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. 2013 Jan 11;288(2):1114-24.
doi: 10.1074/jbc.M112.417071. Epub 2012 Nov 21.

The Neuroendocrine Protein 7B2 Suppresses the Aggregation of Neurodegenerative Disease-Related Proteins

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Free PMC article

The Neuroendocrine Protein 7B2 Suppresses the Aggregation of Neurodegenerative Disease-Related Proteins

Michael Helwig et al. J Biol Chem. .
Free PMC article

Abstract

Neurodegenerative diseases such as Alzheimer (AD) and Parkinson (PD) are characterized by abnormal aggregation of misfolded β-sheet-rich proteins, including amyloid-β (Aβ)-derived peptides and tau in AD and α-synuclein in PD. Correct folding and assembly of these proteins are controlled by ubiquitously expressed molecular chaperones; however, our understanding of neuron-specific chaperones and their involvement in the pathogenesis of neurodegenerative diseases is limited. We here describe novel chaperone-like functions for the secretory protein 7B2, which is widely expressed in neuronal and endocrine tissues. In in vitro experiments, 7B2 efficiently prevented fibrillation and formation of Aβ(1-42), Aβ(1-40), and α-synuclein aggregates at a molar ratio of 1:10. In cell culture experiments, inclusion of recombinant 7B2, either in the medium of Neuro-2A cells or intracellularly via adenoviral 7B2 overexpression, blocked the neurocytotoxic effect of Aβ(1-42) and significantly increased cell viability. Conversely, knockdown of 7B2 by RNAi increased Aβ(1-42)-induced cytotoxicity. In the brains of APP/PSEN1 mice, a model of AD amyloidosis, immunoreactive 7B2 co-localized with aggregation-prone proteins and their respective aggregates. Furthermore, in the hippocampus and substantia nigra of human AD- and PD-affected brains, 7B2 was highly co-localized with Aβ plaques and α-synuclein deposits, strongly suggesting physiological association. Our data provide insight into novel functions of 7B2 and establish this neural protein as an anti-aggregation chaperone associated with neurodegenerative disease.

Figures

FIGURE 1.
FIGURE 1.
7B2 co-localizes with amyloid plaque pathology. A, the hippocampus of a 12-month-old mutant APP/PSEN1 mouse strongly stained for Aβ1–42 immunoreactivity. A composite image shows significant overlap between Aβ1–42 (green)and 7B2 (red) immunoreactivity, resulting in a yellow color in the merged image (arrows). Scale bars = 100 μm. B, staining showing significant co-localization (overlap resulting in purple hue) of 7B2 (red) with Aβ1–42 (green) and genuine extracellular plaques (blue) in the cortices of APP/PSEN1 mice. Plaques were visualized by staining with the in vivo amyloid-imaging fluorophore methoxy-X04.
FIGURE 2.
FIGURE 2.
Co-localization of 7B2 with extra- and intracellular protein aggregates in AD hippocampus and PD Lewy bodies. A, AD brain: schematic representation of 7B2 immunoreactivity (red stars) and Aβ-positive plaques (green stars) as detected throughout the extent of the human brain sample at the level of the hippocampus. Upper panels, low magnification images provide an overview of the areas of Aβ-immunoreactive deposits (red) and 7B2 expression (green) within in the hippocampus. Lower panel, high magnification image of representative amyloid plaques within the hippocampus confirms a high degree of co-localization (arrowhead) of 7B2 immunoreactivity with Aβ immunoreactivity. B, PD brain: 7B2 immunoreactivity (red stars) was found throughout the mesencephalon, whereas Lewy bodies were confined to the substantia nigra (green stars). Upper panels, low magnification images provide an overview of the areas of α-synuclein-immunoreactive deposits in Lewy bodies (green) and 7B2 expression (red) within the substantia nigra. Lower panel, high magnification image of representative Lewy bodies within the substantia nigra confirms a high degree of co-localization (arrowhead) of 7B2 immunoreactivity with α-synuclein immunoreactivity. The majority of 7B2 immunoreactivity was confined to areas near the nucleus, suggesting intracellular localization. C, 7B2 immunoreactivity was detected in a human control brain sample. Shown are representative images of the hippocampus in a non-diseased control brain. Although only limited Aβ immunoreactivity (red) and no plaque burden were detected, we observed significant 7B2 immunoreactivity (green) that was confined to areas near cell nuclei, suggesting intracellular localization. CA, cerebral aqueduct; CT, corticopontine tract; Hp, hippocampus; LN, lentiform nucleus; MRF, mesencephalic reticular formation; PT, pyramidal tract; RN, red nucleus; SN, substantia nigra; TH; thalamus. Scale bars = 10 μm.
FIGURE 3.
FIGURE 3.
7B2 decreases Aβ-induced cell death in Neuro-2A cells. A, Neuro-2A cells were treated with 10 μm1–42 for 48 h to induce cell death in the presence or absence of 7B2. Left panel, quantification of Aβ-induced cell death by the WST-1 cell viability assay. Right panels, representative photomicrographs showing viable calcein AM-stained Neuro-2A cells following treatment with Aβ1–42 with or without 7B2. ns, not significant. B, quantification of endogenous 7B2 levels in Neuro-2A cells by radioimmunoassay following either adenoviral (AV) overexpression or RNAi-mediated knockdown. cont., control. C, Aβ1–42-induced cell death following manipulation of intracellular 7B2 levels was monitored using the WST-1 cell viability assay. D, exogenously added recombinant His-tagged 7B2 (green arrows) was internalized and co-localized with Alexa Fluor-labeled Aβ1–42 (red) in Neuro-2A cells, indicating co-uptake into the cytosol of Neuro-2A cells (yellow arrows). Anti-His tag antiserum was used for the experiment in D.
FIGURE 4.
FIGURE 4.
Structure-function analysis of 7B2 proteins in suppressing Aβ fibrillation. A, upper, amino acid sequence of rat 7B2 including the N-terminal signal peptide and the C-terminal inhibitory peptide domain. Putative post-translational modification sites are marked (red P, known phosphorylation site; blue P, hypothetical phosphorylation site; black S, sulfation; S-S, disulfide bond); the C-terminal cleavage site is underlined; and the minimal amino acid sequence required for PC2 activation is boxed. The first and last three amino acids of the 7B2 fragments used in this study are indicated in boldface. Lower, domain structure of 7B2 and schematic representation of the N-terminal deletions and peptides used in this study. B, Aβ1–42 (20 μm) was incubated with either full-length 7B2 (27 kDa; red) or truncated proteins and peptides (2 μm). Protein fibrillation was monitored using a ThT fibrillation assay. C, the inhibition of Aβ1–42 aggregation in the presence of 27-kDa 7B2 was dose-dependent and was most effective at a 7B2:Aβ1–42 molar ratio of 1:10. D, quantification of supernatant (soluble Aβ1–42) versus pellet (insoluble Aβ1–42) dot intensities revealed a ratio shift (supernatant:pellet) toward the soluble Aβ1–42 species following the addition of 7B2. AU, absorbance units. E, quantification of Aβ1–42 fibril formation observed after 72 h of incubation in reactions with or without 7B2 by transmission electron microscopy. ***, p < 0.001.
FIGURE 5.
FIGURE 5.
7B2 does not disintegrate preformed mature Aβ1–42 fibrils but suppresses Aβ1–40 and α-synuclein fibrillation. A, Aβ1–42 (20 μm) was incubated at 37 °C, followed by the addition of 2 μm 27- or 21-kDa 7B2 at the time point indicated by the arrow. Protein aggregation was monitored with the ThT fibrillation assay. Further Aβ1–42 aggregation was inhibited once 7B2 was added; however, preformed mature fibrils were not affected (n = 3/group). Aβ1–40 (20 μm) (B) and α-synuclein (αSyn; 44 μm) (C) were incubated with full-length 7B2 (27 kDa; red) or 21-kDa 7B2 (blue), respectively, and fibrillation was monitored by the ThT assay. D, dose dependence relationship for inhibition of α-synuclein fibrillation by 27-kDa 7B2.
FIGURE 6.
FIGURE 6.
7B2 does not possess chaperone-like refolding activity. Unfolded and inactive firefly luciferase (40 μm) was incubated with either 21- or 27-kDa 7B2 (4 μm), followed by measurement of regained (refolded) luciferase enzyme activity, determined by luciferin bioluminescence assay.

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