Defective autophagy in neurons and astrocytes from mice deficient in PI(3,5)P2

Abstract

Mutations affecting the conversion of PI3P to the signaling lipid PI(3,5)P(2) result in spongiform degeneration of mouse brain and are associated with the human disorders Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis (ALS). We now report accumulation of the proteins LC3-II, p62 and LAMP-2 in neurons and astrocytes of mice with mutations in two components of the PI(3,5)P(2) regulatory complex, Fig4 and Vac14. Cytoplasmic inclusion bodies containing p62 and ubiquinated proteins are present in regions of the mutant brain that undergo degeneration. Co-localization of p62 and LAMP-2 in affected cells indicates that formation or recycling of the autolysosome is impaired. These results establish a role for PI(3,5)P(2) in autophagy in the mammalian central nervous system (CNS) and demonstrate that mutations affecting PI(3,5)P(2) can contribute to inclusion body disease.

Figure 1.

Markers of autophagy are elevated in brain from Fig4^−/− brain. Total brain homogenates were examined by western blotting with antibodies to components of the autophagy pathway. (A) Accumulation of p62 in mutant brain and spinal cord (left and middle panels); quantitative comparison (right panel). (B) qRT-PCR analysis of p62 transcript, relative to the Tata-binding protein transcript, in total brain RNA. Mean ± SD (n = 3 mice of each genotype, assayed in quadruplicate). (C) Normal levels of p62 protein in non-neural tissues. (D) Elevation of LC3-II in mutant brain. (E) Normal level of LC3-I in mutant brain [short exposure of the film in (D)]. (A)–(C) 30 µg protein per lane; (D)–(E) 50 µg protein per lane.

Figure 2.

Normal levels of proteins involved in initiation and mTOR-mediated regulation of autophagy. (A) beclin-1; (B) mTOR; (C) Ser2448 phospho-mTOR; (D) phosphorylated p70 S6 kinase, a downstream target of mTORC1. Thirty micrograms of protein per lane.

Figure 3.

p62 is co-localized with ubiquitin in intracellular inclusion bodies. (A) Two-color immunofluorescence of cortical sections from Fig4^−/− mouse demonstrates co-localization of ubiquitin and p62. (B and C) Peroxidase immunostaining detects immunodense cytoplasmic inclusion bodies containing p62 and ubiquitin in mutant brain that are not present in wild-type controls. (D) Ultrastructure (TEM) of Fig4^−/− brainstem at 7 days of age. (E) Western blot of soluble protein and 15 000g pellet from brain homogenates prepared in radioimmunoprecipitation buffer. Thirty micrograms of protein per lane.

Figure 4.

Accumulation of p62 in reactive astrocytes and neurons of Fig4^−/− brain. (A) Immunostaining of cortical sections demonstrates co-localization of p62 and GFAP, an astrocyte marker. (B) p62 accumulation in GFAP-positive Bergmann glia in mutant cerebellum; see also Supplementary Material, Fig. S2. (C) Western blot demonstrates increased abundance of full-length GFAP and degradation products in mutant brain; 30 µg protein per lane. (D) Lack of p62 accumulation in astrocytes in the CA1 region of the hippocampus from Fig4^+/+ epileptic brain. (E) Neurons were identified with antibody to the nuclear marker NeuN. Only a small number of NeuN-labeled nuclei are associated with cytoplasmic p62 (insert, arrowheads). (F) Most of the p62 in this section is localized in GFAP-positive astrocytes, as also seen in (A).

Figure 5.

p62-containing astrocytes in Fig4^−/− brain at 1 week of age. (A) Accumulation of p62 in GFAP-positive astrocytes in the thalamus, which exhibits spongiform degeneration at 1 week of age (3). (B) Accumulation of p62 in white matter tracts around the cerebellar nuclei, a site of extensive spongiform degeneration in older animals. (C) The cortex is unaffected at P7 (3).

Figure 6.

Accumulation of LAMP-2 in Fig4^−/− brain. LAMP-2 is a major component of lysosomal membranes, and is also present in late endosomes and autophagosomes. (A) Western blot demonstrates accumulation of LAMP-2 in mutant brain (left panel) with quantitative comparison (right panel). (B) LAMP-2 is concentrated in GFAP-positive astrocytes and not in NeuN-poistive neurons. (C) Co-localization of LAMP-2 and p62. (D) High-resolution confocal imaging (×180) demonstrates subcellular co-localization of LAMP-2 and p62. (E) In cultured cerebellar granule neurons from Fig4^−/− mice, soma and processes (insert) are filled with LAMP-2-positive vacuoles.

Figure 7.

Impaired autophagy and astrocytosis in a second mouse mutant affecting PI(3,5)P₂. (A) The protein VAC14 binds FIG4 and FAB1 kinase in the molecular complex that regulates PI(3,5)P₂ synthesis. The Vac14-L156R mutation (red) in the ingls mutant mouse results in destabilization of the complex and reduced levels of PI(3,5)P₂ (2). (B) Elevated levels of p62 and LC3-II in brain homogenates from Vac14-mutant mice. Each lane contains 30 µg of protein. (C) Co-localization of p62 and ubiquitin in ingls cortex. (D) Accumulation of p62 in astrocytes and neurons. (E) Accumulation of LAMP-2 in ingls brain; 30 µg protein per lane.

Figure 8.

Schematic depiction of maturation and resolution of the autophagosome. (A) Proteins that accumulate in brain of pale tremor (Fig4) and ingls (Vac14) mutant mice. (B) Model of maturation of autophagic vesicles, with two potential sites of slowdown or blockage that could explain the accumulation of autophagosome and lysosome marker proteins.