Intracellular accumulation of amyloidogenic fragments of amyloid-beta precursor protein in neurons with Niemann-Pick type C defects is associated with endosomal abnormalities

Abstract

Niemann-Pick type C disease (NPC) is characterized by neurodegeneration secondary to impaired cholesterol trafficking and excessive glycosphingolipid storage. Abnormal cholesterol and ganglioside metabolism may influence the generation and aggregation of amyloidogenic fragments (ie, C99 and Abeta) from amyloid-beta precursor protein (APP), crucial factors causing neurodegeneration in Alzheimer's disease. To reveal whether abnormal accumulation and aggregation of APP fragments also occurs in NPC, we studied their expression in cultured cortical neurons treated with U18666A, a compound widely used to induce NPC defects, and also in brain tissues from NPC patients. U18666A treatment resulted in increased intraneuronal levels of C99 and insoluble Abeta42, which were distributed among early and late endosomes, in compartments distinct from where endogenous cholesterol accumulates. Analyses of NPC brains revealed that C99 or other APP C-terminal fragments (APP-CTF), but not Abeta42, accumulated in Purkinje cells, mainly in early endosomes. In contrast, in hippocampal pyramidal neurons, the major accumulated species was Abeta42, in late endosomes. Similar to what has been shown in Alzheimer's disease, cathepsin D, a lysosomal hydrolase, was redistributed to early endosomes in NPC Purkinje cells, where it co-localized with C99/APP-CTF. Our results suggest that endosomal abnormalities related to abnormal lipid trafficking in NPC may contribute to abnormal APP processing and Abeta42/C99/APP-CTF deposition.

Figure 1

U18666A treatment increases the level of C99 and insoluble Aβ42 in cultured cortical neurons but not in MC65 neuroblastoma cells expressing C99. A: Cortical neurons from newborn mice were infected with Adv-APP695 on day 4 in culture and treated with U18666A (3 μg/ml) on day 5 for 24 hours (APP+U18666A). MC65 cells were cultured in serum-free Opti-MEM medium without tetracycline to induce C99 expression for 24 hours, followed by U18666A (3 μg/ml) treatment (MC65+U18666A). For control cultures (labeled as APP and MC65), equal volumes of solvent were added. Equal amounts (10 μg from cortical neurons and 5 μg from MC65 cells) of proteins from cell homogenates were subjected to bicine SDS gel electrophoresis and Western blot analysis using 6E10 and B994 antibodies. The locations of C99, Aβ40, and Aβ42 bands are indicated. The Aβ standard contains a mixture of synthetic Aβ40 and Aβ42. Note that in bicine SDS-polyacrylamide gel electrophoresis, Aβ42 has a paradoxically lower apparent molecular mass than Aβ40. MC65 cells expressed relatively less endogenous APP; however, on longer exposure of the film, the APP bands recognized by 6E10 in MC65 samples became apparent. The experiment was performed three times with similar results. B: Same but independent experiment using cortical neurons was performed as described in A. The proteins were separated by 10 to 20% gradient Tris-HCL gel electrophoresis and the blot analyzed by 22C11 antibody. Shown are cultured neurons infected with Adv-cont (Cont) or Adv-APP695 (APP) without or with treatment of U18666A. C: Cortical neurons expressing APP695 with or without U18666A were sequentially extracted in RIPA followed by formic acid (FA), and the extracts were subjected to sandwich ELISA assay for quantification of Aβ40 and Aβ42. Depicted are mean values from two independent experiments, each done with duplicate samples. Error bars indicate 1 SD.

Figure 2

Aβ-positive deposits reside in endosomal compartments distinct from cholesterol-rich compartment in U18666A-treated neurons. Confocal microscopy was performed on double-fluorescence-labeled U18666A-treated neurons to determine the subcellular location of Aβ-positive deposits. Neurons were cultured and treated as described in Figure 1. Cells were then fixed and double labeled with filipin and 4G8 in LBPA (A) and Lamp1 (B). The arrow in A points to a large 4G8-positive granule. In C, neurons were double labeled with paired antibodies to Aβ (4G8 or anti-Aβ42) and to organelle markers (Lamp1, LBPA, Rab7, and Rab5a) in a manner that a monoclonal antibody was paired with a polyclonal antibody. Shown here are merged images. The color of fluorescence applied to each primary antibody is indicated. The yellow color in the merged images indicates co-localization. Scale bars, 4 μm.

Figure 3

Increased levels of C99 in NPC cerebellum and Αβ42 in NPC hippocampus. Equal amounts of protein in homogenates of cerebral cortex (Cx), cerebellum (Cb), and hippocampus (Hp) from a juvenile NPC subject (11 years old), an adult NPC subject (31 years old), and a normal control (23 years old) were subjected to bicine SDS-polyacrylamide gel electrophoresis and Western blot analysis using 6E10 (left). Because of the limitation of available brain homogenates, the blot was then stripped by boiling in acidic pH buffer, neutralized, and reprobed with B994 antibody (right). The positions of the C99, Aβ40, and Aβ42 bands are indicated.

Figure 4

C99 accumulates in the early endosomes of NPC Purkinje cells and Aβ42 accumulates in NPC hippocampal pyramidal neurons, cerebral blood vessels, and co-localizes with LBPA. A: Paraffin-embedded sections from control and NPC cerebellums were immunostained with 4G8. Color development was then obtained using a DAB staining kit and Meyer’s hematoxylin counterstain. Presented are cerebellar sections from a 23-year-old NPC subject and an age-matched control. Original magnifications are indicated. B: Confocal microscopic images of a representative Purkinje cell in NPC cerebellar sections double labeled with 4G8 and B994. C: Confocal microscopic images of a representative Purkinje cell in NPC cerebellar sections double labeled with 4G8 and endosomal markers Rab5a and Rab7. Shown are merged images. The color of fluorescence applied to each primary antibody is indicated. The yellow color in the merged images indicates co-localization. D: Paraffin-embedded sections from control and NPC hippocampus were immunostained with anti-Aβ42. Color development was then obtained using red chromogen 3-amino-9-ethyl carbazole (AEC) and Meyer’s hematoxylin counterstain. Arrows point to Aβ42 deposits in the blood vessel wall. Original magnifications are indicated. E: Confocal microscopic images of a representative pyramidal neuron in NPC CA1 double labeled with antibodies to Aβ42 and LBPA. The yellow color in the merged images indicates co-localization. Scale bars, 4 μm.

Figure 5

CatD is redistributed to early endosomes and co-localizes with C99. A: Confocal microscopic images of control and NPC Purkinje cells double labeled with antibodies to CatD and Lamp1 or EEA1. The arrow points to an enlarged CatD- and EEA1-positive vesicle. B: Confocal microscopic images of control and NPC Purkinje cells double labeled with 4G8 and antibodies to CatD and Lamp1. Shown are merged images. The color of fluorescence applied to each primary antibody is indicated. The yellow color in the merged images indicates co-localization. The arrow points to an enlarged CatD- and 4G8-positive vesicle. C: Control and NPC cerebellar sections were immunostained with Rab5a; high-power images of 25 randomly selected Purkinje cells were taken and the total area of immunopositive objects per cell was obtained using Metamorph software. Values presented are mean percentage of cell area occupied by Rab5a-positive early endosomes in each individual brain. The horizontal line indicates the mean value of patients in each group. Statistical analysis using Student’s t-test revealed a two-tailed P value of 0.06 for comparison between NPC and controls. Scale bars, 4 μm.