Sandra I. Vieira

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

BackgroundRetrograde 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 Alzheimers 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 Alzheimers 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.ResultsThrough 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.ConclusionsWe 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.

Synthesis, mechanical and biological characterization of ionic doped carbonated hydroxyapatite/β-tricalcium phosphate mixtures

The influence of ionic substituents in calcium phosphates intended for bone and tooth replacement biomedical applications is an important research topic, owing to the essential roles played by trace elements in biological processes. The present study investigates the mechanical and biological evaluation of ionic doped hydroxyapatite/β-tricalcium phosphate mixtures which have been prepared by a simple aqueous precipitation method. Heat treating the resultant calcium phosphates in a carbonated atmosphere led to the formation of ionic doped carbonated hydroxyapatite/β-tricalcium phosphate mixtures containing the essential ions of biological apatite. The structural analysis determined by Rietveld refinement confirmed the presence of hydroxyapatite as the main phase, together with a considerable amount of β-tricalcium phosphate. Such phase assemblage is essentially due to the influence of substituted ions during synthesis. The results from mechanical tests proved that carbonate substitutions are detrimental for the mechanical properties of apatite-based ceramics. In vitro proliferation assays of osteoblastic-like cells (MC3T3-E1 cell line) to powders revealed that carbonate incorporation can either delay or accelerate MC3T3 proliferation, although reaching the same proliferation levels as control cells after 2 weeks in culture. Further, the powders enable pre-osteoblastic differentiation in a similar manner to control cells, as indirectly measured by ALP activity and Type-I collagen medium secretion.

Aβ promotes Alzheimer’s disease‐like cytoskeleton abnormalities with consequences to APP processing in neurons

J. Neurochem. (2010) 113, 761–771.

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 653YTSI656 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 APP695-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.

Enhanced generation of Alzheimer’s amyloid‐β following chronic exposure to phorbol ester correlates with differential effects on alpha and epsilon isozymes of protein kinase C

Alzheimer’s amyloid precursor protein (APP) sorting and processing are modulated through signal transduction mechanisms regulated by protein phosphorylation. Notably, protein kinase C (PKC) appears to be an important component in signaling pathways that control APP metabolism. PKCs exist in at least 11 conventional and unconventional isoforms, and PKCα and PKCε isoforms have been specifically implicated in controlling the generation of soluble APP and amyloid‐β (Aβ) fragments of APP, although identification of the PKC substrate phospho‐state‐sensitive effector proteins remains challenging. In the current study, we present evidence that chronic application of phorbol esters to cultured cells in serum‐free medium is associated with several phenomena, namely: (i) PKCα down‐regulation; (ii) PKCε up‐regulation; (iii) accumulation of APP and/or APP carboxyl‐terminal fragments in the trans Golgi network; (iv) disappearance of fluorescence from cytoplasmic vesicles bearing a green fluorescent protein tagged form of APP; (v) insensitivity of soluble APP release following acute additional phorbol application; and (vi) elevated cellular APP mRNA levels and holoprotein, and secreted Aβ. These data indicate that, unlike acute phorbol ester application, which is accompanied by lowered Aβ generation, chronic phorbol ester treatment causes differential regulation of PKC isozymes and increased Aβ generation. These data have implications for the design of amyloid‐lowering strategies based on modulating PKC activity.

Signal transduction therapeutics: relevance for Alzheimer's disease.

It is now widely accepted that abnormal processing of the Alzheimer’s amyloid precursor protein (APP) can contribute significantly to Alzheimer’s disease (AD). APP can be processed proteolytically to give rise to several fragments, including toxic β-amyloid (Aβ) fragments that are subsequently deposited as amyloid plaques in brains of AD patients. Data from several groups have revealed that APP processing can be regulated by phosphorylation and phosphorylation-dependent events. Consequently, the key players controlling such signal transduction cascades, the protein kinases and phosphatases, as well as their corresponding regulatory proteins, take on added importance. By characterizing how altered cell signaling might contribute to APP processing, one can identify potential targets for signal transduction therapeutics. Here, we review APP phosphorylation and phosphorylation-dependent events in APP processing, with particular focus on phosphatases that impact on APP processing, and their binding and regulatory proteins. Particular attention is given to protein phosphatase 1 (PP1), as it seems to have a central role not only in the regulation of APP cleavage events but also in the molecular control of neurotransmission and in age-related memory deterioration. The development of specific drugs targeting protein phosphatase binding proteins would constitute potential therapeutic agents with a high degree of specificity. The identification of such targets provides novel therapeutic avenues for normal aging and for neurodegenerative conditions such as AD.

Tyr687 dependent APP endocytosis and Abeta production.

The neurotoxic Abeta peptide is derived by proteolytic processing from the Alzheimer’s amyloid precursor protein (APP), whose short cytoplasmic domain contains several phosphorylatable amino acids. The latter can be phosphorylated ‘in vitro’ and ‘in vivo,’ and in some cases phosphorylation appears to be associated with the disease condition. Using APP-GFP fusion proteins to monitor APP processing, the role of Tyr687 was addressed by mimicking its constitutive phosphorylation (Y687E) and dephosphorylation (Y687F). Contrasting effects on subcellular APP distribution were observed. Y687E-APP-GFP was targeted to the membrane but could not be detected in transferrin containing vesicular structures, and exhibited a concomitant and dramatic decrease in Abeta production. In contrast, Y687F-APP-GFP was endocytosed similarly to wild type APP, but was relatively favoured for beta-secretase cleavage. Overall, Tyr687 appears to be a critical residue determining APP targeting and processing via different pathways, including endocytosis and retrograde transport. Significantly, from a disease perspective, mimicking Tyr687 phosphorylation resulted in a hitherto undescribed inhibition of Abeta production. Our results provide novel insights into the role of direct APP phosphorylation on APP targeting, processing and Abeta production.

In Vitro performance assessment of new brushite-forming Zn- and ZnSr-substituted β-TCP bone cements

The present study investigated the in vitro performance of brushite-forming Zn- and ZnSr-substituted beta-TCP bone cements in terms of wet mechanical strength and biological response. Quantitative phase analysis and structural refinement of the powdered samples were performed by X-ray powder diffraction and Rietveld refinement technique. Initial and final setting times of the cement pastes, measured using Gilmore needles technique, showed that ZnSrCPC sets faster than ZnCPC. The measured values of the wet strength after 48 h of immersion in PBS solution at 37 degrees C showed that ZnSrCPC cements are stronger than ZnCPC cements. Human osteosarcoma-derived MG63 cell line proved the nontoxicity of the cement powders, using the resazurin metabolic assay.

Tyrosine 687 Phosphorylated Alzheimer’s Amyloid Precursor Protein Is Retained Intracellularly and Exhibits a Decreased Turnover Rate

Tyrosine 687 (Y687) of the Alzheimer’s amyloid precursor protein (APP) was shown to be phosphorylated in the brains of Alzheimer’s disease patients. This residue lies within a typical endocytosis consensus motif commonly found in molecules with receptor functions, strongly suggesting a potential role for APP in signal transduction. Consequently, the work here described addressed how phosphorylation of Y687 may be affecting APP in terms of its proteolytic cleavage and subcellular distribution. Our data show that the APP mutant mimicking constitutive dephosphorylation of Y687 had a faster turnover rate, both in terms of maturation and metabolism, when compared to Wt-APP-GFP and even more so when compared to the mutant mimicking constitutive phosphorylation. Thus, the mutant mimicking constitutively phosphorylated Y687 had a much higher t1/2 and was significantly retained both in the ER and TGN. Additionally, this mutant was not incorporated into visible vesicular structures, with a concomitant dramatic decrease in Aβ production. Our findings point to the direct phosphorylation of APP on Y687 as an important regulatory mechanism in terms of determining the subcellular localization of APP and modulating its processing via different proteolytic pathways.

A Model System to Study Intracellular Trafficking and Processing of the Alzheimer's Amyloid Precursor Protein

The occurrence of consensus phosphorylation sites in the intracellular domain of the Alzheimer’s amyloid precursor protein (APP), coupled with observations of their in vivo phosphorylation, prompted several workers to investigate the effects that phosphorylation of such sites could have on APP metabolism and subsequent Aβ production. However, hitherto all attempts to dissect the role played by such phosphorylation events failed to reveal substantial effects. Having decided to revisit this problem, our new approach was based on the following vectors: (1) site-directed mutagenesis of the target amino acids to mimic a specific phosphorylation state, (2) expression of wild-type and mutant APP-GFP (green fluorescent protein) fusion proteins for ease of visualization, (3) controlled low level expression to avoid ‘flooding’ cellular pathways, and (4) the use of cycloheximide to inhibit de novo protein synthesis. Using this method we were able to detect specific differences in APP processing that were correlated with the mimicked phosphorylation state of several phosphorylation sites. New combined methodologies, like the one described here, allow for the detailed analysis of key control points in the cellular metabolism of specific proteins that are central to neurodegenerative diseases and may be under the control of specific posttranslational modifications, such as reversible phosphorylation.