Expression of Dystrophin Dp71 Splice Variants Is Temporally Regulated During Rodent Brain Development.

Dystrophin Dp71 is the major product of the Duchenne muscular dystrophy (DMD) gene in the brain, and its loss in DMD patients and mouse models leads to cognitive impairments. Dp71 is expressed as a range of proteins generated by alternative splicing of exons 71 to 74 and 78, classified in the main Dp71d and Dp71f groups that contain specific C-terminal ends. However, it is unknown whether each isoform has a specific role in distinct cell types, brain regions, and/or stages of brain development. In the present study, we characterized the expression of Dp71 isoforms during fetal (E10.5, E15.5) and postnatal (P1, P7, P14, P21 and P60) mouse and rat brain development. We finely quantified the expression of several Dp71 transcripts by RT-PCR and cloning assays in samples from whole-brain and distinct brain structures. The following Dp71 transcripts were detected: Dp71d, Dp71d∆71, Dp71d∆74, Dp71d∆71,74, Dp71d∆71-74, Dp71f, Dp71f∆71, Dp71f∆74, Dp71f∆71,74, and Dp71fΔ71-74. We found that the Dp71f isoform is the main transcript expressed at E10.5 (> 80%), while its expression is then progressively reduced and replaced by the expression of isoforms of the Dp71d group from E15.5 to postnatal and adult ages. This major finding was confirmed by third-generation nanopore sequencing. In addition, we found that the level of expression of specific Dp71 isoforms varies as a function of postnatal stages and brain structure. Our results suggest that Dp71 isoforms have different and complementary roles during embryonic and postnatal brain development, likely taking part in a variety of maturation processes in distinct cell types.

AAV-Mediated Restoration of Dystrophin-Dp71 in the Brain of Dp71-Null Mice: Molecular, Cellular and Behavioral Outcomes.

A deficiency in the shortest dystrophin-gene product, Dp71, is a pivotal aggravating factor for intellectual disabilities in Duchenne muscular dystrophy (DMD). Recent advances in preclinical research have achieved some success in compensating both muscle and brain dysfunctions associated with DMD, notably using exon skipping strategies. However, this has not been studied for distal mutations in the DMD gene leading to Dp71 loss. In this study, we aimed to restore brain Dp71 expression in the Dp71-null transgenic mouse using an adeno-associated virus (AAV) administrated either by intracardiac injections at P4 (ICP4) or by bilateral intracerebroventricular (ICV) injections in adults. ICP4 delivery of the AAV9-Dp71 vector enabled the expression of 2 to 14% of brain Dp71, while ICV delivery enabled the overexpression of Dp71 in the hippocampus and cortex of adult mice, with anecdotal expression in the cerebellum. The restoration of Dp71 was mostly located in the glial endfeet that surround capillaries, and it was associated with partial localization of Dp71-associated proteins, α1-syntrophin and AQP4 water channels, suggesting proper restoration of a scaffold of proteins involved in blood-brain barrier function and water homeostasis. However, this did not result in significant improvements in behavioral disturbances displayed by Dp71-null mice. The potential and limitations of this AAV-mediated strategy are discussed. This proof-of-concept study identifies key molecular markers to estimate the efficiencies of Dp71 rescue strategies and opens new avenues for enhancing gene therapy targeting cognitive disorders associated with a subgroup of severely affected DMD patients.

The unconditioned fear response in vertebrates deficient in dystrophin.

Dystrophin loss due to mutations in the Duchenne muscular dystrophy (DMD) gene is associated with a wide spectrum of neurocognitive comorbidities, including an aberrant unconditioned fear response to stressful/threat stimuli. Dystrophin-deficient animal models of DMD demonstrate enhanced stress reactivity that manifests as sustained periods of immobility. When the threat is repetitive or severe in nature, dystrophinopathy phenotypes can be exacerbated and even cause sudden death. Thus, it is apparent that enhanced sensitivity to stressful/threat stimuli in dystrophin-deficient vertebrates is a legitimate cause of concern for patients with DMD that could impact neurocognition and pathophysiology. This review discusses our current understanding of the mechanisms and consequences of the hypersensitive fear response in preclinical models of DMD and the potential challenges facing clinical translatability.

Networking to Optimize Dmd exon 53 Skipping in the Brain of mdx52 Mouse Model.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that disrupt the open reading frame and thus prevent production of functional dystrophin proteins. Recent advances in DMD treatment, notably exon skipping and AAV gene therapy, have achieved some success aimed at alleviating the symptoms related to progressive muscle damage. However, they do not address the brain comorbidities associated with DMD, which remains a critical aspect of the disease. The mdx52 mouse model recapitulates one of the most frequent genetic pathogenic variants associated with brain involvement in DMD. Deletion of exon 52 impedes expression of two brain dystrophins, Dp427 and Dp140, expressed from distinct promoters. Interestingly, this mutation is eligible for exon skipping strategies aimed at excluding exon 51 or 53 from dystrophin mRNA. We previously showed that exon 51 skipping can restore partial expression of internally deleted yet functional Dp427 in the brain following intracerebroventricular (ICV) injection of antisense oligonucleotides (ASO). This was associated with a partial improvement of anxiety traits, unconditioned fear response, and Pavlovian fear learning and memory in the mdx52 mouse model. In the present study, we investigated in the same mouse model the skipping of exon 53 in order to restore expression of both Dp427 and Dp140. However, in contrast to exon 51, we found that exon 53 skipping was particularly difficult in mdx52 mice and a combination of multiple ASOs had to be used simultaneously to reach substantial levels of exon 53 skipping, regardless of their chemistry (tcDNA, PMO, or 2'MOE). Following ICV injection of a combination of ASO sequences, we measured up to 25% of exon 53 skipping in the hippocampus of treated mdx52 mice, but this did not elicit significant protein restoration. These findings indicate that skipping mouse dystrophin exon 53 is challenging. As such, it has not yet been possible to answer the pertinent question whether rescuing both Dp427 and Dp140 in the brain is imperative to more optimal treatment of neurological aspects of dystrophinopathy.

Delayed postnatal brain development and ontogenesis of behavior and cognition in a mouse model of intellectual disability.

Intellectual disability (ID) is a neurodevelopmental disorder associated with impaired cognitive and adaptive behaviors and represents a major medical issue. Although ID-patients develop behavioral problems and are diagnosed during childhood, most behavioral studies in rodent models have been conducted in adulthood, missing precocious phenotypes expressed during this critical time-window characterized by intense brain plasticity. Here, we selectively assessed postnatal ontogenesis of behavioral and cognitive processes, as well as postnatal brain development in the male Rsk2-knockout mouse model of the Coffin-Lowry syndrome, an X-linked disorder characterized by ID and neurological abnormalities. While Rsk2-knockout mice were born healthy, a longitudinal MRI study revealed a transient secondary microcephaly and a persistent reduction of hippocampal and cerebellar volumes. Specific behavioral parameters from postnatal day 4 (P4) unveiled delayed acquisition of sensory-motor functions and alterations of spontaneous and cognitive behaviors during adolescence, which together, represent hallmarks of neurodevelopmental disorders. Together, our results suggest for the first time that RSK2, an effector of the MAPK signaling pathways, plays a crucial role in brain and cognitive postnatal development. This study also provides new relevant measures to characterize postnatal cognitive development of mouse models of ID and to design early therapeutic approaches.

Partial restoration of brain dystrophin by tricyclo-DNA antisense oligonucleotides alleviates emotional deficits in mdx52 mice.

The mdx52 mouse model recapitulates a frequent mutation profile associated with brain involvement in Duchenne muscular dystrophy. Deletion of exon 52 impedes expression of two dystrophins (Dp427, Dp140) expressed in brain, and is eligible for therapeutic exon-skipping strategies. We previously showed that mdx52 mice display enhanced anxiety and fearfulness, and impaired associative fear learning. In this study, we examined the reversibility of these phenotypes using exon 51 skipping to restore exclusively Dp427 expression in the brain of mdx52 mice. We first show that a single intracerebroventricular administration of tricyclo-DNA antisense oligonucleotides targeting exon 51 restores 5%-15% of dystrophin protein expression in the hippocampus, cerebellum, and cortex, at stable levels between 7 and 11 week after injection. Anxiety and unconditioned fear were significantly reduced in treated mdx52 mice and acquisition of fear conditioning appeared fully rescued, while fear memory tested 24 h later was only partially improved. Additional restoration of Dp427 in skeletal and cardiac muscles by systemic treatment did not further improve the unconditioned fear response, confirming the central origin of this phenotype. These findings indicate that some emotional and cognitive deficits associated with dystrophin deficiency may be reversible or at least improved by partial postnatal dystrophin rescue.

Investigating the Impact of Delivery Routes for Exon Skipping Therapies in the CNS of DMD Mouse Models.

Nucleic acid-based therapies have demonstrated great potential for the treatment of monogenetic diseases, including neurologic disorders. To date, regulatory approval has been received for a dozen antisense oligonucleotides (ASOs); however, these chemistries cannot readily cross the blood-brain barrier when administered systemically. Therefore, an investigation of their potential effects within the central nervous system (CNS) requires local delivery. Here, we studied the brain distribution and exon-skipping efficacy of two ASO chemistries, PMO and tcDNA, when delivered to the cerebrospinal fluid (CSF) of mice carrying a deletion in exon 52 of the dystrophin gene, a model of Duchenne muscular dystrophy (DMD). Following intracerebroventricular (ICV) delivery (unilateral, bilateral, bolus vs. slow rate, repeated via cannula or very slow via osmotic pumps), ASO levels were quantified across brain regions and exon 51 skipping was evaluated, revealing that tcDNA treatment invariably generates comparable or more skipping relative to that with PMO, even when the PMO was administered at higher doses. We also performed intra-cisterna magna (ICM) delivery as an alternative route for CSF delivery and found a biased distribution of the ASOs towards posterior brain regions, including the cerebellum, hindbrain, and the cervical part of the spinal cord. Finally, we combined both ICV and ICM injection methods to assess the potential of an additive effect of this methodology in inducing efficient exon skipping across different brain regions. Our results provide useful insights into the local delivery and associated efficacy of ASOs in the CNS in mouse models of DMD. These findings pave the way for further ASO-based therapy application to the CNS for neurological disease.

Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion.

Oxytocin (OT) is a prominent regulator of many aspects of mammalian social behavior and stored in large dense-cored vesicles (LDCVs) in hypothalamic neurons. It is released in response to activity-dependent Ca2+ influx, but is also dependent on Ca2+ release from intracellular stores, which primes LDCVs for exocytosis. Despite its importance, critical aspects of the Ca2+-dependent mechanisms of its secretion remain to be identified. Here we show that lysosomes surround dendritic LDCVs, and that the direct activation of endolysosomal two-pore channels (TPCs) provides the critical Ca2+ signals to prime OT release by increasing the releasable LDCV pool without directly stimulating exocytosis. We observed a dramatic reduction in plasma OT levels in TPC knockout mice, and impaired secretion of OT from the hypothalamus demonstrating the importance of priming of neuropeptide vesicles for activity-dependent release. Furthermore, we show that activation of type 1 metabotropic glutamate receptors sustains somatodendritic OT release by recruiting TPCs. The priming effect could be mimicked by a direct application of nicotinic acid adenine dinucleotide phosphate, the endogenous messenger regulating TPCs, or a selective TPC2 agonist, TPC2-A1-N, or blocked by the antagonist Ned-19. Mice lacking TPCs exhibit impaired maternal and social behavior, which is restored by direct OT administration. This study demonstrates an unexpected role for lysosomes and TPCs in controlling neuropeptide secretion, and in regulating social behavior.

Retinal dystrophins and the retinopathy of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by X-linked inherited or de novo DMD gene mutations predominantly affecting males who develop early-onset muscle degeneration, severely affecting their quality of life and leading to reduced life expectancy. DMD patients may also develop proliferative retinopathy, cataract, ERG abnormalities, altered contrast sensitivity, color vision losses, and elevated flash detection thresholds during dark adaptation. Depending on the position of the genetic alteration in the large DMD gene, it is associated with a lack of the full-length dystrophin protein possibly with an additional loss of one or several other dystrophins, which are normally transcribed from internal promoters in retina and crystalline lens. During the last decades, the properties of the dystrophins have been characterized in patients with different genetic alterations and in genetic mouse models of DMD. The complex expression pattern of the dystrophins in photoreceptors, Müller glial cells and astrocytes, likely influences synaptic transmission, ionic balance and vascular integrity of the retina. However, the specific function of each retinal dystrophin remains largely unknown. This review describes the current knowledge on dystrophin expression, the putative molecular, structural, and physiological properties of retinal dystrophins, and the main clinical implications associated with the loss of dystrophins in DMD patients and mouse models. Current data and working hypotheses warrant future research on retinal dystrophins to increase our understanding of dystrophin function in the central nervous system in general and to unveil new retinal mechanisms and therapeutic avenues for retinal diseases.

Tissue- and cell-specific whole-transcriptome meta-analysis from brain and retina reveals differential expression of dystrophin complexes and new dystrophin spliced isoforms.

The large DMD gene encodes a group of dystrophin proteins in brain and retina, produced from multiple promoters and alternative splicing events. Dystrophins are core components of different scaffolding complexes in distinct cell types. Their absence may thus alter several cellular pathways, which might explain the heterogeneous genotype-phenotype relationships underlying central comorbidities in Duchenne muscular dystrophy (DMD). However, the cell-specific expression of dystrophins and associated proteins (DAPs) is still largely unknown. The present study provides a first RNA-Seq-based reference showing tissue- and cell-specific differential expression of dystrophins, splice variants and DAPs in mouse brain and retina. We report that a cell type may express several dystrophin complexes, perhaps due to expression in separate cell subdomains and/or subpopulations, some of which with differential expression at different maturation stages. We also identified new splicing events in addition to the common exon-skipping events. These include a new exon within intron 51 (E51b) in frame with the flanking exons in retina, as well as inclusions of intronic sequences with stop codons leading to the presence of transcripts with elongated exons 40 and/or 41 (E40e, E41e) in both retina and brain. PCR validations revealed that the new exons may affect several dystrophins. Moreover, immunoblot experiments using a combination of specific antibodies and dystrophin-deficient mice unveiled that the transcripts with stop codons are translated into truncated proteins lacking their C-terminus, which we called N-Dp427 and N-Dp260. This study thus uncovers a range of new findings underlying the complex neurobiology of DMD.

Abnormal Expression of Synaptic and Extrasynaptic GABAA Receptor Subunits in the Dystrophin-Deficient mdx Mouse.

Duchenne muscular dystrophy (DMD) is a neurodevelopmental disorder primarily caused by the loss of the full-length Dp427 dystrophin in both muscle and brain. The basis of the central comorbidities in DMD is unclear. Brain dystrophin plays a role in the clustering of central gamma-aminobutyric acid A receptors (GABAARs), and its loss in the mdx mouse alters the clustering of some synaptic subunits in central inhibitory synapses. However, the diversity of GABAergic alterations in this model is still fragmentary. In this study, the analysis of in vivo PET imaging of a benzodiazepine-binding site radioligand revealed that the global density of central GABAARs is unaffected in mdx compared with WT mice. In contrast, semi-quantitative immunoblots and immunofluorescence confocal imaging in tissue sections revealed complex and differential patterns of alterations of the expression levels and/or clustered distribution of a variety of synaptic and extrasynaptic GABAAR subunits in the hippocampus, cerebellum, cortex, and spinal cord. Hence, dystrophin loss not only affects the stabilization of synaptic GABAARs but also influences the subunit composition of GABAARs subtypes at both synaptic and extrasynaptic sites. This study provides new molecular outcome measures and new routes to evaluate the impact of treatments aimed at compensating alterations of the nervous system in DMD.

Partial Restoration of Brain Dystrophin and Behavioral Deficits by Exon Skipping in the Muscular Dystrophy X-Linked (mdx) Mouse.

OBJECTIVES: Duchenne muscular dystrophy is associated with various degrees of cognitive impairment and behavioral disturbances. Emotional and memory deficits also constitute reliable outcome measures to assess efficacy of treatments in the mdx mouse lacking the muscle and neuronal full-length dystrophins. The present study aimed to evaluate whether these deficits could be alleviated by the restoration of brain dystrophin. METHODS: We performed intracerebroventricular administration of a new potent tricyclo-DNA antisense oligonucleotide (tcDNA-ASO) containing a full phosphodiester backbone conjugated to a palmitic acid moiety (tcDNA-ASO), designed to skip the mutated exon 23 of mdx mice. RESULTS: We first show that the tcDNA-ASO rescues expression of brain dystrophin to 10-30% of wild-type levels and significantly reduces the abnormal unconditioned fear responses in mdx mice in a dose-dependent manner, 5 weeks post-injection. Exon skipping efficiency, ASO biodistribution, protein restoration and effect on the fear response were optimal with a dose of 400 µg at 6-7 weeks post-injection, with synaptic-like expression in brain tissues such as the hippocampus and amygdala. Furthermore, this dose of tcDNA-ASO restored long-term memory retention of mdx mice in an object recognition task, but only had minor effects on fear conditioning. INTERPRETATION: These results suggest for the first time that postnatal re-expression of brain dystrophin could reverse or at least alleviate some cognitive deficits associated with Duchenne muscular dystrophy. ANN NEUROL 2022;92:213-229.

Disruption of amygdala Tsc2 in adolescence leads to changed prelimbic cellular activity and generalized fear responses at adulthood in rats.

Adolescence constitutes a period of vulnerability in the emergence of fear-related disorders (FRD), as a massive reorganization occurs in the amygdala-prefrontal cortex network, critical to regulate fear behavior. Genetic and environmental factors during development may predispose to the emergence of FRD at the adult age, but the underlying mechanisms are poorly understood. In the present study, we tested whether a partial knock-down of tuberous sclerosis complex 2 (Tsc2, Tuberin), a risk gene for neurodevelopmental disorders, in the basolateral amygdala (BLA) from adolescence could alter fear-network functionality and create a vulnerability ground to FRD appearance at adulthood. Using bilateral injection of a lentiviral vector expressing a miRNA against Tsc2 in the BLA of early (PN25) or late adolescent (PN50) rats, we show that alteration induced specifically from PN25 resulted in an increased c-Fos activity at adulthood in specific layers of the prelimbic cortex, a resistance to fear extinction and an overgeneralization of fear to a safe, novel stimulus. A developmental dysfunction of the amygdala could thus play a role in the vulnerability to FRD emergence at adulthood. We propose our methodology as an alternative to model the developmental vulnerability to FRD, especially in its comorbidity with TSC2-related autism syndrome.

Palmitic acid conjugation enhances potency of tricyclo-DNA splice switching oligonucleotides.

Tricyclo-DNA (tcDNA) is a conformationally constrained oligonucleotide analog that has demonstrated great therapeutic potential as antisense oligonucleotide (ASO) for several diseases. Like most ASOs in clinical development, tcDNA were modified with phosphorothioate (PS) backbone for therapeutic purposes in order to improve their biodistribution by enhancing association with plasma and cell protein. Despite the advantageous protein binding properties, systemic delivery of PS-ASO remains limited and PS modifications can result in dose limiting toxicities in the clinic. Improving extra-hepatic delivery of ASO is highly desirable for the treatment of a variety of diseases including neuromuscular disorders such as Duchenne muscular dystrophy. We hypothesized that conjugation of palmitic acid to tcDNA could facilitate the delivery of the ASO from the bloodstream to the interstitium of the muscle tissues. We demonstrate here that palmitic acid conjugation enhances the potency of tcDNA-ASO in skeletal and cardiac muscles, leading to functional improvement in dystrophic mice with significantly reduced dose of administered ASO. Interestingly, palmitic acid-conjugated tcDNA with a full phosphodiester backbone proved effective with a particularly encouraging safety profile, offering new perspectives for the clinical development of PS-free tcDNA-ASO for neuromuscular diseases.

Emotional behavior and brain anatomy of the mdx52 mouse model of Duchenne muscular dystrophy.

The exon-52-deleted mdx52 mouse is a critical model of Duchenne muscular dystrophy (DMD), as it features a deletion in a hotspot region of the DMD gene, frequently mutated in patients. Deletion of exon 52 impedes expression of several brain dystrophins (Dp427, Dp260 and Dp140), thus providing a key model for studying the cognitive impairment associated with DMD and testing rescuing strategies. Here, using in vivo magnetic resonance imaging and neurohistology, we found no gross brain abnormalities in mdx52 mice, suggesting that the neural dysfunctions in this model are likely at the level of brain cellular functionalities. Then, we investigated emotional behavior and fear learning performance of mdx52 mice compared to mdx mice that only lack Dp427 to focus on behavioral phenotypes that could be used in future comparative preclinical studies. mdx52 mice displayed enhanced anxiety and a severe impairment in learning an amygdala-dependent Pavlovian association. These replicable behavioral outcome measures are reminiscent of the internalizing problems reported in a quarter of DMD patients, and will be useful for preclinical estimation of the efficacy of treatments targeting brain dysfunctions in DMD.

Altered visual processing in the mdx52 mouse model of Duchenne muscular dystrophy.

The mdx52 mouse model of Duchenne muscular dystrophy (DMD) is lacking exon 52 of the DMD gene that is located in a hotspot mutation region causing cognitive deficits and retinal anomalies in DMD patients. This deletion leads to the loss of the dystrophin proteins, Dp427, Dp260 and Dp140, while Dp71 is preserved. The flash electroretinogram (ERG) in mdx52 mice was previously characterized by delayed dark-adapted b-waves. A detailed description of functional ERG changes and visual performances in mdx52 mice is, however, lacking. Here an extensive full-field ERG repertoire was applied in mdx52 mice and WT littermates to analyze retinal physiology in scotopic, mesopic and photopic conditions in response to flash, sawtooth and/or sinusoidal stimuli. Behavioral contrast sensitivity was assessed using quantitative optomotor response (OMR) to sinusoidally modulated luminance gratings at 100% or 50% contrast. The mdx52 mice exhibited reduced amplitudes and delayed implicit times in dark-adapted ERG flash responses, particularly in their b-wave and oscillatory potentials, and diminished amplitudes of light-adapted flash ERGs. ERG responses to sawtooth stimuli were also diminished and delayed for both mesopic and photopic conditions in mdx52 mice and the first harmonic amplitudes to photopic sine-wave stimuli were smaller at all temporal frequencies. OMR indices were comparable between genotypes at 100% contrast but significantly reduced in mdx52 mice at 50% contrast. The complex ERG alterations and disturbed contrast vision in mdx52 mice include features observed in DMD patients and suggest altered photoreceptor-to-bipolar cell transmission possibly affecting contrast sensitivity. The mdx52 mouse is a relevant model to appraise the roles of retinal dystrophins and for preclinical studies related to DMD.

Dp71 contribution to the molecular scaffold anchoring aquaporine-4 channels in brain macroglial cells.

Intellectual disability in Duchenne muscular dystrophy has been associated with the loss of dystrophin-protein 71, Dp71, the main dystrophin-gene product in the adult brain. Dp71 shows major expression in perivascular macroglial endfeet, suggesting that dysfunctional glial mechanisms contribute to cognitive impairments. In the present study, we investigated the molecular alterations induced by a selective loss of Dp71 in mice, using semi-quantitative immunogold analyses in electron microscopy and immunofluorescence confocal analyses in brain sections and purified gliovascular units. In macroglial pericapillary endfeet of the cerebellum and hippocampus, we found a drastic reduction (70%) of the polarized distribution of aquaporin-4 (AQP4) channels, a 50% reduction of ß-dystroglycan, and a complete loss of α1-syntrophin. Interestingly, in the hippocampus and cortex, these effects were not homogeneous: AQP4 and AQP4ex isoforms were mostly lost around capillaries but preserved in large vessels corresponding to pial arteries, penetrating cortical arterioles, and arterioles of the hippocampal fissure, indicating the presence of Dp71-independent pools of AQP4 in these vascular structures. In conclusion, the depletion of Dp71 strongly alters the distribution of AQP4 selectively in macroglial perivascular endfeet surrounding capillaries. This effect likely affects water homeostasis and blood-brain barrier functions and may thus contribute to the synaptic and cognitive defects associated with Dp71 deficiency.

Dystroglycan Mediates Clustering of Essential GABAergic Components in Cerebellar Purkinje Cells.

Muscle dystrophin-glycoprotein complex (DGC) links the intracellular cytoskeleton to the extracellular matrix. In neurons, dystroglycan and dystrophin, two major components of the DGC, localize in a subset of GABAergic synapses, where their function is unclear. Here we used mouse models to analyze the specific role of the DGC in the organization and function of inhibitory synapses. Loss of full-length dystrophin in mdx mice resulted in a selective depletion of the transmembrane ß-dystroglycan isoform from inhibitory post-synaptic sites in cerebellar Purkinje cells. Remarkably, there were no differences in the synaptic distribution of the extracellular α-dystroglycan subunit, of GABAA receptors and neuroligin 2. In contrast, conditional deletion of the dystroglycan gene from Purkinje cells caused a disruption of the DGC and severely impaired post-synaptic clustering of neuroligin 2, GABAA receptors and scaffolding proteins. Accordingly, whole-cell patch-clamp analysis revealed a significant reduction in the frequency and amplitude of spontaneous IPSCs recorded from Purkinje cells. In the long-term, deletion of dystroglycan resulted in a significant decrease of GABAergic innervation of Purkinje cells and caused an impairment of motor learning functions. These results show that dystroglycan is an essential synaptic organizer at GABAergic synapses in Purkinje cells.

Long-Term Efficacy of AAV9-U7snRNA-Mediated Exon 51 Skipping in mdx52 Mice.

Gene therapy and antisense approaches hold promise for the treatment of Duchenne muscular dystrophy (DMD). The advantages of both therapeutic strategies can be combined by vectorizing antisense sequences into an adeno-associated virus (AAV) vector. We previously reported the efficacy of AAV-U7 small nuclear RNA (U7snRNA)-mediated exon skipping in the mdx mouse, the dys - /utr - mouse, and the golden retriever muscular dystrophy (GRMD) dog model. In this study, we examined the therapeutic potential of an AAV-U7snRNA targeting the human DMD exon 51, which could be applicable to 13% of DMD patients. A single injection of AAV9-U7 exon 51 (U7ex51) induces widespread and sustained levels of exon 51 skipping, leading to significant restoration of dystrophin and improvement of the dystrophic phenotype in the mdx52 mouse. However, levels of dystrophin re-expression are lower than the skipping levels, in contrast with previously reported results in the mdx mouse, suggesting that efficacy of exon skipping may vary depending on the targeted exon. Additionally, while low levels of exon skipping were measured in the brain, the dystrophin protein could not be detected, in line with a lack of improvement of their abnormal behavioral fear response. These results thus confirm the high therapeutic potential of the AAV-mediated exon-skipping approach, yet the apparent discrepancies between exon skipping and protein restoration levels suggest some limitations of this experimental model.