- Open Access
Activation of Pro-survival CaMK4β/CREB and Pro-death MST1 signaling at early and late times during a mouse model of prion disease
© Shott et al.; licensee BioMed Central Ltd. 2014
- Received: 5 June 2014
- Accepted: 29 August 2014
- Published: 2 September 2014
The signaling pathways most critical to prion disease pathogenesis are as yet incompletely characterized. We have developed a kinomics approach to identify signaling pathways that are dysregulated during prion pathogenesis. The approach is sensitive and specific enough to detect signaling pathways dysregulated in a simple in vitro model of prion pathogenesis. Here, we used this approach to identify signaling pathways dysregulated during prion pathogenesis in vivo.
Mice intraperitoneally infected with scrapie (strain RML) were euthanized at 70, 90, 110, 130 days post-infection (dpi) or at terminal stages of disease (155–190 dpi). The levels of 139 protein kinases in brainstem-cerebellum homogenates were analyzed by multiplex Western blots, followed by hierarchical clustering and analyses of activation states.
Hierarchical and functional clustering identified CaMK4β and MST1 signaling pathways as potentially dysregulated. Targeted analyses revealed that CaMK4β and its downstream substrate CREB, which promotes neuronal survival, were activated at 70 and 90 dpi in cortical, subcortical and brainstem-cerebellum homogenates from scrapie-infected mice. The activation levels of CaMK4β/CREB signaling returned to those in mock-infected mice at 110 dpi, whereas MST1, which promotes neuronal death, became activated at 130 dpi.
Pro-survival CaMK4β/CREB signaling is activated in mouse scrapie at earlier times and later inhibited, whereas pro-death MST1 signaling is activated at these later times.
- Prion disease
- Protein kinase
- Multiplex Western blots
Prion diseases are a family of invariably lethal chronic neurodegenerative diseases that affect humans (kuru; Creutzfeldt-Jakob disease, CJD; Gerstmann-Sträussler-Scheinker disease, GSS; fatal familial insomnia, FFI), and other species such as cattle (bovine spongiform encephalopathy, BSE), goat, sheep (scrapie), deer, elk and moose (chronic wasting disease, CWD)[1, 2]. Human prion diseases can be infectious (acquired), inherited (genetic), or sporadic. The latter are the most common, accounting for approximately 85% of cases. Whereas the inherited and acquired cases may be suspected, from the risk facts, before the onset of clinical symptom, the sporadic cases can only be diagnosed after the onset of clinical symptoms.
The neuropathology of prion diseases is characterized by gliosis, spongiform degeneration, and neuronal death. The conversion of the cellular prion protein (PrPC) into its pathological conformation (PrPSc) is essential for pathogenesis. Neuronal death and disease progression were prevented in scrapie-infected mice by conditional ablation of PrPC at the time when PrPSc was first detected. Inhibition of PrPC conversion to PrPSc is thus a validated target for therapeutic intervention. Many compounds have been identified to inhibit PrP conversion or the accumulation of PrPSc in vitro[6, 7]. Only one, however, the diphenyl pyrazole derivative anle138b [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole], prolonged survival of scrapie-infected mice when treatment was started after the onset of clinical signs of disease. Unfortunately, none affected survival of patients with CJD, GSS, or FFI[9–18]. Another compound that prolonged survival of scrapie-infected mice after the onset of clinical disease is the calcineurin/ protein phosphatase 3 inhibitor FK506. FK506, however, did not affect the levels of PrPC or the accumulation of PrPSc, indicating that FK506 acts downstream from the accumulation of PrPSc. As FK506 is a known (calcineurin) signaling inhibitor, these results suggest that dysregulated signaling downstream of PrP conversion is an alternative therapeutic target against prion diseases.
Protein kinases (and phosphatases) modulate by reversible phosphorylation the function, localization, or activities of approximately one-third of cellular proteins. Protein kinases are therefore critical regulators of signal transduction. Their dysregulation is implicated in the pathogenesis of many chronic diseases, including neurodegenerative diseases such as Alzheimer’s and Parkinson’s[21–23]. Consequently, protein kinases are major therapeutic targets. It is estimated that up to 30% of the research and development budget of the pharmaceutical industry is invested in protein kinase inhibitors[24, 25]. For example, protein kinase inhibitors are the largest group of new cancer therapeutics. Thirty-one protein kinase inhibitors are in clinical use, over 500 are involved in approximately 2,700 clinical trials, and thousands more are in various stages of pre-clinical development ([26–28] and summary of). Protein kinase inhibitors therefore constitute a rapidly growing group of clinical drugs that have the potential to considerably impact treatment of chronic diseases.
Considering the critical roles that protein kinases play in the pathogenesis of other chronic neurodegenerative diseases, it is not surprising that they also participate in the pathogenesis of prion diseases. For example, the activation of vascular endothelial growth factor receptor (VEGFR) inhibited death of cultured neurons treated with the neurotoxic prion peptide PrP106-126. Death of PrP106-126-treated cultured neurons was also inhibited by Abelson leukemia oncogene cellular homolog (c-Abl) knockdown and treatment with the glycogen synthase kinase 3 (GSK3) inhibitor lithium. Scrapie-infected mice treated with the protein kinase R-like endoplasmic reticulum kinase (PERK) inhibitor GSK2606414 survived longer than vehicle-treated mice. The phosphoinositide-dependent kinase 1 (PDK1) inhibitor BX912 also prolonged survival of scrapie-infected mice. Protein kinase inhibitors may have good potential in prion disease therapeutics. Unfortunately, the signaling pathways most critical to prion disease pathogenesis have yet to be fully identified.
We have developed a kinomics approach to identify signaling pathways dysregulated during prion disease pathogenesis (Shott et al., companion paper). We initially tested the approach in a simplified in vitro model of prion disease pathogenesis (Shott et al., companion paper). Here, we applied the approach to mice infected with mouse-adapted scrapie. We identified two signaling pathways dysregulated during scrapie pathogenesis. The calcium/calmodulin-dependent protein kinase, beta isoform (CaMK4β)/cAMP response element-binding protein (CREB) signaling pathway, which promotes neuronal survival, was activated at earlier times but its activation state returned to that in mock-infected mice later on. Mammalian STE20-like protein kinase 1 (MST1) signaling, which promotes neuronal death, was, in contrast, activated at these later times. The dysregulation of CaMK4β/CREB and MST1 signaling pathways may therefore be critical to the neurodegeneration in scrapie infected mice.
PrPres is first detected in scrapie-infected mice at 130 dpi
Mock-infected mice or mice infected intraperitoneally with scrapie (mouse-adapted strain RML) were euthanized at 70, 90, 110, 130 days post-infection (dpi), or at terminal stages of disease (155–190 dpi). Brains were dissected into (i) cortical (cerebrum), (ii) subcortical (including thalamus, hypothalamus and hippocampus) and (iii) brainstem-cerebellum as described.
Primary kinomic screens identified two signaling pathways of potential interest, which are involved in neuronal death and survival
Primary screens of brainstem-cerebellum homogenates from scrapie-infected mice therefore identified two signaling pathways which may be dysregulated during pathogenesis. The NMDAR-regulated CaMK4β signaling pathway promotes neuronal survival and the expression levels of the protein kinases involved changed (increased) the most at earlier times. Conversely, the MST1 signaling pathway promotes neuronal death, and the expression levels of involved protein kinases were most affected (decreased) at later times.
CaMK4β/CREB signaling is activated at early stages of scrapie in mice
We next performed targeted tertiary (phosphorylation state-specific) analyses to characterize the activation state of (NMDAR-regulated) CaMK4β/CREB signaling in scrapie-infected mice. The activation of Lyn and RSK1 involves autophosphorylation on Y396 or S380, respectively[47–50]. Activated RSK1 (P-S380) inhibits nNOS by phosphorylation on S847. Activated p38 (P-T180/Y182) phosphorylates PSD-95 on S290. Activated CaMK4β (P-T196; corresponding to human T200) phosphorylates CREB on S133[51, 52]. Phosphorylated CREB promotes transcription of genes that encode proteins involved in neuronal survival[53, 54]. There is no antibody specific for T180/Y182 phosphorylation on only p38γ. We therefore used an antibody that detects T180/Y182 phosphorylation on all p38 isoforms (p38α, p38β, p38γ, p38δ). In summary, we analyzed the levels of activating phosphorylation of p38 (P-T180/Y182), Lyn (P-Y396), RSK1 (P-S380), CaMK4β (P-T196), and CREB (P-S133), and the levels of inhibitory phosphorylation of nNOS (P-S847). No antibody specific for p38-phosphorylated PSD-95 (P-S290) was available.
In summary, CaMK4β/CREB signaling was activated in scrapie-infected mice at early times. The expression levels and the levels of phosphorylated CREB (P-S133) and activated CaMK4β (P-T196) were higher in scrapie- than in mock-infected mice at 70 and 90 dpi.
MST1 is activated at late stages of mouse scrapie
To test this model, we performed targeted (phosphorylation state-specific) tertiary Western blots to characterize the activation state of MST1/FOXO3 signaling. MST1 is activated by autophosphorylation on T183 (P-T183). Activated MST1 phosphorylates FOXO3 in the cytosol on S208 (corresponding to S207 in human). Phosphorylated FOXO3 (P-S208) then translocates to the nucleus where it activates transcription of genes that encode proteins involved in neuronal death. Activated MST1 is further activated by caspase-3 cleavage. Cleaved MST1 further promotes cell death[57–60]. We analyzed the levels of activating phosphorylation of MST1 (P-T183) and FOXO3 (P-S207) and activating cleavage of MST1. MST1 activation is promoted by JNK-mediated phosphorylation on S82 and active JNK2 is phosphorylated on T183/Y185. As no antibody available was specific for P-T183/Y185 of only JNK2 or P-S82 of MST1, we used antibodies that detect T183/Y185 phosphorylation on all JNK isoforms (JNK1, JNK2, JNK3; p46 [JNK1α1, JNK1β1, JNK2α1, JNK2β1, JNK3α1] and p54 [JNK1α2, JNK1β2, JNK2α2, JNK2β2, JNK3β2]) or autophosphorylated MST1 (P-T183).
In summary, MST1 signaling was activated in scrapie-infected mice at late times. Levels of cleaved MST1 were higher at 130 dpi, when total levels of MST1 and FOXO3 were lower. At terminal stages of disease, levels of activated MST1 (P-T183) were higher in scrapie- than in mock-infected mice.
The activation of CaMK4β/CREB signaling at pre-clinical stages of prion disease had not been described. CREB is critical to neuronal survival. CREB/cAMP response element modulator (CREM) double knockout mice, or mice in which CREB is inhibited by overexpression of a dominant negative mutant or inhibitory peptides, suffer extensive neuronal loss[62–64]. Active CREB promotes neuronal survival by regulating transcription of “activity-regulated inhibitor of death” (AID) genes, including gadd45 β, gadd45 γ, btg2, npas4, nr4a1, inhba, atf3, ifi202b, serpinb2[53, 54]. CREB also regulates the transcription of miR132-3p[65, 66], which modulates synapse morphology. High-throughput analyses have identified changes in many mRNAs and miRNAs, including miR132-3p, in scrapie-infected mice prior to the accumulation of PrPres or the onset of clinical disease[68–73]. Elevated levels of miR132-3p and AID genes (gadd45 β, gadd45 γ, btg2, npas4, nr4a1) were observed at 70–110 dpi after infection with the same scrapie strain and by the same route of inoculation as in the current experiments. Activated CaMK4β/CREB signaling may well promote neuronal survival early in prion infection by upregulating the expression of miR132-3p and AID genes.
CaMK4β/CREB signaling returned to the activation levels of mock-infected mice at 110 dpi. Calcineurin regulates the activity of CaMK4 and CREB, and elevated calcineurin activity was observed in scrapie-infected mice at clinical stages of disease. Moreover, the calcineurin inhibitor FK506 prolonged survival of scrapie-infected mice and inhibited neuronal death in cultured neurons treated with PrP106-126[19, 76, 77]. CaMK4β/CREB signaling is therefore most likely neuroprotective in scrapie-infected mice. Decreased levels of activated CaMK4β (P-T196) and phosphorylated CREB (P-S133) were associated with decreased expression levels, suggesting that degradation may be involved in downregulation of this pathway. CaMK4 and CREB are degraded by calpain, a calcium-dependent protease[78, 79]. Active calpain degraded CaMK4 and CREB in cultured neurons treated with hydrogen peroxide after CREB (P-S133) dephosphorylation. Increased levels of calpain have been observed in in vitro and in vivo models of prion disease[81, 82], and calpain inhibition limited neuronal death in prion-infected cultured organotypic cerebellar slices or cultured neurons treated with PrP106-126[81, 83, 84]. CaMK4β/CREB signaling activation may be inhibited in scrapie-infected mice after 90 days by dephosphorylation and degradation mediated by calpain. CaMK4 and CREB are also dephosphorylated by protein phosphatase 2A (PP2A)[85–87], the activity of which has yet to be evaluated during scrapie infection.
MST1 signaling was activated at 130 dpi, which had not been described in prion disease. MST1 has been shown to be involved in other neurodegenerative diseases. Genetically modified mice that model amyotrophic lateral sclerosis (ALS) lose fewer neurons and survive longer if they are knocked out of MST1. Active MST1 (P-T183) is cleaved by active (cleaved) caspase-3[57–60], the levels of which are elevated in scrapie-infected mice (before the accumulation of PrPres) and in cultured neurons or neuroblastoma cells exposed to PrP106-126 or PrPSc[89–92]. We observed elevated levels of cleaved MST1 at 130 dpi, suggesting that it is also cleaved by caspase-3 in vivo. Although cleaved MST1 retains the T183 phosphorylation site, T183 phosphorylation is not required for cleavage[56, 93], and we did not detect cleaved MST1 with the phosphorylation-specific antibody. Caspase-cleaved MST1 translocates to the nucleus and induces chromatin condensation by inducing phosphorylation of histone H2B on S14. Chromatin condensation has been observed previously in scrapie-infected GT1 cells and mice[95–98]. However, caspase-3 activation is not required for MST1 activation or neuronal death in prion disease[92, 99, 100].
MST1 activates FOXO3, which is otherwise maintained in the cytosol in an inactive state by interaction with 14-3-3 proteins. Cleaved MST1 predominately localizes to the nucleus and is therefore unable to phosphorylate FOXO3[102, 103]. The levels of phosphorylated FOXO3 (P-S208) might have been higher (but were not statistically different) after the levels of cleaved MST1 had returned to those in mock-infected mice. Active FOXO3 upregulates transcription of genes encoding pro-apoptotic proteins including Bim (bcl-2 interacting mediator of cell death; bcl2l11), Puma (Bcl-2-binding component 3; bbc3) and Noxa (phorbol-12-myristate-13-acetate-induced protein 1; pmaip1)[104–106]. The levels of Bim, Puma, bcl2l11, bbc3 and pmaip1 are elevated in scrapie-infected mice at late stages of disease progression[72, 107, 108], suggesting that neuronal death mediated by MST1 signaling could involve FOXO3.The CaMK4β/CREB and MST1 signaling pathways were identified in our screens because the expression levels of the involved protein kinases changed coordinately during prion disease progression (Figures 5 and7). The CaMK4β/CREB signaling pathway promotes neuronal survival. CaMK4β and CREB were expressed to higher levels and activated at earlier stages of disease. Neurons may activate this pathway to protect themselves from prion-mediated death. Later (at 110 dpi), the neuroprotective CaMK4β/CREB signaling was lost and MST1 signaling was activated. Activation of MST1 signaling was associated with lower levels of full length MST1 and FOXO3. The decrease in full length MST1 was two-fold greater than the increase in the cleaved form (detected in the same blots with the same antibody). Cleaved MST1 may be less stable than full length MST, or full length MST1 may be processed by caspase-dependent and independent pathways.
The opposing changes in expression and activation state suggest an attempt to prevent the neuronal death that would result from activated MST1 signaling. The levels of upstream kinases DLK, MKK7, and JNK2 were also lower in scrapie-infected mice at 130 dpi, albeit only in the brainstem-cerebellum (Figure 7). The cerebellum (in the brainstem-cerebellum) may contain ~50% of all neurons in an adult mouse brain and loses the most neurons in RML-infected mice, as indicated by nuclear DNA fragmentation. The changes in the total levels of proteins involved in the MST1 signaling pathway in each brain region may reflect the differences in the number of affected neurons in each brain region.
The different numbers of affected neurons in each region may also be reflected in the higher levels of total and phosphorylated CaMK4β in the brainstem-cerebellum than in the subcortical or cortical regions. Regardless of absolute numbers, higher levels of total and phosphorylated CREB were expressed in all brain regions. Although CaMK4 (P-T196) could also be responsible for CREB (P-S133) phosphorylation, there were no differences in the levels of activated CaMK4 (P-T196) in scrapie- versus mock-infected mice (data not shown). CREB is also phosphorylated on S133 by other protein kinases, including RSK, cAMP-dependent protein kinase (PKA), and mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) (for a review, see). There was no correlation between the levels of active RSK1 (P-S380) and phosphorylated CREB (P-S133). We did not evaluate other protein kinases upstream of CREB because they were not identified in the primary screening. However, CREB may well be activated independently of CaMK4β in the cortical region or subcortical region of scrapie-infected mice.
The data presented cannot discriminate whether the observed changes in signaling pathways are a cause or a consequence of the pathology. Neither can discriminate whether they result from a loss of PrPC function, a gain of PrPres function, or an altered signaling function resulting from the progressive PrPC misfolding into PrPres. Moreover, the dysregulation could have been directly triggered by PrPres or PrPC acting on the neurons, or mediated by the glial alterations that occur during disease progression. The data presented does not resolve either whether the observed signaling changes are specific for prion diseases, or common to other neurodegenerative diseases. Future work will have to address these possibilities.
We used a kinomics approach to identify two dysregulated signaling pathways, involved in neuronal survival/death, in scrapie-infected mice. Their dysregulation at different times during disease is temporally consistent with the neuronal loss during prion disease. Our findings identified novel signaling involved in prion-mediated neuronal death in vivo and identify potential targets for intervention. It is possible to test now the roles of these signaling pathways in prion disease, as well-characterized inhibitors of several of these proteins are available.
All of the procedures involving live animals were approved by the Canadian Science Centre for Human and Animal Health – Animal Care Committee (CSCHAH-ACC) or the University of British Columbia Animal Care Committee according to the guidelines set by the Canadian Council on Animal Care. The approval identifications for this study were animal use document (AUD)#H-08-009 and AUD#H-11-020.
Animals and sample collection
CD1 mice (Charles River Laboratories) between 4–6 weeks of age were infected intraperitoneally with the Rocky Mountain Laboratory (RML) mouse-adapted strain of scrapie. The inoculums consisted of 200 μl of 1% brain homogenate in PBS from either clinically ill or normal control mice. Animals were sacrificed at 70, 90, 110, 130 days post infection (dpi) and terminal disease (155–190 dpi). Clinical signs depicting terminal disease consisted of kyphosis, dull ruffled coat, weight loss of 20% or more and ataxia. Brain samples were collected and macrodissected into three sections, (i) cortical, (ii) subcortical (including thalamus, hypothalamus and hippocampus) and (iii) brainstem-cerebellum. Each section was flash frozen using a dry ice/methanol mixture and stored at −80°C until processing. A total of 3 scrapie- and 3 mock-infected samples were collected per time point.
All procedures were performed at 4°C or on ice. Weighed frozen brainstem-cerebellum, subcortical, and cortical regions from mock- or scrapie-infected mice were homogenized in 3 mL of freshly prepared lysis buffer (20 mM MOPS [pH 7.0], 2 mM EGTA, 5 mM EDTA, 1% Nonidet P-40, 0.01% phosphatase inhibitor cocktail [Pierce, Rockford, Illinois, USA], 0.02% protease inhibitor cocktail [Sigma-Aldrich, St. Louis, Missouri, USA], 10 mM DTT, pH 7.2) per 250 mg of brain, using a tissue homogenizer with disposable tips (TH and hard tissue OMNI tip, respectively; OMNI International, Kennesaw, Georgia, USA). Brain homogenates were passed twice through a 21 gauge needle, sonicated five times for 20 s intervals at 88 W output (431C1 cup horn probe, S-4000 sonicator; Qsonica, Newtown, Connecticut, USA), and pre-cleared by centrifugation for 30 min at 14,000 × g (JLA 16.250 BC rotor, Avanti J-E centrifuge; Beckman/Coulter, Brea, California, USA). Approximately 200 μL aliquots of supernatant were aliquoted into 1.5 mL tubes, snap frozen in liquid nitrogen and stored at −80°C.
Protein concentration was determined by Bradford’s assay (Bio-Rad Laboratories, Hercules, California, USA). Protein concentration and equal sample loading was re-tested in preliminary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Brain homogenates were mixed with an equal volume of 2X SDS loading buffer (125 mM Tris-Cl [pH 6.8], 20% glycerol, 4% SDS, 0.005% bromophenol blue, 260 mM DTT) and denatured by incubation at 100°C for 10 min. Afterward, 15-well 8% SDS-PAGE gels (Mini-PROTEAN; Bio-Rad Laboratories) were loaded with 40 μg of denatured protein per linear cm of well (running buffer; 190 mM glycine, 24.8 mM Tris, 0.1% SDS, pH 8.3). Proteins were run through the stacking gel at 50 V, and then resolved for 90 min at 100 V, always at room temperature. Proteins were stained with Coomassie blue G-250 (Bio-Safe Coomassie; Bio-Rad Laboratories) according to the manufacturer’s instructions. Signal from Coomassie-stained protein was detected using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, Nebraska, USA) in the 700 nm channel and quantitated using Odyssey 3.0 software (LI-COR Biosciences). Protein amounts were calculated relative to a pre-quantitated standard brain homogenate.
Sodium phosphotungstic acid precipitation
Sodium phosphotungstic acid (NaPTA) precipitation was adapted from[112, 113]. One milligram of mouse brain homogenate was mixed with an equal volume of 4% sodium lauroylsarcosine (sarkosyl) in PBS. Samples were incubated for 10 min at 37°C with constant agitation (1000 rpm, Thermomixer; Eppendorf, Hamburg, Germany). A 37°C NaPTA solution (PBS, 4% NaPTA, 170 mM MgCl2, pH 7.4) was then added to a final concentration of 0.3% NaPTA. Samples were incubated for 60 min at 37°C with constant agitation (1000 rpm, Thermomixer; Eppendorf), then centrifuged at 37°C for 30 min at 16,000 × g (FA-45-18-11 rotor, 5418 microfuge; Eppendorf). Pellets were resuspended in 5 μL of 0.1% sarkosyl in PBS and digested with 20 μg of proteinase K (PK; in 0.01 M CaCl2) (Roche, Indianapolis, Indiana, USA) for 30 min at 37°C with a brief vortex after 15 min. Digestion was stopped and PK-resistant protein was denatured by quickly adding an approximately equal volume 5X SDS loading buffer (300 mM Tris-Cl [pH 6.8], 50% glycerol, 25% β-mercaptoethanol, 10% SDS, 1% bromophenol blue), and immediately incubating at 100°C for 10 min. Samples were resolved by SDS-PAGE and analyzed by Western blot.
All procedures were performed at room temperature and all washes were performed using gentle rocking, unless otherwise indicated.
PrPres was analyzed in 1.0 mg of NaPTA-enriched, PK-treated mouse brain homogenate using 15-well 12% SDS-PAGE gels (NuPAGE Novex Bis-Tris; Life Technologies Inc., Carlsbad, California, USA). The running buffer (50 mM MOPS, 50 mM Tris, 1 mM EDTA, 0.1% SDS, pH 7.7) within the upper (cathode) chamber contained 5 mM sodium bisulfite. Proteins were run through the stacking gel at 60 V, and then resolved for 2.5 h at 120 V. Afterward, polyvinylidene fluoride (PVDF) membranes (Immuno-Blot, 0.2 μm; Bio-Rad Laboratories) were soaked in methanol for 2 min, then equilibrated in transfer buffer (190 mM glycine, 24.5 mM Tris, 10% methanol) for 20 min. Filter paper (2 sheets/membrane) were equilibrated in transfer buffer for 5 min. Proteins were transferred at 4°C for 2 h at 30 V. After transfer, membranes were dried, soaked in methanol for 2 min and washed twice for 10 min each in Tris-buffered saline (TBS; 140 mM NaCl, 3 mM KCl, 25 mM Tris, pH 7.6). Membranes were blocked for 1 h in TBST (TBS/0.1% Tween-20) with 5% milk, then probed with PrP primary antibody (clone SAF83; a kind gift from Dr. Deborah McKenzie, University of Alberta) diluted to 1:10,000 in TBST with 5% milk for 18 h at 4°C. Afterward, membranes were washed with TBST once for 5 min and thrice for 10 min each. Membranes were incubated with goat anti-mouse horseradish peroxidase (HRP)-labeled secondary antibody (Bio-Rad Laboratories) diluted to 1:40,000 in TBST with 5% milk for 1 h. Membranes were washed in TBST once for 5 min and thrice for 15 min each, incubated for 5 min with enhanced chemiluminescent substrate (SuperSignal West Femto; Pierce) and then exposed to film (Super RX; Fujifilm, Tokyo, Japan). Exposed film was developed and scanned (CanoScan LiDE 200; Canon, Tokyo, Japan). Signal was quantitated using ImageJ (Version 1.47c; National Institutes of Health, Bethesda, Maryland, USA).
To analyze GFAP and total PrP, brain homogenates were mixed with a one-fourth volume of 5X SDS loading buffer. Then, 100 μg of denatured protein was loaded per linear cm onto 15-well 12% SDS-PAGE gels (Mini-PROTEAN; Bio-Rad Laboratories). Proteins were run through the stacking gel at 50 V, and then resolved for 100 min at 100 V. Afterward, gels were equilibrated in transfer buffer (384 mM glycine, 49.6 mM Tris, 20% methanol, 0.01% SDS) for 30 min. PVDF membranes and filter paper (2 sheets/membrane) were also equilibrated in transfer buffer for 20 and 5 min, respectively. Proteins were transferred for 23 h at 4°C; 1 h at 54 mA, 4 h at 189 mA, 8 h at 270 mA and 10 h at 378 mA. Membranes were blocked with 10% blocking buffer (Sigma-Aldrich) for 1 h and probed simultaneously with primary antibodies specific for PrP (clone SAF83) and GFAP (Abcam Inc., Cambridge, Massachusetts, USA) diluted to 1:10,000 and 1:20,000 in 10% blocking buffer with 0.1% Tween-20, respectively, for 18 h at 4°C. Afterward, membranes were washed with TBST once for 5 min and thrice for 10 min each. Membranes were then incubated for 1 h with donkey anti-mouse IRDye 680- (LI-COR Biosciences) and donkey anti-rabbit IRDye 800- (LI-COR Biosciences) labeled secondary antibodies diluted to 1:20,000 in 10% blocking buffer with 0.1% Tween-20 and 0.01% SDS. Afterward, membranes were washed in TBST thrice for 10 min each, and once with TBS for 5 min. Signal from pre-stained protein standards and IRDye 680-labeled secondary antibody was detected at 700 nm using an Odyssey infrared imaging system (LI-COR Biosciences). Signal from IRDye 800-labeled secondary antibody was detected at 800 nm. Signal was quantitated using Odyssey 3.0 software (LI-COR Biosciences). Membranes were then stained with Coomassie blue R-250 (Bio-Rad Laboratories) for 10 min before destaining with 40% methanol in 10% glacial acetic acid thrice for 10 min each, or until excess stain was removed. Signal from Coomassie-stained protein was detected at 700 nm using the Odyssey and quantitated using Odyssey 3.0 software.
For multiplex Western blots, brain homogenates were mixed with an equal volume of 2X, or one-fifth volume of 6X (375 mM Tris-Cl [pH 6.8], 60% glycerol, 12% SDS, 0.015% bromophenol blue, 780 mM DTT), SDS-PAGE loading buffer, and then 200 μg of denatured protein was loaded per linear cm of single-well 8% SDS-PAGE gels (Mini-PROTEAN; Bio-Rad Laboratories). Proteins were resolved as described for protein quantitation and transferred as described for Western blots of GFAP and total PrP. Dried membranes were probed immediately or stored at −30°C. All multiplex Western blots were performed in three sets, each composed of one membrane from a mock- and one from a scrapie-infected mouse euthanized at 70, 90, 110, 130 dpi, or at terminal stage of disease. Membranes were blocked for 1 h with 10% blocking buffer (Sigma-Aldrich) for evaluation of total protein levels, or in 3% BSA (Rockland, Gilbertsville, Pennsylvania, USA) for evaluation of phosphorylation levels. Membranes were rinsed briefly with TBS and positioned within a 24-lane multi-screen apparatus (MPX; LI-COR Biosciences). Combinations of primary antibodies were diluted in 10% blocking buffer or 3% BSA, as appropriate, with 0.1% Tween-20. One hundred sixty microliters of each antibody dilution was loaded in each lane of the multi-screen apparatus. After incubation for 18 h at 4°C, membranes were briefly washed once with TBST within the multi-screen apparatus then removed from the apparatus and further washed in TBST, once for 5 min and four times for 15 min each. Membranes were incubated with secondary antibody diluted to 1:20,000 in 10% blocking buffer or 3% BSA, as appropriate, with 0.1% Tween-20 and 0.01% SDS for 1 h. Mouse monoclonal primary antibodies were detected with donkey anti-mouse IRDye 680-labeled secondary antibody. Rabbit or goat primary polyclonal primary antibodies were detected with donkey anti-rabbit or donkey anti-goat IRDye 800-labeled secondary antibody (LI-COR Biosciences), respectively. Membranes were washed in TBST four times for 15 min each and once with TBS for 5 min. Signal from IRDye 680- and IRDye 800-labeled secondary antibody was detected at 700 and 800 nm, respectively, using the Odyssey system. Signal was quantitated using Odyssey 3.0 software.
Membranes from scrapie-infected mice were stripped (only once) together and in parallel with the membranes from the mock-infected mice from the same set, under conditions to minimize protein loss. Membranes were stripped with mild stripping buffer (25 mM glycine, 1% SDS, pH 2.0) once for 5 min and four times for 15 min each, then with harsh stripping buffer (50 mM Tris-Cl [pH 7.0], 2% SDS, 50 mM DTT) once for 15 min at 37°C. Afterward, stripped membranes were washed with TBST once and TBS once for 5 min each, then blocked and reprobed with a second set of primary antibodies as described.
Hierarchical cluster analysis
For each set, the densitometric data from the primary multiplex Western blots of brainstem-cerebellum homogenates of scrapie-infected mice was normalized to that from the mock-infected mice. Relative protein kinase expression levels were then log2 transformed and analyzed with Gene Cluster 3.0 using Euclidean distance and complete linkage. Java Treeview was used to present the clusters.
All data was analyzed using Prism (Version 5.0f; GraphPad Software Inc., La Jolla, California, USA). The targeted Western blots were performed in three experimental sets on different days. Each set encompassed one scrapie- and one mock-infected mouse from each time point. The ratios from each set were thus analyzed by paired t-test. The ratios were log transformed before analyses to transform the increased and decreased ratios (ratios greater or smaller than 1, respectively) to proportional values. For nonlinear regression analyses, curves of the normalized total and phosphorylation protein levels in scrapie-infected mice were compared to no changes (a line with intercept = 1, slope = 0), representing the levels in mock-infected mice, using a replicates test for lack-of-fit.
This work was supported by PrioNet Canada and Alberta Prion Research Institute grants to LMS, a Burroughs Wellcome Fund Award to LMS. LMS is a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease. RHS was supported by NSERC, 75th Anniversary Graduate Student Award (Faculty of Medicine and Dentistry, University of Alberta) and Queen Elizabeth II Doctoral Scholarship (Government of the Province of Alberta). We would like to thank Nicole Beausoleil and the staff at the NML animal house for the maintenance of mice used in this study.
- Prusiner S: Prions. Proc Natl Acad Sci U S A 1998, 95: 13363-13383. 10.1073/pnas.95.23.13363PubMedPubMed CentralGoogle Scholar
- Baeten L, Powers B, Jewell J, Spraker T, Miller M: A natural case of chronic wasting disease in a free-ranging moose (Alces alces shirasi). J Wildl Dis 2007, 43: 309-314. 10.7589/0090-3558-43.2.309PubMedGoogle Scholar
- Wadsworth J, Hill A, Beck J, Collinge J: Molecular and clinical classification of human prion disease. Br Med Bull 2003, 66: 241-254. 10.1093/bmb/66.1.241PubMedGoogle Scholar
- Paterson R, Torres-Chae C, Kuo A, Ando T, Nguyen E, Wong K, Dearmond S, Haman A, Garcia P, Johnson D, Miller B, Geschwind M: Differential diagnosis of jakob-creutzfeldt disease. Arch Neurol 2012, 69: 1578-1582. 10.1001/2013.jamaneurol.79PubMedPubMed CentralGoogle Scholar
- Mallucci G, Dickinson A, Linehan J, Klohn P, Brandner S, Collinge J: Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis. Science 2003, 302: 871-874. 10.1126/science.1090187PubMedGoogle Scholar
- Sakaguchi S: Systematic review of the therapeutics for prion diseases. Brain Nerve 2009, 61: 929-938.PubMedGoogle Scholar
- Sim V: Prion disease: chemotherapeutic strategies. Infect Disord Drug Targets 2012, 12: 144-160. 10.2174/187152612800100161PubMedGoogle Scholar
- Wagner J, Ryazanov S, Leonov A, Levin J, Shi S, Schmidt F, Prix C, Pan-Montojo F, Bertsch U, Mitteregger-Kretzschmar G, Geissen M, Eiden M, Leidel F, Hirschberger T, Deeg A, Krauth J, Zinth W, Tavan P, Pilger J, Zweckstetter M, Frank T, Bahr M, Weishaupt J, Uhr M, Urlaub H, Teichmann U, Samwer M, Botzel K, Groschup M, Kretzschmar H, et al.: Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson’s disease. Acta Neuropathol 2013, 125: 795-813. 10.1007/s00401-013-1114-9PubMedPubMed CentralGoogle Scholar
- Haik S, Marcon G, Mallet A, Tettamanti M, Welaratne A, Giaccone G, Azimi S, Pietrini V, Fabreguettes J, Imperiale D, Cesaro P, Buffa C, Aucan C, Lucca U, Peckeu L, Suardi S, Tranchant C, Zerr I, Houillier C, Redaelli V, Vespignani H, Campanella A, Sellal F, Krasnianski A, Seilhean D, Heinemann U, Sedel F, Canovi M, Gobbi M, Di Fede G, et al.: Doxycycline in Creutzfeldt-Jakob disease: a phase 2, randomised, double-blind, placebo-controlled trial. Lancet Neurol 2014, 13: 150-158. 10.1016/S1474-4422(13)70307-7PubMedGoogle Scholar
- Geschwind M, Kuo A, Wong K, Haman A, Devereux G, Raudabaugh B, Johnson D, Torres-Chae C, Finley R, Garcia P, Thai J, Cheng H, Neuhaus J, Forner S, Duncan J, Possin K, Dearmond S, Prusiner S, Miller B: Quinacrine treatment trial for sporadic Creutzfeldt-Jakob disease. Neurology 2013, 81: 2015-2023. 10.1212/WNL.0b013e3182a9f3b4PubMedPubMed CentralGoogle Scholar
- Honda H, Sasaki K, Minaki H, Masui K, Suzuki S, Doh-Ura K, Iwaki T: Protease-resistant PrP and PrP oligomers in the brain in human prion diseases after intraventricular pentosan polysulfate infusion. Neuropathology 2012, 32: 124-132. 10.1111/j.1440-1789.2011.01245.xPubMedGoogle Scholar
- Terada T, Tsuboi Y, Obi T, Doh-ura K, Murayama S, Kitamoto T, Yamada T, Mizoguchi K: Less protease-resistant PrP in a patient with sporadic CJD treated with intraventricular pentosan polysulphate. Acta Neurol Scand 2010, 121: 127-130. 10.1111/j.1600-0404.2009.01272.xPubMedGoogle Scholar
- Masullo C, Macchi G, Xi Y, Pocchiari M: Failure to ameliorate Creutzfeldt-Jakob disease with amphotericin B therapy. J Infect Dis 1992, 165: 784-785. 10.1093/infdis/165.4.784PubMedGoogle Scholar
- Haik S, Brandel J, Salomon D, Sazdovitch V, Delasnerie-Laupretre N, Laplanche J, Faucheux B, Soubrie C, Boher E, Belorgey C, Hauw J, Alperovitch A: Compassionate use of quinacrine in Creutzfeldt-Jakob disease fails to show significant effects. Neurology 2004, 63: 2413-2415. 10.1212/01.WNL.0000148596.15681.4DPubMedGoogle Scholar
- Collinge J, Gorham M, Hudson F, Kennedy A, Keogh G, Pal S, Rossor M, Rudge P, Siddique D, Spyer M, Thomas D, Walker S, Webb T, Wroe S, Darbyshire J: Safety and efficacy of quinacrine in human prion disease (PRION-1 study): a patient-preference trial. Lancet Neurol 2009, 8: 334-344. 10.1016/S1474-4422(09)70049-3PubMedPubMed CentralGoogle Scholar
- Benito-Leon J: Combined quinacrine and chlorpromazine therapy in fatal familial insomnia. Clin Neuropharmacol 2004, 27: 201-203. 10.1097/01.wnf.0000134853.36429.0ePubMedGoogle Scholar
- Whittle I, Knight R, Will R: Unsuccessful intraventricular pentosan polysulphate treatment of variant Creutzfeldt-Jakob disease. Acta Neurochir (Wien) 2006, 148: 677-679. 10.1007/s00701-006-0772-yGoogle Scholar
- Tsuboi Y, Doh-Ura K, Yamada T: Continuous intraventricular infusion of pentosan polysulfate: clinical trial against prion diseases. Neuropathology 2009, 29: 632-636. 10.1111/j.1440-1789.2009.01058.xPubMedGoogle Scholar
- Mukherjee A, Morales-Scheihing D, Gonzalez-Romero D, Green K, Taglialatela G, Soto C: Calcineurin inhibition at the clinical phase of prion disease reduces neurodegeneration, improves behavioral alterations and increases animal survival. PLoS Pathog 2010, 6: e1001138. 10.1371/journal.ppat.1001138PubMedPubMed CentralGoogle Scholar
- Johnson S, Hunter T: Kinomics: methods for deciphering the kinome. Nat Methods 2005, 2: 17-25. 10.1038/nmeth731PubMedGoogle Scholar
- Kinase-Disease association http://www.cellsignal.com/common/content/content.jsp?id=science-tables-kinase-disease
- Martin L, Latypova X, Wilson C, Magnaudeix A, Perrin M, Yardin C, Terro F: Tau protein kinases: involvement in Alzheimer’s disease. Ageing Res Rev 2013, 12: 289-309. 10.1016/j.arr.2012.06.003PubMedGoogle Scholar
- Wood-Kaczmar A, Gandhi S, Wood N: Understanding the molecular causes of Parkinson’s disease. Trends Mol Med 2006, 12: 521-528. 10.1016/j.molmed.2006.09.007PubMedGoogle Scholar
- Fabbro D, Cowan-Jacob S, Mobitz H, Martiny-Baron G: Targeting cancer with small-molecular-weight kinase inhibitors. Methods Mol Biol 2012, 795: 1-34. 10.1007/978-1-61779-337-0_1PubMedGoogle Scholar
- Bamborough P: System-based drug discovery within the human kinome. Expert Opin Drug Discov 2012, 7: 1053-1070.PubMedGoogle Scholar
- Knight Z, Lin H, Shokat K: Targeting the cancer kinome through polypharmacology. Nat Rev Cancer 2010, 10: 130-137. 10.1038/nrc2787PubMedPubMed CentralGoogle Scholar
- Cohen P, Alessi D: Kinase drug discovery–what’s next in the field? ACS Chem Biol 2013, 8: 96-104. 10.1021/cb300610sPubMedPubMed CentralGoogle Scholar
- Clinical Trials of Protein Kinase Inhibitors http://www.researchandmarkets.com/reports/1196697/protein_kinase_inhibitors_in_oncology_drug
- Protein Kinase Inhibitors in Oncology Drug Pipeline Update 2014. http://www.researchandmarkets.com/reports/1196697/protein_kinase_inhibitors_in_oncology_drug
- Arsenault R, Li Y, Potter A, Griebel P, Kusalik A, Napper S: Induction of ligand-specific PrP (C) signaling in human neuronal cells. Prion 2012, 6: 477-488. 10.4161/pri.21914PubMedPubMed CentralGoogle Scholar
- Pan B, Yang LF, Wang J, Wang YS, Wang JH, Zhou XM, Yin XM, Zhang ZQ, Zhao DM: c-Abl Tyrosine Kinase Mediates Neurotoxic Prion Peptide-Induced Neuronal Apoptosis via Regulating Mitochondrial Homeostasis. Mol Neurobiol 2014, 49: 1102-1116. 10.1007/s12035-014-8646-4PubMedGoogle Scholar
- Perez M, Rojo AI, Wandosell F, Diaz-Nido J, Avila J: Prion peptide induces neuronal cell death through a pathway involving glycogen synthase kinase 3. Biochem J 2003, 372: 129-136. 10.1042/BJ20021596PubMedPubMed CentralGoogle Scholar
- Moreno JA, Halliday M, Molloy C, Radford H, Verity N, Axten JM, Ortori CA, Willis AE, Fischer PM, Barrett DA, Mallucci GR: Oral Treatment Targeting the Unfolded Protein Response Prevents Neurodegeneration and Clinical Disease in Prion-Infected Mice. Sci Transl Med 2013,5(206):206ra138.PubMedGoogle Scholar
- Pietri M, Dakowski C, Hannaoui S, Alleaume-Butaux A, Hernandez-Rapp J, Ragagnin A, Mouillet-Richard S, Haik S, Bailly Y, Peyrin J, Launay J, Kellermann O, Schneider B: PDK1 decreases TACE-mediated alpha-secretase activity and promotes disease progression in prion and Alzheimer’s diseases. Nat Med 2013, 19: 1124-1131. 10.1038/nm.3302PubMedGoogle Scholar
- Allen Brain Atlas http://www.brain-map.org
- Sakaguchi S, Katamine S, Shigematsu K, Nakatani A, Moriuchi R, Nishida N, Kurokawa K, Nakaoke R, Sato H, Jishage K: Accumulation of proteinase K-resistant prion protein (PrP) is restricted by the expression level of normal PrP in mice inoculated with a mouse-adapted strain of the Creutzfeldt-Jakob disease agent. J Virol 1995, 69: 7586-7592.PubMedPubMed CentralGoogle Scholar
- Lasmezas C, Deslys J, Demaimay R, Adjou K, Hauw J, Dormont D: Strain specific and common pathogenic events in murine models of scrapie and bovine spongiform encephalopathy. J Gen Virol 1996, 77: 1601-1609. 10.1099/0022-1317-77-7-1601PubMedGoogle Scholar
- Jendroska K, Heinzel F, Torchia M, Stowring L, Kretzschmar H, Kon A, Stern A, Prusiner S, DeArmond S: Proteinase-resistant prion protein accumulation in Syrian hamster brain correlates with regional pathology and scrapie infectivity. Neurology 1991, 41: 1482-1490. 10.1212/WNL.41.9.1482PubMedGoogle Scholar
- Jang B, Kim E, Choi J, Jin J, Kim J, Ishigami A, Maruyama N, Carp R, Kim Y, Choi E: Accumulation of citrullinated proteins by up-regulated peptidylarginine deiminase 2 in brains of scrapie-infected mice: a possible role in pathogenesis. Am J Pathol 2008, 173: 1129-1142. 10.2353/ajpath.2008.080388PubMedPubMed CentralGoogle Scholar
- Baldauf E, Beekes M, Diringer H: Evidence for an alternative direct route of access for the scrapie agent to the brain bypassing the spinal cord. J Gen Virol 1997, 78: 1187-1197.PubMedGoogle Scholar
- Kimberlin R, Walker C: Pathogenesis of mouse scrapie: patterns of agent replication in different parts of the CNS following intraperitoneal infection. J R Soc Med 1982, 75: 618-624.PubMedPubMed CentralGoogle Scholar
- Cole S, Kimberlin R: Pathogenesis of mouse scrapie: dynamics of vacuolation in brain and spinal cord after intraperitoneal infection. Neuropathol Appl Neurobiol 1985, 11: 213-227. 10.1111/j.1365-2990.1985.tb00019.xPubMedGoogle Scholar
- Mabuchi T, Kitagawa K, Kuwabara K, Takasawa K, Ohtsuki T, Xia Z, Storm D, Yanagihara T, Hori M, Matsumoto M: Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo. J Neurosci 2001, 21: 9204-9213.PubMedGoogle Scholar
- Kalia L, Salter M: Interactions between Src family protein tyrosine kinases and PSD-95. Neuropharmacology 2003, 45: 720-728. 10.1016/S0028-3908(03)00313-7PubMedGoogle Scholar
- Song T, Sugimoto K, Ihara H, Mizutani A, Hatano N, Kume K, Kambe T, Yamaguchi F, Tokuda M, Watanabe Y: p90 RSK-1 associates with and inhibits neuronal nitric oxide synthase. Biochem J 2007, 401: 391-398. 10.1042/BJ20060580PubMedPubMed CentralGoogle Scholar
- Sabio G, Reuver S, Feijoo C, Hasegawa M, Thomas G, Centeno F, Kuhlendahl S, Leal-Ortiz S, Goedert M, Garner C, Cuenda A: Stress- and mitogen-induced phosphorylation of the synapse-associated protein SAP90/PSD-95 by activation of SAPK3/p38gamma and ERK1/ERK2. Biochem J 2004, 380: 19-30. 10.1042/BJ20031628PubMedPubMed CentralGoogle Scholar
- Kmiecik T, Shalloway D: Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell 1987, 49: 65-73. 10.1016/0092-8674(87)90756-2PubMedGoogle Scholar
- Kmiecik T, Johnson P, Shalloway D: Regulation by the autophosphorylation site in overexpressed pp60c-src. Mol Cell Biol 1988, 8: 4541-4546.PubMedPubMed CentralGoogle Scholar
- Vik T, Ryder J: Identification of serine 380 as the major site of autophosphorylation of Xenopus pp90rsk. Biochem Biophys Res Commun 1997, 235: 398-402. 10.1006/bbrc.1997.6794PubMedGoogle Scholar
- Dalby K, Morrice N, Caudwell F, Avruch J, Cohen P: Identification of regulatory phosphorylation sites in mitogen-activated protein kinase (MAPK)-activated protein kinase-1a/p90rsk that are inducible by MAPK. J Biol Chem 1998, 273: 1496-1505. 10.1074/jbc.273.3.1496PubMedGoogle Scholar
- Matthews R, Guthrie C, Wailes L, Zhao X, Means A, McKnight G: Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression. Mol Cell Biol 1994, 14: 6107-6116. 10.1128/MCB.14.9.6107PubMedPubMed CentralGoogle Scholar
- Ribar T, Rodriguiz R, Khiroug L, Wetsel W, Augustine G, Means A: Cerebellar defects in Ca2+/calmodulin kinase IV-deficient mice. J Neurosci 2000, 20: RC107.PubMedGoogle Scholar
- Zhang S, Zou M, Lu L, Lau D, Ditzel D, Delucinge-Vivier C, Aso Y, Descombes P, Bading H: Nuclear calcium signaling controls expression of a large gene pool: identification of a gene program for acquired neuroprotection induced by synaptic activity. PLoS Genet 2009, 5: e1000604. 10.1371/journal.pgen.1000604PubMedPubMed CentralGoogle Scholar
- Tan Y, Zhang S, Hoffmann T, Bading H: Increasing levels of wild-type CREB up-regulates several activity-regulated inhibitor of death (AID) genes and promotes neuronal survival. BMC Neurosci 2012, 13: 48. 10.1186/1471-2202-13-48PubMedPubMed CentralGoogle Scholar
- Lehtinen M, Yuan Z, Boag P, Yang Y, Villen J, Becker E, DiBacco S, de la Iglesia N, Gygi S, Blackwell T, Bonni A: A conserved MST-FOXO signaling pathway mediates oxidative-stress responses and extends life span. Cell 2006, 125: 987-1001. 10.1016/j.cell.2006.03.046PubMedGoogle Scholar
- Glantschnig H, Rodan G, Reszka A: Mapping of MST1 kinase sites of phosphorylation. Activation and autophosphorylation. J Biol Chem 2002, 277: 42987-42996. 10.1074/jbc.M208538200PubMedGoogle Scholar
- Kakeya H, Onose R, Osada H: Caspase-mediated activation of a 36-kDa myelin basic protein kinase during anticancer drug-induced apoptosis. Cancer Res 1998, 58: 4888-4894.PubMedGoogle Scholar
- Graves J, Gotoh Y, Draves K, Ambrose D, Han D, Wright M, Chernoff J, Clark E, Krebs E: Caspase-mediated activation and induction of apoptosis by the mammalian Ste20-like kinase Mst1. EMBO J 1998, 17: 2224-2234. 10.1093/emboj/17.8.2224PubMedPubMed CentralGoogle Scholar
- Graves J, Draves K, Gotoh Y, Krebs E, Clark E: Both phosphorylation and caspase-mediated cleavage contribute to regulation of the Ste20-like protein kinase Mst1 during CD95/Fas-induced apoptosis. J Biol Chem 2001, 276: 14909-14915. 10.1074/jbc.M010905200PubMedGoogle Scholar
- Lee K, Ohyama T, Yajima N, Tsubuki S, Yonehara S: MST, a physiological caspase substrate, highly sensitizes apoptosis both upstream and downstream of caspase activation. J Biol Chem 2001, 276: 19276-19285. 10.1074/jbc.M005109200PubMedGoogle Scholar
- Bi W, Xiao L, Jia Y, Wu J, Xie Q, Ren J, Ji G, Yuan Z: c-Jun N-terminal kinase enhances MST1-mediated pro-apoptotic signaling through phosphorylation at serine 82. J Biol Chem 2010, 285: 6259-6264. 10.1074/jbc.M109.038570PubMedPubMed CentralGoogle Scholar
- Mantamadiotis T, Lemberger T, Bleckmann S, Kern H, Kretz O, Martin Villalba A, Tronche F, Kellendonk C, Gau D, Kapfhammer J, Otto C, Schmid W, Schutz G: Disruption of CREB function in brain leads to neurodegeneration. Nat Genet 2002, 31: 47-54. 10.1038/ng882PubMedGoogle Scholar
- Jancic D, Lopez de Armentia M, Valor L, Olivares R, Barco A: Inhibition of cAMP response element-binding protein reduces neuronal excitability and plasticity, and triggers neurodegeneration. Cereb Cortex 2009, 19: 2535-2547. 10.1093/cercor/bhp004PubMedGoogle Scholar
- Ao H, Ko S, Zhuo M: CREB activity maintains the survival of cingulate cortical pyramidal neurons in the adult mouse brain. Mol Pain 2006, 2: 15. 10.1186/1744-8069-2-15PubMedPubMed CentralGoogle Scholar
- Vo N, Klein M, Varlamova O, Keller D, Yamamoto T, Goodman R, Impey S: A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis. Proc Natl Acad Sci U S A 2005, 102: 16426-16431. 10.1073/pnas.0508448102PubMedPubMed CentralGoogle Scholar
- Nudelman A, DiRocco D, Lambert T, Garelick M, Le J, Nathanson N, Storm D: Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo. Hippocampus 2010, 20: 492-498.PubMedPubMed CentralGoogle Scholar
- Magill S, Cambronne X, Luikart B, Lioy D, Leighton B, Westbrook G, Mandel G, Goodman R: microRNA-132 regulates dendritic growth and arborization of newborn neurons in the adult hippocampus. Proc Natl Acad Sci U S A 2010, 107: 20382-20387. 10.1073/pnas.1015691107PubMedPubMed CentralGoogle Scholar
- Hwang D, Lee I, Yoo H, Gehlenborg N, Cho J, Petritis B, Baxter D, Pitstick R, Young R, Spicer D, Price N, Hohmann J, Dearmond S, Carlson G, Hood L: A systems approach to prion disease. Mol Syst Biol 2009, 5: 252.PubMedPubMed CentralGoogle Scholar
- Booth S, Bowman C, Baumgartner R, Sorensen G, Robertson C, Coulthart M, Phillipson C, Somorjai R: Identification of central nervous system genes involved in the host response to the scrapie agent during preclinical and clinical infection. J Gen Virol 2004, 85: 3459-3471. 10.1099/vir.0.80110-0PubMedGoogle Scholar
- Moody L, Herbst A, Yoo H, Vanderloo J, Aiken J: Comparative prion disease gene expression profiling using the prion disease mimetic, cuprizone. Prion 2009, 3: 99-109. 10.4161/pri.3.2.9059PubMedPubMed CentralGoogle Scholar
- Brown A, Rebus S, McKimmie C, Robertson K, Williams A, Fazakerley J: Gene expression profiling of the preclinical scrapie-infected hippocampus. Biochem Biophys Res Commun 2005, 334: 86-95. 10.1016/j.bbrc.2005.06.060PubMedGoogle Scholar
- Majer A, Medina S, Niu Y, Abrenica B, Manguiat K, Frost K, Philipson C, Sorensen D, Booth S: Early mechanisms of pathobiology are revealed by transcriptional temporal dynamics in hippocampal CA1 neurons of prion infected mice. PLoS Pathog 2012, 8: e1003002. 10.1371/journal.ppat.1003002PubMedPubMed CentralGoogle Scholar
- Kim H, Snyder G, Blazey T, Race R, Chesebro B, Skinner P: Prion disease induced alterations in gene expression in spleen and brain prior to clinical symptoms. Adv Appl Bioinform Chem 2008, 1: 29-50.PubMedPubMed CentralGoogle Scholar
- Kasahara J, Fukunaga K, Miyamoto E: Differential effects of a calcineurin inhibitor on glutamate-induced phosphorylation of Ca2+/calmodulin-dependent protein kinases in cultured rat hippocampal neurons. J Biol Chem 1999, 274: 9061-9067. 10.1074/jbc.274.13.9061PubMedGoogle Scholar
- Bito H, Deisseroth K, Tsien R: CREB phosphorylation and dephosphorylation: a Ca(2+)- and stimulus duration-dependent switch for hippocampal gene expression. Cell 1996, 87: 1203-1214. 10.1016/S0092-8674(00)81816-4PubMedGoogle Scholar
- Agostinho P, Oliveira C: Involvement of calcineurin in the neurotoxic effects induced by amyloid-beta and prion peptides. Eur J Neurosci 2003, 17: 1189-1196. 10.1046/j.1460-9568.2003.02546.xPubMedGoogle Scholar
- Nakagaki T, Satoh K, Ishibashi D, Fuse T, Sano K, Kamatari Y, Kuwata K, Shigematsu K, Iwamaru Y, Takenouchi T, Kitani H, Nishida N, Atarashi R: FK506 reduces abnormal prion protein through the activation of autolysosomal degradation and prolongs survival in prion-infected mice. Autophagy 2013,9(9):1386-1394. 10.4161/auto.25381PubMedGoogle Scholar
- McGinnis K, Whitton M, Gnegy M, Wang K: Calcium/calmodulin-dependent protein kinase IV is cleaved by caspase-3 and calpain in SH-SY5Y human neuroblastoma cells undergoing apoptosis. J Biol Chem 1998, 273: 19993-20000. 10.1074/jbc.273.32.19993PubMedGoogle Scholar
- Watt F, Molloy P: Specific cleavage of transcription factors by the thiol protease, m-calpain. Nucleic Acids Res 1993, 21: 5092-5100. 10.1093/nar/21.22.5092PubMedPubMed CentralGoogle Scholar
- See V, Loeffler J: Oxidative stress induces neuronal death by recruiting a protease and phosphatase-gated mechanism. J Biol Chem 2001, 276: 35049-35059. 10.1074/jbc.M104988200PubMedGoogle Scholar
- Guo Y, Gong H, Zhang J, Xie W, Tian C, Chen C, Shi Q, Wang S, Xu Y, Zhang B, Dong X: Remarkable reduction of MAP2 in the brains of scrapie-infected rodents and human prion disease possibly correlated with the increase of calpain. PLoS One 2012, 7: e30163. 10.1371/journal.pone.0030163PubMedPubMed CentralGoogle Scholar
- O’Donovan C, Tobin D, Cotter T: Prion protein fragment PrP-(106–126) induces apoptosis via mitochondrial disruption in human neuronal SH-SY5Y cells. J Biol Chem 2001, 276: 43516-43523. 10.1074/jbc.M103894200PubMedGoogle Scholar
- Falsig J, Sonati T, Herrmann U, Saban D, Li B, Arroyo K, Ballmer B, Liberski P, Aguzzi A: Prion pathogenesis is faithfully reproduced in cerebellar organotypic slice cultures. PLoS Pathog 2012, 8: e1002985. 10.1371/journal.ppat.1002985PubMedPubMed CentralGoogle Scholar
- Lopes J, Oliveira C, Agostinho P: Role of cyclin-dependent kinase 5 in the neurodegenerative process triggered by amyloid-Beta and prion peptides: implications for Alzheimer’s disease and prion-related encephalopathies. Cell Mol Neurobiol 2007, 27: 943-957. 10.1007/s10571-007-9224-3PubMedGoogle Scholar
- Wadzinski B, Wheat W, Jaspers S, Peruski LJ, Lickteig R, Johnson G, Klemm D: Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 1993, 13: 2822-2834.PubMedPubMed CentralGoogle Scholar
- Park I, Soderling T: Activation of Ca2+/calmodulin-dependent protein kinase (CaM-kinase) IV by CaM-kinase kinase in Jurkat T lymphocytes. J Biol Chem 1995, 270: 30464-30469. 10.1074/jbc.270.51.30464PubMedGoogle Scholar
- Westphal R, Anderson K, Means A, Wadzinski B: A signaling complex of Ca2 + −calmodulin-dependent protein kinase IV and protein phosphatase 2A. Science 1998, 280: 1258-1261. 10.1126/science.280.5367.1258PubMedGoogle Scholar
- Lee J, Shin J, Hwang S, Gwag B, McKee A, Lee J, Kowall N, Ryu H, Lim D, Choi E: MST1 functions as a key modulator of neurodegeneration in a mouse model of ALS. Proc Natl Acad Sci U S A 2013, 110: 12066-12071. 10.1073/pnas.1300894110PubMedPubMed CentralGoogle Scholar
- Jamieson E, Jeffrey M, Ironside J, Fraser J: Activation of Fas and caspase 3 precedes PrP accumulation in 87 V scrapie. Neuroreport 2001, 12: 3567-3572. 10.1097/00001756-200111160-00039PubMedGoogle Scholar
- Hetz C, Russelakis-Carneiro M, Maundrell K, Castilla J, Soto C: Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein. EMBO J 2003, 22: 5435-5445. 10.1093/emboj/cdg537PubMedPubMed CentralGoogle Scholar
- Corsaro A, Thellung S, Villa V, Principe D, Paludi D, Arena S, Millo E, Schettini D, Damonte G, Aceto A, Schettini G, Florio T: Prion protein fragment 106–126 induces a p38 MAP kinase-dependent apoptosis in SH-SY5Y neuroblastoma cells independently from the amyloid fibril formation. Ann N Y Acad Sci 2003, 1010: 610-622. 10.1196/annals.1299.114PubMedGoogle Scholar
- White A, Guirguis R, Brazier M, Jobling M, Hill A, Beyreuther K, Barrow C, Masters C, Collins S, Cappai R: Sublethal concentrations of prion peptide PrP106-126 or the amyloid beta peptide of Alzheimer’s disease activates expression of proapoptotic markers in primary cortical neurons. Neurobiol Dis 2001, 8: 299-316. 10.1006/nbdi.2001.0386PubMedGoogle Scholar
- Praskova M, Khoklatchev A, Ortiz-Vega S, Avruch J: Regulation of the MST1 kinase by autophosphorylation, by the growth inhibitory proteins, RASSF1 and NORE1, and by Ras. Biochem J 2004, 381: 453-462. 10.1042/BJ20040025PubMedPubMed CentralGoogle Scholar
- Cheung W, Ajiro K, Samejima K, Kloc M, Cheung P, Mizzen C, Beeser A, Etkin L, Chernoff J, Earnshaw W, Allis C: Apoptotic phosphorylation of histone H2B is mediated by mammalian sterile twenty kinase. Cell 2003, 113: 507-517. 10.1016/S0092-8674(03)00355-6PubMedGoogle Scholar
- Schatzl H, Laszlo L, Holtzman D, Tatzelt J, DeArmond S, Weiner R, Mobley W, Prusiner S: A hypothalamic neuronal cell line persistently infected with scrapie prions exhibits apoptosis. J Virol 1997, 71: 8821-8831.PubMedPubMed CentralGoogle Scholar
- Siso S, Puig B, Varea R, Vidal E, Acin C, Prinz M, Montrasio F, Badiola J, Aguzzi A, Pumarola M, Ferrer I: Abnormal synaptic protein expression and cell death in murine scrapie. Acta Neuropathol 2002, 103: 615-626. 10.1007/s00401-001-0512-6PubMedGoogle Scholar
- Giese A, Groschup M, Hess B, Kretzschmar H: Neuronal cell death in scrapie-infected mice is due to apoptosis. Brain Pathol 1995, 5: 213-221. 10.1111/j.1750-3639.1995.tb00597.xPubMedGoogle Scholar
- Williams A, Lucassen P, Ritchie D, Bruce M: PrP deposition, microglial activation, and neuronal apoptosis in murine scrapie. Exp Neurol 1997, 144: 433-438. 10.1006/exnr.1997.6424PubMedGoogle Scholar
- Saez-Valero J, Angeretti N, Forloni G: Caspase-3 activation by beta-amyloid and prion protein peptides is independent from their neurotoxic effect. Neurosci Lett 2000, 293: 207-210. 10.1016/S0304-3940(00)01532-9PubMedGoogle Scholar
- Engelstein R, Grigoriadis N, Greig N, Ovadia H, Gabizon R: Inhibition of P53-related apoptosis had no effect on PrP(Sc) accumulation and prion disease incubation time. Neurobiol Dis 2005, 18: 282-285. 10.1016/j.nbd.2004.10.008PubMedGoogle Scholar
- Brunet A, Bonni A, Zigmond M, Lin M, Juo P, Hu L, Anderson M, Arden K, Blenis J, Greenberg M: Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999, 96: 857-868. 10.1016/S0092-8674(00)80595-4PubMedGoogle Scholar
- Ura S, Masuyama N, Graves J, Gotoh Y: Caspase cleavage of MST1 promotes nuclear translocation and chromatin condensation. Proc Natl Acad Sci U S A 2001, 98: 10148-10153. 10.1073/pnas.181161698PubMedPubMed CentralGoogle Scholar
- Anand R, Kim A, Brent M, Marmorstein R: Biochemical analysis of MST1 kinase: elucidation of a C-terminal regulatory region. Biochemistry 2008, 47: 6719-6726. 10.1021/bi800309mPubMedPubMed CentralGoogle Scholar
- You H, Pellegrini M, Tsuchihara K, Yamamoto K, Hacker G, Erlacher M, Villunger A, Mak T: FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal. J Exp Med 2006, 203: 1657-1663. 10.1084/jem.20060353PubMedPubMed CentralGoogle Scholar
- Czymai T, Viemann D, Sticht C, Molema G, Goebeler M, Schmidt M: FOXO3 modulates endothelial gene expression and function by classical and alternative mechanisms. J Biol Chem 2010, 285: 10163-10178. 10.1074/jbc.M109.056663PubMedPubMed CentralGoogle Scholar
- Obexer P, Geiger K, Ambros P, Meister B, Ausserlechner M: FKHRL1-mediated expression of Noxa and Bim induces apoptosis via the mitochondria in neuroblastoma cells. Cell Death Differ 2007, 14: 534-547. 10.1038/sj.cdd.4402017PubMedGoogle Scholar
- Hetz C, Lee A, Gonzalez-Romero D, Thielen P, Castilla J, Soto C, Glimcher L: Unfolded protein response transcription factor XBP-1 does not influence prion replication or pathogenesis. Proc Natl Acad Sci U S A 2008, 105: 757-762. 10.1073/pnas.0711094105PubMedPubMed CentralGoogle Scholar
- Steele A, King O, Jackson W, Hetz C, Borkowski A, Thielen P, Wollmann R, Lindquist S: Diminishing apoptosis by deletion of Bax or overexpression of Bcl-2 does not protect against infectious prion toxicity in vivo. J Neurosci 2007, 27: 13022-13027. 10.1523/JNEUROSCI.3290-07.2007PubMedPubMed CentralGoogle Scholar
- Fu Y, Rusznak Z, Herculano-Houzel S, Watson C, Paxinos G: Cellular composition characterizing postnatal development and maturation of the mouse brain and spinal cord. Brain Struct Funct 2013, 218: 1337-1354. 10.1007/s00429-012-0462-xPubMedGoogle Scholar
- Johannessen M, Delghandi M, Moens U: What turns CREB on? Cell Signal 2004, 16: 1211-1227. 10.1016/j.cellsig.2004.05.001PubMedGoogle Scholar
- Pelech S, Sutter C, Zhang H: Kinetworks protein kinase multiblot analysis. Methods Mol Biol 2003, 218: 99-111.PubMedGoogle Scholar
- Safar J, Wille H, Itri V, Groth D, Serban H, Torchia M, Cohen F, Prusiner S: Eight prion strains have PrP(Sc) molecules with different conformations. Nat Med 1998, 4: 1157-1165. 10.1038/2654PubMedGoogle Scholar
- Wadsworth J, Joiner S, Hill A, Campbell T, Desbruslais M, Luthert P, Collinge J: Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet 2001, 358: 171-180. 10.1016/S0140-6736(01)05403-4PubMedGoogle Scholar
- Kumar R, McClain D, Young R, Carlson G: Cholesterol transporter ATP-binding cassette A1 (ABCA1) is elevated in prion disease and affects PrPC and PrPSc concentrations in cultured cells. J Gen Virol 2008, 89: 1525-1532. 10.1099/vir.0.83358-0PubMedPubMed CentralGoogle Scholar
- Otter T, King S, Witman G: A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins. Anal Biochem 1987, 162: 370-377. 10.1016/0003-2697(87)90406-4PubMedGoogle Scholar
- Yeung Y, Stanley E: A solution for stripping antibodies from polyvinylidene fluoride immunoblots for multiple reprobing. Anal Biochem 2009, 389: 89-91. 10.1016/j.ab.2009.03.017PubMedPubMed CentralGoogle Scholar
- Harlow E, Lane D: Using Antibodies: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 1999.Google Scholar
- de Hoon M, Imoto S, Nolan J, Miyano S: Open source clustering software. Bioinformatics 2004, 20: 1453-1454. 10.1093/bioinformatics/bth078PubMedGoogle Scholar
- Saldanha A: Java Treeview–extensible visualization of microarray data. Bioinformatics 2004, 20: 3246-3248. 10.1093/bioinformatics/bth349PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.