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.

Stomach clusterin as a gut-derived feeding regulator.

The stomach has emerged as a crucial endocrine organ in the regulation of feeding since the discovery of ghrelin. Gut-derived hormones, such as ghrelin and cholecystokinin, can act through the vagus nerve. We previously reported the satiety effect of hypothalamic clusterin, but the impact of peripheral clusterin remains unknown. In this study, we administered clusterin intraperitoneally to mice and observed its ability to suppress fasting-driven food intake. Interestingly, we found its synergism with cholecystokinin and antagonism with ghrelin. These effects were accompanied by increased c-fos immunoreactivity in nucleus tractus solitarius, area postrema, and hypothalamic paraventricular nucleus. Notably, truncal vagotomy abolished this response. The stomach expressed clusterin at high levels among the organs, and gastric clusterin was detected in specific enteroendocrine cells and the submucosal plexus. Gastric clusterin expression decreased after fasting but recovered after 2 hours of refeeding. Furthermore, we confirmed that stomachspecific overexpression of clusterin reduced food intake after overnight fasting. These results suggest that gastric clusterin may function as a gut-derived peptide involved in the regulation of feeding through the gut-brain axis. [BMB Reports 2024; 57(3): 149-154].

Zinc Acts Synergistically with Berberine for Enhancing Its Efficacy as an Anti-cancer Agent by Inducing Clusterin-Dependent Apoptosis in HT-29 Colorectal Cancer Cells.

It is now widely accepted that anti-cancer medications are most effective when administered in combination. Zinc is an essential micronutrient whilst berberine is a well-known natural phytochemical, both having multiple molecular mechanisms of action. The present study aimed to determine the combinatorial effect of zinc and berberine on the human adenocarcinoma HT-29 cancer cell line. The anti-proliferative activity of berberine and zinc was determined by cell viability and colony-forming assays. The combination index and drug reduction index values of zinc and berberine co-treatments were estimated by suitable software. Flow cytometry was used to analyse cell cycle distribution and Annexin V/PI staining. The expression of apoptosis and zinc signalling markers were analysed by RT-qPCR and immunoblot analysis. Berberine decreased the viability of colon cancer cells in a dose-dependent manner whilst zinc alone had no significant influence on it. However, zinc and berberine co-treatment resulted in a synergistic anti-cancer action which was demonstrated by G2/M phase arrest of cell growth at a lower dose of berberine. Annexin V assay demonstrated that the synergistic impact of zinc and berberine boosted the number of apoptotic cells. Gene expression analysis at both transcriptional and translational levels showed the upregulation of apoptotic (caspase-3 and caspase-8) and a zinc-sensing receptor (GPR39) gene with concomitant downregulation of anti-apoptotic genes like proliferating cell nuclear antigen (PCNA) and clusterin. Our findings showed that the combination of zinc and berberine has synergistic anti-cancer efficacy and thus could be used as a potential chemopreventive option for colon cancer.

Effects of leptin treatment immediately after neonatal seizures on serum clusterin and VEGF levels and brain oxidative stress-related proteins and neurobehavioral phenotypes.

The developing infant brain has a different response mechanism and repair potential for injury than the adult brain. There is an urgent need for new anticonvulsants to effectively control neonatal seizures while minimizing the drug's toxic damage to the developing brain. Leptin protects neuronal plasma membrane integrity, while it has clinical advantages in terms of anticonvulsant properties as well. This study aimed to evaluate the effect of immediate leptin treatment on the serum concentration of clusterin and vascular endothelial growth factor (VEGF), neuronal plasma membrane integrity-related proteins, and the neurobehavioral phenotypes following neonatal seizures. Leptin was injected i.p at a dose of 4 mg/kg 1 hour after daily 30 minutes prolonged seizures for consecutive 10 days. The serum biomarkers (clusterin and VEGF), and brain protein expression of ATF-4/GRP78/autophagy axis were measured by enzyme-linked immunosorbent assay and western blot in the acute phase (24 hours after the last seizures), respectively. Behavioral and histopathological phenotypes and seizure threshold were conducted from P23 to P34, respectively. There were rapid elevation of serum VEGF and clusterin as well as upregulated protein expression of ATF-4, GRP78, Beclin-1, and LC3 in the cerebral cortex and hippocampus following a neonatal seizure, which was restored by immediate treatment with leptin after seizures. In addition, leptin improved seizure-induced impaired neuropsychological, and cognitive functioning. Furthermore, leptin succeeded in ameliorating markers of neuronal excitability, including seizure threshold and hippocampal mossy fiber sprouting. In conclusion, this study verified that immediate treatment with leptin after neonatal seizures restored both rapid elevation of serum clusterin as well as upregulated protein expression of ATF-4/GRP78/autophagy axis in the cerebral cortex and hippocampus, which contributes to the recovery of neurological function.

Exercise as a non-pharmacological intervention to protect pancreatic beta cells in individuals with type 1 and type 2 diabetes.

AIMS/HYPOTHESIS: Diabetes is characterised by progressive loss of functional pancreatic beta cells. None of the therapeutic agents used to treat diabetes arrest this process; preventing beta cell loss remains a major unmet need. We have previously shown that serum from eight young healthy male participants who exercised for 8 weeks protected human islets and insulin-producing EndoC-ßH1 cells from apoptosis induced by proinflammatory cytokines or the endoplasmic reticulum (ER) stressor thapsigargin. Whether this protective effect is influenced by sex, age, training modality, ancestry or diabetes is unknown. METHODS: We enrolled 82 individuals, male or female, non-diabetic or diabetic, from different origins, in different supervised training protocols for 8-12 weeks (including training at home during the COVID-19 pandemic). EndoC-ßH1 cells were treated with 'exercised' serum or with the exerkine clusterin to ascertain cytoprotection from ER stress. RESULTS: The exercise interventions were effective and improved [Formula: see text] values in both younger and older, non-obese and obese, non-diabetic and diabetic participants. Serum obtained after training conferred significant beta cell protection (28% to 35% protection after 4 and 8 weeks of training, respectively) from severe ER stress-induced apoptosis. Cytoprotection was not affected by the type of exercise training or participant age, sex, BMI or ancestry, and persisted for up to 2 months after the end of the training programme. Serum from exercised participants with type 1 or type 2 diabetes was similarly protective. Clusterin reproduced the beneficial effects of exercised sera. CONCLUSIONS/INTERPRETATION: These data uncover the unexpected potential to preserve beta cell health by exercise training, opening a new avenue to prevent or slow diabetes progression through humoral muscle-beta cell crosstalk.

Low-dose brain irradiation normalizes TSPO and CLUSTERIN levels and promotes the non-amyloidogenic pathway in pre-symptomatic TgF344-AD rats.

Preclinical studies have recently evaluated the impact of low-dose brain radiation therapy (LD-RT) in animal models of Alzheimer's disease (AD) showing anti-amyloid and anti-inflammatory effects of this treatment. Its effectiveness varied, however, depending on the LD-RT protocol used and the stage when the treatment was applied. In this study, we aimed to evaluate the therapeutic potential of 10 Gy delivered in five daily fractions of 2 Gy (a protocol previously shown to induce an improvement of cognitive performances) in 9-month-old TgF344-AD rats, modeling at a pre-symptomatic stage of the disease. We showed that at an early stage, LD-RT was able to lower levels of the 18-kDa translocator protein (TSPO)-mediated neuroinflammation to normal ranges in addition to the secreted CLUSTERIN, another inflammatory protein also involved in Aß aggregation. In addition, we demonstrated that LD-RT reduces all amyloid forms (~ - 60 to - 80%, P < 0.01; soluble and aggregated forms of Aß40, Aß42, and Aßoligomers). Interestingly, we showed for the first time that sAPPα levels were improved by the treatment, showing a higher activation of the non-amyloidogenic pathway, that could favor neuronal survival. The current evidence confirms the capacity of LD-RT to successfully modulate two pathological hallmarks of AD, namely amyloid and neuroinflammation, when applied before symptoms onset.

Novel insight into the role of clusterin on intraocular pressure regulation by modifying actin polymerization and extracellular matrix remodeling in the trabecular meshwork.

This study provides comprehensive mechanistic evidence for the role of clusterin, a stress-response secretory chaperone protein, in the modulation of intraocular pressure (IOP) by regulating the trabecular meshwork (TM) actin cytoskeleton and the extracellular matrix (ECM). The pathological stressors on TM known to elevate IOP significantly lowered clusterin protein levels indicating stress-related clusterin function loss. Small interfering RNA-mediated clusterin loss in human TM cells in vitro induced actin polymerization and stabilization via protein kinase D1, serine/threonine-protein kinase N2 (PRK2), and LIM kinase 1 (LIMK1), and the recruitment and activation of adhesome proteins including paxillin, vinculin, and integrin αV and ß5. A complete loss of clusterin as seen in clusterin knockout mice (Clu-/- ) led to significant IOP elevation at postnatal Day 70. Contrarily, constitutive clusterin expression using adenovirus (AdCLU) in HTM cells resulted in the loss of actin polymerization via decreased PRK2, and LIMK1 and negative regulation of integrin αV and ß5. Furthermore, we found that AdCLU treatment in HTM cells significantly decreased the ECM protein expression and distribution by significantly increasing matrix metalloprotease 2 (MMP2) activity and lowering the levels of pro-fibrotic proteins such as transforming growth factor-ß2 (TGFß2), thrombospondin-1 (TSP-1), and plasminogen activator inhibitor-1 (PAI-1). Finally, we found that HTM cells supplemented with recombinant human clusterin attenuated the pro-fibrotic effects of TGFß2. For the first time this study demonstrates the importance of clusterin in the regulation of TM actin cytoskeleton - ECM interactions and the maintenance of IOP, thus making clusterin an interesting target to reverse elevated IOP.

Inhibition of Clusterin Represses Proliferation by Inducing Cellular Senescence in Pancreatic Cancer.

BACKGROUND: The outcome of pancreatic ductal adenocarcinoma (PDAC) is unsatisfactory, and the identification of novel therapeutic targets is urgently needed. Clinical studies on the antisense oligonucleotide that targets clusterin (CLU) expression have been conducted and have shown efficacy in other cancers. We aimed to investigate the effects of CLU in PDAC and the underlying mechanisms with a view to the clinical application of existing drugs. METHODS: We knocked down CLU in PDAC cells and evaluated changes in cell proliferation. To elucidate the mechanism responsible for these changes, we performed western blot analysis, cell cycle assay, and senescence-associated ß-galactosidase (SA-ß-gal) staining. To evaluate the clinical significance of CLU, immunohistochemistry was performed, and CLU expression was analyzed in specimens resected from PDAC patients not treated with preoperative chemotherapy. RESULTS: Knockdown of CLU significantly decreased cell proliferation and did not induce apoptosis, but did induce cellular senescence by increasing the percentage of G1-phase and SA-ß-gal staining-positive cells. A marker of DNA damage such as γH2AX and factors related to cellular senescence, such as p21 and the senescence-associated secretory phenotype, were upregulated by knockdown of CLU. CLU expression in resected PDAC specimens was located in the cytoplasm of tumor cells and revealed significantly better recurrence-free survival and overall survival in the CLU-low group than in the CLU-high group. CONCLUSIONS: We identified that CLU inhibition leads to cellular senescence in PDAC. Our findings suggest that CLU is a novel therapeutic target that contributes to the prognosis of PDAC by inducing cellular senescence.

Clusterin in Alzheimer's disease: An amyloidogenic inhibitor of amyloid formation?

Clusterin is a heterodimeric glycoprotein (α- and ß-chain), which has been described as an extracellular molecular chaperone. In humans, clusterin is an amyloid-associated protein, co-localizing with fibrillar deposits in several amyloidoses, including Alzheimer's disease. To clarify its potential implication in amyloid formation, we located aggregation-prone regions within the sequence of clusterin α-chain, via computational methods. We had peptide-analogues, which correspond to each of these regions, chemically synthesized and experimentally demonstrated that all of them can form amyloid-like fibrils. We also provide evidence that the same peptide-analogues can inhibit amyloid-ß fibril formation, potentially making them appropriate drug candidates for Alzheimer's disease. At the same time, our findings hint that the respective aggregation-prone clusterin regions may be implicated in the molecular mechanism in which clusterin inhibits amyloid formation. Furthermore, we suggest that molecular chaperones with amyloidogenic properties might have a role in the regulation of amyloid formation, essentially acting as functional amyloids.

Apolipoprotein J Attenuates Vascular Restenosis by Promoting Autophagy and Inhibiting the Proliferation and Migration of Vascular Smooth Muscle Cells.

This research investigated the mechanism of CLU in vascular restenosis by regulating vascular smooth muscle cell (VSMC) proliferation and migration. Firstly, rat models of balloon injury (BI) were established, followed by the assessment of the injury to the common carotid artery. The effect of CLU on the intimal hyperplasia of BI rats was measured after the intervention in CLU, in addition to the evaluation of proliferation, migration, and autophagy of VSMCs. Moreover, the interaction between ATG and LC3 was analyzed, followed by validation of the role of autophagy in CLU's regulation on the proliferation and migration of VSMCs. It was found that CLU was highly expressed in BI rats. Altogether, our findings indicated that CLU was highly expressed in vascular restenosis, and CLU over-expression promoted the binding between ATG3 and LC3, thus facilitating VSMC autophagy and eventually attenuating intimal hyperplasia and vascular restenosis.

Clusterin inhibits Aß42 aggregation through a "strawberry model" as detected by FRET-FCS.

Extracellular plaque deposits of ß-amyloid peptide (Aß) are one of the main pathological features of Alzheimer's disease (AD). The aggregation of Aß42 species, especially Aß42 oligomers, is still an active research field in AD pathogenesis. Secretory clusterin protein (sCLU), an extracellular chaperone, plays an important role in AD pathogenesis. Although sCLU interacts directly with Aß42 in vitro and in vivo, the mechanism is not clear. In this paper, His-tagged sCLU (sCLU-His) was cloned, expressed and purified, and we applied florescence resonance energy transfer-fluorescence correlation spectroscopy (FRET-FCS) to investigate the direct interaction of sCLU-His and Aß42 at the single-molecule fluorescence level in vitro. Here, we chose four different fluorescently labeled Aß42 oligomers to form two different groups of aggregation models, easy or difficult to aggregate. The results showed that sCLU-His could form complexes with both aggregation models, and sCLU-His inhibited the aggregation of Aß42/RB  ~ Aß42/Atto647 (easy to aggregate model). The complexes were produced as the Aß42/Label adhered to the sCLU-His, which is similar to a "strawberry model," as strawberry seeds are dotted on the outer surface of strawberries. This work provided additional insight into the interaction mechanism of sCLU and Aß42 .

Over-expression of Apolipoprotein J Inhibits Cholesterol Crystal-Induced Inflammatory Responses via Suppressing NLRP3 Inflammasome Activation in THP-1 Macrophages.

Apolipoprotein J (clusterin) is a component of high-density lipoproteins, the high level of which is reversely correlated with the risk of coronary heart disease. In addition, it exerts anti-inflammatory and anti-apoptotic effects on endothelial cells and inhibits smooth muscle cell migration and proliferation, indicating that it may play a protective role in cardiovascular disease. However, the exact mechanisms by which this occurs remain unclear. This study aimed to clarify these underlying protective mechanisms by researching the inhibitory effects of apolipoprotein J via the NOD-like receptor protein 3 pathway on the inflammation induced by cholesterol crystals in THP­1 macrophages. In culture, THP-1 macrophages were infected with adenoviral vectors containing apolipoprotein J genes and subsequently treated with cholesterol crystals. The inflammatory cytokines interleukin­1ß, interleukin 18 and tumour necrosis factor α were quantitatively measured with ELISA kits. NOD-like receptor protein 3, cysteinyl aspartate specific proteinase 1 and interleukin 1ß were evaluated by Western blot and PCR analysis. As a result, apolipoprotein J expression was found to remarkably decrease the levels of inflammatory cytokines, including tumour necrosis factor α, interleukin 18 and interleukin 1ß, secreted by THP­1 macrophages. It was also found capable of inhibiting the levels of NOD-like receptor protein 3, cysteinyl aspartate-specific proteinase 1 and interleukin 1ß both at the protein and mRNA levels. In the current study, we revealed that over-expression of apolipoprotein J attenuated the inflammation induced by cholesterol crystals through inhibition of the NOD-like receptor protein 3 inflammasome pathway.

Clusterin Protects Lipotoxicity-Induced Apoptosis via Upregulation of Autophagy in Insulin-Secreting Cells.

BACKGROUND: There is a great need to discover factors that could protect pancreatic ß-cells from apoptosis and thus prevent diabetes mellitus. Clusterin (CLU), a chaperone protein, plays an important role in cell protection in numerous cells and is involved in various cellular mechanisms, including autophagy. In the present study, we investigated the protective role of CLU through autophagy regulation in pancreatic ß-cells. METHODS: To identify the protective role of CLU, mouse insulinoma 6 (MIN6) cells were incubated with CLU and/or free fatty acid (FFA) palmitate, and cellular apoptosis and autophagy were examined. RESULTS: Treatment with CLU remarkably upregulated microtubule-associated protein 1-light chain 3 (LC3)-II conversion in a doseand time-dependent manner with a significant increase in the autophagy-related 3 (Atg3) gene expression level, which is a mediator of LC3-II conversion. Moreover, co-immunoprecipitation and fluorescence microscopy experiments showed that the molecular interaction of LC3 with Atg3 and p62 was markedly increased by CLU. Stimulation of LC3-II conversion by CLU persisted in lipotoxic conditions, and FFA-induced apoptosis and dysfunction were simultaneously improved by CLU treatment. Finally, inhibition of LC3-II conversion by Atg3 gene knockdown markedly attenuated the cytoprotective effect of CLU. CONCLUSION: Taken together, these findings suggest that CLU protects pancreatic ß-cells against lipotoxicity-induced apoptosis via autophagy stimulation mediated by facilitating LC3-II conversion. Thus, CLU has therapeutic effects on FFA-induced pancreatic ß-cell dysfunction.

Crucial role of the terminal complement complex in chondrocyte death and hypertrophy after cartilage trauma.

OBJECTIVE: Innate immune response and particularly terminal complement complex (TCC) deposition are thought to be involved in the pathogenesis of posttraumatic osteoarthritis. However, the possible role of TCC in regulated cell death as well as chondrocyte hypertrophy and senescence has not been unraveled so far and was first addressed using an ex vivo human cartilage trauma-model. DESIGN: Cartilage explants were subjected to blunt impact (0.59 J) and exposed to human serum (HS) and cartilage homogenate (HG) with or without different potential therapeutics: RIPK1-inhibitor Necrostatin-1 (Nec), caspase-inhibitor zVAD, antioxidant N-acetyl cysteine (NAC) and TCC-inhibitors aurintricarboxylic acid (ATA) and clusterin (CLU). Cell death and hypertrophy/senescence-associated markers were evaluated on mRNA and protein level. RESULTS: Addition of HS resulted in significantly enhanced TCC deposition on chondrocytes and decrease of cell viability after trauma. This effect was potentiated by HG and was associated with expression of RIPK3, MLKL and CASP8. Cytotoxicity of HS could be prevented by heat-inactivation or specific inhibitors, whereby combination of Nec and zVAD as well as ATA exhibited highest cell protection. Moreover, HS+HG exposition enhanced the gene expression of CXCL1, IL-8, RUNX2 and VEGFA as well as secretion of IL-6 after cartilage trauma. CONCLUSIONS: Our findings imply crucial involvement of the complement system and primarily TCC in regulated cell death and phenotypic changes of chondrocytes after cartilage trauma. Inhibition of TCC formation or downstream signaling largely modified serum-induced pathophysiologic effects and might therefore represent a therapeutic target to maintain the survival and chondrogenic character of cartilage cells.

Low-density lipoprotein aggregation is inhibited by apolipoprotein J-derived mimetic peptide D-[113-122]apoJ.

Mimetic peptides are promising therapeutic agents for atherosclerosis prevention. A 10-residue class G* peptide from apolipoprotein J (apoJ), namely, D-[113-122]apoJ, possesses anti-inflammatory and anti-atherogenic properties. This prompted us to determine its effect on the aggregation process of low-density lipoprotein (LDL) particles, an early event in the development of atherosclerosis. LDL particles with and without [113-122]apoJ peptide were incubated at 37 °C with sphingomyelinase (SMase) or were left to aggregate spontaneously at room temperature. The aggregation process was analyzed by size-exclusion chromatography (SEC), native gradient gel electrophoresis (GGE), absorbance at 405 nm, dynamic light scattering (DLS), and transmission electronic microscopy (TEM). In addition, circular dichroism was used to determine changes in the secondary structure of apoB, and SDS-PAGE was performed to assess apoB degradation. At an equimolar ratio of [113-122]apoJ peptide to apoB-100, [113-122]apoJ inhibited both SMase-induced or spontaneous LDL aggregation. All methods showed that [113-122]apoJ retarded the progression of SMase-induced LDL aggregation at long incubation times. No effect of [113-122]apoJ on apoB secondary structure was observed. Binding experiments showed that [113-122]apoJ presents low affinity for native LDL but binds readily to LDL during the first stages of aggregation. Laurdan fluorescence experiments showed that mild aggregation of LDL resulted in looser lipid packaging, which was partially prevented by D-[113-122]apoJ. These results demonstrate that [113-122]apoJ peptide prevents SMase-induced LDL aggregation at an equimolar ratio and opens the possibility for the use of this peptide as a therapeutic tool.

Clusterin Impairs Hepatic Insulin Sensitivity and Adipocyte Clusterin Associates With Cardiometabolic Risk.

OBJECTIVE: Components of the adipose tissue (AT) extracellular matrix (ECM) are recently discovered contributors to obesity-related cardiometabolic disease. We identified increased adipocyte expression of ECM-related clusterin (apolipoprotein J) in obese versus lean women by microarray. Our objective was to determine 1) whether subcutaneous AT adipocyte (SAd) clusterin and serum clusterin are associated with insulin resistance (IR) and known markers of cardiometabolic risk and 2) how clusterin may contribute to increased risk. RESEARCH DESIGN AND METHODS: We validated increased clusterin expression in adipocytes from a separate group of 18 lean and 54 obese individuals. The relationship of clusterin gene expression and plasma clusterin with IR, cardiovascular biomarkers, and risk of cardiovascular disease (CVD) was then determined. Further investigations in human cultured cells and in aged LDLR-/- mice prone to development of obesity-associated complications were performed. RESULTS: SAd clusterin correlated with IR, multiple CVD biomarkers, and CVD risk, independent of traditional risk factors. Circulating human clusterin exhibited similar associations. In human adipocytes, palmitate enhanced clusterin secretion, and in human hepatocytes, clusterin attenuated insulin signaling and APOA1 expression and stimulated hepatic gluconeogenesis. LRP2 (megalin), a clusterin receptor, highly expressed in liver, mediated these effects, which were inhibited by LRP2 siRNA. In response to Western diet feeding, an increase in adipocyte clusterin expression was associated with a progressive increase in liver fat, steatohepatitis, and fibrosis in aged LDLR-/- mice. CONCLUSIONS: Adipocyte-derived clusterin is a novel ECM-related protein linking cardiometabolic disease and obesity through its actions in the liver.

Clusterin Reduces Cold Ischemia-Reperfusion Injury in Heart Transplantation Through Regulation of NF-kB Signaling and Bax/Bcl-xL Expression.

BACKGROUND/AIMS: Ischemia-reperfusion (I/R) injury is an unavoidable event occurring during heart transplantation and is a key factor in graft failure and the long-term survival rate of recipients. Therefore, there is an urgent need for the development of new therapies to prevent I/R injury. Clusterin is a hetero-dimeric glycoprotein with an antiapoptotic function. In this study, we investigated whether clusterin was cardioprotective in heart transplantation against I/R injury using an in vivo rat model and an in vitro cell culture system, and examined the underlying mechanisms of I/R injury. METHODS: Heart grafts from wild-type C57BL/6 mice were preserved in UW solution (control) or UW solution containing recombinant human apolipoprotein-J (hr clusterin) for 24 h. The preserved hearts were implanted into recipient mice of the same strain as the donors for 72 h, and the heart grafts were then taken for histopathological and gene expression analyses. An in vitro ischemia reperfusion model using H9C2 cells or H9C2/clusterin cDNA cells was constructed. The expression of clusterin, p65, Bax, Bcl-xL, IL-1ß, and TNF-α protein and mRNA in heart tissue and H9C2 cells was detected by western blot, reverse transcription-polymerase chain reaction (RT-PCR), and quantitative RT-PCR assays; IL-1ß and TNF-α protein was detected by enzyme-linked immunosorbent assays; NF-kB activity was detected by an electrophoretic mobility shift assay; cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and flow cytometric analyses. RESULTS: Cold I/R caused severe morphologic myocardial injury to heart grafts from wild-type C57BL/6 mice, whereas grafts from hr clusterin preservation showed less damage, as demonstrated by decreased cell apoptosis/death, decreased neutrophil infiltration, and the preservation of the normal structure of the heart. Clusterin reduced the expression of p65, pre-inflammatory IL-1ß, and TNF-α, and the pro-apoptotic gene Bax, while it enhanced the expression of the anti-apoptotic gene Bcl-xL in vitro and in vivo. Clusterin inhibited cell apoptosis/death and reduced pre-inflammatory. CONCLUSION: Clusterin is a promising target for preventing cold I/R injury in heart transplantation. This study also shows that the resultant protective effects of clusterin are mediated by NF-κB signaling and Bax/Bcl-xL expression.

Intraventricular infusion of clusterin ameliorated cognition and pathology in Tg6799 model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by the deposition of amyloid-ß (Aß) in brain parenchyma and cerebral blood vessels as cerebral amyloid angiopathy (CAA). Clusterin, a chaperon protein associated with Aß aggregation, toxicity and transport through blood-brain barrier, may play a key role in the development of AD. Recently, clusterin peptide D-[113-122] was shown to mimic clusterin's function and exerted therapeutic effect in atherosclerosis. In this study, we investigated whether this clusterin peptide also affected (Aß) deposition in AD transgenic mouse. RESULTS: Using a micropump, synthetic peptide 113-122 of clusterin protein (20 µg/200 µl) was infused into the lateral ventricle of 8-month 5 × FAD transgenic mouse model (Tg6799), for 2 weeks. Water-maze testing showed an improved cognitive function of the Tg6799 mice treated with clusterin. Immunocytochemistry and quantitative analysis revealed that intraventricular (icv) administration of clusterin peptide in Tg6799 mouse reduced Aß plaques as well the severity of cerebral amyloid angiopathy. Enzyme-linked immunosorbent assay demonstrated a decreased in the soluble levels of Aß (Aß40 and Aß42) in the brain. Western-blot revealed an increased level of LRP-2 after clusterin peptide treatment. CONCLUSION: These results suggest that icv infusion of clusterin peptide D-[113-122] offers a promising therapeutic approach to reduce Aß deposition as well as CAA. The LRP2-mediated clearance system might be involved in the mechanism of these effects.

Regulatory effects of simvastatin and apoJ on APP processing and amyloid-ß clearance in blood-brain barrier endothelial cells.

Amyloid-ß peptides (Aß) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aß metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aß. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aß metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aß oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aß clearance across the pBCEC monolayer. Treatment of pBCEC with Aß(1-40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3×Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aß oligomers and reduced Aß uptake and cell-associated Aß oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aß processing and clearance at the BBB.

Therapeutic Strategies for Restoring Tau Homeostasis.

Normal tau homeostasis is achieved when the synthesis, processing, and degradation of the protein is balanced. Together, the pathways that regulate tau homeostasis ensure that the protein is at the proper levels and that its posttranslational modifications and subcellular localization are appropriately controlled. These pathways include the enzymes responsible for posttranslational modifications, those systems that regulate mRNA splicing, and the molecular chaperones that control tau turnover and its binding to microtubules. In tauopathies, this delicate balance is disturbed. Tau becomes abnormally modified by posttranslational modification, it loses affinity for microtubules, and it accumulates in proteotoxic aggregates. How and why does this imbalance occur? In this review, we discuss how molecular chaperones and other components of the protein homeostasis (e.g., proteostasis) network normally govern tau quality control. We also discuss how aging might reduce the capacity of these systems and how tau mutations might further affect this balance. Finally, we discuss how small-molecule inhibitors are being used to probe and perturb the tau quality-control systems, playing a particularly prominent role in revealing the logic of tau homeostasis. As such, there is now interest in developing these chemical probes into therapeutics, with the goal of restoring normal tau homeostasis to treat disease.