Clusterin in Alzheimer's Disease: Mechanisms, Genetics, and Lessons From Other Pathologies

Abstract

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 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.

Keywords: DKK1; Wnt signaling; amyloid; cell death; neurodegeneration; neuroprotection; oxidative stress.

FIGURE 1

CLU gene and protein structure. CLU is a single copy gene containing nine exons. Exon 1 is a non-coding exon and two translational start sites have been identified, located in exons 2 and 3. An additional ATG has been predicted to be found in exon 1 of alternative variants of CLU, but their biological relevance is unclear (Prochnow et al., 2013). The main CLU mRNA transcript is transcript NM_001831.3 and typically translation begins at the translational start site located in exon 2. This produces an immature preproprotein (NP_001822.3) that contains exons 2–8 and the coding portion of exon 9 and includes an endoplasmic reticulum (ER)-signal peptide located in exon 2 that enables this immature protein to be processed, modified and cleaved in the traditional secretory pathway to produce mature sCLU. During the production of mature sCLU, the ER-signal peptide is cleaved and removed, and an additional cleavage event takes place between amino acid residues 227 and 228 resulting in the formation of an α-chain and a β-chain linked by disulphide bonds. Glycosylation occurs at 6 sites (indicated in red) on both the β-chain (sites 86, 103, and 145) and the α-chain (sites 291, 354, and 374). Two phosphorylation sites are also known (indicated in pink) at residues 133 and 396. Due to discrepancies in the literature, positioning of the α-chain and a β-chain, amino acid annotations, and length of the ER-signal peptide have been drawn based on current annotations provided by NCBI for the clusterin preproprotein NP_001822.3.

FIGURE 2

The biogenesis of mature, secreted clusterin involves the traditional secretory pathway common to other secreted proteins. CLU is transcribed into the mRNA NM_001831.3, containing exons 1–9. Within exon 2 is a 22-amino acid signal peptide that targets the preproprotein (NP_001822.3) to the endoplasmic reticulum (ER) and the Golgi apparatus. This immature preproprotein undergoes a series of modifications including phosphorylation, cleavage, and extensive glycosylation. Finally, mature secreted clusterin (sCLU) is produced and secreted from cells. Although traditionally referred to as a secreted protein, a number of reports have shown the existence of clusterin inside the cell, referred to as intracellular clusterin. In comparison to secreted clusterin, which has a well described biogenesis, the origin and production of intracellular clusterin is much less clear. Although originally believed that intracellular clusterin arose from a distinct CLU mRNA transcript to sCLU, it is now extensively believed that these two proteins share the common mRNA transcript NM_001831.3 and that intracellular clusterin may exist due to altered localization of secreted clusterin. Stress may impair a cell’s ability to secrete sCLU, resulting in its localization within the cell. Alternatively, sCLU may not undergo canonical biogenesis, resulting in incomplete glycosylation and/or cleavage, and this may lead immature sCLU to escape the secretory pathway and remain inside the cell. Additionally, secreted clusterin may be taken into cells by uptake mechanisms resulting in the presence of mature, glycosylated sCLU within the cell. Alternative theories suggest that CLU mRNA may undergo stress-induced alternative splicing and non-canonical translation to produce a truncated, non-glycosylated clusterin protein that lacks exon 2 and therefore does not become secreted, but instead accumulates within the cell. Research from oncology indicates a pro-survival function of secreted clusterin and a pro-apoptotic function of intracellular clusterin. Secreted clusterin interacts with the BAX-Ku70 complex, stabilizing it thereby inhibiting the translocation of BAX to the mitochondria where it would promote apoptotic pathways. In contrast, intracellular clusterin competes with BAX for binding with Ku70 and thereby inhibiting their complex formation, resulting in increased free BAX that can translocate to the mitochondria. Secreted clusterin is also thought to interact with Bcl-xl proteins promoting its anti-apoptotic function, whereas intracellular clusterin interacts with this same protein to reduce its anti-apoptotic function. Intracellular clusterin has also been shown to interact with DNA-PK complexes and thereby inhibit DNA repair, resulting in DNA damage and cell stress and death. This figure was produced using icons in the icon library of the Reactome database that are free for download and modification: (https://reactome.org/icon-lib) Accessed August 28, 2018; (Sidiropoulos et al., 2017).

FIGURE 3

A multitude of SNPs have been identified in CLU, both intronic and exonic. The figure highlights a number of these SNPs identified in AD GWAS.