Limits...
Neuronal function and dysfunction of Drosophila dTDP.

Lin MJ, Cheng CW, Shen CK - PLoS ONE (2011)

Bottom Line: In contrast, overexpression of dTDP in the motor neurons also resulted in reduced larval and adult locomotor activities, but this was accompanied by a decrease of the number of boutons and axon branches at NMJ.On the other hand, constitutive mushroom body-specific knockdown of dTDP expression did not affect the structure of the mushroom bodies, but it impaired the learning ability of the flies, albeit moderately.The effects of mis-expression of dTDP on Drosophila NMJ suggest that eukaryotic TDP-43 guards against over development of the synapses.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.

ABSTRACT

Background: TDP-43 is an RNA- and DNA-binding protein well conserved in animals including the mammals, Drosophila, and C. elegans. In mammals, the multi-function TDP-43 encoded by the TARDBP gene is a signature protein of the ubiquitin-positive inclusions (UBIs) in the diseased neuronal/glial cells of a range of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U).

Methodology/principal findings: We have studied the function and dysfunction of the Drosophila ortholog of the mammalian TARDBP gene, dTDP, by genetic, behavioral, molecular, and cytological analyses. It was found that depletion of dTDP expression caused locomotion defect accompanied with an increase of the number of boutons at the neuromuscular junctions (NMJ). These phenotypes could be rescued by overexpression of Drosophila dTDP in the motor neurons. In contrast, overexpression of dTDP in the motor neurons also resulted in reduced larval and adult locomotor activities, but this was accompanied by a decrease of the number of boutons and axon branches at NMJ. Significantly, constitutive overexpression of dTDP in the mushroom bodies caused smaller axonal lobes as well as severe learning deficiency. On the other hand, constitutive mushroom body-specific knockdown of dTDP expression did not affect the structure of the mushroom bodies, but it impaired the learning ability of the flies, albeit moderately. Overexpression of dTDP also led to the formation of cytosolic dTDP (+) aggregates.

Conclusion/significance: These data together demonstrate the neuronal functions of dTDP, and by implication the mammalian TDP-43, in learning and locomotion. The effects of mis-expression of dTDP on Drosophila NMJ suggest that eukaryotic TDP-43 guards against over development of the synapses. The conservation of the regulatory pathways of functions and dysfunctions of Drosophila dTDP and mammalian TDP-43 also shows the feasibility of using the flies as a model system for studying the normal TDP-43 function and TDP-43 proteinopathies in the vertebrates including human.

Show MeSH

Related in: MedlinePlus

Pathologies of flies with motor neuron-specific overexpression of dTDP.(A) Whole-mount immunostaining analysis of the larval ventral nerve cords. (a), control flies D42>+; (b) D42>dTDP#18-1; (c) D42>dTDP#5-1; (d) magnified pictures of the boxed area in (c). Red, dTDP; blue, DAPI; green, mCD8::GFP. Note the predominant nuclear localization of dTDP in (a). (b), in the low-level dTDP-overexpressing flies (D42>dTDP#18-1), dTDP was distributed in both the nucleus and cytosol. (c) and (d), in the high-level dTDP-overexpressing flies (D42>dTDP#5-1), dTDP became predominantly localized in the cytosol (arrows in d) and often formed dTDP (+) aggregates (arrowheads in d). (B) Histograph of the densities of the bouton numbers. NMJ staining of the third instar larvae was carried out for D42>+, D42>dTDP#5-1, and D42>dTDP#18-1, and the densities of the bouton numbers were counted and compared. Note that a decrease of the bouton number was observed only in the high-level dTDP-overexpressing fly line (D42>dTDP#5-1), **, p<0.001. The averages of the total bouton numbers are: D42>+, 80±4 (N = 20); D42>dTDP#5-1, 46±15 (N = 12); D42>dTDP#18-1, 74±17 (N = 20). The means of the muscle areas are: D42>+, 75,925 µm2; D42>dTDP#5-1, 56,177 µm2; D42>dTDP#18-1, 71,189 µm2. (C) Larvae movement assay. Parallel to data of (B), only the larvae with high dTDP overexpression (D42>dTDP#5-1) showed severe movement defect. (D) and (E) Pathogenesis of adult flies with motor neuron-specific overexpression of dTDP. (D) Movement defect of fly line D42>dTDP#18-1. Phototaxis was performed at three different ages of the adult flies and the movement indexes were normalized to that of the control line D42>+. Note the age-dependent declines of the performance of the dTDP-overexpressing flies. N = 5, **, p<0.001. (E) Whole-mount immunostaining of the thoracic ganglia of day-9 adult flies. Red, dTDP; blue, DAPI; green, mCD8::GFP. (a) Control flies (D42>+). Note the predominant nuclear localization of dTDP. (b) D42>dTDP#18-1 flies. Two regions under the confocal microscope are shown. Note the presence of cells with mainly cytoplasmic distribution of dTDP, as exemplified by cells pointed with the arrows. The dTDP (+) aggregates in the cytoplasm are exemplified by the arrowheads. (F) Western blotting analysis of soluble and insoluble proteins in the heads and thoraces of 13-day old D42>dTDP#18-1 flies with the motor neuron-specific overexpression of dTDP. Different fractions of the protein extracts were isolated from the heads and thoraces as described in the Materials and Methods, and analyzed by Western blotting with use of anti-dTDP and anti-tubulin. T, total protein; R, RIPA-soluble fraction; U, urea-soluble fraction. Note the presence of dTDP in the urea-soluble fraction from D42>dTDP#18-1 but not D42>+.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105987&req=5

pone-0020371-g005: Pathologies of flies with motor neuron-specific overexpression of dTDP.(A) Whole-mount immunostaining analysis of the larval ventral nerve cords. (a), control flies D42>+; (b) D42>dTDP#18-1; (c) D42>dTDP#5-1; (d) magnified pictures of the boxed area in (c). Red, dTDP; blue, DAPI; green, mCD8::GFP. Note the predominant nuclear localization of dTDP in (a). (b), in the low-level dTDP-overexpressing flies (D42>dTDP#18-1), dTDP was distributed in both the nucleus and cytosol. (c) and (d), in the high-level dTDP-overexpressing flies (D42>dTDP#5-1), dTDP became predominantly localized in the cytosol (arrows in d) and often formed dTDP (+) aggregates (arrowheads in d). (B) Histograph of the densities of the bouton numbers. NMJ staining of the third instar larvae was carried out for D42>+, D42>dTDP#5-1, and D42>dTDP#18-1, and the densities of the bouton numbers were counted and compared. Note that a decrease of the bouton number was observed only in the high-level dTDP-overexpressing fly line (D42>dTDP#5-1), **, p<0.001. The averages of the total bouton numbers are: D42>+, 80±4 (N = 20); D42>dTDP#5-1, 46±15 (N = 12); D42>dTDP#18-1, 74±17 (N = 20). The means of the muscle areas are: D42>+, 75,925 µm2; D42>dTDP#5-1, 56,177 µm2; D42>dTDP#18-1, 71,189 µm2. (C) Larvae movement assay. Parallel to data of (B), only the larvae with high dTDP overexpression (D42>dTDP#5-1) showed severe movement defect. (D) and (E) Pathogenesis of adult flies with motor neuron-specific overexpression of dTDP. (D) Movement defect of fly line D42>dTDP#18-1. Phototaxis was performed at three different ages of the adult flies and the movement indexes were normalized to that of the control line D42>+. Note the age-dependent declines of the performance of the dTDP-overexpressing flies. N = 5, **, p<0.001. (E) Whole-mount immunostaining of the thoracic ganglia of day-9 adult flies. Red, dTDP; blue, DAPI; green, mCD8::GFP. (a) Control flies (D42>+). Note the predominant nuclear localization of dTDP. (b) D42>dTDP#18-1 flies. Two regions under the confocal microscope are shown. Note the presence of cells with mainly cytoplasmic distribution of dTDP, as exemplified by cells pointed with the arrows. The dTDP (+) aggregates in the cytoplasm are exemplified by the arrowheads. (F) Western blotting analysis of soluble and insoluble proteins in the heads and thoraces of 13-day old D42>dTDP#18-1 flies with the motor neuron-specific overexpression of dTDP. Different fractions of the protein extracts were isolated from the heads and thoraces as described in the Materials and Methods, and analyzed by Western blotting with use of anti-dTDP and anti-tubulin. T, total protein; R, RIPA-soluble fraction; U, urea-soluble fraction. Note the presence of dTDP in the urea-soluble fraction from D42>dTDP#18-1 but not D42>+.

Mentions: Two independent lines (D42>dTDP#5-1 and D42>dTDP#18-1) with dTDP overexpression in the motor neurons were also generated with use of the GAL4-UAS system. Immunostaining was then used to examine the levels and distribution patterns of dTDP in the larval ventral nerve cords of these two mutant lines in comparison to the D42>+ control flies. As seen in Fig. 5A-a, the endogenous dTDP of the wild type was of relatively low level and mostly in the nucleus. On the other hand, both overexpressing lines had elevated levels of dTDP than the control (compared b and c to a of Fig. 5A). Furthermore, in the two dTDP-overexpressing lines, the dTDP molecules appeared to be translocated from nucleus to the cytoplasm (Fig. 5A-b and c) in a dTDP dose-dependent manner. In D42>dTDP#18-1, the dTDP signals were present in both the nucleus and cytoplasm (Fig. 5A-b); in line D42>dTDP#5-1 which had a higher level of dTDP overexpression in the motor neurons (Fig. 5A-c), the dTDP signals were mostly detected in the cytoplasm (arrows, Fig. 5A-d) and some cells even contained dTDP (+) aggregates (arrowheads, Fig. 5A-d). Interestingly, the survivals of these flies were also dTDP dose-dependent, with all of the flies of D42>dTDP#5-1 dying at early pupal stage while the average life span of the D42>dTDP#18-1 flies being 18 days.


Neuronal function and dysfunction of Drosophila dTDP.

Lin MJ, Cheng CW, Shen CK - PLoS ONE (2011)

Pathologies of flies with motor neuron-specific overexpression of dTDP.(A) Whole-mount immunostaining analysis of the larval ventral nerve cords. (a), control flies D42>+; (b) D42>dTDP#18-1; (c) D42>dTDP#5-1; (d) magnified pictures of the boxed area in (c). Red, dTDP; blue, DAPI; green, mCD8::GFP. Note the predominant nuclear localization of dTDP in (a). (b), in the low-level dTDP-overexpressing flies (D42>dTDP#18-1), dTDP was distributed in both the nucleus and cytosol. (c) and (d), in the high-level dTDP-overexpressing flies (D42>dTDP#5-1), dTDP became predominantly localized in the cytosol (arrows in d) and often formed dTDP (+) aggregates (arrowheads in d). (B) Histograph of the densities of the bouton numbers. NMJ staining of the third instar larvae was carried out for D42>+, D42>dTDP#5-1, and D42>dTDP#18-1, and the densities of the bouton numbers were counted and compared. Note that a decrease of the bouton number was observed only in the high-level dTDP-overexpressing fly line (D42>dTDP#5-1), **, p<0.001. The averages of the total bouton numbers are: D42>+, 80±4 (N = 20); D42>dTDP#5-1, 46±15 (N = 12); D42>dTDP#18-1, 74±17 (N = 20). The means of the muscle areas are: D42>+, 75,925 µm2; D42>dTDP#5-1, 56,177 µm2; D42>dTDP#18-1, 71,189 µm2. (C) Larvae movement assay. Parallel to data of (B), only the larvae with high dTDP overexpression (D42>dTDP#5-1) showed severe movement defect. (D) and (E) Pathogenesis of adult flies with motor neuron-specific overexpression of dTDP. (D) Movement defect of fly line D42>dTDP#18-1. Phototaxis was performed at three different ages of the adult flies and the movement indexes were normalized to that of the control line D42>+. Note the age-dependent declines of the performance of the dTDP-overexpressing flies. N = 5, **, p<0.001. (E) Whole-mount immunostaining of the thoracic ganglia of day-9 adult flies. Red, dTDP; blue, DAPI; green, mCD8::GFP. (a) Control flies (D42>+). Note the predominant nuclear localization of dTDP. (b) D42>dTDP#18-1 flies. Two regions under the confocal microscope are shown. Note the presence of cells with mainly cytoplasmic distribution of dTDP, as exemplified by cells pointed with the arrows. The dTDP (+) aggregates in the cytoplasm are exemplified by the arrowheads. (F) Western blotting analysis of soluble and insoluble proteins in the heads and thoraces of 13-day old D42>dTDP#18-1 flies with the motor neuron-specific overexpression of dTDP. Different fractions of the protein extracts were isolated from the heads and thoraces as described in the Materials and Methods, and analyzed by Western blotting with use of anti-dTDP and anti-tubulin. T, total protein; R, RIPA-soluble fraction; U, urea-soluble fraction. Note the presence of dTDP in the urea-soluble fraction from D42>dTDP#18-1 but not D42>+.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105987&req=5

pone-0020371-g005: Pathologies of flies with motor neuron-specific overexpression of dTDP.(A) Whole-mount immunostaining analysis of the larval ventral nerve cords. (a), control flies D42>+; (b) D42>dTDP#18-1; (c) D42>dTDP#5-1; (d) magnified pictures of the boxed area in (c). Red, dTDP; blue, DAPI; green, mCD8::GFP. Note the predominant nuclear localization of dTDP in (a). (b), in the low-level dTDP-overexpressing flies (D42>dTDP#18-1), dTDP was distributed in both the nucleus and cytosol. (c) and (d), in the high-level dTDP-overexpressing flies (D42>dTDP#5-1), dTDP became predominantly localized in the cytosol (arrows in d) and often formed dTDP (+) aggregates (arrowheads in d). (B) Histograph of the densities of the bouton numbers. NMJ staining of the third instar larvae was carried out for D42>+, D42>dTDP#5-1, and D42>dTDP#18-1, and the densities of the bouton numbers were counted and compared. Note that a decrease of the bouton number was observed only in the high-level dTDP-overexpressing fly line (D42>dTDP#5-1), **, p<0.001. The averages of the total bouton numbers are: D42>+, 80±4 (N = 20); D42>dTDP#5-1, 46±15 (N = 12); D42>dTDP#18-1, 74±17 (N = 20). The means of the muscle areas are: D42>+, 75,925 µm2; D42>dTDP#5-1, 56,177 µm2; D42>dTDP#18-1, 71,189 µm2. (C) Larvae movement assay. Parallel to data of (B), only the larvae with high dTDP overexpression (D42>dTDP#5-1) showed severe movement defect. (D) and (E) Pathogenesis of adult flies with motor neuron-specific overexpression of dTDP. (D) Movement defect of fly line D42>dTDP#18-1. Phototaxis was performed at three different ages of the adult flies and the movement indexes were normalized to that of the control line D42>+. Note the age-dependent declines of the performance of the dTDP-overexpressing flies. N = 5, **, p<0.001. (E) Whole-mount immunostaining of the thoracic ganglia of day-9 adult flies. Red, dTDP; blue, DAPI; green, mCD8::GFP. (a) Control flies (D42>+). Note the predominant nuclear localization of dTDP. (b) D42>dTDP#18-1 flies. Two regions under the confocal microscope are shown. Note the presence of cells with mainly cytoplasmic distribution of dTDP, as exemplified by cells pointed with the arrows. The dTDP (+) aggregates in the cytoplasm are exemplified by the arrowheads. (F) Western blotting analysis of soluble and insoluble proteins in the heads and thoraces of 13-day old D42>dTDP#18-1 flies with the motor neuron-specific overexpression of dTDP. Different fractions of the protein extracts were isolated from the heads and thoraces as described in the Materials and Methods, and analyzed by Western blotting with use of anti-dTDP and anti-tubulin. T, total protein; R, RIPA-soluble fraction; U, urea-soluble fraction. Note the presence of dTDP in the urea-soluble fraction from D42>dTDP#18-1 but not D42>+.
Mentions: Two independent lines (D42>dTDP#5-1 and D42>dTDP#18-1) with dTDP overexpression in the motor neurons were also generated with use of the GAL4-UAS system. Immunostaining was then used to examine the levels and distribution patterns of dTDP in the larval ventral nerve cords of these two mutant lines in comparison to the D42>+ control flies. As seen in Fig. 5A-a, the endogenous dTDP of the wild type was of relatively low level and mostly in the nucleus. On the other hand, both overexpressing lines had elevated levels of dTDP than the control (compared b and c to a of Fig. 5A). Furthermore, in the two dTDP-overexpressing lines, the dTDP molecules appeared to be translocated from nucleus to the cytoplasm (Fig. 5A-b and c) in a dTDP dose-dependent manner. In D42>dTDP#18-1, the dTDP signals were present in both the nucleus and cytoplasm (Fig. 5A-b); in line D42>dTDP#5-1 which had a higher level of dTDP overexpression in the motor neurons (Fig. 5A-c), the dTDP signals were mostly detected in the cytoplasm (arrows, Fig. 5A-d) and some cells even contained dTDP (+) aggregates (arrowheads, Fig. 5A-d). Interestingly, the survivals of these flies were also dTDP dose-dependent, with all of the flies of D42>dTDP#5-1 dying at early pupal stage while the average life span of the D42>dTDP#18-1 flies being 18 days.

Bottom Line: In contrast, overexpression of dTDP in the motor neurons also resulted in reduced larval and adult locomotor activities, but this was accompanied by a decrease of the number of boutons and axon branches at NMJ.On the other hand, constitutive mushroom body-specific knockdown of dTDP expression did not affect the structure of the mushroom bodies, but it impaired the learning ability of the flies, albeit moderately.The effects of mis-expression of dTDP on Drosophila NMJ suggest that eukaryotic TDP-43 guards against over development of the synapses.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.

ABSTRACT

Background: TDP-43 is an RNA- and DNA-binding protein well conserved in animals including the mammals, Drosophila, and C. elegans. In mammals, the multi-function TDP-43 encoded by the TARDBP gene is a signature protein of the ubiquitin-positive inclusions (UBIs) in the diseased neuronal/glial cells of a range of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U).

Methodology/principal findings: We have studied the function and dysfunction of the Drosophila ortholog of the mammalian TARDBP gene, dTDP, by genetic, behavioral, molecular, and cytological analyses. It was found that depletion of dTDP expression caused locomotion defect accompanied with an increase of the number of boutons at the neuromuscular junctions (NMJ). These phenotypes could be rescued by overexpression of Drosophila dTDP in the motor neurons. In contrast, overexpression of dTDP in the motor neurons also resulted in reduced larval and adult locomotor activities, but this was accompanied by a decrease of the number of boutons and axon branches at NMJ. Significantly, constitutive overexpression of dTDP in the mushroom bodies caused smaller axonal lobes as well as severe learning deficiency. On the other hand, constitutive mushroom body-specific knockdown of dTDP expression did not affect the structure of the mushroom bodies, but it impaired the learning ability of the flies, albeit moderately. Overexpression of dTDP also led to the formation of cytosolic dTDP (+) aggregates.

Conclusion/significance: These data together demonstrate the neuronal functions of dTDP, and by implication the mammalian TDP-43, in learning and locomotion. The effects of mis-expression of dTDP on Drosophila NMJ suggest that eukaryotic TDP-43 guards against over development of the synapses. The conservation of the regulatory pathways of functions and dysfunctions of Drosophila dTDP and mammalian TDP-43 also shows the feasibility of using the flies as a model system for studying the normal TDP-43 function and TDP-43 proteinopathies in the vertebrates including human.

Show MeSH
Related in: MedlinePlus