Gain of toxic function by long-term AAV9-mediated SMN overexpression in the sensorimotor circuit.

The neurodegenerative disease spinal muscular atrophy (SMA) is caused by deficiency in the survival motor neuron (SMN) protein. Currently approved SMA treatments aim to restore SMN, but the potential for SMN expression beyond physiological levels is a unique feature of adeno-associated virus serotype 9 (AAV9)-SMN gene therapy. Here, we show that long-term AAV9-mediated SMN overexpression in mouse models induces dose-dependent, late-onset motor dysfunction associated with loss of proprioceptive synapses and neurodegeneration. Mechanistically, aggregation of overexpressed SMN in the cytoplasm of motor circuit neurons sequesters components of small nuclear ribonucleoproteins, leading to splicing dysregulation and widespread transcriptome abnormalities with prominent signatures of neuroinflammation and the innate immune response. Thus, long-term SMN overexpression interferes with RNA regulation and triggers SMA-like pathogenic events through toxic gain-of-function mechanisms. These unanticipated, SMN-dependent and neuron-specific liabilities warrant caution on the long-term safety of treating individuals with SMA with AAV9-SMN and the risks of uncontrolled protein expression by gene therapy.

Intragenic complementation of amino and carboxy terminal SMN missense mutations can rescue Smn null mice.

Spinal muscular atrophy is caused by reduced levels of SMN resulting from the loss of SMN1 and reliance on SMN2 for the production of SMN. Loss of SMN entirely is embryonic lethal in mammals. There are several SMN missense mutations found in humans. These alleles do not show partial function in the absence of wild-type SMN and cannot rescue a null Smn allele in mice. However, these human SMN missense allele transgenes can rescue a null Smn allele when SMN2 is present. We find that the N- and C-terminal regions constitute two independent domains of SMN that can be separated genetically and undergo intragenic complementation. These SMN protein heteromers restore snRNP assembly of Sm proteins onto snRNA and completely rescue both survival of Smn null mice and motor neuron electrophysiology demonstrating that the essential functional unit of SMN is the oligomer.

Protective effects of butyrate-based compounds on a mouse model for spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a childhood-onset degenerative disease resulting from the selective loss of motor neurons in the spinal cord. SMA is caused by the loss of SMN1 (survival motor neuron 1) but retention of SMN2. The number of copies of SMN2 modifies disease severity in SMA patients as well as in mouse models, making SMN2 a target for therapeutics development. Sodium butyrate (BA) and its analog (4PBA) have been shown to increase SMN2 expression in SMA cultured cells. In this study, we examined the effects of BA, 4PBA as well as two BA prodrugs-glyceryl tributyrate (BA3G) and VX563-on the phenotype of SMNΔ7 SMA mice. Treatment with 4PBA, BA3G and VX563 but not BA beginning at PND04 significantly improved the lifespan and delayed disease end stage, with administration of VX563 also improving the growth rate of these mice. 4PBA and VX563 improved the motor phenotype of SMNΔ7 SMA mice and prevented spinal motor neuron loss. Interestingly, neither 4PBA nor VX563 had an effect on SMN expression in the spinal cords of treated SMNΔ7 SMA mice; however, they inhibited histone deacetylase (HDAC) activity and restored the normal phosphorylation states of Akt and glycogen synthase kinase 3ß, both of which are altered by SMN deficiency in vivo. These observations show that BA-based compounds with favorable pharmacokinetics ameliorate SMA pathology possibly by modulating HDAC and Akt signaling.

Gemin5 Binds to the Survival Motor Neuron mRNA to Regulate SMN Expression.

Reduced expression of SMN causes spinal muscular atrophy, a severe neurodegenerative disease. Despite the importance of maintaining SMN levels, relatively little is known about the mechanisms by which SMN levels are regulated. We show here that Gemin5, the snRNA-binding protein of the SMN complex, binds directly to the SMN mRNA and regulates SMN expression. Gemin5 binds with high specificity, both in vitro and in vivo, to sequence and structural elements in the SMN mRNA 3'-untranslated region that are reminiscent of the snRNP code to which Gemin5 binds on snRNAs. Reduction of Gemin5 redistributes the SMN mRNA from heavy polysomes to lighter polysomes and monosomes, suggesting that Gemin5 functions as an activator of SMN translation. SMN protein is not stoichiometrically present on the SMN mRNA with Gemin5, but the mRNA-binding activity of Gemin5 is dependent on SMN levels, providing a feedback mechanism for SMN to regulate its own expression via Gemin5. This work both reveals a new autoregulatory pathway governing SMN expression, and identifies a new mechanism through which SMN can modulate specific mRNA expression via Gemin5.

Spinal Muscular Atrophy Biomarker Measurements from Blood Samples in a Clinical Trial of Valproic Acid in Ambulatory Adults.

BACKGROUND: Clinical trials of therapies for spinal muscular atrophy (SMA) that are designed to increase the expression the SMN protein ideally include careful assessment of relevant SMN biomarkers. OBJECTIVE: In the SMA VALIANT trial, a recent double-blind placebo-controlled crossover study of valproic acid (VPA) in ambulatory adult subjects with SMA, we investigated relevant pharmacodynamic biomarkers in blood samples from SMA subjects by direct longitudinal measurement of histone acetylation and SMN mRNA and protein levels in the presence and absence of VPA treatment. METHODS: Thirty-three subjects were randomized to either VPA or placebo for the first 6 months followed by crossover to the opposite arm for an additional 6 months. Outcome measures were compared between the two treatments (VPA and placebo) using a standard crossover analysis. RESULTS: A significant increase in histone H4 acetylation was observed with VPA treatment (p = 0.005). There was insufficient evidence to suggest a treatment effect with either full length or truncated SMN mRNA transcript levels or SMN protein levels. CONCLUSIONS: These measures were consistent with the observed lack of change in the primary clinical outcome measure in the VALIANT trial. These results also highlight the added benefit of molecular and pharmacodynamic biomarker measurements in the interpretation of clinical trial outcomes.

U1A regulates 3' processing of the survival motor neuron mRNA.

Insufficient expression of the survival motor neuron (SMN) protein causes spinal muscular atrophy, a neurodegenerative disease characterized by loss of motor neurons. Despite the importance of maintaining adequate SMN levels, little is known about factors that control SMN expression, particularly 3' end processing of the SMN pre-mRNA. In this study, we identify the U1A protein as a key regulator of SMN expression. U1A, a component of the U1 snRNP, is known to inhibit polyadenylation upon direct binding to mRNA. We show that U1A binds directly and with high affinity and specificity to the SMN 3'-UTR adjacent to the polyadenylation site, independent of the U1 snRNP (U1 small nuclear ribonucleoprotein). Binding of U1A inhibits polyadenylation of the SMN pre-mRNA by specifically inhibiting 3' cleavage by the cleavage and polyadenylation specificity factor. Expression of U1A in excess of U1 snRNA causes inhibition of SMN polyadenylation and decreases SMN protein levels. This work reveals a new mechanism for regulating SMN levels and provides new insight into the roles of U1A in 3' processing of mRNAs.

Spliceosomal small nuclear ribonucleoprotein biogenesis defects and motor neuron selectivity in spinal muscular atrophy.

The SMN protein is essential and participates in the assembly of macromolecular complexes of RNA and protein in all cells. The best-characterized function of SMN is as an assembler of spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN performs this function as part of a complex with several other proteins called Gemins. snRNPs are assembled in the cytoplasm in a stepwise manner and then are imported to the nucleus where they participate globally in the splicing of pre-mRNA. Mutations in the SMN1 gene result in the motor neuron disease, spinal muscular atrophy (SMA). Most of these mutations result in a reduction in the expression levels of the SMN protein, which, in turn, results in a reduction in snRNP assembly capacity. This review highlights current studies that have investigated the mechanism of SMN-dependent snRNP assembly, as well as the downstream effects on pre-mRNA splicing that result from a decrease in SMN. This article is part of a Special Issue entitled "RNA-Binding Proteins".

A SMN missense mutation complements SMN2 restoring snRNPs and rescuing SMA mice.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease. Loss of the survival motor neuron (SMN1) gene, in the presence of the SMN2 gene causes SMA. SMN functions in snRNP assembly in all cell types, however, it is unclear how this function results in specifically motor neuron cell death. Lack of endogenous mouse SMN (Smn) in mice results in embryonic lethality. Introduction of two copies of human SMN2 results in a mouse with severe SMA, while one copy of SMN2 is insufficient to overcome embryonic lethality. We show that SMN(A111G), an allele capable of snRNP assembly, can rescue mice that lack Smn and contain either one or two copies of SMN2 (SMA mice). The correction of SMA in these animals was directly correlated with snRNP assembly activity in spinal cord, as was correction of snRNA levels. These data support snRNP assembly as being the critical function affected in SMA and suggests that the levels of snRNPs are critical to motor neurons. Furthermore, SMN(A111G) cannot rescue Smn-/- mice without SMN2 suggesting that both SMN(A111G) and SMN from SMN2 undergo intragenic complementation in vivo to function in heteromeric complexes that have greater function than either allele alone. The oligomer composed of limiting full-length SMN and SMN(A111G) has substantial snRNP assembly activity. Also, the SMN(A2G) and SMN(A111G) alleles in vivo did not complement each other leading to the possibility that these mutations could affect the same function.

Neuronal SMN expression corrects spinal muscular atrophy in severe SMA mice while muscle-specific SMN expression has no phenotypic effect.

Spinal muscular atrophy (SMA) is caused by loss of the survival motor neuron gene (SMN1) and retention of the SMN2 gene. The copy number of SMN2 affects the amount of SMN protein produced and the severity of the SMA phenotype. While loss of mouse Smn is embryonic lethal, two copies of SMN2 prevents this embryonic lethality resulting in a mouse with severe SMA that dies 5 days after birth. Here we show that expression of full-length SMN under the prion promoter (PrP) rescues severe SMA mice. The PrP results in high levels of SMN in neurons at embryonic day 15. Mice homozygous for PrP-SMN with two copies of SMN2 and lacking mouse Smn survive for an average of 210 days and lumbar motor neuron root counts in these mice were normal. Expression of SMN solely in skeletal muscle using the human skeletal actin (HSA) promoter resulted in no improvement of the SMA phenotype or extension of survival. One HSA line displaying nerve expression of SMN did affect the SMA phenotype with mice living for an average of 160 days. Thus, we conclude that expression of full-length SMN in neurons can correct the severe SMA phenotype in mice. Furthermore, a small increase of SMN in neurons has a substantial impact on survival of SMA mice while high SMN levels in mature skeletal muscle alone has no impact.

Survival motor neuron function in motor axons is independent of functions required for small nuclear ribonucleoprotein biogenesis.

Spinal muscular atrophy (SMA) is a motor neuron degenerative disease caused by low levels of the survival motor neuron (SMN) protein and is linked to mutations or loss of SMN1 and retention of SMN2. How low levels of SMN cause SMA is unclear. SMN functions in small nuclear ribonucleoprotein (snRNP) biogenesis, but recent studies indicate that SMN may also function in axons. We showed previously that decreasing Smn levels in zebrafish using morpholinos (MO) results in motor axon defects. To determine how Smn functions in motor axon outgrowth, we coinjected smn MO with various human SMN RNAs and assayed the effect on motor axons. Wild-type SMN rescues motor axon defects caused by Smn reduction in zebrafish. Consistent with these defects playing a role in SMA, SMN lacking exon 7, the predominant form from the SMN2 gene, and human SMA mutations do not rescue defective motor axons. Moreover, the severity of the motor axon defects correlates with decreased longevity. We also show that a conserved region in SMN exon 7, QNQKE, is critical for motor axon outgrowth. To address the function of SMN important for motor axon outgrowth, we determined the ability of different SMN forms to oligomerization and bind Sm protein, functions required for snRNP biogenesis. We identified mutations that failed to rescue motor axon defects but retained snRNP function. Thus, we have dissociated the snRNP function of SMN from its function in motor axons. These data indicate that SMN has a novel function in motor axons that is relevant to SMA and is independent of snRNP biosynthesis.