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. Homozygous absence of the telomeric copy (SMN1) correlates with development of SMA because differential splicing of the centromeric copy (SMN2) predominantly produces a biologically inactive protein isoform. The low amounts of SMN produced from SMN2 are adequate for a fetus to develop, but are insufficient to maintain healthy motor neurons throughout life. One of the biological functions of SMN is to catalyze the biosynthesis of components of the spliceosome, an enzymatic machinery that removes introns from pre-mRNAs. Indeed using animal models it was demonstrated that purified building blocks of the spliceosome are sufficient to rescue developmental defect caused by reduced levels of SMN. These experiments demonstrated that elevated levels of the splicing machinery are essential for proper differentiation of stem cells into motor neurons. Based on these observations we hypothesize that alternative pre-mRNA splicing of several genes set in stone a developmental pathway that imprints unique gene expression features throughout motor neuron differentiation and maintenance. In this application we propose to test this hypothesis by analyzing genome-wide alternative pre-mRNA splicing during the differentiation of human embryonic stem cells into motor neurons. 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 and other neurological disease 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 in part because the state of California actively supports 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.
SYNOPSIS: This is a proposal from Klemens Hertel at UC Irvine to test the hypothesis that alternative splcing events or cascades carried out early during differentiation from stem cells dictate the the fate and longevity of motoneurons. The experiments will involve Real-time PCR and microarray assessments of hESC cells that are being differentiated into motoneurons. The work will be carried out in conjunction with the UC Irvine Stem Cell Centers that will provide the cells for the analysis, derived mostly from the cell lines isolated by Doug Melton at Harvard. SIGNIFICANCE AND INNOVATION: This is an important and innovative proposal. Although the cause of spinal muscular atrophy (SMA) is well known to be due to an abnormal gene, the mechanisms controlling expression of motoneuronal degeneration is not well-understood. The applicant, who holds an NIH grant to study alternative splicing mechanisms of mRNA, proposes a very interesting hypothesis that alternative splicing contributes to the fate and longevity of motoneurons. If true, this will not only provide significant therapeutic targets for SMA but potentially also for many other diseases. The study of the mRNA of hESC cells during their differentiation and maturation into motoneurons is the logical and best way to test this hypothesis. SMA is the second most common autosomal recessive disease (with a carrier frequency of 1 in 50) and the leading cause of inherited infant mortality. For this reason, clues as to how mutations in SMN1 cause SMA are important in understanding the disease and its potential treatment. A recent report demonstrated that motor neuron degeneration in zebrafish caused by low levels of SMN is rescued by an injection of purified snRNPs suggesting that a defect in the pre-mRNA splicing from an SMN1 mutation perturbs the normal stem cell differentiation into motor neurons. For this reason, it is important to clarify the role of pre-mRNA splicing in the normal differentiation of hESCs into motor neurons. This knowledge may also be important in improving methods to efficiently and accurately differentiate hESCs into motor neurons. The focus of this proposal on the role of pre-mRNA splicing on hESC differentiation into motor neurons is a novel one. STRENGTHS: This is a well-written application with a feasible experimental plan that tests an exciting new hypothesis for a devastating genetic disease. The use of hESCs rather than mouse SCs is of special value in studies of SMA since there is only one SMN gene in mice. The PI is an experienced investigator with a strong track record of productivity and has assembled excellent experienced collaborators: Hans Keirstead, Center for Genomics Microarray Facility, Stem Cell Center. Other strengths of the proposal are the following. The proposal has a novel focus on the potential role of alternative pre-mRNA splicing on motor neuron differentiation from hESCs. There is interesting and informative description of alternative pre-mRNA splicing during motor neuron differentiation from hESCs - it may be that the products of certain mRNA isoforms will be useful in protocols for hESC differentiation. WEAKNESSES: The rationale for looking at an alteration in the accuracy of pre-mRNA splicing during the differentiation of motor neurons from hESCs was not clear. The PI may find a correlation between isoforms and differentiation states, but the planned studies will not be able to make a convincing case for a cause-effect relationship between alternative splicing and motor neuron differentiation from hESCs. It would have been valuable for the PI to have designed experiments to more directly see the effect of altering or decreasing splicing on motor neuron differentiation. What would happen if there was a knock-down in SMN in hESC differentiation and would this be rescued by snRNPs? It would have been helpful if the PI had provided anticipated pitfalls and plans to overcome difficulties that might arise. DISCUSSION: While it is valuable to look at different isoforms during differentiation, it is unclear what the rationale is for this proposal. It is clear that 70% of genes are alternatively spliced, but whether differences in splicing are related to differentiation in unknown. Reviewers questioned what is the basis for the hypothesis? Despite the zebrafish experiment with SMN1 defects and rescue by injecting purified snRNP, there doesn't appear to be a convincing case presented for alternative splicing being involved in motor neuron differentiation. Is there a test for cause and effect? SMA could be a disease of mRNA trafficking as opposed to splicing. In particular, this grant has essentially nothing to do with SMA and is not really a disease-related grant; thus the enthusiasm was tempered. Reviewers didn't understand the relevance of studying the UBAS2 gene (which has no real splice regulation?) to see if different isoforms would be maintained in different states of differentiation.