The Journal of Neuroscience, December 6, 2006, ():
GGA1 Is Expressed in the Human Brain and Affects the Generation of Amyloid ß-Peptide
J. Neurosci. Wahle et al.
26: 12838
Supplemental Data
Files in this Data Supplement:
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Supplementary table S1: Characterization of human brain material.
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Supplementary Table S3: GGA1 expression in the normal aged human brain and in AD.
Cases with a post-mortem interval of 12-24 hours and 24-48 hours did not significantly show differences in the staining pattern and the staining intensity.
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Supplementary Figure S2: Expression of GGA1 in neuronal cells in normal human brain.
A: The hippocampal formation exhibits a distinct diffuse neuropil staining for GGA1 of the neuropil of the pyramidal cell layer of the subiculum, the Ammon’s horn sectors CA1-CA3 as well as in the molecular layer of the dentate gyrus (arrows). The presubilcular region, the temporal isocortex (TI) and the CA4 region exhibit lower levels of GGA1. The white matter did not exhibit a significant GGA1 staining. B-G: In addition to the neuropil neurons were stained with anti-GGA1 in cell type specific manner. Dentate granule cells (B, E) did not show a significant GGA1 expression in the parikaryon whereas CA4 dentate hilar cells were almost entirely filled with strongly GGA1 positive cytoplasmic material (C, F) and CA1 pyramidal cells contained small amounts of GGA1-positive cytoplasmic material (D, G). Glial cells did not show GGA1 in this case. H: Similar as in the hippocampal formation various amounts of neuropil associated GGA1-positive material was observed in the medulla oblongata. The inferior olivary nucleus (arrows) und the arcuate nucleus (arrowheads) exhibited a strong neuropil staining whereas other brain stem nuclei such as the nuclei of the VIIIth cranial nerve (arrows with star) showed only a slight neuropil staining. The neurons of the brain stem nuclei showed varying degrees of cytoplasmic GGA1-positive material (I-L). In the reticular formation large cells of the intermediate reticular zone exhibited a homogenous weak staining in the cytoplasm (J, K). In contrast, small and medium sized reticular neurons exhibited a strong, Nissl substance-related staining pattern (J, K) indicating that different types of neurons differ in their GGA1 expression. TI = temporal isocortex; WM = white matter; GCL = granule cell layer of the dentate gyrus; ML = molecular layer of the fascia dentate; IRZ = intermediate reticular zone; Ro = raphe obscurus nucleus; SolN = solitary nucleus; IN = intercalated nucleus of the medulla oblongata; X = nucleus of the Xth cranial nerve; XII = nucleus of the XIIth cranial nerve. Calibration bar in L: A = 800µm; B, C, D = 230µm; E-F = 25µm; H = 400µm, I = 140µm, J = 20µm, K, L = 4µm
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Supplementary Table S4: Expression of GGA1 in glial cells in normal human brain.
A: Glial cells of the subpial zone of the molecular layer of the occipital cortex exhibited a significant cytoplasmatic staining with anti-GGA1 antibodies. B: The higher magnification levels clearly indicated the cytoplasmatic location. Nearly the entire cytoplasm contained GGA1. C: Double label immunohistochemistry showed the astrocytic nature of the GGA1 positive cells (brown) by coexpressing GFAP (blue-black). Calibration bar in C: A= 40µm, B,B= 5µm.
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Supplementary Figure S5: Expression of GGA1 variants did not affect total protein secretion, total activity and maturation of BACE1, and production of intracellular AICD.
A) Analysis of total protein secretion. Cells were starved at 37°C in methionine-free, serum-free medium for 45 min and then labeled with 35Smethionine/35Scysteine (MP Biomedicals Inc.) at 37°C for two hrs. Cells were then washed with phosphate-buffered saline (PBS) and chased in medium supplemented with 10% fetal calf serum and excess amounts of unlabeled methionine for additional two hrs. Aliquots of the conditioned chase media of radiolabeled cells was applied to 2 x 2 cm paper squares (Whatman, 3MM) soaked with 10% trichloroacetic acid (w/v), and dried. The filter papers were washed in 5% trichloroacetic acid (w/v), absolute ethanol, and acetone (twice each). Radioactivity in precipitated proteins was determined by scintillation counting.Values represent the means of three independent experiments ± s.d. B) Endogenous BACE1 activity in cell lysates of HEK293 cells stably overexpressing APP alone or together with GGA1 or GGA1 DN was measured by a fluorometric assay (see Methods). C) HEK293 cells stably overexpressing BACE1 (B1) alone or together with GGA1 or GGA1 DN were labeled with 35Smethionine for 15 min and chased for the indicated time periods. BACE1 was immunoprecipitated from cell lysates, separated by SDS-PAGE, and detected by phosphorimaging. After the pulse BACE1 is detected in the immature form, which undergoes maturation during the chase period. After 4 hrs chase, BACE1 is predominantly detected in its mature form. Values represent the means of three independent experiments ± s.d. D) The soluble APP intracellular domain (AICD) generated by γ-secretase cleavage was detected in lysates of the indicated cell lines by western immunoblotting with antibody 140 that recognizes the C-terminus of APP.
