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Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons.

Beazely MA, Weerapura M, MacDonald JF - Mol Brain (2008)

Bottom Line: Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons.This mimics the effects of PDGF receptor activation in these neurons.This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, University of Toronto, ON, Canada. mbeazely@uwaterloo.ca

ABSTRACT

Background: We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA) currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl), control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood.

Results: Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation.

Conclusion: This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

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Abl kinase immunoreactivity is decreased in the S2 and S3 fractions after PDGFβ receptor activation. A) Hippocampal slices were treated for 10 minutes with 10 ng/mL PDGF-BB, were homogenized, and S2, S3, and P3 fractions were prepared as described in the methods section. Equal amounts of total protein from each fraction were resolved on SDS gels, transferred to nitrocellulose membranes, and immunoblotted with anti-Abl or anti-PSD-95 antibodies (n = 6). B) PDGF-BB treated fractions were normalized to control for each fractionation sample. Data represent the mean +/- standard error for 6 independent experiments. PDGF-BB treatment significantly decreased Abl kinase immunoreactivity in the S2 and S3 fractions, * p < 0.05, Student's unpaired t-test.
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Figure 3: Abl kinase immunoreactivity is decreased in the S2 and S3 fractions after PDGFβ receptor activation. A) Hippocampal slices were treated for 10 minutes with 10 ng/mL PDGF-BB, were homogenized, and S2, S3, and P3 fractions were prepared as described in the methods section. Equal amounts of total protein from each fraction were resolved on SDS gels, transferred to nitrocellulose membranes, and immunoblotted with anti-Abl or anti-PSD-95 antibodies (n = 6). B) PDGF-BB treated fractions were normalized to control for each fractionation sample. Data represent the mean +/- standard error for 6 independent experiments. PDGF-BB treatment significantly decreased Abl kinase immunoreactivity in the S2 and S3 fractions, * p < 0.05, Student's unpaired t-test.

Mentions: Abl kinase is localized in both the synaptosomal and post-synaptic density fractions in adult rat brain homogenates [34] and Abl immunoreactivity is detected pre- and post-synaptically [18]. Upon stimulation of hippocampal slices with PDGF-BB, Abl kinase immunoreactivity decreased in both the S2 (cytosolic) and S3 (light membrane) fractions (Figure 3A, B). There was a trend towards an increase in immunoreactivity for Abl kinase in the triton-insoluble fraction (Figure 3A, B), suggesting that Abl kinase is moving from the cytosolic and light membrane fraction to the triton-insoluble fraction. Conversely, it is possible that Abl is being rapidly degraded in the S2 and S3 fractions. The NR1 subunit of the NMDA receptor was not significantly altered with respect to subcellular localization (Table 1).


Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons.

Beazely MA, Weerapura M, MacDonald JF - Mol Brain (2008)

Abl kinase immunoreactivity is decreased in the S2 and S3 fractions after PDGFβ receptor activation. A) Hippocampal slices were treated for 10 minutes with 10 ng/mL PDGF-BB, were homogenized, and S2, S3, and P3 fractions were prepared as described in the methods section. Equal amounts of total protein from each fraction were resolved on SDS gels, transferred to nitrocellulose membranes, and immunoblotted with anti-Abl or anti-PSD-95 antibodies (n = 6). B) PDGF-BB treated fractions were normalized to control for each fractionation sample. Data represent the mean +/- standard error for 6 independent experiments. PDGF-BB treatment significantly decreased Abl kinase immunoreactivity in the S2 and S3 fractions, * p < 0.05, Student's unpaired t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651131&req=5

Figure 3: Abl kinase immunoreactivity is decreased in the S2 and S3 fractions after PDGFβ receptor activation. A) Hippocampal slices were treated for 10 minutes with 10 ng/mL PDGF-BB, were homogenized, and S2, S3, and P3 fractions were prepared as described in the methods section. Equal amounts of total protein from each fraction were resolved on SDS gels, transferred to nitrocellulose membranes, and immunoblotted with anti-Abl or anti-PSD-95 antibodies (n = 6). B) PDGF-BB treated fractions were normalized to control for each fractionation sample. Data represent the mean +/- standard error for 6 independent experiments. PDGF-BB treatment significantly decreased Abl kinase immunoreactivity in the S2 and S3 fractions, * p < 0.05, Student's unpaired t-test.
Mentions: Abl kinase is localized in both the synaptosomal and post-synaptic density fractions in adult rat brain homogenates [34] and Abl immunoreactivity is detected pre- and post-synaptically [18]. Upon stimulation of hippocampal slices with PDGF-BB, Abl kinase immunoreactivity decreased in both the S2 (cytosolic) and S3 (light membrane) fractions (Figure 3A, B). There was a trend towards an increase in immunoreactivity for Abl kinase in the triton-insoluble fraction (Figure 3A, B), suggesting that Abl kinase is moving from the cytosolic and light membrane fraction to the triton-insoluble fraction. Conversely, it is possible that Abl is being rapidly degraded in the S2 and S3 fractions. The NR1 subunit of the NMDA receptor was not significantly altered with respect to subcellular localization (Table 1).

Bottom Line: Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons.This mimics the effects of PDGF receptor activation in these neurons.This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, University of Toronto, ON, Canada. mbeazely@uwaterloo.ca

ABSTRACT

Background: We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA) currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl), control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood.

Results: Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation.

Conclusion: This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

Show MeSH
Related in: MedlinePlus