Pikfyve Is Essential for Lysosomal Homeostasis in Macrophages

The biogenesis and degradative functions of the lysosome in macrophages and other cells is critically dependent on the membrane trafficking along the endolysosomal system. This trafficking is tightly regulated by several signaling molecules including the endosomal phosphoinositide PtdIns(3,5)P2, which is synthesized from PtdIns(3)P in mammals by the lipid kinase, PIKfyve. Although PtdIns(3,5)P2 deficiency is associated with neurodegeneration in mice and humans, the critical importance of PIKfyve to normal macrophage function is poorly understood. To address this, we genetically engineered mice to lack PIKfyve in their macrophages by pairing PIKfyve^fl/fl mice with mice that are transgenic for the myeloid-specific LysM-Cre (PIKfyve^fl/fl LysM-Cre). PIKfyve^fl/fl LysM-Cre mice develop weight gain and massive hepatosplenomegaly. Immunohistochemical analyses of the liver and spleen from PIKfyve^fl/fl LysM-Cre mice demonstrate tissue infiltration of mononuclear cells with numerous cytoplasmic vacuoles that stain with the macrophage-specific marker, anti-F4/80 antibody. To verify that these vacuolated cells result from PIKfyve ablation, we utilized genetic lineage tracing by crossing PIKfyve^fl/fl LysM-Cre mice with mice transgenic for a Cre-dependent β-galactosidase reporter. Histochemical analyses using a chromogenic substrate, x-gal, reveal β-galactosidase activity in cells with small cytoplasmic vacuoles, demonstrating Cre expression and therefore PIKfyve ablation in these cells. However, β-galactosidase activity is absent in cells with large vacuoles, indicating that these cells do not express Cre and still contain PIKfyve. These findings suggest that the cytoplasmic vacuolation resulted from both cell-autonomous and non-cell-autonomous effects of PIKfyve deletion. To investigate the macrophage-specific effects of PIKfyve ablation in the lysosomal biogenesis and functions, we isolated macrophages from the bone marrow and spleen of PIKfyve^fl/fl LysM-Cre mice using magnetic beads bound to anti-F4/80 antibody. Immunoblot analysis of F4/80+ PIKfyve-null macrophages reveal increased expression of lysosomal proteins including Cathepsin D, Cathepsin S, and LAMP-1, demonstrating that PIKfyve is essential for lysosome homeostasis in macrophages.

Recent studies showed that the transcription factor TFEB is a master regulator of lysosomal biogenesis and function. When TFEB is phosphorylated by mTORC1 on the lysosomal membrane, TFEB is retained in the cytosol, and therefore unable to promote gene transcription. However, when TFEB is de-phosphorylated, TFEB translocates to the nucleus where it is transcriptionally active. Since PtdIns(3,5)P2 was previously shown to regulate the recruitment of mTORC1 to the lysosome, we hypothesized that the synthesis of PtdIns(3,5)P2 by PIKfyve regulates TFEB phosphorylation and indirectly controls TFEB transcriptional activity. To test this hypothesis, we studied the expression of TFEB in the F4/80+ macrophages isolated from the control and PIKfyve^fl/fl LysM-Cre mice. While wild-type macrophages express TFEB in both phosphorylated and dephosphorylated forms, PIKfyve-null macrophages primarily express the dephosphorylated form of TFEB. This suggests that PIKfyve is required for the phosphorylation of TFEB and consequently PIKfyve prevents TFEB’s ability to transcribe lysosomal genes. Thus, PIKfyve ablation within macrophages upregulates the transcription of lysosomal genes via TFEB activation. Our study suggests that PIKfyve is a critical regulator of lysosomal homeostasis and demonstrates a previously unrecognized link between PIKfyve, PtdIns(3,5)P2 synthesis, and the TFEB pathway in the regulation of lysosomal biogenesis and function within macrophages.