Deletion of Clusterin Protects Cochlear Hair Cells against Hair Cell Aging and Ototoxicity.

Hearing loss is a debilitating disease that affects 10% of adults worldwide. Most sensorineural hearing loss is caused by the loss of mechanosensitive hair cells in the cochlea, often due to aging, noise, and ototoxic drugs. The identification of genes that can be targeted to slow aging and reduce the vulnerability of hair cells to insults is critical for the prevention of sensorineural hearing loss. Our previous cell-specific transcriptome analysis of adult cochlear hair cells and supporting cells showed that Clu, encoding a secreted chaperone that is involved in several basic biological events, such as cell death, tumor progression, and neurodegenerative disorders, is expressed in hair cells and supporting cells. We generated Clu-null mice (C57BL/6) to investigate its role in the organ of Corti, the sensory epithelium responsible for hearing in the mammalian cochlea. We showed that the deletion of Clu did not affect the development of hair cells and supporting cells; hair cells and supporting cells appeared normal at 1 month of age. Auditory function tests showed that Clu-null mice had hearing thresholds comparable to those of wild-type littermates before 3 months of age. Interestingly, Clu-null mice displayed less hair cell and hearing loss compared to their wildtype littermates after 3 months. Furthermore, the deletion of Clu is protected against aminoglycoside-induced hair cell loss in both in vivo and in vitro models. Our findings suggested that the inhibition of Clu expression could represent a potential therapeutic strategy for the alleviation of age-related and ototoxic drug-induced hearing loss.

Sterol O-acyltransferase 2 chaperoned by apolipoprotein J facilitates hepatic lipid accumulation following viral and nutrient stresses.

The risks of non-alcoholic fatty liver disease (NAFLD) include obese and non-obese stresses such as chronic hepatitis C virus (HCV) infection, but the regulatory determinants remain obscure. Apolipoprotein J (ApoJ) served as an ER-Golgi contact-site chaperone near lipid droplet (LD), facilitating HCV virion production. We hypothesized an interplay between hepatic ApoJ, cholesterol esterification and lipid deposit in response to NAFLD inducers. Exposures of HCV or free-fatty acids exhibited excess LDs along with increased ApoJ expression, whereas ApoJ silencing alleviated hepatic lipid accumulation. Both stresses could concomitantly disperse Golgi, induce closer ApoJ and sterol O-acyltransferase 2 (SOAT2) contacts via the N-terminal intrinsically disordered regions, and increase cholesteryl-ester. Furthermore, serum ApoJ correlated positively with cholesterol and low-density lipoprotein levels in normal glycaemic HCV patients, NAFLD patients and in mice with steatosis. Taken together, hepatic ApoJ might activate SOAT2 to supply cholesteryl-ester for lipid loads, thus providing a therapeutic target of stress-induced steatosis.

Serum complement proteomics reveal biomarkers for hypertension disorder of pregnancy and the potential role of Clusterin.

INTRODUCTION: Hypertension disorder of pregnancy (HDP) is one of the leading causes of maternal and foetal illness. The aim of the current study was to identify and verify novel serum markers for HDP. METHODS: A label-free LC-MS/MS method was used to establish the serum proteomic profiles of 12 pre-HDP (before clinical diagnosis of HDP) pregnancies and verify prioritized candidates in the verification set of 48 pre-HDP pregnancies. These biomarkers were revalidated by ELISA in an independent cohort of 88 pre-HDP pregnancies. Subsequently, the candidate biomarkers were histologically analysed by immunohistochemistry, and function was evaluated in TEV-1 cells. RESULTS: We identified 33 proteins with significantly increased abundance and 14 with decreased abundance (peptide FDR ≤ 1%, P < 0.05). Complement was one of the top enriched components in the pre-HDP group compared with the control group. Three complement factors (CLU, CFHR5, and CRP) were significantly increased in the three sets, of which CLU was a critical factor for the development of HDP (OR = 1.22, P < 0.001). When these three factors and body weight were combined, the AUC was 0.74, with a sensitivity of 0.67 and specificity of 0.68 for HDP prediction compared with normal pregnancy. In addition, inflammation-induced CLU could inhibit the invasion of TEV-1 cells. CONCLUSIONS: Complement proteins may play an essential role in the occurrence of HDP by acting on trophoblast cells. CLU may be a high-risk factor for HDP, and the models combining candidates show reasonable screening efficiency of HDP in the first half of pregnancy.

Exploring the Role of CLU in the Pathogenesis of Alzheimer's Disease.

Alzheimer's disease (AD) is a chronic and devastating neurodegenerative disorder that is affecting elderly people at an increasing rate. Clusterin (CLU), an extracellular chaperone, is an ubiquitously expressed protein that can be identified in various body fluids and tissues. Expression of CLU can lead to various processes including suppression of complement system, lipid transport, chaperone function, and also controlling neuronal cell death and cell survival mechanisms. Studies have confirmed that the level of CLU expression is increased in AD. Furthermore, CLU also decreased the toxicity and aggregation of amyloid beta (Aß). However when the Aß level was far greater than CLU, then the amyloid generation was increased. CLU was also found to incorporate in the amyloid aggregates, which were more harmful as compared with the Aß42 aggregates alone. Growing evidence indicates that CLU plays roles in AD pathogenesis via various processes, including aggregation and clearance of Aß, neuroinflammation, lipid metabolism, Wnt signaling, copper homeostasis, and regulation of neuronal cell cycle and apoptosis. In this article, we represent the critical interaction of CLU and AD based on recent advances. Furthermore, we have also focused on the Aß-dependent and Aß-independent mechanisms by which CLU plays a role in AD pathogenesis.

Clusterin suppresses spermatogenic cell apoptosis to alleviate diabetes-induced testicular damage by inhibiting autophagy via the PI3K/AKT/mTOR axis.

BACKGROUND INFORMATION: Diabetes-induced testicular dysfunction is characterised by abnormal apoptosis of spermatogenic cells, but the underlying mechanism is poorly understood. This study aimed to investigate the roles of clusterin (CLU) in testicular damage associated with diabetes pathogenesis, as well as the molecular mechanism. A rat diabetes model was established using streptozocin, and the mouse spermatogenic cell line GC-1 spg was treated with high glucose as a cellular model. CLU was overexpressed in GC-1 spg cells, followed by detection of serum testosterone, cell proliferation, cell apoptosis and autophagy. RESULTS: CLU expression was significantly reduced and LC3 expression was elevated in testis tissues in the rat diabetes model and high glucose-treated GC-1 spg cells. High glucose led to suppressed viability, enhanced apoptosis, reduced Bcl-2 expression, elevated Bax expression and cleavage of Caspase-3/-9 in GC-1 spg cells, and these effects were abrogated by CLU overexpression. Additionally, CLU overexpression repressed LC3 and Beclin-1 expression, reduced the LC3II/LC3I ratio and promoted p62 expression in GC-1 spg cells in the presence of high glucose, and these effects were all mitigated by rapamycin treatment. Inhibition of PI3K/AKT/mTOR signalling with LY294002 activated autophagy in CLU-overexpressing GC-1 spg cells under high glucose conditions. CLU overexpression repressed autophagy and alleviated testicular damage in diabetic rats, which was also abrogated by LY294002 treatment. CONCLUSIONS: CLU expression is suppressed during diabetes-induced testicular damage, whereas CLU overexpression alleviates diabetes-induced testicular damage by activating PI3K/AKT/mTOR signalling to inhibit autophagy and further repress spermatogenic cell apoptosis.

Glomerular clusterin expression is increased in diabetic nephropathy and protects against oxidative stress-induced apoptosis in podocytes.

Clusterin, a glycoprotein encoded by the CLU gene, is expressed in many tissues, including the kidney, and clusterin expression is upregulated in the glomeruli of patients with various forms of kidney disease. Here, we investigated the role of clusterin in diabetic nephropathy (DN). In this study, we found that glomerular clusterin expression was increased in both patients with DN and streptozotocin-induced diabetic mice and that it co-localised with the podocyte marker WT1, indicating clusterin is expressed in podocytes. In our in vitro analysis, we found no significant change in CLU mRNA expression in podocytes following stimulation with high glucose and angiotensin II; in contrast, CLU mRNA expression was significantly upregulated following methylglyoxal stimulation. Methylglyoxal treatment also significantly decreased the mRNA expression of the slit diaphragm markers ZO-1 and NEPH1 and significantly increased the mRNA expression of the oxidative stress marker HO-1. Lastly, we showed that pre-incubating podocytes with recombinant human clusterin protein increased podocyte survival, prevented slit diaphragm damage, and reduced oxidative stress‒induced apoptosis following methylglyoxal stimulation. Taken together, our results indicate that glomerular clusterin is upregulated in DN, and this increase in clusterin expression may protect against oxidative stress-induced apoptosis in podocytes, providing a possible new therapeutic target for DN and other kidney diseases.

Quantitative proteomics decodes clusterin as a critical regulator of paternal factors responsible for impaired compensatory metabolic reprogramming in recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is a perplexing problem experienced with two or more consecutive miscarriages wherein the cause remains unexplained in >50% of cases. However, despite several evidences of involvement of paternal factors on early embryogenesis and placental development, its contribution towards RPL has been largely unexplored. There is augmented lipid peroxidation, protein carbonylation, thionylation and enhanced histone retention in spermatozoa of RPL patients. Differentially expressed proteins in the spermatozoa of RPL patients may contribute towards aberrant embryo development and pregnancy loss. The present study comprised of male partners of RPL patients (n = 16) with the absence of any female factor abnormality and age-matched fertile healthy donors (n = 20). Pooled sperm samples from each group were subjected to high-throughput liquid chromatography-tandem mass spectrophotometry (LC-MS/MS) and subsequent bioinformatic analysis that identifies key proteins to be differentially expressed (DEPs). A total of 23 DEPs were identified with ≥2.0 fold change were considered to be significant. A key finding of the study was clusterin (CLUS), a predominant oxidative stress protein that takes part in an array of pre- and post-fertilisation molecular processes, found to be underexpressed as it was confirmed by Western blot analysis. This pilot study supports contributions of paternal oxidative predominance in RPL and encourages further investigation.

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 contributes to early stage of Alzheimer's disease pathogenesis.

While clusterin is reportedly involved in Alzheimer's disease (AD) pathogenesis, how clusterin interacts with amyloid-ß (Aß) to cause Aß neurotoxicity remains unclear in vivo. Using 5×FAD transgenic mice, which develop robust AD pathology and memory deficits when very young, we detected interactions between clusterin and Aß in the mouse brains. The two proteins were concurrently upregulated and bound or colocalized with each other in the same complexes or in amyloid plaques. Neuropathology and cognitive performance were assessed in the progeny of clusterin-null mice crossed with 5×FAD mice, yielding clu-/- ;5×FAD and clu+/+ ;5×FAD. We found far less of the various pools of Aß proteins, most strikingly soluble Aß oligomers and amyloid plaques in clu-/- ;5×FAD mice at 5 months of age. At that age, those mice also had higher levels of neuronal and synaptic proteins and better motor coordination, spatial learning and memory than age-matched clu+/+ ;5×FAD mice. However, at 10 months of age, these differences disappeared, with Aß and plaque deposition, neuronal and synaptic proteins and impairment of behavioral and cognitive performance similar in both groups. These findings demonstrate that clusterin is necessarily involved in early stages of AD pathogenesis by enhancing toxic Aß pools to cause Aß-directed neurodegeneration and behavioral and cognitive impairments, but not in late stage.

A Role for Clusterin in Exfoliation Syndrome and Exfoliation Glaucoma?

The multifunctional protein clusterin (CLU) is a secreted glycoprotein ubiquitously expressed throughout the body, including in the eye. Its primary function is to act as an extracellular molecular chaperone, preventing the precipitation and aggregation of misfolded extracellular proteins. Clusterin is commonly identified at fluid-tissue interfaces, and has been identified in most body fluids. It is a component of exfoliation material, and CLU mRNA is reduced in eyes with exfoliation syndrome compared with controls. SNPs located in the CLU genomic region have been associated with Alzheimer disease (AD) at the genome-wide level and several CLU SNPs located in an apparent regulatory region have been nominally associated with XFS/XFG in Caucasians with European ancestry and in south Indians. Interestingly, clusterin associates with altered elastic fibers in human photoaged skin and prevents UV-induced elastin aggregation in vitro. In light of the known geographic risk factors for XFS/XFG, which could include UV light, investigations of CLU-geographic interactions could be of interest. Future studies investigating rare CLU variation and other complex interactions including gene-gene interactions in XFS/XFG cases and controls may also be fruitful. Although CLU has been considered as a therapeutic target in AD, cancer and dry eye, a role for clusterin in XFS/XFG needs to be better defined before therapeutic approaches involving CLU can be entertained.

Germ cell-specific apoptosis by extracellular clusterin in cryptorchid dog testes.

Mammalian testes are maintained at a relatively lesser temperature than the abdominal region so that normal spermatogenesis can occur. Germ cell apoptosis has resulted in heat-damaged testes that occurs as a result of cryptorchidism, but the mechanism is not yet fully understood. To elucidate the cause of germ-cell death by cryptorchidism, cryptorchidism was surgically induced in dog testes and histological and molecular analyses were performed. Histological data indicated that the seminiferous tubules of cryptorchid testes and epididymis contained fewer germ cells. Total RNA sequencing was performed to screen for overexpressed genes in cryptorchid dog testes. Clusterin RNA was in greater abundance (approximately 12.8-fold) in cryptorchid testes than in normal testes. In addition, cleaved caspase-3 and -8 were detected in greater abundance in cryptorchid dog testes. Real time RT-PCR and western blotting analysis indicated there was a greater abundance of clusterin in cryptorchid dog testes. Furthermore, clusterin was detected in extracellular regions of cryptorchid dog testes during the 4 weeks after surgery. Thus, germ-cell specific apoptosis and expression of clusterin genes occur with a resulting presence of this protein in extracellular regions of cryptorchid dog testes. This result will facilitate further study of spermatogenesis and the specific mechanisms by which cryptorchidism results in male infertility.

The cytoprotective protein clusterin is overexpressed in hypergastrinemic rodent models of oxyntic preneoplasia and promotes gastric cancer cell survival.

The cytoprotective protein clusterin is often dysregulated during tumorigenesis, and in the stomach, upregulation of clusterin marks emergence of the oxyntic atrophy (loss of acid-producing parietal cells)-associated spasmolytic polypeptide-expressing metaplasia (SPEM). The hormone gastrin is important for normal function and maturation of the gastric oxyntic mucosa and hypergastrinemia might be involved in gastric carcinogenesis. Gastrin induces expression of clusterin in adenocarcinoma cells. In the present study, we examined the expression patterns and gastrin-mediated regulation of clusterin in gastric tissue from: humans; rats treated with proton pump (H+/K+-ATPase) inhibitors and/or a gastrin receptor (CCK2R) antagonist; H+/K+-ATPase ß-subunit knockout (H/K-ß KO) mice; and Mongolian gerbils infected with Helicobacter pylori and given a CCK2R antagonist. Biological function of secretory clusterin was studied in human gastric cancer cells. Clusterin was highly expressed in neuroendocrine cells in normal oxyntic mucosa of humans and rodents. In response to hypergastrinemia, expression of clusterin increased significantly and its localization shifted to basal groups of proliferative cells in the mucous neck cell-chief cell lineage in all animal models. That shift was partially inhibited by antagonizing the CCK2R in rats and gerbils. The oxyntic mucosa of H/K-ß KO mice contained areas with clusterin-positive mucous cells resembling SPEM. In gastric adenocarcinomas, clusterin mRNA expression was higher in diffuse tumors containing signet ring cells compared with diffuse tumors without signet ring cells, and clusterin seemed to be secreted by tumor cells. In gastric cancer cell lines, gastrin increased secretion of clusterin, and both gastrin and secretory clusterin promoted survival after starvation- and chemotherapy-induced stress. Overall, our results indicate that clusterin is overexpressed in hypergastrinemic rodent models of oxyntic preneoplasia and stimulates gastric cancer cell survival.

Clusterin in the eye: An old dog with new tricks at the ocular surface.

The multifunctional protein clusterin (CLU) was first described in 1983 as a secreted glycoprotein present in ram rete testis fluid that enhanced aggregation ('clustering') of a variety of cells in vitro. It was also independently discovered in a number of other systems. By the early 1990s, CLU was known under many names and its expression had been demonstrated throughout the body, including in the eye. Its homeostatic activities in proteostasis, cytoprotection, and anti-inflammation have been well documented, however its roles in health and disease are still not well understood. CLU is prominent at fluid-tissue interfaces, and in 1996 it was demonstrated to be the most highly expressed transcript in the human cornea, the protein product being localized to the apical layers of the mucosal epithelia of the cornea and conjunctiva. CLU protein is also present in human tears. Using a preclinical mouse model for desiccating stress that mimics human dry eye disease, the authors recently demonstrated that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration in the tears. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to LGALS3 (galectin-3), a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. CLU depletion from the ocular surface epithelia is seen in a variety of inflammatory conditions in humans and mice that lead to squamous metaplasia and a keratinized epithelium. This suggests that CLU might have a specific role in maintaining mucosal epithelial differentiation, an idea that can now be tested using the mouse model for desiccating stress. Most excitingly, the new findings suggest that CLU could serve as a novel biotherapeutic for dry eye disease.

A potential endophenotype for Alzheimer's disease: cerebrospinal fluid clusterin.

Genome-wide association studies have associated clusterin (CLU) variants with Alzheimer's disease (AD). However, the role of CLU on AD pathogenesis is not totally understood. We used cerebrospinal fluid (CSF) and plasma CLU levels as endophenotypes for genetic studies to understand the role of CLU in AD. CSF, but not plasma, CLU levels were significantly associated with AD status and CSF tau/amyloid-beta ratio, and highly correlated with CSF apolipoprotein E (APOE) levels. Several loci showed almost genome-wide significant associations including LINC00917 (p = 3.98 × 10(-7)) and interleukin 6 (IL6, p = 9.94 × 10(-6), in the entire data set and in the APOE ε4- individuals p = 7.40 × 10(-8)). Gene ontology analyses suggest that CSF CLU levels may be associated with wound healing and immune response which supports previous functional studies that demonstrated an association between CLU and IL6. CLU may play a role in AD by influencing immune system changes that have been observed in AD or by disrupting healing after neurodegeneration.

Apolipoprotein-J prevents angiotensin II-induced apoptosis in neonatal rat ventricular cells.

BACKGROUND: Up-regulation of angiotensin II (AngII) occurs in cardiac diseases, such as congestive heart failure, cardiac hypertrophy, myocardial ischemia and atrial fibrillation, which represent major health problems. Evidence from in vivo studies suggests that the level of Apolipoprotein-J (ApoJ) is also elevated but plays a protective role in cardiovascular disease. This study aimed to evaluate the protective effects of ApoJ against cytotoxicity of AngII in neonatal rat ventricular cells (NRVCs). METHODS AND RESULTS: In culture, NRVCs were damaged by exposure to AngII, and ApoJ overexpression using an adenovirus vector significantly reduced the AngII-induced cell injury. ApoJ also prevented AngII from augmenting Nox2/gp91(phox) expression. The reactive oxygen species (ROS) scavenger, Mn(III)TBAP, showed similar results of attenuating AngII-induced cell damage. Furthermore, ApoJ overexpression increased phosphorylation of Akt, and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 diminished the antioxidant effects of ApoJ, and prevented the protective effect of ApoJ against the cytotoxicity of AngII. Moreover, upregulation of nuclear factor κB (NF-κB) p65 expression and phosphorylation of p38 mitogen-activated protein kinase (MAPK) mediated by AngII in cultured NRVCs were significantly inhibited by overexpression of ApoJ. The p38 MAPK inhibitor SB203580 and the NF-κB inhibitor PDTC protected NRVCs from injury caused by AngII. CONCLUSIONS: ApoJ serves as a cytoprotective protein in NRVCs against cytotoxicity of AngII through the PI3K-Akt-ROS and MAPK/ NF-κB pathways.

Alzheimer's disease is associated with altered expression of genes involved in immune response and mitochondrial processes in astrocytes.

Alzheimer's disease (AD) is characterized by deficits in cerebral metabolic rates of glucose in the posterior cingulate (PC) and precuneus in AD subjects, and in APOEε4 carriers, decades before the onset of measureable cognitive deficits. However, the cellular and molecular basis of this phenotype remains to be clarified. Given the roles of astrocytes in energy storage and brain immunity, we sought to characterize the transcriptome of AD PC astrocytes. Cells were laser capture microdissected from AD (n = 10) and healthy elderly control (n = 10) subjects for RNA sequencing. We generated >5.22 billion reads and compared sequencing data between controls and AD patients. We identified differentially expressed mitochondria-related genes including TRMT61B, FASTKD2, and NDUFA4L2, and using pathway and weighted gene coexpression analyses, we identified differentially expressed immune response genes. A number of these genes, including CLU, C3, and CD74, have been implicated in beta amyloid generation or clearance. These data provide key insights into astrocyte-specific contributions to AD, and we present this data set as a publicly available resource.

Impacts of PICALM and CLU variants associated with Alzheimer's disease on the functional connectivity of the hippocampus in healthy young adults.

PICALM rs3851179 and CLU rs11136000 have been recently associated with Alzheimer's disease (AD). Investigating the effects of these genetic variants on the resting-state functional connectivity (rsFC) of the hippocampus may provide new insight into AD pathogenesis. We investigated the main effects and interactions of these two genetic variants on hippocampal rsFC in 283 healthy young adults. The hippocampus showed positive rsFC with the default mode network and negative rsFC with the fronto-parietal network. Risk PICALM G-allele carriers showed weaker negative rsFC compared with AA carriers, whereas risk CLU-CC carriers exhibited stronger positive and negative rsFC than T-allele carriers. There existed complex interactions between PICALM and CLU on the negative rsFC of the hippocampus. Moreover, we found an allele-dependent effect of CLU on hippocampal connectivity when an additive genetic model was applied to CLU. Most of these effects remained significant even after controlling for individual ApoE status. Our results suggest that PICALM and CLU risk genotypes exert differential impacts on the hippocampal rsFC in healthy young subjects. The complex interactions between PICALM and CLU should be considered when investigating the impact of these two genetic variants on the brain.

Induction of clusterin expression by neuronal cell death in Zebrafish.

Clusterin, a protein associated with multiple functions, is expressed in a wide variety of mammalian tissues. Although clusterin is known to be involved in neurodegenerative diseases, ageing, and tumorigenesis, a detailed analysis of the consequences of gain- or loss-of-function approaches has yet to be performed to understand the underlying mechanisms of clusterin functions. Since clusterin levels change in neurological diseases, it is likely that clusterin contributes to cell death and degeneration in general. Zebrafish was investigated as a model system to study human diseases. During development, zebrafish clusterin was expressed in the notochord and nervous system. Embryonic overexpression of clusterin by mRNA microinjection did not affect axis formation, whereas its knock-down by anti-sense morpholino treatment resulted in neuronal cell death. To analyze the function of clusterin in neurodegeneration, a transgenic zebrafish was investigated, in which nitroreductase expression is regulated under the control of a neuron-specific huC promoter which is active between the stages of early neuronal precursors and mature neurons. Nitroreductase turns metronidazole into a cytotoxic agent that induces cell death within 12 h. After metronidazole treatment, transgenic zebrafish showed neuron-specific cell death. Interestingly, we also observed a dramatic induction of clusterin expression in the brain and spinal cord in these fish, suggesting a direct or indirect role of clusterin in neuronal cell death and thus, more generally, in neurodegeneration.

The role of clusterin in amyloid-ß-associated neurodegeneration.

IMPORTANCE: Converging evidence indicates that clusterin, a chaperone glycoprotein, influences Alzheimer disease neurodegeneration. However, the precise role of clusterin in Alzheimer disease pathogenesis is still not well understood. OBJECTIVE: To elucidate the relationship between clusterin, amyloid-ß (Aß), phosphorylated tau (p-tau), and the rate of brain atrophy over time among nondemented older individuals. DESIGN, SETTING, AND PARTICIPANTS: This longitudinal cohort included cognitively normal older participants and individuals with mild cognitive impairment assessed with baseline lumbar puncture and longitudinal structural magnetic resonance imaging. We examined 241 nondemented older individuals from research centers across the United States and Canada (91 participants with a Clinical Dementia Rating score of 0 and 150 individuals with a Clinical Dementia Rating score of 0.5). MAIN OUTCOMES AND MEASURES: Using linear mixed-effects models, we investigated interactions between cerebrospinal fluid (CSF) clusterin, CSF Aß1-42, and CSF p-tau at threonine 181 (p-tau181p) on the atrophy rate of the entorhinal cortex and hippocampus. RESULTS: Across all participants, we found a significant interaction between CSF clusterin and CSF Aß1-42 on the entorhinal cortex atrophy rate but not on the hippocampal atrophy rate. Cerebrospinal fluid clusterin was associated with the entorhinal cortex atrophy rate among CSF Aß1-42-positive individuals but not among CSF Aß1-42-negative individuals. In secondary analyses, we found significant interactions between CSF Aß1-42 and CSF clusterin, as well as CSF Aß1-42 and CSF p-tau181p, on the entorhinal cortex atrophy rate. We found similar results in subgroup analyses within the mild cognitive impairment and cognitively normal cohorts. CONCLUSIONS AND RELEVANCE: In nondemented older individuals, Aß-associated volume loss occurs in the presence of elevated clusterin. The effect of clusterin on Aß-associated brain atrophy is not confounded or explained by p-tau. These findings implicate a potentially important role for clusterin in the earliest stages of the Alzheimer disease neurodegenerative process and suggest independent effects of clusterin and p-tau on Aß-associated volume loss.