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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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Characteristics of the dTDP imprecise excision mutant dTDPex26.(A) Organization of the six-exon dTDP gene in the dTDPex26 line. This semi-lethal dTDP was generated by imprecise excision of the P-element KG08578 leading to a 932 bp-deletion (the hatched block) in the 5′ end of the dTDP gene. The primers used for PCR in the RT-PCR analysis are indicated. Also shown is the most neighboring gene CG4585 located at upstream of dTDP. Lower panels, RT-PCR analysis of dTDP gene expression in the wild type (yw, left panel) and homozygous dTDPex26 (right panel). Note the absence of dTDP signal in the dTDPex26 samples. (B) Immunostaining of the third instar larval segment A3 muscle 6/7 (a,b) and the third instar larval ventral nerve cords (c,d) from yw control and dTDPex26 mutant with anti-dTDP antibody (green). The nuclei were visualized by DAPI (blue), and the actin cytoskeleton was labeled by phalloidin (magenta). The neurons of the ventral nerve cords were also stained with anti-elav (red). Panel rows a and c show the nuclear localization of dTDP in the yw control and panel rows b and d show the lack of anti-dTDP signals in the dTDPex26 mutant. (C) Larval movement assay. The movement of the wandering third instar larvae on the agar plate was monitored for 2 minutes and the average distance of the larval trails was calculated. Note the severe movement defect of the homozygous dTDPex26 mutant in comparison to the yw control, and the partial rescue of this phenotype in dTDPex26; D42>dTDP#18-1 (N = 15, ***, p<0.0001). (D) Comparison of the bouton number/muscle area of yw, dTDPex26 and dTDPex26; D42>dTDP#18-1. The A3 NMJs on muscles 6/7 of the late third instar larvae were co-stained with antibodies against HRP (red) and Synapsin (green), respectively, as exemplified in the right 2 panels. The dTDPex26 mutant had higher bouton number per unit area of the muscle than the yw control, and this phenotype of dTDPex26 was rescued in dTDPex26; D42>dTDP#18-1. *, p<0.05. The averages of the total bouton numbers are: yw, 67±17 (N = 18); dTDPex26, 94±14 (N = 16); dTDPex26; D42>dTDP#18-1, 75±8 (N = 12). The means of the muscle areas are: yw, 63,187 µm2; dTDPex26, 62,565 µm2; dTDPex26; D42>dTDP#18-1, 64,310 µm2.
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pone-0020371-g001: Characteristics of the dTDP imprecise excision mutant dTDPex26.(A) Organization of the six-exon dTDP gene in the dTDPex26 line. This semi-lethal dTDP was generated by imprecise excision of the P-element KG08578 leading to a 932 bp-deletion (the hatched block) in the 5′ end of the dTDP gene. The primers used for PCR in the RT-PCR analysis are indicated. Also shown is the most neighboring gene CG4585 located at upstream of dTDP. Lower panels, RT-PCR analysis of dTDP gene expression in the wild type (yw, left panel) and homozygous dTDPex26 (right panel). Note the absence of dTDP signal in the dTDPex26 samples. (B) Immunostaining of the third instar larval segment A3 muscle 6/7 (a,b) and the third instar larval ventral nerve cords (c,d) from yw control and dTDPex26 mutant with anti-dTDP antibody (green). The nuclei were visualized by DAPI (blue), and the actin cytoskeleton was labeled by phalloidin (magenta). The neurons of the ventral nerve cords were also stained with anti-elav (red). Panel rows a and c show the nuclear localization of dTDP in the yw control and panel rows b and d show the lack of anti-dTDP signals in the dTDPex26 mutant. (C) Larval movement assay. The movement of the wandering third instar larvae on the agar plate was monitored for 2 minutes and the average distance of the larval trails was calculated. Note the severe movement defect of the homozygous dTDPex26 mutant in comparison to the yw control, and the partial rescue of this phenotype in dTDPex26; D42>dTDP#18-1 (N = 15, ***, p<0.0001). (D) Comparison of the bouton number/muscle area of yw, dTDPex26 and dTDPex26; D42>dTDP#18-1. The A3 NMJs on muscles 6/7 of the late third instar larvae were co-stained with antibodies against HRP (red) and Synapsin (green), respectively, as exemplified in the right 2 panels. The dTDPex26 mutant had higher bouton number per unit area of the muscle than the yw control, and this phenotype of dTDPex26 was rescued in dTDPex26; D42>dTDP#18-1. *, p<0.05. The averages of the total bouton numbers are: yw, 67±17 (N = 18); dTDPex26, 94±14 (N = 16); dTDPex26; D42>dTDP#18-1, 75±8 (N = 12). The means of the muscle areas are: yw, 63,187 µm2; dTDPex26, 62,565 µm2; dTDPex26; D42>dTDP#18-1, 64,310 µm2.

Mentions: The function of dTDP in Drosophila development was examined by analysis of fly mutants, with -expression of dTDP. For this, deletion mutations of the dTDP gene in the KG08578 insertion line were generated by imprecise excision. Twelve out of the ninety stocks with excision of the P element from KG08578 could not survive to the adult stage. One of these recessive lethal dTDP lines, dTDPex26, was analyzed in details. dTDPex26 consisted of an imprecise excision of the P-element leading to a 932 bp-deletion in the 5′ end of dTDP gene, and no dTDP expression could be detected by RT-PCR analysis at all developmental stages of the homozygous mutant dTDPex26 flies obtained with use of the green balancer (Fig. 1A). The deletion in dTDPex26 mutant did not affect the RNA level from the gene CG4585 at upstream of dTDP (RT-PCR data not shown). The homozygous dTDPex26 line was semi-lethal with most of the flies viable from embryonic to early pupal stage but few of them (approximately 10%) eclosed to the adult stage. Most of the flies were trapped in the pupal cases and the survived ones showed weakness in their legs and severe movement defects.


Neuronal function and dysfunction of Drosophila dTDP.

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

Characteristics of the dTDP imprecise excision mutant dTDPex26.(A) Organization of the six-exon dTDP gene in the dTDPex26 line. This semi-lethal dTDP was generated by imprecise excision of the P-element KG08578 leading to a 932 bp-deletion (the hatched block) in the 5′ end of the dTDP gene. The primers used for PCR in the RT-PCR analysis are indicated. Also shown is the most neighboring gene CG4585 located at upstream of dTDP. Lower panels, RT-PCR analysis of dTDP gene expression in the wild type (yw, left panel) and homozygous dTDPex26 (right panel). Note the absence of dTDP signal in the dTDPex26 samples. (B) Immunostaining of the third instar larval segment A3 muscle 6/7 (a,b) and the third instar larval ventral nerve cords (c,d) from yw control and dTDPex26 mutant with anti-dTDP antibody (green). The nuclei were visualized by DAPI (blue), and the actin cytoskeleton was labeled by phalloidin (magenta). The neurons of the ventral nerve cords were also stained with anti-elav (red). Panel rows a and c show the nuclear localization of dTDP in the yw control and panel rows b and d show the lack of anti-dTDP signals in the dTDPex26 mutant. (C) Larval movement assay. The movement of the wandering third instar larvae on the agar plate was monitored for 2 minutes and the average distance of the larval trails was calculated. Note the severe movement defect of the homozygous dTDPex26 mutant in comparison to the yw control, and the partial rescue of this phenotype in dTDPex26; D42>dTDP#18-1 (N = 15, ***, p<0.0001). (D) Comparison of the bouton number/muscle area of yw, dTDPex26 and dTDPex26; D42>dTDP#18-1. The A3 NMJs on muscles 6/7 of the late third instar larvae were co-stained with antibodies against HRP (red) and Synapsin (green), respectively, as exemplified in the right 2 panels. The dTDPex26 mutant had higher bouton number per unit area of the muscle than the yw control, and this phenotype of dTDPex26 was rescued in dTDPex26; D42>dTDP#18-1. *, p<0.05. The averages of the total bouton numbers are: yw, 67±17 (N = 18); dTDPex26, 94±14 (N = 16); dTDPex26; D42>dTDP#18-1, 75±8 (N = 12). The means of the muscle areas are: yw, 63,187 µm2; dTDPex26, 62,565 µm2; dTDPex26; D42>dTDP#18-1, 64,310 µm2.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020371-g001: Characteristics of the dTDP imprecise excision mutant dTDPex26.(A) Organization of the six-exon dTDP gene in the dTDPex26 line. This semi-lethal dTDP was generated by imprecise excision of the P-element KG08578 leading to a 932 bp-deletion (the hatched block) in the 5′ end of the dTDP gene. The primers used for PCR in the RT-PCR analysis are indicated. Also shown is the most neighboring gene CG4585 located at upstream of dTDP. Lower panels, RT-PCR analysis of dTDP gene expression in the wild type (yw, left panel) and homozygous dTDPex26 (right panel). Note the absence of dTDP signal in the dTDPex26 samples. (B) Immunostaining of the third instar larval segment A3 muscle 6/7 (a,b) and the third instar larval ventral nerve cords (c,d) from yw control and dTDPex26 mutant with anti-dTDP antibody (green). The nuclei were visualized by DAPI (blue), and the actin cytoskeleton was labeled by phalloidin (magenta). The neurons of the ventral nerve cords were also stained with anti-elav (red). Panel rows a and c show the nuclear localization of dTDP in the yw control and panel rows b and d show the lack of anti-dTDP signals in the dTDPex26 mutant. (C) Larval movement assay. The movement of the wandering third instar larvae on the agar plate was monitored for 2 minutes and the average distance of the larval trails was calculated. Note the severe movement defect of the homozygous dTDPex26 mutant in comparison to the yw control, and the partial rescue of this phenotype in dTDPex26; D42>dTDP#18-1 (N = 15, ***, p<0.0001). (D) Comparison of the bouton number/muscle area of yw, dTDPex26 and dTDPex26; D42>dTDP#18-1. The A3 NMJs on muscles 6/7 of the late third instar larvae were co-stained with antibodies against HRP (red) and Synapsin (green), respectively, as exemplified in the right 2 panels. The dTDPex26 mutant had higher bouton number per unit area of the muscle than the yw control, and this phenotype of dTDPex26 was rescued in dTDPex26; D42>dTDP#18-1. *, p<0.05. The averages of the total bouton numbers are: yw, 67±17 (N = 18); dTDPex26, 94±14 (N = 16); dTDPex26; D42>dTDP#18-1, 75±8 (N = 12). The means of the muscle areas are: yw, 63,187 µm2; dTDPex26, 62,565 µm2; dTDPex26; D42>dTDP#18-1, 64,310 µm2.
Mentions: The function of dTDP in Drosophila development was examined by analysis of fly mutants, with -expression of dTDP. For this, deletion mutations of the dTDP gene in the KG08578 insertion line were generated by imprecise excision. Twelve out of the ninety stocks with excision of the P element from KG08578 could not survive to the adult stage. One of these recessive lethal dTDP lines, dTDPex26, was analyzed in details. dTDPex26 consisted of an imprecise excision of the P-element leading to a 932 bp-deletion in the 5′ end of dTDP gene, and no dTDP expression could be detected by RT-PCR analysis at all developmental stages of the homozygous mutant dTDPex26 flies obtained with use of the green balancer (Fig. 1A). The deletion in dTDPex26 mutant did not affect the RNA level from the gene CG4585 at upstream of dTDP (RT-PCR data not shown). The homozygous dTDPex26 line was semi-lethal with most of the flies viable from embryonic to early pupal stage but few of them (approximately 10%) eclosed to the adult stage. Most of the flies were trapped in the pupal cases and the survived ones showed weakness in their legs and severe movement defects.

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