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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 (Aβ) has been of great interest to the AD field, including clusterin's apparent role in altering Aβ aggregation and/or clearance. Additionally, clusterin has been more recently identified as a mediator of Aβ toxicity, as evidenced by the neuroprotective effect of knockdown and knockout in rodent and human iPSC-derived neurons. is also the third most significant genetic risk factor for late onset AD and several variants have been identified in . Although the exact contribution of these variants to altered AD risk is unclear, some have been linked to altered 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.

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 β-amyloid (Aβ) toxicity. Mutations in the clusterin gene (CLU), a major genetic AD risk factor, are known to have important roles in Aβ processing. Here we investigate how CLU mediates Aβ-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 Aβ-mediated neurodegeneration in cortical neurons differentiated from wild type and CLU knockout iPSCs. We measured response to Aβ 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 Aβ peptides and Aβ oligomers, in a dose dependent manner, and that intracellular levels of clusterin are increased following Aβ treatment. However, in CLU knockout neurons Aβ 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 Aβ in neurons with the CLU deletion. Four of the five genes previously identified as downstream to Aβ and Dickkopf-1 (DKK1) proteins in an Aβ-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 Aβ treated neurons. The absence of neurodegeneration in the CLU knockout neurons in response to Aβ compared to the wild type neurons supports the role of clusterin in Aβ-mediated AD pathogenesis.

MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate gene expression through post-transcriptional repression of target genes. They have been shown to be implicated in the pathophysiology of Alzheimer’s disease (AD) and proposed as disease biomarkers. In the present work, we have studied the expression levels of 754 miRNAs in cerebrospinal fluid (CSF) from AD patients and control subjects. We have explored a first screening cohort ( N  = 20) and selected 12 miRNAs to be further tested in a second independent validation cohort ( N  = 69). We have found a significant upregulation of miR-222 and miR-125b in AD CSF. Of these, the association of miR-222 with AD is novel and reported here for the first time whereas upregulation of miR-125b has been previously reported in AD brain. Yet we do not find association with other miRNAs which were previously linked to AD. Our results shed light on potential underlying pathophysiological processes of AD and also point out the need for consensus procedures in CSF miRNA detection and data analysis.

Alzheimer’s disease (AD) is the most common of the neurodegenerative diseases. Recent diagnostic criteria have defined a preclinical disease phase during which neuropathological substrates are thought to be present in the brain. There is an urgent need to find measurable alterations in this phase as well as a good peripheral biomarker in the blood. We selected a cohort of 100 subjects (controls = 47; preclinical AD = 11; patients with AD = 42) and analyzed whole blood expression of 20 genes by quantitative polymerase chain reaction. The selected genes belonged to calcium signaling, senescence and autophagy, and mitochondria/oxidative stress pathways. Additionally, two genes associated with an increased risk of developing AD (clusterin ( CLU ) and bridging integrator 1 ( BIN1 )) were also analyzed. We detected significantly different gene expressions of BECN1 and PRKCB between the control and the AD groups and of CDKN2A between the control and the preclinical AD groups. Notably, these three genes are also considered tumor suppressor ( CDKN2A and BECN1 ) or tumor promoter ( PRKCB ) genes. Gene-gene expression Pearson correlations were computed separately for controls and patients with AD. The significant correlations ( p  < 0.001) were represented in a network analysis with Cytoscape tool, which suggested an uncoupling of mitochondria-related genes in AD group. Whole blood is emerging as a valuable tissue in the study of the physiopathology of AD.

Clusterin ( CLU ) is one of the most significant genetic risk factors for late onset Alzheimer’s disease (AD). However, the mechanisms by which CLU contributes to AD development and pathogenesis remain unclear. Studies have demonstrated that the trafficking and localisation of glycosylated CLU proteins is altered by CLU -AD mutations and amyloid-β (Aβ), which may contribute to AD pathogenesis. However, the roles of non-glycosylated and glycosylated CLU proteins in mediating Aβ toxicity have not been studied in human neurons. iPSCs with altered CLU trafficking were generated following the removal of CLU exon 2 by CRISPR/Cas9 gene editing. Neurons were generated from control (CTR) and exon 2 −/− edited iPSCs and were incubated with aggregated Aβ peptides. Aβ induced changes in cell death and neurite length were quantified to determine if altered CLU protein trafficking influenced neuronal sensitivity to Aβ. Finally, RNA-Seq analysis was performed to identify key transcriptomic differences between CLU exon 2  −/− and CTR neurons. The removal of CLU exon 2, and the endoplasmic reticulum (ER)-signal peptide located within, abolished the presence of glycosylated CLU and increased the abundance of intracellular, non-glycosylated CLU. While non-glycosylated CLU levels were unaltered by Aβ 25–35 treatment, the trafficking of glycosylated CLU was altered in control but not exon 2  −/− neurons. The latter also displayed partial protection against Aβ-induced cell death and neurite retraction. Transcriptome analysis identified downregulation of multiple extracellular matrix (ECM) related genes in exon 2  −/− neurons, potentially contributing to their reduced sensitivity to Aβ toxicity. This study identifies a crucial role of glycosylated CLU in facilitating Aβ toxicity in human neurons. The loss of these proteins reduced both, cell death and neurite damage, two key consequences of Aβ toxicity identified in the AD brain. Strikingly, transcriptomic differences between exon 2  −/− and control neurons were small, but a significant and consistent downregulation of ECM genes and pathways was identified in exon 2  −/− neurons. This may contribute to the reduced sensitivity of these neurons to Aβ, providing new mechanistic insights into Aβ pathologies and therapeutic targets for AD.

Alzheimer's disease (AD) is the most common of the neurodegenerative diseases. Recent diagnostic criteria have defined a preclinical disease phase during which neuropathological substrates are thought to be present in the brain. There is an urgent need to find measurable alterations in this phase as well as a good peripheral biomarker in the blood. We selected a cohort of 100 subjects (controls = 47; preclinical AD = 11; patients with AD = 42) and analyzed whole blood expression of 20 genes by quantitative polymerase chain reaction. The selected genes belonged to calcium-signaling, senescence and autophagy, and mitochondria/oxidative stress pathways. Additionally, two genes associated with an increased risk of developing AD (CLU and BIN1) were also analyzed. We detected significantly different gene expressions of BECN1 and PRKCB between the control and the AD groups; and, of CDKN2A between the control and the preclinical AD groups. Notably, these three genes are also considered tumor suppressor (CDKN2A and BECN1) or tumor promoter (PRKCB) genes. Gene-gene expression Pearson correlations were computed separately for controls and patients with AD. The significant correlations (p<0.001) were represented in a network analysis with Cytoscape tool, which suggested an uncoupling of mitochondriarelated genes in AD group. Whole blood is emerging as a valuable tissue in the study of the physiopathology of AD.

Treatment of neurons with β-amyloid peptide (Aβ1-42) has been widely used as a model to interrogate the cellular and molecular mechanisms underlying Alzheimer’s disease, and as an assay system to identify drugs that reverse or block disease phenotype. Prior studies have largely relied on high content imaging (HCI) to extract cellular features such as neurite length or branching, but these have not offered a robust/comprehensive means of relating readout to Aβ1-42 concentrations. Here, we use a deep learning-based cell profiling technique to directly measure the impact of Aβ1-42 on primary murine cortical neurons. The deep learning model achieved approximately 80% accuracy, compared to 54% for the cell phenotypic feature-based approach. The deep learning model could distinguish subtle neuronal morphological changes induced by a range of Aβ1-42 concentration. When tested on a separate dataset, the accuracy remained comparable and dropped by only 2%. Our study demonstrates that deep learning-based cell profiling is superior to HCI-based feature extraction on neuronal morphology and it provides an alternative to a dose/response curve, where the modality of the response does not have to be pre-determined. Moreover, this approach could form the basis of a screening tool that can be applied to any cellular model where appropriate phenotypic markers based on genotypes and/or pathological insults are available.