APOE2 enhances neuroprotection against Alzheimer's disease through multiple molecular mechanisms.

The common APOE2 gene variant is neuroprotective against Alzheimer's disease (AD) and reduces risk by nearly 50%. However, the mechanisms by which APOE2 confers neuroprotection are largely unknown. Here we showed that ApoE protein abundance in human postmortem cortex follows an isoform-dependent pattern (E2>E3>E4). We also identified a unique downstream transcriptional profile determined by microarray and characterized by downregulation of long-term potentiation (LTP) related transcripts and upregulation of extracellular matrix (ECM)/integrin-related transcripts in E2 cases and corroborated this finding at the protein level by demonstrating increases in ECM collagens and laminins. In vivo studies of healthy older individuals demonstrated a unique and advantageous biomarker signature in E2 carriers. APOE2 also reduced the risk of mild cognitive impairment to AD conversion by half. Our findings suggest that ApoE2 protein abundance, coupled with its inability to bind to LDLRs, may act to increase amyloid-beta (Ab) clearance. In addition, increased ECM and reduced LTP-related expression results in diminished activity-dependent Ab secretion and/or excitotoxicity, and thus also promotes neuroprotection.

Molecular signatures in post-mortem brain tissue of younger individuals at high risk for Alzheimer's disease as based on APOE genotype.

Alzheimer's disease (AD) is a neurodegenerative condition characterized histopathologically by neuritic plaques and neurofibrillary tangles. The objective of this transcriptional profiling study was to identify both neurosusceptibility and intrinsic neuroprotective factors at the molecular level, not confounded by the downstream consequences of pathology. We thus studied post-mortem cortical tissue in 28 cases that were non-APOE4 carriers (called the APOE3 group) and 13 cases that were APOE4 carriers. As APOE genotype is the major genetic risk factor for late-onset AD, the former group was at low risk for development of the disease and the latter group was at high risk for the disease. Mean age at death was 42 years and none of the brains had histopathology diagnostic of AD at the time of death. We first derived interregional difference scores in expression between cortical tissue from a region relatively invulnerable to AD (primary somatosensory cortex, BA 1/2/3) and an area known to be susceptible to AD pathology (middle temporal gyrus, BA 21). We then contrasted the magnitude of these interregional differences in between-group comparisons of the APOE3 (low risk) and APOE4 (high risk) genotype groups. We identified 70 transcripts that differed significantly between the groups. These included EGFR, CNTFR, CASP6, GRIA2, CTNNB1, FKBPL, LGALS1 and PSMC5. Using real-time quantitative PCR, we validated these findings. In addition, we found regional differences in the expression of APOE itself. We also identified multiple Kyoto pathways that were disrupted in the APOE4 group, including those involved in mitochondrial function, calcium regulation and cell-cycle reentry. To determine the functional significance of our transcriptional findings, we used bioinformatics pathway analyses to demonstrate that the molecules listed above comprised a network of connections with each other, APOE, and APP and MAPT. Overall, our results indicated that the abnormalities that we observed in single transcripts and in signaling pathways were not the consequences of diagnostic plaque and tangle pathology, but preceded it and thus may be a causative link in the long molecular prodrome that results in clinical AD.

Transduction of human GAD67 cDNA into immortalized striatal cell lines using an Epstein-Barr virus-based plasmid vector increases GABA content.

The M213-20 and M213-1L cell lines were immortalized from rat striatum using the tsA58 allele of the SV40 large T antigen, contain the GAD enzyme, and produce GABA (Giordano et al., 1994, Exp. Neurol. 124:395-400). Cell lines that produce large amounts of GABA may be useful for transplantation into the brain in conditions such as Huntington's disease or epilepsy, where localized application of GABA may be of therapeutic value. We have explored the potential use of the pREP10 plasmid vector, which replicates episomally, to increase GAD expression and GABA production in M213-20 and M213-1L cells. Human GAD(67) cDNA was transfected into M213-20 and M213-1L, and subclones were isolated with hygromycin selection. Immunochemical studies showed increased GAD(67) expression compared to the parent M213-20 and M213-1L cell lines. Staining for the EBNA antigen and Southern blots demonstrated that the pREP10 plasmid was stably maintained in the cells for at least 12-15 months in culture. Several clones were isolated in which GABA concentrations were increased by as much as 4-fold (M213-1L) or 44-fold (M213-20) compared to the parent cell lines or 12-fold (M213-1L) and 94-fold (M213-20) greater than rat striatal tissue (1.678 +/- 0.4 micromol/g prot). The ability of these cells to continue to produce large amounts of GABA while being maintained in culture for extended periods suggests that similar methods might be used with human cell lines to produce cells that can be transplanted into the brain to deliver GABA for therapeutic purposes.