Background: Identifying physiologically relevant binding partners of amyloid-β (Aβ) that modulate in vivo fibril formation may yield new insights into Alzheimer’s disease (AD) etiology. Human cathelicidin peptide, LL-37, is an innate immune effector and modulator, ubiquitous in human tissues and expressed in myriad cell types.Objective:We present in vitro experimental evidence and discuss findings supporting a novel hypothesis that LL-37 binds to Aβ42 and can modulate Aβ fibril formation.Methods:Specific interactions between LL-37 and Aβ (with Aβ in different aggregation states, assessed by capillary electrophoresis) were demonstrated by surface plasmon resonance imaging (SPRi). Morphological and structural changes were investigated by transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy. Neuroinflammatory and cytotoxic effects of LL-37 alone, Aβ42 alone, and LL-37/Aβ complexes were evaluated in human microglia and neuroblastoma cell lines (SH-SY5Y).Results:SPRi shows binding specificity between LL-37 and Aβ, while TEM shows that LL-37 inhibits Aβ42 fibril formation, particularly Aβ’s ability to form long, straight fibrils characteristic of AD. CD reveals that LL-37 prevents Aβ42 from adopting its typical β-type secondary structure. Microglia-mediated toxicities of LL-37 and Aβ42 to neurons are greatly attenuated when the two peptides are co-incubated prior to addition. We discuss the complementary biophysical characteristics and AD-related biological activities of these two peptides.Conclusion:Based on this body of evidence, we propose that LL-37 and Aβ42 may be natural binding partners, which implies that balanced (or unbalanced) spatiotemporal expression of the two peptides could impact AD initiation and progression.

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Alzheimer's disease is likely to be caused by copathogenic factors including aggregation of Aβ peptides into oligomers and fibrils, neuroinflammation and oxidative stress. To date, no effective treatments are available and because of the multifactorial nature of the disease, it emerges the need to act on different and simultaneous fronts. Despite the multiple biological activities ascribed to curcumin as neuroprotector, its poor bioavailability and toxicity limit the success in clinical outcomes. To tackle Alzheimer's disease on these aspects, the curcumin template was suitably modified and a small set of analogues was attained. In particular, derivative 1 turned out to be less toxic than curcumin. As evidenced by capillary electrophoresis and transmission electron microscopy studies, 1 proved to inhibit the formation of large toxic Aβ oligomers, by shifting the equilibrium towards smaller non-toxic assemblies and to limit the formation of insoluble fibrils. These findings were supported by molecular docking and steered molecular dynamics simulations which confirmed the superior capacity of 1 to bind Aβ structures of different complexity. Remarkably, 1 also showed in vitro anti-inflammatory and anti-oxidant properties. In summary, the curcumin-based analogue 1 emerged as multipotent compound worth to be further investigated and exploited in the Alzheimer's disease multi-target context.

Free solution capillary electrophoresis with UV detection is here used to retrieve information on the conformational changes of wild-type β2 -microglobulin and a series of naturally and artificially created variants known to have different stability and amyloidogenic potential. Under nondenaturing conditions, the resolution of at least two folding conformers at equilibrium is obtained and a third species is detected for the less stable isoforms. Partial denaturation by using chaotropic agents such as acetonitrile or trifluoroethanol reveals that the separated peaks are at equilibrium, as the presence of less structured species is either enhanced or induced at the expenses of the native form. Reproducible CE data allow to obtain an interesting semiquantitative correlation between the peak areas observed and the protein stability. Thermal unfolding over the range 25-42°C is induced inside the capillary for the two pathogenic proteins (wtβ2 -microglobulin and D76N variant): the large differences observed upon small temperature variation draw attention on the robustness of analytical methods when dealing with proteins prone to misfolding and aggregation.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is not restricted to the neuronal compartment but includes important interactions with immune cells, including microglia. Protein aggregates, common pathological hallmarks of AD, bind to pattern recognition receptors on microglia and trigger an inflammatory response, which contributes to disease progression and severity. In this context, curcumin is emerging as a potential drug candidate able to affect multiple key pathways implicated in AD, including neuroinflammation. Therefore, we studied the effect of curcumin and its structurally related analogues cur6 and cur16 on amyloid-β (Aβ)-induced microglia activation and neuronal cell death, as well as their effect on the modulation of Aβ aggregation. Primary cortical microglia and neurons were exposed to two different populations of Aβ42 oligomers (Aβ42Os) where the oligomeric state had been assigned by capillary electrophoresis and ultrafiltration. When stimulated with high molecular weight Aβ42Os, microglia released proinflammatory cytokines that led to early neuronal cell death. The studied compounds exerted an anti-inflammatory effect on high molecular weight Aβ42O-stimulated microglia and possibly inhibited microglia-mediated neuronal cell toxicity. Furthermore, the tested compounds demonstrated antioligomeric activity during the process of in vitro Aβ42 aggregation. These findings could be investigated further and used for the optimization of multipotent candidate molecules for AD treatment

Nat. Acad. Sci. US 112 (2015) 5407). The effect of the metal ions can be followed by bulk fluorescence measurements as well as by FCS.