Mice deficient in Epg5 exhibit selective neuronal vulnerability to degeneration

Abstract

The molecular mechanism underlying the selective vulnerability of certain neuronal populations associated with neurodegenerative diseases remains poorly understood. Basal autophagy is important for maintaining axonal homeostasis and preventing neurodegeneration. In this paper, we demonstrate that mice deficient in the metazoan-specific autophagy gene Epg5/epg-5 exhibit selective damage of cortical layer 5 pyramidal neurons and spinal cord motor neurons. Pathologically, Epg5 knockout mice suffered muscle denervation, myofiber atrophy, late-onset progressive hindquarter paralysis, and dramatically reduced survival, recapitulating key features of amyotrophic lateral sclerosis (ALS). Epg5 deficiency impaired autophagic flux by blocking the maturation of autophagosomes into degradative autolysosomes, leading to accumulation of p62 aggregates and ubiquitin-positive inclusions in neurons and glial cells. Epg5 knockdown also impaired endocytic trafficking. Our study establishes Epg5-deficient mice as a model for investigating the pathogenesis of ALS and indicates that dysfunction of the autophagic-endolysosomal system causes selective damage of neurons associated with neurodegenerative diseases.

Figure 1.

Epg5^−/− mice show motor deficits. (A) Scheme for generating Epg5 knockout mice. HSVTK, herpes simplex virus thymidine kinase; Neo, neomycin. (B) Epg5 protein and the wild-type copy of Epg5 are absent in Epg5^−/− mice. There is an intervening lane between Epg5^+/− and Epg5^−/− lanes in the Western blot. (C) Body weight curves of Epg5^+/− and Epg5^−/− males. Means ± SEM of 12 mice are shown. (D) Time spent on a rotarod. The horizontal lines indicate mean times. (E) Epg5^−/− males show gradually worsening limb-clasping reflexes. (F) The hindquarters of Epg5^−/− mice are completely paralyzed at 10 mo of age. (G) Spinal deformation in an Epg5^−/− male at 10 mo of age. (H) Epg5^−/− females gradually develop the limb-clasping phenotype.

Figure 2.

Defects in brain and spinal cord in Epg5^−/− mice. (A) Nissl staining of the fifth layer of the cortex in 10-mo-old Epg5^+/− and Epg5^−/− mice. (B) The number of pyramidal cells in the fifth layer per millimeters squared. (C and D) H&E staining of the hippocampus shows reduced pyramidal cell numbers in Epg5^−/− mice. Means ± SEM of three mice are shown in B and D. (E) The normally developed cerebellum in Epg5^−/− mice. (F) The thickness of the molecular layer in the cerebellum in mutant and control mice. (G and H) Purkinje cells, stained by calbindin, are not reduced in number in Epg5^−/− mice. Means ± SEM of five mice are shown in F and H. (I) Arrows indicate Pyknotic neurons in the anterior horn of Epg5^−/− mice. (J) The number of motor neurons in the spinal cord (SC) in Epg5^+/− and Epg5^−/− mice. (K) The numbers of Nissl-stained interneurons in the spinal cord are similar in control and Epg5^−/− mice. Means ± SEM of three mice are shown in J and K. (L) Eosinophilic spheroids (arrows) accumulate in the thoracic spinal cord of Epg5^−/− mice. (M) Eosinophilic spheroids are swollen degenerating axons (arrow). (N) EM pictures showing degenerated axons (arrows) in the spinal cord of Epg5^−/− mice. (O and P) GFAP staining in Epg5^+/− and Epg5^−/− mice. DCST, dorsal corticospinal tract. Bars: (A, C, L, and O) 50 µm; (E) 500 µm; (G) 20 µm; (I) 100 µm; (M and P) 10 µm; (N) 2 µm.

Figure 3.

Accumulation of LC3-II, p62 aggregates, and ubiquitin-positive inclusions in Epg5 knockout mice. (A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5^+/− and Epg5^−/− mice. SC, spinal cord. (B) Polyubiquitinated proteins in detergent-soluble (S) and -insoluble (I) fractions from brain and spinal cord homogenates of Epg5^+/− and Epg5^−/− mice. (C) p62 aggregates and ubiquitin-positive aggregates are absent from Epg5^+/− mice but dramatically accumulate and are colocalized (arrows) in Epg5^−/− mice. (D–F) p62 aggregates are detected in neurons (arrows; D) and oligodendrocytes (arrows; E) but absent from astrocytes (F) in Epg5^−/− mice. (G) Cytoplasmic TDP-43 aggregates accumulate in motor neurons in the anterior horn of spinal cord in Epg5^−/− mice. Arrows show cytoplasmic TDP-43 aggregates. DSCT, dorsal corticospinal tract. (H) Percentages of cytoplasmic TDP-43 aggregate-positive motor neurons in the fifth layer of cortex and anterior horn of the spinal cord in Epg5^+/− and Epg5^−/− mice. Means ± SEM of three mice are shown. LC, lateral column. Bars, 10 µm.

Figure 4.

Epg5 knockout mice show muscle atrophy. (A and B) Electromyography of the gastrocnemius muscle from a 3-mo-old Epg5^−/− mouse showing fibrillations (A) and positive sharp waves (B). These defects were absent in control mice. (C) At 10 mo, Epg5^−/− mice showed high-amplitude and long-duration action potentials when conducting MUAP tests. The blue lines show the start and end of an action potential. Dur, duration; Amp, amplitude. (D–F) H&E staining of gastrocnemius muscles showed features of muscle degeneration in Epg5^−/− mice. The arrow in E indicates centrally nucleated fibers. (G) The transcription levels of the atrophy-related genes were up-regulated in gastrocnemius muscles of Epg5^−/− mice. (H and I) Electron micrographs of Epg5^+/− and Epg5^−/− muscles. The arrows indicate abnormal enlarged mitochondria. (J) Levels of LC3-II and p62 in muscle extracts from Epg5^+/− and Epg5^−/− mice. (K) Percentage of p62 aggregate-positive myofibers in Epg5^+/− and Epg5^−/− mice. Means ± SEM of three mice are shown. (L) Accumulation and colocalization of p62 and ubiquitin aggregates in the gastrocnemius muscles of Epg5^−/− mice but not controls. Bars: (D–F and L) 10 µm; (H and I) 1 µm.

Figure 5.

Epg5 deficiency causes a defect in autophagosome maturation and impairs endocytic trafficking. (A) Number of LC3 puncta in Epg5^+/− (control) and Epg5^−/− MEFs. (B) Western blot showing levels of LC3-I and LC3-II in control and Epg5^−/− MEFs upon indicated treatment (Starv., starvation; Rapa., rapamycin; Bafilo., bafilomycin A1). (C) Proteinase K (ProK) protection assay in different MEFs. PNS, postnuclear supernatant; P, pellet fractions. (D) Colocalization ratio of LC3 and LAMP-1 in control and Epg5^−/− MEFs. 50 cells were examined per time point in A and D. (E) Colocalization of LC3 puncta and lysosomes in control and Epg5^−/− MEFs 4 h after starvation. (F) Under nutrient repletion conditions, almost no autophagic elements are detected in Epg5^+/− MEFs. RER, rough ER; M, mitochondrion; N, nucleus. The arrowhead indicates a vacuole with a late residual body-like appearance (aAV-III). (G) Under nutrient repletion conditions, Epg5^−/− MEFs accumulate a large number of autophagic vacuoles. Red arrow, a likely autophagosome; red arrowheads, complex vacuoles of the aAV-I type; white arrowheads, aAV-II vacuoles. (H and I) High magnification of autophagic elements in unstarved Epg5^−/− MEFs. (H) The red arrow indicates a complex autophagic vacuole containing multilayered membrane structures (white arrowheads) and a lipid-like region (L). (I) The red arrow indicates an autophagosome containing cytoplasm and ribosomes. The red arrowhead points to an autophagosome containing a mitochondrion and cytoplasmic material with membranous structures. The white arrow shows an aAV-III vacuole. (J) Compared with control cells, Rab5-labeled early endosomes are significantly enlarged in Epg5 siRNA-treated cells. (K and L) EGFR degradation in control and Epg5 knockdown cells. (M and N) Distribution of Alexa Fluor 488–conjugated transferrin at different time points in control siRNA- and Epg5 siRNA-treated cells. Higher magnification views are shown in the insets. Error bars indicate SEMs. Bars: (E, J, M and N, main images) 10 µm; (F and G) 2 µm; (H and I) 100 nm; (J, M, and N, insets) 1 µm.