PhD Candidate Florey Institute of Neuroscience and Mental Health
Rationale: Aim 1: To quantify A) total SCN2A and B) mutant SCN2A protein levels using selected reaction monitoring (SRM) in three heterozygous mouse models of SCN2A encephalopathy, each representing a unique clinical phenotype seen in the SCN2A disease population. Aim 2: To establish and study the proteome in several models of SCN2A encephalopathy using TMT labelling mass spectrometry techniques, with a focus on detection of SCN2A peptides. The models investigated were knock-in transgenic mice, NGN2 derived neurons from patient induced pluripotent stem cells (iPSCs), and human brain and cerebrospinal fluid (CSF). BACKGROUND: SCN2A encodes a voltage-gated sodium channel and de novo variants are the cause of autism and a spectrum of developmental and epileptic encephalopathies (DEEs). There is growing evidence to support that the different phenotypes correlate with the resulting functional consequence of the mutations, i.e., whether the protein is loss or gain-of-function. Methods: Brain samples were collected from WT mice and mice heterozygous for SCN2A R854Q, S1759R, or R1883Q at postnatal (P) day 16-20. NGN2 neurons were collected 21 days after differentiation and consisted of mutated and an isogenic corrected control lines. Human pooled CSF samples were obtained commercially from Lee Biosciences from healthy individuals. All samples were homogenized, and disulfide bridges were reduced and alkylated. Samples were then digested with trypsin/LysC and analysed on an Agilent 6495 triple quadrupole (QQQ) mass spectrometer or samples were further treated with TMT labelling reagents, pooled, fractionated, and ran on a Thermofisher Orbitrap mass spectrometer. Proteins that showed a significant change between conditions were validated using commercially available ELISA kits where possible. Results: No significant difference was measured in total SCN2A levels between R1883Q and WT mice. Tau protein was significantly increased in R1883Q mice compared to WT controls. Several SCN2A peptides can be measured in human brain and mouse brain but are present at undetectable levels in cerebrospinal fluid. Conclusions: Using the Agilent 6495 QQQ and Thermofisher Q-Exactive Orbitrap mass spectrometer instruments we were able to detect expression of several proteins in SCN2A disease models and quantify changes in expression levels between mutant and WT controls. The expression of SCN2A protein was unchanged between untreated WT and mutant R1882Q mice, indicating that the severe phenotype seen in the mouse models is purely due to functional effects and firing properties rather than changes in protein expression. Funding: Please list any funding that was received in support of this abstract.: Funding for this project was by RogCon Biosciences.