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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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Related in: MedlinePlus

Age associated arrhythmicity is dependent on ena function.(A) Rescue of ENA function on the PDF circuit restores behavioral rhythmicity in aged flies. Recombinant pdf-gal4, enarev carrying one copy of UAS-ena were indistinguishable from control UAS-ena. Representative double plotted actograms for aged (24–28d) flies are shown. Overexpression of ENA in young flies do not affect locomotor activity rhythms (data not shown). (B) The percentage of rhythmicity for aged flies for each strain is shown. pdf-gal4, enarev/++ is significantly different from both UAS-ena/+ (control) and pdf-gal4, enarev/UAS-ena (*** p<0.001). (C) Representative actograms of young (3d) and aged (21d) flies carrying one or two copies of enarev, along with the transheterozygotes enarev/enaGC5. Both homozygous enarev and enarev/enaGC5 exhibit a decline on rhythm strength. (D) Summary of behavioral data for flies of the indicated genotypes. Control enarev/+ flies stay rhythmic throughout lifespan. Aged enarev is significantly different from its younger counterpart (* p<0.05). Both aged enarev and enarev/enaGC5 are different from old enarev/+ (* p<0.05). Experiments summarized in B and D were repeated at least 3 times. (E) Whole mount brain immunofluorescense of 10 day-old adult y w flies were stained with a specific antibody against ENA. A general staining of neuropils in the adult brain was observed. Single confocal planes (2 µm thick) are shown at two depths (8 and 22 µm) to highlight different brain areas. Some of the neuropils labeled with ENA are the outer (o me) and inner medulla (i me), lobula (lo) and lobula plate (lo p) within the optic lobe, the protocerebral bridge (pr br) in the central complex as well as other regions in the protocerebrum such as the lateral horn (l ho). Other structures, as the protolateral deutocerebrum (p l deu), superior lateral protocerebrum (s l pr), the peduncles of the mushroom body (pe), pars intercerebralis (pars in), suboesophageal ganglion (su oes g) and oesophagus (oes) are also shown in the figure. (F) ena levels are reduced in enarev mutants compared to the control y w. Images were taken with the same confocal settings for direct comparison; projections of 2.3 µm depth are shown. ENA immunohistochemistry was repeated at least three times. (G) RT-PCR analysis was performed in adult enarev, enarev/+ and control total RNA. The ratio between ena and actin expression levels for each genotype is shown. Quantification of RNA levels showed significant changes in enarev homozygous (* p<0.05) whereas a minor (non significant) decrease was seen in enarev/+ heterozygous when compared to the y w control line. The experiment was repeated three times employing independent RNA preparations.
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pone-0003332-g004: Age associated arrhythmicity is dependent on ena function.(A) Rescue of ENA function on the PDF circuit restores behavioral rhythmicity in aged flies. Recombinant pdf-gal4, enarev carrying one copy of UAS-ena were indistinguishable from control UAS-ena. Representative double plotted actograms for aged (24–28d) flies are shown. Overexpression of ENA in young flies do not affect locomotor activity rhythms (data not shown). (B) The percentage of rhythmicity for aged flies for each strain is shown. pdf-gal4, enarev/++ is significantly different from both UAS-ena/+ (control) and pdf-gal4, enarev/UAS-ena (*** p<0.001). (C) Representative actograms of young (3d) and aged (21d) flies carrying one or two copies of enarev, along with the transheterozygotes enarev/enaGC5. Both homozygous enarev and enarev/enaGC5 exhibit a decline on rhythm strength. (D) Summary of behavioral data for flies of the indicated genotypes. Control enarev/+ flies stay rhythmic throughout lifespan. Aged enarev is significantly different from its younger counterpart (* p<0.05). Both aged enarev and enarev/enaGC5 are different from old enarev/+ (* p<0.05). Experiments summarized in B and D were repeated at least 3 times. (E) Whole mount brain immunofluorescense of 10 day-old adult y w flies were stained with a specific antibody against ENA. A general staining of neuropils in the adult brain was observed. Single confocal planes (2 µm thick) are shown at two depths (8 and 22 µm) to highlight different brain areas. Some of the neuropils labeled with ENA are the outer (o me) and inner medulla (i me), lobula (lo) and lobula plate (lo p) within the optic lobe, the protocerebral bridge (pr br) in the central complex as well as other regions in the protocerebrum such as the lateral horn (l ho). Other structures, as the protolateral deutocerebrum (p l deu), superior lateral protocerebrum (s l pr), the peduncles of the mushroom body (pe), pars intercerebralis (pars in), suboesophageal ganglion (su oes g) and oesophagus (oes) are also shown in the figure. (F) ena levels are reduced in enarev mutants compared to the control y w. Images were taken with the same confocal settings for direct comparison; projections of 2.3 µm depth are shown. ENA immunohistochemistry was repeated at least three times. (G) RT-PCR analysis was performed in adult enarev, enarev/+ and control total RNA. The ratio between ena and actin expression levels for each genotype is shown. Quantification of RNA levels showed significant changes in enarev homozygous (* p<0.05) whereas a minor (non significant) decrease was seen in enarev/+ heterozygous when compared to the y w control line. The experiment was repeated three times employing independent RNA preparations.

Mentions: To determine whether ena down-regulation by itself could be responsible for the progressive arrhythmicity two complementary approaches were carried out. To assess whether increasing ena expression within the GAL4-mediated hypomorph is sufficient to rescue wild type behavior, a copy of UAS-ena was introduced in pdf>enarev. Restoring ENA levels reduced the arrhythmicity of aged pdf>enarev which became undistinguishable from control flies (UAS-ena/+, Fig. 4A–B).


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)

Age associated arrhythmicity is dependent on ena function.(A) Rescue of ENA function on the PDF circuit restores behavioral rhythmicity in aged flies. Recombinant pdf-gal4, enarev carrying one copy of UAS-ena were indistinguishable from control UAS-ena. Representative double plotted actograms for aged (24–28d) flies are shown. Overexpression of ENA in young flies do not affect locomotor activity rhythms (data not shown). (B) The percentage of rhythmicity for aged flies for each strain is shown. pdf-gal4, enarev/++ is significantly different from both UAS-ena/+ (control) and pdf-gal4, enarev/UAS-ena (*** p<0.001). (C) Representative actograms of young (3d) and aged (21d) flies carrying one or two copies of enarev, along with the transheterozygotes enarev/enaGC5. Both homozygous enarev and enarev/enaGC5 exhibit a decline on rhythm strength. (D) Summary of behavioral data for flies of the indicated genotypes. Control enarev/+ flies stay rhythmic throughout lifespan. Aged enarev is significantly different from its younger counterpart (* p<0.05). Both aged enarev and enarev/enaGC5 are different from old enarev/+ (* p<0.05). Experiments summarized in B and D were repeated at least 3 times. (E) Whole mount brain immunofluorescense of 10 day-old adult y w flies were stained with a specific antibody against ENA. A general staining of neuropils in the adult brain was observed. Single confocal planes (2 µm thick) are shown at two depths (8 and 22 µm) to highlight different brain areas. Some of the neuropils labeled with ENA are the outer (o me) and inner medulla (i me), lobula (lo) and lobula plate (lo p) within the optic lobe, the protocerebral bridge (pr br) in the central complex as well as other regions in the protocerebrum such as the lateral horn (l ho). Other structures, as the protolateral deutocerebrum (p l deu), superior lateral protocerebrum (s l pr), the peduncles of the mushroom body (pe), pars intercerebralis (pars in), suboesophageal ganglion (su oes g) and oesophagus (oes) are also shown in the figure. (F) ena levels are reduced in enarev mutants compared to the control y w. Images were taken with the same confocal settings for direct comparison; projections of 2.3 µm depth are shown. ENA immunohistochemistry was repeated at least three times. (G) RT-PCR analysis was performed in adult enarev, enarev/+ and control total RNA. The ratio between ena and actin expression levels for each genotype is shown. Quantification of RNA levels showed significant changes in enarev homozygous (* p<0.05) whereas a minor (non significant) decrease was seen in enarev/+ heterozygous when compared to the y w control line. The experiment was repeated three times employing independent RNA preparations.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553195&req=5

pone-0003332-g004: Age associated arrhythmicity is dependent on ena function.(A) Rescue of ENA function on the PDF circuit restores behavioral rhythmicity in aged flies. Recombinant pdf-gal4, enarev carrying one copy of UAS-ena were indistinguishable from control UAS-ena. Representative double plotted actograms for aged (24–28d) flies are shown. Overexpression of ENA in young flies do not affect locomotor activity rhythms (data not shown). (B) The percentage of rhythmicity for aged flies for each strain is shown. pdf-gal4, enarev/++ is significantly different from both UAS-ena/+ (control) and pdf-gal4, enarev/UAS-ena (*** p<0.001). (C) Representative actograms of young (3d) and aged (21d) flies carrying one or two copies of enarev, along with the transheterozygotes enarev/enaGC5. Both homozygous enarev and enarev/enaGC5 exhibit a decline on rhythm strength. (D) Summary of behavioral data for flies of the indicated genotypes. Control enarev/+ flies stay rhythmic throughout lifespan. Aged enarev is significantly different from its younger counterpart (* p<0.05). Both aged enarev and enarev/enaGC5 are different from old enarev/+ (* p<0.05). Experiments summarized in B and D were repeated at least 3 times. (E) Whole mount brain immunofluorescense of 10 day-old adult y w flies were stained with a specific antibody against ENA. A general staining of neuropils in the adult brain was observed. Single confocal planes (2 µm thick) are shown at two depths (8 and 22 µm) to highlight different brain areas. Some of the neuropils labeled with ENA are the outer (o me) and inner medulla (i me), lobula (lo) and lobula plate (lo p) within the optic lobe, the protocerebral bridge (pr br) in the central complex as well as other regions in the protocerebrum such as the lateral horn (l ho). Other structures, as the protolateral deutocerebrum (p l deu), superior lateral protocerebrum (s l pr), the peduncles of the mushroom body (pe), pars intercerebralis (pars in), suboesophageal ganglion (su oes g) and oesophagus (oes) are also shown in the figure. (F) ena levels are reduced in enarev mutants compared to the control y w. Images were taken with the same confocal settings for direct comparison; projections of 2.3 µm depth are shown. ENA immunohistochemistry was repeated at least three times. (G) RT-PCR analysis was performed in adult enarev, enarev/+ and control total RNA. The ratio between ena and actin expression levels for each genotype is shown. Quantification of RNA levels showed significant changes in enarev homozygous (* p<0.05) whereas a minor (non significant) decrease was seen in enarev/+ heterozygous when compared to the y w control line. The experiment was repeated three times employing independent RNA preparations.
Mentions: To determine whether ena down-regulation by itself could be responsible for the progressive arrhythmicity two complementary approaches were carried out. To assess whether increasing ena expression within the GAL4-mediated hypomorph is sufficient to rescue wild type behavior, a copy of UAS-ena was introduced in pdf>enarev. Restoring ENA levels reduced the arrhythmicity of aged pdf>enarev which became undistinguishable from control flies (UAS-ena/+, Fig. 4A–B).

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