c-Abl Activation Linked to Autophagy-Lysosomal Dysfunction Contributes to Neurological Impairment in Niemann-Pick Type A Disease

Abstract

Niemann-Pick type A (NPA) disease is a fatal lysosomal neurodegenerative disorder caused by the deficiency in acid sphingomyelinase (ASM) activity. NPA patients present severe and progressive neurodegeneration starting at an early age. Currently, there is no effective treatment for this disease and NPA patients die between 2 and 3 years of age. NPA is characterized by an accumulation of sphingomyelin in lysosomes and dysfunction in the autophagy-lysosomal pathway. Recent studies show that c-Abl tyrosine kinase activity downregulates autophagy and the lysosomal pathway. Interestingly, this kinase is also activated in other lysosomal neurodegenerative disorders. Here, we describe that c-Abl activation contributes to the mechanisms of neuronal damage and death in NPA disease. Our data demonstrate that: 1) c-Abl is activated in-vitro as well as in-vivo NPA models; 2) imatinib, a clinical c-Abl inhibitor, reduces autophagy-lysosomal pathway alterations, restores autophagy flux, and lowers sphingomyelin accumulation in NPA patient fibroblasts and NPA neuronal models and 3) chronic treatment with nilotinib and neurotinib, two c-Abl inhibitors with differences in blood-brain barrier penetrance and target binding mode, show further benefits. While nilotinib treatment reduces neuronal death in the cerebellum and improves locomotor functions, neurotinib decreases glial activation, neuronal disorganization, and loss in hippocampus and cortex, as well as the cognitive decline of NPA mice. Our results support the participation of c-Abl signaling in NPA neurodegeneration and autophagy-lysosomal alterations, supporting the potential use of c-Abl inhibitors for the clinical treatment of NPA patients.

Keywords: Niemann-Pick disease; autophay-lysosomal pathway; c-Abl kinase; lysosomal storage disorder (LSD); neurodegeneration.

FIGURE 1

c-Abl activation regulates autophagy and lysosomal alterations in NPA fibroblasts. (A) Human fibroblasts homogenates from NPA patient and healthy control (HC) subjects (50 μg protein/lane) were used to measure p-c-Abl levels. The graph shows quantifications of p-c-Abl levels normalized by GAPDH and c-Abl expression. The data shown are from three independent experiments. Student’s t-test: *p < 0.05. (B) HC and NPA fibroblasts were fixed and immunostained using an anti-p-c-Abl Tyr412 antibody (green) and Hoechst staining for nucleus (blue). For each condition, n = 15 cells were measured by experiment; three independent experiments were performed. Student’s t-test: ****p < 0.0001. (C) HC and NPA fibroblasts were incubated with BODIPY-SM to confirm sphingomyelin accumulation. The images were taken with a × 40 objective lens. (D) Homogenates from fibroblasts from NPA patient and healthy control (HC) subject (50 μg protein/lane) were used to measure p62 and LC3II levels. The graph shows quantifications of protein levels normalized by GAPDH and LC3I expression, respectively. The image is representative of five independent experiments. Student’s t-test: **p < 0.01; ***p < 0.001; (E) HC and NPA fibroblasts were treated with imatinib (10 μM) for 24 h, fixed, and immunostained using an anti-p62 antibody (green) and (F) anti-Lamp1 (green). Hoechst staining for the nucleus (blue). For each condition, n = 10–18 cells were measured by experiment; three independent experiments were performed. ANOVA, Tukey post-hoc: ***p < 0.001; ****p < 0.0001. In the box-and-whisker plots, the center line denotes the median value, edges are upper and lower quartiles and whiskers show minimum and maximum values.

FIGURE 2

c-Abl inhibition improves autophagy flux and decreases sphingomyelin accumulation in NPA neuronal models. (A) p-c-Abl levels were measured in Neural Stem Cells (NSCs) extracts by Western blot. Images representative from four independent experiments are shown. Student’s t-test: *p < 0.05. (B) BODIPY-SM staining indicates sphingomyelin accumulation. Fluorescent microscopic images of NPA NSCs treated imatinib 0.001 µM by 24 h. For each condition, n = 150 cells were measured by experiment; three independent experiments were performed. Student’s t-test: **p < 0.01. (C) NPA NSCs expressing mRFP-GFP-LC3 were treated with imatinib 0.001 µM by 24 h or vehicle. Graph shows the rate between RFP intensity and GFP intensity corresponding to autolysosomes. For each condition, n = 50 cells were measured by experiment; three different experiments were performed. Student’s t-test: *p < 0.05. (D) Primary neurons were 7 days in vitro, fixed, and immunostained using anti-p-c-Abl Tyr412 antibody (red) and Hoechst staining for nucleus (blue). For each condition, n = 10–20 neurons were measured by experiment; three independent experiments were performed. Student’s t-test: *p < 0.05. (E) Primary hippocampal neurons were treated with imatinib 5 µM by 24 h. Sphingomyelin accumulation was analyzed by BODIPY-SM. For each condition, n = 10–20 neurons were measured by experiment; three independent experiments were performed. ANOVA, Tukey post-hoc: ****p < 0.0001. In the box-and-whisker plots, the center line denotes the median value, edges are upper and lower quartiles and whiskers show minimum and maximum values.

FIGURE 3

Acute imatinib treatment decreases neuronal death in the cerebellum and improves locomotor function in NPA mice. (A) WT and NPA brain homogenates (50 μg protein/lane) from mice at 4 weeks old were analyzed by Western blot. The graph shows quantifications of p-c-Abl levels normalized by GAPDH and c-Abl levels. The number of animals was: WT = 3; NPA = 4; Student’s t-test: *p < 0.05. (B) WT and NPA mice were i.p. injected with imatinib (12.5 mg/kg) or vehicle from 3 weeks of age until 7 weeks of age. The Purkinje neuron marker calbindin was analyzed by immunohistochemistry. A quantification of calbindin-immunoreactive Purkinje cell bodies in cerebellar sections is shown. (C) CD68 marker was evaluated by immunofluorescence analysis in the cerebellum from WT and NPA mice. For (B,C), the number of animals was WT control = 3; NPA control = 5; NPA imatinib = 4. Images were taken with × 4 objective. ANOVA, Tukey post-hoc: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (D) Mice were treated for 4 weeks and motor coordination was assessed weekly by the hanging test. Data are shown as mean ± SEM. ANOVA, Tukey post-hoc: *p < 0.05, NPA control is statistically different from WT control and NPA with imatinib. The following number of animals was used: WT control = 11; WT imatinib = 10; NPA control = 8; NPA imatinib = 9. In the box-and-whisker plots, the center line denotes the median value, edges are upper and lower quartiles, whiskers show minimum and maximum values and points are individual experiments.

FIGURE 4

Chronic c-Abl inhibition treatment delays locomotor impairment in NPA mice. WT and NPA mice received nilotinib (200 ppm; 30 mg/kg) and neurotinib (67 ppm; 10 mg/kg) supplemented diets or control diet starting at p21 until 11 months of age. (A) p-c-Abl protein levels were evaluated in cerebellum homogenates from WT and NPA mice of 5 months of age by Western blot. The number of animals was three by condition. ANOVA, Tukey post-hoc:*p < 0.05. (B) Motor coordination was assessed weekly by the Hanging test. Data are shown as mean ± SEM. ANOVA, Tukey post-hoc: ****p < 0.0001; NPA control is statistically different from WT control and nilotinib NPA. (C) Deterioration curve of mice was performed using percent of mice with a score equal to or above 3. For (B,C), the following number of animals was used: WT control (Ctrl) = 10; NPA control (Ctrl) = 10; NPA nilotinib (Nilo) = 11; NPA neurotinib (Neuro) = 10. (D) Purkinje neuron marker Calbindin was analyzed by immunohistochemistry. Calbindin intensity was quantified. A representative image by condition is shown (n = 3 mice/group). Images were taken with × 2 objective. ANOVA, Tukey post-hoc: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. In the box-and-whisker plots, the center line denotes the median value, edges are upper and lower quartiles, whiskers show minimum and maximum values and points are the number of animals used.

FIGURE 5

Chronic c-Abl inhibition treatment improves cognitive decline and decreases brain neuronal disorganization in NPA mice. WT and NPA mice received nilotinib (200 ppm) and neurotinib (67 ppm) supplemented diets or control diet starting at p21 until 7 months of age. (A) Memory flexibility test was used to evaluate cognitive functions. Graphs show the number of trials every day during the test (A) and the average among 4 days of test (B). The number of animals was: WT control (Ctrl) diet = 4; WT nilotinib (Nilo) diet = 5; WT neurotinib (Neuro) diet = 6; NPA control diet = 6; NPA nilotinib (Nilo) diet = 5; NPA neurotinib (Neuro) diet = 5. ANOVA, Tukey post-hoc: *p < 0.05; **p < 0.01; ***p < 0.001 (C) Coronal sections of WT and NPA brain were stained with anti-NeuN antibody and 3,39-diaminobenzidine as chromogen. The rectangles in the first photo indicate where the magnification shown in the following photos comes from. Arrows point to actual discontinuities. Graph bars indicating discontinuity differences between WT and NPA mice with treatment in hippocampal subfields CA1, CA2, and CA3. Data are shown as the number of discontinuities/animal. The following number of animals was used: WT control = 4; NPA control = 4; NPA nilotinib = 3; NPA neurotinib = 4. ANOVA, Tukey post-hoc: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (D) Coronal sections were stained with calbindin which is a member of the large EF-hand family of calcium-binding proteins. These staining methods evidenced well-defined layers in the cortex and hippocampus structure. Neuronal disorganization with a decreased number of neurons is evident. The rectangles in the first photo indicate where the magnification shown in the following photos comes from. Image representative is shown. The following number of animals was used: WT control = 3; NPA control = 3; NPA nilotinib = 3; NPA neurotinib = 3. ANOVA, Tukey post-hoc: *p < 0.05; **p < 0.01; ****p < 0.0001. (E) Cortex neurons of the brains from WT and NPA mice were stained with NeuN antibody and 3,39-diaminobenzidine as chromogen. The area of the neuronal body was measured. 50 cells were measured by each mouse. The following number of animals was used: WT control (Ctrl) = 4; NPA control (Ctrl) = 3; NPA nilotinib = 3; NPA neurotinib = 4. ANOVA, Tukey post-hoc: *p < 0.05; **p < 0.01. (F) Slices were stained with Filipin staining to evaluate lipid accumulation. The following number of animals was used: WT control (Ctrl) = 3; NPA control (Ctrl) = 3; NPA nilotinib = 3; NPA neurotinib = 3. Image representative. ANOVA, Tukey post-hoc: *p < 0.05; ****p < 0.0001. Scale bar = 50 μm. In the box-and-whisker plots, the center line denotes the median value, edges are upper and lower quartiles, whiskers show minimum and maximum values and points are individual experiments.

FIGURE 6

Chronic c-Abl inhibition decreases glial activation in NPA mice. WT and NPA mice received nilotinib (200 ppm; 30 mg/kg) and neurotinib (67 ppm; 10 mg/kg) supplemented diets or control diet starting at p21 until 7 months of age. (A) Markers of astrocyte (GFAP) and microglia (Iba-1) were analyzed by immunofluorescence in slices of brain from WT and NPA mice. Confocal images were obtained of the cortex for each condition. A representative image of the cortex is shown in the first row; astrocytes (cyan hot), microglia (orange hot), and nucleus (grays). Representative images to visualize astrocyte and microglial shape are shown in the second and third row, respectively. (B) Astrocyte area was measured from GFAP positive cells. For each condition, n = 10 cells were measured by animal; three mice/group. ANOVA, Tukey post-hoc: **p < 0.01, ***p < 0.001. (C) Microglia shape was determined from Iba-1 fluorescence. For each condition, n = 5–10 cells were measured by animal; three mice/group. ANOVA, Tukey post-hoc: **p < 0.01, ****p < 0.0001. In the box-and-whisker plots, the center line denotes the median value, edges are upper and lower quartiles and whiskers show minimum and maximum values.