BRD7 facilitates ferroptosis via modulating clusterin promoter hypermethylation and suppressing AMPK signaling in diabetes-induced testicular damage.

BACKGROUND: Diabetes mellitus (DM)-induced testicular damage is associated with sexual dysfunction and male infertility in DM patients. However, the pathogenesis of DM-induced testicular damage remains largely undefined. METHODS: A streptozotocin (STZ)-induced diabetic model and high glucose (HG)-treated in vitro diabetic model were established. The histological changes of testes were assessed by H&E staining. Serum testosterone, iron, MDA and GSH levels were detected using commercial kits. Cell viability and lipid peroxidation was monitored by MTT assay and BODIPY 581/591 C11 staining, respectively. qRT-PCR, immunohistochemistry (IHC) or Western blotting were employed to detect the levels of BRD7, Clusterin, EZH2 and AMPK signaling molecules. The associations among BRD7, EZH2 and DNMT3a were detected by co-IP, and the transcriptional regulation of Clusterin was monitored by methylation-specific PCR (MSP) and ChIP assay. RESULTS: Ferroptosis was associated with DM-induced testicular damage in STZ mice and HG-treated GC-1spg cells, and this was accompanied with the upregulation of BRD7. Knockdown of BRD7 suppressed HG-induced ferroptosis, as well as HG-induced Clusterin promoter methylation and HG-inactivated AMPK signaling in GC-1spg cells. Mechanistical studies revealed that BRD7 directly bound to EZH2 and regulated Clusterin promoter methylation via recruiting DNMT3a. Knockdown of Clusterin or inactivation of AMPK signaling reverses BRD7 silencing-suppressed ferroptosis in GC-1spg cells. In vivo findings showed that lack of BRD7 protected against diabetes-induced testicular damage and ferroptosis via increasing Clusterin expression and activating AMPK signaling. CONCLUSION: BRD7 suppressed Clusterin expression via modulating Clusterin promoter hypermethylation in an EZH2 dependent manner, thereby suppressing AMPK signaling to facilitate ferroptosis and induce diabetes-associated testicular damage.

Protective effect of clusterin against interleukin-1ß-induced apoptosis and inflammation in human knee osteoarthritis chondrocytes.

Chondrocyte apoptosis is recognized as one of the pathological features involved in cartilage degeneration driving the onset and progression of knee osteoarthritis (OA). This study aimed to determine the molecular mechanism underlying the effect of clusterin (CLU), anti-apoptotic molecule, in human knee OA chondrocytes. Primary knee OA chondrocytes were isolated from the cartilage of knee OA patients and divided into five groups: (1) the cells treated with interleukin (IL)-1ß, (2) CLU alone, (3) a combination of IL-1ß and CLU, (4) LY294002 (PI3K inhibitor) along with IL-1ß and CLU, and (5) the untreated cells. Production of apoptotic, inflammatory, anabolic, and catabolic mediators in knee OA chondrocytes was determined after treatment for 24 h. Our in vitro study uncovered that CLU significantly suppressed the production of inflammatory mediators [nitric oxide (NO), IL6, and tumor necrosis factor (TNF)-α] and apoptotic molecule (caspase-3, CASP3). CLU significantly upregulated messenger ribonucleic acid (mRNA) expressions of anabolic factors [SRY-box transcription factor-9 (SOX9) and aggrecan (ACAN)], but significantly downregulated mRNA expressions of IL6, nuclear factor kappa-B (NF-κB), CASP3, and matrix metalloproteinase-13 (MMP13). Anti-apoptotic and anti-inflammatory effects of CLU were mediated through activating PI3K/Akt signaling pathway. The findings suggest that CLU might have beneficial effects on knee OA chondrocytes by exerting anti-apoptotic and anti-inflammatory functions via PI3K/Akt pathway, making CLU a promising target for potential therapeutic interventions in knee OA.

Method to Assess the Intracellular Fate and Bioavailability of Clusterin Using Live Cell Confocal Microscopy Imaging.

Clusterin, also known as apolipoprotein J, is an ATP-independent holdase chaperone protein. Clusterin is involved in various functions including protein quality control and lipid transport. Though clusterin is secreted upon stress, the intracellular fate of clusterin after a stress response is not well understood. The protocol described here utilizes clusterin tagged to fluorescent proteins like green fluorescent protein and red fluorescent protein to understand the intracellular fate of clusterin.

Methionine oxidation of clusterin in Alzheimer's disease and its effect on clusterin's binding to beta-amyloid.

Clusterin is a secreted glycoprotein that participates in multiple physiological processes through its chaperon function. In Alzheimer's disease, the brain functions under an increased oxidative stress condition that causes an elevation of protein oxidation, resulting in enhanced pathology. Accordingly, it is important to determine the type of human brain cells that are mostly prone to methionine oxidation in Alzheimer's disease and specifically monitoring the methionine-oxidation levels of clusterin in human and mice brains and its effect on clusterin's function. We analyzed the level of methionine sulfoxide (MetO)-clusterin in these brains, using a combination of immunoprecipitation and Western-blott analyses. Also, we determine the effect of methionine oxidation on clusterin ability to bind beta-amyloid, in vitro, using calorimetric assay. Our results show that human neurons and astrocytes of Alzheimer's disease brains are mostly affected by methionine oxidation. Moreover, MetO-clusterin levels are elevated in postmortem Alzheimer's disease human and mouse brains in comparison to controls. Finally, oxidation of methionine residues of purified clusterin reduced its binding efficiency to beta-amyloid. In conclusion, we suggest that methionine oxidation of brain-clusterin is enhanced in Alzheimer's disease and that this oxidation compromises its chaperon function, leading to exacerbation of beta-amyloid's toxicity in Alzheimer's disease.

Decoding CLU (Clusterin): Conquering cancer treatment resistance and immunological barriers.

One major obstacle in the treatment of cancer is the presence of proteins resistant to cancer therapy, which can impede the effectiveness of traditional approaches such as radiation and chemotherapy. This resistance can lead to disease progression and cause treatment failure. Extensive research is currently focused on studying these proteins to create tailored treatments that can circumvent resistance mechanisms. CLU (Clusterin), a chaperone protein, has gained notoriety for its role in promoting resistance to a wide range of cancer treatments, including chemotherapy, radiation therapy, and targeted therapy. The protein has also been discovered to have a role in regulating the immunosuppressive environment within tumors. Its ability to influence oncogenic signaling and inhibit cell death bolster cancer cells resistant against treatments, which poses a significant challenge in the field of oncology. Researchers are actively investigating to the mechanisms by which CLU exerts its resistance-promoting effects, with the ultimate goal of developing strategies to circumvent its impact and enhance the effectiveness of cancer therapies. By exploring CLU's impact on cancer, resistance mechanisms, tumor microenvironment (TME), and therapeutic strategies, this review aims to contribute to the ongoing efforts to improve cancer treatment outcomes.

Therapeutic Potential of Clusterin Inhibition in Human Cancer.

Clusterin (CLU) protein is involved in various pathophysiological processes including carcinogenesis and tumor progression. In recent years, the role of the secretory isoform has been demonstrated in tumor cells, where it inhibits apoptosis and favors the acquisition of resistance to conventional treatments used to treat cancer. To determine the possible therapeutic potential of inhibiting this protein, numerous studies have been carried out in this field. In this article, we present the existing knowledge to date on the inhibition of this protein in different types of cancer and analyze the importance it could have in the development of new therapies targeted against this disease.

The correlation between human seminal plasma sialoproteins and ejaculate parameters.

Sialic acids are negatively charged carbohydrates that are components of saccharide chains covalently linked to macromolecules. Sialylated glycoproteins are important for most biological processes, including reproduction, where they are associated with spermatogenesis, sperm motility, immune responses, and fertilization. Changes in the glycoprotein profile or sialylation in glycoproteins are likely to affect the quality of ejaculate. The aim of this study was to determine differences in the degree of sialylation between normozoospermic ejaculates and ejaculates with a pathological spermiogram using two lectins, Sambucus nigra (SNA) and Maackia amurensis (MAL II/MAA) recognizing α-2,6 or α-2,3 linkage of Sia to galactosyl residues. Our results show a close relationship between seminal plasma (SP) sialoproteins and the presence of anti-sperm antibodies in the ejaculate, apoptotic spermatozoa, and ejaculate quality. Using mass spectrometry, we identified SP sialoproteins such as, semenogelins, glycodelin, prolactin-inducible protein, lactotransferrin, and clusterin that are associated with spermatozoa and contribute to the modulation of the immune response and sperm apoptosis. Our findings suggest a correlation between the degree of SP glycoprotein sialylation and the existence of possible pathological states of spermatozoa and reproductive organs. Glycoproteins sialylation represents a potential parameter reflecting the overall quality of ejaculate and could potentially be utilised in diagnostics.

CLU (clusterin) and PPARGC1A/PGC1α coordinately control mitophagy and mitochondrial biogenesis for oral cancer cell survival.

Mitophagy involves the selective elimination of defective mitochondria during chemotherapeutic stress to maintain mitochondrial homeostasis and sustain cancer growth. Here, we showed that CLU (clusterin) is localized to mitochondria to induce mitophagy controlling mitochondrial damage in oral cancer cells. Moreover, overexpression and knockdown of CLU establish its mitophagy-specific role, where CLU acts as an adaptor protein that coordinately interacts with BAX and LC3 recruiting autophagic machinery around damaged mitochondria in response to cisplatin treatment. Interestingly, CLU triggers class III phosphatidylinositol 3-kinase (PtdIns3K) activity around damaged mitochondria, and inhibition of mitophagic flux causes the accumulation of excessive mitophagosomes resulting in reactive oxygen species (ROS)-dependent apoptosis during cisplatin treatment in oral cancer cells. In parallel, we determined that PPARGC1A/PGC1α (PPARG coactivator 1 alpha) activates mitochondrial biogenesis during CLU-induced mitophagy to maintain the mitochondrial pool. Intriguingly, PPARGC1A inhibition through small interfering RNA (siPPARGC1A) and pharmacological inhibitor (SR-18292) treatment counteracts CLU-dependent cytoprotection leading to mitophagy-associated cell death. Furthermore, co-treatment of SR-18292 with cisplatin synergistically suppresses tumor growth in oral cancer xenograft models. In conclusion, CLU and PPARGC1A are essential for sustained cancer cell growth by activating mitophagy and mitochondrial biogenesis, respectively, and their inhibition could provide better therapeutic benefits against oral cancer.

Secreted clusterin inhibits tumorigenesis by modulating tumor cells and macrophages in human meningioma.

BACKGROUND: Meningioma is the most common primary intracranial tumor with a high frequency of postoperative recurrence, yet the biology of the meningioma malignancy process is still obscure. METHODS: To identify potential therapeutic targets and tumor suppressors, we performed single-cell transcriptome analysis through meningioma malignancy, which included 18 samples spanning normal meninges, benign and high-grade in situ tumors, and lung metastases, for extensive transcriptome characterization. Tumor suppressor candidate gene and molecular mechanism were functionally validated at the animal model and cellular levels. RESULTS: Comprehensive analysis and validation in mice and clinical cohorts indicated clusterin (CLU) had suppressive function for meningioma tumorigenesis and malignancy by inducing mitochondria damage and triggering type 1 interferon pathway dependent on its secreted isoform, and the inhibition effect was enhanced by TNFα as TNFα also induced type 1 interferon pathway. Meanwhile, both intra- and extracellular CLU overexpression enhanced macrophage polarization towards M1 phenotype and TNFα production, thus promoting tumor killing and phagocytosis. CONCLUSIONS: CLU might be a key brake of meningioma malignance by synchronously modulating tumor cells and their microenvironment. Our work provides comprehensive insights into meningioma malignancy and a potential therapeutic strategy.

Clusterin: a marker and mediator of chemoresistance in colorectal cancer.

Intra-tumoural heterogeneity and cancer cell plasticity in colorectal cancer (CRC) have been key challenges to effective treatment for patients. It has been suggested that a subpopulation of LGR5-expressing cancer stem cells (CSCs) is responsible for driving tumour relapse and therapy resistance in CRC. However, studies have revealed that the LGR5+ve CSC population is highly sensitive to chemotherapy. It has been hypothesised that another subset of tumour cells can phenotypically revert to a stem-like state in response to chemotherapy treatment which replenishes the LGR5+ve CSC population and maintains tumour growth. Recently, a unique stem cell population marked by enriched clusterin (CLU) expression and termed the revival stem cell (RevSC) was identified in the regenerating murine intestine. This CLU-expressing cell population is quiescent during homeostasis but has the ability to survive and regenerate other stem cells upon injury. More recently, the CLU+ve signature has been implicated in several adverse outcomes in CRC, including chemotherapy resistance and poor patient survival; however, the mechanism behind this remains undetermined. In this review, we discuss recent insights on CLU in CRC and its roles in enhancing the plasticity of cells and further consider the implications of CLU as a prospective target for therapeutic intervention.

Secretory Clusterin Inhibits Dopamine Neuron Apoptosis in MPTP Mice by Preserving Autophagy Activity.

Secretory clusterin (sCLU) plays an important role in the research progress of nervous system diseases. However, the physiological function of sCLU in Parkinson's disease (PD) are unclear. The purpose of this study was to examine the effects of sCLU-mediated autophagy on cell survival and apoptosis inhibition in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. We found that MPTP administration induced prolonged pole-climbing time, shortened traction time and rotarod time, significantly decreased TH protein expression in the SN tissue of mice. In contrast, sCLU -treated mice took less time to climb the pole and had an extended traction time and rotating rod time. Meanwhile, sCLU intervention induced increased expression of the TH protein in the SN of mice. These results indicated that sCLU intervention could reduce the loss of dopamine neurons in the SN area and alleviate dyskinesia in mice. Furthermore, MPTP led to suppressed viability, enhanced apoptosis, an increased Bax/Bcl-2 ratio, and cleaved caspase-3 in the SN of mice, and these effects were abrogated by sCLU intervention. In addition, MPTP increased the levels of P62 protein, decreased Beclin1 protein, decreased the ratio of LC3B-II/LC3B-I, and decreased the numbers of autophagosomes and autophagolysosomes in the SN tissues of mice. These effects were also abrogated by sCLU intervention. Activation of PI3K/AKT/mTOR signaling with MPTP inhibited autophagy in the SN of MPTP mice; however, sCLU treatment activated autophagy in MPTP-induced PD mice by inhibiting PI3K/AKT/mTOR signaling. These data indicated that sCLU treatment had a neuroprotective effect in an MPTP-induced model of PD.

Clusterin is upregulated by erastin, a ferroptosis inducer and exerts cytoprotective effects in pancreatic adenocarcinoma cells.

Ferroptosis is a novel form of cell death, which is distinguished from apoptosis and necrosis, and characterized by accumulation of lipid-based reactive oxygen species (ROS) in an iron-dependent manner. Erastin, a small molecule, was widely reported to trigger ferroptosis in various kinds of cancer cells, including pancreatic cancer cells by inducing ROS accumulation. However, how erastin treatment exerts cytotoxicity is not still fully understood. In this study, the effects of erastin in causing pancreatic cancer cell death via inducing ferroptosis and apoptosis are investigated. As expected, erastin treatment caused ROS accumulation, increase in iron concentration and non-apoptotic cell death, which is different from that of induced by apoptosis inducer, staurosporine. Interestingly, erastin treatment caused the upregulation of clusterin, which contributes to the regulation of malignant behaviors of pancreatic cancer, including preventing apoptosis and inducing chemoresistance. Without erastin treatment, overexpressed clusterin significantly promoted cell proliferation, which is consistent with its cytoprotective roles. After erastin treatment, overexpressed clusterin decreased erastin-induced ROS accumulation and cell death. By measuring iron concentration, reduced glutathione (GSH) and glutathione peroxidase 4 (GPX4), it is revealed that clusterin caused resistance to erastin-induced ferroptosis potentially via maintaining the enzymatic activity of GPX4, without disturbing GSH amount. Thus, ferroptosis inducer, erastin, may crosstalk with apoptotic cell death via regulating clusterin, indicating a more complex regulatory network between ferroptosis and apoptosis.

Clusterin protects mature dendritic cells from reactive oxygen species mediated cell death.

Dendritic cells (DCs) play a key role in the induction of the adaptive immune response. They capture antigens in peripheral tissues and prime naïve T lymphocytes, triggering the adaptive immune response. In the course of inflammatory processes DCs face stressful conditions including hypoxia, low pH and high concentrations of reactive oxygen species (ROS), among others. How DCs survive under these adverse conditions remain poorly understood. Clusterin is a protein highly expressed by tumors and usually associated with bad prognosis. It promotes cancer cell survival by different mechanisms such as apoptosis inhibition and promotion of autophagy. Here, we show that, upon maturation, human monocyte-derived DCs (MoDCs) up-regulate clusterin expression. Clusterin protects MoDCs from ROS-mediated toxicity, enhancing DC survival and promoting their ability to induce T cell activation. In line with these results, we found that clusterin is expressed by a population of mature LAMP3+ DCs, called mregDCs, but not by immature DCs in human cancer. The expression of clusterin by intratumoral DCs was shown to be associated with a transcriptomic profile indicative of cellular response to stress. These results uncover an important role for clusterin in DC physiology.

Assessment of MMP14, CAV2, CLU and SPARCL1 expression profiles in endometriosis.

Endometriotic cells exhibit a notable degree of invasiveness and some characteristics of tissue remodeling underlying lesion formation. In this regard, do matrix metalloproteinases 14 (MMP14) and other related genes such as SPARC-like protein 1 (SPARCL1), caveolin 2 (CAV2), and clusterin (CLU) exert any significant influence in the processes of endometriosis development and pathophysiology is not apparent. We aim to assess whether these genes could serve as potential diagnostic biomarkers in endometriosis. Microarray-based gene expression analysis was performed on total RNA extracted from endometriotic tissue samples treated with and without gonadotropin-releasing hormone agonist (GnRHa). The GnRHa untreated patients were considered the control group. The validation of genes was performed using quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR analysis showed significant downregulation in the expression of MMP14 (p = 0.024), CAV2 (p = 0.017), and upregulation of CLU (p = 0.005) in endometriosis patients treated with GnRHa. SPARCL1 did not show any significant (p = 0.30) change in the expression compared to the control group. These data have the potential to contribute to the comprehension of the molecular pathways implicated in the remodeling of the extracellular matrix, which is a vital step for the physiology of the endometrium. Based on the result, it is concluded that changes in the expression of MMP14, CAV2, and CLU post-treatment imply their role in the pathophysiology of endometriosis and may serve as a potential diagnostic biomarker of endometriosis in response to GnRHa treatment in patients with ovarian endometrioma.

Clusterin Expression in Colorectal Carcinomas.

Colorectal cancer is the third most diagnosed cancer, behind only breast and lung cancer. In terms of overall mortality, it ranks second due to, among other factors, problems with screening programs, which means that one of the factors that directly impacts survival and treatment success is early detection of the disease. Clusterin (CLU) is a molecular chaperone that has been linked to tumorigenesis, cancer progression and resistance to anticancer treatments, which has made it a promising drug target. However, it is still necessary to continue this line of research and to adjust the situations in which its use is more favorable. The aim of this paper is to review the current genetic knowledge on the role of CLU in tumorigenesis and cancer progression in general, and discuss its possible use as a therapeutic target in colorectal cancer.

Role of clusterin gene 3'-UTR polymorphisms and promoter hypomethylation in the pathogenesis of pseudoexfoliation syndrome and pseudoexfoliation glaucoma.

Pseudoexfoliation (PEX) is a multifactorial age-related disease characterized by the deposition of extracellular fibrillar aggregates in the anterior ocular tissues. This study aims to identify the genetic and epigenetic contribution of clusterin (CLU) in PEX pathology. CLU is a molecular chaperone upregulated in PEX and genetically associated with the disease. Sequencing of a 2.9 kb region encompassing the previously associated rs2279590 in 250 control and 313 PEX [(207 pseudoexfoliation syndrome (PEXS) and 106 pseudoexfoliation glaucoma (PEXG)] individuals identified three single nucleotide polymorphisms (SNPs), rs9331942, rs9331949 and rs9331950, in the 3'-UTR of CLU of which rs9331942 and rs9331949 were found to be significantly associated with PEXS and PEXG as risk factors. Following in silico analysis, in vitro luciferase reporter assays in human embryonic kidney cells revealed that risk alleles at rs9331942 and rs9331949 bind to miR-223 and miR-1283, respectively, suggesting differential regulation of clusterin in the presence of risk alleles at the SNPs. Further, through bisulfite sequencing, we also identified that CLU promoter is hypomethylated in DNA from blood and lens capsules of PEX patients compared to controls that correlated with decreased expression of DNA methyltransferase 1 (DNMT1). Promoter demethylation of CLU using DNMT inhibitor, 5'-aza-dC, in human lens epithelial cells increased CLU expression. Chromatin immunoprecipitation assays showed that the demethylated CLU promoter provides increased access to the transcription factor, Sp1, which might lead to enhanced expression of CLU. In conclusion, this study highlights the different molecular mechanisms of clusterin regulation in pseudoexfoliation pathology.

Differential Sperm Proteomics Reveals the Significance of Fatty Acid Synthase and Clusterin in Idiopathic Recurrent Pregnancy Loss.

Recurrent pregnancy loss (RPL) is a pervasive health issue affecting a large number of couples globally, which leads to increased emotional and financial strain on the affected families. While female factors have been extensively studied and are well known, the contribution of male factors to RPL remains largely unknown. As high as 40% of RPL cases are unexplained, which are termed as idiopathic RPL (iRPL), necessitating the investigation of male factors. The role of spermatozoa in early embryonic development is now well established, and recent research studies have shown that oxidative stress and DNA fragmentation in sperm cells are linked to RPL. The aim of this study was to identify proteomic markers of iRPL in human spermatozoa using tandem mass spectrometry. A label-free method quantified a total of 1820 proteins, and statistical analysis identified 359 differentially expressed proteins, the majority of which were downregulated in iRPL samples (344). Bioinformatics analysis revealed that proteomic alterations were mainly associated with biological processes such as response to stress, protein folding, chromatin organization, DNA conformation change, oxidative phosphorylation, and electron transport chain. In coherence with past studies, we determined fatty acid synthase (FASN) and clusterin (CLU) to be the most potential sperm markers for iRPL and confirmed their expression changes in iRPL by western blotting. Conclusively, we believe that FASN and CLU might serve as potential markers of iRPL and suggest exploratory functional studies to identify their specific role in pregnancy loss.

Reelin Plasma Levels Identify Cognitive Decline in Alcohol Use Disorder Patients During Early Abstinence: The Influence of APOE4 Expression.

BACKGROUND: Apolipoprotein E (APOE)-4 isoform, reelin, and clusterin share very-low-density liporeceptor and apolipoprotein E receptor 2 receptors and are related to cognition in neuropsychiatric disorders. These proteins are expressed in plasma and brain, but studies involving plasma expression and cognition are scarce. METHODS: We studied the peripheral expression (plasma and peripheral blood mononuclear cells) of these proteins in 24 middle-aged patients with alcohol use disorder (AUD) diagnosed at 4 to 12 weeks of abstinence (t = 0) and 34 controls. Cognition was assessed using the Test of Detection of Cognitive Impairment in Alcoholism. In a follow-up study (t = 1), we measured reelin levels and evaluated cognitive improvement at 6 months of abstinence. RESULTS: APOE4 isoform was present in 37.5% and 58.8% of patients and controls, respectively, reaching similar plasma levels in ε4 carriers regardless of whether they were patients with AUD or controls. Plasma reelin and clusterin were higher in the AUD group, and reelin levels peaked in patients expressing APOE4 (P < .05, η2 = 0.09), who showed reduced very-low-density liporeceptor and apolipoprotein E receptor 2 expression in peripheral blood mononuclear cells. APOE4 had a negative effect on memory/learning mainly in the AUD group (P < .01, η2 = 0.15). Multivariate logistic regression analyses identified plasma reelin as a good indicator of AUD cognitive impairment at t = 0. At t = 1, patients with AUD showed lower reelin levels vs controls along with some cognitive improvement. CONCLUSIONS: Reelin plasma levels are elevated during early abstinence in patients with AUD who express the APOE4 isoform, identifying cognitive deterioration to a great extent, and it may participate as a homeostatic signal for cognitive recovery in the long term.

Plexina4 and cell survival in the developing zebrafish hindbrain.

BACKGROUND: Growth factors are important in the developing and mature nervous system to support the survival of neurons. Developmental signaling molecules are known for their roles in controlling neurogenesis and neural circuit formation. Whether or not these molecules also have roles in cell survival in the developing nervous system is poorly understood. Plexins are a family of transmembrane receptors that bind Semaphorin ligands and are known to function in the guidance of developing axons and blood vessels. RESULTS: In embryonic zebrafish, plexina4 is expressed widely in the brain, becoming largely restricted to the hindbrain as neurogenesis and differentiation proceed. Apoptosis is increased in the embryonic hindbrain of a plexina4ca307/ca307 CRISPR mutant. Based on the literature, we tested the secreted heat shock protein, Clusterin, as a candidate ligand to mediate cell survival through Plexina4. clusterin is expressed by the floor plate of the embryonic zebrafish hindbrain, in proximity to plexina4-expressing hindbrain cells. Morpholino-mediated knockdown of Clusterin increases cell apoptosis in the hindbrain, with additional cell death observed in epistasis experiments where Clusterin is knocked down in a plexina4 mutant background. CONCLUSIONS: Our data suggest that Plexina4 promotes cell survival in the developing zebrafish hindbrain, likely through a pathway independent of Clusterin.

GABAergic signaling abnormalities in a novel CLU mutation Alzheimer's disease mouse model.

The CLU rs11136000C mutation (CLUC) is the third most common risk factor for Alzheimer's disease (AD). However, the mechanism by which CLUC leads to abnormal GABAergic signaling in AD is unclear. To address this question, this study establishes the first chimeric mouse model of CLUC AD. Examination of grafted CLUC medial ganglionic eminence progenitors (CLUC hiMGEs) revealed increased GAD65/67 and a high frequency of spontaneous releasing events. CLUC hiMGEs also impaired cognition in chimeric mice and caused AD-related pathologies. The expression of GABA A receptor, subunit alpha 2 (Gabrα2) was higher in chimeric mice. Interestingly, cognitive impairment in chimeric mice was reversed by treatment with pentylenetetrazole, which is a GABA A receptor inhibitor. Taken together, these findings shed light on the pathogenesis of CLUC AD using a novel humanized animal model and suggest sphingolipid signaling over-activation as a potential mechanism of GABAergic signaling disorder.