University of Cambridge
Mutational and Structural Investigation of SARM1, a Protein that Mediates Axon Loss
Abstract
dc:description.abstractSARM1 is a pro-degenerative NADase that executes the programmed axon degeneration pathway after nerve injury and in diseases including polyneuropathies. The ARM domain of SARM1 plays a crucial role in regulating its NADase activity. By attenuating this activity, axon degeneration is delayed, making SARM1 an important target for drug development. This thesis characterises activation at the ARM domain allosteric site, where NMN and NAD bind in competition, respectively activating SARM1 and blocking its activation. In addition, this thesis tests whether a rare, natural ARM domain mutant SARM1W253C in a patient with a complex disorder with developmental and degenerative symptoms, confers a gain-of-function consistent with it having a causative role. Finally, this thesis uses a novel SARM1 protein purification method to recapitulate SARM1 Cryo-EM structures from literature. Site-directed mutagenesis was used to modify the ARM domain allosteric site with artificial mutants, or to introduce the SARM1W253C natural mutant. Mutants were expressed in HEK293T cells to determine their influence on NAD levels and mutant proteins isolated using immunoprecipitation for NADase assays of basal and NMN-induced activity. Selected ARM mutants and SARM1W253C were further characterised by microinjecting SCG neurons with mutant DNA and comparing the degree of cell death and axon degeneration to those induced by previously characterised gain-of-function SARM1Δ229-235 and/or WT after removing NAD precursor nicotinamide riboside (NR) or addition of Vacor, which normally leads to SARM1 activation. All artificial mutants in the SARM1 ARM domain allosteric site influence NAD levels in transfected HEK293T cells and alter basal and/or induced SARM1 NADase activity. Residues in the D317 mobile loop in the allosteric site in particular are important in regulating SARM1 activation, providing a new target for how to block activation therapeutically. The SARM1W253C natural mutant has shown high constitutive NADase activity similar to those reported previously in ALS gain-of-function variants, and SCG neurons expressing SARM1W253C protein degenerate after NR removal, similar to ALS highly active variants. Data are consistent with SARM1W253C conferring gain-of-function, a finding that is now being extended using patient iPSC-derived neurons.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2024
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Hopkins, Eleanor
- Advisor dc:contributor.advisor
-
- Coleman, Michael
Subjects
dc:subject × 4Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.117956
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/383685