Publications by authors named "Che-Kun James Shen"

42 Publications

Cabozantinib promotes erythroid differentiation in K562 erythroleukemia cells through global changes in gene expression and JNK activation.

Cancer Gene Ther 2021 Jun 11. Epub 2021 Jun 11.

Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei, Taiwan.

Cabozantinib is a potent tyrosine kinase inhibitor with multiple targets including MET, VEGFR2, RET, KIT, and FLT3. Cabozantinib is widely used for the treatment of medullary thyroid cancer and renal cell carcinoma. We recently suggested cabozantinib as a potential therapeutic alternative for acute myeloid leukemia (AML) patients with FLT3-internal tandem duplication (FLT3-ITD). Here, we report that cabozantinib can promote differentiation in erythroid leukemia cells. We found that K562 erythroid leukemia cells treated with 1 μM cabozantinib for 72 h underwent erythroid lineage differentiation. Transcriptomic analysis revealed that various pathways associated with heme biosynthesis, hemoglobin production, and GATA1 targets were upregulated, whereas cell survival pathways were downregulated. Further examination revealed that cabozantinib-induced erythroid differentiation is at least in part regulated by JNK activation and phosphorylation. Levels of phosphorylated BCR-ABL, AKT, STAT5, ERK, and p38 also decreased following cabozantinib treatment. Therefore, we indicate that cabozantinib has dual functions. First, it induces K562 cell differentiation toward the erythroid lineage by upregulating heme biosynthesis, globin synthesis, and erythroid-associated reactions. Second, cabozantinib inhibits K562 cell proliferation by inhibiting the phosphorylation of BCR-ABL and the downstream MAPK, PI3K-AKT, and JAK-STAT signaling pathways.
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http://dx.doi.org/10.1038/s41417-021-00358-wDOI Listing
June 2021

Negative Regulation of the Differentiation of Flk2 CD34 LSK Hematopoietic Stem Cells by EKLF/KLF1.

Int J Mol Sci 2020 Nov 10;21(22). Epub 2020 Nov 10.

Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan.

Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2 CD34 Lin Sca1 c-Kit (LSK)-HSC. In an interesting correlation, is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor β subunit (CSF2RB). As a result, Flk2 CD34 LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2 CD34 LSK-HSCs.
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http://dx.doi.org/10.3390/ijms21228448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697791PMC
November 2020

Potent and orally active purine-based fetal hemoglobin inducers for treating β-thalassemia and sickle cell disease.

Eur J Med Chem 2021 Jan 16;209:112938. Epub 2020 Oct 16.

Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Rd., Zhunan Town, Miaoli Country, 35053, Taiwan, ROC. Electronic address:

Reactivation of fetal hemoglobin (HbF) expression by therapeutic agents has been suggested as an alternative treatment to modulate anemia and the related symptoms of severe β-thalassemia and sickle cell disease (SCD). Hydroxyurea (HU) is the first US FDA-approved HbF inducer for treating SCD. However, approximately 25% of the patients with SCD do not respond to HU. A previous study identified TN1 (1) as a small-molecule HbF inducer. However, this study found that the poor potency and oral bioavailability of compound 1 limits the development of this inducer for clinical use. To develop drug-like compounds, further structure-activity relationship studies on the purine-based structure of 1 were conducted. Herein, we report our discovery of a more potent inducer, compound 13a, that can efficiently induce γ-globin gene expression at non-cytotoxic concentrations. The molecular mechanism of 13a, for the regulation HbF expression, was also investigated. In addition, we demonstrated that oral administration of 13a can ameliorate anemia and the related symptoms in SCD mice. The results of this study suggest that 13a can be further developed as a novel agent for treating hemoglobinopathies, such as β-thalassemia and SCD.
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http://dx.doi.org/10.1016/j.ejmech.2020.112938DOI Listing
January 2021

A robust TDP-43 knock-in mouse model of ALS.

Acta Neuropathol Commun 2020 01 21;8(1). Epub 2020 Jan 21.

Institute of Molecular Biology, Academia Sinica, Taipei, 11529, Taiwan, Republic of China.

Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset degenerative disorder of motor neurons. The diseased spinal cord motor neurons of more than 95% of amyotrophic lateral sclerosis (ALS) patients are characterized by the mis-metabolism of the RNA/DNA-binding protein TDP-43 (ALS-TDP), in particular, the presence of cytosolic aggregates of the protein. Most available mouse models for the basic or translational studies of ALS-TDP are based on transgenic overexpression of the TDP-43 protein. Here, we report the generation and characterization of mouse lines bearing homologous knock-in of fALS-associated mutation A315T and sALS-associated mutation N390D, respectively. Remarkably, the heterozygous TDP-43 (N390D/+) mice but not those heterozygous for the TDP-43 (A315T/+) mice develop a full spectrum of ALS-TDP-like pathologies at the molecular, cellular and behavioral levels. Comparative analysis of the mutant mice and spinal cord motor neurons (MN) derived from their embryonic stem (ES) cells demonstrates that different ALS-associated TDP-43 mutations possess critical ALS-causing capabilities and pathogenic pathways, likely modified by their genetic background and the environmental factors. Mechanistically, we identify aberrant RNA splicing of spinal cord Bcl-2 pre-mRNA and consequent increase of a negative regulator of autophagy, Bcl-2, which correlate with and are caused by a progressive increase of TDP-43, one of the early events associated with ALS-TDP pathogenesis, in the spinal cord of TDP-43 (N390D/+) mice and spinal cord MN derived from their ES cells. The TDP-43 (N390D/+) knock-in mice appear to be an ideal rodent model for basic as well as translational studies of ALS- TDP.
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http://dx.doi.org/10.1186/s40478-020-0881-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6975031PMC
January 2020

TDP-43 facilitates milk lipid secretion by post-transcriptional regulation of Btn1a1 and Xdh.

Nat Commun 2020 01 17;11(1):341. Epub 2020 Jan 17.

State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 650223, Kunming, China.

Milk lipid secretion is a critical process for the delivery of nutrition and energy from parent to offspring. However, the underlying molecular mechanism is less clear. Here we report that TDP-43, a RNA-binding protein, underwent positive selection in the mammalian lineage. Furthermore, TDP-43 gene (Tardbp) loss induces accumulation of large lipid droplets and severe lipid secretion deficiency in mammary epithelial cells to outside alveolar lumens, eventually resulting in lactation failure and pup starvation within three weeks postpartum. In human milk samples from lactating women, the expression levels of TDP-43 is positively correlated with higher milk output. Mechanistically, TDP-43 exerts post-transcriptional regulation of Btn1a1 and Xdh mRNA stability, which are required for the secretion of lipid droplets from epithelial cells to the lumen. Taken together, our results highlights the critical role of TDP-43 in milk lipid secretion, providing a potential strategy for the screening and intervention of clinical lactation insufficiency.
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http://dx.doi.org/10.1038/s41467-019-14183-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969145PMC
January 2020

TDP-43 Regulates Coupled Dendritic mRNA Transport-Translation Processes in Co-operation with FMRP and Staufen1.

Cell Rep 2019 Dec;29(10):3118-3133.e6

Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan. Electronic address:

Tightly regulated transport of messenger ribonucleoprotein (mRNP) granules to diverse locations of dendrites and axons is essential for appropriately timed protein synthesis within distinct sub-neuronal compartments. Perturbations of this regulation lead to various neurological disorders. Using imaging and molecular approaches, we demonstrate how TDP-43 co-operates with two other RNA-binding proteins, FMRP and Staufen1, to regulate the anterograde and retrograde transport, respectively, of Rac1 mRNPs in mouse neuronal dendrites. We also analyze the mechanisms by which TDP-43 mediates coupled mRNA transport-translation processes in dendritic sub-compartments by following in real-time the co-movement of RNA and endogenous fluorescence-tagged protein in neurons and by simultaneous examination of transport/translation dynamics by using an RNA biosensor. This study establishes the pivotal roles of TDP-43 in transporting mRNP granules in dendrites, inhibiting translation inside those granules, and reactivating it once the granules reach the dendritic spines.
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http://dx.doi.org/10.1016/j.celrep.2019.10.061DOI Listing
December 2019

TDP-43 is Required for Mammary Gland Repopulation and Proliferation of Mammary Epithelial Cells.

Stem Cells Dev 2019 07 11;28(14):944-953. Epub 2019 Jun 11.

2State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China.

Mammary gland stem cells (MaSCs), assumed to be the original cells of breast cancer, play essential roles in regulating mammary gland homeostasis and development. Previously, we identified a crucial regulatory role of TAR DNA-binding protein 43 (TDP-43), an RNA-binding protein, in the progression of triple-negative breast cancer. However, the function of TDP-43 in MaSCs is unclear. Based on single-cell data analysis of the mammary gland, TDP-43 showed potential involvement in the regulation of MaSCs. We therefore investigated the effects of TDP-43 on the mammary gland development. Our data both in vitro and in vivo demonstrated that TDP-43 was required for the mammary gland repopulation, which suggested the potential role in the regulation of MaSCs. Knockdown of TDP-43 inhibited proliferation of mammary epithelial cells (MECs) and mammary morphogenesis. RNA-seq data and other experiments identified that loss of TDP-43 induced the upregulation of genes related to the cell cycle, providing a possible mechanism for TDP-43 in regulating mammary gland repopulation. Thus, our findings indicate a previously unknown role of TDP-43 in MECs.
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http://dx.doi.org/10.1089/scd.2019.0011DOI Listing
July 2019

Znf179 E3 ligase-mediated TDP-43 polyubiquitination is involved in TDP-43- ubiquitinated inclusions (UBI) (+)-related neurodegenerative pathology.

J Biomed Sci 2018 Nov 8;25(1):76. Epub 2018 Nov 8.

Graduate Institute of Neural Regenerative Medicine, College of Medical Science and Technology/Center for Neurotrauma and Neuroregeneration, Taipei Medical University, Taipei, 115, Taiwan.

Background: The brain predominantly expressed RING finger protein, Znf179, is known to be important for embryonic neuronal differentiation during brain development. Downregulation of Znf179 has been observed in motor neurons of adult mouse models for amyotrophic lateral sclerosis (ALS), yet the molecular function of Znf179 in neurodegeneration has never been previously described. Znf179 contains the classical C3HC4 RING finger domain, and numerous proteins containing C3HC4 RING finger domain act as E3 ubiquitin ligases. Hence, we are interested to identify whether Znf179 possesses E3 ligase activity and its role in ALS neuropathy.

Methods: We used in vivo and in vitro ubiquitination assay to examine the E3 ligase autoubiquitination activity of Znf179 and its effect on 26S proteasome activity. To search for the candidate substrates of Znf179, we immunoprecipitated Znf179 and subjected to mass spectrometry (MS) analysis to identify its interacting proteins. We found that ALS/ FTLD-U (frontotemporal lobar degeneration (FTLD) with ubiquitin inclusions)-related neurodegenerative TDP-43 protein is the E3 ligase substrate of Znf179. To further clarify the role of E3 ubiquitin ligase Znf179 in neurodegenerative TDP-43-UBI (ubiquitinated inclusions) (+) proteinopathy, the effect of Znf179-mediated TDP-43 polyubiquitination on TDP-43 protein stability, aggregate formation and nucleus/cytoplasm mislocalization were evaluated in vitro cell culture system and in vivo animal model.

Results: Here we report that Znf179 is a RING E3 ubiquitin ligase which possesses autoubiquitination feature and regulates 26S proteasome activity through modulating the protein expression levels of 19S/20S proteasome subunits. Our immunoprecipitation assay and MS analysis results revealed that the neuropathological TDP-43 protein is one of its E3 ligase substrate. Znf179 interactes with TDP-43 protein and mediates polyubiquitination of TDP-43 in vitro and in vivo. In neurodegenerative TDP-43 proteinopathy, we found that Znf179-mediated polyubiquitination of TDP-43 accelerates its protein turnover rate and attenuates insoluble pathologic TDP-43 aggregates, while knockout of Znf179 in mouse brain results in accumulation of insoluble TDP-43 and cytosolic TDP-43 inclusions in cortex, hippocampus and midbrain regions.

Conclusions: Here we unveil the important role for the novel E3 ligase Znf179 in TDP-43-mediated neuropathy, and provide a potential therapeutic strategy for combating ALS/ FTLD-U neurodegenerative pathologies.
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http://dx.doi.org/10.1186/s12929-018-0479-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223059PMC
November 2018

DNA Demethylation by DNMT3A and DNMT3B in vitro and of Methylated Episomal DNA in Transiently Transfected Cells.

Biochim Biophys Acta Gene Regul Mech 2018 11 6;1861(11):1048-1061. Epub 2018 Oct 6.

Institute of Molecular Biology, Academia Sinica, Taipei City 115, Taiwan. Electronic address:

The DNA methylation program in vertebrates is an essential part of the epigenetic regulatory cascade of development, cell differentiation, and progression of diseases including cancer. While the DNA methyltransferases (DNMTs) are responsible for the in vivo conversion of cytosine (C) to methylated cytosine (5mC), demethylation of 5mC on cellular DNA could be accomplished by the combined action of the ten-eleven translocation (TET) enzymes and DNA repair. Surprisingly, the mammalian DNMTs also possess active DNA demethylation activity in vitro in a Ca- and redox conditions-dependent manner, although little is known about its molecular mechanisms and occurrence in a cellular context. In this study, we have used LC-MS/MS to track down the fate of the methyl group removed from 5mC on DNA by mouse DNMT3B in vitro and found that it becomes covalently linked to the DNA methylation catalytic cysteine of the enzyme. We also show that Ca homeostasis-dependent but TET1/TET2/TET3/TDG-independent demethylation of methylated episomal DNA by mouse DNMT3A or DNMT3B can occur in transfected human HEK 293 and mouse embryonic stem (ES) cells. Based on these results, we present a tentative working model of Ca and redox conditions-dependent active DNA demethylation by DNMTs. Our study substantiates the potential roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation during Ca-dependent physiological processes.
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http://dx.doi.org/10.1016/j.bbagrm.2018.09.009DOI Listing
November 2018

A placental growth factor is silenced in mouse embryos by the zinc finger protein ZFP568.

Science 2017 05;356(6339):757-759

The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

Insulin-like growth factor 2 (IGF2) is the major fetal growth hormone in mammals. We identify zinc finger protein 568 (ZFP568), a member of the rapidly evolving Kruppel-associated box-zinc finger protein (KRAB-ZFP) family linked primarily to silencing of endogenous retroelements, as a direct repressor of a placental-specific transcript (designated ) in mice. Loss of , which causes gastrulation failure, or mutation of the ZFP568-binding site at the promoter causes inappropriate activation. Deletion of can completely rescue gastrulation phenotypes through late gestation. Our data highlight the exquisite selectivity with which members of the KRAB-ZFP family repress their targets and identify an additional layer of transcriptional control of a key growth factor regulating fetal and placental development.
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http://dx.doi.org/10.1126/science.aah6895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309218PMC
May 2017

Epigenetic Enhancement of the Post-replicative DNA Mismatch Repair of Mammalian Genomes by a Hemi-CpG-Np95-Dnmt1 Axis.

Sci Rep 2016 11 25;6:37490. Epub 2016 Nov 25.

Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan.

DNA methylation at C of CpG dyads (CpG) in vertebrate genomes is essential for gene regulation, genome stability and development. We show in this study that proper functioning of post-replicative DNA mismatch repair (MMR) in mammalian cells relies on the presence of genomic CpG, as well as on the maintenance DNA methyltransferase Dnmt1 independently of its catalytic activity. More importantly, high efficiency of mammalian MMR surveillance is achieved through a hemi-CpG-Np95(Uhrf1)-Dnmt1 axis, in which the MMR surveillance complex(es) is recruited to post-replicative DNA by Dnmt1, requiring its interactions with MutSα, as well as with Np95 bound at the hemi-methylated CpG sites. Thus, efficiency of MMR surveillance over the mammalian genome in vivo is enhanced at the epigenetic level. This synergy endows vertebrate CpG methylation with a new biological significance and, consequently, an additional mechanism for the maintenance of vertebrate genome stability.
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http://dx.doi.org/10.1038/srep37490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122852PMC
November 2016

Therapeutic effect of berberine on TDP-43-related pathogenesis in FTLD and ALS.

J Biomed Sci 2016 Oct 21;23(1):72. Epub 2016 Oct 21.

Graduate Institute of Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.

Background: In the central nervous system regions of the sporadic and familial FTLD and ALS patients, TDP-43 has been identified as the major component of UBIs inclusions which is abnormally hyperphosphorylated, ubiquitinated, and cleaved into C-terminal fragments to form detergent-insoluble aggregates. So far, the effective drugs for FTLD and ALS neurodegenerative diseases are yet to be developed. Autophagy has been demonstrated as the major metabolism route of the pathological TDP-43 inclusions, hence activation of autophagy is a potential therapeutic strategy for TDP-43 pathogenesis in FTLD and ALS. Berberine, a traditional herbal medicine, is an inhibitor of mTOR signal and an activator for autophagy. Berberine has been implicated in several kinds of diseases, including the neuronal-related pathogenesis, such as Parkinson's, Huntington's and Alzheimer's diseases. However, the therapeutic effect of berberine on FTLD or ALS pathology has never been investigated.

Results: Here we studied the molecular mechanism of berberine in cell culture model with TDP-43 proteinopathies, and found that berberine is able to reverse the processing of insoluble TDP-43 aggregates formation through deregulation of mTOR/p70S6K signal and activation of autophagic degradation pathway. And inhibition of autophagy by specific autophagosome inhibitor, 3-MA, reverses the effect of berberine on reducing the accumulation of insoluble TDP-43 and aggregates formation. These results gave us the notion that inhibition of autophagy by 3-MA reverses the effect of berberine on TDP-43 pathogenesis, and activation of mTOR-regulated autophagy plays an important role in berberine-mediated therapeutic effect on TDP-43 proteinopathies.

Conclusion: We supported an important notion that the traditional herb berberine is a potential alternative therapy for TDP-43-related neuropathology. Here we demonstrated that berberine is able to reverse the processing of insoluble TDP-43 aggregates formation through deregulation of mTOR/p70S6K signal and activation of autophagic degradation pathway. mTOR-autophagy signals plays an important role in berberine-mediated autophagic clearance of TDP-43 aggregates. Exploring the detailed mechanism of berberine on TDP-43 proteinopathy provides a better understanding for the therapeutic development in FTLD and ALS.
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http://dx.doi.org/10.1186/s12929-016-0290-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5073438PMC
October 2016

Co-regulation of mRNA translation by TDP-43 and Fragile X Syndrome protein FMRP.

Acta Neuropathol 2016 11 12;132(5):721-738. Epub 2016 Aug 12.

Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, 115, Taiwan.

For proper mammalian brain development and functioning, the translation of many neuronal mRNAs needs to be repressed without neuronal activity stimulations. We have discovered that the expression of a subclass of neuronal proteins essential for neurodevelopment and neuron plasticity is co-regulated at the translational level by TDP-43 and the Fragile X Syndrome protein FMRP. Using molecular, cellular and imaging approaches, we show that these two RNA-binding proteins (RBP) co-repress the translation initiation of Rac1, Map1b and GluR1 mRNAs, and consequently the hippocampal spinogenesis. The co-repression occurs through binding of TDP-43 to mRNA(s) at specific UG/GU sequences and recruitment of the inhibitory CYFIP1-FMRP complex by its glycine-rich domain. This novel regulatory scenario could be utilized to silence a significant portion of around 160 common target mRNAs of the two RBPs. The study establishes a functional/physical partnership between FMRP and TDP-43 that mechanistically links several neurodevelopmental disorders and neurodegenerative diseases.
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http://dx.doi.org/10.1007/s00401-016-1603-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5073124PMC
November 2016

G-CSF-mobilized Bone Marrow Mesenchymal Stem Cells Replenish Neural Lineages in Alzheimer's Disease Mice via CXCR4/SDF-1 Chemotaxis.

Mol Neurobiol 2017 Oct 5;54(8):6198-6212. Epub 2016 Oct 5.

Institute of Basic Medical Science, National Cheng Kung University, Tainan, Taiwan.

Recent studies reported granulocyte colony-stimulating factor (G-CSF) treatment can improve the cognitive function of Alzheimer's disease (AD) mice, and the mobilized hematopoietic stem cells (HSCs) or bone marrow mesenchymal stem cells (BM-MSCs) are proposed to be involved in this recovery effect. However, the exact role of mobilized HSC/BM-MSC in G-CSF-based therapeutic effects is still unknown. Here, we report that C-X-C chemokine receptor type 4 (CXCR4)/stromal cell-derived factor 1 (SDF-1) chemotaxis was a key mediator in G-CSF-based therapeutic effects, which was involved in the recruitment of repair-competent cells. Furthermore, we found both mobilized HSCs and BM-MSCs were able to infiltrate into the brain, but only BM-MSCs replenished the neural lineage cells and contributed to neurogenesis in the brains of AD mice. Together, our data show that mobilized BM-MSCs are involved in the replenishment of neural lineages following G-CSF treatment via CXCR4/SDF-1 chemotaxis and further support the potential use of BM-MSCs for further autogenically therapeutic applications.
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http://dx.doi.org/10.1007/s12035-016-0122-xDOI Listing
October 2017

Structural analysis of disease-related TDP-43 D169G mutation: linking enhanced stability and caspase cleavage efficiency to protein accumulation.

Sci Rep 2016 Feb 17;6:21581. Epub 2016 Feb 17.

Institute of Molecular Biology, Academia Sinica, Taipei, 11529, Taiwan.

The RNA-binding protein TDP-43 forms intracellular inclusions in amyotrophic lateral sclerosis (ALS). While TDP-43 mutations have been identified in ALS patients, how these mutations are linked to ALS remains unclear. Here we examined the biophysical properties of six ALS-linked TDP-43 mutants and found that one of the mutants, D169G, had higher thermal stability than wild-type TDP-43 and that it was cleaved by caspase 3 more efficiently, producing increased levels of the C-terminal 35 kD fragments (TDP-35) in vitro and in neuroblastoma cells. The crystal structure of the TDP-43 RRM1 domain containing the D169G mutation in complex with DNA along with molecular dynamics simulations reveal that the D169G mutation induces a local conformational change in a β turn and increases the hydrophobic interactions in the RRM1 core, thus enhancing the thermal stability of the RRM1 domain. Our results provide the first crystal structure of TDP-43 containing a disease-linked D169G mutation and a disease-related mechanism showing that D169G mutant is more susceptible to proteolytic cleavage by caspase 3 into the pathogenic C-terminal 35-kD fragments due to its increased stability in the RRM1 domain. Modulation of TDP-43 stability and caspase cleavage efficiency could present an avenue for prevention and treatment of TDP-43-linked neurodegeneration.
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http://dx.doi.org/10.1038/srep21581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756693PMC
February 2016

Rapamycin alleviates pathogenesis of a new Drosophila model of ALS-TDP.

J Neurogenet 2015 25;29(2-3):59-68. Epub 2015 Sep 25.

a Institute of Molecular Medicine, College of Medicine, National Taiwan University , Taipei , Taiwan.

TDP-43 is a multi-functional RNA/DNA-binding protein, well-conserved among many species including mammals and Drosophila. However, it is also a major component of the pathological inclusions associated with degenerating motor neurons of amyotrophic lateral sclerosis (ALS). Further, TDP-43 is a signature protein in one subtype of frontotemporal degeneration, FTLD-U. Currently, there are no effective drugs for these neurodegenerative diseases. We describe the generation and characterization of a new fly model of ALS-TDP with transgenic expression of the Drosophila ortholog of TDP-43, dTDP, in adult flies under the control of a temperature-sensitive motor neuron-specific GAL4, thus bypassing the deleterious effect of dTDP during development. Diminished lifespan as well as impaired locomotor activities of the flies following induction of dTDP overexpression have been observed. Dissection of the T1/T2 region of the thoracic ganglia has revealed loss of these neurons. To counter the defects in this fly model of ALS-TDP, we have examined the therapeutic effects of the autophagy activator, rapamycin. Although harmful to the control flies, administration of 400 μM rapamycin before the induction of dTDP overexpression can significantly reduce the number of neurons bearing dTDP (+) aggregates, as well as partially rescue the diminished lifespan and locomotive defects of the ALS-TDP flies. Furthermore, we identify S6K, a downstream mediator of the TOR pathway, as one genetic modifier of dTDP. In sum, this Drosophila model of ALS-TDP under temporal and spatial control presents a useful new genetic tool for the screening and validation of therapeutic drugs for ALS. Furthermore, the data support our previous finding that autophagy activators including rapamycin are potential therapeutic drugs for the progression of neurodegenerative diseases with TDP-43 proteinopathies.
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http://dx.doi.org/10.3109/01677063.2015.1077832DOI Listing
August 2016

Pharmacological Induction of Human Fetal Globin Gene in Hydroxyurea-Resistant Primary Adult Erythroid Cells.

Mol Cell Biol 2015 Jul 18;35(14):2541-53. Epub 2015 May 18.

Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

Pharmacological induction of the fetal γ globin gene and the consequent formation of HbF (α2/γ2) in adult erythroid cells are one feasible therapeutic strategy for sickle cell disease (SCD) and severe β-thalassemias. Hydroxyurea (HU) is the current drug of choice for SCD, but serious side effects limit its clinical use. Moreover, 30 to 50% of patients are irresponsive to HU treatment. We have used high-throughput screening to identify benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one and its derivatives (compounds I to VI) as potent γ globin inducers. Of the compounds, I to V exert superior γ globin induction and have better therapeutic potential than HU, likely because of their activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway and modulation of expression levels and/or chromosome binding of γ globin gene regulators, including BCL11A, and chromatin structure over the γ globin promoter. Unlike sodium butyrate (NaB), the global levels of acetylated histones H3 and H4 are not changed by compound II treatment. Remarkably, compound II induces the γ globin gene in HU-resistant primary human adult erythroid cells, the p38 signaling pathway of which appears to be irresponsive to HU and NaB as well as compound II. This study provides a new framework for the development of new and superior compounds for treating SCD and severe β-thalassemias.
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http://dx.doi.org/10.1128/MCB.00035-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475914PMC
July 2015

Spermidine on neurodegenerative diseases.

Cell Cycle 2015 ;14(5):697-8

a Institute of Life Science ; National Defense Medical Center ; Taipei , Taiwan.

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http://dx.doi.org/10.1080/15384101.2015.1006551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614629PMC
May 2016

Disruption of the nuclear membrane by perinuclear inclusions of mutant huntingtin causes cell-cycle re-entry and striatal cell death in mouse and cell models of Huntington's disease.

Hum Mol Genet 2015 Mar 14;24(6):1602-16. Epub 2014 Nov 14.

Developmental Biology Center, Department of Developmental and Cell Biology

Accumulation of N-terminal fragments of mutant huntingtin (mHTT) in the cytoplasm, nuclei and axons of neurons is a hallmark of Huntington's disease (HD), although how these fragments negatively impact neurons remains unclear. We followed the distribution of mHTT in the striata of transgenic R6/2-J2 HD mice as their motor function declined. The fraction of cells with diffuse, perinuclear or intranuclear mHTT changed in parallel with decreasing motor function. In transgenic mice, medium spiny neurons (MSNs) that exhibited perinuclear inclusions expressed cell-cycle markers typically not seen in the striata of normal mice, and these cells are preferentially lost as disease progresses. Electron microscopy reveals that perinuclear inclusions disrupt the nuclear envelope. The progression of perinuclear inclusions being accompanied by cell-cycle activation and culminating in cell death was also observed in 1° cortical neurons. These observations provide a strong correlation between the subcellular location of mHTT, disruption of the nucleus, re-entry into the cell-cycle and eventual neuronal death. They also highlight the fact that the subcellular distribution of mHTT is highly dynamic such that the distribution of mHTT observed depends greatly on the stage of the disease being examined.
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http://dx.doi.org/10.1093/hmg/ddu574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381756PMC
March 2015

Active DNA demethylation of the vertebrate genomes by DNA methyltransferases: deaminase, dehydroxymethylase or demethylase?

Epigenomics 2014 Jun;6(3):353-63

Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan.

Vertebrate DNA methyltransferases (DNMTs) have been thought to primarily function to covalently add a methyl group to the 5-position of cytosine. However, recent discovery of the DNA demethylation and dehydroxymethylation activities of DNMTs in vitro suggest new routes to complete the dynamic cycle of DNA methylation-demethylation of the vertebrate genomes. The in vitro reaction conditions suggest that vertebrate DNMTs can switch from DNA methylases to DNA dehydroxymethylases under oxidative stress and to DNA demethylases in the presence of calcium ion under nonreducing conditions. These environmental parameters provide clues regarding the choices in vivo of DNMT activities utilized in different physiological systems. In particular, the nature of these parameters suggest that the DNA demethylation and dehydroxymethylation activities of the vertebrate DNMTs play essential roles in multiple biological processes including early embryo development, regulation of neuronal plasticity, tumorigenesis and hormone-regulated transcription.
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http://dx.doi.org/10.2217/epi.14.21DOI Listing
June 2014

Metabolism and mis-metabolism of the neuropathological signature protein TDP-43.

J Cell Sci 2014 Jul 23;127(Pt 14):3024-38. Epub 2014 May 23.

Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan Graduate Institute of Neural Regenerative Medicine, College of Medical Science and Technology/Center for Neurotrauma and Neuroregeneration, Taipei Medical University, Taipei, Taiwan

TDP-43 (also known as TARDBP) is a pathological signature protein of neurodegenerative diseases, with TDP-43 proteinopathies including frontotemporal lobar degeneration (FTLD)-TDP and amyotrophic lateral sclerosis (ALS)-TDP. These TDP-43 proteinopathies are characterized by cytoplasmic insoluble TDP-43-positive aggregates in the diseased cells, the formation of which requires the seeding of TDP-25 fragment generated by caspase cleavage of TDP-43. We have investigated the metabolism and mis-metabolism of TDP-43 in cultured cells and found that endogenous and exogenously overexpressed TDP-43 is degraded not only by the ubiquitin proteasome system (UPS) and macroautophagy, but also by the chaperone-mediated autophagy (CMA) mediated through an interaction between Hsc70 (also known as HSPA8) and ubiquitylated TDP-43. Furthermore, proteolytic cleavage of TDP-43 by caspase(s) is a necessary intermediate step for degradation of the majority of the TDP-43 protein, with the TDP-25 and TDP-35 fragments being the main substrates. Finally, we have determined the threshold level of the TDP-25 fragment that is necessary for formation of the cytosolic TDP-43-positive aggregates in cells containing the full-length TDP-43 at an elevated level close to that found in patients with TDP-43 proteinopathies. A comprehensive model of the metabolism and mis-metabolism of TDP-43 in relation to these findings is presented.
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http://dx.doi.org/10.1242/jcs.136150DOI Listing
July 2014

Tight regulation of a timed nuclear import wave of EKLF by PKCθ and FOE during Pro-E to Baso-E transition.

Dev Cell 2014 Feb;28(4):409-22

Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 115, ROC. Electronic address:

Erythropoiesis is a highly regulated process during which BFU-E are differentiated into RBCs through CFU-E, Pro-E, PolyCh-E, OrthoCh-E, and reticulocyte stages. Uniquely, most erythroid-specific genes are activated during the Pro-E to Baso-E transition. We show that a wave of nuclear import of the erythroid-specific transcription factor EKLF occurs during the Pro-E to Baso-E transition. We further demonstrate that this wave results from a series of finely tuned events, including timed activation of PKCθ, phosphorylation of EKLF at S68 by P-PKCθ(S676), and sumoylation of EKLF at K74. The latter EKLF modifications modulate its interactions with a cytoplasmic ankyrin-repeat-protein FOE and importinβ1, respectively. The role of FOE in the control of EKLF nuclear import is further supported by analysis of the subcellular distribution patterns of EKLF in FOE-knockout mice. This study reveals the regulatory mechanisms of the nuclear import of EKLF, which may also be utilized in the nuclear import of other factors.
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http://dx.doi.org/10.1016/j.devcel.2014.01.007DOI Listing
February 2014

Misregulated progesterone secretion and impaired pregnancy in Cyp11a1 transgenic mice.

Biol Reprod 2013 Oct 17;89(4):91. Epub 2013 Oct 17.

Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.

Normal pregnancy is supported by increased levels of progesterone (P4), which is secreted from ovarian luteal cells via enzymatic steps catalyzed by P450scc (CYP11A1) and HSD3B. The development and maintenance of corpora lutea during pregnancy, however, are less well understood. Here we used Cyp11a1 transgenic mice to delineate the steps of luteal cell differentiation during pregnancy. Cyp11a1 in a bacterial artificial chromosome was injected into mouse embryos to generate transgenic mice with transgene expression that recapitulated endogenous Cyp11a1 expression. Cyp11a1 transgenic females displayed reduced pregnancy rate, impaired implantation and placentation, and decreased litter size in utero, although they produced comparable numbers of blastocysts. The differentiation of transgenic luteal cells was delayed during early pregnancy as shown by the delayed activation of genes involved in steroidogenesis and cholesterol availability. Luteal cell mitochondria were elongated, and their numbers were reduced, with morphology and numbers similar to those observed in granulosa cells. Transgenic luteal cells accumulated lipid droplets and secreted less progesterone during early pregnancy. The progesterone level returned to normal on gestation day 9 but was not properly withdrawn at term, leading to delayed stillbirth. P4 supplementation rescued the implantation rates but not the ovarian defects. Thus, overexpression of Cyp11a1 disrupts normal development of the corpus luteum, leading to progesterone insufficiency during early pregnancy. Misregulation of the progesterone production in Cyp11a1 transgenic mice during pregnancy resulted in aberrant implantation, anomalous placentation, and delayed parturition.
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http://dx.doi.org/10.1095/biolreprod.113.110833DOI Listing
October 2013

Similar dose-dependence of motor neuron cell death caused by wild type human TDP-43 and mutants with ALS-associated amino acid substitutions.

J Biomed Sci 2013 May 30;20:33. Epub 2013 May 30.

Institute of Molecular Medicine, National Taiwan University, Taipei, Taiwan.

Background: TDP-43, a multi-functional DNA/ RNA-binding protein encoded by the TARDBP gene, has emerged as a major patho-signature factor of the ubiquitinated intracellular inclusions (UBIs) in the diseased cells of a range of neurodegenerative diseases. Mutations in at least 9 different genes including TARDBP have been identified in ALS with TDP-43 (+)-UBIs. Thus far, the pathogenic role(s) of the more than 30 ALS-associated mutations in the TARDBP gene has not been well defined.

Results: By transient DNA transfection studies, we show that exogenously expressed human TDP-43 (hTDP-43), either wild type (WT) or 2 different ALS mutant (MT) forms, could cause significantly higher apoptotic death rate of a mouse spinal motor neuron-like cell line (NSC34) than other types of cells, e.g. mouse neuronal Neuro2a and human fibroblast HEK293T cells. Furthermore, at the same plasmid DNA dose(s) used for transfection, the percentages of NSC34 cell death caused by the 2 exogenously expressed hTDP-43 mutants are all higher than that caused by the WT hTDP-43. Significantly, the above observations are correlated with higher steady-state levels of the mutant hTDP-43 proteins as well as their stabilities than the WT.

Conclusions: Based on these data and previous transgenic TDP-43 studies in animals or cell cultures, we suggest that one major common consequence of the different ALS-associated TDP-43 mutations is the stabilization of the hTDP-43 polypeptide. The resulting elevation of the steady state level of hTDP-43 in combination with the relatively low tolerance of the spinal motor neurons to the increased amount of hTDP-43 lead to the neurodegeneration and pathogenesis of ALS, and of diseases with TDP-43 proteinopathies in general.
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http://dx.doi.org/10.1186/1423-0127-20-33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3684520PMC
May 2013

Modulation of mGluR-dependent MAP1B translation and AMPA receptor endocytosis by microRNA miR-146a-5p.

J Neurosci 2013 May;33(21):9013-20

Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan, Republic of China.

The translation of dendritic microtubule-associated protein 1B (MAP1B) is exaggerated upon group I mGluR activation leading to AMPA receptor (AMPAR) endocytosis and consequent long-term depression. However, the mechanisms of regulation of MAP1B protein synthesis in the mature dendrites remain unclear. Here we have identified miR-146a-5p that targets the 3' UTR of MAP1B mRNA and represses its translation. Inhibition of the endogenous miR-146a-5p in mouse cultured hippocampal neurons triggers an increase of the dendritic MAP1B protein as well as the internalization of AMPARs, resulting in a decline in synaptic transmission. Conversely, enforced expression of miR-146a-5p inhibits MAP1B translation and attenuates group I mGluR-induced AMPAR endocytosis. Moreover, siRNA-mediated knockdown of MAP1B recovers the impairment of synaptic transmission caused by inhibition of miR-146a-5p. These results reveal that miR-146a-5p modulates the synaptic plasticity via repression of MAP1B protein synthesis.
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http://dx.doi.org/10.1523/JNEUROSCI.5210-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705019PMC
May 2013

DNA 5-methylcytosine demethylation activities of the mammalian DNA methyltransferases.

J Biol Chem 2013 Mar 7;288(13):9084-91. Epub 2013 Feb 7.

Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan.

Methylation at the 5-position of DNA cytosine on the vertebrate genomes is accomplished by the combined catalytic actions of three DNA methyltransferases (DNMTs), the de novo enzymes DNMT3A and DNMT3B and the maintenance enzyme DNMT1. Although several metabolic routes have been suggested for demethylation of the vertebrate DNA, whether active DNA demethylase(s) exist has remained elusive. Surprisingly, we have found that the mammalian DNMTs, and likely the vertebrates DNMTs in general, can also act as Ca(2+) ion- and redox state-dependent active DNA demethylases. This finding suggests new directions for reinvestigation of the structures and functions of these DNMTs, in particular their roles in Ca(2+) ion-dependent biological processes, including the genome-wide/local DNA demethylation during early embryogenesis, cell differentiation, neuronal activity-regulated gene expression, and carcinogenesis.
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http://dx.doi.org/10.1074/jbc.M112.445585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3610981PMC
March 2013

Autophagy activation ameliorates neuronal pathogenesis of FTLD-U mice: a new light for treatment of TARDBP/TDP-43 proteinopathies.

Autophagy 2013 Feb 29;9(2):239-40. Epub 2012 Oct 29.

Institute of Life Science, National Defense Medical Center, Taipei, Taiwan.

The administration of rapamycin, an MTOR-dependent autophagy activator, for the treatment of neurodegenerative diseases has been tested in several animal models. Thus, whether autophagy activation would lead to the clearance of abnormal accumulation of aggregated proteins in neurodegenerative diseases is worthy of exploration. We have recently shown that rapamycin administration at the early pathological stage of a mouse model with frontotemporal lobar dementia (FTLD-U) characterized with cytoplasmic TARDBP/TDP-43(+)/ubiquitin(+) inclusions (UBIs) in the diseased neurons could rescue the learning/memory deficiency and the abnormal motor function disorder of the mice. This was accompanied by a decreased level of CASP3/caspase-3 and a reduction of the neuronal loss in the mouse forehead. Moreover, autophagy activation at a late pathological stage also could improve motor function, which was accompanied by a reduction of the TARDBP(+) UBIs. This study has set the principal for therapy of neurodegenerative diseases with the TARDBP protein, i.e., amyotrophic lateral sclerosis (ALS)-TDP and FTLD-TDP43, with the use of autophagy activators.
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http://dx.doi.org/10.4161/auto.22526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3552888PMC
February 2013

Targeted disruption in mice of a neural stem cell-maintaining, KRAB-Zn finger-encoding gene that has rapidly evolved in the human lineage.

PLoS One 2012 10;7(10):e47481. Epub 2012 Oct 10.

Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, Republic of China.

Understanding the genetic basis of the physical and behavioral traits that separate humans from other primates is a challenging but intriguing topic. The adaptive functions of the expansion and/or reduction in human brain size have long been explored. From a brain transcriptome project we have identified a KRAB-Zn finger protein-encoding gene (M003-A06) that has rapidly evolved since the human-chimpanzee separation. Quantitative RT-PCR analysis of different human tissues indicates that M003-A06 expression is enriched in the human fetal brain in addition to the fetal heart. Furthermore, analysis with use of immunofluorescence staining, neurosphere culturing and Western blotting indicates that the mouse ortholog of M003-A06, Zfp568, is expressed mainly in the embryonic stem (ES) cells and fetal as well as adult neural stem cells (NSCs). Conditional gene knockout experiments in mice demonstrates that Zfp568 is both an NSC maintaining- and a brain size-regulating gene. Significantly, molecular genetic analyses show that human M003-A06 consists of 2 equilibrated allelic types, H and C, one of which (H) is human-specific. Combined contemporary genotyping and database mining have revealed interesting genetic associations between the different genotypes of M003-A06 and the human head sizes. We propose that M003-A06 is likely one of the genes contributing to the uniqueness of the human brain in comparison to other higher primates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047481PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3468564PMC
February 2013

Phosphorylation-dependent SUMOylation of the transcription factor NF-E2.

PLoS One 2012 10;7(9):e44608. Epub 2012 Sep 10.

Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.

Nuclear factor erythroid-derived 2 (NF-E2), a heterodimer composed of p45 and p18, is a transcriptional activator in hematopoietic progenitors. The transcriptional activity of NF-E2 is not only upregulated by SUMOylation but also stimulated by the cAMP-dependent protein kinase A (PKA). However, the relationship between SUMOylation and phosphorylation in the activation of NF-E2 is unclear. In the present studies, we have demonstrated that PKA enhances NF-E2 SUMOylation in an in vitro system using purified proteins, suggesting a possible mechanism for PKA-dependent activation of the NF-E2 transcription factor through SUMOylation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044608PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3438180PMC
March 2013

Autophagy activators rescue and alleviate pathogenesis of a mouse model with proteinopathies of the TAR DNA-binding protein 43.

Proc Natl Acad Sci U S A 2012 Sep 29;109(37):15024-9. Epub 2012 Aug 29.

Institute of Life Science, National Defense Medical Center, Taipei, Taiwan 11490.

TDP-43 is a multifunctional DNA/RNA-binding protein that has been identified as the major component of the cytoplasmic ubiquitin (+) inclusions (UBIs) in diseased cells of frontotemporal lobar dementia (FTLD-U) and amyotrophic lateral sclerosis (ALS). Unfortunately, effective drugs for these neurodegenerative diseases are yet to be developed. We have tested the therapeutic potential of rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) and three other autophagy activators (spermidine, carbamazepine, and tamoxifen) in a FTLD-U mouse model with TDP-43 proteinopathies. Rapamycin treatment has been reported to be beneficial in some animal models of neurodegenerative diseases but not others. Furthermore, the effects of rapamycin treatment in FTLD-U have not been investigated. We show that rapamycin treatment effectively rescues the learning/memory impairment of these mice at 3 mo of age, and it significantly slows down the age-dependent loss of their motor function. These behavioral improvements upon rapamycin treatment are accompanied by a decreased level of caspase-3 and a reduction of neuron loss in the forebrain of FTLD-U mice. Furthermore, the number of cells with cytosolic TDP-43 (+) inclusions and the amounts of full-length TDP-43 as well as its cleavage products (35 kDa and 25 kDa) in the urea-soluble fraction of the cellular extract are significantly decreased upon rapamycin treatment. These changes in TDP-43 metabolism are accompanied by rapamycin-induced decreases in mTOR-regulated phospho-p70 S6 kinase (P-p70) and the p62 protein, as well as increases in the autophagic marker LC3. Finally, rapamycin as well as spermidine, carbamazepine, and tamoxifen could also rescue the motor dysfunction of 7-mo-old FTLD-U mice. These data suggest that autophagy activation is a potentially useful route for the therapy of neurodegenerative diseases with TDP-43 proteinopathies.
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http://dx.doi.org/10.1073/pnas.1206362109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443184PMC
September 2012
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