Reduced expression of the inhibitory synapse scaffolding protein gephyrin in Alzheimer's disease.

Excitotoxicity may contribute to neuronal and synaptic loss in Alzheimer's disease (AD). Aberrant levels of gephyrin, a post-synaptic receptor-stabilizing protein, could affect the inhibitory modulation of excitatory impulses. We assayed gephyrin protein in two brain areas susceptible to neuronal loss in AD, and in a spared area, in autopsy tissue from normal subjects (n=15) and AD patients (n=5). Quantification was by in-gel immunodetection against known concentrations of a recombinant truncated gephyrin standard. Gephyrin abundance was significantly reduced (P<0.01) in AD. Area-wise analysis showed that gephyrin levels were reduced in both spared and susceptible regions, indicating a global phenomenon. When samples were categorized on an index of pathological severity, gephyrin levels decreased with increasing severity until a moderate index was reached, and then increased, suggesting that higher gephyrin levels might compensate for excitotoxic damage in late stages of the disease. AD males showed a more pronounced reduction in gephyrin levels than AD females cf same-sex controls. A major splice variant of gephyrin was detected in all cases and in all three brain areas. This is the first study of gephyrin expression in AD.

Upregulation of beta-catenin levels in superior frontal cortex of chronic alcoholics.

BACKGROUND: Chronic and excessive alcohol misuse results in neuroadaptive changes in the brain. The complex nature of behavioral, psychological, emotional, and neuropathological characteristics associated with alcoholism is likely a reflection of the network of proteins that are affected by alcohol-induced gene expression patterns in specific brain regions. At the molecular level, however, knowledge remains limited regarding alterations in protein expression levels affected by chronic alcohol abuse. Thus, novel techniques that allow a comprehensive assessment of this complexity will enable the simultaneous assessment of changes across a group of proteins in the relevant neural circuitry. METHODS: A proteomics analysis was performed using antibody microarrays to determine differential protein levels in superior frontal cortices between chronic alcoholics and age- and gender-matched control subjects. Seventeen proteins related to the catenin signaling pathway were analyzed, including alpha-, beta-, and delta-catenins, their upstream activators cadherin-3 (type I cadherin) and cadherin-5 (type II cadherin), and 5 cytoplasmic regulators c-Src, CK1 epsilon, GSK-3beta, PP2A-C alpha, and APC, as well as the nuclear complex partner of beta-catenin CBP and 2 downstream genes Myc and cyclin D1. ILK, G(alpha1), G(beta1), and G(beta2), which are activity regulators of GSK-3beta, were also analyzed. RESULTS: Both alpha- and beta-catenin showed significantly increased levels, while delta-catenin did not change significantly, in chronic alcoholics. In addition, the level of the beta-catenin downstream gene product Myc was significantly increased. Average levels of the catenin regulators c-Src, CK1 epsilon, and APC were also increased in chronic alcoholics, but the changes were not statistically significant. CONCLUSION: Chronic and excessive alcohol consumption leads to an upregulation of alpha- and beta-catenin levels, which in turn increase downstream gene expressions such as Myc that is controlled by beta-catenin signaling. This study showed that the beta-catenin signal transduction pathway was upregulated by chronic alcohol abuse, and prompts further investigation of mechanisms underlying the upregulation of alpha- and beta-catenins in alcoholism, which may have considerable pathogenic and therapeutic relevance.

Development of DNA aptamers targeting low-molecular-weight amyloid-ß peptide aggregates in vitro.

We have developed a novel functional nucleic acid aptamer to amyloid-ß peptide 1-40 (Aß1-40) and investigated its potential to detect Aß peptide fragments in neuropathologically confirmed Alzheimer brain hippocampus tissues samples. Our results demonstrate that the aptamer candidate RNV95 could detect tetrameric/pentameric low-molecular-weight Aß aggregates in autopsy hippocampal tissue from two neuropathologically confirmed Alzheimer disease cases. Although these are preliminary observations, detailed investigations are under way. This is the first demonstration of aptamer-Aß binding in Alzheimer brain tissues.

Selective loss of synaptic proteins in Alzheimer's disease: evidence for an increased severity with APOE varepsilon4.

A pathological feature of Alzheimer's disease (AD) is an area-specific neuronal loss that may be caused by excitotoxicity-related synaptic dysfunction. Relative expression levels of synaptophysin, dynamin I, complexins I and II, N-cadherin, and alphaCaMKII were analysed in human brain tissue from AD cases and controls in hippocampus, and inferior temporal and occipital cortices. Synaptophysin and dynamin I are presynaptic terminal proteins not specific to any neurotransmitter system whereas complexin II, N-cadherin, and alphaCaMKII are specific for excitatory synapses. Complexin I is a presynaptic protein localised to inhibitory synapses. There were no significant differences in synaptophysin, dynamin I, N-cadherin, or alphaCaMKII protein levels between AD cases and controls. The complexin proteins were both markedly lower in AD cases than in controls (P < 0.01). Cases were also categorised by APOE genotype. Averaged across areas there was a 36% lowering of presynaptic proteins in AD cases carrying at least one epsilon4 allele compared with in AD cases lacking the epsilon4 allele. We infer that synaptic protein level is not indicative of neuronal loss, but the synaptic dysfunction may result from the marked relative loss of the complexins in AD, and lower levels of presynaptic proteins in AD cases with the APOE epsilon4 allele.

The identification and characterization of excitotoxic nerve-endings in Alzheimer disease.

Regionally specific neuronal loss is a distinguishing feature of Alzheimer disease (AD). Excitotoxicity is a mechanism commonly invoked to explain this. We review the accumulating evidence for such a hypothesis, particularly the altered expression and pharmacology of glutamate receptors and transporters in pathologically susceptible regions of the AD brain. Loss of neurons would be expected to lead to the retrograde degeneration of their afferents, which should be reflected in a loss of presynaptic markers such as synaptophysin. We discuss the possibility that neurons may be destroyed locally, but that glutamatergic presynaptic terminals may remain, or even re-proliferate. The reduced glutamate uptake site density in AD brain may signify a loss of the transporters on otherwise intact terminals, rather than the loss of glutamatergic afferents. Neuronal death may follow if cells are exposed to excessive amounts of glutamate; the loss of transporters from functioning, but defective, glutamate terminals would mean they could continue to release glutamate to exacerbate excitotoxicity. We discuss experimental methods to quantitate synapses, which are crucial for deciding between the various possibilities.

Cofilin rods and aggregates concur with tau pathology and the development of Alzheimer's disease.

BACKGROUND: Imaging of human brain as well as cellular and animal models has highlighted a role for the actin cytoskeleton in the development of cell pathology in Alzheimer's disease (AD). Rods and aggregates of the actin-associated protein cofilin are abundant in grey matter of postmortem AD brain and rods are found inside neurites in animal and cell models of AD. OBJECTIVE: We sought further understanding of the significance of cofilin rods/aggregates to the disease process: Do rods/aggregates correlate with AD progression and the development of hallmark neurofibrillary tangles and neuropil threads? Are cofilin rods/aggregates found in the same neurites as hyperphosphorylated tau? METHODS: The specificity of rods/aggregates to AD compared with general aging and their spatial relationship to tau protein was examined in postmortem human hippocampus, inferior temporal cortex, and anterior cingulate cortex. RESULTS: The presence of cofilin rods/aggregates correlated with the extent of tau pathology independent of patient age. Densities of rods/aggregates were fourfold greater in AD compared with aged-matched control brains and rods/aggregates were significantly larger in AD brain. We did not find evidence for our hypothesis that intracellular cofilin rods are localized to tau-positive neuropil threads. Instead, data suggest the involvement of microglia in the clearance of cofilin rods/aggregates and/or in their synthesis in and around amyloid plaques and surrounding neuropil. CONCLUSION: Cofilin rods and aggregates signify events initiated early in the pathological cascade. Further definition of the mechanisms leading to their formation in the human brain will provide insights into the cellular causes of AD.

Nucleic acid aptamers as novel class of therapeutics to mitigate Alzheimer's disease pathology.

Deposition of amyloid-ß (Aß) peptides in the brain is a central event in the pathogenesis of Alzheimer's disease (AD), which makes Aß peptides a crucial target for therapeutic intervention. Significant efforts have been made towards the development of ligands that bind to Aß peptides with a goal of early detection of amyloid aggregation and the neutralization of Aß toxicity. Short single-stranded oligonucleotide aptamers bind with high affinity and specificity to their targets. Aptamers that specifically bind to Aß monomers, specifically the 40 and 42 amino acid species (Aß(1-40) and Aß(1- 42)), fibrils and plaques have a great potential for diagnostic applications and the treatment of AD. Herein, we review the aptamers that bind to the various forms of Aß peptides for use in diagnosis and to inhibit plaque formation.

Regional expression of dopamine D1 and D2 receptor proteins in the cerebral cortex of asphyxic newborn infants.

Dopamine D(1) and D(2) receptor protein expression was examined by Western blotting in newborn infants dying from cerebral asphyxia between 31 and 42 weeks' gestation, and matched controls. Frontal, occipital, temporal, and motor cortex tissue samples were obtained at autopsy (median postmortem interval 35 hours) and frozen for storage at -80 degrees C. A total of 2 immunoreactive bands were detected with each primary antibody in infant brain, whereas a single band was present in adult human and rat tissue. Immunoreactivity varied between cortical areas for both receptors, but their regional patterns differed significantly. D(1) protein levels were higher in motor and temporal cortex than in frontal or occipital cortex. D(2) protein showed graded expression frontal > motor > occipital > temporal cortex. Asphyxia cases showed lower expression of the upper D(2) immunoreactive band, but no difference in regional pattern. Lower D(2) receptor expression may attenuate stress responses and underlie increased vulnerability to hypoxia at birth.