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Cytoplasmic mislocalization of RNA splicing factors and aberrant neuronal gene splicing in TDP-43 transgenic pig brain.

Wang G, Yang H, Yan S, Wang CE, Liu X, Zhao B, Ouyang Z, Yin P, Liu Z, Zhao Y, Liu T, Fan N, Guo L, Li S, Li XJ, Lai L - Mol Neurodegener (2015)

Bottom Line: We found that transgenic TDP-43 is also distributed in the cytoplasm of neuronal cells in the spinal cord and brain.The cytoplasmic localization of PSF and NeuN as well as abnormal PSF-related neuronal RNA splicing was also found in ALS patient brains.Our findings from a large mammalian model suggest that cytoplasmic mutant TDP-43 could reduce the nuclear function of RNA splicing factors, contributing to neuropathology.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China. ghwang85@gmail.com.

ABSTRACT

Background: TAR DNA-binding protein 43 (TDP-43) is a nuclear protein, but it is redistributed in the neuronal cytoplasm in both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Because small transgenic animal models often lack cytoplasmic TDP-43, how the cytoplasmic accumulation of TDP-43 contributes to these diseases remains unclear. The current study is aimed at studying the mechanism of cytoplasmic pathology of TDP-43.

Results: We established transgenic pigs expressing mutant TDP-43 (M337V). This pig model shows severe phenotypes and early death. We found that transgenic TDP-43 is also distributed in the cytoplasm of neuronal cells in the spinal cord and brain. Transgenic TDP-43 interacts with PSF, an RNA splicing factor that associates with NeuN to regulate neuronal RNA splicing. The interaction of TDP-43, PSF and NeuN causes PSF and NeuN mislocalize into the neuronal cytoplasm in transgenic pigs. Consistently, abnormal PSF-related neuronal RNA splicing is seen in TDP-43 transgenic pigs. The cytoplasmic localization of PSF and NeuN as well as abnormal PSF-related neuronal RNA splicing was also found in ALS patient brains.

Conclusion: Our findings from a large mammalian model suggest that cytoplasmic mutant TDP-43 could reduce the nuclear function of RNA splicing factors, contributing to neuropathology.

No MeSH data available.


Related in: MedlinePlus

TDP-43 interacts with PSF and NeuN. a Silver-staining of 4-12 % SDS-PAGE gel showing GST-TDP-43 pull-down from pig cortex lysates. GST served as a control. The indicated band was analyzed by mass spectrometry and found to contain PSF. b Western blotting with anti-PSF verifying the interaction of GST-TDP-43 and PSF in pig brain cortex lysates. Two GST and GST-TDP-43 pull-down proteins were analyzed. c Purified His-PSF was incubated with transgenic pig brain cortex lysates, and the bound proteins were subjected to western blot, which shows the in vitro direct interaction of endogenous pig TDP-43 and transgenic human TDP-43 with His-PSF. Input is 5 % of purified His-PSF, and 40 % of pulldown was loaded into the SDS gel. d PSF contains N-terminal, RRM1, RRM2, and C-terminal domains. The N-terminal domain is a glycine/proline/arginine rich domain, the C-terminal and the NOPS regions of PSF were found to bind to TDP-43. TDP-43 contains N-terminal, RRM1, RRM2, and C-terminal (G-rich) domains. e Purification of His-PSF containing different domains as indicated with the corresponding amino acid positions. f In vitro GST-pull down identifying that the C-terminal regions containing amino acids 444–707 of PSF bind to C-terminal GST-TDP-43 (amino acids 264–414), but not N-terminal TDP-43 (amino acids 1–251)
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Fig5: TDP-43 interacts with PSF and NeuN. a Silver-staining of 4-12 % SDS-PAGE gel showing GST-TDP-43 pull-down from pig cortex lysates. GST served as a control. The indicated band was analyzed by mass spectrometry and found to contain PSF. b Western blotting with anti-PSF verifying the interaction of GST-TDP-43 and PSF in pig brain cortex lysates. Two GST and GST-TDP-43 pull-down proteins were analyzed. c Purified His-PSF was incubated with transgenic pig brain cortex lysates, and the bound proteins were subjected to western blot, which shows the in vitro direct interaction of endogenous pig TDP-43 and transgenic human TDP-43 with His-PSF. Input is 5 % of purified His-PSF, and 40 % of pulldown was loaded into the SDS gel. d PSF contains N-terminal, RRM1, RRM2, and C-terminal domains. The N-terminal domain is a glycine/proline/arginine rich domain, the C-terminal and the NOPS regions of PSF were found to bind to TDP-43. TDP-43 contains N-terminal, RRM1, RRM2, and C-terminal (G-rich) domains. e Purification of His-PSF containing different domains as indicated with the corresponding amino acid positions. f In vitro GST-pull down identifying that the C-terminal regions containing amino acids 444–707 of PSF bind to C-terminal GST-TDP-43 (amino acids 264–414), but not N-terminal TDP-43 (amino acids 1–251)

Mentions: TDP-43 is involved in gene transcription and RNA splicing via its interactions with other proteins. To identify any pig proteins that could interact with TDP-43 and are involved in TDP-43-mediated toxicity in transgenic pigs, we generated GST-TDP-43 and used it to pull down pig brain cortex proteins. The GST pull-down uncovered a band that is specific to GST-TDP-43 pull-down (Fig. 5a). Via mass spectrometry, this band was identified as the splicing factor proline-glutamine rich (SFPQ/PSF) or PSF, an essential pre-mRNA splicing factor required in spliceosome formation [32–34]. Western blot analysis confirmed the interaction of PSF in pig brain with GST-TDP-43 (Fig. 5b). We also generated His-tagged PSF and purified this recombined protein from bacteria. In vitro binding showed that His-PSF could bind endogenous pig TDP-43 and transgenic hTDP-43 in the pig brain cortex lysates (Fig. 5c). Like TDP-43, PSF contains N-terminal, RRM1, RRM2 and C-terminal domains. To define the binding region in TDP-43, we generated GST fusion proteins containing different TDP-43 fragments (1–251, 1–414, and 264–414 amino acids) and used these GST fusion proteins to incubate with His-PSF proteins containing different regions (Fig. 5d). The results revealed that C-terminal PSF (444–707 amino acids) binds GST fusion protein containing C-terminal (264–414 amino acids) TDP-43 (Fig. 5e and f).Fig. 5


Cytoplasmic mislocalization of RNA splicing factors and aberrant neuronal gene splicing in TDP-43 transgenic pig brain.

Wang G, Yang H, Yan S, Wang CE, Liu X, Zhao B, Ouyang Z, Yin P, Liu Z, Zhao Y, Liu T, Fan N, Guo L, Li S, Li XJ, Lai L - Mol Neurodegener (2015)

TDP-43 interacts with PSF and NeuN. a Silver-staining of 4-12 % SDS-PAGE gel showing GST-TDP-43 pull-down from pig cortex lysates. GST served as a control. The indicated band was analyzed by mass spectrometry and found to contain PSF. b Western blotting with anti-PSF verifying the interaction of GST-TDP-43 and PSF in pig brain cortex lysates. Two GST and GST-TDP-43 pull-down proteins were analyzed. c Purified His-PSF was incubated with transgenic pig brain cortex lysates, and the bound proteins were subjected to western blot, which shows the in vitro direct interaction of endogenous pig TDP-43 and transgenic human TDP-43 with His-PSF. Input is 5 % of purified His-PSF, and 40 % of pulldown was loaded into the SDS gel. d PSF contains N-terminal, RRM1, RRM2, and C-terminal domains. The N-terminal domain is a glycine/proline/arginine rich domain, the C-terminal and the NOPS regions of PSF were found to bind to TDP-43. TDP-43 contains N-terminal, RRM1, RRM2, and C-terminal (G-rich) domains. e Purification of His-PSF containing different domains as indicated with the corresponding amino acid positions. f In vitro GST-pull down identifying that the C-terminal regions containing amino acids 444–707 of PSF bind to C-terminal GST-TDP-43 (amino acids 264–414), but not N-terminal TDP-43 (amino acids 1–251)
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Fig5: TDP-43 interacts with PSF and NeuN. a Silver-staining of 4-12 % SDS-PAGE gel showing GST-TDP-43 pull-down from pig cortex lysates. GST served as a control. The indicated band was analyzed by mass spectrometry and found to contain PSF. b Western blotting with anti-PSF verifying the interaction of GST-TDP-43 and PSF in pig brain cortex lysates. Two GST and GST-TDP-43 pull-down proteins were analyzed. c Purified His-PSF was incubated with transgenic pig brain cortex lysates, and the bound proteins were subjected to western blot, which shows the in vitro direct interaction of endogenous pig TDP-43 and transgenic human TDP-43 with His-PSF. Input is 5 % of purified His-PSF, and 40 % of pulldown was loaded into the SDS gel. d PSF contains N-terminal, RRM1, RRM2, and C-terminal domains. The N-terminal domain is a glycine/proline/arginine rich domain, the C-terminal and the NOPS regions of PSF were found to bind to TDP-43. TDP-43 contains N-terminal, RRM1, RRM2, and C-terminal (G-rich) domains. e Purification of His-PSF containing different domains as indicated with the corresponding amino acid positions. f In vitro GST-pull down identifying that the C-terminal regions containing amino acids 444–707 of PSF bind to C-terminal GST-TDP-43 (amino acids 264–414), but not N-terminal TDP-43 (amino acids 1–251)
Mentions: TDP-43 is involved in gene transcription and RNA splicing via its interactions with other proteins. To identify any pig proteins that could interact with TDP-43 and are involved in TDP-43-mediated toxicity in transgenic pigs, we generated GST-TDP-43 and used it to pull down pig brain cortex proteins. The GST pull-down uncovered a band that is specific to GST-TDP-43 pull-down (Fig. 5a). Via mass spectrometry, this band was identified as the splicing factor proline-glutamine rich (SFPQ/PSF) or PSF, an essential pre-mRNA splicing factor required in spliceosome formation [32–34]. Western blot analysis confirmed the interaction of PSF in pig brain with GST-TDP-43 (Fig. 5b). We also generated His-tagged PSF and purified this recombined protein from bacteria. In vitro binding showed that His-PSF could bind endogenous pig TDP-43 and transgenic hTDP-43 in the pig brain cortex lysates (Fig. 5c). Like TDP-43, PSF contains N-terminal, RRM1, RRM2 and C-terminal domains. To define the binding region in TDP-43, we generated GST fusion proteins containing different TDP-43 fragments (1–251, 1–414, and 264–414 amino acids) and used these GST fusion proteins to incubate with His-PSF proteins containing different regions (Fig. 5d). The results revealed that C-terminal PSF (444–707 amino acids) binds GST fusion protein containing C-terminal (264–414 amino acids) TDP-43 (Fig. 5e and f).Fig. 5

Bottom Line: We found that transgenic TDP-43 is also distributed in the cytoplasm of neuronal cells in the spinal cord and brain.The cytoplasmic localization of PSF and NeuN as well as abnormal PSF-related neuronal RNA splicing was also found in ALS patient brains.Our findings from a large mammalian model suggest that cytoplasmic mutant TDP-43 could reduce the nuclear function of RNA splicing factors, contributing to neuropathology.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China. ghwang85@gmail.com.

ABSTRACT

Background: TAR DNA-binding protein 43 (TDP-43) is a nuclear protein, but it is redistributed in the neuronal cytoplasm in both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Because small transgenic animal models often lack cytoplasmic TDP-43, how the cytoplasmic accumulation of TDP-43 contributes to these diseases remains unclear. The current study is aimed at studying the mechanism of cytoplasmic pathology of TDP-43.

Results: We established transgenic pigs expressing mutant TDP-43 (M337V). This pig model shows severe phenotypes and early death. We found that transgenic TDP-43 is also distributed in the cytoplasm of neuronal cells in the spinal cord and brain. Transgenic TDP-43 interacts with PSF, an RNA splicing factor that associates with NeuN to regulate neuronal RNA splicing. The interaction of TDP-43, PSF and NeuN causes PSF and NeuN mislocalize into the neuronal cytoplasm in transgenic pigs. Consistently, abnormal PSF-related neuronal RNA splicing is seen in TDP-43 transgenic pigs. The cytoplasmic localization of PSF and NeuN as well as abnormal PSF-related neuronal RNA splicing was also found in ALS patient brains.

Conclusion: Our findings from a large mammalian model suggest that cytoplasmic mutant TDP-43 could reduce the nuclear function of RNA splicing factors, contributing to neuropathology.

No MeSH data available.


Related in: MedlinePlus