Postsynaptic Targeting and Mobility of Membrane Surface-Localized hASIC1a.

Acid-sensing ion channels (ASICs), the main H+ receptors in the central nervous system, sense extracellular pH fluctuations and mediate cation influx. ASIC1a, the major subunit responsible for acid-activated current, is widely expressed in brain neurons, where it plays pivotal roles in diverse functions including synaptic transmission and plasticity. However, the underlying molecular mechanisms for these functions remain mysterious. Using extracellular epitope tagging and a novel antibody recognizing the hASIC1a ectodomain, we examined the membrane targeting and dynamic trafficking of hASIC1a in cultured cortical neurons. Surface hASIC1a was distributed throughout somata and dendrites, clustered in spine heads, and co-localized with postsynaptic markers. By extracellular pHluorin tagging and fluorescence recovery after photobleaching, we detected movement of hASIC1a in synaptic spine heads. Single-particle tracking along with use of the anti-hASIC1a ectodomain antibody revealed long-distance migration and local movement of surface hASIC1a puncta on dendrites. Importantly, enhancing synaptic activity with brain-derived neurotrophic factor accelerated the trafficking and lateral mobility of hASIC1a. With this newly-developed toolbox, our data demonstrate the synaptic location and high dynamics of functionally-relevant hASIC1a on the surface of excitatory synapses, supporting its involvement in synaptic functions.

Cell-Type Identification in the Autonomic Nervous System.

The autonomic nervous system controls various internal organs and executes crucial functions through sophisticated neural connectivity and circuits. Its dysfunction causes an imbalance of homeostasis and numerous human disorders. In the past decades, great efforts have been made to study the structure and functions of this system, but so far, our understanding of the classification of autonomic neuronal subpopulations remains limited and a precise map of their connectivity has not been achieved. One of the major challenges that hinder rapid progress in these areas is the complexity and heterogeneity of autonomic neurons. To facilitate the identification of neuronal subgroups in the autonomic nervous system, here we review the well-established and cutting-edge technologies that are frequently used in peripheral neuronal tracing and profiling, and discuss their operating mechanisms, advantages, and targeted applications.

The acid-sensing ion channel ASIC1a mediates striatal synapse remodeling and procedural motor learning.

Acid-sensing ion channel 1a (ASIC1a) is abundant in multiple brain regions, including the striatum, which serves as the input nucleus of the basal ganglia and is critically involved in procedural learning and motor memory. We investigated the functional role of ASIC1a in striatal neurons. We found that ASIC1a was critical for striatum-dependent motor coordination and procedural learning by regulating the synaptic plasticity of striatal medium spiny neurons. Global deletion of Asic1a in mice led to increased dendritic spine density but impaired spine morphology and postsynaptic architecture, which were accompanied by the decreased function of N-methyl-d-aspartate (NMDA) receptors at excitatory synapses. These structural and functional changes caused by the loss of ASIC1a were largely mediated by reduced activation (phosphorylation) of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated protein kinases (ERKs). Consequently, Asic1a null mice exhibited poor performance on multiple motor tasks, which was rescued by striatal-specific expression of either ASIC1a or CaMKII. Together, our data reveal a previously unknown mechanism mediated by ASIC1a that promotes the excitatory synaptic function underlying striatum-related procedural learning and memory.

Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling.

Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system.

Acid-sensing ion channels: trafficking and pathophysiology.

Acid-sensing ion channels (ASICs) are proton-gated cation channels that are widely expressed in both the peripheral and central nervous systems. ASICs contribute to a variety of pathophysiological conditions that involve tissue acidosis, such as ischemic stroke, epileptic seizures and multiple sclerosis. Although much progress has been made in researching the structure-function relationship and pharmacology of ASICs, little is known about the trafficking of ASICs and its contribution to ASIC function. The recent identification of the mechanism of membrane insertion and endocytosis of ASIC1a highlights the emerging role of ASIC trafficking in regulating its pathophysiological functions. In this review, we summarize the recent advances and discuss future directions on this topic.

Molecular mechanism of constitutive endocytosis of Acid-sensing ion channel 1a and its protective function in acidosis-induced neuronal death.

Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the peripheral and CNSs, which critically contribute to a variety of pathophysiological conditions that involve tissue acidosis, such as ischemic stroke and epileptic seizures. However, the trafficking mechanisms of ASICs and the related proteins remain largely unknown. Here, we demonstrate that ASIC1a, the main ASIC subunit in the brain, undergoes constitutive endocytosis in a clathrin- and dynamin-dependent manner in both mouse cortical neurons and heterologous cell cultures. The endocytosis of ASIC1a was inhibited by either the small molecular inhibitor tyrphostin A23 or knockdown of the core subunit of adaptor protein 2 (AP2) µ2 using RNA interference, supporting a clathrin-dependent endocytosis of ASIC1a. In addition, the internalization of ASIC1a was blocked by dominant-negative dynamin1 mutation K44A and the small molecular inhibitor dynasore, suggesting that it is also dynamin-dependent. We show that the membrane-proximal residues (465)LCRRG(469) at the cytoplasmic C terminus of ASIC1a are critical for interaction with the endogenous adaptor protein complex and inhibition of ASIC1a internalization strongly exacerbated acidosis-induced death of cortical neurons from wild-type but not ASIC1a knock-out mice. Together, these results reveal the molecular mechanism of ASIC1a internalization and suggest the importance of endocytic pathway in functional regulation of ASIC1a channels as well as neuronal damages mediated by these channels during neurodegeneration.

PI3-kinase/Akt pathway-regulated membrane insertion of acid-sensing ion channel 1a underlies BDNF-induced pain hypersensitivity.

Central neural plasticity plays a key role in pain hypersensitivity. This process is modulated by brain-derived neurotrophic factor (BDNF) and also involves the type 1a acid-sensing ion channel (ASIC1a). However, the interactions between the BDNF receptor, tropomyosin-related kinase B (TrkB), and ASIC1a are unclear. Here, we show that deletion of ASIC1 gene suppressed the sustained mechanical hyperalgesia induced by intrathecal BDNF application in mice. In both rat spinal dorsal horn neurons and heterologous cell cultures, the BDNF/TrkB pathway enhanced ASIC1a currents via phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB/Akt) cascade and phosphorylation of cytoplasmic residue Ser-25 of ASIC1a, resulting in enhanced forward trafficking and increased surface expression. Moreover, in both rats and mice, this enhanced ASIC1a activity was required for BDNF-mediated hypersensitivity of spinal dorsal horn nociceptive neurons and central mechanical hyperalgesia, a process that was abolished by intrathecal application of a peptide representing the N-terminal region of ASIC1a encompassing Ser-25. Thus, our results reveal a novel mechanism underlying central sensitization and pain hypersensitivity, and reinforce the critical role of ASIC1a channels in these processes.

Highly conserved salt bridge stabilizes rigid signal patch at extracellular loop critical for surface expression of acid-sensing ion channels.

Acid-sensing ion channels (ASICs) are non-selective cation channels activated by extracellular acidosis associated with many physiological and pathological conditions. A detailed understanding of the mechanisms that govern cell surface expression of ASICs, therefore, is critical for better understanding of the cell signaling under acidosis conditions. In this study, we examined the role of a highly conserved salt bridge residing at the extracellular loop of rat ASIC3 (Asp(107)-Arg(153)) and human ASIC1a (Asp(107)-Arg(160)) channels. Comprehensive mutagenesis and electrophysiological recordings revealed that the salt bridge is essential for functional expression of ASICs in a pH sensing-independent manner. Surface biotinylation and immunolabeling of an extracellular epitope indicated that mutations, including even minor alterations, at the salt bridge impaired cell surface expression of ASICs. Molecular dynamics simulations, normal mode analysis, and further mutagenesis studies suggested a high stability and structural constrain of the salt bridge, which serves to separate an adjacent structurally rigid signal patch, important for surface expression, from a flexible gating domain. Thus, we provide the first evidence of structural requirement that involves a stabilizing salt bridge and an exposed rigid signal patch at the destined extracellular loop for normal surface expression of ASICs. These findings will allow evaluation of new strategies aimed at preventing excessive excitability and neuronal injury associated with tissue acidosis and ASIC activation.

[Detecting human chromosome anomalies with primed in situ labeling (PRINS)].

Numerical chromosome anomaly was one of the most important kinds of human chromosome diseases by inducing pregnancy loss, miscarriage, infant death, congenital malformations and nerve damage. The present study was to establish a rapid, reliable and reasonable multicolor primed in situ labeling (PRINS) protocol for diagnosing numerical anomaly in human chromosome. First, nuclei of cultured lymphocytes and sperms were labeled with the method of PRINS, and then nuclei of cultured lymphocytes, sperms and other specimen were labeled with the method of updated non-ddNTP-blocking multicolor PRINS technique. The labeling effect of different target sequences and the feature of different fluorochromes were evaluated by experiment. Meanwhile, several parameters of PRINS were optimized to obtain more homogeneous and stable labeling effect. At last, the applicative value of PRINS was evaluated by comparing the clinical effect and labeling characteristics between FISH probe and PRINS. In the present study, several chromosomes were simultaneously marked successfully in the same sperm nucleus within 2.5 hours. And the frequency of one-color-labeling reached 99%. The many advantages, compared with FISH, make PRINS become the first choice in diagnosing diseases related to numerical anomaly in human chromosome.

[Triple-color primed in situ labeling protocol of human metaphase chromosomes].

OBJECTIVE: To study the feasibility of simultaneous detection for several chromosomes with optimized triple-color primed in situ labelling (PRINS) protocol in cultured peripheral blood lymphocytes. METHODS: Pre-test of gonosome detection with dual-color PRINS protocol was performed to explore and optimize the order and condition of PRINS primers. A peripheral blood sample from a Klinefelter's syndrome patient (47, XXY) had also been studied with optimized triple-color PRINS to prove the correspondence between the number of signals and chromosomes. RESULTS: Chromosome 18, X and Y had been simultaneously and specifically marked within 3 hours. The frequency of successful labeling reached 90% both in dual-color and triple-color test. Two chromosome X had been correctly showed in lymphocyte sample of Klinerfelter's syndrome. CONCLUSION: Numerical chromosome anomalies could be rapidly and exactly detected with this non-ddNTP-blocking multicolor PRINS protocol in peripheral blood lymphocytes. The results of in situ labeling are much clearer with inner control.