The isotropic fractionator provides evidence for differential loss of hippocampal neurons in two mouse models of Alzheimer's disease
© Brautigam et al.; licensee BioMed Central Ltd. 2012
Received: 2 May 2012
Accepted: 18 September 2012
Published: 22 November 2012
The accumulation of amyloid beta (Aβ) oligomers or fibrils is thought to be one of the main causes of synaptic and neuron loss, believed to underlie cognitive dysfunction in Alzheimer’s disease (AD). Neuron loss has rarely been documented in amyloid precursor protein (APP) transgenic mouse models. We investigated whether two APP mouse models characterized by different folding states of amyloid showed different neuronal densities using an accurate method of cell counting.
We examined total cell and neuronal populations in Swedish/Indiana APP mutant mice (TgCRND8) with severe Aβ pathology that includes fibrils, plaques, and oligomers, and Dutch APP mutant mice with only Aβ oligomer pathology. Using the isotropic fractionator, we found no differences from control mice in regional total cell populations in either TgCRND8 or Dutch mice. However, there were 31.8% fewer hippocampal neurons in TgCRND8 compared to controls, while no such changes were observed in Dutch mice.
We show that the isotropic fractionator is a convenient method for estimating neuronal content in milligram quantities of brain tissue and represents a useful tool to assess cell loss efficiently in transgenic models with different types of neuropathology. Our data support the hypothesis that TgCRND8 mice with a spectrum of Aβ plaque, fibril, and oligomer pathology exhibit neuronal loss whereas Dutch mice with only oligomers, showed no evidence for neuronal loss. This suggests that the combination of plaques, fibrils, and oligomers causes more damage to mouse hippocampal neurons than Aβ oligomers alone.
KeywordsAlzheimer’s disease Mouse models Amyloid beta (Aβ) Isotropic fractionator Neuronal loss
Summary of cell and neuron counts from different studies according to brain region and counting method
Cell or neuron number
Age of animals and transgene(s) harbored
100.10 ± 7.90 x 106
6 months; K670M/N671L/V717F APP
108.90 ± 16.25 x 106
43.92 ± 2.78 x 106
6 months; K670M/N671L/V717F APP
49.17 ± 5.32 x 106
Isotropic Fractionator/Flow Cytometry
44.03 ± 0.42 x 106
31.8% decrease in neurons
6 months; K670M/N671L/V717F APP
No decrease in neurons
18 month Dutch E693Q APP mice
~25% decrease in neurons
14-18 month-old TgAPP23 (K670M/N671L) mice
APP/ PS1M146L (CBA - 12.5% × C57Bl6 - 87.5%)
~30% decrease in neurons
17 month APP (751 SL ) and presenilin 1 (PS1M146L) mice
(C57BL/6 50%-CBA 25%–129SV 25%)
~50% decrease in neurons
10 month APPSLPS1KI (APP K670N/M671L, and V717I; PS1 M233T/L235P knock-in)
33% decrease in neurons
6 month APP/PS1KI
(APP K670N/M671L, and V717I; PS1 M233T/L235P knock-in)
Aberrant accumulation of brain Aβ over decades has been proposed to result in synaptic and neuronal loss associated with the progression of AD (for review see 13). Evidence indicates that accumulation of soluble oAβ relates to cognitive impairment in AD patients 14and AD mouse models 2315. However, the possibility that the severity of neurotoxicity and neuronal loss may be related to levels of Aβ solubility or confirmation (oAβ or fAβ) remains unclear. Using high-resolution 3D reconstructions, intraneuronal accumulation of fAβ has been observed by some investigators who proposed that this fAβ leads to the disruption and degeneration of spines and neurites, and ultimately to neuronal death 16, even though other investigators have reported that oAβ alone is sufficient for behavioral dysfunction 15.
Importantly, other studies have demonstrated reduced neuron densities in various lines of APP mice following onset of plaque pathology. These observations were primarily derived from the hippocampus and are similar in magnitude to the loss we report in TgCRND8 mice, albeit using different counting methods (Table 1). Using a stereologic approach, Calhoun et al. (1998) found a ~25% loss of CA1 neurons in TgAPP23 mice, while Schmitz et al. (2004) found a ~30% loss of neurons in the CA1-3 regions in an APP/PS1 double mutant mouse, and Breyhan et al. (2009) found a 33% loss of CA1 pyramidal neurons in APP/PS1 knock-in mice. Our results indicate that, in wildtype mice, estimates derived from the isotropic fractionator are generally comparable to stereologic estimates. Most importantly, we observed an apparent neuronal loss in TgCRND8 mice that form oAβ, insoluble fAβ, and amyloid plaques, but not in Dutch mice that produce only oAβ. It is evident that both oAβ and fAβ are toxic and implicated in the neurodegeneration process. However, various Aβ species may impair neuronal function via different mechanisms, and clarification of this point merits further investigation.
Eight 6-month-old TgCRND8 APPK670N/M671L/V717F mice and seven wildtype littermates, as well as nine 18-month-old Dutch APPE639Q mice and eight of their littermates were used in this study. These ages were chosen as previous studies demonstrated clear behavioral impairments and amyloid deposition in the TgCRND8 mice and intracellular accumulation in the Dutch mice at these time points 35. All mice used in the current study were group-housed, given ad libitum access to food and water, and housed under a 12-hour light/dark cycle. All animal procedures were conducted in accordance with the National Institute of Health Guidelines for the Care and Use of Experimental Animals and were approved by the Institutional Animal Care and Use Committee of the Icahn School of Medicine at Mount Sinai.
Animals were perfused as previously described 17. Brains were removed from the skull and postfixed in 4% PFA at 4°C. Brains were weighed, cut into half, and further dissected into three regions, cerebellum, hippocampus, and neocortex and weighed. Results were multiplied by 2 to obtain estimates of cell numbers for the entire brain. In addition, brains were immunostained with the anti-amyloid monoclonal antibody 6E10 (Covance) as described in 15.
The isotropic fractionator is a fast and reliable technique for determining total cell and neuronal counts and cell densities in the brain. The procedure involves processing of highly anisotropic brain structures into homogeneous isotropic suspensions of cell nuclei that can be easily quantified 6. Because the estimates of cell counts are obtained separately from the determination of brain volume, the two measurements can be used in comparative studies of brain-volume variation (i.e., comparing across species or between different mouse lines 6). Briefly, postfixed samples were mechanically dissociated and homogenized in a solution of 40 mM sodium citrate and 1% Triton X-100 as previously described 6. The homogenates were collected and the homogenizer washed at least twice to collect any residual cells. To visualize nuclei and obtain total cell counts, 1 μl of 4’,6-diamidino-2-phenylindole (Sigma) was added to the cell suspension (volumes according to brain region; 10 ml for cerebellum and neocortex, 3 ml for hippocampus. The volume for each brain region was optimized so that at least 500 nuclei could be counted) To determine total neuron counts, 1 ml of each cell suspension was removed, washed with PBS, and centrifuged for 10 minutes at 4000 x g. Cells were then incubated in anti-NeuN antibody (1:200; Millipore) for 2 hours followed by incubation in a secondary anti-mouse IgG-Alexa-Fluor 594 for 1.5 hours (1:200; Millipore). Cells were then counted with a hemocytometer using a 20X/0.45 N.A., Plan-Apochromat objective on a Zeiss Axiophot microscope equipped with a motorized stage. All four quadrants in both the upper and lower grids of the hemocytometer were counted and averaged. Total cell numbers and neuronal numbers were calculated by multiplying the number of cells/ml by final volume. Densities were calculated by dividing the number of cells/ml by the mass of brain region.
All statistical analyses were performed using SPSS v19.0. Independent samples t-tests were used for comparison of TgCRND8 mutant APP or Dutch mutant APP mice to their respective littermates. Levene's test for homogeneity of variance was used for inclusion in parametric tests (p > 0.05). Significance for t-tests are reported with a p value < 0.05 using two-tailed tests with an α level of 0.05. Values represent mean ± SEM.
Amyloid precursor protein
This work was supported by NIH grants P50 AG05138 (DLD, SG, PRH), P01 AG10491 (SG), and F31 AG039890 (HB). Support was also provided by the Canadian Institutes of Health Research (MOP-115056, PEF and PHH), Alberta Heritage Foundation for Medical Research (DW), and the Wellcome Trust, Medical Research Council, Howard Hughes Medical Institute, and Alzheimer Society of Ontario (PHH). We would like to thank the members of the Ehrlich, Gandy, and Hof laboratories for help and discussion, and Dr Suzana Herculano-Houzel (Federal University of Rio de Janeiro) for introducing us to the isotropic fractionator. We would like to acknowledge Dr Camilla Butti (Icahn School of Medicine at Mount Sinai) for her help with optimizing the isotropic fractionator protocol.
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