Neonatal systemic gene therapy restores cardiorespiratory function in a rat model of Pompe disease.

Absence of functional acid-α-glucosidase (GAA) leads to early-onset Pompe disease with cardiorespiratory and neuromuscular failure. A novel Pompe rat model ( ) was used to test the hypothesis that neonatal gene therapy with adeno-associated virus serotype 9 (AAV9) restores cardiorespiratory neuromuscular function across the lifespan. Temporal vein administration of AAV9-DES-GAA or sham (saline) injection was done on post-natal day 1; rats were studied at 6-12 months old.

HSP90 Inhibitor, NVP-AUY922, Improves Myelination in Vitro and Supports the Maintenance of Myelinated Axons in Neuropathic Mice.

Hereditary demyelinating neuropathies linked to peripheral myelin protein 22 (PMP22) involve the disruption of normal protein trafficking and are therefore relevant targets for chaperone therapy. Using a small molecule HSP90 inhibitor, EC137, in cell culture models, we previously validated the chaperone pathway as a viable target for therapy development. Here, we tested five commercially available inhibitors of HSP90 and identified BIIB021 and AUY922 to support Schwann cell viability and enhance chaperone expression.

Locomotor and skeletal muscle abnormalities in trembler J neuropathic mice.

Introduction: Patients with hereditary peripheral neuropathies exhibit characteristic deformities of the hands and feet and have difficulty ambulating. To examine to what extent neuropathic animals recapitulate these deficits, we studied trembler J (TrJ) mice, which model early-onset demyelinating neuropathy.

Methods: A cohort of 4-month-old female wild type and neuropathic mice were evaluated for locomotor measurements, neuromuscular function, and skeletal muscle proteolysis and morphometry.

C/P MRS Metabolic Biomarkers of Disease Progression and Response to AAV Delivery of hGAA in a Mouse Model of Pompe Disease.

The development of therapeutic clinical trials for glycogen storage disorders, including Pompe disease, has called for non-invasive and objective biomarkers. Glycogen accumulation can be measured in vivo with C MRS. However, clinical implementation remains challenging due to low signal-to-noise.

Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe Disease.

Pompe disease is a progressive neuromuscular disorder caused by lysosomal accumulation of glycogen from a deficiency in acid alpha-glucosidase (GAA). Replacement of the missing enzyme is available by repeated protein infusions; however, efficacy is limited by immune response and inability to restore enzymatic function in the central nervous system. An alternative therapeutic option is adeno-associated virus (AAV)-mediated gene therapy, which results in widespread gene transfer and prolonged transgene expression.

Comparative impact of AAV and enzyme replacement therapy on respiratory and cardiac function in adult Pompe mice.

Pompe disease is an autosomal recessive genetic disorder characterized by a deficiency of the enzyme responsible for degradation of lysosomal glycogen (acid α-glucosidase (GAA)). Cardiac dysfunction and respiratory muscle weakness are primary features of this disorder. To attenuate the progressive and rapid accumulation of glycogen resulting in cardiorespiratory dysfunction, adult Gaa (-/-) mice were administered a single systemic injection of rAAV2/9-DES-hGAA (AAV9-DES) or bimonthly injections of recombinant human GAA (enzyme replacement therapy (ERT)).

Correcting Neuromuscular Deficits With Gene Therapy in Pompe Disease.

Objective: We have recently reported on the pathology of the neuromuscular junction (NMJ) in Pompe disease, reflecting disruption of neuronal and muscle homeostasis as a result of glycogen accumulation. The aim of this study was to examine how the alteration of NMJ physiology contributes to Pompe disease pathology; we performed molecular, physiological, and histochemical analyses of NMJ-related measures of the tibialis anterior muscles of young-, mid-, and late-stage alpha-glucosidase (GAA)-deficient mice.

Methods: We performed intramuscular injection of an adeno-associated virus (AAV)9 vector expressing GAA (AAV9-hGAA) into the tibialis anterior muscle of Gaa(-/-) mice at early, mid, and severe pathological time points.

Peripheral nerve and neuromuscular junction pathology in Pompe disease.

Pompe disease is a systemic metabolic disorder characterized by lack of acid-alpha glucosidase (GAA) resulting in ubiquitous lysosomal glycogen accumulation. Respiratory and ambulatory dysfunction are prominent features in patients with Pompe yet the mechanism defining the development of muscle weakness is currently unclear. Transgenic animal models of Pompe disease mirroring the patient phenotype have been invaluable in mechanistic and therapeutic study.

Rapamycin improves peripheral nerve myelination while it fails to benefit neuromuscular performance in neuropathic mice.

Charcot--Marie-Tooth disease type 1A (CMT1A) is a hereditary peripheral neuropathy characterized by progressive demyelination and distal muscle weakness. Abnormal expression of peripheral myelin protein 22 (PMP22) has been linked to CMT1A and is modeled by Trembler J (TrJ) mice, which carry the same leucine to proline substitution in PMP22 as affected pedigrees. Pharmacologic modulation of autophagy by rapamycin in neuron-Schwann cell explant cultures from neuropathic mice reduced PMP22 aggregate formation and improved myelination.

Dilysine motifs in exon 2b of SMN protein mediate binding to the COPI vesicle protein α-COP and neurite outgrowth in a cell culture model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder that stems from low levels of survival of motor neuron (SMN) protein. The processes that cause motor neurons and muscle cells to become dysfunctional are incompletely understood. We are interested in neuromuscular homeostasis and the stresses put upon that system by loss of SMN.

COPI transport complexes bind to specific RNAs in neuronal cells.

Our fundamental understanding of how several thousand diverse RNAs are recognized in the soma, sorted, packaged, transported and localized within the cell is fragmentary. The COPa and COPb proteins of the coatomer protein I (COPI) vesicle complex were reported to interact with specific RNAs and represent a candidate RNA sorting and transport system. To determine the RNA-binding profile of Golgi-derived COPI in neuronal cells, we performed formaldehyde-linked RNA immunoprecipitation, followed by high-throughput sequencing, a process we term FLRIP-Seq (FLRIP, formaldehyde-cross-linked immunoprecipitation).

Using mini-genes to identify factors that modulate alternative splicing.

Many genetic mutations result in the disruption of (alternative) splicing. Prime examples are the SMN1 and SMN2 genes: a silent mutation in SMN2 leads to the skipping of the constitutive exon 7 in the majority of SMN2 transcripts, while this exon is generally included in SMN1 transcripts. Lack of SMN is embryonic lethal and loss of SMN1 genes leads to a severe decrease in SMN protein and is associated with spinal muscular atrophy.

Mutations in the survival motor neuron (SMN) protein alter the dynamic nature of nuclear bodies.

The childhood disorder spinal muscular atrophy (SMA) is caused by reduced expression of the survival motor neuron (SMN) protein. SMN is a multifunctional protein that has been implicated in the production, processing and transport of RNA and ribonucleoproteins (RNPs). Within the nucleus, SMN is predominantly targeted to Cajal bodies (CB), which are involved in the maturation and processing of several subclasses of RNPs.

SMN, Gemin2 and Gemin3 associate with beta-actin mRNA in the cytoplasm of neuronal cells in vitro.

Childhood spinal muscular atrophy is caused by a reduced expression of the survival motor neuron (SMN) protein. SMN has been implicated in the axonal transport of beta-actin mRNA in both primary and transformed neuronal cell lines, and loss of this function could account, at least in part, for spinal muscular atrophy onset and pathological specificity. Here we have utilised a targeted screen to identify mRNA associated with SMN, Gemin2 and Gemin3 in the cytoplasm of a human neuroblastoma cell line, SHSY5Y.

Analysis of SMN-neurite granules: Core Cajal body components are absent from SMN-cytoplasmic complexes.

Childhood spinal muscular atrophy (SMA) is caused by a reduction in survival motor neuron (SMN) protein. SMN is a ubiquitously expressed house keeping protein that is involved in RNA production and processing. However, although SMN is expressed in every cell type, only the lower motor neurons of the spinal cord are degraded in SMA.

SMN and the Gemin proteins form sub-complexes that localise to both stationary and dynamic neurite granules.

Childhood spinal muscular atrophy (SMA) is caused by a reduction in survival motor neuron (SMN) protein. SMN is expressed in every cell type, but it is predominantly the lower motor neurones of the spinal cord that degenerate in SMA. SMN has been linked to the axonal transport of beta-actin mRNA, a breakdown in which could trigger disease onset.

Identification of a tripartite import signal in the Ewing Sarcoma protein (EWS).

The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals.

Identification and characterisation of a nuclear localisation signal in the SMN associated protein, Gemin4.

Gemin4 is a ubiquitously expressed multifunctional protein that is involved in U snRNP assembly, apoptosis, nuclear/cytoplasmic transportation, transcription, and RNAi pathways. Gemin4 is one of the core components of the Gemin-complex, which also contains survival motor neuron (SMN), the seven Gemin proteins (Gemin2-8), and Unrip. Mutations in the SMN1 gene cause the autosomal recessive disorder spinal muscular atrophy (SMA).

Targeting of SMN to Cajal bodies is mediated by self-association.

The childhood autosomal recessive disorder spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron (SMN) gene. SMN localizes diffusely in the cytoplasm and in distinct nuclear structures called Cajal bodies. Cajal bodies are believed to be the storage and processing sites of several ribonucleoproteins.