Reduced secreted clusterin as a mechanism for Alzheimer-associated CLU mutations.
ABSTRACT: The clusterin (CLU) gene has been identified as an important risk locus for Alzheimer's disease (AD). Although the actual risk-increasing polymorphisms at this locus remain to be identified, we previously observed an increased frequency of rare non-synonymous mutations and small insertion-deletions of CLU in AD patients, which specifically clustered in the ?-chain domain of CLU. Nonetheless the pathogenic nature of these variants remained unclear. Here we report a novel non-synonymous CLU mutation (p.I360N) in a Belgian Alzheimer patient and have explored the pathogenic nature of this and 10 additional CLU mutations on protein localization and secretion in vitro using immunocytochemistry, immunodetection and ELISAs.Three patient-specific CLU mutations in the ?-chain (p.I303NfsX13, p.R338W and p.I360N) caused an alteration of the subcellular CLU localization and diminished CLU transport through the secretory pathway, indicative of possible degradation mechanisms. For these mutations, significantly reduced CLU intensity was observed in the Golgi while almost all CLU protein was exclusively present in the endoplasmic reticulum. This was further confirmed by diminished CLU secretion in HEK293T and HEK293 FLp-In cell lines.Our data lend further support to the contribution of rare coding CLU mutations in the pathogenesis of neurodegenerative diseases. Functional analyses suggest reduced secretion of the CLU protein as the mode of action for three of the examined CLU mutations. One of those is a frameshift mutation leading to a loss of secreted protein, and the other two mutations are amino acid substitutions in the disulfide bridge region, possibly interfering with heterodimerization of the ?- and ?-chain of CLU.
Project description:BACKGROUND:We have followed-up on the recent genome-wide association (GWA) of the clusterin gene (CLU) with increased risk for Alzheimer disease (AD), by performing an unbiased resequencing of all CLU coding exons and regulatory regions in an extended Flanders-Belgian cohort of Caucasian AD patients and control individuals (n = 1930). Moreover, we have replicated genetic findings by targeted resequencing in independent Caucasian cohorts of French (n = 2182) and Canadian (n = 573) origin and by performing meta-analysis combining our data with previous genetic CLU screenings. RESULTS:In the Flanders-Belgian cohort, we identified significant clustering in exons 5-8 of rare genetic variations leading to non-synonymous substitutions and a 9-bp insertion/deletion affecting the CLU ?-chain (p = 0.02). Replicating this observation by targeted resequencing of CLU exons 5-8 in 2 independent Caucasian cohorts of French and Canadian origin identified identical as well as novel non-synonymous substitutions and small insertion/deletions. A meta-analysis, combining the datasets of the 3 cohorts with published CLU sequencing data, confirmed that rare coding variations in the CLU ?-chain were significantly enriched in AD patients (OR(MH) = 1.96 [95% CI = 1.18-3.25]; p = 0.009). Single nucleotide polymorphisms (SNPs) association analysis indicated the common AD risk association (GWA SNP rs11136000, p = 0.013) in the 3 combined datasets could not be explained by the presence of the rare coding variations we identified. Further, high-density SNP mapping in the CLU locus mapped the common association signal to a more 5' CLU region. CONCLUSIONS:We identified a new genetic risk association of AD with rare coding CLU variations that is independent of the 5' common association signal identified in the GWA studies. At this stage the role of these coding variations and their likely effect on the ?-chain domain and CLU protein functioning remains unclear and requires further studies.
Project description:Clusterin (CLU) is one of the risk genes most associated with late onset Alzheimer's disease (AD), and several genetic variants in CLU are associated with AD risk. However, the functional role of known AD risk genetic variants in CLU has been little explored. We investigated the effect of an AD risk variant (rs7982) in the 5th exon of CLU on alternative splicing by using an integrative approach of brain-tissue-based RNA-Seq and whole genome sequencing data from Accelerating Medicines Partnership-Alzheimer's Disease (AMP-AD). RNA-Seq data were generated from three regions in the temporal lobe of the brain-the temporal cortex, superior temporal gyrus, and parahippocampal gyrus. The rs7982 was significantly associated with intron retention (IR) of the 5th exon of CLU; as the number of alternative alleles (G) increased, the IR rates decreased more significantly in females than in males. Our results suggest a sex-dependent role of rs7982 in AD pathogenesis via splicing regulation.
Project description: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 ?4- 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.
Project description:Clusterin (CLU) or APOJ is a multifunctional glycoprotein that has been implicated in several physiological and pathological states, including Alzheimer's disease (AD). With a prominent extracellular chaperone function, additional roles have been discussed for clusterin, including lipid transport and immune modulation, and it is involved in pathways common to several diseases such as cell death and survival, oxidative stress, and proteotoxic stress. Although clusterin is normally a secreted protein, it has also been found intracellularly under certain stress conditions. Multiple hypotheses have been proposed regarding the origin of intracellular clusterin, including specific biogenic processes leading to alternative transcripts and protein isoforms, but these lines of research are incomplete and contradictory. Current consensus is that intracellular clusterin is most likely to have exited the secretory pathway at some point or to have re-entered the cell after secretion. Clusterin's relationship with amyloid beta (A?) has been of great interest to the AD field, including clusterin's apparent role in altering A? aggregation and/or clearance. Additionally, clusterin has been more recently identified as a mediator of A? toxicity, as evidenced by the neuroprotective effect of CLU knockdown and knockout in rodent and human iPSC-derived neurons. CLU is also the third most significant genetic risk factor for late onset AD and several variants have been identified in CLU. Although the exact contribution of these variants to altered AD risk is unclear, some have been linked to altered CLU expression at both mRNA and protein levels, altered cognitive and memory function, and altered brain structure. The apparent complexity of clusterin's biogenesis, the lack of clarity over the origin of the intracellular clusterin species, and the number of pathophysiological functions attributed to clusterin have all contributed to the challenge of understanding the role of clusterin in AD pathophysiology. Here, we highlight clusterin's relevance to AD by discussing the evidence linking clusterin to AD, as well as drawing parallels on how the role of clusterin in other diseases and pathways may help us understand its biological function(s) in association with AD.
Project description:BACKGROUND:We examined the effect of the novel Alzheimer's disease (AD) risk variant rs11136000 single nucleotide polymorphism in the clusterin gene (CLU) on longitudinal changes in resting state regional cerebral blood flow (rCBF) during normal aging and investigated its influence on cognitive decline in presymptomatic stages of disease progression. METHODS:Subjects were participants in the Baltimore Longitudinal Study of Aging. A subset of 88 cognitively normal older individuals had longitudinal (15)O-water positron emission tomography measurements of rCBF at baseline and up to eight annual follow-up visits. We also analyzed trajectories of cognitive decline among CLU risk carriers and noncarriers in individuals who remained cognitively normal (n = 599), as well as in those who subsequently converted to mild cognitive impairment or AD (n = 95). RESULTS:Cognitively normal carriers of the CLU risk allele showed significant and dose-dependent longitudinal increases in resting state rCBF in brain regions intrinsic to memory processes. There were no differences in trajectories of memory performance between CLU risk carriers and noncarriers who remained cognitively normal. However, in cognitively normal individuals who eventually converted to mild cognitive impairment or AD, CLU risk carriers showed faster rates of decline in memory performance relative to noncarriers in the presymptomatic stages of disease progression. CONCLUSIONS:The AD risk variant CLU influences longitudinal changes in brain function in asymptomatic individuals and is associated with faster cognitive decline in presymptomatic stages of disease progression. These results suggest mechanisms underlying the role of CLU in AD and may be important in monitoring disease progression in at-risk elderly.
Project description:Our understanding of the molecular processes underlying Alzheimer's disease (AD) is still limited, hindering the development of effective treatments, and highlighting the need for human-specific models. Advances in identifying components of the amyloid cascade are progressing, including the role of the protein clusterin in mediating ?-amyloid (A?) toxicity. Mutations in the clusterin gene (CLU), a major genetic AD risk factor, are known to have important roles in A? processing. Here we investigate how CLU mediates A?-driven neurodegeneration in human induced pluripotent stem cell (iPSC)-derived neurons. We generated a novel CLU-knockout iPSC line by CRISPR/Cas9-mediated gene editing to investigate A?-mediated neurodegeneration in cortical neurons differentiated from wild type and CLU knockout iPSCs. We measured response to A? using an imaging assay and measured changes in gene expression using qPCR and RNA sequencing. In wild type neurons imaging indicated that neuronal processes degenerate following treatment with A?25-35 peptides and A?1-42 oligomers, in a dose dependent manner, and that intracellular levels of clusterin are increased following A? treatment. However, in CLU knockout neurons A? exposure did not affect neurite length, suggesting that clusterin is an important component of the amyloid cascade. Transcriptomic data were analyzed to elucidate the pathways responsible for the altered response to A? in neurons with the CLU deletion. Four of the five genes previously identified as downstream to A? and Dickkopf-1 (DKK1) proteins in an A?-driven neurotoxic pathway in rodent cells were also dysregulated in human neurons with the CLU deletion. AD and lysosome pathways were the most significantly dysregulated pathways in the CLU knockout neurons, and pathways relating to cytoskeletal processes were most dysregulated in A? treated neurons. The absence of neurodegeneration in the CLU knockout neurons in response to A? compared to the wild type neurons supports the role of clusterin in A?-mediated AD pathogenesis.
Project description:The molecular chaperone Clusterin (CLU) impacts the amyloid pathway in Alzheimer's disease (AD) but its role in tau pathology is unknown. We observed CLU co-localization with tau aggregates in AD and primary tauopathies and CLU levels were upregulated in response to tau accumulation. To further elucidate the effect of CLU on tau pathology, we utilized a gene delivery approach in CLU knock-out (CLU KO) mice to drive expression of tau bearing the P301L mutation. We found that loss of CLU was associated with exacerbated tau pathology and anxiety-like behaviors in our mouse model of tauopathy. Additionally, we found that CLU dramatically inhibited tau fibrilization using an in vitro assay. Together, these results demonstrate that CLU plays a major role in both amyloid and tau pathologies in AD.
Project description:OBJECTIVE:Type 2 diabetes mellitus (T2DM) is related to an elevated risk of mild cognitive impairment (MCI). Plasma clusterin is reported associated with the early pathology of Alzheimer's disease (AD) and longitudinal brain atrophy in subjects with MCI. The rs11136000 single nucleotide polymorphism within the clusterin (CLU) gene is also associated with the risk of AD. We aimed to investigate the associations among plasma clusterin, rs11136000 genotype and T2DM-associated MCI. METHODS:A total of 231 T2DM patients, including 126 MCI and 105 cognitively healthy controls were enrolled in this study. Demographic parameters were collected and neuropsychological tests were conducted. Plasma clusterin and CLU rs11136000 genotype were examined. RESULTS:Plasma clusterin was significantly higher in MCI patients than in control group (p = 0.007). In subjects with MCI, plasma clusterin level was negatively correlated with Montreal cognitive assessment and auditory verbal learning test_delayed recall scores (p = 0.027 and p = 0.020, respectively). After adjustment for age, educational attainment, and gender, carriers of rs11136000 TT genotype demonstrated reduced risk for MCI compared with the CC genotype carriers (OR = 0.158, ?(2) = 4.113, p = 0.043). Multivariable regression model showed that educational attainment, duration of diabetes, high-density lipoprotein cholesterol (HDL-c), and plasma clusterin levels are associated with MCI in T2DM patients. CONCLUSIONS:Plasma clusterin was associated with MCI and may reflect a protective response in T2DM patients. TT genotype exhibited a reduced risk of MCI compared to CC genotype. Further investigations should be conducted to determine the role of clusterin in cognitive decline. Trial registration Advanced Glycation End Products Induced Cognitive Impairment in Diabetes: BDNF Signal Meditated Hippocampal Neurogenesis ChiCTR-OCC-15006060; http://www.chictr.org.cn/showproj.aspx?proj=10536.
Project description:Alzheimer's disease (AD) is characterized by amyloid-? (A?) peptide deposition in brain parenchyma as plaques and in cerebral blood vessels as cerebral amyloid angiopathy (CAA). CAA deposition leads to several clinical complications, including intracerebral hemorrhage. The underlying molecular mechanisms that regulate plaque and CAA deposition in the vast majority of sporadic AD patients remain unclear. The clusterin (CLU) gene is genetically associated with AD and CLU has been shown to alter aggregation, toxicity, and blood-brain barrier transport of A?, suggesting it might play a key role in regulating the balance between A? deposition and clearance in both brain and blood vessels. Here, we investigated the effect of CLU on A? pathology using the amyloid precursor protein/presenilin 1 (APP/PS1) mouse model of AD amyloidosis on a Clu+/+ or Clu-/- background. We found a marked decrease in plaque deposition in the brain parenchyma but an equally striking increase in CAA within the cerebrovasculature of APP/PS1;Clu-/- mice. Surprisingly, despite the several-fold increase in CAA levels, APP/PS1;Clu-/- mice had significantly less hemorrhage and inflammation. Mice lacking CLU had impaired clearance of A? in vivo and exogenously added CLU significantly prevented A? binding to isolated vessels ex vivo. These findings suggest that in the absence of CLU, A? clearance shifts to perivascular drainage pathways, resulting in fewer parenchymal plaques but more CAA because of loss of CLU chaperone activity, complicating the potential therapeutic targeting of CLU for AD.
Project description:<h4>Background</h4>Genome-wide association studies have established clusterin (CLU) as a genetic modifier for late-onset Alzheimer's disease (AD). Both protective and risk alleles have been identified which may be associated with its expression levels. However, the physiological function of clusterin in the central nervous system remains largely unknown.<h4>Methods</h4>We examined Clu expression in mouse brains by immunohistochemistry and high-resolution imaging. We performed electrophysiological recordings and morphological analysis of dendritic spines in wild-type and Clu knockout mice. We tested synaptic function of astrocytic Clu using neuron-glia co-cultures and by AAV-mediated astroglial Clu expression in vivo. Finally, we investigated the role of astrocytic Clu on synaptic properties and amyloid pathology in 5xFAD transgenic mouse model of AD.<h4>Results</h4>We show that astrocyte secreted Clu co-localizes with presynaptic puncta of excitatory neurons. Loss of Clu led to impaired presynaptic function and reduced spine density in vivo. Neurons co-cultured with Clu-overexpressing astrocytes or treated with conditioned media from HEK293 cells transfected with Clu displayed enhanced excitatory neurotransmission. AAV-mediated astroglial Clu expression promoted excitatory neurotransmission in wild-type mice and rescued synaptic deficits in Clu knockout mice. Overexpression of Clu in the astrocytes of 5xFAD mice led to reduced A? pathology and fully rescued the synaptic deficits.<h4>Conclusion</h4>We identify Clu as an astrocyte-derived synaptogenic and anti-amyloid factor; the combination of these activities may influence the progression of late-onset AD.