Involvement of protein kinase D in uridine diphosphate-induced microglial macropinocytosis and phagocytosis

Area of Science:

• Neuroscience
• Cell Biology
• Immunology

Background:

• Microglia are essential for brain homeostasis, clearing tissue debris through phagocytosis.
• Extracellular nucleotides, like uridine diphosphate (UDP), activate microglial P2Y(6) receptors, initiating dynamic responses.
• The intracellular signaling pathways downstream of microglial P2Y(6) receptor activation remain largely unknown.

Purpose of the Study:

• To elucidate the intracellular mechanisms regulating microglial responses to UDP stimulation via P2Y(6) receptors.
• To investigate the role of protein kinase D (PKD) in UDP-induced microglial membrane dynamics and phagocytosis.

Main Methods:

• Stimulation of primary microglia with UDP.
• Assessment of membrane dynamics and macropinocytosis using fluorescent dextran.
• Pharmacological inhibition of protein kinase D (PKD) and protein kinase C (PKC) using specific inhibitors (Gö6976, CID755673, Gö6983).
• Quantification of microsphere and soluble beta-amyloid uptake.

Main Results:

• UDP stimulation induced immediate membrane motility and macropinocytosis, characterized by vacuole formation.
• PKD inhibitors (Gö6976, CID755673) suppressed UDP-induced vacuole formation, membrane motility, and uptake of dextran, beta-amyloid, and microspheres.
• UDP triggered rapid PKD phosphorylation and membrane translocation, a process inhibited by PKC inhibition (Gö6983), although Gö6983 did not fully block microsphere uptake, suggesting a PKC-independent PKD role.

Conclusions:

• Protein kinase D (PKD) plays a critical role in regulating UDP-induced microglial membrane dynamics and macropinocytosis.
• A PKC-independent function of PKD is involved in enhancing the uptake of extracellular fluid and particulate matter by microglia.
• These findings reveal a novel intracellular signaling pathway crucial for microglial surveillance and debris clearance in the brain.