Abstract
Heparan sulfate (HS)-containing, S-nitrosylated (SNO) glypican-1 (Gpc-1) releases anhydromannose-containing HS (anMan-HS) by SNO-catalyzed autodegradation in endosomes. Transport of anMan-HS to the nucleus requires processing of the amyloid precursor protein (APP) to amyloid beta peptides (Aβ). To further examine the relationship between APP and Gpc-1 processing in normal fibroblasts we have suppressed Gpc-1 autodegradation by aminoguanidine inhibition of NO synthesis and prevented lysosomal degradation of anMan-HS by using chloroquine. Deconvolution immunofluorescence microscopy and SDS-PAGE using anMan- and APP/Aβ-specific antibodies and markers for nuclei and autophagosomes were used to identify subcellular localization of Aβ and its oligomeric state. Wild-type mouse embryonic fibroblasts (WT MEF) grown during NO-deprivation accumulated 95–98 % of Aβ as oligomers in the nucleus. WT MEF treated with chloroquine accumulated both anMan-HS and Aβ, first in the nucleus then in autophagosomes. Maximal nuclear anMan-HS and Aβ accumulation was obtained after 4 and 7 h of growth, respectively. Both yielded similar banding patterns on SDS-PAGE which were also similar to the Aβ oligomers obtained after NO-deprivation. Nuclear Aβ accumulation was marginally increased (from 54 to 58 %) by suppression of both release and degradation of anMan-HS. Nuclear exit of Aβ, accumulated during growth in aminoguanidine, was enhanced by ascorbate-induced reactivation of anMan-HS production. Transgenic Alzheimer disease mouse (Tg2576) MEF, which produces excess amount of Aβ was used for comparison. Overall, nuclear Aβ exit and lysosomal degradation was compromised by inhibition of the autophagosome-lysosome pathway in both WT and Tg2576 MEF, while only WT MEF was sensitive to suppression of Gpc-1 autodegradation.
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Abbreviations
- AD:
-
Alzheimer’s disease
- Aβ:
-
Amyloid beta
- anMan/AM:
-
Anhydromannose
- APP:
-
Amyloid precursor protein
- CTF:
-
C-terminal fragment
- DAPI:
-
4,6-diamidino-2-phenylindole
- Gpc-1:
-
Glypican-1
- HS:
-
Heparan sulfate
- mAb:
-
Monoclonal antibody
- MEF:
-
Mouse embryonic fibroblast
- NO:
-
Nitric oxide
- pAb:
-
Polyclonal antibody
- SNO:
-
S-nitrosothiol
- Tg2576:
-
Transgenic Alzheimer disease mouse
- WT:
-
Wild-type
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Acknowledgments
This work was supported by grants from the Swedish Research Council, the Swedish Cancer Society, Alfred Österlund, Kock, Åhlen, Stohnes, Längmanska kulturfond, Gamla Tjänarinnor, and Olle Engkvist Foundations.
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Cheng, F., Fransson, LÅ. & Mani, K. Suppression of glypican-1 autodegradation by NO-deprivation correlates with nuclear accumulation of amyloid beta in normal fibroblasts. Glycoconj J 32, 675–684 (2015). https://doi.org/10.1007/s10719-015-9616-4
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DOI: https://doi.org/10.1007/s10719-015-9616-4