Journal: bioRxiv

Article Title: Endothelial Adgrl2 expression and alternative splicing controls the cerebrovasculature

doi: 10.1101/2025.10.24.682442

Figure Lengend Snippet: Expression of the glutamatergic neuronal isoform of Adgrl2 in endothelial cells results in BBB permeability changes and ectopic synapse formation. ( A ) Hypothesis of Adgrl2 isoform switching in endothelial cells. While wild-type mice express the Adgrl2 isoform in endothelial cells that lack key alternatively spliced exons (-Exon9/14/23/28), the isoform specific Adgrl2 knock-in line ( Adgrl2 KI ) expresses the isoform with their inclusion (+Exon9/14/23/28). Shown is a schematic representation of the Adgrl2 protein structure, with domains affected by alternative splicing. ( B ) Summary graph of BBB function assessed by NaFl permeability assay in wild-type (wt) and homozygous Adgrl2 KI/KI mice (n = 9 mice; P30-35). ( C ) Representative images of mouse brain vasculature in the hippocampal CA1 region visualized using CD31 immunohistochemistry. ( D ) Summary graphs of vascular architecture metrics including density (left), vessel widths (middle), and branch points (right) between wild-type (n = 8 mice; P30-35) and Adgrl2 KI/KI mice (n = 7 mice; P30-35). ( E ) Confocal image from hippocampal CA1 stratum lacunosum-moleculare (SLM) region, with immunohistochemistry labeled endothelial cells (CD31) co-labelled with the excitatory presynaptic marker vGlut2. ( F ) Left, Magnified images of example vGlut1 contact points with endothelial cells in the CA1-SLM region. Right , summary graphs of vGlut1 contact point densities on CD31 labeled endothelial surfaces in wild-type and homozygous Adgrl2 KI/KI mice (n = 7 mice; P30-35). ( G ) Similar as described for (F), but for vGlut2 contact points with endothelial cells (n = 8 mice; P30-35). ( H ) Similar as described for (F), but for vGat contact points with endothelial cells (n = 7 mice; P30-35). ( I ) Left, Magnified images of pericyte marker Cspg4 in contact with endothelial cells. Right , summary graphs of Cspg4 surface coverage of CD31 signal in wild-type and Adgrl2 KI/KI mice (n = 3 mice; P30-35). ( J ) Left, Overview and magnified images of astrocyte marker Aqp4 in contact with CD31. Right, summary graphs of Aqp4 surface coverage of CD31 signal in wild-type and Adgrl2 KI/KI mice (n = 7 mice; P30-35). Data shown are means ± SEM. Statistical analysis was performed by Student’s t-test (*p < 0.05).

Article Snippet: The following primary antibodies were used: CD31 (1:1000; rat polyclonal; 553370; BD Pharmingen), Cspg4 (1:200; rabbit polyclonal; 55027-1-AP; Proteintech), Aqp4 (1:200; rabbit polyclonal; #59678T; Cell Signaling Technology), VGLUT1 (1:500; guinea pig polyclonal; 135-318; Synaptic Systems), vGlut2 (1:1000; guinea pig polyclonal; 135-404; Synaptic Systems), vGat (1:1000; rabbit polyclonal; 131-004; Synaptic Systems), GFP (1:500; Rabbit polyclonal; Cat# A-11122; Invitrogen).

Techniques: Expressing, Permeability, Knock-In, Alternative Splicing, Immunohistochemistry, Labeling, Marker