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Keywords:

  • Alzheimer's disease;
  • β-amyloid;
  • clioquinol;
  • imaging;
  • zinc

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Disclosure
  9. References

Neocortical β-amyloid (Aβ) aggregates in Alzheimer's disease (AD) are enriched in transition metals that mediate assembly. Clioquinol (CQ) targets metal interaction with Aβ and inhibits amyloid pathology in transgenic mice. Here, we investigated the binding properties of radioiodinated CQ ([125I]CQ) to different in vitro and in vivo Alzheimer models. We observed saturable binding of [125I]CQ to synthetic Aβ precipitated by Zn2+ (Kd = 0.45 and 1.40 nm for Aβ1-42 and Aβ1-40, respectively), which was fully displaced by free Zn2+, Cu2+, the chelator DTPA (diethylene triamine pentaacetic acid) and partially by Congo red. Sucrose density gradient of post-mortem AD brain indicated that [125I]CQ concentrated in a fraction enriched for both Aβ and Zn, which was modulated by exogenous addition of Zn2+ or DTPA. APP transgenic (Tg2576) mice injected with [125I]CQ exhibited higher brain retention of tracer compared to non-Tg mice. Autoradiography of brain sections of these animals confirmed selective [125I]CQ enrichment in the neocortex. Histologically, both thioflavine-S (ThS)-positive and negative structures were labeled by [125I]CQ. A pilot SPECT study of [123I]CQ showed limited uptake of the tracer into the brain, which did however, appear to be more rapid in AD patients compared to age-matched controls. These data support metallated Aβ species as the neuropharmacological target of CQ and indicate that this drug class may have potential as in vivo imaging agents for Alzheimer neuropathology.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Disclosure
  9. References

Accumulation of β-amyloid (Aβ) in the brain as diffuse and mature plaques as well as cerebrovascular deposits are hallmarks of Alzheimer's disease (AD) (Glenner & Wong, 1984; Masters et al., 1985). However, the in vivo dynamics of the formation of these proteinaceous structures as well as their exact contribution to AD progression are not fully understood. Therefore, amyloid-imaging agents that allow observation of the in vivo amyloid formation and load are expected to improve the understanding of this pathology and assist in diagnosis. Recently, thioflavine-T derivatives have been developed, which specifically stain amyloid aggregates enriched in β-sheet structure (Klunk et al., 2003, 2004). Because many different proteins form amyloid, it is important to develop detection agents that are selective for Aβ aggregates.

There is increasing evidence that Aβ deposition in AD is influenced by endogenous cerebral transition metals (Bush, 2003). Aβ is a copper/zinc metalloprotein (Bush et al., 1994b; Atwood et al., 1998) that becomes redox-active and toxic (Huang et al., 1999; Opazo et al., 2002) when inappropriately bound to these metals. Zinc (Zn), copper (Cu) and iron (Fe) accumulate in senile plaques (SP) in Alzheimer's disease (Lovell et al., 1998; Suh et al., 2000) but only Zn and Cu have been found to coordinate Aβ directly in AD-affected brain (Opazo et al., 2002; Dong et al., 2003). Cu/Zn chelators dissolve Aβ deposits from AD-affected post-mortem brain tissue (Cherny et al., 1999). The Tg2576 transgenic mouse model for AD also develops Aβ plaques containing high concentrations of Zn (Lee et al., 1999), which are largely (≈80%) induced by Zn2+ released during neurotransmission and are concentrated in synaptic vesicles through the activity of Zn2+ transporter 3 (ZnT3) (Lee et al., 2002).

Clioquinol (iodochlorohydroxyquinoline – CQ) is a quinoline chelator that crosses the blood–brain barrier (Padmanabhan et al., 1989) and inhibits the precipitation of Aβ by Cu2+ and Zn2+ (Cherny et al., 2001), rescuing the toxicity of Aβ in neuronal cell culture (Abramov et al., 2003). Delivered orally, it markedly inhibits cerebral Aβ accumulation in the Tg2576 mouse and improves the neurological scores of the treated animals (Cherny et al., 2001). A recent pilot phase 2, double-blind, placebo-controlled clinical study showed that cognitive decline in AD patients administered CQ orally was significantly slowed compared with placebo control. These effects correlated with changes in plasma Zn and Aβ levels (Ritchie et al., 2003). CQ has also been recently reported to halt cognitive deterioration and improve brain glucose metabolism monitored by positron emission tomography (PET) in subjects with familial AD (Ibach et al., 2005).

While difficulties in large-scale chemical synthesis have been an obstacle to the escalation of clinical trials of CQ, new compounds interdicting the metal-binding properties of Aβ are emerging through transgenic mouse studies (Lee et al., 2004) and early-phase clinical trials (http://www.pranabio.com/research/). Meanwhile, CQ remains an important compound for characterizing the biochemistry of AD in preclinical models. Neurochemical studies to date suggest further that CQ might be adapted as an imaging probe to monitor the progression of Aβ pathology. Here we report that [125I]CQ specifically binds to Aβ-Zn synthetic complexes and concentrates with the Aβ from AD post-mortem brain fractions in a manner which is modulated by Zn2+. The brain and blood pharmacokinetics of [125I]CQ were different in Tg2576 mice compared to control mice. [125I]CQ retention was greater in the brains of Tg2576 mice and was associated with [125I]CQ binding to neocortical structures in brain sections that was not observed in non-Tg mice. Both preamyloid and ThS-positive amyloid aggregates were identified as targets of [125I]CQ. These data support zinc-metallated Aβ as the primary target of CQ neuropharmacological activity and indicate that [125I]CQ could be a prototype for a new class of AD imaging agent.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Disclosure
  9. References

In vitro binding affinity of [125I]CQ for Aβ–Zn aggregates

We carried out saturation binding experiments with [125I]CQ by incubating Aβ1-40-Zn and Aβ1-42-Zn aggregates in the presence of increasing concentrations of [125I]CQ up to 2 nm (2125 Ci mmol−1). Under these conditions, we observed one affinity class of saturable binding site of CQ for each type of aggregate (Fig. 1A), but the [125I]CQ affinity was higher for Aβ1-42-Zn (Kd = 0.45 nm) than for Aβ1-40-Zn (Kd = 1.40 nm) complexes (Fig. 1A). Moreover, Aβ1-42-Zn exhibited nearly double the [125I]CQ Bmax compared to binding to Aβ1-40-Zn aggregates (Bmax of 58.7 and 37.7 pm, respectively) (Fig. 1A). Because CQ has a low partitioning coefficient in aqueous solution, it was not technically feasible to use unlabelled CQ in a competition analysis. Also, because the [125I]CQ forms a presumed ternary complex with Zn2+-Aβ, analysis had to be performed under conditions where the Zn2+ is not dissociated from the peptide by the chelating action of the CQ. Since CQ and Aβ have similar affinities for Zn2+ (high nanomolar) (Padmanabhan et al., 1989; Bush et al., 1994a), the maximum concentration of CQ used in the saturation assay was ≤ 1% of the concentration of Aβ:Zn2+ present.

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Figure 1. In vitro binding affinity of [125I]CQ for Aβ-Zn aggregates. (A) Scatchard plots of [125I]CQ binding to Aβ1-40-Zn (open circled) and Aβ1-42-Zn (filled circles) aggregates. Kd values of 1.40 pm and 0.45 pm were calculated for Aβ1-40-Zn and Aβ1-42-Zn complexes, respectively. (B) Effect of different compounds on the [125I]CQ binding to Aβ42-Zn complexes. Results are means ± SEM.

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We next tested the [125I]CQ binding for the Aβ1-42-Zn aggregates in the presence of classical amyloid probes (Congo red and thioflavine-T), ZnCl2, CuCl2, and metal chelators glycine (which weakly chelates Zn2+, log K1 = 5.16) and diethylene triamine pentaacetic acid (DTPA) (a high-affinity Zn2+ chelator, log K1 = 18.7). DTPA (Ki = 30.8 ± 2.6 µm), ZnCl2 (Ki = 14.8 ± 3.8 µm) and CuCl2 (Ki = 9.7 ± 0.1 µm) were the most effective competitors for [125I]CQ, followed by Congo red (Ki = 71.4 ± 18.2 µm) (Fig. 1B). ThT and glycine did not interfere with [125I]CQ binding of Aβ1-42-Zn aggregates. These data indicate that CQ binding on Aβ-Zn complexes reflects a specific interaction modulated by the colocalization of Zn2+. The CQ binding site for Aβ-Zn complexes either partially overlaps with the Congo red binding site, or Congo red has some metal binding affinity. However, the CQ binding site on Aβ: Zn2+ is totally separate from the Aβ binding site occupied by thioflavine-T.

Zinc and β-amyloid levels modulate the interaction of [125I]CQ with brain extracts

We analysed the interactions of [125I]CQ with human cortex homogenates in sucrose gradients to gauge the potential of the tracer to identify native Aβin vivo. Because the average levels of Zn in the neocortex are ≈100 µm (Frederickson et al., 2005), unlabeled CQ (75 µm total, 99% labeled +1% unlabeled) was added to the cortical homogenates so that the tracer would not be overwhelmed by the Zn background and so be unable to form ternary complexes with the metallated peptide. Ultracentrifugation of the postnuclear homogenate (frontal gray matter) through a sucrose gradient revealed a differential profile of [125I]CQ between AD and age-matched normal control (AC) samples (Fig. 2A). For AD samples, the [125I]CQ signal was markedly enriched compared to AC samples in four fractions (numbers 4–7) comprising the center of the sucrose gradient (0.8–1.3 M sucrose).

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Figure 2. In vitro interaction of [125I]CQ with human brain extracts. (A) [125I]CQ sedimentation profile in the presence of AD vs. AC brain samples. (B) Total levels of Zn2+ in brain postnuclear samples from AD and control samples. (C) Levels of Zn2+ in sucrose fraction number 4 of AD and control samples. Results are means ± SE (N = 3; *P < 0.05). (D) [125I]CQ sedimentation profile is modulated by Zn2+ and DTPA. The arrow indicates the reference value. Results are means ± SEM (N = 4; *P < 0.05, ***P < 0.001). (E) Correlation of CQ/Aβ content in fraction 4. One AD sample and one AC sample from the experiment in Fig. 2D was assayed in triplicate for CQ and Aβ concentrations to determine whether the Aβ levels correlated as the Zn2+ levels were manipulated by DTPA (the lowest [125I]CQ and Aβ value in each set of three) or additional Zn2+ (the highest [125I]CQ and Aβ value in each set of three). Values are means ± SD.

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The AD postnuclear samples were found to have higher levels of total Zn compared to the AC samples (Fig. 2B), as well as a twofold increase in the levels of Aβ peptide (data not shown). The levels of other metals (Cu, Fe, Mn, or Al) were not significantly different in the postnuclear homogenate (data not shown). After sucrose gradient separation, AD samples had a significant enrichment in the relative levels of Zn present in fraction 4 (Fig. 2C), the fraction with greatest [125I]CQ differences between AD and control samples (Fig. 2A). Levels of Cu, Fe, Mn and Al were again not different in fraction 4. Because of the specific association between the enrichments of both [125I]CQ and Zn in this fraction, we studied it further to determine whether external manipulation in the levels of Zn2+ could change the CQ profile. We preincubated homogenized human brain samples with [125I]-CQ (18 Ci mol−1) in the absence or presence of additional Zn2+ (+75 µm) or DTPA (750 µm). The samples were then refractionated by ultracentrifugation on a sucrose gradient as described previously. We observed that coincubation with Zn2+ increased the levels of [125I]CQ in fraction 4 of both the AD samples (P < 0.03) and the AC samples (P < 0.04), while DTPA decreased [125I]-CQ binding to baseline values in both AD (P < 0.02) and AC (P < 0.001) samples (Fig. 2D). Furthermore, Zn2+ induced a more marked increase in [125I]CQ retention in this fraction for AD compared to AC samples (approximately twofold higher, P < 0.01), probably because the levels of Aβ in fraction 4, were also approximately twofold higher in the AD samples.

In contrast, DTPA was equally effective in abolishing [125I]CQ binding to fraction 4 in both AD and AC samples, decreasing the levels of [125I]CQ to similar values (P < 0.11). These data are compatible with Zn2+ being a major mediator for the targeting of CQ to Aβ.

To estimate the selectivity of [125I]CQ binding to Aβ in the brain samples, we quantified the [125I]CQ and Aβ levels by Western blotting in AC and AD samples treated, as before (see Fig. 2D) with additional Zn2+ or DTPA to chelate the Zn2+. Both the [125I]CQ and Aβ levels covaried with the available Zn2+ levels in the sample, so that the [125I]CQ and Aβ levels correlated (Fig. 2E). However, the concentration of [125I]CQ was at least 1000-fold greater than Aβ. Therefore, it is likely that Aβ only represents a small proportion of the available protein ligands for the Zn2+ in the sample (which itself is present at much higher, micromolar, levels). Nevertheless, the relationship of CQ to Aβ is clearly modulated by the available Zn2+. The shift in the regression curve to the right in the AD samples compared to the AC samples is most likely explained by the presence of increased Aβ in those samples.

Pharmacokinetics and binding of [125I]CQ in Tg2576

To investigate the interactions of CQ with brain Aβ plaques in vivo, we injected Tg2576 mice and non-Tg littermate controls i.p. with [125I]CQ (25 nmoles; 0.5 µCi). Tg2576 and non-Tg animals were sacrificed 1 h postinjection and the radioactivity entering the brain counted. Tg2576 mice showed 36% higher [125I]CQ brain retention than non-Tg mice at this time (P < 0.05, Fig. 3A). This difference was greater (≈65% increase in Tg mice at 15 min postinjection, P < 0.01) when [125I]CQ (2125 Ci mmol−1) was administered via the lateral tail vein in nonanesthetized animals (Fig. 3B). At 5 min postinjection, [125I]CQ is rapidly incorporated in the brain of both Tg2576 and non-Tg animals (Fig. 3A). There was no difference between Tg2576 and non-Tg mice in brain [125I]CQ levels at 5 min post–IP injection (Fig. 3A), or in brain [125I]CQ levels normalized for blood [125I]CQ levels (Fig. 3D). Therefore the increase in [125I]CQ levels observed in Tg mice at 1 h (Fig. 3A) is unlikely to be due to increased permeability of the blood–brain barrier in the transgenics (Ujiie et al., 2003). At 2–3 h postinjection > 80% of the [125I]CQ had exited the brains of both Tg and non-Tg mice (Fig. 3A,B), indicating a fast clearance of CQ from the brain. We also observed that the rate of CQ clearance from the blood was twofold faster in Tg than non-Tg mice (P < 0.05, Fig. 3C). This may reflect the same gain of function in metal clearance previously observed in both APP Tg mice and in CT100 Tg mice, linked to the overexpression of Aβ/APP (Maynard et al., 2002; Bayer et al., 2003; Phinney et al., 2003).

image

Figure 3. Pharmacokinetics of [125I]CQ in Tg2576 and non-Tg mice mice. (A) Brain [125I]CQ retention in animals i.p. administered with [125I]CQ. Data are means ± SEM from Tg (black circles) (5 min, N = 3; 60 min, N = 4; 120 min, N = 6; 180 min, N = 3) and non-Tg mice (white circles) (5 min, N = 3; 60 min, N = 5; 120 min, N = 5; 180 min, N = 3) (*P < 0.05). (B) Brain [125I]CQ retention in animals i.v. administered [125I]CQ. Data are from Tg2576 (black circles) (15 min, N = 4; 60 min, N = 3; 120 min, N = 3) and non-Tg mice (white circles) (15 min, N = 4; 60 min, N = 3; 120 min, N = 3) (**P < 0.01). (C) Blood [125I]CQ levels same animals studied in A. (D) [125I]CQ brain/blood ratio same animals studied in A.

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To further characterize the retention of [125I]CQ into brain, we calculated the [125I]CQ brain/blood ratio over time after IP injection (Fig. 3D). While the CQ brain/blood ratio for non-Tg mice decreased over time, the CQ brain/blood ratio for Tg mice remained higher than non-Tg mice over the same interval, indicating a significant difference in brain/blood ratio of CQ between the groups that was maximal (+55.6%) at 2 h (P < 0.05). By 3 h postinjection there was no difference in the CQ brain/blood ratio between Tg and non-Tg animals, as brain CQ levels approached the limits of detection. These data are consistent with the presence of metallated Aβ species in Tg2576 brain that accelerate the kinetics of CQ retention and then retard its clearance. Confirming this interpretation, we detected [125I]CQ incorporation into unfixed Tg brain sections by emulsion autoradiography of animals injected with the tracer in vivo (Fig. 4A), which was absent in the control brain (Fig. 4B). [125I]CQ binding was principally associated with punctiform deposits in the neocortex, in the known distribution territory of amyloid plaques. We were also able to detect similar punctate deposits in the hippocampal formation of Tg mice administered with [125I]CQ by the same protocol (Fig. 4C), which was absent in the nontransgenic hippocampal region (Fig. 4D). These data suggest that CQ might interact directly with Aβ amyloid aggregates present in the brains of Tg2576 mice.

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Figure 4. In vivo binding of [125I]CQ to brain structure. Film-emulsion autoradiography staining of [125I]CQ in unfixed coronal brain sections of Tg2576 (A, cortex; C, hippocampus) and non-Tg mice (B, cortex; D, hippocampus) i.v. administered [125I]CQ in vivo. The images are coronal sections representative of three different sections.

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To investigate the colocalization of [125I]CQ with Aβ deposits, we studied post-mortem brain serial sections from double transgenic mice (APP/PS1-dE9) and non-Tg mice that had received intravenous injections of [125I]CQ (Fig. 5). We also studied serial sections of post-mortem brain from double transgenic mice (APP/PS1-dE9) and non-Tg mice incubated with [125I]CQ (0.1 nm) or thioflavine-S (ThS, 0.5%) (Fig. 6). Because aldehyde and other cross-linking fixatives denature metal binding sites, and would disrupt the ability of [125I]CQ to form a ternary complex with Zn2+:Aβ, our histological studies were performed on unfixed tissue which limited the discrimination of the probe colocalization with amyloid structures. However, with both approaches it was apparent that only some but not all amyloid deposits bound [125I]CQ, and conversely [125I]CQ frequently bound to cortex lacking Aβ immunoreactivity or ThS stain. Both [125I]CQ binding and ThS staining were absent in non-Tg control cortex. These data indicate that CQ does not invariably form a stable complex with plaque amyloid. This is not surprising since we would expect that the chelator may be able to distract Zn2+ from Aβ whereupon both the CQ and Aβ may dissipate into cellular compartments or into the periphery. Furthermore, an alternative in vivo target of CQ may possibly be oligomeric forms of preamyloid or diffuse deposit, since not all forms of metal-induced Aβ aggregate are fibrillar (Huang et al., 1997; Yoshiike et al., 2001).

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Figure 5. In vivo localization of [125I]CQ and Aβ-immunoreactive aggregates in brain structures. Representative unfixed coronal brain sections from non-Tg (A) and TgAPP-PS1 (B–D) mice administered with i.v. [125I]CQ in vivo were costained with an Aβ-antibody (1E8). Brown positive aggregates correspond to Aβ aggregates and black positive aggregates correspond to the film-emulsion autoradiography staining of [125I]CQ. Magnification ×40. Six transgenic animals were examined.

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Figure 6. In vitro localization of [125I]CQ and ThS positive amyloid aggregates in Tg brain. Adjacent unfixed coronal hippocampal sections from TgAPP-PS1 mice were stained in vitro with [125I]CQ (0.1 nm) (A) or ThS (0.5%) (C). (B) and (D) corresponds to a magnification of the area delimited by the red square showed in (A) and (C), respectively. Aggregates costained by ThS/[125I]CQ are enclosed in circles. Aggregates stained by ThS (D, white arrow) but not stained by [125I]CQ are indicated (B, black arrow). (A) and (C), magnification ×10. (B) and (D), magnification ×20. Six transgenic mice were examined.

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Pilot [123I]CQ SPECT studies in humans

A pilot study was performed on human volunteers exhibiting mild AD (N = 3) or healthy aging (AC, N = 3), who were injected with [123I]CQ followed by SPECT analysis to show retention of the tracer in the brain. The tracer signal was found to be too low to permit imaging. Tracer counting was possible in the cerebral hemispheres although counts were not sufficiently high to achieve reliable data from other brain regions such as the cerebellum. The movement of tracer into the human brain (Fig. 7) was generally similar to [125I]CQ brain distribution in mice (Fig. 3A,B), with an epoch of rapid increase into the cerebrum (up to 25 min), followed by an epoch of rapid decline (to 90 min), and a relatively long-lasting trough level epoch. While too small a study for statistical comparison, mean maximal brain retention was 65% greater (≈2% ID/L, P < 0.05) in AD than in AC subjects in the rapid retention epoch (0–25 min). Furthermore, tracer levels were 25% lower in AD than in AC subjects in the trough epoch (90–135 min, Fig. 7) suggesting altered distribution of [123I]CQ in AD.

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Figure 7. In vivo SPECT retention of [123I]CQ into human brain. Retention of [123I]CQ over time in the brains of subjects exhibiting mild AD (N = 3) or healthy aging (AC, N = 3) as measured by SPECT.

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No differences were observed in [123I]CQ plasma clearance. At 90 min postinjection, 65% of the radioactivity in plasma corresponded to unmetabolized [123I]CQ. Whole body biodistribution studies showed that [123I]CQ is metabolized in liver and excreted through bile and urine.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Disclosure
  9. References

The therapeutic approach represented by CQ is based upon the metallochemistry of Aβ peptide, thought by some to be central to the neurodegeneration observed in AD (Bush, 2003). We and others have characterized Aβ as a metalloprotein able to coordinate Cu2+, Zn2+ and Fe3+ (Bush et al., 1994b; Atwood et al., 2000; Yoshiike et al., 2001; Opazo et al., 2002; Dong et al., 2003). CQ is an orally bioavailable chelator able to prevent Aβ precipitation by Zn2+ (Cherny et al., 2001) and to rescue Cu2+-mediated Aβ toxicity in cell culture (Abramov et al., 2003). These interactions have been the presumptive mechanism of action of CQ treatment in clearing amyloid deposition in Tg2576 mice (Cherny et al., 2001), in decreasing plasma Aβ1-42 and in increasing plasma Zn in AD patients (Ritchie et al., 2003). Our current findings provide the first evidence of a direct interaction of CQ with metallated Aβin vivo. [125I]CQ showed a saturable binding affinity for Aβ1-40-Zn and Aβ1-42-Zn aggregates of Kd = 1.40 and 0.45 nm, respectively (Fig. 1A). These Kd values are similar to the Kd values reported for other Aβ labelling agents, 125I-tagged styrylbenzene (Zhuang et al., 2001) and 125I-tagged thioflavin derivatives, as well as [3H]BTA1 (Klunk et al., 2003).

The different Kd and Bmax values of [125I]CQ for Aβ1-42-Zn and Aβ1-40-Zn aggregates indicate that there are significant structural differences between these two aggregates. The inhibition studies show that only ZnCl2, CuCl2, and a chelator, DTPA (Ki 14.8 ± 3.8, 9.7 ± 0.1 µm and 30.8 ± 2.6 µm, respectively) were effective competitors for the binding of CQ to Aβ1-42-Zn aggregates. While Congo red was able to partially inhibit CQ binding at high concentrations, ThT did not inhibit the CQ binding to Aβ1-42-Zn aggregates (Fig. 1B). These results suggest that the CQ binding site on Aβ1-42-Zn aggregates partly overlaps with the binding site for Congo red. The Bmax of [125I]CQ was in the pm range, indicating that the maximum of CQ that bound was no more than ≈5% of the maximum amount of CQ present (2 nm). This is probably because the saturable binding we observe involves a [125I]CQ-Zn2+-Aβ ternary complex, and the Zn2+ can dissociate from the peptide as the CQ concentration rises. One mole of Zn2+ binds to Aβ with a Kd of ≈100 nm, and a second mole binds with Kd = 5 µm (Bush et al., 1994a). CQ affinity for Zn2+ is ≈100 nm, with a 2 : 1 stoichiometry (Padmanabhan et al., 1989; Di Vaira et al., 2004). CQ will readily distract the lower affinity bound Zn2+ from Aβ, and therefore the low Bmax probably reflects Zn2+ dissociating from Aβ.

The observation that both a chelator and excess Zn2+ inhibit [125I]CQ binding to Aβ (Fig. 1B) is explained if we consider that the competition analysis compares the effects of a competitor against [125I]CQ after it has formed a precipitated ternary complex with Aβ and Zn (Aβ-Zn-[125I]CQ). [125I]CQ did not bind to Aβ aggregates except in the presence of Zn, indicating that Zn2+ is acting as an ionic ligating bridge. The DTPA must be inhibiting the CQ association to Aβ aggregates by dissociating the Zn2+ ligand into the soluble phase. Indeed, if we had been using CQ at greater than tracer concentrations, it too would cause the Zn2+ to dissociate and so would not appear to label the Zn-Aβ complex.

Another way of competing the [125I]CQ off the Aβ-Zn-CQ ternary complex was to increase the concentration of Zn2+ in the solution. This distracted the [125I]CQ away from binding to Zn-Aβ aggregates, forming free [125I]CQ-Zn complexes that are soluble. As the zinc concentration rises, the metal binding sites present on the Aβ peptide become saturated. After saturation, as the zinc concentration rises further, the excess soluble Zn2+ competes with the [125I]CQ bound to the Aβ-Zn-[125I]CQ ternary complex, from where the [125I]CQ dissociates into the soluble phase forming Zn-[125I]CQ complexes.

We also observed a different sedimentation profile of [125I]CQ labeling in centrifuged brain homogenates from AD cases compared to AC samples (Fig. 2A). Four sucrose gradient fractions (0.8–1.3 M sucrose) containing Aβ peptide were enriched in [125I]CQ levels. This pattern correlated with the higher levels of Aβ and Zn2+ present in AD brain fractions compared to AC samples. Moreover, addition of DTPA and Zn2+ to the human brain samples strongly modulated the sedimentation profile of [125I]CQ (Fig. 2D). These data support the targeting of CQ to metallated human brain Aβ.

Cu, Fe and Zn are enriched in Aβ plaques (Lovell et al., 1998; Suh et al., 2000), but Zn2+ may be most responsible for Aβ amyloid assembly because genetic ablation of the synaptic zinc transporter ZnT3 markedly inhibits both parenchymal and vascular amyloid in Tg2576 mice (Lee et al., 2002; Friedlich et al., 2004). While our findings support the interaction of CQ with Zn2+-mediated Aβ assemblies, the contribution of Cu and Fe to the CQ interactions with Aβ cannot be excluded. Indeed, Cu2+ was able to compete for the binding of CQ to Aβ1-42-Zn aggregates in the in vitro binding assays (Fig. 1B).

According to previous and present in vivo data, three principal steps are envisaged in the mechanism of action of CQ. First, CQ enters to the brain and forms a ternary complex with metallated Aβ (Targeting, fast step); second, CQ abstracts the Cu2+/Zn2+ from Aβ (Transfer, slow step); third, CQ delivers Cu2+/Zn2+ to other metal binding ligands or proteins (Redistribution, fast step) (Cherny et al., 2001). After this, CQ is cleared into the plasma without necessarily promoting the excretion of metal (Ritchie et al., 2003).

The normal mouse brain took up more CQ tracer than could be accounted for by the volume of blood (Fig. 3D). Similarly, tissue from healthy non-Tg mouse brains (Fig. 3), and in vivo tracer analysis in healthy aged human volunteers (Fig. 7) also indicated that CQ enters normal brain tissue. Therefore, CQ may, to some extent, interact with normal tissue components. However, it is clear that the APP transgenic mice enrich the CQ in the neocortex and hippocampus (Fig. 4), further supporting the ability of CQ to selectively interact with Aβ accumulations. Histology indicated that the [125I]CQ bound to both plaques and nonplaque structures (Figs 5 and 6). This is not surprising since CQ was identified on the basis of its ability to prevent metal-mediated Aβ precipitation and radical formation, rather than to prevent fibril formation. Unlike Aβ fibrils, metal-induced Aβ aggregates are readily dissociable (Huang et al., 1997; Cherny et al., 1999, 2001), and therefore [125I]CQ may be accompanying Zn2+ off the amyloid target and into cells during the periods of our studies. The structure of fibrils in plaques is likely to be relatively more static and therefore fibril ligands are likely to be more easily colocalized with plaque. On the other hand, radioactive fibril ligands may be unable to detect the dissociable Aβ conformers that [125I]CQ detects.

By comparison with recent candidate styrylbenzoxazole probes for Aβ tested on a different strain of APP transgenic mice (Okamura et al., 2004), [125I]CQ only had one-tenth the percentage ID g−1 brain retention. The rapid blood–brain barrier (BBB) penetration observed for [125I]CQ in the early minutes post i.v. injection, is compatible with its low theoretical value for topological polar surface area (TPSA, 33.12 Å2) (Ertl et al., 2000). Molecules with values less than 70 Å2 readily cross the BBB (e.g. diazepam, which has a TPSA of 32.7 Å2). Despite this, we found that less than 1% of an injected dose of CQ enters the brain of the Tg2576 transgenic mouse (Fig. 3B). Possibly, blood binding factors like albumin (Hobara & Taketa, 1976) may retard the entry of CQ across the BBB. Despite the relatively inefficient brain retention that we observed, oral CQ treatment markedly decreases cerebral Aβ deposition in this model (Cherny et al., 2001), suggesting that its biochemical effects are potent. Therefore, the efficacy of CQ or similar metal-targeted drug candidates may be improved by pharmacological strategies that increase the compound's brain penetration (e.g. decrease hepatic metabolism, increase blood–brain barrier permeability).

Our pilot data from human subjects with AD and healthy controls (Fig. 7) revealed also that [123I]CQ is taken up and cleared from the brain at rates that were reminiscent of the retention into amyloid-bearing transgenic and normal mouse brain (Fig. 3). While the retention levels were too low to permit SPECT imaging, the differentiation of radiolabeled CQ retention by both APP transgenic mice and in AD subjects is concordant with the tracer interacting with Aβ neuropathology. Greater brain retention for imaging may be enabled by strategies to increase tracer delivery, or modifying formulation. Even if the efficiency of brain uptake of CQ cannot be safely improved, our findings establish the principles of developing the class of compound represented by CQ as potential Aβ ligands for in vivo imaging.

These data also suggest that CQ represents a potential new class of therapeutic because it does not act through a nonspecific brain metal depletion mechanism, such as that involved in chelation therapy. Indeed, treatment of Tg2576 mice with a ‘traditional’ high-affinity metal chelator, TETA, had no effect on brain Aβ levels (Cherny et al., 2001). Instead, the salutory effects of CQ may include the reestablishment of normal metal homeostasis (Ritchie et al., 2003). Supporting this possibility, CQ normalizes brain Cu and Zn levels by elevating levels of these metals by ≈10% in Tg2576 mice (Cherny et al., 2001).

While the clinical utility of CQ remains to be determined, the molecule's ability to be radioiodinated makes it a useful reagent for the exploration of basic neuropathological mechanisms in AD models. Compared to current amyloid-targeting imaging agents, the class of molecule represented by CQ interacts with an alternative, metal-associated, biochemical target, which may be useful in the differentiation of AD pathology.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Disclosure
  9. References

Mice

All mice were housed according to standard animal care protocols, fed ad libitum, and maintained in a pathogen-free environment at MHRI. The transgenic status of all animals was confirmed by PCR of tail snips, using the 3′UTR of the hamster cosmid PrP vector as a hybridization probe (Hsiao et al., 1996), and for overexpression of the Aβ peptide by Western blot (WO2) of post-mortem brain tissue (Cherny et al., 1999). The Tg2576 colony was maintained by crossing female Tg (HuAPP695.SWE)2576 (Tg2576) with C57BL6/SJL F1 males (Jackson Laboratories, Riverside, CA, USA). Non-Tg littermates were used as controls. All single transgenic (Tg2576) and control male mice used in this study were 11–13 months old. Double transgenic mice (APP/PS1-dE9) expressing mutant human presenilin 1 (DeltaE9) and HuAPP695.SWE (Jankowsky et al., 2004) were also obtained from Jackson Laboratories. All double APP/PS1-dE9 and their control counterparts were used at 9 months old.

Binding assay

[125I]CQ was generated by chemical exchange of 125I for the sole iodine atom on CQ (SigmaAldrich, Sydney, Australia) according to published methods (Toyokura et al., 1975). When synthesized in-house, the [125I]CQ product generally achieved specific activity of 18 Ci mol−1. Higher specific activity [125I]CQ (2125 Ci mmol−1) was provided by ANSTO (Sydney, Australia). Aβ peptides 1–40 and 1–42 were synthesized by the W. Keck Laboratory, Yale University, New Haven, Connecticut. Aβ peptide stock solutions were prepared in water and protein concentrations quantified according to established procedures (Huang et al., 1997). Aβ peptide 1–40 or 1–42 (25 µm) were induced to aggregate in the presence of ZnCl2 (25 µm) in buffer PBS (pH 7.4) while stirring at 20 °C for 1 h. To ensure the aggregation of Aβ peptides, the mixtures were incubated at 37 °C for 48 h. Binding studies were carried out in 12 mm × 75 mm borosilicate glass tubes. Aβ-Zn aggregates (200 nm) were added to a mixture containing 0.01–2.0 nm of [125I]CQ (2125 Ci mmol−1) in buffer Tris-HCl (pH 7.35; final volume of 1 mL). The final concentration of ethanol was 10%. Nonspecific binding was defined in the presence of 500 µm DTPA to abolish metal binding to peptide. The mixture was incubated at 20 °C for 1 h, and the bound and free radioactivities were separated by vacuum filtration through Whatman GF/B filters using a Millipore system (Sigma) followed by 3 × 3 mL washes of Tris-HCl containing 10% ethanol at 20 °C. Filters containing the bound [125I]CQ were counted in a gamma counter (LKB 1272, Wallac, Gaithersburg, MD, USA). Specificity of the binding was determined in the presence of different compounds (thioflavine-T, Congo red, DTPA, ZnCl2 CuCl2 and glycine, Sigma, St Louis, MO, USA). Ki values were estimated for each compound.

Biodistribution studies

Intraperitoneal administration of [125I]CQ

Male Tg2576 and non-Tg mice were injected i.p. with 0.5 µCi (5 µL, 100% DMSO) of [125I]CQ (specific activity 18 Ci mol−1), synthesized as described previously (Ando et al., 1974). Sodium pentobarbital was used for anesthesia (40 mg kg−1). The animals were sacrificed at intervals and a blood sample was taken by cardiac puncture. The different organs were dissected, weighed and radioactivity was assessed in a gamma counter.

Intravenous administration of [125I]CQ

Male Tg2576 and non-Tg mice were injected in the tail vein with 1 µCi (20 µL, 60% ethanol/40% 0.1 m H2PO4/HCO3, pH 6.9) of [125I]CQ (specific activity of 2125 Ci mol−1). The animals were sacrificed at intervals and a blood sample was taken by cardiac puncture. The different organs were dissected out, weighed and radioactivity assessed in a gamma counter.

[125I]CQ in vivo autoradiography in Tg and non-Tg mice

Male Tg2576 and non-Tg mice were injected in the jugular vein with 50 µCi of [125I]CQ (specific activity of 2125 Ci mol−1) under sodium pentobarbital anaesthesia (40 mg kg−1). After 10 min, the animals were sacrificed and the brains were dissected out, immersed in isopentane and cooled with liquid nitrogen for 5 min. Brain sections of 40 µm were then cut in a cryostat at −20 °C. The brain sections were adhered to silanized microscope slides and kept frozen at −20 °C for 12 h. The sections were then treated with LM-1 emulsion (Amersham Pharmacia Biotech, UK) for 30 s and dried for 1 h. They were then placed on X-ray film, and kept under dark conditions for 7 days at 22 °C. The films were developed and images digitized using the computer program Image v1.30 (NIH, USA).

[125I]CQ and Aβ immuno-staining in brain mouse sections in vivo

Male TgAPP-PS1 and non-Tg mice were injected in the jugular vein with 50 µCi of [125I]CQ (specific activity 2125 Ci mol−1) under anesthesia (as before). Brains were removed, frozen and cryosectioned as before. The sections were stained with a monoclonal antibody (1E8) to detect Aβ immunoreactive aggregates as previously described (McLean et al., 1999). Radiolabelling was imaged and assessed as before.

[125I]CQ and thioflavine-S staining in brain mouse sections in vitro

Brain cryosections from TgAPP-PS1 and non-Tg mice were prepared as previously. Before the staining procedures were performed, the sections were left at 22 °C for 1 h. The sections were then treated with [125I]CQ (0.1 nm) (specific activity of 2125 Ci mol−1), for 1 h in buffer Tris-HCl (pH 7.35; final volume of 25 mL) with ethanol (10% v/v). The sections were washed five times with the same buffer, dried at 20 °C for 4 h and kept frozen at −20 °C for 12 h. The sections were treated with LM-1 emulsion (Amersham Bioscience, Buckinghamshire, UK) for 30 s, dried for 1 h, then placed on X-ray film and kept under dark conditions for 1 day at 22 °C. The sections were developed and images photographed under a light microscope. Adjacent sections were stained with thioflavine-S (0.5% w/v) for 10 min at 22 °C. Sections were washed two times in 80% ethanol. These sections were analysed and photographed under a fluorescence microscope.

Human tissue selection

Post-mortem tissues, stored at −80 °C, were obtained from the Brain Bank at the University of Melbourne (supported by the National Health and Medical Research Council) together with accompanying histopathological and clinical data. AD was assessed according to Consortium to Establish a Register for Alzheimer's Disease (CERAD) criteria (Mirra et al., 1991). In order to examine the chemical architecture of the Aβ deposition that is observed in non-AD aged brain, Aβ immunohistochemistry was used to select age-matched control (AC) cases who did not reach CERAD criteria and in which amyloid deposition, if present, was detectable only in the form of diffuse plaques but not neuritic plaques.

Sample preparation and sucrose gradient

Cortical meninges were removed, and gray matter (0.25 g) was homogenized using a glass-teflon homogenizer (Sigma Aldrich, St Louis, MO, USA) for 3 min, in 1 mL of ice-cold 0.25 m sucrose in 10 mm Tris-HCl (pH 7.4), containing a mixture of protease inhibitors (Bio-Rad, Hercules, CA, USA), without EDTA. To obtain the postnuclear cytosolic and membrane fraction, the homogenate was centrifuged at 1000 g for 10 min, and the supernatant was removed and divided into 100 µL aliquots. A sample of postnuclear fraction (100 µg) was incubated with [125I]CQ (75 µm) in the absence or presence of ZnCl2 (75 µm) and DTPA (750 µm) for 1 h at 20 °C. Fifty microliter (50 µL) of each sample was loaded on a discontinuous sucrose gradient comprised of 0.9 mL of 2 m sucrose, 1 mL of 1.3 m sucrose, 1 mL of 1.16 m sucrose, 0.8 mL of 0.8 m sucrose and 0.8 mL of 0.25 M sucrose, each in 10 mm Tris-HCl, pH 7.4. The gradients were centrifuged for 2.5 h at 100 000 g in a Beckman SW50Ti rotor (Beckman Coulter, Buckinghamshire, UK) (Greenfield et al., 1999). Nine fractions of 0.485 mL were collected from above each interface and assessed for radioactivity in a gamma counter. The protein concentration was monitored by absorbance at 280 nm.

[123I]CQ human SPECT studies

Written informed consent for participation in this study was obtained prior to the scan from either the individual subjects or their caregivers. Further approval for AD patients was obtained through the Victorian Civil and Administrative Tribunal (VCAT). Approval for the study was obtained from the Austin Health Human Research Ethics Committee, Austin Radiation Subcommittee and the Victorian Department of Human Services Radiation Safety Unit.

[123I]CQ was synthesized and prepared according to recently developed protocols (Papazian et al., 2005). Three male normal volunteers (AC), aged 68.4 ± 3.3 years with normal neurological and cognitive examination (Mini-mental State Examination, MMSE 28.7 ± 0.6), and three mild AD patients (1 female and 2 males) aged 78.4 ± 8.1 years (MMSE 22.7 ± 7.5) underwent dynamic SPECT imaging on a triple-headed SPECT camera (IRIX® Picker, Picker International, Cleveland, OH, USA) with low-energy, high-resolution (LEHR) collimators, for 120 min (4 × 5′; 4 × 10′; 3 × 20′, respectively) after intravenous injection of 3.73 ± 1.5 mCi (138 ± 57 MBq) of [123I]CQ (specific activity > 5000 mCi µmol−1). Images were reconstructed by filtered back projection using a Metz filter and Chang attenuation correction. Planar whole body scans were obtained at 0.5, 3, 6, and 24 h after injection of [123I]CQ in AC. Venous blood samples were obtained throughout the study for assessment of plasma radioactivity and metabolite analysis.

Regions of interest were selected from brain images and decay-corrected time activity curves were generated. Brain kinetics of the radiotracer were calculated by fitting the radioactivity concentration values at the different time points to a triple exponential equation:

  • E(t) = Ae−αt + Be−βt + Ce−λt

where E is the brain radioactivity concentration and A, B, and C are coefficients for the initial rapid distribution (α), distribution/disposition (β), and elimination (λ) phases, respectively, with the constraint E(0) = 0. The clearance t1/2 was calculated as: t1/2 = 0.693/λ.

Statistical analysis

Data represent mean ± SEM. Specific comparisons were assessed by t-test or anova for each data set. Linear regression and analysis of variance was used for analyzing relationships between two selected variables. A P-value less than 0.05 was considered significant.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Disclosure
  9. References

The authors are grateful to Roberto Cappai, Gaik Kok and Tina Cardamone (University of Melbourne); Thomas Dyrks and Andrea Lippoldt (Schering AG.); Andrew Katsifis (ANSTO, Australia); Kathy Novakovic, Bridget Chappell, Gordon Chan, Julia Ellis, Kenneth Young, Paul U, Kerryn Dickinson, Graeme O’Keefe, Tim Saunder. (Austin Hospital); Andrew J. Mackinnon and Geoff Pavey (MHRI). Supported by funds from National Health and Medical Research Council of Australia (to R.A.C., A.I.B. and C.L.M.), Schering AG (to C.L.M.), Alzheimer's Association, American Health Assistance Foundation and the Australian Research Council (to A.I.B.), and Prana Biotechnology Ltd (to C.L.M.).

Disclosure

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Disclosure
  9. References

Dr Cherny is a paid consultant for in Prana Biotechnology Ltd, which has an interest in clioquinol. Dr Bush is a member of the scientific advisory board, paid consultant for and shareholder in Prana Biotechnology Ltd. Dr Masters is a member of the scientific advisory board, a director and a shareholder in Prana Biotechnology Ltd.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Disclosure
  9. References
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