Sonogenetic Modulation of Cellular Activities Using an Engineered Auditory-Sensing Protein

Abstract

Biomolecules that respond to different external stimuli enable the remote control of genetically modified cells. Chemogenetics and optogenetics, two tools that can control cellular activities via synthetic chemicals or photons, respectively, have been widely used to elucidate underlying physiological processes. These methods are, however, very invasive, have poor penetrability, or low spatiotemporal precision, attributes that hinder their use in therapeutic applications. We report herein a sonogenetic approach that can manipulate target cell activities by focused ultrasound stimulation. This system requires an ultrasound-responsive protein derived from an engineered auditory-sensing protein prestin. Heterogeneous expression of mouse prestin containing two parallel amino acid substitutions, N7T and N308S, that frequently exist in prestins from echolocating species endowed transfected mammalian cells with the ability to sense ultrasound. An ultrasound pulse of low frequency and low pressure efficiently evoked cellular calcium responses after transfecting with prestin(N7T, N308S). Moreover, pulsed ultrasound can also non-invasively stimulate target neurons expressing prestin(N7T, N308S) in deep regions of mice brains. Our study delineates how an engineered auditory-sensing protein can cause mammalian cells to sense ultrasound stimulation. Moreover, owing to the great penetration of low-frequency ultrasound (~400 mm in depth), our sonogenetic tools will serve as new strategies for non-invasive therapy in deep tissues of large animals like primates.

Sonogenetic control of mammalian cells using exogenous Transient Receptor Potential A1 channels

“…We previously showed that exogenous expression of the C. elegans TRP-4 mechanoreceptor enables ultrasound-sensitivity in neurons that are otherwise unresponsive to ultrasound stimulation 23 . Similar ultrasound-sensitivity has also been observed in vitro in cells induced to express proteins belonging to the mechanosensitive (MSC) 24 , Piezo 25 , Prestin 26 , transient receptor potential (TRP) 21 , and TREK 27 families. We therefore hypothesized that proteins from these ion channel families and others hypothesized to have mechanosensitivity might confer ultrasound sensitivity to mammalian cells.…”

“…Finally, we demonstrate that hs TRPA1 can be used to selectively activate a specific cell population in vivo with ultrasound pulses (1-100 ms) from a 6.91 MHz transducer. We have not tested additional ultrasound frequencies, which may explain why our screen did not identify previously published candidates 24–26 . Our data hint that ultrasound might not act as a simple stretch force on the membrane, and that channels that likewise sense other perturbations, including lipid bilayer changes, could be good candidates for sonogenetics.…”

“…We previously showed that overexpressing the mechanosensory receptor TRP-4 (a TRP-N homolog) in C. elegans neurons renders them sensitive to short pulses of ultrasound, identifying the first putative sonogenetic candidate 23 . Multiple groups have since identified additional ultrasound-sensitive candidates including MscL 24 , Prestin 26 , Piezo 25 , TREK 27 , MEC-4 22 , TRPC1, TRPP2, and TRPM4 21 using in vitro assays, but demonstration of their activity in vivo has been elusive. We identified hs TRPA1 and its mammalian homologs as top hits in an unbiased screen from a curated library of candidate proteins, emphasizing the unique nature of this protein.…”

Sonogenetics for noninvasive and cellular-level neuromodulation in rodent brain

“…Past research effort has been focused on developing sonogenetics based on mechanosensitive ion channels. One most recent study showed that an engineered auditory-sensing protein, prestin, successfully stimulated neurons in deep regions of mouse brains as evaluated by c-fos staining of ex vivo brain slices 16 . However, FUS-evoked calcium influx in HEK293T cells expressing this protein lasted for more than 100 s after a single FUS stimulation with no evidence to show the calcium influx could be turned off.…”

“…Other mechanosensitive ion channels, for example, TREK-1 11 , MscL 12 , Piezo1 13,14 , and TRPA1 15 have been proposed as potential sonogenetic tools based on in vitro cell culture studies without in vivo validation. A most recent study showed that an engineered auditory-sensing protein (prestin) caused neurons in the mouse brain to sense ultrasound stimulation 16 ; however, this protein, which is not an ion channel, lacks the ability to control neuron activity in a temporally precise fashion. To our knowledge, there is so far no study that has identified ultrasoundsensitive ion channels for spatiotemporally precise modulation of neural activity in mammalian brains.…”