Duchenne Muscular Dystrophy Mouse Research Articles

Autophagy as a new therapeutic target in Duchenne muscular dystrophy

A resolutive therapy for Duchene muscular dystrophy, a severe degenerative disease of the skeletal muscle, is still lacking. Because autophagy has been shown to be crucial in clearing dysfunctional organelles and in preventing tissue damage, we investigated its pathogenic role and its suitability as a target for new therapeutic interventions in Duchenne muscular dystrophy (DMD). Here we demonstrate that autophagy is severely impaired in muscles from patients affected by DMD and mdx mice, a model of the disease, with accumulation of damaged organelles. The defect in autophagy was accompanied by persistent activation via phosphorylation of Akt, mammalian target of rapamycin (mTOR) and of the autophagy-inhibiting pathways dependent on them, including the translation-initiation factor 4E-binding protein 1 and the ribosomal protein S6, and downregulation of the autophagy-inducing genes LC3, Atg12, Gabarapl1 and Bnip3. The defective autophagy was rescued in mdx mice by long-term exposure to a low-protein diet. The treatment led to normalisation of Akt and mTOR signalling; it also reduced significantly muscle inflammation, fibrosis and myofibre damage, leading to recovery of muscle function. This study highlights novel pathogenic aspects of DMD and suggests autophagy as a new effective therapeutic target. The treatment we propose can be safely applied and immediately tested for efficacy in humans.

Dystrophin and utrophin “double knockout” dystrophic mice exhibit a spectrum of degenerative musculoskeletal abnormalities

Duchenne muscular dystrophy (DMD) is a degenerative muscle disorder characterized by the lack of dystrophin expression at the sarcolemma of muscle fibers. In addition, DMD patients acquire osteopenia, fragility fractures, and scoliosis indicating that a deficiency in skeletal homeostasis coexists but little is known about the effects of DMD on bone and other connective tissues within the musculoskeletal system. Recent evidence has emerged implicating adult stem cell dysfunction in DMD myopathogenesis. Given the common mesenchymal origin of muscle and bone, we sought to investigate bone and other musculoskeletal tissues in a DMD mouse model. Here, we report that dystrophin-utrophin double knockout (dko) mice exhibit a spectrum of degenerative changes, outside skeletal muscle, in bone, articular cartilage, and intervertebral discs, in addition to reduced lifespan, muscle degeneration, spinal deformity, and cardiomyopathy previously reported. We also report these mice to have a reduced capacity for bone healing and exhibit spontaneous heterotopic ossification in the hind limb muscles. Therefore, we propose the dko mouse as a model for premature musculoskeletal aging and posit that a similar phenomenon may occur in patients with DMD.

T.P.34 FHL1 reduces muscle degeneration in dystrophin-deficient mdx mice through the sarcolemmal recruitment of utrophin

Abstract Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations, resulting in loss of dystrophin from the sarcolemma. Utrophin is a therapeutic target for DMD due to its functional compensation for dystrophin, by forming the protective utrophin–glycoprotein complex (UGC) at the sarcolemma. In adult muscle fibres utrophin localizes to the neuromuscular junction, therefore the challenge is to identify factors which increase utrophin expression and sarcolemmal recruitment. Calcineurin/NFATc1 is a utrophin regulatory pathway and we reported that FHL1 coactivates NFATc1. We crossed our skeletal muscle FHL1 transgenic mice with the mdx model of DMD. FHL1 ameliorated degeneration in multiple mdx muscles at 4- and 16-weeks of age. Relative to mdx mice, mdx /FHL1 mice also exhibited decreased serum creatine kinase (2.5-fold) and a reduction in muscle fibres stained for serum IgM, further indicating the protective effect of FHL1. Chronic myofibre damage in DMD and mdx mice induces pronounced muscle infiltration with macrophages, which were reduced in mdx /FHL1 mice. The benefit of FHL1 was sustained in older mdx /FHL1 mice (9months) as shown by a significant reduction (2-fold) in diaphragm fibrosis compared to mdx mice. In functional studies the tibialis anterior from mdx /FHL1 mice was also protected against contraction-induced injury, indicating FHL1 stabilizes muscle fibres during repetitive muscle contractions. We identified the mechanism by which FHL1 reduced muscle degeneration; luciferase assays revealed FHL1 potentiated NFATc1-activation of the utrophin A promoter and utrophin mRNA and protein were increased in mdx /FHL1 mice. Immunofluorescence analysis revealed utrophin localized to the sarcolemma in adult muscle fibres of mdx /FHL1 mice, where it directed formation of the UGC by recruiting α- and β-dystroglycan, α- and γ-sarcoglycan and syntrophin. This study identifies FHL1 as a utrophin regulatory protein and therefore a potential therapeutic target for DMD.

Engineering Multiple U7snRNA Constructs to Induce Single and Multiexon-skipping for Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD but still faces personalized medicine challenges as different mutations found in DMD patients require skipping of different exons. However, 70% of DMD patients harbor dystrophin gene deletions in a mutation-rich area or "hot-spot" in the central genomic region. In this study, we have developed 11 different U7 small-nuclear RNA, to shuttle antisense sequences designed to mask key elements involved in the splicing of exons 45 to 55. We demonstrate that these constructs induce efficient exon skipping both in vitro in DMD patients' myoblasts and in vivo in human DMD (hDMD) mice and that they can be combined into a single vector to achieve a multi skipping of at least 3 exons. These very encouraging results provide proof of principle that efficient multiexon-skipping can be achieved using adeno-associated viral (AAV) vectors encoding multiple U7 small-nuclear RNAs (U7snRNAs), offering therefore very promising tools for clinical treatment of DMD.

Treatment with a nitric oxide‐donating NSAID counteracts functional muscle ischemia in dystrophin‐deficient mdx mice

The dystrophin‐deficient muscles of boys with Duchenne muscular dystrophy (DMD) and mdx mice, a model of DMD, are susceptible to ischemia during exercise due to loss of neuronal nitric oxide synthase (nNOS) from the sarcolemma. We hypothesized that treatment with a NO‐donating drug would compensate for nNOS deficiency and counteract functional muscle ischemia. We fed mdx mice a standard diet containing 1% soybean oil (vehicle) or a low (15 mg/kg) or high (45 mg/kg) dose of a NO‐releasing derivative of the NSAID flurbiprofen (n = 12/group). After 1 month of treatment, we compared vasoconstrictor responses to intra‐arterial norepinephrine (NE) in resting and contracting hindlimbs. In vehicle‐treated mice, the usual effect of muscle contraction to attenuate NE‐mediated vasoconstriction was impaired as shown by similar decreases in femoral vascular conductance in contracting vs resting hindlimbs (attenuation ratio = 0.88 ± 0.03). NE‐induced ischemia was also seen in the contracting hindlimbs of mice treated with low dose drug (0.92 ± 0.04; P > 0.05 vs vehicle), but was markedly attenuated in mice treated with high dose drug (0.22 ± 0.03; P < 0.05 vs vehicle or low dose). The beneficial effect of the high dose was maintained with treatment up to 3 months. These data demonstrate a robust anti‐ischemic effect of a NO‐donating drug in mdx mice and suggest a potential use in the treatment of DMD patients. Supported by NicOx, 801130.

Letter by Fayssoil Regarding Article, “Early Treatment With Lisinopril and Spironolactone Preserves Cardiac and Skeletal Muscle in Duchenne Muscular Dystrophy Mice”

To the Editor: I read with great interest the article by Rafael-Fortney et al1 about the effect of early treatment with lisinopril and spironolactone on cardiac and skeletal muscle in Duchenne muscular dystrophy mice. This article raised the concerns stated below. The authors have …

Response to Letter Regarding Article, “Early Treatment With Lisinopril and Spironolactone Preserves Cardiac and Skeletal Muscle in Duchenne Muscular Dystrophy Mice”

Thank you for the insightful comments regarding our work.1 We have not yet explored the level of renin-angiotensin-aldosterone system activation in this model, which might help shed light on underlying mechanisms by which the drugs studied produce their benefits. However, one study of muscle biopsies from patients with Duchenne muscular dystrophy shows upregulation of angiotensin-converting enzyme in dystrophic in comparison with normal muscle.2 Because …

Negamycin analogue with readthrough-promoting activity as a potential drug candidate for duchenne muscular dystrophy.

A series of (+)-negamycin 1 analogues were synthesized, and their readthrough-promoting activity was evaluated for nonsense mutations in Duchenne muscular dystrophy (DMD). A structure-activity relationship study indicated that 11b was the most potent drug candidate. Immunohistochemical analyses suggested that treatment with 11b restored dystrophin expression in mdx mice, a DMD mouse model. Furthermore, 11b decreased serum creatine kinase (CK) levels, an indicator of muscle fiber destruction. Most importantly, 11b demonstrated lower toxicity than 1, and thus, it could be a useful candidate for long-term treatment of DMD.

The different impact of a high fat diet on dystrophic mdx and control C57Bl/10 mice.

The absence of functional dystrophin protein in patients with Duchenne muscular dystrophy (DMD) and dystrophic mdx mice leads to fragile myofibre membranes and cycles of myofibre necrosis and regeneration. It is proposed that both DMD patients and mdx mice have an altered metabolism and impaired energy status and that nutritional supplementation may reduce the severity of dystropathology. This research compares the in vivo responses of dystrophic mdx and normal control C57Bl/10 mice to a high protein (50%) or a high fat (16%) diet. Consumption of a high protein diet had minimal effects on the body composition or muscle morphology in both strains of mice. In contrast, differences between the strains were seen in response to the high fat diet; this response also varied between mdx mice aged <24 weeks, and mdx mice aged 24 - 40 weeks. C57Bl/10 mice demonstrated many negative side effects after consuming the high fat diet, including weight gain, increased body fat, and elevated inflammatory cytokines. In contrast, after consuming the high fat diet for 16 weeks the mdx mice (< 24 weeks) remained lean with minimal fat deposition and were resistant to changes in body composition. These results support the proposal that energy metabolism in dystrophic mdx mice is altered compared to normal C57Bl/10 mice and this enables the mdx mice to better metabolise the high fat diet and avoid fat deposition. However, older mdx mice (24 – 40-week-old), with increased energy intake, exhibited some mild adverse effects of a high fat diet but to a far lesser extent than age-matched C57Bl/10 mice. Benefits of the high fat diet on dystrophic muscles of young mice were demonstrated by the significantly increased running ability (km) of voluntarily exercised mdx mice and significantly reduced myofibre necrosis in 24-week-old sedentary mdx mice. These novel data clearly identify an ‘altered’ response to a high fat diet in dystrophic mdx compared to normal C57Bl/10 mice. Our data indicate that the high fat diet may better meet the energy needs of mdx mice to reduce muscle damage and improve muscle function.

AAV6-mediated Systemic shRNA Delivery Reverses Disease in a Mouse Model of Facioscapulohumeral Muscular Dystrophy

Treatment of dominantly inherited muscle disorders remains a difficult task considering the need to eliminate the pathogenic gene product in a body-wide fashion. We show here that it is possible to reverse dominant muscle disease in a mouse model of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a common form of muscular dystrophy associated with a complex cascade of epigenetic events following reduction in copy number of D4Z4 macrosatellite repeats located on chromosome 4q35. Several 4q35 genes have been examined for their role in disease, including FRG1. Overexpression of FRG1 causes features related to FSHD in transgenic mice and the FRG1 mouse is currently the only available mouse model of FSHD. Here we show that systemic delivery of RNA interference expression cassettes in the FRG1 mouse, after the onset of disease, led to a dose-dependent long-term FRG1 knockdown without signs of toxicity. Histological features including centrally nucleated fibers, fiber size reduction, fibrosis, adipocyte accumulation, and inflammation were all significantly improved. FRG1 mRNA knockdown resulted in a dramatic restoration of muscle function. Through RNA interference (RNAi) expression cassette redesign, our method is amenable to targeting any pathogenic gene offering a viable option for long-term, body-wide treatment of dominant muscle disease in humans.

Human Muscle-Derived Cell Populations Isolated by Differential Adhesion Rates: Phenotype and Contribution to Skeletal Muscle Regeneration in Mdx/SCID Mice

Muscle-derived stem cells (MDSCs) isolated from murine skeletal tissue by the preplate method have displayed the capability to commit to the myogenic lineage and regenerate more efficiently than myoblasts in skeletal and cardiac muscle in murine Duchenne Muscular Dystrophy mice (mdx). However, until now, these studies have not been translated to human muscle cells. Here, we describe the isolation, by a preplate technique, of candidate human MDSCs, which exhibit myogenic and regenerative characteristics similar to their murine counterparts. Using the preplate isolation method, we compared cells that adhere faster to the flasks, preplate 2 (PP2), and cells that adhere slower, preplate 6 (PP6). The human PP6 cells express several markers of mesenchymal stem cells and are distinct from human PP2 (a myoblast-like population) based on their expression of CD146 and myogenic markers desmin and CD56. After transplantation to the gastrocnemius muscle of mdx/SCID mice, we observe significantly higher levels of PP6 cells participating in muscle regeneration as compared with the transplantation of PP2 cells. This study supports some previous findings related to mouse preplate cells, and also identifies some differences between mouse and human muscle preplate cells.

Early treatment with lisinopril and spironolactone preserves cardiac and skeletal muscle in Duchenne muscular dystrophy mice.

Nearly universal cardiomyopathy in Duchenne muscular dystrophy (DMD) contributes to heart failure and death. Because DMD patients show myocardial fibrosis well before functional impairment, we postulated that earlier treatment using drugs with antifibrotic effect may be beneficial. Three groups of 10 utrn(+/-);mdx, or "het" mice, deficient for dystrophin and haploinsufficient for utrophin with skeletal myopathy and cardiomyopathy that closely mimics clinical DMD were studied. One het group received spironolactone and lisinopril starting at 8 weeks of life (het-treated-8); a second received the same starting at 4 weeks of life (het-treated-4), and the third het group was untreated. At 20 weeks, all mice had normal ejection fractions though circumferential strain rate was abnormal (-0.21±0.08) in untreated hets. This improved to -0.40±0.07 in het-treated-8 mice (P=0.003) and further improved to -0.56±0.10 in het-treated-4 mice (P=0.014 for het-treated-4 versus het-treated-8). Treated mice showed less cardiomyocyte damage, with a 44% reduction in intracardiomyocyte serum immunoglobulin G localization in het-treated-8 mice (P<0.0001) and a further 53% reduction in het-treated-4 mice (P=0.0003 versus het-treated-8); matrix metalloproteinases were similarly reduced. Cardiac, limb, and diaphragm function by ex vivo muscle testing remained at 80% of normal with early treatment compared to a decline to 40% of normal skeletal muscle function without treatment. These findings offer clinically available medications with proven antifibrotic effect as a new therapeutic strategy in DMD. Early initiation greatly attenuated myocardial disease and, for the first time with these drugs, improved skeletal myopathy. Thus, early initiation of such agents warrants further clinical evaluation to maintain ambulatory, respiratory, and cardiac function for patients with DMD and related myopathies.

1H MRS Measurements Of Myocellular Lipids After Injury To Healthy And Dystrophic Muscles

Duchenne muscular dystrophy (DMD) and mdx mice (an animal model for human DMD) are considered homologous based on the lack of dystrophin. The parallels in damage between injured and dystrophic skeletal muscles are striking; both are characterized by inflammation, enzyme efflux, fiber splitting, regeneration, etc. Interestingly, there is little evidence of fatty infiltration in the muscles of mdx mice when compared to patients with DMD, and the role of fat post injury is unclear. Fat is stored inside fibers as droplets (intra-myocellular lipids, IMCL) or in adipocytes between myofibers (extra-myocellular lipids, EMCL) and can be identified using magnetic resonance spectroscopy. PURPOSE: To examine the effects of injury on IMCL and EMCL in muscles of healthy mice and mdx mice. We hypothesized that muscles from mdx and injured mice would have increased fatty content after injury. METHODS: Unilateral injury to the tibialis anterior muscle (TA) was induced by 15 lengthening contractions in mdx mice (n=4) and control mice (n=4). High resolution proton density-weighted (100 μm × 100 μm × 750 µm) and in vivo localized 1H MRS were acquired on a Bruker 7T animal MRI system. Oil red O (ORO) staining of sections was used to identify lipid depositions. RESULTS: Mdx and control muscles generated a similar amount of absolute force, but mdx were significantly more susceptible to injury (85% vs 42% loss of force, respectively, p<0.01). EMCL to total metabolic peaks (EMCL/total) ratio significantly increased in the injured TA (0.23/0.60) compared to uninjured TA (0.05/0.08) (p<0.05). IMCL/total ratio also significantly increased in the injured TA (0.03/0.04) compared to the uninjured TA (0.06/0.17). For mdx muscles, the EMCL peak did not change (injured leg 0.05/0.06, uninjured 0.02/0.18). IMCL ratio was 0, but increased to 0.07/0.36 after injury. ORO staining confirmed the changes quantified by the 1H MRS findings. CONCLUSIONS: The data suggest that 1H MRS can be used as a biomarker to determine metabolic alterations in skeletal muscle associated with injury and/or disease. In the control muscles, EMCL and IMCL ratios are associated with the amount of force loss, but the mdx muscles present a more complex picture. This work was supported by grants to RML from the NIH (K01AR053235 and 1R01AR059179) and from the Muscular Dystrophy Association

Mechanisms inducing low bone density in duchenne muscular dystrophy in mice and humans

Patients affected by Duchenne muscular dystrophy (DMD) and dystrophic MDX mice were investigated in this study for their bone phenotype and systemic regulators of bone turnover. Micro–computed tomographic (µCT) and histomorphometric analyses showed reduced bone mass and higher osteoclast and bone resorption parameters in MDX mice compared with wild-type mice, whereas osteoblast parameters and mineral apposition rate were lower. In a panel of circulating pro-osteoclastogenic cytokines evaluated in the MDX sera, interleukin 6 (IL-6) was increased compared with wild-type mice. Likewise, DMD patients showed low bone mineral density (BMD) Z-scores and high bone-resorption marker and serum IL-6. Human primary osteoblasts from healthy donors incubated with 10% sera from DMD patients showed decreased nodule mineralization. Many osteogenic genes were downregulated in these cultures, including osterix and osteocalcin, by a mechanism blunted by an IL-6-neutralizing antibody. In contrast, the mRNAs of osteoclastogenic cytokines IL6, IL11, inhibin-βA, and TGFβ2 were increased, although only IL-6 was found to be high in the circulation. Consistently, enhancement of osteoclastogenesis was noted in cultures of circulating mononuclear precursors from DMD patients or from healthy donors cultured in the presence of DMD sera or IL-6. Circulating IL-6 also played a dominant role in osteoclast formation because ex vivo wild-type calvarial bones cultured with 10% sera of MDX mice showed increase osteoclast and bone-resorption parameters that were dampen by treatment with an IL-6 antibody. These results point to IL-6 as an important mediator of bone loss in DMD and suggest that targeted anti-IL-6 therapy may have a positive impact on the bone phenotype in these patients. © 2011 American Society for Bone and Mineral Research

Impaired respiratory function in mdx and mdx/utrn+/− mice

Muscle fibrosis is a prominent pathological feature that directly causes muscle dysfunction in Duchenne muscular dystrophy (DMD). The DMD mouse models, mdx mice and mdx mice with haploinsufficiency of the utrophin gene (mdx/utrn(+/-) ), display progressive diaphragm fibrosis. We performed unrestrained whole-body plethysmography (WBP) in mdx and mdx/utrn(+/-) mice, and compared them with wild-type controls. Respiratory function gauged by respiratory frequency, tidal volume, minute volume, peak inspiratory flow, and peak expiratory flow was significantly impaired in the mdx mice. Consistent with more severe diaphragm fibrosis in the mdx/utrn(+/-) mice, respiratory impairment was worse than in mdx mice at 6 months. WBP is useful for monitoring in vivo respiratory function of mdx and mdx/utrn(+/-) mice, and it may serve as an outcome measurement for therapies that target diaphragm fibrosis. The mdx/utrn(+/-) mouse model may be better than the mdx model for testing antifibrotic therapies, especially at the severe stage.

Marginal Level Dystrophin Expression Improves Clinical Outcome in a Strain of Dystrophin/Utrophin Double Knockout Mice

Inactivation of all utrophin isoforms in dystrophin-deficient mdx mice results in a strain of utrophin knockout mdx (uko/mdx) mice. Uko/mdx mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD) patients. Here we tested the hypothesis that marginal level dystrophin expression may improve the clinical outcome of uko/mdx mice. It is well established that mdx3cv (3cv) mice express a near-full length dystrophin protein at ∼5% of the normal level. We crossed utrophin-null mutation to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies.

Short Telomeres and Stem Cell Exhaustion Model Duchenne Muscular Dystrophy in mdx/mTR Mice

In Duchenne muscular dystrophy (DMD), dystrophin mutation leads to progressive lethal skeletal muscle degeneration. For unknown reasons, dystrophin deficiency does not recapitulate DMD in mice (mdx), which have mild skeletal muscle defects and potent regenerative capacity. We postulated that human DMD progression is a consequence of loss of functional muscle stem cells (MuSC), and the mild mouse mdx phenotype results from greater MuSC reserve fueled by longer telomeres. We report that mdx mice lacking the RNA component of telomerase (mdx/mTR) have shortened telomeres in muscle cells and severe muscular dystrophy that progressively worsens with age. Muscle wasting severity parallels a decline in MuSC regenerative capacity and is ameliorated histologically by transplantation of wild-type MuSC. These data show that DMD progression results, in part, from a cell-autonomous failure ofMuSC to maintain the damage-repair cycle initiated by dystrophin deficiency. The essential role of MuSC function has therapeutic implications for DMD.

Overexpression of SERCA1a in the mdx Diaphragm Reduces Susceptibility to Contraction-Induced Damage

Although the precise pathophysiological mechanism of muscle damage in dystrophin-deficient muscle remains disputed, calcium appears to be a critical mediator of the dystrophic process. Duchenne muscular dystrophy patients and mouse models of dystrophin deficiency exhibit extensive abnormalities of calcium homeostasis, which we hypothesized would be mitigated by increased expression of the sarcoplasmic reticulum calcium pump. Neonatal adeno-associated virus gene transfer of sarcoplasmic reticulum ATPase 1a to the mdx diaphragm decreased centrally located nuclei and resulted in reduced susceptibility to eccentric contraction-induced damage at 6 months of age. As the diaphragm is the mouse muscle most representative of human disease, these results provide impetus for further investigation of therapeutic strategies aimed at enhanced cytosolic calcium removal.

BMP antagonists enhance myogenic differentiation and ameliorate the dystrophic phenotype in a DMD mouse model

Duchenne Muscular Dystrophy (DMD) is an X-linked lethal muscle wasting disease characterized by muscle fiber degeneration and necrosis. The progressive pathology of DMD can be explained by an insufficient regenerative response resulting in fibrosis and adipose tissue formation. BMPs are known to inhibit myogenic differentiation and in a previous study we found an increased expression of a BMP family member BMP4 in DMD myoblasts. The aim of the current study was therefore to investigate whether inhibition of BMP signaling could be beneficial for myoblast differentiation and muscle regeneration processes in a DMD context. All tested BMP inhibitors, Noggin, dorsomorphin and LDN-193189, were able to accelerate and enhance myogenic differentiation. However, dorsomorphin repressed both BMP and TGFβ signaling and was found to be toxic to primary myoblast cell cultures. In contrast, Noggin was found to be a potent and selective BMP inhibitor and was therefore tested in vivo in a DMD mouse model. Local adenoviral-mediated overexpression of Noggin in muscle resulted in an increased expression of the myogenic regulatory genes Myog and Myod1 and improved muscle histology. In conclusion, our results suggest that repression of BMP signaling may constitute an attractive adjunctive therapy for DMD patients.

A deficit of brain dystrophin 71 impairs hypothalamic osmostat

Patients with Duchenne muscular dystrophy (DMD) and mdx mice, devoid of dystrophin proteins, show altered ionic homeostasis. To clarify dystrophin's involvement in the central control of osmotic stimuli, we investigated the effect of the disruption of Dp71, the major form of dystrophin in the brain, on the hypothalamoneurohypophysis system (HNHS) osmoregulatory response. Dp71 and Dp140 are the principal DMD gene products in the supraoptic nucleus (SON) and neurohypophysis (NH). They are present in astrocyte and pituicyte end-feet, suggesting involvement in both intrinsic osmosensitivity of the SON and vasopressin (AVP) release from the NH. In Dp71-null mice, the cellular distribution of Dp140 was modified, this protein being detected on the membrane of magnocellular soma. The plasma osmolality of Dp71-null mice was lower than that of wild-type mice under normal conditions, and this difference was maintained after salt loading, indicating a change in the set point for osmoregulation in the absence of Dp71. The increase in AVP levels detected in the SON and NH of the wild-type was not observed in Dp71-null mice following salt loading, and the increase in AVP mRNA levels in the SON was smaller in Dp71-null than in wild-type mice. This suggests that Dp71 may be involved in the functional activity of the HNHS. Its astrocyte end-feet localization emphasizes the importance of neuronal-vascular-glial interactions for the central detection of osmolality. In the SON, Dp71 may be involved in osmosensitivity and definition of the "osmostat," whereas, in the neurohypophysis, it may be involved in fine-tuning AVP release.