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Enhanced neuronal Met signalling levels in ALS mice delay disease onset.

Genestine M, Caricati E, Fico A, Richelme S, Hassani H, Sunyach C, Lamballe F, Panzica GC, Pettmann B, Helmbacher F, Raoul C, Maina F, Dono R - Cell Death Dis (2011)

Bottom Line: Enhancing Met levels in neurons does not affect either motor neuron (MN) development or maintenance.Thus, our studies highlight the properties of RTKs to counteract toxic signals in a disease characterized by dysfunction of multiple cell types by acting in MNs.Moreover, they emphasize the relevance of genetically assessing the effectiveness of agents targeting neurons during ALS evolution.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology Institute of Marseille-Luminy, UMR 6216, CNRS-Inserm-Université de la Méditerranée, Campus de Luminy-Case 907, Marseille Cedex 09, France.

ABSTRACT
Signalling by receptor tyrosine kinases (RTKs) coordinates basic cellular processes during development and in adulthood. Whereas aberrant RTK signalling can lead to cancer, reactivation of RTKs is often found following stress or cell damage. This has led to the common belief that RTKs can counteract degenerative processes and so strategies to exploit them for therapy have been extensively explored. An understanding of how RTK stimuli act at cellular levels is needed, however, to evaluate their mechanism of therapeutic action. In this study, we genetically explored the biological and functional significance of enhanced signalling by the Met RTK in neurons, in the context of a neurodegenerative disease. Conditional met-transgenic mice, namely Rosa26(LacZ-stop-Met), have been engineered to trigger increased Met signalling in a temporal and tissue-specific regulated manner. Enhancing Met levels in neurons does not affect either motor neuron (MN) development or maintenance. In contrast, increased neuronal Met in amyotrophic lateral sclerosis (ALS) mice prolongs life span, retards MN loss, and ameliorates motor performance, by selectively delaying disease onset. Thus, our studies highlight the properties of RTKs to counteract toxic signals in a disease characterized by dysfunction of multiple cell types by acting in MNs. Moreover, they emphasize the relevance of genetically assessing the effectiveness of agents targeting neurons during ALS evolution.

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Temporal and tissue-specific Met transgene activation using Cre-mediated recombination. (a) The R26LacZ−stop−Met construct targeted in the Rosa26 locus is shown. The targeting construct consists of the CMV-enhancer and the chicken β-actin promoter (CMV/β-actin) driving the β-geo reporter and a Met chimeric cDNA. The reporter gene is followed by three copies of the SV40 polyadenylation signal (3xpA) and flanked by loxP sites. The Met chimeric cDNA consists of a 5′ portion encoding the mouse extracellular domain fused to a 3′ region coding for the intracellular human portion. Positions of the probes used for Southern analysis (a and b) as well as primers used for PCR (1, 2, 3) are indicated. (b) Southern blots of neo-resistant ES clones analyzed with probe a (EcoRI digestion; top) and probe b (EcoRV digestion; bottom). The 15.5 Kb band corresponds to the wild-type allele whereas the 5.5 Kb band depicts the recombinant allele in clones 1 and 2. (c) PCR of mouse-tail genomic DNAs showing the mutant allele before and after Cre recombination (top). The presence of an amplicon in the Nes-R26Met mice with the 1+2 primers is detected because genotypes are performed using DNA extracted from tails, which include tissues with and without recombination. PCR showing the genotypes of heterozygous and homozygous Nes-R26Met mice (bottom). (d) Western blots showing the expression of the Met chimeric protein (using anti-human Met antibodies) in brain and spinal cord extracts from embryos (E15.5) and newborn mice (P7) with the indicated genotypes. (e) Quantification analysis of Met chimeric protein levels versus endogenous Met. Western blot analysis of protein extracts from dissected brains and spinal cords at different developmental stages using antibodies recognizing the Met kinase domain (MetKD). (f) Quantification analyses revealed that levels of Met chimeric proteins were at least five- and sevenfolds increased in brains and spinal cords of heterozygous Nes-R26Met, respectively, when compared with the endogenous mouse Met. Met chimeric levels were double in homozygous mice. Numbers on columns indicate fold of increase in Met protein levels over the endogenous Met. In the graph, Nes-R26Met and Nes-R26Met/Met genotypes are indicated as M/+ and M/M, respectively. Values are expressed as means±S.E.M. Tub, tubulin; hMet, human-Met
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fig1: Temporal and tissue-specific Met transgene activation using Cre-mediated recombination. (a) The R26LacZ−stop−Met construct targeted in the Rosa26 locus is shown. The targeting construct consists of the CMV-enhancer and the chicken β-actin promoter (CMV/β-actin) driving the β-geo reporter and a Met chimeric cDNA. The reporter gene is followed by three copies of the SV40 polyadenylation signal (3xpA) and flanked by loxP sites. The Met chimeric cDNA consists of a 5′ portion encoding the mouse extracellular domain fused to a 3′ region coding for the intracellular human portion. Positions of the probes used for Southern analysis (a and b) as well as primers used for PCR (1, 2, 3) are indicated. (b) Southern blots of neo-resistant ES clones analyzed with probe a (EcoRI digestion; top) and probe b (EcoRV digestion; bottom). The 15.5 Kb band corresponds to the wild-type allele whereas the 5.5 Kb band depicts the recombinant allele in clones 1 and 2. (c) PCR of mouse-tail genomic DNAs showing the mutant allele before and after Cre recombination (top). The presence of an amplicon in the Nes-R26Met mice with the 1+2 primers is detected because genotypes are performed using DNA extracted from tails, which include tissues with and without recombination. PCR showing the genotypes of heterozygous and homozygous Nes-R26Met mice (bottom). (d) Western blots showing the expression of the Met chimeric protein (using anti-human Met antibodies) in brain and spinal cord extracts from embryos (E15.5) and newborn mice (P7) with the indicated genotypes. (e) Quantification analysis of Met chimeric protein levels versus endogenous Met. Western blot analysis of protein extracts from dissected brains and spinal cords at different developmental stages using antibodies recognizing the Met kinase domain (MetKD). (f) Quantification analyses revealed that levels of Met chimeric proteins were at least five- and sevenfolds increased in brains and spinal cords of heterozygous Nes-R26Met, respectively, when compared with the endogenous mouse Met. Met chimeric levels were double in homozygous mice. Numbers on columns indicate fold of increase in Met protein levels over the endogenous Met. In the graph, Nes-R26Met and Nes-R26Met/Met genotypes are indicated as M/+ and M/M, respectively. Values are expressed as means±S.E.M. Tub, tubulin; hMet, human-Met

Mentions: To enhance Met signalling levels in a temporally and spatially regulated manner, we generated mice carrying a conditional mouse–human chimeric met transgene (mettg). In particular, we engineered a mouse strain in which a cytomegalovirus (CMV)-enhancer/β-actin-promoter controls the expression of a floxed-β-geo-reporter gene followed by a mettg. Such a strategy was chosen to keep the mettg silent, unless the stop cassette is excised by cre-mediated recombination. To avoid integration site effects and favour loxP-site accessibility, the construct was inserted into the Rosa26 locus (Rosa26LacZ−stop−Met, referred to R26LacZ−stop−Met; Figures 1a–c).


Enhanced neuronal Met signalling levels in ALS mice delay disease onset.

Genestine M, Caricati E, Fico A, Richelme S, Hassani H, Sunyach C, Lamballe F, Panzica GC, Pettmann B, Helmbacher F, Raoul C, Maina F, Dono R - Cell Death Dis (2011)

Temporal and tissue-specific Met transgene activation using Cre-mediated recombination. (a) The R26LacZ−stop−Met construct targeted in the Rosa26 locus is shown. The targeting construct consists of the CMV-enhancer and the chicken β-actin promoter (CMV/β-actin) driving the β-geo reporter and a Met chimeric cDNA. The reporter gene is followed by three copies of the SV40 polyadenylation signal (3xpA) and flanked by loxP sites. The Met chimeric cDNA consists of a 5′ portion encoding the mouse extracellular domain fused to a 3′ region coding for the intracellular human portion. Positions of the probes used for Southern analysis (a and b) as well as primers used for PCR (1, 2, 3) are indicated. (b) Southern blots of neo-resistant ES clones analyzed with probe a (EcoRI digestion; top) and probe b (EcoRV digestion; bottom). The 15.5 Kb band corresponds to the wild-type allele whereas the 5.5 Kb band depicts the recombinant allele in clones 1 and 2. (c) PCR of mouse-tail genomic DNAs showing the mutant allele before and after Cre recombination (top). The presence of an amplicon in the Nes-R26Met mice with the 1+2 primers is detected because genotypes are performed using DNA extracted from tails, which include tissues with and without recombination. PCR showing the genotypes of heterozygous and homozygous Nes-R26Met mice (bottom). (d) Western blots showing the expression of the Met chimeric protein (using anti-human Met antibodies) in brain and spinal cord extracts from embryos (E15.5) and newborn mice (P7) with the indicated genotypes. (e) Quantification analysis of Met chimeric protein levels versus endogenous Met. Western blot analysis of protein extracts from dissected brains and spinal cords at different developmental stages using antibodies recognizing the Met kinase domain (MetKD). (f) Quantification analyses revealed that levels of Met chimeric proteins were at least five- and sevenfolds increased in brains and spinal cords of heterozygous Nes-R26Met, respectively, when compared with the endogenous mouse Met. Met chimeric levels were double in homozygous mice. Numbers on columns indicate fold of increase in Met protein levels over the endogenous Met. In the graph, Nes-R26Met and Nes-R26Met/Met genotypes are indicated as M/+ and M/M, respectively. Values are expressed as means±S.E.M. Tub, tubulin; hMet, human-Met
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101808&req=5

fig1: Temporal and tissue-specific Met transgene activation using Cre-mediated recombination. (a) The R26LacZ−stop−Met construct targeted in the Rosa26 locus is shown. The targeting construct consists of the CMV-enhancer and the chicken β-actin promoter (CMV/β-actin) driving the β-geo reporter and a Met chimeric cDNA. The reporter gene is followed by three copies of the SV40 polyadenylation signal (3xpA) and flanked by loxP sites. The Met chimeric cDNA consists of a 5′ portion encoding the mouse extracellular domain fused to a 3′ region coding for the intracellular human portion. Positions of the probes used for Southern analysis (a and b) as well as primers used for PCR (1, 2, 3) are indicated. (b) Southern blots of neo-resistant ES clones analyzed with probe a (EcoRI digestion; top) and probe b (EcoRV digestion; bottom). The 15.5 Kb band corresponds to the wild-type allele whereas the 5.5 Kb band depicts the recombinant allele in clones 1 and 2. (c) PCR of mouse-tail genomic DNAs showing the mutant allele before and after Cre recombination (top). The presence of an amplicon in the Nes-R26Met mice with the 1+2 primers is detected because genotypes are performed using DNA extracted from tails, which include tissues with and without recombination. PCR showing the genotypes of heterozygous and homozygous Nes-R26Met mice (bottom). (d) Western blots showing the expression of the Met chimeric protein (using anti-human Met antibodies) in brain and spinal cord extracts from embryos (E15.5) and newborn mice (P7) with the indicated genotypes. (e) Quantification analysis of Met chimeric protein levels versus endogenous Met. Western blot analysis of protein extracts from dissected brains and spinal cords at different developmental stages using antibodies recognizing the Met kinase domain (MetKD). (f) Quantification analyses revealed that levels of Met chimeric proteins were at least five- and sevenfolds increased in brains and spinal cords of heterozygous Nes-R26Met, respectively, when compared with the endogenous mouse Met. Met chimeric levels were double in homozygous mice. Numbers on columns indicate fold of increase in Met protein levels over the endogenous Met. In the graph, Nes-R26Met and Nes-R26Met/Met genotypes are indicated as M/+ and M/M, respectively. Values are expressed as means±S.E.M. Tub, tubulin; hMet, human-Met
Mentions: To enhance Met signalling levels in a temporally and spatially regulated manner, we generated mice carrying a conditional mouse–human chimeric met transgene (mettg). In particular, we engineered a mouse strain in which a cytomegalovirus (CMV)-enhancer/β-actin-promoter controls the expression of a floxed-β-geo-reporter gene followed by a mettg. Such a strategy was chosen to keep the mettg silent, unless the stop cassette is excised by cre-mediated recombination. To avoid integration site effects and favour loxP-site accessibility, the construct was inserted into the Rosa26 locus (Rosa26LacZ−stop−Met, referred to R26LacZ−stop−Met; Figures 1a–c).

Bottom Line: Enhancing Met levels in neurons does not affect either motor neuron (MN) development or maintenance.Thus, our studies highlight the properties of RTKs to counteract toxic signals in a disease characterized by dysfunction of multiple cell types by acting in MNs.Moreover, they emphasize the relevance of genetically assessing the effectiveness of agents targeting neurons during ALS evolution.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology Institute of Marseille-Luminy, UMR 6216, CNRS-Inserm-Université de la Méditerranée, Campus de Luminy-Case 907, Marseille Cedex 09, France.

ABSTRACT
Signalling by receptor tyrosine kinases (RTKs) coordinates basic cellular processes during development and in adulthood. Whereas aberrant RTK signalling can lead to cancer, reactivation of RTKs is often found following stress or cell damage. This has led to the common belief that RTKs can counteract degenerative processes and so strategies to exploit them for therapy have been extensively explored. An understanding of how RTK stimuli act at cellular levels is needed, however, to evaluate their mechanism of therapeutic action. In this study, we genetically explored the biological and functional significance of enhanced signalling by the Met RTK in neurons, in the context of a neurodegenerative disease. Conditional met-transgenic mice, namely Rosa26(LacZ-stop-Met), have been engineered to trigger increased Met signalling in a temporal and tissue-specific regulated manner. Enhancing Met levels in neurons does not affect either motor neuron (MN) development or maintenance. In contrast, increased neuronal Met in amyotrophic lateral sclerosis (ALS) mice prolongs life span, retards MN loss, and ameliorates motor performance, by selectively delaying disease onset. Thus, our studies highlight the properties of RTKs to counteract toxic signals in a disease characterized by dysfunction of multiple cell types by acting in MNs. Moreover, they emphasize the relevance of genetically assessing the effectiveness of agents targeting neurons during ALS evolution.

Show MeSH
Related in: MedlinePlus