Adrian G. Todd

SMN, Gemin2 and Gemin3 Associate with β-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. Importantly, we have provided the first direct evidence that beta-actin mRNA is present in SMN cytoplasmic complexes in SHSY5Y cells.

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). We demonstrate that COPa co-immunoprecipitates a specific set of RNAs that are enriched in G-quadruplex motifs and fragile X mental retardation protein-associated RNAs and that encode factors that predominantly localize to the plasma membrane and cytoskeleton and function within signaling pathways. These data support the novel function of COPI in inter-compartmental trafficking of RNA.

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. It is known that SMN is present in transport ribonucleoproteins (RNPs) granules that also contain Gemin2 and Gemin3. To further characterise these granules we have performed live cell imaging of GFP-tagged SMN, GFP-Gemin2, GFP-Gemin3, GFP-Gemin6 and GFP-Gemin7. In all, we have made two important observations: (1) SMN granules appear metamorphic; and (2) the SMN-Gemin complex(es) appears to localise to two distinct subsets of bodies in neurites; stationary bodies and smaller dynamic bodies. This study provides an insight into the neuronal function of the SMN complex.

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. Here, we demonstrate significant pathological alterations at neuromuscular junctions (NMJs) of the diaphragm and tibialis anterior muscle as prominent features of disease pathology in Gaa knockout mice. Postsynaptic defects including increased motor endplate area and fragmentation were readily observed in Gaa(-/-) but not wild-type mice. Presynaptic neuropathic changes were also evident, as demonstrated by significant reduction in the levels of neurofilament proteins, and alterations in axonal fiber diameter and myelin thickness within the sciatic and phrenic nerves. Our data suggest the loss of NMJ integrity is a primary contributor to the decline in respiratory and ambulatory function in Pompe and arises from both pre- and postsynaptic pathology. These observations highlight the importance of systemic phenotype correction, specifically restoration of GAA to skeletal muscle and the nervous system for treatment of Pompe disease.

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. Here we asked whether rapamycin administration by food supplementation, or intraperitoneal injection, could alleviate the neuropathic phenotype of affected mice and improve neuromuscular performance. Cohorts of male and female wild type (Wt) and TrJ mice were assigned to placebo or rapamycin treatment starting at 2 or 4months of age and tested monthly on the rotarod. While neither long-term feeding (8 or 10months) on rapamycin-enriched diet, or short-term injection (2months) of rapamycin improved locomotor performance of the neuropathic mice, both regimen benefited peripheral nerve myelination. Together, these results indicate that while treatment with rapamycin benefits the myelination capacity of neuropathic Schwann cells, this intervention does not improve neuromuscular function. The observed outcome might be the result of the differential response of nerve and skeletal muscle tissue to rapamycin.

Correcting Neuromuscular Deficits With Gene Therapy in Pompe Disease.

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.

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)). Assessment of cardiac function and morphology was measured 1 and 3 months after initiation of treatment while whole-body plethysmography and diaphragmatic contractile function was evaluated at 3 months post-treatment in all groups. Gaa–/– animals receiving either AAV9-DES or ERT demonstrated a significant improvement in cardiac function and diaphragmatic contractile function as compared to control animals. AAV9-DES treatment resulted in a significant reduction in cardiac dimension (end diastolic left ventricular mass/gram wet weight; EDMc) at 3 months postinjection. Neither AAV nor ERT therapy altered minute ventilation during quiet breathing (eupnea). However, breathing frequency and expiratory time were significantly improved in AAV9-DES animals. These results indicate systemic delivery of either strategy improves cardiac function but AAV9-DES alone improves respiratory parameters at 3 months post-treatment in a murine model of Pompe disease.

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. Both enzyme replacement therapy (ERT) and gene therapy can elicit anti-GAA immune reactions that dampen their effectiveness and pose life-threatening risks to patient safety. To modulate the immune responses related to gene therapy, we show that a human codon-optimized GAA (coGAA) driven by a liver-specific promoter (LSP) using AAV9 is capable of promoting immune tolerance in a Gaa(-/-) mouse model. Copackaging AAV9-LSP-coGAA with the tissue-restricted desmin promoter (AAV9-DES-coGAA) demonstrates the necessary cell autonomous expression in cardiac muscle, skeletal muscle, peripheral nerve, and the spinal cord. Simultaneous high-level expression in liver led to the expansion of GAA-specific regulatory T-cells (Tregs) and induction of immune tolerance. Transfer of Tregs into naïve recipients prevented pathogenic allergic reactions after repeated ERT challenges. Copackaged AAV9 also attenuated preexisting humoral and cellular immune responses, which enhanced the biochemical correction. Our data present a therapeutic design in which simultaneous administration of two copackaged AAV constructs may provide therapeutic benefit and resolve immune reactions in the treatment of multisystem disorders.

Identification of a self-association domain in the Ewing’s sarcoma protein: a novel function for arginine-glycine-glycine rich motifs?

The Ewings sarcoma (EWS) protein is a ubiquitously expressed RNA chaperone. The EWS protein localizes predominantly to the nucleus. Previous reports have suggested that the EWS protein is capable of dimerizing. However, to date this has not been confirmed. Here, using a novel panel of recombinant proteins, we have performed an in vitro biomolecular interaction analysis of the EWS protein. We have demonstrated that all three arginine-glycine-glycine (RGG) motifs are capable of binding directly to the survival motor neuron protein, a Tudor domain containing EWS binding partner. We have also confirmed EWS is capable of self-associating, and we have mapped this binding domain to the RGG motifs. We have also found that self-association may be required for EWS nuclear import. This is the first direct evidence of RGG domains being involved in self-association and has implications on all RGG-containing proteins.

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. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.