The mechanosensitive ion channel Piezo1 contributes to ultrasound neuromodulation.

Transcranial low-intensity ultrasound is a promising neuromodulation modality, with the advantages of noninvasiveness, deep penetration, and high spatiotemporal accuracy. However, the underlying biological mechanism of ultrasonic neuromodulation remains unclear, hindering the development of efficacious treatments. Here, the well-known Piezo1 was studied through a conditional knockout mouse model as a major mediator for ultrasound neuromodulation ex vivo and in vivo. We showed that Piezo1 knockout (P1KO) in the right motor cortex of mice significantly reduced ultrasound-induced neuronal calcium responses, limb movement, and muscle electromyogram (EMG) responses. We also detected higher Piezo1 expression in the central amygdala (CEA), which was found to be more sensitive to ultrasound stimulation than the cortex was. Knocking out the Piezo1 in CEA neurons showed a significant reduction of response under ultrasound stimulation, while knocking out astrocytic Piezo1 showed no-obvious changes in neuronal responses. Additionally, we excluded an auditory confound by monitoring auditory cortical activation and using smooth waveform ultrasound with randomized parameters to stimulate P1KO ipsilateral and contralateral regions of the same brain and recording evoked movement in the corresponding limb. Thus, we demonstrate that Piezo1 is functionally expressed in different brain regions and that it is an important mediator of ultrasound neuromodulation in the brain, laying the ground for further mechanistic studies of ultrasound.

Modulation of deep neural circuits with sonogenetics.

Noninvasive control of neuronal activity in the deep brain can be illuminating for probing brain function and treating dysfunctions. Here, we present a sonogenetic approach for controlling distinct mouse behavior with circuit specificity and subsecond temporal resolution. Targeted neurons in subcortical regions were made to express a mutant large conductance mechanosensitive ion channel (MscL-G22S), enabling ultrasound to trigger activity in MscL-expressing neurons in the dorsal striatum and increase locomotion in freely moving mice. Ultrasound stimulation of MscL-expressing neurons in the ventral tegmental area could activate the mesolimbic pathway to trigger dopamine release in the nucleus accumbens and modulate appetitive conditioning. Moreover, sonogenetic stimulation of the subthalamic nuclei of Parkinson's disease model mice improved their motor coordination and mobile time. Neuronal responses to ultrasound pulse trains were rapid, reversible, and repeatable. We also confirmed that the MscL-G22S mutant is more effective to sensitize neurons to ultrasound compared to the wild-type MscL. Altogether, we lay out a sonogenetic approach which can selectively manipulate targeted cells to activate defined neural pathways, affect specific behaviors, and relieve symptoms of neurodegenerative disease.

Blocking Stemness and Metastatic Properties of Ovarian Cancer Cells by Targeting p70S6K with Dendrimer Nanovector-Based siRNA Delivery.

Metastasis is the cause of most (>90%) cancer deaths and currently lacks effective treatments. Approaches to understanding the biological process, unraveling the most effective molecular target(s), and implementing nanotechnology to increase the therapeutic index are expected to facilitate cancer therapy against metastasis. Here, we demonstrate the potential advantages of bringing these three approaches together through the rational design of a small interfering RNA (siRNA) that targets p70S6K in cancer stem cells (CSCs) in combination with dendrimer nanotechnology-based siRNA delivery. Our results demonstrated that the generation 6 (G6) poly(amidoamine) dendrimer can be used as a nanovector to effectively deliver p70S6K siRNA by forming uniform dendriplex nanoparticles that protect the siRNA from degradation. These nanoparticles were able to significantly knock down p70S6K in ovarian CSCs, leading to a marked reduction in CSC proliferation and expansion without obvious toxicity toward normal ovarian surface epithelial cells. Furthermore, treatment with the p70S6K siRNA/G6 dendriplexes substantially decreased mesothelial interaction, migration and invasion of CSCs in vitro, as well as tumor growth and metastasis in vivo. Collectively, these results suggest that p70S6K constitutes a promising therapeutic target, and the use of siRNA in combination with nanotechnology-based delivery may constitute a new approach for molecularly targeted cancer therapy to treat metastasis.

PINK1/Parkin-Mediated Mitophagy Promotes Resistance to Sonodynamic Therapy.

BACKGROUND/AIMS: Sonodynamic therapy (SDT), based on the synergistic effect of low-intensity ultrasound and sonosensitizer, is a potential approach for non-invasive treatment of cancers. In SDT, mitochondria played a crucial role in cell fate determination. However, mitochondrial activities and their response to SDT remain elusive. The purpose of this study was to examine the response of mitochondria to SDT in tumor cells. METHODS: A human breast adenocarcinoma cell line - MCF-7 cells were subjected to 5-aminolevulinic acid (ALA)-SDT, with an average ultrasonic intensity of 0.25W/cm2. Mitochondrial dynamics and redox balance were examined by confocal immunofluorescence microscopy and western blot. The occurrence of mitophagy was determined by confocal immunofluorescence microscopy. RESULTS: Our results showed that ALA-SDT could induce mitochondrial dysfunction through mitochondrial depolarization and fragmentation and lead to mitophagy. The Parkin-dependent signaling pathway was involved and promoted resistance to ALA-SDT induced cell death. Finally, excessive production of ROS was found to be necessary for the initiation of mitophagy. CONCLUSION: Taken together, we conclude that ROS produced by 5-ALA-SDT could initiate PINK1/Parkin-mediated mitophagy which may exert a protective effect against 5-ALA-SDT-induced cell death in MCF-7 cells.

The mechanosensitive ion channel Piezo1 modulates the migration and immune response of microglia.

Microglia are the brain's resident immune cells, performing surveillance to promote homeostasis and healthy functioning. While microglial chemical signaling is well-studied, mechanical cues regulating their function are less well-understood. Here, we investigate the role of the mechanosensitive ion channel Piezo1 in microglia migration, pro-inflammatory cytokine production, and stiffness sensing. In Piezo1 knockout transgenic mice, we demonstrated the functional expression of Piezo1 in microglia and identified genes whose expression was consequently affected. Functional assays revealed that Piezo1 deficiency in microglia enhanced migration toward amyloid ß-protein, and decreased levels of pro-inflammatory cytokines produced upon stimulation by lipopolysaccharide, both in vitro and in vivo. The phenomenon could be mimicked or reversed chemically using a Piezo1-specific agonist or antagonist. Finally, we also showed that Piezo1 mediated the effect of substrate stiffness-induced migration and cytokine expression. Altogether, we show that Piezo1 is an important molecular mediator for microglia, its activation modulating microglial migration and immune responses.

Photonic Nanojet-Mediated Optogenetics.

Optogenetics has become a widely used technique in neuroscience research, capable of controlling neuronal activity with high spatiotemporal precision and cell-type specificity. Expressing exogenous opsins in the selected cells can induce neuronal activation upon light irradiation, and the activation depends on the power of incident light. However, high optical power can also lead to off-target neuronal activation or even cell damage. Limiting the incident power, but enhancing power distribution to the targeted neurons, can improve optogenetic efficiency and reduce off-target effects. Here, the use of optical lenses made of polystyrene microspheres is demonstrated to achieve effective focusing of the incident light of relatively low power to neighboring neurons via photonic jets. The presence of microspheres significantly localizes and enhances the power density to the target neurons both in vitro and ex vivo, resulting in increased inward current and evoked action potentials. In vivo results show optogenetic stimulation with microspheres that can evoke significantly more motor behavior and neuronal activation at lowered power density. In all, a proof-of-concept of a strategy is demonstrated to increase the efficacy of optogenetic neuromodulation using pulses of reduced optical power.

Behavioral and Functional Assessment of Ultrasound Neuromodulation on Caenorhabditis Elegans.

Ultrasound brain stimulation is a promising modality for probing brain function and treating brain diseases. However, its mechanism is as yet unclear, and in vivo effects are not well-understood. Here, we present a top-down strategy for assessing ultrasound bioeffects in vivo, using Caenorhabditis elegans. Behavioral and functional changes of single worms and of large populations upon ultrasound stimulation were studied. Worms were observed to significantly increase their average speed upon ultrasound stimulation, adapting to it upon continued treatment. Worms also generated more reversal turns when ultrasound was ON, and within a minute post-stimulation, they performed significantly more reversal and omega turns than prior to ultrasound. In addition, in vivo calcium imaging showed that the neural activity in the worms' heads and tails was increased significantly by ultrasound stimulation. In all, we conclude that ultrasound can directly activate the neurons of worms in vivo, in both of their major neuronal ganglia, and modify their behavior.

Protocol for the sonogenetic stimulation of mouse brain by non-invasive ultrasound.

Manipulating specific neural activity by targeted ultrasound intervention is a powerful method to gain causal insight into brain functions and treat brain disorders. The technique of sonogenetics enables controlling of cells that are genetically modulated with ultrasound-sensitive ion channels. Here, we detail the preparations, surgical procedures, ultrasound stimulation process, and simultaneous electromyogram (EMG) measurement necessary for successful sonogenetic stimulation in mice. For complete details on the use and execution of this protocol, please refer to Qiu et al. (2020).

Precise Ultrasound Neuromodulation in a Deep Brain Region Using Nano Gas Vesicles as Actuators.

Ultrasound is a promising new modality for non-invasive neuromodulation. Applied transcranially, it can be focused down to the millimeter or centimeter range. The ability to improve the treatment's spatial resolution to a targeted brain region could help to improve its effectiveness, depending upon the application. The present paper details a neurostimulation scheme using gas-filled nanostructures, gas vesicles (GVs), as actuators for improving the efficacy and precision of ultrasound stimuli. Sonicated primary neurons display dose-dependent, repeatable Ca2+ responses, closely synced to stimuli, and increased nuclear expression of the activation marker c-Fos in the presence of GVs. GV-mediated ultrasound triggered rapid and reversible Ca2+ responses in vivo and could selectively evoke neuronal activation in a deep-seated brain region. Further investigation indicate that mechanosensitive ion channels are important mediators of this effect. GVs themselves and the treatment scheme are also found not to induce significant cytotoxicity, apoptosis, or membrane poration in treated cells. Altogether, this study demonstrates a simple and effective method to achieve enhanced and better-targeted neurostimulation with non-invasive low-intensity ultrasound.

Biogenic nanobubbles for effective oxygen delivery and enhanced photodynamic therapy of cancer.

Tumor hypoxia is believed to be a factor limiting successful outcomes of oxygen-consuming cancer therapy, thereby reducing patient survival. A key strategy to overcome tumor hypoxia is to increase the prevalence of oxygen at the tumor site. Oxygen-containing microbubbles/nanobubbles have been developed to supply oxygen and enhance the effects of therapies such as radiotherapy and photodynamic therapy. However, the application of these bubbles is constrained by their poor stability, requiring major workarounds to increase their half-lives. In this study, we explore the potential of biogenic gas vesicles (GVs) as a new kind of oxygen carrier to alleviate tumor hypoxia. GVs, which are naturally formed, gas-filled, protein-shelled compartments, were modified on the surface of their protein shells by a layer of liposome. A substantial improvement of oxygen concentration was observed in hypoxic solution, in hypoxic cells, as well as in subcutaneous tumors when lipid-GVs(O2) were added/tail-injected. Significant enhancement of tumor cell apoptosis and necrosis was also observed during photodynamic therapy (PDT) in the presence of lipid-GVs(O2) both in vitro and in vivo. Lipid-GVs(O2) alone induced no obvious change in cell viability in vitro or any apparent pathological abnormalities after mice were tail-injected with them. In all, lipid-GVs exhibited promising performance for intravenous gas delivery, enhanced PDT efficacy and low toxicity, a quality that may be applied to alleviate hypoxia in cancers, as well as hypoxia-related clinical treatments. STATEMENT OF SIGNIFICANCE: The development of stable oxygen-filled micro/nanobubbles capable of delivering oxygen to tumor sites is a major hurdle to enhancing the efficacy of cancer therapy. Currently, micro/nanobubbles are limited by their instability when oxygen is encapsulated, creating a large pressure gradient and surface tension. To improve stability, we modified the surfaces of GVs, a biogenic stable nanoscale hollow structure, as a new class of oxygen carriers. Lipid-coated GVs were found to be stable in solution and effective O2 carriers. This will overcome the limitations of coalescence, short circulation time of synthetic bubbles during application. Our surface-modified GVs demonstrated low toxicity in vitro cell in vivo, while also being able to overcome hypoxia-associated therapy resistance when combined with photodynamic therapy.

Surface-modified GVs as nanosized contrast agents for molecular ultrasound imaging of tumor.

Nanobubbles, as a kind of new ultrasound contrast agent (UCAs), have shown promise to penetrate tumor vasculature to allow for targeted imaging. However, their inherent physical instability is an ongoing concern that could weaken their imaging ability with ultrasound. Gas vesicles (GVs), which are genetically encoded, naturally stable nanostructures, have been developed as the first ultrasonic biomolecular reporters which showed strong contrast enhancement. However, further development of tumor imaging with GVs is limited by the quick clearance of GVs by the reticuloendothelial system (RES). Here, we developed PEGylated HA-GVs (PH-GVs) for in-tumor molecular ultrasound imaging by integrating polyethylene glycol (PEG) and hyaluronic acid (HA) in GV shells. PH-GVs were observed to accumulate around CD44-positive cells (SCC7) but not be internalized by macrophage cell line RAW 264.7. Green fluorescence from PH-GVs was found around cell nuclei in the tumor site after 6 h and the signal was sustained over 48 h following tail injection, demonstrating PH-GVs' ability to escape the clearance from the RES and to penetrate tumor vasculature through enhanced permeability and retention (EPR) effects. Further, PH-GVs produced strong ultrasound contrast in the tumor site in vivo, with no obvious side-effects detected following intravenous injection. Thus, we demonstrate the potential of PH-GVs as novel, nanosized and targeted UCAs for efficient and specific molecular tumor imaging, paving the way for the application of GVs in precise and personalized medicine.

Targeted Neurostimulation in Mouse Brains with Non-invasive Ultrasound.

Recently developed brain stimulation techniques have significantly advanced our ability to manipulate the brain's function. However, stimulating specific neurons in a desired region without significant surgical invasion remains a challenge. Here, we demonstrate a neuron-specific and region-targeted neural excitation strategy using non-invasive ultrasound through activation of heterologously expressed mechanosensitive ion channels (MscL-G22S). Low-intensity ultrasound is significantly better at inducing Ca2+ influx and neuron activation in vitro and at evoking electromyogram (EMG) responses in vivo in targeted cells expressing MscL-G22S. Neurons in the cerebral cortex or dorsomedial striatum of mice are made to express MscL-G22S and stimulated ultrasonically. We find significant upregulation of c-Fos in neuron nuclei only in the regions expressing MscL-G22S compared with the non-MscL controls, as well as in various other regions in the same brain. Thus, we detail an effective approach for activating specific regions and cell types in intact mouse brains by sensitizing them to ultrasound using a mechanosensitive ion channel.

The Mechanosensitive Ion Channel Piezo1 Significantly Mediates In Vitro Ultrasonic Stimulation of Neurons.

Ultrasound brain stimulation is a promising modality for probing brain function and treating brain disease non-invasively and with high spatiotemporal resolution. However, the mechanism underlying its effects remains unclear. Here, we examine the role that the mouse piezo-type mechanosensitive ion channel component 1 (Piezo1) plays in mediating the in vitro effects of ultrasound in mouse primary cortical neurons and a neuronal cell line. We show that ultrasound alone could activate heterologous and endogenous Piezo1, initiating calcium influx and increased nuclear c-Fos expression in primary neurons but not when pre-treated with a Piezo1 inhibitor. We also found that ultrasound significantly increased the expression of the important proteins phospho-CaMKII, phospho-CREB, and c-Fos in a neuronal cell line, but Piezo1 knockdown significantly reduced this effect. Our findings demonstrate that the activity of mechanosensitive ion channels such as Piezo1 stimulated by ultrasound is an important contributor to its ability to stimulate cells in vitro.

Combination of dendrimer-nanovector-mediated small interfering RNA delivery to target Akt with the clinical anticancer drug paclitaxel for effective and potent anticancer activity in treating ovarian cancer.

The recently discovered small interfering RNA (siRNA) holds great promise in cancer therapy. However, efficient and safe delivery systems are required for the development of new therapeutic paradigms. Ovarian cancer has the highest mortality of all gynecologic tumors, and there is an urgent need for specific and effective therapies. The phosphatidylinositol 3-kinase/Akt pathway, which is strongly implicated in the biology of ovarian cancer, constitutes an attractive therapeutic target. In this study, we describe a triethanolamine-core poly(amidoamine) dendrimer which forms stable nanoparticles with the Akt siRNA, protects siRNA against RNase digestion, and is highly effective for initiating Akt target-gene silencing both in vitro and in vivo, while being minimally toxic. Most importantly, it could potentiate the antitumor effect of the anticancer drug paclitaxel. These results represent the proof-of-concept, demonstrating that dendrimer-mediated Akt siRNA delivery, in combination with a chemotherapeutic regimen, may constitute a promising nanomedicine approach in cancer therapy.