Mice
PKCα-M489V Mouse Generation. C57BL/6NTac-Prkca mice containing the M489V mutation in Prkca were generated by Taconic Biosciences GmbH for Cure Alzheimer’s Fund as previously described 34.
APPswe + PKCα-M489V Mouse Generation. This mouse was generated by Taconic Biosciences GmbH for Cure Alzheimer’s Fund by an initial intercross of C57BL/6NTac-PrkcaM489V homozygous mice and the transgenic APPswe mice (B6;SJL-Tg(APPSWE)2576Kha, model 1349, Taconic) 41. From this intercross, PrkcaM489V heterozygous mice carrying the transgenic APPswe mice (HET;APP) and PrkcaM489V heterozygous mice not carrying the transgenic APPswe (HET;NON-APP) were obtained. Then, the offspring (HET;APP x HET;NON-APP) was intercrossed to generate the first cohort. Afterwards, HET;APP x HET;NON-APP breeding pairs were maintained to generate all the cohorts used for the experimental studies.
All procedures involving animals were approved by The Scripps Research Institute’s Institutional Animal Care and Usage Committee (IACUC) and the University of California San Diego IACUC, and met the guidelines of the National Institute of Health detailed in the Guide for the Care and Use of Laboratory Animals 76.
Behavioral tests
Barnes maze test. This is a spatial memory test 77, 78, 79 sensitive to impaired hippocampal function 80. Mice learn to find an escape tunnel among 20 possibilities below an elevated, brightly lit and noisy platform using cues placed around the room. Spatial learning and memory are assessed across trials and then directly analyzed on the final probe trial in which the tunnel is removed and the time spent in each quadrant is determined; the percent time spent in the target quadrant (the one originally containing the escape box) is compared with the average percent time in the other three quadrants. This is a direct test of spatial memory as there is no potential for local cues to be used in the mouse’s behavioral decision.
Locomotor activity test. Locomotor activity was measured in polycarbonate cages (42 x 22 x 20 cm) placed into frames (25.5 x 47 cm) mounted with two levels of photocell beams at 2 and 7 cm above the bottom of the cage (San Diego Instruments, San Diego, CA). These two sets of beams allowed for the recording of both horizontal (locomotion) and vertical (rearing) behavior. A thin layer of bedding material was applied to the bottom of the cage. Mice were tested for 120 minutes and data were collected in 5-minute intervals.
Light/dark test. The light/dark transfer procedure has been used to assess anxiety-like behavior in mice by capitalizing on the conflict between exploration of a novel environment and the avoidance of a brightly lit open field 81. The apparatus is a rectangular box made of Plexiglas divided by a partition into two environments. One compartment (14.5x27x26.5 cm) is dark (8–16 lux) and the other compartment (28.5x27x26.5 cm) is highly illuminated (400–600 lux) by a 60 W light source located above it. The compartments are connected by an opening (7.5x7.5 cm) located at floor level in the center of the partition. The time spent in the light compartment is used as a predictor of anxiety-like behavior, i.e., a greater amount of time in the light compartment is indicative of decreased anxiety-like behavior. Mice were placed in the dark compartment to start the 5-minute test.
ANOVA was used for the statistical analyses of behavioral results, followed by post hoc Student's t-tests as appropriate (*p < 0.05, **p < 0.01, ***p < 0.001).
Electrophysiology
Organotypic slice cultures. Organotypic hippocampal slices were prepared from P5-P7 mice pups as previously described 82. Slice cultures were maintained by changing media every two days, and 18–24 h prior to electrophysiological experiments, slices were infected with Sindbis viruses to express the APP derived peptides CT84 and CT100 as previously described 50.
Electrophysiological recordings. Hippocampal organotypic slices were used for electrophysiological recordings shown in Fig. 7. Slices made from PKCα WT and M489V littermates were interleaved. Simultaneous whole-cell recordings were obtained from two neurons, one infected and one neighboring control CA1 pyramidal neurons under visual guidance using differential interference contrast and fluorescence microscopy. One stimulating electrode (contact Pt/Ir cluster electrodes (Frederick Haer)) was placed between 100 and 300 µm down the apical dendrite. Whole-cell recordings were obtained with Axopatch-1D amplifiers (Molecular Devices) using 3 to 5 MΩ pipettes with an internal solution containing 115 mM cesium methanesulfonate, 20 mM CsCl, 10 mM HEPES, 2.5 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM sodium phosphocreatine (Sigma), and 0.6 mM EGTA (Amresco), at pH 7.25. External perfusion consisted of artificial cerebrospinal fluid containing 119 mM NaCl, 2.5 mM KCl, 4 mM CaCl2, 4 mM MgCl2, 26 mM NaHCO3, 1 mM NaH2PO4, 11 mM glucose, 0.004 mM 2-chloroadenosine (Sigma), and 0.1 mM picrotoxin (Sigma) (pH 7.4), and gassed with 5% CO2/95% O2 at 27°C. The AMPAR-mediated excitatory post-synaptic current (EPSC) was measured as peak inward current at a holding potential of -60 mV. Evoked responses were analyzed by averaging 30–100 sweeps using Igor Pro software, blind to experimental conditions.
Mass Spectrometry – Phosphoproteomics
Murine brain tissue lysis. 3-month-old WT and homozygous M489V mice, and 4.5 and 6-month-old WT and homozygous M489V mice with or without the APP transgene were sacrificed and hemibrains were obtained and immediately snap-frozen. Frozen tissue was thawed on ice and homogenized via bead beating in a buffer containing 3% sodium dodecyl sulfate (SDS), 75 mM NaCl, 1 mM NaF, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1X complete EDTA-free protease inhibitor cocktail from Roche (Basel, Switzerland) and 50 mM HEPES, pH 8.5 83. Tissues were sonicated with a probe sonicator to ensure full lysis, insoluble cellular debris was removed via centrifugation (16,000 x g, 10 min, 4°C), and resultant supernatants were used for downstream processing.
Protein Digestion. Proteins were denatured by addition of urea (4 M final concentration) then reduced with dithiothreitol (DTT) and alkylated with iodoacetamide (IAA) 84. Proteins were then precipitated with methanol/chloroform as previously described 84 and dried on a heat block at 56°C. Dried protein pellets were resolubilized in 1 M urea in 50 mM HEPES, pH 8.5 for digestion in a two-step process (LysC for 16 h at room temperature (RT) followed by Trypsin for 6 h at 37°C). Digests were acidified by addition of trifluoroacetic acid (TFA), and digested peptides were desalted with C18 Sep-Paks 85. Desalted peptides were dried, re-suspended in 50% acetonitrile/5% formic acid and quantified using the Pierce™ Quantitative Colorimetric Peptide Assay. Peptides from matched samples were aliquoted for both standard proteomics (50 µg) and phosphoproteomics (4 mg) and lyophilized.
Phosphopeptide Enrichment. Phosphopeptides were enriched by TiO2 beads as previously described 86, 87. Peptides were resuspended in binding buffer (2 M lactic acid, 50% acetonitrile) and incubated with TiO2 beads that were pre-washed 1X with binding buffer, 1X with elution buffer (50 mM KH2PO4, pH 10) and 2X with binding buffer. Enrichment was conducted at a ratio of 1:4 (peptides:beads) for 1 h at RT. Peptide:bead complexes were washed 3X with binding buffer and 3X with wash buffer (50% acetonitrile/0.1% trifluoracetic acid) to remove non-specific binding. Phosphopeptides were then eluted from the beads using 2X 5 min incubations in elution buffer while vortexing at RT. Enriched phosphopeptides were desalted and lyophilized prior to TMT labeling.
Tandem Mass Tag (TMT) Labeling. For both standard and phosphoproteomics, peptides were labeled for quantitation using TMT 10-plex reagents 88, 89. TMT reagents were resuspended in dry acetonitrile to a concentration of 20 µg/µl. Lyophilized peptides were resuspended in 30% acetonitrile in 200 mM HEPES, pH 8.5 and mixed with 8 µl of the appropriate TMT reagent. The TMT126 reagent in each 10plex was reserved for a bridge channel, which consists of an equal amount of each sample pooled together, and the remaining TMT reagents were used to label individual sample digests. The bridge channel served to control for experimental variation between individual 10plex experiments. TMT labeling was conducted for one hour at RT, quenched with 9 µl of 5% hydroxylamine for 15 min at RT, then acidified with 50 µl of 1% TFA and pooled. The multiplexed samples were desalted as above to remove unreacted TMT reagents, then lyophilized.
Basic Reverse-phase Liquid Chromatography Fractionation (bRPLC). Multiplexed samples were fractionated by bRPLC with fraction combining as previously described 85. Samples were resuspended in 5% formic acid in 5% acetonitrile and separated on a 4.6 mm x 250 mm C18 column using an Ultimate 3000 HPLC into 96 fractions. The resultant fractions were then combined into 24 fractions and lyophilized prior to LC-MS3 analysis.
LC-MS3 Analysis. Samples were resuspended in 5% acetonitrile/5% formic acid and separated on an Easy-nanoLC 1000 in-line with an Orbitrap Fusion Tribrid mass spectrometer. Samples were loaded onto a glass capillary column (length: 30 cm, I.D. 100 µm, O.D. 350 µm) pulled and packed in-house with 0.5 cm of 5 µm C4 resin followed by 0.5 cm of 3 µm C18 resin, with the remainder of the column packed with 1.8 µm of C18 resin. Once the sample was loaded, peptides were eluted using a gradient ranging from 11–30% acetonitrile in 0.125% formic acid over 180 min at a flow rate of 300 nl/minute. The column was heated to 60°C and electrospray ionization was achieved by applying of 2,000 V of electricity through a T-junction at the inlet of the column.
All data were centroided and collected in data-dependent mode. An MS1 survey scan was performed over a mass to charge (m/z) range of 500–1200 at a resolution of 60,000 in the Orbitrap. Automatic gain control (AGC) was set to 200,000 with a maximum ion inject time of 100 ms and a lower threshold for ion intensity of 50,000. Ions selected for MS2 analysis were isolated with a width of 0.5 m/z in the quadrupole and fragmented using collision induced dissociation (CID) with a normalized collision energy of 30%. Ion fragments were detected in the linear ion trap with the rapid scan rate setting with an AGC of 10,000 and a maximum inject time of 35 ms. MS3 analysis was conducted using the synchronous precursor selection (SPS) to simultaneously isolate 10 ions (regular proteomics) or 3 ions (phosphoproteomics) to maximize TMT sensitivity 90. TMT reporter ions were fragmented off the peptides with higher energy collision induced dissociation (normalized energy of 50%) and MS3 fragment ions were analyzed in the Orbitrap at a resolution of 60,000. The AGC was set to 50,000 with a maximum ion injection time of 150 ms. MS2 ions 40 m/z below and 15 m/z above the MS1 precursor ion were excluded from MS3 selection.
Data Processing and Analysis. Resultant mass spectrometry data files were analyzed using Proteome Discoverer 2.1. MS2 spectra were queried against the Uniprot human protein database (downloaded: 05/2017) using the Sequest search algorithm 91. A decoy search was also conducted with sequences in reverse to estimate false-discovery rate (FDR) 92, 93, 94. A mass tolerance of 50 ppm was used for MS1 spectra and a tolerance of 0.6 Da was used for MS2 spectra. TMT 10-plex reagents on lysine and peptide n-termini and carbamidomethylation of cysteines were included as static modifications. Oxidation of methionine and, for the phosphoproteomics experiments, phosphorylation of serine, threonine and tyrosine residues, were also included in the search parameters as variable modifications. The target-decoy strategy was used to filter results to a 1% FDR at the peptide and protein levels 92, 93, 94. Reporter ion intensities extracted from MS3 spectra were used for quantitative analysis. For the regular proteomics, protein-level abundance values were calculated by summing signal to noise values for all peptides per protein meeting the specified filters. Data were normalized as previously described 95. Phosphopeptide abundance was normalized similarly, except quantitation was summed to the unique phosphopeptide level then normalized to the total protein level. Phosphosite localization was performed using the PhosphoRS node within Proteome Discoverer. The PTMphinder R package was used to localize phosphorylated residues in the context of full-length proteins 96. Prior to direct statistical comparisons, K-means clustering was used to group all quantified phosphopeptides with similar expression profiles. Gene ontology analysis was used to identify enriched pathways in clustered phosphopeptides through the DAVID server 97, 98. K-means clustering was used to group all quantified phosphopeptides with similar expression profiles, prior to direct statistical comparisons. STRING-db was utilized to generate functional protein association networks of proteins of interest 99. Connections were limited to high confidence (0.7) with a maximum of 20 connections for the second shell. First shell of interactions was restricted to the query only.
Spine density analysis
Brain Slice Preparation. Mice were deeply anesthetized with ketamine, and perfused with 0.9% w/v Sodium Chloride, then perfused with 4% paraformaldehyde (PFA) in phosphate buffer (PB). The brain tissues were removed and post-fixed in 4% PFA in PB for 30 minutes. Using a vibratome, 100 µm coronal sections were sliced and stored in 1x dPBS. Alexa Fluor® 594 Hydrazide (Thermo Fisher Scientific A10438) was injected into CA1 pyramidal neurons to follow neuronal projections. Injected slices were post-fixed for 15 minutes on 4% PFA in PB prior to mounting with Aqua-Poly/Mount (Polysciences Inc. 18606-20).
Confocal Microscopy and Dendritic Spine Analysis. Immunofluorescent images of hippocampal neurons were acquired with a Leica DMI6000 inverted microscope equipped with a Yokogawa Nipkow Spinning disk confocal head, an Orca ER High Resolution black and white cooled CCD camera (6.45 µm/pixel at 1×) (Hamamatsu), Plan Apochromat 63×/1.4 numerical aperture objective, and Andor 100 mW 561 nm laser. Confocal z-stacks were acquired in all experiments and all imaging was acquired in the dynamic range of 8-bit acquisition (0–255 pixel intensity units, respectively) with Volocity (PerkinElmer) imaging software. Imaged dendrites from one secondary dendrite per cell (after 1 branch) at a distance of 40–80 µm from the soma were straightened using ImageJ. We estimated spine density as the number of manually counted spines (length 2 experimental conditions). Statistical significance was determined using unpaired Student’s t-tests.
Western Blot analysis
Human brains (frontal cortex) were provided by the ADRC Neuropathology Core at UCSD. Mice were euthanized and brain samples were obtained and snap-frozen immediately after collection. All procedures involving animals were approved by The Scripps Research Institute’s Institutional Animal Care and Usage Committee (IACUC) and the UCSD IACUC, and met the guidelines of the National Institute of Health detailed in the Guide for the Care and Use of Laboratory Animals 76.
Frozen brain tissues were lysed and homogenized in a Dounce tissue grinder with RIPA buffer (50 mM Tris, pH 7.4, 1% Triton X-100, 1% NaDOC, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 10 mM NaF, 1 mM DTT, 1 mM Na3VO4, 1 mM PMSF, 50 µg/mL leupeptin, 1 µM microcystin, and 2 mM benzamidine). Homogenates were sonicated and protein was quantified using a BCA protein assay kit (Thermo Fisher Scientific). Fifty micrograms of protein were separated by standard SDS/PAGE and transferred to PVDF membranes (BioRad). Membranes were blocked with 5% BSA or 5% milk for one hour at room temperature and analyzed by immunoblotting with specific antibodies. Detection of immunoreactive bands was performed via chemiluminescence on a FluorChemQ imaging system (Alpha Innotech).
Antibodies: anti-PKCα antibody (610108) was from BD Transduction Laboratories. β-Actin antibody was purchased from Sigma-Aldrich (A2228), total SAP97 was from Enzo Life Sciences (clone RPI 197.4, ADI-VAM-PS005), phospho-MARCKS (sc-12971-R) and total MARCKS (sc-6454) were obtained from Santa Cruz Biotechnology. GAPDH (2118), vinculin (4650), phospho-(Ser) PKC substrate (2261S), phospho-ERK1/2 (9101) and total ERK1/2 (9102) antibodies were purchased from Cell Signaling Technology. The pSAP97 (T656) antibody was previously described 32.
Amyloid-β ELISA analysis
Brain tissues from APP transgenic mice harboring WT or mutant M489V PKCα were snap-frozen in liquid nitrogen and stored at -80°C until use. Frozen brains were then homogenized in a Dounce tissue grinder and proteins sequentially extracted as previously described 100. Aβ levels in 2% SDS and formic acid extracts were quantified using the Human/Rat Aβ-40 and Aβ-42 ELISA kits from Wako (#294-62501 and #292-64501 respectively), following the manufacturer’s recommendations.
Statistical analysis
For the statistical analyses of behavioral results, analysis of variance (ANOVA) was used followed by post hoc Student's t-tests as appropriate. For spine density statistical analysis and immunoblots statistical analysis unpaired Student's t-tests were used. To assess statistical significance of dual-patch recordings, paired t-tests were used, and to compare CT100-induced depression in WT versus M489V mice unpaired Student's t-tests were used