Mouse Models of PI(3,5)P2 Deficiency with Impaired Lysosome Function

Abstract

The endo-lysosomal system and autophagy are essential components of macromolecular turnover in eukaryotic cells. The low-abundance signaling lipid PI(3,5)P2 is a key regulator of this pathway. Analysis of mouse models with defects in PI(3,5)P2 biosynthesis have revealed the unique dependence of the mammalian nervous system on this signaling pathway. This insight led to the discovery of the molecular basis for several human neurological disorders, including Charcot-Marie-Tooth Disease and Yunis-Varon Syndrome. Spontaneous mutants, conditional knockouts, transgenic lines and gene-trap alleles of Fig4, Vac14 and Pikfyve (Fab1) in the mouse have provided novel information regarding the role of PI(3,5)P2 in vivo. This review summarizes what has been learned from mouse models and highlights the utility of manipulating complex signaling pathways in vivo.

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

“…The localization and levels of each PtdInsP depends on the targeting and activity of numerous lipid kinases, phosphatases and phospholipases that mediate the synthesis and turnover of each PtdInsP 3,4 . Hence, misregulation of the PtdInsP regulatory machinery can perturb cell function, leading to diseases such as cancer and degenerative diseases 2,5,6 . To fully understand the roles and functions of PtdInsPs and their regulatory machinery, both microscopybased and biochemical-based techniques have been developed to track and quantify PtdInsPs.…”

“…Atg18 is also a negative regulator of PtdIns(3,5)P 2 since the deletion of its gene, ATG18, causes a 10 to 20-fold increase in PtdIns(3,5)P 2 levels 29,30 . Overall, changes in the levels of PtdIns(3,5)P 2 severely impacts the function of the yeast vacuole and the mammalian lysosomes, consequently affecting processes such as membrane trafficking, phagosome maturation, autophagy and ion transport 6,19,21,31 . This article describes the process of radioisotope labeling of PtdInsPs with 3 H-myo-inositol in yeast to detect the relative levels of PtdIns(3,5)P 2 in wild-type, vac14Δ and atg18Δ yeast strains.…”

The Protein Complex of Neurodegeneration-related Phosphoinositide Phosphatase Sac3 and ArPIKfyve Binds the Lewy Body-associated Synphilin-1, Preventing Its Aggregation

“…The rationale for this prediction stems from findings for the removal of Sph1 aggregates by constitutive autophagy in SH-SY5Y cells (42) and for arrested autophagic progression in Sac3-deficient cells (7). Moreover, the ArPIKfyve-Sac3 complex, alone or with PIKfyve, has been reported to be essential for the proper performance of the endolysosomal system (5).…”

“…This prevents rapid clearance of Sac3 by the ubiquitin-proteasome system, extending the half-life and increasing the steady-state levels of the phosphatase (6). Concordantly, knockdown or knock-out of ArPIKfyve results in reduced or undetectable levels of Sac3 (1,(5)(6)(7). Furthermore, work by us and others has found that the primary molecular defect underlying the pathogenesis of hereditary demyelinating neuropathy CMT4J, a disease caused by compound heterozygosity with one null Sac3 allele and one missense Ile 41 -to-Thr mutation, is a failure in the ArPIKfyvedependent protection mechanism toward the Sac3…”

“…liver, kidney, lung, heart, etc. (7). By contrast, pikfyve hypomorphic mice, which also die soon after birth, have prominent defects not only in the brain but also in all organs tested, including heart, lung, kidney etc., whereas a complete pikfyve systemic disruption results in preimplantation mouse embryo death (17,20).…”