Copper (Cu) has been implicated in the progression of Alzheimer’s disease (AD), and aggregation of Cu and amyloid β peptide (Aβ) are considered key pathological features of AD. Metal chelators are considered to be potential therapeutic agents for AD because of their capacity to reduce metal ion-induced Aβ aggregation through the regulation of metal ion distribution. Here, we used phage display technology to screen, synthesize, and evaluate a novel Cu(II)-binding peptide that specifically blocked Cu-triggered Aβ aggregation. The Cu(II)-binding peptide (S-A-Q-I-A-P-H, PCu) identified from the phage display heptapeptide library was used to explore the mechanism of PCu inhibition of Cu²⁺-mediated Aβ aggregation and Aβ production. In vitro experiments revealed that PCu directly inhibited Cu²⁺-mediated Aβ aggregation and regulated copper levels to reduce biological toxicity. Furthermore, PCu reduced the production of Aβ by inhibiting Cu²⁺-induced BACE1 expression and improving Cu(II)-mediated cell oxidative damage. Cell culture experiments further demonstrated that PCu had relatively low toxicity. This Cu(II)-binding peptide that we have identified using phage display technology provides a potential therapeutic approach to prevent or treat AD.

Zn²⁺ has been implicated in the progression of Alzheimer's disease (AD), as amyloid-β protein (Aβ) aggregation and neurotoxicity are mediated by zinc ions. Therefore, development of metal chelators for inhibiting and regulating metal-triggered Aβ aggregation has received attention as a strategy for treating AD. Here, we used an approach based on phage display to screen for a Zn(II)-binding peptide that specifically blocks Zn-triggered Aβ aggregation. A fixed Zn(II) resin was prepared using Ni-IDA affinity resin, and the target Zn(II) was screened by interaction with a heptapeptide phage library. After negative biopanning against IDA and four rounds of positive biopanning against Zn(II), high specificity Zn(II)-binding phages were obtained. Through DNA sequencing and ELISA, 15 sets of Zn(II)-binding peptides with high histidine contents were identified. We chose a highly specific peptide against Zn(II) with the sequence of H-M-Q-T-N-H-H, and its abilities to chelate Zn²⁺ and inhibit Zn²⁺-mediated Aβ aggregation were assessed in vitro. We loaded the Zn(II)-binding peptide onto PEG-modified chitosan nanoparticles (NPs) to improve the stability and the bioavailability of the Zn(II) binding peptide. PEG-modified chitosan NPs loaded with Zn(II)-binding peptide (PEG/PZn-CS NPs) reduced Zn²⁺ concentrations and Aβ secretion in mouse neuroblastoma (N)2a cells stably over-expressing the APP Swedish mutation (N2aswe). Zn²⁺-Induced neurotoxicity, oxidative stress, and apoptosis were attenuated by PEG/PZn-CS NPs. Intranasal administration of PEG/PZn-CS NPs improved the cognitive ability of APPswe/PS1d9 (APP/PS1) double-transgenic mice and reduced Aβ plaques in the mouse brain. This study indicated that a Zn(II)-binding peptide and its NPs have promise as a potential anti-AD agent.

Alzheimer’s disease (AD) is a type of neurodegenerative disease that is associated with the accumulation of amyloid plaques. Increasing non-amyloidogenic processing and/or manipulating amyloid precursor protein signaling could reduce AD amyloid pathology and cognitive impairment. D-penicillamine (D-Pen) is a water-soluble metal chelator and can reduce the aggregation of amyloid-β (Aβ) with metals in vitro. However, the potential mechanism of D-Pen for treating neurodegenerative disorders remains unexplored. In here, a novel type of chitosan-based hydrogel to carry D-Pen was designed and the D-Pen-CS/β-glycerophosphate hydrogel were characterized by scanning electron microscopy and HPLC. Behavior tests investigated the learning and memory levels of APP/PS1 mice treated through the D-Pen hydrogel nasal delivery. In vivo and in vitro findings showed that nasal delivery of D-Pen-CS/β-GP hydrogel had properly chelated metal ions that reduced Aβ deposition. Furthermore, D-Pen mainly regulated A disintegrin and metalloprotease 10 (ADAM10) expression via melatonin receptor 1 (MTNR1α) and the downstream PKA/ERK/CREB pathway. The present data demonstrated D-Pen significantly improved the cognitive ability of APP/PS1 mice and reduced Aβ generation through activating ADAM10 and accelerating non-amyloidogenic processing. Hence, these findings indicate the potential of D-Pen as a promising agent for treating AD.