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Lewy bodies, the pathological hallmark of dementia with Lewy bodies (DLB), are large juxtanuclear inclusions of aggregated alpha-synuclein. However, the small number of cortical Lewy bodies relative to the total neuron count does not correlate with the extent of cognitive impairment. In contrast to dopaminergic neurons in Parkinson's disease, nerve cell loss is usually less prevalent in the cortex of DLB, suggesting a different mechanism of neurodegeneration. Because antibodies used for immunodetection per se do not generally differentiate the aggregated from the physiological and monomeric isoform of alpha-synuclein, we developed the paraffin-embedded tissue (PET) blot and the protein aggregate filtration (PAF) assay for the sensitive and selective detection of alpha-synuclein aggregates in tissue slides and brain homogenates, respectively. In contrast to common immunohistochemistry, the PET blot detected an enormous number of small alpha-synuclein aggregates, which, in contrast to the few Lewy bodies, may explain the cognitive impairment in DLB. Using the PAF assay, we demonstrate that the absolute majority of alpha-synuclein aggregates are located at presynaptic terminals, suggesting a severe pathological impact on synaptic function. Indeed, parallel to the massive presynaptic accumulation of alpha-synuclein aggregates, we observed significant synaptic pathology with almost complete loss of dendritic spines at the postsynaptic area. Our results provide strong evidence for a novel concept of neurodegeneration for DLB in which synaptic dysfunction is caused by presynaptic accumulation of alpha-synuclein aggregates. This concept may also be valid for Parkinson's disease.

NMDA receptor hypofunction in schizophrenia has been inferred by a large number of clinical and preclinical observations; however, whether and how NMDA receptors are exactly involved in the pathogenesis of schizophrenia are still unknown and subject to interpretation. Here we show, in two independent samples of brains from patients with schizophrenia, a significant decrease in the phosphorylation level at serine 897 (S897) of the NMDA receptor type 1 (NR1) subunit. Our finding, together with a previous report that antipsychotics increase phosphorylation of NR1 at S897 in vivo, strongly suggests that insufficient phosphorylation at S897 may contribute to the neuronal pathology underlying schizophrenia.

It has been shown that cytoplasmic RNA oxidation occurs to a great extent in the brains of Alzheimer's disease (AD) patients. The goal of this study was to isolate and identify oxidized RNA species in AD. We show that significant amounts of poly(A)+ mRNAs are oxidized in AD brains. RNA oxidation is not random but highly selective. Importantly, many identified oxidized mRNA species have been implicated in the pathogenesis of AD. Quantitative analysis revealed that some mRNA species are more susceptible to oxidative damage. We also investigated the biological consequence of oxidatively damaged mRNAs by expressing them in cell lines. Our data indicated that abnormal processing of proteins occurred to the oxidized mRNAs. This may implicate the potential contribution of RNA oxidation in the pathogenesis of AD.

2-(4'-methylaminophenyl)benzothiazole (BTA-1) is an uncharged derivative of thioflavin-T that has high affinity for Abeta fibrils and shows very good brain entry and clearance. In this study, we asked whether BTA-1, at concentrations typical of those achieved during positron emission tomography (PET) studies, could specifically bind to amyloid deposits in the complex milieu of human brain or whether amyloid binding was overshadowed by nonspecific binding, found even in brains that did not contain amyloid deposits. We quantitatively assessed [3H]BTA-1 binding to crude homogenates of postmortem brain obtained from nine Alzheimer's disease (AD) subjects, eight controls, and six subjects with non-AD dementia. BTA-1 binding was >10-fold higher in AD brain, and the majority (94%) of the binding was specific (displaceable). High-affinity [3H]BTA-1 was observed only in AD brain gray matter and was not present in control brain gray matter, AD brain white matter, or cerebellum. The K(d) of [3H]BTA-1 for binding to AD brain (5.8 +/- 0.90 nm) was very similar to the K(d) for binding to synthetic Abeta fibrils. In addition, the K(i) of various BTA analogs for inhibition of [3H]BTA-1 binding to AD brain homogenates was very similar to their K(i) for inhibition of [3H]BTA-1 binding to synthetic Abeta fibrils. Nanomolar concentrations of [3H]BTA-1 did not appear to bind to neurofibrillary tangles. Finally, BTA-1 did not appear to bind significantly to common neuroreceptors or transporter sites. These data suggest that the binding of BTA-1 to AD brain is dominated by a specific interaction with Abeta amyloid deposits.

Selective blockers of the norepinephrine transporter (NET) inhibit dopamine uptake in the prefrontal cortex. This suggests that dopamine in this region is normally cleared by the somewhat promiscuous NET. We have tested this hypothesis by comparing the effects of inhibitors selective for the three monoamine transporters with those of a nonspecific inhibitor, cocaine, on uptake of 3H-dopamine into synaptosomes from frontal cortex, caudate nucleus, and nucleus accumbens from wild-type, NET, and dopamine transporter (DAT) knock-out mice. Dopamine uptake was inhibited by cocaine and nisoxetine, but not by GBR12909, in frontal cortex synaptosomes from wild-type or DAT knock-out mice. At transporter-specific concentrations, nisoxetine and GBR12909 failed to block dopamine uptake into frontal cortex synaptosomes from NET knock-out mice. The efficacy of cocaine at the highest dose (1 mm) was normal in DAT knock-out mice but reduced by 70% in NET knock-out mice. Nisoxetine inhibited dopamine uptake by 20% in caudate and nucleus accumbens synaptosomes from wild-type and DAT knock-out mice but had no effect in those from NET knock-out mice. Cocaine failed to block dopamine uptake into caudate or nucleus accumbens synaptosomes from DAT knock-out mice. Cocaine and GBR12909 each inhibited dopamine uptake into caudate synaptosomes from NET knock-out mice, but cocaine effectiveness was reduced in the case of nucleus accumbens synaptosomes. Thus, whereas dopamine uptake in caudate and nucleus accumbens depends primarily on the DAT, dopamine uptake in frontal cortex depends primarily on the NET. These data underscore the fact that which transporter clears dopamine from a given region depends on both the affinities and the local densities of the transporters.

The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA) is a drug of abuse and has been shown to be neurotoxic to 5-HT terminals in many species. MDMA-engendered neurotoxicity has been shown to be affected by both ambient temperature and core body temperature. We now report that small (2 degreesC) changes in ambient temperature produce changes in core temperature in MDMA-treated rats, but the same changes in ambient temperature do not affect core temperature of saline-treated animals. Furthermore, increases in core temperature of MDMA-treated animals increase neurotoxicity. Rats were given MDMA (20 or 40 mg/kg) or saline and placed in an ambient temperature of 20, 22, 24, 26, 28, or 30 degreesC using a novel temperature measurement apparatus that controls ambient temperature +/-0.5 degrees C. Two weeks after MDMA treatment, the rats were killed, and regional 5-HT and 5-hydroxyindole acetic acid levels were analyzed as a measure of neurotoxicity. Rats treated with MDMA at 20 and 22 degrees C showed a hypothermic core temperature response. Treatment with MDMA at 28 and 30 degreesC produced a hyperthermic response. At ambient temperatures of 20-24 degrees C, neurotoxicity was not observed in the frontal cortex, somatosensory cortex, hippocampus, or striatum. At ambient temperatures of 26-30 degrees C, neurotoxicity was seen and correlated with core temperature in all regions examined. These data indicate that ambient temperature has a significant affect on MDMA neurotoxicity, core temperature, and thermoregulation in rats. This finding has implications on both the temperature dependence of the mechanism of MDMA neurotoxicity and human use because fatal hyperthermia is associated with MDMA use in humans.

Abnormal protein processing and modification is associated with Alzheimer's disease (AD) pathology. The role of phosphorylation in AD has been studied extensively because the presumed abnormal phosphorylation of tau protein is believed to play a role in the formation of paired helical filaments. Glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues is a dynamic protein modification of intracellular proteins, and it shares similar features with protein phosphorylation. In this study, O-GlcNAc glycosylation of proteins from autopsied human brains with confirmed AD and non-AD age-matched controls was examined. O-GlcNAcylation was demonstrated by labeling protein extracts with [3H]galactose in the presence of galactosyltransferase and subsequent analyses of saccharide-protein linkage and saccharide structure. The number of O-GlcNAc-containing proteins and the overall O-GlcNAc level do not appear to be different between AD and control brain tissues. The only significant change observed is a marked reduction of O-GlcNAcylated clathrin assembly protein-3 (AP-3) in AD. The reduction is more evident in brain neocortical regions, and there appears to be a negative correlation between O-glycosylated AP-3 and the density of neurofibrillary tangles. These data suggest a possible association between the O-glycosylated AP-3 and AD pathology.