Dystrophins In The Nervous System: From Neurophysiology to Molecular Therapy – DYSther

The DYSther project combines expertise of four teams, from molecular to behavior, forming a multidisciplinary network to tackle emerging questions that link fundamental research to technological challenges aimed at improving research tools and translational rescue strategies. Our experimental approach, undertaken in mouse models of the human disease, is based on converging and complementary methods including cellular and molecular biology, biochemistry, neuroanatomy, brain and cell imaging, pharmacology, electrophysiology and behavioral exploration. We also use models in which specific genes facilitate cell imaging or enable activation of specific neuronal networks by optogenetic technology, or modulate inflammation of nervous tissues to study underlying risk factors in this process.
The numerous data generated by this approach enable identification of specific biomarkers to be used for relevant evaluation of the efficacy of the molecular tools that we develop and produce in large amounts for therapeutical strategies. To help these molecules to cross barriers and diffuse in all affected regions of the central nervous system, we pair them with particular chemistries or we include them in specific vectors selected for their potential to target specific cell types.

Identification of the behavioral defects due to the loss of Dp427 or Dp71 dystrophins shows that in addition to learning and memory deficits, emotional disturbances and executive dysfunctions may be present, depending on the type brain dystrophins that is missing. The Dp427-deficient mdx mouse displays enhanced stress reactivity, altered social behavior and deficient long-term memory. The molecular tools developed to treat muscular dystrophy through Dp427 rescue have been optimized so that they can also produce beneficial effects in brain. Enhanced stress reactivity constitutes a relevant marker for rapid evaluation of treatment effects on mdx mice behavior. Dp71, a short form of dystrophin associated with severe intellectual disturbances in certain patients, is mainly expressed in glial cells and plays a role in the mechanisms that maintain brain ion equilibrium and regulate the capacity of circulating molecules to penetrate nervous tissues. Our recent studies uncovered major alterations in the number and morphology of glial cells and in the organization of the vascular network in the retina. The modifications due to the loss of Dp71 in the brain are in part similar to those reported in retina (work in progress). We also have selected a gene-transfer vector aimed at rescuing Dp71 expression in glial cells to compensate these alterations. The efficacy of this tool has recently been validated in our studies in retinal tissues. The dysfunctions identified in these models will serve as markers to evaluate efficacy of the molecular tools developed for therapeutic approaches. Optimal doses of therapeutic molecules and vectors required for this work have been charaterized and their functional and therapeutical effects are under investigation.

The DYSther project will bring new knowledge on central nervous system functioning, on the mechanisms shared by retina and brain and on the determination of novel markers to test treatment efficacy on central dysfunctions in muscular dystrophy. Our main goal is to identify and produce molecular tools bringing gene therapy closer to a full compensation of symptoms in muscular dystrophy patients. We also aim at identifying the most reliable molecular tools and optimal conditions to correct defective genes or to compensate their dysfunction by gene therapy. This reasearch should therefore have a wide impact on numerous genetic diseases involving central nervous system dysfunctions leading to sensory, cognitive and neuropsychiatric disturbances.

We found that absence of the full-length neuronal form of dystrophin (Dp427) in mdx mice leads to altered emotional responses and social behavior that may underlie part of the cognitive and neuropsychiatric disturbances in muscular dystrophy (Miranda et al., Mol Autism, 2015; Vaillend and Chaussenot, Hum Mol Genet, 2017). The molecular tools developed for therapeutic intervention in this model have been optimized (Relizani et al., submitted). The glial role of Dp71 has neen further detailed in retina (Giocanti-Aurégan et al., Glia, 2015). Distinct Dp71 isoforms are detected in brain and retina, suggesting different functions in these tissues (Aragon et al., Mol Neurobiol, 2017). Our new vector enables to rescue Dp71 expression and function in retina (Vacca et al., Hum Mol Genet, 2016). This work also led to several communications in international congresses or within science popularization interventions.

Recent advances in our understanding of dystrophin-dependent physiological mechanisms responsible for Duchenne muscular dystrophy (DMD) led to the development of innovative tools for molecular therapies. Much less is known, however, on their potency to alleviate the brain and cognitive impairments associated with DMD. We previously showed that gene-therapy strategies, such as antisense-mediated exon skipping, hold realistic prospects of restoring both muscle and brain functions in the dystrophin(Dp427)-deficient mdx mouse. However, their efficacy to correct behavioral deficits was not estimated, as viral vectors precluded body-wide gene delivery. Our progresses in developing new chemistries of oligonucleotide analogs and new generations of adeno-associated vectors should overcome such limitations, as attested by our preliminary results in mdx mice. However, the intellectual disabilities in DMD are associated with mutations that lead to a cumulative loss of several dystrophin products encoded by distinct internal promoters, such as the Dp71 protein, which loss is a pivotal aggravating factor for cognitive status in DMD. A major challenge to achieve a full compensation of motor and cognitive dysfunctions in DMD therefore involves identification of reliable biomarkers and target mechanisms affected by the loss of both Dp427 and Dp71 and the development of appropriate molecular tools to rescue their respective functions.
The DYSther project combines expertise of four teams, from the molecular to the physiological and behavioral levels, forming a multidisciplinary network to tackle emerging questions that link fundamental research to technological challenges aimed at improving research tools and translational rescue strategies. DYSther is aimed at better understanding the multiple roles of dystrophin products in the CNS and to validate biomarkers for molecular therapies. Task 1 will characterize the deficits in high-order cognitive functions in mice lacking Dp427 or Dp71, with behavioral tests refined from a translational dialogue with clinicians. This will constitute the behavioral read-outs of the efficacy of gene therapy tools, including exon-skipping using AAV-U7 vectors and/or new generations of antisense oligonucleotide analogs that cross over the blood-brain barrier, and cell-specific re-expression of Dp71. These vectors will also serve to understand the implication of various brain structures and cellular subtypes in establishing the phenotype, through local delivery and use of cell-specific promoters. Task 2 will bring together concepts from brain and retina studies to decipher the role of Dp71 in glial-vascular interactions in normal and challenged conditions. Task3 will determine the impact of Dp71 deficiency on glial physiology in cerebellum, a major structure involved in the cognitive impairment in DMD. Functional similarities between retinal Müller and cerebellar Bergmann glial cells suggest that most hypotheses derived from retina studies could be tested in cerebellar tissues. We will particularly focus on the Dp71-dependent mechanisms whereby astrocytes control extracellular K+ concentration and water balance in both normal and ischemic conditions. This will be completed by in vivo magnetic resonance imaging (MRI) studies of the dynamics and recovery of water accumulation during ischemia/hypoxia and brain edema. These two tasks will also generate many new read-outs that we will use to assess therapeutic rescue and modulation of Dp71 expression in both normal and pathological conditions.
The DYSther project will thus bring fundamental knowledge on the roles of dystrophins in CNS, while bringing gene therapy closer to a full compensation of symptoms in Duchenne patients. Expected breakthroughs in the mechanisms underlying glial-vascular functions, as well as in the technological capacity of new molecular tools to cross the nervous system barriers, is also of critical interest for several pathologies other than DMD.