Poster abstracts

Poster number 2 submitted by Anton Blatnik

Identification of Mutant Suppressors of SMN Loss of Function

Anton J Blatnik III (The Ohio State Biochemistry Program, Department of Biological Chemistry & Pharmacology, The Ohio State University Wexner Medical Center ), Vicki L. McGovern, Thanh T. Le, Chitra C. Iyer, Kaitlyn M. Corlett (Department of Biological Chemistry & Pharmacology, The Ohio State University Wexner Medical Center), Aurelie Massoni-Laporte, Narasimhan Madabusi (Department of Biological Chemistry & Pharmacology, The Ohio State University Wexner Medical Center), Shibi Likhite (Center for Gene Therapy, The Research Institute at Nationwide Childrens Hospital), Brian K. Kaspar (Center for Gene Therapy, The Research Institute at Nationwide Childrens Hospital; Department of Pediatrics, Department of Neuroscience , The Ohio State University Wexner Medical Center), Arthur H M Burghes (Department of Biological Chemistry & Pharmacology, The Ohio State University Wexner Medical Center)

Abstract:
Spinal Muscular Atrophy (SMA) is a pediatric neurodegenerative disorder caused by deletion or mutation of SMN1 and retention of SMN2, resulting in Survival of Motor Neuron (SMN) protein deficiency (1-3). A ubiquitously expressed, essential protein, how deficiency of SMN results in specific motoneuron loss remains illusive (3). SMN replacement therapies are proving clinically efficacious but downstream targets of SMN deficiency in motoneurons are not known (3-6). The SMN oligomeric complex functions in assembly of spliceosomal snRNPs, thus it is plausible splicing of motoneuron-specific pre-mRNAs is altered (3, 7-11). However, SMN has been proposed to have additional function in transport of particular mRNAs down axons (12-19). To determine how these two pathways contribute to SMA, we are identifying suppressors that restore function in both cases. In SMN axonal function, overexpression of HuD has been shown to rescue axonal defects in culture and zebrafish (17-19). We are constructing scAAV9-HuD to overexpress HuD in SMA mouse motoneurons and will assay for recovery of motor unit electrophysiology. In mice, we have found SMN missense mutants are nonfunctional in the absence of full length SMN (20-21). Therefore, to examine SMN function in snRNP assembly, we have developed a cell-based suppressor screen in which endogenous SMN can be conditionally deleted by activating Cre, whilst expressing SMN missense mutants. We are investigating mutants SMNE134K and SMNT274I—the former disrupts binding of the RG rich tails of SmD1 and SmD3 (22-23), whereas the function disrupted by the latter remains unknown (24-26). We have mutagenized these cells, screened >600 clones for deletion of SMN, and have obtained 15 growing lines. We have sequenced 5 of these lines for retention of SMNE134K mutant. No mutations were found in plausible targets SmD1 and SmD3. We have since sent these clones for whole genome sequencing to identify the mutant suppressor. Following identification, the mutant suppressor(s) will be confirmed by cloning it/them into lentivirus and adding back into the SMN E134K parental cell lines. Furthermore, mutant suppressors will be cloned into scAAV9 and tested in the SMN E134K mouse to determine contribution to SMA pathogenesis. This work will clarify the role each pathway plays in the motoneuron phenotype of SMA.

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Keywords: spinal muscular atrophy, missense mutations, mutant suppressor