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.

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.

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.

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.

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.

Rescue of Defective Electroretinographic Responses in Dp71-Null Mice With AAV-Mediated Reexpression of Dp71.

Purpose: To study the potential effect of a gene therapy, designed to rescue the expression of dystrophin Dp71 in the retinas of Dp71-null mice, on retinal physiology. Methods: We recorded electroretinograms (ERGs) in Dp71-null and wild-type littermate mice. In dark-adapted eyes, responses to flashes of several strengths were measured. In addition, flash responses on a 25-candela/square meters background were measured. On- and Off-mediated responses to sawtooth stimuli and responses to photopic sine-wave modulation (3-30 Hz) were also recorded. After establishing the ERG phenotype, the ShH10-GFP adeno-associated virus (AAV), which has been previously shown to target specifically Müller glial cells (MGCs), was delivered intravitreously with or without (sham therapy) the Dp71 coding sequence under control of a CBA promoter. ERG recordings were repeated three months after treatment. Real-time quantitative PCR and Western blotting analyses were performed in order to quantify Dp71 expression in the retinas. Results: Dp71-null mice displayed reduced b-waves in dark- and light-adapted flash ERGs and smaller response amplitudes to photopic rapid-on sawtooth modulation and to sine-wave stimuli. Three months after intravitreal injections of the ShH10-GFP-2A-Dp71 AAV vector, ERG responses were completely recovered in treated eyes of Dp71-null mice. The functional rescue was associated with an overexpression of Dp71 in treated retinas. Conclusions: The present results show successful functional recovery accompanying the reexpression of Dp71. In addition, this experimental model sheds light on MGCs influencing ERG components, since previous reports showed that aquaporin 4 and Kir4.1 channels were mislocated in MGCs of Dp71-null mice, while their distribution could be normalized following intravitreal delivery of the same ShH10-GFP-2A-Dp71 vector.

Evidence of the involvement of dystrophin Dp71 in corneal angiogenesis.

Purpose: The aim of this study was to define the role of dystrophin Dp71 in corneal angiogenesis. Methods: Inflammation-induced corneal neovascularization experiments were performed in Dp71-null mice and C57BL/6J wild-type mice. Results: The corneal neovascular area covered by neovascularization was larger in the injured corneas of the Dp71-null mice compared to the corneas of the wild-type mice: 40.72% versus 26.33%, respectively (p<0.005). Moreover, increased angiogenesis was associated with a high expression of vascular endothelial growth factor (VEGF). Similarly, aortic ring assays showed a significant enhancement of the neovascular area. Conclusions: These results suggest that dystrophin Dp71 could play an important role as a negative regulator of corneal angiogenesis.

Expression and localization of dystrophins and ß-dystroglycan in the hypothalamic supraoptic nuclei of rat from birth to adulthood.

Dystrophins (Dps) are the sub-membranous proteins that work via the dystrophin-associated proteins complex, which comprises ß-dystroglycan (ß-DG), a cell surface receptor for extracellular matrix. Recently, we have revealed ß-DG decrease and central function impairment of supraoptic nucleus (SON) in Dp71 deficient adult mice, opening the question on the profiles of Dps and ß-DG during SON development. At birth and the age of 10, 20 and 60 days, we examined the expression by RT-PCR and Western-blotting, and the distribution by immunohistochemistry of Dps and ß-DG. Also, we analyzed, by immunohistochemistry and Western-blotting, the neuropeptide, arginine vasopressin (AVP), in the SON at the different ages. At birth, Dp71 and to a lesser extends, Dp140 and Dp427, and also ß-DG are revealed in the SON. They are localized in the magnocellular neurons (MCNs), astrocytes and vessels. From birth to adulthood, the AVP raise in the SON coincides with the progressive increase of Dp71 level while the level of Dp140 and Dp427 increased only at D20, D10 post-natal development, respectively, and ß-DG expression did not change. Moreover, the location of Dps or/and ß-DG in the cell compartments was modified during development: at D10, Dps appeared in the astrocytes end-feet surrounding MCNs, and at D20, Dps and ß-DG codistributed in the astrocytes end-feet, surrounding MCNs and vessels. Such a distribution marks the first steps of post-natal SON development and may be considered essential in the establishment of structural plasticity mechanisms in SON, where astrocyte end-feet, vessels, magnocellular neurons, are physiologically associated. The disappearance of ß-DG in the MCNs nucleus marks the adulthood SON and suggests that the complex of Dps associating ß-DG is required for the nucleoskeleton function in the post-natal development.

Dystrophin Dp71 Isoforms Are Differentially Expressed in the Mouse Brain and Retina: Report of New Alternative Splicing and a Novel Nomenclature for Dp71 Isoforms.

Multiple dystrophin Dp71 isoforms have been identified in rats, mice, and humans and in several cell line models. These Dp71 isoforms are produced by the alternative splicing of exons 71 to 74 and 78 and intron 77. Three main groups of Dp71 proteins are defined based on their C-terminal specificities: Dp71d, Dp71f, and Dp71e. Dp71 is highly expressed in the brain and retina; however, the specific isoforms present in these tissues have not been determined to date. In this work, we explored the expression of Dp71 isoforms in the mouse brain and retina using RT-PCR assays followed by the cloning of PCR products into the pGEM-T Easy vector, which was used to transform DH5α cells. Dp71-positive colonies were later analyzed by PCR multiplex and DNA sequencing to determine the alternative splicing. We thus demonstrated the expression of Dp71 transcripts corresponding to Dp71, Dp71a, Dp71c, Dp71b, Dp71ab, Dp71 Δ110, and novel Dp71 isoforms spliced in exon 74; 71 and 74; 71, 73 and 74; and 74 and 78, which we named Dp71d Δ74 , Dp71d Δ71,74 , Dp71d Δ71,73-74 , and Dp71f Δ74 , respectively. Additionally, we demonstrated that the Dp71d group of isoforms is highly expressed in the brain, while the Dp71f group predominates in the retina, at both the cDNA and protein levels. These findings suggest that distinct Dp71 isoforms may play different roles in the brain and retina.

The 3D Retinal Capillary Circulation in Pigs Reveals a Predominant Serial Organization.

Purpose: To establish a model of the retinal capillary circulation in pigs, which in many aspects is close to the human retina. Methods: Using high density confocal microscopy image stacks of immunolabeled porcine retinal whole mounts, microvessels close to the optic nerve head were traced in three dimensions. The direction of flow of individual capillaries was deduced from their arteriolar and/or venous connections. Results: From major arteries, second-order arteries traversed the nerve fiber layer and resolved exclusively into the superficial vascular plexus (SVP), which dichotomized the blood flow between radial peripapillary capillaries (RPCs) on one side and the intermediate (IVP) and deep vascular plexus (DVP) on the other. Each RPC was supplied by one or several capillaries from the SVP and drained to the IVP or DVP. The DVP was a mosaic of approximately 300 to 600 µm wide anastomotic watersheds, each drained by one or two venules connected to major veins. A presumptive direction of flow could be determined for >90% of capillaries. These results suggest a model of the capillary circulation in which the three microvessel layers are serially organized with RPCs are in parallel between the SVP and IVP or DVP. Conclusions: In the peripapillary retina of pigs, microvascular layers have a serial arrangement, with RPCs emerging from the SVP and draining to the IVP or DVP; hence, connected in parallel of this scheme. The bulk of flow, therefore, traverses the SVP and DVP successively. This organization contributes to the higher oxygen saturation in the SVP and RPCs than in the DVP. Physiopathologic implications of this model regarding retinal diseases are discussed.

Protection of Glial Müller Cells by Dexamethasone in a Mouse Model of Surgically Induced Blood-Retinal Barrier Breakdown.

Purpose: Breakdown of the inner blood-retinal barrier (iBRB) occurs in many retinal disorders and may cause retinal edema often responsible for vision loss. Dexamethasone is used in clinical practice to restore iBRB. The aim of this study was to characterize the impact of a surgically induced iBRB breakdown on retinal homeostatic changes due to dystrophin Dp71, aquaporin-4 (AQP4), and Kir4.1 alterations in Müller glial cells (MGC) in a mouse model. The protective effect of dexamethasone was assessed in this model. Moreover, retinal explants were used to control MGC exposure to a hypoosmotic solution containing barium. Methods: Partial lens surgery was performed in C57BL6/J mice. Dystrophin Dp71, AQP4, and Kir4.1 expression was analyzed by quantitative RT-PCR, Western blot, and immunohistochemistry. Twenty-four hours after surgery, mice received a single intravitreal injection of dexamethasone or of vehicle. Results: After partial lens surgery, iBRB permeability increased while Dp71 and AQP4 were downregulated and Kir4.1 was delocalized. These effects were partially prevented by dexamethasone injection. In the retinal explant model, MGC were swollen and Dp71, AQP4, and Kir4.1 were downregulated after exposure to a hypoosmotic solution containing barium, but not in the presence of dexamethasone. Heat shock factor protein 1 (HSF1) was overexpressed in dexamethasone-treated retinas. Conclusions: Partial lens surgery induces iBRB breakdown and molecular changes in MGC, including a downregulation of Dp71 and AQP4 and the delocalization of Kir4.1. Dexamethasone seems to protect retina from these molecular changes by upregulating HSF1.

AAV-mediated gene therapy in Dystrophin-Dp71 deficient mouse leads to blood-retinal barrier restoration and oedema reabsorption.

Dystrophin-Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells that provide a mechanical link at the Müller cell membrane by direct binding to actin and a transmembrane protein complex. Its absence has been related to blood-retinal barrier (BRB) permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels (1). We have previously shown that the adeno-associated virus (AAV) variant, ShH10, transduces Müller cells in the Dp71-null mouse retina efficiently and specifically (2,3). Here, we use ShH10 to restore Dp71 expression in Müller cells of Dp71 deficient mouse to study molecular and functional effects of this restoration in an adult mouse displaying retinal permeability. We show that strong and specific expression of exogenous Dp71 in Müller cells leads to correct localization of Dp71 protein restoring all protein interactions in order to re-establish a proper functional BRB and retina homeostasis thus preventing retina from oedema. This study is the basis for the development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular oedema such as diabetic retinopathy (DR).

Altered astrocyte morphology and vascular development in dystrophin-Dp71-null mice.

Understanding retinal vascular development is crucial because many retinal vascular diseases such as diabetic retinopathy (in adults) or retinopathy of prematurity (in children) are among the leading causes of blindness. Given the localization of the protein Dp71 around the retinal vessels in adult mice and its role in maintaining retinal homeostasis, the aim of this study was to determine if Dp71 was involved in astrocyte and vascular development regulation. An experimental study in mouse retinas was conducted. Using a dual immunolabeling with antibodies to Dp71 and anti-GFAP for astrocytes on retinal sections and isolated astrocytes, it was found that Dp71 was expressed in wild-type (WT) mouse astrocytes from early developmental stages to adult stage. In Dp71-null mice, a reduction in GFAP-immunopositive astrocytes was observed as early as postnatal day 6 (P6) compared with WT mice. Using real-time PCR, it was showed that Dp71 mRNA was stable between P1 and P6, in parallel with post-natal vascular development. Regarding morphology in Dp71-null and WT mice, a significant decrease in overall astrocyte process number in Dp71-null retinas at P6 to adult age was found. Using fluorescence-conjugated isolectin Griffonia simplicifolia on whole mount retinas, subsequent delay of developing vascular network at the same age in Dp71-null mice was found. An evidence that the Dystrophin Dp71, a membrane-associated cytoskeletal protein and one of the smaller Duchenne muscular dystrophy gene products, regulates astrocyte morphology and density and is associated with subsequent normal blood vessel development was provided.

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease.

Müller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling Müller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms. There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5 has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label Müller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

Dystrophin Dp71 gene deletion induces retinal vascular inflammation and capillary degeneration.

We have previously shown that the deletion of the dystrophin Dp71 gene induces a highly permeable blood-retinal barrier (BRB). Given that BRB breakdown is involved in retinal inflammation and the pathophysiology of many blinding eye diseases, here we investigated whether the absence of Dp71 brings out retinal vascular inflammation and vessel loss by using specific Dp71-null mice. The expression of vascular endothelial growth factor (VEGF), quantified by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay methods, was higher in the retina of Dp71-null mice than in wild-type mice. In contrast, no differences were observed in VEGFR-2 and tumor necrosis factor-α expression. Moreover, mRNA expression of water channel, aquaporin 4 (AQP4) was increased after Dp71 deletion. The Dp71 deletion was also associated with the overexpression of intercellular adhesion molecule 1, which is expressed on endothelial cells surface to recruit leukocytes. Consistent with these findings, the total number of adherent leukocytes per retina, assessed after perfusion with fluorescein isothiocyanate-conjugated concanavalin A, was increased in the absence of Dp71. Finally, a significant increase in capillary degeneration quantified after retinal trypsin digestion was observed in mice lacking Dp71. These data illustrate for the first time that the deletion of Dp71 was associated with retinal vascular inflammation, vascular lesions with increased leukocyte adhesion and capillary degeneration. Thus, dystrophin Dp71 could play a critical role in retinal vascular inflammation disease, and therefore represent a potential therapeutic target.

AAV-mediated gene delivery in Dp71-null mouse model with compromised barriers.

Formation and maintenance of the blood-retinal barrier (BRB) is required for proper vision and breaching of this barrier contributes to the pathology in a wide variety of retinal conditions such as retinal detachment and diabetic retinopathy. Dystrophin Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells, its absence has been related to BRB permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels. Dp71-null mouse is thus an excellent model to approach the study of retinal pathologies showing blood-retinal barrier permeability. We aimed to investigate the participation of Müller cells in the BRB and in the inner limiting membrane of Dp71-null mice compared with wild-type mice in order to understand how these barriers work in this model of permeable BRB. To this aim, we used an Adeno-associated virus (AAV) variant, ShH10-GFP, engineered to target Müller cells specifically. ShH10 coding GFP was introduced by intravitreal injection and Müller cell transduction was studied in Dp71-null mice in comparison to wild-type animals. We show that Müller cell transduction follows a significantly different pattern in Dp71-null mice indicating changes in viral cell-surface receptors as well as differences in the permeability of the inner limiting membrane in this mouse line. However, the compromised BRB of the Dp71-null mice does not lead to virus leakage into the bloodstream when the virus is injected intravitreally - an important consideration for AAV-mediated retinal gene therapy.