Regional differences in Alzheimer's disease pathology confound behavioural rescue after amyloid-β attenuation

Abstract

Failure of Alzheimer's disease clinical trials to improve or stabilize cognition has led to the need for a better understanding of the driving forces behind cognitive decline in the presence of active disease processes. To dissect contributions of individual pathologies to cognitive function, we used the TgF344-AD rat model, which recapitulates the salient hallmarks of Alzheimer's disease pathology observed in patient populations (amyloid, tau inclusions, frank neuronal loss, and cognitive deficits). scyllo-Inositol treatment attenuated amyloid-β peptide in disease-bearing TgF344-AD rats, which rescued pattern separation in the novel object recognition task and executive function in the reversal learning phase of the Barnes maze. Interestingly, neither activities of daily living in the burrowing task nor spatial memory in the Barnes maze were rescued by attenuating amyloid-β peptide. To understand the pathological correlates leading to behavioural rescue, we examined the neuropathology and in vivo electrophysiological signature of the hippocampus. Amyloid-β peptide attenuation reduced hippocampal tau pathology and rescued adult hippocampal neurogenesis and neuronal function, via improvements in cross-frequency coupling between theta and gamma bands. To investigate mechanisms underlying the persistence of spatial memory deficits, we next examined neuropathology in the entorhinal cortex, a region whose input to the hippocampus is required for spatial memory. Reduction of amyloid-β peptide in the entorhinal cortex had no effect on entorhinal tau pathology or entorhinal-hippocampal neuronal network dysfunction, as measured by an impairment in hippocampal response to entorhinal stimulation. Thus, rescue or not of cognitive function is dependent on regional differences of amyloid-β, tau and neuronal network dysfunction, demonstrating the importance of staging disease in patients prior to enrolment in clinical trials. These results further emphasize the need for combination therapeutic approaches across disease progression.

Keywords: Alzheimer’s disease; amyloid-β; cognition; hippocampal-entorhinal circuitry; tau.

Figure 1

Amyloid-β attenuation rescues pattern separation and executive function but not activities of daily living and spatial memory. Tg and NTg rats untreated or treated to attenuate amyloid-β (Tg-SI, NTg-SI), were tested on a battery of behavioural tasks. (A) TgF344-AD rats exhibit deficits in the burrowing task that were not rescued by treatment (n = 16). (B) TgF344-AD rats have impaired pattern separation in the novel object recognition task, which was rescued by amyloid attenuation (from n = 16, rats were excluded for not performing the task: NTg = 12; NTg-SI = 13; Tg = 11; Tg-SI = 10). (C and D) TgF344-AD rats, regardless of treatment, exhibit a trend to deficits in latency to escape and significant deficits in search strategies used in the Barnes maze spatial memory probe (n = 16). (E) Reversal learning phase demonstrated NTg (−9.72 ± 1.70), NTg-SI (−8.97 ± 1.96) and Tg-SI (−7.56 ± 1.97) rats decreased escape latency across 10 trials, whereas Tg rats (−3.24 ± 1.70) did not (n = 16). (F and G) Search strategies utilized by the NTg, NTg-SI and Tg-SI rats significantly improved, whereas Tg rats did not improve (n = 16). Mean ± standard error of mean (SEM) or 95% confidence interval (CI) (linear regression), one-way (A–D) or repeated measures (E and F) ANOVA with Holm-Sidak post hoc test, *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Therapeutic administration of scyllo-inositol reduced amyloid-β load. (A) Tg-SI rats visually demonstrate decreased amyloid-β coverage and plaque size in the hippocampus. Scale bar = 100 μm. (B) The percentage of hippocampal area covered by amyloid-β plaques is significantly attenuated in Tg-SI, and (C) plaques of all sizes are reduced. (D) Levels of plasma amyloid-β_40/42 are unchanged indicating that clearance pathways are maintained. n = 6 per group (B and C) and n = 10–12 per group (D): amyloid-β₄₀ Tg: n = 11; amyloid-β₄₂ Tg: n = 10; amyloid-β_40/42 Tg-SI n = 12 (mean ± SEM, two-sided unpaired t-test, **P < 0.01).

Figure 3

Deficits in differentiation and survival of newborn neurons rescued by amyloid-β attenuation. Untreated and treated NTg and TgF344-AD rats were injected with EdU and BrdU to label proliferating and surviving cells, respectively. (A) Tg rats, regardless of treatment, exhibit a significant increase in EdU+ proliferating cells. (B) However, Tg rats have significantly fewer newborn cells expressing a neuronal phenotype (EdU+DCX+). This deficit is rescued in Tg-SI rats. (C) GFAP (white), EdU (green), and DCX (red) labelling in the subgranular zone (SGZ) and granule cell layer (GCL) of NTg, NTg-SI, Tg and Tg-SI rats, demonstrate deficits in neuronal differentiation and migration in Tg rats, which were rescued in Tg-SI rats. (D) BrdU (red), NeuN (blue) and GFAP (white) staining demonstrate the integration of surviving newborn neurons into the granule cell layer. (E) No genotype or treatment differences were detected in number of BrdU+ surviving cells. (F) Tg rats exhibit significant deficits in survival of newborn neurons, (BrdU+NeuN+), which were rescued by treatment. (C and D) Dashed lines separate subgranular zone and inner and outer granule cell layers. Scale bars = 20 μm. NTg and NTg-SI n = 7; Tg and Tg-SI n = 8 (mean ± SEM, one-way ANOVA with Holm-Sidak post hoc test, *P < 0.05; **P < 0.01; ***P < 0.001).

Figure 4

Rescue of hippocampal neuronal density and function in treated TgF344-AD rats. To determine if loss of hippocampal neurons and/or function contribute to the behavioural phenotypes, we determined neuronal density and cross-frequency coupling by in vivo electrophysiology. (A) Tg rats display a significant loss of NeuN (black) staining in the hippocampus, compared to NTg rats. Scale bar = 400 μm. (B) The density of NeuN staining in the granule cell layer (GCL) of Tg rats is significantly decreased by 11 ± 3.6 %, compared to NTgs, and is rescued by amyloid-β attenuation (n = 8). (C) TgF344-AD rats have impaired modulation between theta low-gamma and between theta high-gamma bands in the hippocampus, which are rescued by amyloid-β attenuation (NTg n = 8; Tg/Tg-SI n = 7). (D) 3D maps of phase amplitude coupling in the hippocampus (representative rat for each cohort) were generated by computing the modulation index as a measure of the coupling between the phase of the theta band with the amplitude of the gamma band. Modulation index values were encoded by colour and plotted according to theta and gamma frequency. 3D maps demonstrate a deficit in untreated Tg compared to NTg rats, and rescue by amyloid-β attenuation (mean ± SEM (indicated by a red line in C], one-way ANOVA with Holm-Sidak post hoc test (B), or with t-test with false discovery rate correction (C), *P < 0.05; ***P < 0.001.

Figure 5

Amyloid-β attenuation decreases tau pathology in a brain region-dependent manner. Tau pathology was assessed in TgF344-AD rats (Tg, Tg-SI) by immunostaining with PHF1 to label hyperphosphorylated tau at S396/404 and Thioflavin-S (Thio-S) to label amyloid-β plaques. (A) PHF1+/Thio-S+ staining in the dentate gyrus (DG) of a Tg rat. Approximately 80–85% of PHF1+ inclusions (red) associate with amyloid-β plaques (green), representing dystrophic neurites. [A(i)] The remainder of the tau inclusions are non-plaque associated, representing pretangle inclusions. (B) PHF1+ inclusions were separated into plaque and non-plaque associated, and quantified by immunohistochemistry (Supplementary Fig. 7) in the dentate gyrus. Amyloid-β attenuation significantly decreased plaque-associated, but not non-plaque associated inclusions. [C and C(i)] PHF1+/Thio-S+ staining in the entorhinal cortex of a Tg rat indicating plaque-associated inclusions and non-plaque associated inclusions. (D) There was no effect of treatment on either plaque-associated or non-plaque associated inclusions in the entorhinal cortex (EC) (E), despite a reduction of amyloid-β plaques in the region. (F) No significant differences were detected in the number of total entorhinal cortical neurons across genotype and treatment (G); however, Tg and Tg-SI rats exhibit a decrease of neurons within layer II of the entorhinal cortex. Scale bars = 20 μm in A and C; 10 μm in A(i) and C(i). n = 6, mean ± SEM, two-sided unpaired t-test, *P < 0.05; ^**P < 0.01; ***P < 0.001.

Figure 6

Impaired connectivity in entorhinal cortical-hippocampal circuit persists after amyloid-β attenuation. We assessed in vivo cross-frequency coupling in the entorhinal cortex, and connectivity with the hippocampus. (A) Representative 3D maps of phase amplitude coupling in the entorhinal cortex were generated by computing the modulation index as a measure of the coupling between the phase of the theta band with the amplitude of the gamma band. Modulation index values were encoded by colour and plotted according to theta and gamma frequency. 3D maps demonstrate a deficit in untreated Tg compared to NTg rats, and rescue by amyloid-β attenuation. (B) Modulation index of theta low-gamma and theta high-gamma bands in entorhinal cortex are impaired in Tg rats and rescued after treatment. To evaluate neuronal circuitry, we stimulated the entorhinal cortex and recorded hippocampal responses. (C) Tg rats, regardless of treatment, exhibit a significantly decreased hippocampal response to entorhinal cortical stimulation at 500 mV and 1000 mV, compared to NTg rats. (D) Representative neuronal trace demonstrating impaired hippocampal response to 500 mV entorhinal stimulation in Tg and Tg-SI rats. Black arrow indicates stimulation onset. NTg n = 8; Tg/Tg-SI n = 7. Red line indicates mean ± SEM, one-way ANOVA with t-test with false discovery rate correction (B) or a Kruskal-Wallis H test followed by Wilcoxon signed-rank test with false discovery rate correction (C). ***P < 0.001.