U1 snRNP is mislocalized in ALS patient fibroblasts bearing NLS mutations in FUS and is required for motor neuron outgrowth in zebrafish.

Mutations in FUS cause amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to neurodegeneration remain obscure. We previously found that U1 snRNP is the most abundant FUS interactor. Here, we report that components of the U1 snRNP core particle (Sm proteins and U1 snRNA), but not the mature U1 snRNP-specific proteins (U1-70K, U1A and U1C), co-mislocalize with FUS to the cytoplasm in ALS patient fibroblasts harboring mutations in the FUS nuclear localization signal (NLS). Similar results were obtained in HeLa cells expressing the ALS-causing FUS R495X NLS mutation, and mislocalization of Sm proteins is RRM-dependent. Moreover, as observed with FUS, knockdown of any of the U1 snRNP-specific proteins results in a dramatic loss of SMN-containing Gems. Significantly, knockdown of U1 snRNP in zebrafish results in motor axon truncations, a phenotype also observed with FUS, SMN and TDP-43 knockdowns. Our observations linking U1 snRNP to ALS patient cells with FUS mutations, SMN-containing Gems, and motor neurons indicate that U1 snRNP is a component of a molecular pathway associated with motor neuron disease. Linking an essential canonical splicing factor (U1 snRNP) to this pathway provides strong new evidence that splicing defects may be involved in pathogenesis and that this pathway is a potential therapeutic target.

The splicing factor U1-70K interacts with the SMN complex and is required for nuclear gem integrity.

The nuclear SMN complex localizes to specific structures called nuclear gems. The loss of gems is a cellular marker for several neurodegenerative diseases. Here, we identify that the U1-snRNP-specific protein U1-70K localizes to nuclear gems, and we show that U1-70K is necessary for gem integrity. Furthermore, we show that the interaction between U1-70K and the SMN complex is RNA independent, and we map the SMN complex binding site to the unstructured N-terminal tail of U1-70K. Consistent with these results, the expression of the U1-70K N-terminal tail rescues gem formation. These findings show that U1-70K is an SMN-complex-associating protein, and they suggest a new function for U1-70K in the formation of nuclear gems.

Decreased number of Gemini of coiled bodies and U12 snRNA level in amyotrophic lateral sclerosis.

Disappearance of TAR-DNA-binding protein 43 kDa (TDP-43) from the nucleus contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS), but the nuclear function of TDP-43 is not yet fully understood. TDP-43 associates with nuclear bodies including Gemini of coiled bodies (GEMs). GEMs contribute to the biogenesis of uridine-rich small nuclear RNA (U snRNA), a component of splicing machinery. The number of GEMs and a subset of U snRNAs decrease in spinal muscular atrophy, a lower motor neuron disease, suggesting that alteration of U snRNAs may also underlie the molecular pathogenesis of ALS. Here, we investigated the number of GEMs and U11/12-type small nuclear ribonucleoproteins (snRNP) by immunohistochemistry and the level of U snRNAs using real-time quantitative RT-PCR in ALS tissues. GEMs decreased in both TDP-43-depleted HeLa cells and spinal motor neurons in ALS patients. Levels of several U snRNAs decreased in TDP-43-depleted SH-SY5Y and U87-MG cells. The level of U12 snRNA was decreased in tissues affected by ALS (spinal cord, motor cortex and thalamus) but not in tissues unaffected by ALS (cerebellum, kidney and muscle). Immunohistochemical analysis revealed the decrease in U11/12-type snRNP in spinal motor neurons of ALS patients. These findings suggest that loss of TDP-43 function decreases the number of GEMs, which is followed by a disturbance of pre-mRNA splicing by the U11/U12 spliceosome in tissues affected by ALS.

Minor splicing pathway is not minor any more: implications for the pathogenesis of motor neuron diseases.

To explore the molecular pathogenesis of amyotrophic lateral sclerosis (ALS), the nuclear function of TAR-DNA binding protein 43 kDa (TDP-43) must be elucidated. TDP-43 is a nuclear protein that colocalizes with Cajal body or Gem in cultured cells. Several recent studies have reported that the decreasing number of Gems accompanied the depletion of the causative genes for ALS, TDP-43 and FUS. Gems play an important role in the pathogenesis of spinal muscular atrophy. Gems are the sites of the maturation of spliceosomes, which are composed of uridylate-rich (U) snRNAs (small nuclear RNAs) and protein complex, small nuclear ribonuclearprotein (snRNP). Spliceosomes regulate the splicing of pre-mRNA and are classified into the major or minor classes, according to the consensus sequence of acceptor and donor sites of pre-mRNA splicing. Although the major class of spliceosomes regulates most pre-mRNA splicing, minor spliceosomes also play an important role in regulating the splicing or global speed of pre-mRNA processing. A mouse model of spinal muscular atrophy, in which the number of Gems is decreased, shows fewer subsets U snRNAs. Interestingly, in the central nervous system, U snRNAs belonging to the minor spliceosomes are markedly reduced. In ALS, the U12 snRNA is decreased only in the tissue affected by ALS and not in other tissues. Although the molecular mechanisms underlying the decreased U12 snRNA resulting in cell dysfunction and cell death in motor neuron diseases remain unclear, these findings suggest that the disturbance of nuclear bodies and minor splicing may underlie the common molecular pathogenesis of motor neuron diseases.

Triptolide increases transcript and protein levels of survival motor neurons in human SMA fibroblasts and improves survival in SMA-like mice.

BACKGROUND AND PURPOSE: Spinal muscular atrophy (SMA) is a progressive neuromuscular disease. Since disease severity is related to the amount of survival motor neuron (SMN) protein, up-regulated functional SMN protein levels from the SMN2 gene are considered a major SMA drug-discovery strategy. In this study, we investigated the possible effects of triptolide, a diterpene triepoxide purified from Tripterygium wilfordii Hook. F., as a new compound for increasing SMN protein. EXPERIMENTAL APPROACH: The effects and mechanisms of triptolide on the production of SMA protein were determined by cell-based assays using the motor neuronal cell line NSC34 and skin fibroblasts from SMA patients. Wild-type (Smn(+/+) SMN2(-/-) , C57BL/6) and SMA-like (Smn(-/-) SMN2) mice were injected with triptolide (0.01 or 0.1 mg·kg(-1) ·day(-1) , i.p.) and their survival rate and level of change in SMN protein in neurons and muscle tissue measured. KEY RESULTS: In NSC34 cells and human SMA fibroblasts, pM concentrations of triptolide significantly increased SMN protein expression and the levels of SMN complex component (Gemin2 and Gemin3). In human SMA fibroblasts, triptolide increased SMN-containing nuclear gems and the ratio of full-length transcripts (FL-SMN2) to SMN2 transcripts lacking exon 7 (SMN2Δ7). Furthermore, in SMA-like mice, triptolide significantly increased SMN protein levels in the brain, spinal cord and gastrocnemius muscle. Furthermore, triptolide treatment increased survival and reduced weight loss in SMA-like mice. CONCLUSION AND IMPLICATIONS: Triptolide enhanced SMN protein production by promoting SMN2 activation, exon 7 inclusion and increasing nuclear gems, and increased survival in SMA mice, which suggests triptolide might be a potential candidate for SMA therapy.