Tissue-specific variation in nonsense mutant transcript level and drug-induced read-through efficiency in the Cln1(R151X) mouse model of INCL.

About 10% of inherited diseases are caused by nonsense mutations [Trends Mol Med 18 (2012) 688], and nonsense suppression drug therapy promoting translation through premature stop codons is an emerging therapeutic approach. Infantile neuronal ceroid lipofuscinosis (INCL), a childhood neurodegenerative disease, results from mutations in the CLN1 gene encoding the lysosomal enzyme, palmitoyl-protein thioesterase 1 (PPT1) [Biochim Biophys Acta 1832 (2013) 1806, Hum Mutat (2012) 63, Biochim Biophys Acta 1832 (2013) 1881]. The nonsense mutation p.R151X is the most common disease-causing CLN1 mutation Hum Mutat (2012) 63. In the novel Cln1(R151X) mouse model of INCL, we found large, tissue-specific variations in Cln1(R151X) mRNA level and PPT1 residual enzyme activity. These tissue-specific differences strongly influenced the read-through efficiency of ataluren (PTC124), a well-known nonsense suppression drug. A two-day treatment with ataluren (10 mg/kg) increased PPT1 enzyme activity in the liver and muscle, but not in any other tissue examined. Our study identifies a new challenge/hurdle for read-through drug therapy: variable efficiency of read-through therapy in the different tissues/organs because of tissue-specific variations in nonsense mutant transcript levels.

The novel Cln1(R151X) mouse model of infantile neuronal ceroid lipofuscinosis (INCL) for testing nonsense suppression therapy.

The neuronal ceroid lipofuscinoses (NCLs), also known as Batten disease, are a group of autosomal recessive neurodegenerative disorders in children characterized by the progressive onset of seizures, blindness, motor and cognitive decline and premature death. Patients with mutations in CLN1 primarily manifest with infantile NCL (INCL or Haltia-Santavuori disease), which is second only to congenital NCL for its age of onset and devastating progression. CLN1 encodes a lysosomal enzyme, palmitoyl-protein thioesterase 1 (PPT1). Nonsense mutations in CLN1 account for 52.3% of all disease causing alleles in infantile NCL, the most common of which worldwide is the p.R151X mutation. Previously, we have shown how nonsense-mediated decay is involved in the degradation of CLN1 mRNA transcripts containing the p.R151X mutation in human lymphoblast cell lines. We have also shown how the read-through drugs gentamicin and ataluren (PTC124) increase CLN1 (PPT1) enzyme activity. Here, we provide the initial characterization of the novel Cln1(R151X) mouse model of infantile neuronal ceroid lipofuscinosis that we have generated. This nonsense mutation model recapitulates the molecular, histological and behavioral phenotypes of the human disease. Cln1(R151X) mice showed a significant decrease in Cln1 mRNA level and PPT1 enzyme activity, accumulation of autofluorescent storage material, astrocytosis and microglial activation in the brain. 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 tests. We also show how the read-through compound ataluren (PTC124) increases PPT1 enzyme activity and protein level in Cln1(R151X) mice in a proof-of-principle study.

Nonsense-mediated decay in genetic disease: friend or foe?

Eukaryotic cells utilize various RNA quality control mechanisms to ensure high fidelity of gene expression, thus protecting against the accumulation of nonfunctional RNA and the subsequent production of abnormal peptides. Messenger RNAs (mRNAs) are largely responsible for protein production, and mRNA quality control is particularly important for protecting the cell against the downstream effects of genetic mutations. 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 these aberrant transcripts, NMD acts to prevent the production of truncated proteins that could otherwise harm the cell through various insults, such as dominant negative effects or the ER stress response. Although NMD functions to protect the cell against the deleterious effects of aberrant mRNA, 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 that NMD plays in genetic disease can undermine current therapeutic strategies aimed at increasing the production of full-length functional protein from genes harboring nonsense mutations. Here, we review the normal function of this RNA surveillance pathway and how it is regulated, provide current evidence for the role that it plays in modulating genetic disease phenotypes, and how NMD can be used as a therapeutic target.

A novel c.776_777insA mutation in CLN1 leads to infantile neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses are the most common autosomal recessive neurodegenerative disorders in children, with a worldwide incidence of 1 in 100,000 live births. Multiple clinical variants are caused by more than 400 mutations in at least 14 different genes. These progressive genetic disorders primarily manifest in the central nervous system because of an extensive loss of neurons, specifically in the cerebral and cerebellar cortices. Patients with mutations in CLN1, which encodes palmitoyl-protein thioesterase 1 (PPT1), primarily manifest with infantile neuronal ceroid lipofuscinosis (Haltia-Santavuori disease). Affected children usually present between 1 and 2 years of age and typically die by 8 to 13 years of age. We describe a patient with infantile neuronal ceroid lipofuscinosis with a novel c.776_777insA mutation in CLN1. This insertion induces a frameshift and a premature stop codon late within the CLN1 messenger RNA (mRNA) transcript which is likely recognized by nonsense-mediated translation repression, decreasing PPT1 abundance.

A novel c.1135_1138delCTGT mutation in CLN3 leads to juvenile neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinosis is the most common childhood neurodegenerative disorder in the world, with an incidence of 1 in 100,000 live births. More than 400 mutations in at least 14 different genes are linked to multiple clinical variants. These progressive genetic disorders primarily manifest in the central nervous system due to an extensive loss of neurons, primarily in the cerebral and cerebellar cortices. Juvenile neuronal ceroid lipofuscinosis is the most common form and is primarily due to mutations in CLN3, which encodes a protein of unknown function. The most common such mutation in CLN3 is a 1.02-kb deletion that results in a frameshift and subsequent premature termination codon. Here we describe a patient with juvenile neuronal ceroid lipofuscinosis who has a novel c.1135_1138delCTGT mutation in CLN3. This deletion induces a frameshift and premature termination codon in CLN3 messenger ribonucleic acid that is likely recognized by nonsense-mediated decay and degraded, subsequently leading to decreased CLN3 protein abundance.

The role of nonsense-mediated decay in neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinosis (NCL), commonly referred to as Batten disease, is 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 14 different genes, leading to five overlapping clinical variants of NCL. A large portion of these mutations lead to premature stop codons (PTCs) and are predicted to predispose mRNA transcripts to nonsense-mediated decay (NMD). Nonsense-mediated decay is associated with a number of other genetic diseases and is an important regulator of disease pathogenesis. We contend that NMD targets PTCs in NCL gene transcripts for degradation. A number of PTC mutations in CLN1, CLN2 and CLN3 lead to a significant decrease in mRNA transcripts and a corresponding decrease in protein levels and function in patient-derived lymphoblast cell lines. Inhibiting NMD leads to an increased transcript level, and where protein function is known, increased activity. Treatment with read-through drugs also leads to increased protein function. Thus, NMD provides a promising therapeutic target that would allow read-through of transcripts to enhance protein function and possibly ameliorate Batten disease pathogenesis.