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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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Reduced enabled levels triggers axonal swellings.(A) Genetic interactions between enarev and khc6 suggest that axon transport defects could be underlying the progressive loss of rhythmicity. The percentage of rhythmic flies for each strain is shown. Transheterozygote enarev/ khc6 flies are significantly different from its younger counterpart (* p<0,05 ) and from the heterozygote aged controls (** p<0,01 and * p<0,05 when compared to enarev/+ and khc6/+, respectively). Experiments were repeated at least three times. Additional details are included Table S2. (B) Third-instar larval segmental nerves were stained against CSP, a synaptic vesicle protein. (B1) The whole larval preparation is shown. The dashed box corresponds to the region shown in B2–B4. (B2) Segmental nerves from control larvae exhibit relatively uniform CSP staining. (B3–B4) However, large immunoreactive CSP clusters (arrows) are observed in the segmental nerves of the positive control elav>APP (B3) as well as elav>enarev (B4) larvae. (C) Quantitative analysis on larval segmental nerves was performed by measuring clog density. Fourteen to thirty five brains were examined. elav>enarev was significantly different from the wild type control (* p<0.05), similarly to what was seen for elav>APP (*** p<0.001). (D) Representative images of TUNEL staining on the indicated genotypes. (E) Quantitative analysis of TUNEL staining showing the extent of neuronal death in elav>enarev and a positive control, both significantly different from a wild type control (* p<0.05, *** p<0.001).
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pone-0003332-g006: Reduced enabled levels triggers axonal swellings.(A) Genetic interactions between enarev and khc6 suggest that axon transport defects could be underlying the progressive loss of rhythmicity. The percentage of rhythmic flies for each strain is shown. Transheterozygote enarev/ khc6 flies are significantly different from its younger counterpart (* p<0,05 ) and from the heterozygote aged controls (** p<0,01 and * p<0,05 when compared to enarev/+ and khc6/+, respectively). Experiments were repeated at least three times. Additional details are included Table S2. (B) Third-instar larval segmental nerves were stained against CSP, a synaptic vesicle protein. (B1) The whole larval preparation is shown. The dashed box corresponds to the region shown in B2–B4. (B2) Segmental nerves from control larvae exhibit relatively uniform CSP staining. (B3–B4) However, large immunoreactive CSP clusters (arrows) are observed in the segmental nerves of the positive control elav>APP (B3) as well as elav>enarev (B4) larvae. (C) Quantitative analysis on larval segmental nerves was performed by measuring clog density. Fourteen to thirty five brains were examined. elav>enarev was significantly different from the wild type control (* p<0.05), similarly to what was seen for elav>APP (*** p<0.001). (D) Representative images of TUNEL staining on the indicated genotypes. (E) Quantitative analysis of TUNEL staining showing the extent of neuronal death in elav>enarev and a positive control, both significantly different from a wild type control (* p<0.05, *** p<0.001).

Mentions: Fast-axonal transport cargoes, such as vesicle-associated synaptic terminal proteins and mitochondria, can accumulate in axonal swellings derived from mutation of kinesin 1 or dynein [36]–[39]. ENA has been found to directly interact with kinesin heavy chain (Khc), a molecular motor involved in fast axonal transport [40]. To examine the possibility that defective axonal transport could contribute to the progressive behavioral phenotype, genetic interactions between a khc mutant (khc6/+) and heterozygous enarev were examined. Interestingly, transheterozygous enarev/khc6 flies displayed progressive loss of rhythmicity (Fig. 6A), suggesting an impairment at this level. To examine whether ENA down-regulation could give rise to abnormal cargo accumulation, the localization of the synaptic vesicle protein CSP in the larval segmental nerves (Fig. 6B) was examined [25], [41]. Axonal clogs are aggregates of membrane bound cargoes and can be a consequence of defective axonal transport [36]. As a positive control APP was overexpressed (elav>APP), a manipulation that has already been demonstrated to induce axonal clogging [25], [42]. Consistent with this notion, the segmental nerves in elav>APP flies displayed conspicuous clusters of the presynaptic protein CSP (Fig. 6B3), which were absent in wild type controls (Fig. 6B2). Strikingly, reduced ENA levels in elav>enarev also resulted in the development of axonal clogs (Fig. 6B4), suggesting impairment at this level. The density of axonal clogs was then measured; elav>enarev flies were significantly different from wild type controls similarly to what was seen for elav>APP (Fig. 6C). Comparable results were obtained when the localization of the synaptic protein SYT was analyzed (data not shown).


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)

Reduced enabled levels triggers axonal swellings.(A) Genetic interactions between enarev and khc6 suggest that axon transport defects could be underlying the progressive loss of rhythmicity. The percentage of rhythmic flies for each strain is shown. Transheterozygote enarev/ khc6 flies are significantly different from its younger counterpart (* p<0,05 ) and from the heterozygote aged controls (** p<0,01 and * p<0,05 when compared to enarev/+ and khc6/+, respectively). Experiments were repeated at least three times. Additional details are included Table S2. (B) Third-instar larval segmental nerves were stained against CSP, a synaptic vesicle protein. (B1) The whole larval preparation is shown. The dashed box corresponds to the region shown in B2–B4. (B2) Segmental nerves from control larvae exhibit relatively uniform CSP staining. (B3–B4) However, large immunoreactive CSP clusters (arrows) are observed in the segmental nerves of the positive control elav>APP (B3) as well as elav>enarev (B4) larvae. (C) Quantitative analysis on larval segmental nerves was performed by measuring clog density. Fourteen to thirty five brains were examined. elav>enarev was significantly different from the wild type control (* p<0.05), similarly to what was seen for elav>APP (*** p<0.001). (D) Representative images of TUNEL staining on the indicated genotypes. (E) Quantitative analysis of TUNEL staining showing the extent of neuronal death in elav>enarev and a positive control, both significantly different from a wild type control (* p<0.05, *** p<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553195&req=5

pone-0003332-g006: Reduced enabled levels triggers axonal swellings.(A) Genetic interactions between enarev and khc6 suggest that axon transport defects could be underlying the progressive loss of rhythmicity. The percentage of rhythmic flies for each strain is shown. Transheterozygote enarev/ khc6 flies are significantly different from its younger counterpart (* p<0,05 ) and from the heterozygote aged controls (** p<0,01 and * p<0,05 when compared to enarev/+ and khc6/+, respectively). Experiments were repeated at least three times. Additional details are included Table S2. (B) Third-instar larval segmental nerves were stained against CSP, a synaptic vesicle protein. (B1) The whole larval preparation is shown. The dashed box corresponds to the region shown in B2–B4. (B2) Segmental nerves from control larvae exhibit relatively uniform CSP staining. (B3–B4) However, large immunoreactive CSP clusters (arrows) are observed in the segmental nerves of the positive control elav>APP (B3) as well as elav>enarev (B4) larvae. (C) Quantitative analysis on larval segmental nerves was performed by measuring clog density. Fourteen to thirty five brains were examined. elav>enarev was significantly different from the wild type control (* p<0.05), similarly to what was seen for elav>APP (*** p<0.001). (D) Representative images of TUNEL staining on the indicated genotypes. (E) Quantitative analysis of TUNEL staining showing the extent of neuronal death in elav>enarev and a positive control, both significantly different from a wild type control (* p<0.05, *** p<0.001).
Mentions: Fast-axonal transport cargoes, such as vesicle-associated synaptic terminal proteins and mitochondria, can accumulate in axonal swellings derived from mutation of kinesin 1 or dynein [36]–[39]. ENA has been found to directly interact with kinesin heavy chain (Khc), a molecular motor involved in fast axonal transport [40]. To examine the possibility that defective axonal transport could contribute to the progressive behavioral phenotype, genetic interactions between a khc mutant (khc6/+) and heterozygous enarev were examined. Interestingly, transheterozygous enarev/khc6 flies displayed progressive loss of rhythmicity (Fig. 6A), suggesting an impairment at this level. To examine whether ENA down-regulation could give rise to abnormal cargo accumulation, the localization of the synaptic vesicle protein CSP in the larval segmental nerves (Fig. 6B) was examined [25], [41]. Axonal clogs are aggregates of membrane bound cargoes and can be a consequence of defective axonal transport [36]. As a positive control APP was overexpressed (elav>APP), a manipulation that has already been demonstrated to induce axonal clogging [25], [42]. Consistent with this notion, the segmental nerves in elav>APP flies displayed conspicuous clusters of the presynaptic protein CSP (Fig. 6B3), which were absent in wild type controls (Fig. 6B2). Strikingly, reduced ENA levels in elav>enarev also resulted in the development of axonal clogs (Fig. 6B4), suggesting impairment at this level. The density of axonal clogs was then measured; elav>enarev flies were significantly different from wild type controls similarly to what was seen for elav>APP (Fig. 6C). Comparable results were obtained when the localization of the synaptic protein SYT was analyzed (data not shown).

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