J. Neurochem. (2011) 119, 220–230.
Impaired metal ion homeostasis causes synaptic dysfunction and treatments for Alzheimer’s disease (AD) that target metal ions have therefore been developed. The leading compound in this class of therapeutic, PBT2, improved cognition in a clinical trial with AD patients. The aim of the present study was to examine the cellular mechanism of action for PBT2. We show PBT2 induces inhibitory phosphorylation of the α- and β-isoforms of glycogen synthase kinase 3 and that this activity is dependent on PBT2 translocating extracellular Zn and Cu into cells. This activity is supported when Aβ:Zn aggregates are the source of extracellular Zn and adding PBT2 to Aβ:Zn preparations promotes Aβ degradation by matrix metalloprotease 2. PBT2-induced glycogen synthase kinase 3 phosphorylation appears to involve inhibition of the phosphatase calcineurin. Consistent with this, PBT2 increased phosphorylation of other calcineurin substrates, including cAMP response element binding protein and Ca2+/calmodulin-dependent protein kinase. These data demonstrate PBT2 can decrease Aβ levels by sequestering the Zn that promotes extracellular formation of protease resistant Aβ:Zn aggregates, and that subsequent intracellular translocation of the Zn by PBT2 induces cellular responses with synapto-trophic potential. Intracellular translocation of Zn and Cu via the metal chaperone activity of PBT2 may be an important mechanism by which PBT2 improves cognitive function in people with AD.
Bcl-2-associated death promoter
cAMP response element binding protein
extracellular signal-regulated kinases 1/2
glycogen synthase kinase 3
Hank’s buffered saline solution
inductively coupled plasma mass spectrometry
matrix metalloprotease 2
The neurodegenerative disorder Alzheimer’s disease (AD) predominantly affects the elderly, presenting clinically as progressive memory loss and behavioral abnormalities. AD is fatal and there is no cure, and the aging global population means that the large social and economic burden AD already has on our communities and health-care systems is set to increase rapidly. Developing and understanding the mechanism of action of new therapeutics is urgently needed.
Central to the cognitive deficits of AD is the amyloid-β peptide (Aβ), a small amyloidogenic peptide that accumulates in the AD-affected brain (Crouch et al. 2008). Therapeutic strategies have been developed over the years to treat AD by targeting Aβ, but most have ultimately failed to show disease modifying outcomes in large scale trials (see Citron 2010 for review). Such outcomes have raised questions regarding the validity of Aβ as a therapeutic target. However, most therapeutics tested to date include inhibitors of Aβ production, anti-Aβ immunotherapy, and therapeutics that promote Aβ clearance. Consistent to all these approaches is that their mechanism of action is restricted to their direct effect on Aβ levels in the brain. This raises the real possibility that effective therapeutics for AD cannot rely on decreasing Aβ levels in isolation.
An alternate and more holistic AD treatment strategy that may have broader therapeutic value is to target the interaction between Aβ and metal ions (Bush 2003). Interaction with the metal ions zinc (Zn) and copper (Cu) promote Aβ aggregation (Bush et al. 1994; Atwood et al. 1998, 2004) and its neurotoxic potential (Huang et al. 1999a,b; Curtain et al. 2001, 2003; Crouch et al. 2005; Smith et al. 2006, 2007; Deshpande et al. 2009), and in vivo studies show that Aβ accumulation in the brains of mice is dependent on the availability of extracellular pools of Zn (Lee et al. 2002). Preventing Aβ–metal interactions therefore led to the development of a library of metal-protein attenuating compounds of which PBT2, a modified 8-hydroxyquinoline, is the most advanced in clinical testing. In vivo testing of PBT2 has shown oral treatment of transgenic AD model mice restores their cognitive capacity to levels expected for cognitively healthy mice (Adlard et al. 2008), and a phase IIa clinical trial of PBT2 has shown that treatment for 12 weeks at 250 mg/day improved measures of cognitive function in patients with mild AD (Lannfelt et al. 2008, 2009; Faux et al. 2010). Although the initial rationale for using PBT2 was to prevent extracellular Aβ–metal interactions (Bush 2008), the additional capacity for PBT2 to chaperone metal ions across the plasma membrane into cells has been demonstrated (Adlard et al. 2008). This metal chaperone activity for PBT2 has recently been shown to promote neurite extension in vitro, and treatment with PBT2 induces a range of synaptotrophic responses in vivo, including restored dentritic spine density and restored levels of synaptic proteins in an AD mouse model (Adlard et al. 2011). Pre-clinical studies using a diverse range of compounds have shown that treatments capable of increasing intracellular levels of Zn or Cu can inhibit Aβ accumulation (White et al. 2006; Caragounis et al. 2007; Donnelly et al. 2008) and increase cognitive function in AD model mice (Malm et al. 2007; Crouch et al. 2009a). Based on this, the metal chaperone activity of PBT2, in addition to its ability to decrease Aβ levels in interstitial fluid of the brain and cerebrospinal fluid (Adlard et al. 2008; Lannfelt et al. 2008), has been proposed to at least in part contribute to the compound’s therapeutic efficacy. The aim of the present study is to examine cellular effects of the metal chaperone activity of PBT2.
The following is a brief summary of the methods. Further details are included in Appendix S1.
Cell culture and treatments
Neuroblastoma SH-SY5Y cells were maintained in Dulbecco’s modified Eagle’s medium:F12 media supplemented with 10% (v/v) serum. Cells were treated with PBT2 or 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol) (CQ) for 1 h and depending on the experiment, treatments were performed in serum-free media, serum-free media supplemented with 10 μM (NH2)2sar (a cell impermeable metal chelator), or metal-free Hank’s Balanced Saline Solution (HBSS). When PBT2 and metal ion co-treatments were performed PBT2 and metal ion stock solutions were combined (PBT2:metal ion molar ratio of 1:1) prior to adding to treatment media. Alternatively, some experiments examined the ability for PBT2 to utilize Zn2 + bound to aggregated Aβ. To achieve this aggregated Aβ with bound Zn2 + was resuspended in 200 μM PBT2 (or water as a control) before diluting into HBSS and adding to cells.
Aβ degradation assays
The degradation of Aβ1-42 was performed as described previously (Crouch et al. 2009b).
Inductively coupled plasma mass-spectrometry (ICP-MS)
Treated cell cultures were centrifuged (1,000 × g, 3 min) to separate cells from the treatment media. Cellular and extracellular levels of Zn and Cu were then measured using ICP-MS as described previously (White et al. 2006).
Labile intracellular Zn
Changes in labile intracellular Zn were detected by incubating cells with zinquin. Intracellular zinquin fluorescence increases when the fluorophore binds labile Zn (Haase and Beyersmann 1999).
Preparation of cell extracts and Western blot analyses
After treating for 1 h with PBT2 (or CQ) cells were collected and cell extracts prepared using Cytobuster Protein Extraction reagent as described previously (Crouch et al. 2009a).
Glycogen synthase kinase 3 (GSK3) activity in cell extracts was determined by measuring the rate of transfer of 32P from [γ-32P]-ATP into a synthetic GSK3 substrate (Cole and Sutherland 2008).
Calcineurin activity was measured using a Calcineurin Activity Assay kit (Merck).
Caspase 3 activity
Caspase 3 activity was measured using a Caspase 3 Activity Assay kit (R&D Systems).
PBT2 induced changes in GSK3 phosphorylation are dependent on extracellular Zn and Cu
Treating SH-SY5Y cells with PBT2 in serum-free cell culture media induced an increase in cellular levels of GSK3α/β phosphorylated at the inhibitory serine 21/9 residue (ser21/9 on GSK3α/β) (Fig. 1a and b and Figure S1). The absence of a dose-response effect above 2.5 μM indicated that maximal GSK3 phosphorylation had occurred. Consistent with phosphorylation of GSK3α/β at the serine 21/9 residue negatively regulating GSK3 kinase activity, changes to GSK3 activity paralleled the observed changes to GSK3 phosphorylation with maximal inhibition of GSK3 activity at 2.5 μM PBT2 (Fig. 1b). Like PBT2, CQ is a cell permeable 8-hydroxyquinoline derivative capable of binding metal ions and translocating them into cells (White et al. 2006). CQ has also generated positive outcomes in clinical and pre-clinical AD trials (Cherny et al. 2001; Ritchie et al. 2003) possibly due to its metal ionophore activity (White et al. 2006). The data in Fig. 1a and b indicate PBT2 is more effective than CQ in inducing GSK3 phosphorylation.
Given that PBT2-mediated cellular effects are hypothesized to involve PBT2-metal interactions (Adlard et al. 2008, 2011), and because the cell culture media we used contains Zn at 1.5 μM (Manufacturer’s specification sheet), we tested whether extracellular metal ions in the cell culture media affected the capacity for PBT2 to induce intracellular GSK3 phosphorylation. First we examined the effects of PBT2 on extracellular and cellular levels of Zn. Figure 1c shows that treating with 10 μM PBT2 for 1 h significantly decreased extracelular levels of Zn and that this was accompanied by an increase in cellular levels of Zn. The same result was observed for levels of Cu in the media and in the cells (Figure S2). Changes to cellular levels of Cu compared to Zn however were quantitatively minor due to the low (5.2 nM) concentration of Cu in the cell culture media. However, when Cu and Zn were examined at the same concentration, both metals were able to support PBT2 induced GSK3 phosphorylation (Figure S3a), whereas a number of other metal ions could not (Figure S3b).
To confirm that PBT2-mediated translocation of Zn (and Cu) from the extracellular media into the cells contributed to the phosphorylation of GSK3, we pre-treated the cell culture media with (NH2)2sar, a cell-impermeable high affinity, broad spectrum metal ion chelator, before adding PBT2. Figure 1d shows that (NH2)2sar prevented the capacity for PBT2 to induce intracellular GSK3 phosphorylation. Furthermore, when cells were treated in metal ion free HBSS instead of cell culture media, PBT2 alone failed to increase GSK3 phosphorylation, but co-treating with equimolar Zn restored the capacity for PBT2 to induce GSK3 phosphorylation (Fig. 2a and b). The increase in GSK3 phosphorylation in cells treated with PBT2 + Zn correlated with an increase in total cellular levels of Zn (Fig. 2c). The ICP-MS analysis used to generate data shown in Fig. 2c, however, does not differentiate between labile Zn within the cells and Zn bound to cellular proteins. Furthermore, it cannot establish whether Zn is released from the PBT2 once the PBT2–Zn complex enters the cell. To examine whether the PBT2 + Zn treatment increases labile Zn within cells we therefore exposed cells to the Zn fluorophore zinquin after treating with PBT2 + Zn. Data in Fig. 2d show zinquin fluorescence only increases in cells treated with PBT2 + Zn. These data indicate that once inside the cell the PBT2–Zn complex dissociates to increase levels of labile Zn. They also indicate that an increase in labile Zn within the cell is required to induce inhibitory phosphorylation of GSK3 (Fig. 2a).
PBT2 prevents the formation of Zn-induced protease resistant Aβ aggregates
We have previously shown that Zn induces protease resistance in synthetic Aβ1-42 (Crouch et al. 2009b). Here, we have shown that while the presence of Zn makes Aβ1-42 resistant to degradation by the protease matrix metalloprotease 2 (MMP2), the additional presence of PBT2 prevents the formation of this protease resistance (Fig. 3). Notably, the presence of PBT2 in the absence of Zn did not affect the capacity for MMP2 to degrade Aβ1-42 (Fig. 3).
Aβ : Zn aggregates are a sufficient source of Zn for PBT2 to induce GSK3 phosphorylation
We examined whether PBT2 could utilize Zn bound to Aβ to induce phosphorylation of GSK3. To achieve this, aggregated forms of Aβ1-42 with and without bound Zn were prepared, then these Aβ preparations were treated with PBT2 before adding them to cells in culture. Aβ1-42 aggregated in the absence of Zn had no effect on cellular GSK3, regardless of the presence of PBT2 (Fig. 4a and b). Aβ1-42 aggregated in the presence of Zn (Aβ:Zn) also had no effect on cellular GSK3. However, treating the Aβ:Zn with PBT2 before exposing it to cells induced significant GSK3 phosphorylation (Fig. 4a and b), indicating that PBT2 is able to access Zn bound to aggregated Aβ and transport the metal into cells to induce GSK3 phosphorylation. To confirm the role for PBT2-mediated intracellular translocation of Zn in this experiment we again measured cellular Zn levels. PBT2 increased cellular Zn levels when ZnSO4 was the source of extracellular Zn and also when Aβ:Zn aggregates were the source of extracellular Zn (Fig. 4c). Notably, treating cells with Aβ:Zn aggregates in the absence of PBT2 also increased cellular levels of Zn (Fig. 4c). This latter result indicated that increasing cellular Zn per se is not sufficient to induce increased phosphorylation of GSK3, because treating cells with Aβ:Zn aggregates did not alter levels of phosphorylated GSK3 (Fig. 4a and b). To confirm this, we again probed treated cells with zinquin. The zinquin data show that treating cells with PBT2 + Zn strongly increases the pool of labile Zn within the cell (Fig. 4d, as per Fig. 2d). Treating cells with Aβ:Zn aggregates on their own did not increase labile Zn within the cell, but co-treating with PBT2 and Aβ:Zn did increase labile Zn within the cell (Fig. 4d). Compared with PBT2 + Zn, the increase in zinquin fluorescence in cells treated with PBT2 + Aβ:Zn was modest, but this result parallels the obsevered changes in GSK3 phosphorylation (Fig. 4a and b). Consistent with data in Fig. 2, these data indicate that PBT2 increases inhibitory GSK3 phosphorylation by increasing levels of labile Zn within the cell.
PBT2 treatment inhibits the phosphatase calcineurin
To examine the mechanism through which PBT2 induced inhibitory phosphorylation of GSK3, cells were treated in HBSS with PBT2 + Zn. The PBT2 + Zn treatment again increased levels of phosphorylated GSK3, and consistent with their capacity to directly phosphorylate GSK3 at residue ser9, the up-stream kinases protein kinase B (Akt) and extracellular signal-regulated kinases 1/2 (ERK1/2) were both activated (Fig. 5a). However, co-treating cells with inhibitors of Akt and ERK1/2 kinase activity (PD98059 and LY294002) was unable to prevent PBT2 + Zn induced GSK3 phosphorylation, despite decreasing activation of their respective kinase targets (Fig. 5a). This result indicates that although Akt and ERK1/2 activation may contribute to the positive therapeutic effects of PBT2, the mechanism through which PBT2 induces GSK3 phosphorylation under these experimental conditions appears independent of the activation of these up-stream kinases. Additional GSK3 phosphorylating kinases (casein kinase 1, protein kinase A, and protein kinase C) were also examined but these kinases were not affected by the PBT2 + Zn treatment (Figure S4).
In pursuing the mechanism of GSK3 phosphorylation further, we examined the phosphatase calcineurin (protein phosphatase 3, formerly protein phosphatase 2B) because of its ability to dephosphorylate GSK3 at the inhibitory phosphorylation sites (Lee et al. 2005; Kim et al. 2009). Measuring phosphatase activity using a selective calcineurin substrate revealed PBT2 + Zn potently inhibited calcineurin activity (Fig. 5b). These data indicate that increased GSK3 phosphorylation in PBT2-treated cells is caused by inhibition of the phosphatase calcineurin. This result is consistent with a previous study that shows decreasing calcineurin levels by siRNA knockdown increases GSK3 phosphorylation (Kim et al. 2009). Consistent with the GSK3 phosphorylation data in Fig. 2 the inhibition of calcineurin in PBT2-treated cells is dependent on the presence of Zn (Fig. 5b).
Inhibition of calcineurin induces multiple down-stream cellular effects
Calcineurin activity regulates a broad range of cellular processes, many of which are pertinent to AD pathology and neuronal synaptic function. For example, through its capacity to increase inhibitory phosphorylation of GSK3β (Figs 1, 2 and 4) calcineurin inhibition has the potential to decrease tau phosporylation, a widely recognized GSK3 substrate (Hanger et al. 1992; Mandelkow et al. 1992). Consistent with this, we found treating cells with PBT2 + Zn decreased tau phosphorylation at the GSK3 phosphorylation site ser396 (Fig. 6a). Additional calcineurin substrates include the transcription factor cAMP response element binding protein (CREB) and the kinase CaMKII, both of which become activated when phosphorylated. Again consistent with its capacity to inhibit calcineurin activity, cells treated with PBT2 + Zn contained elevated levels of phosphorylated CREB and CaMKII (Fig. 6b and c). Finally, we examine the effects of PBT2 + Zn on caspase 3 activity. Levels of active caspase 3 are regulated in part by the phosphorylation state of Bcl-2-associated death promoter (BAD) (i.e. BAD dephosphorylation is pro-apoptotic). Because calcineurin is responsible for dephosphorylating BAD, calcineurin activity can therefore promote caspase 3 activity via BAD (Wang et al. 1999). Although we were unable to detect PBT2-mediated changes to BAD phosphorylation (not shown), we were able to show PBT2 + Zn significantly decreased caspase 3 activity (Fig. 6d).
Aβ interactions with the metal ions Cu and Zn lead to peptide aggregation (Bush et al. 1994; Atwood et al. 1998, 2004) and the formation of soluble toxic oligomers (Huang et al. 1999a,b; Curtain et al. 2001, 2003; Crouch et al. 2005; Smith et al. 2006, 2007; Deshpande et al. 2009). For this reason, the original rationale for developing a library of metal-protein attenuating compounds as a strategy to treat AD was to inhibit Aβ interactions with Cu and Zn. PBT2 is the lead therapeutic candidate from this library and in a double-blind, placebo-controlled phase IIa clinical trial PBT2 has demonstrated potential as a novel disease modifying therapy for AD (Lannfelt et al. 2008, 2009; Faux et al. 2010). Consistent with the original rationale the data presented in Fig. 3 show that PBT2 is able to inhibit Zn-induced formation of protease resistant Aβ aggregates. Promoting Aβ degradation by preventing Aβ:Zn interactions is consistent with our previous work that shows Aβ:Zn aggregates form a secondary structure in which the MMP2 protease cleavage site becomes inaccessible (Crouch et al. 2009b). In the absence of such Zn-induced secondary structure, the degradation of Aβ by MMP2 (and other proteases) is rapid (Crouch et al. 2009b), and could therefore make the Aβ ammenable to degradation by proteases already present within the brain. This may explain the rapid decrease in brain Aβ levels in mice treated with PBT2 where interstitial Aβ levels dropped by ∼50% 4–8 h after PBT2 administration (Adlard et al. 2008).
The data in Fig. 3 also illustrate a salient attribute of the metal-protein attenuating compound library; the affinity of the molecule for Cu and Zn is high enough that it can inhibit Aβ/metal interactions but not so high that it inhibits the actions of essential metal-dependent enzymes. The MMP2 enzyme that degrades Aβ requires Zn for optimal hydrolytic function (Diaz and Suarez 2007). The data in Fig. 3 therefore demonstrate that although the metal-binding activity of PBT2 was strong enough to inhibit Aβ/Zn interactions it did not impair the normal Zn-dependent activity of MMP2.
The high-affinity membrane-impermeable chelator (NH2)2sar prevents PBT2 from inducing GSK3 phosphorylation (Fig. 1d), indicating that PBT2 requires an interaction with extracellular metals for its effect on GSK3. Consistent with this the titration of Cu and Zn back into metal-free media restored the ability for PBT2 to induce GSK3 phosphorylation (Fig. 2a and b, Figure S3a). Recent studies have highlighted that a range of structurally diverse compounds capable of increasing bioavailable Cu and Zn are able to activate neuroprotective cell signaling cascades (White et al. 2006; Malm et al. 2007; Donnelly et al. 2008; Crouch et al. 2009a). As an 8-hydroxyquinoline PBT2 coordinates Cu and Zn in a 2 : 1 ratio and this is accompanied by a deprotonation of the phenol proton. This results in PBT2 forming a neutral complex with metal ions that is capable of crossing cellular membranes. Consistent with this, the data in Fig. 1c and Figure S2 show that PBT2 can transport Zn and Cu ions into the cell. Once inside cells the moderate affinity that PBT2 has for Cu and Zn means that the metals become bioavailable (i.e. able to dissociate from the PBT2), and this is supported by data in Figs 2d and 4d that shows PBT2 increases labile Zn within the cell. The increase in bioavailable metal initiates a signaling cascade including inhibitory phosphorylation of GSK3 (Figs 1, 2 and 4). There is a rapidly growing body of literature to support GSK3 inhibition as a valid therapeutic target in AD (Martinez and Perez 2008).
Inhibitors of Akt and ERK1/2 activation were ineffective in preventing GSK3 phosphorylation (Fig. 5a), indicating that direct phosphorylation of GSK3 by Akt or ERK1/2 does not contribute to the observed increase in phospho-GSK3 after treating with PBT2. However, these data do not exclude the possibility that Akt and ERK1/2 activation contribute to the in vivo therapeutic effects of PBT2, as the activation of both of these kinases has the potential to confer multiple neurotrophic effects (Read and Gorman 2009; Samuels et al. 2009; Shioda et al. 2009).
The phosphorylation state of any protein reflects a balance between the relative activity of kinases and phosphatases. Given the data shown in Fig. 5a, the effects of PBT2 on calcineurin were examined because calcineurin is a phosphatase that directly dephosphorylates GSK3 at the inhibitory serine residues (Kim et al. 2009). Treating with PBT2 or Zn alone had no effect, but treating with PBT2 + Zn together inhibited calcineurin activity (Fig. 5b). These data therefore indicate PBT2 promotes GSK3 phosphorylation by inhibiting the dephosphorylating activity of calcineurin. The inhibitory effect of PBT2 towards calcineurin activity may involve PBT2-mediated delivery of Zn to an intracellular environment where the Zn is able to directly interact with the phosphatase, as previous studies have already shown the capacity for Zn to directly inhibit calcineurin activity (Takahashi et al. 2003; Tanaka et al. 2005). Indirect activity of the PBT2-delivered Zn on calcineurin, however, cannot be excluded. One possibility in this regard is that PBT2 increases GSK3 phosphorylation by decreasing levels of Aβ. Consistent with this, the presence of Aβ is known to induce GSK3 activation (Resende et al. 2008), and the data presented in Fig. 3 demonstrate PBT2 promotes the degradation of an otherwise protease resistant form of Aβ. It is unclear, however, whether this mechanism contributes significantly to the observed increases in GSK3 phosphorylation in our present work because our experiments used un-transfected SH-SY5Y cells in which Aβ levels are non-detectable.
This study provides new data about the mechanisms that underlie the therapeutic efficacy of AD treatments that increase intracellular levels of bioavailable metals, and suggests that calcineurin inhibition has the potential to attenuate symptoms of AD that are the consequence of decreased synaptic function. Supporting this, we examined a number of potential downstream consequences of calcineurin inhibition and found PBT2 + Zn decreased tau phosphorylation, increased CREB and CaMKII phosphorylation, and decreased caspase 3 activity (Fig. 6). These responses are all directly or indirectly regulated by calcineurin activity (Wu et al. 2007; Yamin 2009). Calcineurin activity is central to many aspects of synaptic plasticity (Groth et al. 2003; Jurado et al. 2010) and as a consequence the inhibition of calcineurin has already been shown to improve synaptic function (Jouvenceau and Dutar 2006).
Evidence for excess calcineurin activity as a causative factor in AD is rapidly expanding. In vitro studies have shown exposure to Aβ increases calcineurin activity in primary cultures of rat brain cortical neurons (Agostinho et al. 2008), and in vivo studies have shown proximity to Aβ plaques in the brain increases neuronal calcineurin activity (Kuchibhotla et al. 2008). Directly pertinent to the potential for excess calcineurin activity to cause synaptic failure in the AD brain, Aβ has been shown to cause extensive morphological changes to neurons (such as dentritic spine loss and neurite dystrophy) via a mechanism involving calcineurin activation (Wu et al. 2010). These studies indicate that Aβ elevates calcineurin activity above basal levels. Moreover, biochemical analyses of post-mortem AD brain have shown altered calcineurin metabolism and markers of elevated calcineurin activity, and these changes correlated directly with pre-mortem measures of cognitive function (Abdul et al. 2009). Furthermore, analyses of brains from subjects with mild cognitive impairment contain elevated levels of cleaved, constitutively active calcineurin, and this same activating cleavage of calcineurin can be induced by oligomeric Aβ (Abdul et al. 2011). Given that Aβ is widely recognized as a primary cause of neurotoxicity and cognitive impairment in AD, it follows that the inhibition of calcineurin activity has been shown to prevent Aβ-induced apoptosis in vitro (Agostinho and Oliveira 2003) and to improve cognitive capacity in an Aβ mouse model for AD (Taglialatela et al. 2009). Furthermore, the calcineurin inhibitor FK506 has recently been shown to promote dentritic branching and spine density in the mouse brain (Spires-Jones et al. 2011). Collectively, these studies provide compelling evidence that excessive calcineurin activity contributes to synaptic failure and that the increased calcineurin activity may be a direct consequence of increased Aβ in the brain. The published data showing the specific calcineurin inhibitor FK506 can generate therapeutic outcomes in vivo in mouse models of AD (Taglialatela et al. 2009) and prion disease (Mukherjee et al. 2010) indicate the therapeutic potential of targeting calcineurin. Nonethless, the validity of calcineurin as a therapeutic target in neurodegenerative disease still requires considerable research attention. Highlighting this is discrepancy in the literature on whether calcineurin activity is decreased or increased in the AD-affected brain. This apparent discrepancy is discussed well by Abdul et al. (2009), drawing attention to additional factors such as sub-cellular location of the calcineurin and the role of endogenous regulators of calcineurin activity. More pertinent to the present study, however, is data showing calcineurin directly dephosphorylates tau (Rahman et al. 2006), indicating that inhibition of calcineurin could potentially exacerbate tau hyperphosphorylation in AD. The data in Fig. 6, however, do not support this possibility because the inhibition of calcineurin in our model with the PBT2 + Zn treatment decreased tau phosphorylation at serine 396. Furthermore, over-expressing constitutively active calcineurin has been shown to increase tau phosphorylation at serine 396 via a mechanism involving dephosphorylation of GSK3 (Kim et al. 2009).
The data presented here provide new mechanistic information on the cellular activity of PBT2, a candidate therapeutic compound for AD (Lannfelt et al. 2008, 2009; Faux et al. 2010). In addition to promoting Aβ degradation by preventing Zn-induced formation of protease resistant Aβ aggregates, the ability for PBT2 to translocate Zn and Cu into cells via its metal chaperone activity activates cellular pathways directly associated with the regulation of synaptic activity. Many of these pathways are activated by inhibition of calcineurin, and are consistent with the ability for PBT2 to promote synaptic long-term potentiation in vitro and to increase markers of synaptic strength in vivo (Adlard et al. 2008, 2011). The salutary properties of PBT2 are related to its moderate affinity for Cu and Zn; the compound has sufficient affinity to inhibit the formation of metal mediated toxic forms of Aβ, and at the same time this affinity is sufficiently low enough that once inside the cell the neutral PBT2–metal complexes are able to make the Cu and Zn bioavailable to initiate neuroprotective cell signaling. In these functions, PBT2 behaves like a metal chaperone able to take Cu and Zn from a location where they are potentially harmful (e.g. in amyloid outside the cell) to a location where they are beneficial. Supporting this, recent work describing the biological function of the amyloid precursor protein as a neuronal ferroxidase has shown that elevated levels of extracellular Zn inhibit amyloid precursor protein ferroxidase activity (Duce et al. 2010). Decreasing excess extracellular Zn with a metal chaperone could be neuroprotective in this context.
To date attempts at dissecting out specific aspects of AD biology and targeting them in isolation have failed. The use of metal chaperones such as PBT2 offers the opportunity to take a more holistic approach to treating AD as we know that these molecules not only promote Aβ degradation, but also inhibit GSK3, tau phosphorylation and calcineurin. Targeting multiple aspects of AD biology holds greater therapeutic potential than methods designed to treat just individual components of this complex disease.
This work was supported by the Australian National Health and Medical Research Council (Program Grant No. 400202). AIB is an Australian Research Council Federation Fellow. KJB is a National Health and Medical Research Council Senior Research Fellow.