Several small molecule ligands for amyloid‐β (Aβ) fibrils deposited in brain have been developed to facilitate radiological diagnosis of Alzheimer’s disease (AD). Recently, the build‐up of Aβ oligomers (AβO) in brain has been recognized as an additional hallmark of AD and may play a more significant role in early stages. Evidence suggests that quantitative assessment of AβO would provide a more accurate index of therapeutic effect of drug trials. Therefore, there is an urgent need to develop methods for efficient identification as well as structural analysis of AβO. We found that some well established amyloid ligands, analogs of Congo red and thioflavin‐T (ThT), bind AβO with high affinity and detect AβO in vitro and in vivo. Binding studies revealed the presence of binding sites for Congo red‐ and thioflavin‐T‐analogs on AβO. Furthermore, these ligands can be used for imaging intracellular AβO in living cells and animals and as positive contrast agent for ultrastructural imaging of AβO, two applications useful for structural analysis of AβO in cells. We propose that by improving the binding affinity of current ligands, in vivo imaging of AβO is feasible by a ‘signal subtraction’ procedure. This approach may facilitate the identification of individuals with early AD.

The blood-brain barrier (BBB), which is formed by adherens and tight junctions (TJs) of endothelial cells, maintains homeostasis of the brain. Disrupted intracellular Ca 2ϩ homeostasis and breakdown of the BBB have been implicated in the pathogenesis of Alzheimer's disease (AD). The receptor for advanced glycation end products (RAGE) is known to interact with amyloid ␤-peptide (A␤) and mediate A␤ transport across the BBB, contributing to the deposition of A␤ in the brain. However, molecular mechanisms underlying A␤-RAGE interaction-induced alterations in the BBB have not been identified. We found that A␤ 1-42 induces enhanced permeability, disruption of zonula occludin-1 (ZO-1) expression in the plasma membrane, and increased intracellular calcium and matrix metalloproteinase (MMP) secretion in cultured endothelial cells. Neutralizing antibodies against RAGE and inhibitors of calcineurin and MMPs prevented A␤ 1-42 -induced changes in ZO-1, suggesting that A␤-RAGE interactions alter TJ proteins through the Ca 2ϩ -calcineurin pathway. Consistent with these in vitro findings, we found disrupted microvessels near A␤ plaque-deposited areas, elevated RAGE expression, and enhanced MMP secretion in microvessels of the brains of 5XFAD mice, an animal model for AD. We have identified a potential molecular pathway underlying A␤-RAGE interaction-induced breakage of BBB integrity. This pathway might play an important role in the pathogenesis of AD.

Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid β (Aβ) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aβ impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aβ accumulation has been detected in the AD brain. However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aβ accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aβ1–42 treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aβ1–42-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aβ1–42 in HT22 cells using Aβ1–42 with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aβ1–42-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aβ1–42 accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aβ1–42-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aβ1–42 accumulation, which mimics the apoptosis process in exogenous Aβ1–42-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aβ1–42 accumulation is the necessary and sufficient condition for Aβ-mediated mitochondria impairments, and leads directly to cellular death rather than along with other Aβ-mediated signaling alterations.

The neurotoxicity of amyloid-b protein (Ab) is widely regarded as one of the fundamental causes of neurodegeneration in Alzheimer's disease (AD). This toxicity is related to Ab aggregation into oligomers, protofibrils and fibrils. Recent studies suggest that intracellular Ab, which causes profound toxicity, could be one of the primary therapeutic targets in AD. So far, no compounds targeting intracellular Ab have been identified. We have investigated the toxicity induced by intracellular Ab in a neuroblastoma MC65 line and found that it was closely related to intracellular accumulation of oligomeric complexes of Ab (Ab-OCs). We further identified a cell-permeable tricyclic pyrone named CP2 that ameliorates this toxicity and significantly reduces the levels of Ab-OCs. In aqueous solution, CP2 attenuates Ab oligomerization and prevents the oligomer-induced death of primary cortical neurons. CP2 analogs represent a new class of promising compounds for the amelioration of Ab toxicities within both intracellular and extracellular sites.

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Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by cognitive deficits, neuroinflammation, and loss of neurons. Recently, it has been shown that ghrelin, a 28 amino acid peptide hormone produced from the stomach and hypothalamus, has been reported as a potential therapeutic agent for several neurological disorders, including Parkinson's disease (PD), stroke, epilepsy, multiple sclerosis, and spinal cord injury. Here we determined the effects of ghrelin on memory impairments and neuropathological changes in an AD mouse model induced by intrahippocampal injection of amyloid-β oligomers (AβO). We report that ghrelin: 1) rescues memory deficits in mice injected with AβO in the hippocampus; 2) decreases AβO-induced microgliosis in hippocampus; 3) attenuates hippocampal neuronal loss mediated by AβO; 4) prevents AβO-associated synaptic degeneration including cholinergic fiber loss. Taken together, our findings demonstrate that ghrelin can ameliorate AβO-induced cognitive impairment associated with neuroinflammation and neuronal loss. These results suggest that ghrelin may be a promising therapeutic agent for the treatment of AD.

By using a highly sensitive technique of atomic force microscopybased single-cell compression, the rigidity of cultured N2a and HT22 neuronal cells was measured as a function of amyloid-β42 (Aβ42) protein treatment. Aβ42 oligomers led to significant cellular stiffening; for example, 90-360% higher force was required to reach 80% deformation for N2a cells. Disaggregated or fibrillar forms of Aβ42 showed much less change. These observations were explained by a combination of two factors: (i) incorporation of oligomer into cellular membrane, which resulted in an increase in the Young's modulus of the membrane from 0.9 ± 0.4 to 1.85 ± 0.75 MPa for N2a cells and from 1.73 ± 0.90 to 5.5 ± 1.4 MPa for HT22 cells, and (ii) an increase in intracellular osmotic pressure (e.g., from 7 to 40 Pa for N2a cells) through unregulated ion influx. These findings and measurements provide a deeper, more characteristic, and quantitative insight into interactions between cells and Aβ42 oligomers, which have been considered the prime suspect for initiating neuronal dysfunction in Alzheimer's disease. atomic force microscopy | Alzheimer's disease | cell mechanics | neuronal dysfunction | Young's moduli

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