Clec16a is Critical for Autolysosome Function and Purkinje Cell Survival

Abstract

CLEC16A is in a locus genetically linked to autoimmune diseases including multiple sclerosis, but the function of this gene in the nervous system is unknown. Here we show that two mouse strains carrying independent Clec16a mutations developed neurodegenerative disease characterized by motor impairments and loss of Purkinje cells. Neurons from Clec16a-mutant mice exhibited increased expression of the autophagy substrate p62, accumulation of abnormal intra-axonal membranous structures bearing the autophagy protein LC3, and abnormal Golgi morphology. Multiple aspects of endocytosis, lysosome and Golgi function were normal in Clec16a-deficient murine embryonic fibroblasts and HeLa cells. However, these cells displayed abnormal bulk autophagy despite unimpaired autophagosome formation. Cultured Clec16a-deficient cells exhibited a striking accumulation of LC3 and LAMP-1 positive autolysosomes containing undigested cytoplasmic contents. Therefore Clec16a, an autophagy protein that is critical for autolysosome function and clearance, is required for Purkinje cell survival.

Figure 1. Mutation of Clec16a in mice induces locomotion deficits.

(a) Image demonstrating normal and aberrant responses hind limb clasping in wild-type and homozygous Clec16a^GT mice. (b) Age of mice during the hind limb test when they either scored a 1 (temporary clasping of one or more hind limb to body <30 seconds) or 2 (sustained clasping of both hind limbs to body <30 seconds). (c) Age of mice at which they dropped from the cage lid within 20 seconds of the lid being inverted. In (b) number of mice/group indicated in each graph; data were analyzed by Log-Rank (Mantel-Cox) test; ****P < 0.0001.

Figure 2. Mutation of Clec16a results in neurodegeneration characterized by loss of Purkinje cells.

(a–d) H & E (a–c) or calbindin immunofluorescence images (d) of brains of 8 week old B6.Clec16a^GT mice (representative of n = 3 mice/group). Arrows indicate loss of Purkinje cells.

Figure 3. Lack of Clec16a results in LC3 and p62 accumulation and ultrastructural abnormalities in cerebellar neurons.

(a) Representative western blot of LC3 protein levels in brain or cerebellum from 8 week old B6.Clec16a^GT mice (representative of n = 3 mice/group). (b–e) Images of immunofluorescence for calbindin and p62 (b) and transmission electron microscopy (c–e) from the deep cerebellar nuclei of 8 week old B6.Clec16a^GT mice (representative of n = 3 mice/group). Images highlighting intact Golgi stacks and enlarged endomembrane compartment in neurons (c,d) and axons (e) in the deep cerebellar nuclei. Golgi stacks and compartment outlined in red dotted lines (d). (f) Images of cryo-immunoelectron microscopy from immunogold labeling for LC3 (12 nm gold) in section of one axon of Purkinje cell of 8 week old B6.Clec16a^GT mice (representative of n = 3 mice/group). Mitochondria are indicated (M).

Figure 4. Increased LC3 and p62 in Clec16a-mutant MEFs and HeLa cells.

(a,b) B6.Clec16a^GT MEFs, untreated or treated with 40 μm chloroquine were imaged by confocal microscopy (a) and the number of LC3+ puncta/cell was quantified (b) (representative of n = 3 experiments, minimum 100 cells/condition quantified/experiment). (c–f). HeLa-CLEC16A cells, untreated or treated with 40 μm chloroquine were imaged by confocal microscopy (c,e) and the number of GFP-LC3+ (d) or p62+ (f) puncta/cell was quantified (representative of n = 3–5 experiments, minimum 100 cells/condition quantified/experiment). (g,h) A representative western blot of p62 in indicated cells starved (Starv) or treated with Bafilomycin A1 (BAF) (g), quantified by p62 levels over tubulin (h) (representative of n = 3 experiments). Data represent mean+/− s.e.m. and are analyzed by unpaired Student’s t-test; *P < 0.5, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns =  not significant. Scale bars indicate 25μm (a,c,e).

Figure 5. Functional endolysosomal system in Clec16a-mutant cells.

(a) Confocal microscopy images of HeLa-CLEC16A cells stained for Rab5 and Rab7 (representative of n = 3 experiments). (b,c) EGF receptor degradation assay in control cells or cells with reduced CLEC16A protein by siRNA transfection (representative of n = 3 experiments). (d) Co-culture assays measuring T-cell proliferation in response to B6.Clec16a^GT macrophages pulsed with Listeria monocytogenes–specific peptide (representative of n = 4 experiments).

Figure 6. Structure and function of the Golgi apparatus in Clec16a-deficient cells.

(a–c) Confocal microscopy images of HeLa-CLEC16A cells stained for GM130, ERGIC-53 (a) and calnexin (c) and quantification of percent of cells/field with dispersed Golgi apparatus morphology (b) (representative of n = 3 experiments, minimum 60 cells/genotype quantified/experiment, data represent mean+/− s.e.m. and were analyzed by unpaired Student’s t-test; ****P < 0.0001). (d,e) A representative western blot for CLEC16A and Rab1b protein in HeLa C1 transfected cells utilized in hGH-GFP secretion assay (d). Remaining intracellular GFP (relative to time = 0) in HeLa C1 cells transfected with control, CLEC16A or RAB1A/B siRNA (E) (representative of n = 2 experiments). (f) A representative western blot of LAMP-1 in EndoH and PNGase digested HeLa-CLEC16A cells (representative of n = 4 experiments).

Figure 7. Clec16a-deficient HeLa cells have increased LAMP-1 and LC3 labeled single membrane bound structures.

(a) Transmission electron microscopy images of HeLa-CLEC16A cells (representative of n = 3 experiments). Black arrow indicates membrane bound structures containing cellular debris. (b) Cryo-immunoelectron microscopy images of immunogold labeling for LAMP-1 (12 nm gold) and LC3 (18 nm gold) in HeLa-CLEC16A cells (representative of n = 3 experiments). Red dotted lines indicate membrane bound structures containing cellular debris. (c,d) HeLa-CLEC16A cells, untreated or treated with 100 nm bafilomycin A1 (BAF), were imaged by confocal microscopy (c) and colocalization of GFP-LC3 and LAMP-1 was evaluated by Pearson’s Correlation Coefficient (d) (representative of n = 5 experiments, data represent mean+/− s.e.m. and were analyzed by unpaired two-tailed Student’s t-test; *P > 0.5, **P > 0.01; ns =  not significant).

Figure 8. Functional lysosomes in CLEC16A-mutant cells.

(a,b) Confocal microscopy images of HeLa-CLEC16A cells stained for LAMP-1 (a) and quantification of LAMP-1+ puncta/cell (b) (representative of n = 5 experiments, minimum 100 cells/genotype quantified/experiment, data represent mean+/− s.e.m. and were analyzed by unpaired Student’s t-test; ns =  not significant). (c) Measurement of lysosomal pH with Lysosensor in untreated or chloroquine-treated B6.Clec16a^GT MEFs (representative of n = 3 experiments, data represent mean+/− s.e.m. and were analyzed by unpaired Student’s t-test; ****P > 0.0001, ns =  not significant). (d,e) Intracellular and extracellular enzyme activity of lysosomal acid hydrolases, α-Mannosidase and β-Hexosaminidase, in HeLa-CLEC16A cells (representative of n = 3 experiments, data represent mean +/− s.e.m. and were analyzed by unpaired Student’s t-test, ns = not significant).