N-methyl-D-aspartate receptor-mediated calcium influx connects amyloid-ß oligomers to ectopic neuronal cell cycle reentry in Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) symptoms reflect synaptic dysfunction and neuron death. Amyloid-ß oligomers (AßOs) induce excess calcium entry into neurons via N-methyl-D-aspartate receptors (NMDARs), contributing to synaptic dysfunction. The study described here tested the hypothesis that AßO-stimulated calcium entry also drives neuronal cell cycle reentry (CCR), a prelude to neuron death in AD. METHODS: Pharmacologic modulators of calcium entry and gene expression knockdown were used in cultured neurons and AD model mice. RESULTS: In cultured neurons, AßO-stimulated CCR was blocked by NMDAR antagonists, total calcium chelation with 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), or knockdown of the NMDAR subunit, NR1. NMDAR antagonists also blocked the activation of calcium-calmodulin-dependent protein kinase II and treatment of Tg2576 AD model mice with the NMDAR antagonist, memantine, prevented CCR. DISCUSSION: This study demonstrates a role for AßO-stimulated calcium influx via NMDAR and CCR in AD and suggests the use of memantine as a disease-modifying therapy for presymptomatic AD.

Rationally designed coiled-coil DNA looping peptides control DNA topology.

Artificial DNA looping peptides were engineered to study the roles of protein and DNA flexibility in controlling the geometry and stability of protein-mediated DNA loops. These LZD (leucine zipper dual-binding) peptides were derived by fusing a second, C-terminal, DNA-binding region onto the GCN4 bZip peptide. Two variants with different coiled-coil lengths were designed to control the relative orientations of DNA bound at each end. Electrophoretic mobility shift assays verified formation of a sandwich complex containing two DNAs and one peptide. Ring closure experiments demonstrated that looping requires a DNA-binding site separation of 310 bp, much longer than the length needed for natural loops. Systematic variation of binding site separation over a series of 10 constructs that cyclize to form 862-bp minicircles yielded positive and negative topoisomers because of two possible writhed geometries. Periodic variation in topoisomer abundance could be modeled using canonical DNA persistence length and torsional modulus values. The results confirm that the LZD peptides are stiffer than natural DNA looping proteins, and they suggest that formation of short DNA loops requires protein flexibility, not unusual DNA bendability. Small, stable, tunable looping peptides may be useful as synthetic transcriptional regulators or components of protein-DNA nanostructures.