Simulation of axoplasmic transport

We have analysed a kinetic model of axonal transport by simulating experimental tracer profiles. The existence of three phases of axoplasmic transport is assumed: fast anterograde, slow anterograde and retrograde. Each phase has its characteristic velocity. Transported materials are postulated to shift between these phases. Also catabolism and sequestration of material is allowed for in our model. Thus, we have set up equations which contain axonal transport, diffusion and cross-over terms. The rate constants of material shifts were determined by computer fitting to experimental data. Best-fitted values of the rate constants for transfer of material between the fast and slow phases were both 2 X 10(-5) sec-1, while the rate constants for transfer between the fast and retrograde phases were both 1 X 10(-5) sec-1. The rate constant of material loss from the slow phase to the extracellular space was 1 X 10(-6) sec-1. The material shift between the slow and retrograde phases was negligibly small. These data show that there is exchange of material between the fast and slow phases and between the fast and retrograde phases. However, there is no significant exchange between the slow and retrograde phases. Diffusion was found to have only a minor effect on the profiles. The velocity of the fast anterograde track in cold-blooded animals was predicted to be around 200 mm/day, or, in other words, to be close to experimentally observed values of the fast anterograde component of axonal transport.     

Differential suppression of axoplasmic transport: Effects of light irradiation to the growth cone of cultured dorsal root ganglion neurons

Summary 1. Growth cones of cultured dorsal root ganglion neurons from mice were irradiated using a mercury lamp.2. The flux of particles of fast retrograde axoplasmic transport decreased promptly after light irradiation without a change in velocity.3. That of anterograde transport decreased as well, but with a significant latency. The decrease in the anterograde flux was attributed to decreased velocity of particles.4. Video-enhanced contrast microscopy of growth cones revealed transient swelling of growth cones and transient stagnation of particles in growth cones.5. The longer the neurite, the larger the latency of the change of the anterograde transport; peripheral information was calculated to be conveyed to the cell body at a speed of 6 µm/min.6. The mechanism of this information conveyance and the export of materials from the cell body are discussed.     

Action of Brefeldin A on Amphibian Neurons: Passage of Newly Synthesized Proteins Through the Golgi Complex Is Not Required for Continued Fast Organelle Transport in Axons

Abstract: The relation between the availability of newly synthesized protein and lipid and the axonal transport of optically detectable organelles was examined in peripheral nerve preparations of amphibia (Rana catesbeiana and Xenopus laevis) in which intracellular traffic from the endo-plasmic reticulum to the Golgi complex was inhibited with brefeldin A (BFA). Accumulation of fast-transported radio-labeled protein or phospholipid proximal to a sciatic nerve ligature was monitored in vitro in preparations of dorsal root ganglia and sciatic nerve. Organelle transport was examined by computer-enhanced video microscopy of single myelinated axons. BFA reduced the amount of radiolabeled protein and lipid entering the fast-transport system of the axon without affecting either the synthesis or the transport rate of these molecules. The time course of the effect of BFA on axonal transport is consistent with an action at an early step in the intrasomal pathway, and with its action being related to the observed rapid (<1 h) disassembly of the Golgi complex. At a concentration of BFA that reduced fast-transported protein by >95%, no effect was observed on the flux or velocity of anterograde or retrograde organelle transport in axons for at least 20 h. Bidirectional axonal transport of organelles was similarly unaffected following suppression of protein synthesis by >99%. The findings suggest that the anterograde flux of transport organelles is not critically dependent on a supply of newly synthesized membrane precursors. The possibilities are considered that anterograde organelles normally arise from membrane components supplied from a post-Golgi storage pool, as well as from recycled retrograde organelles.     

Retrograde Axonal Transport of Phospholipid in Rat Sciatic Nerve

Abstract: Retrograde axonal transport of phospholipid was studied in rat sciatic motoneuron axons by placing collection crushes on the nerve at intervals after injection of [methyl-³H]choline into the lumbosacral spinal cord, and allowing labelled material undergoing anterograde or retrograde movement to accumulate adjacent to the collection crushes. Control experiments showed that the accumulations of label were not a result of local uptake of circulating precursor. The majority of the ³H label was associated with phosphatidylcholine. Accumulation of label at the distal collection crush, representing retrograde transport, was observed subsequent to the anterograde transport of phospholipid. In comparison with previous study on retrograde transport of protein, the following points were noted: (1) onset of retrograde transport occurred at approximately the same time after precursor injection (10–20 h) for both protein and phospholipid; (2) retrograde transport of lipids was more prolonged: maximum retrograde transport occurred later for phospholipid (30 h) than for protein (15–20 h), and declined to half-maximum between 49 and 99 h, compared to a corresponding value of 24–28 h for protein; (3) the proportion of total anterograde-transported activity subsequently undergoing retrograde transport was less in the case of phospholipid, at least over the time interval studied (up to 99 h after precursor injection). The similar times of onset of retrograde transport of phospholipid and protein support the concept of retrograde transport as a recycling mechanism returning to the cell body membrane fragments that were earlier transported into the axon. Coordinated retrograde transport of labelled protein and phospholipid components of the recycled membranes would be predicted. Differences between protein and phospholipid in the subsequent time course and amount of retrograde transport may reflect differences in axonal handling of protein and lipid. Both the more prolonged outflow of labelled lipids from cell body into axon and exchange with a distal pool of unlabelled phospholipid may account for the prolonged time course of retrograde transport of labelled lipid.     

Retrograde Effect of Muscle on Forms of Acetylcholinesterase in Peripheral Nerves

In the peripheral nerves of birds and mammals, acetylcholinesterase (AChE) exists in four main molecular forms (G1, G2, G4, and A12). The two heaviest forms (G4 and A12) are carried by rapid axoplasmic transport, whereas the two lightest forms (G1 and G2) are probably much more slowly transported. Here we report that nerves innervating fast-twitch (F nerves) and slow-twitch (S nerves) muscles of the rabbit differ both in their AChE molecular form patterns and in their anterograde and retrograde axonal transport parameters. Since we had previously shown a selective regulation of this enzyme in fast and slow parts of rabbit semimembranosus muscle, we wondered whether the differences observed in the nerve could be affected by the twitch properties of muscle. The results reported here show that in F nerves that reinnervate slow-twitch muscles, both the AChE molecular form patterns and axonal transport parameters turn into those of the S nerve. These data suggest the existence of a retrograde specific effect exerted by the muscles on their respective motoneurons.     

Nucleotide specificity for the bidirectional transport of membrane-bounded organelles in isolated axoplasm.

Video microscopy of isolated axoplasm from the squid giant axon permits correlated quantitative analyses of membrane-bounded organelle transport both in the intact axoplasm and along individual microtubules. As a result, the effects of experimental manipulations on both anterograde and retrograde movements of membrane-bounded organelles can be evaluated under nearly physiological conditions. Since anterograde and retrograde fast axonal transport are similar but distinct cellular processes, a systematic biochemical analysis is important for a further understanding of the molecular mechanisms for each. In this series of experiments, we employed isolated axoplasm of the squid to define the nucleoside triphosphate specificity for bidirectional organelle motility in the axon. Perfusion of axoplasm with 2-20 mM ATP preserved optimal vesicle velocities in both the anterograde and retrograde directions. Organelle velocities decreased to less than 50% of optimal values when the axoplasm was perfused with 10-20 mM UTP, GTP, ITP, or CTP with simultaneous depletion of endogenous ATP with hexokinase. Under the same conditions, TTP and ATP-gamma-S were unable to support significant levels of transport. None of the NTPs tested had a differential effect on anterograde vs. retrograde movement of vesicles. Surprisingly, several inconsistencies were revealed when a comparison was made between these results and nucleoside triphosphate specificities that have been reported for putative organelle motors by using in vitro assays. These data may be used in conjunction with data from well-defined in vitro assays to develop models for the molecular mechanisms of axonal transport.     

Impairment of anterograde and retrograde neurofilament transport after anti-kinesin and anti-dynein antibody microinjection in chicken dorsal root ganglia

The purpose of the present study was to investigate the participation of the motor proteins kinesin and dynein in axonal transport of neurofilaments (NF) in cultured dorsal root ganglia neurons. Therefore, we performed live-recording studies of the green fluorescent protein-tagged neurofilament M (GFP-NF-M) to assay transport processes in neurons. Co-localization studies revealed that GFP-NF-M was capable to build a functional NF network with other NF subunits, including phosphorylated heavy neurofilaments (NF-H-PH). Time-lapse recordings using confocal laser scanning microscopy exhibited fast transport of NF dots in anterograde and retrograde direction through a photobleached gap. Following microinjection of anti-kinesin antibodies or colchicine treatment an impairment of anterograde as well as retrograde NF transport was observed during live-recording experiments. In contrast, microinjection of anti-dynein antibodies only impaired retrograde transport of NF whereas the anterograde movement of GFP-NF-M was unaffected. Treatment of the cells with unspecific antibodies had no effect.     

Tight Functional Coupling of Kinesin-1A and Dynein Motors in the Bidirectional Transport of Neurofilaments

We have tested the hypothesis that kinesin-1A (formerly KIF5A) is an anterograde motor for axonal neurofilaments. In cultured sympathetic neurons from kinesin-1A knockout mice, we observed a 75% reduction in the frequency of both anterograde and retrograde neurofilament movement. This transport defect could be rescued by kinesin-1A, and with successively decreasing efficacy by kinesin-1B and kinesin-1C. In wild-type neurons, headless mutants of kinesin-1A and kinesin-1C inhibited both anterograde and retrograde movement in a dominant-negative manner. Because dynein is thought to be the retrograde motor for axonal neurofilaments, we investigated the effect of dynein inhibition on anterograde and retrograde neurofilament transport. Disruption of dynein function by using RNA interference, dominant-negative approaches, or a function-blocking antibody also inhibited both anterograde and retrograde neurofilament movement. These data suggest that kinesin-1A is the principal but not exclusive anterograde motor for neurofilaments in these neurons, that there may be some functional redundancy among the kinesin-1 isoforms with respect to neurofilament transport, and that the activities of the anterograde and retrograde neurofilament motors are tightly coordinated.     

Mechanisms of depletion of substance P by capsaicin

Capsaicin is a neurotoxin that can deplete sensory nerves of their content of substance P and interfere with certain sensory functions, such as responses of animals to noxious heat stimuli. In adult guinea pigs, a species that is susceptible to the effects of capsaicin on both substance P content and sensory function, capsaicin induces selective depletion of substance P from dorsal root ganglia and the dorsal spinal cord, sites of the cell bodies and central terminals of primary afferent neurons, respectively. As the onset of thermal analgesia in guinea pigs precedes depletion of substance P, direct neural actions of capsaicin probably account for its effects on sensory function. Capsaicin interferes with the retrograde transport of nerve growth factor (NGF) to the cell bodies of sensory nerves. Decreased availability of NGF at the site of neural protein synthesis leads to decreased synthesis of substance P. After failure of synthesis of substance P, the content of the peptide in sensory nerves gradually decreases until depletion occurs.     

Studies on the mechanism of the reversal of rapid organelle transport in myelinated axons of Xenopus laevis

Rapid organelle transport was studied by computer- and video-enhanced microscopy in the region of localized lesions in single myelinated axons of Xenopus laevis. Localized lesions were created that were either impermeable to small ions in the bathing medium or were permeable to agents with molecular weights up to 10,000. Providing the axons were bathed in a suitable "internal" medium, organelle transport continued to within a few micrometers of the lesion whether the lesion was permeable or not. Organelles undergoing anterograde and retrograde transport reversed their direction of transport on reaching the lesion. In preparations with lesions that were permeable, nonhydrolyzable analogs of ATP inhibited normally directed and reversed organelle transport. In permeable preparations, vanadate and EDTA inhibited retrograde and reversed retrograde transport at different intra-axonal concentrations; anterograde and reversed anterograde transport were also differentially inhibited. Anterograde and retrograde organelle transport were also shown to be inhibited at different intraaxonal concentrations of vanadate and EDTA. The results provide evidence for the existence of two different axonal transport mechanisms in myelinated axons. The two mechanisms can account for the normally directed and reversed transport of individual organelles.     

Regulation of axon transport of hydrocortisone via spinal root fibers and its modification in old rats

The intensity of axon transport (AT) of³H-hydrocortisone via fibers of the ventral (anterograde AT) and dorsal (retrograde AT) roots was shown to depend on the concentration of labelled hormone injected into the gray matter of theL ₅-L₆ spinal segments. The concentration dependence of anterograde flow (axons of motoneurons) was manifested both in the velocity and amount of transported hormone, while only velocity was modified in retrograde AT (in central processes of dorsal root ganglion neurons). In old rats, a concentration-dependent increase in the amount of labelled hormone was observed only within rather narrow range of activity of injected³H-hydrocortisone (from 0.036 to 0.108 MBq/μl). An increase in the activity of labelled hormone to 0.14–0.18 MBq/μl suppressed its uptake into the ventral root fibers. At the same time, modifications of the AT velocity showed direct dependence on the concentration within the entire studied range of³H-hydrocortisone activity. similar modification of the velocity were observed in the dorsal roots of old rats. It is supposed that an increase in the intracellular concentration of a glucocorticoid activates the mechanisms of hormone removal from the motoneurons, and these mechanisms become sharply weaked in old age.     

Retrograde but not anterograde bead movement in intact axons requires dynein

Dynein and kinesin have been implicated as the molecular motors that are responsible for the fast transport of axonal membranous organelles and vesicles. Experiments performed in vitro with partially reconstituted preparations have led to the hypothesis that kinesin moves organelles in the anterograde direction and dynein moves them in the retrograde direction. However, the molecular basis of transport directionality remains unclear. In the experiments described here, carboxylated fluorescent beads were injected into living Mauthner axons of lamprey and the beads were observed to move in both the anterograde and retrograde directions. The bead movement in both directions required intact microtubules, occurred at velocities approaching organelle fast transport in vivo, and was inhibited by vanadate at concentrations that inhibit organelle fast transport. When living axons were injected with micromolar concentrations of vanadate and irradiated at 365 nm prior to bead injections, a treatment that results in the V1 photolysis of dynein, the retrograde movement of the beads was specifically abolished. Neither the ultraviolet irradiation alone nor the vanadate alone produced the retrograde-specific inhibition. These results support the hypothesis that dynein is required for retrograde, but not anterograde, transport in vivo. © 1995 John Wiley & Sons, Inc.     

Products of endocytosis and autophagy are retrieved from axons by regulated retrograde organelle transport

Cellular homeostasis in neurons requires that the synthesis and anterograde axonal transport of protein and membrane be balanced by their degradation and retrograde transport. To address the nature and regulation of retrograde transport in cultured sympathetic neurons, I analyzed the behavior, composition, and ultrastructure of a class of large, phase-dense organelles whose movement has been shown to be influenced by axonal growth (Hollenbeck, P. J., and D. Bray. 1987. J. Cell Biol. 105:2827-2835). In actively elongating axons these organelles underwent both anterograde and retrograde movements, giving rise to inefficient net retrograde transport. This could be shifted to more efficient, higher volume retrograde transport by halting axonal outgrowth, or conversely shifted to less efficient retrograde transport with a larger anterograde component by increasing the intracellular cyclic AMP concentration. When neurons were loaded with Texas red- dextran by trituration, autophagy cleared the label from an even distribution throughout the neuronal cytosol to a punctate, presumably lysosomal, distribution in the cell body within 72 h. During this process, 100% of the phase-dense organelles were fluorescent, showing that they contained material sequestered from the cytosol and indicating that they conveyed this material to the cell body. When 29 examples of this class of organelle were identified by light microscopy and then relocated using correlative electron microscopy, they had a relatively constant ultrastructure consisting of a bilamellar or multilamellar boundary membrane and cytoplasmic contents, characteristic of autophagic vacuoles. When neurons took up Lucifer yellow, FITC-dextran, or Texas red-ovalbumin from the medium via endocytosis at the growth cone, 100% of the phase-dense organelles became fluorescent, demonstrating that they also contain products of endocytosis. Furthermore, pulse-chase experiments with fluorescent endocytic tracers confirmed that these organelles are formed in the most distal region of the axon and undergo net retrograde transport. Quantitative ratiometric imaging with endocytosed 8-hydroxypyrene-1,3,6- trisulfonic acid showed that the mean pH of their lumena was 7.05. These results indicate that the endocytic and autophagic pathways merge in the distal axon, resulting in a class of predegradative organelles that undergo regulated transport back to the cell body.     

Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

The importance of endosome-to–trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport.     

JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors

Regulation of the opposing kinesin and dynein motors that drive axonal transport is essential to maintain neuronal homeostasis. Here, we examine coordination of motor activity by the scaffolding protein JNK-interacting protein 1 (JIP1), which we find is required for long-range anterograde and retrograde amyloid precursor protein (APP) motility in axons. We identify novel interactions between JIP1 and kinesin heavy chain (KHC) that relieve KHC autoinhibition, activating motor function in single molecule assays. The direct binding of the dynactin subunit p150^Glued to JIP1 competitively inhibits KHC activation in vitro and disrupts the transport of APP in neurons. Together, these experiments support a model whereby JIP1 coordinates APP transport by switching between anterograde and retrograde motile complexes. We find that mutations in the JNK-dependent phosphorylation site S421 in JIP1 alter both KHC activation in vitro and the directionality of APP transport in neurons. Thus phosphorylation of S421 of JIP1 serves as a molecular switch to regulate the direction of APP transport in neurons.     

Studies on the mechanism of axoplasmic transport in the crayfish cord

Following the injection of ³H-leucine into a crayfish ganglion, tritiumlabeled proteins were detected remaining in the ganglion and moving at a slow linear rate caudad along the nerve cord. The rate of movement increased linearly with temperature between 5 and 20°C, but ceased at 3° C. The movement was also blocked for a distance around a colchicine-injected ganglion. Both of these observations would be compatible with the involvement of microtubules in slow axoplasmic transport. However, in both instances normal-appearing microtubules were observed by electron microscopy. Gel electropherograms of the denatured labeled proteins showed that the transported proteins are complex and may correspond to most of the axoplasmic proteins including the microtubules.     

Rapid axonal transport in Xenopus nerve in divalent cation free media

An investigation was made of the effects of bathing media low in divalent cations on rapid axonal transport in the sciatic nerve of the amphibian Xenopus laevis. The anterograde transport of a pulse of [35S]methionine proteins was observed using a multiple proportional counter as the detector. Organelles undergoing anterograde and retrograde transport were detected by light microscopy. The structure of nerve fibers was examined by light and electron microscopy. There was no significant difference in the anterograde transport of proteins in nerves bathed in normal medium (NM) containing millimolar Ca2+ and Mg2+ and in those bathed in calcium-free medium (CaFM) containing Mg2+. The anterograde transport of labelled proteins continued at a normal velocity in nerves bathed in divalent cation free medium (DCFM) for at least 14 h. DCFM did cause some alterations in protein transport: the ratio of the plateau (following pulse passage) to the peak radioactivity was increased, the pulse amplitude decreased more rapidly, and the label continued to arrive at the distal end of the nerve for greater than 16 h. Anterograde and retrograde organelle transport continued normally for periods of greater than or equal to 4 h in fibres bathed in DCFM. All myelinated fibres became distorted within 4 h in DCFM. Similar distortion was rare in fibres bathed in CaFM. The results indicate that axonal transport in Xenopus is largely independent of lowered concentrations of divalent cations in the bathing medium. Those alterations in axonal transport that were produced by DCFM may have been secondary to morphological changes in the nerve fibres.     

Capsaicin stimulates glucose uptake in C2C12 muscle cells via the reactive oxygen species (ROS)/AMPK/p38 MAPK pathway

Capsaicin has been reported to regulate blood glucose levels and to ameliorate insulin resistance in obese mice. This study demonstrates that capsaicin increases glucose uptake directly by activating AMP-activated protein kinase (AMPK) in C2C12 muscle cells, which manifested as an attenuation of glucose uptake when compound C, an AMPK inhibitor, was co-administered with capsaicin. However, the insulin signaling molecules insulin receptor substrate-1 (IRS-1) and Akt were not affected by capsaicin. Additional results showed that p38 mitogen-activated protein kinase (MAPK) is also involved in capsaicin-induced glucose transport downstream of AMPK because capsaicin increased p38 MAPK phosphorylation significantly and its specific inhibitor SB203580 inhibited capsaicin-mediated glucose uptake. Treatment with an AMPK inhibitor reduced p38 MAPK phosphorylation, but the p38 MAPK inhibitor had no effect on AMPK. Capsaicin stimulated ROS generation in C2C12 muscle cells, and when ROS were captured using the nonspecific antioxidant NAC, the increase in both capsaicin-induced AMPK phosphorylation and capsaicin-induced glucose uptake was attenuated, suggesting that ROS function as an upstream activator of AMPK. Taken together, these results suggest that capsaicin, independent of insulin, increases glucose uptake via ROS generation and consequent AMPK and p38 MAPK activations.     

In vitro capsaicin-induced cytological changes and alteration in calcium distribution in giant serotonergic neurons of the snail Helix pomatia: a light- and electron-microscopic study

Morphological changes induced by capsaicin were studied in the serotonergic metacerebral giant neurons of the cerebral ganglia of Helix pomatia under in vitro conditions. Capsaicin at a concentration of 10^-4 M caused characteristic structural alterations in the giant serotonergic neurons but did not significantly influence serotonin immunoreactivity in the neurons. At the lightmicroscopic level, the most conspiciuous structural alterations were swelling of the cell bodies, which contained a swollen pale nucleus. Under the electron microscope, the nuclei,mitochondria and the cisternae of the endoplasmic reticulum were swollen in the capsaicin-affected metacerebral giant neurons. Electron-microscopic cytochemical techniques for calcium demonstration revealed electron-dense deposits in the swollen mitochondria and in the cisternae of the endoplasmic reticulum, suggesting an increased Ca²⁺ influx. The serotonergic metacerebral giant neurons could be labelled by cobalt (1 mM) in the presence of capsaicin (10^-4 M) suggesting that capsaicin opens the cation chanels of the capsaicin-sensitive neuronal membrane. The morphological and cytochemical alterations induced by capsaicin in the serotonergic metacerebral giant neurons of Helix pomatia closely resemble those induced in sensory neurons of mammalian dorsal root ganglion.This work was supported by OTKA grants No.: 2477, T016861, T017127 and ETT 587/93     

Capsaicin induces mitochondrial dysfunction and apoptosis in anaplastic thyroid carcinoma cells via TRPV1-mediated mitochondrial calcium overload

Anaplastic thyroid cancer (ATC) is a rare malignancy and has a poor prognosis due to its aggressive behavior and resistance to treatments. Calcium (Ca²⁺) serves as a ubiquitous cellular second messenger and influences several tumor behaviors. Therefore, Ca²⁺ modulation is expected to be a novel therapeutic target in cancers. However, whether Ca²⁺ modulation is effective in ATC therapy remains unknown. In this study, we reported that capsaicin (CAP), a transient receptor potential vanilloid type1 (TRPV1) agonist, inhibited the viability of anaplastic thyroid cancer cells. Capsaicin treatment triggered Ca²⁺ influx by TRPV1 activation, resulting in disequilibrium of intracellular calcium homeostasis. The rapidly increased cytosolic Ca²⁺ concentration was mirrored in the mitochondria and caused a severe condition of mitochondrial calcium overload in ATC cells. In addition, the disruption of mitochondrial calcium homeostasis caused by capsaicin led to mitochondrial dysfunction in ATC cells, as evidenced by the production of mitochondrial reactive oxygen species (ROS), depolarization of mitochondrial membrane potential (ΔΨm), and opening of mitochondrial permeability transition pore (mPTP). Next, the resulting release of cyt c into the cytosol triggered apoptosome assembly and subsequent caspase activation and apoptosis. It was worth noting that both TRPV1 antagonist (capsazepine) and calcium chelator (BAPTA) could attenuate aberrant Ca²⁺ homeostasis, mitochondrial dysfunction and apoptosis induced by capsaicin treatment. Thus, our study demonstrated that capsaicin induced mitochondrial calcium overload and apoptosis in ATC cells through a TRPV1-mediated pathway. The better understanding of the anti-cancer mechanisms of calcium modulation provides a potential target for the ATC therapy.