X-linked SBMA model mice display relevant non-neurological phenotypes and their expression of mutant androgen receptor protein in motor neurons is not required for neuromuscular disease.

X-linked spinal and bulbar muscular atrophy (SBMA; Kennedy's disease) is a rare neuromuscular disorder characterized by adult-onset proximal muscle weakness and lower motor neuron degeneration. SBMA was the first human disease found to be caused by a repeat expansion mutation, as affected patients possess an expanded tract of CAG repeats, encoding polyglutamine, in the androgen receptor (AR) gene. We previously developed a conditional BAC fxAR121 transgenic mouse model of SBMA and used it to define a primary role for skeletal muscle expression of polyglutamine-expanded AR in causing the motor neuron degeneration. Here we sought to extend our understanding of SBMA disease pathophysiology and cellular basis by detailed examination and directed experimentation with the BAC fxAR121 mice. First, we evaluated BAC fxAR121 mice for non-neurological disease phenotypes recently described in human SBMA patients, and documented prominent non-alcoholic fatty liver disease, cardiomegaly, and ventricular heart wall thinning in aged male BAC fxAR121 mice. Our discovery of significant hepatic and cardiac abnormalities in SBMA mice underscores the need to evaluate human SBMA patients for signs of liver and heart disease. To directly examine the contribution of motor neuron-expressed polyQ-AR protein to SBMA neurodegeneration, we crossed BAC fxAR121 mice with two different lines of transgenic mice expressing Cre recombinase in motor neurons, and after updating characterization of SBMA phenotypes in our current BAC fxAR121 colony, we found that excision of mutant AR from motor neurons did not rescue neuromuscular or systemic disease. These findings further validate a primary role for skeletal muscle as the driver of SBMA motor neuronopathy and indicate that therapies being developed to treat patients should be delivered peripherally.

Cancer-associated SMARCAL1 loss-of-function mutations promote alternative lengthening of telomeres and tumorigenesis in telomerase-negative glioblastoma cells.

BACKGROUND: Telomere maintenance mechanisms are required to enable the replicative immortality of malignant cells. While most cancers activate the enzyme telomerase, a subset of cancers uses telomerase-independent mechanisms termed alternative lengthening of telomeres (ALT). ALT occurs via homology-directed-repair mechanisms and is frequently associated with ATRX mutations. We previously showed that a subset of adult glioblastoma (GBM) patients with ATRX-expressing ALT-positive tumors harbored loss-of-function mutations in the SMARCAL1 gene, which encodes an annealing helicase involved in replication fork remodeling and the resolution of replication stress. However, the causative relationship between SMARCAL1 deficiency, tumorigenesis, and de novo telomere synthesis is not understood. METHODS: We used a patient-derived ALT-positive GBM cell line with native SMARCAL1 deficiency to investigate the role of SMARCAL1 in ALT-mediated de novo telomere synthesis, replication stress, and gliomagenesis in vivo. RESULTS: Inducible rescue of SMARCAL1 expression suppresses ALT indicators and inhibits de novo telomere synthesis in GBM and osteosarcoma cells, suggesting that SMARCAL1 deficiency plays a functional role in ALT induction in cancers that natively lack SMARCAL1 function. SMARCAL1-deficient ALT-positive cells can be serially propagated in vivo in the absence of detectable telomerase activity, demonstrating that the SMARCAL1-deficient ALT phenotype maintains telomeres in a manner that promotes tumorigenesis. CONCLUSIONS: SMARCAL1 deficiency is permissive to ALT and promotes gliomagenesis. Inducible rescue of SMARCAL1 in ALT-positive cell lines permits the dynamic modulation of ALT activity, which will be valuable for future studies aimed at understanding the mechanisms of ALT and identifying novel anticancer therapeutics that target the ALT phenotype.

What's new and what's next for gene therapy in Pompe disease?

INTRODUCTION: Pompe disease is an autosomal recessive disorder caused by a deficiency of acid-α-glucosidase (GAA), an enzyme responsible for hydrolyzing lysosomal glycogen. A lack of GAA leads to accumulation of glycogen in the lysosomes of cardiac, skeletal, and smooth muscle cells, as well as in the central and peripheral nervous system. Enzyme replacement therapy has been the standard of care for 15 years and slows disease progression, particularly in the heart, and improves survival. However, there are limitations of ERT success, which gene therapy can overcome. AREAS COVERED: Gene therapy offers several advantages including prolonged and consistent GAA expression and correction of skeletal muscle as well as the critical CNS pathology. We provide a systematic review of the preclinical and clinical outcomes of adeno-associated viral mediated gene therapy and alternative gene therapy strategies, highlighting what has been successful. EXPERT OPINION: Although the preclinical and clinical studies so far have been promising, barriers exist that need to be addressed in gene therapy for Pompe disease. New strategies including novel capsids for better targeting, optimized DNA vectors, and adjuctive therapies will allow for a lower dose, and ameliorate the immune response.

Respiratory dysfunction in a mouse model of spinocerebellar ataxia type 7.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal-dominant neurodegenerative disorder caused by a CAG repeat expansion in the coding region of the ataxin-7 gene. Infantile-onset SCA7 patients display extremely large repeat expansions (>200 CAGs) and exhibit progressive ataxia, dysarthria, dysphagia and retinal degeneration. Severe hypotonia, aspiration pneumonia and respiratory failure often contribute to death in affected infants. To better understand the features of respiratory and upper airway dysfunction in SCA7, we examined breathing and putative phrenic and hypoglossal neuropathology in a knock-in mouse model of early-onset SCA7 carrying an expanded allele with 266 CAG repeats. Whole-body plethysmography was used to measure awake spontaneously breathing SCA7-266Q knock-in mice at baseline in normoxia and during a hypercapnic/hypoxic respiratory challenge at 4 and 8 weeks, before and after the onset of disease. Postmortem studies included quantification of putative phrenic and hypoglossal motor neurons and microglia, and analysis of ataxin-7 aggregation at end stage. SCA7-266Q mice had profound breathing deficits during a respiratory challenge, exhibiting reduced respiratory output and a greater percentage of time in apnea. Histologically, putative phrenic and hypoglossal motor neurons of SCA7 mice exhibited a reduction in number accompanied by increased microglial activation, indicating neurodegeneration and neuroinflammation. Furthermore, intranuclear ataxin-7 accumulation was observed in cells neighboring putative phrenic and hypoglossal motor neurons in SCA7 mice. These findings reveal the importance of phrenic and hypoglossal motor neuron pathology associated with respiratory failure and upper airway dysfunction, which are observed in infantile-onset SCA7 patients and likely contribute to their early death.

Glycogen accumulation in smooth muscle of a Pompe disease mouse model.

Pompe disease is a lysosomal storage disease caused by mutations within the GAA gene, which encodes acid α-glucosidase (GAA)-an enzyme necessary for lysosomal glycogen degradation. A lack of GAA results in an accumulation of glycogen in cardiac and skeletal muscle, as well as in motor neurons. The only FDA approved treatment for Pompe disease-an enzyme replacement therapy (ERT)-increases survival of patients, but has unmasked previously unrecognized clinical manifestations of Pompe disease. These clinical signs and symptoms include tracheo-bronchomalacia, vascular aneurysms, and gastro-intestinal discomfort. Together, these previously unrecognized pathologies indicate that GAA-deficiency impacts smooth muscle in addition to skeletal and cardiac muscle. Thus, we sought to characterize smooth muscle pathology in the airway, vascular, gastrointestinal, and genitourinary in the Gaa-/- mouse model. Increased levels of glycogen were present in smooth muscle cells of the aorta, trachea, esophagus, stomach, and bladder of Gaa-/- mice, compared to wild type mice. In addition, there was an increased abundance of both lysosome membrane protein (LAMP1) and autophagosome membrane protein (LC3) indicating vacuolar accumulation in several tissues. Taken together, we show that GAA deficiency results in subsequent pathology in smooth muscle cells, which may lead to life-threatening complications if not properly treated.

Respiratory pathology in the Optn-/- mouse model of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder that results in death due to respiratory failure. Many genetic defects are associated with ALS; one such defect is a mutation in the gene encoding optineurin (OPTN). Using an optineurin null mouse (Optn-/-), we sought to characterize the impact of optineurin deficiency on respiratory neurodegeneration. Respiratory function was assessed at 6 and 12 mo of age using whole body plethysmography at baseline during normoxia (FiO2: 0.21; N2 balance) and during a respiratory challenge with hypoxia and hypercapnia (FiCO2: 0.07, FiO2: 0.10; N2 balance). Histological analyses to assess motor neuron viability and respiratory nerve integrity were performed in the medulla, cervical spinal cord, hypoglossal nerve, and phrenic nerve. Minute ventilation, peak inspiratory flow, and peak expiratory flow are significantly reduced during a respiratory challenge in 6 mo Optn-/-mice. By 12 mo, tidal volume is also significantly reduced in Optn-/- mice. Furthermore, 12mo Optn-/- mice exhibit hypoglossal motor neuron loss, phrenic and hypoglossal dysmyelination, and accumulated mitochondria in the hypoglossal nerve axons. Overall, these data indicate that Optn-/- mice display neurodegenerative respiratory dysfunction and are a useful model to study the impact of novel therapies on respiratory function for optineurin-deficient ALS patients.

Motor axonopathies in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by deleterious mutations in the DMD gene which encodes the dystrophin protein. Skeletal muscle weakness and eventual muscle degradation due to loss of dystrophin are well-documented pathological hallmarks of DMD. In contrast, the neuropathology of this disease remains understudied despite the emerging evidence of neurological abnormalities induced by dystrophin loss. Using quantitative morphological analysis of nerve sections, we characterize axonopathies in the phrenic and hypoglossal (XII) nerves of mdx mice. We observe dysfunction in these nerves - which innervate the diaphragm and genioglossus respectively - that we propose contributes to respiratory failure, the most common cause of death in DMD. These observations highlight the importance in the further characterization of the neuropathology of DMD. Additionally, these observations underscore the necessity in correcting both the nervous system pathology in addition to skeletal muscle deficits to ameliorate this disease.

The Respiratory Phenotype of Rodent Models of Amyotrophic Lateral Sclerosis and Spinocerebellar Ataxia.

Amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia (SCA) are neurodegenerative disorders that result in progressive motor dysfunction and ultimately lead to respiratory failure. Rodent models of neurodegenerative disorders provide a means to study the respiratory motor unit pathology that results in respiratory failure. In addition, they are important for pre-clinical studies of novel therapies that improve breathing, quality of life, and survival. The goal of this review is to compare the respiratory phenotype of two neurodegenerative disorders that have different pathological origins, but similar physiological outcomes. Manuscripts reviewed were identified using specific search terms and exclusion criteria. We excluded manuscripts that investigated novel therapeutics and only included those manuscripts that describe the respiratory pathology. The ALS manuscripts describe pathology in respiratory physiology, the phrenic and hypoglossal motor units, respiratory neural control centers, and accessory respiratory muscles. The SCA rodent model manuscripts characterized pathology in overall respiratory function, phrenic motor units and hypoglossal motor neurons. Overall, a combination of pathology in the respiratory motor units and control centers contribute to devastating respiratory dysfunction.

The impact of Pompe disease on smooth muscle: a review.

Pompe disease (OMIM 232300) is an autosomal recessive disorder caused by mutations in the gene encoding acid α-glucosidase (GAA) (EC 3.2.1.20), the enzyme responsible for hydrolyzing lysosomal glycogen. The primary cellular pathology is lysosomal glycogen accumulation in cardiac muscle, skeletal muscle, and motor neurons, which ultimately results in cardiorespiratory failure. However, the severity of pathology and its impact on clinical outcomes are poorly described in smooth muscle. The advent of enzyme replacement therapy (ERT) in 2006 has improved clinical outcomes in infantile-onset Pompe disease patients. Although ERT increases patient life expectancy and ventilator free survival, it is not entirely curative. Persistent motor neuron pathology and weakness of respiratory muscles, including airway smooth muscles, contribute to the need for mechanical ventilation by some patients on ERT. Some patients on ERT continue to experience life-threatening pathology to vascular smooth muscle, such as aneurysms or dissections within the aorta and cerebral arteries. Better characterization of the disease impact on smooth muscle will inform treatment development and help anticipate later complications. This review summarizes the published knowledge of smooth muscle pathology associated with Pompe disease in animal models and in patients.