Novel Tools towards Magnetic Guidance of Neurite Growth: (I) Guidance of Magnetic Nanoparticles into Neurite Extensions of Induced Human Neurons and In Vitro Functionalization with RAS Regulating Proteins

Abstract

Parkinson’s disease (PD) is a neurodegenerative disease associated with loss or dysfunction of dopaminergic neurons located in the substantia nigra (SN), and there is no cure available. An emerging new approach for treatment is to transplant human induced dopaminergic neurons directly into the denervated striatal brain target region. Unfortunately, neurons grafted into the substantia nigra are unable to grow axons into the striatum and thus do not allow recovery of the original connectivity. Towards overcoming this general limitation in guided neuronal regeneration, we develop here magnetic nanoparticles functionalized with proteins involved in the regulation of axonal growth. We show covalent binding of constitutive active human rat sarcoma (RAS) proteins or RAS guanine nucleotide exchange factor catalytic domain of son of sevenless (SOS) by fluorescence correlation spectroscopy and multiangle light scattering as well as the characterization of exchange factor activity. Human dopaminergic neurons were differentiated from neural precursor cells and characterized by electrophysiological and immune histochemical methods. Furthermore, we demonstrate magnetic translocation of cytoplasmic γ-Fe2O3@SiO2 core-shell nanoparticles into the neurite extensions of induced human neurons. Altogether, we developed tools towards remote control of directed neurite growth in human dopaminergic neurons. These results may have relevance for future therapeutic approaches of cell replacement therapy in Parkinson’s disease.

Parallelized Manipulation of Adherent Living Cells by Magnetic Nanoparticles-Mediated Forces

“…Such engineered MNPs have been used to modulate the spatial distribution of signaling molecules involved in cellular polarity, thus enabling the local formation of membrane protrusions [ 11 ]. Recent studies have proposed promising strategies with MNPs designed to activate specifically axonal growth in reprogrammed neurons from induced pluripotent stems cells [ 12 , 13 ]. However, these innovative approaches are very complex and will need further investigations before being really effective for therapeutic applications.…”

Parallelized manipulation of adherent living cells by magnetic nanoparticles-mediated forces

“…Such engineered MNPs have been used to modulate the spatial distribution of signaling molecules involved in cellular polarity, thus enabling the local formation of membrane protrusions (Etoc et al 2015). Recent studies have proposed promising strategies with MNPs designed to activate specifically axonal growth in reprogrammed neurons from induced pluripotent stems cells (Jin et al 2019;Schöneborn et al 2019). However, these innovative approaches are very complex and will need further investigations before being really effective for therapeutic applications.…”

Magnetic spatiotemporal control of SOS1 coupled nanoparticles for guided neurite growth in dopaminergic single cells

“…To analyze the in vitro capturing of HT fusion proteins by MNPs, MNPs were measured by multiangle light scattering (MALS) after the addition of purified HT-H-RAS V12 and HT-SOS1cat fusion proteins (Table 1 ), as published recently 26 . The addition of HT-H-RAS V12 to HTL-MNPs significantly increased the hydrodynamic radius (r h ) from 40.3 ± 1.5 nm to 42.5 ± 1.3 nm.…”

“…2 ). To monitor the cytoplasmic mobility of the fusion protein when bound to HTL-MNPs, we cloned a Clover-tagged version of SOS1cat fusion proteins and determined its GEF activity 26 . In order to preserve the C-terminal membrane anchoring mechanism of HT-H-RAS V12 , we chose to generate an internal ribosome entry site (IRES) construct that expressed HT-H-RAS V12 and Clover as separate proteins to detect transfected cells (Fig.…”