Generation of caspase-6 cleaved tau neoepitope monoclonal antibodies
Besides TauC3 (D421), mAbs that recognize specific caspase-cleaved tau epitopes are crucially missing. Here, we developed two neoepitope mAbs against D13 and D402 tau cleavage sites (Fig. 1a) using hybridoma technology. Neoepitope antibodies selectively bound to cleaved tau (1-402 or 14–441) but not full-length tau (1-441) as detected by ELISA and western blot analyses (Fig. S1; S2-a, b; See Supplemental Experimental Procedures). Antibody specificity was also confirmed by immunofluorescence (IF) and antigen competition assays where mAb.D402 and mAb.D13 primary antibodies were preincubated with the cognate peptides used as immunogens (Table S1), resulting in loss of antibody signal (Fig. S2-c). Together with TauC3, mAb.D402, and mAb.D13 neoepitope antibodies were used to identify caspase-mediated pathological changes in postmortem brains and in tauV337M and tauWT iNs.
Tau pathological changes in postmortem brains of FTLD and AD patients
We explored whether tau pathological changes are present in human postmortem brains with tau accumulation, including a case with the same tauV337M mutation as in the iN model described below. We immunostained sections from the temporal cortex of an FTLD tauV337M carrier and an AD patient, both at end-stage disease, and a healthy elderly control using multiplex immunohistochemistry targeting active caspase-6 and caspase-cleaved tau markers (Fig. 1b). We detected active caspase-6 positivity in the cytoplasm and neurites in both disease cases (brown), but not in the healthy control even after prolonged incubation with the chromogen to confirm the absence of signal (Fig. 1b vii-ix). The FTLD tauV337M carrier was also positive for TauC3 and mAb.D402 (red) in the cytoplasm and neurites that partially co-occurred with active caspase-6. AD brain sections showed strong positivity for all caspase-cleaved tau markers, including mAb.D13 that was absent in FTLD. Such variation on tau pathological burden is expected among individuals with different tauopathies. Moreover, neurons in the AD sections showed partial overlap between cleaved-tau and active caspase-6 in cytoplasm and neurites. Taken together, our results show that neoepitope mAbs label pathological tau inclusions in AD and FTLD-tau and corroborate the presence of active caspase-6 in the human brain seen in earlier studies .
Characterization of iPSC-induced neurons
Considering that tauV337M causes FTD in humans, we generated heterozygous tauV337M iNs and isogenic tauWT controls to examine the disease mechanisms involving caspase activation and cleaved tau pathology in a clinically relevant cell culture model. Neuronal induction of a well-characterized human tauWT line was performed using TALEN-based integration of a doxycycline-inducible Ngn2 transgene into the AAVS1 safe harbor as previously described [32, 38]. CRISPR/Cas9 genome editing was used to introduce the tauV337M into tauWT iPSCs to study the mutation’s effects in isolation from the donor’s genetic background. Both iPSC groups had a normal karyotype (Fig. S3-a) and typical colony-type morphology (Fig. 2a). Moreover, genomic DNA sequencing of tauV337M and tauWT iPSCs confirmed the presence of the heterozygous tauV337M in exon 12 (Fig. S3-b), and homogeneous expression of the pluripotency markers NANOG, OCT4, and SOX2 (Fig. S3-c) in the iNs confirmed the absence of NGN2 expression leakage without the addition of doxycycline. Following doxycycline treatment, iNs exhibited neuron-like morphology in 5–7 days and mature neuronal morphology between 3–4 weeks (Fig. 2a).
One month-old iNs were positive for the microtubule-associated protein 2 (MAP2) neuronal marker in the cytoplasm and neurites, and for the deep and upper cortical nuclear markers CTIP2 and SATB2, respectively, as confirmed by IF (Fig. 2b). iNs were also positive for the glutamatergic marker vesicular glutamate transporter 1 (VGlut1) in the cytoplasm and neurites, an anticipated outcome of the NGN2 expression [32, 41]. Overall, our findings demonstrate that at one-month post-differentiation, iNs exhibit neuron-specific morphology and express cortical and glutamatergic markers.
Increased levels of pathological tau in the tau V337M neurons
Since FTLD-tau is characterized by progressive accumulation of toxic tau species and neuronal loss, we examined the presence and temporal course of tau pathological accumulation of the tauV337M in the mutant iNs relative to WT isogenic controls. We compared iNs cultured from 1 to 3 months using western blot and a panel of tau antibodies, including total and oligomeric tau, 3R and 4R tau, and caspase-cleaved tau (Fig. 3).
Protein analysis using the total tau antibody HT7 showed positivity for distinct molecular weight bands corresponding to separate tau isoforms (Fig. 3a). Tau antibody specificity was confirmed by the recombinant human tau ladder included here as an approximate guide of the tau isoform placement as it lacks the tau PTMs present in the cell lysates that could influence tau molecular weight. In agreement with previous studies showing enriched 3R tau levels in early neuronal development [42, 43], we detected predominantly 3R isoform expression in the iNs and minimal 4R tau levels, both in tauV337M and tauWT iNs (Fig. 3a). Antibody specificity for 3R and 4R tau was confirmed by the positivity of the respective isoform bands in the recombinant tau ladder. Total tau levels were comparable between the mutant and control iNs (Fig. S4a), suggesting that any changes in the amount of pathological tau between the two groups are likely MAPT mutation-dependent and not due to changes in the overall tau levels.
Oligomeric tau aggregates could represent highly toxic and pathologically significant tau species in tauopathies . We therefore investigated the presence and temporal changes of oligomeric and misfolded tau aggregates recognized by the antibody T18  and non-denaturing conditions to preserve the original folded state of tau (Fig. 3b). To estimate the molecular weight of the native proteins, we used a protein standard for native electrophoresis stained with Coomassie Blue for band visualization. We detected elevated oligomeric tau levels in tauV337M iNs relative to controls at 2 and 3 months post differentiation. These results were reproduced in three independent experiments (Fig. S4-b-d). The protein standard revealed bands at 480–1,048 kD molecular weight, corresponding to tau oligomers, with no tau detected below that range. Conformational tau species identified by the MC1 antibody  represent one of the most commonly detectable pathological features of tauopathies. Using the same experimental approach, we probed iN lysates using native electrophoresis. We observed a similar band pattern to T18, with elevated levels of conformational tau present after two months of culture only in the mutant iNs (Fig. 3b, S4 b-d). Overall, our results indicate a time-dependent increase of tau toxic species in the form of oligomeric and conformationally-modified tau in the mutant iNs compared to controls at two months post differentiation, but not earlier.
Next, we examined the temporal changes in p-tau and caspase-cleaved tau levels in the iNs using the anti-phospho-tau mAb PHF1 (Ser396/404), an epitope that is phosphorylated early in tau inclusion formation in humans , and three caspase-cleaved tau mAbs, including cleavage sites primarily targeted by caspase-6, D402 and D13, and the D421 site cleaved by multiple caspases [8, 17]. Semi-quantitative analyses of GAPDH-normalized band intensities revealed a 2.5-fold increase of PHF-1 levels in tauV337M relative to controls at three months post differentiation but not earlier (Fig. 3c). Similarly, caspase-cleaved tau levels showed a 2.5 to 3-fold increase of D421 and D402-positive bands and a 2-fold increase of the D13 in tauV337M iNs relative to controls at three months post differentiation. Again, these differences were absent in younger cells (Fig. 3d-f). Altogether, our results demonstrate a significantly higher accumulation of p-tau and caspase-cleaved tau in tauV337M iNs relative to controls that accumulated at three months post differentiation, but not at earlier stages.
Caspase inhibition is neuroprotective against stress-induced cytotoxicity in the tau V337M neurons
To test whether tauV337M iNs are more vulnerable to stressors relative to controls, we treated three-month tauV337M iNs with increasing concentrations of the wide-spectrum kinase inhibitor and apoptosis-inducer staurosporine (STS) for up to 48h. Treatment was followed by detection of cytotoxicity levels measured by lactate dehydrogenase (LDH) release (Fig. S5). We observed a 2-fold increase in cytotoxicity levels in the tauV337M iNs relative to tauWT controls, using 40 µM STS for 48h (Fig. S5-b), and selected this condition for further studies. Cytotoxicity was significantly reduced by treatment with the pan-caspase inhibitor z-VAD-fmk (300 µM for 48h or 4 x 75 µM in 12h intervals; Fig. S5-c), indicating that cell death occurred via apoptosis.
Based on these established conditions (Fig. S5), we exposed iNs to 40 µM STS for 48h and increasing doses of z-VAD-fmk (300 and 600 µM; Fig. 4). TauV337M and tauWT iNs treated with vehicle (DMSO) or z-VAD-fmk (600 µM) in the absence of STS showed low LDH release (Fig. 4); hence, baseline levels of apoptosis were low for iNs cultured for three months. Following STS treatment, however, we observed almost a 5-fold increase in cytotoxicity in the tauV337M iNs compared to a 4-fold increase in control iNs. The mutant group was significantly more vulnerable to cytotoxicity following acute stress by STS (p < 0.001). Moreover, STS co-treatment with z-VAD-fmk significantly reversed cytotoxicity levels in the mutant iNs. Overall, our results demonstrated increased vulnerability to apoptotic cell death in tauV337M iNs that was ameliorated by caspase inhibition.
To assess specific changes in the activity of caspase − 6 and − 3/7 between the tauV337M and tauWT iNs following stress exposure, we treated three-month tauV337M and control iNs with STS and measured caspase activity. Caspase-6 activity was measured by ELISA for cleaved lamin A, a selective substrate of caspase-6 over other caspases , while Caspases-3/7 activity was measured using a DEVD-aminoluciferin substrate assay (Fig. 4). We observed a 4-fold increase in caspase-6 activity in the tauV337M iNs compared to a 1-fold increase in control iNs following 20 µM STS treatment for 48h (Fig. 5a), indicating that the mutant group had significantly higher caspase-6 activity relative to controls (p < 0.01). Notably, STS co-treatment with 10 µM of the caspase-6 inhibitor z-VEID-fmk significantly reversed caspase-6 activity levels in the mutant iNs (p < 0.001, z-VEID-FMK/STS vs STS alone). We also observed a 6-fold increase in active caspase-3/7 levels (Fig. 5b) in the tauV337M and tauWT treated with 40 µM STS for 6h; there was no significant difference between caspase-3/7 activity between tauV337M and tauWT iNs. Caspase activity was reversed to baseline levels after the addition of 300 µM of the pan-caspase inhibitor z-VAD-fmk. Overall, these results demonstrate that STS treatment induced apoptotic cell death and a marked increase in caspase activity in the iNs that was suppressed by caspase inhibition. Caspase-6, but not caspases-3/-7, was preferentially activated in the tauV337M iNs relative to controls.
Next, we performed western blot analyses using lysates of iNs treated with 40 µM STS and 300 µM z-VAD-fmk (Fig. 5c-d). Following STS treatment, we observed a statistically significant 2-fold increase in TauC3 binding in the tauV337M iNs treated with STS compared to vehicle-treated cells (p < 0.001). TauC3 binding was not increased in control iNs treated with STS. In line with the cytotoxicity data (Fig. 4), STS co-treatment with z-VAD-fmk in tauV337M iNs reversed TauC3 binding to baseline levels.
Stress-induced reduction of neurite length is rescued by caspase inhibition
The reduction in neurite length is a morphological indicator of compromised cell viability and neurotoxicity [34, 49]. To examine the phenotypic effects of STS and z-VAD-fmk treatment on neurites in mutant and control iNs, we used immunocytochemistry (ICC) with DAPI to label cell nuclei (blue), and MAP2 (red) to label cytoplasm and neurites (Fig. 6a). Cells were imaged and subjected to automated quantification of neurite length under the same treatment conditions as our cytotoxicity assay (Fig. 4). We observed comparable mean neurite length in untreated iNs; neurite lengths were reduced upon treatment with 40 µM STS by 1.6-fold for tauV337M (p < 0.0001) and 1.3-fold (p < 0.0001) for tauWT (Fig. 6b). Co-treatment of STS with z-VAD-fmk partially restored neurite length and preserved MAP2-positive processes in tauV337M iNs compared to STS-treated cells (p < 0.01). For tauWT iNs, neurite length in STS treated cells was also restored upon the addition of z-VAD-fmk (p < 0.05). These data further demonstrate that neurotoxicity following STS treatment in the iNs was significantly caspase-dependent.