In vitro transgenic models to elucidate the molecular mechanisms of TDP-43 pathology in amyotrophic lateral sclerosis
<p>Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder characterized by loss of upper and lower motor neurons. TDP-43 was identified as a major protein component of the characteristic neuronal inclusions and it has been detected in 90% of ALS cases. Furtherm...
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| Formaat: | Thesis |
| Taal: | English |
| Gepubliceerd in: |
2013
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| _version_ | 1826315835027750912 |
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| author | Mutihac, R |
| author2 | Wade-Martins, R |
| author_facet | Wade-Martins, R Mutihac, R |
| author_sort | Mutihac, R |
| collection | OXFORD |
| description | <p>Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder characterized by loss of upper and lower motor neurons. TDP-43 was identified as a major protein component of the characteristic neuronal inclusions and it has been detected in 90% of ALS cases. Furthermore, pathogenic mutations in the gene encoding TDP-43, <em>TARDBP</em>, were found in both sporadic and familial ALS cases.</p> <p>The aim of this study is to investigate the molecular mechanisms of cellular dysfunction and ultimately death caused by TDP-43 mutations in human cells using established cell lines and human motor neurons derived from induced pluripotent stem cells (iPSCs). We generated a novel <em>in vitro</em> cellular model using a fluorescently tagged human genomic <em>TARDBP</em> locus carrying three ALS-specific mutations, A382T, M337V or Y374X. In site specific bacterial artificial chromosome (BAC) human stable cell lines, TDP M337V mislocalized to the cytoplasm more frequently than wild-type TDP-43 (TDP Ypet) and TDP-A382T, an effect potentiated by oxidative stress. Cytoplasmic mislocalization was significantly higher in TDP M337V cells compared to TDP-Ypet and correlated with cell death. Cells expressing the mislocalized TDP M337V mutant spontaneously developed cytoplasmic punctae, while for TDP-A382T punctae were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin (TG). Lowering Ca2+ concentration in the ER of TDP-Ypet cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca<sup>2+</sup> dysregulation as a potential mediator of pathology. Ca<sup>2+</sup> signaling from the ER was impaired in cells carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca<sup>2+</sup> stores content and delayed Ca<sup>2+</sup> release induced by carbachol compared to TDP-Ypet cells. The deficits in Ca<sup>2+</sup> release correlated with upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored amplitude of Ca<sup>2+</sup> oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 through Bcl-2 regulation of ER Ca<sup>2+</sup> signalling.</p> <p>Preliminary work in iPSC-derived motor neurons transduced with genomic DNA expression TDP-43 vectors using Herpes Simplex Virus type 1 (HSV-1) amplicons showed cytoplasmic redistribution of TDP-43 under high oxidative stress, without significant differences between mutations and wild-type. TDP-43 mutations delivered by HSV-1 amplicons also did not affect survival of iPSC-derived motor neurons. In ALS patient-derived motor neurons carrying <em>C9orf72</em> expansions, TDP-43 pathology was not detected. However, preliminary data indicate that <em>C9orf72</em> MNs present ER Ca<sup>2+</sup> dysregulation with significantly high intracellular Ca<sup>2+</sup> concentration, which correlates with high protein levels of ER stress markers and low levels of Bcl 2.</p> <p>This work highlights a potentially pathogenic role for TDP-43 mutations in the dysregulation of Ca<sup>2+</sup> homeostasis and explores the use of iPS technology to investigate the effects of ALS-associated mutations in healthy and patient-derived motor neurons.</p> |
| first_indexed | 2024-03-06T19:31:17Z |
| format | Thesis |
| id | oxford-uuid:1d8aa081-342f-4994-ac74-12c6ebdc30c6 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:33:27Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | oxford-uuid:1d8aa081-342f-4994-ac74-12c6ebdc30c62024-12-01T16:39:00ZIn vitro transgenic models to elucidate the molecular mechanisms of TDP-43 pathology in amyotrophic lateral sclerosisThesishttp://purl.org/coar/resource_type/c_db06uuid:1d8aa081-342f-4994-ac74-12c6ebdc30c6NeuroscienceMotor neurone degenerative diseaseGenetics (medical sciences)Biology (medical sciences)Stem cells (clinical sciences)NeurogeneticsEnglishOxford University Research Archive - Valet2013Mutihac, RWade-Martins, RTalbot, K<p>Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder characterized by loss of upper and lower motor neurons. TDP-43 was identified as a major protein component of the characteristic neuronal inclusions and it has been detected in 90% of ALS cases. Furthermore, pathogenic mutations in the gene encoding TDP-43, <em>TARDBP</em>, were found in both sporadic and familial ALS cases.</p> <p>The aim of this study is to investigate the molecular mechanisms of cellular dysfunction and ultimately death caused by TDP-43 mutations in human cells using established cell lines and human motor neurons derived from induced pluripotent stem cells (iPSCs). We generated a novel <em>in vitro</em> cellular model using a fluorescently tagged human genomic <em>TARDBP</em> locus carrying three ALS-specific mutations, A382T, M337V or Y374X. In site specific bacterial artificial chromosome (BAC) human stable cell lines, TDP M337V mislocalized to the cytoplasm more frequently than wild-type TDP-43 (TDP Ypet) and TDP-A382T, an effect potentiated by oxidative stress. Cytoplasmic mislocalization was significantly higher in TDP M337V cells compared to TDP-Ypet and correlated with cell death. Cells expressing the mislocalized TDP M337V mutant spontaneously developed cytoplasmic punctae, while for TDP-A382T punctae were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin (TG). Lowering Ca2+ concentration in the ER of TDP-Ypet cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca<sup>2+</sup> dysregulation as a potential mediator of pathology. Ca<sup>2+</sup> signaling from the ER was impaired in cells carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca<sup>2+</sup> stores content and delayed Ca<sup>2+</sup> release induced by carbachol compared to TDP-Ypet cells. The deficits in Ca<sup>2+</sup> release correlated with upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored amplitude of Ca<sup>2+</sup> oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 through Bcl-2 regulation of ER Ca<sup>2+</sup> signalling.</p> <p>Preliminary work in iPSC-derived motor neurons transduced with genomic DNA expression TDP-43 vectors using Herpes Simplex Virus type 1 (HSV-1) amplicons showed cytoplasmic redistribution of TDP-43 under high oxidative stress, without significant differences between mutations and wild-type. TDP-43 mutations delivered by HSV-1 amplicons also did not affect survival of iPSC-derived motor neurons. In ALS patient-derived motor neurons carrying <em>C9orf72</em> expansions, TDP-43 pathology was not detected. However, preliminary data indicate that <em>C9orf72</em> MNs present ER Ca<sup>2+</sup> dysregulation with significantly high intracellular Ca<sup>2+</sup> concentration, which correlates with high protein levels of ER stress markers and low levels of Bcl 2.</p> <p>This work highlights a potentially pathogenic role for TDP-43 mutations in the dysregulation of Ca<sup>2+</sup> homeostasis and explores the use of iPS technology to investigate the effects of ALS-associated mutations in healthy and patient-derived motor neurons.</p> |
| spellingShingle | Neuroscience Motor neurone degenerative disease Genetics (medical sciences) Biology (medical sciences) Stem cells (clinical sciences) Neurogenetics Mutihac, R In vitro transgenic models to elucidate the molecular mechanisms of TDP-43 pathology in amyotrophic lateral sclerosis |
| title | In vitro transgenic models to elucidate the molecular mechanisms of TDP-43 pathology in amyotrophic lateral sclerosis |
| title_full | In vitro transgenic models to elucidate the molecular mechanisms of TDP-43 pathology in amyotrophic lateral sclerosis |
| title_fullStr | In vitro transgenic models to elucidate the molecular mechanisms of TDP-43 pathology in amyotrophic lateral sclerosis |
| title_full_unstemmed | In vitro transgenic models to elucidate the molecular mechanisms of TDP-43 pathology in amyotrophic lateral sclerosis |
| title_short | In vitro transgenic models to elucidate the molecular mechanisms of TDP-43 pathology in amyotrophic lateral sclerosis |
| title_sort | in vitro transgenic models to elucidate the molecular mechanisms of tdp 43 pathology in amyotrophic lateral sclerosis |
| topic | Neuroscience Motor neurone degenerative disease Genetics (medical sciences) Biology (medical sciences) Stem cells (clinical sciences) Neurogenetics |
| work_keys_str_mv | AT mutihacr invitrotransgenicmodelstoelucidatethemolecularmechanismsoftdp43pathologyinamyotrophiclateralsclerosis |