Pro-NGF isolated from the human brain affected by Alzheimer's disease induces neuronal apoptosis mediated by p75NTR - PubMed (original) (raw)

Pro-NGF isolated from the human brain affected by Alzheimer's disease induces neuronal apoptosis mediated by p75NTR

Carlos E Pedraza et al. Am J Pathol. 2005 Feb.

Abstract

The pro-form of nerve growth factor (pro-NGF) has been shown to be a high affinity ligand for p75NTR and to induce apoptosis through this receptor. It has been reported that pro-NGF, rather than mature NGF, is the predominant form of this neurotrophin in human brain. In the present work we studied the potential involvement of pro-NGF purified from human brains affected by Alzheimer's disease (AD), where it is especially abundant, in the neuronal apoptosis observed in this disease. Western blot analysis of human brain tissue showed the existence of several pro-NGF forms. Some of these pro-NGF forms were significantly increased in AD brain cortex in a disease stage-dependent manner. Pro-NGF, purified by chromatography from human AD brains, induced apoptotic cell death in sympathetic neurons and in a p75NTR stably transfected cell line. Blocking p75NTR in cell culture abolished neuronal apoptosis caused by pro-NGF. p75NTR-transfected cells underwent apoptosis in the presence of pro-NGF while control wild-type cells did not. Taken together, these results indicate that pro-NGF purified from AD human brains can induce apoptosis in neuronal cell cultures through its interaction with the p75NTR receptor.

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Figures

Figure 1

Figure 1

The different antibodies anti-pro-NGF and anti-mNGF recognize high molecular weight forms of pro-NGF in human brain tissue and cerebrospinal fluid. Human samples from AD brain tissue or cerebrospinal fluid (CSF) (30 μg per lane) were analyzed by Western blot using two different antibodies directed against either the pro-domain of pro-NGF (C: anti-pre-pro-NGF, Prohormone Sciences; D: anti-pro-NGF, see Materials and Methods) or the mature part of the molecule (A: H20, Santa Cruz; B: anti-mNGF, Cederlane Labs.).

Figure 2

Figure 2

Western blotting analysis of pro-NGF forms in human brain samples from frontal and entorhinal cortex. High molecular weight forms of pro-NGF (53, 37, 32, and 26 kd) were immunodetected with an antibody directed against the pro-domain of pro-NGF (A). Pre-incubation of anti-pro-NGF with the antigenic peptide (1:20) blocks immunoreactivity in human brain samples (B: lane 1, AD; lane 2, antigenic peptide blockage). The content of pro-NGF forms is clearly increased in Alzeimer’s disease (AD)-affected brains (A, lanes AD) compared to controls (A, lanes C). β-actin was used as loading control. Densitometry analysis of anti-pro-NGF immunodetected bands shows a significant increase of several bands in AD-affected tissue in a disease stage-dependent manner. Tissue samples obtained from human frontal and entorhinal brain cortex affected by AD were classified into A, B, and C stages according to Braak and Braak (see Table 1 and Materials and Methods). Bars represent the mean of four stage B and five stage C samples as percentage of the mean of five stage A samples (controls) (B). (*, P < 0.05; **, P < 0.01; Student’s _t_-test).

Figure 3

Figure 3

Pro-NGF immunoreactivity in the frontal cortex (A, D, and F), CA1 area of the hippocampus (B and E), and subcortical white matter of the frontal cortex (C) in control cases. Pro-NGF is expressed in neurons (A, B, D, and E) and in glia (C). Pro-NGF immunoreactivity is present as a fine granular precipitate in the cytoplasm of neurons and main dendritic branches (D and E), but also in scattered nuclei (F). H and I: Double-staining immunohistochemistry showing pro-NGF expression (dark blue precipitate) in GFAP-immunoreactive (brown) astrocytes. G: Anti-pro-NGF blocked immunoreactivity with the antigenic peptide (1:10). A–C, bar in C = 25 μm; D–F, bar in F = 10 μm. G–I, bar in I = 10 μm.

Figure 4

Figure 4

Analysis by Western blots of Pro-NGF isolated from frontal cortex AD-affected tissue. Protein extracts from post-mortem AD-affected tissue were processed for ion-exchange chromatography (see Materials and Methods). In the final purification fraction (hbi-pro-NGF), different pro-NGF isolated forms of 53, 32, and 26 kd (A, lane 2) were detected by Western blotting with anti-pro-NGF antibody. Hbi-pro-NGF blocks anti-pro-NGF immunodetection of pro-NGF forms in human brain tissue homogenates (B, lane 2). B, lane 1: total lysate of human brain AD-affected tissue. The 53-kd band immunodetected in hbi-pro-NGF is a glycosylated form of the pro-neurotrophin as the treatment with N-glycanase (C, lane 2) results in a decrease in its apparent MW (C, lane 1, untreated hbi-pro-NGF).

Figure 5

Figure 5

Trypsin pre-treatment of hbi-pro-NGF generates mature NGF which protects PC12 cells from deprivation-induced apoptotic death. PC12 cells were serum-deprived and treated with NGF (100 ng/ml), hbi-pro-NGF (25 ng/ml), or with trypsin-digested hbi-pro-NGF (25 ng/ml hbi-pro-NGF treated with 50 mg/ml trypsin for 10 seconds at 37°C) for 48 hours. Apoptotic nucleus morphology was detected by Hoechst staining (A). Differentiated cells were counted as positive when neurite extensions were longer than a cell body (B). Results are the mean ± SD of 900 to 1600 cells counted in a representative experiment carried out in triplicate. Statistic was done by comparing between treatments and deprived cells. **, P < 0.05, Student’s _t_-test.

Figure 6

Figure 6

Hbi-pro-NGF induces apoptosis in SCG neurons. Cultured SCG neurons were treated for 24 hours with hbi-pro-NGF (25 ng/ml) and apoptotic cell death was evidenced by means of the TUNEL method and staining with Hoechst (A). Quantification of TUNEL-positive cells shows that the cell death increases with the increased concentration of hbi-pro-NGF (B). Bar = 50 μm.

Figure 7

Figure 7

Anti-pro-NGF antibody blocks hbi-pro-NGF-induced apoptosis in SGC cells and in 3T3-p75st. Induction of apoptotic cell death in SCG cells treated with hbi-pro-NGF is prevented by pre-incubation with anti-p75 antibody (REX, 50 ng/ml), anti-pro-NGF (20 μg/ml), or anti-β-NGF (25 μg/ml) (A). Apoptotic cell death was evidenced by means of the TUNEL assay. Hbi-pro-NGF also induces apoptotic nucleus morphology determined by Hoechst staining in the non-neuronal cell line 3T3-p75st. Apoptotic nuclei reach ∼25% of total cells (B) and the pre-incubation of hbi-pro-NGF with anti-pro-NGF for 2 hours before its addition to cell cultures completely blocks cell death induced by 30-hour treatment with hbi-pro-NGF (25 ng/ml). 3T3 wild-type cells are not affected by the treatment with hbi-pro-NGF (C). **, P < 0.01, Student’s _t_-test.

Figure 8

Figure 8

Hbi-pro-NGF forms separated by gel filtration chromatography induce cell death in 3T3-p75st cells. Three fractions of gel filtration chromatography of hbi-pro-NGF (8 μg total protein of each fraction) containing pro-NGF forms of 54, 32, and 26 kd, were added to 3T3-p75st for 30 hours. Apoptotic nuclei morphology was determined by Hoechst staining. Three μg of Hbi-pro-NGF was used as control of death induction in deprived cells. Apoptotic cell nuclei were counted as percentage of the total cells in each treatment. Bars are median ± SD of triplicates.

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