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A novel Drosophila model of TDP-43 proteinopathies: N-terminal sequences combined with the Q/N domain induce protein functional loss and locomotion defects

View Article: PubMed Central - PubMed

ABSTRACT

Transactive response DNA-binding protein 43 kDa (TDP-43, also known as TBPH in Drosophila melanogaster and TARDBP in mammals) is the main protein component of the pathological inclusions observed in neurons of patients affected by different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). The number of studies investigating the molecular mechanisms underlying neurodegeneration is constantly growing; however, the role played by TDP-43 in disease onset and progression is still unclear. A fundamental shortcoming that hampers progress is the lack of animal models showing aggregation of TDP-43 without overexpression. In this manuscript, we have extended our cellular model of aggregation to a transgenic Drosophila line. Our fly model is not based on the overexpression of a wild-type TDP-43 transgene. By contrast, we engineered a construct that includes only the specific TDP-43 amino acid sequences necessary to trigger aggregate formation and capable of trapping endogenous Drosophila TDP-43 into a non-functional insoluble form. Importantly, the resulting recombinant product lacks functional RNA recognition motifs (RRMs) and, thus, does not have specific TDP-43-physiological functions (i.e. splicing regulation ability) that might affect the animal phenotype per se. This novel Drosophila model exhibits an evident degenerative phenotype with reduced lifespan and early locomotion defects. Additionally, we show that important proteins involved in neuromuscular junction function, such as syntaxin (SYX), decrease their levels as a consequence of TDP-43 loss of function implying that the degenerative phenotype is a consequence of TDP-43 sequestration into the aggregates. Our data lend further support to the role of TDP-43 loss-of-function in the pathogenesis of neurodegenerative disorders. The novel transgenic Drosophila model presented in this study will help to gain further insight into the molecular mechanisms underlying neurodegeneration and will provide a valuable system to test potential therapeutic agents to counteract disease.

No MeSH data available.


Related in: MedlinePlus

Effect of transgene expression on Drosophila lifespan and on external eye structure and/or function. (A) Lifespan is dramatically reduced in flies expressing the AggIn transgene in neurons (elav-Gal4>UAS_5A and elav-Gal4>UAS_2B) versus a control fly not expressing any transgene (elav-Gal4>+) or a transgenic fly line expressing the control protein EGFP (elav-Gal4>UAS_Egfp). Median survival is 18 days for elav-Gal4>UAS_5A; 29 days for elav-Gal4<UAS_2B; 64 days for controls (both elav-Gal4>+ and elav-Gal4>UAS_Egfp). n>120 animals for each genotype; P<0.001 (log-rank test) for all the following genotype pairs: elav-Gal4>+ versus elav-Gal4>UAS_2B; elav-Gal4>+ versus elav-Gal4>UAS_5A; elav-Gal4>UAS_Egfp versus elav-Gal4>UAS_2B; elav-Gal4>UAS_Egfp versus elav-Gal4>UAS_5A; elav-Gal4>UAS_2B versus elav-Gal4>UAS_5A. Time points (days) corresponding to 25%, 50% and 75% survival are also shown in the graph for each genotype and reported in the flanking summary table. (B) External eye phenotype and phototaxis assay of 1-day-old flies. (a,a′) Oregon (wild-type); (b,b′) GMR-Gal4>UAS_Egfp; (c,c′) GMR-Gal4>UAS_2B; (d,d′) GMR-Gal4>UAS_5A. The AggIn expression in the eye, using the GMR-Gal4 driver, did not result in any substantial alteration of the external eye phenotype (upper panel, compare pictures in c and d versus controls in a and b). However, the vision assay (lower panel) revealed that a minor fraction of the AggIn-expressing population of flies (8.5% of GMR-Gal4>UAS_2B, and 20.4% of GMR-Gal4>UAS_5A) exhibited vision defects because they did not reach the light within 1 min of time in a phototaxis assay (compare white bars in c′ and d′ fractions with the negative controls in a′ and b′). Error bars indicate s.e.m. (n=3).
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DMM023382F4: Effect of transgene expression on Drosophila lifespan and on external eye structure and/or function. (A) Lifespan is dramatically reduced in flies expressing the AggIn transgene in neurons (elav-Gal4>UAS_5A and elav-Gal4>UAS_2B) versus a control fly not expressing any transgene (elav-Gal4>+) or a transgenic fly line expressing the control protein EGFP (elav-Gal4>UAS_Egfp). Median survival is 18 days for elav-Gal4>UAS_5A; 29 days for elav-Gal4<UAS_2B; 64 days for controls (both elav-Gal4>+ and elav-Gal4>UAS_Egfp). n>120 animals for each genotype; P<0.001 (log-rank test) for all the following genotype pairs: elav-Gal4>+ versus elav-Gal4>UAS_2B; elav-Gal4>+ versus elav-Gal4>UAS_5A; elav-Gal4>UAS_Egfp versus elav-Gal4>UAS_2B; elav-Gal4>UAS_Egfp versus elav-Gal4>UAS_5A; elav-Gal4>UAS_2B versus elav-Gal4>UAS_5A. Time points (days) corresponding to 25%, 50% and 75% survival are also shown in the graph for each genotype and reported in the flanking summary table. (B) External eye phenotype and phototaxis assay of 1-day-old flies. (a,a′) Oregon (wild-type); (b,b′) GMR-Gal4>UAS_Egfp; (c,c′) GMR-Gal4>UAS_2B; (d,d′) GMR-Gal4>UAS_5A. The AggIn expression in the eye, using the GMR-Gal4 driver, did not result in any substantial alteration of the external eye phenotype (upper panel, compare pictures in c and d versus controls in a and b). However, the vision assay (lower panel) revealed that a minor fraction of the AggIn-expressing population of flies (8.5% of GMR-Gal4>UAS_2B, and 20.4% of GMR-Gal4>UAS_5A) exhibited vision defects because they did not reach the light within 1 min of time in a phototaxis assay (compare white bars in c′ and d′ fractions with the negative controls in a′ and b′). Error bars indicate s.e.m. (n=3).

Mentions: To study the effects of the AggIn expression in neurons, we first analyzed the lifespan of elav-Gal4>UAS_5A and elav-Gal4>UAS_2B flies versus two different control flies: one not expressing any transgene (elav-Gal4>+), and a second transgenic fly line expressing the irrelevant protein EGFP (elav-Gal4>UAS_Egfp). As clearly shown in Fig. 4A, lifespan was dramatically reduced in flies expressing AggIn. Indeed, whereas we observed a median survival of 64 days for control flies (both elav-Gal4>+ and elav-Gal4>UAS_Egfp), we found a median survival of only 18 days for elav-Gal4>UAS_5A and 29 days for elav-Gal4<UAS_2B. Such a significant decline in survival during aging suggests that transgene expression has strong phenotypic consequences, whose intensity is related to transgene expression levels.Fig. 4.


A novel Drosophila model of TDP-43 proteinopathies: N-terminal sequences combined with the Q/N domain induce protein functional loss and locomotion defects
Effect of transgene expression on Drosophila lifespan and on external eye structure and/or function. (A) Lifespan is dramatically reduced in flies expressing the AggIn transgene in neurons (elav-Gal4>UAS_5A and elav-Gal4>UAS_2B) versus a control fly not expressing any transgene (elav-Gal4>+) or a transgenic fly line expressing the control protein EGFP (elav-Gal4>UAS_Egfp). Median survival is 18 days for elav-Gal4>UAS_5A; 29 days for elav-Gal4<UAS_2B; 64 days for controls (both elav-Gal4>+ and elav-Gal4>UAS_Egfp). n>120 animals for each genotype; P<0.001 (log-rank test) for all the following genotype pairs: elav-Gal4>+ versus elav-Gal4>UAS_2B; elav-Gal4>+ versus elav-Gal4>UAS_5A; elav-Gal4>UAS_Egfp versus elav-Gal4>UAS_2B; elav-Gal4>UAS_Egfp versus elav-Gal4>UAS_5A; elav-Gal4>UAS_2B versus elav-Gal4>UAS_5A. Time points (days) corresponding to 25%, 50% and 75% survival are also shown in the graph for each genotype and reported in the flanking summary table. (B) External eye phenotype and phototaxis assay of 1-day-old flies. (a,a′) Oregon (wild-type); (b,b′) GMR-Gal4>UAS_Egfp; (c,c′) GMR-Gal4>UAS_2B; (d,d′) GMR-Gal4>UAS_5A. The AggIn expression in the eye, using the GMR-Gal4 driver, did not result in any substantial alteration of the external eye phenotype (upper panel, compare pictures in c and d versus controls in a and b). However, the vision assay (lower panel) revealed that a minor fraction of the AggIn-expressing population of flies (8.5% of GMR-Gal4>UAS_2B, and 20.4% of GMR-Gal4>UAS_5A) exhibited vision defects because they did not reach the light within 1 min of time in a phototaxis assay (compare white bars in c′ and d′ fractions with the negative controls in a′ and b′). Error bars indicate s.e.m. (n=3).
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Related In: Results  -  Collection

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DMM023382F4: Effect of transgene expression on Drosophila lifespan and on external eye structure and/or function. (A) Lifespan is dramatically reduced in flies expressing the AggIn transgene in neurons (elav-Gal4>UAS_5A and elav-Gal4>UAS_2B) versus a control fly not expressing any transgene (elav-Gal4>+) or a transgenic fly line expressing the control protein EGFP (elav-Gal4>UAS_Egfp). Median survival is 18 days for elav-Gal4>UAS_5A; 29 days for elav-Gal4<UAS_2B; 64 days for controls (both elav-Gal4>+ and elav-Gal4>UAS_Egfp). n>120 animals for each genotype; P<0.001 (log-rank test) for all the following genotype pairs: elav-Gal4>+ versus elav-Gal4>UAS_2B; elav-Gal4>+ versus elav-Gal4>UAS_5A; elav-Gal4>UAS_Egfp versus elav-Gal4>UAS_2B; elav-Gal4>UAS_Egfp versus elav-Gal4>UAS_5A; elav-Gal4>UAS_2B versus elav-Gal4>UAS_5A. Time points (days) corresponding to 25%, 50% and 75% survival are also shown in the graph for each genotype and reported in the flanking summary table. (B) External eye phenotype and phototaxis assay of 1-day-old flies. (a,a′) Oregon (wild-type); (b,b′) GMR-Gal4>UAS_Egfp; (c,c′) GMR-Gal4>UAS_2B; (d,d′) GMR-Gal4>UAS_5A. The AggIn expression in the eye, using the GMR-Gal4 driver, did not result in any substantial alteration of the external eye phenotype (upper panel, compare pictures in c and d versus controls in a and b). However, the vision assay (lower panel) revealed that a minor fraction of the AggIn-expressing population of flies (8.5% of GMR-Gal4>UAS_2B, and 20.4% of GMR-Gal4>UAS_5A) exhibited vision defects because they did not reach the light within 1 min of time in a phototaxis assay (compare white bars in c′ and d′ fractions with the negative controls in a′ and b′). Error bars indicate s.e.m. (n=3).
Mentions: To study the effects of the AggIn expression in neurons, we first analyzed the lifespan of elav-Gal4>UAS_5A and elav-Gal4>UAS_2B flies versus two different control flies: one not expressing any transgene (elav-Gal4>+), and a second transgenic fly line expressing the irrelevant protein EGFP (elav-Gal4>UAS_Egfp). As clearly shown in Fig. 4A, lifespan was dramatically reduced in flies expressing AggIn. Indeed, whereas we observed a median survival of 64 days for control flies (both elav-Gal4>+ and elav-Gal4>UAS_Egfp), we found a median survival of only 18 days for elav-Gal4>UAS_5A and 29 days for elav-Gal4<UAS_2B. Such a significant decline in survival during aging suggests that transgene expression has strong phenotypic consequences, whose intensity is related to transgene expression levels.Fig. 4.

View Article: PubMed Central - PubMed

ABSTRACT

Transactive response DNA-binding protein 43&#8197;kDa (TDP-43, also known as TBPH in Drosophila melanogaster and TARDBP in mammals) is the main protein component of the pathological inclusions observed in neurons of patients affected by different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). The number of studies investigating the molecular mechanisms underlying neurodegeneration is constantly growing; however, the role played by TDP-43 in disease onset and progression is still unclear. A fundamental shortcoming that hampers progress is the lack of animal models showing aggregation of TDP-43 without overexpression. In this manuscript, we have extended our cellular model of aggregation to a transgenic Drosophila line. Our fly model is not based on the overexpression of a wild-type TDP-43 transgene. By contrast, we engineered a construct that includes only the specific TDP-43 amino acid sequences necessary to trigger aggregate formation and capable of trapping endogenous Drosophila TDP-43 into a non-functional insoluble form. Importantly, the resulting recombinant product lacks functional RNA recognition motifs (RRMs) and, thus, does not have specific TDP-43-physiological functions (i.e. splicing regulation ability) that might affect the animal phenotype per se. This novel Drosophila model exhibits an evident degenerative phenotype with reduced lifespan and early locomotion defects. Additionally, we show that important proteins involved in neuromuscular junction function, such as syntaxin (SYX), decrease their levels as a consequence of TDP-43 loss of function implying that the degenerative phenotype is a consequence of TDP-43 sequestration into the aggregates. Our data lend further support to the role of TDP-43 loss-of-function in the pathogenesis of neurodegenerative disorders. The novel transgenic Drosophila model presented in this study will help to gain further insight into the molecular mechanisms underlying neurodegeneration and will provide a valuable system to test potential therapeutic agents to counteract disease.

No MeSH data available.


Related in: MedlinePlus