Investigation of microglia phenotypes in an iPSC model of Huntington’s Disease

Stöberl, Nina 2022. Investigation of microglia phenotypes in an iPSC model of Huntington’s Disease. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Huntington’s disease (HD) is a neurodegenerative disorder caused by a dominantly inherited CAG repeat expansion in the huntingtin gene (HTT). Multiple studies indicate an involvement of neuroinflammation in HD pathology, including several PET studies demonstrating that microglial activation correlates with disease severity in HD patients. Nonetheless, an open question is whether mutant HTT expression leads to cell-autonomous transcriptional and functional changes in microglia, which potentially contribute to the disease pathology. In this thesis, a patient-derived induced pluripotent stem cell (iPSC) model of HD with 109 CAG repeats (Q109), and isogenic controls with a corrected wild-type length of 22 repeats (Q22) were used. Q109 and Q22 iPSC were differentiated to microglia-like cells, as well as astrocytes and neurons. Cell types were characterised using immunocytochemistry as well as flow cytometry. Investigation of CAG repeat size was performed using PCR and capillary electrophoresis. Microglia function was investigated in monoculture using a wide range of assays as well as immunostaining of cellular compartments. Bulk RNA sequencing was performed on microglia-like cells under basal conditions and after pro-inflammatory stimulation. The secretome of microglia-like cell monocultures as well as microglia-astrocyte co-cultures was investigated via mass spectrometry. Both, Q109 and Q22 iPSC successfully differentiated to microglia-like cells, astrocytes and neurons. Q109 microglia-like cells, astrocytes and neurons demonstrated HTT CAG repeat expansion in culture. Q109 microglia-like cells showed impairment in key microglia functions, including phagocytosis and endocytosis and these functional impairments were also evident on a gene expression level. Furthermore, the secretion of pro-inflammatory cytokines was increased in Q109 microglia-like cells. RNA sequencing revealed an upregulation of genes involved in translation and mismatch repair in the comparison of Q109 versus Q22 microglia-like cells. In the investigation of microglia-astrocytes co-culture supernatants, astrocytes played a more prominent role for differentially secreted proteins. These observations demonstrate that iPSC-derived microglia-like cells can be utilized to better understand the role of microglia in HD pathology. The findings indicate a mutant HTT induced change in microglia transcription and cellular function which is independent of the detrimental HD brain environment.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	21 April 2023
Last Modified:	21 Apr 2024 01:30
URI:	https://orca.cardiff.ac.uk/id/eprint/158983

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