Human Dystrophy Research Articles

Microfluidic-assisted cyclic mechanical stimulation affects cellular membrane integrity in a human muscular dystrophy in vitro model

The development of a microfluidic-based cell stretching device allows to investigate membrane permeability during cyclic mechanical stimulation in a human Duchenne Muscular Dystrophy skeletal musclein vitromodel.

Abstract 19370: Myocardial Extracellular Volume is Elevated in Human Duchenne Muscular Dystrophy

Introduction: Duchenne muscular dystrophy (DMD) leads to cardiomyopathy (CM) with variable severity and age of onset. Predicting early CM would alter therapeutic approaches and improve morbidity and mortality. Extracellular volume (ECV) calculated with cardiac MRI (CMR) quantifies extracellular matrix expansion, including myocardial fibrosis, and has never been reported in human DMD. We hypothesized that subjects with DMD would have abnormal ECV and that these values would correlate with other markers of LV function. Methods: 27 DMD subjects were prospectively studied. CMR included LVEF, late gadolinium enhancement (LGE), circumferential strain (ε cc ), and modified Look-Locker (MOLLI) sequences to calculate ECV maps (in-house software using Matlab). ECV calculated for each segment in the short axis at mid-ventricle and compared with LVEF and ε cc using linear regression. Normal values taken from unmatched cohort of healthy male adults with mean ECV of 0.25 ± 0.015 (range 0.23-0.28). Results: Imaging was adequate to calculate ECV maps in 20 DMD subjects. Mean age in years was 14. Mean LVEF was 52% and mean global ε cc was -14.6%; 11 subjects had LVEF < 55% and 6 had negative LGE. Mean ECV was 0.33 ± 0.05 (0.25-0.45); there was significant intersubject and intersegment variability (Figure 1). When compared with highest mean control ECV (0.28), only one subject had a normal ECV in every segment. In subjects with LVEF ≥ 55%, the mean ECV was 0.32 ± 0.07 (0.25-0.45). In subjects with negative LGE, the mean ECV was 0.28 ± 0.04 (0.25-0.36). The ECV of the inferolateral and anterolateral segments correlated with LVEF (p=0.025 and p<0.001) and the inferolateral ECV correlated with mean ε cc (p=0.025). Conclusions: In this cohort, 19/20 DMD subjects have elevated segmental ECV, even with normal LVEF and negative LGE. Segmental ECV correlates with both LVEF and mean ε cc . ECV may be a more subtle biomarker of early myocardial disease than standard measures such as LVEF and LGE in human DMD.

Glycobiology of α-dystroglycan and muscular dystrophy.

Most proteins are modified by glycans, which can modulate the biological properties and functions of glycoproteins. The major glycans can be classified into N-glycans and O-glycans according to their glycan-peptide linkage. This review will provide an overview of the O-mannosyl glycans, one subtype of O-glycans. Originally, O-mannosyl glycan was only known to be present on a limited number of glycoproteins, especially α-dystroglycan (α-DG). However, once a clear relationship was established between O-mannosyl glycan and the pathological mechanisms of some congenital muscular dystrophies in humans, research on the biochemistry and pathology of O-mannosyl glycans has been expanding. Because α-DG glycosylation is defective in congenital muscular dystrophies, which also feature abnormal neuronal migration, these disorders are collectively called α-dystroglycanopathies. In this article, I will describe the structure, biosynthesis and pathology of O-mannosyl glycans.

Asynchronous remodeling is a driver of failed regeneration in Duchenne muscular dystrophy.

We sought to determine the mechanisms underlying failure of muscle regeneration that is observed in dystrophic muscle through hypothesis generation using muscle profiling data (human dystrophy and murine regeneration). We found that transforming growth factor β-centered networks strongly associated with pathological fibrosis and failed regeneration were also induced during normal regeneration but at distinct time points. We hypothesized that asynchronously regenerating microenvironments are an underlying driver of fibrosis and failed regeneration. We validated this hypothesis using an experimental model of focal asynchronous bouts of muscle regeneration in wild-type (WT) mice. A chronic inflammatory state and reduced mitochondrial oxidative capacity are observed in bouts separated by 4 d, whereas a chronic profibrotic state was seen in bouts separated by 10 d. Treatment of asynchronously remodeling WT muscle with either prednisone or VBP15 mitigated the molecular phenotype. Our asynchronous regeneration model for pathological fibrosis and muscle wasting in the muscular dystrophies is likely generalizable to tissue failure in chronic inflammatory states in other regenerative tissues.

Excess SMAD signaling contributes to heart and muscle dysfunction in muscular dystrophy.

Disruption of the dystrophin complex causes muscle injury, dysfunction, cell death and fibrosis. Excess transforming growth factor (TGF) β signaling has been described in human muscular dystrophy and animal models, where it is thought to relate to the progressive fibrosis that characterizes dystrophic muscle. We now found that canonical TGFβ signaling acutely increases when dystrophic muscle is stimulated to contract. Muscle lacking the dystrophin-associated protein γ-sarcoglycan (Sgcg null) was subjected to a lengthening protocol to produce maximal muscle injury, which produced rapid accumulation of nuclear phosphorylated SMAD2/3. To test whether reducing SMAD signaling improves muscular dystrophy in mice, we introduced a heterozygous mutation of SMAD4 (S4) into Sgcg mice to reduce but not ablate SMAD4. Sgcg/S4 mice had improved body mass compared with Sgcg mice, which normally show a wasting phenotype similar to human muscular dystrophy patients. Sgcg/S4 mice had improved cardiac function as well as improved twitch and tetanic force in skeletal muscle. Functional enhancement in Sgcg/S4 muscle occurred without a reduction in fibrosis, suggesting that intracellular SMAD4 targets may be important. An assessment of genes differentially expressed in Sgcg muscle focused on those encoding calcium-handling proteins and responsive to TGFβ since this pathway is a target for mediating improvement in muscular dystrophy. These data demonstrate that excessive TGFβ signaling alters cardiac and muscle performance through the intracellular SMAD pathway.

Motor Physical Therapy Affects Muscle Collagen Type I and Decreases Gait Speed in Dystrophin-Deficient Dogs

Golden Retriever Muscular Dystrophy (GRMD) is a dystrophin-deficient canine model genetically homologous to Duchenne Muscular Dystrophy (DMD) in humans. Muscular fibrosis secondary to cycles of degeneration/regeneration of dystrophic muscle tissue and muscular weakness leads to biomechanical adaptation that impairs the quality of gait. Physical therapy (PT) is one of the supportive therapies available for DMD, however, motor PT approaches have controversial recommendations and there is no consensus regarding the type and intensity of physical therapy. In this study we investigated the effect of physical therapy on gait biomechanics and muscular collagen deposition types I and III in dystrophin-deficient dogs. Two dystrophic dogs (treated dogs-TD) underwent a PT protocol of active walking exercise, 3×/week, 40 minutes/day, 12 weeks. Two dystrophic control dogs (CD) maintained their routine of activities of daily living. At t0 (pre) and t1 (post-physical therapy), collagen type I and III were assessed by immunohistochemistry and gait biomechanics were analyzed. Angular displacement of shoulder, elbow, carpal, hip, stifle and tarsal joint and vertical (Fy), mediolateral (Fz) and craniocaudal (Fx) ground reaction forces (GRF) were assessed. Wilcoxon test was used to verify the difference of biomechanical variables between t0 and t1, considering p<.05. Type I collagen of endomysium suffered the influence of PT, as well as gait speed that had decreased from t0 to t1 (p<.000). The PT protocol employed accelerates morphological alterations on dystrophic muscle and promotes a slower velocity of gait. Control dogs which maintained their routine of activities of daily living seem to have found a better balance between movement and preservation of motor function.

Myofiber damage precedes macrophage infiltration after contraction‐induced injury in dysferlin‐null muscle (731.3)

Mutations in the DYSF gene that encodes the protein, dysferlin, lead to late‐onset, human muscular dystrophies known as dysferlinopathies. The A/J mouse spontaneously develops a mild dysferlinopathy after 6 months of age and at younger ages models the subclinical phase of the human disease. We subjected 3‐4 month old A/J mice to in vivo large‐strain lengthening injury (LSI) from lengthening contractions and studied the role of inflammation in the onset of muscle damage and recovery thereafter. We show that post‐LSI, myofiber damage in the tibialis anterior muscle occurs prior to inflammatory cell infiltration. Indeed, most edema and inflammation, monitored by T2‐weighted MRI and by immunofluorescence labeling of neutrophils and macrophages, respectively, develops 24‐72 hr after LSI, well after the appearance of damaged myofibers. The cytokines IL‐1A, MCP‐1, and MCSF are elevated several‐fold at 72 hr after injury, consistent with extensive macrophage infiltration. Control A/WySnJ mice show much less myofiber damage, much less inflammation and much lower cytokine levels after LSI than A/J. Depletion of monocytes and macrophages by systemic clodronate administration, fails to suppress myofiber damage or to accelerate functional recovery in A/J mice. Our studies suggest that, although macrophage infiltration is prominent in A/J muscle following LSI, it is the consequence and not the primary cause of progressive myofiber damage.$graphic_F38BE44F‐E3E9‐4CCD‐9D37‐186DE40DCC8F$Grant Funding Source: Supported by a grant to JAR from the Jain Foundation Inc.

Endoplasmic reticulum microenvironment and conserved histidines govern ELOVL4 fatty acid elongase activity

Autosomal dominant Stargardt-like macular dystrophy (STGD3) in humans results from mutations in elongation of very long chain FAs-like 4 (ELOVL4), which leads to vision loss in young adults. ELOVL4 is an integral endoplasmic reticulum (ER) protein that mediates the elongation of very long chain (VLC) FAs. Mutations in ELOVL4 lead to truncation and mislocalization of the translated protein from the ER, the site of FA elongation. Little is known about the enzymatic elongation of VLC-FAs by ELOVL4. We over-expressed full-length mouse ELOVL4, an N-glycosylation-deficient mutant, an ER-retention mutant, and mutants of active site histidines to parse their individual roles in VLC-FA elongation. ELOVL4 elongated appropriate precursors to the corresponding VLC-FA species ≥28 carbons. Active site histidine mutants of ELOVL4 did not elongate appropriate precursors, establishing ELOVL4 as the elongase. Displacing ELOVL4 from the ER was sufficient to cause loss of condensation activity, while absence of N-glycosylation was irrelevant for enzyme function. This study shows that ELOVL4 enzymatic activity is governed by individual histidines in its active site and the ER microenvironment, both of which are essential for elongation of VLC-FAs.

The retinal phenotype of Usher syndrome: Pathophysiological insights from animal models

The Usher syndrome (USH) is the most prevalent cause of inherited deaf-blindness. Three clinical subtypes, USH1–3, have been defined, and ten USH genes identified. The hearing impairment due to USH gene defects has been shown to result from improper organisation of the hair bundle, the sound receptive structure of sensory hair cells. In contrast, the cellular basis of the visual defect is less well understood as this phenotype is absent in almost all the USH mouse models that faithfully mimic the human hearing impairment. Structural and molecular interspecies discrepancies regarding photoreceptor calyceal processes and the association with the distribution of USH1 proteins have recently been unravelled, and have led to the conclusion that a defect in the USH1 protein complex-mediated connection between the photoreceptor outer segment and the surrounding calyceal processes (in both rods and cones), and the inner segment (in rods only), probably causes the USH1 retinal dystrophy in humans.

Sonic hedgehog gene therapy increases the ability of the dystrophic skeletal muscle to regenerate after injury

The Hedgehog (Hh) pathway is a crucial regulator of muscle development during embryogenesis. We have previously demonstrated that Sonic hedgehog (Shh) regulates postnatal myogenesis in the adult skeletal muscle both directly, by acting on muscle satellite cells, and indirectly, by promoting the production of growth factors from interstitial fibroblasts. Here, we show that in mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, progression of the dystrophic pathology corresponds to progressive inhibition of the Hh signaling pathway in the skeletal muscle. We also show that the upregulation of the Hh pathway in response to injury and during regeneration is significantly impaired in mdx muscle. Shh treatment increases the proliferative potential of satellite cells isolated from the muscles of mdx mice. This treatment also increases the production of proregenerative factors, such as insulin-like growth factor-1 and vascular endothelial growth factor, from fibroblasts isolated from the muscle of mdx mice. In vivo, overexpression of the Hh pathway using a plasmid encoding the human Shh gene promotes successful regeneration after injury in terms of increased number of proliferating myogenic cells and newly formed myofibers, as well as enhanced vascularization and decreased fibrosis.

Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans

BackgroundLaminopathies are diseases characterized by defects in nuclear envelope structure. A well-known example is Emery-Dreifuss muscular dystrophy, which is caused by mutations in the human lamin A/C and emerin genes. While most nuclear envelope proteins are ubiquitously expressed, laminopathies often affect only a subset of tissues. The molecular mechanisms underlying these tissue-specific manifestations remain elusive. We hypothesize that different functional subclasses of genes might be differentially affected by defects in specific nuclear envelope components.ResultsHere we determine genome-wide DNA association profiles of two nuclear envelope components, lamin/LMN-1 and emerin/EMR-1 in adult Caenorhabditis elegans. Although both proteins bind to transcriptionally inactive regions of the genome, EMR-1 is enriched at genes involved in muscle and neuronal function. Deletion of either EMR-1 or LEM-2, another integral envelope protein, causes local changes in nuclear architecture as evidenced by altered association between DNA and LMN-1. Transcriptome analyses reveal that EMR-1 and LEM-2 are associated with gene repression, particularly of genes implicated in muscle and nervous system function. We demonstrate that emr-1, but not lem-2, mutants are sensitive to the cholinesterase inhibitor aldicarb, indicating altered activity at neuromuscular junctions.ConclusionsWe identify a class of elements that bind EMR-1 but do not associate with LMN-1, and these are enriched for muscle and neuronal genes. Our data support a redundant function of EMR-1 and LEM-2 in chromatin anchoring to the nuclear envelope and gene repression. We demonstrate a specific role of EMR-1 in neuromuscular junction activity that may contribute to Emery-Dreifuss muscular dystrophy in humans.

Autonomic, locomotor and cardiac abnormalities in a mouse model of muscular dystrophy: targeting the renin–angiotensin system

New Findings What is the topic of this review? This symposium report summarizes autonomic, cardiac and skeletal muscle abnormalities in sarcoglycan-δ-deficient mice (Sgcd-/-), a mouse model of limb girdle muscular dystrophy, with emphasis on the roles of autonomic dysregulation and activation of the renin-angiotensin system at a young age. What advances does it highlight? The contributions of the autonomic nervous system and the renin-angiotensin system to the pathogenesis of muscular dystrophy are highlighted. Results demonstrate that autonomic dysregulation precedes and predicts later development of cardiac dysfunction in Sgcd-/- mice and that treatment of young Sgcd-/- mice with the angiotensin type 1 receptor antagonist losartan or with angiotensin-(1-7) abrogates the autonomic dysregulation, attenuates skeletal muscle pathology and increases spontaneous locomotor activity. Muscular dystrophies are a heterogeneous group of genetic muscle diseases characterized by muscle weakness and atrophy. Mutations in sarcoglycans and other subunits of the dystrophin-glycoprotein complex cause muscular dystrophy and dilated cardiomyopathy in animals and humans. Aberrant autonomic signalling is recognized in a variety of neuromuscular disorders. We hypothesized that activation of the renin-angiotensin system contributes to skeletal muscle and autonomic dysfunction in mice deficient in the sarcoglycan-δ (Sgcd) gene at a young age and that this early autonomic dysfunction contributes to the later development of left ventricular (LV) dysfunction and increased mortality. We demonstrated that young Sgcd-/- mice exhibit histopathological features of skeletal muscle dystrophy, decreased locomotor activity and severe autonomic dysregulation, but normal LV function. Autonomic regulation continued to deteriorate in Sgcd-/- mice with age and was accompanied by LV dysfunction and dilated cardiomyopathy at older ages. Autonomic dysregulation at a young age predicted later development of LV dysfunction and higher mortality in Sgcd-/- mice. Treatment of Sgcd-/- mice with the angiotensin type 1 receptor blocker losartan for 8-9 weeks, beginning at 3 weeks of age, decreased fibrosis and oxidative stress in skeletal muscle, increased locomotor activity and prevented autonomic dysfunction. Chronic infusion of the counter-regulatory peptide angiotensin-(1-7) resulted in similar protection. We conclude that activation of the renin-angiotensin system, at a young age, contributes to skeletal muscle and autonomic dysfunction in muscular dystrophy. We speculate that the latter is mediated via abnormal sensory nerve and/or cytokine signalling from dystrophic skeletal muscle to the brain and contributes to age-related LV dysfunction, dilated cardiomyopathy, arrhythmias and premature death. Therefore, correcting the early autonomic dysregulation and renin-angiotensin system activation may provide a novel therapeutic approach in muscular dystrophy.

Angiogenic Impairment of the Vascular Endothelium

Objective— Dystrophin, the missing or defective protein in Duchenne muscular dystrophy, is expressed not only in muscle cells but also in vascular endothelial cells (ECs). In this study, we assessed the effects of dystrophin deficiency on the angiogenic capacities of ECs. Approach and Results— We isolated vascular ECs from mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, and wild-type controls, and we found that mdx -derived ECs have impaired angiogenic properties, in terms of migration, proliferation, and tube formation. They also undergo increased apoptosis in vitro compared with wild-type cells and have increased senescence-associated β-galactosidase activity. Mdx -derived ECs also display reduced ability to support myoblast proliferation when cocultured with satellite cell–derived primary myoblasts. These endothelial defects are mirrored by systemic impairment of angiogenesis in vivo, both on induction of ischemia, stimulation with growth factors in the corneal model and matrigel plug assays, and tumor growth. We also found that dystrophin forms a complex with endothelial NO synthase and caveolin-1 in ECs, and that NO production and cGMP formation are compromised in ECs isolated from mdx mice. Interestingly, treatment with aspirin enhances production of both cGMP and NO in dystrophic ECs, whereas low-dose aspirin improves the dystrophic phenotype of mdx mice in vivo, in terms of resistance to physical exercise, muscle fiber permeability, and capillary density. Conclusions— These findings demonstrate that impaired angiogenesis is a novel player and potential therapeutic target in Duchenne muscular dystrophy.

Structural and Functional Analysis of the Native Peripherin-ROM1 Complex Isolated from Photoreceptor Cells

Peripherin and its homologue ROM1 are retina-specific members of the tetraspanin family of integral membrane proteins required for morphogenesis and maintenance of photoreceptor outer segments, regions that collect light stimuli. Over 100 pathogenic mutations in peripherin cause inherited rod- and cone-related dystrophies in humans. Peripherin and ROM1 interact in vivo and are predicted to form a core heterotetrameric complex capable of creating higher order oligomers. However, structural analysis of tetraspanin proteins has been hampered by their resistance to crystallization. Here we present a simplified methodology for high yield purification of peripherin-ROM1 from bovine retinas that permitted its biochemical and biophysical characterization. Using size exclusion chromatography and blue native gel electrophoresis, we confirmed that the core native peripherin-ROM1 complex exists as a tetramer. Peripherin, but not ROM1, is glycosylated and we examined the glycosylation site and glycan composition of ROM1 by liquid chromatographic tandem mass spectrometry. Mass spectrometry was used to analyze the native complex in detergent micelles, demonstrating its tetrameric state. Our electron microscopy-generated structure solved to 18 Å displayed the tetramer as an elongated structure with an apparent 2-fold symmetry. Finally, we demonstrated that peripherin-ROM1 tetramers induce membrane curvature when reconstituted in lipid vesicles. These results provide critical insights into this key retinal component with a poorly defined function.

Abstract 13: Central Sympathoinhibition Abrogates Skeletal Muscle Fibrosis, Oxidative Stress and Autonomic Dysregulation in a Mouse Model of Muscular Dystrophy

Sarcoglycan mutations cause muscular dystrophy in humans. We recently demonstrated that sarcoglycan delta deficient (Sgcd-/-) mice with muscular dystrophy exhibit autonomic dysregulation [Hypertension 2010]. We hypothesized that excessive sympathetic activity contributes to skeletal muscle pathology, decreased locomotor activity and autonomic dysregulation in young (10-12 wks) Sgcd-/- mice. The centrally-acting sympathoinhibitory drug rilmenidine (RIL) was infused into the brain of control C57BL6 and Sgcd-/- mice by osmotic pump for 7-9 wks beginning at 3 wks of age (42 ng/g/hr, ICV). Separate groups of mice were infused with saline vehicle (VEH). Blood pressure (BP), heart rate (HR) and locomotor activity were measured by telemetry. Cardiac (HR responses to propranolol) and vasomotor (BP response to ganglionic blockade) sympathetic tone were increased in VEH-treated Sgcd-/- mice, and normalized by RIL (Table). The RIL-induced sympathoinhibition in Sgcd-/- mice was accompanied by increases in baroreflex sensitivity (BRS, sequence technique), cardiovagal tone (HR response to atropine) and activity, with no change in BP (Table). RIL also decreased oxidative stress (superoxide) by 56% and fibrosis in Sgcd-/- skeletal muscle. RIL did not affect measured variables in control mice (Table). In summary, RIL-induced sympathoinhibition decreased skeletal muscle pathology, increased locomotor activity and improved autonomic regulation in young Sgcd-/- mice. The results implicate increased sympathetic activity in the pathogenesis of muscular dystrophy, and suggest that targeting the brain to inhibit sympathetic activity may provide a novel therapeutic approach.

P.20.9 Muscle fibrosis in the sgcb-null mouse model versus the mdx model

The Sgcb-null mouse model, with beta-sarcoglycan knocked-down, develops severe muscular dystrophy with early fibrosis like the human limb girdle muscular dystrophy type 2E. The mdx mouse, lacking dystrophin, is the most used model for Duchenne muscular dystrophy (DMD). Unlike DMD, the mdx mouse has mild clinical features and shows little endomysial fibrosis in limb muscles. We have characterized the progression of muscle fibrosis, at molecular and histopathological level, in the Sgcb-null mouse and compared results to findings in the mdx. We evaluated expression of collagen I, III and VI, and of decorin and TGFbeta1 by immunohistochemistry or immunoblotting, and transcript levels by Real-Time PCR, in the quadriceps and diaphragm muscles, at 2, 4, 8, 12, 26 and 52 weeks in the sgcb-null mouse, and at 12, 26 and 52 weeks in the mdx. We found severe histopathological features from 4 weeks on, and concomitant deposition of extracellular matrix components in the Sgcb-null mouse, with collagen I, III and VI significantly increased since early ages both in the quadriceps and diaphragm. When compared to the mdx muscles, histopatological features of both Sgcb-null and age-matched mdx mice were similar at all examined ages except that in Sgcb-null mice the extent of connective tissue was generally greater. This was particularly evident in the quadriceps muscle where the endomysial connective tissue was prominent and the extent of the various collagens was significantly greater in the Sgcb-null mice at all ages compared to mdx. Furthermore, differently than in the Scgb-null mouse, where the amount all of three collagen isoforms increased steadily, in the mdx they remained stable. The Sgcb-null mouse represents a useful model for evaluating the pathogenetic mechanisms of muscle fibrosis and for development of anti-fibrotic treatments.

M.I.1 Mechanism of laminin assembly: Insight for structural repairs of MDC1A

Laminins play a key role in the assembly of sarcolemmal basement membranes by establishing anchors to the cell surface and underlying cytoskeleton, by polymerizing to form a cohesive scaffold, and by forming links to type IV collagen and other extracellular components. Laminin-deficient (MDC1A) congenital muscular dystrophy is caused by structural instability of the basement membrane in which absent or poorly-expressed laminin-211 is replaced by laminins-411 and -511. Laminin-411, the main compensator, is an inadequate substitute for the lost laminin because it adheres poorly to integrins and dystroglycan and cannot polymerize. The first deficiency has been repaired with miniagrin, an engineered protein that binds to the coiled-coil of laminins, anchoring it to dystroglycan. We now find that the second deficiency can be repaired with αLNNd, a protein engineered to bind to the nidogen-binding locus and enable polymerization of laminin-411, promoting its self-assembly. Transgenic αLNNd-expressing mice were bred with dystrophic dy2J (α2LN deletion, non-polymerizing) and dy3K (laminin α2 knockout) mice. The transgene was found to increase the accumulation of compensating laminins and to substantially improve hindlimb muscle histology in dy2J as evidenced by decreased myofiber fibrosis, reduced fraction of central nuclei and increased myofiber cross-sectional area. αLNNd was also found to ameliorate the severe dy3K dystrophy as seen by a ∼2.5-fold increased lifespan with increase body weight, muscle strength, and improved muscle histology. The findings support a critical role of laminin polymerization for stable scaffold structure. They also suggest a new therapeutic basis for structural repair of the human dystrophy.

Distinct and Atypical Intrinsic and Extrinsic Cell Death Pathways between Photoreceptor Cell Types upon Specific Ablation of Ranbp2 in Cone Photoreceptors

Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2), a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1+-apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct retinal spatial signatures as well as with other etiologically distinct neurodegenerative disorders.

Expanding Donor Muscle‐Derived Cells for Transplantation

Studies in mice showed tremendous promise for the eventual clinical utility of myoblast transplantation to treat human muscular dystrophies. Initial attempts to translate the murine studies to humans, however, were not successful, due in part to limited engraftability of expanded donor myoblasts. Conventionally, muscle cells have been cultured on collagen-coated tissue culture-treated polystyrene. However, this promotes lineage progression and differentiation of cells, which limits engraftment potential. This unit describes the isolation of canine muscle-derived cells, ex vivo expansion of cells on plates coated with a modified Notch ligand, and the xenotransplant method used to evaluate engraftment potential. Activation of Notch signaling in freshly isolated canine muscle-derived cells with Delta-1(ext)-IgG inhibits myogenic differentiation, and maintains cells earlier in myogenic lineage progression. Delta-1(ext)-IgG-expanded cells engraft into the regenerating muscle of NOD/SCID mice more effectively than control cells expanded on human IgG, as evidenced by a significant increase in the number of muscle fibers expressing canine dystrophin in recipient murine muscle. Therefore, this protocol provides the basis for further developing culture conditions for ex vivo expansion of donor muscle cells for transplant.

Modifying muscular dystrophy through transforming growth factor‐β

Muscular dystrophy arises from ongoing muscle degeneration and insufficient regeneration. This imbalance leads to loss of muscle, with replacement by scar or fibrotic tissue, resulting in muscle weakness and, eventually, loss of muscle function. Human muscular dystrophy is characterized by a wide range of disease severity, even when the same genetic mutation is present. This variability implies that other factors, both genetic and environmental, modify the disease outcome. There has been an ongoing effort to define the genetic and molecular bases that influence muscular dystrophy onset and progression. Modifier genes for muscle disease have been identified through both candidate gene approaches and genome-wide surveys. Multiple lines of experimental evidence have now converged on the transforming growth factor-β (TGF-β) pathway as a modifier for muscular dystrophy. TGF-β signaling is upregulated in dystrophic muscle as a result of a destabilized plasma membrane and/or an altered extracellular matrix. Given the important biological role of the TGF-β pathway, and its role beyond muscle homeostasis, we review modifier genes that alter the TGF-β pathway and approaches to modulate TGF-β activity to ameliorate muscle disease.