Enhancing prestin oligomerization for superior sonogenetics.

Background: Sonogenetics enables non-invasive control of cellular activity using ultrasound (US)-responsive proteins. Prestin, a motor protein from outer hair cells, can render cells responsive to US when ectopically expressed, offering potential therapeutic applications for neurodegenerative diseases and epilepsy. However, current prestin variants exhibit suboptimal oligomerization, reducing their efficacy in neuromodulation. This study aimed to enhance prestin sonogenetic performance by improving oligomerization. Methods: We screened four echolocating bat prestin orthologs and point mutants of mouse prestin variants for enhanced US responsiveness. Subcellular distribution and oligomerization of prestin variants were evaluated using live-cell imaging and Förster resonance energy transfer. Calcium imaging was used to assess functional responses in HEK293T cells and cultured neurons. In vivo functionality was tested in mice by AAV-mediated expression of prestin variants in hippocampal neurons and motor cortex, followed by transcranial US stimulation and c-Fos staining to assess neuronal activation. To evaluate cross-species efficacy, we expressed prestin variants in ray sensory neurons of Caenorhabditis elegans and quantified US-evoked behavioral responses. Tissue safety was confirmed via histological analysis in mice. Results: Echolocating bat prestins did not improve US responsiveness compared to mouse prestin. In contrast, an engineered variant, Prestin(N548S, V715G), exhibited enhanced oligomerization and punctate membrane localization. This variant triggered stronger Ca²⁺ influx in vitro. In vivo, US stimulation of the cortex expressing Prestin(N548S, V715G) induced robust neuronal activation, as indicated by increased c-Fos expression and evoked tail movement in mice. In Caenorhabditis elegans, expression of Prestin(N548S, V715G) in ray neurons conferred significant US-evoked behavioral responses. No adverse histological effects were observed in mouse tissue. Conclusion: Our findings show that enhancing prestin oligomerization, rather than relying on naturally evolved echolocating variants, significantly improves its sonogenetic efficacy. The Prestin(N548S, V715G) variant represents a potent and biosafe tool for non-invasive neuromodulation and holds promise for future therapeutic applications in neurological disorders.