Impacts of increased α-synuclein on clathrin-mediated endocytosis at synapses:implications for neurodegenerative diseases

α-Synuclein causes synaptic pathologies in several neurodegenerative diseases：Parkinson’s disease（PD）is a neurodegenerative disease that impacts the lives of millions of people worldwide.A pathological hallmark of PD,as well as dementia with Lewy bodies（DLB）and several Alzheimer’s disease variants,is the appearanceof intracellular inclusions called Lewy bodies, which contain high levels of aggregated α-synuclein,

Numerical study of clathrin-mediated endocytosis of nanoparticles by cells under tension

In this study, a three-dimensional mathematical model was used to study the contribution of clathrins during the process of cellular uptake of spherical nanoparticles under different membrane tensions. The clathrin-coated pit (CCP) that forms around the inward budding of the cell membrane was modeled as a vesicle with bending rigidity. An optimization algorithm was proposed for minimizing the total energy of the system, which comprises the deforming nanoparticle, receptor-ligand bonds, cell membrane, and CCP, in which way, the profile of the system is acquired. The results showed that the CCP enable full wrapping of the nanoparticles at various membrane tensions. When the cell membrane tension increases, the total deformation energy also increases, but the ratio of CCP bending to the minimum value of the total energy of the system decreases. The results also showed that the diameter of the endocytic vesicles determined by the competition between the stretching of the cell membrane and confinement of the coated pits are much larger than the nanoparticles, which is quit different as the results in passive endocytosis that is not facilitated by the CCPs. The present results indicate that variations of tension on cell membranes constitutes a biophysical marker for understanding the size distribution of CCPs observed in experiments. The present results also suggest that the early abortion of endocytosis is related to that the receptor-ligand bonds cannot generate adequate force to wrap the nanoparticles into the cell membrane before the clathrins respond to support the endocytic vesicles. Correspondingly, late abortion may relate to the inability of CCPs to confine the nanoparticles until the occurrence of the necking stage of endocytosis.

Osmotic Stress Modulates the Balance between Exocytosis and Clathrin-Mediated Endocytosis in Arabidopsis thaliana

The sessile life style of plants creates the need to deal with an often adverse environment, in which water availability can change on a daily basis, challenging the cellular physiology and integrity. Changes in os- motic conditions disrupt the equilibrium of the plasma membrane： hypoosmotic conditions increase and hyperosmotic environment decrease the cell volume. Here, we show that short-term extracellular osmotic treatments are closely followed by a shift in the balance between endocytosis and exocytosis in root mer- istem cells. Acute hyperosmotic treatments （ionic and nonionic） enhance clathrin-mediated endocytosis simultaneously attenuating exocytosis, whereas hypoosmotic treatments have the opposite effects. In addition to clathrin recruitment to the plasma membrane, components of early endocytic trafficking are essential during hyperosmotic stress responses. Consequently, growth of seedlings defective in elements of clathrin or early endocytic machinery is more sensitive to hyperosmotic treatments. We also found that the endocytotic response to a change of osmotic status in the environment is dominant over the presum- ably evolutionary more recent regulatory effect of plant hormones, such as auxin. These results imply that osmotic perturbation influences the balance between endocytosis and exocytosis acting through clathrin- mediated endocytosis. We propose that tension on the plasma membrane determines the addition or removal of membranes at the cell surface, thus preserving cell integrity.

Trafficking pathway between plasma membrane and mitochondria via clathrin-mediated endocytosis

Endocytosis is a basic cellular process that describes a form of active transport across the plasma membrane into the cell.The endocytic pathway consists of distinct membrane compartments;internalized molecules are delivered to early endosomes,and some of them are recycled back to the surface,whereas other molecules are sent to late endosomes and lysosomes for degradation.However,little is known about how mitochondria are involved in the endocytic pathway.Here,we report that FM dyes, membrane-impermeant fluorescent lipid probes,can traffic to mitochondria directly from the plasma membrane by clathrinmediated endocytosis.FM dye entry into mitochondria uses microtubule-dependent active transport,but the mechanism is different from the classical endocytic pathway.Hence,this study reveals a previously unrealized lipid trafficking pathway from the plasma membrane to mitochondria.

Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis

Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance,currently,there is no effective therapy to treat morphine tolerance.In the current study,we aimed to develop a monoclonal antibody(mAb)precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms.We successfully prepared a mAb targeting MOR,named 3A5C7,by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization,and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation.Treatment of two cell lines,HEK293T and SH-SY5Y,with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2(GRK2)/b-arrestin2-dependent mechanism,as demonstrated by immunofluorescence staining,flow cytometry,Western blotting,coimmunoprecipitation,and small interfering ribonucleic acid(siRNA)-based knockdown.This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR.We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid.Western blot,enzyme-linked immunosorbent assays,and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase,the in vitro biomarker of morphine tolerance,via the GRK2/b-arrestin2 pathway.Furthermore,in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice,and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence.Finally,intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/b-arrestin2 pathway.Collectively,our study provided a therapeutic mAb,3A5C7,targeting MOR to treat morphine tolerance,mediated by enhancing morphine-induced MOR endocytosis.The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.

The endocytosis and intracellular fate of nanomedicines: Implication for rational design

Nanomedicines employ multiple endocytic pathways to enter cells.Their following fate is interesting,but it is not sufficient understood currently.This review introduces the endocytic pathways,presents new technologies to confirm the specific endocytic pathways and discusses factors for pathway selection.In addition,some intriguing implication about nanomedicine design based on endocytosis will also be discussed at the end.This review may provide new thoughts for the design of novel multifunctional nanomedicines.

Nexus of signaling and endocytosis in oncogenesis driven by non-small cell lung cancer-associated epidermal growth factor receptor mutants

Epidermal growth factor receptor(EGFR) controls a wide range of cellular processes, and aberrant EGFR signaling as a result of receptor overexpression and/or mutation occurs in many types of cancer. Tumor cells in non-small cell lung cancer(NSCLC) patients that harbor EGFR kinase domain mutations exhibit oncogene addiction to mutant EGFR, which confers high sensitivity to tyrosine kinase inhibitors(TKIs). As patients invariably develop resistance to TKIs, it is important to delineate the cell biological basis of mutant EGFR-induced cellular transformation since components of these pathways can serve as alternate therapeutic targets to preempt or overcome resistance. NSCLC-associated EGFR mutants are constitutively-active and induce ligandindependent transformation in nonmalignant cell lines. Emerging data suggest that a number of factors are critical for the mutant EGFR-dependent tumorigenicity, and bypassing the effects of TKIs on these pathways promotes drug resistance. For example, activation of downstream pathways such as Akt, Erk, STAT3 and Src is critical for mutant EGFR-mediated biological processes. It is now well-established that the potency and spatiotemporal features of cellular signaling by receptor tyrosine kinases such as EGFR, as well as the specific pathways activated, is determined by the nature of endocytic traffic pathways through which the active receptors traverse. Recent evidence indicates that NSCLCassociated mutant EGFRs exhibit altered endocytic trafficking and they exhibit reduced Cbl ubiquitin ligasemediated lysosomal downregulation. More recent work has shown that mutant EGFRs undergo ligand-independent traffic into the endocytic recycling compartment, a behavior that plays a key role in Src pathway activation and oncogenesis. These studies are beginning to delineate the close nexus between signaling and endocytic traffic of EGFR mutants as a key driver of oncogenicprocesses. Therefore, in this review, we will discuss the links between mutant EGFR signaling and endocytic properties, and introduce potential mechanisms by which altered endocytic properties of mutant EGFRs may alter signaling and vice versa as well as their implications for NSCLC therapy.

Endocytosis unplugged： multiple ways to enter the cell

Endocytosis 在房间表面发生并且与它的成分膜蛋白质和类脂化合物一起包含血浆膜(下午) 的成为主观。Endocytosis 涉及细胞外的周围的采样并且也服务调整在房间表面开始的各种各样的过程。这些包括滋养的举起，从房间表面受体，和为在 metazoans 工作的房间和织物的许多另外的过程必需品发信号。它对下午类脂化合物和蛋白质动态平衡的维护也中央。有成为主观的多重工具并发地操作，在房间表面。与在高分辨率的可视化技术的前进，同时追踪多重 endocytic 货物现在是可能的，揭示在一个单个房间的 endocytic 进程的显著差异。实时房间成像和有效基因操作的联合也帮助了在成为主观的这些机制理解分子的播放器的功能的层次。这里，我们提供各种各样的 endocytic 线路的一篇报道，他们越过数的操作和出现的机制。

Endocytosis of adiponectin receptor I through a clathrin- and Rab5-dependent pathway

在真核细胞的房间，受体 endocytosis 是调整发信号的 transduction 的一个关键事件。Adiponectin 受体属于从 G-protein-coupled 受体是不同的并且在糖尿病和新陈代谢的症候群的致病有关键角色的一个新受体家庭。这里，我们分析了 adiponectin 和 adiponectin 受体的 endocytosis 1 (AdipoR1 ) 并且发现他们两个都被使内在化进跟随类似的交通线路的 transferrin 积极的分隔空间。由表示 Eps15 异种或弄空的 K+ 堵住调停 clathrin 的 endocytosis 在血浆膜套住 AdipoR1，并且 K+ 弄空废除了 adiponectin 成为主观，显示 AdipoR1 和 adiponectin 的 endocytosis 是 clathrin 依赖的。K+ 的弄空和异种提高的 Eps15 的 overexpression 刺激 adiponectin 的激活安培的蛋白质 kinase phosphorylation，建议 AdipoR1 力量 downregulate adiponectin 发信号的 endocytosis。另外，有小 GTPase Rab5 的 AdipoR1 colocalizes，和主导的否定 Rab5 废除 AdipoR1 endocytosis。这些数据显示 AdipoR1 通过一条 clathrin 依赖、 Rab5 依赖的小径被使内在化并且 endocytosis 可以在 adiponectin 发信号的规定起一个作用。

Mechanical force-driven TNFαendocytosis governs stem cell homeostasis

Mesenchymal stem cells(MSCs)closely interact with the immune system,and they are known to secrete inflammatory cytokines in response to stress stimuli.The biological function of MSC-derived inflammatory cytokines remains elusive.Here,we reveal that even under physiological conditions,MSCs produce and release a low level of tumor necrosis factor alpha(TNFα),which is unexpectedly required for preserving the self-renewal and differentiation of MSCs via autocrine/paracrine signaling.Furthermore,TNFαcritically maintains MSC function in vivo during bone homeostasis.Mechanistically,we unexpectedly discovered that physiological levels of TNFαsafeguard MSC homeostasis in a receptor-independent manner through mechanical force-driven endocytosis and that endocytosed TNFαbinds to mammalian target of rapamycin(mTOR)complex 2 and restricts mTOR signaling.Importantly,inhibition of mTOR signaling by rapamycin serves as an effective osteoanabolic therapeutic strategy to protect against TNFαdeficiency and mechanical unloading.Collectively,these findings unravel the physiological framework of the dynamic TNFαshuttlebased mTOR equilibrium that governs MSC and bone homeostasis.

A Numerical Study of Passive Receptor-Mediated Endocytosis of Nanoparticles:The Effect of Mechanical Properties

In this work,a three-dimensional axisymmetric model with nanoparticle,receptor-ligand bonds and cell membrane as a system was used to study the quasi-static receptor-mediated endocytosis process of spherical nanoparticles in drug delivery.The minimization of the system energy function was carried out numerically,and the deformations of nanoparticle,receptor-ligand bonds and cell membrane were predicted.Results show that passive endocytosis may fail due to the rupture of receptor-ligand bonds during the wrapping process,and the size and rigidity of nanoparticles affect the total deformation energy and the terminal wrapping stage.Our results suggest that,in addition to the energy requirement,the success of passive endocytosis also depends on the maximum strength of the receptor-ligand bonds.

Endocytosis of FcαR is clathrin and dynamin dependent, but its cytoplasmic domain is not required

Fc 伪 R，为 IgA 的 Fc 受体，为调停 IgA 的有免疫力的回答是必要的。以前的研究证明了 IgA 和 IgA 免疫者建筑群能是很快由 Fc 伪 R 的 endocytosed。然而，内在的机制仍然保持不清楚。这里，我们在 monocytic 房间线调查了 Fc 伪 R 的 endocytic 小径， U937，那自然地快速的 Fc 伪 R 并且在 transfected 汉语仓鼠卵巢(CHO ) ， COS-7 和 Hela 房间。由使用不同 endocytic 小径的选择化学禁止者， Eps15 的主导否定的异种的 overexpression 并且 clathrin 击倒经由 RNA 干扰的重链(CHC ) ，我们证明 Fc 伪 R 的 endocytosis 通过一条调停 clathrin 的小径。endocytosed Fc 伪 R 走进 Rab5 积极、 Rab11 积极的内涵体。然而， Fc 伪 R 的 endocytosis 不能被 Rab5 的主导否定的异种堵住。我们也证明 Fc 伪 R 的 endocytosis 由 overexpressing 是 dynamin 依赖的 dynamin 的主导否定的异种。为 Fc 伪 R 的潜在的 endocytic 主题也被检验。出人意料地，我们发现 Fc 伪 R 的全部细胞质的域没为 Fc 伪 R 的 endocytic 进程被要求。我们断定 Fc 伪 R 的 endocytosis 是 clathrin-dependent, 并且 dynamin 依赖，但是没被 Rab5，和 endocytic 主题调整不位于 Fc 伪 R 的细胞质的领域。

The relationship between endocytosis of peritoneal macrophages induced by concanavalin A and intracellular free calcium in mo

In this experiment the morphological changes of mouse peritoneal macrophages in the course of their conjugation with colloidal gold-labelled concanavalin A(ConA-Au) i by the surface receptor and then the endocytosis and transport of the ConA were observed

Localization of proteins involved in endocytosis at tubulobulbar complexes in rat testes

Tubulobulbar Complexes (TBCs) are actin-rich structures formed between Sertoli-cells and spermatids at the time of sperm release. The main functions of the TBCs are to remove excess spermatid cytoplasm and acrosomal contents, internalize and recycle junctional complexes by endocytosis prior to spermiation. However, in addition to recycling some of the molecules undergo lysosomal degradation. The molecular machinery involved in endocytosis at the TBCs is not well understood. To bridge this gap localization of various proteins, involved at various steps of endocytosis studied in other systems, was demonstrated in TBCs using testicular fragmented material or sections by immunoblotting and immunofluroscence. The presence of key molecules like Vamp-2, syntaxin and Lamp-2 indicates occurrence of lysosomal degradation in addition to junctional recycling at the TBCs present at the time of sperm release. TBCs are endocytic devices functioning to recycle junctional molecules or remove spermatid cytoplasm that were present between spermatids and Sertoli-cells all through the process of spermatid maturation and in turn regulate male fertility.

Endocytosis and Vesicular Transport of Plasmid DNA in Cells During Electric Field-Mediated Gene Delivery

Pulsed electric field has been used widely as a nonviral approach to improving gene delivery in basic and translational research[1-2].The technique has been called electrotransfection(ET),electroporation,electrogene transfer,and gene electroinjection in the literature [1,3].It has a great potential to improve clinical treatment of diseases through delivery of vaccines and therapeutic genes,genome and epigenome editing,and generation of human induced pluripotent stem cells for tissue engineering[1-3].During ET,extracellular transport of plasmid DNA(pDNA)relies on electrophoresis,which is critical for applications in vivo.However,mechanisms of intracellular transport remain to be understood.The lack of understanding has hindered the translation of ET technology to the clinic.It is well known that pulsed electric field can generate transient hydrophilic pores in the plasma membrane(i.e.,electroporation)that permit membrane-impermeant molecules to enter cells.Although the pores have yet to be visualized directly under a microscope,the electric field-induced membrane permeabilization has been demonstrated through experimental measurements of electrical conductance of synthetic lipid membranes and plasma membranes,direct observation of fluorescent markers crossing the membranes facing both cathode and anode,and numerical simulations of the membrane permeabilization[1,3].Results from the simulations have predicted that the cutoff size of the pores is on the order of a few hundred nanometers,and the lifetime of the pores that are larger than 100 nm is on the order of 10 msec.Although these data provide a solid evidence of the membrane permeabilization,recent studies have demonstrated that the generation of the pores is insufficient for ET[1,4].The reasons are as follows.First,the lifetime of the pores is several orders of magnitude shorter than the time scale for pDNA uptake,which is on the order of 10 min.Second,complex formation between pDNA and plasma membrane is a necessary condition for successful gene transfer.Third,inhibition of clathrin mediated endocytosis or Rac-1 dependent micropinocytosis can reduce the amount of pDNA internalized by cells [1].Finally,we demonstrate that few pDNA molecules can be observed in the cytosol that are not associated with the intracellular vesicles[5],suggesting that pDNA uptake is mediated by endocytosis.In addition to the internalization,ET requires the pDNA in the cytoplasm to reach the nucleus.To understand mechanisms of intracellular trafficking of pDNA,we have examined time-dependent pDNA distributions in cells,quantitatively determined percentages of pDNA molecules associated with different endocytic compartments using transmission electron microscopy(TEM),and investigated different approaches to facilitate cytoplasmic transport and nuclear entry of pDNA.Our data have shown that electrotransfected pDNA is located in different vesicular ultrastructures at or near the plasma membrane at10 min post application of electric pulses[5].In the hard-to-transfect cells(e.g.,4T1),pDNA penetration from the cell surface is less active,and the total number of vesicular structures associated with pDNA is low,compared to those in the easyto-transfect cells(e.g.,COS7).Our data have also shown that macropinocytosis is the most common pathway shared by all types of cells.To investigate how improve pDNA transport in cells,we have photochemically treated cells to non-specifically induce pDNA escape from intracellular vesicles,or blocked endosome and autophagic vacuole maturation through treatment of cells with Bafilomycin Al,an inhibitor of vacuolar H+ATPase.Our data demonstrate that both treatments can lead to reduction of ET efficiency although the treatment for inducing endosomal escape can enhance poly-L-lysine mediated gene delivery.These data suggest that the vesicles play an important role in protecting the naked pDNA during intracellular trafficking.The nuclear envelope is another major barrier to ET.To facilitate the nuclear entry,we have examined three different approaches.One is to synchronize the nuclear envelope breakdown(NEBD)prior to ET;the second approach is to pre-treat cells with a nuclear pore dilating agent(i.e.,trans-1,2-cyclohexanediol);and the third one is to incorporate a nuclear targeting sequence(NTS)(i.e.,SV40)into the pDNA.Our data have shown that the synchronization of the NEBD can significantly improve the ET efficiency without compromising the cell viability.The nuclear pore dilation can improve the ET as well but the dilating agent is cytotoxic.The incorporation of NTS into pDNA can improve the gene delivery efficiency but the improvement is cell-type dependent,suggesting that the NTS has to be screened and optimized for the cells of interest.In summary,the transient pores in the plasma membrane induced by the electric pulses will enable cellular uptake of membrane-impermeant molecules up to the size of small proteins.Larger molecules(e.g.,pDNA)have to be internalized via endocytic processes triggered by the pulsed electric field.Within the cells,pDNA transport is mediated by vesicles and can be blocked by non-specific escape from vesicles or inhibition of vesicle maturation.The nuclear entry of pDNA can be enhanced,without compromising cell viability,through the use of the NTS or the synchronization of the NEBD.

Exocytosis,endocytosis and recycling of secretory vesicles in neuroendocrine cells,and its regulation by cortical actin

The cortical actin network is a mesh of filaments distributed beneath the plasmalemma that dynamically reacts in response to stimuli.This dynamic network of cortical filaments,together with motor myosin partners,adjusts the plasmalemma tension,organizes membrane protein microdomains,remodels the cell surface and drives vesicle motion in order to fine-tune exocytosis,endocytosis and recycling of secretory vesicles.In this review,we discuss how these mechanisms work in secretory cells.

Overexpression of Toll-like receptor 4 contributes to the internalization and elimination of Escherichia coli in sheep by enhancing caveolae-dependent endocytosis

Background:Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health.Toll-like receptor 4(TLR4)plays a crucial role in host defence against Gram-negative bacteria.Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections.In a previous study,we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance.The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood.Results:In the present study,we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model.The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection.This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis.Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4,Src signalling,or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria,weakened the ability of the monocytes to eliminate the bacteria,and increased the pH of the endosomes.Conclusion:Together,our results reveal the effects of TLR4 on the control of E.coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model,providing theoretical support for breeding disease resistance in the future.Furthermore,Src and caveolin 1(CAV1)could be potentially valuable targets for the control of infectious diseases.

High-density lipoprotein endocytosis in endothelial cells

AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.

GPCR/endocytosis/ERK signaling/S2R is involved in the regulation of the internalization,mitochondria-targeting and-activating properties of human salivary histatin 1

Human salivary histatin 1(Hst1)exhibits a series of cell-activating properties,such as promoting cell spreading,migration,and metabolic activity.We recently have shown that fluorescently labeled Hst1(F-Hst1)targets and activates mitochondria,presenting an important molecular mechanism.However,its regulating signaling pathways remain to be elucidated.We investigated the influence of specific inhibitors of G protein-coupled receptors(GPCR),endocytosis pathways,extracellular signal-regulated kinases1/2(ERK1/2)signaling,p38 signaling,mitochondrial respiration and Na+/K+-ATPase activity on the uptake,mitochondria-targeting and-activating properties of F-Hst1.We performed a si RNA knockdown(KD)to assess the effect of Sigma-2 receptor(S2R)/Transmembrane Protein 97(TMEM97)—a recently identified target protein of Hst1.We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1.Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1.The inhibitors of GPCR,ERK1/2,phagocytosis,and clathrin-mediated endocytosis(CME)as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake,which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity.Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1.We further showed the intracellular trafficking and targeting process of F-Hst1,in which early endosome plays an important role.Overall,phagocytosis,CME,GPCR,ERK signaling,and S2R/TMEM97 are involved in the internalization of Hst1,while only CME and S2R/TMEM97 are critical for its subcellular targeting.The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.

S4A-3 The Small GTPase Rac1 Contributes to Extinction of Aversive Memories of Drug Withdrawal by Facilitating GABAA Receptor Endocytosis in the vmPFC

Aim:Extinction of aversive memories associated with drug withdrawal has been proposed as a therapeutic strategy for the treatment of drug addiction.However,the mechanisms underlying extinction of such memory are poorly understood.This study was,therefore,undertaken to investigate the role of Rho GTPase Rac1-mediated GABAAR endocytosis in the vmPFC in extinction of aversive memories associated with drug withdrawal.Methods:conditioned place aversion(CPA)was used as a model for measurement of the aversive memories of opiate withdrawal.Extinction experiments were performed as described in our previous study(Wang et al.,2012).Results:we found that extinction of CPA required activation of Rac1 in the vmPFC in a brain-derived neurotrophic factor(BDNF)-dependent manner,which triggers actin polymerization via Pak1-cofilin signaling pathway,leading to synaptic localization of activity-regulated cytoskeleton-associated protein(Arc)in the vmPFC.The synaptic Arc further determines GABAA receptor(GABAAR)endocytosis that is necessary and sufficient for vmPFC long-term potentiation and CPA extinction.Thus,extinction of an aversive memory associated with drug withdrawal is intriguingly controlled by Rac1-dependent GABAAR endocytosis in the vmPFC,thereby suggesting therapeutic targets to promote extinction of the unwanted memory.Conclusion:BDNF dependent Rac1 GTPase activation in the vmPFC contributes to aversive memory extinction by Arc-mediated GABAA receptor endocytosis.