3 research outputs found
Computational analysis of calcium signaling and membrane electrophysiology in cerebellar Purkinje neurons associated with ataxia
BACKGROUND: Mutations in the smooth endoplasmic reticulum (sER) calcium channel Inositol Trisphosphate Receptor type 1 (IP3R1) in humans with the motor function coordination disorders Spinocerebellar Ataxia Types 15 and 16 (SCA15/16) and in a corresponding mouse model, the IP3R1(delta18/delta18) mice, lead to reduced IP3R1 levels. We posit that increasing IP3R1 sensitivity to IP3 in ataxias with reduced IP3R1 could restore normal calcium response. On the other hand, in mouse models of the human polyglutamine (polyQ) ataxias, SCA2, and SCA3, the primary finding appears to be hyperactive IP3R1-mediated calcium release. It has been suggested that the polyQ SCA1 mice may also show hyperactive IP3R1. Yet, SCA1 mice show downregulated gene expression of IP3R1, Homer, metabotropic glutamate receptor (mGluR), smooth endoplasmic reticulum Ca-ATP-ase (SERCA), calbindin, parvalbumin, and other calcium signaling proteins. RESULTS: We create a computational model of pathological alterations in calcium signaling in cerebellar Purkinje neurons to investigate several forms of spinocerebellar ataxia associated with changes in the abundance, sensitivity, or activity of the calcium channel IP3R1. We find that increasing IP3R1 sensitivity to IP3 in computational models of SCA15/16 can restore normal calcium response if IP3R1 abundance is not too low. The studied range in IP3R1 levels reflects variability found in human and mouse ataxic models. Further, the required fold increases in sensitivity are within experimental ranges from experiments that use IP3R1 phosphorylation status to adjust its sensitivity to IP3. Results from our simulations of polyglutamine SCAs suggest that downregulation of some calcium signaling proteins may be partially compensatory. However, the downregulation of calcium buffer proteins observed in the SCA1 mice may contribute to pathology. Finally, our model suggests that the calcium-activated voltage-gated potassium channels may provide an important link between calcium metabolism and membrane potential in Purkinje cell function. CONCLUSION: Thus, we have established an initial platform for computational evaluation and prediction of ataxia pathophysiology. Specifically, the model has been used to investigate SCA15/16, SCA1, SCA2, and SCA3. Results suggest that experimental studies treating mouse models of any of these ataxias with appropriately chosen peptides resembling the C-terminal of IP3R1 could adjust receptor sensitivity, and thereby modulate calcium release and normalize IP3 response. In addition, the model supports the hypothesis of IP3R1 supersensitivity in SCA1
Computational Analysis of Calcium Signaling and Membrane Electrophysiology in Cerebellar Purkinje Neurons Associated With Ataxia
Background
Mutations in the smooth endoplasmic reticulum (sER) calcium channel Inositol Trisphosphate Receptor type 1 (IP3R1) in humans with the motor function coordination disorders Spinocerebellar Ataxia Types 15 and 16 (SCA15/16) and in a corresponding mouse model, the IP3R1delta18/delta18 mice, lead to reduced IP3R1 levels. We posit that increasing IP3R1 sensitivity to IP3 in ataxias with reduced IP3R1 could restore normal calcium response. On the other hand, in mouse models of the human polyglutamine (polyQ) ataxias, SCA2, and SCA3, the primary finding appears to be hyperactive IP3R1-mediated calcium release. It has been suggested that the polyQ SCA1 mice may also show hyperactive IP3R1. Yet, SCA1 mice show downregulated gene expression of IP3R1, Homer, metabotropic glutamate receptor (mGluR), smooth endoplasmic reticulum Ca-ATP-ase (SERCA), calbindin, parvalbumin, and other calcium signaling proteins. Results
We create a computational model of pathological alterations in calcium signaling in cerebellar Purkinje neurons to investigate several forms of spinocerebellar ataxia associated with changes in the abundance, sensitivity, or activity of the calcium channel IP3R1. We find that increasing IP3R1 sensitivity to IP3 in computational models of SCA15/16 can restore normal calcium response if IP3R1 abundance is not too low. The studied range in IP3R1 levels reflects variability found in human and mouse ataxic models. Further, the required fold increases in sensitivity are within experimental ranges from experiments that use IP3R1 phosphorylation status to adjust its sensitivity to IP3. Results from our simulations of polyglutamine SCAs suggest that downregulation of some calcium signaling proteins may be partially compensatory. However, the downregulation of calcium buffer proteins observed in the SCA1 mice may contribute to pathology. Finally, our model suggests that the calcium-activated voltage-gated potassium channels may provide an important link between calcium metabolism and membrane potential in Purkinje cell function. Conclusion
Thus, we have established an initial platform for computational evaluation and prediction of ataxia pathophysiology. Specifically, the model has been used to investigate SCA15/16, SCA1, SCA2, and SCA3. Results suggest that experimental studies treating mouse models of any of these ataxias with appropriately chosen peptides resembling the C-terminal of IP3R1 could adjust receptor sensitivity, and thereby modulate calcium release and normalize IP3 response. In addition, the model supports the hypothesis of IP3R1 supersensitivity in SCA1
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Modulation of transglutaminase 2 activity in H9c2 cells by protein kinase A and protein kinase C signalling
Transglutaminase 2 (TG2; EC 2.3.2.13) has been shown to protect cardiomyocytes against ischaemia and reperfusion-induced cell death and to mediate cell survival in many cell types. Given the prominent role of PKA and PKC in cardioprotection, this study investigated whether TG2 was involved in the cytoprotection induced by activation of these two kinases in cardiomyocyte-like H9c2 cells.
Cultured H9c2 cells were extracted following stimulation with activators of PKC (phorbol-12-myristate-13-acetate; PMA) and PKA (forskolin; FK). Transglutaminase 2 activity was determined using an amine incorporating (in vitro and in situ) and a protein crosslinking assays. Different protein kinase inhibitors were used to determine the involvement of PKC and PKA in the activation of TG2 in H9c2 cells. To confirm the involvement of TG2 activity via PKC and PKA, TG2 specific (Z-DON and R283) inhibitors were used. Western blot analysis revealed the presence of TG2 and TG1 (TG2 >> TG1) protein, but not TG3. Since the H2O2, a major contributor to reactive oxygen species following damage was used to induce oxidative stress. The role of TG2 in PMA- and forskolin-induced cytoprotection was investigated by monitoring H2O2-induced oxidative stress in H9c2 cells. The identification of TG2 substrates in H9c2 cells was investigated using pull down assay coupled with proteomic analysis techniques.
The PMA and FK-induced time and concentration-dependent increases in TG2 catalysed biotin cadaverine incorporation in H9c2 cells. Forskolin but not PMA also increased TG2 catalysed protein crosslinking. The PKC (Ro-31 8220) and PKA (KT 5720 and Rp-8-Cl-cAMPS) inhibitors, blocked PMA and FK-induced TG2 activity. Immunocytochemistry using ExtrAvidin®-FITC revealed in situ TG2-mediated biotin cadaverine incorporation into protein substrates following stimulation of PMA, FK and their receptor agonists. The TG2 inhibitors Z-DON and R283 attenuated the PMA- and FK-induced increases in TG2 activity. Pre-treatment with PMA and FK reversed H2O2-induced cell death as judged by a MTT reduction assay and the release of cellular LDH. The TG2 inhibitors R283 and Z-DON blocked PMA and FK-induced cytoprotection. Proteomic analysis identified more than 25 proteins that serve as intracellular substrates for TG2 following PMA and FK stimulation. Some of these identified proteins have already been reported as TG2 substrates, but not in H9c2 cells e.g. tubulin while others e.g. α-actinin have not been identified before.
In summary, these data have shown TG2 activity to be stimulated via PKA and PKC-dependent signalling pathways in H9c2 cells and suggest a role for TG2 in cytoprotection-induced via these two protein kinases