SPG302 Reverses Synaptic and Cognitive Deficits Without Altering Amyloid or Tau Pathology in a Transgenic Model of Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is conceptualized as a synaptic failure disorder in which loss of glutamatergic synapses is a major driver of cognitive decline. Thus, novel therapeutic strategies aimed at regenerating synapses may represent a promising approach to mitigate cognitive deficits in AD patients. At present, no disease-modifying drugs exist for AD, and approved therapies are palliative at best, lacking in the ability to reverse the synaptic failure. Here, we tested the efficacy of a novel synaptogenic small molecule, SPG302 - a 3rd-generation benzothiazole derivative that increases the density of axospinous glutamatergic synapses - in 3xTg-AD mice. Daily dosing of 3xTg-AD mice with SPG302 at 3 and 30 mg/kg (i.p.) for 4 weeks restored hippocampal synaptic density and improved cognitive function in hippocampal-dependent tasks. Mushroom and stubby spine profiles were increased by SPG302, and associated with enhanced expression of key postsynaptic proteins - including postsynaptic density protein 95 (PSD95), drebrin, and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) - and increased colocalization of PSD95 with synaptophysin. Notably, SPG302 proved efficacious in this model without modifying Aβ and tau pathology. Thus, our study provides preclinical support for the idea that compounds capable of restoring synaptic density offer a viable strategy to reverse cognitive decline in AD.

Keywords: 3xTg-AD mice; Alzheimer’s disease; Dendritic spines; SPG302; Synaptic deficits; Synaptic markers.

Fig. 1

SPG302 restores behavioral deficits in 3xTg-AD mice. (A) Mice were trained on the spatial reference version of the MWM at 7 months of age. Acquisition curves (A1) are shown for the 5 days of training on the MWM. Two-way ANOVA: trials [F(4,285) = 92.63, p < 0.0001], treatment [F(5,285) = 30.33, p < 0.0001], and interaction [F(20,285) = 2.699, p = 0.0002], Tukey’s multiple comparison test, ****p < 0.0001, ***p < 0.001, *p < 0.05 (significance indicated for 3 × vehicle: * versus WT vehicle, # versus 3 × SPG302 3 mg/kg or 30 mg/kg). (A2) Frequency of WT vehicle, WT SPG302 3 mg/kg, WT SPG302 30 mg/kg, 3 × vehicle, 3 × SPG302 3 mg/kg and 3 × SPG302 30 mg/kg were measured 24 h after the last training session. Frequency is reduced in 3 × vehicle compared to WT vehicle. However, SPG302 treatment rescue frequency values in 3xTg-AD mice to WT mice. One-way ANOVA, ****p < 0.0001, F(5,56) = 7.828, Tukey’s multiple comparisons test, ***p < 0.001, *p < 0.05. (A3) Duration in zone. Time spent in the platform quadrant is reduced in 3x-vehicle vs WT-vehicle group. SPG302 treatment in 3xTg-AD mice increase the duration in zone time, similar to WT mice group. One-way ANOVA, **p = 0.0017, F(5,56) = 4.458, Tukey’s multiple comparisons test, **p < 0.01, *p < 0.05. Distance moved (A4) and velocity (A5) showed no differences between groups. (B) CFC analysis. 3 × vehicle showed a reduction in the inactivity duration which was recovered with SPG302 treatment to WT levels. t-test, *p < 0.05. n = 8–11 per group. The values represent means ± SEM

Fig. 2

SPG302 treatment rescue synaptic puncta density in 3xTg-AD mice. (A) Confocal images of PSD95 and synaptophysin in the stratum radiatum of CA1 hippocampal area. (B) Quantitative analysis of these images revealed a reduction in the number of PSD95 (B1) (one-way ANOVA, **p = 0.0013, F(3,16) = 8.537, Tukey’s multiple comparisons tests, **p < 0.01, *p < 0.05) and SYN (B2) (one-way ANOVA, *p = 0.0134, F(3,16) = 4.894, Tukey’s multiple comparisons tests, *p < 0.05) puncta as well as the colocalization between both markers (B3) (one-way ANOVA, *p = 0.0274, F(3,16) = 3.960, Tukey’s multiple comparisons tests, *p < 0.05) in 3 × vehicle compared to WT vehicle. SPG302 treatment restores PSD95 number of spots and colocalization. However, no changes were observed with the treatment for the presynaptic marker synaptophysin. n = 5 per group. The values represent means ± SEM. Scale bars: 50 μm

Fig. 3

SPG302 improve dendritic spine density in 3xTg-AD mice and synaptic-related proteins. (A) Dendritic spines analysis in WT vehicle, 3 × vehicle, 3 × SPG302 3 mg/kg and 3 × SPG302 30 mg/kg. Light microscopic images of radiatum layer in CA1 subfield (A1) in WT vehicle (A1a), 3 × vehicle (A1b), 3 × SPG302 3 mg/kg (A1c) and 3 × SPG302 30 mg/kg (A1d). (A2) Stereological quantification showed a significant decrease in the density of total dendritic spines in 3x-vehicle group compared to WT-vehicle group (one-way ANOVA, ****p < 0.0001, F(3,12) = 23.28, Tukey’s multiple comparisons tests, ***p < 0.001, **p < 0.01), in mushroom (one-way ANOVA, *p = 0.0249, F(3,12) = 4.482, Tukey’s multiple comparisons tests, **p < 0.01, *p < 0.05) and stubby spines (one-way ANOVA, ***p < 0.0001, F(3,12) = 16.80, Tukey’s multiple comparisons tests, ***p < 0.001, **p < 0.01). Notably, SPG302 treatment in 3xTg-AD mice rescue total, mushroom and stubby spines compared to vehicle (n = 4 per group). (B) Immunoblot analysis of Drebrin, GluA1, p-GluaA1, PSD95, SV2A, Fascin, p-Fascin and synaptophysin in hippocampal synaptosome from WT vehicle, 3 × vehicle, 3 × SPG302 3 mg/kg and 3 × SPG302 30 mg/kg group are shown in alternate lanes (B1). (B2) Quantification normalized to β-tubulin for Drebrin, GluA1, PSD95, SV2A, Fascin and synaptophysin, normalized to GluA1 for p-GluA1, and to Fascin for p-Fascin, and expressed as relative units, showed a significant reduction in Drebrin, p-GluA1 and PSD95 in 3 × vehicle vs WT vehicle group. Notably, a significant increase in Drebrin (3 × SPG302 3 mg/kg), p-GluA1 (3 × SPG302 30 mg/kg) and PSD5 (3 × SPG302 30 mg/kg) is shown in 3 × treated with SPG302 compared to 3x-vehicle group. Unpaired t test, **p < 0.01, *p < 0.05. n = 5 per group. The values represent means ± SEM. Scale bars: 6.25 μm

Fig. 4

SPG302 treatment does not alter Aβ or tau pathology. (A) Light microscopic images stained with 6E10 (A1a-A1c), HT7 (A2a-A2c) and AT180 (A3a-A3c) in 3 × vehicle (A1a, A2a and A3a), 3 × SPG302 3 mg/kg (A1b, A2b and A3b) and SPG302 30 mg/kg (A1c, A2c and A3c). No differences were detected in the staining of these three markers between the different groups. (B) Amyloid (B1), pTau (B2) and Tau (B2) levels measured by ELISA in 3 × vehicle, 3 × SPG302 3 mg/kg and 3 × SPG302 30 mg/kg revealed no differences for any of these markers between the groups. n = 8–10 per group. The values represent means ± SEM. so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; slm, stratum lacunosum-moleculare. Scale bars: 100 μm