Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results in death from respiratory failure. No cure exists for this devastating disease, but therapy that directly targets the respiratory system has the potential to prolong survival and improve quality of life in some cases of ALS. The objective of this study was to enhance breathing and prolong survival by suppressing superoxide dismutase 1 (SOD1) expression in respiratory motor neurons using adeno-associated virus (AAV) expressing an artificial microRNA targeting the SOD1 gene. AAV-miR SOD1 was injected in the tongue and intrapleural space of SOD1 G93A mice, and repetitive respiratory and behavioral measurements were performed until the end stage. Robust silencing of SOD1 was observed in the diaphragm and tongue as well as systemically. Silencing of SOD1 prolonged survival by approximately 50 days, and it delayed weight loss and limb weakness in treated animals compared to untreated controls. Histologically, there was preservation of the neuromuscular junctions in the diaphragm as well as the number of axons in the phrenic and hypoglossal nerves. Although SOD1 suppression improved breathing and prolonged survival, it did not ameliorate the restrictive lung phenotype. Suppression of SOD1 expression in motor neurons that underlie respiratory function prolongs survival and enhances breathing until the end stage in SOD1 G93A ALS mice.

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.

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, spontaneous breathing at baseline in normoxia and during a hypercapnic/hypoxic respiratory challenge at 4 and 8 weeks, before and after 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 have 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 exhibit a reduction in number accompanied by increased microglial activation, indicating neurodegeneration and neuroinflammation. Furthermore, intranuclear ataxin-7 accumulation is 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.

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Spinocerebellar ataxia type 7 (SCA‐7) is a neurodegenerative polyglutamine disease within the family of spinocerebellar ataxias. Classically SCA7 is a disease of ataxia and vision loss. However, patients with SCA also have difficulty breathing, swallowing, coughing and maintaining a stable open airway which often result in fatal respiratory failure. While respiratory measures have been reported in other SCA mouse models, ventilation in a SCA‐7 mouse has not yet been studied. Using the SCA‐7 mouse model (Atxn7266Q/+) we sought to characterize the respiratory pathology of SCA‐7. Whole body plethysmography (WBP) was used to assess respiratory dysfunction in the Atxn7266Q/+ mouse both at baseline breathing (FiO2: 0.21; nitrogen balance) and during a hypercapnic and hypoxic challenge (Fi:CO2: 0.07, FiO2: 0.10; nitrogen balance). Neurobehavioral testing and strength was assessed using the inverted screen test and the grip strength test. Post mortem histological analysis was performed on the phrenic and hypoglossal motor neurons and nerves. At 4 weeks of age, the SCA‐7 mice are indistinguishable from their unaffected littermates. However, by 8 weeks of age, SCA‐7 mice perform poorly on both neurobehavioral and strength tests as compared to their WT littermates. 50% of the SCA‐7 mice died by 9 weeks, while littermate controls thrived. SCA‐7 mice have elevated respiratory rate at baseline, as well as reduced tidal volume, minute ventilation, peak inspiratory flow, peak expiratory flow, and inspiratory time during the respiratory challenge. These results indicate that the SCA‐7 mice have respiratory dysfunction. To understand the respiratory deficits, ongoing studies are histologically analyzing the diaphragm neuromuscular junction, phrenic and hypoglossal nerves, as well as phrenic and hypoglossal motor nuclei. Preliminary nerve analysis of the hypoglossal and phrenic nerves reveals significant axonal pathology. In conclusion, WBP analysis and preliminary histological nerve data reveals respiratory dysfunction in the Atxn7266Q/+ mouse model. Support or Funding Information Funding: R01 HD099486‐01; 1R21NS098131‐01

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder caused by a deleterious CAG repeat expansion in the coding region of the ataxin‐7 gene on chromosome 3. Patients with infantile SCA‐7 have the largest repeat expansion (>200 repeats) and the most severe disease characterized by progressive loss of coordination, dysarthria, dysphagia and retinal degeneration. Death in these infants results from severe hypotonia, aspiration pneumonia and respiratory failure. To understand the mechanism behind aspiration and respiratory failure in SCA7, we comprehensively examined hypoglossal (XII) and phrenic neuropathology of a mouse model of infantile SCA7: the SCA7‐266Q mice. Previously, we showed that SCA7‐266Q mice exhibit progressive respiratory dysfunction along with irregular breathing and prolonged apneic events by 9‐11 weeks of age. In this study, we performed post‐mortem histological analysis of the XII and phrenic motor neurons and nerves in SCA‐7 and WT control mice at 9 weeks of age. Specifically, we studied the presence of glial cells and the degree of neurodegeneration within these motor nuclei. We also performed g‐ratio analysis on semi‐thin sections of the XII and phrenic nerves. Finally, using electron microscopy, we examined the mitochondrial and myelin pathology in XII and phrenic nerves. The phrenic motor neurons exhibit enhanced neurodegeneration, exacerbated astrogliosis and microglial recruitment in the motor neuron pool. In addition, the XII and phrenic nerves of SCA7‐266Q mice have increased g‐ratio compared to WT controls which signifies thinning of myelin sheaths and is consistent with demyelination. Furthermore, EM images of the XII and phrenic nerves demonstrate an increase in size of mitochondria in SCA7‐266Q mice compared to their WT controls. Finally, preliminary qPCR data exhibited an anomaly in the expression of the neuromuscular junction markers in the diaphragm of the SCA7‐266Q mice. Clinically, these findings underscore the importance of respiratory neuropathology that is responsible for the dysphagia, aspiration pneumonia and respiratory failure seen in infantile SCA7.

Duchenne muscular dystrophy (DMD) is the most common X‐linked disease affecting 1 in 3500 male births. DMD is characterized by mutations in the DMD gene, which encodes the protein dystrophin that provides elasticity in skeletal muscle. Mutations in the DMD gene result in a lack of dystrophin which causes muscle fibers to degenerate and leads to inflammation, fibrosis, and muscle atrophy. Boys with DMD have progressive muscle weakness within the diaphragm that leads to respiratory failure in late adolescence/early adulthood. The most common DMD mouse model – the mdx mouse – does not have the same genetic defect as those in humans which makes gene editing impossible in this model. Therefore, a novel mouse model carrying the human gene was created which has the human exon 52 deletion in the dystrophin gene (hDMD/Δ52;mdx). Since respiratory failure is the major cause of morbidity in DMD and needs to be a target for future therapies, we sought to characterize the respiratory pathology in this novel DMD model. Whole body plethysmography (WBP) was used to assess respiration in normoxic air (FiO2: 0.21; nitrogen balance; baseline) and during a challenge with hypercapnic and hypoxic conditions (FiCO2: 0.07, FiO2: 0.10; nitrogen balance). Post mortem studies included immunohistochemistry of the diaphragm and tongue and diaphragm neuromuscular junction analysis. At baseline hDMD/Δ52;mdx mice are indistinguishable from either mdx or wildtype (WT) mice at 2, 6, and 12 months for most measures. However, during the challenge hDMD/Δ52;mdx mice have reduced frequency and minute ventilation by 6 months, which continues to decline at 12 months compared to WT mice. Starting at 6 months of age diaphragm neuromuscular junctions in the hDMD/Δ52;mdx and the mdx mice show similar pathology of decreased colocalization of pre‐ and post‐synaptic endplates. Myofiber atrophy, fibrosis, and regeneration are significant in the diaphragms, but only mild in the tongues of both models. In conclusion, the hDMD/Δ52;mdx exhibits moderate respiratory pathology, and serves as a relevant animal model to study the impact of novel gene editing therapies on respiratory function.

Amyotrophic Lateral Sclerosis (ALS) is a devastating and fatal neurodegenerative disease with no current cure. Patients with ALS die 3–5 years after diagnosis when they ultimately succumb to inadequate ventilation, hypoxia, and respiratory failure. Several genes associated with ALS have recently been discovered. One of these genes encodes optineurin (OPTN) which is associated with neurodegeneration in both ALS and glaucoma. Optineurin has multiple roles in various biochemical pathways such as regulation of inflammation and autophagy. Through these functions, OPTN appears to be neuroprotective but the exact mechanism by which loss of OPTN results in progressive neural degeneration and respiratory failure in ALS is still unclear.Since respiratory pathology is a significant cause of morbidity and mortality in ALS, this study sought to characterize the respiratory pathology in a novel ALS mouse model – the Optn−/− mouse. The hypothesis driving this work is that the Optn−/− mouse has respiratory insufficiency due to degeneration of the respiratory motor unit – motor neuron, nerve, neuromuscular junction and muscle. Whole body plethysmography (WBP) was used to assess breathing at baseline and during a respiratory challenge with hypercapnic and hypoxic conditions (FiCO2: 0.07, FiO2: 0.10; nitrogen balance). During the respiratory challenge, compared to the WT mice, Optn−/− mice had significantly lower peak inspiratory flow and peak expiratory flow. Peak inspiratory flow and peak expiratory flow are indirect measures of inspiratory and expiratory muscle strength. Furthermore, throughout the challenge period Optn−/− mice spent a greater amount of time in apnea indicating pathology in the respiratory control centers. The weakness and pathology in control of breathing during the respiratory challenge progresses as the mice age. Post mortem immunohistochemical studies of the neuromuscular junctions within the diaphragm showed that Optn−/− mice had fragmented synaptotagmin staining indicating presynaptic degeneration. Finally, the hypoglossal nerves of Optn−/− mice had significantly reduced g‐ratio which indicates pathology and decompaction of myelin sheaths. In conclusion, the Optn−/− ALS mouse model displays respiratory insufficiency, aberrant control of breathing, and increased nerve demyelination.Support or Funding InformationR21 NS098131‐02 (MKE) and K08HD077040‐07 (MKE)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.