PICALM modulates autophagy activity and tau accumulation.

Genome-wide association studies have identified several loci associated with Alzheimer's disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.We are grateful for funding from a Wellcome Trust Principal Research Fellowship (D.C.R.), a Wellcome Trust/MRC Strategic Grant on Neurodegeneration (D.C.R., C.J.O’.K.), a Wellcome Trust Strategic Award to Cambridge Institute for Medical Research, Wellcome Trust Studentship (E.Z.), the Alzheimer’s disease Biomedical Research Unit and Addenbrooke’s Hospital, the Tau Consortium, a fellowship from University of Granada (A.L.R.), a V Foundation/Applebee’s Research Grant (D.S.W.) and NCI R01 CA 109281 (D.S.W.).This is the final published version. It is also available from Nature Publishing at http://www.nature.com/ncomms/2014/140922/ncomms5998/full/ncomms5998.html

Human TRIM Gene Expression in Response to Interferons

Tripartite motif (TRIM) proteins constitute a family of proteins that share a conserved tripartite architecture. The recent discovery of the anti-HIV activity of TRIM5α in primate cells has stimulated much interest in the potential role of TRIM proteins in antiviral activities and innate immunity.To test if TRIM genes are up-regulated during antiviral immune responses, we performed a systematic analysis of TRIM gene expression in human primary lymphocytes and monocyte-derived macrophages in response to interferons (IFNs, type I and II) or following FcγR-mediated activation of macrophages. We found that 27 of the 72 human TRIM genes are sensitive to IFN. Our analysis identifies 9 additional TRIM genes that are up-regulated by IFNs, among which only 3 have previously been found to display an antiviral activity. Also, we found 2 TRIM proteins, TRIM9 and 54, to be specifically up-regulated in FcγR-activated macrophages.Our results present the first comprehensive TRIM gene expression analysis in primary human immune cells, and suggest the involvement of additional TRIM proteins in regulating host antiviral activities

Rôle du récepteur FLT3 dans la pathogenèse des leucémies associées aux réarrangements du gène MLL ou à la surexpression des gènes HOXA9 et MEIS1

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

The acute promyelocytic leukemia PML-RARα protein induces hepatic preneoplastic and neoplastic lesions in transgenic mice

International audienceThe PML-RAR alpha hybrid protein generated by the t(15;17) translocation in acute promyelocytic leukemia (APL) is thought to play a central role in the oncogenic process. However, analysis of the oncogenic activity of the fusion protein in tissue culture assays or in mice has been hampered by its apparent toxicity in multiple murine cells. To circumvent this problem, we generated an inducible line of transgenic mice, MT135, in which the expression of PML-RAR alpha is driven by the metallothionein promoter. After 5 days zinc stimulation, 27 out of 54 mice developed hepatic preneoplasia and neoplasia including foci of basophilic hepatocytes, dysplasia and carcinoma with a significantly higher incidence of lesions in females than in males. The rapid onset of liver pathologies was dependent on overexpression of the transgene since it was not detected in noninduced transgenic animals of the same age. The PML-RAR alpha protein was always present in altered tissues at much higher levels than in the surrounding normal liver tissues. In addition, overexpression of PML-RAR alpha resulted in a strong proliferative response in the hepatocytes. We conclude that overexpression of PML-RAR alpha deregulates cell proliferation and can induce tumorigenic changes in vivo

<i>Flt3</i>^−/−<i>MLL-ENL</i> leukemias are sensitive to PKC412.

<p><b>A</b><b> </b>: Cytotoxic dose response of BM blasts from five <i>Flt3</i>^−/−<i>MLL-ENL</i> leukemic mice. Viable cells were counted after 3 days of culture in presence of increasing concentrations of PKC412 and shown as percentage of untreated control cells. Data points represent means of duplicate or triplicate wells, +/− SD. <b>B</b>: Fold expansion of viable cells during 3 days of culture (number of live cells at the end of culture divided by number of cells initially seeded) either in absence or presence of 500 nM PKC412. Mean +/− SD of results from 6 <i>Flt3</i>^−/−<i>MLL-ENL</i> leukemic mice. <b>C</b>: Flow cytometry analysis of Mac-1 and Gr-1 expression after 3 days of culture of samples #54 and #762 either without or with PKC412 (700 nM). The percentages of cells in the quadrants are indicated. Similar induction of maturation was seen in blasts from 5 different <i>Flt3^−/− MLL-ENL</i> leukemic mice.</p

<i>MLL</i> fusion genes transform BM progenitors independently of <i>Flt3 in vitro</i>.

<p>Myeloid colony forming assays were performed with <i>Flt3</i>^+/+ and <i>Flt3</i>^−/− progenitors infected by empty MIE vector, <i>MLL-ENL</i> or <i>MLL-CBP</i>-encoding vectors. Numbers of colonies are shown for 10³ (first passage colonies) or 10⁴ (second to fourth passage colonies) seeded cells. Results shown are the mean +/− SEM of 3 (MLL-CBP and MIE) or 5 (MLL-ENL) independent experiments.</p

The sensitivity of <i>MLL-ENL</i> leukemia cells to PKC412 correlates with their level of Flt3 expression.

<p><b>A</b>: Cytotoxic dose response of BM blasts from five <i>Flt3</i>^+/+<i>MLL-ENL</i> mice. Viable cells were counted after 3 days of culture in the presence of increasing concentrations of PKC412, and shown as a percentage of untreated control cells. The percentage of Flt3 expressing-blasts measured by flow cytometry at the start of the assay is indicated in parantheses. Data points represent means of duplicate or triplicate wells, +/− SD<b>. B</b>: Fold expansion of viable cells during 3 days of culture (number of live cells at the end of culture divided by number of cells initially seeded) either in absence or presence of 500 nM PKC412. Mean +/− SD of results from 7 <i>Flt3</i>^+/+<i>MLL-ENL</i> leukemic mice. <b>C</b>: Flt3 expression determined by flow cytometry in BM sample #243, containing initially 94% of Flt3 positive cells, measured after 3 days of culture in the absence (left) or presence (right) of PKC412 (500 nM). The gray-filled curves show non-specific fluorescence. Data is representative of 3 separate experiments. <b>D</b>: Flow cytometry analysis of Mac-1 and Gr-1 expression and morphology after 3 days of culture of samples #94 and #243 with or without PKC412 (500 nM). The percentages of cells in the quadrants are indicated. Wright Giemsa stain of cytospin preparations are shown. Similar induction of maturation was seen in blasts from 7 different <i>Flt3⁺</i>^/+<i>MLL-ENL</i> leukemic mice.</p

The onset and characteristics of <i>MLL-ENL</i> and <i>MLL-CBP</i> leukemias are not influenced by <i>Flt3</i> expression.

<p><b>A</b>: Engraftment of empty vector MIE-transplanted mice measured as a percentage of GFP-expressing peripheral blood white blood cells (WBC) detected by flow cytometry. Results of 5 separate mice, transplanted with <i>Flt3</i>^+/+ or <i>Flt3</i>^−/− MIE-transduced BM, analyzed at either 104 (diamonds) or 111 (triangles) days post-transplantation. The percentage of GFP expression in the MIE-transduced <i>Flt3</i>^+/+ or <i>Flt3</i>^−/− BM at the time of transplantation was between 65% and 85%. <b>B</b>: Survival curves of mice transplanted with <i>MLL-ENL</i> or <i>MLL-CBP</i>-infected <i>Flt3</i>^+/+ and <i>Flt3</i>^−/− BM (9 to 10 mice per cohort). Control mice injected with empty vector MIE-infected <i>Flt3</i>^+/+ or <i>Flt3</i>^−/− BM remained healthy for more than 300 days. <b>C</b>: Flt3 protein levels detected by Western blot analyses of BM from primary (left panel) as well as secondary (right panel) <i>Flt3</i>^+/+<i>MLL-ENL</i> leukemic mice. The survival, in days, of the primary leukemic mice is shown in parentheses (left panel). BM from a primary <i>Flt3</i>^−/−<i>MLL-ENL</i> leukemic mouse is shown as a negative control in the first lane; asterisks show non-specific bands and arrows point to Flt3 bands (the upper band is glycosylated Flt3). BM protein lysates of secondary transplant mice (labeled TR) and lysates from their corresponding primary donor are loaded in adjacent lanes (right panel). Actin serves as a loading control (lower panels). <b>D</b>: Expression of Mac-1 and Gr-1 in BM blasts harvested from diseased mice. Cells were gated for GFP expression. One representative MLL-ENL and MLL-CBP mouse of each genotype are shown out of 7–8 different mice analyzed for each group. <b>E</b>: Expression of Mac-1 in BM blasts (gated for GFP expression) from leukemic mice determined by flow cytometry. Shown are average +/− SD (n = 7 to 8). *p<0.05. <b>F</b>: Expression of F4/80 in BM blasts (gated for GFP expression) from leukemic mice determined by flow cytometry. Shown are mean +/− SD (n = 7 to 8). <b>G</b>: Survival curves of secondary transplant <i>Flt3</i>^+/+ and <i>Flt3</i>^−/−<i>MLL-ENL</i> leukemias. Graph shows survival of 13 secondary recipient mice of 5 different primary <i>Flt3</i>^−/−<i>MLL-ENL</i> (each transplanted into 2 to 3 hosts) and 20 secondary recipient mice of 7 different primary <i>Flt3</i>^+/+<i>MLL-ENL</i> (each transplanted into 2 to 4 hosts). <b>H</b>: Survival curves of secondary transplant <i>Flt3</i>^+/+ and <i>Flt3</i>^−/−<i>MLL-CBP</i> leukemias. Graph shows survival of 7 secondary recipient mice of 3 different primary <i>Flt3</i>^−/− or <i>Flt3</i>^+/+<i>MLL-CBP</i> (each transplanted into 2 to 3 hosts).</p