Progression of clinical deterioration and pathological changes in patients with Alzheimer disease evaluated at biopsy and autopsy.

OBJECTIVES: To quantify the progression of senile plaques, neurofibrillary tangles, cerebral amyloid angiopathy, and microglial activation in the cortex and white matter of patients with Alzheimer disease evaluated at both biopsy and subsequent autopsy and correlate these changes with the progression of neurologic impairment. SETTING: Academic referral center for patient with Alzheimer disease. PATIENTS: Four patients meeting the clinical criteria for Alzheimer disease, enrolled in a pilot study for the evaluation of response to intracerebroventricular administration of bethanechol chloride. The patients were followed up until death occurred and autopsy was performed. RESULTS: All 4 patients had progressive deterioration from the time of biopsy to autopsy (9-11 years). Pathological investigations showed a striking increase in the density of senile plaques and neurofibrillary tangles in 2 of 4 patients from biopsy to autopsy, and a significant increase in microglial activation in 1 of 4 cases. Severity of cerebral amyloid angiopathy varied significantly among patients, 1 of whom displayed striking amyloid deposition with associated subcortical white matter atrophy. CONCLUSIONS: These unique data demonstrate that the progressive neurologic impairment in Alzheimer disease is accompanied by a significant increase in senile plaque and neurofibrillary tangle counts in the frontal cortex and, possibly in some patients, by increased microglial cell activation. Cerebral amyloid angiopathy was associated with significant white matter disease.

bcl-2 expression decreases methyl mercury-induced free-radical generation and cell killing in a neural cell line.

Methyl mercury neurotoxicity is associated with a broad range of neuropathologic and biochemical disturbances which include induction of oxidative injury. Treatment of the hypothalamic neural cell line GT1-7 with 10 microM methyl mercury (MeHg) for 3 h resulted in increased formation of reactive oxygen species (ROS), and decreased levels of reduced glutathione (GSH), associated with 20% cell death. Cells transfected with an expression construct for the anti-apoptotic proto-oncogene, bcl-2, displayed attenuated ROS induction and negligible cell death. Twenty-four-h exposure to 5 microM MeHg killed 56% of control cells, but only 19% of bcl-2-transfected cells. By using diethyl maleate to deplete cells of GSH, we demonstrate that the differential sensitivity to MeHg was not due solely to intrinsically different GSH levels. The data suggest that MeHg-mediated cell killing correlates more closely with ROS generation than with GSH levels and that bcl-2 protects MeHg-treated cells by suppressing ROS generation.

Rapid cell death induced by methyl mercury in suspension of cerebellar granule neurons.

We have further investigated the cytotoxicity of methyl mercury (MeHg) in cerebellar granule neurons isolated from 5-12-day-old rats. At 20 microM MeHg adenosine triphosphate (ATP) levels were reduced to 30% of control within 15 minutes and 1% of control at three hours (h), while cell viability assayed by trypan blue exclusion was reduced to approximately 80% and 20% of control, respectively. When potassium cyanide (KCN) was used to reduce ATP levels greater than 95%, virtually no change in cell viability was observed during three h incubation. Potassium cyanide combined with cycloheximide and actinomycin D to inhibit ATP and macromolecule synthesis simultaneously caused substantially less cell death than that produced by MeHg. Comparable rates of cell death were obtained when the free-radical generating system, hypoxanthine plus xanthine oxidase, was included with KCN in the incubation. Murine hybridoma MHY206 cells, representing a non-neuronal cell type, were less sensitive to cell killing by MeHg compared to granule neurons at equivalent cell protein concentrations. A three h exposure to 20 microM MeHg resulted in the death of 96% of the granule neurons while only 27% of the hybridoma cells were permeable to trypan blue. The results suggest that additional cytotoxic mechanisms beyond perturbations of the main metabolic pathways are involved in the neurotoxic mechanism of action of MeHg in cerebellar granule neurons. The results also indicate that oxidative or free-radical-generating systems are capable of reproducing the temporal pattern of neuronal cell destruction manifested by MeHg.

Abnormalities of peripheral nerve in patients with human immunodeficiency virus infection.

The purpose of this study was to assess systematically morphology of peripheral nerves from patients with human immunodeficiency virus infection (acquired immunodeficiency syndrome [AIDS] and AIDS-related complex) examined at autopsy. Sural nerve specimens were taken from 25 patients (mean age 44 years) and evaluated by routine procedures used in our laboratory. In 13 cases no detectable abnormality was seen. Twelve patients (48%) showed loss of myelinated fibers with disproportionately greater loss of large myelinated fibers. Three of these patients showed severe myelinated fiber loss; 2 had no documented symptoms and no other known predisposing factors for a peripheral neuropathy. Changes suggestive of wallerian degeneration were occasionally seen, as were epineurial and endoneurial inflammatory infiltrates. Segmental demyelination was not identified in any nerve examined. Electron microscopy revealed thickened basement membranes around small blood vessels, Schwann cells, and fibroblasts. Peripheral nerve abnormalities in patients with AIDS or ARC are frequent and their pathogenesis remains unclear.

Arginine metabolism and urea synthesis in cultured rat skeletal muscle cells.

Skeletal muscle is known to contain arginase, but, because this enzyme is also present in erythrocytes, the exact origin of arginine-derived ornithine in peripheral tissues is uncertain. In the present studies, skeletal muscle cells obtained from regenerating hindlimb muscle of adult rats were grown in primary tissue culture for approximately 3 wk and then studied in regard to changes in medium amino acid concentrations over a 48-h period. The consumption of arginine and serine was observed in parallel with the production of ornithine, proline, citrulline, glycine, and urea. Medium threonine and methionine concentrations were relatively constant over 48 h. Incubation of muscle cells with [U-14C]arginine resulted in the formation of [14C]ornithine and [14C]proline at rates at least 10-fold greater than could be accounted for by enzyme constituents of fetal calf serum. In addition, [guanido-14C]arginine was converted to [14C]urea and [U-14C]serine was converted to [14C]glycine. These studies indicate that cultured skeletal muscle cells contain a high arginase capacity and actively synthesize ornithine and urea from arginine.

Effects of clofibric acid on amino acid metabolism in cultured rat skeletal muscle.

Well-differentiated cultured skeletal muscle cells (myotubes) obtained from adult rats were incubated for up to 48 h in Dulbecco's modified Eagle's medium. Medium glucose decreased from 4.9 +/- 0.1 mM at 0 h to 13 +/- 1 microM by 24 h; approximately 60% of glucose was converted to lactate. Pyruvate, alanine, and citrate were continuously produced, even during the period of 24-48 h when no glucose or lactate utilization was observed. Branched-chain amino acid utilization increased more than fourfold during the incubation period of 24-48 h; during this time, intracellular ATP, pyruvate, alpha-ketoglutarate, malate, and citrate levels were constant despite the absence of glucose or lactate consumption. Incubation of muscle cells with 2 mM clofibric acid resulted in a 76% inhibition of leucine metabolism. Coincident with the drug-induced inhibition of a branched-chain amino acid utilization, alanine and citrate production was blocked, and cell levels of pyruvate, alpha-ketoglutarate, malate, and citrate were markedly reduced. These studies suggest branched-chain amino acids contribute significantly to anaplerotic pathways in cultured skeletal muscle cells and that these pathways lead to the net production of alanine and citrate during periods of minimal carbohydrate utilization.

Branched chain amino acid oxidation in cultured rat skeletal muscle cells. Selective inhibition by clofibric acid.

Leucine metabolism in skeletal muscle is linked to protein turnover. Since clofibrate is known both to cause myopathy and to decrease muscle protein content, the present investigations were designed to examine the effects of acute clofibrate treatment on leucine oxidation. Rat skeletal muscle cells in tissue culture were used in these studies because cultivated skeletal muscle cells, like muscle in vivo, have been shown to actively utilize branched chain amino acids and to produce alanine. The conversion of [1-(14)C]leucine to (14)CO(2) or to the [1-(14)C]keto-acid of leucine (alpha-keto-isocaproate) was linear for at least 2 h of incubation; the production of (14)CO(2) from [1-(14)C]leucine was saturable with a K(m) = 6.3 mM and a maximum oxidation rate (V(max)) = 31 nmol/mg protein per 120 min. Clofibric acid selectively inhibited the oxidation of [1-(14)C]leucine (K(i) = 0.85 mM) and [U-(14)C]isoleucine, but had no effect on the oxidation of [U-(14)C]glutamate, -alanine, -lactate, or -palmitate. The inhibition of [1-(14)C]leucine oxidation by clofibrate was also observed in the rat quarter-diaphragm preparation. Clofibrate primarily inhibited the production of (14)CO(2) and had relatively little effect on the production of [1-(14)C]keto-acid of leucine. A physiological concentration-3.0 g/100 ml-of albumin, which actively binds clofibric acid, inhibited but did not abolish the effects of a 2-mM concentration of clofibric acid on leucine oxidation. Clofibrate treatment stimulated the net consumption of pyruvate, and inhibited the net production of alanine. The drug also increased the cytosolic NADH/NAD(+) ratio as reflected by an increase in the lactate/pyruvate ratio, in association with a decrease in cell aspartate levels. The changes in pyruvate metabolism and cell redox state induced by the drug were delayed compared with the nearly immediate inhibition of leucine oxidation. These studies suggest that clofibric acid, in concentrations that approximate high therapeutic levels of the drug, selectively inhibits branched chain amino acid oxidation, possibly at the level of the branched chain keto-acid dehydrogenase.