Divergent pathways mediate spine alterations and cell death induced by amyloid-beta, wild-type tau, and R406W tau

Abstract

Alzheimer's disease is characterized by synaptic alterations and neurodegeneration. Histopathological hallmarks represent amyloid plaques composed of amyloid-beta (Abeta) and neurofibrillary tangles containing hyperphosphorylated tau. To determine whether synaptic changes and neurodegeneration share common pathways, we established an ex vivo model using organotypic hippocampal slice cultures from amyloid precursor protein transgenic mice combined with virus-mediated expression of EGFP-tagged tau constructs. Confocal high-resolution imaging, algorithm-based evaluation of spines, and live imaging were used to determine spine changes and neurodegeneration. We report that Abeta but not tau induces spine loss and shifts spine shape from mushroom to stubby through a mechanism involving NMDA receptor (NMDAR), calcineurin, and GSK-3beta activation. In contrast, Abeta alone does not cause neurodegeneration but induces toxicity through phosphorylation of wild-type (wt) tau in an NMDAR-dependent pathway. We show that GSK-3beta levels are elevated in APP transgenic cultures and that inhibiting GSK-3beta activity or use of phosphorylation-blocking tau mutations prevented Abeta-induced toxicity of tau. FTDP-17 tau mutants are differentially affected by Abeta. While R406W tau shows increased toxicity in the presence of Abeta, no change is observed with P301L tau. While blocking NMDAR activity abolishes toxicity of both wt and R406W tau, the inhibition of GSK-3beta only protects against toxicity of wt tau but not of R406W tau induced by Abeta. Tau aggregation does not correlate with toxicity. We propose that Abeta-induced spine pathology and tau-dependent neurodegeneration are mediated by divergent pathways downstream of NMDAR activation and suggest that Abeta affects wt and R406W tau toxicity by different pathways downstream of NMDAR activity.

Figure 1.

Spine density of EGFP- and EGFP-tau-expressing neurons in hippocampal slice cultures. A, Confocal image of a whole slice (left; scale bar, 300 μm) after Sindbis virus-mediated expression of EGFP-tau. Note that tau is expressed in every hippocampal subregion with highest efficiency in CA3. Typical morphology of a CA3 pyramidal neuron (right; scale bar, 25 μm) with basal dendrites in stratum oriens and apical dendrites in stratum radiatum region of the hippocampus. B, Representative high-resolution images of 20- to 30-μm-long dendritic fragments of stratum radiatum thick and thin and stratum oriens from CA1 and CA3 neurons after blind deconvolution. Scale bar, 5 μm. C, Spine density in hippocampal CA1 and CA3 neurons from APP transgenic and nontransgenic mice after targeted expression of EGFP or EGFP-tau [n = 19 (EGFP), n = 10 (EGFP-tau)]. Spine density is strongly reduced on APP transgenic background independent of the presence of tau. D, Effect of γ-secretase inhibitor DAPT on spine density of EGFP-tau-expressing neurons from APP transgenic and nontransgenic mice. High-resolution image from CA1 stratum radiatum thick (left; scale bar, 5 μm). For quantitative analysis of spine density (right), data from CA1 and CA3 regions were pooled. DAPT (0.5 μm) completely abolished spine loss, whereas 1 μm DAPT had only a partial albeit still significant effect (n = 41 for 0.5 μm DAPT treatment and n = 40 for 1 μm DAPT-treated and untreated cultures). Analysis of spine density shows a reduction of spine loss by DAPT, which is maximal at 0.5 μm. All values are shown as mean ± SEM (**p < 0.01, ***p < 0.001; one-tailed unpaired Student's t test). DG, Dentate gyrus; str.or., stratum oriens; str.rad., stratum radiatum; non tg., nontransgenic.

Figure 2.

Spine morphology of EGFP- and EGFP-tau-expressing neurons in hippocampal slice cultures. A, Steps of image processing for detection and analysis of spines. Confocal raw image was deconvoluted using 3D blind algorithm (Autodeblur). Medial axis extraction was performed by software 3DMA neuron for identification of dendritic backbone. Spine detection routine allowed determining single spines, which were automatically analyzed for length and volume. Spine shape was classified by 3DMA neuron software to one of the following three types: “mushroom,” “stubby,” or “thin.” B, Spine length in hippocampal CA1 and CA3 pyramidal neurons from APP transgenic and nontransgenic mice after targeted expression of EGFP or EGFP-tau. Spine length is significantly reduced on APP transgenic background independent of tau. C, Spine volume in neurons from APP transgenic and nontransgenic mice after targeted expression of EGFP or EGFP-tau. No difference is observed in spine volume between APP transgenic and nontransgenic cultures. D, Fraction of spines with different shape. Representative high-resolution images (left) show the three different spine types for classification, namely, mushroom (left), stubby (middle), and thin (right). The fraction of mushroom spines is significantly decreased, while stubby spines increase in CA1 and CA3 neurons from APP transgenic mice, independent of the presence of tau. E, Representative images of EGFP-labeled postsynaptic spines with synaptophysin-positive presynaptic boutons. All values are shown as mean ± SEM (*p < 0.05; **p < 0.01; one-tailed unpaired Student's t test). n = 19 (EGFP), n = 20 (EGFP-tau). mush., Mushroom spine; stub., stubby spine; Synaptophys., synaptophysin. Scale bars, 0.5 μm.

Figure 3.

Effect of NMDAR antagonist CPP, calcineurin inhibitor FK-506, and GSK-3β inhibitor TDZD on spines in hippocampal slice cultures. A, Spine density in EGFP-expressing hippocampal CA1 and CA3 neurons from APP transgenic and nontransgenic mice without treatment (top) and after treatment with 20 μm CPP (bottom). Representative high-resolution images of 20- to 30-μm-long dendritic fragments of stratum radiatum thick from CA1 and CA3 neurons after blind deconvolution (left) and quantification of spine density (right) are shown. In untreated slices from APP transgenic mice, spine density is strongly reduced compared with nontransgenic slices (n = 10). After CPP treatment, spine density does not differ between APP transgenic and nontransgenic slices [n = 18 (nontransgenic), n = 17 (APP transgenic)]. Compared with untreated cultures (Fig. 1 C) spine density is reduced on nontransgenic background and increased for APP transgenic mice. B, Spine length and volume after treatment with 20 μm CPP. No difference in spine length is observed between APP transgenic and nontransgenic controls after CPP treatment. Compared with untreated cultures, spine length is significantly reduced in controls. CPP has no effect on spine volume. C, Fraction of spines with different shapes after CPP treatment. CPP increases the fraction of mushroom-shaped spines in APP transgenic cultures to control levels. D, Representative images of dendritic fragments after treatment with 1 μm FK-506 (left) and quantification of spine density (right). FK-506 increases spine density in APP transgenic slices while reducing spine density in controls (n = 14). E, Representative images of dendritic fragments after treatment with 10 μm TDZD (left) and quantification of spine density (right). TDZD completely abolishes spine loss in APP transgenic cultures and does not affect controls (n = 12). (^# p < 0.05 and ^## p < 0.01 indicate a significant decrease and ⁺ p < 0.05; ⁺⁺⁺ p < 0.001 a significant increase compared with untreated cultures; mean ± SEM; one-tailed unpaired Student's t test). mush., Mushroom spine; stub., stubby spine; str.rad., stratum radiatum. Scale bars, 5 μm.

Figure 4.

Effect of CPP, FK-506, and TDZD on the survival of wt tau-expressing neurons in the CA3 region of hippocampal slice cultures. A, Live imaging of EGFP-tau-expressing CA3 neurons from nontransgenic and APP transgenic cultures from day 2 to day 4 postinfection after treatment as indicated. Scale bars, 25 μm. B, Quantification of cell loss on day 3 (top) and day 4 (bottom) postinfection standardized to the respective nontransgenic control. The fraction of nondegenerated neurons as determined by morphological criteria is shown. No difference in cell survival between APP transgenic and nontransgenic cultures expressing only EGFP is observed. EGFP-tau expression results in progressive loss of neurons in cultures from APP transgenic mice. Treatment with γ-secretase inhibitor DAPT, NMDAR antagonist CPP, or GSK-3β inhibitor TDZD but not calcineurin inhibitor FK-506 abolishes tau-dependent neuronal loss on APP transgenic background. C, Effect of TDZD on expression and phosphorylation of GSK-3β in APP and nontransgenic slices as determined by Western blot analysis (left). Quantification of total GSK-3β relative to tubulin (top right) and of phospho-GSK-3β relative to total GSK-3β (bottom, right). Expression of GSK-3β is increased in APP transgenic slices. TDZD treatment increases phospho-GSK-3β (inactive GSK-3β) levels. The experiment was performed in triplicate. Values are shown as mean ± SEM (B) and mean ± SD (C) with *p < 0.05, ***p < 0.001; Student's t test [n = 11 (EGFP), n = 8 (EGFP-tau, nontransgenic), n = 12 (EGFP-tau, APP transgenic), n = 8 (DAPT, nontransgenic), n = 12 (DAPT, APP transgenic), n = 9 (FK-506, nontransgenic), n = 10 (FK-506, APP transgenic), n = 9 (TDZD)].

Figure 5.

Survival of hippocampal CA3 neurons after expression of disease-relevant tau constructs in hippocampal slice cultures. A, Schematic representation of the primary structure of the used tau constructs. B, Western blot showing expression of the different tau constructs (Tau-5) and phosphorylation at the PHF-1 site. Quantification of PHF-1 signal relative to total tau shows increased phosphorylation of wt tau on APP transgenic background. Phosphorylation of R406W tau and P301L tau is reduced compared with wt tau by 65 and 44%, respectively. In contrast to wt tau, phosphorylation of R406W tau and P301L tau is not increased on APP background. Expression levels of the different constructs varied due to different numbers of infected cells. Experiment was performed in triplicate. C, Live imaging of hippocampal CA3 neurons from nontransgenic (top) or APP transgenic mice (bottom) expressing EGFP-tagged tau mutants from day 2 to day 4 postinfection. Scale bars, 25 μm. D, Quantification of cell loss on day 3 (left) and day 4 (right). Cell numbers on days 3 and 4 are shown relative to day 2 (set as 100%) for the respective construct. Strong and progressive cell death is seen for cells expressing PHP tau, independent of transgenic background. Expression of R406W tau causes increased neuronal loss in nontransgenic controls on day 4 compared with wt tau expression and strongly induces cell death in APP transgenic cultures. No difference is seen in Ala tau- and P301L-expressing neurons in APP transgenic cultures and nontransgenic controls. Values are shown as mean ± SD (B) and mean ± SEM (D) with *p < 0.05 and ***p < 0.001; Student's t test [n = 8 (wt tau, nontransgenic), n = 12 (wt tau, APP transgenic), n = 8 (Ala tau), n = 14 (PHP tau, nontransgenic), n = 12 (PHP tau, APP transgenic), n = 8 (R406W tau), n = 13 (P301L tau, nontransgenic), n = 10 (P301L tau, APP transgenic)].

Figure 6.

Effect of CPP, FK-506, and TDZD on the survival of R406W tau-expressing neurons in the CA3 region of hippocampal slice cultures. A, Live imaging of EGFP-R406W tau-expressing CA3 neurons from nontransgenic and APP transgenic cultures from day 2 to day 4 postinfection after treatment as indicated. Scale bars, 25 μm. B, Quantification of cell loss on day 3 (top) and day 4 (bottom) postinfection standardized to the respective nontransgenic control. The fraction of nondegenerated neurons as determined by morphological criteria is shown. Neuronal loss is increased after expression of R406W tau on APP transgenic background compared with nontransgenic control. This effect is abolished by treating cultures with CPP but not with FK-506 or TDZD. Note that treatment with FK-506 and TDZD also decreased neuronal survival of the controls. All values are shown as mean ± SEM with ***p < 0.001; one-tailed unpaired Student's t test; [n = 8 (R406W tau, untreated), n = 9 (R406W tau, nontransgenic, CPP), n = 12 (R406W tau, APP transgenic, CPP), n = 8 (R406W tau, nontransgenic, FK-506), n = 9 (R406W tau, APP transgenic, FK-506), n = 9 (R406W tau, TDZD)].

Figure 7.

Sequential extraction of wt, R406W, and P301L tau from hippocampal slice cultures. Tau solubility profiles from lysates of infected nontransgenic and APP transgenic slices. The extraction was performed using the following buffers of increasing stringency: high salt (HS), 1% Triton (Trit.), RIPA, 2% SDS, and 70% FA. The majority of wt tau protein was found in the HS fraction. In nontransgenic controls, R406W tau and P301L tau is increased in the insoluble fraction to 54–57% compared with wt tau (42%). On the APP transgenic background the insoluble tau fraction is decreased for all constructs (33–39%). Equal amounts of lysates from each extraction step were loaded and stained with Tau-5 antibody against total tau.

Figure 8.

Schematic representation showing the proposed pathways that mediate spine pathology and tau-dependent cell death. The formation of Aβ is central, since blocking Aβ production by treatment with the γ-secretase inhibitor DAPT abolishes spine changes and the induction of tau toxicity. NMDAR activity is required for both induction of spine alterations and Aβ-induced tau toxicity, since blocking NMDAR activity with CPP abolishes both pathologies. Aβ but not tau causes loss of spines, reduction of spine length, and alterations in spine shape, as evidenced by a shift from mushroom to stubby spines. In contrast, Aβ alone is not neurotoxic but requires tau to induce cell death. GSK-3β is activated by Aβ and participates in both cascades but calcineurin is operating only in mediating spine changes. Within spines, calcineurin is upstream of GSK-3β, since calcineurin inhibition mimics the effect of the NMDAR antagonist CPP. In the soma, Aβ activates GSK-3β independent of calcineurin, which is essential for the induction of wt tau toxicity, since blocking GSK-3β activity abolishes cell death caused by wt tau in APP transgenic cultures, whereas calcineurin inhibition has no effect on cell survival. In contrast, induction of R406W tau toxicity by Aβ is GSK-3β independent, suggesting that Aβ affects R406W tau by a different mechanism. Continuous lines show direct effects, and dashed lines show indirect effects with potential intermediate steps.