Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Decreased ENA levels triggers progressive degeneration in the optic lobe.Frontal adult head semi-thin sections (1 µm thick) were stained with methylene blue and examined by light microscopy. Representative sections are included. Two different drivers were employed in order to reduce ENA levels; elav-gal4, a panneuronal driver, and th-gal4, driving GAL4 expression specifically in the dopaminergic neurons. Young (0–3 days) and old flies (30 days) were analyzed for each genotype. Reduction of ENA levels both panneurally and in the dopaminergic system causes degeneration in the same areas of the brain (indicated by a red asterisc within the affected region). elav>enarev flies show age dependent vacuolization in the medulla and the lamina within the optic lobe while the nervous system of the control line (elav-gal4/+) is well preserved throughout the time evaluated. Reduced ENA levels exclusively in dopaminergic neurons (th>enarev) also leads to vacuolization in the optic lobe in aged flies, although to a lower extent. enarev shows specific degeneration within the optic lobe, characterized by the occurrence of vacuoles in the medulla and lamina mainly in older flies. Four to ten heads were analyzed per genotype in 2–5 different experiments. Different regions in the adult brain are indicated as follows: medulla (me), retina (re), lamina (la), lateral horn (l ho), peduncles of the mushroom body (pe), fan-shaped body (fb), ellipsoid body (eb), calix (ca), lobula (lo), suboesophageal ganglion (su oes g) and oesophagus (oes), giant commissure (gc), nodules (no). (B) Progressive expression of enarev reduces the number of PDF neurons. pdf-gal4>GFP and pdf-gal4>GFP; enarev adult brains were dissected and the number of GFP positive small (s-LNvs) and large (l-LNvs) neurons per hemisphere was counted in young (0–3 day old) and aged (30 day old) individuals. Statistical analysis included pairwise comparisons employing Student t test. The s-LNvs showed a small but significant decrease in the number of PDF positive neurons (* p<0.05). No differences in the large LNv cluster were observed. (C) Vacuolization within the optic lobe progressively impairs a behavioral response in elav>enarev. In a longitudinal assay the phototactic response of flies of the indicated genotypes was examined. The performance in this paradigm decreased as the flies aged for all genotypes. As anticipated from the estructural defect elav>enarev flies performed progressively worse, becoming significantly different from both genetic controls by the end of the experiment (* p<0.05). Homozygous enarev flies did not show any clear response in any of the paradigms tested (see also Fig. S2).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2553195&req=5

pone-0003332-g005: (A) Decreased ENA levels triggers progressive degeneration in the optic lobe.Frontal adult head semi-thin sections (1 µm thick) were stained with methylene blue and examined by light microscopy. Representative sections are included. Two different drivers were employed in order to reduce ENA levels; elav-gal4, a panneuronal driver, and th-gal4, driving GAL4 expression specifically in the dopaminergic neurons. Young (0–3 days) and old flies (30 days) were analyzed for each genotype. Reduction of ENA levels both panneurally and in the dopaminergic system causes degeneration in the same areas of the brain (indicated by a red asterisc within the affected region). elav>enarev flies show age dependent vacuolization in the medulla and the lamina within the optic lobe while the nervous system of the control line (elav-gal4/+) is well preserved throughout the time evaluated. Reduced ENA levels exclusively in dopaminergic neurons (th>enarev) also leads to vacuolization in the optic lobe in aged flies, although to a lower extent. enarev shows specific degeneration within the optic lobe, characterized by the occurrence of vacuoles in the medulla and lamina mainly in older flies. Four to ten heads were analyzed per genotype in 2–5 different experiments. Different regions in the adult brain are indicated as follows: medulla (me), retina (re), lamina (la), lateral horn (l ho), peduncles of the mushroom body (pe), fan-shaped body (fb), ellipsoid body (eb), calix (ca), lobula (lo), suboesophageal ganglion (su oes g) and oesophagus (oes), giant commissure (gc), nodules (no). (B) Progressive expression of enarev reduces the number of PDF neurons. pdf-gal4>GFP and pdf-gal4>GFP; enarev adult brains were dissected and the number of GFP positive small (s-LNvs) and large (l-LNvs) neurons per hemisphere was counted in young (0–3 day old) and aged (30 day old) individuals. Statistical analysis included pairwise comparisons employing Student t test. The s-LNvs showed a small but significant decrease in the number of PDF positive neurons (* p<0.05). No differences in the large LNv cluster were observed. (C) Vacuolization within the optic lobe progressively impairs a behavioral response in elav>enarev. In a longitudinal assay the phototactic response of flies of the indicated genotypes was examined. The performance in this paradigm decreased as the flies aged for all genotypes. As anticipated from the estructural defect elav>enarev flies performed progressively worse, becoming significantly different from both genetic controls by the end of the experiment (* p<0.05). Homozygous enarev flies did not show any clear response in any of the paradigms tested (see also Fig. S2).

Mentions: To address whether down-regulated ENA function could lead to degeneration within the brain we employed the panneuronal driver elav [32]. Semi-thin frontal sections stained with methylene blue were analyzed in young and aged flies. Mutant brains (elav>enarev) from old flies displayed significant cortex and neuropil vacuolization (Fig. 5A). This phenomenon was not apparent in parental strains (elav-gal4/+ and heterozygous enarev) or in young elav>enarev flies, revealing an age dependence of the neuropathological phenotype.


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)

(A) Decreased ENA levels triggers progressive degeneration in the optic lobe.Frontal adult head semi-thin sections (1 µm thick) were stained with methylene blue and examined by light microscopy. Representative sections are included. Two different drivers were employed in order to reduce ENA levels; elav-gal4, a panneuronal driver, and th-gal4, driving GAL4 expression specifically in the dopaminergic neurons. Young (0–3 days) and old flies (30 days) were analyzed for each genotype. Reduction of ENA levels both panneurally and in the dopaminergic system causes degeneration in the same areas of the brain (indicated by a red asterisc within the affected region). elav>enarev flies show age dependent vacuolization in the medulla and the lamina within the optic lobe while the nervous system of the control line (elav-gal4/+) is well preserved throughout the time evaluated. Reduced ENA levels exclusively in dopaminergic neurons (th>enarev) also leads to vacuolization in the optic lobe in aged flies, although to a lower extent. enarev shows specific degeneration within the optic lobe, characterized by the occurrence of vacuoles in the medulla and lamina mainly in older flies. Four to ten heads were analyzed per genotype in 2–5 different experiments. Different regions in the adult brain are indicated as follows: medulla (me), retina (re), lamina (la), lateral horn (l ho), peduncles of the mushroom body (pe), fan-shaped body (fb), ellipsoid body (eb), calix (ca), lobula (lo), suboesophageal ganglion (su oes g) and oesophagus (oes), giant commissure (gc), nodules (no). (B) Progressive expression of enarev reduces the number of PDF neurons. pdf-gal4>GFP and pdf-gal4>GFP; enarev adult brains were dissected and the number of GFP positive small (s-LNvs) and large (l-LNvs) neurons per hemisphere was counted in young (0–3 day old) and aged (30 day old) individuals. Statistical analysis included pairwise comparisons employing Student t test. The s-LNvs showed a small but significant decrease in the number of PDF positive neurons (* p<0.05). No differences in the large LNv cluster were observed. (C) Vacuolization within the optic lobe progressively impairs a behavioral response in elav>enarev. In a longitudinal assay the phototactic response of flies of the indicated genotypes was examined. The performance in this paradigm decreased as the flies aged for all genotypes. As anticipated from the estructural defect elav>enarev flies performed progressively worse, becoming significantly different from both genetic controls by the end of the experiment (* p<0.05). Homozygous enarev flies did not show any clear response in any of the paradigms tested (see also Fig. S2).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003332-g005: (A) Decreased ENA levels triggers progressive degeneration in the optic lobe.Frontal adult head semi-thin sections (1 µm thick) were stained with methylene blue and examined by light microscopy. Representative sections are included. Two different drivers were employed in order to reduce ENA levels; elav-gal4, a panneuronal driver, and th-gal4, driving GAL4 expression specifically in the dopaminergic neurons. Young (0–3 days) and old flies (30 days) were analyzed for each genotype. Reduction of ENA levels both panneurally and in the dopaminergic system causes degeneration in the same areas of the brain (indicated by a red asterisc within the affected region). elav>enarev flies show age dependent vacuolization in the medulla and the lamina within the optic lobe while the nervous system of the control line (elav-gal4/+) is well preserved throughout the time evaluated. Reduced ENA levels exclusively in dopaminergic neurons (th>enarev) also leads to vacuolization in the optic lobe in aged flies, although to a lower extent. enarev shows specific degeneration within the optic lobe, characterized by the occurrence of vacuoles in the medulla and lamina mainly in older flies. Four to ten heads were analyzed per genotype in 2–5 different experiments. Different regions in the adult brain are indicated as follows: medulla (me), retina (re), lamina (la), lateral horn (l ho), peduncles of the mushroom body (pe), fan-shaped body (fb), ellipsoid body (eb), calix (ca), lobula (lo), suboesophageal ganglion (su oes g) and oesophagus (oes), giant commissure (gc), nodules (no). (B) Progressive expression of enarev reduces the number of PDF neurons. pdf-gal4>GFP and pdf-gal4>GFP; enarev adult brains were dissected and the number of GFP positive small (s-LNvs) and large (l-LNvs) neurons per hemisphere was counted in young (0–3 day old) and aged (30 day old) individuals. Statistical analysis included pairwise comparisons employing Student t test. The s-LNvs showed a small but significant decrease in the number of PDF positive neurons (* p<0.05). No differences in the large LNv cluster were observed. (C) Vacuolization within the optic lobe progressively impairs a behavioral response in elav>enarev. In a longitudinal assay the phototactic response of flies of the indicated genotypes was examined. The performance in this paradigm decreased as the flies aged for all genotypes. As anticipated from the estructural defect elav>enarev flies performed progressively worse, becoming significantly different from both genetic controls by the end of the experiment (* p<0.05). Homozygous enarev flies did not show any clear response in any of the paradigms tested (see also Fig. S2).
Mentions: To address whether down-regulated ENA function could lead to degeneration within the brain we employed the panneuronal driver elav [32]. Semi-thin frontal sections stained with methylene blue were analyzed in young and aged flies. Mutant brains (elav>enarev) from old flies displayed significant cortex and neuropil vacuolization (Fig. 5A). This phenomenon was not apparent in parental strains (elav-gal4/+ and heterozygous enarev) or in young elav>enarev flies, revealing an age dependence of the neuropathological phenotype.

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