Cognitive changes and modified processing of amyloid precursor protein in the cortical and hippocampal system after cholinergic synapse loss and muscarinic receptor activation.
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A number of in vitro studies have shown that activation of muscarinic receptors by cholinergic agonists stimulates the nonamyloidogenic, alpha-secretase-processing pathway of amyloid precursor protein (APP). To determine whether increased cholinergic neurotransmission can modify the APP processing in vivo, we administered a muscarinic receptor agonist (RS86) to normal or aged rats and rats with severe basal forebrain cholinergic deficits (induced by 192 IgG-saporin). The levels of the cell-associated APP in neocortex, hippocampus, and striatum, as well as the secreted form of APP (APPs) in cerebrospinal fluid, were examined by Western blots. Additionally, we investigated the association between the altered APP levels and behavioral deficits caused by cholinergic lesions. We found that treatment with muscarinic receptor agonist resulted in decreased APP levels in neocortex and hippocampus and increased levels of APPs in cerebrospinal fluid. Regulation of APP processing by the muscarinic agonist treatment occurred not only in normal rats, but also in aged and cholinergic denervated rats that model this aspect of Alzheimer's disease. Interestingly, we found that elevation of APP in neocortex correlated with the cognitive deficits in water-maze testing of rats with cholinergic dysfunction. These data indicate that increased cholinergic neurotransmission can enhance nonamyloidogenic APP processing in intact and lesioned rats and that APP may be involved in cognitive performance. (+info)
Presenilin 1 controls gamma-secretase processing of amyloid precursor protein in pre-golgi compartments of hippocampal neurons.
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Mutations of presenilin 1 (PS1) causing Alzheimer's disease selectively increase the secretion of the amyloidogenic betaA4(1-42), whereas knocking out the gene results in decreased production of both betaA4(1-40) and (1-42) amyloid peptides (De Strooper et al. 1998). Therefore, PS1 function is closely linked to the gamma-secretase processing of the amyloid precursor protein (APP). Given the ongoing controversy on the subcellular localization of PS1, it remains unclear at what level of the secretory and endocytic pathways PS1 exerts its activity on APP and on the APP carboxy-terminal fragments that are the direct substrates for gamma-secretase. Therefore, we have reinvestigated the subcellular localization of endogenously expressed PS1 in neurons in vitro and in vivo using confocal microscopy and fine-tuned subcellular fractionation. We show that uncleaved PS1 holoprotein is recovered in the nuclear envelope fraction, whereas the cleaved PS fragments are found mainly in post-ER membranes including the intermediate compartment (IC). PS1 is concentrated in discrete sec23p- and p58/ERGIC-53-positive patches, suggesting its localization in subdomains involved in ER export. PS1 is not found to significant amounts beyond the cis-Golgi. Surprisingly, we found that APP carboxy-terminal fragments also coenrich in the pre-Golgi membrane fractions, consistent with the idea that these fragments are the real substrates for gamma-secretase. Functional evidence that PS1 exerts its effects on gamma-secretase processing of APP in the ER/IC was obtained using a series of APP trafficking mutants. These mutants were investigated in hippocampal neurons derived from transgenic mice expressing PS1wt or PS1 containing clinical mutations (PS1(M146L) and PS1(L286V)) at physiologically relevant levels. We demonstrate that the APP-London and PS1 mutations have additive effects on the increased secretion of betaA4(1-42) relative to betaA4(1-40), indicating that both mutations operate independently. Overall, our data clearly establish that PS1 controls gamma(42)-secretase activity in pre-Golgi compartments. We discuss models that reconcile this conclusion with the effects of PS1 deficiency on the generation of betaA4(1-40) peptide in the late biosynthetic and endocytic pathways. (+info)
Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE.
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Cerebral deposition of amyloid beta peptide (Abeta) is an early and critical feature of Alzheimer's disease. Abeta generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: beta-secretase and gamma-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of beta-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increased the amount of beta-secretase cleavage products, and these were cleaved exactly and only at known beta-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreased the amount of beta-secretase cleavage products, and purified BACE protein cleaved APP-derived substrates with the same sequence specificity as beta-secretase. Finally, the expression pattern and subcellular localization of BACE were consistent with that expected for beta-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease. (+info)
Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin I and SH3PX1.
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Metalloprotease disintegrins (a disintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin, cysteine-rich proteins (MDC)) are a family of membrane-anchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as proline-rich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain- and phox homology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization. (+info)
The Alzheimer's disease amyloid precursor protein modulates copper-induced toxicity and oxidative stress in primary neuronal cultures.
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The amyloid precursor protein (APP) of Alzheimer's disease can reduce copper (II) to copper (I) in a cell-free system potentially leading to increased oxidative stress in neurons. We used neuronal cultures derived from APP knock-out (APP(-/-)) and wild-type (WT) mice to examine the role of APP in copper neurotoxicity. WT cortical, cerebellar, and hippocampal neurons were significantly more susceptible than their respective APP(-/-) neurons to toxicity induced by physiological concentrations of copper but not by zinc or iron. There was no difference in copper toxicity between APLP2(-/-) and WT neurons, demonstrating specificity for APP-associated copper toxicity. Copper uptake was the same in WT and APP(-/-) neurons, suggesting APP may interact with copper to induce a localized increase in oxidative stress through copper (I) production. This was supported by significantly higher levels of copper-induced lipid peroxidation in WT neurons. Treatment of neuronal cultures with a peptide corresponding to the human APP copper-binding domain (APP142-166) potentiated copper but not iron or zinc toxicity. Incubation of APP142-166 with low-density lipoprotein (LDL) and copper resulted in significantly increased lipid peroxidation compared to copper and LDL alone. Substitution of the copper coordinating histidine residues with asparagines (APP142-166(H147N, H149N, H151N)) abrogated the toxic effects. A peptide corresponding to the zinc-binding domain (APP181-208) failed to induce copper or zinc toxicity in neuronal cultures. These data support a role for the APP copper-binding domain in APP-mediated copper (I) generation and toxicity in primary neurons, a process that has important implications for Alzheimer's disease and other neurodegenerative disorders. (+info)
The amyloid precursor protein of Alzheimer disease in human brain and blood.
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Studies of the metabolism and function of the amyloid precursor protein (APP) and its proteolytic fragment A beta in cultured cells, transgenic mice, and post-mortem brain tissue have advanced our understanding of Alzheimer disease (AD). However, the molecular pathogenesis of the disease is still not clear, and we are a long way from finding a cure for the disease. Studies carried out on human platelets and leukocytes have also helped shed light on APP and A beta metabolism and function. Platelet and leukocyte APP isoforms are processed using mechanisms similar to those in neuronal cells to generate A beta and soluble forms of APP. The activation of platelets and leukocytes leads to the secretion of APP and A beta, resulting in higher levels of these proteins in serum. APP and A beta in the circulation may be involved in the regulation of platelet function and in the modulation of immune responses. Because human platelets and lymphocytes produce all forms of APP and secrete amyloidogenic A beta peptides, these tissues may be useful in monitoring responses to therapeutic interventions directed at APP metabolism. Although not of neuronal origin, further studies on the more accessible ex vivo tissues, including platelets and leukocytes and other blood components, may reveal potential peripheral markers for AD and will further our understanding of the molecular pathogenesis of AD. (+info)
Amyloid precursor protein, although partially detergent-insoluble in mouse cerebral cortex, behaves as an atypical lipid raft protein.
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Lipid rafts are regions of the plasma membrane that are enriched in cholesterol, glycosphingolipids and acylated proteins, and which have been proposed as sites for the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Lipid rafts can be isolated on the basis of their insolubility in Triton X-100 at 4 degrees C, with the resulting low-density, detergent-insoluble glycolipid-enriched fraction (DIG) being isolated by flotation through a sucrose density gradient. The detergent-insolubility of APP in mouse cerebral cortex relative to a variety of DIG marker proteins (alkaline phosphatase, flotillin, F3 protein and prion protein) and non-DIG proteins (alkaline phosphodiesterase I, aminopeptidase A and clathrin) has been examined. Alkaline phosphatase, flotillin, F3 protein and the prion protein were present exclusively in the DIG region of the sucrose gradient over a range of protein/detergent ratios used to solubilize the membranes and displayed a characteristic enrichment in the low-density fraction as the protein/detergent ratio was decreased. In contrast, most of the APP, alkaline phosphodiesterase I, aminopeptidase A and clathrin was effectively solubilized at all of the protein/detergent ratios examined. However, a minor proportion of these latter proteins was detected in DIGs at levels which remained constant irrespective of the protein/detergent ratio. When DIGs were isolated from the sucrose gradients and treated with excess Triton X-100, both the DIG marker proteins and APP, alkaline phosphodiesterase I and clathrin were predominantly resistant to detergent extraction at 37 degrees C. These results show that, although a minor proportion of APP is present in DIGs, where it is detergent-insoluble even at 37 degrees C, it behaves as an atypical lipid raft protein and raises questions as to whether lipid rafts are a site for its proteolytic processing. (+info)
Elimination of the class A scavenger receptor does not affect amyloid plaque formation or neurodegeneration in transgenic mice expressing human amyloid protein precursors.
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The class A scavenger receptor (SR) is expressed on reactive microglia surrounding cerebral amyloid plaques in Alzheimer's disease (AD). Interactions between the SR and amyloid beta peptides (Abeta) in microglial cultures elicit phagocytosis of Abeta aggregates and release of neurotoxins. To assess the role of the SR in amyloid clearance and Abeta-associated neurodegeneration in vivo, we used the platelet-derived growth factor promoter to express human amyloid protein precursors (hAPPs) in neurons of transgenic mice. With increasing age, hAPP mice develop AD-like amyloid plaques. We bred heterozygous hAPP (hAPP(+/-)) mice that were wild type for SR (SR(+/+)) with SR knockout (SR(-/-)) mice. Crosses among the resulting hAPP(+/-)SR(+/-) offspring yielded hAPP(+/-) and hAPP(-/-) littermates that were SR(+/+) or SR(-/-). These second-generation mice were analyzed at 6 and 12 months of age for extent of cerebral amyloid deposition and loss of synaptophysin-immunoreactive presynaptic terminals. hAPP(-/-)SR(-/-) mice showed no lack of SR expression, plaque formation, or synaptic degeneration, indicating that lack of SR expression does not result in significant accumulation of endogenous amyloidogenic or neurotoxic factors. In hAPP(+/-) mice, ablation of SR expression did not alter number, extent, distribution, or age-dependent accumulation of plaques; nor did it affect synaptic degeneration. Our results do not support a critical pathogenic role for microglial SR expression in neurodegenerative alterations associated with cerebral beta amyloidosis. (+info)