ABCA1 Deficiency Affects Basal Cognitive Deficits and Dendritic Density in Mice

Transgenic mice

All experiments followed NIH guidelines for the Care and Use of Laboratory Animals and were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Data was collected and analyzed for all experiments where researchers were blinded to genotypes and/or treatments described below. Abca1^het mice were bred to Abca1^het mice to yield Abca1^ko, Abca1^het, and Abca1^wt littermates. All mice used in this study were purchased from Jackson Laboratory and bred onto a C57BL/6J background for greater than 11 generations. APP/PS1ΔE9 (B6.Cg-Tg(APPswe, PSEN1ΔE9)85Dbo/Mmjax; referred to as APP) transgenic mice, also purchased from Jackson Laboratory, were crossbred to Abca1^het mice to generate APP/Abca1^ko, APP/Abca1^het, APP/Abca1^wt and corresponding non-APP expressing littermates. Non-APP expressing –Abca1^wt littermates are referred here in as WT mice. The progeny were identified by PCR. All reagents purchased through Fisher Scientific unless otherwise stated.

Cannula implantation

To examine the effects of Aβ oligomers on memory, mice were infused with Aβ oligomers directly into the hippocampus through guide cannulas. Following anesthesia with isoflurane, the head was shaven and sterilized with two separate iodine – alcohol washes. A 50% mixture of bupivacaine and lidocaine were applied to the surgical site and ophthalmic ointment applied to the eyes. The head was leveled in a stereotaxic frame and an incision made exposing the dorsal aspect of the skull. Two holes were drilled into the skull (coordinates: P = 2.46 mm, L =±1.50 mm) and 26-gauge guide cannulas (Plastics One) were lowered into the dorsal part of the hippocampi to a depth of 1.30 mm. Cannulas were fixed to the skull with acrylic dental cement attached to two bone anchoring screws and the surgical opening sutured closed. Animals were allowed to recover for 8 days before behavioral testing started.

Aβ oligomer preparation

Under a fume hood, 1 mg of Aβ₄₂ peptide (American Peptide Company) was dissolved in ice cold 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP, Fluka) to obtain a 1 mM solution then vortex for few seconds. The solution was quickly aliquoted into 3 polypropylene vials and dried with a gentle stream of N₂ to obtain a clear peptide film in the bottom of the vials. Prior to use, one film was re-suspended in anhydrous DMSO to form a 5 mM solution, sonicated in water bath for 10 min and diluted 200X with sterile phosphate-buffered saline (PBS). Aβ samples were left at room temperature for 24 h to form oligomer complexes and stored at –20°C until use. The same concentration of scrambled Aβ (AnaSpec) was dissolved in vehicle and utilized as a negative control.

Aβ oligomer characterization

Behavioral testing

Animal tissue processing

Mice were anesthetized with Avertin (250 mg/kg of body weight, i.p.) and blood was drawn from the heart. The mice were perfused transcardially with 25 ml of cold 0.1 m PBS, pH 7.4. One hemisphere was drop fixed in 4% phosphate-buffered paraformaldehyde at 4°C for 48 h before storage in 30% sucrose.

Neurite morphology quantification

Neurite morphometry analysis was performed as described before [13]. Coronal brain sections (30 μm thickness) were obtained with a sliding microtome. Sections were stored in cryoprotectant at –20°C until staining commenced. For immunofluorescence labeling, brain tissue sections were rinsed 3 times in PBS for 10 min and incubated with 1% Triton X-100 (permeabilizing reagent) in PBS solution for 5 h at 4°C. The tissue was washed 3 times in PBS for 10 min each and blocked with 10% serum and 0.3% Triton X-100 in PBS solution for 30 min at room temperature (RT). The sections were incubated for 72 h at 4°C with microtubule-associated protein 2 (MAP2) primary antibody (1 : 2000; Millipore), a cytoskeletal protein that binds to tubulin and stabilizes microtubules and is essential for the development and maintenance of neuronal morphology. Tissue was rinsed 3 times in PBS for 10 min each to remove unreacted primary antibodies. Tissue sections were incubated with Cy3-conjugated anti-sheep antibody (1 : 500; Jackson-ImmunoResearch) secondary antibody for 2 hr at RT. Sections were washed 2 times in PBS for 10 min and counterstained with H33342 nuclear reagent (1 : 3000; Sigma-Aldrich) for 5 min at RT. Sections were washed 3 times in PBS for 10 min each and then, were mounted onto plus-coated slides and coverslipped using gelvatol mounting media. The FilamentTracer module of Imaris (Bitplane), which facilitates 3D neuron reconstruction, was utilized to determine neuronal patterning and connections of hippocampal brain sections. The MAP2 (Cy3) channel was used to quantify total neurite length, the number of segments, and the number of branches in the CA1 and CA2 regions of the hippocampus. 5 sections per animal, starting at the beginning of the dentate gyrus, and every 300 μm were used. For neurite analysis, 4-5 confocal images (considered replicates) were acquired for the CA1 region while 3-4 confocal pictures were captured for the CA2 region. Confocal fluorescence micrographs were obtained using a 60x lens magnification at very high resolution (100 μs exposure). For unbiased examination, the size and the length of the neurites were the only parameters that required manual introduction. For parity, image assessment must use identical grid dimensions. Neurite length was normalized to the number of MAP2 neurons.

Statistical analysis

Data are reported as means±SEM. Statistical differences between mean scores during acquisition phase of training in the MWM were analyzed with two-way ANOVA and t-test was used to determine difference for each trial day. One-way ANOVA followed by Tukey’s post hoc analysis for multiple comparisons was used to analyze the MWM probe trial data. Performance in RWM was assessed with two-way ANOVA Repeated measure (using Genotype or Infusion Group and Trial as sources of variation) followed by Tukey’s post hoc analysis. Significant differences between groups for the rest of the data were determined by t-test. All statistical analyses were performed in GraphPad Prism, version 6.0, and differences were considered significant where p < 0.05.

Abca1 deficiency significantly impairs spatial memory in APP transgenic mice

Recently we showed that lack of Abca1 increases amyloid load and worsens the performance of APP expressing mice in the contextual fear conditioning test [13]. The contextual fear condition paradigm assesses hippocampal-dependent associative learning to unconditional stimulus such as electric shock [5]. To test if ABCA1 deficiency affects other types of memory, such as spatial learning and retention and long-term memory, we employed MWM and the novel object recognition test, respectively. We used 7-month-old APP mice that express wild type Abca1 (APP/Abca1^wt) or Abca1^ko (APP/Abca1^ko) and their non-APP transgenic littermates (WT and Abca1^ko). As illustrated in Fig. 1A and B, during the acquisition phase, APP mice with intact Abca1 performed similarly to WT mice (no significant main effect of genotype); however, there was a statistical difference on the last day of testing (p < 0.05) suggesting impaired acquisition. In contrast, APP/Abca1^ko mice performed much worse than Abca1^ko (significant main effect of genotype, p < 0.001), particularly on the last days of MWM. This suggests that the lack of ABCA1 impairs spatial acquisition in mice expressing human APP. In the MWM probe trial, APP/Abca1^ko mice demonstrated decreased memory retention exemplified by a longer latency time to enter the target zone compared to APP/Abca1^wt and non-transgenic Abca1^ko mice (Fig. 1C). APP/Abca1^ko mice also spent the shortest time in the target quadrant compared to the other genotypes; however, the difference with Abca1^ko was not significant (Fig. 1D). These changes in performance were not due to swim speeds as there was no difference in the swim speeds of the assessed genotypes.

To confirm that the memory impairment is not restricted only to MWM, the mice were also tested using the novel object recognition paradigm as before [16]. Once more, APP/Abca1^ko mice demonstrated the worst performance (Fig. 1E, p < 0.05 compared are APP/Abca1^ko and APP/Abca1^wt), with APP/Abca1^ko mice exploring the novel object far less than the other genotypes.

The behavior experiments presented on Fig. 1 demonstrate that ABCA1 deficiency exaggerates cognitive deficits in APP transgenic mice that could be a result of either increased endogenously formed Aβ oligomers or amyloid plaques present in APP/Abca1^ko mice.

Characterization of Aβ oligomers utilized in this study

It has been reported that Aβ oligomers affect synaptic plasticity and cognitive function [17]. Recently we have shown that amyloid load and A11-positive Aβ oligomers are significantly increased in 7-month-old APP/Abca1^ko mice in comparison with APP mice expressing Abca1^wt [13]. To determine how the acute infusion of Aβ oligomers into the hippocampus affects the memory of WT and Abca1^ko mice, we used Aβ₄₂ peptide and employed several methods to characterize the Aβ oligomers. Fig. 2A shows SDS PAGE followed by western blotting with anti-Aβ 6E10 antibody. As visible, at time 0, Aβ existed only as monomers and low MW oligomers such as dimers, trimers, and tetramers. Upon oligomerization, following 24 h incubation, there was an increase of higher MW oligomers such as 9-mer, 12-mer etc.; however, we were unable to detect oligomers with MW higher than 100 kDa. For comparison, on the right of Fig. 2A is shown western blotting of Aβ fibrils. Aβ oligomers were also confirmed using conformation specific A11 antibody. As seen on Fig. 2B, when the dot blot was probed with A11, there was an increase in the intensity of oligomeric Aβ from time 0 to 48 h incubation. Finally, on Fig. 2C are shown electron micrographs of Aβ at time 0 and 24 h incubation. At time 0 (Fig. 2C-a), disaggregated Aβ₄₂ is present as a monomer.After incubation for 24 h at room temperature, Aβ₄₂ was converted into rod-shaped oligomers with diameter of approximately 10 nm and length between 20–50 nm (Fig. 2C-b).

Infusion of Aβ oligomers into hippocampus differentially affects cognitive deficits in WT and Abca1^ko mice

To test the effect of the oligomers shown on Fig. 2, on cognition we infused WT and Abca1^ko mice, which do not express human APP transgene, with the 24 h oligomeric Aβ preparation. Young 7-month-old mice were implanted with cannulas into both hippocampi (see picture on Fig. 3A) followed by a recovery period of 8 days before behavioral testing. Aβ₄₂ oligomers were infused bilaterally into the hippocampus 30 min prior to each of the RWM training blocks (2–6 trial blocks and 1–3 trial blocks) for a total of 3 infusions. RWM paradigm tests reference memory [5] and were used as a more demanding task, compared to MWM, to detect relatively subtle changes in cognitive function. Control mice were infused with scrambled Aβ. In addition, mice were compared to naïve mice that were not subjected to surgery. As shown on Fig. 3B, in WT mice Aβ₄₂ oligomers significantly affected cognitive performance as compared to the control mice infused with scrambled Aβ (compare purple circles to grey squares). In contrast, there was no statistical difference between naïve (no surgery) and mice infused with scrambled Aβ (Fig. 3B, compare grey squares to black triangles). This experiment suggests that surgical procedures followed by the infusion of scrambled peptide do not affect the memory of WT mice.

We next examined how Aβ oligomers infused into the hippocampus would impact cognitive performance of age-matched Abca1^ko mice and compared them to controls of Abca1^ko and WT mice infused with scrambled Aβ. Surprisingly, we found a statistically significant difference between WT and Abca1^ko infused with control scrambled Aβ (see Fig. 3C, compare black to grey squares) suggesting that Abca1^ko mice are vulnerable to the effect of stress/trauma induced by surgery and infusion of scrambled Aβ. Due to the worsened performance of the control Abca1^ko mice (injected with scrambled Aβ), there was no statistical difference between their behavior and Abca1^ko mice infused with Aβ oligomers (compare black squares to red circles). Our conclusion is that Abca1^ko mice have basal cognitive deficits that prevent them from coping with additional stressors that do not impact performance of normal healthy WT mice.

Deficiency of Abca1 significantly impairs neurite morphology in CA1 but not in CA2 region of the hippocampus

Recently we have demonstrated that Abca1 as well as Apoe deficiency significantly affects dendrite architecture in human APP transgenic mice [13]. The behavior tests shown on Fig. 2 prompted us to examine the dendritic parameters of Abca1^ko mice that do not express human APP transgene. To test the effect of Abca1 deficiency we used 7-month-old Abca1^ko mice and compared them to WT mice (the same age as in Fig. 2). For neurite morphometry of pyramidal hippocampal neurons, we used MAP2 stained brain sections and performed analysis in two different regions (CA1 and CA2) of medial hippocampal sections. Since neurites form complex tree-like structures, 3D image reconstruction is necessary to obtain accurate measurements. Examination of neurites in the CA1 region (Fig. 4A) revealed a significant decrease in neurite length, number of neurite segments and number of branches (Fig. 4A, B; p < 0.05) in Abca1^ko mice when compared to WT. These changes in neurite architecture were restricted to the CA1 region of the hippocampus as we did not observe any changes in the CA2 region when comparing Abca1^ko to WT mice (Fig. 4C, D). The data were normalized to the number of neurons and there was no significant change in the numbers of counted nuclei (not shown) when comparing Abca1^ko mice and WT mice in either region of the hippocampus. Our data demonstrate that the genetically engineered deletion of Abca1 disrupts neurite morphology in the hippocampus, and this effect is specific for CA1 region.