Cytotoxicity and internalization properties of novel alpha7 nicotinic receptor selective peptides.

Abstract

Cell-penetrating peptides (CPPs) contain ≤30 residues and have the unique ability to enter cells, making them attractive agents for Central Nervous System (CNS)-targeted drug delivery. Examples are CPPs derived from the rabies virus glycoprotein (RVG). RVG-derived peptides make use of a nicotinic acetylcholine receptor (nAChR)-mediated mechanism to enter the mouse CNS and deliver cargo. However, previously developed RVG-derived peptides lack selectivity for distinct nAChR subtypes, which are associated with different neurological diseases. As an example, dysregulation of the homomeric α7-nAChR is involved in Alzheimer's disease and depression. In an effort to develop an α7-nAChR-selective CPP, we hypothesized that combining regions of the RVG with those of a protein known to specifically interact with the α7-nAChR subtype could create a chimeric peptide with CPP properties that is α7-nAChR selective. However, insertion of protein segments into RVG brings possibility for altering cytotoxic and cell internalization properties. To investigate these changes for our RVG-chimeric peptides, we cultured and transfected neuronal-like cells with α7 subunits and the nAChR chaperone NACHO, and verified expression using fluorescent tags. We treated cells with RVG or fusion peptides using a range of concentrations to obtain their cytotoxic profiles using a redox indicator for cell viability. Separately, α7-nAChR cells were treated with the lead peptide containing the fluorescent tag FITC and visualized using live cell confocal fluorescence microscopy. We did not observe significant cytotoxic effects of the original RVG or chimeric peptides for concentrations ranging from 0.3 nM to 100 μM. Additionally, our data suggests that the lead chimeric peptide can enter neuronal-like cells by using an α7-nAChR-mediated mechanism. An α7-nAChR subtype-selective therapeutic agent has the potential to aid in depression treatment, without creating unwanted off-target effects. Moreover, it may be useful as vehicle for CNS-targeted drug delivery.