Sibylle Jablonka

Smn, the spinal muscular atrophy–determining gene product, modulates axon growth and localization of β-actin mRNA in growth cones of motoneurons

Spinal muscular atrophy (SMA), a common autosomal recessive form of motoneuron disease in infants and young adults, is caused by mutations in the survival motoneuron 1 (SMN1) gene. The corresponding gene product is part of a multiprotein complex involved in the assembly of spliceosomal small nuclear ribonucleoprotein complexes. It is still not understood why reduced levels of the ubiquitously expressed SMN protein specifically cause motoneuron degeneration. Here, we show that motoneurons isolated from an SMA mouse model exhibit normal survival, but reduced axon growth. Overexpression of Smn or its binding partner, heterogeneous nuclear ribonucleoprotein (hnRNP) R, promotes neurite growth in differentiating PC12 cells. Reduced axon growth in Smn-deficient motoneurons correlates with reduced β-actin protein and mRNA staining in distal axons and growth cones. We also show that hnRNP R associates with the 3′ UTR of β-actin mRNA. Together, these data suggest that a complex of Smn with its binding partner hnRNP R interacts with β-actin mRNA and translocates to axons and growth cones of motoneurons.

Stiff person syndrome-associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition

Synaptic inhibition is a central factor in the fine tuning of neuronal activity in the central nervous system. Symptoms consistent with reduced inhibition such as stiffness, spasms and anxiety occur in paraneoplastic stiff person syndrome with autoantibodies against the intracellular synaptic protein amphiphysin. Here we show that intrathecal application of purified anti-amphiphysin immunoglobulin G antibodies induces stiff person syndrome-like symptoms in rats, including stiffness and muscle spasms. Using in vivo recordings of Hoffmann reflexes and dorsal root potentials, we identified reduced presynaptic GABAergic inhibition as an underlying mechanism. Anti-amphiphysin immunoglobulin G was internalized into neurons by an epitope-specific mechanism and colocalized in vivo with presynaptic vesicular proteins, as shown by stimulation emission depletion microscopy. Neurons from amphiphysin deficient mice that did not internalize the immunoglobulin provided additional evidence of the specificity in antibody uptake. GABAergic synapses appeared more vulnerable than glutamatergic synapses to defective endocytosis induced by anti-amphiphysin immunoglobulin G, as shown by increased clustering of the endocytic protein AP180 and by defective loading of FM 1-43, a styryl dye used to label cell membranes. Incubation of cultured neurons with anti-amphiphysin immunoglobulin G reduced basal and stimulated release of γ-aminobutyric acid substantially more than that of glutamate. By whole-cell patch-clamp analysis of GABAergic inhibitory transmission in hippocampus granule cells we showed a faster, activity-dependent decrease of the amplitude of evoked inhibitory postsynaptic currents in brain slices treated with antibodies against amphiphysin. We suggest that these findings may explain the pathophysiology of the core signs of stiff person syndrome at the molecular level and show that autoantibodies can alter the function of inhibitory synapses in vivo upon binding to an intraneuronal key protein by disturbing vesicular endocytosis.

A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy

5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA.

Defective Ca2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy

Proximal spinal muscular atrophy (SMA) is a motoneuron disease for which there is currently no effective treatment. In animal models of SMA, spinal motoneurons exhibit reduced axon elongation and growth cone size. These defects correlate with reduced β-actin messenger RNA and protein levels in distal axons. We show that survival motoneuron gene (Smn)–deficient motoneurons exhibit severe defects in clustering Cav2.2 channels in axonal growth cones. These defects also correlate with a reduced frequency of local Ca2+ transients. In contrast, global spontaneous excitability measured in cell bodies and proximal axons is not reduced. Stimulation of Smn production from the transgenic SMN2 gene by cyclic adenosine monophosphate restores Cav2.2 accumulation and excitability. This may lead to the development of new therapies for SMA that are not focused on enhancing motoneuron survival but instead investigate restoration of growth cone excitability and function.

Bag1 is essential for differentiation and survival of hematopoietic and neuronal cells

Bag1 is a cochaperone for the heat-shock protein Hsp70 that interacts with C-Raf, B-Raf, Akt, Bcl-2, steroid hormone receptors and other proteins. Here we use targeted gene disruption in mice to show that Bag1 has an essential role in the survival of differentiating neurons and hematopoietic cells. Cells of the fetal liver and developing nervous system in Bag1−/− mice underwent massive apoptosis. Lack of Bag1 did not disturb the primary function of Akt or Raf, as phosphorylation of the forkhead transcription factor FKHR and activation of extracellular signal–regulated kinase (Erk)-1/2 were not affected. However, the defect was associated with the disturbance of a tripartite complex formed by Akt, B-Raf and Bag1, in addition to the absence of Bad phosphorylation at Ser136. We also observed reduced expression of members of the inhibitor of apoptosis (IAP) family. Our data show that Bag1 is a physiological mediator of extracellular survival signals linked to the cellular mechanisms that prevent apoptosis in hematopoietic and neuronal progenitor cells.

Missense mutation in the tubulin-specific chaperone E (Tbce) gene in the mouse mutant progressive motor neuronopathy, a model of human motoneuron disease

Progressive motor neuronopathy (pmn) mutant mice have been widely used as a model for human motoneuron disease. Mice that are homozygous for the pmn gene defect appear healthy at birth but develop progressive motoneuron disease, resulting in severe skeletal muscle weakness and respiratory failure by postnatal week 3. The disease starts at the motor endplates, and then leads to axonal loss and finally to apoptosis of the corresponding cell bodies. We localized the genetic defect in pmn mice to a missense mutation in the tubulin-specific chaperone E (Tbce) gene on mouse chromosome 13. The human orthologue maps to chromosome 1q42.3. The Tbce gene encodes a protein (cofactor E) that is essential for the formation of primary α-tubulin and β-tubulin heterodimeric complexes. Isolated motoneurons from pmn mutant mice exhibit shorter axons and axonal swelling with irregularly structured β-tubulin and tau immunoreactivity. Thus, the pmn gene mutation provides the first genetic evidence that alterations in tubulin assembly lead to retrograde degeneration of motor axons, ultimately resulting in motoneuron cell death.

Adenosine receptor A2A-R contributes to motoneuron survival by transactivating the tyrosine kinase receptor TrkB

Neurotrophins are potent survival factors for developing and injured neurons. However, they are not being used to treat neurodegenerative diseases because of difficulties in administration and numerous side effects that have been encountered in previous clinical trials. Their biological activities use Trk (tropomyosin-related kinase) transmembrane tyrosine kinases. Therefore, one alternative approach is to use transactivation pathways such as adenosine 2A receptor agonists, which can activate Trk receptor signaling independent of neurotrophin binding. However, the relevance in vivo and applicability of these transactivation events during neurodegenerative and injury conditions have never been extensively studied. Here we demonstrate that motoneuron survival after facial nerve lesioning is significantly enhanced by transactivation of Trk receptor tyrosine kinases by adenosine agonists. Moreover, survival of motoneurons directly required the activation of the BDNF receptor TrkB and an increase in Akt (AKT8 virus oncogene cellular homolog) activity. The ability of small molecules to activate a trophic response by using Trk signaling provides a unique mechanism to promote survival signals in motoneurons and suggests new strategies for using transactivation in neurodegenerative diseases.

The spinal muscular atrophy disease protein SMN is linked to the rho-kinase pathway via profilin

Spinal muscular atrophy (SMA), a frequent neurodegenerative disease, is caused by reduced levels of functional survival of motoneuron (SMN) protein. SMN is involved in multiple pathways, including RNA metabolism and splicing as well as motoneuron development and function. Here we provide evidence for a major contribution of the Rho-kinase (ROCK) pathway in SMA pathogenesis. Using an in vivo protein interaction system based on SUMOylation of proteins, we found that SMN is directly interacting with profilin2a. Profilin2a binds to a stretch of proline residues in SMN, which is heavily impaired by a novel SMN2 missense mutation (S230L) derived from a SMA patient. In different SMA models, we identified differential phosphorylation of the ROCK-downstream targets cofilin, myosin-light chain phosphatase and profilin2a. We suggest that hyper-phosphorylation of profilin2a is the molecular link between SMN and the ROCK pathway repressing neurite outgrowth in neuronal cells. Finally, we found a neuron-specific increase in the F-/G-actin ratio that further support the role of actin dynamics in SMA pathogenesis.

Isolation and enrichment of embryonic mouse motoneurons from the lumbar spinal cord of individual mouse embryos

Cultured spinal motoneurons are a valuable tool for studying the basic mechanisms of axon and dendrite growth and also for analyses of pathomechanisms underlying diseases like amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). As motoneurons in the developing spinal cord of mice constitute only a minor population of neurons, these cells need to be enriched in order to study them in the absence of contaminating neuronal and non-neuronal cells. Here, we describe a protocol for the isolation and in vitro cultivation of embryonic primary motoneurons from individual mouse embryos. Tissue dissection, cell isolation and a p75NTR-antibody-based panning technique, which highly enriches motoneurons within <8 h are described. This protocol is aimed to provide an alternative to the established FACS-based protocols describing p75NTR-based enrichments of neurons. This protocol will help in facilitating the research on molecular mechanisms underlying motoneuron development, survival and disease mechanisms.

Neuromuscular defects and breathing disorders in a new mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron (SMN) protein leading to muscle paralysis and respiratory failure. In mouse, introducing the human SMN2 gene partially rescues Smn(-)(/)(-) embryonic lethality. However current models were either too severe or nearly unaffected precluding convenient drug testing for SMA. We report here new SMN2;Smn(-/-) lines carrying one to four copies of the human SMN2 gene. Mice carrying three SMN2 copies exhibited an intermediate phenotype with delayed appearance of motor defects and developmental breathing disorders reminiscent of those found in severe SMA patients. Although normal at birth, at 7 days of age respiratory rate was decreased and apnea frequency was increased in SMA mice in parallel with the appearance of neuromuscular junction defects in the diaphragm. With median survival of 15 days and postnatal onset of neurodegeneration, these mice could be an important tool for evaluating new therapeutics.