Inactivation of the SMN Complex by Oxidative Stress

Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6148, USA.
Molecular cell (Impact Factor: 14.46). 07/2008; 31(2):244-54. DOI: 10.1016/j.molcel.2008.06.004
Source: PubMed

ABSTRACT The SMN complex is essential for the biogenesis of small nuclear ribonucleoproteins (snRNPs), the major constituents of the spliceosome. Deficiency in functional SMN protein causes spinal muscular atrophy, a common motor neuron degenerative disease of severity commensurate with SMN levels and, correspondingly, snRNP assembly decreases. We developed a high-throughput screen for snRNP assembly modifiers and discovered that reactive oxygen species (ROS) inhibit SMN-complex activity in a dose-dependent manner. ROS-generating compounds, e.g., the environmental toxins menadione and beta-lapachone (in vivo IC(50) = 0.45 muM) also cause intermolecular disulfide crosslinking of SMN. Both the oxidative inactivation and SMN crosslinking can be reversed by reductants. We identified two cysteines that form SMN-SMN disulfide crosslinks, defining specific contact points in oligomeric SMN. Thus, the SMN complex is a redox-sensitive assemblyosome and an ROS target, suggesting that it may play a role in oxidative stress pathophysiology, which is associated with many degenerative diseases.

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Available from: Elizabeth Ottinger, Feb 03, 2014
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    • "The splicing changes that we uncovered in SMN2 transgene transcripts during late stage SMA were not described in these studies, although in other studies, SMN2 splicing was shown to decrease in type I mice at P1 (Jodelka et al. 2010; Ruggiu et al. 2012). Dying mice likely suffer from hypoxia; hypoxic stress induces reactive oxygen species generation, which inactivates the SMN complex (Wan et al. 2008). This complex plays fundamental roles in assembling snRNPs, which are required for splicing (Burghes and Beattie 2009), and low SMN levels result in decreased SMN2 exon 7 splicing through a feedback loop (Jodelka et al. 2010; Ruggiu et al. 2012). "
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    ABSTRACT: Antisense oligonucleotides (ASOs) are versatile molecules that can be designed to specifically alter splicing patterns of target pre-mRNAs. Here we exploit this feature to phenocopy a genetic disease. Spinal muscular atrophy (SMA) is a motor neuron disease caused by loss-of-function mutations in the SMN1 gene. The related SMN2 gene expresses suboptimal levels of functional SMN protein due to alternative splicing that skips exon 7; correcting this defect-e.g., with ASOs-is a promising therapeutic approach. We describe the use of ASOs that exacerbate SMN2 missplicing and phenocopy SMA in a dose-dependent manner when administered to transgenic Smn(-/-) mice. Intracerebroventricular ASO injection in neonatal mice recapitulates SMA-like progressive motor dysfunction, growth impairment, and shortened life span, with α-motor neuron loss and abnormal neuromuscular junctions. These SMA-like phenotypes are prevented by a therapeutic ASO that restores correct SMN2 splicing. We uncovered starvation-induced splicing changes, particularly in SMN2, which likely accelerate disease progression. These results constitute proof of principle that ASOs designed to cause sustained splicing defects can be used to induce pathogenesis and rapidly and accurately model splicing-associated diseases in animals. This approach allows the dissection of pathogenesis mechanisms, including spatial and temporal features of disease onset and progression, as well as testing of candidate therapeutics.
    Genes & development 08/2012; 26(16):1874-84. DOI:10.1101/gad.197418.112 · 12.64 Impact Factor
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    • "This is particularly well demonstrated in ALS, where both sporadic and familial forms share the same symptoms and pathological phenotype, that are recapitulated in available animal and cell models, with a prominent role for mitochondrial damage and resulting oxidative stress (for an extended Review, see [25]). Oxidative stress is reported also in SMA [26] and reactive oxygen species (ROS) inhibit assembly and activity of SMN complex in a dose-dependent manner [27]. Mitochondrial damage seems to be invariably present in neurodegenerative conditions [28] including SMA [29] [30] [31] [32] and SBMA [33], in which mitochondrial dysfunction may be due to the interaction between AR and cytochrome c oxidase subunit Vb (COXVb) [34]. "
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    ABSTRACT: Motor neuron diseases (MNDs) are a rather heterogeneous group of diseases, with either sporadic or genetic origin or both, all characterized by the progressive degeneration of motor neurons. At the cellular level, MNDs share features such as protein misfolding and aggregation, mitochondrial damage and energy deficit, and excitotoxicity and calcium mishandling. This is particularly well demonstrated in ALS, where both sporadic and familial forms share the same symptoms and pathological phenotype, with a prominent role for mitochondrial damage and resulting oxidative stress. Based on recent data, however, altered control of gene expression seems to be a most relevant, and previously overlooked, player in MNDs. Here we discuss which may be the links that make pathways apparently as different as altered gene expression, mitochondrial damage, and oxidative stress converge to generate a similar motoneuron-toxic phenotype.
    International Journal of Cell Biology 05/2012; 2012:908724. DOI:10.1155/2012/908724
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    • "Unbiased global RNA sequencing approaches have recently identified TDP-43-binding sites in a large number of mRNAs including those that are involved in regulating synaptic function, RNA metabolism, neuronal development as well as neurodegeneration including FUS/ TLS and TDP-43 itself (Polymenidou et al, 2011; Sephton et al, 2011; Tollervey et al, 2011). Further supporting a role in RNA processing came recently from studies showing that TDP-43 localizes to punctate neuronal granules and cytoplasmic stress granules (SGs) in primary neurons and cultured cells exposed to various forms of stress (Wang et al, 2008; Colombrita et al, 2009; Freibaum et al, 2010; Liu-Yesucevitz et al, 2010; Dewey et al, 2011; McDonald et al, 2011). Although the significance of TDP-43 re-localization is not yet clear, SGs represent cytoplasmic hubs regulating mRNA expression, processing, and sorting that may be critical for neuronal survival. "
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    ABSTRACT: TDP-43 is the major disease protein in ubiquitin-positive inclusions of amyotrophic lateral sclerosis and frontotemporal lobar degeneration (FTLD) characterized by TDP-43 pathology (FTLD-TDP). Accumulation of insoluble TDP-43 aggregates could impair normal TDP-43 functions and initiate disease progression. Thus, it is critical to define the signalling mechanisms regulating TDP-43 since this could open up new avenues for therapeutic interventions. Here, we have identified a redox-mediated signalling mechanism directly regulating TDP-43. Using in vitro and cell-based studies, we demonstrate that oxidative stress promotes TDP-43 cross-linking via cysteine oxidation and disulphide bond formation leading to decreased TDP-43 solubility. Biochemical analysis identified several cysteine residues located within and adjacent to the second RNA-recognition motif that contribute to both intra- and inter-molecular interactions, supporting TDP-43 as a target of redox signalling. Moreover, increased levels of cross-linked TDP-43 species are found in FTLD-TDP brains, indicating that aberrant TDP-43 cross-linking is a prominent pathological feature of this disease. Thus, TDP-43 is dynamically regulated by a redox regulatory switch that links oxidative stress to the modulation of TDP-43 and its downstream targets.
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