The Rogdi knockout mouse is a model for Kohlschütter-Tönz syndrome.

Kohlschütter-Tönz syndrome (KTS) is a rare autosomal recessive disorder characterized by severe intellectual disability, early-onset epileptic seizures, and amelogenesis imperfecta. Here, we present a novel Rogdi mutant mouse deleting exons 6-11- a mutation found in KTS patients disabling ROGDI function. This Rogdi-/- mutant model recapitulates most KTS symptoms. Mutants displayed pentylenetetrazol-induced seizures, confirming epilepsy susceptibility. Spontaneous locomotion and circadian activity tests demonstrate Rogdi mutant hyperactivity mirroring patient spasticity. Object recognition impairment indicates memory deficits. Rogdi-/- mutant enamel was markedly less mature. Scanning electron microscopy confirmed its hypomineralized/hypomature crystallization, as well as its low mineral content. Transcriptomic RNA sequencing of postnatal day 5 lower incisors showed downregulated enamel matrix proteins Enam, Amelx, and Ambn. Enamel crystallization appears highly pH-dependent, cycling between an acidic and neutral pH during enamel maturation. Rogdi-/- teeth exhibit no signs of cyclic dental acidification. Additionally, expression changes in Wdr72, Slc9a3r2, and Atp6v0c were identified as potential contributors to these tooth acidification abnormalities. These proteins interact through the acidifying V-ATPase complex. Here, we present the Rogdi-/- mutant as a novel model to partially decipher KTS pathophysiology. Rogdi-/- mutant defects in acidification might explain the unusual combination of enamel and rare neurological disease symptoms.

AAV-delivered diacylglycerol kinase DGKk achieves long-term rescue of fragile X syndrome mouse model.

Fragile X syndrome (FXS) is the most frequent form of familial intellectual disability. FXS results from the lack of the RNA-binding protein FMRP and is associated with the deregulation of signaling pathways downstream of mGluRI receptors and upstream of mRNA translation. We previously found that diacylglycerol kinase kappa (DGKk), a main mRNA target of FMRP in cortical neurons and a master regulator of lipid signaling, is downregulated in the absence of FMRP in the brain of Fmr1-KO mouse model. Here we show that adeno-associated viral vector delivery of a modified and FMRP-independent form of DGKk corrects abnormal cerebral diacylglycerol/phosphatidic acid homeostasis and FXS-relevant behavioral phenotypes in the Fmr1-KO mouse. Our data suggest that DGKk is an important factor in FXS pathogenesis and provide preclinical proof of concept that its replacement could be a viable therapeutic strategy in FXS.

Behavioral Testing Design for Evaluation of Cognitive Disabilities.

Intellectual disabilities (ID) constitute a class of human neurodevelopmental diseases and are a major medical and socioeconomic problem owing to their high incidence and enormous burden to the families of those affected. In the past three decades, mutant mouse technologies have provided powerful tools for elucidating the pathophysiological mechanisms underlying behavioral and developmental alterations related to IDs and for addressing new therapeutic strategies, and major progress has been made revealing previously unidentified genes involved in ID. However, the pathological hallmarks of IDs are very heterogeneous in regard to both the functional deficits observed and the severity of the phenotype, even within the same mutation types. For this reason, an appropriate experimental design is required to reduce the risk of false negatives and positives in animal functional genomic studies. This experimental design should address functions important to evaluate, tests, and the appropriate workflow. Here, we propose an extensive behavioral screen with detailed protocols, which was successfully used in a systematic mouse functional genomic approach to gain pathway-based insights into mechanisms leading to cognitive dysfunction in humans. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Assessment of circadian activity and ingestive behavior Basic Protocol 2: Assessment of neurological reflexes and motor abilities using the grip and rotarod tests Basic Protocol 3: Evaluation of anxiety-related behavior using the elevated plus maze Basic Protocol 4: Evaluation of recognition memory using the object recognition task Basic Protocol 5: Evaluation of social behavior using the social recognition test Basic Protocol 6: Evaluation of working memory using the Y-maze spontaneous alternation test Basic Protocol 7: Evaluation of emotional learning and memory using the fear conditioning test.

Translation of GGC repeat expansions into a toxic polyglycine protein in NIID defines a novel class of human genetic disorders: The polyG diseases.

Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disease characterized by the presence of intranuclear inclusions of unknown origin. NIID is caused by an expansion of GGC repeats in the 5' UTR of the NOTCH2NLC (N2C) gene. We found that these repeats are embedded in a small upstream open reading frame (uORF) (uN2C), resulting in their translation into a polyglycine-containing protein, uN2CpolyG. This protein accumulates in intranuclear inclusions in cell and mouse models and in tissue samples of individuals with NIID. Furthermore, expression of uN2CpolyG in mice leads to locomotor alterations, neuronal cell loss, and premature death of the animals. These results suggest that translation of expanded GGC repeats into a novel and pathogenic polyglycine-containing protein underlies the presence of intranuclear inclusions and neurodegeneration in NIID.

A new mouse model of ARX dup24 recapitulates the patients' behavioral and fine motor alterations.

The aristaless-related homeobox (ARX) transcription factor is involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopmental disorders in humans, among which the most frequent, a 24 bp duplication in the polyalanine tract 2 (c.428_451dup24), gives rise to intellectual disability, fine motor defects with or without epilepsy. To understand the functional consequences of this mutation, we generated a partially humanized mouse model carrying the c.428_451dup24 duplication (Arxdup24/0) that we characterized at the behavior, neurological and molecular level. Arxdup24/0 males presented with hyperactivity, enhanced stereotypies and altered contextual fear memory. In addition, Arxdup24/0 males had fine motor defects with alteration of reaching and grasping abilities. Transcriptome analysis of Arxdup24/0 forebrains at E15.5 showed a down-regulation of genes specific to interneurons and an up-regulation of genes normally not expressed in this cell type, suggesting abnormal interneuron development. Accordingly, interneuron migration was altered in the cortex and striatum between E15.5 and P0 with consequences in adults, illustrated by the defect in the inhibitory/excitatory balance in Arxdup24/0 basolateral amygdala. Altogether, we showed that the c.428_451dup24 mutation disrupts Arx function with a direct consequence on interneuron development, leading to hyperactivity and defects in precise motor movement control and associative memory. Interestingly, we highlighted striking similarities between the mouse phenotype and a cohort of 33 male patients with ARX c.428_451dup24, suggesting that this new mutant mouse line is a good model for understanding the pathophysiology and evaluation of treatment.

Increased H3K9 methylation and impaired expression of Protocadherins are associated with the cognitive dysfunctions of the Kleefstra syndrome.

Kleefstra syndrome, a disease with intellectual disability, autism spectrum disorders and other developmental defects is caused in humans by haploinsufficiency of EHMT1. Although EHMT1 and its paralog EHMT2 were shown to be histone methyltransferases responsible for deposition of the di-methylated H3K9 (H3K9me2), the exact nature of epigenetic dysfunctions in Kleefstra syndrome remains unknown. Here, we found that the epigenome of Ehmt1+/- adult mouse brain displays a marked increase of H3K9me2/3 which correlates with impaired expression of protocadherins, master regulators of neuronal diversity. Increased H3K9me3 was present already at birth, indicating that aberrant methylation patterns are established during embryogenesis. Interestingly, we found that Ehmt2+/- mice do not present neither the marked increase of H3K9me2/3 nor the cognitive deficits found in Ehmt1+/- mice, indicating an evolutionary diversification of functions. Our finding of increased H3K9me3 in Ehmt1+/- mice is the first one supporting the notion that EHMT1 can quench the deposition of tri-methylation by other Histone methyltransferases, ultimately leading to impaired neurocognitive functioning. Our insights into the epigenetic pathophysiology of Kleefstra syndrome may offer guidance for future developments of therapeutic strategies for this disease.

Fasudil treatment in adult reverses behavioural changes and brain ventricular enlargement in Oligophrenin-1 mouse model of intellectual disability.

Loss of function mutations in human Oligophrenin1 (OPHN1) gene are responsible for syndromic intellectual disability (ID) associated with cerebellar hypoplasia and cerebral ventricles enlargement. Functional studies in rodent models suggest that OPHN1 linked ID is a consequence of abnormal synaptic transmission and shares common pathophysiological mechanisms with other cognitive disorders. Variants of this gene have been also identified in autism spectrum disorder and schizophrenia. The advanced understanding of the mechanisms underlying OPHN1-related ID, allowed us to develop a therapeutic approach targeting the Ras homolog gene family, member A (RHOA) signalling pathway and repurpose Fasudil- a well-tolerated Rho Kinase (ROCK) and Protein Kinase A (PKA) inhibitor- as a treatment of ID. We have previously shown ex-vivo its beneficial effect on synaptic transmission and plasticity in a mouse model of the OPHN1 loss of function. Here, we report that chronic treatment in adult mouse with Fasudil, is able to counteract vertical and horizontal hyperactivities, restores recognition memory and limits the brain ventricular dilatation observed in Ophn1-/y However, deficits in working and spatial memories are partially or not rescued by the treatment. These results highlight the potential of Fasudil treatment in synaptopathies and also the need for multiple therapeutic approaches especially in adult where brain plasticity is reduced.

An Early Postnatal Oxytocin Treatment Prevents Social and Learning Deficits in Adult Mice Deficient for Magel2, a Gene Involved in Prader-Willi Syndrome and Autism.

BACKGROUND: Mutations of MAGEL2 have been reported in patients presenting with autism, and loss of MAGEL2 is also associated with Prader-Willi syndrome, a neurodevelopmental genetic disorder. This study aimed to determine the behavioral phenotype of Magel2-deficient adult mice, to characterize the central oxytocin (OT) system of these mutant mice, and to test the curative effect of a peripheral OT treatment just after birth. METHODS: We assessed the social and cognitive behavior of Magel2-deficient mice, analyzed the OT system of mutant mice treated or not by a postnatal administration of OT, and determined the effect of this treatment on the brain. RESULTS: Magel2 inactivation induces a deficit in social recognition and social interaction and a reduced learning ability in adult male mice. In these mice, we reveal anatomical and functional modifications of the OT system and show that these defects change from birth to adulthood. Daily administration of OT in the first postnatal week was sufficient to prevent deficits in social behavior and learning abilities in adult mutant male mice. We show that this OT treatment partly restores a normal OT system. Thus, we report that an alteration of the OT system around birth has long-term consequences on behavior and on cognition. Importantly, an acute OT treatment of Magel2-deficient pups has a curative effect. CONCLUSIONS: Our study reveals that OT plays a crucial role in setting social behaviors during a period just after birth. An early OT treatment in this critical period could be a novel therapeutic approach for the treatment of neurodevelopmental disorders such as Prader-Willi syndrome and autism.

The homeodomain factor Gbx1 is required for locomotion and cell specification in the dorsal spinal cord.

Dorsal horn neurons in the spinal cord integrate and relay sensory information to higher brain centers. These neurons are organized in specific laminae and different transcription factors are involved in their specification. The murine homeodomain Gbx1 protein is expressed in the mantle zone of the spinal cord at E12.5-13.5, correlating with the appearance of a discernable dorsal horn around E14 and eventually defining a narrow layer in the dorsal horn around perinatal stages. At postnatal stages, Gbx1 identifies a specific subpopulation of GABAergic neurons in the dorsal spinal cord. We have generated a loss of function mutation for Gbx1 and analyzed its consequences during spinal cord development. Gbx1 (-/-) mice are viable and can reproduce as homozygous null mutants. However, the adult mutant mice display an altered gait during forward movement that specifically affects the hindlimbs. This abnormal gait was evaluated by a series of behavioral tests, indicating that locomotion is impaired, but not muscle strength or motor coordination. Molecular analysis showed that the development of the dorsal horn is not profoundly affected in Gbx1 (-/-) mutant mice. However, analysis of terminal neuronal differentiation revealed that the proportion of GABAergic inhibitory interneurons in the superficial dorsal horn is diminished. Our study unveiled a role for Gbx1 in specifying a subset of GABAergic neurons in the dorsal horn of the spinal cord involved in the control of posterior limb movement.

Reliability, robustness, and reproducibility in mouse behavioral phenotyping: a cross-laboratory study.

Establishing standard operating procedures (SOPs) as tools for the analysis of behavioral phenotypes is fundamental to mouse functional genomics. It is essential that the tests designed provide reliable measures of the process under investigation but most importantly that these are reproducible across both time and laboratories. For this reason, we devised and tested a set of SOPs to investigate mouse behavior. Five research centers were involved across France, Germany, Italy, and the UK in this study, as part of the EUMORPHIA program. All the procedures underwent a cross-validation experimental study to investigate the robustness of the designed protocols. Four inbred reference strains (C57BL/6J, C3HeB/FeJ, BALB/cByJ, 129S2/SvPas), reflecting their use as common background strains in mutagenesis programs, were analyzed to validate these tests. We demonstrate that the operating procedures employed, which includes open field, SHIRPA, grip-strength, rotarod, Y-maze, prepulse inhibition of acoustic startle response, and tail flick tests, generated reproducible results between laboratories for a number of the test output parameters. However, we also identified several uncontrolled variables that constitute confounding factors in behavioral phenotyping. The EUMORPHIA SOPs described here are an important start-point for the ongoing development of increasingly robust phenotyping platforms and their application in large-scale, multicentre mouse phenotyping programs.