Cpne1 deficiency preserves sperm motility under Ca2+ channel blockade.

Calcium ions (Ca2+) play crucial roles in sperm motility and fertilization. The copine (CPNE) family comprises several Ca2+-dependent phospholipid-binding proteins. Of these, CPNE1 is extensively expressed in mammalian tissues; however, its precise role in testicular development and spermatogenesis is yet to be fully characterized. In this study, we used proteomics to analyze testicular biopsies and found that levels of CPNE1 were significantly reduced in patients with non-obstructive azoospermia (defective spermatogenesis) compared to those in patients with obstructive azoospermia (physiological spermatogenesis). In mice, CPNE1 is expressed at various stages of germ cell development and is associated with the Golgi apparatus. Ultimately, CPNE1 is expressed in the flagella of mature sperms. To further examine the role of CPNE1, we developed a Cpne1 knockout mouse model. Analysis showed that the loss of Cpne1 did not impair testicular development, spermatogenesis, or sperm morphology and motility in physiological conditions. When treated with gadolinium (III) chloride or 2-aminoethoxydiphenyl borate, known inhibitors of store-operated Ca2+ entry, Ca2+ signals and sperm motility were significantly compromised in wild-type mice; however, both mechanisms were conserved in KO mice. These results suggested that CPNE1 is dispensable for testicular development, spermatogenesis or sperm motility in physiological conditions. In addition, CPNE1 may represent a target of Ca2+ channel inhibitors and may therefore be implicated in the regulation of Ca2+ signaling and sperm motility.

Exploring novel protein biomarkers for early-stage diagnosis and prognosis of T-acute lymphoblastic leukemia (T-ALL).

BACKGROUND: Efficient classification of T-acute lymphoblastic leukemia (T-ALL) involves considering various factors, such as age, white blood cell count, and chromosomal alterations. However, studying protein markers are crucial to improving T-ALL patients' diagnosis and treatment. A study analyzing the expression of proteomes was conducted to identify promising early-stage biomarkers for T-ALL patients METHODS: Label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze the blood proteins of both patients and healthy individuals to identify new biomarkers for T-ALL. The findings were validated by RT-PCR, ELISA and computational analysis RESULTS: The study identified 1467 proteins in the blood, of which nine were upregulated and 35 were downregulated by more than 2-fold. T-ALL patients showed a significant increase in specific disease-related proteins, such as eleven-nineteen lysine-rich leukemia protein, triggering receptor expressed on myeloid cells 1, cisplatin resistance-associated-overexpressed protein, X-ray radiation resistance-associated protein 1, tumor necrosis factor receptor superfamily member 10D, protein S100-A8, and copine-4, by more than 3-fold CONCLUSION: The findings of this study provide a valuable protein map of leukemic cells and identify potential biomarkers for leukemic aggressiveness. However, further studies using larger T-ALL patient samples must confirm these preliminary results.

Copines, a Family of Calcium Sensor Proteins and Their Role in Brain Function.

The Copines are a family of evolutionary conserved calcium-binding proteins found in most eukaryotic organisms from protists to humans. They share a unique architecture and contain tandem C2 domains and a Von Willebrand factor type A (VWA) domain. C2 domains in Copines bind calcium, phospholipids, and other proteins and mediate the transient association of these proteins with biological membranes at elevated calcium levels. The VWA domain also binds calcium and is involved in protein-protein interactions. Here, we provide a comprehensive review of the sequences, structures, expression, targeting, and function of the entire family of known Copine proteins (Copine 1-9 in mammals) with a particular emphasis on their functional roles in the mammalian brain. Neuronal Copines are implicated in a wide array of processes from cell differentiation to synaptic transmission and plasticity and are also linked to several pathological conditions from cancers to brain diseases. This review provides the most up-to-date insights into the structure and function of Copines, with an emphasis on their role in brain function.

Copine C plays a role in adhesion and streaming in Dictyostelium.

Copines are a family of calcium-dependent membrane-binding proteins. To study these proteins, anull mutant for cpnC was created in Dictyostelium, which has six copines genes (cpnA-cpnF). During development, cpnC- cells were able to aggregate, but did not form streams. Once aggregated into mounds, they formed large ring structures. cpnC- cells were less adherent to plastic substrates, but more adherent to other cells. These phenotypes correlated with changes in adhesion protein expression with decreased expression of SibA and increased expression of CsaA in developing cpnC- cells. We also measured the expression of RegA, a cAMP phosphodiesterase, and found that cpnC- cells have reduced RegA expression. The reduced RegA expression in cpnC- cells is most likely responsible for the observed phenotypes.

Cpne7 deficiency induces cellular senescence and premature aging of dental pulp.

Once tooth development is complete, odontoblasts and their progenitor cells in the dental pulp play a major role in protecting tooth vitality from external stresses. Hence, understanding the homeostasis of the mature pulp populations is just as crucial as understanding that of the young, developing ones for managing age-related dentinal damage. Here, it is shown that loss of Cpne7 accelerates cellular senescence in odontoblasts due to oxidative stress and DNA damage accumulation. Thus, in Cpne7-null dental pulp, odontoblast survival is impaired, and aberrant dentin is extensively formed. Intraperitoneal or topical application of CPNE7-derived functional peptide, however, alleviates the DNA damage accumulation and rescues the pathologic dentin phenotype. Notably, a healthy dentin-pulp complex lined with metabolically active odontoblasts is observed in 23-month-old Cpne7-overexpressing transgenic mice. Furthermore, physiologic dentin was regenerated in artificial dentinal defects of Cpne7-overexpressing transgenic mice. Taken together, Cpne7 is indispensable for the maintenance and homeostasis of odontoblasts, while promoting odontoblastic differentiation of the progenitor cells. This research thereby introduces its potential in oral disease-targeted applications, especially age-related dental diseases involving dentinal loss.

Copine7 deficiency leads to hepatic fat accumulation via mitochondrial dysfunction.

Objective: Mitochondrial dysfunction affects hepatic lipid homeostasis and promotes ROS generation. Copine7 (CPNE7) belongs to the ubiquitous copine family of calcium-dependent phospholipid binding proteins. CPNE7 has a high calcium ion binding affinity and the capacity to scavenge reactive oxygen species (ROS). A recent study reported that abnormalities in fatty acid and lipid metabolism were linked to the gene variant of CPNE7. Therefore, the purpose of this study is to examine the role of Cpne7 in hepatic lipid metabolism based on mitochondrial function. Methods: Lipid metabolism, mitochondrial function, and ROS production were investigated in high-fat diet (HFD)-fed Cpne7-/- mice and H2O2-damaged HepG2 hepatocytes following CPNE7 silencing or overexpression. Results: Cpne7 deficiency promoted severe hepatic steatosis in the HFD-induced NAFLD model. More importantly, mitochondrial dysfunction was observed along with an imbalance of mitochondrial dynamics in the livers of HFD-fed Cpne7-/-mice, resulting in high ROS levels. Similarly, CPNE7-silenced HepG2 hepatocytes showed high ROS levels, mitochondrial dysfunction, and increased lipid contents. On the contrary, CPNE7-overexpressed HepG2 cells showed low ROS levels, enhanced mitochondrial function and decreased lipid contents under H2O2-induced oxidative stress. Conclusions: In the liver, Cpne7 deficiency causes excessive ROS formation and mitochondrial dysfunction, which aggravates lipid metabolism abnormalities. These findings provide evidence that Cpne7 deficiency contributes to the pathogenesis of NAFLD, suggesting Cpne7 as a novel therapeutic target for NAFLD.

Copine-6 is a Ca2+ sensor for activity-induced AMPA receptor exocytosis.

The recruitment of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors underlies the strengthening of neuronal connectivity during learning and memory. This process is triggered by N-methyl-D-aspartate (NMDA) receptor-dependent postsynaptic Ca2+ influx. Synaptotagmin (Syt)-1 and -7 have been proposed as Ca2+ sensors for AMPA receptor exocytosis but are functionally redundant. Here, we identify a cytosolic C2 domain-containing Ca2+-binding protein, Copine-6, that forms a complex with AMPA receptors. Loss of Copine-6 expression impairs activity-induced exocytosis of AMPA receptors in primary neurons, which is rescued by wild-type Copine-6 but not Ca2+-binding mutants. In contrast, Copine-6 loss of function does not affect steady-state expression or tetrodotoxin-induced synaptic upscaling of surface AMPA receptors. Loss of Syt-1/Syt-7 significantly reduces Copine-6 protein expression. Interestingly, overexpression of wild-type Copine-6, but not the Ca2+-binding mutants, restores activity-dependent exocytosis of AMPA receptors in Syt-1/Syt-7 double-knockdown neurons. We conclude that Copine-6 is a postsynaptic Ca2+ sensor that mediates AMPA receptor exocytosis during synaptic potentiation.

Potential role of TREM2 in high cholesterol­induced cell injury and metabolic dysfunction in SH­SY5Y cells.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an important member of the immunoglobulin family of inflammatory stimulating receptors and is involved in a number of pathophysiological processes. The present study aimed to investigate the role of TREM2 in neurotoxicity induced by high cholesterol levels in SH-SY5Y cells and explore the potential mechanism. SH-SY5Y cells were routinely cultured and stimulated with a range of cholesterol concentrations. Cell viability was assessed using an MTT assay, morphological changes were observed, and the cell cycle distribution was measured using flow cytometry. Lipid deposition was measured by Oil red O staining, and the mRNA and protein expression levels of SRBEP-1 and SRBEP-2 were detected by quantitative PCR and western blotting, respectively. Moreover, the protein expression levels of BDNF, Copine-6, TREM1, TREM2, and key molecules of the Wnt signaling pathways were detected by western blotting. Finally, TREM2 was overexpressed to investigate its potential role in high cholesterol-induced neurotoxicity. The results showed that cell viability was significantly decreased in SH-SY5Y cells stimulated with cholesterol (0.1~100 µM) in a dose- and time-dependent manner. Stimulation with 100 µM cholesterol for 24 h resulted in morphological injuries, increased the proportion of SH-SY5Y cells at G0/G1, the degree of lipid accumulation, and the protein expression levels of sterol regulatory element binding protein (SREBP)1 and SREBP2, markedly decreased the protein expression levels of BDNF, Copine-6, and TREM2, and the p-ß-catenin/ß-catenin ratio, and increased the expression levels of nesfatin-1, TREM1 and the p-GSK3ß/GSK3ß ratio. Furthermore, the imbalanced expression of BDNF, Copine-6, nesfatin-1, and p-GSK3ß induced by high cholesterol levels was reversed after overexpression of TREM2. These results suggest that a high concentration of cholesterol could induce cell injury and lipid deposition in SH-SY5Y cells and that the underlying mechanism may be associated with imbalanced TREM2 expression.

CPNE1 promotes non-small cell lung cancer progression by interacting with RACK1 via the MET signaling pathway.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the most lethal tumour worldwide. Copine 1 (CPNE1) was identified as a novel oncogene in NSCLC in our previous study. However, its specific function and relative mechanisms remain poorly understood. METHODS: The biological role of CPNE1 and RACK1 in NSCLC was investigated using gene expression knockdown and overexpression, cell proliferation assays, clonogenic assays, and Transwell assays. The expression levels of CPNE1, RACK1 and other proteins were determined by western blot analysis. The relationship between CPNE1 and RACK1 was predicted and investigated by mass spectrometry analysis, immunofluorescence staining, and coimmunoprecipitation. NSCLC cells were treated with a combination of a MET inhibitor and gefitinib in vitro and in vivo. RESULTS: We found that CPNE1 facilitates tumorigenesis in NSCLC by interacting with RACK1, which further induces activation of MET signaling. CPNE1 overexpression promoted cell proliferation, migration, invasion and MET signaling in NSCLC cells, whereas CPNE1 knockdown produced the opposite effects. In addition, the suppression of the enhancing effect of CPNE1 overexpression on tumorigenesis and MET signaling by knockdown of RACK1 was verified. Moreover, compared to single-agent treatment, dual blockade of MET and EGFR resulted in enhanced reductions in the tumour volume and downstream signaling in vivo. CONCLUSIONS: Our findings show that CPNE1 promotes tumorigenesis by interacting with RACK1 and activating MET signaling. The combination of a MET inhibitor with an EGFR-TKI attenuated tumour growth more significantly than either single-drug treatment. These findings may provide new insights into the biological function of CPNE1 and the development of novel therapeutic strategies for NSCLC. Video Abstract.

Prognostic Value of Copine 1 in Patients With Renal Cell Carcinoma.

BACKGROUND/AIM: Renal cell carcinoma (RCC) is among the most common renal malignancies and requires reliable biomarkers for optimum diagnosis and prognosis. Copines are a family of calcium-dependent phospholipid-binding proteins that were reported to be associated with various cancers. We aimed to investigate the prognostic value of Copines 1 and 3 in RCC patients. MATERIALS AND METHODS: Copines 1 and 3 bioinformatics analysis and immunohistochemical (IHC) staining were performed on patients with RCC. RESULTS: The findings revealed significant association between Copine 1 expression and the patients' age, nuclear grade, and tumor stage. Bioinformatics analysis showed a similar trend for the mRNA expression of CPNE1, the gene that encodes Copine 1. Interestingly, results revealed a positive association between Copine 1 and both EphA and Ki-67 expression levels. Noteworthy, there was no significant association between Copine 3 expression and any parameters. CONCLUSION: Copine 1 may be used as an independent biomarker or in combination with both EphA2 and Ki-67 to predict disease outcome.

CPNE3 interaction with RACK1 protects against myocardial ischemia/reperfusion injury.

Copine 3 (CPNE3) and receptor for activated C kinase 1 (RACK1) have been determined to be risk factors for patients with acute myocardial ischemia/reperfusion (I/R). The present study aimed to evaluate the role of CPNE3 and its interaction with RACK1 in myocardial (I/R) injury. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting were performed to detect CPNE3 and RACK1 expression levels in H9c2 cells before and after the transfection of CPNE3 overexpression plasmid or small interfering RNA-RACK1. Cell viability was detected using a Cell Counting Kit-8 assay, and immunoprecipitation assays were performed to determine the interaction between CPNE3 and RACK1. A commercial kit was used to examine lactate dehydrogenase (LDH) levels. The expression levels of inflammatory cytokines were detected via RT-qPCR and western blotting. Cell apoptosis was assessed via TUNEL staining and western blotting. The results demonstrated that the expression levels of CPNE3 and RACK1 were decreased in hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocytes, which was consistent with the expression levels observed in the myocardial I/R injury rat model. It was found that CPNE3 overexpression upregulated RACK1 expression, increased cell viability and suppressed the release of LDH in H/R-induced H9c2 cells. Furthermore, CPNE3 overexpression inhibited the release of inflammatory cytokines and decreased cell apoptosis in H/R-induced cardiomyocytes by activating RACK1 expression. The present study suggested that CPNE3 served an important role in preventing I/R injury by interacting with RACK1, providing novel insight into the prevention of myocardial I/R injury, as well as the treatment and care of patients with myocardial I/R.

Copine-7 is required for REM sleep regulation following cage change or water immersion and restraint stress in mice.

Sleep is affected by the environment. In rodents, changes in the amount of rapid eye movement sleep (REMS) can precede those of other sleep/wake stages. The molecular mechanism underlying the dynamic regulation of REMS remains poorly understood. Here, we focused on the sublaterodorsal nucleus (SLD), located in the pontine tegmental area, which plays a crucial role in the regulation of REMS. We searched for genes selectively expressed in the SLD and identified copine-7 (Cpne7), whose involvement in sleep was totally unknown. We generated Cpne7-Cre knock-in mice, which enabled both the knockout (KO) of Cpne7 and the genetic labeling of Cpne7-expressing cells. While Cpne7-KO mice exhibited normal sleep under basal conditions, the amount of REMS in Cpne7-KO mice was larger compared to wildtype mice following cage change or water immersion and restraint stress, both of which are conditions that acutely reduce REMS. Thus, it was suggested that copine-7 is involved in negatively regulating REMS under certain conditions. In addition, chemogenetically activating Cpne7-expressing neurons in the SLD reduced the amount of REMS, suggesting that these neurons negatively regulate REMS. These results identify copine-7 and Cpne7-expressing neurons in the SLD as candidate molecular or neuronal components of the regulatory system that controls REMS.

Are Genome-Wide Association Study Identified Single-Nucleotide Polymorphisms Associated With Sprint Athletic Status? A Replication Study With 3 Different Cohorts.

PURPOSE: To replicate previous genome-wide association study identified sprint-related polymorphisms in 3 different cohorts of top-level sprinters and to further validate the obtained results in functional studies. METHODS: A total of 240 Japanese, 290 Russians, and 593 Brazilians were evaluated in a case-control approach. Of these, 267 were top-level sprint/power athletes. In addition, the relationship between selected polymorphisms and muscle fiber composition was evaluated in 203 Japanese and 287 Finnish individuals. RESULTS: The G allele of the rs3213537 polymorphism was overrepresented in Japanese (odds ratio [OR]: 2.07, P = .024) and Russian (OR: 1.93, P = .027) sprinters compared with endurance athletes and was associated with an increased proportion of fast-twitch muscle fibers in Japanese (P = .02) and Finnish (P = .041) individuals. A meta-analysis of the data from 4 athlete cohorts confirmed that the presence of the G/G genotype rather than the G/A+A/A genotypes increased the OR of being a sprinter compared with controls (OR: 1.49, P = .01), endurance athletes (OR: 1.79, P = .001), or controls + endurance athletes (OR: 1.58, P = .002). Furthermore, male sprinters with the G/G genotype were found to have significantly faster personal times in the 100-m dash than those with G/A+A/A genotypes (10.50 [0.26] vs 10.76 [0.31], P = .014). CONCLUSION: The rs3213537 polymorphism found in the CPNE5 gene was identified as a highly replicable variant associated with sprinting ability and the increased proportion of fast-twitch muscle fibers, in which the homozygous genotype for the major allele (ie, the G/G genotype) is preferable for performance.

CPNE1-mediated neuronal differentiation can be inhibited by HAX1 expression in HiB5 cells.

We previously demonstrated that CPNE1 induces neuronal differentiation and identified two binding proteins of CPNE1 (14-3-3γ and Jab1) as potential regulators of CPNE1-mediated neuronal differentiation in hippocampal progenitor cells. To better understand the cellular processes in which CPNE1 participates in neuronal differentiation, we here carried out a yeast two-hybrid screening to find another CPNE1 binding protein. Among the identified proteins, HCLS1-related protein X-1 (HAX1) directly interacts with CPNE1. Immunostaining experiments showed that a fraction of CPNE1 and HAX1 co-localized in the cytosol, particularly in the plasma membrane. In addition, the physical interaction as well as the specific binding regions between CPNE1 and HAX1 were confirmed in vitro and in vivo. Moreover, AKT phosphorylation, Tuj1 (neuronal marker protein) expression, and neurite outgrowth are all reduced in CPNE1/HAX1 overexpressing cells compared to CPNE1 only overexpressing HiB5 cells. Conversely, the HAX1 mutant that does not bind to CPNE1 was unable to inhibit the CPNE1-mediated neuronal differentiation. Together these results indicate that HAX1 is a binding partner of CPNE1 and CPNE1-mediated neuronal differentiation is negatively affected through the binding of HAX1, especially its N-terminal region, with CPNE1.

Corrigendum: Quercetin Alleviates LPS-Induced Depression-Like Behavior in Rats via Regulating BDNF-Related Imbalance of Copine 6 and TREM1/2 in the Hippocampus and PFC.

[This corrects the article DOI: 10.3389/fphar.2019.01544.].

Copine A regulates the size and exocytosis of contractile vacuoles and postlysosomes in Dictyostelium.

Copines are a family of cytosolic proteins that associate with membranes in a calcium-dependent manner and are found in many eukaryotic organisms. Dictyostelium discoideum has six copine genes (cpnA-cpnF), and cells lacking cpnA(cpnA- ) have defects in cytokinesis, chemotaxis, adhesion, and development. CpnA has also been shown to associate with the plasma membrane, contractile vacuoles (CV), and organelles of the endolysosomal pathway. Here, we use cpnA- cells to investigate the role of CpnA in CV function and endocytosis. When placed in water, cpnA- cells made abnormally large CVs that took longer to expel. Visualization of CVs with the marker protein GFP-dajumin indicated that cpnA- cells had fewer CVs that sometimes refilled before complete emptying. In endocytosis assays, cpnA- cells took up small fluorescent beads by macropinocytosis at rates similar to parental cells. However, cpnA- cells reached a plateau sooner than parental cells and had less fluorescence at later time points. p80 antibody labeling of postlysosomes (PL) indicated that there were fewer and smaller PLs in cpnA- cells. In dextran pulse-chase experiments, the number of PLs peaked earlier in cpnA- cells, and the PLs did not become as large and disappeared sooner as compared to parental cells. PLs in cpnA- cells were also shown to have more actin coats, suggesting CpnA may play a role in actin filament disassembly on PL membranes. Overall, these results indicate that CpnA is involved in the regulation of CV size and expulsion, and the maturation, size, and exocytosis of PLs.

Resveratrol Reduces Glucolipid Metabolic Dysfunction and Learning and Memory Impairment in a NAFLD Rat Model: Involvement in Regulating the Imbalance of Nesfatin-1 Abundance and Copine 6 Expression.

Resveratrol (RES) is a polyphenolic compound, and our previous results have demonstrated its neuroprotective effect in a series of animal models. The aim of this study was to investigate its potential effect on a nonalcoholic fatty liver disease (NAFLD) rat model. The parameters of liver function and glucose and lipid metabolism were measured. Behavior performance was observed via the open field test (OFT), the sucrose preference test (SPT), the elevated plus maze (EPM), the forced swimming test (FST), and the Morris water maze (MWM). The protein expression levels of Copine 6, p-catenin, catenin, p-glycogen synthase kinase-3beta (GSK3ß), GSK3ß, and cyclin D1 in the hippocampus and prefrontal cortex (PFC) were detected using Western blotting. The results showed that RES could reverse nesfatin-1-related impairment of liver function and glucolipid metabolism, as indicated by the decreased plasma concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), glucose, insulin, and nesfatin-1; increase the plasma level of high-density lipoprotein cholesterol (HDL-C); and reduce hepatocyte steatosis in NAFLD rats. Although there was no significant difference among groups with regard to performance in the OFT, EPM, and FST tasks, RES-treated NAFLD rats showed an increased sucrose preference index in the SPT and improved learning and memory ability in the MWM task. Furthermore, the imbalanced protein expression levels of Copine 6, p-catenin, and p-GSK3ß in the hippocampus and PFC of NAFLD rats were also restored to normal by treatment with RES. These results suggested that four consecutive weeks of RES treatment not only ameliorated glucolipid metabolic impairment and liver dysfunction in the NAFLD rat model but also mitigated the attendant behavioral and cognitive impairments. In addition to the mediating role of nesfatin-1, the mechanism underlying the therapeutic effect of RES on NAFLD might be associated with its ability to regulate the imbalanced expression level of Copine 6 and the Wnt signaling pathway in the hippocampus and PFC.

Copine A Interacts with Actin Filaments and Plays a Role in Chemotaxis and Adhesion.

Copines make up a family of calcium-dependent, phospholipid-binding proteins found in numerous eukaryotic organisms. Copine proteins consist of two C2 domains at the N-terminus followed by an A domain similar to the von Willebrand A domain found in integrins. We are studying copine protein function in the model organism, Dictyostelium discoideum, which has six copine genes, cpnA-cpnF. Previous research showed that cells lacking the cpnA gene exhibited a cytokinesis defect, a contractile vacuole defect, and developmental defects. To provide insight into the role of CpnA in these cellular processes, we used column chromatography and immunoprecipitation to isolate proteins that bind to CpnA. These proteins were identified by mass spectrometry. One of the proteins identified was actin. Purified CpnA was shown to bind to actin filaments in a calcium-dependent manner in vitro. cpnA- cells exhibited defects in three actin-based processes: chemotaxis, cell polarity, and adhesion. These results suggest that CpnA plays a role in chemotaxis and adhesion and may do so by interacting with actin filaments.

Differential expression and subcellular localization of Copines in mouse retina.

Combinatorial expression of Brn3 transcription factors is required for the development of cell-specific morphologies in retinal ganglion cells (RGCs). The molecular mechanisms by which Brn3s regulate RGC type specific features are largely unexplored. We previously identified several members of the Copine (Cpne) family of molecules as potential targets of Brn3 transcription factors in the retina. We now use in situ hybridization and immunohistochemistry to characterize Copine expression in the postnatal and adult mouse retina. We find that Cpne5, 6, and 9 are expressed in the ganglion cell layer (GCL) and inner nuclear layer (INL) in both amacrine cells and RGCs. Cpne4 expression is restricted to one amacrine cell population of the INL, but is specifically expressed in RGCs in the GCL. Cpne4 expression in RGCs is regulated by Brn3b both cell autonomously (in Brn3b+ RGCs) and cell nonautonomously (in Brn3b- RGCs). Copines exhibit a variety of subcellular distributions when overexpressed in tissue culture cells (HEK293), and can induce the formation of elongated processes reminiscent of neurites in these non-neuronal cells. Our results suggest that Copines might be involved in a combinatorial fashion in Brn3b-dependent specification of RGC types. Given their expression profile and previously proven role as Ca2+ sensors, they may participate in the morphogenetic processes that shape RGC dendrite and axon formation at early postnatal ages.

Quercetin Alleviates LPS-Induced Depression-Like Behavior in Rats via Regulating BDNF-Related Imbalance of Copine 6 and TREM1/2 in the Hippocampus and PFC.

Quercetin is a polyphenol with multiple biological activities, and results of our preliminary study showed that it could shorten the immobility time of mice in the forced swimming test and tail suspending test. The aim of this study was to investigate its effects on the behavioral performance of lipopolysaccharide (LPS)-challenged rats and explore the potential mechanism. The results showed that intragastrical administration of quercetin (40 mg/kg) could improve the bodyweight gain of LPS-challenged rats, increase the saccharin preference index in the saccharin preference test and the novel arm preference index in the Y-maze, and decrease the immobility time in the FST. However, it showed no significant effect on the performance of LPS-challenged rats in the Morris water maze and the plasma concentrations of nesfatin-1, C-reactive protein (CRP), and IL-6. Results of western blot showed that the expression levels of BDNF, Copine 6, p-TrkB, and the triggering receptors expressed on myeloid cells (TREM) 1 were decreased in both the hippocampus and the prefrontal cortex (PFC) of LPS-challenged rats, while the expression of TREM2 was increased. The protein expression of synapsin-1 was decreased in the hippocampus without significant changes in the PFC. These imbalance protein expressions could be balanced by treatment with quercetin. The results suggested that quercetin could alleviate LPS-induced depression-like behaviors and impairment of learning and memory in rats, the mechanism of which might be involved with regulating the BDNF-related imbalance expression of Copine 6 and TREM1/2 in the hippocampus and the PFC.