Break Down of the Complexity and Inconsistency Between Levels of Matriglycan and Disease Phenotype in FKRP-Related Dystroglycanopathies: A Review and Model of Interpretation.

Dystroglycanopathies are a group of muscle degenerative diseases characterized with significant reduction in matriglycan expression critical in disease pathogenesis. Missense point mutations in the Fukutin-related protein (FKRP) gene cause variable reduction in the synthesis of matriglycan on alpha-dystroglycan (α-DG) and a wide range of disease severity. Data analyses of muscle biopsies from patients fail to show consistent correlation between the levels of matriglycan and clinical phenotypes. By reviewing clinical reports in conjunction with analysis of clinically relevant mouse models, we identify likely causes for the confusion. Nearly all missense FKRP mutations retain variable, but sufficient function for the synthesis of matriglycan during the later stage of muscle development and periods of muscle regeneration. These factors lead to a highly heterogenous pattern of matriglycan expression in diseased muscles, depending on age and stages of muscle regeneration. The limited size in clinical biopsy samples from different parts of even a single muscle tissue at different time points of disease progression may well mis-represent the residual function (base-levels) of the mutated FKRPs and phenotypes. We propose to use a simple Multi Point tool from ImageJ to more accurately measure the signal intensity of matriglycan expression on fiber membrane for assessing mutant FKRP function and therapeutic efficacy. A robust and sensitive immunohistochemical protocol would further improve reliability and comparability for the detection of matriglycan.

Improved efficacy of FKRP AAV gene therapy by combination with ribitol treatment for LGMD2I.

Mutations in the fukutin-related protein (FKRP) gene cause dystroglycanopathy, with disease severity ranging from mild LGMD2I to severe congenital muscular dystrophy. Recently, considerable progress has been made in developing experimental therapies, with adeno-associated virus (AAV) gene therapy and ribitol treatment demonstrating significant therapeutic effect. However, each treatment has its strengths and weaknesses. AAV gene therapy can achieve normal levels of transgene expression, but it requires high doses, with toxicity concerns and variable distribution. Ribitol relies on residual FKRP function and restores limited levels of matriglycan. We hypothesized that these two treatments can work synergistically to offer an optimized therapy with efficacy and safety unmatched by each treatment alone. The most effective treatment is the combination of high-dose (5e-13 vg/kg) AAV-FKRP with ribitol, whereas low dose (1e-13 vg/kg) AAV-FKRP combined with ribitol showed a 22.6% increase in positive matriglycan fibers and the greater improvement in pathology when compared to low-dose AAV-FKRP alone. Together, our results support the potential benefits of combining ribitol with AAV gene therapy for treating FKRP-related muscular dystrophy. The fact that ribitol is a metabolite in nature and has already been tested in animal models and clinical trials in humans without severe side effects provides a safety profile for it to be trialed in combination with AAV gene therapy.

Ribitol dose-dependently enhances matriglycan expression and improves muscle function with prolonged life span in limb girdle muscular dystrophy 2I mouse model.

Limb Girdle Muscular Dystrophy 2I (LGMDR9) is one of the most common LGMD characterized by defects in glycosylation of α-dystroglycan (matriglycan) resulting from mutations of Fukutin-related protein (FKRP). There is no effective therapy currently available. We recently demonstrated that ribitol supplement increases levels of matriglycan in cells in vitro and in FKRP-P448L (P448L) mutant mouse model through drinking water administration. To be clinically relevant, we have now conducted a dose-escalating efficacy study by gavage in P448L mutant mice. Six months of ribitol treatment daily significantly rescued functions of skeletal, respiratory, and cardiac muscles dose-dependently. This was associated with a dose dependent increase in matriglycan and improvement in muscle pathology with reductions in muscle degeneration, inflammatory infiltration and fibrosis. Importantly, ribitol significantly increased life span and muscle functions of the female animals receiving treatment from 10 months of age. The only observed side effect was gastrointestinal tract bloating with loose stool and this effect is also dose dependent. The results validate the mechanism that ribitol as a pre-substrate of glycosyltransferase is able to compensate for the decreased function of mutant FKRP with restoration of matriglycan expression and provide a guidance for future clinical trial design.

Ribitol enhances matriglycan of α-dystroglycan in breast cancer cells without affecting cell growth.

The laminin-binding glycan (matriglycan) on α-dystroglycan (α-DG) enables diverse roles, from neuronal development to muscle integrity. Reduction or loss of matriglycan has also been implicated in cancer development and metastasis, and specifically associated with high-grade tumors and poor prognoses in breast cancers. Hyperglycosylation of α-DG with LARGE overexpression is shown to inhibit cancer cell growth and tumorigenicity. We recently demonstrated that ribitol, considered to be a metabolic end-product, enhances matriglycan expression in dystrophic muscles in vivo. In the current study, we tested the hypothesis that ribitol could also enhance matriglycan expression in cancer cells. Our results showed for the first time that ribitol is able to significantly enhance the expression of matriglycan on α-DG in breast cancer cells. The ribitol effect is associated with an increase in levels of CDP-ribitol, the substrate for the ribitol-5-phosphate transferases FKRP and FKTN. Direct use of CDP-ribitol is also effective for matriglycan expression. Ribitol treatment does not alter the expression of FKRP, FKTN as well as LARGEs and ISPD which are critical for the synthesis of matriglycan. The results suggest that alteration in substrates could also be involved in regulation of matriglycan expression. Interestingly, expression of matriglycan is related to cell cycle progression with highest levels in S and G2 phases and ribitol treatment does not alter the pattern. Although matriglycan up-regulation does not affect cell cycle progression and proliferation of the cancer cells tested, the novel substrate-mediated treatment opens a new approach easily applicable to experimental systems in vivo for further exploitation of matriglycan expression in cancer progression and for therapeutic potential.

ISPD Overexpression Enhances Ribitol-Induced Glycosylation of α-Dystroglycan in Dystrophic FKRP Mutant Mice.

Dystroglycanopathy, a subgroup of muscular dystrophies, is characterized by hypoglycosylation of α-dystroglycan (α-DG), which reduces its laminin-binding activity to extracellular matrix proteins, causing progressive loss of muscle integrity and function. Mutations in the fukutin-related protein (FKRP) gene are the most common causes of dystroglycanopathy. FKRP transfers ribitol-5-phosphate to the O-mannosyl glycan on α-DG from substrate cytidine diphosphate (CDP)-ribitol, which is synthesized by isoprenoid synthase domain-containing protein (ISPD). We previously reported that oral administration of ribitol restores therapeutic levels of functional glycosylation of α-DG (F-α-DG) in a FKRP mutant mouse model. Here we examine the contribution of adeno-associated virus (AAV)-mediated overexpression of ISPD to the levels of CDP-ribitol and F-α-DG with and without ribitol supplementation in the disease model. ISPD overexpression alone and in combination with ribitol improves dystrophic phenotype. Furthermore, the combined approach of ribitol and ISPD acts synergistically, increasing F-α-DG up to 40% of normal levels in cardiac muscle and more than 20% in limb and diaphragm. The results suggest that low levels of substrate limit production of CDP-ribitol, and endogenous ISPD also becomes a limiting factor in the presence of a supraphysiological concentration of ribitol. Our data support further investigation of the regulatory pathway for enhancing efficacy of ribitol supplement to FKRP-related dystroglycanopathy.

Ribitol restores functionally glycosylated α-dystroglycan and improves muscle function in dystrophic FKRP-mutant mice.

O-mannosylated α-dystroglycan (α-DG) serves as receptors for cell-cell and cell-extracellular matrix adhesion and signaling. Hypoglycosylation of α-DG is involved in cancer progression and underlies dystroglycanopathy with aberrant neuronal development. Here we report that ribitol, a pentose alcohol with previously unknown function in mammalian cells, partially restores functional O-mannosylation of α-DG (F-α-DG) in the dystroglycanopathy model containing a P448L mutation in fukutin-related protein (FKRP) gene, which is clinically associated with severe congenital muscular dystrophy. Oral administration of ribitol increases levels of ribitol-5-phosphate and CDP-ribitol and restores therapeutic levels of F-α-DG in skeletal and cardiac muscles. Furthermore, ribitol, given before and after the onset of disease phenotype, reduces skeletal muscle pathology, significantly decreases cardiac fibrosis and improves skeletal and respiratory functions in the FKRP mutant mice. Ribitol treatment presents a new class, low risk, and easy to administer experimental therapy to restore F-α-DG in FKRP-related muscular dystrophy.

Skeletal, cardiac, and respiratory muscle function and histopathology in the P448Lneo- mouse model of FKRP-deficient muscular dystrophy.

BACKGROUND: Fukutin-related protein (FKRP) mutations are the most common cause of dystroglycanopathies known to cause both limb girdle and congenital muscular dystrophy. The P448Lneo- mouse model has a knock-in mutation in the FKRP gene and develops skeletal, respiratory, and cardiac muscle disease. METHODS: We studied the natural history of the P448Lneo- mouse model over 9 months and the effects of twice weekly treadmill running. Forelimb and hindlimb grip strength (Columbus Instruments) and overall activity (Omnitech Electronics) assessed skeletal muscle function. Echocardiography was performed using VisualSonics Vevo 770 (FujiFilm VisualSonics). Plethysmography was performed using whole body system (ADInstruments). Histological evaluations included quantification of inflammation, fibrosis, central nucleation, and fiber size variation. RESULTS: P448Lneo- mice had significantly increased normalized tissue weights compared to controls at 9 months of age for the heart, gastrocnemius, soleus, tibialis anterior, quadriceps, and triceps. There were no significant differences seen in forelimb or hindlimb grip strength or activity monitoring in P448Lneo- mice with or without exercise compared to controls. Skeletal muscles demonstrated increased inflammation, fibrosis, central nucleation, and variation in fiber size compared to controls (p < 0.05) and worsened with exercise. Plethysmography showed significant differences in respiratory rates and decreased tidal and minute volumes in P448Lneo- mice (p < 0.01). There was increased fibrosis in the diaphragm compared to controls (p < 0.01). Echocardiography demonstrated decreased systolic function in 9-month-old mutant mice (p < 0.01). There was increased myocardial wall thickness and mass (p < 0.001) with increased fibrosis in 9-month-old P448Lneo- mice compared to controls (p < 0.05). mRNA expression for natriuretic peptide type A (Nppa) was significantly increased in P448Lneo- mice compared to controls at 6 months (p < 0.05) and for natriuretic peptide type B (Nppb) at 6 and 9 months of age (p < 0.05). CONCLUSIONS: FKRP-deficient P448Lneo- mice demonstrate significant deficits in cardiac and respiratory functions compared to control mice, and this is associated with increased inflammation and fibrosis. This study provides new functional outcome measures for preclinical trials of FKRP-related muscular dystrophies.

Long-Term Treatment of Tamoxifen and Raloxifene Alleviates Dystrophic Phenotype and Enhances Muscle Functions of FKRP Dystroglycanopathy.

The third most common form of limb-girdle muscular dystrophies is caused by mutations of the Fukutin-related protein (FKRP) gene, with no effective therapy available. Selective estrogen receptor modulators, tamoxifen and raloxifene, have been widely used for human conditions for their anti-inflammatory, antifibrosis, prevention of bone loss, and muscle building effects (essential features for muscular dystrophy therapies). We evaluated therapeutic values of tamoxifen and raloxifene in FKRPP448L mutant mouse with severe dystrophic phenotype. The mice were treated with the drugs for 1 year through daily gavage. We demonstrate that tamoxifen and raloxifene significantly ameliorated the disease progression. The improvement includes increase in grip force production, extended running time and distance in treadmill test, and enhancement in cardiac and respiratory functions. Significant reduction in muscle pathology includes diminished fibrosis and fiber degeneration. Tamoxifen and raloxifene also significantly mitigated bone loss. Tamoxifen, but not raloxifene, caused severe adverse effects on male reproductive organs. The results demonstrate that tamoxifen and raloxifene hold significant potential for treating FKRP-related muscular dystrophy and probably other muscular dystrophies. Sex-related differential effects of the drugs call for a careful consideration for the drug and dosage selection in male and female patient populations.

Distinct expression of functionally glycosylated alpha-dystroglycan in muscle and non-muscle tissues of FKRP mutant mice.

The glycosylation of alpha-dystroglycan (α-DG) is crucial in maintaining muscle cell membrane integrity. Dystroglycanopathies are identified by the loss of this glycosylation leading to a breakdown of muscle cell membrane integrity and eventual degeneration. However, a small portion of fibers expressing functionally glycosylated α-DG (F-α-DG) (revertant fibers, RF) have been identified. These fibers are generally small in size, centrally nucleated and linked to regenerating fibers. Examination of different muscles have shown various levels of RFs but it is unclear the extent of which they are present. Here we do a body-wide examination of muscles from the FKRP-P448L mutant mouse for the prevalence of RFs. We have identified great variation in the distribution of RF in different muscles and tissues. Triceps shows a large increase in RFs and together with centrally nucleated fibers whereas the pectoralis shows a reduction in revertant but increase in centrally nucleated fibers from 6 weeks to 6 months of age. We have also identified that the sciatic nerve with near normal levels of F-α-DG in the P448Lneo- mouse with reduced levels in the P448Lneo+ and absent in LARGEmyd. The salivary gland of LARGEmyd mice expresses high levels of F-α-DG. Interestingly the same glands in the P448Lneo-and to a lesser degree in P448Lneo+ also maintain considerable amount of F-α-DG, indicating the non-proliferating epithelial cells have a molecular setting permitting glycosylation.

Progressive Dystrophic Pathology in Diaphragm and Impairment of Cardiac Function in FKRP P448L Mutant Mice.

Mutations in the gene for fukutin-related protein represent a subset of muscular dystrophies known as dystroglycanopathies characterized by loss of functionally-glycosylated-alpha-dystroglycan and a wide range of dystrophic phenotypes. Mice generated by our lab containing the P448L mutation in the fukutin-related protein gene demonstrate the dystrophic phenotype similar to that of LGMD2I. Here we examined the morphology of the heart and diaphragm, focusing on pathology of diaphragm and cardiac function of the mutant mice for up to 12 months. Both diaphragm and heart lack clear expression of functionally-glycosylated-alpha-dystroglycan throughout the observed period. The diaphragm undergoes progressive deterioration in histology with increasing amount of centranucleation and inflammation. Large areas of mononuclear cell infiltration and fibrosis of up to 60% of tissue area were detected as early as 6 months of age. Despite a less severe morphology with only patches of mononuclear cell infiltration and fibrosis of ~5% by 12 months of age in the heart, cardiac function is clearly affected. High frequency ultrasound reveals a smaller heart size up to 10 months of age. There are significant increases in myocardial thickness and decrease in cardiac output through 12 months. Dysfunction in the heart represents a key marker for evaluating experimental therapies aimed at cardiac muscle.

Glucocorticoid Steroid and Alendronate Treatment Alleviates Dystrophic Phenotype with Enhanced Functional Glycosylation of α-Dystroglycan in Mouse Model of Limb-Girdle Muscular Dystrophy with FKRPP448L Mutation.

Fukutin-related protein-muscular dystrophy is characterized by defects in glycosylation of α-dystroglycan with variable clinical phenotypes, most commonly as limb-girdle muscular dystrophy 2I. There is no effective therapy available. Glucocorticoid steroids have become the standard treatment for Duchenne and other muscular dystrophies with serious adverse effects, including excessive weight gain, immune suppression, and bone loss. Bisphosphonates have been used to treat Duchenne muscular dystrophy for prevention of osteoporosis. Herein, we evaluated prednisolone and alendronate for their therapeutic potential in the FKRPP448L-mutant mouse representing moderate limb-girdle muscular dystrophy 2I. Mice were treated with prednisolone, alendronate, and both in combination for up to 6 months. Prednisolone improved muscle pathology with significant reduction in muscle degeneration, but had no effect on serum creatine kinase levels and muscle strength. Alendronate treatment did not ameliorate muscle degeneration, but demonstrated a limited enhancement on muscle function test. Combined treatment of prednisolone and alendronate provided best improvement in muscle pathology with normalized fiber size distribution and significantly reduced serum creatine kinase levels, but had limited effect on muscle force generation. The use of alendronate significantly mitigated the bone loss. Prednisolone alone and in combination with alendronate enhance functionally glycosylated α-dystroglycan. These results, for the first time, demonstrate the efficacy and feasibility of this alliance treatment of the two drugs for fukutin-related protein-muscular dystrophy.

Dystroglycanopathy muscles lacking functional glycosylation of alpha-dystroglycan retain regeneration capacity.

In dystroglycanopathies, lack of glycosylated alpha-dystroglycan (α-DG) alters membrane fragility leading to fiber damage and repetitive cycles of muscle degeneration and regeneration. However the effect of the glycosylation of α-DG on muscle regeneration is not clearly understood. In this study, we examined the regenerative capacity of dystrophic muscles in vivo in FKRP mutant and LARGE(myd) mice with little and complete lack of functionally glycosylated α-DG (F-α-DG) respectively. The number of regenerating fibers expressing embryonic myosin heavy chain (eMyHC) in the diseased muscles up to the age of 10 months is higher than or at similar levels to wild type muscle after notexin and polyethyleminine insults. The process of fiber maturation is not significantly affected by the lack of F-α-DG assessed by size distribution. The earlier appearance of a larger number of regenerating fibers after injury is consistent with the observation that the populations of myogenic satellite cells are increased and being readily activated in the dystroglycanopathy muscles. F-α-DG is expressed at trace amounts in undifferentiated myoblasts, but increases in differentiated myotubes in vitro. We therefore conclude that muscle regeneration is not impaired in the early stage of the dystroglycanopathies, and F-α-DG does not play a significant role in myogenic cell proliferation and fiber formation and maturation.

Restoration of Functional Glycosylation of α-Dystroglycan in FKRP Mutant Mice Is Associated with Muscle Regeneration.

Mutations in fukutin-related protein (FKRP) gene are characterized with lack of functionally glycosylated α-dystroglycan (F-α-DG). Surprisingly, a few muscle fibers express strong F-α-DG. Herein, we investigated the restoration of F-α-DG in the FKRP mutant muscles and showed that the restoration of glycosylation is associated with muscle regeneration and dependent on the expression of both like-glycosyltransferase (LARGE) and partially functional FKRP. F-α-DG in the regenerating fibers reaches up to normal levels and lasts for >4 weeks, but no up-regulation of the LARGE and FKRP is detected during the regeneration process. The FKRP protein with P448L mutation is sufficient for functional glycosylation of α-DG in regenerating fibers, but not in mature fibers. Thus, factors other than FKRP enable regenerating fibers to produce functional α-DG, compensating for the defect in FKRP function. Identification of factors other than LARGE and FKRP could generate new approaches for restoration of F-α-DG in mature muscle fibers with defects in FKRP functions.

Mouse models of fukutin-related protein mutations show a wide range of disease phenotypes.

Dystroglycanopathies are characterized by a reduction in the glycosylation of alpha-dystroglycan (α-DG). A common cause for this subset of muscular dystrophies is mutations in the gene of fukutin-related protein (FKRP). FKRP mutations have been associated with a wide spectrum of clinical severity from severe Walker-Warburg syndrome and muscle-eye-brain disease with brain and eye defects to mild limb-girdle muscular dystrophy 2I with myopathy only. To examine the affects of FKRP mutations on the severity of the disease, we have generated homozygous and compound heterozygous mouse models with human mutations in the murine FKRP gene. P448Lneo+ and E310delneo+ mutations result in severe dystrophic and embryonic lethal phenotypes, respectively. P448Lneo+/E310delneo+ compound heterozygotes exhibit brain defects and severe muscular dystrophies with near absence of α-DG glycosylation. Removal of the Neo(r) cassette from the P448Lneo+ homozygous mice eliminates overt brain and eye defects, and reduces severity of dystrophic phenotypes. Furthermore, introduction of the common L276I mutation to generate transgenic L276Ineo+ homozygous and L276Ineo+/P448Lneo+ and L276Ineo+/E310delneo+ compound heterozygotes results in mice displaying milder dystrophies with reduced α-DG glycosylation and no apparent brain defects. Limited sampling and variation in functionally glycosylated α-DG levels between and within muscles may explain the difficulties in correlating FKRP expression levels with phenotype in clinics. The nature of individual mutations, expression levels and status of muscle differentiation all contribute to the phenotypic manifestation. These mutant FKRP mice are useful models for the study of disease mechanism(s) and experimental therapies.

Aged B lymphocytes retain their ability to express surface markers but are dysfunctional in their proliferative capability during early activation events.

BACKGROUND: Ageing is associated with dysfunction in the humoral response leading to decreased protection against infectious diseases. Defects in T cell function due to age have been well characterized but it is unclear if dysfunctions in antibody responses are due to deficiencies in a helper environment or intrinsic B cell defects. Previous studies from our laboratory have shown that aged B lymphocytes are able to differentiate into high affinity antibody-secreting cells at a frequency similar to their young counterparts. However, expansion of B cells in vivo was reduced in aged animals when compared to young. METHODS: To further investigate the cause of this reduced expansion, we have now examined early activation events of aged B cells in response to anti-CD40 monoclonal antibody (mAb) and lipopolysaccharide (LPS) stimulation in vitro. To do this spleen cells were harvested from young, middle-aged and aged quasi-monoclonal (QM) mice and cultured in complete RPMI for 24 and 48 hours. Cultures contained either LPS or anti-CD40 mAb and murine IL-4. Cells were collected and analyzed using flow cytometry. To examine the proliferative capacity of aged B cells spleen cells were collected as before and cultured in 96 well microtiter plates with either LPS or anti-CD40 mAb and murine IL-4 for 24 hours. Tritiated thymidine ([3H]-Tdr) was added to each well and incubated for another 24 hours after which cells were collected and analyzed using a scintillation counter. RESULTS: Resting aged B cells exhibited similar levels of CD40 expression when compared to young cells and efficiently up-regulated CD86 and CD69 and also down-regulated CD38 upon stimulation. However, aged B cells proliferated less than young B cells and showed a consistent, but not statistically significant, reduction in their ability to form blast cells. CONCLUSION: Aged B cells exhibited a reduced response in some early activation events but produced at least a partial response in all cases. Thus, therapeutic intervention may be possible, despite intrinsically different responses in aged B cells.