Synergistic effects of amyloid-beta and wild-type human tau on dendritic spine loss in a floxed double transgenic model of Alzheimer's disease

Abstract

Synapse number is the best indicator of cognitive impairment In Alzheimer's disease (AD), yet the respective contributions of Aβ and tau, particularly human wild-type tau, to synapse loss remain undefined. Here, we sought to elucidate the Aβ-dependent changes in wild-type human tau that trigger synapse loss and cognitive decline in AD by generating two novel transgenic mouse models. The first overexpresses floxed human APP with Swedish and London mutations under the thy1 promoter, and recapitulates important features of early AD, including accumulation of soluble Aβ and oligomers, but no plaque formation. Transgene excision via Cre-recombinase reverses cognitive decline, even at 18-months of age. Secondly, we generated a human wild-type tau-overexpressing mouse. Crossing of the two animals accelerates cognitive impairment, causes enhanced accumulation and aggregation of tau, and results in reduction of dendritic spines compared to single transgenic hTau or hAPP mice. These results suggest that Aβ-dependent acceleration of wild-type human tau pathology is a critical component of the lasting changes to dendritic spines and cognitive impairment found in AD.

Keywords: Alzheimer's disease; Dendritic spines; Transgenic; Wild-type tau.

Figure 1. Novel hAPP_SL mouse model exhibits increasing Aβ, oligomers and cognitive deficits without plaque deposition

A, Schematic diagram of hAPP_SL transgene with Swedish**(KM670/671NL) and London*(V717I) mutations indicated. B, Western blot of founder A3 line compared to nontransgenic with an antibody that recognizes a conserved epitope between human and mouse APP (CT20) demonstrates approximately 4-fold overexpression of the hAPP_SL protein. C, Comparison of human APP expression levels in the hemizygous hAPP_SL mice versus homozygous 3xTg-AD mice shows greater expression in 3xTg-AD mice in the hippocampus, but comparable levels in the cortex at 12 months of age. D, OC-positive pre-fibrillar oligomer levels increase with age (n=8 per group, *p<0.05 compared to 6m). E, Immunofluorescence staining of 18-month old hAPP_SL mice for full-length APP (6E10, red) and C-terminal fragments (CT20, green) demonstrates accumulation pf APP and Aβ in CA1 pyramidal neurons. Intracellular Aβ, which is labeled by 6E10 but not CT20, is indicated by arrowheads. F, hAPP_SL mice accumulate increasing levels of soluble and insoluble Aβ as measured by ELISA from cortical samples (n=8 per group, *p<0.05 compared to 3m, **p<0.01 compared to 3m). G, Despite the increases in Aβ and Aβ oligomers, no plaque deposition occurs as late as 18 months in hemizygous mice, as seen by staining with Congo Red compared to 3xTg-AD. H, At 12 months of age, hAPP_SL mice show cognitive deficits in multiple cognitive tasks, including novel object recognition (n=8 per group). I, Despite the cognitive deficits, no changes are observed in the synaptic markers synaptophysin and PSD-95 with age in hAPP_SL mice. However, there is an increase in NR2B subunits at 12 months, and a trend toward increased phosphorylated NR2B at 18 months (n=4 per group, *p<0.05 compared to 3m). Error bars +1 SEM for all graphs.

Figure 2. Knockdown of hAPP_SL transgene expression by AAV-mediated delivery of Cre rescues cognition in aged mice

A, Study design. B–E, Five weeks post-injection, 18-month old hAPP_SL mice injected with vehicle show robust APP expression in CA1, while mice injected with AAV expressing Cre recombinase have a remarkable reduction in human APP expression in the dorsal hippocampus (B and D- vehicle, C and E- Cre). F, Quantification of steady states human APP levels shows almost complete reduction of APP in CA1 pyramidal cells, but no change in cortical areas. G, Biochemical analysis of APP levels in microdissected hippocampal lysates shows a 50% reduction of human APP. H, hAPP_SL mice injected with either vehicle or Cre virus were tested for cognition by Barnes Maze. By days 3 and 4, hAPP_SL/Cre mice learned to find the escape hole significantly faster than hAPP_SL/Veh mice, performing almost at wild-type levels. I, Memory for the escape hole location was assessed 24-hours after the final learning trial. hAPP_SL/Cre mice showed strong memory for the platform location, finding the escape hole in approximately 1 minute, while hAPP_SL/Veh mice never attempted to enter the correct hole. N=6–10 per group, *p<0.05 and **p<0.01 compared to nTg Veh. Error bars +1 SEM for all graphs.

Figure 3. Generation of hTau mouse to study Aβ-dependent and independent effects on wild-type tau pathology

A, Schematic design of full-length human tau, 4R/2N, transgene. B, Total tau levels (HT7) are trending toward an increase in hemizygous hTau mice compared to homozygous 3xTg-AD mice at 4 months. Levels of tau phosphorylated at Thr231/Ser235 (AT180) are increased in 3xTg-AD, which is expected due to the presence of the human APP transgene. 3xTg-AD mice harbor 4R/0N tau with the P301L mutation. CȃD, Somatodendritc, phosphorylated tau is evident in multiple brain regions such as the amygdala, hippocampus and cortex. Here, we show that tau mislocalization and hyperphosphorylation increase with age in hTau mice, with approximately 85% of tau positive cells containing hyperphosphorylated PHF-1 tau by 18 months in CA1 neurons. E, The first observed memory deficits occur at 18-months in hTau mice. These mice exhibit memory deficits in novel object recognition compared to age-matched nontransgenic mice, corresponding with the time point of enhanced dendritic tau localization and decreased PSD-95. (n=8 per group) F–G, Steady state levels of total tau remain constant with age, but there is a significant decrease in the level of postsynaptic marker PSD-95. Interestingly, there is corresponding increase in the both the levels and phosphorylated state of NMDAR NR2B subunits. (n=4–5 per group, *p<0.05 compared to 6m hTau) H–I, Interestingly, insoluble tau levels are significantly increased in 18-month hTau mice compared to 12-month hTau mice in cortical samples. No differences were seen in levels of tau phosphorylation in the insoluble fraction. Scale bars represent 20 µm in all images.

Figure 4. Acceleration of cognitive deficits in double transgenic hAPP_SL/hTau mice

A, Importantly, no differences in motor skills were found between all groups of nontransgenic, single transgenic and double transgenic mice, as tested by five 5-minute trials (T1-T5) on the Rotarod. B, Mice were also analyzed for hippocampus-independent cognition using novel object recognition. Nontransgenic, hAPP_SL, and hTau groups all displayed preference for the novel object, indicating they remembered the old object from training, while hAPP_SL/hTau showed no preference and therefore no memory for the old object. C–E, All groups were tested for hippocampus dependent memory using Morris water maze. No differences were found in swim speed (C). hAPP_SL/hTau mice showed significant deficits in learning the platform location compared to nTg mice on Day 4 (D), and also showed memory deficits for the platform location on the 24-hour probe trial (E). A trend toward increased latency to the platform location was also found in hAPP_SL/hTau mice compared to nTg. (n=10 nTg, n=6 hAPP, n=8 hTau, n=4 hAPP_SL/hTau)

Figure 5. Human APP expression augments wild-type tau pathology

A–D, Staining for total human tau and human APP revealed an increased in hTau expression in the hippocampus of hAPP_SL/hTau mice, specifically in the granule cells of the dentate gyrus. No differences were observed in levels of APP. E, Quantification of protein levels by western blot analysis of hippocampal lysates confirmed the increase in human tau, but not human APP in double transgenic mice. F, Interestingly, no changes were seen in levels of phosphorylated tau (not shown), or in the major tau kinases GSK3β, CDK5 or JNK in soluble hippocampal lysates (n=4 per group) G–H, However, levels of total tau and phosphorylated tau, in monomeric, aggregated, and cleaved forms, were increased in the insoluble fraction of hippocampal lysates in hAPP_SL/hTau mice compared to single transgenic hTau mice.

Figure 6. Decreased spine density but no overt neuronal loss in hAPP_SL/hTau mice

A–C, Golgi staining revealed a dramatic loss in the number of spines in hAPP_SL/hTau mice in the stratum radiatum layer of the hippocampus compared to all other groups at 8 months of age (B). A representative dendrite is shown from each group (A). Classification of these spines suggests that the majority of spine loss in hAPP_SL/hTau mice is due to a loss of mature mushroom spines. D–H, To further investigate synaptic changes, sections from each group were stained for PSD-95 (shown) and synaptophysin (not shown), and the number of puncta in the stratum radiatum was counted in each section. Interestingly, hAPP_SL/hTau mice showed a 60% decrease in PSD-95 puncta (G–H), while no differences in synaptophysin were observed. I–J, Biochemical analysis of hippocampal lysates confirmed the decrease in PSD-95, and also demonstrated a significant increase in the protein kinase Fyn, which has been implicated in AD synaptotoxicity. Despite the marked loss of spines, NR2B and pNR2B-1472 still trended to increase in hAPP_SL/hTau mice. K–M, To check if the observed spine loss could be due to a loss of CA1 pyramidal cells, hAPP_SL/hTau and nontransgenic mice were stained with NeuN. No difference in the intensity of the stain was observed. (*p<0.05, ***p<0.001 compared to nTg, n=4 per group)