Autophagy and Cell-Autonomy in Mouse Models of PI(3,5)P2 Deficiency

Abstract

FIG4 is the 5-phosphatase required for synthesis and turnover of the membrane signaling lipid PI(3,5)P2 (phosphatidylinositol-(3,5)-bisphosphate). Mutation of Fig4 causes neurodegeneration in mice and the neuropathy Charcot-Marie-Tooth type 4J (CMT4J) in humans. Fig4 null mice (pale tremor) exhibit juvenile lethality and a severe movement disorder due to spongiform neurodegeneration of the CNS. To examine disease pathogenesis at the subcellular level, we investigated macroautophagy (autophagy), a lysosomal pathway for degradation of long-lived cytoplasmic substrates that requires the PI(3,5)P2 precursor PI3P and is critical for neuronal survival. We observed accumulation of the autophagy markers p62, LC3-II, LAMP-1, LAMP-2 and ubiquitin in brain of Fig4 null mice with PI(3,5)P2 deficiency. These markers were colocalized within cytoplasmic inclusions in many reactive astrocytes and within a small number of neurons. Accumulation of the tumor-suppressor p53 in an aggregated conformation was also observed. Ultrastructural characterization of Fig4 null cortex revealed cells with striking accumulation of heterogeneous, electron-dense autophagic vacuoles, and other cells with apparently empty cytoplasmic vacuoles. Accumulation of undigested material does not result from defective acidification of lysosomes in Fig4 null mice. Knockout of p62 (Sqstm1) did not reduce lethality or prevent inclusion formation in Fig4/p62 double null mice. The observed late-stage blockade in bulk autophagy demonstrates a novel role for PI(3,5)P2 in the lysosomal clearance of autophagy substrates. To distinguish between cell-autonomous effects of PI(3,5)P2 deficiency in neurons and astrocytes, we generated transgenic Fig4 null mice with FIG4 expression driven by the neuron-specific enolase (NSE) promoter or the astrocyte-specific GFAP promoter. Neuronal expression of FIG4 rescued the juvenile lethality of Fig4 null mice and prevented spongiform degeneration and motor dysfunction. Hypomyelination of the CNS and peripheral nerves was also rescued. Expression of FIG4 in astrocytes prevented the accumulation of autophagy intermediates but did not prevent neurodegeneration or motor dysfunction. Infiltration of microglia was reduced by expression of FIG4 in either astrocytes or neurons. The data demonstrate that PI(3,5)P2 has cell-autonomous functions in both neurons and glia, but neurons are responsible for the major pathological effects in Fig4 mutation in mice. The primary role of neuronal Fig4 expression has implications for therapeutic approaches to CMT4J.