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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.


Expression levels of transgene in two Drosophila lines. (A) Western blot analysis of total protein extracts from fly heads of GMR-Gal4>UAS_5A and GMR-Gal4>UAS_2B and (B) elav-Gal4>UAS_5A and elav-Gal4>UAS_2B lines. Eye-specific and pan-neuronal expression of the AggIn construct was achieved using the GMR- and elav-Gal4 drivers, respectively. Western blot densitometry was performed using the ImageJ software and the normalized expression of the transgenic protein is reported in the graphs (mean±s.e.m., n=3).
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DMM023382F3: Expression levels of transgene in two Drosophila lines. (A) Western blot analysis of total protein extracts from fly heads of GMR-Gal4>UAS_5A and GMR-Gal4>UAS_2B and (B) elav-Gal4>UAS_5A and elav-Gal4>UAS_2B lines. Eye-specific and pan-neuronal expression of the AggIn construct was achieved using the GMR- and elav-Gal4 drivers, respectively. Western blot densitometry was performed using the ImageJ software and the normalized expression of the transgenic protein is reported in the graphs (mean±s.e.m., n=3).

Mentions: To create a novel animal model for TDP-43 aggregation, we cloned the AggIn construct in the pUASTattB vector, under the control of the upstream activating sequence (UAS). After embryo injection, five different fly lines were obtained and screened for transgene expression by using the GMR-Gal4 driver. Although four of these lines expressed the transgene at comparable levels, the fifth demonstrated a higher expression level (data not shown). Therefore, in the subsequent steps of our study, we focused our attention on two of these transgenic lines: the one expressing the transgene at the top expression level (UAS_5A) and one out of the four expressing the transgene at comparable level (UAS_2B) (Fig. 3A).Fig. 3.


A novel Drosophila model of TDP-43 proteinopathies: N-terminal sequences combined with the Q/N domain induce protein functional loss and locomotion defects
Expression levels of transgene in two Drosophila lines. (A) Western blot analysis of total protein extracts from fly heads of GMR-Gal4>UAS_5A and GMR-Gal4>UAS_2B and (B) elav-Gal4>UAS_5A and elav-Gal4>UAS_2B lines. Eye-specific and pan-neuronal expression of the AggIn construct was achieved using the GMR- and elav-Gal4 drivers, respectively. Western blot densitometry was performed using the ImageJ software and the normalized expression of the transgenic protein is reported in the graphs (mean±s.e.m., n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920146&req=5

DMM023382F3: Expression levels of transgene in two Drosophila lines. (A) Western blot analysis of total protein extracts from fly heads of GMR-Gal4>UAS_5A and GMR-Gal4>UAS_2B and (B) elav-Gal4>UAS_5A and elav-Gal4>UAS_2B lines. Eye-specific and pan-neuronal expression of the AggIn construct was achieved using the GMR- and elav-Gal4 drivers, respectively. Western blot densitometry was performed using the ImageJ software and the normalized expression of the transgenic protein is reported in the graphs (mean±s.e.m., n=3).
Mentions: To create a novel animal model for TDP-43 aggregation, we cloned the AggIn construct in the pUASTattB vector, under the control of the upstream activating sequence (UAS). After embryo injection, five different fly lines were obtained and screened for transgene expression by using the GMR-Gal4 driver. Although four of these lines expressed the transgene at comparable levels, the fifth demonstrated a higher expression level (data not shown). Therefore, in the subsequent steps of our study, we focused our attention on two of these transgenic lines: the one expressing the transgene at the top expression level (UAS_5A) and one out of the four expressing the transgene at comparable level (UAS_2B) (Fig. 3A).Fig. 3.

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.