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A functional misexpression screen uncovers a role for enabled in progressive neurodegeneration.

Rezával C, Berni J, Gorostiza EA, Werbajh S, Fagilde MM, Fernández MP, Beckwith EJ, Aranovich EJ, Sabio y García CA, Ceriani MF - PLoS ONE (2008)

Bottom Line: One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain.Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant.Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Genética del Comportamiento, Fundación Instituto Leloir, Instituto de Investigaciones Bioquímicas-Buenos Aires (IIB-BA, CONICET), Buenos Aires, Argentina.

ABSTRACT
Drosophila is a well-established model to study the molecular basis of neurodegenerative diseases. We carried out a misexpression screen to identify genes involved in neurodegeneration examining locomotor behavior in young and aged flies. We hypothesized that a progressive loss of rhythmic activity could reveal novel genes involved in neurodegenerative mechanisms. One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain. Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant. Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor. Thus, this mutant recapitulates two important features of human neurodegenerative diseases, i.e., vulnerability of certain neuronal populations and progressive degeneration, offering a unique scenario in which to unravel the specific mechanisms in an easily tractable organism.

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Plasmid rescue and RT-PCR analysis identifies ena as the affected locus.(A) Schematic diagram depicting the position of the P[UAS] transposon within the DNA region trapped by the insertion (to scale, in kb). The P[UAS]117 is located within the first exon of ena upstream of the ATG. It also landed within the first intron of CG15118 and near CG15111. Arrows indicate the direction of transcription for each gene. A–E refers to the different splicing variants in each locus. (B) To test the GAL4 mediated effect on each locus an ubiquitous heat shock (hs) GAL4 driver was employed. RT-PCR analysis was performed in hs>P[UAS]117 total larval RNA after a heat-shock stimulus (+hs). A non-pulsed control was used as baseline (-hs) along with a wild type control (y w). Semi quantitative RT-PCR products were analyzed on agarose gels stained with ethidium bromide (the image reflects ena levels at cycle 28). actin levels were compared for quality control of the independent RNA preparations and normalization. (C) The ratio between each independent gene to actin levels highlights significant changes only in the ena locus (* p<0.05). The insertion per se significantly reduces ena levels (* p<0.05). Statistical analysis included a Student t test. The experiment was repeated 3–4 times using independent RNA preparations.
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pone-0003332-g003: Plasmid rescue and RT-PCR analysis identifies ena as the affected locus.(A) Schematic diagram depicting the position of the P[UAS] transposon within the DNA region trapped by the insertion (to scale, in kb). The P[UAS]117 is located within the first exon of ena upstream of the ATG. It also landed within the first intron of CG15118 and near CG15111. Arrows indicate the direction of transcription for each gene. A–E refers to the different splicing variants in each locus. (B) To test the GAL4 mediated effect on each locus an ubiquitous heat shock (hs) GAL4 driver was employed. RT-PCR analysis was performed in hs>P[UAS]117 total larval RNA after a heat-shock stimulus (+hs). A non-pulsed control was used as baseline (-hs) along with a wild type control (y w). Semi quantitative RT-PCR products were analyzed on agarose gels stained with ethidium bromide (the image reflects ena levels at cycle 28). actin levels were compared for quality control of the independent RNA preparations and normalization. (C) The ratio between each independent gene to actin levels highlights significant changes only in the ena locus (* p<0.05). The insertion per se significantly reduces ena levels (* p<0.05). Statistical analysis included a Student t test. The experiment was repeated 3–4 times using independent RNA preparations.

Mentions: Plasmid rescue analysis revealed that P[UAS]117 is inserted within the first exon of enabled (ena) upstream of the ATG, and thus it interrupts four out of the five splice variants predicted (Fig. 3A). The P element is located in reverse orientation with regard to transcription at this locus, potentially driving transcription of an antisense RNA in a GAL4-dependent manner. Such possibility is not unprecedented [18]. P[UAS]117 also interrupts the long splice variant of the predicted gene CG15118; it is located within its first intron, upstream of the exon containing the ATG in the same orientation. The transcriptional start sites of the three remaining splice variants lie nearly 5 kb downstream, and therefore it is unlikely that they will be affected. Within this region there is a third predicted gene (CG15111) that runs in the opposite orientation to P[UAS]117 but it is not physically interrupted by it.


A functional misexpression screen uncovers a role for enabled in progressive neurodegeneration.

Rezával C, Berni J, Gorostiza EA, Werbajh S, Fagilde MM, Fernández MP, Beckwith EJ, Aranovich EJ, Sabio y García CA, Ceriani MF - PLoS ONE (2008)

Plasmid rescue and RT-PCR analysis identifies ena as the affected locus.(A) Schematic diagram depicting the position of the P[UAS] transposon within the DNA region trapped by the insertion (to scale, in kb). The P[UAS]117 is located within the first exon of ena upstream of the ATG. It also landed within the first intron of CG15118 and near CG15111. Arrows indicate the direction of transcription for each gene. A–E refers to the different splicing variants in each locus. (B) To test the GAL4 mediated effect on each locus an ubiquitous heat shock (hs) GAL4 driver was employed. RT-PCR analysis was performed in hs>P[UAS]117 total larval RNA after a heat-shock stimulus (+hs). A non-pulsed control was used as baseline (-hs) along with a wild type control (y w). Semi quantitative RT-PCR products were analyzed on agarose gels stained with ethidium bromide (the image reflects ena levels at cycle 28). actin levels were compared for quality control of the independent RNA preparations and normalization. (C) The ratio between each independent gene to actin levels highlights significant changes only in the ena locus (* p<0.05). The insertion per se significantly reduces ena levels (* p<0.05). Statistical analysis included a Student t test. The experiment was repeated 3–4 times using independent RNA preparations.
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Related In: Results  -  Collection

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pone-0003332-g003: Plasmid rescue and RT-PCR analysis identifies ena as the affected locus.(A) Schematic diagram depicting the position of the P[UAS] transposon within the DNA region trapped by the insertion (to scale, in kb). The P[UAS]117 is located within the first exon of ena upstream of the ATG. It also landed within the first intron of CG15118 and near CG15111. Arrows indicate the direction of transcription for each gene. A–E refers to the different splicing variants in each locus. (B) To test the GAL4 mediated effect on each locus an ubiquitous heat shock (hs) GAL4 driver was employed. RT-PCR analysis was performed in hs>P[UAS]117 total larval RNA after a heat-shock stimulus (+hs). A non-pulsed control was used as baseline (-hs) along with a wild type control (y w). Semi quantitative RT-PCR products were analyzed on agarose gels stained with ethidium bromide (the image reflects ena levels at cycle 28). actin levels were compared for quality control of the independent RNA preparations and normalization. (C) The ratio between each independent gene to actin levels highlights significant changes only in the ena locus (* p<0.05). The insertion per se significantly reduces ena levels (* p<0.05). Statistical analysis included a Student t test. The experiment was repeated 3–4 times using independent RNA preparations.
Mentions: Plasmid rescue analysis revealed that P[UAS]117 is inserted within the first exon of enabled (ena) upstream of the ATG, and thus it interrupts four out of the five splice variants predicted (Fig. 3A). The P element is located in reverse orientation with regard to transcription at this locus, potentially driving transcription of an antisense RNA in a GAL4-dependent manner. Such possibility is not unprecedented [18]. P[UAS]117 also interrupts the long splice variant of the predicted gene CG15118; it is located within its first intron, upstream of the exon containing the ATG in the same orientation. The transcriptional start sites of the three remaining splice variants lie nearly 5 kb downstream, and therefore it is unlikely that they will be affected. Within this region there is a third predicted gene (CG15111) that runs in the opposite orientation to P[UAS]117 but it is not physically interrupted by it.

Bottom Line: One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain.Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant.Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Genética del Comportamiento, Fundación Instituto Leloir, Instituto de Investigaciones Bioquímicas-Buenos Aires (IIB-BA, CONICET), Buenos Aires, Argentina.

ABSTRACT
Drosophila is a well-established model to study the molecular basis of neurodegenerative diseases. We carried out a misexpression screen to identify genes involved in neurodegeneration examining locomotor behavior in young and aged flies. We hypothesized that a progressive loss of rhythmic activity could reveal novel genes involved in neurodegenerative mechanisms. One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain. Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant. Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor. Thus, this mutant recapitulates two important features of human neurodegenerative diseases, i.e., vulnerability of certain neuronal populations and progressive degeneration, offering a unique scenario in which to unravel the specific mechanisms in an easily tractable organism.

Show MeSH
Related in: MedlinePlus