Genetic Dissection of the role of Piga and Pgap2 in the embryonic mouse brain

Glycosylphosphatidylinositol (GPI) anchors are a post-translational modification of over 150 proteins. These GPI-anchored proteins are enriched in lipid rafts in the plasma membrane and serve a variety of functions including acting as receptors and cell adhesion molecules. Human pathogenic variants in GPI biosynthesis pathway enzymes are collectively called inherited GPI deficiencies and lead to several brain anomalies such as global developmental delay, hypomyelination, cerebellar hypoplasia, and premature death. PIGA is the first catalytic enzyme in the GPI anchor biosynthesis cascade, which creates the backbone of all GPI anchors, and PGAP2 is one of the last enzymes in the GPI-anchor biosynthesis pathway and modifies the protein-bound anchor for trafficking to the cell membrane. A Nestin-Cre mediated deletion of Piga in the mouse led to early postnatal death and significant structural brain malformations, similar to pathogenic human PIGA variants. While this model provides an opportunity to develop treatments for these neurological phenotypes, we still lack a deep understanding of GPI-anchor functions in brain development. Additionally, there are no studies on Pgap2 in the brain to date. We extended these studies with a series of genetic ablations to precisely determine the role of Piga and Pgap2 in the forebrain, oligodendrocytes, and cerebellum. We find Piga expression in the hindbrain is absolutely required for survival while Pgap2 ablations were much less deleterious. These studies broaden our knowledge on the brain region-specific requirements of GPI-anchor biosynthesis enzymes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/692975v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@18eb4feorg.highwire.dtl.DTLVardef@cb152org.highwire.dtl.DTLVardef@14d7e84org.highwire.dtl.DTLVardef@ad2d9a_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementIn this study, we examine the roles of the GPI-anchor biosynthesis enzymes Piga and Pgap2 to understand the poorly understood mechanisms underlying brain anomalies seen in the inherited GPI deficiencies. Using Cre/lox conditional mouse genetic models, we deleted Piga and Pgap2 from various brain regions, including the forebrain, oligodendrocytes, and cerebellum, and observed brain abnormalities. We find that Piga ablations mimic many brain phenotypes seen in human inherited GPI deficiencies, while Pgap2 ablations do not grossly disrupt neurodevelopment. Additionally, we demonstrate that Piga expression is essential in the cerebellum for survival. These findings reveal mechanisms underlying several inherited GPI deficiency brain phenotypes and will inform targeted therapeutic strategies.