BackgroundInfants and children with dominant de novo mutations in GNAO1 exhibit movement disorders, epilepsy, or both. Children with loss-of-function (LOF) mutations exhibit Epileptiform Encephalopathy 17 (EIEE17). Gain-of-function (GOF) mutations or those with normal function are found in patients with Neurodevelopmental Disorder with Involuntary Movements (NEDIM). There is no animal model with a human mutant GNAO1 allele.ObjectivesHere we develop a mouse model carrying a human GNAO1 mutation (G203R) and determine whether the clinical features of patients with this GNAO1 mutation, which includes both epilepsy and movement disorder, would be evident in the mouse model.MethodsA mouse Gnao1 knock-in GOF mutation (G203R) was created by CRISPR/Cas9 methods. The resulting offspring and littermate controls were subjected to a battery of behavioral tests. A previously reported GOF mutant mouse knock-in (Gnao1+/G184S), which has not been found in patients, was also studied for comparison.ResultsGnao1+/G203R mutant mice are viable and gain weight comparably to controls. Homozygotes are non-viable. Grip strength was decreased in both males and females. Male Gnao1+/G203R mice were strongly affected in movement assays (RotaRod and DigiGait) while females were not. Male Gnao1+/G203R mice also showed enhanced seizure propensity in the pentylenetetrazole kindling test. Mice with a G184S GOF knock-in also showed movement-related behavioral phenotypes but females were more strongly affected than males.ConclusionsGnao1+/G203R mice phenocopy children with heterozygous GNAO1 G203R mutations, showing both movement disorder and a relatively mild epilepsy pattern. This mouse model should be useful in mechanistic and preclinical studies of GNAO1-related movement disorders.

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Understanding the interplay between diabetes and biofilms may lead to novel treatments and better clinical management of chronic wounds.

Neurodevelopmental disorder with involuntary movements (NEDIM, OMIM: 617493) is a severe, early onset neurological condition characterized by a delay in psychomotor development, hypotonia, and hyperkinetic involuntary movements. Heterozygous de novo mutations in the GNAO1 gene cause NEDIM. Gαo, the gene product of GNAO1, is the alpha subunit of Go, a member of the heterotrimeric Gi/o family of G-proteins. Go is found abundantly throughout the brain but the pathophysiological mechanisms linking Gαo functions to clinical manifestations of GNAO1-related disorders are still poorly understood. One of the most common mutant alleles among the GNAO1 encephalopathies is the c.626G>A or p.Arg209His (R209H) mutation. We developed heterozygous knock-in Gnao1+/R209H mutant mice using CRISPR/Cas9 methodology to assess whether a mouse model could replicate aspects of the NEDIM clinical pattern. Mice carrying the R209H mutation exhibited increased locomotor activity and a modest gait abnormality at 8-12 weeks. In contrast to mice carrying other mutations in Gnao1, the Gnao1+/R209H mice did not show enhanced seizure susceptibility. Levels of protein expression in multiple brain regions were unchanged from WT mice but the nucleotide exchange rate of mutant R209H Gαo was 9 times faster than WT. The atypical neuroleptic risperidone has shown efficacy in a patient with the R209H mutation. It also alleviated the hyperlocomotion phenotype observed in our mouse model but suppressed locomotion in WT mice as well. In this study, we show that Gnao1+/R209H mice mirror elements of the patient phenotype and respond to an approved pharmacological agent.

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

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Patients with de novo mutations in the gene for Gao (GNAO1) exhibit either early infantile epileptic encephalopathy (EIEE17) or neurodevelopmental delay with involuntary movements (NEDIM). Gao is the a subunit of the most abundant heterotrimeric G protein in brain. As with other Gi/o family G proteins, it can signal directly to effectors or by release of free Gbg resulting in inhibition of adenylyl cyclase or modulation of Ca++ and K+ ion channels. Loss‐of‐Function (LOF) alleles in GNAO1 result in epilepsy while GOF mutations result in movement disorder with or without seizures. Here we report the first mouse model of a human GNAO1 encephalopathy. Heterozygous GOF mutant mice (Gnao1+/G203R) were created using CRISPR/Cas9 to determine whether they replicate the clinical pattern of children with de novo GNAO1+/G203R mutations who display epilepsy as well as severe choreoathetosis and dystonia. There was a striking sex difference in the observed phenotypes. Male Gnao1+/G203R mice displayed significant deficits in open field, Rotarod®, Digigait®, and grip strength assessments. Also, they had enhanced seizure susceptibility in pentylenetrazol (PTZ) kindling studies. None of these differences were seen in female mice. Unlike children with the GNAO1 G203R mutation, there were no obvious dystonic or choreoathetotic movements or postures at baseline. After administration of oxotremorine, however, male Gnao1+/G203R mice displayed more forelimb raising and hindlimb splaying than was observed in WT mice. This novel model phenocopies the combined epilepsy and movement disorder phenotype of children with GNAO1 G203R mutations. These mice should facilitate studies of neural mechanisms and permit preclinical drug repurposing.Support or Funding InformationSupported by grants from the MSU Clinical and Translational Science Institute and The Bow Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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