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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC Apr 13, 2011.
Published in final edited form as:
PMCID: PMC2988475
NIHMSID: NIHMS244777
Apolipoprotein E4 Causes Age- and Tau-Dependent Impairment of GABAergic Interneurons, Leading to Learning and Memory Deficits in Mice
Yaisa Andrews-Zwilling,1,5 Nga Bien-Ly,1,3 Qin Xu,1,2 Gang Li,1,5 Aubrey Bernardo,1 Seo Yeon Yoon,1 Daniel Zwilling,1,5 Tonya Xue Yan,1 Ligong Chen,1 and Yadong Huang1,2,3,4,5
1Gladstone Institute of Neurological Disease, University of California, San Francisco, California 94158, USA
2Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California 94158, USA
3Biomedical Sciences Graduate Program, University of California, San Francisco, California 94158, USA
4Department of Pathology University of California, San Francisco, California 94158, USA
5Department of Neurology, University of California, San Francisco, California 94158, USA
Address for correspondence Yadong Huang, MD, PhD Gladstone Institute of Neurological Disease 1650 Owens Street San Francisco, CA 94158 Tel: 415-734-2511 Fax: 415-355-0824 ; yhuang/at/gladstone.ucsf.edu
Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease. However, the underlying mechanisms are unclear. We found that female apoE4 knock-in (KI) mice had an age-dependent decrease in hilar GABAergic interneurons that correlated with the extent of learning and memory deficits, as determined in the Morris water maze, in aged mice. Treating apoE4-KI mice with daily peritoneal injections of the GABAA receptor potentiator pentobarbital at 20 mg/kg for 4 weeks rescued the learning and memory deficits. In neurotoxic apoE4 fragment transgenic mice, hilar GABAergic interneuron loss was even more pronounced and also correlated with the extent of learning and memory deficits. Neurodegeneration and tauopathy occurred earliest in hilar interneurons in apoE4 fragment transgenic mice; eliminating endogenous Tau prevented hilar GABAergic interneuron loss and the learning and memory deficits. The GABAA receptor antagonist picrotoxin abolished this rescue, while pentobarbital rescued learning deficits in the presence of endogenous Tau. Thus, apoE4 causes age- and Tau-dependent impairment of hilar GABAergic interneurons, leading to learning and memory deficits in mice. Consequently, reducing tau and enhancing GABA signaling are potential strategies to treat or prevent apoE4-related Alzheimer's disease.
Keywords: Alzheimer's disease, apoE, GABAergic interneuron, learning, memory, proteolysis, Tau
Alzheimer's disease, the most common age-dependent neurodegenerative disorder, is characterized by progressive memory loss and cognitive decline (Hardy and Selkoe, 2002; Perrin et al., 2009). The neuropathological hallmarks of Alzheimer's disease are amyloid plaques, extracellular deposits of amyloid–β (Aβ) peptides, and neurofibrillary tangles, which are intraneuronal filaments composed of hyperphosphorylated Tau (Brunden et al., 2009; Crowther, 1993; Selkoe, 1991; Tanzi and Bertram, 2001). Apolipoprotein (apo) E4, the major known genetic risk factor for Alzheimer's disease (Corder et al., 1993; Strittmatter et al., 1993), colocalizes with amyloid plaques and neurofibrillary tangles (Namba et al., 1991; Strittmatter et al., 1993; Wisniewski and Frangione, 1992). ApoE is a 34-kDa protein composed of 299 amino acids, which exists in three major isoforms (apoE2, apoE3 and apoE4) in humans (Mahley, 1988; Weisgraber, 1994). They differ at one or two positions in their primary sequence. ApoE3 has Cys-112 and Arg-158, whereas apoE4 has arginines at both positions, and apoE2 has cysteines (Mahley, 1988; Weisgraber, 1994). ApoE4, found in 65–80% of cases of sporadic and familial Alzheimer's disease (Farrer et al., 1997), increases the occurrence and lowers the age of onset of the disease in a gene dose–dependent manner (Corder et al., 1993; Farrer et al., 1997). ApoE4 is also associated with cognitive impairment in humans (Caselli et al., 2009) and causes learning and memory deficits in mice (Bour et al., 2008; Hartman et al., 2001; Raber et al., 1998; Raber et al., 2000). Although biochemical, cell biological, transgenic animal, and clinical studies have suggested potential explanations for apoE4's contribution to the pathogenesis of Alzheimer's disease (for reviews see Huang, 2006a, b; Huang, 2010; Mahley et al., 2006; Kim et al., 2009; Zhong and Weisgraber, 2009; Irizarry et al., 2000; Bell and Zlokovic, 2009; Bu, 2009; Herz and Beffert, 2000; Herz, 2009; Hoe and Rebeck, 2008), the mechanisms are still unclear.
We previously reported that apoE4 impairs GABAergic interneurons in the hilus of the dentate gyrus in human apoE knock-in (KI) mice (Li et al., 2009), likely due to increased Tau phosphorylation mediated by neurotoxic apoE4 fragments (Huang, 2006b; Huang, 2010; Li et al., 2009). Interestingly, apoE4 is associated with subclinical epileptiform activity under stress (Palop and Mucke, 2009) and increased brain activity at rest and in response to memory tasks in humans (Filippini et al., 2009), probably reflecting impaired GABAergic inhibitory neuronal functions. There is evidence of GABAergic interneuron impairment in Alzheimer's disease patients, including a decrease in GABA or somatostatin levels in brains and cerebrospinal fluid (CSF) (Bareggi et al., 1982; Davies et al., 1980; Hardy et al., 1987; Seidl et al., 2001; Zimmer et al., 1984) that is exacerbated by apoE4 (Grouselle et al., 1998). Importantly, learning triggers a rapid increase in inhibitory synaptogenesis in the cortex and a lasting increase in GABA release from hippocampal interneurons in mice, suggesting that learning normally involves an increase in inhibitory synaptic plasticity and GABA release (Cui et al., 2008; Nitz and McNaughton, 2004; Perrin et al., 2009). We hypothesize that apoE4 causes hippocampal GABAergic interneuron impairment, leading to learning and memory deficits. In the present study, we tested this hypothesis in different mouse models of apoE.
Reagents and Cell Culture
Minimal essential medium (MEM), Opti-MEM, and fetal bovine serum were from Invitrogen. Primary hippocampal neuronal cultures were prepared from P0 pups of apoE4(Δ272–299)mE−/−Tau+/+, apoE4(Δ272–299)mE−/−Tau−/−, mE−/−Tau+/+, mE−/−Tau−/− and wildtype mice, as reported (Li et al., 2009). Hippocampi were isolated on postnatal day 0, and dissociated cells were plated at 125,000 cells/ml in Neurobasal medium supplemented with B27, 100 U/ml penicillin G, and 100 μg/ml streptomycin. The genotypes of cultures were determined by PCR analysis of the tails of the pups from which the cells were obtained. After 14 days in vitro, the cultures were fixed in 4% paraformaldehyde in phosphate-buffered saline (135 mM NaCl, 2.7 mM KCl, 43 mM Na2HPO4, 14 mM KH2PO4, pH 7.4) for 30 min at room temperature. After permeabilization in phosphate-buffered saline with 0.1% Triton for 10 min, cells were placed in blocking buffer (phosphate-buffered saline with 10% normal serum from the same species that produced the secondary antibody and 0.01% Triton) for 30 min. Primary anti-GAD67 (1:250; Chemicon) were applied overnight at 4°C and visualized with anti-mouse IgG conjugated with Alexa Fluor 488. Cells were counter-stained with DAPI. To measure GABAergic neuronal survival in hippocampal neuron cultures, GAD67-positive neurons were counted in 15–30 random fields under a fluorescence microscope at 200x magnification (Li et al., 2009).
Mice and Treatments
Wildtype mice and human apoE3-KI and apoE4-KI mice (Sullivan et al., 2004) were from Taconic. Tau−/− mice (Dawson et al., 2001) were backcrossed onto the mE−/− background, and mE−/−Tau−/− mice were crossed with apoE4(Δ272–299)mE−/−Tau+/+ mice. Studies were conducted on female mice at 1, 3, 6, 12, 16, or 21 months of age. All mice were on a C57BL/6 genetic background. Some apoE4-KI and apoE4(Δ272–299)mE−/−Tau+/+ mice received daily intraperitoneal injections of pentobarbital (20 mg/kg) or saline in their home cages for 21 days before the first day of the Morris water maze training and 1 h after daily training. Some apoE4(Δ272–299)mE−/−Tau−/− mice were given daily injections of picrotoxin (1 mg/kg) or saline intraperitoneally in their home cages for 3 days before the Morris water maze training and 30 min before daily training. Brain tissues were collected after a 1-min transcardial perfusion with 0.9% NaCl. One hemibrain from each mouse was fixed in 4% paraformaldehyde, sectioned (30 μm) with a microtome, and immunostained as described below. All experiments were performed in accordance with NIH and institutional guidelines.
Immunohistochemistry and Image Collection
Sliding microtome sections (30 μm) were immunostained with the following primary antibodies: polyclonal goat anti-human apoE (1:8000 for fluorescence; Calbiochem), rabbit anti-neuropeptide Y (1:8000 for DAB; ImmunoStar), rat anti-somatostatin (1:100 for DAB; Chemicon), mouse anti-GAD67 (1:1000 for DAB; Chemicon), mouse anti-MAP2 (1:500 for fluorescence; Sigma), mouse anti-synaptophysin (1:500 for fluorescence; DakoCytomation), and phosphorylation-dependent monoclonal antibody AT8 (p-Ser202; 1:100 for DAB; Endogen). Primary antibodies were detected with biotinylated goat anti-rabbit or goat anti-rat IgG (both 1:200; Vector Laboratories), Alexa Fluor 488-labeled goat anti-rabbit IgG (1:2000; Invitrogen), or Alexa Fluor 594–labeled donkey anti-mouse IgG (1:2000; Invitrogen). Stained sections were examined with a Radiance 2000 laser-scanning confocal system (Bio-Rad) mounted on a Nikon Optiphot-2 microscope. Images were processed with Photoshop CS (Adobe Systems).
Quantitative Analyses of Immunostained Brain Sections
GABAergic interneurons in the hilus of the dentate gyrus were quantified by counting GAD67-, NPY-, and somatostatin-positive cells in every tenth serial coronal section throughout the rostrocaudal extent of the hippocampus by an investigator blinded to genotype and treatment (Li et al., 2009). Results are presented as the total number of positive cells counted per hemibrain, multiplied by two (for both hemibrains), and then by 10 (for every tenth serial section).
Morris Water Maze
The water maze pool (diameter, 122 cm) contained opaque water (22–23°C) with a platform 10 cm in diameter. The platform was submerged 1.5 cm during hidden platform sessions (Harris et al., 2003; Raber et al., 1998; Roberson et al., 2007) and marked with black-and-white-striped mast (15 cm high) during cued training sessions. Mice were trained to locate the hidden platform (hidden days 1–5) and the cued platform (visible days 1–3) in two daily sessions (3.5 h apart), each consisting of two 60-s trials (hidden and cued training) with a 15-min intertrial interval. The platform location remained constant in the hidden platform sessions but was changed for each cued platform session. Entry points were changed semirandomly between trials. Twenty-four, 72, and 96 hours after the last hidden platform training, a 60-s probe trial (platform removed) was performed. Entry points for the probe trial were in the west quadrant, and the target quadrant was in the southeast quadrant. Performance was monitored with an EthoVision video-tracking system (Noldus Information Technology). The data were presented as percent time spent and platform crossings. Percent time spent compares the time spent in the target quadrant to the average time spent in the other three quadrants. Platform crossings compare the number of crossings over the position of the target platform to the average number of crossings over the equivalent position of the platform in the other three quadrants.
Elevated Plus Maze
The elevated plus maze tests “emotionality” and unconditioned anxiety-related behaviors that involve a conflict between the rodent's desire to explore a novel environment and anxiogenic elements such as elevation and an unfamiliar, brightly illuminated area (Roberson et al., 2007). The maze consists of two open arms and two closed arms equipped with rows of infrared photo-cells interfaced with a computer (Hamilton). Mice were placed individually into the center of the maze and allowed to explore for 10 min. The number of beam breaks was recorded to calculate the amount of time spent and distance moved in each arm and the number of entries into the open and closed arms. After each mouse was tested, the maze was cleaned with 70% ethanol to standardize odors.
Electrophysiology
Wildtype, ApoE3-KI, and apoE4-KI mice were sacrificed and processed for slice preparation as described (Li et al., 2009). Brains were quickly removed into an ice-cold solution containing (in mM) 110 choline chloride, 2.5 KCl, 1.3 KH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 10 dextrose, 1.3 sodium ascorbate, and 0.6 sodium pyruvate (300–305 mOsm). Horizontal slices (350 μm thick) were cut with a Vibratome, maintained in continuously oxygenated external solution (in mM: 125 NaCl, 2.5 KCl, 1.3 KH2PO4, 25 NaHCO3, 2 CaCl2, 1.3 MgCl2, 1.3 sodium ascorbate, 0.6 sodium pyruvate, 10 dextrose, pH 7.4) at 30°C for at least 40 min, and incubated at room temperature for at least 60 min before recording. Whole-cell voltage-clamp recordings from dentate gyrus granule cells were obtained with an infrared differential interference contrast video microscopy system. Patch electrodes (3–6 MΩ) were pulled from borosilicate glass capillary tubing (World Precision Instruments) on a horizontal Flaming-Brown microelectrode puller (model P-97, Sutter Instruments). Intracellular patch pipette solution contained (in mM) 120 Cs-gluconate, 10 HEPES, 0.1 EGTA, 15 CsCl2, 4 MgCl2, 4 Mg-ATP, and 0.3 Na2-GTP, pH 7.25 (285– 290 mOsm). To measure mIPSCs, slices were perfused with artificial cerebrospinal fluid containing 20 μ M DNQX, 50 μ M D-AP5, and 1 μM TTX. Whole-cell voltage-clamp data were low-pass filtered at 6 kHz (–3 dB, eight-pole Bessel), digitally sampled at 10 kHz with a Multiclamp 700A amplifier (Axon Instruments), and acquired with a Digidata-1322 digitizer and pClamp 9.2 software (Axon Instruments). Whole-cel l access resistance was monitored throughout the recording, and cells were rejected if values changed by >25%. mIPSCs were analyzed with a program provided by Dr. John Huguenard (Stanford University).
Statistical Analyses
Values are expressed as mean ± SEM or mean ± SD. Statistical analyses were performed with GraphPad Prism, Statview 5.0 (SAS Institute) or SPSS-10 (SPSS). Differences between means were assessed by t test, Mann-Whitney U test, one-factor ANOVA, or two-factor ANOVA, followed by Bonferroni, Tukey-Kramer or Fisher's PLSD post hoc tests. P < 0.05 was considered statistically significant.
Age-dependent decrease in GABAergic interneurons in the hilus of dentate gyrus of female apoE4-KI mice
To assess the effect of aging and apoE4 on GABAergic interneurons in the hippocampus, we first quantified their numbers in the hilus of dentate gyrus of female apoE3-KI and apoE4-KI mice at 1, 3, 6, 12, 16, and 21 months of age. We studied female mice because they are susceptible to apoE4-induced learning and memory deficits (Raber et al., 1998; Raber et al., 2000). Anti-GAD67 and anti-somatostatin immunostaining, as shown representatively in 16-month-old apoE3-KI and apoE4-KI mice (Fig. 1A–D), revealed a significantly greater age-dependent decrease in GABAergic interneurons in the hilus of the dentate gyrus of female apoE4-KI mice than in age- and sex-matched apoE3-KI mice (ANOVA; GAD67, age, F5 = 22.65, p < 0.001; apoE genotype, F1 = 21.74, p < 0.001; interaction, F5 = 0.4601, p > 0.05; somatostatin, age, F5 = 4.972, p < 0.001; apoE genotype, F1 = 29.6, p < 0.001; interaction, F5 = 1.335, p > 0.05) (Fig. 1E,F). The significant difference between apoE4-KI and apoE3-KI mice was first observed at 6 months (two-tailed t test; GAD67, t6 = 2.963, p < 0.05; somatostatin, t6 = 5.455, p < 0.05) and was most pronounced at 16 (two-tailed t test; GAD67, t20 = 2.628, p < 0.01; somatostatin, t20 = 4.334, p < 0.01) and 21 (two-tailed t test; GAD67, t14 = 3.503, p < 0.01; somatostatin, t14 = 3.637, p < 0.01) months (Fig. 1E,F). ApoE3-KI mice had a milder age-dependent decrease in hilar GABAergic interneurons (Fig. 1E,F). Interestingly, the number of GABAergic interneurons in the hippocampal CA1 area did not differ in apoE3-KI and apoE4-KI mice at 16 months (Fig. 1G–I), suggesting a region-specific detrimental effect of apoE4 on GABAergic interneurons. As controls, female wildtype mice at 3, 12, and 16 months of age were also included in the study. At all three ages, the numbers of hilar GABAergic interneurons in wildtype mice were similar to those in apoE3-KI mice (Fig. 1J–M). Thus, as compared to mouse apoE, human apoE3 has no effect, but human apoE4 has detrimental effect, on hilar GABAergic interneurons.
Figure 1
Figure 1
Age-dependent significant decrease in numbers of GABAergic interneurons in the hilus of dentate gyrus of female apoE4-KI mice. A–D, Representative photomicrographs (200x) from female apoE3-KI and apoE4-KI mice at 16 months of age show GABAergic (more ...)
Presynaptic GABAergic input onto granule cells is reduced in female apoE4-KI mice
The axonal termini of GABAergic interneurons on granule cells in the dentate gyrus of female wildtype, apoE3-KI, and apoE4-KI mice at 16 months of age were assessed by anti-GAD67 and anti-synaptophysin double immunofluorescence staining and confocal imaging analysis. The GABAergic axonal termini on granule cells were significantly decreased at the absolute level (GAD67 fluorescence intensity) and relative to the presynaptic marker synaptophysin (GAD67/synaptophysin ratio) in apoE4-KI mice as compared to wildtype (two-tailed t test; GAD67, t16 = 3.112, p < 0.005; GAD67/Syn, t16 = 3.076, p < 0.005) and apoE3-KI (two-tailed t test; GAD67, t20 = 5.677, p < 0.005; GAD67/Syn, t20 = 5.678, p < 0.005) mice (Fig. 2A–K). To assess the functional consequence of this finding, we performed whole-cell patch-clamp recordings from granule cells; glutamate currents were blocked with 6,7-dinitroquinoxaline-2,3-dione (DNQX) (20 μM) and D-(-)-2-amino-5-phosphonovaleric acid (D-AP5) (50 μM), and action potential–mediated GABA release was blocked with tetrodotoxin (TTX) (1 μM). Consistent with the above findings, the frequency of miniature inhibitory postsynaptic currents (mIPSCs) was ~40% lower in apoE4-KI mice than in wildtype (two-tailed t test; t19 = 2.816, p < 0.01) and apoE3-KI (two-tailed t test; t18 = 2.712, p < 0.01) mice (Fig. 2L–O). The mIPSC amplitude and membrane resistance were not altered significantly (Fig. 2P,Q). These results suggest that apoE4-KI mice have fewer functional GABAergic synapses onto granule cells.
Figure 2
Figure 2
Presynaptic GABAergic input onto granule cells is reduced in female apoE4-KI mice. A–I, Representative confocal images of the granule cell layer of the dentate gyrus of female wildtype (A–C), apoE3-KI (D–F), and apoE4-KI (G–I) (more ...)
Hilar GABAergic interneuron impairment precedes learning and memory deficits in female apoE4-KI mice
Next, we tested spatial learning and memory of female wildtype, apoE3-KI, and apoE4-KI mice at 12, 16, and 21 months of age in the Morris water maze. At 12 months, wildtype, apoE3-KI, and apoE4-KI mice performed equally well in the hidden platform and probe trials (not shown), suggesting normal learning and memory in all three groups. At 16 months, wildtype and apoE3-KI mice quickly learned to find the hidden platform, which requires spatial learning, but apoE4-KI mice showed deficits (repeated measures ANOVA; F(2,28) = 9.217, p < 0.01; post-hoc comparisons; E4-KI vs E3-KI, t20 = 3.521, p < 0.01; E4-KI vs WT, t19 = 2.927, p < 0.01) (Fig. 3A). Similar learning deficits were found in apoE4-KI mice at 21 months of age (Fig. 4D). Swim speeds did not differ among the three groups of mice (Supplemental Fig. 1A), indicating that the impairment was not due to motor deficits. Wildtype, apoE3-KI, and apoE4-KI mice performed equally well in visible platform trials (Fig. 3A and Fig. 4D). In the probe trial 96 h after the last hidden platform trial, 16-month-old apoE4-KI mice had a deficit potentially in memory retention (Fig. 3B, probe 3), although they performed as well as wildtype and apoE3-KI mice in probe trials at 24 (Supplemental Fig. 1B) and 72 h (Fig. 3B, probe 2). Thus, hilar GABAergic interneuron impairment, first observed at 6 months of age, precedes the learning and memory deficits, which were first observed at 16 months of age, in apoE4-KI mice.
Figure 3
Figure 3
Correlation of hilar GABAergic interneuron impairment with spatial learning deficits in apoE4-KI mice. A, Nine wildtype, ten apoE3-KI, and 12 apoE4-KI female mice were tested at 16 months of age in the Morris water maze. Points represent averages of daily (more ...)
Figure 4
Figure 4
GABAA receptor potentiator pentobarbital rescues spatial learning and memory deficits in apoE4-KI mice. A, Female 16-month-old apoE4-KI mice were treated with pentobarbital (PB, 20 mg/kg i.p.) or saline (n = 6–13 mice per group) for 21 days before (more ...)
Hilar GABAergic interneuron impairment correlates with spatial learning deficits in female apoE4-KI mice
In days 1–5 of the hidden platform trials, the number of hilar GABAergic interneurons correlated inversely with escape latency of apoE4-KI mice (GAD67, R2 = 0.385, p < 0.05, n =12; somatostatin, R2 = 0.516, p < 0.01, n =12), but not apoE3-KI and wildtype mice, at 16 months of age (Fig. 3C–F and Supplemental Fig. 2A,B); no correlation was observed in visible platform trials (Supplemental Fig. 2C,D and Supplemental Fig. 3A–D). Similar results were obtained in apoE3-KI and apoE4-KI mice at 21 months of age (Fig. 3G,H and Supplemental Fig. 3E,F). Interestingly, at both ages, all apoE3-KI and wildtype mice had more than 2500 hilar GABAergic interneurons (Fig. 3D,F,H and Supplemental Fig. 2), whereas ~50% of the apoE4-KI mice had fewer than 2500 (Fig. 3C,E,G) and had greater learning deficits in the hidden platform trials (Fig. 3C,E,G).
We then looked at the individual numbers of hilar GABAergic interneurons in female apoE4-KI mice at 6 or 12 months of age, when they also had, on average, significantly fewer hilar GABAergic interneurons than apoE3-KI and wildtype mice at similar ages (Fig. 1E,F,L,M). Interestingly, none of those mice had fewer than 2500 hilar GABAergic interneurons, and none of those mice had learning and memory deficits at 12 months, as mentioned above. Thus, 2500 might be the threshold number of hilar GABAergic interneurons that determines normal versus impaired learning performance of female mice in the Morris water maze.
Pentobarbital rescues spatial learning and memory deficits in female apoE4-KI mice
To determine whether the loss of GABAergic interneurons contributes directly to the learning and memory deficits, we treated 16-month-old female apoE4-KI mice with daily peritoneal injections of the GABAA receptor potentiator pentobarbital at 20 mg/kg for 4 weeks. This treatment rescued the learning and memory deficits (Fig. 4A,B) but did not alter the number of hilar GABAergic interneurons (Fig. 4C). The learning deficit was also rescued in 21-month-old female apoE4-KI mice by pentobarbital treatment (Fig. 4D).
Alzheimer's disease-like neurodegeneration occurs in transgenic mice expressing low levels of apoE4(Δ272–299)
We reported that neurons under stress, including neurons cultured in vitro (Harris et al., 2004b; Xu et al., 2008), express apoE and that neuronal apoE undergoes proteolytic cleavage to generate neurotoxic fragments, with apoE4 being more susceptible to the cleavage than apoE3 (Brecht et al., 2004; Harris et al., 2003; Huang et al., 2001). In primary hippocampal neuronal cultures, apoE4 impairs the survival of GABAergic interneurons by generating more neurotoxic apoE fragments and increasing the levels of phosphorylated Tau (p-Tau) (Li et al., 2009).
To assess the contributions of apoE4 fragments and p-Tau to hilar GABAergic interneuron impairment and behavioral deficits in vivo, we studied transgenic mice expressing low levels of apoE4(Δ272–299), a major neurotoxic fragment in mouse and Alzheimer's disease brains (Brecht et al., 2004; Harris et al., 2003), under the control of the neuron-specific Thy-1 promoter. These mice develop Alzheimer's disease-like neurodegeneration and spatial learning and memory deficits (Harris et al., 2003). To eliminate confounding effects of mouse apoE, we crossed the original apoE4(Δ272–299) transgenic line with apoE knockout (mE−/−) mice to generate apoE4(Δ272–299)mE−/−Tau+/+ mice. To assess the effect of tau removal on Alzheimer's disease-like neuronal and behavioral deficits caused by apoE4 fragments, we crossed apoE4(Δ272–299)mE−/−Tau+/− mice with mE−/−Tau+/− mice to generate littermates of apoE4(Δ272–299)mE−/−Tau+/+, apoE4(Δ272–299)mE−/−Tau−/−, mE−/−Tau+/+, and mE−/−Tau−/− mice. Eliminating endogenous tau did not alter the expression levels of apoE4(Δ272–299) (not shown). Age- and sex-matched wildtype mice were included as controls.
Morphological studies revealed neuronal deficits in the hippocampus of 12-month-old apoE4(Δ272–299)mE−/−Tau+/+ mice, including presynaptic accumulation of apoE4 fragments as determined by anti-apoE and anti-synaptophysin (a presynaptic marker) or anti-MAP2 (a dendritic marker) double immunostaining (Fig. 5A–F), neurodegeneration as determined by hematoxylin/eosin and anti-MAP2 immunostaining (Fig. 5G–J), and tau pathology as determined by anti-p-Tau (AT8 monoclonal antibody) immunostaining (Fig. 5L,M,O,P) in the hilus of the dentate gyrus, the hippocampal CA3 area, and the subiculum. Strikingly, neurodegeneration and Tau pathology occurred earliest in the hilus (Fig. 5G–J,L).
Figure 5
Figure 5
Localization of apoE4(Δ272–299) in the hippocampus and its effects on neurodegeneration and Tau pathology in the presence and absence of Tau. A–D, Double immunofluorescence staining for apoE (green) and NeuN (red) in the hippocampus (more ...)
Tau removal prevents loss of hilar GABAergic interneurons in female apoE4(Δ272–299) mice
Immunostaining for GAD67 (Fig. 6A–E), neuropeptide Y (NPY) (Fig. 6F–J), and somatostatin (Fig. 6K–O) revealed 40–50% fewer GABAergic interneurons in the hilus of apoE4(Δ272–299)mE−/−Tau+/+ mice than in mE−/−Tau+/+ mice (two-tailed t test; GAD67, t22 = 5.536, p < 0.005; NPY, t14 = 4.738, p < 0.005; somatostatin, t14 = 3.964, p < 0.005) or wildtype controls (two-tailed t test; GAD67, t13 = 5.193, p < 0.005; NPY, t13 = 5.897, p < 0.005; somatostatin, t13 = 4.031, p < 0.005) (Fig. 6P–R). Eliminating Tau prevented neuronal deficits in apoE4 fragment transgenic mice, including loss of GABAergic interneurons in the hilus (two-tailed t test; GAD67, t20 = 5.083, p < 0.005; NPY, t20 = 8.685, p < 0.005; somatostatin, t20 = 3.991, p < 0.005) (Fig. 6), neurodegeneration (compare Fig. 5K to 5I,J), and Tau pathology in hilar interneurons (compare Fig. 5N to 5L,M).
Figure 6
Figure 6
Loss of GABAergic interneurons in the hilus of the dentate gyrus of apoE4(Δ272–299)mE−/−Tau+/+ mice and rescue by Tau removal. The brains of 14 mE−/−Tau+/+, 10 apoE4(Δ272–299)mE−/− (more ...)
In 14-day primary hippocampal neuronal cultures, immunostaining for GAD67 (Fig. 7A,B) revealed ~50% fewer GABAergic neurons in cultures from apoE4(Δ272–299)mE−/−Tau+/+ mice than from mE−/−Tau+/+ controls (two-tailed t test; t24 = 2.595, p < 0.01) (Fig. 7E) and markedly lower GAD67 immunoreactivity in neurites of surviving GABAergic neurons (compare Fig. 7B to 7A). Tau removal increased the survival of GABAergic neurons from apoE4(Δ272–299)mE−/−Tau−/− mice to levels higher than in mE−/−Tau+/+ mice (two-tailed t test; t23 = 3.49, p < 0.01) (Fig. 7A–C,E). Removing Tau also increased the survival of GABAergic neurons from mE−/−Tau−/− mice to levels higher than those of neurons from mE−/−Tau+/+ mice (two-tailed t test; t21 = 3.145, p < 0.01) (Fig. 7A,D,E). Thus, eliminating endogenous Tau rescues apoE4 fragment-caused GABAergic interneuron impairment both in mice and in primary hippocampal neuronal cultures.
Figure 7
Figure 7
Eliminating Tau prevents the neurotoxic effect of apoE4 fragments on primary hippocampal GABAergic neurons. A–D, Primary hippocampal neurons from individual P0 pups (mE−/−Tau+/+, apoE4(Δ272–299)mE−/− (more ...)
Tau removal prevents spatial learning and memory deficits in female apoE4(Δ272–299) mice
To assess effects of Tau removal on learning and memory deficits induced by apoE4 fragments, we tested 12-month-old female mice in the Morris water maze. In the hidden platform trial, mE−/−Tau+/+ and wildtype mice quickly learned the task, but apoE4(Δ272–299)mE−/−Tau+/+ mice showed a deficit (repeated measures ANOVA; F(4,55) = 10.24, p < 0.001; post-hoc comparisons; apoE4(Δ272–299)mE−/−Tau+/+ vs other groups, the smallest t20 = 3.537, the biggest p < 0.01) (Fig. 8A). Swim speeds of the mice did not differ (Supplemental Fig. 4). ApoE4(Δ272–299)mE−/−Tau−/− mice performed as well as mE−/−Tau+/+ and wildtype mice in the hidden platform trial (Fig. 8A). Thus, Tau removal prevented the apoE4 fragment-induced learning deficit. In subsequent visible platform trials, all groups of mice performed equally well (Fig. 8A). In the probe trial 24 h after the last hidden platform trial, apoE4(Δ272–299)mE−/−Tau+/+ mice had deficits in the target crossing and target quadrant tests that were eliminated by Tau removal (Fig. 8B,C). Interestingly, in the elevated plus maze, which assesses hippocampus-independent anxiety, apoE4(Δ272–299)mE−/−Tau+/+ mice had increased anxiety that was unaffected by Tau removal (Supplemental Fig. 5A), suggesting that elimination of Tau specifically affects hippocampus-dependent learning and memory performance.
Figure 8
Figure 8
Spatial learning and memory deficits in apoE4(Δ272–299)mE−/−Tau+/+ mice and rescue by Tau removal. A, Fourteen mE−/−Tau+/+, 10 apoE4(Δ272–299)mE−/−Tau+/+, 12 apoE4(Δ272–299)mE (more ...)
Hilar GABAergic interneuron impairment correlates with spatial learning deficits in female apoE4(Δ272–299) mice with Tau
In apoE4(Δ272–299)mE−/−Tau+/+ mice, the number of GABAergic interneurons in the hilus correlated inversely with escape latency on days 1–5 of the hidden platform test (GAD67, R2 = 0.515, p < 0.05, n =10; somatostatin, R2 = 0.526, p < 0.05, n =10; NPY, R2 = 0.410, p < 0.05, n =10) (Fig. 8D–F). Importantly, as in apoE4-KI mice (Fig. 3C,E,G), apoE4(Δ272–299)mE−/−Tau+/+ mice with fewer than 2500 hilar GABAergic interneurons had greater learning deficits in the hidden platform trials (Fig. 8D–F), consistent with a threshold of ~2500 hilar GABAergic interneurons for normal versus impaired learning performance in the Morris water maze. The number of hilar GABAergic interneurons did not correlate with performance in visible platform trials in apoE4(Δ272–299)mE−/−Tau+/+ mice (Supplemental Fig. 5B–D).
Tau removal prevents apoE4-induced learning and memory deficits by protecting against hilar GABAergic interneuron impairment
Finally, we determined whether the rescue of learning and memory deficits by Tau removal reflects protection against GABAergic interneuron impairment. ApoE4(Δ272–299)mE−/−Tau−/− mice were treated with a subthreshold dose (1 mg/kg) of picrotoxin, a GABAA receptor antagonist, to block GABA signaling. The rescue was abolished (Fig. 8G,H), but the number of hilar GABAergic interneurons was unaltered (Supplemental Fig. 5E). Picrotoxin at this dose did not alter learning and memory in wildtype or mE−/−Tau+/+ mice (Supplemental Fig. 5F,G). In contrast, treatment of apoE4(Δ272–299)mE−/−Tau+/+ mice with pentobarbital, a GABAA receptor potentiator, rescued the learning deficit (Supplemental Fig. 5H). Evidently, Tau removal rescues apoE4 fragment-induced learning and memory deficits by preventing the loss of GABAergic interneurons.
This study shows that female apoE4-KI mice have a significant age-dependent decrease in hilar GABAergic interneurons that correlates with the extent of apoE4-induced learning and memory deficits in aged mice. In neurotoxic apoE4 fragment transgenic mice, the interneuron loss was even more pronounced and correlated with the extent of learning and memory deficits. The interneuron loss and learning and memory deficits in these mice were prevented by Tau removal, and the prevention was abolished by blocking GABA signaling with picrotoxin. In both groups of mice, the GABAA receptor potentiator pentobarbital rescued the learning and memory deficits. Thus, apoE4 causes age- and Tau-dependent hilar GABAergic interneuron impairment, leading to learning and memory deficits in mice.
Roles of apoE4-caused GABAergic interneuron impairment in learning and memory deficits and Alzheimer's disease pathogenesis
The GABAergic system appears to be important in the neuronal control of learning and memory. Learning triggers a rapid increase in inhibitory synaptogenesis and the GABA content of inhibitory synapses (Jasinska et al., 2010), accompanied by long-lasting enhancement of synaptic inhibition onto excitatory neurons in mice (Brosh and Barkai, 2009). A novel environment and spatial learning also trigger lasting increase in GABA release from hippocampal GABAergic interneurons in mice (Cui et al., 2008; Nitz and McNaughton, 2004). Conversely, decreasing GABA levels in the hippocampus by overexpressing GABA transport 1 (GAT1), which is responsible for GABA reuptake after its synaptic release, impairs learning and memory in mice (Hu et al., 2004). Thus, learning appears to involve an increase in inhibitory synaptic plasticity and GABA release.
Dysfunction of the GABAergic system might contribute to cognitive impairment in humans. Alzheimer's disease patients have decreased GABA or somatostatin levels in the brain and CSF (Bareggi et al., 1982; Davies et al., 1980; Hardy et al., 1987; Seidl et al., 2001; Zimmer et al., 1984) that are exacerbated by apoE4 (Grouselle et al., 1998). A single nucleotide polymorphism (SNP) of the somatostatin gene is associated with increased risk for Alzheimer's disease in apoE4, but not apoE3, carriers (Vepsalainen et al., 2007; Xue et al., 2009). Furthermore, GABA levels in human CSF decrease with age (Bareggi et al., 1982)—the strongest risk factor for Alzheimer's disease.
We found a significant age-dependent decrease in hilar GABAergic interneurons in apoE4-KI mice that correlated with apoE4-induced learning deficits in aged mice. Interestingly, a threshold number of hilar GABAergic interneurons (~2500) appeared to distinguish normal versus impaired learning performance in the Morris water maze. Importantly, in apoE4-KI mice, hilar GABAergic interneuron impairment preceded learning and memory deficits, and the deficits were rescued by treatment with the GABAA receptor potentiator pentobarbital. Thus, it is conceivable that apoE4 causes age-dependent impairment of hilar GABAergic interneurons, leading to learning and memory deficits in mice. Consequently, enhancing GABA signaling might be a strategy to treat Alzheimer's disease related to apoE4. Interestingly, apoE4 is associated with subclinical epileptiform activity under stress (Palop and Mucke, 2009) and increased brain activity at rest and in response to memory tasks in humans (Dennis et al., 2009; Filippini et al., 2009), probably reflecting impaired GABAergic inhibitory neuronal functions in humans. Thus, the importance of apoE4-caused GABAergic interneuron impairment in the pathogenesis of Alzheimer's disease warrants further studies in humans.
How does impairment of GABAergic interneurons by apoE4 lead to learning and memory deficits? Since learning and memory normally involve an increase in inhibitory synaptic plasticity and GABA release, apoE4-caused GABAergic interneuron impairment could contribute directly to learning and memory deficits. On the other hand, apoE4-caused GABAergic interneuron dysfunction can also lead to impairment of adult hippocampal neurogenesis in mice (Li et al., 2009). Adult hippocampal neurogenesis has been implicated in learning and memory processes (Zhao et al., 2008). Thus, the other possibility is that apoE4-caused GABAergic interneuron dysfunction leads to impaired adult hippocampal neurogenesis, which indirectly contributes to learning and memory deficits. Interestingly, boosting GABA signaling with pentobarbital also rescues apoE4-casued impairment of hippocampal neurogenesis in apoE4-KI mice (Li et al., 2009). Clearly, these two possibilities are not mutually exclusive and warrant further studies to dissect the mechanisms in detail.
It should be emphasized that we only tested female mice in the current study because they are more susceptible than male mice to apoE4-induced learning and memory deficits (Raber et al., 1998; Raber et al., 2000). Likewise, in humans, women with apoE4 have higher risk than men with apoE4 to develop Alzheimer's disease (Farrer et al., 1997). These raise the question as to whether male apoE4-KI mice have a similar or less severe impairment of hilar GABAergic interneurons. Experimentally addressing this question in future studies could help to understand better the gender difference in developing Alzheimer's disease in humans with apoE4.
Importance of Tau in GABAergic interneuron impairment and learning and memory deficits caused by apoE4 and its fragment
Tau removal rescued the learning and memory deficits in mice by preventing apoE4 fragment-caused GABAergic interneuron impairment in the hilus of the hippocampus. This highlights the importance of Tau (or p-Tau) in apoE4's contribution to Alzheimer's disease pathogenesis. ApoE is found in neurofibrillary tangles in Alzheimer's disease brains (Crowther, 1993; Huang, 2006b; Huang, 2010). ApoE3 and apoE4 appear to have different effects on Tau phosphorylation and aggregation (Brecht et al., 2004; Huang, 2006a, b; Huang, 2010; Mahley et al., 2006; Strittmatter et al., 1994). In transgenic mice, expression of human apoE4 in neurons, but not in astrocytes, increases Tau phosphorylation (Brecht et al., 2004; Tesseur et al., 2000a; Tesseur et al., 2000b), consistent with a neuron-specific effect (Huang, 2006a, b; Huang, 2010; Mahley et al., 2006). Previously, we showed that apoE4 and its C-terminal-truncated fragments stimulate Tau phosphorylation and formation of intracellular neurofibrillary tangle-like inclusions in transgenic mice (Brecht et al., 2004; Harris et al., 2003; Huang et al., 2001). In primary hippocampal neuronal cultures, apoE4 impairs the survival of GABAergic interneurons by generating more neurotoxic apoE fragments and increasing the levels of p-Tau in GABAergic interneurons (Li et al., 2009). Here, Tau removal prevented GABAergic interneuron impairment and learning and memory deficits caused by apoE4 fragments in transgenic mice, providing direct in vivo evidence that apoE4 acts upstream of Tau in Alzheimer's disease pathogenesis, as suggested (Brecht et al., 2004; Harris et al., 2004a; Small and Duff, 2008).
Aβ peptides may act upstream of tau, stimulating tau phosphorylation and neurofibrillary tangle formation in human mutant Tau transgenic mice overproducing or injected with Aβ peptides (Götz et al., 2001; Lewis et al., 2001). Knockout of Tau also abolishes Aβ-induced neurotoxicity in primary neuronal cultures and behavioral deficits in transgenic mice, suggesting that Tau is essential for Aβ-related detrimental effects both in vitro and in vivo (Ittner et al., 2010; Rapoport et al., 2002; Roberson et al., 2007). Thus, acting downstream of apoE4 and Aβ peptides, Tau or p-Tau might be a general causative factor in Alzheimer's disease pathogenesis. Therefore, reducing Tau may be an effective strategy to treat or prevent Alzheimer's disease.
In addition to Tau as a key mediator, other reported loss-of-function or gain-of-negative functions of apoE4 might also contribute to the GABAergic interneuron impairment and learning and memory deficits. These include the dysregulation of neuronal signaling pathways, impairment of glucose metabolism and mitochondrial integrity and function, and decreased protein levels of apoE4 in brains (for reviews see Huang, 2006a, b; Huang, 2010; Mahley et al., 2006; Kim et al., 2009; Bu, 2009; Herz, 2009; Hoe and Rebeck, 2008). Clearly, further studies are needed to determine the potential contributions of other apoE4-related loss-of-function or gain-of-negative functions to GABAergic interneuron impairment and learning and memory deficits.
Supplementary Material
Supp1
Acknowledgements
This work was supported in part by the J. David Gladstone Institutes and National Institutes of Health Grants P01 AG022074 and C06RR18928. We thank Drs. M. Vitek and H. Dawson at Duke University for providing tau knockout mice, Dr. N. Devidze and I. Lo and P. Hampto at the Gladstone Behavioral Core for assisting on behavioral tests, and Dr. B. Halabisky for assisting on electrophysiology. We also thank J. Carroll, C. Goodfellow, and A. Wilson for graphics, S. Ordway and G. Howard for editorial assistance, and L. Turney for manuscript preparation.
Dr. Yadong Huang has received funding from Merck for other research on apoE4 and its role in neurodegenerative disorders.
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