Talk Abstracts

Talk on Friday 04:15-04:30pm submitted by Geneva LaForce

Pre-clinical models of PCH10 link CLP1 p.R140H mutation with mRNA misprocessing in motor neuron degeneration

Geneva R. LaForce (Department of Genetics and Genome Sciences, Case Western Reserve University), Jordan S. Farr, Cydni Akesson (Department of Genetics and Genome Sciences, Case Western Reserve University), Evren Gumus (Department of Medical Genetics, Faculty of Medicine, University of Harran), Eric J. Wagner (Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston), Polyxeni Philippidou (Department of Neurosciences, Case Western Reserve University), Ashleigh E. Schaffer (Department of Genetics and Genome Sciences, Case Western Reserve University)

Abstract:
Pontocerebellar Hypoplasia Type 10 (PCH10) is a pediatric neurodegenerative disorder caused by a homozygous p.R140H mutation in CLP1 that leads to hypoplasia of the cerebellum and brainstem with motor neuron degeneration^1,2. Here, we present new clinical data from PCH10 patients showing severe frontotemporal cortical and motor neuron degeneration, with minor cerebellar atrophy. These findings suggest phenotypic variability in the penetrance of brain, but not motor neuron features. To date, the pathogenic mechanism of neurodegeneration in PCH10 is unknown. To study the disease pathogenesis of PCH10 in vivo, we developed a mouse model harboring the p.R140H mutation. Mutant mice present with spasticity and seizures. Analysis of the brain revealed no change in cerebellar morphology or cell density, but a decrease in cell density in the anterior isocortex in CLP1 mutant mice compared to controls. Further, we found smaller neuromuscular junctions without overt changes in phrenic motor neuron innervation in the diaphragms of mutant mice. These findings are consistent with the described clinical features of PCH10. To determine the molecular consequence of CLP1 p.R140H in motor neuron disease pathogenesis, we generated iPSC-derived motor neurons from PCH10 patients and an unaffected relative. Characterization of mature motor neurons revealed a phrenic-like identity. During differentiation, we found reduced cell density in PCH10 motor neurons compared to control motor neurons, possibly akin to motor neuron degeneration. CLP1 is a multifunctional RNA kinase important for tRNA and mRNA maturation³. To determine if tRNA or mRNA processing is altered in PCH10 motor neurons, Northern blot and RNA-sequencing analysis of iPSC-derived motor neurons found defects in mRNA processing resulting in differential gene expression, alternative splicing events, and alternative polyadenylation, without changes in tRNA biogenesis. Our data expand the clinical spectrum of PCH10, develop accurate pre-clinical models for the disease, and link p.R140H CLP1 mutation with dysregulation of mRNA processing leading to motor neuron degeneration.

References:
1. Schaffer, A.E. et al. CLP1 founder mutation links tRNA splicing and maturation to cerebellar development and neurodegeneration. Cell 157, 651-63 (2014).
2. Karaca, E. et al. Human CLP1 mutations alter tRNA biogenesis, affecting both peripheral and central nervous system function. Cell 157, 636-50 (2014).
3. Paushkin, S.V., Patel, M., Furia, B.S., Peltz, S.W. & Trotta, C.R. Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 3' end formation. Cell 117, 311-21 (2004).

Keywords: CLP1, Pontocerebellar Hypoplasia Type 10, Neurodegeneration