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Dordick, J.

Publications and source records attributed to Dordick, J..

2 recordsLinked to original sources
bioRxiv2023

Alternating Magnetic Fields Drive Stimulation of Gene Expression via Generation of Reactive Oxygen Species

Magnetogenetics represents a method for remote control of cellular function. Despite successful use of magnetic fields to control gene expression in vitro and in vivo, the mechanism underlying magnetogenetics is largely unknown, thereby hindering further development and applications. Previous work suggests a chemical-based mechanism involving the generation of reactive oxygen species (ROS) as a platform initiator and downstream signaling molecule. Herein, a chemical biology approach was used to elucidate further the mechanism of radio frequency-alternating magnetic field (RF-AMF) stimulation of a TRPV1-ferritin magnetogenetics platform that leads to Ca2+ gating. RF-AMF stimulation of HEK 293T cells expressing TRPV1-Ferritin resulted in a [~]30% and [~]140% increase in intra- and extracellular ROS levels, respectively. Mutations to specific cysteine residues in TRPV1 responsible for ROS sensitivity eliminated RF-AMF driven Ca2+-dependent transcription of secreted embryonic alkaline phosphatase (SEAP). Using a non-tethered (to TRPV1) ferritin also eliminated RF-AMF driven SEAP production. These results suggest ferritin-dependent ROS activation of TRPV1 plays a key role in the initiation of magnetogenetics. Furthermore, inhibition of IP3R-based endoplasmic reticulum (ER) Ca2+ release with Xestospongin C, inhibition of protein kinase C (PKC) activity with Go 6983, or inhibition of NADPH oxidase (NOX) isoforms 1/2 gp91phoxcytochrome with GSK 2795039 eliminated short-term RF-AMF potentiation of SEAP production with capsaicin. Similarly, Go 6983 significantly reduced long-term RF-AMF capsaicin potentiation of SEAP production. These results suggest that ROS-activated TRPV1 signaling to increase intracellular Ca2+ includes pathways involving PKC, NOX, and the ER. Together, these findings fill in current knowledge gaps in the mechanism of magnetogenetics, which may lead to translational applications in medicine and biotechnology.

bioengineering↗
bioRxiv2023

Magnetogenetic cell activation using endogenous ferritin

The ability to precisely control the activity of defined cell populations enables studies of their physiological roles and may provide therapeutic applications. While prior studies have shown that magnetic activation of ferritin-tagged ion channels allows cell-specific modulation of cellular activity, the large size of the constructs made the use of adeno-associated virus, AAV, the vector of choice for gene therapy, impractical. In addition, simple means for generating magnetic fields of sufficient strength have been lacking. Toward these ends, we first generated a novel anti-ferritin nanobody that when fused to transient receptor potential cation channel subfamily V member 1, TRPV1, enables direct binding of the channel to endogenous ferritin in mouse and human cells. This smaller construct can be delivered in a single AAV and we validated that it robustly enables magnetically induced cell activation in vitro. In parallel, we developed a simple benchtop electromagnet capable of gating the nanobody-tagged channel in vivo. Finally, we showed that delivering these new constructs by AAV to pancreatic beta cells in combination with the benchtop magnetic field delivery stimulates glucose-stimulated insulin release to improve glucose tolerance in mice in vivo. Together, the novel anti-ferritin nanobody, nanobody-TRPV1 construct and new hardware advance the utility of magnetogenetics in animals and potentially humans.

bioengineering↗