Evidence for lysosomal processing of amyloid ?-protein precursor in cultured cells

Cole, Gregory M.; Huynh, Tuan V.; Saitoh, Tsunao

doi:10.1007/bf00965926

Cited by 113 publications

(39 citation statements)

References 27 publications

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“…Identical results were obtained for cells expressing only APP (data not shown). This observation is consistent with previous studies showing that alkalization of intracellular compartments alters the turnover of APP and its proteolytic fragments (30,31). However, this treatment did not have any significant effect on the relative levels of either full-length PS1 or its endoproteolytic fragments.…”

Section: Inhibiting Degradation Of the App C-terminal Fragment-supporting

confidence: 93%

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Inhibiting Amyloid Precursor Protein C-terminal Cleavage Promotes an Interaction with Presenilin 1

Verdile

Martins

Duthie

et al. 2000

Journal of Biological Chemistry

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Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-␤ protein, which is central to the pathogenesis of Alzheimer's disease. It has been demonstrated that PS1 regulates the ␥-secretase proteolysis of the amyloid precursor protein (APP) C-terminal fragment (APP-C100), which is the final step in amyloid-␤ protein production. The mechanism and detailed pathway of this PS1 activity has yet to be fully resolved, but it may be due to a presenilin-controlled trafficking of the APP fragment or possibly an inherent PS1 proteolytic activity. We have investigated the possibility of a direct interaction of PS1 and the APP-C100 within the high molecular mass presenilin complex. However, the APP-C100 is rapidly degraded, and if it forms, then any PS1⅐APP complex is likely to be very transitory. To circumvent this problem, we have utilized the protease inhibitor N-acetyl-leucyl-norleucinal (LLnL) and the lysosomotropic agent NH 4 Cl, which inhibits the turnover of the APP-C100. Under these conditions, levels of the fragment increased appreciably, and as shown by glycerol gradient analysis, the APP-C100 shifted to a higher molecular mass complex that overlapped with PS1. Immunoprecipitation studies demonstrated that a significant population of the APP-C100 co-precipitated with PS1. These findings suggest that PS1 may mediate the shuttling of APP fragments and/or facilitate their presentation for ␥-secretase cleavage through a direct interaction. Alzheimer's disease (AD)1 is characterized by extensive neuronal loss culminating in the presentation of dementia. One of the key histopathological elements of the disease is the abnormal deposition of amyloid plaques (for review, see Ref. 1). The major protein component of these plaques is an ϳ4-kDa peptide (ranging from 39 to 43 residues in length) termed the amyloid-␤ protein (A␤) (2). A␤ is derived by proteolytic processing of the type 1 transmembrane amyloid precursor protein (APP). A␤ is released by the secretase proteases, which initially cleave APP at the N-terminal ␤-secretase site contained within the extracellular domain of the precursor. A ␤-secretase candidate gene has recently been independently identified by several groups (3-5). The residual 14-kDa C-terminal APP fragment containing A␤, as well as the transmembrane and intracellular domains, is subsequently cleaved by a putative enzyme ␥-secretase (6). The ␥-secretase cleavage occurs at two principle sites to produce either the A␤ residues 1-40 (A␤ 40 ) or a longer, more amyloidogenic form containing residues 1-42 (A␤ 42 ) (7-9).Presenilin 1 (PS1) accounts for the majority of inherited forms of early onset familial AD (for review, see Ref. 10). Although the precise function of PS1 and its relationship to AD have yet to be resolved, it has been clearly demonstrated that FAD-linked PS1 mutations result in a relative increase in the level of A␤ 42 as compared with A␤ 40 (11)(12)(13)(14). These findings indicate that PS1 is intimately involved in A␤ production, which is further supported by the significan...

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Section: Inhibiting Degradation Of the App C-terminal Fragment-supporting

confidence: 93%

“…been shown to play a role in the processing of the APP-C100 fragment to generate A␤ (30,31). As with LLnL, the NH 4 Cl treatment increases the cellular level of the APP-C100 fragment (Fig.…”

Section: Inhibiting Degradation Of the App C-terminal Fragment-mentioning

confidence: 90%

Inhibiting Amyloid Precursor Protein C-terminal Cleavage Promotes an Interaction with Presenilin 1

Verdile

Martins

Duthie

et al. 2000

Journal of Biological Chemistry

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“…In an alternative pathway involving the endosomal/lysosomal system, APP is processed into C-terminal fragments that contain the entire ␤A4 domain (Cole et al, 1989;Golde et al, 1992;Haass et al, 1992a). Although indicating the potential importance of this part in amyloidogenic processing, these fragments also have been proposed to be intermediates of the final degradation of APP in lysosomes .…”

Section: Abstract: Alzheimer's Disease; App Processing; ␤-Amyloid; Cmentioning

confidence: 99%

“…␤A4 derives from the larger amyloid precursor protein (APP) by the activity of two proteases termed ␤-and ␥-secretase (Kang et al, 1987). During its transport through the constitutive secretory pathway a proportion of APP is secreted by cleavage within the ␤A4 sequence, thereby precluding the generation of ␤A4 (Weidemann et al, 1989;Esch et al, 1990).In an alternative pathway involving the endosomal/lysosomal system, APP is processed into C-terminal fragments that contain the entire ␤A4 domain (Cole et al, 1989;Golde et al, 1992;Haass et al, 1992a). Although indicating the potential importance of this part in amyloidogenic processing, these fragments also have been proposed to be intermediates of the final degradation of APP in lysosomes .…”

mentioning

confidence: 99%

Late Compartments of Amyloid Precursor Protein Transport in SY5Y Cells Are Involved in β-Amyloid Secretion

1997

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Amyloid plaques, composed mainly of the 39-43 amino acid ␤A4 peptide, are a characteristic feature of Alzheimer's disease. Generation of ␤A4 by proteolytic processing of the amyloid precursor protein (APP) is thought to occur in a pathway that includes the activity of two as yet unknown proteases, with ␤-secretase cleaving at the N terminus and ␥-secretase releasing the C terminus of ␤A4. Inhibition studies and the finding that cell surface APP can serve as a direct precursor of ␤A4 suggest that the endosomal/lysosomal compartment is involved in the proteolysis of APP into ␤A4.In this study we targeted APP695 chimeric proteins directly into the endosomal/lysosomal compartment. This decreased the amount of released ␤A4, while the generation of the ␤A4 N terminus continued. APP695 proteins were constructed also, which carried sorting signals responsible for recycling between the trans-Golgi network (TGN) and the cell surface. These proteins were processed into secreted ␤A4 at even higher levels than wild-type APP695. Moreover, retention of APP695 proteins in the endoplasmic reticulum led to neither ␤A4 secretion nor to processing by ␤-secretase in human SH-SY5Y neuroblastoma cells.These data suggest that a ␤-cleavage activity resides in a late endosomal compartment and that a ␥-cleavage occurs in early endosomes, resulting in the generation of ␤A4 peptides with the majority ending at residue 40. Key words: Alzheimer's disease; APP processing; ␤-amyloid; chimeric proteins; endosomal/lysosomal compartment; transportA major histopathological marker of Alzheimer's disease is the presence of cerebral amyloid plaques, which are composed mainly of the insoluble 4 kDa ␤A4 peptide (Glenner and Wong, 1984;Masters et al., 1985). ␤A4 derives from the larger amyloid precursor protein (APP) by the activity of two proteases termed ␤-and ␥-secretase (Kang et al., 1987). During its transport through the constitutive secretory pathway a proportion of APP is secreted by cleavage within the ␤A4 sequence, thereby precluding the generation of ␤A4 (Weidemann et al., 1989;Esch et al., 1990).In an alternative pathway involving the endosomal/lysosomal system, APP is processed into C-terminal fragments that contain the entire ␤A4 domain (Cole et al., 1989;Golde et al., 1992;Haass et al., 1992a). Although indicating the potential importance of this part in amyloidogenic processing, these fragments also have been proposed to be intermediates of the final degradation of APP in lysosomes . However, various agents that interfere with pH gradients reduce the generation of ␤A4 drastically (Haass et al., 1992b;Shoji et al., 1992), supporting the idea that an acidic compartment is necessary for ␤A4 formation.To analyze the involvement of the endosomal/lysosomal system in the processing of APP695 into ␤A4, we created APP695 chimeras by exchanging the cytoplasmic domain of APP695 with that of the human lysosomal-associated membrane protein-1 (hLAMP-1) and the human cation-dependent mannose 6-phosphate receptor (CD-MPR). These APP hybrids were expected...

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“…APP can be either secreted, a process that would cleave APP within the (3-AP and prevent S-AP formation (18)(19)(20)(21), or processed through an endosomal-lysosomal pathway (22,23), which could presumably generate 3-AP (24)(25)(26)(27)(28). In humans, several alternatively spliced transcripts ofthe APP gene have been found in different tissues (29); however, the normal function of the various proteins encoded by the APP and APP-related genes is poorly understood.…”

mentioning

confidence: 99%

apl-1, a Caenorhabditis elegans gene encoding a protein related to the human beta-amyloid protein precursor.

Daigle

1993

Proc. Natl. Acad. Sci. U.S.A.

197

140

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The major component of senile plaques found in the brains of Alzheimer disease patients is the 3-amyloid peptide, which is derived from a larger amyloid precursor protein (APP). Recently, a number of APP and APP-related proteins have been identified in different organisms and constitute the family of APP proteins. We have isolated several cDNAs encoding an APP-related protein in the nematode Caenorhabdiis elegans and have designated the corresponding gene as apl-i. The apl-i transcripts undergo two forms of posttranscriptional modification: trans-splicing and alternative polyadenylylation. In vitro translation of an apl-i cDNA results in a protein of approximately the expected size. Similar to the Drosophila, human, and mouse APP-related proteins, APL-1 does not appear to contain the 13-amyloid peptide. Because APP-related proteins seem to be conserved through evolution, the apl-i gene from C. elegans should be important for determining the normal function of human APP. ', where Y is T or C, N is G, A, T, or C, and R is A or G) corresponding to a highly conserved cytoplasmic region between APP and APP-related proteins. Prehybridization and hybridization were carried out at 37°C. Filters were washed at 40°C and exposed to film using intensifying screens at -700C.Positive clones were isolated and excised from the AZAP vector according to the manufacturer's protocol (Stratagene) to yield pBluescript plasmids. DNA sequencing using the double-stranded dideoxynucleotide method (38) identified three APP-related clones (APL3.2, -6.2, and -1.2.2). To isolate additional clones, the cDNA library was rescreened with APL3.2, which was 32P-labeled by random priming. For this second screen, prehybridization and hybridization were done at 650C in 6x standard saline citrate (SSC)/5x Denhardt's solution/1% SDS/0.05% sodium pyrophosphate/ salmon sperm DNA (100 ,ug/ml). Filters were then washed in 2x SSC/0.1% SDS/0.05% sodium pyrophosphate for 30 min at room temperature, followed by two 15-min washes at 680C in 0.lx SSC/0.1% SDS/0.05% sodium pyrophosphate. Hybridizing plaques were visualized by autoradiography. Isolated clones were excised and sequenced as described above.

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Evidence for lysosomal processing of amyloid ?-protein precursor in cultured cells

Cited by 113 publications

References 27 publications

Inhibiting Amyloid Precursor Protein C-terminal Cleavage Promotes an Interaction with Presenilin 1

Inhibiting Amyloid Precursor Protein C-terminal Cleavage Promotes an Interaction with Presenilin 1

Late Compartments of Amyloid Precursor Protein Transport in SY5Y Cells Are Involved in β-Amyloid Secretion

apl-1, a Caenorhabditis elegans gene encoding a protein related to the human beta-amyloid protein precursor.

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