Appoptosin-Mediated Caspase Cleavage of Tau Contributes to Progressive Supranuclear Palsy Pathogenesis

Abstract

Progressive supranuclear palsy (PSP) is a movement disorder characterized by tau neuropathology where the underlying mechanism is unknown. An SNP (rs1768208 C/T) has been identified as a strong risk factor for PSP. Here, we identified a much higher T-allele occurrence and increased levels of the pro-apoptotic protein appoptosin in PSP patients. Elevations in appoptosin correlate with activated caspase-3 and caspase-cleaved tau levels. Appoptosin overexpression increased caspase-mediated tau cleavage, tau aggregation, and synaptic dysfunction, whereas appoptosin deficiency reduced tau cleavage and aggregation. Appoptosin transduction impaired multiple motor functions and exacerbated neuropathology in tau-transgenic mice in a manner dependent on caspase-3 and tau. Increased appoptosin and caspase-3-cleaved tau were also observed in brain samples of patients with Alzheimer's disease and frontotemporal dementia with tau inclusions. Our findings reveal a novel role for appoptosin in neurological disorders with tau neuropathology, linking caspase-3-mediated tau cleavage to synaptic dysfunction and behavioral/motor defects.

Figure 1. Elevated Appoptosin Levels Associate with Higher T-Allele Occurrence at SNP rs1768208 and Correlate with Increased Caspase-3 Activation and Tau Cleavage in PSP Brains

(A and B) The frequency and occurrence of the minor T- allele at SNP rs1768208 (C/T) in control and PSP patients. T occurrence: number of cases with T-allele/number of total cases (Control, n=22; PSP, n=26). Odds ratio is calculated with R statistical software. (C) Relative appoptosin mRNA levels in control and PSP brain samples (Control, n=15; PSP, n=23). (D) Western blot analysis of appoptosin, c-caspase-3, c-tau, PHF-1 and tau levels in the frontal cortex of PSP and control individuals (Control, n=15; PSP, n=23). (E) Immunohistochemistry analysis of appoptosin, c-caspase-3 and c-tau and PHF-1 in the cortex of PSP and control individuals (n=4). Scale bar, 100 μm. Red arrows indicate some of the cells double stained appoptosin and c-tau in adjacent sections. (F) Relative appoptosin protein levels in SNP rs1768208 T allele carriers and non-T allele (C) carriers (n=19 per group). (G and H) The correlation between c-caspase-3 and appoptosin (G), and between c-tau and appoptosin (H) protein levels (Regression analysis, n=38). Data represent mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001 by nonparametric t-test. See also Figure S1.

Figure 2. Appoptosin Regulates Tau Cleavage through Caspase-3 Activation

(A) Change in c-caspase-3, c-tau and tau levels in neuronal cultures following appoptosin transduction as determined by western blot (n=3 per group, two-tailed Student’s t-test). (B) Western blot analysis of c-tau, c-caspase3, tau and PSD95 in cortical neuronal cultures from appoptosin knockout mouse embryos or littermate controls (n=3 per group, two-tailed Student’s t-test). (C) Change in c-tau and cleaved-PARP (c-PARP) levels in neuronal cultures after appoptosin overexpression and exposure to DMSO (−), caspase-3 inhibitor (C3I), or caspase-9 inhibitor (C9I) (n=3 per group, repeated-measures one-way ANOVA with Dunnett’s post hoc analysis). Data represent mean ± SD. *P < 0.05, **P < 0.01, and #P < 0.05. See also Figure S2.

Figure 3. Overexpression of Appoptosin Reduces Tau Association with Microtubules and Promotes Tau Cleavage

(A) Change in tau found in microtubule-bound and unbound fractions following appoptosin overexpression and treatment with C3I (n=3 per group, repeated-measures One-way ANOVA with Dunnett’s post hoc analysis). (B) Quantification of SDS-insoluble tau aggregates in neuronal cultures following appoptosin transduction as determined by filter/trap assays. SDS-soluble tau was used as a control (n=3 per group, two-tailed Student’s t-test). (C) Staining of c-tau and MAP2 in neurons transduced with AAV-appoptosin or AAV-GFP. Green color represents GFP without staining, which was used to identify transduced neurons (n=15 per group, two-tailed Student’s t-test). Scale bar, 20 μm. (D) c-Tau puncta in dendrites of appoptosin KO or littermate control neurons were detected by immunocytochemistry (n=15 per group, two-tailed Student’s t-test). Scale bar, 20 μm. Data represent mean ± SD. *P < 0.05, **P < 0.01, and #P < 0.05. Data represent mean ± SD.

Figure 4. Overexpression of Appoptosin Promotes c-Tau Distribution to Post-Synaptic Compartments, Disrupts Synaptic Structure and Depletes Cell Surface Glutamate Receptors

(A) Distribution of c-tau, c-caspase-3, and total tau in postnuclear (S1), synaptosomal (Syn) and PSD fractions after appoptosin overexpression (n=3 per group, two-tailed Student’s t-test). (B and C) DIV (days in vitro) 14 neurons were transfected or transduced with AAV-appoptosin. Numbers of dendritic spines (B) and Synapsin-I/PSD95 double-positive puncta (C) in neurons with appoptosin overexpression and caspase-3 inhibition (n=15 per group, one-way ANOVA with Tukey’s post hoc analysis). Scale bar, 10 μm. (D) Cell surface levels of NR1, NR2A, NR2B, GluR1, GluR2 and Na⁺/K⁺ATPase (as a control) in neurons with appoptosin overexpression, as determined by cell surface biotinylation assays (n=3 per group, two-tailed Student’s t test). Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and #P < 0.05. See also Figure S3.

Figure 5. Overexpression of Appopotosin Causes PSP-like Motor Deficits and Induces Tau Pathology

(A) Schematic workflow of stereotaxic AAV injection into the globus pallidus of JNPL3 mice. LV, lateral ventricle; CPu, caudate putamen; GP: globus pallidus. (B–F) Behavioral and histological analysis of JNPL3 mice injected with AAV-appoptosin or AAV-GFP (control). (B) Confirmation of appoptosin overexpression within the injected area (n=8 per group, two-tailed Student’s t test). Scale bar, 30 μm. (C) Catwalk analysis: comparison of stride length of each limb (RF, right front; RH, right hind; LF, left front; LH, left hind) of experimental mice (n=8 per group, two-way ANOVA with Sidak’s post hoc analysis). (D) Accelerating rotarod test: latency of experimental mice on the rotor at days after training as indicated (n=8 per group, two-way ANOVA with Sidak’s post hoc analysis). (E) Number of slips observed in experimental mice in balance beam tests (n=8 per group, two-tailed Student’s t test). (F) The expression of GFP, c-caspase-3, c-tau, PHF-1 tau, and tau in the injected area in experimental mice. n=8, Scale bar, 30 μm. Data represent mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. See also Figure S4.

Figure 6. Caspase Inhibition and Tau Knockout Reduces Detrimental Effect Caused by Appoptosin Overexpression

(A, B and C) Behavioral and histological analysis of JNPL3 mice injected with AAV-appoptosin or AAV-GFP (control), and treated with or without caspase-3 inhibitor. (A) Comparison of stride length of each limb (RF, right front; RH, right hind; LF, left front; LH, left hind) of experimental mice (n=11 per group, two-way ANOVA with Tukey’s post hoc analysis). (B) Immunostaining to show expression of c-Tau, PHF1, NeuN, GFAP, and MAP2 in the infection area. (C) Quantification of immunostaining from JNPL3 mice (n=8 per group, one-way ANOVA with Dunnett’s post hoc analysis) (D and E) Behavioral and histological analysis of wild type (WT) mice and tau knockout (KO) mice injected with AAV-appoptosin or AAV-GFP. (D) Number of errors made by experimental mice during the round beam test (n=12 for WT and Tau KO mice, n=9 for WT-appoptosin mice, n=11 for Tau KO-appoptosin mice, two-way ANOVA with Sidak’s post hoc analysis). (E) Expression of NeuN, GFAP, MAP2 and c-caspase-3 in the injection area (n=11 for WT and Tau KO-appoptosin mice, n=9 for WT-appoptosin mice, n=10 for Tau KO mice, two-way ANOVA with Sidak’s post hoc analysis). Scale bar, 50 μm. Data represent mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. See also Figure S4G.

Figure 7. Appoptosin Is Upregulated in Tauopathic Disorders, and Drives Pathogenesis through Caspase Activation, Tau Cleavage and Aggregation

(A) Analysis of appoptosin and c-tau expression in the frontal cortex of AD, PD, control, FTD-T and HD individuals (AD, n=8; PD, n=3; control, n=3; FTD-T, n=3; HD, n=3). Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test. (B) Appoptosin potentially drives PSP pathogenesis through caspase-3 mediated tau cleavage and NFT formation. Appoptosin expression is increased in PSP individuals through a C/T polymorphism at the SNP rs1768208 or stress. Increased appoptosin protein increases heme synthesis, and thus activates the intrinsic caspase pathway and caspase-mediated tau cleavage. Cleaved tau (c-tau) is prone to aggregate to form neurofibrillary tangles (NFT), which is deleterious to neurons. c-Tau also can be mis-sorted into the postsynaptic compartment to trigger synaptic dysfunction. Together with caspase activation, these events eventually lead to tau pathology and neurodegeneration in PSP.