THE CONDITIONAL MUTATED RAD21 KNOCK-IN MOUSE MODEL FOR CHRONIC INTESTINAL PSEUDO-OBSTRUCTION: NEUROCHEMICAL AND FUNCTIONAL ANALYSIS

Introduction: RAD21encodes for cohesin complex molecules and acts in DNA repair of various cells, including central, peripheral and enteric neurons. We previously identified a novel missense RAD21mutation (p.Ala622Thr) in a consanguineous family with multiple patients affected by a recessive form of chronic intestinal pseudo-obstruction (CIPO). This is one of the worst motility disorders characterized by recurrent sub-obstructive episodes without mechanical causes.
Aims & Methods: We aimed at characterizing the in vivo and ex vivo effects of the CIPO-causing RAD21 variant on gut motility, and the density of enteric cholinergic neurons and synaptic vesicles in our conditional knock-in mouse. For this purpose, a genetically re-constructed rad21KI(Wnt1-Cre x rad21fl/fl) mouse, carrying the p.A626T missense variant was developed (in the mouse the p.A626 position is the human amino acid p.A622homologue).The mutation p.A626T was introduced in rad21 to be expressed in response to Cre-driven recombination of the floxed alleles, inducing its expression into enteric neuron subsets in response to Wnt1 expression at any developmental phases.Homozygous rad21KI vs. wild-type (WT) mice were compared. Intestinal transit time was evaluated with carmine red dye gavage. Recordings of isometric tension using organ-bath techniques was performed to evaluate myogenic and nerve-mediated motility in the large intestine. Immunolabelling was performed in whole-mount myenteric plexus with antibodies against the pan-neuronalmarker HuC/D, choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS) and synaptophysin (Syn) in the small and large intestine.
Results: Intestinal transit time was increased in rad21KI vs. WT (260.5±20.85 min vs. 190±12.25 min; P=0.0213). In the organ bath experiments, motility index and mean active force (both indexes of myoelectric activity), showed a significant reduction in rad21KI vs. WT mice (rad21KI 3.007±0.279 vs. WT 5.261±0.297, P=0.0022); rad21KI 0.4529±0.02599 vs. WT 0.9413±0.1445, P=0.0087, respectively). A significant reduction of HuC/D/ChAT-positive myenteric neurons/field was detected in the small and large intestine of rad21KI mice, vs. WT (rad21KI 19.33±2.94 vs. WT 35.56±4.10, P=0.015; rad21KI 20.14±2.66 vs. WT 40.94±7.52, P=0.0260, respectively).The synaptic vesicle analysis showed a significant decrease of Syn immunoreactivity in the rad21KI small and large intestine vs. WT (22.12±1.65 vs. 34.11±2.07; P=0.0126 and 22.53±3.31 vs.36.54±3.54; P=0.0115, respectively).
Conclusion: rad21KI mice showed a significantly delayed intestinal transit and reduced motility associated with decreased number of ChAT positive myenteric neurons along with a lower synaptic density. Taken together, these findings are reminiscent of CIPO phenotype and support the rad21KI mouse as a model for genetically-driven dysmotility.