- Email: [email protected]
Mis-splicing of Tau Exon 2 and Exon 10 in myotonic dystrophy brain would result from different mechanisms
Poster Presentations P3
P3-120
MIS-SPLICING OF TAU EXON 2 AND EXON 10 IN MYOTONIC DYSTROPHY BRAIN WOULD RESULT FROM DIFFERENT MECHANISMS
Marie-Laure Caillet-Boudin1, Claire-Marie Dhaenens2, MarieLise Frandemiche2, Helene Tran2, Celine Carpentier2, FranciscoJose Fernandez-Gomez2, Andone Sistiaga3, Maria Goicoechea3, Edwige Van Brussel2, Helene Obriot2, Sabiha Eddarkaoui2, Marie-Helene Gavaert4, Bernard Sablonniere2, Adolfo Lopez-de-munain5, Luc Buee1, Susanna Schraen2, Nicolas Sergeant2, 1Inserm-Univ. Lille, Lille, France; 2 Inserm, U837-1, Univ. Lille Nord de France, IMPRT, Centre Jean-Pierre Aubert, Lille, France, Lille, France; 3Neurological Department and Experimental Unit, Donostia Hospital, San Sebastian, Spain, San Sebastian, Spain; 4Neuropathological Department, Regional University Hospital Centre, Lille, France., Lille, France; 5Neurological Department and Experimental Unit, Donostia Hospital, San Sebastian, Spain, Lille, France. Background: The mis-splicing of Tau is instrumental to the development of neurofibrillary degeneration (NFD) naturopathies such as FTDP-17 in which MAPTmutations lead to a defective splicing of Tau. A mis-splicing of Tau is also observed in the brain and muscle of patients affected by myotonic dystrophy of type I (DM1). Moreover, this Tau mis-splicing is associated with the development of NFD. DM1 is a neuromuscular disease characterized by an unstable CTG expansion. This mutation is responsible for a toxic gain of RNA-function, leading to the gain or loss of function of several splicing factors, including the CELF and MBNL families, respectively. It ultimately leads to an indirect mis-splicing of numerous transcripts. In regards to Tau mis-splicing, exon-2 inclusion is highly decreased in DM1 brains. In contrast, a mis-splicing of Tau exon-10 is seldom observed in DM1 but the mechanisms triggering this mis-splicing event remain unknown. Methods: We herein aimed to gain further insights into these molecular mechanisms involved in normal and pathological splicing of Tau exons 2 and 10 using cell-based models. Results: Two among the brains of 5 DM1 patients showed an exon-10 mis-splicing whereas exon-2 inclusion was reduced in all DM1 patients. The over-expression of long stretches of CTGrepeats promoted the exclusion of both Tau exon 2 and exon 10. Interestingly, MBNL1 loss of expression efficiently repressed Tau exon 2 inclusion but failed to modulate Tau exon-10 splicing. Analyzing the splicing functions of three major CELF splicing factors, we showed that CELF 4 acts as a repressor for exon-2 or as an enhancer for exon-10 inclusion. In sharp contrast, CELF2 repressed both exons 2 and 10 inclusions whereas CELF1 was inefficient to modulate Tau splicing. In addition, 2D electrophoresis coupled to western-blotting suggest a possible change inbrain CELF2 activity induced by its hyperphosphorylation in DM1. Conclusions: Altogether, we showed that MBNL1 and CELF2 are implicated in the mis-splicing of Tau-exons 2 and 10, respectively, but not CELF1. Moreover, our Tau splicing study also suggests that two different mechanisms may contribute to the mis-splicing of several targets in DM1.
months. In this study, we assessed whether behavioral impairments occurred in those mice, and whether the premature neurodegeneration phenotype occurs in multiple strains of p73+/- mice or p73+/- mice crossed with other mice with Alzheimer’s disease-like phenotypes such as Pin1-/-. Furthermore, we performed a more extensive analysis of tau phosphorylation at multiple time points, and examined whether p73 might function in part to prevent neurodegeneration by suppressing the activity of other tau kinases such as GSK3b and Cdk5. Methods: We crossed p73+/- mice into two different genetic backgrounds, Balb/c129 and C57 BL6. Aged mice were used at 18 months old. To analyze the potential involvement of p73 in Alzheimer’s disease, we crossed p73+/- mice with the transgenic CRND8 (TgCRND8+/ø) mouse model of Alzheimer’s disease. We analyzed these crosses at 1.5-2 months, an early time point of plaque formation. We also analyzed the effect of p73 heterozygosity in a second model of neurodegeneration, Pin1-/- mice, which show a neurodegenerative phenotype with tau phosphorylation at 9 months of age. Tau phosphorylation status was assessed using several antibodies against phosphorylated tau (AT8, AT100 and AT180) and activated forms of Cdk5 (phospho-Tyr15 Cdk5), GSK3b (phospho-Tyr 216 GSK3b, phospho-Ser9 GSK3b) and c-Abl (phosphoTyr412 c-Abl). The effect of p73 haploinsufficiency on behavioral and motor impairments was assessed using the Morris water maze and limb clasping test. Results: We found enhanced tau phosphorylation and PHFlike structures in aged p73+/- mice animals from both genetic backgrounds. The phosphorylation of GSK3b and Cdk5 was enhanced in the CNS of these mice, as was the phosphorylation of c-Abl, a minor tau kinase but important in the control of Cdk5 activity. In p73+/- mice crossed with CRND8 mice, tau phosphorylation and the phosphorylation of GSK3b, Cdk5 and c-Abl was enhanced at 2 months as compared to p73+/+ 3 CRND8 mice. Similarly, the phosphorylation of GSK3b, Cdk5 and c-Abl was elevated in p73+/- 3 Pin1-/- mice. These data suggest that loss of p73 causes increases the activity of the tau kinases, GSK3b, Cdk5, JNK and c-Abl, which may account for the enhanced tau phosphorylation in the p73+/- and p73+/mice crossed with CRND8 or Pin1-/- mice. Finally, to evaluate whether tau phosphorylation induced by p73 haploinsufficiency induce behavioral impairments at 2 months of age, we performed a Morris water maze test. Our preliminary findings indicate high latency escape values during water maze training and poor performance in the probe test in p73+/- 3 CRND8 mice compared to p73+/+ 3 CRND8 mice at 2 months of age. Conclusions: We extend and confirm our previous findings showing that p73 haploinsufficiency induces tau phosphorylation, and results in much earlier appearance of tau phosphorylation in two mouse models of neurodegeneration. The increase in tau phosphorylation may be due to disregulation of the activity of the tau kinases GSK3b, Cdk5, c-Abl and JNK. These results, together with our previous work, suggest that in mice, the levels of p73 are an important determinant in regulating the onset and severity of neurodegeneration and Alzheimer’s disease-like phenotypes. P3-122
P3-121
THE REGULATION OF TAU PHOSPHORYLATION AND TAU KINASE ACTIVITY BY THE P53 FAMILY MEMBER P73 IN ALZHEIMER’S DISEASE ANIMAL MODELS
Gonzalo Cancino1, Adeliade Yiu2, Sheena Josselyn1, Freda Miller1, David Kaplan1, 1The Hospital for Sick Children, Toronto, Ontario, Canada; 2 University of Toronto, Toronto, Ontario, Canada. Background: The cellular mechanisms that regulate nervous system degeneration during aging or neurodegenerative disorders are poorly understood. Recent work has shown that p53 family members participate in the regulation of cell death in several neurodegenerative disorders and in aging of the nervous system. In previous work from our laboratory, we reported that haploinsufficiency of the p53 family member p73 in aged mice results in premature neurodegeneration, behavioral impairments, neuronal loss, activation of JNK, and tau phosphorylation. P73+/- mice crossed to the transgenic CRND8 (TgCRND8+/ø) mouse model of Alzheimer’s disease that develop plaques, but not tangles, by 3 months of age and do not display neuronal loss, exhibited neuronal loss and tau phosphorylation at 1.5-2
S553
PRESENCE OF SET IN THE CYTOPLASM IN TWO IN VITRO MODELS IS NOT ALWAYS ASSOCIATED WITH ITS CLEAVAGE BUT INDUCES TAU HYPERPHOSPHORYLATION
Stephanie Chasseigneaux, Christine Clamagirand, Patricia Facchinetti, Christiane Rose, Bernadette Allinquant, INSERM U894 and Universite Paris-Descartes, Paris, France. Background: We previously observed in an in vitro model of neuronal death induced by over expression of the juxtamembranar cytoplasmic domain of amyloid precursor protein (APP) unmasked after caspase cleavage (Jcasp), that SET is an actor of the cell death and that it delocalizes from the nucleus to the cytoplasm. We wanted to check if the presence of SET in the cytoplasm in this model or directly after internalization of the SET recombinant protein requires its cleavage and what are the consequences in Tau phosphorylation. Methods: We used mouse embryonic primary neurons 5 days in vitro and adult mouse brain slices maintained in oxygenated physiological buffer for 6h. We over expressed through the help of a cell permeable peptide, either the Jcasp peptide or the whole recombinant SET protein from 3h to 6h. Then the cells or tissues were investigated for SET and Tau
P3-120
MIS-SPLICING OF TAU EXON 2 AND EXON 10 IN MYOTONIC DYSTROPHY BRAIN WOULD RESULT FROM DIFFERENT MECHANISMS
Marie-Laure Caillet-Boudin1, Claire-Marie Dhaenens2, MarieLise Frandemiche2, Helene Tran2, Celine Carpentier2, FranciscoJose Fernandez-Gomez2, Andone Sistiaga3, Maria Goicoechea3, Edwige Van Brussel2, Helene Obriot2, Sabiha Eddarkaoui2, Marie-Helene Gavaert4, Bernard Sablonniere2, Adolfo Lopez-de-munain5, Luc Buee1, Susanna Schraen2, Nicolas Sergeant2, 1Inserm-Univ. Lille, Lille, France; 2 Inserm, U837-1, Univ. Lille Nord de France, IMPRT, Centre Jean-Pierre Aubert, Lille, France, Lille, France; 3Neurological Department and Experimental Unit, Donostia Hospital, San Sebastian, Spain, San Sebastian, Spain; 4Neuropathological Department, Regional University Hospital Centre, Lille, France., Lille, France; 5Neurological Department and Experimental Unit, Donostia Hospital, San Sebastian, Spain, Lille, France. Background: The mis-splicing of Tau is instrumental to the development of neurofibrillary degeneration (NFD) naturopathies such as FTDP-17 in which MAPTmutations lead to a defective splicing of Tau. A mis-splicing of Tau is also observed in the brain and muscle of patients affected by myotonic dystrophy of type I (DM1). Moreover, this Tau mis-splicing is associated with the development of NFD. DM1 is a neuromuscular disease characterized by an unstable CTG expansion. This mutation is responsible for a toxic gain of RNA-function, leading to the gain or loss of function of several splicing factors, including the CELF and MBNL families, respectively. It ultimately leads to an indirect mis-splicing of numerous transcripts. In regards to Tau mis-splicing, exon-2 inclusion is highly decreased in DM1 brains. In contrast, a mis-splicing of Tau exon-10 is seldom observed in DM1 but the mechanisms triggering this mis-splicing event remain unknown. Methods: We herein aimed to gain further insights into these molecular mechanisms involved in normal and pathological splicing of Tau exons 2 and 10 using cell-based models. Results: Two among the brains of 5 DM1 patients showed an exon-10 mis-splicing whereas exon-2 inclusion was reduced in all DM1 patients. The over-expression of long stretches of CTGrepeats promoted the exclusion of both Tau exon 2 and exon 10. Interestingly, MBNL1 loss of expression efficiently repressed Tau exon 2 inclusion but failed to modulate Tau exon-10 splicing. Analyzing the splicing functions of three major CELF splicing factors, we showed that CELF 4 acts as a repressor for exon-2 or as an enhancer for exon-10 inclusion. In sharp contrast, CELF2 repressed both exons 2 and 10 inclusions whereas CELF1 was inefficient to modulate Tau splicing. In addition, 2D electrophoresis coupled to western-blotting suggest a possible change inbrain CELF2 activity induced by its hyperphosphorylation in DM1. Conclusions: Altogether, we showed that MBNL1 and CELF2 are implicated in the mis-splicing of Tau-exons 2 and 10, respectively, but not CELF1. Moreover, our Tau splicing study also suggests that two different mechanisms may contribute to the mis-splicing of several targets in DM1.
months. In this study, we assessed whether behavioral impairments occurred in those mice, and whether the premature neurodegeneration phenotype occurs in multiple strains of p73+/- mice or p73+/- mice crossed with other mice with Alzheimer’s disease-like phenotypes such as Pin1-/-. Furthermore, we performed a more extensive analysis of tau phosphorylation at multiple time points, and examined whether p73 might function in part to prevent neurodegeneration by suppressing the activity of other tau kinases such as GSK3b and Cdk5. Methods: We crossed p73+/- mice into two different genetic backgrounds, Balb/c129 and C57 BL6. Aged mice were used at 18 months old. To analyze the potential involvement of p73 in Alzheimer’s disease, we crossed p73+/- mice with the transgenic CRND8 (TgCRND8+/ø) mouse model of Alzheimer’s disease. We analyzed these crosses at 1.5-2 months, an early time point of plaque formation. We also analyzed the effect of p73 heterozygosity in a second model of neurodegeneration, Pin1-/- mice, which show a neurodegenerative phenotype with tau phosphorylation at 9 months of age. Tau phosphorylation status was assessed using several antibodies against phosphorylated tau (AT8, AT100 and AT180) and activated forms of Cdk5 (phospho-Tyr15 Cdk5), GSK3b (phospho-Tyr 216 GSK3b, phospho-Ser9 GSK3b) and c-Abl (phosphoTyr412 c-Abl). The effect of p73 haploinsufficiency on behavioral and motor impairments was assessed using the Morris water maze and limb clasping test. Results: We found enhanced tau phosphorylation and PHFlike structures in aged p73+/- mice animals from both genetic backgrounds. The phosphorylation of GSK3b and Cdk5 was enhanced in the CNS of these mice, as was the phosphorylation of c-Abl, a minor tau kinase but important in the control of Cdk5 activity. In p73+/- mice crossed with CRND8 mice, tau phosphorylation and the phosphorylation of GSK3b, Cdk5 and c-Abl was enhanced at 2 months as compared to p73+/+ 3 CRND8 mice. Similarly, the phosphorylation of GSK3b, Cdk5 and c-Abl was elevated in p73+/- 3 Pin1-/- mice. These data suggest that loss of p73 causes increases the activity of the tau kinases, GSK3b, Cdk5, JNK and c-Abl, which may account for the enhanced tau phosphorylation in the p73+/- and p73+/mice crossed with CRND8 or Pin1-/- mice. Finally, to evaluate whether tau phosphorylation induced by p73 haploinsufficiency induce behavioral impairments at 2 months of age, we performed a Morris water maze test. Our preliminary findings indicate high latency escape values during water maze training and poor performance in the probe test in p73+/- 3 CRND8 mice compared to p73+/+ 3 CRND8 mice at 2 months of age. Conclusions: We extend and confirm our previous findings showing that p73 haploinsufficiency induces tau phosphorylation, and results in much earlier appearance of tau phosphorylation in two mouse models of neurodegeneration. The increase in tau phosphorylation may be due to disregulation of the activity of the tau kinases GSK3b, Cdk5, c-Abl and JNK. These results, together with our previous work, suggest that in mice, the levels of p73 are an important determinant in regulating the onset and severity of neurodegeneration and Alzheimer’s disease-like phenotypes. P3-122
P3-121
THE REGULATION OF TAU PHOSPHORYLATION AND TAU KINASE ACTIVITY BY THE P53 FAMILY MEMBER P73 IN ALZHEIMER’S DISEASE ANIMAL MODELS
Gonzalo Cancino1, Adeliade Yiu2, Sheena Josselyn1, Freda Miller1, David Kaplan1, 1The Hospital for Sick Children, Toronto, Ontario, Canada; 2 University of Toronto, Toronto, Ontario, Canada. Background: The cellular mechanisms that regulate nervous system degeneration during aging or neurodegenerative disorders are poorly understood. Recent work has shown that p53 family members participate in the regulation of cell death in several neurodegenerative disorders and in aging of the nervous system. In previous work from our laboratory, we reported that haploinsufficiency of the p53 family member p73 in aged mice results in premature neurodegeneration, behavioral impairments, neuronal loss, activation of JNK, and tau phosphorylation. P73+/- mice crossed to the transgenic CRND8 (TgCRND8+/ø) mouse model of Alzheimer’s disease that develop plaques, but not tangles, by 3 months of age and do not display neuronal loss, exhibited neuronal loss and tau phosphorylation at 1.5-2
S553
PRESENCE OF SET IN THE CYTOPLASM IN TWO IN VITRO MODELS IS NOT ALWAYS ASSOCIATED WITH ITS CLEAVAGE BUT INDUCES TAU HYPERPHOSPHORYLATION
Stephanie Chasseigneaux, Christine Clamagirand, Patricia Facchinetti, Christiane Rose, Bernadette Allinquant, INSERM U894 and Universite Paris-Descartes, Paris, France. Background: We previously observed in an in vitro model of neuronal death induced by over expression of the juxtamembranar cytoplasmic domain of amyloid precursor protein (APP) unmasked after caspase cleavage (Jcasp), that SET is an actor of the cell death and that it delocalizes from the nucleus to the cytoplasm. We wanted to check if the presence of SET in the cytoplasm in this model or directly after internalization of the SET recombinant protein requires its cleavage and what are the consequences in Tau phosphorylation. Methods: We used mouse embryonic primary neurons 5 days in vitro and adult mouse brain slices maintained in oxygenated physiological buffer for 6h. We over expressed through the help of a cell permeable peptide, either the Jcasp peptide or the whole recombinant SET protein from 3h to 6h. Then the cells or tissues were investigated for SET and Tau