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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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dTDP in the adult brains of 3-day old wild type and mutant Drosophila.(A) Distribution of the endogenous dTDP proteins in the mushroom bodies of the OK107>+ flies as analyzed by immunostaining with anti-dTDP (red), DAPI (blue), and mCD8::GFP (green). (a) a low-magnification photo; (b), higher magnification photo of the boxed area in (a); (c), higher magnification photo of the boxed area in (b). Note the predominant nuclear localization of dTDP. (B) Western blot of the total head protein extracts from the adult flies. The blot was probed with anti-dTDP and anti-tubulin. Note the decreases, by approximately 46% and 23%, of the dTDP levels in the heads of OK107>38377 (lane 2) and OK107>38379 (lane 3), respectively, with mushroom body-specific dsRNA-knockdown of dTDP. Also, dTDP levels in the dTDP-overexpressing lines OK107>dTDP#5-1 (lane 4) and OK107>dTDP#18-1 (lane 5) were 7- and 3 fold, respectively, higher than the OK107>+ control (lane 1). (C) GFP-lighted morphologies of the mushroom bodies in OK107>+ (a), OK107>38377 (b), OK107>dTDP#5-1 (c), and OK107>dTDP#18-1 (d). The α, α′, β, β′, and γ lobes were indicated in (a). Note the smaller lobes in (c) and (d). (D) Immunostaining of dTDP in the brains of OK107>38377 flies with mushroom body-specific knockdown of dTDP expression. Note the significantly reduced signals of dTDP in the mushroom bodies when compared to the yw control in Fig. 2A-a. (E) Subcellular distribution of dTDP in the mushroom bodies of the dTDP-overexpressing line OK107>dTDP#5-1. (a) a set of low magnification pictures; (b) and (c), high magnification pictures of the immunostaining patterns of the OK107>dTDP#5-1 mushroom body, with (b) from the boxed area in (a) and (c) from the boxed area in (b). Note the presence of cells with mainly cytoplasmic dTDP (the arrow in b) and cells with cytoplasmic dTDP (+) aggregates (the arrowheads). (F) Western blotting analysis of soluble and insoluble proteins in the heads of 3-day old dTDP-overexpressing flies. Different fractions of protein extracts were isolated from 3 days-old fly heads as described in Materials and Methods, and analyzed by Western blotting with use of anti-dTDP and anti-tubulin. Note the presence of high molecular weight dTDP species in the urea-soluble fraction of OK107>dTDP#5-1. T, total protein; R, RIPA-soluble fraction; U, urea-soluble fraction.
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pone-0020371-g002: dTDP in the adult brains of 3-day old wild type and mutant Drosophila.(A) Distribution of the endogenous dTDP proteins in the mushroom bodies of the OK107>+ flies as analyzed by immunostaining with anti-dTDP (red), DAPI (blue), and mCD8::GFP (green). (a) a low-magnification photo; (b), higher magnification photo of the boxed area in (a); (c), higher magnification photo of the boxed area in (b). Note the predominant nuclear localization of dTDP. (B) Western blot of the total head protein extracts from the adult flies. The blot was probed with anti-dTDP and anti-tubulin. Note the decreases, by approximately 46% and 23%, of the dTDP levels in the heads of OK107>38377 (lane 2) and OK107>38379 (lane 3), respectively, with mushroom body-specific dsRNA-knockdown of dTDP. Also, dTDP levels in the dTDP-overexpressing lines OK107>dTDP#5-1 (lane 4) and OK107>dTDP#18-1 (lane 5) were 7- and 3 fold, respectively, higher than the OK107>+ control (lane 1). (C) GFP-lighted morphologies of the mushroom bodies in OK107>+ (a), OK107>38377 (b), OK107>dTDP#5-1 (c), and OK107>dTDP#18-1 (d). The α, α′, β, β′, and γ lobes were indicated in (a). Note the smaller lobes in (c) and (d). (D) Immunostaining of dTDP in the brains of OK107>38377 flies with mushroom body-specific knockdown of dTDP expression. Note the significantly reduced signals of dTDP in the mushroom bodies when compared to the yw control in Fig. 2A-a. (E) Subcellular distribution of dTDP in the mushroom bodies of the dTDP-overexpressing line OK107>dTDP#5-1. (a) a set of low magnification pictures; (b) and (c), high magnification pictures of the immunostaining patterns of the OK107>dTDP#5-1 mushroom body, with (b) from the boxed area in (a) and (c) from the boxed area in (b). Note the presence of cells with mainly cytoplasmic dTDP (the arrow in b) and cells with cytoplasmic dTDP (+) aggregates (the arrowheads). (F) Western blotting analysis of soluble and insoluble proteins in the heads of 3-day old dTDP-overexpressing flies. Different fractions of protein extracts were isolated from 3 days-old fly heads as described in Materials and Methods, and analyzed by Western blotting with use of anti-dTDP and anti-tubulin. Note the presence of high molecular weight dTDP species in the urea-soluble fraction of OK107>dTDP#5-1. T, total protein; R, RIPA-soluble fraction; U, urea-soluble fraction.

Mentions: Since human TDP-43 was the major component of the UBIs found in the diseased cells of the central nervous system (CNS) of the ALS and the FTLD-U patients [11], we investigated whether dTDP played a role in the functioning of the Drosophila CNS, in particular the mushroom body supporting the olfactory learning of the fruit flies [25], [26]. With use of a home-made anti-dTDP antibody for immunostaining and membrane-targeted GFP (mCD8::GFP) driven by OK107-GAL4 to mark the mushroom bodies, we found that dTDP, similar to the mammalian TDP-43, was broadly distributed in the adult brain with most dTDP molecules located in the cell body, especially in the nucleus (Fig. 2A). We then generated fly lines with mushroom body-specific, dsRNA-mediated knockdown of dTDP expression (OK107>38377 and OK107>38379) and with overexpression of dTDP in the mushroom bodies (OK107>dTDP#5-1 and OK107>dTDP#18-1), respectively, with use of the GAL4-UAS system (Fig. 2).


Neuronal function and dysfunction of Drosophila dTDP.

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

dTDP in the adult brains of 3-day old wild type and mutant Drosophila.(A) Distribution of the endogenous dTDP proteins in the mushroom bodies of the OK107>+ flies as analyzed by immunostaining with anti-dTDP (red), DAPI (blue), and mCD8::GFP (green). (a) a low-magnification photo; (b), higher magnification photo of the boxed area in (a); (c), higher magnification photo of the boxed area in (b). Note the predominant nuclear localization of dTDP. (B) Western blot of the total head protein extracts from the adult flies. The blot was probed with anti-dTDP and anti-tubulin. Note the decreases, by approximately 46% and 23%, of the dTDP levels in the heads of OK107>38377 (lane 2) and OK107>38379 (lane 3), respectively, with mushroom body-specific dsRNA-knockdown of dTDP. Also, dTDP levels in the dTDP-overexpressing lines OK107>dTDP#5-1 (lane 4) and OK107>dTDP#18-1 (lane 5) were 7- and 3 fold, respectively, higher than the OK107>+ control (lane 1). (C) GFP-lighted morphologies of the mushroom bodies in OK107>+ (a), OK107>38377 (b), OK107>dTDP#5-1 (c), and OK107>dTDP#18-1 (d). The α, α′, β, β′, and γ lobes were indicated in (a). Note the smaller lobes in (c) and (d). (D) Immunostaining of dTDP in the brains of OK107>38377 flies with mushroom body-specific knockdown of dTDP expression. Note the significantly reduced signals of dTDP in the mushroom bodies when compared to the yw control in Fig. 2A-a. (E) Subcellular distribution of dTDP in the mushroom bodies of the dTDP-overexpressing line OK107>dTDP#5-1. (a) a set of low magnification pictures; (b) and (c), high magnification pictures of the immunostaining patterns of the OK107>dTDP#5-1 mushroom body, with (b) from the boxed area in (a) and (c) from the boxed area in (b). Note the presence of cells with mainly cytoplasmic dTDP (the arrow in b) and cells with cytoplasmic dTDP (+) aggregates (the arrowheads). (F) Western blotting analysis of soluble and insoluble proteins in the heads of 3-day old dTDP-overexpressing flies. Different fractions of protein extracts were isolated from 3 days-old fly heads as described in Materials and Methods, and analyzed by Western blotting with use of anti-dTDP and anti-tubulin. Note the presence of high molecular weight dTDP species in the urea-soluble fraction of OK107>dTDP#5-1. T, total protein; R, RIPA-soluble fraction; U, urea-soluble fraction.
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pone-0020371-g002: dTDP in the adult brains of 3-day old wild type and mutant Drosophila.(A) Distribution of the endogenous dTDP proteins in the mushroom bodies of the OK107>+ flies as analyzed by immunostaining with anti-dTDP (red), DAPI (blue), and mCD8::GFP (green). (a) a low-magnification photo; (b), higher magnification photo of the boxed area in (a); (c), higher magnification photo of the boxed area in (b). Note the predominant nuclear localization of dTDP. (B) Western blot of the total head protein extracts from the adult flies. The blot was probed with anti-dTDP and anti-tubulin. Note the decreases, by approximately 46% and 23%, of the dTDP levels in the heads of OK107>38377 (lane 2) and OK107>38379 (lane 3), respectively, with mushroom body-specific dsRNA-knockdown of dTDP. Also, dTDP levels in the dTDP-overexpressing lines OK107>dTDP#5-1 (lane 4) and OK107>dTDP#18-1 (lane 5) were 7- and 3 fold, respectively, higher than the OK107>+ control (lane 1). (C) GFP-lighted morphologies of the mushroom bodies in OK107>+ (a), OK107>38377 (b), OK107>dTDP#5-1 (c), and OK107>dTDP#18-1 (d). The α, α′, β, β′, and γ lobes were indicated in (a). Note the smaller lobes in (c) and (d). (D) Immunostaining of dTDP in the brains of OK107>38377 flies with mushroom body-specific knockdown of dTDP expression. Note the significantly reduced signals of dTDP in the mushroom bodies when compared to the yw control in Fig. 2A-a. (E) Subcellular distribution of dTDP in the mushroom bodies of the dTDP-overexpressing line OK107>dTDP#5-1. (a) a set of low magnification pictures; (b) and (c), high magnification pictures of the immunostaining patterns of the OK107>dTDP#5-1 mushroom body, with (b) from the boxed area in (a) and (c) from the boxed area in (b). Note the presence of cells with mainly cytoplasmic dTDP (the arrow in b) and cells with cytoplasmic dTDP (+) aggregates (the arrowheads). (F) Western blotting analysis of soluble and insoluble proteins in the heads of 3-day old dTDP-overexpressing flies. Different fractions of protein extracts were isolated from 3 days-old fly heads as described in Materials and Methods, and analyzed by Western blotting with use of anti-dTDP and anti-tubulin. Note the presence of high molecular weight dTDP species in the urea-soluble fraction of OK107>dTDP#5-1. T, total protein; R, RIPA-soluble fraction; U, urea-soluble fraction.
Mentions: Since human TDP-43 was the major component of the UBIs found in the diseased cells of the central nervous system (CNS) of the ALS and the FTLD-U patients [11], we investigated whether dTDP played a role in the functioning of the Drosophila CNS, in particular the mushroom body supporting the olfactory learning of the fruit flies [25], [26]. With use of a home-made anti-dTDP antibody for immunostaining and membrane-targeted GFP (mCD8::GFP) driven by OK107-GAL4 to mark the mushroom bodies, we found that dTDP, similar to the mammalian TDP-43, was broadly distributed in the adult brain with most dTDP molecules located in the cell body, especially in the nucleus (Fig. 2A). We then generated fly lines with mushroom body-specific, dsRNA-mediated knockdown of dTDP expression (OK107>38377 and OK107>38379) and with overexpression of dTDP in the mushroom bodies (OK107>dTDP#5-1 and OK107>dTDP#18-1), respectively, with use of the GAL4-UAS system (Fig. 2).

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