Aim, design and setting of the study
The aim of this study was to assess the reversibility of Casp6-mediated age-dependent cognitive deficits in vivo with MB treatment. MB is a non-toxic, blood brain barrier permeable inhibitor of caspases  and is already being tested in human clinical AD trials [17, 54]. The ACL Caspase-6 KI/Cre mice are cognitively impaired at 15 months of age . MB was administered 3 months after the onset of cognitive deficits to assess reversibility of well-established cognitive defects. Cognitive status was assessed at 18 months, and again at 19 months of age, after 1 month of treatment. Additional treatment and cognitive assessments were planned every month thereafter until 24 months of age or until an effect was observed. Since reversibility of cognitive deficits was observed within 1 month of treatment, animals were sacrificed at 19 months of age and submitted to electrophysiology of acute hippocampal slices, immunohistochemistry of brain sections and biochemical analyses of brain tissues.
Human Casp6 transgenic mice
C57BL/6 Casp6 transgenic knock-in ACL (type I) mice (KI/Cre) express a self-activated form of human Casp6 lacking the pro-domain under Cre/loxP recombination. A floxed STOP sequence between human Casp6 and the CMV immediate early enhancer/chicken β-actin promoter in the knock-in gene was excised by Ca++/calmodulin kinase IIα (CAMKIIα)-regulated Cre expression, allowing the expression of human Casp6 in the hippocampal CA1 pyramidal cells [29, 50]. Since the transgene was inserted in the hypoxanthine-guanine phosphoribosyl transferase locus of chromosome X (HPRT), only males were used in all experiments to avoid lionization effects. The wild type (WT/WT), KI transgenics without Cre (KI/WT), or Cre only (WT/Cre) mouse littermates were used as controls. Casp6 Knock-Out (KO) mice were obtained from Jackson Laboratories (Bar Harbour, ME, USA). The type II ACL/G mice resulted from CAMKIIα-Cre recombinase gene expression in testis and deletion of the STOP sequence of the floxed transgene, allowing male germ line transmission of the STOP-excised transgene and thus whole body expression of a transgene in the F2 progeny . Treatments and behavioral tests were done blinded to the ACL mice type and genotype. All animal procedures followed the Canadian Council on animal care guidelines, which were approved by the McGill University Animal Care Committees. Mice were bred and maintained in the Goodman Cancer Research Centre Mouse Transgenic Facility at McGill University and transferred to the Lady Davis Institute for experiments. Mice were housed at 22 °C, in 55% humidity, and on a reversed 12 h light/dark cycle with ad libitum food and water.
Oral administration of MB
Mice of 18 months of age were used for the study since ACL human Casp6 transgenic mice displayed episodic and spatial memory impairment at the age of 15 months  and 18 month-old mice are considered old according to Jackson Laboratories. Eighteen-month-old littermates from KI/Cre (ACL/G n = 27, ACL n = 17), KI/WT (ACL/G n = 8, ACL n = 23), WT/Cre (n = 38), and WT/WT (n = 40) were administered approximately 20 mg/kg/day 3,7-bis(Dimethylamino) phenazathionium chloride (MB), (Sigma-Aldrich, St. Louis, MO, USA) and 2 mM saccharin (Sigma), or 2 mM saccharin (vehicle) only in drinking water for 38 days (including the 8-day behavioral tests). During treatment, 1 vehicle-treated WT/WT mouse died, 2 MB-treated mice died (ACL/G KI/Cre and WT/WT). This was expected for aged mice. The dose was chosen based on a previously published paper , where MB was given orally to test its ability to prevent Tau aggregation. The oral administration of MB to C57Bl6 mice was shown to be well-tolerated and led to peak concentrations in plasma and brain within 2 h of treatment, and a half-life of 4.7 h in plasma and 30.6 h in brain . Oral bioavailability was ~ 18% and much better than that delivered by i.v. treatment. MB was delivered in drinking water based on a 1 mL/10 g water consumption rate of an adult mouse ad libitum, and actual water consumption rate was measured for each cage (1–3 mice per cage) every day to confirm that the mice drank the drug. MB-treated mice (body weight = 44.43 ± 1.48 g) consumed 4.25 ± 0.21 mL/day, and vehicle-treated mice (body weight = 44.95 ± 1.46 g) consumed 4.51 ± 0.13 mL/day. The body weights of animals were measured every week and did not change over the time of treatment.
After habituation to the experimenter for 1–2 weeks, KI/Cre (ACL/G n = 18, ACL n = 8), KI/WT (ACL/G n = 8, ACL n = 14), WT/Cre (n = 27), and WT/WT (n = 24) mice were sequentially tested in open field, novel object recognition, and Barnes maze tasks before and after treatment. Equipment was wiped with 70% ethanol to eliminate odour cues between each mouse.
Open field test
Each mouse was placed in a corner of a 40 cm × 40 cm × 30 cm cuboid acrylic plexiglass box, and was allowed to freely explore for 5 min. The HVS 2100 automated video tracking system (HVS Image, Hamptom, UK) divided the field in 4 rows × 4 columns yielding 16 virtual zones and analyzed the total distance travelled, the percentage of time moving, the number of entries into virtual zones, the time spent in each virtual zones, and the percentage of used zones.
Novel object recognition (NOR)
NOR task was performed 24 h after the open field test. Two identical objects were placed in the northwest and northeast corner at 5 cm from the walls and separated by 30 cm. Mice were placed in the box, where they encountered both objects. After 5 min, mice were replaced in their homecage. After 2 h, mice were returned to the NOR box for 5 min where a familiar object was replaced with a novel object. The position of the novel object was changed between each animal to avoid any bias related to a preference in the location of the new object and the use of possible confounding spatial cues. In addition to the tracking record by the HVS 2100 system, the experimenter recorded the number of times mice touched each object. Two WT/WT mice (vehicle- and MB-treated) and three ACL/G KI/Cre (1 vehicle- and 2 MB-treated) were excluded because they froze during the test. The discrimination index was calculated as the (number of time touching the novel object – number of time touching the familiar object)/(number of time touching both object).
The Barnes maze apparatus consisted of a round 90 cm diameter table with 20 equidistant 5 cm-diameter holes on the edge. All the holes were blocked except for the target hole, which had an escape hatch under the table. The four walls around the table were pasted with high contrast visual cues to allow spatial memory. Mice were placed on the center of the table under a black box. When the trial started, the black box was taken away, and a strong light and buzzer served as the stimuli for mice to search for an escape. Once mice escaped successfully, the light and buzzer were turned off. The Barnes maze task was administered in three phases: adaptation (day 0), spatial acquisition training (day 1–4) and probe test (day 5). On the day of adaptation, mice were allowed to explore the table for 60 s, and stay in the hatch for 120 s. On days 1–4 of training for spatial acquisition, the escape latency and the number of errors made before finding the escape hatch were recorded. If the mouse could not escape within 180 s, the experimenter gently led it into the escape hatch and allowed it to remain there for 60 s. Mice were given four trials per day with an inter-trial interval of 15 min. On day 5, mice were given a single probe test, in which the target hole was blocked. The mouse was allowed to explore for 90 s, and the primary latency and primary errors to reach the escape hole, and the number of visits to each hole were recorded. The HVS 2100 automated video software tracked the animals during all trials. Two WT/Cre (vehicle- and MB-treated) and one MB-treated ACL KI/Cre were excluded because they froze during the test.
After behavioral analyses, mice were anaesthetized by isoflurane (Thermo Fisher Scientific, Waltham, MA, USA), and perfused transcardially with 50 ml ice cold 0.9% saline and 200 ml 4% paraformaldehyde in 0.2 M Phosphate Buffer using a peristaltic pump (Thermo Fisher Scientific). Brains were removed and post-fixed in 10% formalin in Phosphate Buffer (Thermo Fisher Scientific) for 24 h and then dehydrated in 70% ethanol for 24 h. Fixed brains were paraffin embedded and cut with a microtome at the Institute for Research in Immunology and Cancer histology centre (University de Montreal, Montreal, Canada). Brains were serially sectioned at 4 μm through the anterior hippocampus between bregma − 1.22 μm and bregma − 1.52 μm. In total, 75 consecutive sections (3 sequential sections per slide) were collected to obtain 25 slides per mouse brain. Every fifth slide, corresponding to an interval of 60 μm, were used for immunohistochemical analysis of microglial Iba1, astrocyte GFAP, and Casp6 activity TubΔCasp6 (EEVGVD438) immunomarkers. The last slide was used for human Casp6 analysis. After deparaffinization in 100% xylene (Thermo Fisher Scientific) twice for 5 min each and rehydration in 100% ethanol twice for 5 min each, 95% ethanol once for 5 min, and MilliQ water for 5 min, slides were incubated in antigen retrieval buffer (10 mM Tris-base, 1 mM EDTA, 0.5% Tween-20, pH 9 for Iba1, human Casp6, and TubΔCasp6 and 0.01 M Tris-Na citrate, pH 6 for GFAP) at 97 °C for 20 min in the Pascal Dako Cytomation (Dako, Burlington, ON, Canada). Immunohistochemical staining was automatically performed by the automated Dako Autostainer Plus slide processor using the EnVision Flex system (Dako). Briefly, slides were treated with 0.03% hydrogen peroxide for 15 min, washed 3 times with Wash Buffer (Dako), blocked with Serum-Free Protein Block (Dako) for 30 min, and incubated with either 1/2000 anti-Iba1 (019-19741Wako, Richmond, VA, USA), 1/8000 anti-GFAP (z-0034 Dako), 1/5000 anti-TubΔCasp6 (GN20622, Laboratory made ), or 1/5000 anti-human Casp6 (LS-B477 Lifespan Bioscience, Seattle, USA), diluted in EnVision Flex Antibody Diluent (Dako) for 30 min. After rinsing in Wash Buffer, brain sections were incubated with anti-rabbit-HRP or anti-mouse-HRP for 30 min. Staining was visualized with 3,3′-diaminobenzidine substrate-chromogen for 10 min and counterstained with hematoxylin (Dako) for 5 min. After dehydrating the slides in MilliQ water for 5 min, 95% ethanol for 3 min, 100% ethanol twice for 5 min each, 100% xylene twice for 3 min each, slides were mounted with Permount mounting medium (Thermo Fisher Scientific).
Slides were scanned using the Mirax Scan (Zeiss, Germany). Three to five micrographs at 40x magnification were taken in the stratum oriens, pyramidal cell layer, stratum radiatum and stratum lacunosum molecular of each hippocampal CA1 region, corpus callosum, fimbria and fornix. Image J software was used to measure the positive immunoreactive area over the total area in square microns. The number of Iba1-positive subtype I-IV microglia were counted in each region by the experimenter blinded to genotype and treatments based on semi quantitative scoring schemes .
Excision of the STOP sequence with CaMKII-α Cre recombinase
Genomic DNA from hippocampus or tail was extracted as described . Twenty ng of genomic DNA was used as template to amplify the recombined or Cre-excised recombined HPRT locus using primers designed by Genoway: Forward 5′-TGCTTCAGTCCCATGTTTGGCAAGG-3′ (GX5889) and Reverse 5′-AAATCTGTGCGGAGCCGAAATCTGG-3′ (GX2752). The recombined HPRT locus and Cre-excised recombined HPRT locus generate a 4195 and 2812 bp amplicon, respectively. The amplicon was achieved using Q5 DNA polymerase with 35 cycles of 98 °C for 10 s, 61 °C for 30 s, 72 °C for 150 s, followed by a 72 °C extension period of 2 min.
Twenty ng of genomic DNA was used to amplify the CaMKII-α Cre using primers: Forward 5′- GACTAAGTTTGTTCGCATCCC − 3′ and Reverse 5′ – ATCCAGGTTACGGATAT AGT-3′. The ~ 1650 bp amplicon was achieved using Q5 DNA polymerase with 35 cycles of 95 °C for 30 s, 45 °C for 30 s, 72 °C for 60 s, followed by a 72 °C extension period of 10 min.
RT-PCR and quantitative PCR (qRT-PCR)
Total RNA was extracted from mouse hippocampus, cortex and cerebellum with the miRNeasy Mini Kit (Qiagen, QC, CA) according to the manufacturer’s protocol. During RNA purification, genomic DNA was digested with the RNase-Free DNase in this kit. One μg of total RNA was converted to cDNA with the avian myeloblastosis reverse transcriptase (Roche, Mannheim, Germany). PCR amplification of a 229 bp human Casp6 amplicon was achieved with Taq DNA polymerase (New England Biolabs, Whitby, ON, Canada) and the 5′-CGATGTGCCAGTCATTCCTT-3′ and 5′-CTCTAAGGAGGAGCCATAT-3′ primers, 30 cycles of 95 °C for 30 s, 61.1 °C for 30 s, 68 °C for 60 s, followed by a 68 °C extension period of 10 min. The 904 bp murine Casp6 amplicon was amplified with 5′- CTCAGGGCTAGGACACCGGTGGGA-3′ and 5′- ATATATGTAGCAAGACAGATGGCC-3′ primers, 35 cycles of 95 °C for 30 s, 64 °C for 1 min, 68 °C for 1 min 30 s, followed by a 68 °C extension period of 5 min. The 151 bp 18S amplicon was obtained with the 5′-GTAACCCGTTGAACCCCAT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′ primers, 27 cycles of 95 °C for 30 s, 58 °C for 30 s, 68 °C for 60 s, followed by a 68 °C extension period for 5 min.
Quantitative RT-PCR was performed with SYBR Green Taq Mastermix (Quanta BioSciences, Gaithersburg, MD, USA) on the Applied Biosystems 7500 Fast Real-Time PCR apparatus (Applied Biosystems, Foster City, CA, USA). Murine Casp6 cDNA was detected with 5′-CATGCAGAAACCGATGGCTTCTA-3′ and 5′-GGACGCAGCATCCACCTGGGTCAC-3′ primers. 18S cDNA was amplified with 5′- GTAACCCGTTGAACCCCAT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′ primers. Results are expressed as fold-induction values normalized to the 18S reference gene using Pfaffl’s method .
Mouse hippocampus, cortex and cerebellum were dissected and frozen at − 80 °C until use. Proteins were extracted by homogenizing (TH Omni International, Marietta, GA) the tissue in 5 volumes of Tris-Triton lysis buffer (10 mM Tris pH 7.4, 100 mM NaCl, 0.1% SDS, 1 mM EDTA, 1% Triton X-100, and10% glycerol with freshly added 0.5% sodium deoxycholate, 300 μM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 2.4 μM pepstatin A, 2 μM leupeptin, 0.8 μM tosyl-L-lysyl-chloromethane hydrochloride, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, and 1 mM sodium orthovanadate). After centrifugation at 13000 x g for 20 min, the supernatant protein concentration was quantified by Bradford assay (Bio-Rad, Canada). Five to twenty μg of protein were separated on 12% or 15% SDS-PAGE (7.5% for Tubulin) and transferred to PVDF membranes (Bio-Rad). Membranes were probed with either 1/1000 anti-mouse and human Casp6 (#9762, Cell Signalling Tech, USA), 1/5000 anti-human Casp6 (LS-B477, Lifespan Bioscience), 1/3000 anti-GFAP (z0334 Dako), or 1/5000 β-Actin (A5441, Sigma) in 5% non-fat milk in TBST (0.1 M Tris HCl, 1.5 M NaCl, 0.5% Tween-20) overnight at 4 °C. Incubation of horseradish peroxidase-linked anti-rabbit secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) at room temperature for 1 h were followed with enhanced chemiluminescence (GE Healthcare Life Sciences, QC, CA). Light emission from the immunopositive protein bands was visualized on X-ray films (Kodak, Rochester, NY, USA). Alkaline phosphatase-linked anti-mouse secondary antibody was used with NBT/BCIP substrate (Promega, WI, USA) for chromogenic detection of β-actin after the chemiluminescent detection. Quantifications were performed with the ImageJ software (NIH, Bethesda, MD, USA).
Extracellular field recordings
After anaesthesia with isoflurane, 19-month-old vehicle-treated KI/Cre (ACL/G n = 4, ACL n = 1), ACL KI/WT (n = 6), WT/Cre (n = 6), and WT/WT (n = 6), and MB-treated KI/Cre (ACL/G n = 3, ACL n = 3), ACL KI/WT (n = 3), WT/Cre (n = 5), and WT/WT (n = 5) mice were perfused transcardially with 5 mL ice-cold N-Methyl-D-glucamine (NMDG, Sigma) artificial cerebrospinal fluid (ACSF; 93 mM NMDG, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 5 mM Na-ascorbate, 3 mM Na-pyruvate, 12 mM N-Acetyl-L-cysteine, 10 mM MgCl2, 0.5 mM CaCl2, 25 mM glucose, pH 7.2, ~ 338 mOsm) saturated with 5% CO2/95% O2 . Brains were removed quickly, submerged in ice-cold NMDG-ACSF, and cut into transverse 300 μm hippocampal slices with a 5000mz-2 vibratome (Campden Instruments Ltd., England). Brain slices were initially recovered in 32 °C NMDG-ACSF for 10 min, transferred to 32 °C normal ACSF (125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 1 mM MgCl2, 2 mM CaCl2, 45 mM glucose, ~ 338 mOsm) for 20 min, and then placed in normal ACSF under 5% CO2/95% O2 for 30 min at room temperature.
All experiments were performed at 31–33 °C in normal ACSF saturated with 5% CO2/95% O2. The recording pipettes of 2–4 MΩ resistance were filled with normal ACSF and placed in the CA1 stratum radiatum. The stimulation electrode was also placed in the CA1 stratum radiatum 200 μm away from the recording pipette. Field recordings were boosted at 100x gain using a BVC-700A amplifier (Dagan Corporation, Minneapolis, MN) with low-pass filtering set to 5 kHz, and then recorded at 10 kHz with a PCI-6229 digitization board (National Instruments, Austin, TX) using custom scripts in Igor Pro 7 (WaveMetrics Inc., Lake Oswego, OR) running on a SuperLogics computer (Natick, MA). For baseline responses, Schaffer collaterals were recruited with 100-μs-long biphasic constant-voltage pulses, repeated once every 20 s, using a BSI-950 stimulus isolation unit (Dagan Corporation, Minneapolis, MN). Minimal and maximal stimulation levels were first determined in the 10 to 50 V range. Schaffer collaterals were then activated by a fixed intermediate voltage for the remainder of the experiment. LTP induction was achieved using a theta-burst protocol consisting of 12 trains of 4 pulses at 100 Hz delivered at 5 Hz and repeated three times at 0.1 Hz .
Recordings with unstable baseline were rejected, as assessed using a t-test of Pearson’s r . Off-line analysis was performed using in-house software  running in Igor Pro 7 (WaveMetrics Inc., Lake Oswego, OR, USA). The slope of fEPSPs was normalized to the initial 20-min-long baseline period before averaging across recordings. Three or four LTP experiments were carried out in each animal.
Neuronal Casp6 activity assay by FLICA
Acute brain slices from 19-month-old mice were obtained as described in the “Extracellular field recordings section. Brain slices were incubated with 1x Casp6-FLICA (FAM-VEID-FMK, ImmunoChemistry Tec., CA) in normal ACSF for 1 h at room temperature with 5% CO2/95% O2, and rinsed three times in fresh ACSF for 10 min. We tested 3–4 slices for each animal for vehicle-treated KI/Cre (ACL/G n = 1, ACL n = 2), MB-treated KI/Cre (ACL n = 3), or vehicle-treated WT/WT (n = 3) mice, and 1 slice for each animal treated with z-VEID-FMK-treated KI/Cre (ACL/G n = 1, ACL n = 2) mice.
Two-photon excitation was achieved using a Chameleon XR Ti:Sa laser (Coherent, Santa Clara, CA, USA) tuned to 820 nm for carboxyfluorescein (FAM). The two-photon microscope was custom-built as previously described . Imaging data were acquired using ScanImage v3.7 running in Matlab (The MathWorks, Natick, MA, USA) via PCI-6110 boards (National Instruments). Images of hippocampal CA1 regions at depths between 0 and 80 μm from the brain slice surface were taken from maximum intensity projections of two-photon laser scanning microscopy image stacks. Imaging areas were scanned at a rate of 2 ms/line and a resolution of 512 × 512 pixels with a slice separation of 2 μm. Each slice was an average of three green-channel frames. FLICA-positive neurons in CA1 regions were quantified at depths of 0, 10, 20 and 30 μm from the brain slice surface.
Statistical analyses of data were performed using Igor Pro 7 or GraphPad prism 7 (GraphPad Software, CA, USA). Comparison between two groups was done using the unpaired Student’s two-tailed t-test. Analysis comparing more than two groups with equal variance was done by one-way ANOVA. Brown-Forsythe’s ANOVA was used if Bartlett’s test indicated heteroscedasticity at the p < 0.05 level. Tukey’s post-hoc or Dunnett’s post-hoc test as indicated in figure legends was used when ANOVA indicated p < 0.05. Analysis of three or more groups at different time points (Barnes maze spatial acquisition training) was done using repeated-measures two-way ANOVA with Dunnett’s post-hoc test.