**4. CLU in the brain: mechanisms of action**

Of the known CLU isoforms, mCLU is by far the most studied and has been described as a chaperone-like protein that clears misfolded proteins, cellular debris, and protein aggregates from the cytosol and extracellular space [106–113]. However, the nCLU and icCLU isoforms remain relatively uncharacterized. Several reports have suggested that nCLU and icCLU exhibit solely proapoptotic characteristics; however, results vary across laboratories and are inconsistent [41, 42, 114–116]. This section reviews the available literature pertaining to CLU isoforms in the brain with particular emphasis on the molecular mechanisms by which CLU protein isoforms regulate amyloid homeostasis, inflammation, and apoptosis.

#### **4.1. CLU and Aβ homeostasis**

In the early 1990s, CLU mRNA and protein levels were found to be significantly elevat‐ ed in AD brain, specifically in the frontal cortex and hippocampus of post-mortem AD brain tissue [117, 118]. Shortly after these discoveries, McGeer et al. demonstrated robust CLU immunoreactivity within senile plaques [119]. It was further demonstrated that mCLUbound soluble Aβ proteins in the cerebral spinal fluid (CSF) [120] and that CLU expres‐ sion increased the solubility of Aβ and prevented Aβ aggregation [121]. These data strongly suggested that CLU may play an important role in the pathogenesis of AD via regula‐ tion of brain amyloid burden. However, contrary to these findings, it has also been demonstrated that increased CLU expression exacerbated Aβ-induced neurotoxicity [122]. Moreover, DeMattos et al. demonstrated that Aβ plaque formation was facilitated by CLU in an animal model of AD suggesting that CLU exerts a negative impact on the brain in the development of AD pathology [123]. These literary contradictions continued to persist until 2007 when a study by Yerbery and colleagues provided a possible explanation for the simultaneously pro- and anti-amyloidogenic effects associated with mCLU [124]. This study indicated that the pro-amyloidogenic effects of mCLU were restricted to conditions in which Aβ was present in a very large molar excess. Under these conditions, mCLU, which functions as a chaperone-like protein to temporarily stabilize misfolded proteins [125], bound to and stabilized Aβ thereby facilitating Aβ aggregation. Alternatively, when mCLU was present at much higher but still substoichiometric levels (i.e. a molar ratio of clusterin:Aβ = 1:10), mCLU provided substantial anti-amyloidogenic effects by inhibiting plaque formation [124]. These data suggest that CLU may exhibit neuroprotective charac‐ teristics in preclinical or early stages of AD when brain amyloid burden is significantly

lower. Alternatively, CLU may exert a negative impact during later stages of AD when brain amyloid burden is extensive, though this hypothesis is yet to be tested. Parallel to this notion, a more recent study performed in rat brains indicated that mCLU prevented Aβ42-induced learning and memory impairments, reduced Aβ42-induced glia inflamma‐ tion, and reduced Aβ42-mediated neuronal degeneration when Aβ42 oligomers were incubated with mCLU prior to brain injection. However, these effects were not observed in rats injected with pre-formed Aβ42 oligomers and mCLU without pre-incubation indicating that mCLU does prevent Aβ42-induced neurotoxicity prior to extensive Aβ42 oligomerization [126]. In addition to these studies, mCLU has been shown to impact the rate of Aβ42 clearance. A study by Bell and colleagues demonstrated that the rate of Aβ clearance was increased by as much as 83% when bound to CLU. This same study further demonstrated that CLU-bound Aβ is transported across the blood-brain barrier specifical‐ ly through LRP2-mediated transport, while APOE-bound Aβ was transported through LRP-1 [127]. While the regulation of Aβ by mCLU is relatively well characterized, one question that remains unanswered is whether alternative CLU isoforms exert some impact on amyloid homeostasis. It has been demonstrated that Aβ toxicity induces the expres‐ sion of intracellular CLU (icCLU) in neurons; however, the physiological impact of increased icCLU expression was not determined in this study [128]. At present, no literature specifically implicates a role for nCLU/icCLU isoforms in the regulation of Aβ; however, as nCLU/icCLU isoforms are reportedly induced by cellular stress in multiple peripheral cell lines and nCLU is induced upon treatment with exogenous Aβ, it is likely that nCLU/ icCLU isoforms mediate some effect on amyloid homeostasis in the brain; however, more research is needed before a conclusion can be made.

#### **4.2. CLU and inflammation**

**Study and year of publication** 

330 Update on Dementia

**Fluid analyzed** 

**Table 2.** CLU as an AD biomarker (2010–2016).

**4.1. CLU and Aβ homeostasis**

**4. CLU in the brain: mechanisms of action**

**Study design and subjects** 

*Control cases: n* = 50

**Diagnoses criteria Major findings** 

*Control criteria:* Cognitively healthy spouses of AD and

VaD patients

Of the known CLU isoforms, mCLU is by far the most studied and has been described as a chaperone-like protein that clears misfolded proteins, cellular debris, and protein aggregates from the cytosol and extracellular space [106–113]. However, the nCLU and icCLU isoforms remain relatively uncharacterized. Several reports have suggested that nCLU and icCLU exhibit solely proapoptotic characteristics; however, results vary across laboratories and are inconsistent [41, 42, 114–116]. This section reviews the available literature pertaining to CLU isoforms in the brain with particular emphasis on the molecular mechanisms by which CLU

In the early 1990s, CLU mRNA and protein levels were found to be significantly elevat‐ ed in AD brain, specifically in the frontal cortex and hippocampus of post-mortem AD brain tissue [117, 118]. Shortly after these discoveries, McGeer et al. demonstrated robust CLU immunoreactivity within senile plaques [119]. It was further demonstrated that mCLUbound soluble Aβ proteins in the cerebral spinal fluid (CSF) [120] and that CLU expres‐ sion increased the solubility of Aβ and prevented Aβ aggregation [121]. These data strongly suggested that CLU may play an important role in the pathogenesis of AD via regula‐ tion of brain amyloid burden. However, contrary to these findings, it has also been demonstrated that increased CLU expression exacerbated Aβ-induced neurotoxicity [122]. Moreover, DeMattos et al. demonstrated that Aβ plaque formation was facilitated by CLU in an animal model of AD suggesting that CLU exerts a negative impact on the brain in the development of AD pathology [123]. These literary contradictions continued to persist until 2007 when a study by Yerbery and colleagues provided a possible explanation for the simultaneously pro- and anti-amyloidogenic effects associated with mCLU [124]. This study indicated that the pro-amyloidogenic effects of mCLU were restricted to conditions in which Aβ was present in a very large molar excess. Under these conditions, mCLU, which functions as a chaperone-like protein to temporarily stabilize misfolded proteins [125], bound to and stabilized Aβ thereby facilitating Aβ aggregation. Alternatively, when mCLU was present at much higher but still substoichiometric levels (i.e. a molar ratio of clusterin:Aβ = 1:10), mCLU provided substantial anti-amyloidogenic effects by inhibiting plaque formation [124]. These data suggest that CLU may exhibit neuroprotective charac‐ teristics in preclinical or early stages of AD when brain amyloid burden is significantly

protein isoforms regulate amyloid homeostasis, inflammation, and apoptosis.

It is well established that persistent inflammation likely caused by the deposition of neurotoxic protein aggregates in the brain is a key component of LOAD [129]. Early studies suggest that CLU inhibits the activation of the complement system in the brain [31, 130–132]. For instance, several early publications indicated that CLU (then referred to as SP-40,40) prevented the formation of the membrane attack complex (MAC), suggesting that increased CLU would suppress initiation of acute inflammation. However, these data were contradicted by a more recent study that demonstrated CLU-mediated activation of the major histocompatibility complex class II (MHC II) antigen in primary cultures of rat microglia. This same study showed that administration of exogenous CLU resulted in the direct activation of microglia in the brain and the subsequent secretion of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α) indicating that increased CLU expression induces the acute inflammatory response [49]. These findings were corroborated by another study that demonstrated increased CLU staining within reactive microglia in the cortices of rats following cerebral ischemia [133]. Collectively, these studies suggest that increased CLU expression results in the activation of glial cells and the subsequent secretion of pro-inflammatory mediators. Therefore, it is possible that the increased secretion of cytokines such as TNF-α could contribute to chronic inflam‐ mation in AD brain; however, this hypothesis requires further testing.

#### **4.3. CLU and apoptosis**

Several studies performed in human cancer cell lines have demonstrated that mCLU and nCLU exhibit opposing effects on cell death pathways. mCLU has been shown to protect cells from oxidative stress and inhibit intrinsic apoptosis by interacting with and stabilizing the KU-70- Bax protein complex [134–138]. In contrast, nCLU is suggested to initiate intrinsic apoptotic pathways resulting in rapid cell death [115, 136]. The contrasting functions of mCLU and nCLU appear to also exist in the brain; however, unlike cancer-focused studies, relatively few brainbased investigations have included an examination of the apoptotic characteristics of nCLU. An early study by Schreiber et al. demonstrated that CLU (then referred to as SGP-2) mRNA expression was rapidly and transiently increased in astrocytes, but not CA3 and CA1 neurons, following administration of kainic acid (KA), a neurotoxic seizure-inducing compound [139]. Another study performed in WT, human CLU overexpressing (hCLU-OE) mice and Cluknockout (Clu-/-) mice subjected to middle cerebral artery occlusion (MCAO) indicated that CLU overexpression resulted in reduced brain injury. Specifically, this study demonstrated a 30–50% increase in CLU mRNA expression 7 days post-ischemia in the ischemic brain hemisphere specifically in the penumbral area (the area that separates necrotic from normal brain tissue). Morphometric analysis of the ischemic hemisphere revealed that the penumbra was significantly thinner in hCLU-OE mice and significantly thicker in Clu-/- mice when compared with WT mice indicating an inverse relationship between CLU mRNA expression and brain injury [140]. Collectively, these two studies strongly support a neuroprotective role for CLU in the brain following significant brain injury. In contrast, ethanol-mediated toxicity has been shown to significantly increase CLU expression in the cortex and amygdala. This upregulated CLU, which was shown to interact with Bcl-XL, was translocated to the nucleus upon exposure to ethanol, and was associated with increased cell death suggesting that these effects were mediated by nCLU [135]. Another study performed in neonatal mice subjected to hypoxic-ischemic brain injury indicated that CLU accumulated in dying neurons following brain injury. Moreover, this study indicated that CLU-deficient mice exhibited 50% less brain injury when compared to wild-type controls indicating that CLU expression exacerbates neuronal cell death following brain injury [141]. Collectively, these studies indicate that nCLU protein expression may be associated with increased cell death following traumatic brain injury or in response to cytotoxic stimuli.

#### **5. Future perspectives**

CLU is currently the third most significant genetic risk factor for the development of LOAD; however, an extensive gap exists in the literature in understanding the neurophysiological and neuropathological functions of CLU. Moreover, the bulk of brain-based CLU research refers to CLU as a single protein with few studies including a characterization of its isoforms. As CLU isoforms appear to mediate different physiological processes, the tendency to focus on the effects of CLU as a singular protein could lead to conflicting reports in the literature that are currently unresolved. Therefore, before researchers can fully ascertain the therapeutic potential of CLU from a clinical perspective, it is vital that these key deficiencies are addressed at the molecular level.

**4.3. CLU and apoptosis**

332 Update on Dementia

injury or in response to cytotoxic stimuli.

**5. Future perspectives**

Several studies performed in human cancer cell lines have demonstrated that mCLU and nCLU exhibit opposing effects on cell death pathways. mCLU has been shown to protect cells from oxidative stress and inhibit intrinsic apoptosis by interacting with and stabilizing the KU-70- Bax protein complex [134–138]. In contrast, nCLU is suggested to initiate intrinsic apoptotic pathways resulting in rapid cell death [115, 136]. The contrasting functions of mCLU and nCLU appear to also exist in the brain; however, unlike cancer-focused studies, relatively few brainbased investigations have included an examination of the apoptotic characteristics of nCLU. An early study by Schreiber et al. demonstrated that CLU (then referred to as SGP-2) mRNA expression was rapidly and transiently increased in astrocytes, but not CA3 and CA1 neurons, following administration of kainic acid (KA), a neurotoxic seizure-inducing compound [139]. Another study performed in WT, human CLU overexpressing (hCLU-OE) mice and Cluknockout (Clu-/-) mice subjected to middle cerebral artery occlusion (MCAO) indicated that CLU overexpression resulted in reduced brain injury. Specifically, this study demonstrated a 30–50% increase in CLU mRNA expression 7 days post-ischemia in the ischemic brain hemisphere specifically in the penumbral area (the area that separates necrotic from normal brain tissue). Morphometric analysis of the ischemic hemisphere revealed that the penumbra was significantly thinner in hCLU-OE mice and significantly thicker in Clu-/- mice when compared with WT mice indicating an inverse relationship between CLU mRNA expression and brain injury [140]. Collectively, these two studies strongly support a neuroprotective role for CLU in the brain following significant brain injury. In contrast, ethanol-mediated toxicity has been shown to significantly increase CLU expression in the cortex and amygdala. This upregulated CLU, which was shown to interact with Bcl-XL, was translocated to the nucleus upon exposure to ethanol, and was associated with increased cell death suggesting that these effects were mediated by nCLU [135]. Another study performed in neonatal mice subjected to hypoxic-ischemic brain injury indicated that CLU accumulated in dying neurons following brain injury. Moreover, this study indicated that CLU-deficient mice exhibited 50% less brain injury when compared to wild-type controls indicating that CLU expression exacerbates neuronal cell death following brain injury [141]. Collectively, these studies indicate that nCLU protein expression may be associated with increased cell death following traumatic brain

CLU is currently the third most significant genetic risk factor for the development of LOAD; however, an extensive gap exists in the literature in understanding the neurophysiological and neuropathological functions of CLU. Moreover, the bulk of brain-based CLU research refers to CLU as a single protein with few studies including a characterization of its isoforms. As CLU isoforms appear to mediate different physiological processes, the tendency to focus on the effects of CLU as a singular protein could lead to conflicting reports in the literature that are currently unresolved. Therefore, before researchers can fully ascertain the therapeutic

First, it is crucial that current and future studies strive to examine CLU isoforms individually, with particular emphasis on separating the nCLU and mCLU isoforms. Of the studies published pertaining to CLU in the brain, approximately five studies include an examination of nCLU. While it is possible that nCLU does function to regulate apoptosis, recent findings from our laboratory indicate roughly equivalent expression levels of both mCLU and nCLU in healthy primary cortical neurons suggesting that nCLU may be integrally involved in cellular homeostasis. Moreover, our recent data indicate that a nCLU or icCLU isoform is localized to the mitochondria suggesting that these alternative CLU isoforms may play an important role in the regulation of brain mitochondria function. While these studies are still underway, future work should focus on identifying the exact CLU isoforms expressed in other types of brain cells including astrocytes and microglia. Moreover, these studies should examine the cellular distribution, key protein modulators, and the neurophysiological function of each nCLU/icCLU isoform.

An emerging topic in the study of AD is the impact of sex on the development and progression of LOAD. As previously discussed, the female population is more susceptible to developing LOAD and the risk conferred by genetic factors, such as APOE, is greater in females. Moreover, our recent analyses have demonstrated that CLU expression is significantly reduced in the early aging of female but not male brain during a time that corresponds with the onset of reproductive senescence [28]. These data strongly suggest that CLU expression is modulated, in part, by sex hormone signaling pathways in the brain. Parallel to these findings, our recent studies have revealed that brain CLU isoform expression is regulated via estrogen receptor (ER) signaling. Additionally, we find that testosterone (TT) differentially regulates mCLU and nCLU expression; TT increases mCLU expression and decreases nCLU expression. An extension of these studies revealed that TT-mediated upregulation of mCLU expression results from the aromatization of TT to 17β-estradiol (E2). These data are particularly interesting when considered in the context of sex hormone changes between men and women throughout the aging process. It is well established that menopausal onset results in a significant and irrever‐ sible decline in ovarian sex hormones, such as E2. However, TT levels in males gradually decline with age at a rate of approximately 2% per year [142]. Therefore, it is possible that TTmediated upregulation of the neuroprotective mCLU isoform may, in part, contribute to the reduced incidence of AD in men. Likewise, the significant reduction in E2 levels in menopausal and/or postmenopausal women may result in significantly reduced mCLU levels thereby contributing to female vulnerability. While more research is needed to fully elucidate the interactions between sex hormones and neuronal CLU isoforms, these data underscore the importance of including sex as a variable in the study of risk factors that mediate the devel‐ opment of LOAD.

It is particularly interesting that two of the top five genetic risk factors associated with the development of LOAD are members of the apolipoprotein family: APOE and CLU. Therefore, another avenue of research to be considered in the AD field is the possibility of intersecting or overlapping risk pathways mediated by these two genetic factors. Studies have shown that APOE and CLU share a number of important physiological properties. For instance, they are among the few proteins associated with brain lipoproteins [143, 144]. They interact with a shared set of cell-surface receptors [108] and both APOE and CLU promote neurite outgrowth [145, 146]. Moreover, elimination of either protein in an AD mouse model results in increased accumulation of Aβ [147]. Furthermore, presence of the C allele of the CLU AD-risk SNP has been shown to exacerbate the APOE ε4-mediated decrease in brain activity during executive attention tasks in young healthy dementia-free adults [148]. In addition, the genetic variance that results in increased AD risk from both genes is also associated with compromised or reduced protein expression and/or binding capabilities. Our data indicate that APOE protein expression levels are significantly increased in 6-month-old female Clu-/- mice. However, mCLU expression levels are significantly reduced in 6-month-old female human APOE ε4 gene targeted-replacement mice when compared to APOE ε3 mice indicating that reduced CLU expression may contribute to APOE ε4-mediated AD risk. Collectively, these studies indicate that APOE and CLU could share common risk pathways that contribute to the development of LOAD. Delineation of such pathways will potentially provide valuable insights for an increased understanding of the etiology of LOAD and ultimately help to devise therapeutic strategies to prevent or reduce the risk of developing the disease.
