Simulative Investigation of the Effects of a Phase Center Shift onto a Log-Periodic Dipole Array

Alzheimer's disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD

Suggested conserved amino acid sequence for reversal of α3β4 nAChR inhibition by Aβ.

<p>Suggested conserved amino acid sequence for reversal of α3β4 nAChR inhibition by Aβ.</p

IQ and selected analogues reverse Aβ40 inhibition of nAChRs in PC12 cells.

<p>(A) Current responses (normalized by the maximal current evoked by 0.2 mM CCh) of neuronal-differentiated PC12 cells exposed for 2 s to 0.2 mM CCh plus 200 nM Aβ40 in all experimental conditions, except for the control measurement with CCh alone, and, as indicated, 500 nM of different IQ analogues. Bars represent means ± S.D. of at least 3 replicate measurements performed in 4–6 different cells (**, p<0.01; *** p<0.001 in the comparison with the control current evoked by CCh alone).</p

IQ makes Aβ40 inhibition of α3β4 nAChR currents in transformed HEK cells reversible.

<p>HEK cells expressing α3β4 nAChRs received consecutive shots (at 5 min intervals) of 0.2 mM CCh plus 200 nM Aβ, in the absence or presence of IQ (0.5 µM) as indicated. Shots 1–3 contained 0.2 mM CCh alone (white bars), 0.2 mM CCh plus 200 nM Aβ (light grey bars), 0.2 mM CCh plus 200 nM Aβ and 0.5/2 µM IQ (grey bars) or 0.5 µM SQI (black bars), used as an inactive control. Shots 4–6 contained 0.2 mM CCh alone for evaluation of reversibility of receptor inhibition. Bars represent mean values ± S.D. of at least 3 replicate measurements (normalized by the maximal current evoked by 0.2 mM CCh) obtained from 4–6 different cells. (***, p<0.001, in comparison with 0.2 mM CCh plus 200 nM Aβ).</p

Effects of IQ and analogues on Aβ40 inhibition of α3β4 nAChRs in transformed HEK cells.

<p>(A) HEK cells expressing recombinant α3β4 nAChRs received 3 consecutive shots (at 4 min intervals) of 0.2 mM CCh plus 200 nM Aβ40 in the absence or presence of 0.5 and 2 µM IQTTWSR. QI and SQI (500 nM) were used as ineffective control peptides. Recovery of current response was evaluated after washout and 3 shots of CCh alone. (B) HEK cells expressing recombinant α3β4 nAChRs received 3 consecutive shots of 0.2 mM CCh plus 200 nM Aβ40 in the absence or presence of 500 nM TTWS, TWSR or IQTTASR. QI and SQI (500 nM) were used as ineffective control peptides. Recovery of current response was evaluated after washout and 3 shots of CCh alone. Bars represent mean values ± S.D. of current responses (normalized by the maximal current evoked by 0.2 mM CCh) of at least 3 measurements performed in at least 3 different cells. (***, p<0.001).</p

TNF-α mediates PKR-dependent memory impairment and brain IRS-1 inhibition induced by Alzheimer's β-Amyloid oligomers in mice and monkeys

Alzheimer's disease (AD) and type 2 diabetes appear to share similar pathogenic mechanisms. dsRNA-dependent protein kinase (PKR) underlies peripheral insulin resistance in metabolic disorders. PKR phosphorylates eukaryotic translation initiation factor 2α (eIF2α-P), and AD brains exhibit elevated phospho-PKR and eIF2α-P levels. Whether and how PKR and eIF2α-P participate in defective brain insulin signaling and cognitive impairment in AD are unknown. We report that β-amyloid oligomers, AD-associated toxins, activate PKR in a tumor necrosis factor α (TNF-α)-dependent manner, resulting in eIF2α-P, neuronal insulin receptor substrate (IRS-1) inhibition, synapse loss, and memory impairment. Brain phospho-PKR and eIF2α-P were elevated in AD animal models, including monkeys given intracerebroventricular oligomer infusions. Oligomers failed to trigger eIF2α-P and cognitive impairment in PKR(-/-) and TNFR1(-/-) mice. Bolstering insulin signaling rescued phospho-PKR and eIF2α-P. Results reveal pathogenic mechanisms shared by AD and diabetes and establish that proinflammatory signaling mediates oligomer-induced IRS-1 inhibition and PKR-dependent synapse and memory loss

An anti-diabetes agent protects the mouse brain from defective insulin signaling caused by Alzheimer’s disease–associated Aβ oligomers

Defective brain insulin signaling has been suggested to contribute to the cognitive deficits in patients with Alzheimer’s disease (AD). Although a connection between AD and diabetes has been suggested, a major unknown is the mechanism(s) by which insulin resistance in the brain arises in individuals with AD. Here, we show that serine phosphorylation of IRS-1 (IRS-1pSer) is common to both diseases. Brain tissue from humans with AD had elevated levels of IRS-1pSer and activated JNK, analogous to what occurs in peripheral tissue in patients with diabetes. We found that amyloid-β peptide (Aβ) oligomers, synaptotoxins that accumulate in the brains of AD patients, activated the JNK/TNF-α pathway, induced IRS-1 phosphorylation at multiple serine residues, and inhibited physiological IRS-1pTyr in mature cultured hippocampal neurons. Impaired IRS-1 signaling was also present in the hippocampi of Tg mice with a brain condition that models AD. Importantly, intracerebroventricular injection of Aβ oligomers triggered hippocampal IRS-1pSer and JNK activation in cynomolgus monkeys. The oligomer-induced neuronal pathologies observed in vitro, including impaired axonal transport, were prevented by exposure to exendin-4 (exenatide), an anti-diabetes agent. In Tg mice, exendin-4 decreased levels of hippocampal IRS-1pSer and activated JNK and improved behavioral measures of cognition. By establishing molecular links between the dysregulated insulin signaling in AD and diabetes, our results open avenues for the investigation of new therapeutics in AD