Pharmacological modification of ATP-dependent microglial activation in a disease model of ALS

Acute or chronic damage of neuronal tissue leads to microglial (MG) activation which is a multistep process involving morphological changes, release of pro-inflammatory substances, proliferation and migration to the affected sites of the brain. Purines as soluble factors are implicated as candidates for the chemotactic attraction of microglia by activating ionotropic P2X and metabotropic P2Y receptors (R). Earlier experiments on transgenic mice carrying the human mSOD1 gene presented evidence that MG cells significantly contribute to disease progression in the later phase of disease and coinciding upregulation of P2X4-R on MG cells may be a relevant factor in this inflammatory response.

Here, we studied the purinergic influence on MG motility and migration in the disease model of ALS. We used time-lapse 2-photon laser-scanning microscopy on spinal cord slices of SOD1^G93A mice with EGFP-labelled MG cells to capture the MG motility. Furthermore, we employed the patch-clamp technique in combination with Ca2+-imaging.

Under saline conditions, an increased number as well as morphologically transformed MG were observed in slices of clinically affected SOD mice compared to controls. Furthermore, baseline motility of mSOD MG branches was significantly increased. Upon superfusion with ATP, numerous MG branches extended to the slice surface indicating a rapid response to this chemotactic stimulus. The ATP-effect was blocked in controls by P2X7-R- and P2X4-R antagonist while in mSOD MG P2X-R inhibition was only partially effective. In addition on a functional level, ATP-application evoked an inward current and mediated intracellular [Ca²⁺] rises. Our results indicate that (a) upregulation of P2X-R in mSOD1 MG cells is functional and coincides with MG overactivation, (b) the MG response to external ATP is predominantly mediated by P2X-R while P2Y-R are not substantially involved. Therefore, upregulation of P2X-R is an important factor in initiating and sustaining MG overactivation during disease progression.