Stasimon/Tmem41b is required for cell proliferation and adult mouse survival.

Stasimon/Tmem41b is a transmembrane protein with phospholipid scrambling activity that resides in the endoplasmic reticulum and has been implicated in autophagy, lipid metabolism, and viral replication. Stasimon/Tmem41b has also been linked to the function of sensory-motor circuits and the pathogenesis of spinal muscular atrophy. However, the early embryonic lethality of constitutive knockout in mice has hindered the analysis of spatial and temporal requirements of Stasimon/Tmem41b in vivo. To address this, we developed a novel mouse line harboring a conditional knockout allele of the Stasimon/Tmem41b gene in which exon 4 has been flanked by loxP sites (Stas/Tmem41bCKO). Cre-mediated recombination of Stas/Tmem41bCKO generates a functionally null allele (Stas/Tmem41bΔ4) resulting in loss of protein expression and embryonic lethality in the homozygous mouse mutant. Here, using a ubiquitously expressed, tamoxifen inducible Cre recombinase in the homozygous Stas/Tmem41bCKO mice, we demonstrate that postnatal depletion of Stasimon/Tmem41b rapidly arrests weight gain in adult mice and causes motor dysfunction and death approximately three weeks after tamoxifen treatment. Moreover, we show that depletion of Stasimon/Tmem41b severely affects cell proliferation in mouse embryonic fibroblasts. This study provides new insights into the essential requirement of Stasimon/Tmem41b for cellular and organismal fitness and expands the experimental toolkit to investigate its functions in the mammalian system.

Evaluation of the orally bioavailable 4-phenylbutyrate-tethered trichostatin A analogue AR42 in models of spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a leading genetic cause for infant death in the world and results from the selective loss of motor neurons in the spinal cord. SMA is a consequence of low levels of SMN protein and small molecules that can increase SMN expression are of considerable interest as potential therapeutics. Previous studies have shown that both 4-phenylbutyrate (4PBA) and trichostatin A (TSA) increase SMN expression in dermal fibroblasts derived from SMA patients. AR42 is a 4PBA-tethered TSA derivative that is a very potent histone deacetylase inhibitor. SMA patient fibroblasts were treated with either AR42, AR19 (a related analogue), 4PBA, TSA or vehicle for 5 days and then immunostained for SMN localization. AR42 as well as 4PBA and TSA increased the number of SMN-positive nuclear gems in a dose-dependent manner while AR19 did not show marked changes in gem numbers. While gem number was increased in AR42-treated SMA fibroblasts, there were no significant changes in FL-SMN mRNA or SMN protein. The neuroprotective effect of this compound was then assessed in SMNΔ7 SMA (SMN2+/+;SMNΔ7+/+;mSmn-/-) mice. Oral administration of AR42 prior to disease onset increased the average lifespan of SMNΔ7 SMA mice by ~ 27% (20.1 ± 1.6 days for AR42-treated mice vs. 15.8 ± 0.4 days for vehicle-treated mice). AR42 treatment also improved motor function in these mice. AR42 treatment inhibited histone deacetylase (HDAC) activity in treated spinal cord although it did not affect SMN protein expression in these mice. AKT and GSK3ß phosphorylation were both significantly increased in SMNΔ7 SMA mouse spinal cords. In conclusion, presymptomatic administration of the HDAC inhibitor AR42 ameliorates the disease phenotype in SMNΔ7 SMA mice in a SMN-independent manner possibly by increasing AKT neuroprotective signaling.

Nusinersen mitigates neuroinflammation in severe spinal muscular atrophy patients.

BACKGROUND: Neuroinflammation contributes to the onset and progression of neurodegenerative diseases, but has not been specifically investigated in patients affected by severe and milder forms of spinal muscular atrophy (SMA). METHODS: In this two-center retrospective study, we investigated signatures of neuroinflammation in forty-eight pediatric male and female SMA1 (n = 18), male and female SMA2 (n = 19), and female SMA3 (n = 11) patients, as well as in a limited number of male and female non-neurological control subjects (n = 4). We employed a Bio-Plex multiplex system based on xMAP technology and performed targeted quantitative analysis of a wide range of pro- and anti-inflammatory cytokines (chemokines, interferons, interleukins, lymphokines and tumor necrosis factors) and neurotrophic factors in the cerebrospinal fluid (CSF) of the study cohort before and after Nusinersen treatment at loading and maintenance stages. RESULTS: We find a significant increase in the levels of several pro-inflammatory cytokines (IL-6, IFN-γ, TNF-α, IL-2, IL-8, IL-12, IL-17, MIP-1α, MCP-1, and Eotaxin) and neurotrophic factors (PDGF-BB and VEGF) in the CSF of SMA1 patients relative to SMA2 and SMA3 individuals, who display levels in the range of controls. We also find that treatment with Nusinersen significantly reduces the CSF levels of some but not all of these neuroinflammatory molecules in SMA1 patients. Conversely, Nusinersen increases the CSF levels of proinflammatory G-CSF, IL-8, MCP-1, MIP-1α, and MIP-1ß in SMA2 patients and decreases those of anti-inflammatory IL-1ra in SMA3 patients. CONCLUSIONS: These findings highlight signatures of neuroinflammation that are specifically associated with severe SMA and the neuro-immunomodulatory effects of Nusinersen therapy.

Spinal muscular atrophy (SMA) is an inherited disorder which leads to muscle weakening. Three therapies have recently been developed, including Nusinersen. However, the effect of SMA on the immune system and how this could be affected by Nusinersen is unknown. The immune system protects the body from infection and, in some disorders, misfunctions and damages the body in the absence of infection. Here, we analyze components of the immune system in body fluids from SMA patients before and after treatment with Nusinersen. The immune system was found to be more active in patients with more severe disease. Treatment with Nusinersen reduced the levels of some, but not all of these, components of the immune system. Thus, treatments that impact the immune system might improve symptoms in patients with SMA.

Nusinersen Induces Disease-Severity-Specific Neurometabolic Effects in Spinal Muscular Atrophy.

Intrathecal delivery of Nusinersen-an antisense oligonucleotide that promotes survival motor neuron (SMN) protein induction-is an approved therapy for spinal muscular atrophy (SMA). Here, we employed nuclear magnetic resonance (NMR) spectroscopy to longitudinally characterize the unknown metabolic effects of Nusinersen in the cerebrospinal fluid (CSF) of SMA patients across disease severity. Modulation of amino acid metabolism is a common denominator of biochemical changes induced by Nusinersen, with distinct downstream metabolic effects according to disease severity. In severe SMA1 patients, Nusinersen stimulates energy-related glucose metabolism. In intermediate SMA2 patients, Nusinersen effects are also related to energy homeostasis but involve ketone body and fatty acid biosynthesis. In milder SMA3 patients, Nusinersen mainly modulates amino acid metabolism. Moreover, Nusinersen modifies the CSF metabolome of a more severe clinical group towards the profile of untreated SMA patients with milder disease. These findings reveal disease severity-specific neurometabolic signatures of Nusinersen treatment, suggesting a selective modulation of peripheral organ metabolism by this CNS-directed therapy in severe SMA patients.

SMN controls neuromuscular junction integrity through U7 snRNP.

The neuromuscular junction (NMJ) is an essential synapse whose loss is a key hallmark of the neurodegenerative disease spinal muscular atrophy (SMA). Here, we show that activity of the SMA-determining SMN protein in the assembly of U7 small nuclear ribonucleoprotein (snRNP)-which functions in the 3'-end processing of replication-dependent histone mRNAs-is required for NMJ integrity. Co-expression of U7-specific Lsm10 and Lsm11 proteins selectively enhances U7 snRNP assembly, corrects histone mRNA processing defects, and rescues key structural and functional abnormalities of neuromuscular pathology in SMA mice-including NMJ denervation, decreased synaptic transmission, and skeletal muscle atrophy. Furthermore, U7 snRNP dysfunction drives selective loss of the synaptic organizing protein Agrin at NMJs innervating vulnerable muscles of SMA mice. These findings reveal a direct contribution of U7 snRNP dysfunction to neuromuscular pathology in SMA and suggest a role for histone gene regulation in maintaining functional synaptic connections between motor neurons and muscles.

Neuromuscular denervation and deafferentation but not motor neuron death are disease features in the Smn2B/- mouse model of SMA.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of motor neurons and skeletal muscle atrophy which is caused by ubiquitous deficiency in the survival motor neuron (SMN) protein. Several cellular defects contribute to sensory-motor circuit pathology in SMA mice, but the underlying mechanisms have often been studied in one mouse model without validation in other available models. Here, we used Smn2B/- mice to investigate specific behavioral, morphological, and functional aspects of SMA pathology that we previously characterized in the SMNΔ7 model. Smn2B/- SMA mice on a pure FVB/N background display deficits in body weight gain and muscle strength with onset in the second postnatal week and median survival of 19 days. Morphological analysis revealed severe loss of proprioceptive synapses on the soma of motor neurons and prominent denervation of neuromuscular junctions (NMJs) in axial but not distal muscles. In contrast, no evidence of cell death emerged from analysis of several distinct pools of lumbar motor neurons known to be lost in the disease. Moreover, SMA motor neurons from Smn2B/- mice showed robust nuclear accumulation of p53 but lack of phosphorylation of serine 18 at its amino-terminal, which selectively marks degenerating motor neurons in the SMNΔ7 mouse model. These results indicate that NMJ denervation and deafferentation, but not motor neuron death, are conserved features of SMA pathology in Smn2B/- mice.

TGS1 impacts snRNA 3'-end processing, ameliorates survival motor neuron-dependent neurological phenotypes in vivo and prevents neurodegeneration.

Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5'-monomethylguanosine cap of small nuclear RNAs (snRNAs) to a trimethylguanosine cap. Here, we show that loss of TGS1 in Caenorhabditis elegans, Drosophila melanogaster and Danio rerio results in neurological phenotypes similar to those caused by survival motor neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3' tails that are often uridylated. snRNAs with defective 3' terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.

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.

Author Correction: A systems view of spliceosomal assembly and branchpoints with iCLIP.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Minor snRNA gene delivery improves the loss of proprioceptive synapses on SMA motor neurons.

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder caused by reduced expression of the survival motor neuron (SMN) protein. SMN has key functions in multiple RNA pathways, including the biogenesis of small nuclear ribonucleoproteins that are essential components of both major (U2-dependent) and minor (U12-dependent) spliceosomes. Here we investigated the specific contribution of U12 splicing dysfunction to SMA pathology through selective restoration of this RNA pathway in mouse models of varying phenotypic severity. We show that virus-mediated delivery of minor snRNA genes specifically improves select U12 splicing defects induced by SMN deficiency in cultured mammalian cells, as well as in the spinal cord and dorsal root ganglia of SMA mice without increasing SMN expression. This approach resulted in a moderate amelioration of several parameters of the disease phenotype in SMA mice, including survival, weight gain, and motor function. Importantly, minor snRNA gene delivery improved aberrant splicing of the U12 intron-containing gene Stasimon and rescued the severe loss of proprioceptive sensory synapses on SMA motor neurons, which are early signatures of motor circuit dysfunction in mouse models. Taken together, these findings establish the direct contribution of U12 splicing dysfunction to synaptic deafferentation and motor circuit pathology in SMA.

Stasimon Contributes to the Loss of Sensory Synapses and Motor Neuron Death in a Mouse Model of Spinal Muscular Atrophy.

Reduced expression of the survival motor neuron (SMN) protein causes the neurodegenerative disease spinal muscular atrophy (SMA). Here, we show that adeno-associated virus serotype 9 (AAV9)-mediated delivery of Stasimon-a gene encoding an endoplasmic reticulum (ER)-resident transmembrane protein regulated by SMN-improves motor function in a mouse model of SMA through multiple mechanisms. In proprioceptive neurons, Stasimon overexpression prevents the loss of afferent synapses on motor neurons and enhances sensory-motor neurotransmission. In motor neurons, Stasimon suppresses neurodegeneration by reducing phosphorylation of the tumor suppressor p53. Moreover, Stasimon deficiency converges on SMA-related mechanisms of p53 upregulation to induce phosphorylation of p53 through activation of p38 mitogen-activated protein kinase (MAPK), and pharmacological inhibition of this kinase prevents motor neuron death in SMA mice. These findings identify Stasimon dysfunction induced by SMN deficiency as an upstream driver of distinct cellular cascades that lead to synaptic loss and motor neuron degeneration, revealing a dual contribution of Stasimon to motor circuit pathology in SMA.

A systems view of spliceosomal assembly and branchpoints with iCLIP.

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we used spliceosome iCLIP, which immunoprecipitates SmB along with small nuclear ribonucleoprotein particles and auxiliary RNA binding proteins, to map spliceosome engagement with pre-messenger RNAs in human cell lines. This revealed seven peaks of spliceosomal crosslinking around branchpoints (BPs) and splice sites. We identified RNA binding proteins that crosslink to each peak, including known and candidate splicing factors. Moreover, we detected the use of over 40,000 BPs with strong sequence consensus and structural accessibility, which align well to nearby crosslinking peaks. We show how the position and strength of BPs affect the crosslinking patterns of spliceosomal factors, which bind more efficiently upstream of strong or proximally located BPs and downstream of weak or distally located BPs. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.

Functional characterization of SMN evolution in mouse models of SMA.

Spinal Muscular Atrophy (SMA) is a monogenic neurodegenerative disorder and the leading genetic cause of infantile mortality. While several functions have been ascribed to the SMN (survival motor neuron) protein, their specific contribution to the disease has yet to be fully elucidated. We hypothesized that some, but not all, SMN homologues would rescue the SMA phenotype in mouse models, thereby identifying disease-relevant domains. Using AAV9 to deliver Smn homologs to SMA mice, we identified a conservation threshold that marks the boundary at which homologs can rescue the SMA phenotype. Smn from Danio rerio and Xenopus laevis significantly prevent disease, whereas Smn from Drosophila melanogaster, Caenorhabditis elegans, and Schizosaccharomyces pombe was significantly less efficacious. This phenotypic rescue correlated with correction of RNA processing defects induced by SMN deficiency and neuromuscular junction pathology. Based upon the sequence conservation in the rescuing homologs, a minimal SMN construct was designed consisting of exons 2, 3, and 6, which showed a partial rescue of the SMA phenotype. While a significant extension in survival was observed, the absence of a complete rescue suggests that while the core conserved region is essential, additional sequences contribute to the overall ability of the SMN protein to rescue disease pathology.

FTO controls reversible m6Am RNA methylation during snRNA biogenesis.

Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m1 and m2, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m1 isoform with a single-methylated adenosine (2'-O-methyladenosine, Am), which is then converted to a dimethylated m2 isoform (N6,2'-O-dimethyladenosine, m6Am). The relative m1 and m2 isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m2 isoform. We show FTO is inhibited by the oncometabolite D-2-hydroxyglutarate, resulting in increased m2-snRNA levels. Furthermore, cells that exhibit high m2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.

Stasimon/Tmem41b localizes to mitochondria-associated ER membranes and is essential for mouse embryonic development.

Stasimon (also known as Tmem41b) is an evolutionarily conserved transmembrane protein first identified for its contribution to motor system dysfunction in animal models of the childhood neurodegenerative disease spinal muscular atrophy (SMA). Stasimon was shown to be required for normal neurotransmission in the motor circuit of Drosophila larvae and proper development of motor axons in zebrafish embryos as well as to suppress analogous neuronal phenotypes in SMA models of these organisms. However, the subcellular localization and molecular functions of Stasimon are poorly understood. Here, we combined immunoprecipitation with mass spectrometry to characterize the Stasimon interactome in mammalian cells, which reveals association with components of the endoplasmic reticulum (ER), mitochondria, and the COPI vesicle trafficking machinery. Expanding on the interaction results, we used subcellular fractionation studies and super-resolution microscopy to identify Stasimon as an ER-resident protein that localizes at mitochondria-associated ER membranes (MAM), functionally specialized contact sites between ER and mitochondria membranes. Lastly, through characterization of novel knockout mice, we show that Stasimon is an essential gene for mouse embryonic development. Together, these findings identify Stasimon as a novel transmembrane protein component of the MAM with an essential requirement for mammalian development.

Dysregulation of Mdm2 and Mdm4 alternative splicing underlies motor neuron death in spinal muscular atrophy.

Ubiquitous deficiency in the survival motor neuron (SMN) protein causes death of motor neurons-a hallmark of the neurodegenerative disease spinal muscular atrophy (SMA)-through poorly understood mechanisms. Here, we show that the function of SMN in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) regulates alternative splicing of Mdm2 and Mdm4, two nonredundant repressors of p53. Decreased inclusion of critical Mdm2 and Mdm4 exons is most prominent in SMA motor neurons and correlates with both snRNP reduction and p53 activation in vivo. Importantly, increased skipping of Mdm2 and Mdm4 exons regulated by SMN is necessary and sufficient to synergistically elicit robust p53 activation in wild-type mice. Conversely, restoration of full-length Mdm2 and Mdm4 suppresses p53 induction and motor neuron degeneration in SMA mice. These findings reveal that loss of SMN-dependent regulation of Mdm2 and Mdm4 alternative splicing underlies p53-mediated death of motor neurons in SMA, establishing a causal link between snRNP dysfunction and neurodegeneration.

Mild SMN missense alleles are only functional in the presence of SMN2 in mammals.

Spinal muscular atrophy (SMA) is caused by reduced levels of full-length SMN (FL-SMN). In SMA patients with one or two copies of the Survival Motor Neuron 2 (SMN2) gene there are a number of SMN missense mutations that result in milder-than-predicted SMA phenotypes. These mild SMN missense mutation alleles are often assumed to have partial function. However, it is important to consider the contribution of FL-SMN as these missense alleles never occur in the absence of SMN2. We propose that these patients contain a partially functional oligomeric SMN complex consisting of FL-SMN from SMN2 and mutant SMN protein produced from the missense allele. Here we show that mild SMN missense mutations SMND44V, SMNT74I or SMNQ282A alone do not rescue mice lacking wild-type FL-SMN. Thus, missense mutations are not functional in the absence of FL-SMN. In contrast, when the same mild SMN missense mutations are expressed in a mouse containing two SMN2 copies, functional SMN complexes are formed with the small amount of wild-type FL-SMN produced by SMN2 and the SMA phenotype is completely rescued. This contrasts with SMN missense alleles when studied in C. elegans, Drosophila and zebrafish. Here we demonstrate that the heteromeric SMN complex formed with FL-SMN is functional and sufficient to rescue small nuclear ribonucleoprotein assembly, motor neuron function and rescue the SMA mice. We conclude that mild SMN missense alleles are not partially functional but rather they are completely non-functional in the absence of wild-type SMN in mammals.

Converging Mechanisms of p53 Activation Drive Motor Neuron Degeneration in Spinal Muscular Atrophy.

The hallmark of spinal muscular atrophy (SMA), an inherited disease caused by ubiquitous deficiency in the SMN protein, is the selective degeneration of subsets of spinal motor neurons. Here, we show that cell-autonomous activation of p53 occurs in vulnerable but not resistant motor neurons of SMA mice at pre-symptomatic stages. Moreover, pharmacological or genetic inhibition of p53 prevents motor neuron death, demonstrating that induction of p53 signaling drives neurodegeneration. At late disease stages, however, nuclear accumulation of p53 extends to resistant motor neurons and spinal interneurons but is not associated with cell death. Importantly, we identify phosphorylation of serine 18 as a specific post-translational modification of p53 that exclusively marks vulnerable SMA motor neurons and provide evidence that amino-terminal phosphorylation of p53 is required for the neurodegenerative process. Our findings indicate that distinct events induced by SMN deficiency converge on p53 to trigger selective death of vulnerable SMA motor neurons.

A Stem Cell Model of the Motor Circuit Uncouples Motor Neuron Death from Hyperexcitability Induced by SMN Deficiency.

In spinal muscular atrophy, a neurodegenerative disease caused by ubiquitous deficiency in the survival motor neuron (SMN) protein, sensory-motor synaptic dysfunction and increased excitability precede motor neuron (MN) loss. Whether central synaptic dysfunction and MN hyperexcitability are cell-autonomous events or they contribute to MN death is unknown. We addressed these issues using a stem-cell-based model of the motor circuit consisting of MNs and both excitatory and inhibitory interneurons (INs) in which SMN protein levels are selectively depleted. We show that SMN deficiency induces selective MN death through cell-autonomous mechanisms, while hyperexcitability is a non-cell-autonomous response of MNs to defects in pre-motor INs, leading to loss of glutamatergic synapses and reduced excitation. Findings from our in vitro model suggest that dysfunction and loss of MNs result from differential effects of SMN deficiency in distinct neurons of the motor circuit and that hyperexcitability does not trigger MN death.