Journal: bioRxiv
Article Title: Rescue of neuropsychiatric phenotypes in a mouse model of 16p11.2 duplication syndrome by genetic correction of an epilepsy network hub
doi: 10.1101/2022.11.09.515866
Figure Lengend Snippet: To identify drivers of pathophysiology in the 16p11.2 dup/+ mouse model, we used gene set analysis in combination with constraint metrics and disease associations to predict the potential for each gene to impact the epilepsy subnetwork. (A) Functionally associated co-expression networks for each gene were compared to proteins in the epilepsy subnetwork identified by MS in 16p11.2 dup/+ mouse model to identify potential network hubs. (B) Bubble plot showing enrichment results from gene set analysis of A on the y-axis. PRRT2 and SEZ6L2 have the highest enrichment scores suggesting they are potential network drivers. The bubble size is scaled by the constraint metric (pLI) for that gene according to the gnomAD database 109 and colored by disease association. Higher pLI scores indicate more intolerance to mutation. Genetic associations are defined as genes with at least one de novo single nucleotide polymorphisms (dnSNVs) in a coding region, identified by exome sequencing studies in EE, ID, ASD or SZ, or genes associated with a brain-related disorder in the OMIM database. PRRT2 is the only epilepsy gene in these datasets, associated with benign familial infantile seizures in OMIM (MIM: 605751). (C) Schematic showing the process of immunoaffinity purification combined with mass spectrometry (IAP-MS) used to identify the PRRT2 interactome. (D) Volcano plot of proteins identified in the PRRT2 immunoaffinity purification-mass spectrometry (IAP-MS) experiment (n= 3 independent experiments). All proteins with spectral counts enriched >2 fold over IgG and p < 0.1 (one-tailed t-test) were considered part of the interactome. A fold change of log 10 (2) indicates that the protein was not identified in the control IgG IAP. Colored protein names indicate epilepsy-associated genes (E) Gene ontology analysis of the PRRT2 interactome identifies a highly enriched set of synaptic regulators (F) Epilepsy and intellectual disability associated proteins (from the OMIM database) are enriched in the PRRT2 interactome dataset. Data shows percentage enrichment and p -values from a hypergeometic test. (G) 72% of PRRT2-interacting proteins are dysregulated in the neocortex of the 16p11.2 dup/+ mouse model (H) Enrichment values from a hypergeometric test of G. (I) PRRT2 is a network hub in the dysregulated epilepsy subnetwork identified in 16p11.2 dup/+ mice. Novel interactions identified in the PRRT2 interactome were mapped onto the proteomic subnetwork identified in . Grey edges represent retrieved protein interactions whereas PRRT2 interactions identified in the IAP-MS experiment are colored as purple edges/nodes. Nodes are scaled by betweenness centrality ( C B ) score to highlight nodes with strong influence on the protein interaction network. Abbreviations: Epi, epilepsy; ID, intellectual disability; SZ, schizophrenia; ASD, autism spectrum disorder; DD, developmental disorder; P corr , Benjamini- Hochberg corrected p -value.
Article Snippet: For western blotting of proteins encoded in the 16p11.2 region, cortical lysates were prepared by solubilizing in cold RIPA buffer (50mM Tris pH 7.4, 150mM NaCl, 5mM EDTA, 1% Triton X- 100, 0.5% sodium deoxycholate, 0.1% SDS, + protease inhibitors added fresh) for 1 h prior to SDS-PAGE and western blotting with SEZ6L2 (Abcam, ab197058, 1:500), PRRT2 (Sigma, HPA014447, 1:2000), ERK1 (Santa Cruz, sc94, 1:5000), and TAOK1 (Santa Cruz, sc-47447, 1:500) antibodies
Techniques: Expressing, Mutagenesis, Sequencing, Immunoaffinity Purification, Mass Spectrometry, One-tailed Test
