Intracellular Calcium Deficits in Drosophila Cholinergic Neurons Expressing Wild Type or FAD-Mutant Presenilin

Much of our current understanding about neurodegenerative diseases can be attributed to the study of inherited forms of these disorders. For example, mutations in the presenilin 1 and 2 genes have been linked to early onset familial forms of Alzheimer's disease (FAD). Using the Drosophila central nervous system as a model we have investigated the role of presenilin in one of the earliest cellular defects associated with Alzheimer's disease, intracellular calcium deregulation. We show that expression of either wild type or FAD-mutant presenilin in Drosophila CNS neurons has no impact on resting calcium levels but does give rise to deficits in intracellular calcium stores. Furthermore, we show that a loss-of-function mutation in calmodulin, a key regulator of intracellular calcium, can suppress presenilin-induced deficits in calcium stores. Our data support a model whereby presenilin plays a role in regulating intracellular calcium stores and demonstrate that Drosophila can be used to study the link between presenilin and calcium deregulation

The Roles of Presenilin and FKBP14 in Drosophila Development and Notch Signalling

The Roles of Presenilin and FKBP14 in Drosophila Development and Notch Signalling; Diana L. van de Hoef, Department of Molecular Genetics, University of Toronto, 2008. The multimolecular gamma-secretase complex cleaves type 1 transmembrane proteins such as Notch and one of the genes targeted in Alzheimer’s disease known as APP. This complex comprises four components, known as anterior pharynx defective 1, presenilin enhancer 2, nicastrin and presenilin. Presenilin is an aspartyl protease that comprises the catalytic core of gamma-secretase, and mutated forms of presenilin cause early-onset familial Alzheimer’s disease. To further define the role of Drosophila Presenilin (Psn), I performed a genetic modifier screen to identify Psn-interacting genes. One of the genes that was identified, known as FKBP14, encodes a peptidyl-prolyl isomerase that may be involved in protein folding in the ER. I demonstrate that an immunosuppressant drug known as FK506, which binds FKBPs and abrogates their function, reduced Psn, anterior pharynx defective 1 and presenilin enhancer 2 protein levels in vivo. I also show that FKBP14 colocalized with anterior pharynx defective 1 and Psn in the ER, suggesting a role in gamma-secretase stability. Consistent with this, I demonstrate that FKBP14 binds with Psn and mediates Psn stability and Notch signalling in vivo. To further characterize the role of FKBP14 in development, I analyzed its expression pattern and phenotypes of an FKBP14 null mutant. I show that FKBP14 localized to embryonic hemocytes and larval tissues, in addition to being expressed in developing egg chambers. FKBP14 function is required during development, since FKBP14 null mutants are recessive lethal. These mutants exhibited defects in larval disc development that resulted in eye, wing and notum phenotypes reminiscent of Psn dominant-negative and Notch-dependent phenotypes. Furthermore, FKBP14 mutants displayed enhanced apoptosis in larval tissues, suggesting a possible involvement in apoptosis regulation. I then examined the effects of FKBP14 overexpression, and observed enhanced Psn protein levels in vivo. Interestingly, co-expression of FKBP14 and Psn resulted in synergistic bristle phenotypes, suggesting a role for FKBP14 function in the Notch signalling pathway. Consistent with this, FKBP14 mutants enhanced Notch loss-of-function phenotypes in the wing. Altogether, my data demonstrate an essential role for FKBP14 during development, particularly in Psn protein maintenance and Notch signalling.Ph

<em>Plasmodium falciparum</em>-Derived Uric Acid Precipitates Induce Maturation of Dendritic Cells

<div><p>Malaria is characterized by cyclical fevers and high levels of inflammation, and while an early inflammatory response contributes to parasite clearance, excessive and persistent inflammation can lead to severe forms of the disease. Here, we show that <em>Plasmodium falciparum</em>-infected erythrocytes contain uric acid precipitates in the cytoplasm of the parasitophorous vacuole, which are released when erythrocytes rupture. Uric acid precipitates are highly inflammatory molecules that are considered a danger signal for innate immunity and are the causative agent in gout. We determined that <em>P. falciparum-</em>derived uric acid precipitates induce maturation of human dendritic cells, increasing the expression of cell surface co-stimulatory molecules such as CD80 and CD86, while decreasing human leukocyte antigen-DR expression. In accordance with this, uric acid accounts for a significant proportion of the total stimulatory activity induced by parasite-infected erythrocytes. Moreover, the identification of uric acid precipitates in <em>P. falciparum</em>- and <em>P. vivax</em>-infected erythrocytes obtained directly from malaria patients underscores the <em>in vivo</em> and clinical relevance of our findings. Altogether, our data implicate uric acid precipitates as a potentially important contributor to the innate immune response to <em>Plasmodium</em> infection and may provide a novel target for adjunct therapies.</p> </div

Immunolocalization of uric acid in <i>P. falciparum</i> cytoplasm.

<p>(A and B) Immunogold staining of intravacuolar <i>P. falciparum</i> demonstrates cytoplasmic distribution of precipitated uric acid. Arrows mark representative uric acid precipitates in the cytoplasm of iRBC. Aggregates of uric acid are also detected at the parasite periphery (A, inset). One hundred sections of labeled parasites, which included >70 parasitophorous vacuoles, were analyzed. RBC, uninfected erythrocytes; ER, endoplasmic reticulum; FV, food vacuole; n, nucleus. Bar is 200 nm. (C) Purified intact parasitophorous vacuoles were obtained from infected erythrocytes at the schizont stage (iRBC) using saponin treatment. After low-speed centrifugation, the purified parasite fraction was collected as a pellet. Pellets were lysed and ultracentrifugation was performed to obtain supernatant (SN) and pellet fractions. Uninfected erythrocyte (RBC) lysates were processed in parallel. Quantitation of uric acid was performed using a colorimetric absorbance assay. Data are displayed as mean ± SEM (n = 3 independent experiments; **p<0.01, ***p<0.001 relative to RBC controls).</p

Uric acid precipitates from purified <i>P. falciparum</i> schizonts induce maturation of dendritic cells.

<p>(A) Dendritic cells enriched from human peripheral blood mononuclear cells were co-cultured with <i>P. falciparum</i> lysate fractions for 42 h. RBC fractions were used as control. Surface expression levels of CD11c, CD80, CD86 and HLA-DR were analyzed in dendritic cells gated for HLA-DR+. <i>P. falciparum</i> pellet fractions (iRBC pellet) induce significantly high CD11c, CD80 and CD86 surface marker expression levels, and reduced HLA-DR expression, relative to dendritic cells alone and to RBC fractions (n = 7 independent human blood donors and n = 6 independent iRBC lysates; *p<0.05; **p<0.01; ***p<0.001, one-way ANOVA with Fisher’s post hoc test). DC, dendritic cells; E, Eluate; Hz, hemozoin; LPS, lipopolysaccharides; SN, supernatant. (B) Dendritic cells from 3 independent donors (D1–3) were co-cultured with different fractions. <i>Top panels</i>: dendritic cells alone (grey filled lines) and co-cultured with lysates of purified iRBC pellet (red), iRBC pellet treated with DNase (green) and iRBC pellet from cultures grown in low exogenous hypoxanthine conditions and treated with uricase (blue). <i>Bottom panels</i>: dendritic cells co-cultured with LPS (light grey) and MSU (red). Control dendritic cells alone (grey filled lines) show baseline expression levels, similar to dendritic cells co-cultured with fractions derived from control uninfected erythrocytes (data not shown). Shown are results from three independent donors of peripheral blood co-cultured with three independent iRBC pellet fractions and two independent iRBC Hx- (0 µM exogenous hypoxanthine) pellet fractions.</p

Formation of uric acid precipitates in <i>P. falciparum</i>-infected erythrocytes is dependent on exogenous hypoxanthine.

<p><i>P. falciparum</i>-infected erythrocytes were cultured in hypoxanthine free (A) and hypoxanthine-supplemented media as the unique purine source (B, 5 µM; C, 250 µM, D, 500 µM). Uric acid antibodies label precipitates within infected parasites <i>in vitro</i>. DAPI labels nuclei (blue). Bar is 5 µm. (E) Quantitation of infected erythrocytes that are positive for uric acid immunostaining. Data are displayed as mean ± SEM (n>100 schizonts in duplicate, from three independent cultures). (F) Quantitation of uric acid in lysate fractions of purified <i>P. falciparum</i> schizonts cultured in increasing hypoxanthine concentrations: Hx- [0 µM Hx] (light grey), 250 µM Hx (dark grey) and 500 µM Hx (black). Ultracentrifugation was performed on lysates to obtain supernatant (SN) and pellet fractions. Uninfected erythrocytes (RBC; white) were processed in parallel. Quantitation of uric acid using a colorimetric absorbance assay is shown. Data are displayed as mean ± SEM (n = 3 independent cultures; *p<0.05 relative to Hx- [0 µM Hx] pellet control).</p

Uric acid precipitates accumulate in murine <i>Plasmodium</i>-infected red blood cells <i>in vivo</i>.

<p>Purified <i>P. yoelii</i> (A) and <i>P. berghei</i> (B) schizonts from infected mice, labeled with anti-uric acid antibodies, show characteristic uric acid immunostaining patterns (red). DAPI (blue) labels nuclei. Bar is 5 µm. (C) Quantitation of uric acid levels in supernatant (SN) and pellet fractions of lysates of purified <i>P. yoelii</i> schizonts (iRBC) or control uninfected erythrocytes (RBC). (D) Quantitation of uric acid plasma levels, using a colorimetric absorbance assay, in uninfected control and <i>P. yoelii</i>-infected mice at day 9 of infection. Data in C and D are displayed as mean ± SEM (n = 6 control and n = 6 <i>P</i>. <i>yoelii</i>-infected mice in each experiment; **p<0.01, ***p<0.001 relative to control).</p

Alzheimer's disease research and development: a call for a new research roadmap.

Epidemiological projections of the prevalence of Alzheimer's disease (AD) and related dementias, the rapidly expanding population over the age of 65, and the enormous societal consequence on health, economics, and community foretell of a looming global public health crisis. Currently available treatments for AD are symptomatic, with modest effect sizes and limited impact on longer term disease outcomes. There have been no newly approved pharmaceutical treatments in the last decade, despite enormous efforts to develop disease-modifying treatments directed at Alzheimer's-associated pathology. An unprecedented collaborative effort of government, regulators, industry, academia, and the community at-large is needed to address this crisis and to develop an actionable plan for rapid progress toward successfully developing effective treatments. Here, we map out a course of action in four key priority areas, including (1) addressing the fundamental mechanisms of disease, with the goal of developing a core set of research tools, a framework for data sharing, and creation of accessible validated and replicated disease models; (2) developing translational research that emphasizes rapid progress in disease model development and better translation from preclinical to clinical stages, deploying leading technologies to more accurately develop predictive models; (3) preventing AD through the development of robust methods and resources to advance trials and creating fundamental resources such as continuous adaptive trials, registries, data repositories, and instrument development; and (4) innovating public/private partnerships and global collaborations, with mechanisms to incentivize collaborations and investments, develop larger precompetitive spaces, and more rapid data sharing

A potential role for plasma uric acid in the endothelial pathology of Plasmodium falciparum malaria.

BACKGROUND: Inflammatory cytokinemia and systemic activation of the microvascular endothelium are central to the pathogenesis of Plasmodium falciparum malaria. Recently, 'parasite-derived' uric acid (UA) was shown to activate human immune cells in vitro, and plasma UA levels were associated with inflammatory cytokine levels and disease severity in Malian children with malaria. Since UA is associated with endothelial inflammation in non-malaria diseases, we hypothesized that elevated UA levels contribute to the endothelial pathology of P. falciparum malaria. METHODOLOGY/PRINCIPAL FINDINGS: We measured levels of UA and soluble forms of intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), E-selectin (sE-Selectin), thrombomodulin (sTM), tissue factor (sTF) and vascular endothelial growth factor (VEGF) in the plasma of Malian children aged 0.5-17 years with uncomplicated malaria (UM, n = 487) and non-cerebral severe malaria (NCSM, n = 68). In 69 of these children, we measured these same factors once when they experienced a malaria episode and twice when they were healthy (i.e., before and after the malaria transmission season). We found that levels of UA, sICAM-1, sVCAM-1, sE-Selectin and sTM increase during a malaria episode and return to basal levels at the end of the transmission season (p<0.0001). Plasma levels of UA and these four endothelial biomarkers correlate with parasite density and disease severity. In children with UM, UA levels correlate with parasite density (r = 0.092, p = 0.043), sICAM-1 (r = 0.255, p<0.0001) and sTM (r = 0.175, p = 0.0001) levels. After adjusting for parasite density, UA levels predict sTM levels. CONCLUSIONS/SIGNIFICANCE: Elevated UA levels may contribute to malaria pathogenesis by damaging endothelium and promoting a procoagulant state. The correlation between UA levels and parasite densities suggests that parasitized erythrocytes are one possible source of excess UA. UA-induced shedding of endothelial TM may represent a novel mechanism of malaria pathogenesis, in which activated thrombin induces fibrin deposition and platelet aggregation in microvessels. This protocol is registered at clinicaltrials.gov (NCT00669084)

Alzheimer's disease research and development: a call for a new research roadmap.

Epidemiological projections of the prevalence of Alzheimer's disease (AD) and related dementias, the rapidly expanding population over the age of 65, and the enormous societal consequence on health, economics, and community foretell of a looming global public health crisis. Currently available treatments for AD are symptomatic, with modest effect sizes and limited impact on longer term disease outcomes. There have been no newly approved pharmaceutical treatments in the last decade, despite enormous efforts to develop disease-modifying treatments directed at Alzheimer's-associated pathology. An unprecedented collaborative effort of government, regulators, industry, academia, and the community at-large is needed to address this crisis and to develop an actionable plan for rapid progress toward successfully developing effective treatments. Here, we map out a course of action in four key priority areas, including (1) addressing the fundamental mechanisms of disease, with the goal of developing a core set of research tools, a framework for data sharing, and creation of accessible validated and replicated disease models; (2) developing translational research that emphasizes rapid progress in disease model development and better translation from preclinical to clinical stages, deploying leading technologies to more accurately develop predictive models; (3) preventing AD through the development of robust methods and resources to advance trials and creating fundamental resources such as continuous adaptive trials, registries, data repositories, and instrument development; and (4) innovating public/private partnerships and global collaborations, with mechanisms to incentivize collaborations and investments, develop larger precompetitive spaces, and more rapid data sharing