APP and Aβ impact upon metal homeostasis: a possible metal transport mechanism
The high energy, positively cooperative hexameric structure adopted by Aβ when binding Cu2+ and Zn2+ (Curtain et al. 2001; Curtain et al. 2003) implies that the complex subserves a possible physiological function. There is an interdependent relationship between Cu levels, APP expression and Aβ production supporting a role for APP and Aβ in Cu efflux. The initial finding showed that the APP and amyloid precursor-like protein 2 (APLP2) knockout mice have specific elevations in brain and liver Cu levels (White et al. 1999b). APLP2 shares a homologous N-terminal Cu-binding domain with APP but does not produce Aβ (Bush et al. 1993; Hesse et al. 1994). Primary cortical neurones and embryonic fibroblasts from APP and APLP2 double knockout mice also display significantly elevated Cu levels (Bellingham et al. 2004a). Conversely, overexpression of APP has been consistently shown to result in decreased Cu levels in three independent transgenic mouse lines (Maynard et al. 2002; Bayer et al. 2003; Phinney et al. 2003) and in primary cortical neurones (Bellingham et al. 2004a).
Overexpression of APP-C100, which contains Aβ but not the N-terminal Zn2+-and Cu2+-binding domain of APP, results in decreased brain Cu levels as well as Fe levels (Maynard et al. 2002). This suggests a role for Aβ in both Cu and Fe homeostasis. The effect of Aβ on Fe levels supports the previous proposal that the APP/Aβ system may play a role in the removal of excess Fe from the cell (Bush 2003). This stems from the identification of an iron-regulatory element (IRE-Type II) in the 5′-untranslated region of APP (Rogers et al. 2002). Alternatively, the decrease in Fe may reflect a homeostatic adjustment to the reduction in Cu levels.
Although Cu is the trace metal most strikingly affected in these transgenic and knockout mouse models, other less pronounced metal level changes may have some importance. The Cu deficit in Tg2576 mice was accompanied by a small but significant decrease in Zn levels (Maynard et al. 2002), whereas the Cu increases in APP–/– mice were accompanied by nonsignificant increases in Zn (White et al. 1999b). Interestingly, in both studies, the molar Zn changes were of similar magnitude to the molar Cu changes. However, because of the greater abundance of Zn in the brain, these changes corresponded to a smaller percentage of total Zn than Cu. The effects on Zn could therefore be a direct result of APP/Aβ expression.
Both APPsw (Tg2576) and APP-C100 mice displayed significant elevations in Mn levels (Maynard et al. 2002). Because Mn does not appreciably interact with Aβ or APP (Bush et al. 1993; Bush et al. 1994a; Bush et al. 1994b; Atwood et al. 1998), the increased Mn levels are more likely a secondary effect of altered metal homeostasis. Elevated Mn levels have also been found in AD brain (Rao et al. 1999). Most Mn in the brain is bound to metalloproteins such as glutamine synthetase and mitochondrial Mn superoxide dismutase (SOD2). A portion of Mn also exists in the synaptic vesicles of glutamatergic neurones and is released into the synaptic cleft along with glutamate, participating in the regulation of synaptic neurotransmission (Takeda 2003).
Abnormally high concentrations of brain Mn have been shown to cause an irreversible neurological syndrome similar to Parkinson's disease (Aschner 1997), hence, the elevated Mn in AD brain may contribute to the pathology. Decreased Cu and increased Mn levels have been observed in other neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Creutzfeldt–Jakob disease (CJD) (Kapaki et al. 1997; Wong et al. 2001). Brain tissue from CJD patients displays decreased Cu and increased Mn levels. Furthermore, prion protein extracted from CJD brain displays a decreased Cu and increased Mn contents (Wong et al. 2001). Interestingly, replacement of Cu by Mn facilitates misfolding of the prion protein in favour of higher β-sheet content, protease resistance and loss of antioxidant function (Brown et al. 2000). However, Mn-induced aggregation of Aβ has not been demonstrated by in vitro studies using synthetic peptides (Bush et al. 1994b; Atwood et al. 1998). Decreased Cu and increased Mn levels have also been found in the serum and CSF (Mn increase nonsignificantly) of ALS patients (Kapaki et al. 1997). Although the relationship between Cu and Mn changes is not well understood, opposing changes in the levels of Cu and Mn are emerging as a common trait of neurological diseases.
The Cu-binding domain of APP shows structural homology to Cu chaperones (Barnham et al. 2003b), supporting the notion that the APP (and APLP2) Cu-binding domain functions as a neuronal metal transporter and/or chaperone to modulate Cu homeostasis. Furthermore, APP gene expression has recently been shown to be downregulated by Cu depletion (Bellingham et al. 2004b), suggesting a negative feedback mechanism evolved to preserve intracellular Cu levels. The Cu stores depleted by APP and Aβ overexpression remain unknown, as is the mechanism by which this occurs.
The main problem with distinguishing whether the effects on Cu homeostasis are elicited by the APP N-terminal Cu-binding domain or Aβ, both of which bind and catalyse the reduction of Cu2+, is that most of the models studied to date utilize the overexpression or ablation of APP, resulting in the joint alterations in the levels of both APP and Aβ. The only direct evidence for Aβ causing Cu efflux, independently of APP, is that Cu levels are decreased in mice overexpressing APP-C100 (Maynard et al. 2002). Furthermore, the magnitude of the Cu elevation in APP–/– mice is greater than that in APLP2–/– mice (White et al. 1999b), suggesting that the increased Cu may be due to the joint effects of the APP N-terminal Cu-binding domain and Aβ rather than the action of the N-terminal copper-binding domain alone, because this domain is homologous in both APP and APLP2. No consensus has been reached as to whether APP expression is altered in AD, except in cases resulting from Down's syndrome or head trauma. The majority of genetic and biochemical evidence links increased the production of Aβ or Aβ42 with AD, rather than increased APP expression. Hence, APP-overexpressing models may not be an accurate reflection of the Cu depletion observed in AD. These models do however increase our understanding of the roles of APP and Aβ in metal homeostasis.
Cu promotes the nonamyloidogenic APP processing pathway
An important link between Cu levels and amyloid formation has recently been unveiled by two independent and complementary studies using two different transgenic APP mouse models. Both studies reported decreased constitutive brain Cu levels (Bayer et al. 2003; Phinney et al. 2003) in agreement with earlier findings (Maynard et al. 2002). Elevation of brain Cu levels, either by dietary Cu supplementation (Bayer et al. 2003) or by the introduction of a mutant allele of the CuATPase7b Cu transporter (Phinney et al. 2003), improved the survival of the mice and resulted in a marked decrease in Aβ and amyloid plaque load. These findings suggest that elevated Cu may drive nonamyloidogenic processing of APP, as demonstrated previously in vitro (Borchardt et al. 1999).
This is important, as it suggests that the decreased Cu levels in AD brain could further increase Aβ production, perpetuating a pathogenic cascade of events. However, if low Cu availability in the brain signals the downregulation of APP expression as occurs in vitro (Bellingham et al. 2004b), this may provide a protective mechanism to limit the amount of Aβ production in an environment where amyloidogenic APP processing is favoured. However, in the transgenic mouse models, which use non-APP promotors, this protective mechanism is absent. Hence, Cu supplementation in humans may not be expected to exert such a profound effect on Aβ levels and amyloid formation. Replenishment of deficient Cu stores in humans may decrease the proportion of APP that undergoes amyloidogenic processing; however, concurrent upregulation of APP expression may further add to the net Aβ burden (Figure 1).
Figure 1. Proposed mechanism for copper (Cu) depletion and β-amyloid formation in humans. Increasing age leads to the accumulation of Cu and iron (Fe) deposits, elevated oxidative stress and reduced pH in the brain, all of which may promote the aggregation of Aβ. Oxidative stress and other metabolic stresses may also promote Aβ production by jointly upregulating amyloid precursor protein (APP) expression and driving amyloidogenic processing of APP (Misonou et al. 2000; Paola et al. 2000; Atwood et al. 2003; Cheng & Trombetta 2004). Higher APP expression and elevated Aβ levels cause greater than required Cu export, leading to increased Cu in cerebrospinal fluid (CSF) and serum, and an intracellular (IC) Cu deficiency in the brain. Cu-deficient superoxide dismutase (SOD1) contributes to the reduced antioxidant capacity of the brain, allowing further oxidative stress. Unlike in transgenic mouse models, decreased intracellular Cu levels may downregulate APP expression (Bellingham et al. 2004a) in an attempt to conserve Cu levels. In Alzheimer's disease (AD) brain, this negative feedback must be insufficient to counteract other factors that promote APP expression, Aβ production and Aβ accumulation. Consequently, a Cu deficiency develops along with the classic Aβ-amyloid pathology of AD. Zn – zinc.
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When attempting to predict the effects of Cu supplementation on human AD patients, the potential benefits of restoring the apparent Cu deficiency on enzyme activities and perhaps nonamyloidogenic processing of APP must be weighed against the potential risk of exacerbating the condition by increasing the availability of Cu for the formation of toxic Aβ oligomers and the generation of ROS, as well as possibly increasing the APP expression. The correlation of the findings obtained from mice to humans is limited not only by the non-native regulation of transgenic APP expression and the lack of the full spectrum of AD pathology in transgenic APP mice but also by the differences between humans and mice in their metal-regulatory machinery.
A recent clinical trial of the Cu/Zn chelator clioquinol has revealed promising effects, with a modest slowing of cognitive decline and a parallel decrease in serum Aβ42 (Ritchie et al. 2003). The effects of oral clioquinol treatment on Tg2576 mice were a striking reduction in brain Aβ levels and amyloid plaque load (Cherny et al. 2001), as has been since achieved with another hydrophobic chelator DP-109 (Lee et al. 2004a). Although clioquinol treatment results in decreased Cu, Fe and cobalt levels in nontransgenic mice (Yassin et al. 2000), Tg2576 mice treated with clioquinol displayed paradoxical elevations in brain Cu and Zn levels (Cherny et al. 2001). A plausible explanation for the findings is that clioquinol treatment loosens amyloid plaques and aggregates and solubilizes smaller Aβ oligomers that would otherwise exert toxicity and become sequestered into amyloid deposits. The liberation of Aβ into more soluble forms allows more efficient clearance of Aβ. Hence, with prolonged treatment, much of the existing amyloid burden has been cleared, and the continued growth of amyloid deposits is prevented. The observation that Cu and Zn levels were elevated suggests that by achieving lower total Aβ levels, the excessive Cu export that occurs in Tg2576 mice is attenuated. The elevation in Zn levels, however, cannot be explained by the reduction in Aβ levels, as APP-C100 mice do not display decreased Zn levels (Maynard et al. 2002). The Cu and Zn elevations could be due in part to Cu and Zn ions liberated from amyloid plaques that are still bound to clioquinol in the brain. However, the net increase in Cu and Zn levels in the brain in the presence of fewer amyloid deposits indicates either greater uptake or reduced export. Hence, the Cu and Zn elevations may be a result of favourable changes to metal homeostasis resulting from the clioquinol treatment. Approximately, 15% of plasma Zn in mice is in communication with Zn released in cortical synapses associated with ZnT3 activity (Friedlich et al. 2004). The normalization of plasma zinc (rising from below normal baseline levels) as a result of clioquinol treatment in AD patients might be explained by the dissolution of parenchymal and cerebrovascular amyloid permitting the re-establishment of communication between plasma and synaptic Zn (Ritchie et al. 2003).