Enduring Elevations of Hippocampal Amyloid Precursor Protein and Iron Are Features of β-Amyloid Toxicity and Are Mediated by Tau
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The amyloid cascade hypothesis of Alzheimer’s disease (AD) positions tau protein as a downstream mediator of β-amyloid (Aβ) toxicity This is largely based on genetic cross breeding, which showed that tau ablation in young (3–7-month-old) transgenic mice overexpressing mutant amyloid precursor protein (APP) abolished the phenotype of the APP AD model. This evidence is complicated by the uncertain impact of overexpressing mutant APP, rather than Aβ alone, and for potential interactions between tau and overexpressed APP. Cortical iron elevation is also implicated in AD, and tau promotes iron export by trafficking APP to the neuronal surface. Here, we utilized an alternative model of Aβ toxicity by directly injecting Aβ oligomers into the hippocampus of young and old wild-type and tau knockout mice. We found that ablation of tau protected against Aβ-induced cognitive impairment, hippocampal neuron loss, and iron accumulation. Despite injected human Aβ being eliminated after 5 weeks, enduring changes, including increased APP levels, tau reduction, tau phosphorylation, and iron accumulation, were observed. While the results from our study support the amyloid cascade hypothesis, they also suggest that downstream effectors of Aβ, which propagate toxicity after Aβ has been cleared, may be tractable therapeutic targets.
KeywordsAlzheimer’s disease Iron Tau β-Amyloid Aging Neuroprotection
The defining pathologies of Alzheimer’s disease (AD)—senile plaques and neurofibrillary tangles—are thought to be linked in a molecular pathway involving β-amyloid (Aβ) and tau protein, the key constituents of the respective pathologies. Proponents of the amyloid cascade hypothesis posit that Aβ recruits tau protein as a downstream effector of toxicity. Aβ-induced cytotoxicity was shown to be tau-dependent in primary neuronal culture: the neurons are protected when tau is lowered by either genetic ablation [knockout (KO)], or by leaving them undifferentiated (where they express lower tau levels than differentiated neurons) [1, 2]. Ex vivo studies of tau KO neurons revealed that the Aβ-induced axonal transport deficits and impairment of hippocampal long-term potentiation were mediated by tau [3, 4]. Reduction of endogenous tau was also shown to ameliorate cognitive behavioral deficits and the death rate of amyloid precursor protein (APP) transgenic mice [5, 6, 7]. Thus, lowering tau is a promising therapeutic concept for AD.
However, chronic loss of tau itself causes impairment to long-term potentiation and long-term depression [8, 9, 10], and tau KO mice exhibit cognitive deficits as early as 4–6 months of age [8, 10]. Aged tau KO mice (≥12 months old) have motor and cognitive impairment that accompanies brain atrophy [11, 12, 13], caused by brain iron [11, 14]. It is thus unclear how the reduction of tau can both precipitate cognitive decline and protect against Aβ-induced toxicity. To further complicate this picture, young (4–7 months old) human APP (hAPP) transgenic mice crossbred with KO mice (hAPP/tau−/− mice) exhibit better cognitive function than APP transgenic mice that express tau normally [5, 6, 7]; however, aged (12 months old) hAPP/tau−/− mice showed enhanced neurodegeneration than the single APP transgenic mice .
We considered whether these results might be explained by altered interactions between APP and tau, rather than tau and Aβ. It has not yet been shown that loss of tau protects against Aβ induced (as opposed to APP-induced) toxicity in an animal model, and it is not known how Aβ recruits tau to cause toxicity. This distinction between APP and Aβ is important in understanding the molecular pathway of AD. It has previously been shown that tau binds to APP , and we showed that tau is necessary for the trafficking of APP to the neuronal surface . In this role, tau facilitates the export of iron as APP stabilizes surface ferroportin, which is the obligate iron export protein . As a result, tau KO mice develop iron-dependent neurodegeneration after the age of 6 months, which can be rescued by iron chelation [11, 14]. This is important because cortical iron elevation has also been implicated in AD pathogenesis .
To test whether toxicity of Aβ itself is influenced by the presence of tau in vivo, we utilized an Aβ injection model . This model precludes potential interactions between tau and APP, which could have confounded previous findings in tau KO mice attributed to Aβ. We injected preprepared Aβ oligomers into the brains of tau KO mice (C57/Bl6 background) at 3 months of age or 12 months of age, and studied the behavioral and biochemical consequences. We found that tau KO mice at both ages are protected against Aβ oligomer toxicity.
Mice and Mice Tissue Preparation
All mice were housed in a conventional animal facility according to standard animal care protocols and fed standard laboratory chow (Code 102108, Barastoc; Ridley AgriProducts, Melbourne, Australia) and tap water ad libitum. All animal procedures were approved by the Florey Institute’s animal ethics committee (13–023) and were performed in accordance with the National Health and Medical Research Council guidelines. Tau KO mice were backcrossed for 10 generations onto a Bl6 background, and mutants were backcrossed to the parental inbred strain every 3 generations . To obtain the mouse brain, mice were euthanized with an overdose of sodium pentobarbitone (Lethabarb, 100 mg/kg, Virbac (Australia) Pty. Ltd; Milperra, NSW, Australia) and perfused with ice-cold saline. The right brain hemisphere was microdissected and stored at −80 °C until required. The left brain hemisphere was fixed either in 4 % paraformaldehyde for 24 h, and then transferred to 30 % sucrose plus phosphate-buffered saline (PBS; pH 7.4), or in 10 % neutral-buffered formalin for immunohistochemistry.
β-Amyloid Oligomer Preparation
Aβ oligomer preparation was performed as previously described . Briefly, Aβ1–42 oligomers were prepared by diluting 5 mM human Aβ1–42 (with 95 % purity by reverse phase high-performance liquid chromatography; Chinapeptides, Shanghai, China) in dimethyl sulfoxide to 100 μM in ice-cold cell culture medium (phenol red-free Ham’s F-12), immediately vortexing for 30 s, and incubating at 4 °C for 24 h. The solution was then centrifuged at 14,000 × g for 10 min at 4 °C to remove insoluble aggregates, and the supernatants containing soluble Aβ1–42 were transferred to clean tubes for injection. Each preparation of Aβ oligomer was freshly made prior to injection.
Mice (70 % female) were injected intraperitoneally with anesthetic and analgesic before the stereotactic injection. Mice were anesthetized, and sedation was maintained with an anesthesia gas mask administering isofluorane and oxygen within the stereotaxic instrument (MD3000; Basi, West Lafayette, IN, USA). PBS or Aβ1–42 oligomer (2 μl, 0.44 mg/ml peptide, 0.5 μl/min) was injected (25-gauge injection needle connected by polyethylene tubing to 5-μl Hamilton microsyringes) into the hippocampal dorsal CA1 area bilaterally. The coordinates from bregma used for CA1 region were as follows: anteroposterior, −2.3 mm; mediolateral, −2.0 mm; dorsoventral, −2.0 mm. The injection needle was inserted 0.5 mm beyond the tip of the cannula. Mice were allowed to recover on a heating pad and returned to the animal facility.
The Y-maze test was performed as previously described . Briefly, all mice were subjected to a 2-trial Y-maze test separated by a 1-h intertrial interval to assess spatial recognition memory, with all testing performed during the light phase of the circadian cycle. Behaviors were recorded on video during a 5-min trial and Ethovision video-tracking system (Noldus, Wageningen, the Netherlands) was used for blinded analysis. Data are expressed as the percentage of frequency for novel arm entries made during the 5-min trial.
Novel Objective Recognition Test
Twenty-four h prior to the test, the mice were habituated to the arenas (50 cm × 50 cm plastic container) for 5 min without objects. Arenas were cleaned with ethanol between each habituation period. The day after the mice re-entered the arenas from the same starting point of the arena (facing the bottom left corner) and were granted 10 min to familiarize themselves with the objects (50 ml tubes × 2 spaced 10 cm apart). After each familiarization period the arena and objects were cleaned with ethanol. Exactly 1 h after the familiarization period, 1 of the 50-ml tubes was replaced with a T75 tissue culture flask as the “novel” object, and the mice were granted 10 min to explore both objects. This recall period was recorded on camera for subsequent blinded analysis on the TopScan Cleversys suite. Four mice were tested in each run and 3 batches of 4 mice were tested every hour. TopScan Cleversys was used to measure the frequency and duration the mice sniffed the objects within the 10-min recall period.
Paraformaldehyde fixed frozen sections were air-dried for 30 min prior to staining. The slides were then placed into a slide rack and immersed in 1 % Neutral Red for 2 min. The slides were then rinsed, placed into a series of ethanol concentrations, ranging from 50 % to 100 %, and immersed into xylene twice for 5 min each. The slides were then coverslipped using DPX mounting medium (BDH, Trajan Scientific Australia Pty Ltd, Ringwood, VIC, Australia) and air-dried.
Frozen, paraformaldehyde-fixed sections were air-dried, and a wax pen was used to circle around each group of sections. The slides were blocked for 30 min at room temperature (RT; blocking solution: 6 % normal goat serum, 3 % Triton X 10 %, in PBS), then incubated with primary antibody [1E8 for Aβ (in house); Anti Neu-N, clone A-60 (Millipore, Darmstadt, Germany), 1:200 in 1 % normal goat serum, 3 % Triton X 10 %, and PBS] for 48 h at 4 °C in a humidified staining chamber. The slides were then washed and incubated with secondary antibody (biotinylated sheep antimouse immunoglobulins, 1:400; Chemicon, Temecula, CA, USA) for 2 h at RT. After incubation with secondary antibody, the slides were incubated with avidin peroxidase solution (0.08 % avidin peroxidase, 0.75 % Triton X 10 %, in 0.1 M phosphate buffer) for 1 h at RT and 3,3’-diaminobenzidine solution (1 % 3,3’-diaminobenzidine, 2.5 % of 1 % cobalt chloride, and 2 % ammonium nickel sulfate, in 0.1 M phosphate buffer) for 20 min rocking at RT. Three precent H2O2 were added onto the slides for chromogen development.
Hippocampal sections from mice were sectioned using a Leica Cryostat set at 50 μ thickness (Leica Microsystems, Milton Keynes, UK). The analysis was performed by 2 independent investigators (blinded to mouse identity) by using the color threshold function of Image J 1.49 m software (National Institutes of Health, Bethesda, MD, USA). Six sections corresponding to the brain region of interest (Bregma −2.8 mm to −3.4 mm) per mouse analyzed. Nissl-stained and NeuN-stained brain sections were photographed by a Leica DFC295camera coupled to a Leica DM IRE2 microscope with Leica Application Suite Version 4.1.0 image analysis software (Leica Microsystems).
Enzyme-Linked Immunosorbent Assay for Aβ Detection
Endogenous mouse Aβ levels were determined using the DELFIA Double Capture (Perkin Elmer, Melbourne, Australia) enzyme-linked immunosorbent assay (ELISA), as previously described . Plates were coated with G210 (for Aβ40) antibody, then blocked with 0.5 % (w/v) casein/PBS buffer (pH 7.4). After washing the plates with buffer containing PBS/Tween-20 (PBST) 0.05 %, 1E8-biotin (epitope Aβ17-22) was diluted in 0.25 % casein/PBST (0.025 %) and added to the wells. Aβ1–40 peptide standards (Keck ERI Amyloid Laboratory, Oxford, CT, USA), diluted in 0.25 % casein/PBST (0.025 %) and sample [15 μl of brain homogenate (1 % sodium dodecyl sulfate/PBS) diluted into 185 μl 0.25 % casein/PBST (0.025 %)] were assayed in triplicate and incubated overnight at 4 °C. The plates were washed, labeled with europium, streptavidin added, and then developed with enhancement solution. Analysis was carried out using the Wallac Victor2 1420 Multilabel Plate Reader (Perkin Elmer) with excitation at 340 nm and emission at 613 nm.
Samples from each experiments were homogenized in PBS (pH 7.4) with ethylenediaminetetraacetic acid-free protease inhibitor cocktail (1:50; Roche, Indianapolis, IN, USA) + phosphatase inhibitors I and II (1:1000) and centrifuged at 40,000 × g for 30 min. Protein concentration was determined by BCA protein assay (Pierce, Rockford, IL, USA). Aliquots of homogenate with equal protein concentrations were separated in 4–12 % bis-Tris gels with NuPAGE MES running buffer (Invitrogen, Carlsbad, CA, USA), and transferred to nitrocellulose membranes by iBlot (Invitrogen). The membranes were blocked with milk (5 % w/v) and probed with appropriate primary and secondary IgG–horseradish peroxidase conjugated antibodies (Dako, Glostrup, Denmark). Enhanced chemiluminescence detection system (GE Healthcare, Little Chalfont, UK) was used for development and Fujifilm LAS-3000 for visualization (Fujifilm, Tokyo, Japan). Densitometry quantification of immunoreactive signals was performed by Image J (1.49 m; National Institutes of Health) and normalized to the relative amount of β-actin and expressed as a percentage of the mean of the control group. The following antibodies were used: anti-β-actin (Sigma, St. Louis, MO, USA); 22C11 (in house); anti-tau (Dako); anti-pTau396 (Invitrogen); 1E8 (in house); WO2 (in house).
Metal content was measured as previously described . Briefly, samples from each experimental condition were freeze dried and then re-suspended in 65 % nitric acid (Suprapur; Merck, Darmstadt, Germany) overnight. The samples were then heated for 20 min at 90 °C, and the equivalent volume of H2O2 (30 % Aristar; BDH) was added for further 15 min incubation at 70 °C. The samples were diluted in double-distilled water and assayed by inductively coupled plasma mass spectrometer (Agilent 7700; Agilent Technologies, Santa Clara, CA, USA). Each sample was measured in triplicate and the concentrations determined from the standard curve were normalized to tissue wet weight.
Statistical analysis was carried out in Prism 6 (GraphPad Software Inc., La Jolla, CA, USA). All tests were 2-tailed, with the level of significance set at 0.05. Detailed tests used in each experiment are described in the text.
Three-Month-Old Tau KO Mice Are Resistant to Aβ Toxicity
In contrast, Aβ1–42 injection had no impact in either cognitive test of 3-month-old tau KO mice for the duration of the experiment (Fig. 1a–f), indicating that Aβ-induced memory loss is mediated by tau. Vehicle-treated tau KO mice were not impaired compared with WT mice (Fig. 1a–f), which reinforces our report and other findings that young tau KO mice have normal cognitive function [5, 6, 13].
Twelve-month-old Tau KO Mice are Resistant to Aβ Toxicity
Increased Hippocampal Iron Induced by Aβ
Tau and Phosphorylated Tau Levels in Aβ-Injected Hippocampus
Aβ injection was previously reported to trigger intracellular Aβ aggregation in transgenic animals, and nonhuman primates, both of which express human sequence Aβ [33, 34]. Mice and rats have a different sequence of Aβ that does not precipitate, even in the presence of zinc . We found no evidence for endogenous mouse Aβ aggregation in the human Aβ injection model (Figs. 4a, b and 7a), consistent with previous findings [36, 37].
We observed that Aβ injection caused cognitive deficits that persisted even after the peptide was cleared from the injection site. This suggests that downstream intracellular toxic events propagate the toxicity of Aβ after the initial insult, and is in accord with the human disease progression where Aβ is elevated decades before symptom onset . We found that one of the persistent effects of Aβ intoxication was increased local iron levels (Fig. 8), which is also pathology of AD [28, 39]. Lowering brain iron has been shown to reduce memory deficits and neuronal loss in AD animal models and in a clinical trial [11, 14, 40, 41, 42, 43]. We recently reported that elevated brain iron, as reflected by elevated cerebrospinal fluid ferritin levels, is associated with accelerated cognitive decline , highlighting the role of iron in AD progression. The current study raises the possibility that Aβ intoxication promotes toxic iron accumulation in the disease.
We previously found that tau reduction causes iron accumulation, which resulted in age- dependent neurodegeneration, manifesting in cognitive deficits and brain atrophy [11, 12, 13]. Tau KO mice have impaired neuronal iron efflux. Brain iron levels are unaffected in young tau KO mice, probably because of compensatory changes, but brain iron levels rise with age , and this rise is exaggerated in tau KOs where it contributes to gray matter atrophy . Here, in an Aβ injection model, we found that cognitive deficits (Figs. 1 and 5) and neuron loss in the hippocampus (Figs. 2, 3 and 6) accompanied the persistent reduction of tau protein levels (Fig. 9) and concomitant iron accumulation (Fig. 8). Iron elevation as a consequence of Aβ toxicity, therefore, could be caused by the suppression of tau protein (without adequate compensatory changes to correct iron elevation). A role for tau in contributing to iron elevation after Aβ injection is supported by our observation that iron levels did not rise in intoxicated tau KO mice. In tau KO mice (where the animal has benefitted from compensatory homeostatic changes since conception), iron was not elevated in response to Aβ injection because tau levels could not be acutely depressed.
In both 3-month-old (Fig. 4) and 12-month-old (Fig. 7) WT mice, an Aβ injection induced a persistent (5-week) elevation in APP. This could reflect a homeostatic adjustment to hippocampal iron elevation. Iron induces the increased translation of APP , and is trafficked by tau to the neuronal surface where it stabilizes ferroportin to promote iron efflux [11, 45, 46]. As iron is still elevated at 5 weeks, clearly the increase in APP expression is insufficient to compensate fully for the perturbance. This would be consistent with the drop of tau being an upstream event induced by Aβ, as tau KO neurons in culture also have a similar increased expression in APP, yet the APP does not reach the neuronal surface to stabilize ferroportin . In tau KO mice, none of these downstream changes are possible as the tau–APP trafficking mechanism as a means of controlling cytoplasmic iron has been absent from conception.
Alternatively, or concurrently, increased tau phosphorylation after Aβ intoxication might also contribute to the neurotoxicity we observed (which cannot occur in tau KO mice). In humans, abnormally phosphorylated tau but not amyloid can be detected as early as young adulthood , indicating a toxicity pathway for tau independent of Aβ. We only observed an increase in the ratio of phospho-tau to tau protein after Aβ injection, not an increase in the total levels of phospho-tau. Taken together, our study supports therapeutic targeting of downstream effectors of Aβ toxicity, such as phospho-tau or iron, which continue to propagate neurotoxicity after Aβ has been cleared.
This work was supported by funds from the Australian Research Council, the National Health and Medical Research Council (NHMRC) of Australia, the Cooperative Research Center for Mental Health, and the Alzheimer’s Australia Dementia Research Foundation. The Florey Institute of Neuroscience and Mental Health acknowledges the strong support from the Victorian Government and, in particular, the funding from the Operational Infrastructure Support Grant.
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