O. A. B. da Cruz e Silva

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.

The Physiological Relevance of Protein Phosphatase 1 and its Interacting Proteins to Health and Disease

Protein phosphorylation is a major regulatory mechanism of signal transduction cascades in eukaryotic cells, catalysed by kinases and reversed by protein phosphatases (PPs). Sequencing of entire genomes has revealed that ~3% of all eukaryotic genes encode kinases or PPs. Surprisingly, there appear to be 2-5 times fewer PPs than kinases. Over the past two decades it has become apparent that the diversity of Ser/Thr-specific PPs (STPP) was achieved not only by the evolution of new catalytic subunits, but also by the ability of a single catalytic subunit to interact with multiple interacting proteins. PP1, a STPP, is involved in the control of important cellular mechanisms. Several isoforms of PP1 are known in mammals: PP1α, PP1β and PP1γ. The various isoforms are highly similar, except for the N- and C-termini. The current view is that since PPs possess exquisite specificities in vivo, the key control mechanism must reside in the nature of the PP1 Interacting Protein (PIP) to which they bind. An increasing number of PIPs have been identified that are responsible for regulating the catalytic activity of PPs. Indeed, the diversity of such PIPs explains the need for relatively few catalytic subunit types, and makes them attractive targets for pharmacological intervention. This review will summarize the PIPs identified using the Yeast Two Hybrid methodology and alternative techniques, for instance bioinformatic and proteomic approaches. Further, it compiles 129 PP1-PIP relevant physiological interactions that are well documented in the literature. Finally, the use of PIPs as therapeutic targets will be addressed.

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.

Αβ Hinders Nuclear Targeting of AICD and Fe65 in Primary Neuronal Cultures

The intracellular domain of the Alzheimer’s amyloid precursor protein (AICD) has been described as an important player in the transactivation of specific genes. It results from proteolytic processing of the Alzheimer’s amyloid precursor protein (APP), as does the neurotoxic Aβ peptide. Although normally produced in cells, Aβ is typically considered to be a neurotoxic peptide, causing devastating effects. By exposing primary neuronal cultures to relatively low Aβ concentrations, this peptide was shown to affect APP processing. Our findings indicate that APP C-terminal fragments are increased with concomitant reduction in the expression levels of APP itself. AICD nuclear immunoreactivity detected under control conditions was dramatically reduced in response to Aβ exposure. Additionally, intracellular protein levels of Fe65 and GSK3 were also decreased in response to Aβ. APP nuclear signaling is altered by Aβ, affecting not only AICD production but also its nuclear translocation and complex formation with Fe65. In effect, Aβ can trigger a physiological negative feedback mechanism that modulates its own production.

Understanding fatty acid metabolism through an active learning approach

A multi‐method active learning approach (MALA) was implemented in the Medical Biochemistry teaching unit of the Biomedical Sciences degree at the University of Aveiro, using problem‐based learning as the main learning approach. In this type of learning strategy, students are involved beyond the mere exercise of being taught by listening. Less emphasis is placed on transmitting information and the focus is shifted toward developing higher order thinking (analysis, synthesis, and evaluation). However, MALA should always involve clearly identified objectives and well‐defined targets. Understanding fatty acid metabolism was one of the proposed goals of the Medical Biochemistry unit. To this end, students were challenged with a variety of learning strategies to develop skills associated with group conflict resolution, critical thinking, information access, and retrieval, as well as oral and written communication skills. Overall, students and learning facilitators were highly motivated by the diversity of learning activities, particularly due to the emphasis on correlating theoretical knowledge with human health and disease. As a quality control exercise, the students were asked to answer a questionnaire on their evaluation of the whole teaching/learning experience. Our initial analysis of the learning outcomes permits us to conclude that the approach undertaken yields results that surpass the traditional teaching methods.

Injectable MnSr-doped brushite bone cements with improved biological performance

Good mechanical properties and high injectability are the major requirements to ensure widespread application of calcium phosphate cements (CPCs) as bone substitutes in minimally invasive surgeries. However, obtaining CPCs that exhibit a good compromise between these two properties as well as good biological performance is still a great challenge. This study presents novel solutions to improve these properties, which include (i) co-doping β-tricalcium phosphate (β-TCP) powder with Sr and Mn, and (ii) adding small amounts of saccharides (sucrose or fructose) to the setting-liquid solution. The combination of these two strategies enabled full injectability and significantly increased the wet compressive strength of CPCs in comparison to undoped or solely Sr-doped CPCs. Furthermore, the proliferative response of human MG63 osteoblastic cells, their rate of collagen-I secretion, and particularly their growth behaviour on the cement surfaces were also enhanced. The overall improved relevant properties of Mn/Sr co-doped CPCs with added sucrose, including in vitro biological performance, renders them very promising materials for bone regeneration and tissue engineering.

APP and its secreted fragment sAPP in SH-SY5Y neuronal-like migration

During adult neurogenesis, neural stem cells produce neural progenitors that migrate toward their final location, in a process highly regulated via signaling cues and cellular sensors[1]. The Alzheimer‟s Amyloid Precursor Protein (APP) is an ubiquitously expressed transmembranar protein that has been implicated in cell migration, together with its secreted proteolytic fragment, sAPP [2]. Although the latter was observed to be motogenic for keratinocytes, via inducing cell polarity and by increasing both the migrating keratinocytes population and their migration velocity[3], less is known of its role in neuronal migration. In order to study the paracrine role of sAPP in neuronal-like cells migration, we performed Scratch Wound Healing (SWH) assays in SH-SY5Y neuroblastoma cells. Migration of non-transfected (NTf) cells was monitored in different backgrounds: a) in the vicinity of GFP (Green Fluorescent Protein) transiently transfected cells (EGFP-N1); b) in the vicinity of cells transfected with wild-type APP cDNA constructs fused to GFP [4] (Wt APP-GFP); and c) in the vicinity of GFP transfected cells in an sAPP-enriched medium (EGFP-N1+sAPP). The “healing” of the “wound gap” by cell migration was monitored by imaging both wound edges at every hour, during 8h (t0-t8), and in the following day (t24), as depicted in phase contrast (PhC) microphotographs of Fig.1A. Cell migration efficiency was evaluated by determination of the following parameters: 1) the migrating cells score (number of NTf cells observed at the wound area with time); 2) their fold increase over time (reflecting the recruitment of new cells to the migratory process), and 3) the final distance migrated by the NTf‟s leading edge (distance measured in a direct line, from the t0 edge of the wound – red line in Fig.1A – to the average line passing by ten random leading edge cells at 24h). Results show that the presence of higher sAPP levels that gradually accumulated in the media via Wt APPGFP proteolysis (Fig.1B, „APP-GFP‟) did not increase but rather slightly decreased the absolute number of initial NTf migrating cells (Fig.2A). Nonetheless, the final number of these migratory cells equaled the control EGFP-N1 condition (Fig.2A, 24h), since cells in the Wt APP-GFP condition suffered a delayed but enhanced recruitment for migration from 4h on (Fig.2B, arrow), as demonstrated by its highest fold increases thereafter (Fig.2B, 4h on). Intriguingly, NTf cells in the EGFP-N1+sAPP condition generally presented the lowest number of migrating cells (Fig.2A), despite its intermediate time pattern of cell recruitment (Fig.2B). These data suggest that sAPP may be responsible for the late enhanced cell recruitment to migration seen in Wt APP-GFP. However, at higher doses (EGFP-N1+sAPP) it decreases the number of cells migrating into the wound area, what may result from sAPP-induced negative feedbacks and/or desensitization mechanisms. Since in this condition there is no real sAPP gradient (it is homogenously distributed throughout cells media), this may also add a negative effect on directed cells migration. In this case cells would be activated to migrate but in a random manner and not towards the center of the gap. Findings that in the EGFPN1+sAPP condition the NTf cells leading edge migrated shorter distances over time (data not shown), support the „loss of migration directionality‟ hypothesis, with this being further pursued by us. With this work we conclude that sAPP may have a paracrine role recruiting neuronal cells for migration, but possibly in a doseand gradient-dependent manner.

Immunolocalization of PPP1C Isoforms in SH-SY5Y Cells During the Cell Cycle

Phosphoprotein phosphatase 1 (PPP1) is the most widely expressed and abundant serine/threonine protein phosphatase. PPP1 regulates a variety of cellular functions such as glycogen metabolism, mitosis and meiosis, cell-cycle arrest, apoptosis, dynamics of actin cytoskeleton, protein synthesis among others. The versatility of the PPP1 catalytic subunit (PPP1C) is achieved by associating with different regulatory subunits that target PPP1C to a particular subcellular compartment determining its substrate specificity and activity. PPP1C is expressed in mammals in three isoforms: PPP1CA, PPP1CB and PPP1CC, each encoded by distinct genes. The PPP1C gene undergoes alternative splicing to originate PPP1CC1 and PPP1CC2 variants. PPP1A and PPP1C1 are expressed virtually in all tissues but are particularly enriched in the brain. The subcellular localization of the endogenous PPP1C isoforms is not fully elucidated, but all isoforms are found in the nucleus and cytoplasm, despite some isoform-specific differences in intranuclear distribution. Furthermore, it is known that PPP1 plays a key role in mitosis where PPP1C isoforms are differentially targeted to specific subcellular structures. However, in previous studies, non-neuronal cells were used as a model system to study PPP1 distribution during the cell cycle. In the studies here described, we used the neuronal-like cell line SH-SY5Y since PPP1 is a crucial protein in several neuronal functions.

Abeta Induces Abnormal Cytoskeletal Dynamics which are Reversible Upon Peptide Removal

A. G. Henriques, S. I. Vieira and O. A. B. da Cruz e SilvaNeuroscience Lab, Center for Cell Biology, Health Sciences Dept. and Biology Dept, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, PortugalAlzheimer´s disease (AD) is characterized by extensive neuronal loss in brain areas related to memory and cognitive functions. Central to the neurodegenerative process is a peptide termed Abeta. The latter is the main component of senile plaques, one of the histopathological hallmarks of AD, and derives from proteolytic processing of the Alzheimer´s amyloid precursor protein (APP). Among the alterations induced by Abeta is increased cellular oxidative stress, imbalanced protein phosphorylation and cytoskeletal abnormalities, all factors that contribute to neuronal death.Previous studies from our laboratory have shown that Abeta leads to intracellular accumulation of the secreted form of APP (sAPP). Consequently we addressed how Abeta may affect cellular morphology or intracellular structures and lead to this phenomenon. Interestingly, alterations in the cytoskeletal network were clearly evident [1, 2]. Namely, sAPP was seen to cluster in cytoskeletal-associated vesicular-like structures, which accumulate close to the plasma membrane [2].Furthermore, and as revealed by microscopy analyses, Abeta incubation for 24 hr also led to rearrangements in the cytoskeletal network in HeLa cells. The re-organization (Figure 1, Abeta) is striking as detected by the phalloidin staining, with F-actin polymerization being markedly increased.Cytoskeletal abnormalities compromise neuronal transport of neurotransmitters and other substances,important to neuronal survival and signalling. Thus, Abeta induced cytoskeletal anomalies are consistent with the cognitive deficits associated with Alzheimer’s disease. Of note, Abeta removal in the last 3 hr of the incubation period (Figure 1, Abeta-Abeta) was able to reverse the effects. These novel findings significant as they show for the first time that the Abeta-induced effects on are cytoskeletal reorganization can be reversed by the simple removal of the toxic Abeta peptide. The mode of action needs to be closely addressed, however, it is relevant to consider that Abeta has been shown to inhibit phosphoprotein phosphate 1 (PPP1) activity and the latter is implicated in cytoskeletal dynamics [3]. Further, as cytoskeleton impairment may mediate part of Abeta toxic effects, drugs targeting cytoskeletal network may also be of therapeutic value in Alzheimer’s disease.Acknowledgements: This work was supported by the Fundacao para a Ciencia e Tecnologia of the Portuguese Ministry of Science and Technology, the European Union (cNeupro), Center for CellBiology UA, REEQ/1023/BIO/2005, PTDC/QUI-BIQ/101317/2008, POCTI/BIA-BCM/58469/2004, and the scholarship awarded to AGH - SFRH/BPD/45611/2008.