Basic Biology II
Mutations in one of the duplicated Survival of Motor Neuron (SMN) genes lead to the progressive loss of motor neurons and subsequent development of Spinal Muscular Atrophy (SMA), a common, usually fatal, pediatric disease. Recent experimental evidence strongly supports the notion that reduced levels of SMN result in a general pre-mRNA splicing defect. The proposed study tests the hypothesis that low levels of SMN result in perturbed pre-mRNA splicing and mRNA transcript levels during motor neuron differentiation, growth, and sprouting, thus reducing the life span of motor neurons as is characteristic for SMA. To test this hypothesis, we plan to use high throughput sequencing technologies to evaluate gene expression and alternative splicing profiles throughout motor neurons differentiation of stem cells derived from normal or SMA patients. These genome-wide surveys will be complemented with experimental verification and computational analyses to gain insights into fluctuations of pre-mRNA splicing networks during the differentiation of stem cells into motor neurons. The results from the proposed experiments will provide expression profiles and an alternative splicing maps that will permit to determine how motor neuron differentiation is perturbed in SMA patients and perhaps other neurological diseases. The verification of the hypothesis will provide new insights into the biological processes that specify longevity of motor neurons and direct future research to identify alternative targets for SMA therapy.
Statement of Benefit to California:
Recent experiments demonstrated that pre-mRNA splicing is required to establish gene expression profiles that dictate the longevity of developing motor neurons. The ability to differentiate human embryonic stem cells into motor neurons in cell culture permits an evaluation of gene expression and alternative pre-mRNA splicing throughout the differentiation process. This unique opportunity is available because the state of California has actively supported research using human embryonic stem cells. The results obtained from this proposal will establish gene expression and alternative splicing maps that link defined developmental events with gene expression and motor neuron differentiation. The resulting signature profiles will enable future studies investigating motor neuron longevity and they will likely identity new molecular targets to battle various motor neuron diseases that have in common premature motor neuron death. As such, the information gained from the proposed experiments would maintain California’s leading status in stem cell research and may provide the intellectual framework for the development of new therapeutic developments in the private sector. An additional benefit to California citizens could be the availability of cutting edge technologies in clinical trials carried out at California's research centers.
EXECUTIVE SUMMARY This proposal focuses on spinal muscular atrophy (SMA), a common human motor neuron disease with a known genetic basis. This disease is caused by mutations in the Survival of Motor Neuron 1 (SMN1) protein and is characterized by the degeneration of motor neurons. Recent data supports the idea that the loss of SMN1 protein leads to aberrant mRNA splicing, which eventually causes motor neuron death by an unknown mechanism. The applicant proposes to investigate potential mechanisms for motor neuron loss in SMA using patient-derived induced pluripotent stem cells (iPSCs) and genome-wide sequencing technologies. In Aim 1, the applicant plans to examine variations in RNA splicing and gene expression at various stages during the differentiation of normal and SMA patient-derived iPSCs into motor neurons. In Aim 2, the applicant proposes to determine whether expression of mRNA splicing regulators is altered in motor neurons differentiated from SMA patient-derived iPSCs. Reviewers agreed that the potential significance of this proposal is high, as SMA is a common and devastating disease. However, they were not convinced that the proposed approach would have a major impact in the field since the work will focus on identifying differential features in gene expression and RNA splicing rather than uncovering or understanding a biological mechanism. A broad body of prior work, from this group and from others, already demonstrates that the SMA gene product is involved in the splicing process. A defective SMA protein will likely generate an extensive repertoire of aberrant gene transcripts and it was unclear to some reviewers how identification of multiple aberrant transcripts would shed light on the basic biology of motor neuron development or on the pathology of SMA. Reviewers were unsure how the applicant would make the analytic leap from descriptive dataset to mechanistic insight. Reviewers found the research plan to be simply designed and based on sound preliminary data. However, they raised a few concerns that detracted from their enthusiasm. The first concern was that the plan is narrowly focused. Reviewers noted that SMA is a heterogeneous disease, with patients classified into three disease types corresponding to disease severity and age of onset; this was not addressed by the applicant. Reviewers felt that the proposal would be stronger had it included study of iPSCs derived from patients of all three types of SMA. The reviewers’ second major concern was the feasibility of the research plan. The stages of motor neuron differentiation, particularly the intermediate stages, may have a substantial degree of cell type heterogeneity that would render the interpretation of genome-wide sequencing data problematic. Methods to assess the purity of stage-specific samples were not presented, nor were alternative approaches that consider the problem of heterogeneity during differentiation. Finally, reviewers noted that if the SMN1 mutation alters the rate of differentiation or affects apoptosis it may be difficult to determine if differences in splicing are due to the direct effects of SMN1 deficiency or indirect effects on the population of cells analyzed. In other words, the proposed experiments may not allow for distinguishing passengers from drivers in the etiology of SMA. The reviewers agreed that the Principal Investigator (PI) has an excellent track record and extensive expertise in RNA splicing. They appreciated the presence of a key collaborator to assist with the hESC work. In general, they found the research team well-suited to carry out the proposed studies. Overall, while reviewers appreciated the significance of the proposed research and praised the PI and research team, concerns regarding the potential impact and the research plan design and feasibility detracted from their enthusiasm.