R. K. Tannenberg

Selective loss of synaptic proteins in Alzheimer's disease: Evidence for an increased severity with APOE ɛ4

A pathological feature of Alzheimers disease (AD) is an area-specific neuronal loss that may be caused by excitotoxicity-related synaptic dysfunction. Relative expression levels of synaptopbysin, dynamin I, complexins I and II, N-cadherin, and alpha CaMKII 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 alpha CaMKII 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 alpha CaMKII 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 epsilon 4 allele compared with in AD cases lacking the epsilon 4 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 epsilon 4 allele. (c) 2006 Elsevier Ltd. All rights reserved.

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.

Reduced Expression of the Inhibitory Synapse Scaffolding Protein Gephyrin in Alzheimer's Disease

Excitotoxicity may contribute to neuronal and synaptic loss in Alzheimers 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 β‐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.

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 Alzheimers 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-β (Aβ) peptides in the brain is a central event in the pathogenesis of Alzheimers disease (AD), which makes Aβ peptides a crucial target for therapeutic intervention. Significant efforts have been made towards the development of ligands that bind to Aβ peptides with a goal of early detection of amyloid aggregation and the neutralization of Aβ toxicity. Short single-stranded oligonucleotide aptamers bind with high affinity and specificity to their targets. Aptamers that specifically bind to Aβ monomers, specifically the 40 and 42 amino acid species (Aβ(1-40) and Aβ(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 Aβ peptides for use in diagnosis and to inhibit plaque formation.

Genetic modulation of amino acid transmitter receptor subunit expression in alcoholic human brain

Early P MRS studies using a novel superfusion system demonstrated that cortical slices respond differently to the two principal elements of ischaemic challenge, hypoxia and hypoglycaemia. In the former, ATP levels are maintained as phosphocreatine (PCr) levels fall, whereas in the latter ATP and PCr levels fall in concert. In both cases P saturation transfer NMR demonstrates an increase in the creatine kinase mediated exchange between ATP and PCr. The use of F NMR in conjunction with the selective calcium chelator 5FBAPTA enables levels of intracellular Ca to be determined in the same preparation. Neither hypoxia nor hypoglycaemia lead to an increase in intracellular Ca whereas in combination a large increase is observed. Previous exposure to the combination of hypoxia and hypoglycaemia (ischaemic pre-conditioning) abolishes the increase in intracellular Ca during subsequent ischaemic challenge. Interestingly, excitotoxic challenge both increases intracellular Ca and releases endogenous Zn . C MRS has demonstrated that, in severe hypoxia, glycerol 3-phosphate is produced as an alternative to the regeneration of NAD by conversion of pyruvate to lactate. C MRS has also proved an ideal tool to study neuronal/glial metabolic interactions in model systems and in man. An increase in TCA cycle rate on visual activation of about 50% has been found similar to the increases in CMRglc reported in PET studies, but suggests in contrast to these studies, that cerebral glucose is metabolised oxidatively. The data have also been used to determine the rate of the glutamate/glutamine cycle via which much of the neurotransmitter glutamate is recycled. Such measurements will be substantially improved at higher fields (7T and up). PL2 Magnetic resonance neurospectroscopy, from test tube to clinic

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

We have developed a novel functional nucleic acid aptamer to amyloid-β peptide 1-40 (Aβ1-40) and investigated its potential to detect Aβ 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 Aβ 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-Aβ binding in Alzheimer brain tissues.

Cell damage/Excitotoxicity: Excitotoxicity and neurodegenerative disease

Excitotoxicity is an established mechanism of neuronal killing. Many neurodegenerative diseases, including epilepsy, have implicated excitotoxicity in their pathogenesis. Studies characterizing excitotoxic mechanisms in these diseases have subsequently focused on studying aspects of glutamate transporters and/or receptors. In this article, we describe a novel approach to delineate the excitatory synapse and its parts. Synaptic dysfunction is often the direct cause of symptoms observed throughout the courses of these diseases. Recent results from our laboratory suggest excitatory synaptic dysfunction in Alzheimers disease, which appears similar to synaptic protein changes observed in epileptic models. Unraveling the dysfunctional synapse into excitatory, inhibitory, pre- and/or postsynaptic subtypes may lead to new avenues of treatment for epilepsy and other neurodegenerative diseases.

Gephyrin in Alzheimer's disease

P-01 DISORGANIZATION OF NEUROFILAMENTS IN NEURONS RESULTS IN NEUROPATHOLOGICAL CHANGES IN YOUNG MICE WITH DISRUPTION OF EXPRESSION OF NEUROFILAMENT LIGHT SUBUNIT Wu, Y.J., He, B.P. Division of Life Science and Technology, Ocean University of China, 5 Yushan Road, Qingdao, Shandong, China 266003 Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, Block MD 10, Singapore 117597