Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCFSlmb degron.

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1 Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers.

Plastin-3 extends survival and reduces severity in mouse models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a leading genetic cause of infantile death and is caused by the loss of survival motor neuron-1 (SMN1). Importantly, a nearly identical gene is present called SMN2; however, the majority of SMN2-derived transcripts are alternatively spliced and encode a truncated, dysfunctional protein. Recently, several compounds designed to increase SMN protein have entered clinical trials, including antisense oligonucleotides (ASOs), traditional small molecules, and gene therapy. Expanding beyond SMN-centric therapeutics is important, as it is likely that the breadth of the patient spectrum and the inherent complexity of the disease will be difficult to address with a single therapeutic strategy. Several SMN-independent pathways that could impinge upon the SMA phenotype have been examined with varied success. To identify disease-modifying pathways that could serve as stand-alone therapeutic targets or could be used in combination with an SMN-inducing compound, we investigated adeno-associated virus-mediated (AAV-mediated) gene therapy using plastin-3 (PLS3). Here, we report that AAV9-PLS3 extends survival in an intermediate model of SMA mice as well as in a pharmacologically induced model of SMA using a splice-switching ASO that increases SMN production. PLS3 coadministration improves the phenotype beyond the ASO, demonstrating the potential utility of combinatorial therapeutics in SMA that target SMN-independent and SMN-dependent pathways.

Astrocytes influence the severity of spinal muscular atrophy.

Systemically low levels of survival motor neuron-1 (SMN1) protein cause spinal muscular atrophy (SMA). α-Motor neurons of the spinal cord are considered particularly vulnerable in this genetic disorder and their dysfunction and loss cause progressive muscle weakness, paralysis and eventually premature death of afflicted individuals. Historically, SMA was therefore considered a motor neuron-autonomous disease. However, depletion of SMN in motor neurons of normal mice elicited only a very mild phenotype. Conversely, restoration of SMN to motor neurons in an SMA mouse model had only modest effects on the SMA phenotype and survival. Collectively, these results suggested that additional cell types contribute to the pathogenesis of SMA, and understanding the non-autonomous requirements is crucial for developing effective therapies. Astrocytes are critical for regulating synapse formation and function as well as metabolic support for neurons. We hypothesized that astrocyte functions are disrupted in SMA, exacerbating disease progression. Using viral-based restoration of SMN specifically to astrocytes, survival in severe and intermediate SMA mice was observed. In addition, neuromuscular circuitry was improved. Astrogliosis was prominent in end-stage SMA mice and in post-mortem patient spinal cords. Increased expression of proinflammatory cytokines was partially normalized in treated mice, suggesting that astrocytes contribute to the pathogenesis of SMA.

Defining the therapeutic window in a severe animal model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the loss of a single gene, Survival Motor Neuron-1 (SMN1). Administration of a self-complementary Adeno-Associated Virus vector expressing full-length SMN cDNA (scAAV-SMN) has proven an effective means to rescue the SMA phenotype in SMA mice, either by intravenous (IV) or intracerebroventricular (ICV) administration at very early time points. We have recently shown that ICV delivery of scAAV9-SMN is more effective than a similar dose of vector administered via an IV injection, thereby providing an important mechanism to examine a timeline for rescuing the disease and determining the therapeutic window in a severe model of SMA. In this report, we utilized a relatively severe mouse model of SMA, SMNΔ7. Animals were injected with scAAV9-SMN vector via ICV injection on a single day, from P2 through P8. At each delivery point from P2 through P8, scAAV9-SMN decreased disease severity. A near complete rescue was obtained following P2 injection while a P8 injection produced a ∼ 40% extension in survival. Analysis of the underlying neuromuscular junction (NMJ) pathology revealed that late-stage delivery of the vector failed to provide protection from NMJ defects despite robust SMN expression in the central nervous system. While our study demonstrates that a maximal benefit is obtained when treatment is delivered during pre-symptomatic stages, significant therapeutic benefit can still be achieved after the onset of disease symptoms.

Restoration of SMN to Emx-1 expressing cortical neurons is not sufficient to provide benefit to a severe mouse model of Spinal Muscular Atrophy.

Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is a leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). However, low, but essential, levels of SMN protein are produced by a nearly identical copy gene called SMN2. Detailed analysis of neuromuscular junctions in SMA mice has revealed a selective vulnerability in a subset of muscle targets, suggesting that while SMN is reduced uniformly, the functional deficits manifest sporadically. Additionally, in severe SMA models, it is becoming increasing apparent that SMA is not restricted solely to motor neurons. Rather, additional tissues including the heart, vasculature, and the pancreas contribute to the complete SMA-associated pathology. Recently, transgenic models have been utilized to examine the tissue-specific requirements of SMN, including selective depletion and restoration of SMN in motor neurons. To determine whether the cortical neuronal populations expressing the Emx-1 promoter are involved in SMA pathology, we generated a novel SMA mouse model in which SMN expression was specifically induced in Emx-1 expressing cortical neurons utilizing an Emx-1-Cre transgene. While SMN expression was robust in the central nervous system as expected, SMA mice did not live longer. Weight and time-to-right motor function were not significantly improved.

Development and characterization of an SMN2-based intermediate mouse model of Spinal Muscular Atrophy.

Spinal Muscular Atrophy (SMA) is due to the loss of the survival motor neuron gene 1 (SMN1), resulting in motor neuron (MN) degeneration, muscle atrophy and loss of motor function. While SMN2 encodes a protein identical to SMN1, a single nucleotide difference in exon 7 causes most of the SMN2-derived transcripts to be alternatively spliced resulting in a truncated and unstable protein (SMNΔ7). SMA patients retain at least one SMN2 copy, making it an important target for therapeutics. Many of the existing SMA models are very severe, with animals typically living less than 2 weeks. Here, we present a novel intermediate mouse model of SMA based upon the human genomic SMN2 gene. Genetically, this model is similar to the well-characterized SMNΔ7 model; however, we have manipulated the SMNΔ7 transgene to encode a modestly more functional protein referred to as SMN read-through (SMN(RT)). By introducing the SMN(RT) transgene onto the background of a severe mouse model of SMA (SMN2(+/+);Smn(-/-)), disease severity was significantly decreased based upon a battery of phenotypic parameters, including MN pathology and a significant extension in survival. Importantly, there is not a full phenotypic correction, allowing for the examination of a broad range of therapeutics, including SMN2-dependent and SMN-independent pathways. This novel animal model serves as an important biological and therapeutic model for less severe forms of SMA and provides an in vivo validation of the SMN(RT) protein.

Partial restoration of cardio-vascular defects in a rescued severe model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a leading genetic cause of infantile death. Loss of a gene called Survival Motor Neuron 1 (SMN1) and, as a result, reduced levels of the Survival Motor Neuron (SMN) protein leads to SMA development. SMA is characterized by the loss of functional motor neurons in the spinal cord. However, accumulating evidence suggests the contribution of other organs to the composite SMA phenotype and disease progression. A growing number of congenital heart defects have been identified in severe SMA patients. Consistent with the clinical cases, we have recently identified developmental and functional heart defects in two SMA mouse models, occurring at embryonic stage in a severe SMA model and shortly after birth in a less severe model (SMN∆7). Our goal was to examine the late stage cardiac abnormalities in untreated SMN∆7 mice and to determine whether gene replacement therapy restores cardiac structure/function in rescued SMN∆7 model. To reveal the extent of the cardiac structural/functional repair in the rescued mice, we analyzed the heart of untreated and treated SMN∆7 model using self-complementary Adeno-associated virus (serotype 9) expressing the full-length SMN cDNA. We examined the characteristics of the heart failure such as remodeling, fibrosis, oxidative stress, and vascular integrity in both groups. Our results clearly indicate that fibrosis, oxidative stress activation, vascular remodeling, and a significant decrease in the number of capillaries exist in the SMA heart. The cardiac structural defects were improved drastically in the rescued animals, however, the level of impairment was still significant compared to the age-matched wildtype littermates. Furthermore, functional analysis by in vivo cardiac magnetic resonance imaging (MRI) revealed that the heart of the treated SMA mice still exhibits functional defects. In conclusion, cardiac abnormalities are only partially rescued in post-birth treated SMA animals and these abnormalities may contribute to the premature death of vector-treated SMA animals with seemingly rescued motor function but an average life span of less than 70 days as reported in several studies.

Direct central nervous system delivery provides enhanced protection following vector mediated gene replacement in a severe model of spinal muscular atrophy.

Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). SMA, however, is not due to complete absence of SMN, rather a low level of functional full-length SMN is produced by a nearly identical copy gene called SMN2. Despite SMN's ubiquitous expression, motor neurons are preferentially affected by low SMN levels. Recently gene replacement strategies have shown tremendous promise in animal models of SMA. In this study, we used self-complementary Adeno Associated Virus (scAAV) expressing full-length SMN cDNA to compare two different routes of viral delivery in a severe SMA mouse model. This was accomplished by injecting scAAV9-SMN vector intravenously (IV) or intracerebroventricularly (ICV) into SMA mice. Both routes of delivery resulted in a significant increase in lifespan and weight compared to untreated mice with a subpopulation of mice surviving more than 200days. However, the ICV injected mice gained significantly more weight than their IV treated counterparts. Likewise, survival analysis showed that ICV treated mice displayed fewer early deaths than IV treated animals. Collectively, this report demonstrates that route of delivery is a crucial component of gene therapy treatment for SMA.

Decreasing disease severity in symptomatic, Smn(-/-);SMN2(+/+), spinal muscular atrophy mice following scAAV9-SMN delivery.

Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disorder, is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). In humans, a nearly identical copy gene is present, SMN2. SMN2 is retained in all SMA patients and encodes the same protein as SMN1. However, SMN1 and SMN2 differ by a silent C-to-T transition at the 5' end of exon 7, causing alternative splicing of SMN2 transcripts and low levels of full-length SMN. SMA is monogenic and therefore well suited for gene-replacement strategies. Recently, self-complementary adeno-associated virus (scAAV) vectors have been used to deliver the SMN cDNA to an animal model of disease, the SMNΔ7 mouse. In this study, we examine a severe model of SMA, Smn(-/-);SMN2(+/+), to determine whether gene replacement is viable in a model in which disease development begins in utero. Using two delivery paradigms, intracerebroventricular injections and intravenous injections, we delivered scAAV9-SMN and demonstrated a two to four fold increase in survival, in addition to improving many of the phenotypic parameters of the model. This represents the longest extension in survival for this severe model for any therapeutic intervention and suggests that postsymptomatic treatment of SMA may lead to significant improvement of disease severity.

Delivery of therapeutic agents through intracerebroventricular (ICV) and intravenous (IV) injection in mice.

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.