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.

Adeno-Associated Virus-Mediated Gene Therapy in the Mashlool, Atp1a3Mashl/+, Mouse Model of Alternating Hemiplegia of Childhood.

Alternating Hemiplegia of Childhood (AHC) is a devastating autosomal dominant disorder caused by ATP1A3 mutations, resulting in severe hemiplegia and dystonia spells, ataxia, debilitating disabilities, and premature death. Here, we determine the effects of delivering an extra copy of the normal gene in a mouse model carrying the most common mutation causing AHC in humans, the D801N mutation. We used an adeno-associated virus serotype 9 (AAV9) vector expressing the human ATP1A3 gene under the control of a human Synapsin promoter. We first demonstrated that intracerebroventricular (ICV) injection of this vector in wild-type mice on postnatal day 10 (P10) results in increases in ouabain-sensitive ATPase activity and in expression of reporter genes in targeted brain regions. We then tested this vector in mutant mice. Simultaneous intracisterna magna and bilateral ICV injections of this vector at P10 resulted, at P40, in reduction of inducible hemiplegia spells, improvement in balance beam test performance, and prolonged survival of treated mutant mice up to P70. Our study demonstrates, as a proof of concept, that gene therapy can induce favorable effects in a disease caused by a mutation of the gene of a protein that is, at the same time, an ATPase enzyme, a pump, and a signal transduction factor.

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.

Intralingual Administration of AAVrh10-miRSOD1 Improves Respiratory But Not Swallowing Function in a Superoxide Dismutase-1 Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by degeneration of motor neurons and muscles, and death is usually a result of impaired respiratory function due to loss of motor neurons that control upper airway muscles and/or the diaphragm. Currently, no cure for ALS exists and treatments to date do not significantly improve respiratory or swallowing function. One cause of ALS is a mutation in the superoxide dismutase-1 (SOD1) gene; thus, reducing expression of the mutated gene may slow the progression of the disease. Our group has been studying the SOD1G93A transgenic mouse model of ALS that develops progressive respiratory deficits and dysphagia. We hypothesize that solely treating the tongue in SOD1 mice will preserve respiratory and swallowing function, and it will prolong survival. At 6 weeks of age, 11 SOD1G93A mice (both sexes) received a single intralingual injection of gene therapy (AAVrh10-miRSOD1). Another 29 mice (both sexes) were divided into two control groups: (1) 12 SOD1G93A mice that received a single intralingual vehicle injection (saline); and (2) 17 non-transgenic littermates. Starting at 13 weeks of age, plethysmography (respiratory parameters) at baseline and in response to hypoxia (11% O2) + hypercapnia (7% CO2) were recorded and videofluoroscopic swallow study testing were performed twice monthly until end-stage disease. Minute ventilation during hypoxia + hypercapnia and mean inspiratory flow at baseline were significantly reduced (p < 0.05) in vehicle-injected, but not AAVrh10-miRSOD1-injected SOD1G93A mice as compared with wild-type mice. In contrast, swallowing function was unchanged by AAVrh10-miRSOD1 treatment (p > 0.05). AAVrh10-miRSOD1 injections also significantly extended survival in females by ∼1 week. In conclusion, this study indicates that intralingual AAVrh10-miRSOD1 treatment preserved respiratory (but not swallowing) function potentially via increasing upper airway patency, and it is worthy of further exploration as a possible therapy to preserve respiratory capacity in ALS patients.

The Respiratory Phenotype of Pompe Disease Mouse Models.

Pompe disease is a glycogen storage disease caused by a deficiency in acid α-glucosidase (GAA), a hydrolase necessary for the degradation of lysosomal glycogen. This deficiency in GAA results in muscle and neuronal glycogen accumulation, which causes respiratory insufficiency. Pompe disease mouse models provide a means of assessing respiratory pathology and are important for pre-clinical studies of novel therapies that aim to treat respiratory dysfunction and improve quality of life. This review aims to compile and summarize existing manuscripts that characterize the respiratory phenotype of Pompe mouse models. Manuscripts included in this review were selected utilizing specific search terms and exclusion criteria. Analysis of these findings demonstrate that Pompe disease mouse models have respiratory physiological defects as well as pathologies in the diaphragm, tongue, higher-order respiratory control centers, phrenic and hypoglossal motor nuclei, phrenic and hypoglossal nerves, neuromuscular junctions, and airway smooth muscle. Overall, the culmination of these pathologies contributes to severe respiratory dysfunction, underscoring the importance of characterizing the respiratory phenotype while developing effective therapies for patients.

Comparisons of Infant and Adult Mice Reveal Age Effects for Liver Depot Gene Therapy in Pompe Disease.

Pompe disease is caused by the deficiency of lysosomal acid α-glucosidase (GAA). It is expected that gene therapy to replace GAA with adeno-associated virus (AAV) vectors will be less effective early in life because of the rapid loss of vector genomes. AAV2/8-LSPhGAA (3 × 1010 vector genomes [vg]/mouse) was administered to infant (2-week-old) or adult (2-month-old) GAA knockout mice. AAV vector transduction in adult mice significantly corrected GAA deficiency in the heart (p < 0.0001), diaphragm (p < 0.01), and quadriceps (p < 0.001) for >50 weeks. However, in infant mice, the same treatment only partially corrected GAA deficiency in the heart (p < 0.05), diaphragm (p < 0.05), and quadriceps (p < 0.05). The clearance of glycogen was much more efficient in adult mice compared with infant mice. Improved wire hang test latency was observed for treated adults (p < 0.05), but not for infant mice. Abnormal ventilation was corrected in both infant and adult mice. Vector-treated female mice demonstrated functional improvement, despite a lower degree of biochemical correction compared with male mice. The relative vector dose for infants was approximately 3-fold higher than adults, when normalized to body weight at the time of vector administration. Given these data, the dose requirement to achieve similar efficacy will be higher for the treatment of young patients.

Pompe disease gene therapy: neural manifestations require consideration of CNS directed therapy.

Pompe disease is a neuromuscular disease caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase leading to lysosomal and cytoplasmic glycogen accumulation in neurons and striated muscle. In the decade since availability of first-generation enzyme replacement therapy (ERT) a better understanding of the clinical spectrum of disease has emerged. The most severe form of early onset disease is typically identified with symptoms in the first year of life, known as infantile-onset Pompe disease (IOPD). Infants are described at floppy babies with cardiac hypertrophy in the first few months of life. A milder form with late onset (LOPD) of symptoms is mostly free of cardiac involvement with slower rate of progression. Glycogen accumulation in the CNS and skeletal muscle is observed in both IOPD and LOPD. In both circumstances, multi-system disease (principally motoneuron and myopathy) leads to progressive weakness with associated respiratory and feeding difficulty. In IOPD the untreated natural history leads to cardiorespiratory failure and death in the first year of life. In the current era of ERT clinical outcomes are improved, yet, many patients have an incomplete response and a substantial unmet need remains. Since the neurological manifestations of the disease are not amenable to peripheral enzyme replacement, we set out to better understand the pathophysiology and potential for treatment of disease manifestations using adeno-associated virus (AAV)-mediated gene transfer, with the first clinical gene therapy studies initiated by our group in 2006. This review focuses on the preclinical studies and clinical study findings which are pertinent to the development of a comprehensive gene therapy strategy for both IOPD and LOPD. Given the advent of newborn screening, a significant focus of our recent work has been to establish the basis for repeat administration of AAV vectors to enhance neuromuscular therapeutic efficacy over the life span.

Reduction of Autophagic Accumulation in Pompe Disease Mouse Model Following Gene Therapy.

BACKGROUND: Pompe disease is a fatal neuromuscular disorder caused by a deficiency in acid α-glucosidase, an enzyme responsible for glycogen degradation in the lysosome. Currently, the only approved treatment for Pompe disease is enzyme replacement therapy (ERT), which increases patient survival, but does not fully correct the skeletal muscle pathology. Skeletal muscle pathology is not corrected with ERT because low cation-independent mannose-6-phosphate receptor abundance and autophagic accumulation inhibits the enzyme from reaching the lysosome. Thus, a therapy that more efficiently targets skeletal muscle pathology, such as adeno-associated virus (AAV), is needed for Pompe disease. OBJECTIVE: The goal of this project was to deliver a rAAV9-coGAA vector driven by a tissue restrictive promoter will efficiently transduce skeletal muscle and correct autophagic accumulation. METHODS: Thus, rAAV9-coGAA was intravenously delivered at three doses to 12-week old Gaa-/- mice. 1 month after injection, skeletal muscles were biochemically and histologically analyzed for autophagy-related markers. RESULTS: At the highest dose, GAA enzyme activity and vacuolization scores achieved therapeutic levels. In addition, resolution of autophagosome (AP) accumulation was seen by immunofluorescence and western blot analysis of autophagy-related proteins. Finally, mice treated at birth demonstrated persistence of GAA expression and resolution of lysosomes and APs compared to those treated at 3 months. CONCLUSION: In conclusion, a single systemic injection of rAAV9-coGAA ameliorates vacuolar accumulation and prevents autophagic dysregulation.

Macroglossia, Motor Neuron Pathology, and Airway Malacia Contribute to Respiratory Insufficiency in Pompe Disease: A Commentary on Molecular Pathways and Respiratory Involvement in Lysosomal Storage Diseases.

The authors of the recently published, "Molecular Pathways and Respiratory Involvement in Lysosomal Storage Diseases", provide an important review of the various mechanisms of lysosomal storage diseases (LSD) and how they culminate in similar clinical pathologies [...].

Systemic Delivery of AAVB1-GAA Clears Glycogen and Prolongs Survival in a Mouse Model of Pompe Disease.

Pompe disease is an autosomal recessive glycogen storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption in muscle and the central nervous system (CNS). Adeno-associated virus (AAV) gene therapy is ideal for Pompe disease, since a single systemic injection may correct both muscle and CNS pathologies. Using the Pompe mouse (B6;129-GaaTm1Rabn/J), this study sought to explore if AAVB1, a newly engineered vector with a high affinity for muscle and CNS, reduces systemic weakness and improves survival in adult mice. Three-month-old Gaa-/- animals were injected with either AAVB1 or AAV9 vectors expressing GAA and tissues were harvested 6 months later. Both AAV vectors prolonged survival. AAVB1-treated animals had a robust weight gain compared to the AAV9-treated group. Vector genome levels, GAA enzyme activity, and histological analysis indicated that both vectors transduced the heart efficiently, leading to glycogen clearance, and transduced the diaphragm and CNS at comparable levels. AAVB1-treated mice had higher GAA activity and greater glycogen clearance in the tongue. Finally, AAVB1-treated animals showed improved respiratory function comparable to wild-type animals. In conclusion, AAVB1-GAA offers a promising therapeutic option for the treatment of muscle and CNS in Pompe 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.

Training Community Members in Public Health Research: Development and Implementation of a Community Participatory Research Pilot Project.

Purpose: Community-based training in public health research can build capacity for community-based participatory research (CBPR) and foster health partnerships between academics and stakeholders. We describe a community-academic partnership developed from a 15-week program, the Community Research Fellows Training (CRFT), designed to increase research literacy and facilitate equitable relationships in community/researcher collaborations and partnerships. The article provides a description of a community and faculty collaboration to conduct a participatory pilot research project that followed program completion. Methods: Four CRFT program alumni formed a community research team and selected a faculty mentor. After a request for proposal release, the team developed a pilot research proposal that addressed a concern for mental health among women experiencing economic stress. After completion of the pilot research, the community researchers elected to participate in two dissemination efforts, including a manuscript reflecting on their research experience. Team successes, challenges, and recommendations for future training are discussed. Results: Each member of the CRFT pilot research team reflects on how training prepared community members to conduct CBPR research through development and implementation of a pilot research project. Community researchers gained experience in grant proposal development, choosing appropriate health interventions, conducting in-person surveys and telephone interviews, and disseminating study findings. Conclusions: Providing training in public health research before community/researcher collaboration can increase community capacity to engage in research as equitable partners in research question development, study design, and data interpretation and dissemination. The project success suggests that this and similar programs maximize the potential of community-academic health partnerships to address health disparities.

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.

Predictors of performance on the National Board of Chiropractic Examiners Parts I and II.

Objective : The purpose of this study was to examine predictors for success on Parts I and II of the National Board of Chiropractic Examiners (NBCE) written examinations. Methods : Two validity studies were conducted to examine the criterion validity of Logan College assessments for Part I and II NBCE scores. Both studies consisted of a longitudinal design to examine the validity of entrance grade point average (GPA), in-program chiropractic course content GPA, and an institutional practice exam on Parts I and II of the NBCE. Results : Analyses revealed that Part I GPA and practice exam scores combined accounted for 72% of the variance within Part I NBCE scores. Furthermore, every subtest of the Part I NBCE could be reliably predicted by course performance. In the 2nd study, Part I GPA, Part I NBCE score, and Part II GPA accounted for 75% of the variance within Part II NBCE scores. Conclusions : Internal training and educational assessments (eg, course grades and practice exams) proved to be strong determinants of NBCE performance above and beyond initial levels of preparedness, thus validating the impact of the chiropractic curriculum on NBCE test achievement.

Adeno-associated virus-mediated gene therapy for metabolic myopathy.

Metabolic myopathies are a diverse group of rare diseases in which impaired breakdown of stored energy leads to profound muscle dysfunction ranging from exercise intolerance to severe muscle wasting. Metabolic myopathies are largely caused by functional deficiency of a single gene and are generally subcategorized into three major types of metabolic disease: mitochondrial, lipid, or glycogen. Treatment varies greatly depending on the biochemical nature of the disease, and unfortunately no definitive treatments exist for metabolic myopathy. Since this group of diseases is inherited, gene therapy is being explored as an approach to personalized medical treatment. Adeno-associated virus-based vectors in particular have shown to be promising in the treatment of several forms of metabolic myopathy. This review will discuss the most recent advances in gene therapy efforts for the treatment of metabolic myopathies.

Idiopathic, isolated fovea plana with bilateral off-centre multifocal ERGs.

BACKGROUND: The purpose of this paper is to report a case of idiopathic isolated fovea plana showing asymmetry in the multifocal electroretinogram (mfERG). METHODS: We carried out optical coherence tomography (OCT) imaging, macular pigment density measurement, genetic testing and electrophysiological testing with visual evoked potentials and mfERGs on a young, highly myopic female of Pakistani origin, who had good visual acuity and no nystagmus. RESULTS: OCT imaging revealed a complete absence of any foveal pit in either eye. Macular pigment density was normal and visual evoked potentials indicated normal chiasmal crossings, excluding albinism. Genetic testing revealed normal PAX6 coding data, excluding aniridia as a cause. mfERGs showed asymmetry consistent with off-centre fixation to the temporal side of the fovea in both eyes, but were otherwise normal. CONCLUSION: Lack of a foveal pit is a well-known finding in conditions such as oculocutaneous albinism and PAX6 gene-related aniridia. Isolated fovea plana is less common, and this case illustrates that the absence of a foveal pit does not necessarily result in a poor visual outcome. The finding of asymmetry in the mfERG in such a case is novel, and may indicate a functional adaptation to the structure of the fovea.