Do Buyers Deserve the Same Fate as child traffickers?

While China has tough laws to deter child trafficking with punishment ranging from up to 10 years in jail to even the death sentence, those who buy trafficked children get off lightly. Under the current law, the maximum punishment they face is three years behind bars.

Unveiling Global Human Trafficking Trends: A Comprehensive Analysis

This paper presents a comprehensive analysis of global human trafficking trends over a twenty-year period, leveraging a robust dataset from the Counter Trafficking Data Collaborative (CTDC). The study unfolds in a systematic manner, beginning with a detailed data collection phase, where ethical and legal standards for data usage and privacy are strictly observed. Following collection, the data undergoes a rigorous preprocessing stage, involving cleaning, integration, transformation, and normalization to ensure accuracy and consistency for analysis. The analytical phase employs time-series analysis to delineate historical trends and utilizes predictive modeling to forecast future trajectories of human trafficking using the advanced analytical capabilities of Power BI. A comparative analysis across regions—Africa, the Americas, Asia, and Europe—is conducted to identify and visualize the distribution of human trafficking, dissecting the data by victim demographics, types of exploitation, and duration of victimization. The findings of this study not only offer a descriptive and predictive outlook on trafficking patterns but also provide insights into the regional nuances that influence these trends. The article underscores the prevalence and persistence of human trafficking, identifies factors contributing to its evolution, and discusses the implications for policy and law enforcement. By integrating a methodological approach with quantitative analysis, this research contributes to the strategic planning and resource allocation for combating human trafficking. It highlights the necessity for continued research and international cooperation to effectively address and mitigate this global issue. The implications of this research are significant, offering actionable insights for policymakers, law enforcement, and advocates in the ongoing battle against human trafficking.

Optic nerve injury-induced regeneration in the adult zebrafish is accompanied by spatiotemporal changes in mitochondrial dynamics

Axonal regeneration in the central nervous system is an energy-intensive process.In contrast to mammals,adult zebrafish can functionally recover from neuronal injury.This raises the question of how zebrafish can cope with this high energy demand.We previously showed that in adult zebrafish,subjected to an optic nerve crush,an antagonistic axon-dendrite interplay exists wherein the retraction of retinal ganglion cell dendrites is a prerequisite for effective axonal repair.We postulate a‘dendrites for regeneration’paradigm that might be linked to intraneuronal mitochondrial reshuffling,as ganglion cells likely have insufficient resources to maintain dendrites and restore axons simultaneously.Here,we characterized both mitochondrial distribution and mitochondrial dynamics within the different ganglion cell compartments(dendrites,somas,and axons)during the regenerative process.Optic nerve crush resulted in a reduction of mitochondria in the dendrites during dendritic retraction,whereafter enlarged mitochondria appeared in the optic nerve/tract during axonal regrowth.Upon dendritic regrowth in the retina,mitochondrial density inside the retinal dendrites returned to baseline levels.Moreover,a transient increase in mitochondrial fission and biogenesis was observed in retinal ganglion cell somas after optic nerve damage.Taken together,these findings suggest that during optic nerve injury-induced regeneration,mitochondria shift from the dendrites to the axons and back again and that temporary changes in mitochondrial dynamics support axonal and dendritic regrowth after optic nerve crush.

Structural determinants specific for retromer protein sorting nexin 5 in regulating subcellular retrograde membrane trafficking

The endosomal trafficking of signaling membrane proteins, such as receptors, transporters and channels, is mediated by the retromer-mediated sorting machinery, composed of a cargo-selective vacuolar protein sorting trimer and a membrane-deforming subunit of sorting nexin proteins. Recent studies have shown that the isoforms, sorting nexin 5 (SNX5) and SNX6, have played distinctive regulatory roles in retrograde membrane trafficking. However, the molecular insight determined functional differences within the proteins remains unclear. We reported that SNX5 and SNX6 had distinct binding affinity to the cargo protein vesicular monoamine transporter 2 (VMAT2). SNX5, but not SNX6, specifically interacted with VMAT2 through the Phox domain, which contains an alpha-helix binding motif. Using chimeric mutagenesis, we identified that several key residues within this domain were unique in SNX5, but not SNX6, and played an auxiliary role in its binding to VMAT2. Importantly, we generated a set of mutant SNX6, in which the corresponding key residues were mutated to those in SNX5. In addition to the gain in binding affinity to VMAT2, their overexpression functionally rescued the altered retrograde trafficking of VMAT2 induced by siRNA-mediated depletion of SNX5. These data strongly suggest that SNX5 and SNX6 have different functions in retrograde membrane trafficking, which is determined by the different structural elements within the Phox domain of two proteins. Our work provides a new information on the role of SNX5 and SNX6 in the molecular regulation of retrograde membrane trafficking and vesicular membrane targeting in monoamine neurotransmission and neurological diseases.

Mitochondria in Huntington’s disease:implications in pathogenesis and mitochondrial-targeted therapeutic strategies

Huntington’s disease is a genetic disease caused by expanded CAG repeats on exon 1 of the huntingtin gene located on chromosome 4.Compelling evidence implicates impaired mitochondrial energetics,altered mitochondrial biogenesis and quality control,disturbed mitochondrial trafficking,oxidative stress and mitochondrial calcium dyshomeostasis in the pathogenesis of the disorder.Unfortunately,conventional mitochondrial-targeted molecules,such as cysteamine,creatine,coenzyme Q10,or triheptanoin,yielded negative or inconclusive results.However,future therapeutic strategies,aiming to restore mitochondrial biogenesis,improving the fission/fusion balance,and improving mitochondrial trafficking,could prove useful tools in improving the phenotype of Huntington’s disease and,used in combination with genome-editing methods,could lead to a cure for the disease.

Organ Donation and Obstacles: University Student Survey

Background: Organ transplantation has helped improve the quality of life of patients with lethal terminal organ failure. This success is owed to the progress made in many fields such as surgery, immunology…However, in our country Morocco, we are faced with reluctance to donate. The study’s objective is to evaluate the perception of organ donation among university students. Methods: We conducted this cross-sectional study with descriptive and analytical aims. Our target population consisted of 991 university students from eight higher education structures. An anonymous questionnaire was distributed to those students by a single interviewer. The questions of the survey answered four main themes. Thus, two types of studies were done. The first was a descriptive study of the socio-demographic characteristics of the selected population and their knowledge and attitudes about organ donation. The second was an analytical study of the correlation between the socio-demographic characteristics, type and level of education of the target population and their knowledge and attitudes towards organ donation. Results: 97.2% of the respondents have already heard about organ donation. If 836 of the students (84.4%) thought that transplantation could be an effective therapeutic alternative, 155 of the students (15.6%) were not aware of this possibility. Furthermore, 298 students, which means 30.1% of the students, did not know that organ transplantation was practiced in Morocco. The causes of refusal found in our study were numerous. 68.5% of students blamed the lack of information as the main cause of their reluctance. 64.7% were afraid of organ trafficking. 41.1% refused the idea of mutilating the body of the deceased, which could impact the funeral. 37.3% thought that donation would be a violation of the human body according to religious values. 33.9% were against donation because they hoped that the brain-dead patient could wake up. The main results of our survey showed the very favorable attitude towards organ donation and transplantation, despite the lack of knowledge on the subject. Conclusion: This study revealed the absence of information on the practice of organ transplantation in Morocco and the underestimation of the number of people waiting for transplantation. On the other hand, like the data found in the literature, our study highlights the religious obstacles and the lack of confidence in the legislation governing donation and transplantation in our country.

Identification and characterization of a novel bipartite nuclear localization signal in the hepatitis B virus polymerase

AIM:To characterize the nuclear import of hepatitis B virus(HBV)polymerase(P)and its relevance for the viral life cycle.METHODS:Sequence analysis was performed to predict functional motives within P.Phosphorylation of P was analyzed by in vitro phosphorylation.Phosphorylation site and nuclear localization signal(NLS)were destroyed by site directed mutagenesis.Functionality of the identified NLS was analyzed by confocal fluorescence microscopy and characterizing the karyopherin binding.Relevance of the structural motives for viral life cycle was studied by infection of primary Tupaia hepatocytes with HBV.RESULTS:We identified by sequence alignment and functional experiments a conserved bipartite NLS containing a casein kinaseⅡ(CKⅡ)phosphorylation site located within the terminal protein domain(TP)of the HBV polymerase.Inhibition of CKⅡimpairs the functionality of this NLS and thereby prevents the nuclear import of the polymerase.Binding of the import factor karyopherin-α2 to the polymerase depends on its CKⅡ-mediated phosphorylation of the bipartite NLS.In HBV-infected primary Tupaia hepatocytes CKⅡinhibition in the early phase(post entry phase)of the infection process prevents the establishment of the infection.CONCLUSION:Based on these data it is suggested that during HBV infection the final import of the genome complex into the nucleus is mediated by a novel bipartite NLS localized in the TP domain of HBV polymerase.

Modulation of cell surface GABA B receptors by desensitization,trafficking and regulated degradation

Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system,and mediates its effects via two classes of receptors:the GABA A and GABA B receptors.GABA A receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission.GABA B receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission.The extent of inhibitory neurotransmission is determined by a variety of factors,such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g.,phosphorylation),as well as by the number of receptors present in the plasma membrane available for signal transduction.The level of GABA B receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation.In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA B receptors in the plasma membrane,and thereby signaling strength.

Important relationships between Rab and MICAL proteins in endocytic trafficking

The internalization of essential nutrients,lipids and receptors is a crucial process for all eukaryotic cells.Accordingly,endocytosis is highly conserved across cell types and species.Once internalized,small cargocontaining vesicles fuse with early endosomes(also known as sorting endosomes),where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway.Although the mechanisms that regulate this sorting are still poorly understood,some receptors are directed to late endosomes and lysosomes for degradation,whereas other receptors are recycled back to the plasma membrane;either directly or through recycling endosomes.The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways.Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membraneassociated proteins,as a consequence of the activity of multiple specific GTPase-activating proteins(GAPs) and GTP exchange factors(GEFs).Once bound to GTP,Rabs interact with a multitude of effector proteins that carry out Rab-specific functions.Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology(EHD) proteins,which are also intimately involved in endocytic regulation.A particularly interesting example of common Rab-EHD interaction partners is the MICALlike protein,MICAL-L1.MICAL-L1 and its homolog,MICAL-L2,belong to the larger MICAL family of proteins,and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins,as well as controlling cytoskeletal rearrangement and neurite outgrowth.In this review,we summarize the functional roles of MICAL and Rab proteins,and focus on the significance of their interactions and the implications for endocytic transport.

Regulatory role of sorting nexin 5 in protein stability and vesicular targeting of vesicular acetylcholine transporter to synaptic vesicle-like vesicles in PC12 cells

Accurate targeting of vesicular acetylcholine transporter(VAChT)to synaptic vesicles(SVs)is indispensable for efficient cholinergic transmission.Previous studies have suggested that the dileucine motif within the C-terminus of the transporter is sufficient for its targeting to SVs.However,the cytosolic machinery underlying specific regulation of VAChT trafficking and targeting to SVs is still unclear.Here we used the C-terminus of VAChT as a bait in a yeast two-hybrid screen to identify sorting nexin 5(SNX5)as its novel interacting protein.SNX5 was detected in the SVs enriched LP2 subcellular fraction of rat brain homogenate and showed strong colocalization with VAChT in both brain sections and PC12 cells.Binding assays suggested that the C-terminal domain of VAChT can interact with both BAR and PX domain of SNX5.Depletion of SNX5 enhanced the degradation of VAChT and the process was mediated through the lysosomal pathway.More importantly,we found that,in PC12 cells,the depletion of SNX5 expression significantly decreased the synaptic vesicle-like vesicles(SVLVs)localization of VAChT.Therefore,the results suggest that SNX5 is a novel regulator for both stability and SV targeting of VAChT.

Promoting axon regeneration in the central nervous system by increasing PI3-kinase signaling

Much research has focused on the PI3-kinase and PTEN signaling pathway with the aim to stimulate repair of the injured central nervous system.Axons in the central nervous system fail to regenerate,meaning that injuries or diseases that cause loss of axonal connectivity have life-changing consequences.In 2008,genetic deletion of PTEN was identified as a means of stimulating robust regeneration in the optic nerve.PTEN is a phosphatase that opposes the actions of PI3-kinase,a family of enzymes that function to generate the membrane phospholipid PIP_(3) from PIP_(2)(phosphatidylinositol(3,4,5)-trisphosphate from phosphatidylinositol(4,5)-bisphosphate).Deletion of PTEN therefore allows elevated signaling downstream of PI3-kinase,and was initially demonstrated to promote axon regeneration by signaling through mTOR.More recently,additional mechanisms have been identified that contribute to the neuron-intrinsic control of regenerative ability.This review describes neuronal signaling pathways downstream of PI3-kinase and PIP3,and considers them in relation to both developmental and regenerative axon growth.We briefly discuss the key neuron-intrinsic mechanisms that govern regenerative ability,and describe how these are affected by signaling through PI3-kinase.We highlight the recent finding of a developmental decline in the generation of PIP_(3) as a key reason for regenerative failure,and summarize the studies that target an increase in signaling downstream of PI3-kinase to facilitate regeneration in the adult central nervous system.Finally,we discuss obstacles that remain to be overcome in order to generate a robust strategy for repairing the injured central nervous system through manipulation of PI3-kinase signaling.

Luminal/extracellular domains of chimeric CI-M6PR-C proteins interfere with their retrograde endosome-to-TGN trafficking in the transient expression system

The membrane trafficking of cation-independent mannose 6-phosphate receptor（CI-M6PR） between the transGolgi network（TGN） and endosomal compartments is not only critical for maintaining lysosomal function but also a well-known event for understanding molecular and cellular mechanisms in retrograde endosome-to-TGN trafficking.Although it has been well established in literature that the C-terminus of bovine CI-M6PR determines its retrograde trafficking,it remains unclear whether the luminal domain of the protein plays a role on these sorting events.In this study,we found that partial deletion of luminal domain of human CI-M6PR mistargeted the mutant protein to nonTGN compartments.Moreover,replacing the luminal domain of both bovine and human CI-M6PR with that from irrelevant membrane proteins such as CD8 or Tac also altered the TGN targeting of the chimeric proteins.On the other hand,only short sequence from HA fused with the transmembrane domain and C-terminus of the receptor,HA-hCIM6PR-tail,resulted in its preferential targeting to TGN as for the full length receptor,strongly suggesting that sorting of the receptor may be influenced by luminal sequence.Furthermore,using this luminal truncated form of HA-hCIM6 PR as a model cargo,we found that the trafficking of the chimeric protein was regulated by the retromer complex through interacting with SNX5.In conclusion,our study strongly suggested that the disrupted luminal domain from hCI-M6PR or other irrelevant membrane proteins interfere with the process of membrane trafficking and TGN targeting of CI-M6PR.

The Heat Shock Protein Story—From Taking mTORC1,2 and Heat Shock Protein Inhibitors as Therapeutic Measures for Treating Cancers to Development of Cancer Vaccines

Heat shock proteins (HSPs) serve to correct proteins’ conformation, send the damaged proteins for degradation (quality control function). Heat shock factors (HSFs) are their transcription factors. The protein complexes mTOR1 and 2 (with the same core mTOR), the phosphoinositide-dependent protein kinase-1 (PDK1), the seine/threonine-specific protein kinase (Akt), HSF1, plus their associated proteins form a network participating in protein synthesis, bio-energy generation, signaling for apoptosis with the help of HSPs. A cancer cell synthesizes proteins at fast rate and needs more HSPs to work on quality control. Shutting down this network would lead to cell death. Thus inhibitors of mTOR (mTORI) and inhibitors of HSPs (HSPI) could drive cancer cell to apoptosis—a “passive approach”. On the other hand, HSPs form complexes with polypeptides characteristic of the cancer cells;on excretion from the cell, they becomes antigens for the immunity cells, eventually leading to maturation of the cytotoxic T cells, forming the basic principle of preparing cancer-specific, person-specific vaccine. Recent finding shows that HSP70 can penetrate cancer cell and expel its analog to extracellular region, giving the hope to prepare a non-person-specific vaccine covering a variety of cancers. Activation of anti-cancer immunity is the “active approach”. On the other hand, mild hyperthermia, with increase of intracellular HSPs, has been found to activate the immunity response, and demonstrate anti-cancer effects. There are certain “mysteries” behind the mechanisms of the active and passive approaches. We analyze the mechanisms involved and provide explanations to some mysteries. We also suggest future research to improve our understanding of these two approaches, in which HSPs play many roles.

CAR T Cell Immunotherapy That Revolutionary Breakthrough in Human Oncology Treatment: A Review

The discovery of CAR T cell immunotherapy, also known as chimeric antigen receptor (CAR) T cell immunotherapy, has added a new dimension to the world of cancer treatment. This is a gene-based treatment in which T cells from the patient’s body are taken and genetically engineered in the lab to grow receptors. T cells containing this receptor are then injected into the patient’s body to bind to the antigen on the surface area of the cancer cell and kill the cancer cell. Structurally, the co-stimulatory domain added to CAR T cells has now reached the 5th GEN of chimeric antigen receptor T cells. Chimeric antigen T cell immunotherapy is the first FDA-approved treatment for hematological malignancies that is both safe and effective. However, due to some challenges such as a lack of safety control, an immunosuppressive tumor microenvironment, ineffective T cell trafficking, and so on, CAR-T immunotherapy treatment for solid malignancy is still in the clinical phase. In the result and discussion, we have presented a survey of CAR T cell therapy with a combination of pharmacological drugs. The things we mentioned are that CAR T cell immunotherapy is innovative, suitable, elegant, and also controls synergistic anti-cancer effects. A better understanding of combinatory CAR T cell therapies provides fundamental information for improvement of those therapies, in addition to the article highlighting future opportunities, commercial advancements, and various applications of CAR T cell therapy in different cancer cells. In the entire review article, we have highlighted the neck and crop of CAR T cell therapy, from which it is easy to understand the therapy and the need for this therapy in cancer prevention and its progress.

Retrograde trafficking of VMAT2 and its role in protein stability in non-neuronal cells

Increasing evidence suggests that the impaired neuroprotection of vesicular monoamine transporter 2（VMAT2）contributes to the pathogenesis of Parkinson＇s disease.That has been linked to aberrant subcellular retrograde trafficking as strongly indicated by recent genomic studies on familial Parkinson＇s diseases.However,whether VMAT2 function is regulated by retrograde trafficking is unknown.By using biochemistry and cell biology approaches,we have shown that VMAT2 was stringently localized to the trans-Golgi network and underwent retrograde trafficking in non-neuronal cells.The transporter also interacted with the key component of retromer,Vps35,biochemically and subcellularly.Using specific siRNA,we further showed that Vps35 depletion altered subcellular localization of VMAT2.Moreover,siRNA-mediated Vps35 knockdown also decreased the stability of VMAT2 as demonstrated by the reduced half-life.Thus,our work suggested that altered vesicular trafficking of VMAT2 may play a vital role in neuroprotection of the transporter as well as in the pathogenesis of Parkinson＇s disease.

Distinctive roles of Rac1 and Rab29 in LRRK2 mediated membrane trafficking and neurite outgrowth

Parkinson＇s disease（PD） associated leucine-rich repeat kinase 2（LRRK2） mutants have shown pathogenic effects on a variety of subcellular processes. Two small GTPases Rac1 and Rab29 have been indicated as possible downstream effectors participating in LRRK2 signaling but their detail mechanisms remain unclear. In this study, we have used biochemical and cell biology approaches to address whether two GTPases interact with LRRK2 and hence function differently in LRRK2 mediated pathogenesis. Here we show that Rac1 and Rab29 specifically interact with LRRK2 with higher affinity for Rab29 and with different preference in functional domain binding. Mutant Rab29 but not Racl alters the endosome-to-TGN retrograde trafficking of a cargo protein cation-independent mannose-6-phosphate receptor（CI-M6 PR） and its stability. On the other hand, overexpressed wild type Rab29 but not Racl rescued the altered retrograde membrane trafficking induced by the pathogenic mutant LRRK2^（G2019 S）. Furthermore,both Rac1 and Rab29 rescued neurite shortening in differentiated SH-SY5 Y cells induced by LRRK2^（G2019 S）. Our study strongly suggests that Rac1 and Rab29 are involved in distinct functions as downstream effectors in LRRK2 signaling pathways.

AAV8 transduction capacity is reduced by prior exposure to endosome-like pH conditions

Adeno-associated virus(AAV)is an essential instrument in the neuroscientist’s toolkit,which allows delivery of DNA to provide labeling with fluorescent proteins or genetic instructions to regulate gene expression.In the field of neural regeneration,the transduction of neurons enables the observation and regulation of axon growth and regeneration,and in the future will likely be a mechanism for delivering molecular therapies to promote sprouting and regeneration after central nervous system injury.Traditional formulations of AAV preparations permit efficient viral transduction under physiologic conditions,but an improved understanding of the mechanistic limitations of AAV transduction may facilitate production of more resilient AAV strains for investigative and therapeutic purposes.We studied AAV transduction in the context of prior exposure of AAV serotype 8(AAV8)to environmental pH within the range encountered during endosomal endocytosis(pH 7.4 to pH 4.4),during which low pH-triggered structural and autoproteolytic changes to the viral capsid are believed to be necessary for endosome escape and virus uncoating.Due to the fundamental nature of these processes,we hypothesized that premature exposure of AAV8 particles to acidic pH would decrease viral transduction of HT1080 cells in vitro,as measured by fluorescent reporter gene expression using high-content imaging analysis.We found that increasingly acidic incubation conditions were associated with concomitant reductions in transduction efficiency,and that quantitative levels of reporter gene expression in transduced cells were similarly decreased.The biggest decrease in transduction occurred between pH 7.4 and pH 6.4,suggesting the possible co-occurrence of a pH-associated event and viral inactivation within that range.Taken together,these findings indicate that exposure of AAV8 to acidic pH for as little as 1 hour is deleterious to transduction ability.Future studies are necessary to understand the pH-associated causative mechanisms involved.This study was approved by the University of Miami Institutional Animal Care and Use Committee,USA(Protocol#18-108-LF)on July 12,2018.

Rabs and axonal regeneration

Membrane trafficking processes are presumably vital for axonal regeneration after injury, but mechanistic understanding in this regard has been sparse. A recent loss-of-function screen had been carried out for factors important for axonal regeneration by cultured cortical neurons and the results suggested that the activity of a number of Rab GTPases might act to restrict axonal regeneration. A loss of Rab27b, in particular, is shown to enhance axonal regeneration in vitro, as well as in C. elegans and mouse central nervous system injury models in vivo. Possible mechanisms underlying this new finding, which has important academic and translational implication, are discussed.

Glycosylated and non-glycosylated quantum dot-displayed peptides trafficked indiscriminately inside lung cancer cells but discriminately sorted in normal lung cells: An indispensable part in nanoparticle-based intracellular drug delivery

Difference in sub-cellular trafficking of glycosylated and naked peptides, between normal and lung cancer cells, was established. Normal lung tissue discriminately sorted glycosylated from non-glycosylated peptides by allowing golgi localization of the glycosylated peptides while restricting golgi entry of the naked peptides. This mechanism was surprisingly not observed in its cancer cell counterpart. Lung cancer cells tend to allow unrestricted localization of both glycosylated and naked peptides in the golgi apparatus. This newly discovered difference in sub-cellular trafficking between normal and lung cancer cells could potentially be used as an effective strategy in targeted intracellular delivery, especially targeting golgi-resident enzymes for possible treatment of diseases associated with glycans and glycoproteins, such as, congenital disease of glycosylation(CDG). This very important detail in intracellular trafficking inside normal and cancer cells is an indispensable part in nanoparticle-based intracellular drug delivery.

Diazepam reduces synaptic GABA type areceptor availability via multiple trafficking mechanisms

OBJECTIVE Investigate the effects of diazepam(DZP) on γ2 subunit containing GABA type A receptor(GABA A R) trafficking.METHODS Immunofluorescence microscopy measured surface GABA A Rs and gephyrin in rat cortical neurons after 24 h exposure of 1.0 μmol·L^(-1) DZP.Biochemical studies of mice injected with 10 mg·kg^(-1) DZP vs vehicle were assessed for γ2 subunit and total gephyrin cortical levels 12 h post-injection.Ubiquitination of the γ2 subunit was studied by immunoprecipitation after 12 h of 1.0 μmol·L^(-1) DZP exposure.A γ2 subunit encoding an N terminal fluorogen-activating peptide and pH-sensitive green fluorescent protein(γ2 pH FAP) measured lysosomal targeting of γ2 containing GABA A Rs.RFP-gephyrin and γ2 pH FAP synaptic diffusion rates were examined using fluorescence recovery after photobleaching(FRAP).RESULTS Extrasynaptic levels of γ2 GABA A Rs decreased by 12.2%,while synaptic gephyrin S270 phosphorylation increased by 18.3% in DZP-treated neurons after 24 h compared to control(P<0.05).Dendritic levels of gephyrin were also reduced to 74.1% of control,while S270 phosphorylation was elevated by 25.2%(P<0.05;P<0.01).Mice 12 h post-DZP injection demonstrated a 12.7% and 26.1% decrease in total γ2 and gephyrin levels,respectively(P<0.05;P<0.01).12 h DZP treatment enhanced γ2 subunit ubiquitination 1.13-fold relative to control(P<0.05).Internalized γ2 pH FAP GABA A Rs associated with lysosomes was 8.0% higher in neurons treated with 12-16 h DZP compared to control.Pilot FRAP experiments suggest gephyrin and γ2 have increased mobility and turnover at synapses following DZP.CONCLUSION DZP treatment decreases γ2 GABA A R levels and gephyrin scaffolding function after one day of exposure,which may contribute to the formation of DZP tolerance.