Mature Amyloid Precursor Protein Research Articles

Inhibition of 37/67kDa Laminin-1 Receptor Restores APP Maturation and Reduces Amyloid-β in Human Skin Fibroblasts from Familial Alzheimer's Disease.

Alzheimer’s disease (AD) is a fatal neurodegenerative disorder caused by protein misfolding and aggregation, affecting brain function and causing dementia. Amyloid beta (Aβ), a peptide deriving from amyloid precursor protein (APP) cleavage by-and γ-secretases, is considered a pathological hallmark of AD. Our previous study, together with several lines of evidence, identified a strict link between APP, Aβ and 37/67kDa laminin receptor (LR), finding the possibility to regulate intracellular APP localization and maturation through modulation of the receptor. Here, we report that in fibroblasts from familial AD (fAD), APP was prevalently expressed as an immature isoform and accumulated preferentially in the transferrin-positive recycling compartment rather than in the Golgi apparatus. Moreover, besides the altered mitochondrial network exhibited by fAD patient cells, the levels of pAkt and pGSK3 were reduced in respect to healthy control fibroblasts and were accompanied by an increased amount of secreted Aβ in conditioned medium from cell cultures. Interestingly, these features were reversed by inhibition of 37/67kDa LR by NSC47924 a small molecule that was able to rescue the “typical” APP localization in the Golgi apparatus, with consequences on the Aβ level and mitochondrial network. Altogether, these findings suggest that 37/67kDa LR modulation may represent a useful tool to control APP trafficking and Aβ levels with implications in Alzheimer’s disease.

APP Maturation and Intracellular Localization Are Controlled by a Specific Inhibitor of 37/67 kDa Laminin-1 Receptor in Neuronal Cells.

Amyloid precursor protein (APP) is processed along both the nonamyloidogenic pathway preventing amyloid beta peptide (Aβ) production and the amyloidogenic pathway, generating Aβ, whose accumulation characterizes Alzheimer’s disease. Items of evidence report that the intracellular trafficking plays a key role in the generation of Aβ and that the 37/67 kDa LR (laminin receptor), acting as a receptor for Aβ, may mediate Aβ-pathogenicity. Moreover, findings indicating interaction between the receptor and the key enzymes involved in the amyloidogenic pathway suggest a strong link between 37/67 kDa LR and APP processing. We show herein that the specific 37/67 kDa LR inhibitor, NSC48478, is able to reversibly affect the maturation of APP in a pH-dependent manner, resulting in the partial accumulation of the immature APP isoforms (unglycosylated/acetylated forms) in the endoplasmic reticulum (ER) and in transferrin-positive recycling endosomes, indicating alteration of the APP intracellular trafficking. These effects reveal NSC48478 inhibitor as a novel small molecule to be tested in disease conditions, mediated by the 37/67 kDa LR and accompanied by inactivation of ERK1/2 (extracellular signal-regulated kinases) signalling and activation of Akt (serine/threonine protein kinase) with consequent inhibition of GSK3β.

The non-peptidic δ-opioid receptor agonist Tan-67 mediates neuroprotection post-ischemically and is associated with altered amyloid precursor protein expression, maturation and processing in mice.

Tan-67 is a selective non-peptidic δ-opioid receptor (DOR) agonist that confers neuroprotection against cerebral ischemia/reperfusion (I/R)-caused neuronal injury in pre-treated animals. In this study, we examined whether post-ischemic administration of Tan-67 in stroke mice is also neuroprotective and whether the treatment affects expression, maturation and processing of the amyloid precursor protein (APP). A focal cerebral I/R model in mice was induced by middle cerebral artery occlusion for 1h and Tan-67 (1.5, 3 or 4.5mg/kg) wasadministered via the tail vein at 1h after reperfusion. Alternatively, naltrindole, a selective DOR antagonist (5mg/kg), was administered 1h before Tan-67 treatment. Our results showed that post-ischemic administration of Tan-67 (3mg/kg or 4.5mg/kg) was neuroprotective as shown by decreased infarct volume and neuronal loss following I/R. Importantly, Tan-67 improved animal survival and neurobehavioral outcomes. Conversely, naltrindole abolished Tan-67 neuroprotection in infarct volume. Tan-67 treatment also increased APP expression, maturation and processing in the ipsilateral penumbral area at 6h but decreased APP expression and maturation in the same brain area at 24h after I/R. Tan-67-induced increase in APP expression was also seen in the ischemic cortex at 24h following I/R. Moreover, Tan-67 attenuated BACE-1 expression, β-secretase activity and the BACE cleavage of APP in the ischemic cortex at 24h after I/R, which was abolished by naltrindole. Our data suggest that Tan-67 is a promising DOR-dependent therapeutic agent for treating I/R-caused disorder and that Tan-67-mediated neuroprotection may be mediated via modulating APP expression, maturation and processing, despite an uncertain causative relationship between the altered APP and the outcomes observed.

Prostaglandin J2 promotes O-GlcNAcylation raising APP processing by α- and β-secretases: relevance to Alzheimer's disease

Regulation of the amyloid precursor protein (APP) processing by α- and β-secretases is of special interest to Alzheimer's disease (AD), as these proteases prevent or mediate amyloid beta formation, respectively. Neuroinflammation is also implicated in AD. Our data demonstrate that the endogenous mediator of inflammation prostaglandin J2 (PGJ2) promotes full-length APP (FL-APP) processing by α- and β-secretases. The decrease in FL-APP was independent of proteasomal, lysosomal, calpain, caspase, and γ-secretase activities. Moreover, PGJ2-treatment promoted cleavage of secreted APP, specifically sAPPα and sAPPβ, generated by α and β-secretase, respectively. Notably, PGJ2-treatment induced caspase-dependent cleavage of sAPPβ. Mechanistically, PGJ2-treatment selectively diminished mature (O- and N-glycosylated) but not immature (N-glycosylated only) FL-APP. PGJ2-treatment also increased the overall levels of protein O-GlcNAcylation, which occurs within the nucleocytoplasmic compartment. It is known that APP undergoes O-GlcNAcylation and that the latter protects proteins from proteasomal degradation. Our results suggest that by increasing protein O-GlcNAcylation levels, PGJ2 renders mature APP less prone to proteasomal degradation, thus shunting APP toward processing by α- and β-secretases.

E3 ligase mahogunin (MGRN1) influences amyloid precursor protein maturation and secretion.

Altered processing of the Amyloid Precursor Protein (APP) is a well-recognized central pathogenic mechanism in Alzheimer's Disease (AD), and regulation of APP processing is a major focus of research in the AD field. However, how age-associated cellular and molecular changes contribute to changes in the amyloidogenic processing of APP have not been extensively clarified so far. We here provide evidence that the processing of APP is influenced by the e3 ubiquitin ligase Mahogunin (MGRN1), a neuroprotective molecule whose levels decrease with aging. Specifically, the expression of MGRN1 inhibits the maturation of APP by sequestering it in the secretory pathway. This sequestration significantly delayed the proteolytic processing of APP, resulting in a reduced β-amyloid (Aβ) peptide release into the extracellular environment. Accordingly, a reduction of MGRN1 levels in hippocampal neurons, as it occurs during physiological aging, leads to an increased Aβ40 and Aβ42 release. We therefore propose that age contributes to the amyloidogenic processing of APP by altering its intracellular trafficking along the secretory pathway due in part to the down-regulation of MGRN1.

CHARACTERIZATION OF A SMALL-MOLECULE APP TRANSLATION INHIBITOR FOR ALZHEIMER’S DISEASE PREVENTION AND THERAPY

Progressive accumulation of β-amyloid (Aβ) in the brain is a critical upstream event in Alzheimer's disease (AD) pathogenesis and considered as a prime target for disease-modifying therapy of AD. Aβ is generated during proteolytic cleavage of the amyloid precursor protein (APP) and substantial effort has been focused on targeting APP β- and γ-secretases to limit Aβ production. However, since biological effects of these enzymes are not solely limited to APP, undesired off target effects are one of the main drawbacks in the implementation of their inhibitors in clinical practice. As an alternative to inhibiting APP processing, Aβ production can also be reduced by lowering APP expression level through targeting its translation with a small molecule translational inhibitor. We characterized in vitro and in vivo properties of 2-[(pyridin-2-ylmethyl)-amino]-phenol (2-PMAP; MW=200.2 Da), which inhibitory effect on APP translation was identified through a small molecule screen. In CHO APP751WT and CHO APP751SW lines, 2-PMAP lowered in a dose-response manner the steady-state APP level and inhibited Aβx-40 and Aβx-42 production with a minimum effective concentration <0.5μM. Its effect on APP translation was highly specific <25μM concentration. 2-PMAP does not alter APP mRNA level, while its dose-dependent effect on APP translation was directly showed in pulse-chase studies, which also confirmed absence of 2-PMAP effect on APP maturation, processing, and degradation. 2-PMAP has no effect on APP cleavage as shown using specific functional assays of APP secretases. 2-PMAP is BBB penetrant after oral and intravenous dosing and when given systemically to APPSW/PS1dE9 AD transgenic mice it reduced levels of full length APP, APP C-terminal fragments (CTFs) and levels of soluble Aβx-40 and Aβx-42 in the brain. A chronic, four-month treatment of APPSW/PS1dE9 mice with 2-PMAP produced significant reduction in Aβ brain deposition and did not reveal any evidence of observable toxicity, while rescuing the mice from memory deficit. 2-PMAP is a small molecule therapeutic effectively ameliorating Aβ pathology through targeting APP translation. 2-PMAP is unlikely to evoke off target effects associated with APP secretases inhibition and provides additional benefit in lowering levels of cytotoxic APP CTFs.

Mitochondria are devoid of amyloid β-protein (Aβ)-producing secretases: Evidence for unlikely occurrence within mitochondria of Aβ generation from amyloid precursor protein

Mitochondrial dysfunction is implicated in the pathological mechanism of Alzheimer's disease (AD). Amyloid β-protein (Aβ), which plays a central role in AD pathogenesis, is reported to accumulate within mitochondria. However, a question remains as to whether Aβ is generated locally from amyloid precursor protein (APP) within mitochondria. We investigated this issue by analyzing the expression patterns of APP, APP-processing secretases, and APP metabolites in mitochondria separated from human neuroblastoma SH-SY5Y cells and those expressing Swedish mutant APP. APP, BACE1, and PEN-2 protein levels were significantly lower in crude mitochondria than microsome fractions while those of ADAM10 and the other γ-secretase complex components (presenilin 1, nicastrin, and APH-1) were comparable between fractions. The crude mitochondrial fraction containing substantial levels of cathepsin D, a lysosomal marker, was further separated via iodixanol gradient centrifugation to obtain mitochondria- and lysosome-enriched fractions. Mature APP, BACE1, and all γ-secretase complex components (in particular, presenilin 1 and PEN-2) were scarcely present in the mitochondria-enriched fraction, compared to the lysosome-enriched fraction. Moreover, expression of the β-C-terminal fragment (β-CTF) of APP was markedly low in the mitochondria-enriched fraction. Additionally, immunocytochemical analysis showed very little co-localization between presenilin 1 and Tom20, a marker protein of mitochondria. In view of the particularly low expression levels of BACE1, γ-secretase complex proteins, and β-CTF in mitochondria, we propose that it is unlikely that Aβ generation from APP occurs locally within this organelle.

Amyloid beta plaque: a culprit for neurodegeneration.

Increasing life expectancy has resulted in an increase in neurodegenerative disorders like Alzheimer's disease. None of the hypothesis proposed till date explains the exact pathobiology of the disease. It is therefore imperative to understand the underlying mechanisms. Amyloid beta (Aβ) is regarded as the main culprit and maximum therapeutic efforts are centered towards Aβ. This review will discuss about the biosynthesis, the physiological role of Aβ including the pathogenic aggregation of Aβ resulting neurodegenerative cognitive disabilities. Most studies of Alzheimer's disease have focused on the biochemical mechanisms involved in the neurodegenerative processes triggered by Aβ aggregates. Aβ is generated from mature amyloid precursor protein being metabolized by two competing pathways, α-secretase pathway (non-amyloidogenic pathway) and β-secretase (amyloidogenic pathway). The physiological roles of Aβ reported in neurotrophic properties, neurogenesis, synaptic plasticity, metal ion sequestration and specificity of blood brain barrier. The neuronal injury is the result of Aβ oligomerization and it is reported that oligomerization of Aβ contributes to neurodegeneration in Alzheimer's disease. The physiological role of Aβ must be considered in the development of medications that intended to decrease its oligomerization forming plaques in a disease like Alzheimer's disease. The biosynthetic pathways for transport and accumulation of Aβ need to be ascertained as an attempt to develop future strategies for prevention of neurodegenerative disorders.

Increased Alzheimer's disease–like pathology in the APP/ PS1ΔE9 mouse model lacking Nrf2 through modulation of autophagy

The presence of senile plaques is one of the major pathologic hallmarks of the brain with Alzheimer's disease (AD). The plaques predominantly contain insoluble amyloid β-peptide, a cleavage product of the larger amyloid precursor protein (APP). Two enzymes, named β and γ secretase, generate the neurotoxic amyloid-β peptide from APP. Mature APP is also turned over endogenously by autophagy, more specifically by the endosomal-lysosomal pathway. A defective lysosomal system is known to be pathogenic in AD. Modulation of NF-E2 related factor 2 (Nrf2) has been shown in several neurodegenerative disorders, and Nrf2 has become a potential therapeutic target for various neurodegenerative disorders, including AD, Parkinson's disease, and amyotrophic lateral sclerosis. In the current study, we explored the effect of genetic ablation of Nrf2 on APP/Aβ processing and/or aggregation as well as changes in autophagic dysfunction in APP/PS1 mice. There was a significant increase in inflammatory response in APP/PS1 mice lacking Nrf2. This was accompanied by increased intracellular levels of APP, Aβ (1–42), and Aβ (1–40), without a change total full-length APP. There was a shift of APP and Aβ into the insoluble fraction, as well as increased poly-ubiquitin conjugated proteins in mice lacking Nrf2. APP/PS1-mediated autophagic dysfunction is also enhanced in Nrf2-deficient mice. Finally, neurons in the APP/PS1/Nrf2−/− mice had increased accumulation of multivesicular bodies, endosomes, and lysosomes. These outcomes provide a better understanding of the role of Nrf2 in modulating autophagy in an AD mouse model and may help design better Nrf2 targeted therapeutics that could be efficacious in the treatment of AD.

The familial Alzheimer's disease APPV717I mutation alters APP processing and Tau expression in iPSC-derived neurons

Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized by extracellular plaques containing amyloid β (Aβ)-protein and intracellular tangles containing hyperphosphorylated Tau protein. Here, we describe the generation of inducible pluripotent stem cell lines from patients harboring the London familial AD (fAD) amyloid precursor protein (APP) mutation (V717I). We examine AD-relevant phenotypes following directed differentiation to forebrain neuronal fates vulnerable in AD. We observe that over differentiation time to mature neuronal fates, APP expression and levels of Aβ increase dramatically. In both immature and mature neuronal fates, the APPV717I mutation affects both β- and γ-secretase cleavage of APP. Although the mutation lies near the γ-secretase cleavage site in the transmembrane domain of APP, we find that β-secretase cleavage of APP is elevated leading to generation of increased levels of both APPsβ and Aβ. Furthermore, we find that this mutation alters the initial cleavage site of γ-secretase, resulting in an increased generation of both Aβ42 and Aβ38. In addition to altered APP processing, an increase in levels of total and phosphorylated Tau is observed in neurons with the APPV717I mutation. We show that treatment with Aβ-specific antibodies early in culture reverses the phenotype of increased total Tau levels, implicating altered Aβ production in fAD neurons in this phenotype. These studies use human neurons to reveal previously unrecognized effects of the most common fAD APP mutation and provide a model system for testing therapeutic strategies in the cell types most relevant to disease processes.

OP07 Neuroprotective effect of hydrogen sulfide: Regulation of amyloidosis and inflammation in SH-SY5Y neuroblastoma and BV-2 microglia cells

The excessive accumulation of amyloid peptide and chronic neuroinflammation have been shown to play indisputable roles in pathogenesis of Alzheimer’s disease (AD). In the process of ongoing neurological damage in AD, increased extracellular levels of ATP and its metabolites (e.g. adenosine) in brain worsen the scenario by evoking the cascade of harmful events. The goal of this project was to investigate anti-amyloidogenic and anti-inflammatory effects of hydrogen sulfide (H 2 S) in various in vitro experimental models used for studying AD pathology. We found that NaHS attenuated HENECA (selective A2A adenosinergic receptor agonist) induced amyloid β -42 (A β 42) production in SH-SY5Y cells. NaHS interfered with the production and post-translational modification of amyloid precursor protein (APP) by inhibiting APP maturation. While NaHS did not affect β -secretase activity, it significantly inhibited γ -secretase activity. NaHS induced-reductions were accompanied by similar decreases in intracellular cAMP levels and phosphorylation of cAMP responsive element binding protein (CREB). Moreover, studies using forskolin (adenylyl cyclase agonist) and IBMX (phosphodiesterase inhibitor) suggested that NaHS may preferentially suppress adenylyl cyclase activity when it is stimulated. Therefore, H 2 S protects SH-SY5Y cells against HENECA induced amyloidogenesis by inhibiting γ -secretase via cAMP dependent pathway. Consistent with its effect in SH-SY5Y cells, NaHS significantly reduced ATP induced APP holoprotein production and A β 42 levels in murine microglial BV-2 cells. We found that NaHS suppressed ATP induced production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF- α ) and interleukin - 1 beta (IL-1 β ). NaHS reversed the upregulation of oxidative stress markers like nitric oxide (NO) and reactive oxygen species (ROS) accompanied by decreases in the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, NaHS suppressed ATP augmentation of extracellular signal-regulated protein kinases 1 and 2 (ERK 1/2) expression as P2 purinergic receptor couple to the ERK signaling pathway. We also found that inhibition of ERK pathway by NaHS blocked specific serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) transcription factor which regulates genes participating in inflammation and amyloidogenesis. To this end, we found that H 2 S inhibits ATP induced inflammation and amyloidogenesis in BV-2 cells via regulating ERK1/2 and STAT3 signaling pathway. Overall, our results demonstrate that by combination of anti-amyloidogenic and anti-inflammatory properties, H 2 S can be of immense therapeutic value in treatment of AD.

Amyloid precursor proteins are constituents of the presynaptic active zone

The amyloid precursor protein (APP) and its mammalian homologs, APLP1, APLP2, have been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a cell surface-localized pool. In the brain, all APPs are restricted to neurons; however, their precise localization at the plasma membrane remained enigmatic. Employing a variety of subcellular fractionation steps, we isolated two synaptic vesicle (SV) pools from rat and mouse brain, a pool consisting of synaptic vesicles only and a pool comprising SV docked to the presynaptic plasma membrane. Immunopurification of these two pools using a monoclonal antibody directed against the 12 membrane span synaptic vesicle protein2 (SV2) demonstrated unambiguously that APP, APLP1 and APLP2 are constituents of the active zone of murine brain but essentially absent from free synaptic vesicles. The specificity of immunodetection was confirmed by analyzing the respective knock-out animals. The fractionation experiments further revealed that APP is accumulated in the fraction containing docked synaptic vesicles. These data present novel insights into the subsynaptic localization of APPs and are a prerequisite for unraveling the physiological role of all mature APP proteins in synaptic physiology.

Statins Reduce Amyloid β-Peptide Production by Modulating Amyloid Precursor Protein Maturation and Phosphorylation Through a Cholesterol-Independent Mechanism in Cultured Neurons

Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been reported to attenuate amyloid-β peptide (Aβ) production in various cellular models. However, the mechanisms by which statins affect neuronal Aβ production have not yet been clarified. Here, we investigated this issue in rat primary cortical neurons using two statins, pitavastatin (PV) and atorvastatin (AV). Treatment of neurons with 0.2-2.5μM PV or AV for 4days induced a concentration- and time-dependent reduction in the secretion of both Aβ40 and Aβ42. Moreover, Western blot analyses of cell lysates showed that treatment with PV or AV significantly reduced expression levels of the mature form of amyloid precursor protein (APP) and Thr668-phosphorylated APP (P-APP), but not immature form of APP; the decreases in P-APP levels were more notable than those of mature APP levels. The statin treatment did not alter expression of BACE1 (β-site APP-cleaving enzyme 1) or γ-secretase complex proteins (presenilin 1, nicastrin, APH-1, and PEN-2). In neurons overexpressing APP via recombinant adenoviruses, PV or AV similarly reduced Aβ secretion and the levels of mature APP and P-APP. Statins also markedly reduced cellular cholesterol content in neurons in a concentration-dependent manner. Co-treatment with mevalonate reversed the statin-induced decreases in Aβ secretion and mature APP and P-APP levels, whereas co-treatment with cholesterol did not, despite recovery of cellular cholesterol levels. Finally, cell-surface biotinylation experiments revealed that both statins significantly reduced the levels of cell-surface P-APP without changing those of cell surface mature APP. These results suggest that statins reduce Aβ production by selectively modulating APP maturation and phosphorylation through a mechanism independent of cholesterol reduction in cultured neurons.

Retention in endoplasmic reticulum 1 (RER1) modulates amyloid-β (Aβ) production by altering trafficking of γ-secretase and amyloid precursor protein (APP).

Aβ production is influenced by intracellular trafficking of secretases and amyloid precursor protein (APP). Retention in endoplasmic reticulum 1 (RER1) regulates the trafficking of γ-secretase and APP, thereby influences Aβ production. RER1, an ER retention/retrieval factor for γ-secretase and APP, modulates Aβ production. RER1 and its influence on γ-secretase and APP may be implicated for a safe strategy to target Aβ production. The presence of neuritic plaques containing aggregated amyloid-β (Aβ) peptides in the brain parenchyma is a pathological hallmark of Alzheimer disease (AD). Aβ is generated by sequential cleavage of the amyloid β precursor protein (APP) by β- and γ-secretase, respectively. As APP processing to Aβ requires transport through the secretory pathway, trafficking of the substrate and access to the secretases are key factors that can influence Aβ production (Thinakaran, G., and Koo, E. H. (2008) Amyloid precursor protein trafficking, processing, and function. J. Biol. Chem. 283, 29615-29619). Here, we report that retention in endoplasmic reticulum 1 (RER1) associates with γ-secretase in early secretory compartments and regulates the intracellular trafficking of γ-secretase. RER1 overexpression decreases both γ-secretase localization on the cell surface and Aβ secretion and conversely RER1 knockdown increases the level of cell surface γ-secretase and increases Aβ secretion. Furthermore, we find that increased RER1 levels decrease mature APP and increase immature APP, resulting in less surface accumulation of APP. These data show that RER1 influences the trafficking and localization of both γ-secretase and APP, thereby regulating the production and secretion of Aβ peptides.

Ubiquilin-1 regulates amyloid precursor protein maturation and degradation by stimulating K63-linked polyubiquitination of lysine 688

The pathogenesis of Alzheimer's disease (AD) is associated with proteolytic processing of the amyloid precursor protein (APP) to an amyloidogenic peptide termed Aβ. Although mutations in APP and the secretase enzymes that mediate its processing are known to result in familial forms of AD, the mechanisms underlying the more common sporadic forms of the disease are still unclear. Evidence suggests that the susceptibility of APP to amyloidogenic processing is related to its intracellular localization, and that secretase-independent degradation may prevent the formation of cytotoxic peptide fragments. Recently, single nucleotide polymorphisms in the UBQLN1 gene have been linked to late-onset AD, and its protein product, ubiquilin-1, may regulate the maturation of full-length APP. Here we show that ubiquilin-1 inhibits the maturation of APP by sequestering it in the early secretory pathway, primarily within the Golgi apparatus. This sequestration significantly delayed the proteolytic processing of APP by secretases and the proteasome. These effects were mediated by ubiquilin-1-stimulated K63-linked polyubiquitination of lysine 688 in the APP intracellular domain. Our results reveal the mechanistic basis by which ubiquilin-1 regulates APP maturation, with important consequences for the pathogenesis of late-onset AD.

LDLR-related protein 10 (LRP10) regulates amyloid precursor protein (APP) trafficking and processing: evidence for a role in Alzheimer’s disease

BackgroundThe Aβ peptide that accumulates in Alzheimer’s disease (AD) is derived from amyloid precursor protein (APP) following proteolysis by β- and γ-secretases. Substantial evidence indicates that alterations in APP trafficking within the secretory and endocytic pathways directly impact the interaction of APP with these secretases and subsequent Aβ production. Various members of the low-density lipoprotein receptor (LDLR) family have been reported to play a role in APP trafficking and processing and are important risk factors in AD. We recently characterized a distinct member of the LDLR family called LDLR-related protein 10 (LRP10) that shuttles between the trans-Golgi Network (TGN), plasma membrane (PM), and endosomes. Here we investigated whether LRP10 participates in APP intracellular trafficking and Aβ production.ResultsIn this report, we provide evidence that LRP10 is a functional APP receptor involved in APP trafficking and processing. LRP10 interacts directly with the ectodomain of APP and colocalizes with APP at the TGN. Increased expression of LRP10 in human neuroblastoma SH-SY5Y cells induces the accumulation of mature APP in the Golgi and reduces its presence at the cell surface and its processing into Aβ, while knockdown of LRP10 expression increases Aβ production. Mutations of key motifs responsible for the recycling of LRP10 to the TGN results in the aberrant redistribution of APP with LRP10 to early endosomes and a concomitant increase in APP β-cleavage into Aβ. Furthermore, expression of LRP10 is significantly lower in the post-mortem brain tissues of AD patients, supporting a possible role for LRP10 in AD.ConclusionsThe present study identified LRP10 as a novel APP sorting receptor that protects APP from amyloidogenic processing, suggesting that a decrease in LRP10 function may contribute to the pathogenesis of Alzheimer’s disease.

DC2 and Keratinocyte-associated Protein 2 (KCP2), Subunits of the Oligosaccharyltransferase Complex, Are Regulators of the γ-Secretase-directed Processing of Amyloid Precursor Protein (APP)

The oligosaccharyltransferase complex catalyzes the transfer of oligosaccharide from a dolichol pyrophosphate donor en bloc onto a free asparagine residue of a newly synthesized nascent chain during the translocation in the endoplasmic reticulum lumen. The role of the less known oligosaccharyltransferase (OST) subunits, DC2 and KCP2, recently identified still remains to be determined. Here, we have studied DC2 and KCP2, and we have established that DC2 and KCP2 are substrate-specific, affecting amyloid precursor protein (APP), indicating that they are not core components required for N-glycosylation and OST activity per se. We show for the first time that DC2 and KCP2 depletion affects APP processing, leading to an accumulation of C-terminal fragments, both C99 and C83, and a reduction in full-length mature APP. This reduction in mature APP levels was not due to a block in secretion because the levels of sAPPα secreted into the media were unaffected. We discover that DC2 and KCP2 depletion affects only the γ-secretase complex, resulting in a reduction of the PS1 active fragment blocking Aβ production. Conversely, we show that the overexpression of DC2 and KCP2 causes an increase in the active γ-secretase complex, particularly the N-terminal fragment of PS1 that is generated by endoproteolysis, leading to a stimulation of Aβ production upon overexpression of DC2 and KCP2. Our findings reveal that components of the OST complex for the first time can interact with the γ-secretase and affect the APP processing pathway.

LRAD3, A Novel Low-Density Lipoprotein Receptor Family Member That Modulates Amyloid Precursor Protein Trafficking

We have identified a novel low-density lipoprotein (LDL) receptor family member, termed LDL receptor class A domain containing 3 (LRAD3), which is expressed in neurons. The LRAD3 gene encodes an ∼50 kDa type I transmembrane receptor with an ectodomain containing three LDLa repeats, a transmembrane domain, and a cytoplasmic domain containing a conserved dileucine internalization motif and two polyproline motifs with potential to interact with WW-domain-containing proteins. Immunohistochemical analysis of mouse brain reveals LRAD3 expression in the cortex and hippocampus. In the mouse hippocampal-derived cell line HT22, LRAD3 partially colocalizes with amyloid precursor protein (APP) and interacts with APP as revealed by coimmunoprecipitation experiments. To identify the portion of APP that interacts with LRAD3, we used solid-phase binding assays that demonstrated that LRAD3 failed to bind to a soluble APP fragment (sAPPα) released after α-secretase cleavage. In contrast, C99, the β-secretase product that remains cell associated, coprecipitated with LRAD3, confirming that regions within this portion of APP are important for associating with LRAD3. The association of LRAD3 with APP increases the amyloidogenic pathway of APP processing, resulting in a decrease in sAPPα production and increased Aβ peptide production. Pulse-chase experiments confirm that LRAD3 expression significantly decreases the cellular half-life of mature APP. These results reveal that LRAD3 influences APP processing and raises the possibility that LRAD3 alters APP function in neurons, including its downstream signaling.

Inhibition of acyl‐coenzyme A: cholesterol acyl transferase modulates amyloid precursor protein trafficking in the early secretory pathway

Amyloid beta-peptide (Abeta) has a central role in the pathogenesis of Alzheimer's disease (AD). Cellular cholesterol homeostasis regulates endoproteolytic generation of Abeta from the amyloid precursor protein (APP). Previous studies have identified acyl-coenzyme A: cholesterol acyltransferase (ACAT), an enzyme that regulates subcellular cholesterol distribution, as a potential therapeutic target for AD. Inhibition of ACAT activity decreases Abeta generation in cell- and animal-based models of AD through an unknown mechanism. Here we show that ACAT inhibition retains a fraction of APP molecules in the early secretory pathway, limiting the availability of APP for secretase-mediated proteolytic processing. ACAT inhibitors delayed the trafficking of immature APP molecules from the endoplasmic reticulum (ER) as shown by metabolic labeling and live-cell imaging. This resulted in partial ER retention of APP and enhanced ER-associated degradation of APP by the proteasome, without activation of the unfolded protein response pathway. The ratio of mature APP to immature APP was reduced in brains of mice treated with ACAT inhibitors, and strongly correlated with reduced brain APP-C99 and cerebrospinal fluid Abeta levels in individual animals. Our results identify a novel ACAT-dependent mechanism that regulates secretory trafficking of APP, likely contributing to decreased Abeta generation in vivo.

S655 phosphorylation enhances APP secretory traffic

Cellular protein phosphorylation regulates proteolytic processing of the Alzheimer's Amyloid Precursor Protein (APP). This appears to occur both indirectly and directly via APP phosphorylation at residues within cytoplasmic motifs related to targeting and protein-protein interactions. The sorting signal (653)YTSI(656) comprises the S655 residue that can be phosphorylated by PKC, particularly in mature APP molecules. The YTSI domain has been associated with APP internalization and Golgi polarized sorting, but no functional significance has been attributed to S655 phosphorylation thus far. Using APP(695)-GFP S655 phosphomutants we show that S655 phosphorylation is a signal that positively modulates APP secretory traffic. The phosphomimicking and dephosphomimicking S655 mutants exhibited contrasting Golgi dynamics, which correlated with differential Golgi vesicular exit and secretory cleavage to sAPP. The role of S655 phosphorylation in APP trafficking at sorting stations, such as the Golgi, its contribution toward cytoprotective alpha sAPP production, and implications for Alzheimer's disease are discussed.