Rapamycin nanoparticles target defective autophagy in muscular dystrophy to enhance both strength and cardiac function.

Duchenne muscular dystrophy in boys progresses rapidly to severe impairment of muscle function and death in the second or third decade of life. Current supportive therapy with corticosteroids results in a modest increase in strength as a consequence of a general reduction in inflammation, albeit with potential untoward long-term side effects and ultimate failure of the agent to maintain strength. Here, we demonstrate that alternative approaches that rescue defective autophagy in mdx mice, a model of Duchenne muscular dystrophy, with the use of rapamycin-loaded nanoparticles induce a reproducible increase in both skeletal muscle strength and cardiac contractile performance that is not achievable with conventional oral rapamycin, even in pharmacological doses. This increase in physical performance occurs in both young and adult mice, and, surprisingly, even in aged wild-type mice, which sets the stage for consideration of systemic therapies to facilitate improved cell function by autophagic disposal of toxic byproducts of cell death and regeneration.

Strength and corticosteroid responsiveness of mdx mice is unchanged by RAG2 gene knockout.

Corticosteroids improve muscle function in boys with Duchenne muscular dystrophy and mdx mice possibly via effects on T-cell and B-cells. We quantified T-cell/B-cell functional effects and refined prednisolone's therapeutic mechanism in mdx mice. RAG2(-/-) mice, which produce no T-cells or B-cells, were crossed with mdx mice, which lack dystrophin protein. Strength testing (3-36 weeks) was performed on treated and control groups of male mdx RAG2(-/-)and mdx RAG2(+/-) mice. Longitudinal grip strength testing and hanging wire testing were assessed. Voluntary wheel running and creatine kinase level were measured. The absence of T-cells/B-cells (RAG2(-/-) mutation) caused no physiologic improvement. Prednisolone improved performance in mdx mice, independent of RAG2 gene expression (+ or -/-). Prednisolone treatment increased the frequency of muscle calcification, while RAG2 genotype had no effect. There was no change in fiber type proportions due to RAG2 genotype or prednisolone treatment. Thus, T-cells and/or B-cells (and immunoglobulins), while demonstrable in mdx mouse muscle, are playing a negligible role in their mdx-related functional outcome. Prednisolone's therapeutic effect is through T-cell/B-cell independent mechanisms in mdx mice.

Brain-derived neurotrophic factor and autoantibodies to neural antigens in sera of children with autistic spectrum disorders, Landau-Kleffner syndrome, and epilepsy.

BACKGROUND: Brain derived neurotrophic factor (BDNF) elevation in newborn sera predicts intellectual/social developmental abnormalities. Other autoantibodies (AAs) to endothelial cells (ECs) and myelin basic protein (MBP) are also elevated in some children. We tested relationships between BDNF, BDNF AAs, and other AAs in children with these disorders. METHODS: BDNF levels and IgG/IgM autoantibodies to BDNF, ECs, MBP, and histones were measured in children with autism, childhood disintegrative disorder (CDD), pervasive developmental delay-not otherwise specified (PDD-nos), acquired epilepsy, Landau-Kleffner syndrome (LKS); healthy children (HC), and children with non-neurological illnesses (NNI). RESULTS: Mean BDNF levels were elevated in children with autism and CDD, (p < or = 0.0002) compared to HC or NNI. Mean IgG and IgM BDNF AAs were elevated in children with autism, CDD and epilepsy (p < or = 0.0005) compared to HC but not to NNI. Mean IgM AA EC titers detected by immunocytochemistry were higher in autism, PDD-NOS, epilepsy, and LKS (p < or = 0.005) compared to HC and NNI. While mean ELISA IgG EC AAs were higher in autism and PPD-NOS (p < 0.005) compared to HC but not NNI, ELISA IgM EC AAs were higher in children with autism, CDD, PDD-NOS, and epilepsy compared to both HC and NNI (p < 0.0005). Mean anti-MBP IgG and IgM titers were higher in all study groups (p < 0.005) except for LKS compared to both HC and NNI. CONCLUSION: Children with developmental disorders and epilepsy have higher AAs to several neural antigens compared to controls. The presence of both BDNF AAs and elevated BDNF levels in some children with autism and CDD suggests a previously unrecognized interaction between the immune system and BDNF.

Complement 3 deficiency and oral prednisolone improve strength and prolong survival of laminin alpha2-deficient mice.

Complement deposition and macrophages are common in biopsies of children with muscular dystrophy. While the presumed roles of complement and macrophages have been those of scavenger to remove and clear necrotic fibers, there is some evidence that they play a primary role in the pathogenesis of these diseases. Here, we explore the role of complement in the pathogenesis of the most severe animal model of congenital dystrophy, the dy-/- mouse, which is laminin alpha2-deficient. We generated animals deficient in both C3 and laminin alpha2. C3 is the third component of the complement cascade and is required for activation of either the classical or alternative pathways. Thirty-three percent of the dy-/-:C3+ mice (n=59) died before 24 weeks while only 14% of the dy-/-:C3-/- (n=29) mice died (p=0.04). Absolute forepaw strength was 25-30% greater for the dy-/-:C3-/- mice up to 20 weeks of age (p<0.05 compared to complement-sufficient). Forepaw strength adjusted for weight also showed significant differences with C3-/- mice being stronger up to 20 weeks (p<0.05). However, by 24 weeks, the two groups did not differ for strength. Next, we treated 20 mice with twice weekly oral prednisolone. Survival at 24 weeks for the prednisolone treated dy-/- mice (C3-/- or C3+) was 90% (p=0.04). This work shows that complement insufficiency and weekly prednisone prolong survival and improve strength of the laminin alpha2-deficient mouse. This work suggests that the complement system may contribute directly to the pathogenesis of this form of dystrophy. Because complement activity may be modified pharmacologically, this work may have implications for treatment of children with congenital muscular dystrophy secondary to laminin alpha2 deficiency.

RAG2 gene knockout in mice causes fatigue.

The effect of a disrupted immune system on the neuromuscular system is poorly characterized. We compared the strength and fatigue of RAG2(-/-) mice, which lack T-cells and B-cells, with immune intact controls. RAG2(-/-) mice demonstrated fatigue with shorter inverted hang-times (HT) and voluntary wheel-running (VWR) distance and total run times; they increased body weight more slowly but had proportionally normal forelimb grip strength (FGS) and VWR speed. Medial rectus femoris histopathology showed no change in fiber type proportions, no variation in type 2b fiber diameter, and no change in the percentage of central nuclei. There was no change in serum creatine kinase (CK) levels. Thus, in RAG2(-/-) mice body weight and fatigue were directly affected by a hypoactive immune system. Whether these effects were centrally or peripherally mediated is unknown. This model may help to explain fatigue in human conditions in which the immune system is suppressed or absent.

Weekly oral prednisolone improves survival and strength in male mdx mice.

Although corticosteroids alleviate weakness in mdx mice, no long-term treatment has determined whether this benefit is maintained. We studied mdx mice forelimb grip strength and fatigue from 3 through 84 weeks and followed survival through 104 weeks. The mdx mice were given twice weekly oral prednisolone (5 mg/kg) beginning at 3 or 4 weeks. Treated mdx mice survived longer than untreated mice. Between 3 and 10 weeks, treated and untreated mdx mice had similar strength. Between 10 and 24 weeks, strength and strength per gram body weight declined more slowly in treated than untreated mdx mice. Between 24 and 84 weeks, treated and untreated mdx mice declined in strength at the same rate, although treated mice remained stronger. Forelimb grip fatigue was present in untreated mdx mice at all time-points compared to wild-type and was not changed significantly by treatment. We have demonstrated long-term benefit of oral prednisolone in the mdx mouse model of Duchenne muscular dystrophy (DMD). As corticosteroids remain the most validated long-term treatment of DMD, this work may allow for better prediction of synergistic treatments likely to translate to effective improvement for boys with this progressive muscular dystrophy.