl‐D‐aspartate receptor complex in postmortem human brain. The effect of glycine on [3H]MK‐801 binding appeared sensitive to duration of terminal coma, and possibly postmortem delay. Thirty percent of the binding occurred in a subfraction of brain tissue and did not show enhancement by glycine and glutamic acid. [3H]Glycine binding to a subfraction free from this component was studied and showed high specific binding. KD and Bmax values showed considerable intersubject variability which did not appear to be due to demographic features or to tissue content of amino acids with an affinity for this site. The pharmacological characteristics of binding in this subfraction and a correlation between Bmax values and the maximal enhancement of [3H]MK‐801 binding by glycine are consistent with [3H]glycine binding occurring to an N‐methyl‐D‐aspartate receptor complex associated site. Further support for this is provided by a significantly lower Bmax value for [3H]glycine binding in subjects with Alzheimers disease and reduced glycine enhancement of [3H]MK‐801 binding. However, the effect of perimortem factors makes it difficult to confidently attribute this solely to a disease‐related change in the receptor. The possible role of the glycine allosteric site in the treatment of neuropsychiatric disorders is discussed.

Postmortem brains reveal similar but not identical amyloid precursor protein-like immunoreactivity in Alzheimer compared with other dementias

Concentrations of amyloid precursor protein (APP)-like immunoreactivity (APPLIR) have been determined by Western blotting in a soluble fraction and two membrane fractions of two areas of brain cortex from patients with Alzheimers disease (AD) and other dementias. There were no significant differences between AD and other cases in species with the Kunitz protease inhibitor domain. However, the total soluble APPLIR was higher in AD and it was hypothesized that this relates to cholinergic hypoactivity.