14-3-3θ Does Not Protect against Behavioral or Pathological Deficits in Alzheimer's Disease Mouse Models

Abstract

Alzheimer's disease (AD) is characterized by progressive cognitive impairment associated with synaptic dysfunction and dendritic spine loss and the pathologic hallmarks of β-amyloid (Aβ) plaques and hyperphosphorylated tau tangles. 14-3-3 proteins are a highly conserved family of proteins whose functions include regulation of protein folding, neuronal architecture, and synaptic function. Additionally, 14-3-3s interact with both Aβ and tau, and reduced levels of 14-3-3s have been shown in the brains of AD patients and in AD mouse models. Here, we examine the neuroprotective potential of the 14-3-3θ isoform in AD models. We demonstrate that 14-3-3θ overexpression is protective and 14-3-3θ inhibition is detrimental against oligomeric Aβ-induced neuronal death in primary cortical cultures. Overexpression of 14-3-3θ using an adeno-associated viral (AAV) vector failed to improve performance on behavioral tests, improve Aβ pathology, or affect synaptic density in the J20 AD mouse model. Similarly, crossing a second AD mouse model, the App^NL-G-F knock-in (APP KI) mouse, with 14-3-3θ transgenic mice failed to rescue behavioral deficits, reduce Aβ pathology, or impact synaptic density in the APP KI mouse model. 14-3-3θ is likely partially insolubilized in the APP models, as demonstrated by proteinase K digestion. These findings do not support increasing 14-3-3θ expression as a therapeutic approach for AD.

Keywords: 14-3-3; Alzheimer’s disease; cortex; hippocampus; mouse model; β-amyloid.

Figure 1.

14-3-3θ reduces oligomeric Aβ toxicity in primary cortical cultures. A, Immunocytochemistry for exogenous HA-tagged 14-3-3θ in nontransgenic (nTg) and 14-3-3θ transgenic mouse neuronal cultures. Scale bar: 100 μm. B, Representative images of primary cortical neurons treated with oligomeric Aβ from nontransgenic or 14-3-3θ mice. Ethidium D (EthD) labels the nuclei of dying cells, while Hoechst 33342 stains the nuclei of all cells. Scale bar: 100 μm. C, Immunocytochemistry for eYFP-difopein in nontransgenic and difopein transgenic mouse neuronal cultures. Scale bar: 100 μm. D, Representative images of primary cortical neurons treated with oligomeric Aβ from nontransgenic or difopein mice. Ethidium D labels the nuclei of dying cells, while Hoechst 33342 stains the nuclei of all cells. Scale bar: 100 μm. E, Western blotting for 14-3-3θ levels in Triton X-100 soluble fractions from hippocampal (H) and cortical (C) cultures from nontransgenic and 14-3-3θ mice. F, Quantification of cell death in primary cortical neurons from nTg or 14-3-3θ littermate mice treated with Aβ oligomers for 24 h. n = 6 per condition; **p ≤ 0.01, ****p ≤ 0.0001 (Tukey’s multiple comparison test). Error bars represent standard error of the mean (SEM). G, Quantification of cell death in primary hippocampal neurons from nTg or 14-3-3θ littermate mice treated with Aβ oligomers for 24 h. n = 7 per condition. Error bars represent SEM. H, Quantification of cell death in primary cortical neurons from nTg or difopein littermate mice treated with Aβ oligomers for 24 h. n = 4 per condition, **p ≤ 0.01, ****p ≤ 0.0001 (Tukey’s multiple comparison test). Error bars represent SEM. I, Quantification of cell death in primary hippocampal neurons from nTg or difopein littermate mice treated with Aβ oligomers for 24 h. n = 4 per condition. Error bars represent SEM.

Figure 2.

14-3-3θ overexpression does not rescue behavioral deficits in J20 mice. A, Representative image of GFP immunostaining of an AAV-GFP-injected nontransgenic (nTg) mouse, indicating correct targeting and expression of the injected virus in the hippocampus of the mice. Scale bar: 100 μm. B, Representative image of V5 immunostaining of an AAV-V5-tagged 14-3-3θ-injected nontransgenic mouse, indicating correct targeting and expression of the injected virus in the hippocampus of the mice. Scale bar: 100 μm. C, Representative image of GFP and 14-3-3θ immunostaining of an AAV-GFP-injected J20 mouse. 14-3-3θ immunostaining detects the endogenous 14-3-3θ expressed in these mice. Scale bar: 100 μm for GFP image and 50 μm for 14-3-3θ image. D, Representative image of V5 and 14-3-3θ immunostaining of an AAV-V5-tagged 14-3-3θ-injected J20 mouse. 14-3-3θ immunostaining detects the endogenous and exogenous 14-3-3θ expressed in these mice, while V5 immunostaining detects only the exogenous V5-tagged 14-3-3θ. Scale bar: 100 μm for GFP image and 50 μm for 14-3-3θ image. E, Quantification of distance traveled in the open field task at 2 MPI. n = 8–10 mice per group; *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001 (Tukey’s multiple comparison test). Error bars represent SEM. F, Quantification of time spent in the open arm on the EPM at 2 MPI. n = 8–10 mice per group. Error bars represent SEM. G, Quantification of latency to platform on the MWM at 2 MPI. n = 7–10 mice per group; ****p < 0.0001 (Tukey’s multiple comparison test). Error bars represent SEM. H, Quantification of the probe trial in the MWM at 2 MPI. n = 7–10 mice per group; *p ≤ 0.05 (Tukey’s multiple comparison test). Error bars represent SEM. I, Quantification of mortality for GFP and 14-3-3θ-injected nTg and J20 mice. J, Quantification of distance traveled in the open field task at 6 MPI. n = 5–10 mice per group; **p ≤ 0.01, ****p ≤ 0.0001 (Tukey’s multiple comparison test). Error bars represent SEM. K, Quantification of time spent in the open arm on the EPM at 6 MPI. n = 5–10 mice per group. Error bars represent SEM. L, Quantification of latency to platform on the MWM at 6 MPI. n = 5–10 mice per group. Error bars represent SEM.

Figure 3.

14-3-3θ overexpression does not modify behavior in APP KI mice. A, Representative images of HA immunostaining in the cortex and hippocampus of APP KI mice demonstrates expression of HA-tagged 14-3-3θ in 14-3-3θ-overexpressing mice (APP KI/14-3-3θ) but not mice without overexpression (APP KI). 14-3-3θ immunostaining detects both endogenous and exogenous 14-3-3θ expressed in APP KI and APP KI/14-3-3θ mice. Scale bar: 100 μm for HA images and 50 μm for 14-3-3θ images. B, Quantification of mortality for nontransgenic, APP KI, and APP KI/14-3-3θ mice. C, Quantification of distance traveled in the open field task at eight to nine months of age. n = 10–16 mice per group. Error bars represent SEM. D, Quantification of time spent in the open arm on the EPM at eight to nine months of age. n = 10–15 mice per group; *p ≤ 0.05, ***p ≤ 0.001 (Tukey’s multiple comparison test). Error bars represent SEM. E, Quantification of latency in crossing to the dark side on day 2 of the passive avoidance task. n = 8–14 mice per group. Error bars represent SEM.

Figure 4.

14-3-3θ overexpression does not reduce Aβ plaque burden in AD mice. A, Representative hippocampal images and quantification of total plaque area of J20 mice injected with either AAV-GFP or AAV-14-3-3θ at 6 MPI. n = 5–6 per group. Error bars represent SEM. Scale bar: 500 μm. B, Representative hippocampal images and quantification of total plaque area of APP KI and APP KI/14-3-3θ mice at eight to nine months of age. n = 10–16 per group. Error bars represent SEM. Scale bar: 500 μm. C, Representative cortical images and quantification of total plaque area of APP KI and APP KI/14-3-3θ mice at eight to nine months of age. n = 10–15 per group. Error bars represent SEM. Scale bar: 500 μm.

Figure 5.

14-3-3θ overexpression does not impact synaptic density in AD mice. A, Representative hippocampal images of synapsin, vGLUT1, and NR2A immunoreactivity and quantification of synapsin immunoreactivity in nontransgenic and J20 mice injected with either AAV-GFP or AAV-14-3-3θ at 6 MPI. n = 5–9 per group. Error bars represent SEM. Scale bars: 50 μm for synapsin images and 10 μm for vGLUT1/NR2A images. B, Representative hippocampal images of synapsin, vGLUT1, and NR2A immunoreactivity and quantification of synapsin immunoreactivity in nontransgenic, APP KI, and APP KI/14-3-3θ mice at eight to nine months of age. n = 9–16 per group. Error bars represent SEM. Scale bars: 50 μm for synapsin images and 10 μm for vGLUT1/NR2A images.

Figure 6.

14-3-3θ immunoreactivity is partially proteinase K-resistant in APP KI mice. Representative images of 14-3-3θ immunostaining in cortical slices with and without proteinase K (PK) treatment from nontransgenic, APP KI, and APP KI/14-3-3θ mice. Scale bar: 50 μm.