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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.

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Effect of transgene expression on TDP-43 target genes in the HEK293 AggIn stable cell line. (A) Upper panel, RT-PCR showing the splicing pattern of the endogenous POLDIP3 gene (exon 3) after tetracycline (tet) induction (+) or not (−) of Flag-tagged wild-type TDP-43 (TDP wt; left-hand two lanes), Flag-tagged TDP-12×Q/N (middle two lanes) or AggIn (right-hand two lanes) in stable HEK293 cell lines. Lower panel, western blot analysis, using anti-POLDIP3 antibody, of total protein lysates extracted from the same samples used in the two right-hand lanes of the upper panel. (B) RT-PCR showing the splicing pattern of endogenous BIM (exon 3) and MADD (exon 31) genes after tetracycline induction (+) or not (−) of the AggIn stable HEK293 cell line.
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DMM023382F2: Effect of transgene expression on TDP-43 target genes in the HEK293 AggIn stable cell line. (A) Upper panel, RT-PCR showing the splicing pattern of the endogenous POLDIP3 gene (exon 3) after tetracycline (tet) induction (+) or not (−) of Flag-tagged wild-type TDP-43 (TDP wt; left-hand two lanes), Flag-tagged TDP-12×Q/N (middle two lanes) or AggIn (right-hand two lanes) in stable HEK293 cell lines. Lower panel, western blot analysis, using anti-POLDIP3 antibody, of total protein lysates extracted from the same samples used in the two right-hand lanes of the upper panel. (B) RT-PCR showing the splicing pattern of endogenous BIM (exon 3) and MADD (exon 31) genes after tetracycline induction (+) or not (−) of the AggIn stable HEK293 cell line.

Mentions: To test the aggregation efficiency of the transgene, we produced a HEK293 AggIn stable cell line. After tetracycline induction, anti-Flag staining showed the presence of many, prevalently cytosolic, aggregates (Fig. 1B, panel B, anti-FLAG +tet). Interestingly, several cell nuclei appeared to be devoid of endogenous TDP-43 (Fig. 1B, Panel B, anti-TDP-43 +tet; empty nuclei marked with asterisks). In order to analyze whether the formation of these aggregates was matched by loss of TDP-43 function, we evaluated the splicing profile of the endogenous gene POLDIP3, whose pre-mRNA processing is determined by TDP-43. In fact, knockdown of TDP-43 causes the exclusion of exon 3 from the mature POLDIP3 mRNA (variant 2) (Fiesel et al., 2012; Shiga et al., 2012). Similar to what happens with overexpression of the TDP-12×Q/N construct (Fig. 2A, upper panel, middle two lanes) (Budini et al., 2015), tetracycline induction of the AggIn protein expression was associated with a strong increase of POLDIP3 variant 2 at both the mRNA (Fig. 2A, upper panel, right-hand two lanes) and protein levels (Fig. 2A, lower panel). This effect is specific for constructs able to induce aggregation and efficient trapping of endogenous TDP-43: in fact, no alteration in the POLDIP3 splicing pattern was observed following overexpression of wild-type TDP-43 (Fig. 2A, upper panel, left-hand two lanes) or, as previously reported, of EGFP-12×Q/N (Budini et al., 2012b, 2015).Fig. 2.


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 TDP-43 target genes in the HEK293 AggIn stable cell line. (A) Upper panel, RT-PCR showing the splicing pattern of the endogenous POLDIP3 gene (exon 3) after tetracycline (tet) induction (+) or not (−) of Flag-tagged wild-type TDP-43 (TDP wt; left-hand two lanes), Flag-tagged TDP-12×Q/N (middle two lanes) or AggIn (right-hand two lanes) in stable HEK293 cell lines. Lower panel, western blot analysis, using anti-POLDIP3 antibody, of total protein lysates extracted from the same samples used in the two right-hand lanes of the upper panel. (B) RT-PCR showing the splicing pattern of endogenous BIM (exon 3) and MADD (exon 31) genes after tetracycline induction (+) or not (−) of the AggIn stable HEK293 cell line.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

DMM023382F2: Effect of transgene expression on TDP-43 target genes in the HEK293 AggIn stable cell line. (A) Upper panel, RT-PCR showing the splicing pattern of the endogenous POLDIP3 gene (exon 3) after tetracycline (tet) induction (+) or not (−) of Flag-tagged wild-type TDP-43 (TDP wt; left-hand two lanes), Flag-tagged TDP-12×Q/N (middle two lanes) or AggIn (right-hand two lanes) in stable HEK293 cell lines. Lower panel, western blot analysis, using anti-POLDIP3 antibody, of total protein lysates extracted from the same samples used in the two right-hand lanes of the upper panel. (B) RT-PCR showing the splicing pattern of endogenous BIM (exon 3) and MADD (exon 31) genes after tetracycline induction (+) or not (−) of the AggIn stable HEK293 cell line.
Mentions: To test the aggregation efficiency of the transgene, we produced a HEK293 AggIn stable cell line. After tetracycline induction, anti-Flag staining showed the presence of many, prevalently cytosolic, aggregates (Fig. 1B, panel B, anti-FLAG +tet). Interestingly, several cell nuclei appeared to be devoid of endogenous TDP-43 (Fig. 1B, Panel B, anti-TDP-43 +tet; empty nuclei marked with asterisks). In order to analyze whether the formation of these aggregates was matched by loss of TDP-43 function, we evaluated the splicing profile of the endogenous gene POLDIP3, whose pre-mRNA processing is determined by TDP-43. In fact, knockdown of TDP-43 causes the exclusion of exon 3 from the mature POLDIP3 mRNA (variant 2) (Fiesel et al., 2012; Shiga et al., 2012). Similar to what happens with overexpression of the TDP-12×Q/N construct (Fig. 2A, upper panel, middle two lanes) (Budini et al., 2015), tetracycline induction of the AggIn protein expression was associated with a strong increase of POLDIP3 variant 2 at both the mRNA (Fig. 2A, upper panel, right-hand two lanes) and protein levels (Fig. 2A, lower panel). This effect is specific for constructs able to induce aggregation and efficient trapping of endogenous TDP-43: in fact, no alteration in the POLDIP3 splicing pattern was observed following overexpression of wild-type TDP-43 (Fig. 2A, upper panel, left-hand two lanes) or, as previously reported, of EGFP-12×Q/N (Budini et al., 2012b, 2015).Fig. 2.

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