**Clusterin (APOJ) in Alzheimer's Disease: An Old Molecule with a New Role**

Sarah K. Woody and Liqin Zhao

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64233

#### **Abstract**

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310 Update on Dementia

SCI.0991-07.2007.

10.1126/science.1063695.

Clusterin (CLU), initially identified in 1983 as a "clustering factor" in ram rete testis fluid, is a multifaceted protein that was re-discovered and subsequently renamed eight times from 1983 to 1992. CLU exists as multiple protein isoforms including the 80 kDa glycosylated mature/secreted form of CLU (mCLU) and the smaller non-modified nuclear and intracellular forms of CLU (nCLU and icCLU, respectively). These isoforms, which are expressed at the highest levels in the brain, are suggested to play distinct roles in various disease processes such as those involving inflammation and apoptosis. Currently, CLU, also known as apolipoprotein J (APOJ) which belongs to the same protein family as apolipoprotein E (APOE), is the third most significant genetic risk factor for the development of late-onset Alzheimer's disease (LOAD); however, an extensive gap exists in the literature in understanding the physiological roles of CLU in normal brain and the pathogenic mechanisms conferred by CLU polymorphisms in the onset of LOAD. In this chapter, we discuss the status of the current knowledge regarding the generation and regulation of CLU protein isoforms, the clinical evidence and possible mechanisms involved in LOAD, and provide our perspectives for future studies.

**Keywords:** late-onset Alzheimer's disease (LOAD), genetic risk factors, clusterin (CLU), apolipoprotein J (APOJ), apolipoprotein E (APOE)

### **1. Introduction**

#### **1.1. Alzheimer's disease: current status and challenges**

Alzheimer's disease (AD) currently affects 35 million people worldwide, including 5.4 million Americans; a number that is estimated to triple by the year 2050 [1]. As the prevalence of AD

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

increases, the AD-associated economic burden will also increase. In 2015, the direct costs associated with the care of AD patients in the United States reached \$226 billion. This number is predicted to reach \$1.1 trillion by the year 2050 making AD one of the most costly chronic illnesses in the world [1]. At present, AD is the sixth leading cause of death in the United States and is the only leading cause of death that cannot be prevented or cured. There are current‐ ly five FDA-approved drugs available to treat AD; however, these drugs do not address the underlying cause of AD and provide only temporary therapeutic relief in a fraction of the patients to whom they are administered. An extensive amount of clinical trials aimed at treating AD have been performed in the last 15 years; all of which have failed [2, 3]. These unanticipated challenges combined with the estimated rapid increases in AD prevalence stress the importance of identifying the underlying AD risk mechanisms that would allow preven‐ tion, risk reduction, and early intervention in the preclinical stage of AD.

#### **1.2. Late-onset AD: complex etiology and risk factors**

There are two types of AD: early-onset familial AD (FAD) and late-onset sporadic AD (LOAD). FAD is rare and mostly caused by inherited genetic mutations that result in abnormal over‐ production of neurotoxic β-amyloid (Aβ) peptides. LOAD, the most common form of AD representing 95% of human cases, develops after age 60 and involves a heterogeneous and multifactorial etiology. It is now widely accepted that a person's risk for developing LOAD is primarily influenced by a combination of complex interactions between genetic and environ‐ mental risk factors. At present, age remains the most predominant risk factor for LOAD. It is estimated that one in nine (11.1%) senior citizens aged 65 or older have been diagnosed with LOAD; a ratio that increases to one in three (33.3%) by age 85 [1]. The National Institute on Aging (NIA) indicates that the risk of developing LOAD doubles every 5 years past the age of 65 [4]. Additionally, epidemiologic studies from the NIA estimate that the total percentage of senior citizens in the United States will increase by 7% by 2030 making senior citizens the fastest-growing age group in the United States and consequently the most at-risk population [4].

Sex also plays a significant role in the development of LOAD. Of the 5.4 million Americans currently living with AD, approximately 65% are women [1]. It was originally postulated that the higher percentage of women living with AD was due to the increased life span of the female population; however, as the average worldwide life expectancy of men and women differs by only 4 years, this presumption is invalid. A meta-analysis of seven sex-specific clinical studies revealed that women are 1.5 times more likely to develop AD than age-matched men, indi‐ cating that the female sex confers AD risk independent of age [5]. In addition to a higher incidence of AD, it is now well established that sex influences both the development and progression of LOAD. For example, female AD patients have been shown to exhibit more severe cognitive decline than men during the progression of AD pathology [6–8]. While the exact mechanisms underlying this sex bias are currently unknown, mounting evidence suggests that female vulnerability to AD is largely associated with the irreversible decline of female sex hormones during the onset of menopause [9–11]. However, despite these findings, the precise molecular mechanisms underlying female vulnerability remain uncharacterized.

Genetic predisposition is another prominent risk factor associated with the development of AD. A long-standing observation in the field of LOAD research is the significantly increased AD risk associated with possession of the human apolipoprotein E ε4 allele (APOE ε4) [12], the most predominant genetic risk factor for LOAD. Possession of the ε4 allele is clinically associated with an increased rate and severity of cognitive decline, a younger age of onset, and altered response to AD treatments [13–16]. Moreover, the ε4 allele has been shown to reduce brain glucose utilization [17], increase neuronal inflammation [18], and is associated with increased Aβ dyshomeostasis [19, 20]. In addition to these data, studies have demonstrated that the APOE ε4-associated AD risk is significantly more pronounced in the female popula‐ tion. For example, a recent clinical study conducted in a cohort of 8084 elderly individuals (healthy controls: *n* = 5496; MCI cases: *n* = 2588) demonstrated that the risk of clinical conversion from healthy aging to MCI or from MCI to AD conferred by the ε4 allele was significantly greater in women than in men, a finding that corresponds with several earlier reports [21–25].

In addition to APOE ε4, two of the largest genome-wide association (GWA) studies ever conducted to date have recently identified several other genetic risk factors that confer a significantly increased risk of developing LOAD [26, 27]. Of the genetic risk factors identified, clusterin (CLU), also known as apolipoprotein J (APOJ), was established as the third most predominant genetic risk factor for LOAD. CLU, which belongs to the same protein family as APOE, has been shown to regulate inflammation, oxidative stress, and amyloid homeostasis in the brain. Moreover, a recent study conducted by our laboratory indicated that CLU mRNA and protein expression levels are significantly reduced specifically in female brain during a time period that likely corresponds to the onset of reproductive senescence. These data suggest that, similar to APOE ε4, CLU is also influenced by sex in the brain aging process and the pathogenesis of LOAD [28]. In the following sections, we summarize the current understand‐ ing of CLU protein isoforms and their biological functions with specific emphasis on the neuroprotective potential of CLU protein isoforms in the brain.

#### **2. Clusterin: from form to function**

#### **2.1. CLU: discovery and nomenclature**

increases, the AD-associated economic burden will also increase. In 2015, the direct costs associated with the care of AD patients in the United States reached \$226 billion. This number is predicted to reach \$1.1 trillion by the year 2050 making AD one of the most costly chronic illnesses in the world [1]. At present, AD is the sixth leading cause of death in the United States and is the only leading cause of death that cannot be prevented or cured. There are current‐ ly five FDA-approved drugs available to treat AD; however, these drugs do not address the underlying cause of AD and provide only temporary therapeutic relief in a fraction of the patients to whom they are administered. An extensive amount of clinical trials aimed at treating AD have been performed in the last 15 years; all of which have failed [2, 3]. These unanticipated challenges combined with the estimated rapid increases in AD prevalence stress the importance of identifying the underlying AD risk mechanisms that would allow preven‐

There are two types of AD: early-onset familial AD (FAD) and late-onset sporadic AD (LOAD). FAD is rare and mostly caused by inherited genetic mutations that result in abnormal over‐ production of neurotoxic β-amyloid (Aβ) peptides. LOAD, the most common form of AD representing 95% of human cases, develops after age 60 and involves a heterogeneous and multifactorial etiology. It is now widely accepted that a person's risk for developing LOAD is primarily influenced by a combination of complex interactions between genetic and environ‐ mental risk factors. At present, age remains the most predominant risk factor for LOAD. It is estimated that one in nine (11.1%) senior citizens aged 65 or older have been diagnosed with LOAD; a ratio that increases to one in three (33.3%) by age 85 [1]. The National Institute on Aging (NIA) indicates that the risk of developing LOAD doubles every 5 years past the age of 65 [4]. Additionally, epidemiologic studies from the NIA estimate that the total percentage of senior citizens in the United States will increase by 7% by 2030 making senior citizens the fastest-growing age group in the United States and consequently the most at-risk population

Sex also plays a significant role in the development of LOAD. Of the 5.4 million Americans currently living with AD, approximately 65% are women [1]. It was originally postulated that the higher percentage of women living with AD was due to the increased life span of the female population; however, as the average worldwide life expectancy of men and women differs by only 4 years, this presumption is invalid. A meta-analysis of seven sex-specific clinical studies revealed that women are 1.5 times more likely to develop AD than age-matched men, indi‐ cating that the female sex confers AD risk independent of age [5]. In addition to a higher incidence of AD, it is now well established that sex influences both the development and progression of LOAD. For example, female AD patients have been shown to exhibit more severe cognitive decline than men during the progression of AD pathology [6–8]. While the exact mechanisms underlying this sex bias are currently unknown, mounting evidence suggests that female vulnerability to AD is largely associated with the irreversible decline of female sex hormones during the onset of menopause [9–11]. However, despite these findings, the precise molecular mechanisms underlying female vulnerability remain uncharacterized.

tion, risk reduction, and early intervention in the preclinical stage of AD.

**1.2. Late-onset AD: complex etiology and risk factors**

[4].

312 Update on Dementia

In 1983, Blaschuk et al. identified a high-molecular weight protein in ram rete testis fluid [29]. Further analyses indicated that this unknown protein was capable of eliciting the "clustering" of Sertoli cells, mouse testis TM-4 cells, and erythrocytes resulting in the name clusterin. In 1984, Griswold and colleagues purified a dimeric acidic glycoprotein (DAG) from the Sertoli cells of rat testes [30]. This abundantly expressed but uncharacterized protein was detected at several molecular weights via reducing chromatography (41 and 29 kDa), western blot (27 and 21 kDa), and immunoprecipitation (70 kDa) [30]. In 1988, another study identified a "novel" protein in human serum. This heterodimeric protein had a molecular mass of 80 kDa, was composed of two 40-kDa chains, and was sequentially unique to all other proteins. Further‐ more, it was concluded that this protein, which was deposited in the renal glomeruli of patients with glomerulonephritis, was integrally involved in kidney health [31]. As a result of these observations, Murphy and colleagues named this protein serum protein 40 kDa,40 kDa (SP-40,40) [31]. In 1990 and the years following, Harmony and colleagues identified and extensively characterized a component of high-density lipoproteins in human plasma which was referred to as apolipoprotein J (APOJ) [32]. However, upon the advent of DNA sequencing technology, it was determined that clusterin, DAG, SP-40,40, and APOJ were in fact the same protein. In the following decade, clusterin was "re-discovered" and subsequently re-labeled with other alternative names including testosterone-repressed prostrate message-2 (TRPM-2) [33], KU70-binding protein 1 (KUB1) [34], complement lysis inhibitor (CLI) [35], and sulphated glycoprotein-2 (SGP2) [36]. In 1992, a forum conducted at Cambridge University officially deemed this diverse protein clusterin (CLU).

#### **2.2. CLU: from gene to protein**

CLU is a single-copy gene located on the short arm of chromosome 8 (8p21-12) [37, 38] where it spans approximately 18,115 base pairs (bp). Upon the splicing of eight introns, this nineexon product spans approximately 2877 bp, and is transcribed into at least two distinct mRNA transcripts. CLU mRNA transcript 1 (NM\_001831.3), the most extensively characterized transcript, is translated into the mature/secreted isoform of CLU (mCLU) that has been predominantly identified and studied in the field of CLU research. CLU mRNA transcript 1 is initially translated into a 449-amino-acid precursor protein (pCLU, 60 kDa) beginning at a canonical translational start site located at base pair 187 in exon 2. This pCLU protein contains an N-terminal 22-amino-acid endoplasmic reticulum (ER)-targeted signaling peptide (amino acids 1–22 or bp 187–252) and two nuclear localization sequences in exon 3 (amino acids 78– 81 or bp 418–429) and exons 8–9 (amino acids 443–447 or bp 1513–1528). The translated pCLU protein is then targeted to the ER where the 22-amino-acid leader sequence (LS) is cleaved. Following LS cleavage, the peptide bond between R227 and S228 is cleaved resulting in the formation of two individual CLU subunits: the alpha subunit (CLUα, 34–37 kDa) and the beta subunit (CLUβ, 36–39 kDa). These two subunits are subsequently linked by five disulphide bonds to form an anti-parallel heterodimer [39]. N-glycosylation at six glycosylation sites is the final step in the generation of mCLU which, under nonreducing conditions, has a molecular weight of 75–80 kDa (**Figure 1A**) [40]. Alternatively, complete removal of exon 2 via alternative splicing results in the fusion of exons 1 and 3, thereby creating CLU mRNA transcript 2. In this secondary transcript, translation is initiated at a canonical translational start site located in exon 3. This results in the production of a CLU protein isoform that lacks the ER LS but retains the nuclear localization sequences. This alternative CLU isoform, which is non-ER targeted and unglycosylated, is shuttled between the cytoplasm and the nucleus and is referred to as "nuclear" CLU (nCLU, 49 kDa, **Figure 1B**). In addition to mCLU and nCLU, emerging evidence indicates that several different splicing variants of CLU also exist. These splicing variants, which are relatively uncharacterized, are suggested to lack portions of exon 2 and/or exon 5 and are generally referred to as "intracellular" isoforms (icCLU, 45, 50, and 53–55 kDa) [41–43].

Initial characterization by Harmony et al. indicated the expression of CLU mRNA in liver, lung, spleen, heart, reproductive tissues, and brain with predominant expression in brain and reproductive tissues [44]. Since this initial characterization, several other research groups, including our own, have detected CLU mRNA and protein expression in many cell lines and tissue types tested. Moreover, CLU appears to be ubiquitously expressed on the subcellular level with multiple studies demonstrating CLU expression in the cytosol [45], nucleus [41], ER, and Golgi apparatus. Within the brain, CLU expression has been detected within neurons [46], astrocytes [46–48], microglia [49], and within the extracellular space [50]. While initial reports indicated that CLU was solely synthesized and secreted from the astrocytes in a manner similar to APOE [51], our recent findings demonstrate that pure cultures of primary neurons express

observations, Murphy and colleagues named this protein serum protein 40 kDa,40 kDa (SP-40,40) [31]. In 1990 and the years following, Harmony and colleagues identified and extensively characterized a component of high-density lipoproteins in human plasma which was referred to as apolipoprotein J (APOJ) [32]. However, upon the advent of DNA sequencing technology, it was determined that clusterin, DAG, SP-40,40, and APOJ were in fact the same protein. In the following decade, clusterin was "re-discovered" and subsequently re-labeled with other alternative names including testosterone-repressed prostrate message-2 (TRPM-2) [33], KU70-binding protein 1 (KUB1) [34], complement lysis inhibitor (CLI) [35], and sulphated glycoprotein-2 (SGP2) [36]. In 1992, a forum conducted at Cambridge University officially

CLU is a single-copy gene located on the short arm of chromosome 8 (8p21-12) [37, 38] where it spans approximately 18,115 base pairs (bp). Upon the splicing of eight introns, this nineexon product spans approximately 2877 bp, and is transcribed into at least two distinct mRNA transcripts. CLU mRNA transcript 1 (NM\_001831.3), the most extensively characterized transcript, is translated into the mature/secreted isoform of CLU (mCLU) that has been predominantly identified and studied in the field of CLU research. CLU mRNA transcript 1 is initially translated into a 449-amino-acid precursor protein (pCLU, 60 kDa) beginning at a canonical translational start site located at base pair 187 in exon 2. This pCLU protein contains an N-terminal 22-amino-acid endoplasmic reticulum (ER)-targeted signaling peptide (amino acids 1–22 or bp 187–252) and two nuclear localization sequences in exon 3 (amino acids 78– 81 or bp 418–429) and exons 8–9 (amino acids 443–447 or bp 1513–1528). The translated pCLU protein is then targeted to the ER where the 22-amino-acid leader sequence (LS) is cleaved. Following LS cleavage, the peptide bond between R227 and S228 is cleaved resulting in the formation of two individual CLU subunits: the alpha subunit (CLUα, 34–37 kDa) and the beta subunit (CLUβ, 36–39 kDa). These two subunits are subsequently linked by five disulphide bonds to form an anti-parallel heterodimer [39]. N-glycosylation at six glycosylation sites is the final step in the generation of mCLU which, under nonreducing conditions, has a molecular weight of 75–80 kDa (**Figure 1A**) [40]. Alternatively, complete removal of exon 2 via alternative splicing results in the fusion of exons 1 and 3, thereby creating CLU mRNA transcript 2. In this secondary transcript, translation is initiated at a canonical translational start site located in exon 3. This results in the production of a CLU protein isoform that lacks the ER LS but retains the nuclear localization sequences. This alternative CLU isoform, which is non-ER targeted and unglycosylated, is shuttled between the cytoplasm and the nucleus and is referred to as "nuclear" CLU (nCLU, 49 kDa, **Figure 1B**). In addition to mCLU and nCLU, emerging evidence indicates that several different splicing variants of CLU also exist. These splicing variants, which are relatively uncharacterized, are suggested to lack portions of exon 2 and/or exon 5 and are generally referred to as "intracellular" isoforms (icCLU, 45, 50, and 53–55 kDa) [41–43].

Initial characterization by Harmony et al. indicated the expression of CLU mRNA in liver, lung, spleen, heart, reproductive tissues, and brain with predominant expression in brain and reproductive tissues [44]. Since this initial characterization, several other research groups,

deemed this diverse protein clusterin (CLU).

**2.2. CLU: from gene to protein**

314 Update on Dementia

**Figure 1.** CLU transcription and translation. In humans, CLU is a single-copy gene located on the short arm of chromo‐ some 8 that is comprised of nine exons spanning approximately 2.8 kb. (**A**) The mCLU isoform is generated from mRNA transcript 1 from a canonical translational start site in exon 2. The resulting precursor protein (pCLU), which contains an N-terminal ER-targeting leader sequence (LS), is transported to the ER where the 22-amino-acid LS is re‐ moved. CLU is then cleaved into the alpha and beta subunits and rapidly disulfide bonded and glycosylated to form an anti-parallel, heterodimeric glycoprotein: mCLU. (**B**) Alternatively, the nCLU isoform is generated from mRNA transcript 2. In this transcript, a splicing event removes exon 2 resulting in the fusion of exons 1 and 3. Translation is initiated at the beginning of exon 3 resulting in a truncated CLU isoform that lacks the ER-targeting LS. Therefore, the nCLU isoform, which retains the nuclear localization sequence, bypasses the ER/Golgi apparatus and is shuttled be‐ tween the cytosol and the nuclear compartment.

mCLU, nCLU, and to a lesser extent icCLU isoforms indicating that neurons are also capable of generating de novo CLU. Though the exact physiological functions of CLU remain a mystery, the nearly ubiquitous nature of CLU indicates the significance of this protein in cellular homeostasis.

### **2.3. CLU: transcriptional regulation**

Though the gene promoter of CLU is highly conserved across species, the transcriptional regulation of CLU is complex as the predominant CLU transcriptional regulators appear to differ between tissue and cell type. However, despite the controversy in the literature, it is generally agreed that CLU is primarily upregulated by cellular injury, cytotoxic insult, and various stress stimuli [52–54]. For instance, Loisen and colleagues demonstrated that the CLU gene promoter contains an MG132 responsive region and a heat-shock element (HSE) indi‐ cating that proteasomal stress directly influences CLU transcription [52]. Another study demonstrated that the CLU gene promoter contains both HSEs and an activator protein-1 (AP-1) response element indicating direct transcriptional regulation by stimuli derived from cellular proliferation and differentiation [54]. In addition to these data, alternative stressrelated transcription factor response elements have been identified in the CLU gene promoter including a cAMP response element (CRE), an AP-2 response element, a specificity protein-1 (SP1) response element, and a glucocorticoid response element (GRE) [33, 53]. It has also been demonstrated that apoptotic stimuli modulates CLU transcription, specifically in cancer. An early study from Cervellera et al. identified a MYB binding site in the 5' flanking region of CLU and that B-MYB, a MYB family member that regulates cellular proliferation and apop‐ tosis, directly bound to and transactivated the CLU gene [55]. CLU transcription is also regulated by several different growth factors including nerve growth factor (NGF) and transforming growth factor beta (TGFβ) [56–58]. For instance, it has been demonstrated that TGFβ induces the upregulation of CLU gene expression by stimulating the interaction between the CLU gene promoter and AP-1 [57]. An extension of these studies demonstrated that TGFβ deficiency resulted in the repression of CLU gene expression via interaction between c-Fos and the CLU gene promoter; an interaction that was abrogated upon cellular stimulation with TGFβ [58].

#### **2.4. CLU: posttranslational modification**

CLU is regulated by several types of posttranslational modification (PTM), the most predom‐ inant type being N-linked glycosylation. As previously indicated, mCLU is N-glycosylated at six different asparagine residues (N86, N103, N145, N291, N354, and N374) during ER-Golgi processing: a modification that comprises approximately 20–25% of the total mass of mCLU [59]. While glycosylation status was originally thought to have little to no impact on CLU function [40, 60], a recent study demonstrated that the chaperone activity of mCLU is depend‐ ent upon mCLU glycosylation [61]. This study also demonstrated that the glycosylation of nCLU did not result in chaperone activity indicating that glycosylation-mediated effects are specific to the mCLU isoform. It has also been established that complete deglycosylation of mCLU results in a 70–90% decrease in mCLU chaperone activity and a significant decrease in the number of α-helices in the secondary structure of CLU. These data suggest that the lack of chaperone activity in deglycosylated mCLU could be, in part, due to the significant changes in secondary structure. Additionally, this study indicates that partially glycosylated mCLU retains chaperone activity suggesting that "core" glycosylation sites are crucial for mCLU function, while peripheral glycosylation may be dispensable [61]. Parallel to these findings, a study by Kang et al. indicated that ER stress, which inhibits protein glycosylation, resulted in rapid retro-translocation of mCLU from the ER yielding several hypo-glycosylated CLU isoforms. These hypo-glycosylated isoforms, which are misfolded and generally nonfunction‐ al, are rapidly poly-ubiquitinated under normal conditions and cleared through proteasomal degradation. However, if the proteasome is chemically inhibited following ER stress, hypoglycosylated CLU accumulates in the cytosolic compartment resulting in cytotoxicity [62]. Collectively, these studies indicate that, contrary to what was originally postulated, glycosy‐ lation is crucial for mCLU chaperone activity.

In addition to N-linked glycosylation, CLU is also a primary target for ubiquitination and phosphorylation. It has been demonstrated that nCLU is a target for K63 ubiquitination through the ubiquitin E3 ligase, a product of von Hippel-Lindau (pVHL). However, contrary to the canonical function of protein ubiquitination, K63-linked ubiquitination of nCLU does not target nCLU for destruction, rather it promotes nCLU nuclear translocation for reasons that are currently unknown [63]. Pertaining to CLU phosphorylation, a recent proteomics study which focused on the identification of the serum phospho-proteome has identified three different phosphorylation sites at residues Thr393, Ser394, and Ser39 within the CLU protein. Additionally, a more recent study indicated that treatment of hepatocytes with 10-mM glucose and fructose significantly increased the levels of mCLU serine phosphorylation. This same study demonstrated increased mCLU serine phosphorylation in both the skeletal muscle and the liver of rats that were orally administered high doses of glucose and/or fructose indicating that phosphorylated CLU may interact with or respond to the activation of glucose-sensitive cellular bioenergetic pathways. In addition to ubiquitination and phosphorylation, an early report indicated that CLU is iodinated at 1 of the 12 tyrosine residues within the CLU protein. This iodination occurs within the apical plasma membrane of thyrocytes and is suggested to serve as a mechanism by which the thyroid gland can conserve iodine, which is relatively rare in the body [64]. It is also suggested that CLU activity is regulated by both sialylation [65] and acetylation [66]; however, definitive acetylation or sialylation sites have not been identified.
