Detailed genetic and functional analysis of the hDMDdel52/mdx mouse model.

Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by reading frame disrupting mutations in the DMD gene leading to absence of functional dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach aimed at restoring the reading frame at the pre-mRNA level, allowing the production of internally truncated partly functional dystrophin proteins. AONs work in a sequence specific manner, which warrants generating humanized mouse models for preclinical tests. To address this, we previously generated the hDMDdel52/mdx mouse model using transcription activator like effector nuclease (TALEN) technology. This model contains mutated murine and human DMD genes, and therefore lacks mouse and human dystrophin resulting in a dystrophic phenotype. It allows preclinical evaluation of AONs inducing the skipping of human DMD exons 51 and 53 and resulting in restoration of dystrophin synthesis. Here, we have further characterized this model genetically and functionally. We discovered that the hDMD and hDMDdel52 transgene is present twice per locus, in a tail-to-tail-orientation. Long-read sequencing revealed a partial deletion of exon 52 (first 25 bp), and a 2.3 kb inversion in intron 51 in both copies. These new findings on the genomic make-up of the hDMD and hDMDdel52 transgene do not affect exon 51 and/or 53 skipping, but do underline the need for extensive genetic analysis of mice generated with genome editing techniques to elucidate additional genetic changes that might have occurred. The hDMDdel52/mdx mice were also evaluated functionally using kinematic gait analysis. This revealed a clear and highly significant difference in overall gait between hDMDdel52/mdx mice and C57BL6/J controls. The motor deficit detected in the model confirms its suitability for preclinical testing of exon skipping AONs for human DMD at both the functional and molecular level.

Using a State-of-the-Art Toolbox to Evaluate Molecular and Functional Readouts of Antisense Oligonucleotide-Induced Exon Skipping in mdx Mice.

Duchenne muscular dystrophy (DMD) is a severe childhood muscle disease primarily caused by the lack of functional dystrophin at the muscle fiber membranes. Multiple therapeutic approaches are currently in (pre)clinical development, aimed at restoring expression of (truncated) dystrophin. Key questions in this phase relate to route of drug administration, dose regimen, and levels of dystrophin required to improve muscle function. A series of studies applying antisense oligonucleotides (AONs) in the mdx mouse model for DMD has been reported over the last two decades, claiming a variable range of exon skipping and increased dystrophin levels correlated to some functional improvement. The aim of this study was to compare the efficacy of subcutaneous (SC) versus intravenous (IV) dosing routes of an mdx-specific AON at both the molecular and functional level, using state-of-the-art quantitative technologies, including digital droplet polymerase chain reaction, capillary Western immunoassay, magnetic resonance imaging, and automated kinematic analysis. The majority of all readouts we quantified, both molecular and functional, showed that IV dosing of the AON had a more pronounced beneficial effect than SC dosing in mdx mice. Last, but not least, the more quantitative molecular and functional data obtained in this study suggest that low levels of dystrophin protein of at least 2.5% of wild type may already have a beneficial effect on muscle leakiness and may improve motor performance of mdx mice.

Effect of Ibuprofen on Skeletal Muscle of Dysferlin-Null Mice.

Ibuprofen, a nonsteroidal anti-inflammatory drug, and nitric oxide (NO) donors have been reported to reduce the severity of muscular dystrophies in mice associated with the absence of dystrophin or α-sarcoglycan, but their effects on mice that are dystrophic due to the absence of dysferlin have not been examined. We have tested ibuprofen, as well as isosorbide dinitrate (ISDN), a NO donor, to learn whether used alone or together they protect dysferlin-null muscle in A/J mice from large strain injury (LSI) induced by a series of high strain lengthening contractions. Mice were maintained on chow containing ibuprofen and ISDN for 4 weeks. They were then subjected to LSI and maintained on the drugs for 3 additional days. We measured loss of torque immediately following injury and at day 3 postinjury, fiber necrosis, and macrophage infiltration at day 3 postinjury, and serum levels of the drugs at the time of euthanasia. Loss of torque immediately after injury was not altered by the drugs. However, the torque on day 3 postinjury significantly decreased as a function of ibuprofen concentration in the serum (range, 0.67-8.2 µg/ml), independent of ISDN. The effects of ISDN on torque loss at day 3 postinjury were not significant. In long-term studies of dysferlinopathic BlAJ mice, lower doses of ibuprofen had no effects on muscle morphology, but reduced treadmill running by 40%. Our results indicate that ibuprofen can have deleterious effects on dysferlin-null muscle and suggest that its use at pharmacological doses should be avoided by individuals with dysferlinopathies.

Hip region muscular dystrophy and emergence of motor deficits in dysferlin-deficient Bla/J mice.

The identification of a dysferlin-deficient animal model that accurately displays both the physiological and behavior aspects of human dysferlinopathy is critical for the evaluation of potential therapeutics. Disease progression in dysferlin-deficient mice is relatively mild, compared to the debilitating human disease which manifests in impairment of particular motor functions. Since there are no other known models of dysferlinopathy in other species, locomotor proficiency and muscular anatomy through MRI (both lower leg and hip region) were evaluated in dysferlin-deficient B6.A-Dysfprmd /GeneJ (Bla/J) mice to define disease parameters for therapeutic assessment. Despite the early and progressive gluteal muscle dystrophy and significant fatty acid accumulation, the emergence of significant motor function deficits was apparent at approximately 1 year of age for standard motor challenges including the rotarod, a marble bury test, grip strength, and swimming speed. Earlier observations of decreased performance for Bla/J mice were evident during extended monitoring of overall exploration and rearing activity. Comprehensive treadmill gait analyses of the Bla/J model indicated significant differences in paw placement angles and stance in relation to speed and platform slope. At 18 months of age, there was no significant difference in the life expectancy of Bla/J mice compared to wild type. Consistent with progressive volume loss and fatty acid accumulation in the hip region observed by MRI, mass measurement of individual muscles confirmed gluteal and psoas muscles were the only muscles demonstrating a significant decrease in muscle mass, which is analogous to hip-girdle weakness observed in human dysferlin-deficient patients. Collectively, this longitudinal analysis identifies consistent disease parameters that can be indicators of efficacy in studies developing treatments for human dysferlin deficiency.

Granulocyte colony stimulating factor attenuates inflammation in a mouse model of amyotrophic lateral sclerosis.

BACKGROUND: Granulocyte colony stimulating factor (GCSF) is protective in animal models of various neurodegenerative diseases. We investigated whether pegfilgrastim, GCSF with sustained action, is protective in a mouse model of amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease with manifestations of upper and lower motoneuron death and muscle atrophy accompanied by inflammation in the CNS and periphery. METHODS: Human mutant G93A superoxide dismutase (SOD1) ALS mice were treated with pegfilgrastim starting at the presymptomatic stage and continued until the end stage. After long-term pegfilgrastim treatment, the inflammation status was defined in the spinal cord and peripheral tissues including hematopoietic organs and muscle. The effect of GCSF on spinal cord neuron survival and microglia, bone marrow and spleen monocyte activation was assessed in vitro. RESULTS: Long-term pegfilgrastim treatment prolonged mutant SOD1 mice survival and attenuated both astro- and microgliosis in the spinal cord. Pegfilgrastim in SOD1 mice modulated the inflammatory cell populations in the bone marrow and spleen and reduced the production of pro-inflammatory cytokine in monocytes and microglia. The mobilization of hematopoietic stem cells into the circulation was restored back to basal level after long-term pegfilgrastim treatment in SOD1 mice while the storage of Ly6C expressing monocytes in the bone marrow and spleen remained elevated. After pegfilgrastim treatment, an increased proportion of these cells in the degenerative muscle was detected at the end stage of ALS. CONCLUSIONS: GCSF attenuated inflammation in the CNS and the periphery in a mouse model of ALS and thereby delayed the progression of the disease. This mechanism of action targeting inflammation provides a new perspective of the usage of GCSF in the treatment of ALS.

Mutant SOD1 from spinal cord of G93A rats is destabilized and binds to inner mitochondrial membrane.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS). Mechanisms of mutant SOD1 toxicity are unknown, but increased SOD1 activity can boost production of reactive oxygen species (ROS) in the mitochondrial intermembrane space (IMS). Using non-reducing SDS-PAGE we found that in G93A-SOD1 rats the mutant SOD1 was prominently destabilized only in the diseased spinal cord, where this mutant enzyme was also up regulated in the IMS with increased ability to bind the inner membrane of isolated non-transgenic mitoplasts. These mitoplasts increased ROS production when exposed to mutant SOD1 from the spinal cord at the presymptomatic stage. The levels of disulfide-reduced SOD1 peaked at the end stage of the disease, whereas protein disulfide isomerase (PDI), a chaperone capable of rearranging disulfide bonds between cysteine residues of SOD1, was increased prior to the end stage. IMS binding and increased ROS production by destabilized SOD1 may contribute to mitochondrial damage in G93A-SOD1 rats.

Deleterious role of superoxide dismutase in the mitochondrial intermembrane space.

This work demonstrates how increased activity of copper-zinc superoxide dismutase (SOD1) paradoxically boosts production of toxic reactive oxygen species (ROS) in the intermembrane space (IMS) of mitochondria. Even though SOD1 is a cytosolic enzyme, a fraction of it is found in the IMS, where it is thought to provide protection against oxidative damage. We found that SOD1 controls cytochrome c-catalyzed peroxidation in vitro when superoxide is available. The presence of SOD1 significantly increased the rate of ROS production in mitoplasts, which are devoid of outer membrane and IMS. In response to inhibition of respiration with antimycin A, isolated mouse wild-type mitochondria increased ROS production, but the mitochondria from mice lacking SOD1 (SOD1(-/-)) did not. Also, lymphocytes isolated from SOD1(-/-) mice produced significantly less ROS than did wild-type cells and were more resistant to apoptosis induced by inhibition of respiration. Moreover, an increased amount of the toxic mutant G93A SOD1 in the IMS increased ROS production. The mitochondrial dysfunction and cell damage paradoxically induced by SOD1-mediated ROS production may be implicated in chronic degenerative diseases.

Pyrrolidine dithiocarbamate activates Akt and improves spatial learning in APP/PS1 mice without affecting beta-amyloid burden.

Pyrrolidine dithiocarbamate (PDTC) is a clinically tolerated inhibitor of nuclear factor-kappaB (NF-kappaB), antioxidant and antiinflammatory agent, which provides protection in brain ischemia models. In neonatal hypoxia-ischemia model, PDTC activates Akt and reduces activation of glycogen synthase kinase 3beta (GSK-3beta). Because chronic inflammation, oxidative stress, and increased GSK-3beta activity are features of Alzheimer's disease (AD) pathology, we tested whether PDTC reduces brain pathology and improves cognitive function in a transgenic animal model of AD. A 7 month oral treatment with PDTC prevented the decline in cognition in AD mice without altering beta-amyloid burden or gliosis. Moreover, marked oxidative stress and activation of NF-kappaB were not part of the brain pathology. Instead, the phosphorylated form of GSK-3beta was decreased in the AD mouse brain, and PDTC treatment increased the phosphorylation of Akt and GSK-3beta. Also, PDTC treatment increased the copper concentration in the brain. In addition, PDTC rescued cultured hippocampal neurons from the toxicity of oligomeric Abeta and reduced tau phosphorylation in the hippocampus of AD mice. Finally, astrocytic glutamate transporter GLT-1, known to be regulated by Akt pathway, was decreased in the transgenic AD mice but upregulated back to the wild-type levels by PDTC treatment. Thus, PDTC may improve spatial learning in AD by interfering with Akt-GSK pathway both in neurons and astrocytes. Because PDTC is capable of transferring external Cu2+ into a cell, and, in turn, Cu2+ is able to activate Akt, we hypothesize that PDTC provides the beneficial effect in transgenic AD mice through Cu2+-activated Akt pathway.

Pyrrolidine dithiocarbamate inhibits induction of immunoproteasome and decreases survival in a rat model of amyotrophic lateral sclerosis.

Pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear transcription factor kappa-B (NF-kappaB) and an antioxidant, has beneficial effects in animal models of various diseases, including arthritis, brain ischemia, spinal cord injury, Alzheimer's disease, and Duchenne muscular dystrophy. Because inflammation and oxidative damage are also hallmarks of amyotrophic lateral sclerosis (ALS), we studied the effect of oral PDTC treatment on G93A-superoxide dismutase 1 (SOD1) transgenic (TG) rat model of human ALS and observed that PDTC treatment significantly decreases the survival. PDTC treatment evoked the end stage of the disease at 121 +/- 21 days, whereas untreated TG animals reached the end stage at 141 +/- 13 days (p < 0.01). The DNA binding activity of NF-kappaB was not altered in G93A-SOD1 TG rats by PDTC treatment. The copper concentration in the spinal cord was increased after PDTC treatment both in G93A-SOD1 TG and wild-type rats, suggesting that increased copper may enhance the neurotoxicity of mutant SOD1. The amount of ubiquitinated proteins were significantly higher and proteasomal activity was decreased in the spinal cords of PDTC-treated TG rats compared with other groups, suggesting that PDTC treatment decreases proteasome function. Immunoblotting and immunocytochemistry showed that the level of immunoproteasome but not constitutive proteasome was increased in glia of G93A-SOD1 TG rats along with disease development. PDTC treatment completely blocked the induction of immunoproteasome expression without affecting constitutive proteasome. These results suggest that PDTC acts as an immunoproteasome inhibitor in mutant SOD1 rats and that immunoproteasome may help the nervous system to cope with deleterious effects of SOD1-G93A mutation.

Antioxidant pyrrolidine dithiocarbamate activates Akt-GSK signaling and is neuroprotective in neonatal hypoxia-ischemia.

Pyrrolidine dithiocarbamate (PDTC), an antioxidant and inhibitor of transcription factor nuclear factor kappa-B (NF-kappaB), has been reported to reduce inflammation and apoptosis. Because PDTC was recently found to protect in various models of adult brain ischemia with a wide therapeutic time window, we tested the effect of PDTC in a rodent model of neonatal hypoxia-ischemia (HI) brain injury. T2-weighed magnetic resonance imaging (T2-MRI) 7 days after the insult showed that a single PDTC (50 mg/kg) injection 2.5 h after the HI reduced the mean brain infarct size by 59%. PDTC reduced the HI-induced dephosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta), expression of cleaved caspase-3, and nuclear translocation of NF-kappaB in the neonatal brain. PDTC targeted directly neurons, as PDTC reduced hypoxia-reoxygenation-induced cell death in pure hippocampal neuronal cultures. It is suggested that in addition to the previously indicated NF-kappaB inhibition as a protective mechanism of PDTC treatment, PDTC may reduce HI-induced brain injury at least partially by acting as an antioxidant, which reduces the Akt-GSK-3beta pathway of apoptotic cell death. The clinically approved PDTC and its analogues may be beneficial after HI insults with a reasonable time window.

beta-Amyloid infusion results in delayed and age-dependent learning deficits without role of inflammation or beta-amyloid deposits.

beta-Amyloid (Abeta) polypeptide plays a critical role in the pathogenesis of Alzheimer's disease (AD), which is characterized by progressive decline of cognitive functions, formation of Abeta deposits and neurofibrillary tangles, and loss of neurons. Increased genetic production or direct intracerebral administration of Abeta in animal models results in Abeta deposition, gliosis, and impaired cognitive functions. Whether aging renders the brain prone to Abeta and whether inflammation is required for Abeta-induced learning deficits is unclear. We show that intraventricular infusion of Abeta1-42 results in learning deficits in 9-month-old but not 2.5-month-old mice. Deficits that become detectable 12 weeks after the infusion are associated with a slight reduction in Cu,Zn superoxide dismutase activity but do not correlate with Abeta deposition and are not associated with gliosis. In rats, Abeta infusion induced learning deficits that were detectable 6 months after the infusion. Approximately 20% of the Abeta immunoreactivity in rats was associated with astrocytes. NMR spectrum analysis of the animals cerebrospinal fluid revealed a strong reduction trend in several metabolites in Abeta-infused rats, including lactate and myo-inositol, supporting the idea of dysfunctional astrocytes. Even a subtle increase in brain Abeta1-42 concentration may disrupt normal metabolism of astrocytes, resulting in altered neuronal functions and age-related development of learning deficits independent of Abeta deposition and inflammation.