Nonsense-mediated decay and neuronal ceroid lipofuscinosis: Utilizing nonsense suppression as a therapeutic tool to enhance functional protein activity

Nonsense-mediated decay (NMD) is an evolutionarily conserved mRNA quality control system in all eukaryotes that degrades transcripts containing premature termination codons (PTCs). By degrading aberrant mRNA transcripts, NMD acts to prevent the production of truncated proteins that could otherwise harm the cell through various insults. There is a growing body of evidence that mutation-, codon-, gene-, cell-, and tissue-specific differences in NMD efficiency can alter the underlying pathology of genetic disease. In addition, the protective role NMD plays in genetic disease could undermine current therapeutic strategies aimed at increasing the production of full-length functional protein from genes harboring PTC's.;The Neuronal ceroid lipofuscinoses (NCLs), commonly referred to as Batten disease, are a group of autosomal recessive neurodegenerative diseases of childhood characterized by seizures, blindness, motor and cognitive decline, and premature death. Currently, there are over 400 known mutations in fourteen different genes that lead to five overlapping clinical variants of NCL. A large portion of these mutations lead to PTC's that are predicted to predispose mRNA transcripts to NMD. We have shown how NMD targets PTC's in the NCL gene transcripts CLN1, CLN2 and CLN3 for degradation. Inhibiting NMD leads to increased transcript level of these genes, and where protein function is known, increased activity. Treatment with read-through drugs also leads to increased protein function in these cell lines.;To further investigate read-through therapy in NCL, we generated and characterized the Cln1R151X mouse model of infantile neuronal ceroid lipofuscinosis (INCL). This model recapitulates the molecular, histological and behavioral phenotype of infantile NCL. Molecular characterization of Cln1R151X mice showed a significant decrease in Cln1 mRNA abundance and PPT1 enzyme activity. This model also shows many histopathological disease features typical of NCL. Behavioral characterization of Cln1 R151X mice at 3 and 5 months of age revealed significant motor deficits as measured by the vertical pole and rotarod test. We also show how the read-through compound ataluren increases PPT1 enzyme activity in Cln1R151X mice, providing additional evidence for the use of read-through drugs in human INCL patients.;In this thesis, I will first review the normal function and regulation of the NMD surveillance pathway, provide current evidence for the role that it plays in modulating genetic disease phenotypes, and also review how NMD can be used as a therapeutic target. Second, I will show how NMD is involved in the degradation of NCL gene transcripts harboring nonsense mutations in human lymphoblast cell lines, and how read-through drugs can increase protein function. Lastly, I will show how NMD is involved in the disease pathogenesis of the novel Cln1R151X mouse model, which recapitulates infantile NCL, and how the read-through drug ataluren increases PPT1 enzyme activity and protein level in both the brain and liver.