Astrocytic Piezo1-mediated mechanotransduction determines adult neurogenesis and cognitive functions.

Adult brain activities are generally believed to be dominated by chemical and electrical transduction mechanisms. However, the importance of mechanotransduction mediated by mechano-gated ion channels in brain functions is less appreciated. Here, we show that the mechano-gated Piezo1 channel is expressed in the exploratory processes of astrocytes and utilizes its mechanosensitivity to mediate mechanically evoked Ca2+ responses and ATP release, establishing Piezo1-mediated mechano-chemo transduction in astrocytes. Piezo1 deletion in astrocytes causes a striking reduction of hippocampal volume and brain weight and severely impaired (but ATP-rescuable) adult neurogenesis in vivo, and it abolishes ATP-dependent potentiation of neural stem cell (NSC) proliferation in vitro. Piezo1-deficient mice show impaired hippocampal long-term potentiation (LTP) and learning and memory behaviors. By contrast, overexpression of Piezo1 in astrocytes sufficiently enhances mechanotransduction, LTP, and learning and memory performance. Thus, astrocytes utilize Piezo1-mediated mechanotransduction mechanisms to robustly regulate adult neurogenesis and cognitive functions, conceptually highlighting the importance of mechanotransduction in brain structure and function.

The mechanosensitive Piezo1 channel is required for bone formation.

Mechanical load of the skeleton system is essential for the development, growth, and maintenance of bone. However, the molecular mechanism by which mechanical stimuli are converted into osteogenesis and bone formation remains unclear. Here we report that Piezo1, a bona fide mechanotransducer that is critical for various biological processes, plays a critical role in bone formation. Knockout of Piezo1 in osteoblast lineage cells disrupts the osteogenesis of osteoblasts and severely impairs bone structure and strength. Bone loss that is induced by mechanical unloading is blunted in knockout mice. Intriguingly, simulated microgravity treatment reduced the function of osteoblasts by suppressing the expression of Piezo1. Furthermore, osteoporosis patients show reduced expression of Piezo1, which is closely correlated with osteoblast dysfunction. These data collectively suggest that Piezo1 functions as a key mechanotransducer for conferring mechanosensitivity to osteoblasts and determining mechanical-load-dependent bone formation, and represents a novel therapeutic target for treating osteoporosis or mechanical unloading-induced severe bone loss.

Author Correction: Structure and mechanogating mechanism of the Piezo1 channel.

In Extended Data Fig. 9a of this Article, the bottom micrographs of mPiezo1-ΔL3-4-IRES-GFP and mPiezo1-ΔL7-8-IRES-GFP (labelled 'permeabilized') are inadvertently the same images. The corrected figure panels are shown in the accompanying Amendment.

A lever-like transduction pathway for long-distance chemical- and mechano-gating of the mechanosensitive Piezo1 channel.

Piezo1 represents a prototype of eukaryotic mechanotransduction channels. The full-length 2547-residue mouse Piezo1 possesses a unique 38-transmembrane-helix (TM) topology and is organized into a three-bladed, propeller-shaped architecture, comprising a central ion-conducting pore, three peripheral blade-like structures, and three 90-Å-long intracellular beam-resembling structures that bridge the blades to the pore. However, how mechanical force and chemicals activate the gigantic Piezo1 machinery remains elusive. Here we identify a novel set of Piezo1 chemical activators, termed Jedi, which activates Piezo1 through the extracellular side of the blade instead of the C-terminal extracellular domain of the pore, indicating long-range allosteric gating. Remarkably, Jedi-induced activation of Piezo1 requires the key mechanotransduction components, including the two extracellular loops in the distal blade and the two leucine residues in the proximal end of the beam. Thus, Piezo1 employs the peripheral blade-beam-constituted lever-like apparatus as a designated transduction pathway for long-distance mechano- and chemical-gating of the pore.

Structure and mechanogating mechanism of the Piezo1 channel.

The mechanosensitive Piezo channels function as key eukaryotic mechanotransducers. However, their structures and mechanogating mechanisms remain unknown. Here we determine the three-bladed, propeller-like electron cryo-microscopy structure of mouse Piezo1 and functionally reveal its mechanotransduction components. Despite the lack of sequence repetition, we identify nine repetitive units consisting of four transmembrane helices each-which we term transmembrane helical units (THUs)-which assemble into a highly curved blade-like structure. The last transmembrane helix encloses a hydrophobic pore, followed by three intracellular fenestration sites and side portals that contain pore-property-determining residues. The central region forms a 90 Å-long intracellular beam-like structure, which undergoes a lever-like motion to connect THUs to the pore via the interfaces of the C-terminal domain, the anchor-resembling domain and the outer helix. Deleting extracellular loops in the distal THUs or mutating single residues in the beam impairs the mechanical activation of Piezo1. Overall, Piezo1 possesses a unique 38-transmembrane-helix topology and designated mechanotransduction components, which enable a lever-like mechanogating mechanism.

A protein interaction mechanism for suppressing the mechanosensitive Piezo channels.

Piezo proteins are bona fide mammalian mechanotransduction channels for various cell types including endothelial cells. The mouse Piezo1 of 2547 residues forms a three-bladed, propeller-like homo-trimer comprising a central pore-module and three propeller-structures that might serve as mechanotransduction-modules. However, the mechanogating and regulation of Piezo channels remain unclear. Here we identify the sarcoplasmic /endoplasmic-reticulum Ca2+ ATPase (SERCA), including the widely expressed SERCA2, as Piezo interacting proteins. SERCA2 strategically suppresses Piezo1 via acting on a 14-residue-constituted intracellular linker connecting the pore-module and mechanotransduction-module. Mutating the linker impairs mechanogating and SERCA2-mediated modulation of Piezo1. Furthermore, the synthetic linker-peptide disrupts the modulatory effects of SERCA2, demonstrating the key role of the linker in mechanogating and regulation. Importantly, the SERCA2-mediated regulation affects Piezo1-dependent migration of endothelial cells. Collectively, we identify SERCA-mediated regulation of Piezos and the functional significance of the linker, providing important insights into the mechanogating and regulation mechanisms of Piezo channels.

Heterologous Expression of the Piezo1-ASIC1 Chimera Induces Mechanosensitive Currents with Properties Distinct from Piezo1.

Piezo1 represents a prototype of the mammalian mechanosensitive cation channel, but its molecular mechanism remains elusive. In a recent study, we showed that C-terminal region, which contains the last two TMs, of 2189-2547 of Piezo1 forms the bona fide pore module, and systematically identified the pore-lining helix and key pore-property-determining residues (Zhao et al., 2016). Furthermore, we have engineered the Piezo1(1-2190)-ASIC1 chimera (fusing the N-terminal region of 1-2190 to the mechano-insensitive ASIC1) that mediated mechanical- and acid-evoked currents in HEK293T cells, indicating the sufficiency of the N-terminal region in mechanotransduction. Now in a Matters Arising, the authors specifically questioned the implication of the chimera data among the many findings shown in our paper. They replicated the chimera-mediated mechanosensitive currents in HEK293T cells that have nearly no detectable expression of endogenous Piezo1, but paradoxically found the chimera to be less effective in Piezo1 knockout HEK293T cells, indicating the involvement of endogenous Piezo1. In this Matters Arising Response, we discuss the chimera results and consider potential interpretations in light of the Matters Arising from Dubin et al. (2017), published concurrently in this issue of Neuron. Please see also the response from Hong et al. (2017), published in this issue.

Ion Permeation and Mechanotransduction Mechanisms of Mechanosensitive Piezo Channels.

Piezo proteins have been proposed as the long-sought-after mechanosensitive cation channels in mammals that play critical roles in various mechanotransduction processes. However, the molecular bases that underlie their ion permeation and mechanotransduction have remained functionally undefined. Here we report our finding of the miniature pore-forming module of Piezo1 that resembles the pore architecture of other trimeric channels and encodes the essential pore properties. We further identified specific residues within the pore module that determine unitary conductance, pore blockage and ion selectivity for divalent and monovalent cations and anions. The non-pore-containing region of Piezo1 confers mechanosensitivity to mechano-insensitive trimeric acid-sensing ion channels, demonstrating that Piezo1 channels possess intrinsic mechanotransduction modules separate from their pore modules. In conclusion, this is the first report on the bona fide pore module and mechanotransduction components of Piezo channels, which define their ion-conducting properties and gating by mechanical stimuli, respectively.

Calstabin 2: An important regulator for learning and memory in mice.

Calstabin2, also named FK506 binding protein 12.6 (FKBP12.6), is a subunit of ryanodine receptor subtype 2 (RyR2) macromolecular complex, which is an intracellular calcium channel and abundant in the brain. Previous studies identified a role of leaky neuronal RyR2 in posttraumatic stress disorder (PTSD). However, the functional role of Calstabin2 in the cognitive function remains unclear. Herein, we used a mouse model of genetic deletion of Calstabin2 to investigate the function of Calstabin2 in cognitive dysfunction. We found that Calstabin2 knockout (KO) mice showed significantly reduced performance in Morris Water Maze (MWM), long-term memory (LTM) contextual fear testing, and rotarod test when compared to wild type (WT) littermates. Indeed, genetic deletion of Calstabin2 reduced long-term potentiation (LTP) at the hippocampal CA3-CA1 connection, increased membrane excitability, and induced RyR2 leak. Finally, we demonstrated that the increase in cytoplasmic calcium activated Ca(2+) dependent potassium currents and led to neuronal apoptosis in KO hippocampal neurons. Thus, these results suggest that neuronal RyR2 Ca(2+) leak due to Calstabin2 deletion contributes to learning deficiency and memory impairment.

Suppression of neuronal excitability by the secretion of the lamprey (Lampetra japonica) provides a mechanism for its evolutionary stability.

Lampreys are one of the most primitive vertebrates still living today. They attach themselves to the body surface of the host fish through their sucker-like mouths and suck blood of the host for days. Recent fossil evidence has indicated that morphology of lampreys in the late Devonian period, over 360 million years ago, already possessed the present day major characteristics, suggesting the evolutionary stability of a highly specialized parasitic feeding habit. Obviously, nociceptive responses and hemostasis of the host are two major barriers to long-term feeding of the parasitic lamprey. It has been found, to counteract hemostasis of the host, that paired buccal glands of lampreys secrete antihemostatic compounds to prevent blood of the host from coagulation. However, it is not known how lampreys make the host lose nociceptive responses. Here, we prepared components of the crude extract from the buccal glands of the lampreys (Lampetra japonica). Then, we show that crude extract and one of its purified components reduce the firing frequency of neuronal action potentials probably through inhibiting the voltage-dependent Na(+) channels. As the voltage-gated Na(+) channels are highly conserved throughout evolution, we argue that the secretion of the lampreys could exert the similar effect on the Na(+) channels of their host fish as well. Therefore, together with its antihemostatic effect, the secretion due to its inhibitory effect on neuronal excitability might provide a mechanism for the parasitic lampreys to keep their evolutionary stability.

Cloning and characterization of a calcium-activated chloride channel in rat uterus.

In a search for genes involved in regulation of uterine contractility, we cloned a novel calcium-activated chloride channel gene, named rat Clca4, from pregnant rat uterus. The gene shares approximately 83% and 70% nucleotide homology with mouse Clca6 and human CLCA4, respectively, and was expressed primarily in rat uterus. The transcripts were upregulated at Gestational Day 22 (prior to parturition), implying a functional involvement in parturition. Western blot analysis showed that rat CLCA4 protein was present in uterus, lung, and heart, but not in any other tissues examined. Confocal microscopy revealed that rat CLCA4 is localized in cell membrane and could not be removed by alkaline or PBS washing. Transient transfection of rat CLCA4-enhanced green fluorescent protein in Chinese hamster ovary cells resulted in production of characteristic Cl(-) currents that could be activated by Ca(2+) and ionomycin but inhibited by niflumic acid, a CLCA-channel blocker. The identification and characterization of rat Clca4 help decipher the contribution of Ca(2+)-activated Cl(-) conductance in myometrial contractility.

Effects of cholesterol levels on the excitability of rat hippocampal neurons.

Changes in the cholesterol levels dynamically alter the microenvironment of the plasma membrane and have been shown to modify functions of ion channels. However, the cellular effect of these modifications is largely unknown. In this report, we demonstrate that cholesterol levels modulate neuronal excitability in rat hippocampal neurons. Reduction of cholesterol levels shortened the duration and increased the firing frequency and peak amplitude of action potentials, while enrichment of cholesterol reversed the effect. Furthermore, we showed that reduction of cholesterol levels increased, while enrichment of cholesterol decreased the amplitude of the delayed rectifier I(K) currents. On the other hand, reduction of cholesterol levels slowed down the inactivation of the fast transient I(A) currents, but enrichment of cholesterol had no significant effect on the I(A) currents. Besides, alteration in cholesterol levels had no significant effect on the action potential in the presence of blockers for both I(K) and I(A) currents. These observations demonstrate that cholesterol levels bi-directionally regulate the neuronal excitability mainly through modifications of the I(K) and I(A) currents, suggesting an optimum level of cholesterol for the optimum excitability of neurons. Alterations in the neuronal cholesterol levels have been associated with aging, cognitive decline, neurodegenerative diseases, etc. Therefore, our findings are important for a deeper understanding of the relationship between the cholesterol level and dysfunctions of the brain at the molecular level.

Activation of a mechanosensitive BK channel by membrane stress created with amphipaths.

Some BK channels are activated in response to membrane stretch. However, it remains largely unknown which membrane component transmits forces to the channel and which part of the channel senses the force. Recently, we have shown that a BK channel cloned from chick heart (named SAKCa channel) is a stretch activated channel, while deletion of a 59 amino acids splice insert (STREX) located in the cytoplasmic side, abolishes its stretch-sensitivity. This finding raised a question whether stress in the bilayer is crucial for the mechanical activation of the channel. To address this question we examined the effects of membrane perturbing amphipaths on the stretch activation of the SAKCa channel and its STREX-deletion mutant. We found that both anionic amphipath trinitrophenol (TNP) and cationic amphipath chlorpromazine (CPZ) could dose-dependently activate the channel by leftward shifting the voltage activation curve when applied alone. In contrast, TNP and CPZ compensated each other's effect when applied sequentially. These results can be understood in the framework of the bilayer couple hypothesis, suggesting that stress in the plasma membrane can activate the SAKCa channel. Interestingly, the STREX-deletion mutant channel has much less sensitivity to the amphipaths, suggesting that STREX acts as an intermediate structure that can indirectly convey stress in the membrane to the gate of the SAKCa channel via an unidentified membrane associated protein(s) that can detect or transmit stress in the membrane.

Inhibitory effect of ganglioside GD1b on K+ current in hippocampal neurons and its involvement in apoptosis suppression.

Gangliosides are endogenous membrane components enriched in neuronal cells. They have been shown to play regulatory roles in many cellular processes. Here, we show for the first time that ganglioside GD1b plays an antiapoptotic role in cultured hippocampal neurons. GD1b inhibited the voltage-dependent outward delayed rectifier current (I(K)) but not the transient outward A-type current in a dose-dependent manner, with an IC50 value of 15.2 microM. This effect appears to be somehow specific, because GD1b, but not GM1, GM2, GM3, GD1a, GD3, or GT1b, was effective in inhibiting I(K). Intracellular application of staurosporine (STS; 0.1 microM) resulted in rapid activation of I(K), which was partially reversed upon addition of the K+ channel blocker tetraethylammonium (TEA; 5 mM) and GD1b (10 microM). Furthermore, GD1b (10 microM) attenuated STS-induced neuronal apoptosis by nearly the same amount as 5 mM TEA. In addition, GD1b suppressed the apoptosis-associated caspase 3 activation that was activated by STS. Collectively, these findings suggest that GD1b plays an antiapoptotic role in cultured hippocampal neurons through its inhibitory effect on the I(K) and caspase activity.