Amyloid Precursor Protein Is Synthesized by Retinal Ganglion Cells, Rapidly Transported to the Optic Nerve Plasma Membrane and Nerve Terminals, and Metabolized

Abstract: We have investigated the synthesis, axonal transport, and processing of the β-amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a f_1/2 of less than 5 h. A significant accumulation of C-terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [³⁵S]- methionine, [³⁵S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is ∼110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.     

The amyloid beta-protein precursor of Alzheimer's disease is degraded extracellularly by a Kunitz protease inhibitor domain-sensitive trypsin-like serine protease in cultures of chick sympathetic neurons.

The amyloid beta-protein precursor (APP) of Alzheimer's disease (AD) is cleaved either by alpha-secretase to generate an N-terminally secreted fragment, or by beta- and gamma-secretases to generate the beta-amyloid protein (Abeta). The accumulation of Abeta in the brain is an important step in the pathogenesis of AD. Alternative mRNA splicing can generate isoforms of APP which contain a Kunitz protease inhibitor (KPI) domain. However, little is known about the physiological function of this domain. In the present study, the metabolic turnover of APP was examined in cultured chick sympathetic neurons. APP was labelled by incubating neurons for 5 h with [35S]methionine and [35S]cysteine. Intracellular labelled APP decayed in a biphasic pattern suggesting that trafficking occurs through two metabolic compartments. The half-lives for APP in each compartment were 1.5 and 5.7 h, respectively. A small fraction (10%) of the total APP was secreted into the culture medium where it was degraded with a half-life of 9 h. Studies using specific protease inhibitors demonstrated that this extracellular breakdown was due to cleavage by a trypsin-like serine protease that was secreted into the culture medium. Significantly, this protease was inhibited by a recombinant isoform of APP (sAPP751), which contains a region homologous to the Kunitz protease inhibitor (KPI) domain. These results suggest that KPI forms of APP regulate extracellular cleavage of secreted APP by inhibiting the activity of a secreted APP-degrading protease.     

The Amyloid Precursor Protein/Protease Nexin 2 Kunitz Inhibitor Domain Is a Highly Specific Substrate of Mesotrypsin

The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-β peptides. The major splice isoforms of APP expressed by most tissues contain a Kunitz protease inhibitor domain; secreted APP containing this domain is also known as protease nexin 2 and potently inhibits serine proteases, including trypsin and coagulation factors. The atypical human trypsin isoform mesotrypsin is resistant to inhibition by most protein protease inhibitors and cleaves some inhibitors at a substantially accelerated rate. Here, in a proteomic screen to identify potential physiological substrates of mesotrypsin, we find that APP/protease nexin 2 is selectively cleaved by mesotrypsin within the Kunitz protease inhibitor domain. In studies employing the recombinant Kunitz domain of APP (APPI), we show that mesotrypsin cleaves selectively at the Arg¹⁵-Ala¹⁶ reactive site bond, with kinetic constants approaching those of other proteases toward highly specific protein substrates. Finally, we show that cleavage of APPI compromises its inhibition of other serine proteases, including cationic trypsin and factor XIa, by 2 orders of magnitude. Because APP/protease nexin 2 and mesotrypsin are coexpressed in a number of tissues, we suggest that processing by mesotrypsin may ablate the protease inhibitory function of APP/protease nexin 2 in vivo and may also modulate other activities of APP/protease nexin 2 that involve the Kunitz domain.     

Age-dependent degradation of amyloid precursor protein in the post-mortem mouse brain cortex

We have examined the degradation of amyloid precursor protein (APP) in the brain cortex of adult (24±2) and old (58±2) mice at different post-mortem time intervals (0, 1.5, 3, 6, 12 and 24 h). The brain cortex extract was prepared and processed for immunoblotting using antibodies against N-terminal 47–62 amino acids (Asp29) and central 301–316 amino acids containing Kunitz protease inhibitor (KPI) domain (Asp45) of APP. Asp29 (N-terminal) recognizes two bands of 140 and 112 kDa. The amount of 140 kDa is relatively higher in adult than old. The level of 112 kDa is 1.6 times lower in adult than old. It shows no remarkable change with varying post-mortem time. On the other hand, Asp45 (KPI) detects two bands of 110 and 116 kDa. While 116 kDa disappears rapidly after death of the animal, 110 kDa shows no remarkable change with different post-mortem periods. Further incubation of the disrupted tissue at 4 °C for 24 h and immunoblot analysis with Asp29 (N-terminal) shows 112 kDa in both ages but 58.5 kDa in adult and 70 kDa in old only. Analysis with Asp45 (KPI) shows only 54 kDa which increases after 3 h in adult but decreases significantly after 1.5 h and becomes undetectable at 24 h in old. Thus the present findings indicate that APP is degraded in a precise pattern and it depends on cellular intactness, post-mortem period and age of the animal.     

Structural features of the KPI domain control APP dimerization, trafficking, and processing

The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz-type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.     

Secreted forms of the amyloid-beta precursor protein are ligands for the class A scavenger receptor

Upon activation, platelets secrete a 120-kDa protein that competes for the binding and internalization of acetyl low density lipoproteins (AcLDL) by macrophages. From the amino-terminal amino acid sequence, amino acid composition, and immunoblot analysis, we identified the active factor in platelet secretion products as sAPP, an alpha-secretase cleavage product of the beta-amyloid precursor protein (APP), that contains a Kunitz-type protease inhibitor (KPI) domain. We showed that both sAPP751 (also called Nexin II) and sAPP695, which does not contain a KPI domain, are ligands for the class A scavenger receptor (SR-A). Chinese hamster ovary cells stably transfected to express the SR-A bound and internalized 4-fold more human platelet-derived sAPP than control cells. The binding and internalization of sAPP were inhibited by the SR-A antagonist fucoidin. In addition, sAPP competed as effectively as fucoidin for SR-A-mediated cell association and degradation of (125)I-AcLDL. To determine if the KPI domain is required for the binding of sAPP to the SR-A, APP751 and APP695 were expressed in Chinese hamster ovary cells, and sAPP751 and sAPP695 purified from the medium were tested for their binding to the SR-A. sAPP751 and sAPP695 were equally effective in competing for the cell association of (125)I-AcLDL by SR-A-expressing cells, demonstrating that the KPI domain is not essential for binding. We also found that sAPP751 is present in extracts of atherosclerotic lesions and that sAPP competes for the SR-A-mediated cell association of oxidized low density lipoprotein. Deletion mutagenesis indicated that a negatively charged region of APP (residues 191-264) contributes to binding to the SR-A. These results suggest that the SR-A contributes to the clearance of sAPP and that sAPP competes for the cell association of other SR-A ligands.     

The Kunitz-protease inhibitor domain in amyloid precursor protein reduces cellular mitochondrial enzymes expression and function

Mitochondrial dysfunction is a prominent feature of Alzheimer’s disease (AD) and this can be contributed by aberrant metabolic enzyme function. But, the mechanism causing this enzymatic impairment is unclear. Amyloid precursor protein (APP) is known to be alternatively spliced to produce three major isoforms in the brain (APP695, APP751, APP770). Both APP770 and APP751 contain the Kunitz Protease Inhibitory (KPI) domain, but the former also contain an extra OX-2 domain. APP695 on the other hand, lacks both domains. In AD, up-regulation of the KPI-containing APP isoforms has been reported. But the functional contribution of this elevation is unclear. In the present study, we have expressed and compared the effect of the non-KPI containing APP695 and the KPI-containing APP751 on mitochondrial function. We found that the KPI-containing APP751 significantly decreased the expression of three major mitochondrial metabolic enzymes; citrate synthase, succinate dehydrogenase and cytochrome c oxidase (COX IV). This reduction lowers the NAD⁺/NADH ratio, COX IV activity and mitochondrial membrane potential. Overall, this study demonstrated that up-regulation of the KPI-containing APP isoforms is likely to contribute to the impairment of metabolic enzymes and mitochondrial function in AD.     

Amyloid Precursor Protein Metabolism in Primary Cell Cultures of Neurons, Astrocytes, and Microglia

Abstract: Amyloid precursor protein (APP) gives rise by proteolytic processing to the amyloid β peptide (Aβ) found abundantly in cerebral senile plaques of individuals with Alzheimer's disease. APP is highly expressed in the brain. To assess the source of cerebral Aβ, the metabolism of APP was investigated in the major cell types of the newborn rat cerebral cortex by pulse/chase labeling and immunoprecipitation of the APP and APP metabolic fragments. We describe a novel C-terminally truncated APP isoform that appears to be made only in neurons. The synthesis, degradation, and metabolism of APP were quantified by phosphorimaging in neurons, astrocytes, and microglia. The results show that although little APP is metabolized through the amyloidogenic pathways in each of the three cultures, neurons appear to generate more Aβ than astrocytes or microglia.     

Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress

We studied the effect of microtubule-associated tau protein on trafficking of vesicles and organelles in primary cortical neurons, retinal ganglion cells, and neuroblastoma cells. Tau inhibits kinesin-dependent transport of peroxisomes, neurofilaments, and Golgi-derived vesicles into neurites. Loss of peroxisomes makes cells vulnerable to oxidative stress and leads to degeneration. In particular, tau inhibits transport of amyloid precursor protein (APP) into axons and dendrites, causing its accumulation in the cell body. APP tagged with yellow fluorescent protein and transfected by adenovirus associates with vesicles moving rapidly forward in the axon (approximately 80%) and slowly back (approximately 20%). Both movements are strongly inhibited by cotransfection with fluorescently tagged tau (cyan fluorescent protein-tau) as seen by two-color confocal microscopy. The data suggests a linkage between tau and APP trafficking, which may be significant in Alzheimer's disease.     

High level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid beta-protein precursor.

The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al. 1988 Nature 311:525-527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.     

Nerve and epidermal growth factors induce the release of the Alzheimer amyloid precursor from PC 12 cell cultures

Antisera against specific sites of the Alzheimer beta Amyloid protein precursor (beta APP) were used to study the effects of nerve and epidermal growth factors on the expression and processing of this protein in PC12 cell cultures. Two major beta APP proteins (140 and 105 kDa) both containing the Kunitz-protease inhibitor insert (KPI), were detected in cell extracts of naive PC12 cells. Treatment of these cultures with nerve growth factor (NGF) induced the release of two beta APP species 125 and 120 kDa, both of which contained the KPI domain and lacked the carboxy-terminal portion of the precursor. The released beta APP contained O-linked sugars. Only one of the released beta APP proteins bound to the lectin Concanavalin A indicating that they differ in their glycosylation. Epidermal growth factor (EGF) also induced the release of beta APP proteins into the culture medium with similar electrophoretic mobilities as those released by NGF.     

Processing of Amyloid Precursor Protein in Human Primary Neuron and Astrocyte Cultures

Abstract: Increased production of amyloid β peptide (Aβ) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because Aβ deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C-terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher-molecular-weight fragments are more abundant. The level of amyloidogenic 4-kDa Aβ exceeds that of nonamyloidogenic 3-kDa Aβ in both neurons and astrocytes. In contrast, microglia make more of the smallest C-terminal fragment and no detectable Aβ. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.     

A dinucleotide deletion in amyloid precursor protein (APP) mRNA associated with sporadic Alzheimer's disease results in efficient secretion of truncated APP isoforms from neuroblastoma cell cultures

Recently, two dinucleotide deletions were detected in the mRNA of the amyloid precursor protein (APP) from cerebral cortex neurons of patients with sporadic Alzheimer's disease (AD) or Down's syndrome. These deletions resulted in truncation of APP, producing an APP isoform with a 38-kDa N-terminus and a novel carboxyl terminus (APP+1). We investigated the subcellular localization and the processing of APP+1 in the neuroblastoma cell line B103. cDNA constructs were generated encoding fusion proteins of APP+1 or full-length APP with the enhanced green fluorescent protein (eGFP). In transient transfection experiments using B103 cells, the APP+1-eGFP fusion protein showed a reticular localization with intense staining in the Golgi complex. Unlike full-length APP fused to eGFP, the APP+1-eGFP fusion protein did not localize to the perinuclear area or to the plasma membrane. Western blot analysis of cell extracts confirmed the translation of the expected fusion proteins. Analysis of the supernatant by western blot indicated that the APP+1-eGFP fusion protein was efficiently secreted from B103 cells, whereas the secreted form of full-length APP fusion protein (APPs) was hardly detectable. Thus, both dinucleotide deletions in the APP mRNA result in truncated APP+1 that is not membrane associated and is readily secreted from neurons.     

Glycosylation of the amyloid peptide precursor containing the Kunitz protease inhibitor domain improves the inhibition of trypsin

The amyloid beta peptide (A beta P) is the major constituent of the amyloid deposits that accumulate extracellularly in the brain of patients with Alzheimer's disease. This peptide is obtained from transmembrane amyloid protein precursors (APP) which sometimes contain a Kunitz protease inhibitor (KPI) insert in their extracellular domain and therefore are able to inhibit serine proteases. Expression of the transmembrane and the secreted APP containing the KPI domain was obtained by transient transfection of COS-1 cells. The overexpressed proteins were detected in immunoblotting experiments and inhibition of trypsin was analyzed using reverse enzymography. Our results indicate that post-translational modifications including glycosylation improve the inhibition of trypsin by the APP containing the KPI domain.     

Synthesis and characterization of the Kunitz protease-inhibitor domain of the beta-amyloid precursor protein.

To understand the pathological process by which amyloid is deposited in Alzheimer's disease, it is important to characterize the proteolytic processing events of the beta-amyloid precursor protein (beta-APP) from which the amyloid-forming fragment is excised. A potentially important component in beta-APP processing is the 57-amino acid (aa) Kunitz serine protease inhibitor (KPI) located within the extracellular domain of both the 751- and 770-aa isoforms of beta-APP. We have synthesized DNA encoding the 57-aa KPI domain as a necessary step in identifying the role of the protease inhibitor in beta-APP processing and amyloid formation. A bacterial secretion system directed by the alkaline phosphatase signal peptide of Escherichia coli linked to a synthetic gene encoding KPI was used to produce soluble, extracellular recombinant KPI (reKPI) protein. The reKPI protein was purified to homogeneity from bacterial supernatants and was biochemically and biologically characterized. Complete aa sequence analysis confirmed the fidelity of the reKPI, and fast-atom bombardment mass-spectral analysis was used to document that reKPI was of the predicted Mr. The reKPI is as active on a molar basis as the inhibitor-containing beta-APP when assayed for inhibition of trypsin activity. Together these data suggest that reKPI protein is properly folded and lacking in modified aa. Hence, this reKPI will be an important reagent in gaining a better understanding of the role of the KPI domain in beta-APP function and metabolism, as well as in the proteolytic events involved in beta-amyloid formation.     

Selective localization of G protein γ5 subunit in the subventricular zone of the lateral ventricle and rostral migratory stream of the adult rat brain

G proteins play important roles in transmembrane signal transduction, and various isoforms of each subunit, alpha, beta and gamma, are highly expressed in the brain. The Ggamma5 subunit is a minor isoform in the adult brain, but we have previously shown it to be highly expressed in the proliferative region of the ventricular zone in the rat embryonic brain. We show here that Ggamma5 is also selectively localized in a proliferative region in the adult rat brain, including the subventricular zone of the lateral ventricle and rostral migratory stream. The Galphai2 subunit colocalized with Ggamma5 in these regions, the two subunits being present in neuronal precursors and ependymal cells but not in proliferating astrocytes. In addition, intense staining of Ggamma5 was seen in axons of the olfactory neurons, which are known to regenerate. These results suggest specific roles for Ggamma5 in precursor cells during neurogenesis so that this isoform might be a useful biological marker.     

Trophic effect of beta-amyloid precursor protein on cerebral cortical neurons in culture

We investigated the effect of human beta-amyloid precursor protein (APP) on rat primary cerebral cortical neurons cultured in a serum-free medium. Two secretory APP species (APP667 and APP592) with and without the protease inhibitor domain were produced by COS-1 cells transfected with APP cDNAs, which encode the N-terminal portions of APP770 and APP695. Both highly purified APP species, when added to the medium, enhanced neuronal survival and neurite extension in a dose-dependent manner with a maximum effect at approximately 100 nM. These results suggest that secreted forms of APP have trophic activity for cerebral cortical neurons.     

Huntingtin associated protein 1 regulates trafficking of the amyloid precursor protein and modulates amyloid beta levels in neurons

J. Neurochem. (2012) 122, 1010-1022. ABSTRACT: Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease. It is axonally transported, endocytosed and sorted to different cellular compartments where amyloid beta (Aβ) is produced. However, the mechanism of APP trafficking remains unclear. We present evidence that huntingtin associated protein 1 (HAP1) may reduce Aβ production by regulating APP trafficking to the non-amyloidogenic pathway. HAP1 and APP are highly colocalized in a number of brain regions, with similar distribution patterns in both mouse and human brains. They are associated with each other, the interacting site is the 371-599 of HAP1. APP is more retained in cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi intermediate compartment in HAP1-/- neurons. HAP1 deletion significantly alters APP endocytosis and reduces the re-insertion of APP into the cytoplasmic membrane. Amyloid precursor protein-YFP(APP-YFP) vesicles in HAP1-/- neurons reveal a decreased trafficking rate and an increased number of motionless vesicles. Knock-down of HAP1 protein in cultured cortical neurons of Alzheimer's disease mouse model increases Aβ levels. Our data suggest that HAP1 regulates APP subcellular trafficking to the non-amyloidogenic pathway and may negatively regulate Aβ production in neurons.     

Alternative splicing generates isoforms of the met receptor tyrosine kinase which undergo differential processing.

The met proto-oncogene is a member of the family of tyrosine kinase growth factor receptors. We describe the isolation and characterization of a cDNA clone (pOK) for the met receptor from a gastric carcinoma cell line. This clone differs from the published cDNA clone by the absence of 54 bp predicted to encode 18 amino acids in the extracellular domain. The pOK cDNA corresponds to the most abundant met RNA species of 8 kb expressed in human cell lines and tissue, and we show that there are in fact two 8-kb met receptor tyrosine kinase (RTK) isoforms that are generated by alternative splicing. This newly described met isoform when transiently expressed in COS cells encodes a protein of 190 kDa which corresponds in size to the p190 met alpha beta heterodimer expressed in human cell lines. Furthermore, we show that the 190-kDa product of pOK consists of the 140-kDa met beta subunit associated with the 50-kDa met alpha subunit. This finding suggests that both the alpha and beta met chains are encoded by this construct and confirms the hypothesis that a single chain precursor is cleaved to produce both subunits of met. In contrast, the previously characterized met isoform corresponds to a minor met RNA species and encodes a protein of 170 kDa that is not cleaved yet is processed in a manner that allows cell surface expression. Both met RTK isoforms are autophosphorylated in the in vitro kinase assay. These results suggest that different isoforms of the met RTK may have distinct biological activities.