CRISPR interference-based functional genetic interaction mapping in FTD/ALS and PD
<p>Parkinson’s disease (PD) and Frontotemporal Dementia/Amyotrophic Lateral Sclerosis (FTD/ALS) are progressive neurodegenerative diseases that are neuropathologically defined by the presence of protein aggregates and the loss of specific subsets of neurons. Both diseases are associated with v...
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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2022
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| _version_ | 1826313438851235840 |
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| author | Humble, SW |
| author2 | Wade-Martins, R |
| author_facet | Wade-Martins, R Humble, SW |
| author_sort | Humble, SW |
| collection | OXFORD |
| description | <p>Parkinson’s disease (PD) and Frontotemporal Dementia/Amyotrophic Lateral Sclerosis (FTD/ALS) are progressive neurodegenerative diseases that are neuropathologically defined by the presence of protein aggregates and the loss of specific subsets of neurons. Both diseases are associated with variants in genes that are implicated in lysosome function and proteostasis. Therefore, it is likely that converging disease pathways exist across these subsets of neurodegeneration, and the interrogation of these pathways can be used both to enhance our understanding of these disorders and lead to innovative therapeutic interventions.</p>
<p>Utilizing a modified i3Neuron platform to facilitate systematic single and pairwise knockdown (KD) of large panels of PD and FTD/ALS-associated genes through CRISPR interference (CRISPRi) to interrogate gene-gene interactions. My efforts focus on generating the first genetic interaction maps between the major genetic causes and risk factors of PD and FTD/ALS. Most notably, our interaction screen has identified several top gene-gene interaction candidates: TARDBP-TMEM106B, CLN8-NBR1, ATP13A2-CLN8, GRN-TMEM106B, and CTSL-GRN.</p>
<p><i>TMEM106B</i> was a component of multiple hits, and a recent study has demonstra- ted that it modifies TDP-43 pathology [1], while overexpression induces cell death and causes the cleavage of TDP-43 [2]. Additionally, it has been implicated as an interactor of <i>GRN</i> in the clinic [3, 4], and as a regulator of lysosomal function and acidification in mice [4, 5]. A remarkable trend was apparent across all interactions with <i>TMEM106B</i>, where the pH is significantly lower than the non-targeting comparison, indicating a postive overall effect on lysosomal function with knockdown.</p>
<p>Strikingly, there is no significant phenotype between CTSL-GRN and non-targeting controls across DQ-BSA assays or the Magic Red assay, despite the severe deficits seen in GRN single KD. Lysosomal pH, however, is decreased significantly, which indicates a potential rescue across all assays. It is known that CTSL cleaves GRN [5], but no current studies exist showing an interaction beyond that, certainly not to the degree of rescue shown by the data in this thesis. This may be an ideal target for follow-up studies as a priority interaction. These promising initial results demonstrate that identifying common pathogenic changes elicited by disease genes can help us understand the underlying converging mechanisms in PD and FTD/ALS. Through these efforts, I hope to help lay the groundwork for improved molecular understanding and therapeutic discovery.</p> |
| first_indexed | 2024-03-07T07:53:48Z |
| format | Thesis |
| id | oxford-uuid:f596e949-c34c-43ea-834b-ca1bbf20591a |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-09-25T04:14:42Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | oxford-uuid:f596e949-c34c-43ea-834b-ca1bbf20591a2024-07-09T12:17:00ZCRISPR interference-based functional genetic interaction mapping in FTD/ALS and PDThesishttp://purl.org/coar/resource_type/c_db06uuid:f596e949-c34c-43ea-834b-ca1bbf20591aNeurobiologyCRISPR (Genetics)Genetic engineeringGeneticsLysosomesMolecular neurobiologyLysosomal storage diseasesEnglishHyrax Deposit2022Humble, SWWade-Martins, RRyan, BWard, MTalbot, KBassett, A<p>Parkinson’s disease (PD) and Frontotemporal Dementia/Amyotrophic Lateral Sclerosis (FTD/ALS) are progressive neurodegenerative diseases that are neuropathologically defined by the presence of protein aggregates and the loss of specific subsets of neurons. Both diseases are associated with variants in genes that are implicated in lysosome function and proteostasis. Therefore, it is likely that converging disease pathways exist across these subsets of neurodegeneration, and the interrogation of these pathways can be used both to enhance our understanding of these disorders and lead to innovative therapeutic interventions.</p> <p>Utilizing a modified i3Neuron platform to facilitate systematic single and pairwise knockdown (KD) of large panels of PD and FTD/ALS-associated genes through CRISPR interference (CRISPRi) to interrogate gene-gene interactions. My efforts focus on generating the first genetic interaction maps between the major genetic causes and risk factors of PD and FTD/ALS. Most notably, our interaction screen has identified several top gene-gene interaction candidates: TARDBP-TMEM106B, CLN8-NBR1, ATP13A2-CLN8, GRN-TMEM106B, and CTSL-GRN.</p> <p><i>TMEM106B</i> was a component of multiple hits, and a recent study has demonstra- ted that it modifies TDP-43 pathology [1], while overexpression induces cell death and causes the cleavage of TDP-43 [2]. Additionally, it has been implicated as an interactor of <i>GRN</i> in the clinic [3, 4], and as a regulator of lysosomal function and acidification in mice [4, 5]. A remarkable trend was apparent across all interactions with <i>TMEM106B</i>, where the pH is significantly lower than the non-targeting comparison, indicating a postive overall effect on lysosomal function with knockdown.</p> <p>Strikingly, there is no significant phenotype between CTSL-GRN and non-targeting controls across DQ-BSA assays or the Magic Red assay, despite the severe deficits seen in GRN single KD. Lysosomal pH, however, is decreased significantly, which indicates a potential rescue across all assays. It is known that CTSL cleaves GRN [5], but no current studies exist showing an interaction beyond that, certainly not to the degree of rescue shown by the data in this thesis. This may be an ideal target for follow-up studies as a priority interaction. These promising initial results demonstrate that identifying common pathogenic changes elicited by disease genes can help us understand the underlying converging mechanisms in PD and FTD/ALS. Through these efforts, I hope to help lay the groundwork for improved molecular understanding and therapeutic discovery.</p> |
| spellingShingle | Neurobiology CRISPR (Genetics) Genetic engineering Genetics Lysosomes Molecular neurobiology Lysosomal storage diseases Humble, SW CRISPR interference-based functional genetic interaction mapping in FTD/ALS and PD |
| title | CRISPR interference-based functional genetic interaction mapping in FTD/ALS and PD |
| title_full | CRISPR interference-based functional genetic interaction mapping in FTD/ALS and PD |
| title_fullStr | CRISPR interference-based functional genetic interaction mapping in FTD/ALS and PD |
| title_full_unstemmed | CRISPR interference-based functional genetic interaction mapping in FTD/ALS and PD |
| title_short | CRISPR interference-based functional genetic interaction mapping in FTD/ALS and PD |
| title_sort | crispr interference based functional genetic interaction mapping in ftd als and pd |
| topic | Neurobiology CRISPR (Genetics) Genetic engineering Genetics Lysosomes Molecular neurobiology Lysosomal storage diseases |
| work_keys_str_mv | AT humblesw crisprinterferencebasedfunctionalgeneticinteractionmappinginftdalsandpd |