Alzheimer's disease-related amyloid-ß1-42 peptide induces the loss of human sperm function.

Characteristically identified as the main component of senile plaques present in patients suffering from Alzheimer's disease, Aß has been detected in human testis and reproductive fluids, but its effect on spermatozoa has not been addressed. The present study evaluated whether the most toxic and aggregant amyloid precursor protein (APP)-proteolytic product, amyloid-ß1-42 (Aß1-42), was capable of affecting sperm functionality. Normozoospermic samples were either exposed to different Aß1-42 doses or to the untreated and scrambled controls for a maximum of 48 h at 37 °C and 5%CO2, and motility, viability and mitochondrial status were evaluated. Additionally, tyrosine phosphorylation was analyzed by immunocytochemistry and acrosomal integrity through PSA-FITC. A shorter treatment period was used to monitor prompt Ca2+ responses. Aß1-42 peptide decreased motility before inducing mitochondrial impairment (p < 0.05; n = 6). Both outcomes became more pronounced with time, reaching their maximal decrease at 48 h, where even 1 µM produced undesirable effects (p < 0.05; n = 6). Aß1-42 peptide also decreased cell survival (p < 0.05; n = 6). Furthermore, although no effects on tyrosine phosphorylation were observed (p > 0.05; n = 6), reduced acrosomal integrity was detected (p < 0.05; n = 7), which was not correlated with viability loss (p > 0.05). In parallel, all Aß1-42 concentrations elicited a [Ca2+]i rise but a significant difference was only observed at 20 µM (p < 0.05; n = 7) and a tendency was obtained with 10 µM (p = 0.053; n = 7). In conclusion, Aß1-42 peptide oligomers impair sperm function in vitro, although further studies are required to determine the clinical relevance of these findings.

Comparison of simple sucrose and percoll based methodologies for synaptosome enrichment.

Synaptosomes are isolated nerve terminals. They represent an extremely attractive in vitro model system to study synaptic physiology since they preserve morphological and functional characteristics of the synapse. As such they have been used to investigate synaptic dysfunctions associated with neuropathologies like Alzheimer's disease. In the present work two simple methodologies for isolating synaptosomal-enriched fractions were compared for the first time. The starting points of both protocols were rat cortical or hippocampal homogenized tissues that underwent several differential centrifugation steps followed by a final purification of synaptosomal-enriched fractions using either a Percoll gradient or a Sucrose gradient. Comparison of the fractions obtained was carried out, using both biochemical and electron microscopy approaches. In the biochemical analysis the protein levels of pre-synaptic, post-synaptic, nuclear and mitochondrial markers were evaluated. Additional characterization of the synaptosomal-enriched fractions was performed using transmission electron microscopy. In summary, the results indicate that under the conditions tested the Sucrose based protocol is more efficient for the isolation of synaptosomal-enriched fractions from both neuronal tissues, being particularly efficient for hippocampus that is a less abundant brain tissue. Further, the sucrose protocol apparently results in a higher yield of viable synaptosomes suitable for further assays, including structural and functional studies of synapses; making this an attractive procedure to study processes associated with neuropathologies.

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 ß-tricalcium phosphate (ß-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.

Complexing Aß prevents the cellular anomalies induced by the Peptide alone.

Retention of intracellular secreted APP (isAPP) can be provoked by the neurotoxic peptide Aß. The latter decreases in the cerebrospinal fluid of Alzheimer's disease (AD) patients, as a consequence of its cerebral accumulation and deposition into senile plaques. Of similar relevance, secreted APP (sAPP) levels can be associated with AD. The studies here presented, reinforce the link between sAPP and Aß and address putative therapeutic strategies. Laminin and gelsolin are potential candidates; both prevent Aß fibril formation by complexing with Aß, thus attenuating its neurotoxicity. We show that preincubation of Aß with laminin and gelsolin has the effect of rendering it less potent to isAPP accumulation in cortical neurons. This appears to be related to a decrease in F-actin polymerization, whereas Aß alone induces the polymerization. Further, Aß decreases gelsolin levels, and the latter is involved in Aß removal. Our data indicates that Aß-laminin and Aß-gelsolin complexes are less neurotoxic and also less potent than fibrillar Aß at inducing isAPP retention. These results validate the potential of these proteins as therapeutic strategies that prevent the Aß-induced effects. In hence, given that Aß decreases the levels of proteins involved in its own clearance, this may contribute to the mechanisms underlying AD pathology.

Synthesis, mechanical and biological characterization of ionic doped carbonated hydroxyapatite/ß-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/ß-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/ß-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 ß-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, PP1ß 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.

Biological responses of brushite-forming Zn- and ZnSr- substituted beta-tricalcium phosphate bone cements.

The core aim of this study was to investigate zinc (Zn)- and zinc and strontium (ZnSr)-containing brushite-forming beta-tricalcium phosphate (TCP) cements for their effects on proliferation and differentiation of osteoblastic-like cells (MC3T3-E1 cell line) as well as for their in vivo behaviour in trabecular bone cylindrical defects in a pilot study. In vitro proliferation and maturation responses of MC3T3-E1 osteoblastic-like cells to bone cements were studied at the cellular and molecular levels. The Zn- and Sr-containing brushite cements were found to stimulate pre-osteoblastic proliferation and osteoblastic maturation. Indeed, MC3T3-E1 cells exposed to the powdered cements had increased proliferative rates and higher adhesiveness capacity, in comparison to control cells. Furthermore, they exhibited higher alkaline phosphatase (ALP) activity and increased Type-I collagen secretion and fibre deposition into the extracellular matrix. Proliferative and collagen deposition properties were more evident for cells grown in cements doped with Sr. The in vivo osteoconductive propertiesof the ZnCPC and ZnSrCPC cements were also pursued. Histological and histomorphometric analyses were performed at 1 and 2 months after implantation, using carbonated apatite cement (Norian SRS) as control. There was no evidence of cement-induced adverse foreign body reactions, and furthermore ZnCPC and ZnSrCPC cements revealed better in vivo performance in comparison to the control apatite cement. Additionally, the presence of both zinc and strontium resulted in the highest rate of new bone formation. These novel results indicate that the investigated ZnCPC and ZnSrCPC cements are both biocompatible and osteoconductive, being good candidate materials to use as bone substitutes.

In vitro performance assessment of new brushite-forming Zn- and ZnSr-substituted beta-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.

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.

Alphabeta 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 Abeta peptide. Although normally produced in cells, Abeta is typically considered to be a neurotoxic peptide, causing devastating effects. By exposing primary neuronal cultures to relatively low Abeta 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 Abeta exposure. Additionally, intracellular protein levels of Fe65 and GSK3 were also decreased in response to Abeta. APP nuclear signaling is altered by Abeta, affecting not only AICD production but also its nuclear translocation and complex formation with Fe65. In effect, Abeta can trigger a physiological negative feedback mechanism that modulates its own production.

Inhibition of protein phosphatase 1 stimulates secretion of Alzheimer amyloid precursor protein.

BACKGROUND: Aberrant metabolism of the Alzheimer amyloid precursor protein (APP) or its amyloidogenic A beta fragment is thought to be centrally involved in Alzheimer's disease. Nonamyloidogenic processing of APP involves its cleavage within the A beta domain by a protease, termed alpha-secretase, and release of the large extracellular domain, termed APPS. Secretion of APPS can be stimulated by phorbol esters, activators of protein kinase C, with concurrent inhibition of A beta production. While the role of protein kinases of APP metabolism has been investigated, considerably less effort has been devoted to elucidating the role played by protein phosphatases. Okadaic acid, a protein phosphatase inhibitor, has been shown to stimulate secretion of APPS, but the identity of the phosphatase involved has not been investigated. MATERIALS AND METHODS: The secretion of APPS from COS-1 cells was measured in the absence or presence of various doses of serine/threonine-specific phosphatase inhibitors. Quantitation of the derived IC50 values was used to determine the identity of the phosphatase involved in the control of APP secretion. RESULTS: The availability of protein phosphatase inhibitors with different relative potencies against the different types of serine/threonine-specific protein phosphatase allowed us to examine which of the four known types of protein phosphatase might be involved in the regulation of APP secretion. Both okadaic acid and calyculin A stimulated the secretion of APP from COS-1 cells in a dose-dependent manner. The half-maximal dose for stimulation of APP secretion was approximately 100-fold higher with okadaic acid than with calyculin A. CONCLUSIONS: The nearly 100-fold difference in the observed IC50 values for okadaic acid and calyculin A implicates a type 1 protein phosphatase in the control of APPS production. Protein phosphatase 1 (PP1) is known to be highly expressed in adult mammalian brain, both in neurons and glia. The identification of a specific phosphatase type in the control of APP secretion opens new avenues to the development of rational therapeutic intervention strategies aimed at the prevention and/or treatment of Alzheimer's Disease.

Regulated cleavage of Alzheimer beta-amyloid precursor protein in the absence of the cytoplasmic tail.

Alzheimer beta-amyloid precursor protein can be phosphorylated on residues Thr654, Ser655 and Thr668 on its cytoplasmic domain. Proteolytic cleavage of the amyloid precursor protein and release of the amyloid precursor protein ectodomain into the medium of cultured cells can be activated by phorbol esters which stimulate protein kinase C. In the present study, using mutated amyloid precursor protein, we show that phosphorylation of cytoplasmic residues is not required for the phorbol ester-activated cleavage and release of the amyloid precursor protein ectodomain. Remarkably, deletion of the entire amyloid precursor protein cytoplasmic tail had no effect on the phorbol ester-activated cleavage/release. The results indicate that activation of amyloid precursor protein cleavage/release by protein kinase C involves phosphorylation of some component of the processing pathway, instead of or in addition to the cytoplasmic tail of the amyloid precursor protein.