Gene variants of the leucine-rich repeat kinase 2 (LRRK2) are associated with susceptibility to Parkinson's disease (PD). Besides brain and periphery, LRRK2 is expressed in various immune cells including dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. However, the function of LRRK2 in the immune system is still incompletely understood. Here, Ca(2+)-signaling was analyzed in DCs isolated from gene-targeted mice lacking lrrk2 (Lrrk2(-/-)) and their wild-type littermates (Lrrk2(+/+)). According to Western blotting, Lrrk2 was expressed in Lrrk2(+/+) DCs but not in Lrrk2(-/-)DCs. Cytosolic Ca(2+) levels ([Ca(2+)]i) were determined utilizing Fura-2 fluorescence and whole cell currents to decipher electrogenic transport. The increase of [Ca(2+)]i following inhibition of sarcoendoplasmatic Ca(2+)-ATPase with thapsigargin (1 µM) in the absence of extracellular Ca(2+) (Ca(2+)-release) and the increase of [Ca(2+)]i following subsequent readdition of extracellular Ca(2+) (SOCE) were both significantly larger in Lrrk2(-/-) than in Lrrk2(+/+) DCs. The augmented increase of [Ca(2+)]i could have been due to impaired Ca(2+) extrusion by K(+)-independent (NCX) and/or K(+)-dependent (NCKX) Na(+)/Ca(2+)-exchanger activity, which was thus determined from the increase of [Ca(2+)]i, (Δ[Ca(2+)]i), and current following abrupt replacement of Na(+) containing (130 mM) and Ca(2+) free (0 mM) extracellular perfusate by Na(+) free (0 mM) and Ca(2+) containing (2 mM) extracellular perfusate. As a result, both slope and peak of Δ[Ca(2+)]i as well as Na(+)/Ca(2+) exchanger-induced current were significantly lower in Lrrk2(-/-) than in Lrrk2(+/+) DCs. A 6 or 24 hour treatment with the LRRK2 inhibitor GSK2578215A (1 µM) significantly decreased NCX1 and NCKX1 transcript levels, significantly blunted Na(+)/Ca(2+)-exchanger activity, and significantly augmented the increase of [Ca(2+)]i following Ca(2+)-release and SOCE. In conclusion, the present observations disclose a completely novel functional significance of LRRK2, i.e., the up-regulation of Na(+)/Ca(2+) exchanger transcription and activity leading to attenuation of Ca(2+)-signals in DCs.-Yan, J., Almilaji, A., Schmid, E., Elvira, B., Shimshek, D. R., van der Putten, H., Wagner, C. A., Shumilina, E., Lang, F. Leucine-rich repeat kinase 2-sensitive Na(+)/Ca2(+) exchanger activity in dendritic cells.
Abstract
Gene variants of the leucine-rich repeat kinase 2 (LRRK2) are associated with susceptibility to Parkinson's disease (PD). Besides brain and periphery, LRRK2 is expressed in various immune cells including dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. However, the function of LRRK2 in the immune system is still incompletely understood. Here, Ca(2+)-signaling was analyzed in DCs isolated from gene-targeted mice lacking lrrk2 (Lrrk2(-/-)) and their wild-type littermates (Lrrk2(+/+)). According to Western blotting, Lrrk2 was expressed in Lrrk2(+/+) DCs but not in Lrrk2(-/-)DCs. Cytosolic Ca(2+) levels ([Ca(2+)]i) were determined utilizing Fura-2 fluorescence and whole cell currents to decipher electrogenic transport. The increase of [Ca(2+)]i following inhibition of sarcoendoplasmatic Ca(2+)-ATPase with thapsigargin (1 µM) in the absence of extracellular Ca(2+) (Ca(2+)-release) and the increase of [Ca(2+)]i following subsequent readdition of extracellular Ca(2+) (SOCE) were both significantly larger in Lrrk2(-/-) than in Lrrk2(+/+) DCs. The augmented increase of [Ca(2+)]i could have been due to impaired Ca(2+) extrusion by K(+)-independent (NCX) and/or K(+)-dependent (NCKX) Na(+)/Ca(2+)-exchanger activity, which was thus determined from the increase of [Ca(2+)]i, (Δ[Ca(2+)]i), and current following abrupt replacement of Na(+) containing (130 mM) and Ca(2+) free (0 mM) extracellular perfusate by Na(+) free (0 mM) and Ca(2+) containing (2 mM) extracellular perfusate. As a result, both slope and peak of Δ[Ca(2+)]i as well as Na(+)/Ca(2+) exchanger-induced current were significantly lower in Lrrk2(-/-) than in Lrrk2(+/+) DCs. A 6 or 24 hour treatment with the LRRK2 inhibitor GSK2578215A (1 µM) significantly decreased NCX1 and NCKX1 transcript levels, significantly blunted Na(+)/Ca(2+)-exchanger activity, and significantly augmented the increase of [Ca(2+)]i following Ca(2+)-release and SOCE. In conclusion, the present observations disclose a completely novel functional significance of LRRK2, i.e., the up-regulation of Na(+)/Ca(2+) exchanger transcription and activity leading to attenuation of Ca(2+)-signals in DCs.-Yan, J., Almilaji, A., Schmid, E., Elvira, B., Shimshek, D. R., van der Putten, H., Wagner, C. A., Shumilina, E., Lang, F. Leucine-rich repeat kinase 2-sensitive Na(+)/Ca2(+) exchanger activity in dendritic cells.
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Yan, Jing