The gut-to-brain axis for toxin-induced defensive responses.

After ingestion of toxin-contaminated food, the brain initiates a series of defensive responses (e.g., nausea, retching, and vomiting). How the brain detects ingested toxin and coordinates diverse defensive responses remains poorly understood. Here, we developed a mouse-based paradigm to study defensive responses induced by bacterial toxins. Using this paradigm, we identified a set of molecularly defined gut-to-brain and brain circuits that jointly mediate toxin-induced defensive responses. The gut-to-brain circuit consists of a subset of Htr3a+ vagal sensory neurons that transmit toxin-related signals from intestinal enterochromaffin cells to Tac1+ neurons in the dorsal vagal complex (DVC). Tac1+ DVC neurons drive retching-like behavior and conditioned flavor avoidance via divergent projections to the rostral ventral respiratory group and lateral parabrachial nucleus, respectively. Manipulating these circuits also interferes with defensive responses induced by the chemotherapeutic drug doxorubicin. These results suggest that food poisoning and chemotherapy recruit similar circuit modules to initiate defensive responses.

Piezo2 channel in nodose ganglia neurons is essential in controlling hypertension in a pathway regulated directly by Nedd4-2.

Baroreflex plays a crucial role in regulation of arterial blood pressure (BP). Recently, Piezo1 and Piezo2, the mechanically-activated (MA) ion channels, have been identified as baroreceptors. However, the underlying molecular mechanism for regulating these baroreceptors in hypertension remains unknown. In this study, we used spontaneously hypertensive rats (SHR) and NG-Nitro-l-Arginine (L-NNA)- and Angiotensin II (Ang II)-induced hypertensive model rats to determine the role and mechanism of Piezo1 and Piezo2 in hypertension. We found that Piezo2 was dominantly expressed in baroreceptor nodose ganglia (NG) neurons and aortic nerve endings in Wistar-Kyoto (WKY) rats. The expression of Piezo2 not Piezo1 was significantly downregulated in these regions in SHR and hypertensive model rats. Electrophysiological results showed that the rapidly adapting mechanically-activated (RA-MA) currents and the responsive neuron numbers were significantly reduced in baroreceptor NG neurons in SHR. In WKY rats, the arterial BP was elevated by knocking down the expression of Piezo2 or inhibiting MA channel activity by GsMTx4 in NG. Knockdown of Piezo2 in NG also attenuated the baroreflex and increased serum norepinephrine (NE) concentration in WKY rats. Co-immunoprecipitation experiment suggested that Piezo2 interacted with Neural precursor cell-expressed developmentally downregulated gene 4 type 2 (Nedd4-2, also known as Nedd4L); Electrophysiological results showed that Nedd4-2 inhibited Piezo2 MA currents in co-expressed HEK293T cells. Additionally, Nedd4-2 was upregulated in NG baroreceptor neurons in SHR. Collectively, our results demonstrate that Piezo2 not Piezo1 may act as baroreceptor to regulate arterial BP in rats. Nedd4-2 induced downregulation of Piezo2 in baroreceptor NG neurons leads to hypertension in rats. Our findings provide a novel insight into the molecular mechanism for the regulation of baroreceptor Piezo2 and its critical role in the pathogenesis of hypertension.

Cannabidiol inhibits febrile seizure by modulating AMPA receptor kinetics through its interaction with the N-terminal domain of GluA1/GluA2.

Cannabidiol (CBD) is a major phytocannabinoid in Cannabis sativa. CBD is being increasingly reported as a clinical treatment for neurological diseases. Febrile seizure is one of the most common diseases in children with limited therapeutic options. We investigated possible therapeutic effects of CBD on febrile seizures and the underlying mechanism. Use of a hyperthermia-induced seizures model revealed that CBD significantly prolonged seizure latency and reduced the severity of thermally-induced seizures. Hippocampal neuronal excitability was significantly decreased by CBD. Further, CBD significantly reduced the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) mediated evoked excitatory postsynaptic currents (eEPSCs) and the amplitude and frequency of miniature EPSCs (mEPSCs). Furthermore, CBD significantly accelerated deactivation in GluA1 and GluA2 subunits. Interestingly, CBD slowed receptor recovery from desensitization of GluA1, but not GluA2. These effects on kinetics were even more prominent when AMPAR was co-expressed with γ-8, the high expression isoform 8 of transmembrane AMPAR regulated protein (TARPγ8) in the hippocampus. The inhibitory effects of CBD on AMPAR depended on its interaction with the distal N-terminal domain of GluA1/GluA2. CBD inhibited AMPAR activity and reduced hippocampal neuronal excitability, thereby improving the symptoms of febrile seizure in mice. The putative binding site of CBD in the N-terminal domain of GluA1/GluA2 may be a drug target for allosteric gating modulation of AMPAR.

Revealing the Inhibitory Effect of Ginseng on Mitochondrial Respiration through Synaptosomal Proteomics.

Ginseng, the active ingredients of which are ginsenosides, is the most popular herbal medicine and has potential merit in the treatment of cerebral disorders. To better understand the function of Ginseng in the cerebral system, we examined changes in the protein expression profiles of synaptosomes extracted from the cerebral cortical and hippocampal tissues of rats administered a high or low dose of Ginseng for 2 weeks. More than 5000 proteins belonging to synaptosomes were simultaneously identified and quantitated by an approach combining tandem mass tags with 2D liquid chromatography-mass spectrometry (LC-MS). Regarding differentially expressed proteins, downregulated proteins were much more highly induced than upregulators in the cerebral cortical and hippocampal synaptosomes, regardless of the dose of Ginseng. Bioinformatic analysis indicated the majority of the altered proteins to be located in the mitochondria, directly or indirectly affecting mitochondrial oxidative respiration. Further functional experiments using the substrate-uncoupler inhibitor titration approach confirmed that three representative ginsenosides were able to inhibit oxidative phosphorylation in mitochondria. Our results demonstrate that Ginseng can regulate the function of mitochondria and alter the energy metabolism of cells, which may be useful for the treatment of central nervous disorders.

Brain circuits for retching-like behavior.

Nausea and vomiting are important defensive responses to cope with pathogens and toxins that invade the body. The nucleus of the solitary tract (NTS) is important for initiating these responses. However, the molecular heterogeneities and cellular diversities of the NTS occlude a better understanding of these defensive responses. Here, we constructed the single-nucleus transcriptomic atlas of NTS cells and found multiple populations of NTS neurons that may be involved in these defensive responses. Among these, we identified Calbindin1-positive (Calb1+) NTS neurons that are molecularly distinct from Tac1+ neurons. These Calb1+ neurons are critical for nausea and retching induced by cereulide; an emetic toxin secreted by Bacillus Cereus. Strikingly, we found that cereulide can directly modulate vagal sensory neurons that innervate Calb1+ NTS neurons, a novel mechanism distinct from that for nausea and retching induced by Staphylococcal enterotoxin A. Together, our transcriptomic atlas of NTS neurons and the functional analyses revealed the neural mechanism for cereulide-induced retching-like behavior. These results demonstrate the molecular and cellular complexities in the brain that underlie defensive responses to the diversities of pathogens and toxins.

Complexin-1 enhances ultrasound neurotransmission in the mammalian auditory pathway.

Unlike megabats, which rely on well-developed vision, microbats use ultrasonic echolocation to navigate and locate prey. To study ultrasound perception, here we compared the auditory cortices of microbats and megabats by constructing reference genomes and single-nucleus atlases for four species. We found that parvalbumin (PV)+ neurons exhibited evident cross-species differences and could respond to ultrasound signals, whereas their silencing severely affected ultrasound perception in the mouse auditory cortex. Moreover, megabat PV+ neurons expressed low levels of complexins (CPLX1-CPLX4), which can facilitate neurotransmitter release, while microbat PV+ neurons highly expressed CPLX1, which improves neurotransmission efficiency. Further perturbation of Cplx1 in PV+ neurons impaired ultrasound perception in the mouse auditory cortex. In addition, CPLX1 functioned in other parts of the auditory pathway in microbats but not megabats and exhibited convergent evolution between echolocating microbats and whales. Altogether, we conclude that CPLX1 expression throughout the entire auditory pathway can enhance mammalian ultrasound neurotransmission.

Potentiation of PIEZO2 mechanically-activated currents in sensory neurons mediates vincristine-induced mechanical hypersensitivity.

Vincristine, a widely used chemotherapeutic agent for treating different cancer, often induces severe peripheral neuropathic pain. A common symptom of vincristine-induced peripheral neuropathic pain is mechanical allodynia and hyperalgesia. However, mechanisms underlying vincristine-induced mechanical allodynia and hyperalgesia are not well understood. In the present study, we show with behavioral assessment in rats that vincristine induces mechanical allodynia and hyperalgesia in a PIEZO2 channel-dependent manner since gene knockdown or pharmacological inhibition of PIEZO2 channels alleviates vincristine-induced mechanical hypersensitivity. Electrophysiological results show that vincristine potentiates PIEZO2 rapidly adapting (RA) mechanically-activated (MA) currents in rat dorsal root ganglion (DRG) neurons. We have found that vincristine-induced potentiation of PIEZO2 MA currents is due to the enhancement of static plasma membrane tension (SPMT) of these cells following vincristine treatment. Reducing SPMT of DRG neurons by cytochalasin D (CD), a disruptor of the actin filament, abolishes vincristine-induced potentiation of PIEZO2 MA currents, and suppresses vincristine-induced mechanical hypersensitivity in rats. Collectively, enhancing SPMT and subsequently potentiating PIEZO2 MA currents in primary afferent neurons may be an underlying mechanism responsible for vincristine-induced mechanical allodynia and hyperalgesia in rats. Targeting to inhibit PIEZO2 channels may be an effective analgesic method to attenuate vincristine-induced mechanical hypersensitivity.

Trigeminal nerve stimulation restores hippocampal dopamine deficiency to promote cognitive recovery in traumatic brain injury.

Cognitive impairment (CI) is a common neurological disease resulting from traumatic brain injury (TBI). Trigeminal nerve stimulation (TNS) is an emerging, non-invasive, and effective neuromodulation therapy especially for patients suffering from brain function disorders. However, the treatment and recovery mechanisms of TNS remain poorly understood. By using combined advanced technologies, we revealed here that the neuroprotective potential of TNS to improve CI caused by TBI. The study results found that 40 Hz TNS treatment has the ability to improve CI in TBI mice and communicates with central nervous system via the trigeminal ganglion (TG). Transsynaptic virus experiments revealed that TG is connected to the hippocampus (HPC) through the corticotropin-releasing hormone (CRH) neurons of paraventricular hypothalamic nucleus (PVN) and the dopamine transporter (DAT) neurons of substantia nigra pars compacta/ventral tegmental area (SNc/VTA). Mechanistically, the data showed that TNS can increase the release of dopamine in the HPC by activating the following neural circuit: TG→CRH+ PVN→DAT+ SNc/VTA → HPC. Bulk RNA sequencing confirmed changes in the expression of dopamine-related genes in the HPC. This work preliminarily explains the efficacy and mechanism of TNS and adds to the increasing evidence demonstrating that nerve stimulation is an effective method to treat neurological diseases.

Influence of human amylin on the membrane stability of rat primary hippocampal neurons.

The two most common aging-related diseases, Alzheimer's disease and type 2 diabetes mellitus, are associated with accumulation of amyloid proteins (ß-amyloid and amylin, respectively). This amylin aggregation is reportedly cytotoxic to neurons. We found that aggregation of human amylin (hAmylin) induced neuronal apoptosis without obvious microglial infiltration in vivo. High concentrations of hAmylin irreversibly aggregated on the surface of the neuronal plasma membrane. Long-term incubation with hAmylin induced morphological changes in neurons. Moreover, hAmylin permeabilized the neuronal membrane within 1 min in a manner similar to Triton X-100, allowing impermeable fluorescent antibodies to enter the neurons and stain intracellular antigens. hAmylin also permeabilized the cell membrane of astrocytes, though more slowly. Under scanning electron microscopy, we observed that hAmylin destroyed the integrity of the cell membranes of both neurons and astrocytes. Additionally, it increased intracellular reactive oxygen species generation and reduced the mitochondrial membrane potential. Thus, by aggregating on the surface of neurons, hAmylin impaired the cell membrane integrity, induced reactive oxygen species production, reduced the mitochondrial membrane potential, and ultimately induced neuronal apoptosis.

Transient Receptor Potential Cation Channel Subfamily Vanilloid 4 and 3 in the Inner Ear Protect Hearing in Mice.

The transient receptor potential cation channel, vanilloid type (TRPV) 3, is a member of the TRPV subfamily that is expressed predominantly in the skin, hair follicles, and gastrointestinal tract. It is also distributed in the organ of Corti of the inner ear and colocalizes with TRPV1 or TRPV4, but its role in auditory function is unknown. In the present study, we demonstrate that TRPV3 is expressed in inner hair cells (HCs) but mainly in cochlear outer HCs in mice, with expression limited to the cytoplasm and not detected in stereocilia. We compared the number of HCs as well as distortion product otoacoustic emissions (DPOAE) and auditory brainstem response (ABR) thresholds between TRPV3 knockout (V3KO) and wild-type (V3WT) mice and found that although most mutants (72.3%) had normal hearing, a significant proportion (27.7%) showed impaired hearing associated with loss of cochlear HCs. Compensatory upregulation of TRPV4 in HCs prevented HC damage and kanamycin-induced hearing loss and preserved normal auditory function in most of these mice. Thus, TRPV4 and TRPV3 in cochlear HCs protect hearing in mice; moreover, the results suggest some functional redundancy in the functions of TRPV family members. Our findings provide novel insight into the molecular basis of auditory function in mammals that can be applied to the development of strategies to mitigate hearing loss.

Mechanosensitive ion channel Piezo1 promotes prostate cancer development through the activation of the Akt/mTOR pathway and acceleration of cell cycle.

Prostate cancer is one of the most common types of cancer affecting men worldwide; however, its etiology and pathological mechanisms remain poorly understood. Mechanical stimulation plays a key role in prostate cancer development. Piezo type mechanosensitive ion channel component 1 (Piezo1), which functions as a cell sensor and transducer of mechanical stimuli, may have a crucial role in the development of prostate cancer. In the present study, the expression of the Piezo1 channel was demonstrated to be significantly elevated in prostate cancer cell lines and in human prostate malignant tumor tissues. Downregulation of Piezo1 significantly suppressed the viability, proliferation and migration of prostate cancer cells in vitro, and inhibited prostate tumor growth in vivo. The activation of the Akt/mTOR pathway or acceleration of cell cycle progression from G0/G1 to S phase may downstream consequences of Piezo 1 signal pathway activation. Downregulation of Piezo1 considerably suppressed Ca2+ signal increments, inhibited the phosphorylation of Akt and mTOR and arrested the cell cycle of prostate cancer cells at G0/G1 phase in while inhibiting the activation of CDK4 and cyclin D1. Taken together, these findings suggest that Piezo1 channels have a crucial role in prostate cancer development and may, therefore, be a novel therapeutic target in the treatment of prostate cancer.

Inhibition of transmembrane member 16A calcium-activated chloride channels by natural flavonoids contributes to flavonoid anticancer effects.

BACKGROUND AND PURPOSE: Natural flavonoids are ubiquitous in dietary plants and vegetables and have been proposed to have antiviral, antioxidant, cardiovascular protective and anticancer effects. Transmembrane member 16A (TMEM16A)-encoded Ca2+ -activated Cl- channels play a variety of physiological roles in many organs and tissues. Overexpression of TMEM16A is also believed to be associated with cancer progression. Therefore, inhibition of TMEM16A current may be a potential target for cancer therapy. In this study, we screened a broad spectrum of flavonoids for their inhibitory activities on TMEM16A currents. EXPERIMENTAL APPROACH: A whole-cell patch technique was used to record the currents. The BrdU assay and transwell technique were used to investigate cell proliferation and migration. KEY RESULTS: At a concentration of 100 µM, 10 of 20 compounds caused significant (>50%) inhibition of TMEM16A currents. The four most potent compounds - luteolin, galangin, quercetin and fisetin - had IC50 values ranging from 4.5 to 15 µM). To examine the physiological relevance of these findings, we also studied the effects of these flavonoids on endogenous TMEM16A currents in addition to cell proliferation and migration in LA795 cancer cells. Among the flavonoids tested, we detected a highly significant correlation between TMEM16A current inhibition and cell proliferation or reduction of migration. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that flavonoids inhibit TMEM16A currents and suggests that flavonoids could have anticancer effects via this mechanism.

Multiple target of hAmylin on rat primary hippocampal neurons.

Alzheimer's disease (AD) and type II diabetes mellitus (DM2) are the most common aging-related diseases and are characterized by ß-amyloid and amylin accumulation, respectively. Multiple studies have indicated a strong correlation between these two diseases. Amylin oligomerization in the brain appears to be a novel risk factor for developing AD. Although amylin aggregation has been demonstrated to induce cytotoxicity in neurons through altering Ca2+ homeostasis, the underlying mechanisms have not been fully explored. In this study, we investigated the effects of amylin on rat hippocampal neurons using calcium imaging and whole-cell patch clamp recordings. We demonstrated that the amylin receptor antagonist AC187 abolished the Ca2+ response induced by low concentrations of human amylin (hAmylin). However, the Ca2+ response induced by higher concentrations of hAmylin was independent of the amylin receptor. This effect was dependent on extracellular Ca2+. Additionally, blockade of L-type Ca2+ channels partially reduced hAmylin-induced Ca2+ response. In whole-cell recordings, hAmylin depolarized the membrane potential. Moreover, application of the transient receptor potential (TRP) channel antagonist ruthenium red (RR) attenuated the hAmylin-induced increase in Ca2+. Single-cell RT-PCR demonstrated that transient receptor potential vanilloid 4 (TRPV4) mRNA was expressed in most of the hAmylin-responsive neurons. In addition, selective knockdown of TRPV4 channels inhibited the hAmylin-evoked Ca2+ response. These results indicated that different concentrations of hAmylin act through different pathways. The amylin receptor mediates the excitatory effects of low concentrations of hAmylin. In contrast, for high concentrations of hAmylin, hAmylin aggregates precipitated on the neuronal membrane, activated TRPV4 channels and subsequently triggered membrane voltage-gated calcium channel opening followed by membrane depolarization. Therefore, our data suggest that TRPV4 is a key molecular mediator for the cytotoxic effects of hAmylin on hippocampal neurons.