β‐Amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for β‐secretase activity in chromaffin vesicles

Molecular misreading of the ␤-amyloid precursor protein (APP) gene generates mRNA with dinucleotide deletions in GAGAG motifs. The resulting truncated and partly frameshifted APP protein (APP ؉1 ) accumulates in the dystrophic neurites and the neurofibrillary tangles in the cortex and hippocampus of Alzheimer patients. In contrast, we show here that neuronal cells transfected with APP ؉1 proficiently secreted APP ؉1 . Because various secretory APP isoforms are present in cerebrospinal fluid (CSF), this study aimed to determine whether APP ؉1 is also a secretory protein that can be detected in CSF. Post-mortem CSF was obtained at autopsy from 50 non-demented controls and 122 Alzheimer patients; all subjects were staged for neuropathology (Braak score). Unexpectedly, we found that the APP ؉1 level in the CSF of non-demented controls was much higher (1.75 ng/ml) than in the CSF of Alzheimer patients (0.51 ng/ml) (p < 0.001), and the level of APP ؉1 in CSF was inversely correlated with the severity of the neuropathology. Moreover the earliest neuropathological changes are already reflected in a significant decrease of the APP ؉1 level in CSF. These data show that APP ؉1 is normally secreted by neurons, preventing intra-neuronal accumulation of APP ؉1 in brains of nondemented controls without neurofibrillary pathology. Alzheimer's disease (AD)1 is a progressive neurodegenerative disease and the most common form of dementia in aged populations (1). The major constituent of the extracellular plaques in the brains of AD patients is amyloid ␤ (A␤), which is cleaved from the ␤-amyloid precursor protein (APP) by ␤ and ␥ secretases. Patients suffering from the hereditary forms of AD either carry a mutation in the APP gene or in one of the presenilin genes. These mutations cause an alteration in the proteolytic processing of APP, resulting in the formation of more A␤ 40 and A␤ 42, which are both prone to aggregate and precipitate in the plaques (2, 3).Genomic APP mutations are not the only source of aberrant APP proteins in AD. We (4) and others (5) have reported that small frameshift mutations occur in APP transcripts near short simple repeats. The observed dinucleotide deletions, such as ⌬GA, in the GAGAG motif of exon 9 or 10 of APP, result in the translation of an aberrant APP protein, i.e. APP ϩ1 , which accumulates in the neurofibrillary tangles, neuropil threads, and dystrophic neurites of the neuritic plaques of AD and Down syndrome patients (4, 6). APP ϩ1 is a truncated APP protein of 348 amino acids with a wild-type N terminus and an aberrant C terminus translated in the ϩ1 reading frame.The different isoforms of full-length APP are type I transmembrane proteins, which are proteolytically cleaved by secretases (7,8) to form secretory APPs and A␤ 40 or A␤ 42 and p3 (9, 10). These secretory APP proteins, sAPP␣ and sAPP␤ (11-13), and A␤ (14) are detectable in cerebrospinal fluid (CSF) as well as in human brain homogenates (15). The present study focuses on APP ϩ1 , which consists of the N-terminal 329 amino acids of t...

Section: Discussionmentioning

confidence: 99%

Frameshifted β-Amyloid Precursor Protein (APP+1) Is a Secretory Protein, and the Level of APP+1 in Cerebrospinal Fluid Is Linked to Alzheimer Pathology

Hol

Dijk

Gerez

et al. 2003

“…This APP species might therefore be a source of A␤ production by soluble proteases of the lysosome, i.e. ␥-secretase-independent proteases, which have been suggested by some authors (62)(63)(64). For example, cathepsin D (the major soluble protease of the lysosome) is capable of cleaving soluble APP fragments at positions 42 and 43 (but not 40) (65).…”

Section: Ps-1 App and Nicastrin Are Resident Lysosomalmentioning

confidence: 91%

Presenilin-1, Nicastrin, Amyloid Precursor Protein, and γ-Secretase Activity Are Co-localized in the Lysosomal Membrane

Pasternak

Bagshaw

Guiral

et al. 2003

276

145

Alzheimer's disease (AD) is caused by the cerebral deposition of ␤-amyloid (A␤), a 38 -43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the ␥-secretase (a complex containing presenilin and nicastrin) to produce A␤ occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the ␥-secretase proteins and a neutral pH optimum of in vitro ␥-secretase assays have challenged this conclusion. We have recently identified nicastrin as a major lysosomal membrane protein. In the present work, we use Western blotting and immunogold electron microscopy to demonstrate that significant amounts of mature nicastrin, presenilin-1, and APP are co-localized with lysosomal associated membrane protein-1 (cAMP-1) in the outer membranes of lysosomes. Furthermore, we demonstrate that these membranes contain an acidic ␥-secretase activity, which is immunoprecipitable with an antibody to nicastrin. These experiments establish APP, nicastrin, and presenilin-1 as resident lysosomal membrane proteins and indicate that ␥-secretase is a lysosomal protease. These data reassert the importance of the lysosomal/endosomal system in the generation of A␤ and suggest a role for lysosomes in the pathophysiology of AD.

“…Significantly, findings in this study showed that the endogenous ␤-secretase activity in A␤-containing regulated secretory vesicles (RSV) possesses high selectivity for cleaving the WT ␤-secretase site but does not cleave the Swedish (Swe) mutant ␤-secretase site. The RSV provide production and synthesis of a major portion of secreted A␤ (8,9). Although many previous studies of ␤-secretase have utilized substrates containing the Swe mutant ␤-secretase site (10 -14) expressed in one extended family (15), it is most relevant to study proteolytic cleavage of the WT ␤-secretase site expressed in the major portion of the AD population.…”

mentioning

confidence: 99%

Inhibitors of Cathepsin B Improve Memory and Reduce β-Amyloid in Transgenic Alzheimer Disease Mice Expressing the Wild-type, but Not the Swedish Mutant, β-Secretase Site of the Amyloid Precursor Protein

Hook

Kindy

Hook

2008

188

218

Elucidation of A␤-lowering agents that inhibit processing of the wild-type (WT) ␤-secretase amyloid precursor protein (APP) site, present in most Alzheimer disease (AD) patients, is a logical approach for improving memory deficit in AD. The cysteine protease inhibitors CA074Me and E64d were selected by inhibition of ␤-secretase activity in regulated secretory vesicles that produce ␤-amyloid (A␤). The regulated secretory vesicle activity, represented by cathepsin B, selectively cleaves the WT ␤-secretase site but not the rare Swedish mutant ␤-secretase site. In vivo treatment of London APP mice, expressing the WT ␤-secretase site, with these inhibitors resulted in substantial improvement in memory deficit assessed by the Morris water maze test. After inhibitor treatment, the improved memory function was accompanied by reduced amyloid plaque load, decreased A␤40 and A␤42, and reduced C-terminal ␤-secretase fragment derived from APP by ␤-secretase. However, the inhibitors had no effects on any of these parameters in mice expressing the Swedish mutant ␤-secretase site of APP. The notable efficacy of these inhibitors to improve memory and reduce A␤ in an AD animal model expressing the WT ␤-secretase APP site present in the majority of AD patients provides support for CA074Me and E64d inhibitors as potential AD therapeutic agents. Alzheimer disease (AD)2 is an age-related neurodegenerative disorder that results in loss of memory and accumulation of neurotoxic ␤-amyloid (A␤) peptides in brain (1-4). Expression of mutant forms of human amyloid precursor protein (APP) in mouse models of AD results in increased A␤ and amyloid plaques in brain, with memory deficits that resemble AD (2, 3, 5-7). Such studies demonstrate that overproduction of A␤ peptides participates as a major factor in the development of AD.A␤ peptides are produced by proteolytic processing of APP, resulting in production of A␤40 and A␤42 (1-40 and 1-42 residues, respectively). Proteases referred to as ␤-secretase and ␥-secretase cleave at the N-and C termini of A␤ within APP to generate A␤ peptides. A␤ peptides then undergo secretion to provide extracellular A␤ that produces neurotoxic effects with aggregation and accumulation in amyloid plaques of AD brains. Compounds that inhibit ␤-secretase activity are particularly attractive as potential therapeutic agents for AD.The majority of AD patients express the wild-type (WT) ␤-secretase site of APP. Therefore, compounds that inhibit cleavage of the WT ␤-secretase site are relevant to the AD population. Significantly, findings in this study showed that the endogenous ␤-secretase activity in A␤-containing regulated secretory vesicles (RSV) possesses high selectivity for cleaving the WT ␤-secretase site but does not cleave the Swedish (Swe) mutant ␤-secretase site. The RSV provide production and synthesis of a major portion of secreted A␤ (8, 9). Although many previous studies of ␤-secretase have utilized substrates containing the Swe mutant ␤-secretase site (10 -14) expressed in one extended family (15), it i...