A major obstacle to pre-symptomatic diagnosis and disease-modifying therapy for Alzheimer's disease (AD) is inadequate understanding of molecular mechanisms of AD pathogenesis. For example, impaired brain insulin signaling is an AD hallmark, but whether and how it might contribute to the synaptic dysfunction and neuron death that underlie memory and cognitive impairment has been mysterious. Neuron death in AD is often caused by cell cycle re-entry (CCR) mediated by amyloid-β oligomers (AβOs) and tau, the precursors of plaques and tangles. We now report that CCR results from AβO-induced activation of the protein kinase complex, mTORC1, at the plasma membrane and mTORC1-dependent tau phosphorylation, and that CCR can be prevented by insulin-stimulated activation of lysosomal mTORC1. AβOs were also shown previously to reduce neuronal insulin signaling. Our data therefore indicate that the decreased insulin signaling provoked by AβOs unleashes their toxic potential to cause neuronal CCR, and by extension, neuron death.
IntroductionAlzheimer's disease (AD) symptoms reflect synaptic dysfunction and neuron death. Amyloid‐β oligomers (AβOs) 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 AβO‐stimulated calcium entry also drives neuronal cell cycle reentry (CCR), a prelude to neuron death in AD.MethodsPharmacologic modulators of calcium entry and gene expression knockdown were used in cultured neurons and AD model mice.ResultsIn cultured neurons, AβO‐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.DiscussionThis study demonstrates a role for AβO‐stimulated calcium influx via NMDAR and CCR in AD and suggests the use of memantine as a disease‐modifying therapy for presymptomatic AD.
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.
further investigate the local nature of these bends, AFM images of HMO1-DNA complexes are imaged to probe the behavior of these complexes as a function of protein concentration. The results show that at lower concentrations, HMO1 preferentially binds to the ends of the double helix and links separate DNA strands. At higher concentrations HMO1 induces formation of a complex network that reorganizes DNA. Although nucleoid associated proteins are under intense investigation, little is known about HMO1. Our studies suggest that HMO1 proteins may facilitate interactions between multiple DNA molecules.
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