Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated

Background

Retrograde transport of several transmembrane proteins from endosomes to the trans-Golgi network (TGN) occurs via Rab 5-containing endosomes, mediated by clathrin and the recently characterized retromer complex. This complex and one of its putative sorting receptor components, SorLA, were reported to be associated to late onset Alzheimer's disease (AD). The pathogenesis of this neurodegenerative disorder is still elusive, although accumulation of amyloidogenic Abeta is a hallmark. This peptide is generated from the sucessive β- and γ- secretase proteolysis of the Alzheimer's amyloid precursor protein (APP), events which are associated with endocytic pathway compartments. Therefore, APP targeting and time of residence in endosomes would be predicted to modulate Abeta levels. However, the formation of an APP- and retromer-containing protein complex with potential functions in retrieval of APP from the endosome to the TGN had, to date, not been demonstrated directly. Further, the motif(s) in APP that regulate its sorting to the TGN have not been characterized.

Results

Through the use of APP-GFP constructs, we show that APP containing endocytic vesicles targeted for the TGN, are also immunoreactive for clathrin-, Rab 5- and VPS35. Further, they frequently generate protruding tubules near the TGN, supporting an association with a retromer-mediated pathway. Importantly, we show for the first time, that mimicking APP phosphorylation at S655, within the APP 653YTSI656 basolateral motif, enhances APP retrieval via a retromer-mediated process. The phosphomimetic APP S655E displays decreased APP lysosomal targeting, enhanced mature half-life, and decreased tendency towards Abeta production. VPS35 downregulation impairs the phosphorylation dependent APP retrieval to the TGN, and decreases APP half-life.

Conclusions

We reported for the first time the importance of APP phosphorylation on S655 in regulating its retromer-mediated sorting to the TGN or lysosomes. Significantly, the data are consistent with known interactions involving the retromer, SorLA and APP. Further, these findings add to our understanding of APP targeting and potentially contribute to our knowledge of sporadic AD pathogenesis representing putative new targets for AD therapeutic strategies.     

Recruitment of the Mint3 Adaptor Is Necessary for Export of the Amyloid Precursor Protein (APP) from the Golgi Complex

The amyloid precursor protein (APP) is a ubiquitously expressed single-pass transmembrane protein that undergoes proteolytic processing by secretases to generate the pathogenic amyloid-β peptide, the major component in Alzheimer plaques. The traffic of APP through the cell determines its exposure to secretases and consequently the cleavages that generate the pathogenic or nonpathogenic peptide fragments. Despite the likely importance of APP traffic to Alzheimer disease, we still lack clear models for the routing and regulation of APP in cells. Like the traffic of most transmembrane proteins, the binding of adaptors to its cytoplasmic tail, which is 47 residues long and contains at least four distinct sorting motifs, regulates that of APP. We tested each of these for effects on the traffic of APP from the Golgi by mutating key residues within them and examining adaptor recruitment at the Golgi and traffic to post-Golgi site(s). We demonstrate strict specificity for recruitment of the Mint3 adaptor by APP at the Golgi, a critical role for Tyr-682 (within the YENPTY motif) in Mint3 recruitment and export of APP from the Golgi, and we identify LAMP1⁺ structures as the proximal destination of APP after leaving the Golgi. Together, these data provide a detailed view of the first sorting step in its route to the cell surface and processing by secretases and further highlight the critical role played by Mint3.     

NGF controls APP cleavage by downregulating APP phosphorylation at Thr668: relevance for Alzheimer's disease

NGF has been implicated in forebrain neuroprotection from amyloidogenesis and Alzheimer's disease (AD). However, the underlying molecular mechanisms are still poorly understood. Here, we investigated the role of NGF signalling in the metabolism of amyloid precursor protein (APP) in forebrain neurons using primary cultures of septal neurons and acute septo‐hippocampal brain slices. In this study, we show that NGF controls the basal level of APP phosphorylation at Thr668 (T668) by downregulating the activity of the Ser/Thr kinase JNK(p54) through the Tyr kinase signalling adaptor SH2‐containing sequence C (ShcC). We also found that the specific NGF receptor, Tyr kinase A (TrkA), which is known to bind to APP, fails to interact with the fraction of APP molecules phosphorylated at T668 (APP^pT668). Accordingly, the amount of TrkA bound to APP is significantly reduced in the hippocampus of ShcC KO mice and of patients with AD in which elevated APP^pT668 levels are detected. NGF promotes TrkA binding to APP and APP trafficking to the Golgi, where APP–BACE interaction is hindered, finally resulting in reduced generation of sAPPβ, CTFβ and amyloid‐beta (1‐42). These results demonstrate that NGF signalling directly controls basal APP phosphorylation, subcellular localization and BACE cleavage, and pave the way for novel approaches specifically targeting ShcC signalling and/or the APP–TrkA interaction in AD therapy.     

A possible role for the Alzheimer amyloid precursor protein in the regulation of epidermal basal cell proliferation

The regulation of epidermal growth involves a number of ions, growth factors and cytokines and possibly additional but as yet unknown factors. Here we report on the potential role of the secretory N-terminal domain (sAPP) of the Alzheimer amyloid precursor protein (APP) in the regulation of keratinocyte proliferation. In human skin APP was detectable predominantly in the basal cell layer of the epidermis whereas the immunocytochemical signal in the underlying mesenchymal tissue was very low. Cultured normal human keratinocytes expressed the three APP isoforms 695, 751 and 770 with highest values for the isoforms 751 and 770. HaCaT cells, a spontaneously immortalized human keratinocyte cell line, exhibited almost identical patterns in the expression of the APP isoforms and in the release of endogenous sAPP. In HaCaT cells, recombinant sAPP (sAPPrec) was found to compete with endogenous sAPP for the same binding sites. Binding of sAPPrec was specific and occurred in microdomains of approximately 0.1 to approximately 0.3 microm in diameter. At 10 nM, sAPPrec binding induced a 2- to 4-fold increase in the rate of cell growth. sAPP concentrations in the conditioned media were found to reach 5-20 nM which is in the mitogenic range of sAPPrec. The proliferative effect of sAPP was inhibited by approximately 50% when antisense oligonucleotides directed against the APP mRNA were applied. The predominant expression of     

Regulation of Secretion of Alzheimer Amyloid Precursor Protein by the Mitogen-Activated Protein Kinase Cascade

Abstract: Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid β-peptide (Aβ). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen-activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12-M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC-independent pathway, increased sAPP secretion mainly through a MAP kinase-independent pathway. Aβ secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for Aβ and sAPP formation.     

Polarized transport of Alzheimer amyloid precursor protein is mediated by adaptor protein complex AP1-1B

Alzheimer amyloid precursor protein (APP) is the precursor for the Abeta peptide involved in pathogenesis of Alzheimer's disease. The soluble ectodomain fragment of APP (sAPP) functions as a growth factor for epithelial cells, suggesting an important function for APP outside neuronal tissue. Previous studies have shown that in polarized epithelial cells, APP is targeted to the basolateral domain. Tyr653 within the cytoplasmic tail of APP mediates the basolateral targeting of APP, but the sorting machinery that binds to this residue has largely remained unknown. In this study, we analyzed the role of adaptor complexes in the polarized sorting of APP. We show that the medium subunit mu1B of the epithelia-specific adaptor protein (AP)-1B binds onto the cytoplasmic tail of APP in a Tyr653-dependent way. Moreover, ectopic expression of mu1B in cells lacking AP-1B resulted in correction of apical missorting of wild-type but not Tyr653Ala APP. Basolateral secretion of sAPP was found to be independent of Tyr653. We propose a model for polarized targeting of APP according to which sorting of APP to basolateral domain is dependent on binding of AP-1B on Tyr653 in basolateral endosomes. This model is in accordance with the current understanding of sorting mechanisms mediating polarized targeting of membrane proteins.     

Binding and selective detection of the secretory N-terminal domain of the alzheimer amyloid precursor protein on cell surfaces.

The secretory N-terminal domain of the Alzheimer amyloid precursor protein (sAPP) evokes specific responses in cells on binding to their surfaces. Because APP is expressed in a large variety of cell types, the localization of sAPP binding requires detection techniques that selectively recognize sAPP as a ligand. For this purpose, we prepared antibodies against recombinant sAPP695 (sAPPrec) previously expressed in E. coli. Such antibodies were found to distinguish between sAPPrec and cellular APP or sAPP, as shown by immunocytochemistry and by immunoblot. In addition, they allowed the selective localization of bound sAPPrec on cell surfaces without any signal from cellular APP or sAPP. Saturation of sAPPrec binding to cell surfaces, as determined radiometrically, was reached at 10 nM [125I]-sAPPrec. Binding was specific because it was almost completely inhibited by a 100-fold excess of unlabeled sAPPrec. This specificity of binding was confirmed by surface plasmon resonance spectroscopy. Binding of sAPPrec to cell surfaces occurred in patches and was dependent on the state of cell differentiation. The sAPPrec used in this study contains heparin binding sites, but enzymatic removal of cell surface associated heparin did not affect sAPPrec binding. Aldehyde fixation of cells strongly inhibited their ability to bind sAPPrec. The data point to a fixation-sensitive sAPPrec binding protein which is detectable in the form of patches and therefore is part of assembled cell surface microdomains.     

Sortilin-related receptor with A-type repeats (SORLA) affects the amyloid precursor protein-dependent stimulation of ERK signaling and adult neurogenesis

Sortilin-related receptor with A-type repeats (SORLA) is a sorting receptor that impairs processing of amyloid precursor protein (APP) to soluble (s) APP and to the amyloid beta-peptide in cultured neurons and is poorly expressed in patients with Alzheimer disease (AD). Here, we evaluated the consequences of Sorla gene defects on brain anatomy and function using mouse models of receptor deficiency. In line with a protective role for SORLA in APP metabolism, lack of the receptor results in increased amyloidogenic processing of endogenous APP and in aggravated plaque deposition when introduced into PDAPP mice expressing mutant human APP. Surprisingly, increased levels of sAPP caused by receptor deficiency correlate with pro-found stimulation of neuronal ERK signaling and with enhanced neurogenesis, providing in vivo support for neurotrophic functions of sAPP. Our data document a role for SORLA not only in control of plaque burden but also in APP-dependent neuronal signaling and suggest a molecular explanation for increased neurogenesis observed in some AD patients.     

Ceramide sphingolipid signaling mediates Tumor Necrosis Factor (TNF)-dependent toxicity via caspase signaling in dopaminergic neurons

Background

The A?? peptide that accumulates in Alzheimer??s disease (AD) is derived from amyloid precursor protein (APP) following proteolysis by ??- and ??-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 A?? 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 A?? production.

Results

In 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 A??, while knockdown of LRP10 expression increases A?? 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 ??-cleavage into A??. Furthermore, expression of LRP10 is significantly lower in the post-mortem brain tissues of AD patients, supporting a possible role for LRP10 in AD.

Conclusions

The 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.     

The secreted Alzheimer-related amyloid precursor protein fragment has an essential role in C. elegans

Mutations in the gene encoding the amyloid precursor protein (APP) or the enzymes that process APP are correlated with familial Alzheimer disease. Alzheimer disease is also associated with insulin resistance (type 2 diabetes). In our recently published study,¹ we obtained genetic evidence that the extracellular fragment of APL-1, the C. elegans ortholog of human APP, may act as a signaling molecule to modulate insulin and nuclear hormone pathways in C. elegans development. In addition, independent of insulin and nuclear hormone signaling, high levels of the extracellular fragment of APL-1 (sAPL-1) leads to a temperature-sensitive embryonic lethality, which is dependent on activity of a predicted receptor protein tyrosine phosphatase (MOA-1/R155.2). Furthermore, this embryonic lethality is enhanced by knockdown of a predicted prion-like protein (pqn-29). The precise molecular mechanisms underlying these processes remain to be determined. Here, we present hypothetical models as to how sAPL-1 signaling influences metabolic and developmental pathways. Together, with previous findings in mammals that the extracellular domain of mammalian APP (sAPP) binds to a death-receptor,² our findings support the model that sAPP signaling affects critical biological processes.     

7-Deoxy-trans-dihydronarciclasine Reduces β-Amyloid and Ameliorates Memory Impairment in a Transgenic Model of Alzheimer’s Disease

The critical pathological feature of Alzheimer’s disease (AD) is the accumulation of β-amyloid (Aβ), the main constituent of amyloid plaques. β-amyloid precursor protein (APP) undergoes amyloidogenic cleavage by β- and γ-secretase generating Aβ at endosomes or non-amyloidogenic processing by α-secretase precluding the production of Aβ at the plasma membrane. Recently, several natural products have been widely researched on the prevention of Aβ accumulation for AD treatment. We previously reported that Lycoris chejuensis K. Tae et S. Ko (CJ), which originated from Jeju Island in Korea, improved the disrupted memory functions and reduced Aβ production in vivo. Here, we further explored the effect of its active component, 7-deoxy-trans-dihydronarciclasine (coded as E144), on Aβ generation and the underlying mechanism. Our results showed that E144 reduced the level of APP, especially its mature form, in HeLa cells overexpressing human APP with the Swedish mutation. Concomitantly, E144 decreased the levels of Aβ, sAPPβ, sAPPα, and C-terminal fragment. In addition, administration of E144 normalized the behavioral deficits in Tg2576 mice, an APP transgenic mouse model of AD. E144 also decreased the Aβ and APP levels in the cerebral cortex of Tg2576 mice. Thus, we propose that E144 could be a potential drug candidate for an anti-amyloid disease-modifying AD therapy.     

BECN1/Beclin 1 sorts cell-surface APP/amyloid β precursor protein for lysosomal degradation

The regulation of plasma membrane (PM)-localized transmembrane protein/receptor trafficking has critical implications for cell signaling, metabolism and survival. In this study, we investigated the role of BECN1 (Beclin 1) in the degradative trafficking of PM-associated APP (amyloid β precursor protein), whose metabolism to amyloid-β, an essential event in Alzheimer disease, is dependent on divergent PM trafficking pathways. We report a novel interaction between PM-associated APP and BECN1 that recruits macroautophagy/endosomal regulatory proteins PIK3C3 and UVRAG. We found that BECN1 promotes surface APP internalization and sorting predominantly to endosomes and endolysosomes. BECN1 also promotes the targeting of a smaller fraction of internalized APP to LC3-positive phagophores, suggesting a role for BECN1-dependent PM macroautophagy in APP degradation. Furthermore, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites (soluble ectodomains, sAPP). The association between APP and BECN1 is dependent on the evolutionarily conserved domain (ECD) of BECN1 (amino acids 267–337). Deletion of a BECN1 ECD subregion (amino acids 285–299) did not impair BECN1- PIK3C3 interaction, PtdIns3K function or macroautophagy, but was sufficient to impair the APP-BECN1 interaction and BECN1's effects on surface APP internalization and degradation, resulting in increased secretion of sAPPs. Interestingly, both the BECN1-APP association and BECN1-dependent APP endocytosis and degradative trafficking were negatively regulated by active AKT. Our results further implicate phosphorylation of the BECN1 Ser295 residue in the inhibition of APP degradation by AKT. Our studies reveal a novel function for BECN1 in the sorting of a plasma membrane protein for endolysosomal and macroautophagic degradation.     

Cab45 is required for Ca2+-dependent secretory cargo sorting at the trans-Golgi network

Ca²⁺ import into the lumen of the trans-Golgi network (TGN) by the secretory pathway calcium ATPase1 (SPCA1) is required for the sorting of secretory cargo. How is Ca²⁺ retained in the lumen of the Golgi, and what is its role in cargo sorting? We show here that a soluble, lumenal Golgi resident protein, Cab45, is required for SPCA1-dependent Ca²⁺ import into the TGN; it binds secretory cargo in a Ca²⁺-dependent reaction and is required for its sorting at the TGN.     

Secreted type of amyloid precursor protein induces glial differentiation by stimulating the BMP/Smad signaling pathway

Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases leading to dementia. Although cytotoxicity of amyloid β peptides has been intensively studied within pathophysiology of AD, the physiological function of amyloid precursor protein (APP) still remains unclarified. We have shown previously that secreted APPα (sAPPα) is associated with glial differentiation of neural stem cells. To elucidate specific mechanisms underlying sAPPα-induced gliogenesis, we examined the potential involvement of bone morphogenic proteins (BMPs). BMPs are one of the factors involved in glial differentiation of neural progenitor cells. When expressions of BMP-2, -4, and -7 were examined, upregulation of BMP-4 expression was solely observed as a result of treatment with sAPPα in a time and dose-dependent manner. Furthermore, the treatment of sAPPα promoted phosphorylation of Smad1/5/8, a downstream signaling mediator of BMP receptors. Interestingly, N-terminal domain of APP (1–205) was sufficient to elevate BMP4 expression, resulting in an increase of glial fibrillary acidic protein (GFAP) expression and phosphorylation of Smad1/5/8. However, the application of APP neutralizing antibody and anti-BMP4 antibody significantly suppressed expression of BMP-4 as well as phosphorylation of Smad1/5/8. Thus, our results indicate that sAPPα-induced gliogenesis is in part mediated by the BMP-4 signaling pathway. We also observed upregulation of BMP-4 and phosphorylation of Smad1/5/8 in APP transgenic mice. It is imperative to unravel the mechanisms underlying the role of BMP-4 during APPα-induced glial differentiation in hope of providing novel prevention or treatment for AD.     

Regulation of amyloid precursor protein (APP) phosphorylation and processing by p35/Cdk5 and p25/Cdk5

The phosphorylation status of amyloid precursor protein (APP) at Thr668 is suggested to play a critical role in the proteolytic cleavage of APP, which generates either soluble APP(beta) (sAPP(beta)) and beta-amyloid peptide (Abeta), the major component of senile plaques in patient brains inflicted with Alzheimer's disease (AD), or soluble APP(alpha) (sAPP(alpha)) and a peptide smaller than Abeta. One of the protein kinases known to phosphorylate APP(Thr668) is cyclin-dependent kinase 5 (Cdk5). Cdk5 is activated by the association with its regulatory partner p35 or its truncated form, p25, which is elevated in AD brains. The comparative effects of p35 and p25 on APP(Thr668) phosphorylation and APP processing, however, have not been reported. In this study, we investigated APP(Thr668) phosphorylation and APP processing mediated by p35/Cdk5 and p25/Cdk5 in the human neuroblastoma cell line SH-SY5Y. Transient overexpression of p35 and p25 elicited distinct patterns of APP(Thr668) phosphorylation, specifically, p35 increasing the phosphorylation of both mature and immature APP, whereas p25 primarily elevated the phosphorylation of immature APP. Despite these differential effects on APP phosphorylation, both p35 and p25 overexpression enhanced the secretion of Abeta, sAPP(beta), as well as sAPP(alpha). These results confirm the involvement of Cdk5 in APP processing, and suggest that p35- and p25-mediated Cdk5 activities lead to discrete APP phosphorylation.     

中药Ⅰ号方对APP/PS1双转基因模型小鼠APP代谢的影响

目的研究中药I号方对APP/PS1双转基因模型小鼠APP代谢的影响。方法将5月龄APP/PS1双转基因模型小鼠随机分为模型组（vehicle）、中药Ⅰ号方低剂量组（0.6 g/kg）、中剂量组（1.2 g/kg）和高剂量组（2.4 g/kg）,并以同窝阴性小鼠作为正常对照组（wild-type,WT）,每组16只,雌雄各半。给药小鼠每天灌胃一次,模型组和正常对照组分别给予等体积的双蒸水灌胃。给药四个月后,用免疫组化和Western blot检测淀粉样前体蛋白（APP）及其代谢产物和分解酶的变化。结果 Western blot结果显示,与模型组相比,治疗组低剂量、中剂量和高剂量给药组能显著降低APP分解酶（ADAM10和BACE1）（P〈0.01）及APP的分解产物的量,如：β-CTF（C99）、α-CTF（C83）、s APPα、s APPβ（P〈0.01）。结论中药I号方通过影响APP的分解过程减少淀粉样蛋白（β-amyloid peptide,Aβ）的生成,减少脑内老年斑的沉积。     

Soluble Amyloid Precursor Protein 770 Is Released from Inflamed Endothelial Cells and Activated Platelets: A NOVEL BIOMARKER FOR ACUTE CORONARY SYNDROME*

Most Alzheimer disease (AD) patients show deposition of amyloid β (Aβ) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid β precursor protein (APP) 770, a different APP isoform from neuronal APP695, and produce Aβ. Since the soluble APP cleavage product, sAPP, is considered to be a possible marker for AD diagnosis, sAPP has been widely measured as a mixture of these variants. We hypothesized that measurement of the endothelial APP770 cleavage product in patients separately from that of neuronal APP695 would enable discrimination between endothelial and neurological dysfunctions. Using our newly developed ELISA system for sAPP770, we observed that inflammatory cytokines significantly enhanced sAPP770 secretion by endothelial cells. Furthermore, we unexpectedly found that sAPP770 was rapidly released from activated platelets. We also found that cerebrospinal fluid mainly contained sAPP695, while serum mostly contained sAPP770. Finally, to test our hypothesis that sAPP770 could be an indicator for endothelial dysfunction, we applied our APP770 ELISA to patients with acute coronary syndrome (ACS), in which endothelial injury and platelet activation lead to fibrous plaque disruption and thrombus formation. Development of a biomarker is essential to facilitate ACS diagnosis in clinical practice. The results revealed that ACS patients had significantly higher plasma sAPP770 levels. Furthermore, in myocardial infarction model rats, an increase in plasma sAPP preceded the release of cardiac enzymes, currently used markers for acute myocardial infarction. These findings raise the possibility that sAPP770 can be a useful biomarker for ACS.     

Ca(2+) signalling in the Golgi apparatus

The Golgi apparatus plays a central role in lipid and protein post-translational modification and sorting. Morphologically the organelle is heterogeneous and it is possible to distinguish stacks of flat cysternae (cis- and medial Golgi), tubular-reticular networks and vesicles (trans-Golgi). These morphological differences parallel a distinct functionality with a selective distribution and complementary roles of the enzymes found in the different compartments.The Golgi apparatus has been also shown to be involved in Ca²⁺ signalling: it is indeed endowed with Ca²⁺ pumps, Ca²⁺ release channels and Ca²⁺ binding proteins and is thought to participate in determining the spatio-temporal complexity of the Ca²⁺ signal within the cell, though this role is still poorly understood.Recently, it has been demonstrated that the organelle is heterogeneous in terms of Ca²⁺ handling and selective reduction of Ca²⁺ concentration, both in vitro and in a genetic human disease, within one of its sub-compartment results in alterations of protein trafficking within the secretory pathway and of the entire Golgi morphology.In this paper we review the available information on the Ca²⁺ toolkit within the Golgi, its heterogeneous distribution in the organelle sub-compartments and discuss the implications of these characteristics for the physiopathology of the Golgi apparatus.     

AlphaLISA-based high-throughput screening assay to measure levels of soluble amyloid precursor protein α

Activation of nonamyloidogenic processing of amyloid precursor protein (APP) has been hypothesized to be a viable approach for Alzheimer’s disease drug discovery. However, until recently, the lack of HTS-compatible assay technologies precluded large scale screening efforts to discover molecules that potentiate nonamyloidogenic pathways. We have developed an HTS-compatible assay based on AlphaLISA technology that quantitatively detects soluble APPα (sAPPα), a marker of nonamyloidogenic processing of APP, released from live cells in low volume, 384-well plates. The assay exhibited good QC parameters (Z′ > 0.5, S/B > 2). A pilot screen of 801 compounds yielded a novel chemotype that increased the release of sAPPα 2-fold at 5 μM. These results suggest that the AlphaLISA-based HTS assay is robust and sensitive and can be used to screen large compound collections to discover molecules that potentiate the release of sAPPα. Additionally, we demonstrated that increase of APP processing by nonamyloidogenic pathways will result in decrease of release of amyloidogenic Aβ40 fragments.