Small molecule structure correctors abolish detrimental effects of apolipoprotein E4 in cultured neurons.

Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC(50) of 23 and 116 nm, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.

Apolipoprotein E4 domain interaction mediates detrimental effects on mitochondria and is a potential therapeutic target for Alzheimer disease.

Apolipoprotein (apo) E4 is the major genetic risk factor for late-onset Alzheimer disease (AD). ApoE4 assumes a pathological conformation through an intramolecular interaction mediated by Arg-61 in the amino-terminal domain and Glu-255 in the carboxyl-terminal domain, referred to as apoE4 domain interaction. Because AD is associated with mitochondrial dysfunction, we examined the effect of apoE4 domain interaction on mitochondrial respiratory function. Steady-state amounts of mitochondrial respiratory complexes were examined in neurons cultured from brain cortices of neuron-specific enolase promoter-driven apoE3 (NSE-apoE3) or apoE4 (NSE-apoE4) transgenic mice. All subunits of mitochondrial respiratory complexes assessed were significantly lower in NSE-apoE4 neurons compared with NSE-apoE3 neurons. However, no significant differences in levels of mitochondrial complexes were detected between astrocytes expressing different apoE isoforms driven by the glial fibrillary acidic protein promoter, leading to our conclusion that the effect of apoE4 is neuron specific. In neuroblastoma Neuro-2A (N2A) cells, apoE4 expression reduced the levels of mitochondrial respiratory complexes I, IV, and V. Complex IV enzymatic activity was also decreased, lowering mitochondrial respiratory capacity. Mutant apoE4 (apoE4-Thr-61) lacking domain interaction did not induce mitochondrial dysfunction in N2A cells, indicating that the effect is specific to apoE4-expressing cells and dependent on domain interaction. Consistent with this finding, treatment of apoE4-expressing N2A cells with a small molecule that disrupts apoE4 domain interaction restored mitochondrial respiratory complex IV levels. These results suggest that pharmacological intervention with small molecules that disrupt apoE4 domain interaction is a potential therapeutic approach for apoE4-carrying AD subjects.

Reactivity of apolipoprotein E4 and amyloid beta peptide: lysosomal stability and neurodegeneration.

We previously demonstrated that apolipoprotein E4 (apoE4) potentiates lysosomal leakage and apoptosis induced by amyloid beta (Abeta) peptide in cultured Neuro-2a cells and hypothesized that the low pH of lysosomes accentuates the conversion of apoE4 to a molten globule, inducing reactive intermediates capable of destabilizing cellular membranes. Here we report that neutralizing lysosomal pH with bafilomycin or NH4Cl abolished the apoE4 potentiation of Abeta-induced lysosomal leakage and apoptosis in Neuro-2a cells. Consistent with these results, apoE4 at acidic pH bound more avidly to phospholipid vesicles and disrupted them to a greater extent than at pH 7.4. Comparison of "Arctic" mutant Abeta, which forms multimers, and GM6 mutant Abeta, which remains primarily monomeric, showed that aggregation is essential for apoE4 to potentiate Abeta-induced lysosomal leakage and apoptosis. Both apoE4 and Abeta1-42 had to be internalized to exert these effects. Blocking the low density lipoprotein receptor-related protein with small interfering RNA abolished the enhanced effects of apoE4 and Abeta on lysosomes and apoptosis. In cultured Neuro-2a cells, Abeta1-42 increased lysosome formation to a greater extent in apoE3- or apoE4-transfected cells than in Neo-transfected cells, as shown by immunostaining for lysosome-associated membrane protein 1. Similarly, in transgenic mice expressing apoE and amyloid precursor protein, hippocampal neurons displayed increased numbers of lysosomes. Thus, apoE4 and Abeta1-42 may work in concert in neurons to increase lysosome formation while increasing the susceptibility of lysosomal membranes to disruption, release of lysosomal enzymes into the cytosol, and neuronal degeneration.

Apolipoprotein E4 potentiates amyloid beta peptide-induced lysosomal leakage and apoptosis in neuronal cells.

We assessed the isoform-specific effects of apolipoprotein (apo) E on the response of Neuro-2a cells to the amyloid beta peptide (Abeta1-42). As determined by the intracellular staining pattern and the release of beta-hexosaminidase into the cytosol, apoE4-transfected cells treated with aggregated Abeta1-42 showed a greater tendency toward lysosomal leakage than neo- or apoE3-transfected cells. Abeta1-42 caused significantly greater cell death and more than 2-fold greater DNA fragmentation in apoE4-secreting than in apoE3-secreting or control cells. H2O2 or staurosporine enhanced cell death and apoptosis in apoE4-transfected cells but not in apoE3-transfected cells. A caspase-9 inhibitor abolished the potentiation of Abeta1-42-induced apoptosis by apoE4. Similar results were obtained with conditioned medium from cells secreting apoE3 or apoE4. Cells preincubated for 4 h with a source of apoE3 or apoE4, followed by removal of apoE from the medium and from the cell surface, still exhibited the isoform-specific response to Abeta1-42, indicating that the potentiation of apoptosis required intracellular apoE, presumably in the endosomes or lysosomes. Studies of phospholipid (dimyristoylphosphatidylcholine) bilayer vesicles encapsulating 5-(and-6)-carboxyfluorescein dye showed that apoE4 remodeled and disrupted the phospholipid vesicles to a greater extent than apoE3 or apoE2. In response to Abeta1-42, vesicles containing apoE4 were disrupted to a greater extent than those containing apoE3. These findings are consistent with apoE4 forming a reactive molecular intermediate that avidly binds phospholipid and may insert into the lysosomal membrane, destabilizing it and causing lysosomal leakage and apoptosis in response to Abeta1-42.