Macroautophagy is defective in mucolipin-1-deficient mouse neurons

Abstract

Mucolipidosis type IV is a neurodegenerative lysosomal disease clinically characterized by psychomotor retardation, visual impairment, and achlorhydria. In this study we report the development of a neuronal cell model generated from cerebrum of Mcoln1(-/-) embryos. Prior functional characterization of MLIV cells has been limited to fibroblast cultures gleaned from patients. The current availability of the mucolipin-1 knockout mouse model Mcoln1(-/-) allows the study of mucolipin-1-defective neurons, which is important since the disease is characterized by severe neurological impairment. Electron microscopy studies reveal significant membranous intracytoplasmic storage bodies, which correlate with the storage morphology observed in cerebral cortex of Mcoln1(-/-) P7 pups and E17 embryos. The Mcoln1(-/-) neuronal cultures show an increase in size of LysoTracker and Lamp1 positive vesicles. Using this neuronal model system, we show that macroautophagy is defective in mucolipin-1-deficient neurons and that LC3-II levels are significantly elevated. Treatment with rapamycin plus protease inhibitors did not increase levels of LC3-II in Mcoln1(-/-) neuronal cultures, indicating that the lack of mucolipin-1 affects LC3-II clearance. P62/SQSTM1 and ubiquitin levels were also increased in Mcoln1(-/-) neuronal cultures, suggesting an accumulation of protein aggregates and a defect in macroautophagy which could help explain the neurodegeneration observed in MLIV. This study describes, for the first time, a defect in macroautophagy in mucolipin-1-deficient neurons, which corroborates recent findings in MLIV fibroblasts and provides new insight into the neuronal pathogenesis of this disease.

Figure 1

Mcoln1^+/+ and Mcoln1^-/- embryonic neuronal cultures. (A-J) Mcoln1^+/+ and Mcoln1^-/- E17 embryonic-derived neuronal cultures after 10 days in vitro, (A and F) Phase contrast microscopy shows neuronal cultures from Mcoln1^+/+ and Mcoln1^-/-, respectively, LysoTracker-stained (red) cells showing presence of larger vesicles in Mcoln1^-/- (G and H in Mcoln1^-/-vs. B and C in Mcoln1^+/+), Lamp1-immunolabelled cells (green) showing presence of larger vesicles in Mcoln1^-/- (I and J in Mcoln1^-/-vs. D and E in Mcoln1^+/+). Bar indicates 50 microns (A, B, D, F, G and I) and 20 microns (C, E, H and J).

Figure 2

Mcoln1^-/- storage bodies in post natal day 7 Mcoln1^-/- pups cortex versus Mcoln1^-/- neuronal cultures. (A and B) (A) Electron microscopy of Mcoln1^-/- embryonic neuronal cultures after 10 days in culture (bar indicates 2 microns) (B) Magnification view of storage body (bar indicates 500 nm). (C and D) (C) Electron microscopy of embryonic cortex from E17 embryos (bar indicate 2 microns) (D) Magnification view of storage body (bar indicates 500 nm). (E and F) (E) Electron microscopy of post natal day 7 pup Mcoln1^-/- brain cortex (bar indicates 2 microns) (F) view of storage body (bar indicates 500 nm).

Figure 3

LC3-II levels in embryonic neuronal cultures (A and C) and human MLIV-affected fibroblasts (B and D). Cell lysates were analyzed by Western blot with anti-LC3A Abgent antibody. LC3-II levels were determined using the average of band densitometry value, divided by β-tubulin (n=4 for primary neuronal cultures and MLIV fibroblasts, n=2 for control fibroblast).

Figure 4

LC3-II turnover in Mcoln1^+/+ (A and B) and Mcoln1^-/- (C and D) neuronal cultures before and after protease inhibitors (Pepstatin and E64) and rapamycin treatments. On graphs B and D, LC3-II levels were determined using the average of band densitometry value, divided by β-tubulin (n=3).

Figure 5

P62/SQSTM1 in detergent-insoluble cell fraction (A and B) and Ubiquitin (C) levels are upregulated in Mcoln1^-/- mouse neuronal cultures. P62/SQSTM1 levels in detergent-soluble and insoluble fractions were determined using the average of each band densitometry value, divided by β-tubulin (n=4).