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Familial amyloid precursor protein mutants cause caspase-6-dependent but amyloid β-peptide-independent neuronal degeneration in primary human neuron cultures.

Although familial Alzheimer disease (AD)-associated autosomal dominant mutants have been extensively studied, little is known about the underlying molecular mechanisms of neurodegeneration induced by these mutants in AD. Wild-type, Swedish or London amyloid precursor protein (APP) transfection in primary human neurons induced neuritic beading, in which several co-expressed proteins, such as enhanced green fluorescent protein, red fluorescent protein (RFP)-tau and RFP-ubiquitin, accumulated. APP-induced neuritic beading was dependent on caspase-6 (Casp6), because it was inhibited with 5 μM z-VEID-fmk or with dominant-negative Casp6. Neuritic beading was independent from APP-mediated amyloid β-peptide (Aβ) production, because the APPM596V (APPMV) mutant, which cannot generate Aβ, still induced Casp6-dependent neuritic beading. However, the beaded neurons underwent Casp6- and Aβ-dependent cell death. These results indicate that overexpression of wild-type or mutant APP causes Casp6-dependent but Aβ-independent neuritic degeneration in human neurons. Because Casp6 is activated early in AD and is involved in axonal degeneration, these results suggest that the inhibition of Casp6 may represent an efficient early intervention against familial forms of AD. Furthermore, these results indicate that removing Aβ without inhibiting Casp6 may have little effect in preventing the progressive dementia associated with sporadic or familial AD.

Strong activity of caspase-6 (Casp6) determined by immunohistochemistry or immunoprecipitation, with neoepitope antisera to the active p20 subunit of Casp6 and to tau (microtubule-associated protein tau) or a-tubulin (Tub) cleaved by Casp6 (tauDCasp6, tau cleaved by Casp6; or TubDCasp6, tubulin cleaved by Casp6), is present in the hippocampus and affected cortices of sporadic and familial forms of Alzheimer disease (AD). [1][2][3][4] Casp6 activity is present in the neuropil threads, neurofibrillary tangles and neuritic senile plaques of the brain of mild cognitively impaired and mild-to-severe AD individuals. 2 Casp6 is also activated in the entorhinal cortex of non-cognitively impaired aged individuals that nevertheless had the lowest global cognitive scores of the group. 2 As the entorhinal cortex is the area first affected in AD brains, these findings suggest that Casp6 could be an early instigator of AD. In contrast to the other two short pro-domain effector caspases (Casps), caspase-3 (Casp3) and caspase-7 (Casp7), Casp6 has not been well investigated.
The high levels of active Casp6 in familial AD brain 1 incite the question of whether familial AD-associated mutant proteins induce Casp6 activation and if this activation depends on Ab. Familial AD occurs either via APP duplication or single point mutations of the amyloid precursor protein (APP) and Presenilin genes. 5,6 No studies have reported the effect of familial AD-associated mutant proteins on Casp6 activity. However, expression of the APP V717I London (Lo) mutant induces Ab-mediated Casp-dependent condensed chromatin in primary cortical neurons, 7 and expression of the APP Lo mutation induces DNA fragmentation in COS or COSlike cell lines. 8,9 Condensed chromatin and DNA fragmentation are both indicative of apoptotic cell death but the exact Casp responsible was not clearly identified in these studies. Expression of the APP Swedish (Sw) mutation increases the vulnerability of transfected PC12 cells to oxidative stress and the cells undergo Casp3-dependent cell death. 10 Because familial mutants increase amyloid b-peptide (Ab) production, Ab is generally thought to cause AD by synaptic or neuronal toxicity (reviewed in Selkoe and Podlisny 11 ). However, it is also possible that yet undiscovered natural functions are disrupted in mutant genes associated with familial AD, and cause undue stress to neurons before or independently from Ab production. Indeed, cellular stresses induce overproduction of Ab in a Casp-dependent manner, 12,13 thus placing Ab downstream of an initial stress.
Pro-Casp-6 (Casp6) is a cytosolic cysteinyl protease zymogen that is activated by self-or exo-proteolytic processing into a p20 and p10 subunit that reassemble to form the hetero-tetrameric active Casp6. 14,15 Casp6 has often been placed downstream of Casp3 and Casp7 in the apoptotic cascade, as it is proteolytically processed and activated by these two enzymes. 16 However, Casp6 is active in the absence of Casp3 and Casp7 in serum-deprived human primary neuronal cultures, and Casp6 activity precedes effector or initiator Casp activation in several cell types. 13,[17][18][19] Unlike the other effector Casps, active Casp6 is not always associated with apoptotic cell death. In AD, active Casp6positive neurons do not show apoptotic morphology. 3 Microinjection of active Casp6, but not Casp3 or Casp7, induces a protracted type of cell death in human neurons. 20 Similarly, activation of Casp6 in HEK293T cells does not induce cell death detectable by MTT reduction, sub-G 1 content assessed by FACS analyses or Casp3 activation. 15 Recently, Casp6 has been recognized as highly important in inducing axonal degeneration, whereas Casp3 induces cell death in APP-mediated death receptor 6 signaling in trophicfactor-deprived commissural, sensory and motor mouse neurons. 21 Although active Casp6 cleaves several neuronal proteins associated with AD, such as APP, tau, 22 and presenilin 1 and 2, 23 Casp6 also cleaves crucial cytoskeleton proteins including a-Tub, and a-actin-regulating post-synaptic density proteins, drebrin, spinophillin, a-actinin-1 and -4. 4 Therefore, the activation of Casp6 may alter the neuronal cytoskeleton and be responsible for the abundant levels of dystrophic neurites present in AD brains.
Dystrophic neurites containing severely altered cytoskeletal organizations are a primary feature of AD pathology. Often characterized by abnormal tau and phosphorylated tau immunoreactivity, dystrophic neurites in AD show axonal swellings, varicosities, densely packed organelles, membranous dense bodies and vesicular profiles. 24,25 Many of these features are present in the brain of aged APP Sw mutant transgenic mice. 26 Overexpression of wild-type and mutant APP associated with familial AD induce axonal transport defects that result in organelle accumulation. [26][27][28][29][30][31] Neuronal degeneration in APP wild-type transgenic mice brains is also associated with memory impairment. 32 Surprisingly, in some animal models, APP-mediated axonopathies are independent of Ab. 26,33 As Casp6 is predominantly activated in sporadic and familial AD brains, in this study, we investigated if APP wild-type or familial AD-associated APP mutants induced Casp6 activity.

Results
Wild-type APP 695 (APP WT ), Sw mutant APP 695 (APP Sw ) or the Lo mutant APP 695 (APP Lo ) induce Casp6-dependent neuritic enhanced green fluorescent protein (EGFP)positive beading in transfected primary human neurons. To assess whether familial AD-associated mutants activate Casp6 in terminally differentiated human neurons, primary cultures were transfected with pBudEGFP/ APP WT , /APP Sw or /APP Lo cDNA constructs. The APP and EGFP are independently expressed from the human elongation factor 1-a and the cytomegalovirus (CMV) promoters, respectively. EGFP, thus, serves as a marker of transfection efficiency. The APP WT represents overexpression of APP associated with trisomy 21 and a few FAD families. 6 The expression of APP from these constructs was confirmed in Neuro-2a (N2a) cells, which in contrast to human neurons can be transfected with high efficiency for biochemical analyses (Supplementary Figure 1). Only 2 mg of total proteins from the transfected cells were loaded on the western blot to avoid endogenous APP detection. Under these conditions, robust expression of APP was detected from each of the APP-expressing constructs, whereas none was observed in EGFP alone or mock-transfected cells; thus confirming APP expression under the CMV promoter of the pBud bigenic vector. As the CMV promoter is active in human primary neurons, these constructs were delivered with a biolistic particle delivery system, the most efficient transfection approach, as human neurons are not amenable to general viral infection systems or overly sensitive to liposomemediated transfections. In the absence of APP co-expression, the EGFP was homogeneously distributed in the cell body and neurites of transfected human primary neurons ( Figure 1a and 1a inset). When APP WT , APP Sw or APP Lo were co-expressed with EGFP, EGFP was often detected in an enlarged cell soma and appeared as beads-ona-string within the neurites (Figure 1b-d). This effect of APP on the EGFP distribution confirms the expected expression of APP from the pBudEGFP/APP constructs. Morphologically, EGFP-positive beads occurred at stochastic intervals and in varying sizes (1-2.5 mm) along the neurites (Figure 1c inset). Nuclear staining with Hoechst 33342 demonstrated that the larger EGFP-positive agglomerates overlapped with nuclei, whereas the smaller beads did not (Figure 1b-d). Under phase contrast, neuritic EGFP-positive beads overlapped with a slight enlargement and darkened area of the neurite resembling varicosities, in the absence of compromised membrane integrity (Figure 1e).
Quantitative analyses demonstrated that 40-60% APP WT -, APP Sw -or APP Lo -transfected neurons developed an EGFPpositive beaded morphology by 48 to 72 h post transfection ( Figure 1f). In contrast, EGFP expression in the absence of APP co-expression showed o10% neuronal beading between 24 to 72 h of transfection. Together, these results indicate that APP WT , APP Sw or APP Lo expression cause an agglomeration of EGFP within neurites of human neurons.
To investigate whether Casp6 activation was involved in neuritic EGFP-positive beading, APP WT -, APP Sw -or APP Lotransfected neurons were treated with 5 mM of the Casp6 peptide inhibitor, z-VEID-fmk. Treatment with the Casp6 inhibitor significantly decreased EGFP-positive beading morphology in APP WT -, APP Sw -or APP Lo -transfected neurons for up to 72 h ( Figure 1f). Casp6 has recently been shown to be associated with neurodegeneration by causing beading of Tub in mouse neurons. 21 We confirmed Casp6dependent Tub beading in human neurons (Supplementary Figure 2). Therefore, these results indicate that APP WT , APP Sw or APP Lo overexpression cause Casp6-dependent neuritic degeneration.
Because peptide inhibitors of Casps can be promiscuous, we confirmed the involvement of Casp6 activity in APP-mediated neuritic beading with the catalytically mutated form of proCasp6, proCasp6C163A. The dominant-negative (DN) nature of proCasp6C163A was first tested on the self-activated form of Casp6 lacking its pro-domain (p20p10Casp6) in the human colon carcinoma HCT116 cell line. In these, the fluorogenic assay revealed that expression of proCasp6C163A completely abrogated p20p10Casp6-mediated VEIDase activity ( Figure 2a). Western blot analyses of the transfected cells showed that proCasp6C163A was expressed as a 32 kDa protein, whereas p20p10Casp6 migrated at 28 kDa, as expected ( Figure 2b). Expression of p20p10Casp6 generated the p20 and p10 subunits of the active Casp6, consistent with the activity detected in Figure 2a. Co-expression of the proCasp6C163A with p20p10Casp6 reduced the level of the proCasp6C163A protein and increased the level of the p20 subunit indicating that proCasp6C163A was slightly processed by the active Casp6, and did not inhibit processing of the p20p10Casp6. These results are consistent with the hypothesis that DN inhibitors inhibit enzymatic Casp activity by generating a hetero-tetrameric enzyme containing catalytically inactive p20 subunits of the cognate Casp, and established the DN nature of proCasp6C163A (DN Casp6). Co-delivery of DN Casp6 with the pBudEGFP/APP Lo in human neurons significantly prevented EGFP-positive neuritic beading, thus confirming the involvement of Casp6 activity in APP-mediated neuritic beading ( Figure 2c).
APP WT -, APP Sw -or APP Lo -induced EGFP-positive beads also contain tau and ubiquitin (Ub) proteins. To assess whether beading was specific to EGFP, red fluorescent protein (RFP)-tau was transfected alone or co-transfected with pBudEGFP, or with pBudEGFP/APP WT , /APP Sw or /APP Lo . RFP-tau was expressed and localized in the cell soma and neurites of neurons ( Figure 3a). In RFP-tau and EGFP co-transfected neurons, both proteins showed an overlapping homogenous distribution in the neurons. In RFPtau neurons co-transfected with pBudEGFP/APP WT , /APP SW or /APP Lo , EGFP-and RFP-positive beads appeared.
As the accumulation of Ub inclusions is a common feature of degenerating neurites, RFP-Ub was also co-transfected with EGFP and APP Lo in neurons. By itself, RFP-Ub evenly distributed in the cell soma and in neurites ( Figure 3b). However, co-transfection of RFP-Ub with EGFP and APP Lo also caused RFP-Ub to accumulate in EGFP-positive beads. Together, the results indicate that the induced neuritic beading was not exclusive to EGFP, as it occurred with RFP-tau and RFP-Ub.
Neuritic beading is independent of Ab production in APP-transfected neurons. To determine whether neuritic beading may be a consequence of Ab overproduction in APP-transfected neurons, we transfected human neurons with the APP MV construct, in which the methionine 596 was mutated to a valine to prevent processing by b-secretase and generation of Ab, as previously demonstrated. 34 The APP MV protein was expressed from the pBudEGFP/APP MV construct at levels similar to those of the APP SW protein (Supplementary Figure 1). In human neurons, pBudEGFP/ APP MV induced EGFP-positive beads at a level consistently observed in pBudEGFP/APP WT -transfected neurons (Figure 4a). Similarly, the presence of the Lo mutation in APP MV also generated 30.05±6.15% beading (n ¼ 444 transfected cells), indicating that the APP MV -mediated beading was not synergistic with the APP Lo -mediated beading. z-VEID-fmk significantly inhibited APP MV -induced EGFP-positive beading, indicating that EGFP-positive beading was dependent on Casp6 activity (Figure 4b). We have previously reported that low levels of Ab 1À42 or Ab 1À40 induce neuritic beading of human neurons after 72 h of treatment and also render neurons vulnerable to a secondary insult. 35 To assess if Ab 1À42 may function synergistically with Ab-independent neuritic beading in pBudEGFP/APP MVtransfected neurons, these were treated with 50 nM Ab 1À42 or control reverse peptide Ab 42À1 . Ab 1À42 increased levels of beading in pBudEGFP/APP MV -transfected neurons after 24 h of incubation, but the increase did not reach statistical significance compared with Ab 42À1 (Figure 4c). However, beading in APP MV -transfected cells treated with Ab 1À42 was significantly inhibited by DN Casp6. These results confirmed  Figure 5B). To confirm that the loss of EGFP-positive neurons was due to cell death, we assessed condensed chromatin with the cell permeable dye Hoechst 33342 at 48 h post transfection. In all, 50, 51 and 39% of pBudEGFP/APP WT -, /APP Sw -or APP Lo -transfected neurons, respectively, exhibited condensed chromatin that was completely inhibited by z-VEID-fmk ( Figure 5C). Likewise, the condensed chromatin in pBudEGFP/APP Lo -transfected neurons was abolished by co-expression of DN Casp6 ( Figure 5D). Additionally, transfected neurons counterstained with terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-Red and Hoechst 33342 showed that beaded APP Lo -transfected neurons had both condensed chromatin and fragmented DNA ( Figure 5E). Collectively, these results indicate that expression of APP WT , APP Sw or APP Lo cause Casp6dependent neuronal cell death.
Cell death is dependent on Ab in APP-transfected neurons. To determine whether cell death may be due to Ab, we measured condensed chromatin in pBudEGFP/ APP MV -transfected human neurons. In contrast to the pBudEGFP/APP WT -transfected neurons, pBudEGFP/ APP MV -transfected human neurons did not show an increase in condensed chromatin after 48 h of transfection (Figure 6a). Similarly, the pBudEGFP/APP MV/Lo -transfected cells showed only 12.2±2.5% condensed chromatin (n ¼ 444 transfected neurons). However, when treated with Ab 1À42 , but not reverse peptide Ab 42À1 , a significant increase in condensed chromatin was observed in the APP MVtransfected neurons (Figure 6b). Condensed chromatin was present in 30-70% of the beaded neurons but rare in nonbeaded neurons (Figure 6c). Furthermore, the DN Casp6 decreased Ab 1À42 -mediated condensed chromatin in beaded neurons indicating that Ab-mediated cell death in human neurons expressing APP MV is also dependent on Casp6 activity (Figure 6c). Together, these results indicate that the Ab generated from APP overexpression in transfected neurons is responsible for cell death.

Discussion
In this study, we showed that conditions associated with familial AD, such as APP WT overexpression or the expression of APP mutants, induce Casp6-dependent but Ab-independent neuritic degeneration in primary cultures of human neurons. Overexpression of APP WT , APP Sw or APP Lo in human neurons resulted in the formation of neuritic swellings or varicosities, wherein proteins such as EGFP, RFP-tau and RFP-Ub accumulated rather than homogeneously distributed within the neurites. Inhibition of APP-mediated neuritic beading by the irreversible z-VEID-fmk peptide inhibitor and DN Casp6, confirmed the importance of Casp6 for neuritic beading. As overexpression of APP WT or APP mutants result in increased levels of Ab, 11,36 it was logical to associate Ab with Casp6-dependent neuritic beading. However, in this study, we showed that Ab was not responsible. The APP MV mutated at the b-secretase site and thus unable to generate Ab, 34 retained its ability to induce Casp6-dependent neuritic beading. Furthermore, the addition of physiologically high levels of Ab 1À42 to the APP MV -transfected neurons did not enhance the level of neuritic beading. That neuritic disruption is distinct from Ab production is consistent with recently published results that showed that axonal defects are not dependent on Ab in an APP overexpression murine model. 33 That Casp6 is responsible for neurodegeneration is entirely consistent with the recently emerging role for Casp6 in neuronal degeneration of mouse primary neurons. 21 This data suggest that Casp6 may also be responsible for the previously reported axonopathy and transport deficits caused by APP WT , APP Sw or APP Lo overexpression in vitro and in vivo. 26,27,29 Together with the present observations, the ability of Casp6 to cleave several important cytoskeletal proteins in human neurons 4 and the strong activity of Casp6 in neuropil threads, neuritic plaques and neurofibrillary tangles of sporadic and familial AD neurons 1-3 suggest that Casp6 has a predominant role in the neurodegenerative events of AD. These findings indicate that inhibition of Casp6 may be an efficient treatment against neurodegeneration in AD.
We also showed that APP WT overexpression or the expression of APP mutants induced Casp6-and Abdependent cell death. Indeed, APP WT , APP Sw or APP Lo expression in primary human neurons caused neuronal dropout, condensed chromatin, and TUNEL staining. This cell death depended on Casp6 activity, occurred uniquely in neurons that presented neuritic beads, and was inhibited by z-VEID-fmk or the DN Casp6. In contrast to neuritic beading, cell death was caused by Ab, as it was absent in APP MVtransfected human neurons, and treatment with 50 nM of extracellular Ab 1À42 re-established cell death in APP MVtransfected beaded neurons. This is entirely consistent with a previous report showing that the APP M596I mutant, which cannot undergo b-secretase cleavage, increases the resistance of APP FAD mutant-expressing cells to apoptosis. 37 Interestingly, in the absence of APP overexpression, concentrations of 100 nM extracellular Ab 1À42 do not induce cell death but reduce the levels of Bcl-2 while increasing the levels of Bax in human neurons, and renders these exposed neurons vulnerable to oxidative stress. 35 These results show that neither APP overexpression nor extracellular Ab are sufficient to initiate cell death in human neurons but a combination of the two lowers the threshold of neuronal resistance to cell death. This phenomenon is consistent with numerous studies, in which APP Sw and APP Lo induced cell death in COS-NK1, PC12, NT2N, rat and mouse primary cortical neurons, although the exact culprit was not identified in these cells. 7,9,38,39 Although overexpression of APP and Ab causes neuritic beading in transgenic mice, it does not produce overwhelming levels of cell death. 40 The ability of Ab to induce cell death in APP-overexpressing neurons in culture but not in transgenic mice may be explained by differences in intrinsic species-specific cell death pathways or neuroprotective effects from supporting cells. In AD, there is an insignificant amount of neuronal loss except early in layer II of the entorhinal cortex and later in the temporal cortex of advanced cases of AD. 41 However, neurodegeneration in the absence of evident cell death, is prominent early in AD, as is synaptic loss which correlates best with the level of dementia. 22 Therefore, the toxicity of Ab on APP-transfected neurons may reflect a non-physiological event that occurs easily in cell cultures but not in AD brains. It is also important to note that Casp6 activity is not associated with acute cell death as observed with the other two effector Casps, Casp3 and Casp7. 15,20 Similarly in AD, the active Casp6 is present in neurons that do not show apoptotic morphology. 3 Furthermore, Casp6 activity causes neuritic degeneration but not cell death in primary mouse neurons. 21 Therefore, the intense Casp6 activity observed in neuropil threads, neurofibrillary tangles and neuritic plaques is more likely associated with neurodegeneration than with cell death in AD. From these results, we conclude that removing Ab without inhibiting Casp6 may have little effect in preventing the progressive dementia associated with sporadic or familial AD.

Materials and Methods
Cell culture. Primary human fetal neurons obtained with ethical approval from the McGill University Institutional Review Board were cultured as described previously. 36. Briefly, 12-week-old fetal cerebrum were dissociated with trypsin, Cloning strategy and site directed mutagenesis of human APP. Full-length APP 695 cDNA (APP WT ) was obtained from Dr. Georges Levesque (Laval University, Quebec, Canada) and subcloned into the bigenic eukaryotic pBudEGFP vector-expressing EGFP under the human elongation factor 1 a-promoter. To mimic the state of duplicated APP in familial AD, APPWT cDNA was subcloned under the human CMV promoter using the HindIII and NotI sites to generate pBudEGFP/APP WT . The KM595/596NL Sw mutation and the V642I Lo mutation were generated by QuikChange site directed mutagenesis (Stratagene, La Jolla, CA, USA) using 5 0 -GATCTCTGAAGTGAATCTGGATGCAGAATTCCG-3 0 and 5 0 -CATAGCGACAGTGATCATCATCACCTTGGTG-3 0 primers to generate pBudEGFP/APP Sw and pBudEGFP/APP Lo vectors, respectively. The histidinetagged DN Casp6 C163A mutant (DN Casp6) was cloned in the pCep4b vector. The b-secretase uncleavable APP MV mutant was a kind gift from Dr. Jannic Boehm (University of Montreal, Quebec, Canada) 34 and was subcloned into the pBudEGFP vector SalI and XbaI restriction sites. The Lo mutation was inserted in the APP MV by Quikchange mutagenesis. The pDs-Red RFP-tau (RFP-tau) construct was a kind gift from Dr. Yasuo Ihara (University of Tokyo, Tokyo, Japan) 42 and the pRFP-C1 monomeric Ub (RFP-Ub) construct was purchased from Addgene (Cambridge, MA, USA). 43 All constructs were confirmed by sequencing at the McGill University and Genome Quebec Innovation Centre.
Transfections. Neurons were plated on poly-L-lysine (20 mg/ml; Sigma-Aldrich, St Louis, MO, USA) coated polychlorotrifluoroethylene Aclar coverslips (Honeywell Aclar Film; http://www51.honeywell.com/sm/aclar/) at a density of 3 Â 10 6 cells/ml and were transfected with the Helios gene gun (Bio-Rad, Mississauga, Ontario, Canada) at a 100 p.s.i. pressure pulse of helium. The cartridges used for transfection were made of 0.033 mg of DNA bound to 4.2 mg of gold microcarrier beads prepared in 1 M calcium chloride and 0.1 ml of 0.05 M spermidine. Approximately 1 mg of DNA coated gold beads/cartridge was delivered to three coverslips of neurons per treatment. When transfecting cells with more than one construct, a ratio of 2 : 1 of RFP-tau or RFP-Ub to pBudEGFP, DN Casp6 and APP MV or 3 : 1 of DN Casp6 to APP Lo was used to prepare cartridges. EGFPpositive cells were counted versus the total number of cells stained with Hoechst 33342 (Sigma-Aldrich) to estimate the transfection efficiency, which was around 0.001%. Primary human neurons resist infection by commonly used viral vectors and lipid-based methods, and therefore, these methods could not be used for these studies. HCT116 cells were plated overnight in a six-well plate at a density of 400 000 cells per well in McCoy's media (Gibco-Invitrogen) supplemented with 10% fetal bovine serum (Fisher, Ottawa, ON, Canada). Transfections were performed on the following day using polyethylenimine (Polysciences Inc., Warrington, PA, USA) at a 2 mg concentration for every 1 mg of DNA. The transfection complex was prepared in 100 ml of serum-free media. At 6 h following transfection, complete media was replenished and cells were incubated for an additional 18 h to allow for expression.
Treatment of APP MV -transfected neurons with Ab peptides. Fibrillar Abs were prepared as described previously. 44 Cells were transfected as described above, incubated for 24 h and then treated with 50 nM Ab 1À42 or Ab 42À1 control reverse peptide (diluted from a 25 mM stock solution in culture media) for 24 h before assessing beading and condensed chromatin. Condensed chromatin. For condensed chromatin, 1 mg/ml of Hoescht 33342 was added to media 20 min before the live assessment, cells were washed twice in PBS pH 7.5 and fresh media was re-applied. EGFP-positive neurons displaying condensed chromatin visualized with Hoescht 33342 were counted.
Tunel. For DNA fragmentation, human neurons were stained with TUNEL-Red (Roche, Laval, Quebec, Canada). Briefly, neurons were fixed for 20 min at room temperature in 4% paraformaldehyde and 4% sucrose, and then permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate before the staining.
Casp6 activity. To assess Casp6 activity in APP WT , APP Sw , APP Lo or APP MVtransfected neurons, neuronal cultures were pre-treated with 5 mM of z-VEID-fmk (Biomol, Plymouth meeting, PA, USA) for 2 h before the gene gun transfections. Media containing the Casp6 inhibitor was changed every 24 h during the live assessment until the 72 h time point. In addition, neurons were co-transfected with the DN Casp6 as described above. To assess Casp6 activity in HCT116 cells, cells were harvested in CHAPS buffer (50 mM HEPES, 0.1% CHAPS, 0.1 mM EDTA, 1 mM DTT) containing protease inhibitors and total protein was quantified by BCA assay (ThermoScientific, Rockford, IL, USA). For measurement of Casp6 activity, fluorogenic Casp activity assays were performed with Stennicke's buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA, 10% sucrose) containing 10 mM Ac-VEID-AFC (Sigma-Aldrich) at 37 1C using a Bio-Rad Fluoromark fluorometer (Hercules, CA, USA) at an excitation wavelength of 390 mM and emission wavelength of 538 nM. Measurements were read every 2 min for 1 h, and released pmol of AFC was calculated from an AFC standard curve run in parallel. The reaction was performed in 50 ml total volume containing 10 mg of total protein lysate.
Western blot analyses of Casp6. Western blotting analysis was performed with 30 mg of HCT116 total protein lysate prepared in Laemmli buffer and eletrophoresed on a 15% SDS-PAGE gel. The following antibodies were used for subsequent detection. Anti-Casp6 polyclonal antiserum from NeoMarkers (Fremont, CA, USA) recognized the full length and p20 subunit of Casp6. The anti-p10Casp6 (Pharmingen) monoclonal antibody recognized the p10 subunit and full-length proCasp6 or proCasp6C163A. The anti-p20Casp6 10630 neoepitope antibody detecting the cleaved p20 subunit of active Casp6 was made in the laboratory. The anti-b-actin monoclonal antibody was raised against the 16 amino acids of the N-terminus of the protein.
Statistical evaluations. Statistical analysis was performed with a one or two way analysis of variance and Tukey, Scheffé or Bonferroni post hoc test using the Statview software (SAS Institute Inc., Cary, NC, USA) or Graphpad Prism software (La Jolla, CA, USA).