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BACKGROUND AND AIMS: Extracellular vesicles (EVs) secreted by cardiosphere-derived cells exert immunomodulatory effects through the transmission of small non-coding RNAs. METHODS: The mechanism and role of yREX3, a small Y RNA abundant in EVs in myocardial injury, was investigated. RESULTS: yREX3 attenuates cardiac ischaemic injury by selective DNA methylation. Synthetic yREX3 encapsulated in lipid nanoparticles triggers broad transcriptomic changes in macrophages, localizes to the nucleus, and mediates epigenetic silencing of protein interacting with C kinase-1 (Pick1) through methylation of upstream CpG sites. Moreover, yREX3 interacts with polypyrimidine tract binding protein 3 (PTBP3) to methylate the Pick1 gene locus in a DNA methyltransferase-dependent manner. Suppression of Pick1 in macrophages potentiates Smad3 signalling and enhances efferocytosis, minimizing heart necrosis in rats with myocardial infarction. Adoptive transfer of Pick1-deficient macrophages recapitulates the cardioprotective effects of yREX3 in vivo. CONCLUSIONS: These findings highlight the role of a small Y RNA mined from EVs with a novel gene-methylating mechanism.

Aims: Islet cell autoantigen 1 (ICA1) is involved in autoimmune diseases and may affect synaptic plasticity as a neurotransmitter. Databases related to Alzheimer's disease (AD) have shown decreased ICA1 expression in patients with AD. However, the role of ICA1 in AD remains unclear. Here, we report that ICA1 expression is decreased in the brains of patients with AD and an AD mouse model. Results: The ICA1 increased the expression of amyloid precursor protein (APP), disintegrin and metalloprotease 10 (ADAM10), and disintegrin and metalloprotease 17 (ADAM17), but did not affect protein half‐life or mRNA levels. Transcriptome sequencing analysis showed that ICA1 regulates the G protein‐coupled receptor signaling pathway. The overexpression of ICA1 increased PKCα protein levels and phosphorylation. Conclusion: Our results demonstrated that ICA1 shifts APP processing to non‐amyloid pathways by regulating the PICK1‐PKCα signaling pathway. Thus, this study suggests that ICA1 is a novel target for the treatment of AD. [ABSTRACT FROM AUTHOR]

Context Metabolic disorders such as obesity represent a major health challenge. Obesity alone has reached epidemic proportions, with at least 2.8 million people worldwide dying annually from diseases caused by overweight or obesity. The brain–metabolic axis is central to maintain homeostasis under metabolic stress via an intricate signaling network of hormones. Protein interacting with C kinase 1 (PICK1) is important for the biogenesis of various secretory vesicles, and we have previously shown that PICK1-deficient mice have impaired secretion of insulin and growth hormone. Objective The aim was to investigate how global PICK1-deficient mice respond to high-fat diet (HFD) and assess its role in insulin secretion in diet-induced obesity. Methods We characterized the metabolic phenotype through assessment of body weight, composition, glucose tolerance, islet morphology insulin secretion in vivo, and glucose-stimulated insulin secretion ex vivo. Results PICK1-deficient mice displayed similar weight gain and body composition as wild-type (WT) mice following HFD. While HFD impaired glucose tolerance of WT mice, PICK1-deficient mice were resistant to further deterioration of their glucose tolerance compared with already glucose-impaired chow-fed PICK1-deficient mice. Surprisingly, mice with β-cell–specific knockdown of PICK1 showed impaired glucose tolerance both on chow and HFD similar to WT mice. Conclusion Our findings support the importance of PICK1 in overall hormone regulation. However, importantly, this effect is independent of the PICK1 expression in the β-cell, whereby global PICK1-deficient mice resist further deterioration of their glucose tolerance following diet-induced obesity. [ABSTRACT FROM AUTHOR]

Long-term potentiation (LTP) is one of the major cellular mechanisms for learning and memory. Activity-dependent increases in surface AMPA receptors (AMPARs) are important for enhanced synaptic efficacy during LTP. Here, we report a novel function of a secretory trafficking protein, ICA69, in AMPAR trafficking, synaptic plasticity, and animal cognition. ICA69 is first identified as a diabetes-associated protein well characterized for its function in the biogenesis of secretory vesicles and trafficking of insulin from ER, Golgi to post-Golgi in pancreatic beta cells. In the brain, ICA69 is found in the AMPAR protein complex through its interaction with PICK1, which binds directly to GluA2 or GluA3 AMPAR subunits. Here, we showed that ICA69 regulates PICK1's distribution in neurons and stability in themouse hippocampus, which in turn can impact AMPAR function in the brain. Biochemical analysis of postsynaptic density (PSD) proteins from hippocampi of mice lacking ICA69 (Ica1 knockout) and their wild-type littermates revealed comparable AMPAR protein levels. Electrophysiological recording and morphological analysis of CA1 pyramidal neurons from Ica1 knockout also showed normal AMPAR-mediated currents and dendrite architecture, indicating that ICA69 does not regulate synaptic AMPAR function and neuron morphology at the basal state. However, genetic deletion of ICA69 in mice selectively impairs NMDA receptor (NMDAR)-dependent LTP but not LTD at Schaffer collateral to CA1 synapses, which correlates with behavioral deficits in tests of spatial and associative learning and memory. Together, we identified a critical and selective role of ICA69 in LTP, linking ICA69-mediated synaptic strengthening to hippocampus-dependent learning and memory. [ABSTRACT FROM AUTHOR]

Aims: To explore the underlying mechanism by which low‐frequency KRAS mutations result in extensive EndMT occurrence. Methods: Exosomes derived from primarily cultured brain arteriovenous malformation (bAVMs) and human umbilical vein endothelial cells (HUVECs) transfected with KRASG12D, KRASWT, or KRASNC lentiviruses were isolated, and their effects on HUVECs were identified by western blotting and immunofluorescence staining. The expression levels of exosomal microRNAs (miRNAs) were evaluated by miRNA microarray, followed by functional experiments on miR‐3131 and detection of its downstream target, and miR‐3131 inhibitor in reversing the EndMT process induced by KRASG12D‐transfected HUVECs and bAVM endothelial cells (ECs) were explored. Results: Exosomes derived from KRASG12D bAVM ECs and KRASG12D‐transfected HUVECs promoted EndMT in HUVECs. MiR‐3131 levels were highest in the exosomes of KRASG12D‐transfected HUVECs, and HUVECs transfected with the miR‐3131 mimic acquired mesenchymal phenotypes. RNA‐seq and dual‐luciferase reporter assays revealed that PICK1 is the direct downstream target of miR‐3131. Exosomal miR‐3131 was highly expressed in KRASG12D bAVMexos compared with non‐KRAS‐mutant bAVMexos or HUVECexos. Finally, a miR‐3131 inhibitor reversed EndMT in HUVECs treated with exosomes or the supernatant of KRASG12D‐transfected HUVECs and KRASG12D bAVM ECs. Conclusion: Exosomal miR‐3131 promotes EndMT in KRAS‐mutant bAVMs, and miR‐3131 might be a potential biomarker and therapeutic target in KRASG12D‐mutant bAVMs. [ABSTRACT FROM AUTHOR]

Muscarinic acetylcholine receptors (mAChRs) have been shown to play significant roles in the regulation of normal cognitive processes in the hippocampus, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are also involved in these processes. This study aims to explore the mAChR-mediated regulation of AMPARs GluA2 trafficking and to reveal the key proteins and the signaling cascade involved in this process. Primary hippocampal neurons, as cell models, were treated with agonist 77-LH-28-1 and antagonist VU0255035, Fsc231, and APV. C57BL/6J male mice were stereotactically injected with 77-LH-28-1 and Fsc231 to obtain hippocampal slices. The trafficking of GluA2 was detected by surface biotinylation and immunostaining. Activation of M1 mAChRs promoted endocytosis and decreased the postsynaptic localization of the AMPA receptor subunit GluA2 and that phosphorylation of GluA2 at Ser880 was increased by M1 mAChR activity. Fsc231 blocked the endocytosis and postsynaptic localization of GluA2 induced by 77-LH-28-1 without affecting the phosphorylation of Ser880. PICK1 was required for M1 mAChR-mediated GluA2 endocytosis and downstream of phosphorylation of GluA2-Ser880, and the PICK1-GluA2 interaction was essential for M1 mAChR-mediated postsynaptic expression of GluA2. Taken together, our results show a functional correlation of M1 mAChRs with GluA2 and the role of PICK1 in their interplay. [ABSTRACT FROM AUTHOR]

Metabotropic glutamate receptor Type 3 (mGlu3) controls the sleep/wake architecture, which plays a role in the glutamatergic pathophysiology of schizophrenia. Interestingly, mGlu3 receptor expression is decreased in the brain of schizophrenic patients. However, little is known about the molecular mechanisms regulating mGlu3 receptors at the cell membrane. Subcellular receptor localization is strongly dependent on protein-protein interactions. Here we show that mGlu3 interacts with PICK1 and that this scaffolding protein is important for mGlu3 surface expression and function in hippocampal primary cultures. Disruption of their interaction via an mGlu3 C-terminal mimicking peptide or an inhibitor of the PDZ domain of PICK1 altered the functional expression of mGlu3 receptors in neurons. We next investigated the impact of disrupting the mGlu3-PICK1 interaction on hippocampal theta oscillations in vitro and in vivo in WT male mice. We found a decreased frequency of theta oscillations in organotypic hippocampal slices, similar to what was previously observed in mGlu3 KO mice. In addition, hippocampal theta power was reduced during rapid eye movement sleep, non-rapid eye movement (NREM) sleep, and wake states after intraventricular administration of the mGlu3 C-terminal mimicking peptide. Targeting the mGlu3-PICK1 complex could thus be relevant to the pathophysiology of schizophrenia. [ABSTRACT FROM AUTHOR]

Abstract SorLA is a member of the Vps10p-domain (Vps10p-D) receptor family of type-I transmembrane proteins conveying neuronal endosomal sorting. The extracellular/luminal moiety of SorLA has a unique mosaic domain composition and interacts with a large number of different and partially unrelated ligands, including the amyloid precursor protein as well as amyloid-β. Several studies support a strong association of SorLA with sporadic and familial forms of Alzheimer’s disease (AD). Although SorLA seems to be an important factor in AD, the large number of different ligands suggests a role as a neuronal multifunctional receptor with additional intracellular sorting capacities. Therefore, understanding the determinants of SorLA’s subcellular targeting might be pertinent for understanding neuronal endosomal sorting mechanisms in general. A number of cytosolic adaptor proteins have already been demonstrated to determine intracellular trafficking of SorLA. Most of these adaptors and several ligands of the extracellular/luminal moiety are shared with the Vps10p-D receptor Sortilin. Although SorLA and Sortilin show both a predominant intracellular and endosomal localization, they are targeted to different endosomal compartments. Thus, independent adaptor proteins may convey their differential endosomal targeting. Here, we hypothesized that Sortilin and SorLA interact with the cytosolic adaptors PSD95 and PICK1 which have been shown to bind the Vps10p-D receptor SorCS3. We observed only an interaction for SorLA and PICK1 in mammalian-two-hybrid, pull-down and cellular recruitment experiments. We demonstrate by mutational analysis that the C-terminal minimal PDZ domain binding motif VIA of SorLA mediates the interaction. Moreover, we show co-localization of SorLA and PICK1 at vesicular structures in primary neurons. Although the physiological role of the interaction between PICK1 and SorLA remains unsolved, our study suggests that PICK1 partakes in regulating SorLA’s intracellular itinerary.

The PDZ family has drawn attention as possible drug targets because of the domains' wide ranges of function and highly conserved binding pockets. The PICK1 PDZ domain has been proposed as a possible drug target because the interactions between the PICK1 PDZ domain and the GluA2 subunit of the AMPA receptor have been shown to progress neurodegenerative diseases. BIO124 has been identified as a sub µM inhibitor of the PICK1–GluA2 interaction. Here, we use all-atom molecular dynamics simulations to reveal the atomic-level interaction pattern between the PICK1 PDZ domain and BIO124. Our simulations reveal three unique binding conformations of BIO124 in the PICK1 PDZ binding pocket, referred to here as state 0, state 1, and state 2. Each conformation is defined by a unique hydrogen bonding network and a unique pattern of hydrophobic interactions between BIO124 and the PICK1 PDZ domain. Interestingly, each conformation of BIO124 results in different dynamic changes to the PICK1 PDZ domain. Unlike states 1 and 2, state 0 induces dynamic coupling between BIO124 and the αA helix. Notably, this dynamic coupling with the αA helix is similar to what has been observed in other PDZ–ligand complexes. Our analysis indicates that the interactions formed between BIO124 and I35 may be the key to inducing dynamic coupling with the αA helix. Lastly, we suspect that the conformational shifts observed in our simulations may affect the stability and thus the overall effectiveness of BIO124. We propose that a physically larger inhibitor may be necessary to ensure sufficient interactions that permit stable binding between a drug and the PICK1 PDZ domain. [ABSTRACT FROM AUTHOR]

Introduction: The PICK1 PDZ domain has been identified as a potential drug target for neurological disorders. After many years of effort, a few inhibitors, such as TAT-C5 and mPD5, have been discovered experimentally to bind to the PDZ domain with a relatively high binding affinity. With the rapid growth of computational research, there is an urgent need for more efficient computational methods to design viable ligands that target proteins., Method: Recently, a newly developed program called AfDesign (part of ColabDesign) at https:// github.com/sokrypton/ColabDesign), an open-source software built on AlphaFold, has been suggested to be capable of generating ligands that bind to targeted proteins, thus potentially facilitating the ligand development process. To evaluate the performance of this program, we explored its ability to target the PICK1 PDZ domain, given our current understanding of it. We found that the designated length of the ligand and the number of recycles play vital roles in generating ligands with optimal properties., Results: Utilizing AfDesign with a sequence length of 5 for the ligand produced the highest comparable ligands to that of prior identified ligands. Moreover, these designed ligands displayed significantly lower binding energy compared to manually created sequences., Conclusion: This work demonstrated that AfDesign can potentially be a powerful tool to facilitate the exploration of the ligand space for the purpose of targeting PDZ domains., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

SorLA is a member of the Vps10p-domain (Vps10p-D) receptor family of type-I transmembrane proteins conveying neuronal endosomal sorting. The extracellular/luminal moiety of SorLA has a unique mosaic domain composition and interacts with a large number of different and partially unrelated ligands, including the amyloid precursor protein as well as amyloid-β. Several studies support a strong association of SorLA with sporadic and familial forms of Alzheimer's disease (AD). Although SorLA seems to be an important factor in AD, the large number of different ligands suggests a role as a neuronal multifunctional receptor with additional intracellular sorting capacities. Therefore, understanding the determinants of SorLA's subcellular targeting might be pertinent for understanding neuronal endosomal sorting mechanisms in general. A number of cytosolic adaptor proteins have already been demonstrated to determine intracellular trafficking of SorLA. Most of these adaptors and several ligands of the extracellular/luminal moiety are shared with the Vps10p-D receptor Sortilin. Although SorLA and Sortilin show both a predominant intracellular and endosomal localization, they are targeted to different endosomal compartments. Thus, independent adaptor proteins may convey their differential endosomal targeting. Here, we hypothesized that Sortilin and SorLA interact with the cytosolic adaptors PSD95 and PICK1 which have been shown to bind the Vps10p-D receptor SorCS3. We observed only an interaction for SorLA and PICK1 in mammalian-two-hybrid, pull-down and cellular recruitment experiments. We demonstrate by mutational analysis that the C-terminal minimal PDZ domain binding motif VIA of SorLA mediates the interaction. Moreover, we show co-localization of SorLA and PICK1 at vesicular structures in primary neurons. Although the physiological role of the interaction between PICK1 and SorLA remains unsolved, our study suggests that PICK1 partakes in regulating SorLA's intracellular itinerary. [ABSTRACT FROM AUTHOR]

Background: FSC231, a PSD‐95/DLG/ZO‐1 (PDZ) domain inhibitor of protein kinase Cα interacting protein 1 (PICK1), has analgesic effects, but the mechanism remains unclear. Methods: The expression level of PICK1 in dorsal root ganglion (DRG) of rats was changed by vector plasmid, and the effect of PICK1 on paclitaxel (PTL)‐induced neuralgia of rats was observed in collaboration with FSC231 treatment. The possible molecular mechanisms were explored by quantitative real‐time polymerase chain reaction (qRT‐PCR), Western Blot and co‐immunoprecipitation (Co‐IP) techniques. Results: PTL treatment can significantly reduce mechanical withdrawal threshold (MWT), shorten thermal withdrawal latency (TWL), promote DRG inflammation and release of substance P (SP), stimulate PICK1 expression, decrease α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid receptor 2 (AMPAR, GluA2) level and increase glycogen synthase kinase‐3β (GSK‐3β) and extracellular regulated protein kinases1/2 (ERK1/2) phosphorylation in rats, while FSC231 treatment can alleviate the above effects induced by PTL. Overexpression of PICK1 can counteract reduced PICK1 level, increased GluA2 level and decreased GSK‐3β and ERK1/2 phosphorylation levels caused by FSC231 treatment. The results of Co‐IP confirmed the interactions between PICK1 and GluA2. Both FSC231 treatment and silent PICK1 improved PTL‐induced MWT reduction, TWL shortening, inflammation, SP release and related gene expression changes, with cumulative effect. Conclusion: FSC231 activates GSK‐3β/ERK1/2 by inhibiting the interaction between PICK1 and GluA2 and alleviates PTL‐induced DRG neuralgia in rats. [ABSTRACT FROM AUTHOR]

Abstract Background Increasing evidence indicates that epithelial-mesenchymal transition (EMT) can be regulated by microRNAs (miRNAs). miR-615-3p was shown to be involved in tumor development. However, the role of miR-615-3p in the metastasis of breast cancer remains largely unknown. Methods The expression of miR-615-3p in breast cancer cells and tissues was assessed by qRT-PCR and situ hybridization assays. Effects of miR-615-3p on tumor metastasis were evaluated with experiments in vitro and mouse model. EMT markers were detected by western blot and immunofluorescence assays. Molecular mechanism of miR-615-3p in the regulation of breast cancer cell metastasis was analyzed by Western Blot, Co-immunoprecipitation, and Luciferase assay. Results In the present study, we found that miR-615-3p was significantly elevated in breast cancer cells and tissues, especially in those with metastasis. In breast cancer cell lines, stable overexpression of miR-615-3p was sufficient to promote cell motility in vitro, and pulmonary metastasis in vivo, accompanied by the reduced expression of epithelial markers and the increased levels of mesenchymal markers. Further studies revealed that the reintroduction of miR-615-3p increased the downstream signaling of TGF-β, the type I receptor (TGFBRI) by targeting the 3′-untranslated regions (3′-UTR) of PICK1. PICK1 inhibits the binding of DICER1 to Smad2/3 and the processing of pre-miR-615-3p to mature miR-615-3p in breast cancer cells, thus exerting a negative feedback loop. Conclusions Our data highlight an important role of miR-615-3p in the molecular etiology of breast cancer, and implicate the potential application of miR-615-3p in cancer therapy.

Abstract Background FSC231, a PSD‐95/DLG/ZO‐1 (PDZ) domain inhibitor of protein kinase Cα interacting protein 1 (PICK1), has analgesic effects, but the mechanism remains unclear. Methods The expression level of PICK1 in dorsal root ganglion (DRG) of rats was changed by vector plasmid, and the effect of PICK1 on paclitaxel (PTL)‐induced neuralgia of rats was observed in collaboration with FSC231 treatment. The possible molecular mechanisms were explored by quantitative real‐time polymerase chain reaction (qRT‐PCR), Western Blot and co‐immunoprecipitation (Co‐IP) techniques. Results PTL treatment can significantly reduce mechanical withdrawal threshold (MWT), shorten thermal withdrawal latency (TWL), promote DRG inflammation and release of substance P (SP), stimulate PICK1 expression, decrease α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid receptor 2 (AMPAR, GluA2) level and increase glycogen synthase kinase‐3β (GSK‐3β) and extracellular regulated protein kinases1/2 (ERK1/2) phosphorylation in rats, while FSC231 treatment can alleviate the above effects induced by PTL. Overexpression of PICK1 can counteract reduced PICK1 level, increased GluA2 level and decreased GSK‐3β and ERK1/2 phosphorylation levels caused by FSC231 treatment. The results of Co‐IP confirmed the interactions between PICK1 and GluA2. Both FSC231 treatment and silent PICK1 improved PTL‐induced MWT reduction, TWL shortening, inflammation, SP release and related gene expression changes, with cumulative effect. Conclusion FSC231 activates GSK‐3β/ERK1/2 by inhibiting the interaction between PICK1 and GluA2 and alleviates PTL‐induced DRG neuralgia in rats.

PDZ domains are modular domains that conventionally bind to C terminal or internal motifs of target proteins to control cellular functions through the regulation of protein complex assemblies. Almost all reported structures of PDZ-target protein complexes rely on fragments or peptides as target proteins. No intact target protein complexed with PDZ was structurally characterized. In this study, we used NMR spectroscopy and other biochemistry and biophysics tools to uncover insights into structural coupling between the PDZ domain of protein interacting with C-kinase 1 (PICK1) and α7 nicotinic acetylcholine receptors (α7 nAChR). Notably, the intracellular domains of both α7 nAChR and PICK1 PDZ exhibit a high degree of plasticity in their coupling. Specifically, the MA helix of α7 nAChR interacts with residues lining the canonical binding site of the PICK1 PDZ, while flexible loops also engage in protein-protein interactions. Both hydrophobic and electrostatic interactions mediate the coupling. Overall, the resulting structure of the α7 nAChR-PICK1 complex reveals an unconventional PDZ binding mode, significantly expanding the repertoire of functionally important PDZ interactions.

Disruption of prefrontal glutamate receptor interacting protein (GRIP), which anchors GluA2‐containing AMPA receptors (AMPARs) into the synaptic membrane, potentiates cue‐induced cocaine seeking in both males and females. Protein interacting with C kinase 1 (PICK1) plays an opposing role to that of GRIP, removing AMPARs from the synapse. Consistent with our hypothesis that disruption of PICK1 in the mPFC would lead to a decrease in addiction‐like behaviour, we found that conditional deletion of PICK1 in the mPFC attenuates cue‐induced cocaine seeking in male mice. However, prefrontal PICK1 deletion had the opposite effect in females, leading to an increase in cue‐induced reinstatement of cocaine seeking. We did not see any effects of PICK1 knockdown on sucrose taking or seeking, suggesting the sex‐specific effects do not generalise to natural reinforcers. These findings suggest the role of PICK1 in the prefrontal cortex of females may not be consistent with its accepted role in males. To determine whether these sex differences were influenced by gonadal hormones, we gonadectomised a cohort of males and found that removal of circulating androgens eliminated the effect of prefrontal PICK1 knockdown. As there was no effect of gonadectomy on its own on any of the behavioural measures collected, our results suggest that androgens may be involved in compensatory downstream effects of PICK1 knockdown. Taken together, these results highlight the need for consideration of sex as a biological variable when examining mechanisms underlying all behaviours, as convergent sex differences can reveal different mechanisms where behavioural sex differences do not exist. [ABSTRACT FROM AUTHOR]

The PDZ family has drawn attention as possible drug targets because of the domains’ wide ranges of function and highly conserved binding pockets. The PICK1 PDZ domain has been proposed as a possible drug target because the interactions between the PICK1 PDZ domain and the GluA2 subunit of the AMPA receptor have been shown to progress neurodegenerative diseases. BIO124 has been identified as a sub µM inhibitor of the PICK1–GluA2 interaction. Here, we use all-atom molecular dynamics simulations to reveal the atomic-level interaction pattern between the PICK1 PDZ domain and BIO124. Our simulations reveal three unique binding conformations of BIO124 in the PICK1 PDZ binding pocket, referred to here as state 0, state 1, and state 2. Each conformation is defined by a unique hydrogen bonding network and a unique pattern of hydrophobic interactions between BIO124 and the PICK1 PDZ domain. Interestingly, each conformation of BIO124 results in different dynamic changes to the PICK1 PDZ domain. Unlike states 1 and 2, state 0 induces dynamic coupling between BIO124 and the αA helix. Notably, this dynamic coupling with the αA helix is similar to what has been observed in other PDZ–ligand complexes. Our analysis indicates that the interactions formed between BIO124 and I35 may be the key to inducing dynamic coupling with the αA helix. Lastly, we suspect that the conformational shifts observed in our simulations may affect the stability and thus the overall effectiveness of BIO124. We propose that a physically larger inhibitor may be necessary to ensure sufficient interactions that permit stable binding between a drug and the PICK1 PDZ domain.

Protein interacting with C-kinase 1 (PICK1) is a 415-aa multidomain scaffold protein encoded by the PICK1 gene. Accumulating evidence suggests that PICK1 is involved in the progression of cancer. However, the role of PICK1 in gastric cancer (GC) remains largely unknown. Using integrated analysis of publicly available GC transcriptome data from the Gene Expression Omnibus (GEO) database and immunohistochemistry analysis of samples obtained from clinical GC patients, we found that PICK1 expression was significantly down-regulated in gastric tumor tissues in comparison with adjacent normal tissues. Our analyses also revealed that decreased expression of PICK1 conferred a disadvantage on overall survival time in GC patients. Additionally, PICK1 expression showed a strong association with the epithelial-mesenchymal transition (EMT) pathway, and PICK1 might represent a functional bridge for EMT. Moreover, PICK1 expression was significantly decreased in the EMT subtype of GC and was negatively correlated with the expression of fibronectin 1 (FN1) and myosin light chain 9 (MYL9) mRNAs. Thus, our study provides evidence that PICK1 is a promising biomarker for the molecular etiology of GC. [ABSTRACT FROM AUTHOR]

PICK1 is a multi-domain scaffolding protein that is uniquely comprised of both a PDZ domain and a BAR domain. While previous experiments have shown that the PDZ domain and the linker positively regulate the BAR domain and the C-terminus negatively regulates the BAR domain, the details of internal regulation mechanisms are unknown. Molecular dynamics (MD) simulations have been proven to be a useful tool in revealing the intramolecular interactions at atomic-level resolution. PICK1 performs its biological functions in a dimeric form which is extremely computationally demanding to simulate with an all-atom force field. Here, we use coarse-grained MD simulations to expose the key residues and driving forces in the internal regulations of PICK1. While the PDZ and BAR domains do not form a stable complex, our simulations show the PDZ domain preferentially interacting with the concave surface of the BAR domain over other BAR domain regions. Furthermore, our simulations show that the short helix in the linker region can form interactions with the PDZ domain. Our results reveal that the surface of the βB-βC loop, βC strand, and αA-βD loop of the PDZ domain can form a group of hydrophobic interactions surrounding the linker helix. These interactions are driven by hydrophobic forces. In contrast, our simulations reveal a very dynamic C-terminus that most often resides on the convex surface of the BAR domain rather than the previously suspected concave surface. These interactions are driven by a combination of electrostatic and hydrophobic interactions.

Regeneration is a unique defense mechanism of liver tissue in response to functional cell loss induced by toxic chemicals or surgical resection. In this study, we found that Islet‐cell autoantigen 69 (Ica69) accelerates liver regeneration in mice. Following 70% partial hepatectomy, both Ica69 mRNA and protein are significantly upregulated in mouse hepatocytes at the early stage of liver regeneration. Compared with the wild‐type mice, Ica69‐deficient mice have more severe liver injury, delayed liver regeneration, and high surgical accidental mortality following hepatectomy. Mechanistically, Ica69 interacts with Pick1 protein to regulate Tgfbr1 protein expression and Tgfβ‐induced Smad2 phosphorylation. Our findings suggest that Ica69 in liver tissue is a new potential target for promoting liver regeneration. [ABSTRACT FROM AUTHOR]

Background: Increasing evidence indicates that epithelial-mesenchymal transition (EMT) can be regulated by microRNAs (miRNAs). miR-615-3p was shown to be involved in tumor development. However, the role of miR-615-3p in the metastasis of breast cancer remains largely unknown. Methods: The expression of miR-615-3p in breast cancer cells and tissues was assessed by qRT-PCR and situ hybridization assays. Effects of miR-615-3p on tumor metastasis were evaluated with experiments in vitro and mouse model. EMT markers were detected by western blot and immunofluorescence assays. Molecular mechanism of miR-615-3p in the regulation of breast cancer cell metastasis was analyzed by Western Blot, Co-immunoprecipitation, and Luciferase assay. Results: In the present study, we found that miR-615-3p was significantly elevated in breast cancer cells and tissues, especially in those with metastasis. In breast cancer cell lines, stable overexpression of miR-615-3p was sufficient to promote cell motility in vitro, and pulmonary metastasis in vivo, accompanied by the reduced expression of epithelial markers and the increased levels of mesenchymal markers. Further studies revealed that the reintroduction of miR-615-3p increased the downstream signaling of TGF-β, the type I receptor (TGFBRI) by targeting the 3′-untranslated regions (3′-UTR) of PICK1. PICK1 inhibits the binding of DICER1 to Smad2/3 and the processing of pre-miR-615-3p to mature miR-615-3p in breast cancer cells, thus exerting a negative feedback loop. Conclusions: Our data highlight an important role of miR-615-3p in the molecular etiology of breast cancer, and implicate the potential application of miR-615-3p in cancer therapy. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is a metabolic disease characterized by an increase in blood glucose levels due to lack of insulin secretion. Previous studies have confirmed that PICK1 is critical for both β-cell function and glucose homeostasis. The aim of this study was to investigate the role of PICK1 in response to high glucose-induced β-cell dysfunction and the molecular mechanism of regulation of PICK1. We found that overexpression of PICK1 in db/db diabetic mice significantly improved glucose tolerance and increased insulin release. High glucose treatment of Min6 cells inhibited PICK1 expression, and overexpression of PICK1 protected against high glucose-induced pancreatic cell dysfunction. Activation of the PI3K/Akt pathway by PICK1 in Min6 cells resulted in increased GLUT2 expression and this increase was abolished by treatment with a PI3K-specific inhibitor. Further, we showed that expression of PICK1 is negatively regulated by miR-139-5p through directly targeting its 3′UTR. These data suggested that PICK1 may participate in the functional protection of pancreatic β-cells through PI3K/Akt signaling, promote insulin secretion, and delay the progression of diabetes, and is negatively regulated by miR-139-5p, further clarifying the regulation of pancreatic β-cell function. • PICK1 improves glucose tolerance and pancreatic β-cell function in HFD-fed mice. • PICK1 inhibits high glucose-induced dysfunction in Min6 pancreatic β-cells. • PICK1 promotes activation of PI3K/Akt pathway and GLUT2 expression. • PICK1 expression is negatively regulated by miR-139-5p. [ABSTRACT FROM AUTHOR]

In vertebrates, the receptor families deleted in colorectal cancer (DCC) and UNC5 mediate responses to the bifunctional guidance cue netrin-1. DCC mediates attraction, whereas a complex of DCC and UNC5 mediates repulsion. Thus, a primary determinant of the responsiveness of an axon to netrin-1 is the presence or absence of UNC5 family members on the cell surface. Currently, little is known about the role of receptor trafficking in regulating neuronal responses to netrin-1. We show that protein interacting with C-kinase 1 (PICK1) recruits activated protein kinase C alpha(PKC alpha) to MycUNC5A at the plasma membrane, stimulating its endocytosis. We identify two PKC alpha phosphorylation sites at serines 408 and 587, as well as dileucine internalization motifs, which are required for this endocytosis. We find that PKC alpha-stimulated internalization of UNC5A alters the functional response of developing hippocampal axons to netrin-1, preventing UNC5A-mediated growth cone collapse and converting netrin-1-stimulated chemorepulsion to attraction. To address whether this conversion in axonal response occurs in neurons expressing endogenous levels of UNC5, we show that mouse cerebellar granule axons exhibit chemorepulsion in a netrin-1 gradient and that this chemorepulsion is converted to chemoattraction after PKC alpha activation. We demonstrate that this repulsion depends on UNC5A because Unc5a-/- axons are not repelled and show this conversion depends on PICK1 because PICK1-/- axons are not converted to chemoattraction after PKC alpha activation. Together, these data provide a potential mechanism to explain how developing neurons alter their responsiveness to netrin-1 at intermediate choice points as they navigate to their targets.

Background: Alzheimer's disease (AD) is a neurodegenerative disease with no effective treatment. Researchers have focused on exploring biomarkers for its early diagnosis, especially on finding a new gene target. Recent studies have shown that protein interacting with C-kinase-1(PICK1) is related to AD through regulating hippocampal synaptic plasticity. PICK1 gene polymorphisms have been identified in psychological and other related disorders.Methods: This study included 133 sporadic AD patients and 173 healthy controls. All coding exons and intron-exon boundaries of the PICK1 gene were amplified by polymerase chain reaction (PCR), which were subsequently sequenced and analyzed.Results: This is the first genetic association study to investigate the association between PICK1 gene and AD risk in Chinese Han population. Seven single nucleotide polymorphisms (SNPs) were found in our research (rs397780637, rs713729, rs2076369, rs58230476, rs7289911, rs149474436; and rs146770324 for patient M1659 only). Frequencies of the T allele (p = 0.002; OR, 0.083; 95%CI, 0.011–0.634) and TT/TC genotypes (p = 0.001) of rs149474436 were lower in AD patients than in the controls. The GG homozygotes of rs397780637 were found to be associated with an increased risk of AD (p = 0.018) in APOEε4 allele carriers, while the frequency of the T allele of rs149474436 was significantly lower among AD patients in APOEε4 non-carriers (p = 0.005).Conclusions: Our results suggest that PICK1 gene SNPs are associated with AD susceptibility in East Asian population, T allele of rs149474436 may play as a protective factor while the rs397780637 GG homozygotes may be associated with an increased risk of AD. Further studies should be considered in a larger cohort of patients with diverse demographics.

The GluA2 subunit of AMPA glutamate receptors (AMPARs) has been shown to be critical for the expression of NMDA receptor (NMDAR)-dependent long-term depression (LTD). However, in young GluA2 knockout (KO) mice, this form of LTD can still be induced in the hippocampus, suggesting that LTD mechanisms may be modified in the presence of GluA2-lacking, Ca2+ permeable AMPARs. In this study, we examined LTD at the CA1 synapse in GluA2 KO mice by using several well-established inhibitory peptides known to block LTD in wild type (WT) rodents. We showed that while LTD in the KO mice is still blocked by the protein interacting with C kinase 1 (PICK1) peptide pepEVKI, it becomes insensitive to the N-ethylmaleimide-sensitive factor (NSF) peptide pep2m. In addition, the effects of actin and cofilin inhibitory peptides were also altered. These results indicate that in the absence of GluA2, LTD expression mechanisms are different from those in WT animals, suggesting that there are multiple molecular processes enabling LTD expression that are adaptable to physiological and genetic manipulations.

α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) has been reported as one of the targets for treatment of epilepsy. Although maladaptive regulation of surface expression of glutamate ionotropic receptor AMPA type subunit 1 (GRIA1) subunit is relevant to the responsiveness to AMPAR antagonists (perampanel and GYKI 52466) in LiCl-pilocarpine-induced chronic epilepsy rats, the underlying mechanisms of refractory seizures to AMPAR antagonists have yet been unclear. In the present study, we found that both AMPAR antagonists restored the up-regulations of GRIA1 surface expression and Src family-mediated glycogen synthase kinase 3β (GSK3β)-Ca2+/cAMP response element-binding protein (CREB) phosphorylations to control levels in responders (whose seizure activities were responsive to AMPAR) but not non-responders (whose seizure activities were uncontrolled by AMPAR antagonists). In addition, 3-chloroacetyl indole (3CAI, an AKT inhibitor) co-treatment attenuated spontaneous seizure activities in non-responders, accompanied by reductions in AKT/GSK3β/CREB phosphorylations and GRIA1 surface expression. Although AMPAR antagonists reduced GRIA2 tyrosine (Y) phosphorylations in responders, they did not affect GRIA2 surface expression and protein interacting with C kinase 1 (PICK1) protein level in both responders and non-responders. Therefore, our findings suggest that dysregulation of AKT/GSK3β/CREB-mediated GRIA1 surface expression may be responsible for refractory seizures in non-responders, and that this pathway may be a potential target to improve the responsiveness to AMPAR antagonists.

To correctly transfer information, neuronal networks need to continuously adjust their synaptic strength to extrinsic stimuli. This ability, termed synaptic plasticity, is at the heart of their function and is, thus, tightly regulated. In glutamatergic neurons, synaptic strength is controlled by the number and function of AMPA receptors at the postsynapse, which mediate most of the fast excitatory transmission in the central nervous system. Their trafficking to, at, and from the synapse, is, therefore, a key mechanism underlying synaptic plasticity. Intensive research over the last 20 years has revealed the increasing importance of interacting proteins, which accompany AMPA receptors throughout their lifetime and help to refine the temporal and spatial modulation of their trafficking and function. In this review, we discuss the current knowledge about the roles of key partners in regulating AMPA receptor trafficking and focus especially on the movement between the intracellular, extrasynaptic, and synaptic pools. We examine their involvement not only in basal synaptic function, but also in Hebbian and homeostatic plasticity. Included in our review are well-established AMPA receptor interactants such as GRIP1 and PICK1, the classical auxiliary subunits TARP and CNIH, and the newest additions to AMPA receptor native complexes. [ABSTRACT FROM AUTHOR]

Insect nicotinic acetylcholine receptors (nAChRs) are not only important neurotransmitter receptors but also effective insecticide targets. The regulation of nAChRs has been mainly studied in vertebrates, especially in mammals. Here, two types of nAChRs were found present in the locust Locusta migratoria manilensis dorsal unpaired median (DUM) neurons, α‐bungarotoxin (α‐Bgt)‐sensitive nAChRs and α‐Bgt‐resistant nAChRs, responding to acetylcholine (ACh) at different concentrations. The homologs to three mammalian nAChR regulators, ubiquilin‐1, CRELD2 (cysteine‐rich with EFG‐like domain 2), and PICK1 (protein interacting with PRKCA 1), were characterized in L. migratoria, and their functions on regulating native nAChRs were investigated via RNAi followed by membrane potential measurement with DiBAC4(3) and agonist‐evoked macroscopic current recording in cultured L. migratoria DUM neurons. Ubiquilin‐1 and PICK1 negatively regulated nAChRs because silencing of ubiquilin‐1 and PICK1 both resulted in increased membrane potential and increased inward currents in DUM neurons, while CRELD2 positively regulated nAChRs as decreased membrane potential and inward currents were observed in DUM neurons. In addition, ubiquilin‐1 regulated both α‐Bgt‐sensitive and α‐Bgt‐resistant types of nAChRs whereas PICK1 and CRELD2 regulated only the α‐Bgt‐resistant nAChRs. The present study broadened our understanding on the regulation of insect nAChRs and will benefit pest management given the important role of nAChRs in insect neurons and insecticide science. Open science badges: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. [ABSTRACT FROM AUTHOR]

Protein interacting with C kinase 1 (PICK1) is an AMPA receptor binding protein that works in conjunction with glutamate receptor interacting protein (GRIP) to balance the number of GluA2-containing AMPARs in the synapse. In male mice, disrupting PICK1 in the medial prefrontal cortex (mPFC) leads to a decrease in cue-induced cocaine seeking and disrupting GRIP in the mPFC has the opposing effect, consistent with other evidence that removal of GluA2-containing AMPARs potentiates reinstatement. However, PICK1 does not seem to play the same role in female mice, as knockdown of either PICK1 or GRIP in the mPFC leads to similar increases in cue-induced cocaine seeking. These previous findings indicate that the role of PICK1 in the prefrontal cortex is sex specific. The goal of the current study was to examine whether ovarian hormones contribute to the effect of prefrontal PICK1 knockdown on reinstatement of cocaine seeking. While we replicated the increased cue-induced cocaine seeking in prefrontal PICK1 knockdown sham mice, we did not see any difference between the GFP control mice and PICK1 knockdowns following ovariectomy. However, this effect was driven primarily by an increase in cocaine seeking in ovariectomized GFP control mice while there was no effect ovariectomy in PICK1 knockdown mice. Taken together, these findings suggest that circulating ovarian hormones interact with the effects of PICK1 on cue-induced reinstatement. [ABSTRACT FROM AUTHOR]

The precise regulation of AMPA receptor (AMPAR) trafficking in neurons is crucial for excitatory neurotransmission, synaptic plasticity and the consequent formation and modification of neural circuits during brain development and learning. Clathrin-mediated endocytosis (CME) is an essential trafficking event for the activity-dependent removal of AMPARs from the neuronal plasma membrane, resulting in a reduction in synaptic strength known as long-term depression (LTD). The regulated AMPAR endocytosis that underlies LTD is caused by specific modes of synaptic activity, most notably stimulation of NMDA receptors (NMDARs) and metabotropic glutamate receptors (mGluRs). Numerous proteins associate with AMPAR subunits, directly or indirectly, to control their trafficking, and therefore the regulation of these protein-protein interactions in response to NMDAR or mGluR signaling is a critical feature of synaptic plasticity. This article reviews the protein-protein interactions that are dynamically regulated during synaptic plasticity to modulate AMPAR endocytosis, focussing on AMPAR binding proteins and proteins that bind the core endocytic machinery. In addition, the mechanisms for the regulation of protein-protein interactions are considered, as well as the functional consequences of these dynamic interactions on AMPAR endocytosis.

Scaffolding proteins serve to assemble protein complexes in dynamic processes by means of specific protein-protein and protein-lipid binding domains. Many of these domains bind either proteins or lipids exclusively; however, it has become increasingly evident that certain domains are capable of binding both. Especially, many PDZ domains, which are highly abundant protein-protein binding domains, bind lipids and membranes. Here we provide an overview of recent large-scale studies trying to generalize and rationalize the binding patterns as well as specificity of PDZ domains towards membrane lipids. Moreover, we review how these PDZ-membrane interactions are regulated in the case of the synaptic scaffolding protein PICK1 and how this might affect cellular localization and function.

The precise regulation of AMPA receptor (AMPAR) trafficking in neurons is crucial for excitatory neurotransmission, synaptic plasticity and the consequent formation and modification of neural circuits during brain development and learning. Clathrin-mediated endocytosis (CME) is an essential trafficking event for the activity-dependent removal of AMPARs from the neuronal plasma membrane, resulting in a reduction in synaptic strength known as long-term depression (LTD). The regulated AMPAR endocytosis that underlies LTD is caused by specific modes of synaptic activity, most notably stimulation of NMDA receptors (NMDARs) and metabotropic glutamate receptors (mGluRs). Numerous proteins associate with AMPAR subunits, directly or indirectly, to control their trafficking, and therefore the regulation of these protein-protein interactions in response to NMDAR or mGluR signaling is a critical feature of synaptic plasticity. This article reviews the protein-protein interactions that are dynamically regulated during synaptic plasticity to modulate AMPAR endocytosis, focussing on AMPAR binding proteins and proteins that bind the core endocytic machinery. In addition, the mechanisms for the regulation of protein-protein interactions are considered, as well as the functional consequences of these dynamic interactions on AMPAR endocytosis. [ABSTRACT FROM AUTHOR]

Parkin functions as a multipurpose E3 ubiquitin ligase, and Parkin loss of function is associated with both sporadic and familial Parkinson's disease (PD). We report that the Bin/Amphiphysin/Rvs (BAR) domain of protein interacting with PRKCA1 (PICK1) bound to the really interesting new gene 1 (RING1) domain of Parkin and potently inhibited the E3 ligase activity of Parkin by disrupting its interaction with UbcH7. Parkin translocated to damaged mitochondria and led to their degradation in neurons, whereas PICK1 robustly inhibited this process. PICK1 also impaired the protective function of Parkin against stresses in SH-SY5Y cells and neurons. The protein levels of several Parkin substrates were reduced in young PICK1-knockout mice, and these mice were resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated toxicity. Taken together, the results indicate that PICK1 is a potent inhibitor of Parkin, and the reduction of PICK1 enhances the protective effect of Parkin. [ABSTRACT FROM AUTHOR]

Summary BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila , disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis. [ABSTRACT FROM AUTHOR]

The role of oxidative stress has been well documented in the development of sepsis-induced acute lung injury (ALI). Protein interaction with C-kinase 1 (PICK1) participates in oxidative stress-related neuronal diseases. However, its function in lung infections and inflammatory diseases is not known. We therefore sought to investigate whether PICK1 is involved in sepsis-induced ALI. Cecal ligation and puncture (CLP) was performed in anesthetized wild type (WT) and PICK1 knock out (KO, PICK1 -/- ) mice with C57BL/6 background. At the time of CLP, mice were given fluid resuscitation. Mouse lungs were harvested at 24 and 72 h for Western blot analysis, qRT-PCR, BALF analysis, Hematoxylin and Eosin staining, TUNEL staining, maleimide staining, flow cytometry analysis, GCL, GSH, GSSG and cysteine levels measurement. A marked elevation of PICK1 mRNA and protein level were demonstrated in lung tissue, which was accompanied by increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and consumption of glutathione (GSH). N-acetylcysteine (NAC), buthionine sulfoximine (BSO) and GSH-monoethyl ester (GSH-MEE) were injected into mice via caudal vein to regulate glutathione (GSH) level in lung. Alterations of lung GSH content induced PICK1 level change after CLP challenge. In PICK1 -/- underwent with CLP, lung injury and survival were significantly aggravated compared with wild-type mice underwent with CLP. Concomitantly, CLP-induced lung cell apoptosis was exacerbated in PICK1 -/- mice. The level of xCT, other than PKCα, in lung tissue was significantly lowered in PICK1 -/- but not in wild type that underwent CLP surgery. Meanwhile, Nrf2 activation, which dominating xCT expression, was inhibited in PICK1-/- but not in wild type mice that underwent CLP surgery, as well. Moreover, higher level of PICK1 was detected in PBMCs of septic patients than healthy controls. Taken together, PICK1 plays a pivotal role in sepsis-induced ALI by regulating GSH synthesis via affecting the substrate-specific subunit of lung cystine/glutamate transporter, xCT. [ABSTRACT FROM AUTHOR]

Overstimulation of glutamate receptors resulting in excessive intracellular calcium concentrations is a major cause of neuronal cell death in epilepsy. The main source of increased calcium concentration during this excitotoxicity is an influx through NMDA subtype of glutamate receptors. The GluR2 (GluA2) hypothesis states that following a neurological insult such as an epileptic seizure, the AMPA receptor subunit GluR2 protein is downregulated. This increases the likelihood of the formation of GluR2-lacking, calcium-permeable AMPA receptor which might further enhance the toxicity of the neurotransmitter, glutamate. The cytosolic protein, PICK1, facilitates the removal of GluA2 subunits from the synaptic plasma membrane. High calcium concentrations may cause PICK1 to bind to the GluA2 subunit of calcium-impermeable AMPARs, leading to an increased internalization of these receptor subunits and a relative increase of GluA2-lacking, calcium-permeable AMPARs. This further escalates the cytosolic calcium concentration. In order to test this hypothesis, we have used kainic acid to induce epilepsy in rats. Using semi-quantitative western blotting combined with univariate and multivariate statistical analyses, we found that both GluA2 and PICK1 were down-regulated in kainate-treated rats for as long as eight weeks after induction of epilepsy. An interesting finding was that statistical analysis indicates that the functional role of PICK1 in our material is to increase GluA2 concentrations in the cells. The observed reduction in PICK1 concentration may thus be an independent contributor to the observed GluA2 reduction. This reduction may possibly be an adaptive mechanism, serving to prevent further loss of GluA2 from the synapses. [ABSTRACT FROM AUTHOR]

Hypoxia is an important feature of nasopharyngeal carcinoma (NPC). "Protein interacting with PRKCA 1" (PICK1) is commonly downregulated in human malignancies and is functionally related to poor prognosis. However, there is a limited understanding of the upstream mechanisms regulating PICK1 currently. PICK1 and HIF-1α expression levels were analyzed by Immunohistochemistry (IHC), western blotting, and quantitative real-time PCR assay. Protein stability and ubiquitin assays were used to investigate PICK1 protein degradation. Immunofluorescence and co-immunoprecipitation assays were used to demonstrate the interaction between RBCK1 and PICK1. Gene knockdown by siRNA transfection was used to investigate the role of HIF-1α and RBCK1 in hypoxia-induced PICK1 degradation. Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EdU) assays and subcutaneous xenograft nude models were used to explore the roles of RBCK1 and PICK1 in NPC cell proliferation. PICK1 expression in NPC tissue was negatively relative to that of HIF-1α. HIF-1α downregulated PICK1 expression by facilitating its ubiquitination by the E3 ligases RANBP2-type and C3HC4-type zinc finger containing 1 (RBCK1), thereby enhancing proteasome-mediated PICK1 degradation. RBCK1 knockdown inhibited NPC cell proliferation, which was ameliorated by double knockdown of RBCK1/PICK1. These data provide evidence for an NPC cell adaptation mechanism to hypoxia, where HIF-1α regulates RBCK1, which targets PICK1 for degradation to promote cell proliferation. • HIF-1α is negatively correlated with PICK1 expression levels in human NPC. • Hypoxia induces ubiquitination degradation of PICK1 protein. • E3 ubiquitin ligase RBCK1 regulates PICK1 degradation under hypoxia. • RBCK1-mediated PICK1 expression down-regulation contributes to the proliferation of NPC cells. [ABSTRACT FROM AUTHOR]

It has been previously identified that α‑amino‑ 3‑hydroxy‑5‑methyl‑4‑isoxazole‑propionic acid receptors (AMPARs) are expressed in pancreatic β cells and regulate exocytosis and insulin release. It is known that protein interacting with C‑kinase 1 (PICK1) regulates trafficking and synaptic targeting of AMPARs in the central nervous system. However, it is unknown whether PICK1 regulates glutamate‑induced insulin release in β cells. The present study demonstrated that glutamate‑induced exocytosis was increased in β cells derived from PICK1‑knockout mice. In agreement with this result, adding PICK1 in β cells reduced glutamate‑induced exocytosis, whereas adding EVKI, a peptide that interrupts the interaction between AMPARs and PICK1, increased the exocytosis of β cells with the application of glutamate. Furthermore, the conductance of ATP‑sensitive potassium (KATP) channels was reduced in PICK1‑knockout mice, which was reversed by the overexpression of PICK1. In addition, PICK1 application reduced voltage oscillation induced by the closure of KATP. Taken together, the results indicate that PICK1 regulates glutamate‑induced exocytosis in β cells. [ABSTRACT FROM AUTHOR]

Previous studies have indicated that presynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) contribute to the regulation of neurotransmitter release. In hippocampal synapses, the presynaptic surface expression of several AMPAR subunits, including GluA2, is regulated in a ligand-dependent manner. However, the molecular mechanisms underlying the presynaptic trafficking of AMPARs are still unknown. Here, using bright-field immunocytochemistry, western blots, and quantitative immunogold electron microscopy of the hippocampal CA1 area from intact adult rat brain, we demonstrate the association of AMPA receptors with the presynaptic active zone and with small presynaptic vesicles, in Schaffer collateral synapses in CA1 of the hippocampus. Furthermore, we show that GluA2 and protein interacting with C kinase 1 (PICK1) are colocalized at presynaptic vesicles. Similar to postsynaptic mechanisms, overexpression of either PICK1 or pep2m, which inhibit the N-ethylmaleimide sensitive fusion protein (NSF)-GluA2 interaction, decreases the concentration of GluA2 in the presynaptic active zone membrane. These data suggest that the interacting proteins PICK1 and NSF act as regulators of presynaptic GluA2-containing AMPAR trafficking between the active zone and a vesicle pool that may provide the basis of presynaptic components of synaptic plasticity. [ABSTRACT FROM AUTHOR]

Lithium (Li + ) is a drug widely employed for treating bipolar disorder, however the mechanism of action is not known. Here we study the effects of Li + in cultured hippocampal neurons on a synaptic complex consisting of δ-catenin, a protein associated with cadherins whose mutation is linked to autism, and GRIP, an AMPA receptor (AMPAR) scaffolding protein, and the AMPAR subunit, GluA2. We show that Li + elevates the level of δ-catenin in cultured neurons. δ-catenin binds to the ABP and GRIP proteins, which are synaptic scaffolds for GluA2. We show that Li + increases the levels of GRIP and GluA2, consistent with Li + -induced elevation of δ-catenin. Using GluA2 mutants, we show that the increase in surface level of GluA2 requires GluA2 interaction with GRIP. The amplitude but not the frequency of mEPSCs was also increased by Li + in cultured hippocampal neurons, confirming a functional effect and consistent with AMPAR stabilization at synapses. Furthermore, animals fed with Li + show elevated synaptic levels of δ-catenin, GRIP, and GluA2 in the hippocampus, also consistent with the findings in cultured neurons. This work supports a model in which Li + stabilizes δ-catenin, thus elevating a complex consisting of δ-catenin, GRIP and AMPARs in synapses of hippocampal neurons. Thus, the work suggests a mechanism by which Li + can alter brain synaptic function that may be relevant to its pharmacologic action in treatment of neurological disease. [ABSTRACT FROM AUTHOR]

Long lasting synaptic plasticity involves both functional and morphological changes, but how these processes are molecularly linked to achieve coordinated plasticity remains poorly understood. Cofilin is a common target of multiple signaling pathways at the synapse and is required for both functional and spine plasticity, but how it is regulated is unclear. In this study, we investigate whether the involvement of cofilin in plasticity is developmentally regulated by examining the role of cofilin in hippocampal long-term depression (LTD) in both young (2 weeks) and mature (2 months) mice. We show that both total protein level of cofilin and its activation undergo significant changes as the brain matures, so that although the amount of cofilin decreases significantly in mature mice, its regulation by protein phosphorylation becomes increasingly important. Consistent with these biochemical data, we show that cofilin-mediated actin reorganization is essential for LTD in mature, but not in young mice. In contrast to cofilin, the GluA2 interactions with NSF and PICK1 appear to be required in both young and mature mice, indicating that AMPAR internalization is a common key mechanism for LTD expression regardless of the developmental stages. These results establish the temporal specificity of cofilin in LTD regulation and suggest that cofilin-mediated actin reorganization may serve as a key mechanism underlying developmental regulation of synaptic and spine plasticity. This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’. [ABSTRACT FROM AUTHOR]

Long-term potentiation (LTP) is one of the major cellular mechanisms for learning and memory. Activity-dependent increases in surface AMPA receptors (AMPARs) are important for enhanced synaptic efficacy during LTP. Here, we report a novel function of a secretory trafficking protein, ICA69, in AMPAR trafficking, synaptic plasticity, and animal cognition. ICA69 is first identified as a diabetes-associated protein well characterized for its function in the biogenesis of secretory vesicles and trafficking of insulin from ER, Golgi to post-Golgi in pancreatic beta cells. In the brain, ICA69 is found in the AMPAR protein complex through its interaction with PICK1, which binds directly to GluA2 or GluA3 AMPAR subunits. Here, we showed that ICA69 regulates PICK1's distribution in neurons and stability in the mouse hippocampus, which in turn can impact AMPAR function in the brain. Biochemical analysis of postsynaptic density (PSD) proteins from hippocampi of mice lacking ICA69 ( Ica1 knockout) and their wild-type littermates revealed comparable AMPAR protein levels. Electrophysiological recording and morphological analysis of CA1 pyramidal neurons from Ica1 knockout also showed normal AMPAR-mediated currents and dendrite architecture, indicating that ICA69 does not regulate synaptic AMPAR function and neuron morphology at the basal state. However, genetic deletion of ICA69 in mice selectively impairs NMDA receptor (NMDAR)-dependent LTP but not LTD at Schaffer collateral to CA1 synapses, which correlates with behavioral deficits in tests of spatial and associative learning and memory. Together, we identified a critical and selective role of ICA69 in LTP, linking ICA69-mediated synaptic strengthening to hippocampus-dependent learning and memory., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chiu, Chen and Huganir.)

A DNA fragment encoding C-terminal BARc region (amino acids 128-416) of rat PICK1 (NP_445912 ) was inserted into a modified vector pMAL-s involving human rhinovirus 3C protease cleavage site to produce a recombinant plasmid, pMAL-s-barc. The construct can express the fusion protein, MBP-BARc in the soluble form in E.coli. To remove the MBP tag, MBP-BARc purified from amylose beads was digested with human rhinovirus 3C protease and the cleavage efficiency is about 95% when the ratio of protein / enzyme (w/w) reaches 50:1, as analyzed on SDS-PAGE. The enzymatic reaction mixture was rapidly separated into two parts, MBP in the supernatant and BARc in the precipitate at the concentration of 1 M ammonium sulfate. In such case, the target protein BARc could be economically produced in a soluble state to be as the sample for measuring its biochemical function, for example, protein-protein interaction and protein-lipid combination.

摘 要： 【目的】 分析圆头精子症患者的遗传学发病机制。 【方法】 回顾分析１例圆头精子症患者，对其进行多种遗传学方法的检测，包括染色体检查、Ｙ染色体微缺失基因检测及通过ａｒｒａｙ－ＣＧＨ芯片技术和Ｓａｎｇｅｒ测序筛查候选基因ＤＰＹ１９Ｌ２、ＰＩＣＫ１、ＳＰＡＴＡ１６。 【结果】 该患者在我中心行两个卵胞浆内单精子显微注射（ＩＣＳＩ）周期后，其妻孕足月剖宫产一对异卵双胞胎女儿。患者染色体核型为４６，ＸＹ、Ｙ染色体微缺失基因检测未见异常，ａｒｒａｙ－ＣＧＨ芯片检测排除了ＤＰＹ１９Ｌ２、ＳＰＡＴＡ１６和ＰＩＣＫ１基因及其他该芯片所涵盖区域的１００ｋｂ以上的片段重复或缺失。Ｓａｎｇｅｒ测序ＰＩＣＫ１、ＳＰＡＴＡ１６基因也未发现点突变和小片段插入缺失等病理性改变。【结论】 圆头精子症患者可通过ＩＣＳＩ方式获得健康后代，本研究排除了已知的候选基因ＤＰＹ１９Ｌ２、ＰＩＣＫ１、ＳＰＡＴＡ１６与该圆头精子症患者的致病相关，可能存在其他致病基因。