Apparent Genetic Rescue of AdultShank3Exon 21 Insertion Mutation Mice Tempered by Appropriate Control Experiments

Abstract

SHANK3 (ProSAP2) is among the most common genes mutated in autism spectrum disorders (ASD) and is the causative gene in Phelan–McDermid syndrome (PMS). We performed genetic rescue of Shank3 mutant phenotypes in adult mice expressing a Shank3 exon 21 insertion mutation (Shank3G). We used a tamoxifen-inducible Cre/loxP system (CreTam) to revert Shank3G to wild-type (WT) Shank3+/+. We found that tamoxifen treatment in adult Shank3GCreTam+ mice resulted in complete rescue of SHANK3 protein expression in the brain and appeared to rescue synaptic transmission and some behavioral differences compared to Shank3+/+CreTam+ controls. However, follow-up comparisons between vehicle-treated, WT Cre-negative mice (Shank3+/+CreTam− and Shank3+/+CreTam+) demonstrated clear effects of CreTam on baseline synaptic transmission and some behaviors, making apparently positive genetic reversal effects difficult to interpret. Thus, while the CreTam tamoxifen-inducible system is a powerful tool that successfully rescues Shank3 expression in our Shank3G/G reversible mutants, one must exercise caution and use appropriate control comparisons to ensure sound interpretation.

Postnatal inactivation of Shank3 expression is insufficient to produce behavioral phenotypes associated with germline deficiency

“…Our ndings add to the growing concern regarding the interpretation of studies utilizing conditional knockout technology to study the plasticity of behavioral phenotypes associated with ASD. Another recent study reported apparent rescue of behavioral phenotypes in vehicle-treated CAGGs-CreER expressing Shank3 mutants, implicating some baseline behavioral effects produced by the Cre transgene (Speed et al, 2019). An alternative potential reason for the negative results presented in the current study is our inclusion of the appropriate Cre-positive mice that did not receive tamoxifen in our control groups, whereas previous studies did not implement all necessary controls.…”

Shank promotes action potential repolarization by recruiting BK channels to calcium nanodomains

“…2C-D). Because CRE expression in muscles produced opposite changes in AP firing patterns in strains containing the shn-1 nu697 and nu652 alleles, these results are unlikely to be caused by toxicity associated with CRE expression (Speed et al, 2019). Collectively, these results suggest that SHN-1 acts in body muscles to control AP duration.…”

Comparison of SHANK3 deficiency in animal models: phenotypes, treatment strategies, and translational implications

“…Avoidance to novelty or inanimate objects was strong in two strains of the model ex4-22|ALL [ 128 , 130 ], which also showed increased escape attempts, but was also consistently described in mice haboring other deletions or mutations such as ex9|ANK [ 82 ], ex11|SH3 [ 123 ], ex13-16|PDZ [ 155 , 160 , 163 , 166 ], ex21|PRO [ 125 , 178 ], and ex21|PRO-InsG3728 [ 126 , 187 ]. It was also observed that targeted KO in somatosensory neurons or cells of the caudal embryo in mice of the strains ex13-16|PDZ-Advillin Cre or -Cdx2 Cre induced such avoidance behavior [ 140 ].…”

“…Sensorimotor function is often assessed in tasks such as the rotarod, which primarily targets coordination, by motor reflexes induced through sensory stimuli such as the righting reflex and geotaxis, and by gait analysis. Abnormalities concerning tasks depending on sensorimotor function have been described in SHANK3-deficient rodents of the deletion models ex4-22|ALL [ 128 , 130 ], ex4-9|ANK [ 78 , 124 , 155 , 167 ], ex11|SH3 [ 123 ], and ex13-16|PDZ [ 83 , 155 , 159 , 183 ], or mice harboring schizophrenia- and ASD-associated mutations (ex21|PRO-R1117X, -InsG3680 [ 127 ] or -InsG3728 [ 126 , 187 ]). Notably, single studies did not observe differences between wildtype and mutant mice of some of these strains [ 80 , 90 , 166 ].…”

“…Often sensorimotor testing in the accelerating rotarod is combined with an analysis of motor learning capability, which should result in an increasing score across multiple trials. Impaired motor learning was found in the deletion model targeting all isoforms of SHANK3 (ex4-22|ALL [ 130 ]), or in models mimicking mutations within exon 21 associated to neuropsychatric disorder (ex21|PRO-InsG3680, -R1117X, -InsG3728 [ 126 , 127 , 187 ]). In addition targeting neocortical excitatory neurons (ex4-22|ALL-NEX Cre ) was sufficient to induce deficitary motor learning, while SHANK3 deficiency in GABAergic neurons of the forebrain, DRD1- or DRD2-positive neurons (ex4-22|ALL-Dlx5/6 Cre , -Drd1 Cre , -Drd2 Cre ) was not [ 129 ].…”