93rd ENMC international workshop: non-5q-spinal muscular atrophies (SMA) – clinical picture (6–8 April 2001, Naarden, The Netherlands)

Spinal Muscular Atrophy: A Deficiency in a Ubiquitous Protein; a Motor Neuron-Specific Disease

AAV9-Mediated Expression of SMN Restricted to Neurons Does Not Rescue the Spinal Muscular Atrophy Phenotype in Mice

The survival of motor neuron (SMN) protein, responsible for the neurodegenerative disease spinal muscular atrophy (SMA), oligomerizes and forms a stable complex with seven other major components, the Gemin proteins. Besides the SMN protein, Gemin2 is a core protein that is essential for the formation of the SMN complex, although the mechanism by which it drives formation is unclear. We have found a novel interaction, a Gemin2 self-association, using the mammalian two-hybrid system and the in vitro pull-down assays. Using in vitro dissociation assays, we also found that the self-interaction of the amino-terminal SMN protein, which was confirmed in this study, became stable in the presence of Gemin2. In addition, Gemin2 knockdown using small interference RNA treatment revealed a drastic decrease in SMN oligomer formation and in the assembly activity of spliceosomal small nuclear ribonucleoprotein (snRNP). Taken together, these results indicate that Gemin2 plays an important role in snRNP assembly through the stabilization of the SMN oligomer/complex via novel self-interaction. Applying the results/techniques to amino-terminal SMN missense mutants that were recently identified from SMA patients, we successfully showed that amino-terminal self-association, Gemin2 binding, the stabilization effect of Gemin2, and snRNP assembly activity were all lowered in the mutant SMN(D44V), suggesting that instability of the amino-terminal SMN self-association may cause SMA in patients carrying this allele.

Bifunctional RNAs Targeting the Intronic Splicing Silencer N1 Increase SMN Levels and Reduce Disease Severity in an Animal Model of Spinal Muscular Atrophy

Splicing Regulation in Neurologic Disease

Dual Masking of Specific Negative Splicing Regulatory Elements Resulted in Maximal Exon 7 Inclusion of SMN2 Gene

A Stem Cell-Based Screening Platform Identifies Compounds that Desensitize Motor Neurons to Endoplasmic Reticulum Stress.

Rare Missense and Synonymous Variants in UBE1 Are Associated with X-Linked Infantile Spinal Muscular Atrophy

Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U snRNPs) is a process facilitated by the macromolecular survival of motor neuron (SMN) complex. This entity promotes the binding of a set of factors, termed LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and unrip have been identified as the major components of the SMN complex. So far, however, only little is known about the architecture of this complex and the contribution of individual components to its function. Here, we present a comprehensive interaction map of all core components of the SMN complex based upon in vivo and in vitro methods. Our studies reveal a modular composition of the SMN complex with the three proteins SMN, Gemin8, and Gemin7 in its center. Onto this central building block the other components are bound via multiple interactions. Furthermore, by employing a novel assay, we were able to reconstitute the SMN complex from individual components and confirm the interaction map. Interestingly, SMN protein carrying an SMA-causing mutation was severely impaired in formation of the SMN complex. Finally, we show that the peripheral component Gemin5 contributes an essential activity to the SMN complex, most likely the transfer of Sm proteins onto the U snRNA. Collectively, the data presented here provide a basis for the detailed mechanistic and structural analysis of the assembly machinery of U snRNPs.

Rapid and Efficient Generation of Functional Motor Neurons From Human Pluripotent Stem Cells Using Gene Delivered Transcription Factor Codes

Within the mammalian central nervous system many forms of neurodegenerative injury are regulated via programmed cell death, a highly conserved program of cellular suicide. Programmed cell death is regulated by multiple signaling pathways, which have been identified within mammalian cells, although several lines of evidence suggest that the intrinsic pathway predominantly regulates the death of motor neurons following acute injury in vivo. We have tested this hypothesis by performing facial axotomies on cytochrome c knock-in mice containing a point mutation in the genomic locus of cytochrome c resulting in a lysine to alanine conversion at position 72 of the protein. The introduced mutation inhibits the ability of cytochrome c to induce the formation of the apoptosome, a protein complex that is principally required for the activation of the intrinsic pathway, but does not alter its function in oxidative phosphorylation. Homozygous cytochrome c knock-in mutants displayed a significant enhancement in motor neuron survival following injury when compared with littermate controls, thus establishing the apoptosome as a viable target for protecting motor neurons from neural injury. However, protection of facial motor neurons differs from that previously reported in mice either overexpressing anti-apoptotic or lacking pro-apoptotic members of the Bcl-2 family, which are thought to regulate several aspects of mitochondrial dysfunction including the release of cytochrome c from the mitochondria to the cytoplasm. Therefore, these results directly demonstrate for the first time the influence of the apoptosome on injury-induced neuronal programmed cell death in vivo isolated from upstream Bcl-2 family-mediated effects.

The neurodegenerative disorder spinal and bulbar muscular atrophy or Kennedy disease is caused by a CAG trinucleotide repeat expansion within the androgen receptor (AR) gene. The resulting expanded polyglutamine tract in the N-terminal region of the receptor renders AR prone to ligand-dependent misfolding and formation of oligomers and aggregates that are linked to neuronal toxicity. How AR misfolding is influenced by post-translational modifications, however, is poorly understood. AR is a target of SUMOylation, and this modification inhibits AR activity in a promoter context-dependent manner. SUMOylation is up-regulated in response to multiple forms of cellular stress and may therefore play an important cytoprotective role. Consistent with this view, we find that gratuitous enhancement of overall SUMOylation significantly reduced the formation of polyglutamine-expanded AR aggregates without affecting the levels of the receptor. Remarkably, this effect requires SUMOylation of AR itself because it depends on intact AR SUMOylation sites. Functional analyses, however, indicate that the protective effects of enhanced AR SUMOylation are not due to alterations in AR transcriptional activity because a branched protein structure in the appropriate context of the N-terminal region of AR is necessary to antagonize aggregation but not for inhibiting AR transactivation. Remarkably, small ubiquitin-like modifier (SUMO) attenuates AR aggregation through a unique mechanism that does not depend on critical features essential for its interaction with canonical SUMO binding motifs. Our findings therefore reveal a novel function of SUMOylation and suggest that approaches that enhance AR SUMOylation may be of clinical use in polyglutamine expansion diseases.

Spliceosome assembly is a dynamic process involving the sequential recruitment and rearrangement of small nuclear ribonucleoproteins (snRNPs) on a pre-mRNA substrate. Here we identify several spliceosome protein interactions with different domains of human splicing factor SPF30 that have the potential to mediate the addition of the tri-snRNP to the prespliceosome. In particular, we show that the C-terminal tails of SmD1, SmD3, and the protein Lsm4 interact with the central Tudor domain of SPF30. We identify a novel interaction between the N-terminal domain of SPF30 and U2AF35, a prespliceosome protein that has a role in recognizing the 3' splice site and recruiting U2 snRNP. We also show that the C terminus of SPF30 interacts with a middle domain of hPrp3, a component of U4/U6 di-snRNP and the tri-snRNP. Importantly, we show that the U2AF35 and hPrp3 interactions with SPF30 can occur simultaneously, thereby potentially linking 3' splice site recognition with tri-snRNP addition. Finally, we note that SPF30 and its partner-interacting domains are not conserved in yeast, suggesting this interaction network may play an important role in the complex splicing observed in higher eukaryotes.

Buying time for infants with spinal muscular atrophy

Polyglutamine expansion within the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA) and is associated with misfolded and aggregated species of the mutant AR. We showed previously that nuclear localization of the mutant AR was necessary but not sufficient for SBMA. Here we show that an interdomain interaction of the AR that is central to its function within the nucleus is required for AR aggregation and toxicity. Ligands that prevent the interaction between the amino-terminal FXXLF motif and carboxyl-terminal AF-2 domain (N/C interaction) prevented toxicity and AR aggregation in an SBMA cell model and rescued primary SBMA motor neurons from 5α-dihydrotestosterone-induced toxicity. Moreover, genetic mutation of the FXXLF motif prevented AR aggregation and 5α-dihydrotestosterone toxicity. Finally, selective androgen receptor modulators, which prevent the N/C interaction, ameliorated AR aggregation and toxicity while maintaining AR function, highlighting a novel therapeutic strategy to prevent the SBMA phenotype while retaining AR transcriptional function.