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Neurobiology of cognitive deficits in murine models of Duchenne muscular dystrophy

Chaussenot R.
Doctoral thesis Life and health sciences. Supported in 2017 at the Paris-Saclay Institute of Neuroscience (Paris-Sud University).
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Abstract

Duchenne muscular dystrophy (DMD) is a neuromuscular syndrome caused by mutations in the dmd gene, leading to the loss of dystrophin proteins, which are normally expressed in various tissues including the brain. Patients exhibit heterogenous cognitive profiles and the presence of intellectual disability depends on the location of the mutation within the gene. This variability can be explained by the complexity of the dmd gene, which includes several internal promoters leading to the cerebral expression of several dystrophins of different sizes. In this thesis work, we focused on two dystrophins : the full-length dystrophin (Dp427) normally expressed in muscle and brain and lost by all DMD patients, and the shortest dystrophin, Dp71, major cerebral product of the dmd gene that is absent in a subgroup of patients. These two dystrophins have distinct cellular functions : Dp427, normally interacting with GABA receptors in inhibitory synapses, plays a role in synaptic plasticity, learning and memory. Its loss leads to mild cognitive deficits. Dp71, mostly expressed in perivascular astrocytes, contributes to the anchoring of ionic channels involved in brain homeostasis and also plays a role in glutamatergic synapses. Dp71 loss strongly aggravate the deficits associated with the loss of Dp427 in patients and lead to severe intellectual disability. Genotype-phenotype relationships need be further specified and it is assumed that beyond deficits severity, the actual nature of cognitive alterations, as well as the presence of sensorial, cognitive, executive and neuropsychiatric disturbances, depend on the specific forms of dystrophin affected by mutations. To study the role of these two dystrophins, we used two mouse models : the mdx mouse that only lacks Dp427, and the Dp71-null mouse that only lacks Dp71. A extensive behavioral study allowed us to better characterize the phenotype associated with the loss of Dp427 and Dp71, detailing integrity of perception and processing of auditory sensory stimuli, of emotional responses and stress reactivity, of learning performance, and of components of executive functions, such like spatial working memory and behavioral flexibility. The work has been completed by collaborative studies aimed at characterizing the role of Dp71 in cortical plasticity and at developing gene therapy approaches to rescue Dp427 function in the mdx mouse. We demonstrate that Dp427 loss perturbs GABAergic functions, stress-induced emotional responses, as well as emotional and long-term memories, without major alterations of sensory and executive functions. We also show that a gene therapy based on systemic injections of antisens oligonucleotides holding specific chemistries and crossing the blood-brain barrier enables Dp427 functional rescue by exon-skipping strategy and alleviates emotional disturbances in mdx mice. The loss of Dp71 has a distinct impact : It alters cortical excitation/inhibition balance and plasticity and disrupt learning, behavioral flexibility and working memory in spatial learning tasks. Our study of these mouse models therefore enabled to clarify the genotype-phenotype relationships and neurobiological bases of this disease, and identified valuable phenotypes to validate treatment efficacy in future brain-targeting preclinical studies.

Relationships linking emotional, motor, cognitive and GABAergic dysfunctions in dystrophin-deficient mdx mice

Vaillend C, Chaussenot R.
Scientific paper Human Molecular Genetics, Volume 26, Issue 6, March 2017, Pages 1041-1055.
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Abstract

Alterations in the Duchenne muscular dystrophy (DMD) gene have been associated with enhanced stress reactivity in vertebrate species, suggesting a role for brain dystrophin in fear-related behavioral and cognitive processes. Because the loss of dystrophin (Dp427) reduces clustering of central γ-aminobutyric acid (GABAA) receptors, it is suspected that local inhibitory tuning and modulation of neuronal excitability are perturbed in a distributed brain circuit that normally controls such critical behavioral functions. In this study, we undertook a large-scale behavioral study to evaluate fear-related behavioral disturbances in dystrophin-deficient mdx mice. We first characterized the behavioral determinants of the enhanced fearfulness displayed by mdx mice following mild acute stress and its association with increased anxiety and altered fear memories. We further demonstrated that this enhanced fearfulness induces long-lasting motor inhibition, suggesting that neurobehavioral dysfunctions significantly influence motor outcome measures in this model. We also found that mdx mice are more sensitive to the sedative and hypnotic effects of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochlorid (THIP), a selective pharmacological activator of extrasynaptic GABAA receptors involved in central tonic inhibition. Our results highlight that information on the emotional aspects of mdx mice are important to better understand the bases of intellectual and neuropsychiatric defects in DMD and to better define valuable functional readouts for preclinical studies. Our data also support the hypothesis that altered spatial localization of GABAA receptors due to Dp427 loss is a pathological mechanism associated with brain dysfunction in DMD, suggesting that extrasynaptic GABAA receptors might be candidate targets for future therapeutic developments.

Cognitive dysfunction in the dystrophin-deficient mouse model of Duchenne muscular dystrophy: A reappraisal from sensory to executive processes

Chaussenot R, Edeline JM, Le Bec B, El Massioui N, Laroche S, Vaillend C.
Scientific paper Neurobiology of Learning and Memory, Volume 124, October 2015, Pages 111-122.
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Abstract

Duchenne muscular dystrophy (DMD) is associated with language disabilities and deficits in learning and memory, leading to intellectual disability in a patient subpopulation. Recent studies suggest the presence of broader deficits affecting information processing, short-term memory and executive functions. While the absence of the full-length dystrophin (Dp427) is a common feature in all patients, variable mutation profiles may additionally alter distinct dystrophin-gene products encoded by separate promoters. However, the nature of the cognitive dysfunctions specifically associated with the loss of distinct brain dystrophins is unclear. Here we show that the loss of the full-length brain dystrophin in mdx mice does not modify the perception and sensorimotor gating of auditory inputs, as assessed using auditory brainstem recordings and prepulse inhibition of startle reflex. In contrast, both acquisition and long-term retention of cued and trace fear memories were impaired in mdx mice, suggesting alteration in a functional circuit including the amygdala. Spatial learning in the water maze revealed reduced path efficiency, suggesting qualitative alteration in mdx mice learning strategy. However, spatial working memory performance and cognitive flexibility challenged in various behavioral paradigms in water and radial-arm mazes were unimpaired. The full-length brain dystrophin therefore appears to play a role during acquisition of associative learning as well as in general processes involved in memory consolidation, but no overt involvement in working memory and/or executive functions could be demonstrated in spatial learning tasks.

Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers

Goyenvalle A, Griffith G, Babbs A, El Andaloussi S, Ezzat K, Avril A, Dugovic B, Chaussenot R, Ferry A, Voit T, Amthor H, Bühr C, Schürch S, Wood MJ, Davies KE, Vaillend C, Leumann C, Garcia L.
Scientific paper Nature Medicine, Volume 21, Issue 3, March 2015, Pages 270-275.
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Abstract

Antisense oligonucleotides (AONs) hold promise for therapeutic correction of many genetic diseases via exon skipping, and the first AON-based drugs have entered clinical trials for neuromuscular disorders. However, despite advances in AON chemistry and design, systemic use of AONs is limited because of poor tissue uptake, and recent clinical reports confirm that sufficient therapeutic efficacy has not yet been achieved. Here we present a new class of AONs made of tricyclo-DNA (tcDNA), which displays unique pharmacological properties and unprecedented uptake by many tissues after systemic administration. We demonstrate these properties in two mouse models of Duchenne muscular dystrophy (DMD), a neurogenetic disease typically caused by frame-shifting deletions or nonsense mutations in the gene encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, respiratory failure and neurocognitive impairment. Although current naked AONs do not enter the heart or cross the blood-brain barrier to any substantial extent, we show that systemic delivery of tcDNA-AONs promotes a high degree of rescue of dystrophin expression in skeletal muscles, the heart and, to a lesser extent, the brain. Our results demonstrate for the first time a physiological improvement of cardio-respiratory functions and a correction of behavioral features in DMD model mice. This makes tcDNA-AON chemistry particularly attractive as a potential future therapy for patients with DMD and other neuromuscular disorders or with other diseases that are eligible for exon-skipping approaches requiring whole-body treatment.

Cerebellar synapse properties and cerebellum-dependent motor and non-motor performance in Dp71-null mice

Helleringer R, Le Verger D, Li X, Izabelle C, Chaussenot R, Belmaati-Cherkaoui M, Dammak R, Decottignies P, Daniel H, Galante G, Vaillend C.
Scientific paper Disease Models & Mechanisms, Volume 11, Issue 6, June 2018.

Abstract

A recent focus has been placed on the role that cerebellar dysfunctions could play in the genesis of cognitive deficits in Duchenne muscular dystrophy (DMD). However, relevant genotype-phenotype analyses are missing to define whether cerebellar defects underlie the severe cases of intellectual deficiency, which have been associated with genetic loss of the smallest product of the dmd gene, the Dp71 dystrophin. To determine for the first time whether Dp71 loss could affect cerebellar physiology and functions, we have used patch-clamp electrophysiological recordings in acute cerebellar slices and a cerebellum-dependent behavioral test battery addressing cerebellum-dependent motor and non-motor functions in Dp71-null transgenic mice. We found that Dp71 deficiency selectively enhances excitatory transmission at glutamatergic synapses formed by climbing fibers (CFs) on Purkinje neurons, but not at those formed by parallel fibers (PFs). Altered basal neurotransmission at CFs was associated with impairments in synaptic plasticity and clustering of the scaffolding post-synaptic density protein PSD-95. At the behavioral level, Dp71-null mice showed some improvements in motor coordination and were unimpaired for muscle force, static and dynamic equilibrium, motivation in high-motor demand and synchronization learning. However, Dp71-null mice displayed altered strategies in goal-oriented navigation tasks, suggesting a deficit in the cerebellum-dependent processing of the procedural components of spatial learning which could contribute to the visuo-spatial deficits identified in this model. In all, the observed deficits suggest that Dp71 loss alters cerebellar synapse function and cerebellum-dependent navigation strategies without being detrimental for motor functions.

Dp71-dystrophin deficiency alters prefrontal cortex excitation-inhibition balance and executive functions

Chaussenot R, Amar M, Fossier P, Vailend C.
Scientific paper Molecular Neurobiology, April 2019.

Abstract

In the Duchenne muscular dystrophy (DMD) syndrome, mutations affecting expression of Dp71, the main dystrophin isoform of the multipromoter dmd gene in brain, have been associated with intellectual disability and neuropsychiatric disturbances. Patients’ profile suggests alterations in prefrontal cortex-dependent executive processes, but the specific dysfunctions due to Dp71 deficiency are unclear. Dp71 is involved in brain ion homeostasis, and its deficiency is expected to increase neuronal excitability, which might compromise the integrity of neuronal networks undertaking high-order cognitive functions. Here, we used electrophysiological (patch clamp) and behavioral techniques in a transgenic mouse that display a selective loss of Dp71 and no muscular dystrophy, to identify changes in prefrontal cortex excitatory/inhibitory (E/I) balance and putative executive dysfunctions. We found prefrontal cortex E/I balance is shifted toward enhanced excitation in Dp71-null mice. This is associated with a selective alteration of AMPA receptor-mediated glutamatergic transmission and reduced synaptic plasticity, while inhibitory transmission is unaffected. Moreover, Dp71-null mice display deficits in cognitive processes that depend on prefrontal cortex integrity, such as cognitive flexibility and sensitivity of spatial working memory to proactive interference. Our data suggest that impaired cortical E/I balance and executive dysfunctions contribute to the intellectual and behavioral disturbances associated with Dp71 deficiency in DMD, in line with current neurobehavioral models considering these functions as key pathophysiological factors in various neurodevelopmental disorders. These new insights in DMD neurobiology also suggest new directions for therapeutic developments targeting excitatory neurotransmission, as well as for guidance of academic environment in severely affected DMD children.

A multi-scale analysis in CD38-/- mice unveils major prefrontal cortex dysfunctions

Martucci L, Amar M, Chaussenot R, Benet G, Bauer O, de Zélicourt A, Nosjean A, Launay JM, Callebert J, Sébrié C, Galione A, Edeline JM, de la Porte S, Fossier P, Granon S, Vaillend C, Cancela JM.
Scientific paper FASEB J., May 2019

Abstract

Autism spectrum disorder (ASD) is characterized by early onset of behavioral and cognitive alterations. Low plasma levels of oxytocin (OT) have also been found in ASD patients; recently, a critical role for the enzyme CD38 in the regulation of OT release was demonstrated. CD38 is important in regulating several Ca2+-dependent pathways, but beyond its role in regulating OT secretion, it is not known whether a deficit in CD38 expression leads to functional modifications of the prefrontal cortex (PFC), a structure involved in social behavior. Here, we report that CD38-/- male mice show an abnormal cortex development, an excitation-inhibition balance shifted toward a higher excitation, and impaired synaptic plasticity in the PFC such as those observed in various mouse models of ASD. We also show that a lack of CD38 alters social behavior and emotional responses. Finally, examining neuromodulators known to control behavioral flexibility, we found elevated monoamine levels in the PFC of CD38-/- adult mice. Overall, our study unveiled major changes in PFC physiologic mechanisms and provides new evidence that the CD38-/- mouse could be a relevant model to study pathophysiological brain mechanisms of mental disorders such as ASD.

A behavioral characterization of executive functions in the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy

Chaussenot R, Laroche S, Vaillend C.
Poster 11th forum of French Neuroscience Society, Lyon, France.

Executive functions in mice lacking the mental retardation-associated dystrophin-Dp71 protein

Chaussenot R, Edeline J-M, Laroche S, Vaillend C.
Poster IFR 144 NeuroSud Paris closing symposium, Gif-sur-Yvette, France.

A behavioral characterization of executive functions in the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy

Chaussenot R, Laroche S, Vaillend C.
Poster Doctoral School 419 (BIO-SIGNE) Day, University Paris-Sud XI, Kremlin-Bîcetre, France.

Major role of dystrophin in fear responses : phenotypic characterization and behavioral rescue by antisens-mediated exon skipping in the mdx mouse

Chaussenot R., Goyenvalle A, Griffith G, Leumann C, Dugovic B, Laroche S, Garcia L, Vaillend C.
Poster FENS-0895, 9th Forum FENS, Milan, Italy.

Major role of dystrophin in fear responses : phenotypic characterization and behavioral rescue by antisens-mediated exon skipping in the mdx mouse

Chaussenot R., Goyenvalle A, Griffith G, Leumann C, Dugovic B, Laroche S, Garcia L, Vaillend C.
Poster Doctoral School 419 (BIO-SIGNE) Day, University Paris-Sud XI, Kremlin-Bîcetre, France.

Neurobiology of cognitive deficits in mouse models of Duchenne muscular dystrophy

Chaussenot R.
Oral Communication University Paris-Sud XI, Orsay, France.

Behavioral phenotype of mouse model Dp71-null

Chaussenot R.
Oral Communication University Paris-Sud XI, Orsay, France.

Is disability an obstacle to life ?

Chaussenot R.
Oral Communication House Of Hope, Paris, France.

Creation of a statistical routine for behavioral analysis with MATLAB

Chaussenot R.
Oral Communication University Paris-Sud XI, Orsay, France.