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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.


Transgene effects on expression of TDP-43 target genes. (A) Western blot for TDP-43-target genes performed in adult transgenic flies. Western blot analysis with anti-SYX and anti-CSP antibodies demonstrates the specific drop in expression of these presynaptic vesicular markers in elav-Gal4>UAS_5A fly heads, as compared to a transgenic line expressing the control protein EGFP (elav-Gal4>UAS_Egfp). Three different time-points were assayed (days 3, 7 and 11). Total protein samples were extracted from adult heads; anti-α-tubulin (TUB) was used as a loading control. The relative expression of target proteins in transgenic lines was calculated by optical densitometry with ImageJ software (Schneider et al., 2012). For each time-point, the percentage expression of SYX and CSP in the 5A line versus the control EGFP was calculated and is reported below the western blot panel. At day 3, the expression of SYX in 5A line was 43±15% that of the Egfp line; at day 7, it was 53±18; at day 11, it was 40±15%. At day 3, the expression of CSP in 5A line was 38±15% that of the Egfp line; at day 7, it was 83±8% s.e.m.; at day 11, it was 73±10% (results are mean±s.e.m.). All results shown are representative of at least three independent experiments. (B) Anti-syntaxin immunofluorescence performed in third-instar larvae NMJs. Confocal microscopy images of NMJs presynaptic boutons in muscle 6 and 7, II segment, are shown. SYX expression (anti-SYX antibody, red) appears to be strongly reduced in the synaptic terminals of elav-Gal4>UAS_5A larvae compared to elav-Gal4>UAS_Egfp control larvae. By contrast, the anti-HRP staining of the neurons is not affected (anti-HRP antibody, green). No significant alteration of the SYX protein expression was detected in elav-Gal4>UAS_Egfp-12×Q/N synaptic terminals. The SYX-normalized expression is quantified in the graph. Results are mean±s.e.m. (n=200). ***P<0.001 (ANOVA).
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DMM023382F7: Transgene effects on expression of TDP-43 target genes. (A) Western blot for TDP-43-target genes performed in adult transgenic flies. Western blot analysis with anti-SYX and anti-CSP antibodies demonstrates the specific drop in expression of these presynaptic vesicular markers in elav-Gal4>UAS_5A fly heads, as compared to a transgenic line expressing the control protein EGFP (elav-Gal4>UAS_Egfp). Three different time-points were assayed (days 3, 7 and 11). Total protein samples were extracted from adult heads; anti-α-tubulin (TUB) was used as a loading control. The relative expression of target proteins in transgenic lines was calculated by optical densitometry with ImageJ software (Schneider et al., 2012). For each time-point, the percentage expression of SYX and CSP in the 5A line versus the control EGFP was calculated and is reported below the western blot panel. At day 3, the expression of SYX in 5A line was 43±15% that of the Egfp line; at day 7, it was 53±18; at day 11, it was 40±15%. At day 3, the expression of CSP in 5A line was 38±15% that of the Egfp line; at day 7, it was 83±8% s.e.m.; at day 11, it was 73±10% (results are mean±s.e.m.). All results shown are representative of at least three independent experiments. (B) Anti-syntaxin immunofluorescence performed in third-instar larvae NMJs. Confocal microscopy images of NMJs presynaptic boutons in muscle 6 and 7, II segment, are shown. SYX expression (anti-SYX antibody, red) appears to be strongly reduced in the synaptic terminals of elav-Gal4>UAS_5A larvae compared to elav-Gal4>UAS_Egfp control larvae. By contrast, the anti-HRP staining of the neurons is not affected (anti-HRP antibody, green). No significant alteration of the SYX protein expression was detected in elav-Gal4>UAS_Egfp-12×Q/N synaptic terminals. The SYX-normalized expression is quantified in the graph. Results are mean±s.e.m. (n=200). ***P<0.001 (ANOVA).

Mentions: SYX (also known as Syx1a) and Cysteine-string protein (CSP) are two presynaptic vesicular proteins. It has been recently reported that their downregulation is an early event of dTDP-43 dysfunction in vivo; in fact, the expression of these proteins was found to be significantly altered in the heads of our dTDP-43- fly model TBPHΔ23 and in neuromuscular junction (NMJ) presynaptic boutons in muscle 6 and 7 of third-instar larvae (Romano et al., 2014). Starting from these observations, we verified the endogenous dTDP-43 function in Drosophila expressing the AggIn transgene. In particular, we compared, by western blotting, the expression of SYX and CSP proteins in the heads of elav-Gal4>UAS_5A versus elav-Gal4>UAS_Egfp control flies. These proteins appeared to be significantly downregulated in our transgenic model (Fig. 7A). Interestingly, the drop in expression was found at all three time points assayed (day 3, 7 and 11), in agreement with the observation that this fly line already has a severe phenotype by 3 days after eclosion.Fig. 7.


A novel Drosophila model of TDP-43 proteinopathies: N-terminal sequences combined with the Q/N domain induce protein functional loss and locomotion defects
Transgene effects on expression of TDP-43 target genes. (A) Western blot for TDP-43-target genes performed in adult transgenic flies. Western blot analysis with anti-SYX and anti-CSP antibodies demonstrates the specific drop in expression of these presynaptic vesicular markers in elav-Gal4>UAS_5A fly heads, as compared to a transgenic line expressing the control protein EGFP (elav-Gal4>UAS_Egfp). Three different time-points were assayed (days 3, 7 and 11). Total protein samples were extracted from adult heads; anti-α-tubulin (TUB) was used as a loading control. The relative expression of target proteins in transgenic lines was calculated by optical densitometry with ImageJ software (Schneider et al., 2012). For each time-point, the percentage expression of SYX and CSP in the 5A line versus the control EGFP was calculated and is reported below the western blot panel. At day 3, the expression of SYX in 5A line was 43±15% that of the Egfp line; at day 7, it was 53±18; at day 11, it was 40±15%. At day 3, the expression of CSP in 5A line was 38±15% that of the Egfp line; at day 7, it was 83±8% s.e.m.; at day 11, it was 73±10% (results are mean±s.e.m.). All results shown are representative of at least three independent experiments. (B) Anti-syntaxin immunofluorescence performed in third-instar larvae NMJs. Confocal microscopy images of NMJs presynaptic boutons in muscle 6 and 7, II segment, are shown. SYX expression (anti-SYX antibody, red) appears to be strongly reduced in the synaptic terminals of elav-Gal4>UAS_5A larvae compared to elav-Gal4>UAS_Egfp control larvae. By contrast, the anti-HRP staining of the neurons is not affected (anti-HRP antibody, green). No significant alteration of the SYX protein expression was detected in elav-Gal4>UAS_Egfp-12×Q/N synaptic terminals. The SYX-normalized expression is quantified in the graph. Results are mean±s.e.m. (n=200). ***P<0.001 (ANOVA).
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DMM023382F7: Transgene effects on expression of TDP-43 target genes. (A) Western blot for TDP-43-target genes performed in adult transgenic flies. Western blot analysis with anti-SYX and anti-CSP antibodies demonstrates the specific drop in expression of these presynaptic vesicular markers in elav-Gal4>UAS_5A fly heads, as compared to a transgenic line expressing the control protein EGFP (elav-Gal4>UAS_Egfp). Three different time-points were assayed (days 3, 7 and 11). Total protein samples were extracted from adult heads; anti-α-tubulin (TUB) was used as a loading control. The relative expression of target proteins in transgenic lines was calculated by optical densitometry with ImageJ software (Schneider et al., 2012). For each time-point, the percentage expression of SYX and CSP in the 5A line versus the control EGFP was calculated and is reported below the western blot panel. At day 3, the expression of SYX in 5A line was 43±15% that of the Egfp line; at day 7, it was 53±18; at day 11, it was 40±15%. At day 3, the expression of CSP in 5A line was 38±15% that of the Egfp line; at day 7, it was 83±8% s.e.m.; at day 11, it was 73±10% (results are mean±s.e.m.). All results shown are representative of at least three independent experiments. (B) Anti-syntaxin immunofluorescence performed in third-instar larvae NMJs. Confocal microscopy images of NMJs presynaptic boutons in muscle 6 and 7, II segment, are shown. SYX expression (anti-SYX antibody, red) appears to be strongly reduced in the synaptic terminals of elav-Gal4>UAS_5A larvae compared to elav-Gal4>UAS_Egfp control larvae. By contrast, the anti-HRP staining of the neurons is not affected (anti-HRP antibody, green). No significant alteration of the SYX protein expression was detected in elav-Gal4>UAS_Egfp-12×Q/N synaptic terminals. The SYX-normalized expression is quantified in the graph. Results are mean±s.e.m. (n=200). ***P<0.001 (ANOVA).
Mentions: SYX (also known as Syx1a) and Cysteine-string protein (CSP) are two presynaptic vesicular proteins. It has been recently reported that their downregulation is an early event of dTDP-43 dysfunction in vivo; in fact, the expression of these proteins was found to be significantly altered in the heads of our dTDP-43- fly model TBPHΔ23 and in neuromuscular junction (NMJ) presynaptic boutons in muscle 6 and 7 of third-instar larvae (Romano et al., 2014). Starting from these observations, we verified the endogenous dTDP-43 function in Drosophila expressing the AggIn transgene. In particular, we compared, by western blotting, the expression of SYX and CSP proteins in the heads of elav-Gal4>UAS_5A versus elav-Gal4>UAS_Egfp control flies. These proteins appeared to be significantly downregulated in our transgenic model (Fig. 7A). Interestingly, the drop in expression was found at all three time points assayed (day 3, 7 and 11), in agreement with the observation that this fly line already has a severe phenotype by 3 days after eclosion.Fig. 7.

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.