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Characterisation of CSMD1, a complement regulator associated with schizophrenia risk

Byrne, Robert 2023. Characterisation of CSMD1, a complement regulator associated with schizophrenia risk. PhD Thesis, Cardiff University.
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CUB and sushi multiple domains 1 (CSMD1) encodes a complex transmembrane protein featuring 15 C-terminal tandem short consensus repeat (SCR) domains, a structural hallmark of complement cascade regulators. CSMD1 SCR17-21 reportedly inhibits the complement classical activation pathway and inhibits membrane attack complex (MAC) assembly. Genetic studies have robustly associated mutations in CSMD1 with schizophrenia risk. Despite this, no further characterisation of CSMD1 has been performed, reliable tools for studying CSMD1 are scarce, and the mechanism linking CSMD1 mutations to schizophrenia remains unknown. Recombinant CSMD1 SCR17-21 and SCR23-26 were expressed as Fc-fusion proteins. Monoclonal antibodies (mAbs) were generated via immunisation of mice and hybridoma fusion, mAbs were characterised and implemented to identify CSMD1-expressing brain cell types via immunofluorescence. Complement regulatory properties of both fragments were examined via various functional assays. Induced pluripotent stem cells (iPSCs) were gene edited to knockout (KO) CSMD1 and introduce schizophrenia risk-associated CSMD1 mutations. CSMD1 mAbs were specific to SCR17-21 and immunostained astrocytes in human temporal cortex and whole mouse brain; immunocytochemistry of human iPSCderived astrocytes and cortical neurons also resulted in positive CSMD1 staining. Both CSMD1 SCR17-21 and SCR23-26 inhibited classical complement activation in haemolytic assays. Both fragments inhibited classical convertase assembly but did not accelerate decay. Neither fragment inhibited MAC assembly in reactive lysis assays, even following pre-incubation with C7 and C8. Candidate CSMD1 KO iPSC lines still expressed CSMD1; two iPSC lines heterozygous for schizophrenia risk mutations were generated. I present a panel of novel mouse mAbs that specifically target CSMD1 SCR17-21 and demonstrate novel evidence of astrocytic CSMD1 expression in human and mouse brain, a finding that may impact schizophrenia pathology. I also demonstrate that CSMD1 possesses two complement regulatory domains, neither of which inhibit MAC assembly. Finally, I have laid groundwork for future iPSC-centric investigation into the role of CSMD1 in schizophrenia.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Medicine
Date of First Compliant Deposit: 26 October 2023
Last Modified: 26 Oct 2023 13:47

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