Alpha-tocopherol (vitamin E) is a fat-soluble antioxidant with neuroprotective properties. There is a gap in research regarding the anti-nociceptive and anti-inflammatory effects of alpha-tocopherol in different tests of nociception as well as its signaling pathways. Therefore, this study aimed to assess the anti-nociceptive and anti-inflammatory effects of alpha-tocopherol in vivo and examine the potential involvement of transient receptor potential vanilloid 1 (TRPV1) and downstream kinase pathways, particularly protein kinases protein kinase A (PKA) and protein kinase C (PKC). Male Swiss albino mice were used in acetic acid-induced writhing, immersion tail-flick, and hot-plate tests for nociception while male Wistar rats were used in carrageenan-induced paw edema assay for inflammation. Alpha-tocopherol (50 mg/kg, i.p.), alone or in combination with TRPV1 antagonist (capsazepine) or PKC/PKA inhibitors (Go 6976, H89, respectively) was administered, followed by conducting the behavioral tests. TRPV1 expression in brain and skin tissues was examined using immunohistochemistry. Alpha-tocopherol significantly reduced writhing responses and extended heat latency in thermal tests. The TRPV1 was involved in the hot-plate and immersion tail-flick assays, while PKC and PKA pathways were implicated in the supraspinal analgesic effect (the hot plate test). Alpha-tocopherol (20 μg) did not exert any significant anti-inflammatory effect in the carrageenan model. Intense TRPV1 immunolabeling was observed in the alpha-tocopherol/ carrageenan condition, particularly in the skin. Alpha-tocopherol possesses a strong anti-nociceptive effect through TRPV1 modulation via PKC/PKA-dependent mechanisms. These findings suggest that alpha-tocopherol can be used as an adjuvant in multimodal pain management protocols.

NR2B-CaMKII signaling in the dentate gyrus driven by astrocytic P2Y1Rs mediates the antidepressant effect of low-dose LPS.

Burn injury frequently leads to heterotopic ossification (HO) during tendon healing, but the underlying molecular mechanisms remain poorly understood. This study investigated the role of the ceramide-protein kinase C zeta (PKCζ) signaling axis in burn-enhanced ectopic calcification during Achilles tendon healing and evaluated the therapeutic potential of PKCζ inhibition. A combined burn injury and Achilles tendon puncture model was established in C57BL/6 mice, with sham control, tendon puncture, and burn combined tendon puncture groups. Micro-computed tomography analysis revealed significantly increased bone volume at the calcaneal insertion site in burn-injured animals. Both total PKCζ and phosphorylated PKCζ expression were markedly elevated in the burn combined injury group. In vitro, C2-ceramide treatment significantly enhanced PKCζ activation and promoted osteogenic differentiation of Achilles tendon stem cells, as evidenced by increased alkaline phosphatase activity, calcium deposition, and upregulation of runt-related transcription factor 2 and osteopontin expression. Co-treatment with 2-acetyl-1,3-cyclopentanedione (ACPD), an atypical PKC (aPKC) inhibitor, effectively blocked ceramide-induced PKCζ activation and osteogenic differentiation. In vivo administration of the aPKC inhibitor significantly reduced ectopic calcification (bone volume on micro-CT) and decreased expression of PKCζ and osteogenic markers. These findings suggest that burn injury enhances ectopic calcification during Achilles tendon healing through activation of the ceramide-PKCζ signaling pathway, promoting aberrant osteogenic differentiation of tendon stem cells. Atypical PKC inhibition represents a potential therapeutic strategy for preventing burn-associated ectopic calcification.

Spontaneous automaticity of sinoatrial node cells (SANCs) is driven by a system that couples ion channels, membrane clock and Ca2+clock, the sarcoplasmic reticulum generated LCRs (local subsarcolemmal Ca2+releases). Although LCRs are critically dependent on high basal cAMP and both PKA (protein kinase A)- and CaMKII (Ca2+/calmodulin dependent protein kinase II)-dependent protein phosphorylation, the link between cAMP and CaMKII remains unclear. Here, we tested a hypothesis that high cAMP activates EPAC (exchange protein directly activated by cAMP) which increases basal CaMKII activity, reinforcing the coupled clock pacemaker system, to boost LCRs and accelerate spontaneous SANC firing. Real-time quantitative polymerase chain reaction, Western blot, immunostaining, whole-cell patch clamp, and line-scan confocal microscopy were employed to study EPAC-dependent regulation of rabbit SANC firing. Both EPAC isoforms were expressed and active in SANC. Selective inhibition of EPAC1 (CE3F4) or EPAC2 (HJC0350) similarly suppressed basal CaMKII activity, CaMKII-dependent phosphorylation of Ca2+-cycling proteins (PLB [phospholamban] and RyR [ryanodine receptors]) and reduced the amplitude of L-type Ca2+current. EPAC1 and EPAC2 inhibitors significantly decreased LCR number, size, and prolonged the LCR period (interval between prior AP-induced Ca2+transient and LCR) reducing spontaneous SANC firing by ≈30%. In contrast, EPAC activator (8-pCPT) increased LCR number and size, shortened the LCR period and accelerated spontaneous firing by≈18%. EPAC-mediated effects were implemented in PKC-dependent manner via EPAC-PLC-PKC-CaMKII signaling pathway, since PKC inhibitor reproduced effects of EPAC inhibition on CaMKII activity, CaMKII-dependent phosphorylation of Ca2+-cycling proteins, LCR parameters, and spontaneous SANC firing. EPAC is an essential component of basal cardiac pacemaker function, which accelerates spontaneous automaticity of SANC via an increase in basal CaMKII-dependent phosphorylation of Ca2+-cycling proteins (PLB, RyR, L-type Ca2+channels, and likely others), leading to amplification of LCR parameters, shortening of LCR timing and resultant spontaneous cycle length. Consequently, EPAC might represent a novel therapeutic target to regulate resting heart rate and treat sinoatrial node dysfunction.

The truncated constitutive active form of protein kinase C (PKC) called protein kinase M (PKM) plays a role in long-term memory maintenance in vertebrate and invertebrate models. Previously we have shown that the Aplysia Kidney/Brain protein (KIBRA) stabilizes the atypical PKM Apl III, but not the classical PKM Apl I in Aplysia neurons. Expression of Aplysia KIBRA with changes in the proposed atypical PKM binding site does not stabilize PKM Apl III and erases forms of plasticity supported by PKM Apl III. Here, we have examined biomolecular fluorescence complementation (BIFC) between KIBRA variants and PKM Apl III in Aplysia neurons. These KIBRA variants include: the KIBRA with changes in the proposed atypical binding site noted above; a splicing variant that stabilizes PKM Apl I, but not PKM Apl III; and several mutations identified in mammalian WW and C2 domain containing protein 3 (WWC3, a member of the chordate-specific expansion of the KIBRA family) associated with cancer or neurodevelopmental disorders. Surprisingly, we find that some KIBRA variants show BIFC with PKM Apl III but do not stabilize PKM Apl III. We used models of protein-protein interactions (AlphaFold 3) to gain insights into the discrepancy between BIFC and stabilization of PKMs by KIBRA and KIBRA variants and suggest a model where stabilization is linked to stable inhibition of PKMs by KIBRA.

Precision modification of heart failure signaling by CRISPR-Cas9 base editing.

Prion propagation, in which the cellular prion protein (PrPC) is conformationally converted into an infectious structure (PrPSc), has been extensively studied. However, the molecular mechanism responsible for the neurotoxicity of prions remains unclear. Synaptic loss is one of the earliest events in both in vivo and in vitro models of prion disease. We previously developed a neuronal cell culture model to analyze the mechanisms of prion-induced synaptic degeneration in a physiologically relevant setting. Using this system, we showed that exposure of hippocampal neurons to PrPSc engages a NMDAR/p38 mitogen-activated protein kinase (MAPK) signaling pathway that results in rapid, PrPC-dependent loss of synaptic transmission and retraction of dendritic spines. To comprehensively identify the components of this synaptotoxic signaling pathway, we measured changes in the phosphoproteome and transcriptome of hippocampal neurons exposed to PrPSc while they were undergoing the process of dendritic spine retraction. We then used these data as input into the L1000 and P100 databases of transcriptomic and proteomic drug signatures, leading to the discovery of 17 compounds that were able to prevent PrPSc-induced spine retraction. These compounds converged on three protein kinase targets: Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and glycogen synthase kinase 3β (GSK3β). Using immunocytochemical staining, we confirmed that PrPSc treatment of hippocampal neurons induced phosphorylation of the three kinases and caused their rapid translocation to dendritic spines. Along with N-methyl-D-aspartate receptors (NMDARs) on the neuronal surface, which trigger an initial influx of Ca2+ in response to PrPSc, these kinases constitute key nodes in a signaling network that mediates prion synaptotoxicity. Taken together, our results provide new insights into the mechanisms of prion neurotoxicity, and they identify novel molecular targets and inhibitory compounds that can be utilized for therapy of prion diseases.

BackgroundPlatelets and certain immune cells contain releasable granules that play essential roles in hemostasis and immune regulation. However, the regulatory mechanisms governing degranulation of platelets versus immune cells remain incompletely understood.ObjectivesThis study aimed to elucidate the distinct regulatory mechanisms underlying granule release in platelets and immune cells.MethodsMouse platelets were freshly isolated from peripheral blood, neutrophils were purified from bone marrow, and mast cells were generated from bone marrow progenitors by in vitro differentiation. The effects of phorbol ester (PMA), ionomycin, and their combination on degranulation of platelets, neutrophils, and mast cells were examined. In addition, pharmacological inhibitors targeting key components of multiple signaling pathways were used to investigate the molecular mechanisms regulating degranulation of these three cell types.ResultsPMA and ionomycin strongly induced α-granule release of platelets, whereas only moderately stimulating dense-granule secretion. For neutrophils, PMA and ionomycin each triggered moderate azurophilic-granule release, whereas their combination markedly enhanced degranulation. In contrast, PMA alone failed to induce mast cell degranulation, ionomycin robustly stimulated granule release, and their combination further amplified ionomycin-induced response. Mechanistically, thrombin-induced platelet degranulation was inhibited exclusively by phospholipase C (PLC) and protein kinase C (PKC) inhibitors. Zymosan-induced neutrophil degranulation was suppressed by inhibitors of phosphoinositide 3 kinase, spleen tyrosine kinase (SYK), Bruton's tyrosine kinase (BTK), mitogen-activated protein kinase 1/2 (MEK1/2) and P38 but was enhanced by PLC and PKC inhibition. Mast cell degranulation induced by IgE and antigen was significantly inhibited by the inhibitors targeting SYK, BTK, PLC, and PKC.ConclusionThese findings reveal substantial differences in the regulatory pathways controlling degranulation in platelets and immune cells. Such distinctions highlight the opportunities for the development of cell type-selective inhibitors to modulate degranulation, providing potential therapeutic strategies for thrombotic diseases and immune-related diseases.

The vicious cycle of hyperglycemia and oxidative stress: Novel mechanistic insights into a pathogenic alliance.

BACKGROUND Irritable bowel syndrome (IBS) is a common disorder of gut-brain interaction and is characterized by chronic abdominal pain and altered bowel habits. Current evidence indicates that immune activation and autoantibody production contribute to IBS pathogenesis. However, the mechanisms by which autoantibodies affect the enteric nervous system and contribute to IBS-related symptoms are poorly understood. AIM To investigate the role of anti-HuD autoantibodies in enteric neuronal apoptosis in an IBS animal model. METHODS A passive-transfer rat model of IBS was generated by intraperitoneal administration of HuD autoantibodies. Gastrointestinal motility, visceral sensitivity, fecal output, and water content were assessed. Enteric neuronal apoptosis in intestinal tissues and primary enteric neurons was analyzed by immunofluorescence. Mechanisms were examined using quantitative reverse transcription polymerase chain reaction, western blotting, and confocal microscopy. Interventions included recombinant HuD, immunoglobulin, 5-hydroxytryptamine receptor modulators, and protein kinase C (PKC) agonists. RESULTS Administration of HuD autoantibodies induced IBS-like phenotypes in rats. We observed increased fecal output, elevated fecal water content, accelerated intestinal transit, and enhanced visceral hypersensitivity. HuD autoantibody exposure significantly increased enteric neuronal apoptosis in vivo and in vitro and suppressed the expression of special AT-rich sequence-binding protein 1 (SATB1). Mechanistically, HuD autoantibodies disrupted HuD-mediated RNA regulation, leading to SATB1 downregulation and inhibition of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. PKC activation restored HuD and SATB1 expression, reactivated PI3K-AKT signaling, and significantly reduced neuronal apoptosis. Treatment with immunoglobulin and 5-hydroxytryptamine receptor agonists showed limited or inconsistent protective effects. CONCLUSION HuD autoantibodies induced enteric neuronal apoptosis through disruption of the HuD-SATB1-PI3K-AKT signaling axis and contributed to IBS-like gastrointestinal dysfunction. Activation of PKC may be a potential therapeutic strategy for IBS.

Pain modulation relies on complex molecular interactions among ion channels, G protein-coupled receptors, and intracellular signaling cascades. The Transient Receptor Potential Vanilloid 1 (TRPV1) channel serves as a polymodal detector and integrator of noxious stimuli, linking sensory transduction with broader neuromodulatory systems. This review delineates the mechanistic crosstalk between TRPV1, endocannabinoid, and opioid pathways in nociceptive regulation. TRPV1 activation by heat, protons, or endogenous lipids induces calcium influx and engages protein kinase C (PKC), protein kinase A (PKA), and mitogen-activated protein kinase (MAPK) pathways that modulate channel phosphorylation and neuronal excitability. Endocannabinoids such as anandamide act as dual CB₁ and TRPV1 agonists, establishing feedback loops that adjust nociceptive thresholds, while μ-opioid receptor activation inhibits adenylate cyclase and TRPV1 sensitization through Gi/o-mediated signaling. Given the recent progress in cryo-electron microscopy and molecular modeling, simulation studies have been possible, revealing key structural determinants underlying key receptor interactions. Integrating pharmacophore modeling, molecular docking, and artificial intelligence-based screening enables rational design of multi-target ligands that exploit TRPV1-endocannabinoid-opioid synergy. This mechanistic framework supports the development of next-generation analgesics that achieve potent, sustained, and safe modulation of nociceptive signaling.

Despite revolutionizing oncology, PD-1/PD-L1-directed immune-checkpoint blockade (ICB) is limited by primary or acquired resistance that is routinely countered-without mechanistic clarity-by empiric rechallenge. Here, we identify substrate-based protein kinase C (PKC) activity as a prognostic biomarker in non-small cell lung cancer and a therapeutic target in anti-PD-1-refractory tumors. Pan-PKC inhibition overcomes anti-PD-1 resistance by inducing Caspase-3/GSDME-dependent immunogenic pyroptotic cell death, promoting tumor-intrinsic PD-L1 degradation via GSK3β activation, and enhancing CD8+ T cell recruitment and effector function through tumor-derived CCL4-CCR5 signaling. Mechanistically, PKC blockade destabilizes XIAP, relieving caspase inhibition, stabilizing PTEN, and suppressing Wnt/β-Catenin-ATF3 signaling to drive CCL4 expression. In resistant models, PKC inhibition synergizes more effectively with anti-CTLA-4 than with anti-PD-1/PD-L1, in association with reduced intratumoral regulatory T cells and reinforcement of CD8+ T cell effector function. These findings define the PKC-XIAP axis as a central regulator of immune resistance and provide a mechanistic rationale for PKC inhibition plus anti-CTLA-4 as a salvage strategy in ICB-refractory cancers.

Decades of research have uncovered the complex signaling network downstream of the opioid receptors and suggested how this signaling could be modulated to improve opioid therapy. In our study, we have found that heat shock protein 90 (Hsp90) regulates downstream opioid signaling oppositely in the brain vs the spinal cord. In the spinal cord, we have found that Hsp90 inhibition enables antinociceptive signaling and disables pronociceptive signaling to enhance opioid pain relief and reduce side effects. We have now extended this study to analyze the contribution of protein kinase C (PKC) to the opioid signaling cascade. We used the Hsp90 inhibitor 17-AAG along with a PKC activator or inhibitor (Go6983) delivered into the spinal cords of male and female CD-1 mice to show that pan-PKC signaling contributes to the enhanced opioid antinociception observed in tail flick and postsurgical pain models. We used Western blot and immunohistochemistry to observe increased pan-PKC phosphorylation across calcitonin gene-related peptide (CGRP) and IB4 nociceptors in the spinal dorsal horn. We then used selective siRNA to identify PKCβ as the active isoform and further found PKCβ to be selectively activated in CGRP neurons by Hsp90 inhibition and morphine combined. Finally, we used cell-type-selective Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to knock down PKCβ in CGRP neurons and showed that this specific isoform in these specific cells was necessary for enhanced opioid antinociception after Hsp90 inhibition.Together, these studies further uncover the novel Hsp90-regulated opioid signaling cascade and suggest how Hsp90 inhibitors could be used to improve opioid therapy by increasing analgesic efficacy and decreasing side effects.

Intermembrane lipid transfer protein VPS13A (VPS13A) is a large protein whose cellular functions are still under investigation. VPS13A gene mutations leading to the absence of protein expression cause Chorea-acanthocytosis (ChAc), an ultra-rare inherited neurodegenerative movement disorder. Although the molecular mechanisms linking VPS13A loss to neuronal dysfunction remain unclear, its role as a bulk lipid transfer protein suggests that impaired lipid distribution may represent a primary pathogenic mechanism leading to neurodegeneration in ChAc. In this study, we investigated the effect of neuronal silencing of VPS13A in a murine model to phenocopy the human disease. Lipidomics analysis revealed an increase in the concentration of several diacylglycerol species induced by VPS13A knockdown. We then explored the downstream molecular pathways related to the altered diacylglycerol levels and found that VPS13A knockdown induces a decrease in protein kinase C (PKC)βII concentration but an increase in PKCα/βII phosphorylation in cultured neurons. Finally, pharmacological inhibition of PKCβII reverted aberrant neuronal morphology and loss of spine density induced by VPS13A KD. These results underscore the importance of VPS13A in regulating neuronal lipid distribution, showing that its absence perturbs the diacylglycerol/PKC signaling pathway, with measurable effects on neuronal structure and synaptic density. Overall, our results underscore a previously underappreciated role for VPS13A in the structural organization of neurons through lipid-mediated signaling mechanisms, providing insight into the cellular dysfunction underlying ChAc.

Deterioration of transverse-axial tubules (t-tubules) contributes to insufficient excitation-contraction coupling in heart failure, yet the key signals and mechanisms remain unclear. Here we aimed to identify the signaling pathways that trigger cardiomyocyte t-tubule loss and its underlying cellular process. Adult rat, rabbit and human ventricular cardiomyocytes and living myocardial slices, were exposed to pharmacological activators of protein kinase C (PKC). PKC activation caused rapid t-tubule loss and impaired Ca²⁺ transients, which were prevented by inhibition of protein kinase D (PKD) or NFκB. RNA-sequencing and phosphoprotein analysis showed activation and crosstalk between PKD-, ERK- and NFκB-dependent pathways, with up-regulation of genes involved in membrane trafficking and endocytosis. Fluorescent dextran uptake assays revealed a clathrin-independent, PI3K- and myosin-I-dependent macropinocytic process whose rate matched the internalization of t-tubule membranes and which was blocked by NFκB inhibition. Constitutive activation of IKK2 in cardiomyocytes of transgenic mice reduced t-tubule density in vivo, confirming that prolonged NFκB activation is sufficient to induce t-tubule remodeling in intact hearts. NFκB inhibition suppressed PKC-induced macropinocytosis also in non-cardiac human cell lines, suggesting that this process represents a conserved cellular response to inflammatory signaling. PKC-PKD-NFκB signaling triggers a macropinocytic form of membrane remodeling that degrades the t-tubule network and impairs excitation-contraction coupling. This identifies a previously unrecognized mechanism linking inflammatory kinase activation to structural and functional decline of cardiomyocytes and suggests that targeting the PKD-NFκB axis could preserve t-tubule integrity and cardiac performance in heart failure.

Polycomb repressive complex 2 (PRC2) regulates the expression of pluripotency genes in embryonic stem cells (ESCs) and suppresses multiple genes associated with development, cell fate determination, and differentiation. Mouse embryonic stem cells (mESCs) derived from protein kinase C inhibition (PKCi) exhibit self-renewal and pluripotency comparable to those ESCs captured by the classical 2iL (CHIR99021, PD0325901, and leukemia inhibitory factor) system. However, the dynamic expression pattern of PRC2 in PKCi-mESCs and its role in regulating pluripotency remain unclear. This study demonstrated that the expression level of the enhancer of zeste 2 gene (Ezh2), of which protein is the catalytic subunit of PRC2 responsible for the trimethylation of lysine 27 on nucleosome histone H3 subunit (H3K27me3), is significantly higher in PKCi-mESCs than in 2iL-mESCs. EZH2 knockdown enhances the self-renewal capacity of PKCi-mESCs, as evidenced by a significant increase in the number of undifferentiated mESCs colonies. The effect of an EZH2 reduced expression is accompanied by the upregulation of specific core pluripotency gene Nanog, along with the general downregulation of differentiational genes representing the three germ layers. Conversely, EZH2 overexpression promotes a significant differentiation of PKCi-mESCs, resulting in the downregulation of pluripotency genes, including core pluripotency genes Nanog and Sox2, as well as naïve pluripotency genes Klf4, Fgf4, and Esrrb, while with a wide upregulation of three germ layer associated genes. Importantly, Cleavage Under Targets and Tagmentation (CUT&Tag) demonstrates that EZH2 directly controls H3K27me3 enrichment at the Nanog promoter near the transcription start site. Thus, EZH2, a core subunit of PRC2, exhibits the distinct regulatory functions orchestrating mESCs at a poised state between self-renewal and differentiation under PKC inhibition. EZH2 exerts histone H3 methyltransferase activity to regulate Nanog expression as one of its key targets, thereby modulating the transcriptional regulatory network that maintains pluripotency and lineage specification in mESCs.

Pheochromocytoma and paraganglioma with a succinate dehydrogenase B subunit (SDHB) pathogenic variant are associated with a significant chance for metastasis. Polyamine pathway inhibitor N1,N11-diethylnorspermine (DENSPM) was previously shown to inhibit cell growth in progenitor cells derived from a human pheochromocytoma (hPheo1). Here, we hypothesized cell death associated with DENSPM treatment due to altered lipid metabolism affects protein kinase C (PKC). From targeted lipidomics analysis, baseline bioactive lipids that are distinct between the hPheo1 WT and SDHB KD cells are PE(P-18:1/16:0), PI(18:1/20:3), LPE(18:0), and LPE(22:4). With DENSPM treatment, the concentrations of multiple plasmanyl phosphatidylethanolamines (PE-O), sphingomyelins (SM), and hexosylceramides (HCER) increased, while the concentrations of several plasmenyl phosphatidylethanolamines (PE-P) were decreased in hPheo1 WT and SDHB KD cells. The differences in PE-Ps, PE-Os, and SMs after DENSPM treatment compared to the vehicle treatment were greater in the SDHB KD cells compared to the hPheo1 WT cells. Basal PKC alpha protein expression was increased in SDHB KD cells compared to hPheo1 WT cells. The protein expression of both PKC alpha and delta was significantly decreased with DENSPM treatment in both cell lines. DENSPM changed pro-caspase-3 and cleaved caspase-3. These data suggest ether phospholipids are biomarkers of DENSPM mediated cell apoptosis through a PKC dependent mechanism.

Differential susceptibility of rat retinal neurons to the Na+/K+-ATPase inhibitor ouabain.

Role of ventrolateral orbital cortex 5-HT2A receptors in formalin-induced secondary mechanical allodynia and hyperalgesia.

Prion propagation, in which the cellular prion protein (PrPC) is conformationally converted into an infectious structure (PrPSc), has been extensively studied. However, the molecular mechanism responsible for the neurotoxicity of prions remains unclear. Synaptic loss is one of the earliest events in both in vivo and in vitro models of prion disease. We previously developed a neuronal cell culture model to analyze the mechanisms of prion-induced synaptic degeneration in a physiologically relevant setting. Using this system, we showed that exposure of hippocampal neurons to PrPSc engages a NMDAR/p38 mitogen-activated protein kinase (MAPK) signaling pathway that results in rapid, PrPC-dependent loss of synaptic transmission and retraction of dendritic spines. To comprehensively identify the components of this synaptotoxic signaling pathway, we measured changes in the phosphoproteome and transcriptome of hippocampal neurons exposed to PrPSc while they were undergoing the process of dendritic spine retraction. We then used these data as input into the L1000 and P100 databases of transcriptomic and proteomic drug signatures, leading to the discovery of 17 compounds that were able to prevent PrPSc-induced spine retraction. These compounds converged on three protein kinase targets: Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and glycogen synthase kinase 3β (GSK3β). Using immunocytochemical staining, we confirmed that PrPSc treatment of hippocampal neurons induced phosphorylation of the three kinases and caused their rapid translocation to dendritic spines. Along with N-methyl-D-aspartate receptors (NMDARs) on the neuronal surface, which trigger an initial influx of Ca2+ in response to PrPSc, these kinases constitute key nodes in a signaling network that mediates prion synaptotoxicity. Taken together, our results provide new insights into the mechanisms of prion neurotoxicity, and they identify novel molecular targets and inhibitory compounds that can be utilized for therapy of prion diseases.