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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.

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The locomotive activity and NMJ staining of larvae with dTDP knockdown in the pan-neurons.(A) Lower locomotive activity of larvae (elav>38377) with knockdown of dTDP than the control (elav>+) (N = 15, *, p<0.05). (B) Quantitative comparison of NMJ bouton numbers of elav>+ and elav>38377 larvae after normalization to the total muscle 6/7 areas. Note the higher bouton density of the knockdown larvae (elav>38377) than the control (elav>+). *, p<0.05. The averages of the total bouton numbers are: elav>+, 95±14; elav>38377, 117±14. The means of the muscle areas are: elav>+, 69,937 µm2; elav>38377, 65,166 µm2. (N = 12 in all cases). (C) Whole-mount immunostaining analysis of the larval ventral nerve cords with dTDP-knockdown in the pan-neurons. (a), control flies elav>+; (b) elav>38377. Red, dTDP; blue, DAPI; green, mCD8::GFP. Note that the pan-neuron-driven dsRNA did not completely knock down the dTDP expression since there were still some dTDP signals in the larval ventral nerve cords of elav>38377.
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pone-0020371-g004: The locomotive activity and NMJ staining of larvae with dTDP knockdown in the pan-neurons.(A) Lower locomotive activity of larvae (elav>38377) with knockdown of dTDP than the control (elav>+) (N = 15, *, p<0.05). (B) Quantitative comparison of NMJ bouton numbers of elav>+ and elav>38377 larvae after normalization to the total muscle 6/7 areas. Note the higher bouton density of the knockdown larvae (elav>38377) than the control (elav>+). *, p<0.05. The averages of the total bouton numbers are: elav>+, 95±14; elav>38377, 117±14. The means of the muscle areas are: elav>+, 69,937 µm2; elav>38377, 65,166 µm2. (N = 12 in all cases). (C) Whole-mount immunostaining analysis of the larval ventral nerve cords with dTDP-knockdown in the pan-neurons. (a), control flies elav>+; (b) elav>38377. Red, dTDP; blue, DAPI; green, mCD8::GFP. Note that the pan-neuron-driven dsRNA did not completely knock down the dTDP expression since there were still some dTDP signals in the larval ventral nerve cords of elav>38377.

Mentions: In view of the loss-of-function phenotype of the locomotive ability of flies with whole-body knockout of dTDP expression (Fig. 1; [23]) and the disease phenotypes caused by overexpression of human hTDP-43 in Drosophila [19], we have tested whether the pathway(s) leading to ALS disease pathology was conserved in the fruit flies by knockdown or overexpression of dTDP in the motor neurons of Drosophila. We first used the motor neuron-specific driver (D42-GAL4) to knockdown the endogenous dTDP in the motor neurons. However, the resulting flies did not exhibit locomotion defect, possibly due to inefficient knockdown of the dTDP. We then used the pan-neuron driver elav-GAL4 to knockdown dTDP. As shown in Fig. 4A, the moving abilities of larvae from elav>38377 were lower than the control larvae (elav>+). Furthermore, similar to the dTDP- fly line dTDPex26 described in Fig. 1, the NMJ boutons of elav>38377 larvae were also higher than the control larvae (Fig. 4B). Although these differences between the control (elav>+) and the dTDP-knockdown larvae (elav>38377) were significant (p<0.05), it was less than those between the control (yw) and the dTDP- mutant dTDPex26 as shown in Fig. 1C and 1D. This might result from the inefficient knock-down of dTDP in the pan-neurons. Indeed, unlike in dTDPex26 (Fig. 1B), some dTDP signals were present in the ventral nerve cords of the elav>38377 flies (Fig. 4C). Finally, we also knocked down dTDP expression in the muscle areas as described in the Materials and Methods, but this step did not cause any change in either the bouton numbers or the larval movement (data not shown).


Neuronal function and dysfunction of Drosophila dTDP.

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

The locomotive activity and NMJ staining of larvae with dTDP knockdown in the pan-neurons.(A) Lower locomotive activity of larvae (elav>38377) with knockdown of dTDP than the control (elav>+) (N = 15, *, p<0.05). (B) Quantitative comparison of NMJ bouton numbers of elav>+ and elav>38377 larvae after normalization to the total muscle 6/7 areas. Note the higher bouton density of the knockdown larvae (elav>38377) than the control (elav>+). *, p<0.05. The averages of the total bouton numbers are: elav>+, 95±14; elav>38377, 117±14. The means of the muscle areas are: elav>+, 69,937 µm2; elav>38377, 65,166 µm2. (N = 12 in all cases). (C) Whole-mount immunostaining analysis of the larval ventral nerve cords with dTDP-knockdown in the pan-neurons. (a), control flies elav>+; (b) elav>38377. Red, dTDP; blue, DAPI; green, mCD8::GFP. Note that the pan-neuron-driven dsRNA did not completely knock down the dTDP expression since there were still some dTDP signals in the larval ventral nerve cords of elav>38377.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105987&req=5

pone-0020371-g004: The locomotive activity and NMJ staining of larvae with dTDP knockdown in the pan-neurons.(A) Lower locomotive activity of larvae (elav>38377) with knockdown of dTDP than the control (elav>+) (N = 15, *, p<0.05). (B) Quantitative comparison of NMJ bouton numbers of elav>+ and elav>38377 larvae after normalization to the total muscle 6/7 areas. Note the higher bouton density of the knockdown larvae (elav>38377) than the control (elav>+). *, p<0.05. The averages of the total bouton numbers are: elav>+, 95±14; elav>38377, 117±14. The means of the muscle areas are: elav>+, 69,937 µm2; elav>38377, 65,166 µm2. (N = 12 in all cases). (C) Whole-mount immunostaining analysis of the larval ventral nerve cords with dTDP-knockdown in the pan-neurons. (a), control flies elav>+; (b) elav>38377. Red, dTDP; blue, DAPI; green, mCD8::GFP. Note that the pan-neuron-driven dsRNA did not completely knock down the dTDP expression since there were still some dTDP signals in the larval ventral nerve cords of elav>38377.
Mentions: In view of the loss-of-function phenotype of the locomotive ability of flies with whole-body knockout of dTDP expression (Fig. 1; [23]) and the disease phenotypes caused by overexpression of human hTDP-43 in Drosophila [19], we have tested whether the pathway(s) leading to ALS disease pathology was conserved in the fruit flies by knockdown or overexpression of dTDP in the motor neurons of Drosophila. We first used the motor neuron-specific driver (D42-GAL4) to knockdown the endogenous dTDP in the motor neurons. However, the resulting flies did not exhibit locomotion defect, possibly due to inefficient knockdown of the dTDP. We then used the pan-neuron driver elav-GAL4 to knockdown dTDP. As shown in Fig. 4A, the moving abilities of larvae from elav>38377 were lower than the control larvae (elav>+). Furthermore, similar to the dTDP- fly line dTDPex26 described in Fig. 1, the NMJ boutons of elav>38377 larvae were also higher than the control larvae (Fig. 4B). Although these differences between the control (elav>+) and the dTDP-knockdown larvae (elav>38377) were significant (p<0.05), it was less than those between the control (yw) and the dTDP- mutant dTDPex26 as shown in Fig. 1C and 1D. This might result from the inefficient knock-down of dTDP in the pan-neurons. Indeed, unlike in dTDPex26 (Fig. 1B), some dTDP signals were present in the ventral nerve cords of the elav>38377 flies (Fig. 4C). Finally, we also knocked down dTDP expression in the muscle areas as described in the Materials and Methods, but this step did not cause any change in either the bouton numbers or the larval movement (data not shown).

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