Sylvia Szucs

Knock‐out mouse for Canavan disease: a model for gene transfer to the central nervous system

Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by deficiency of aspartoacylase (ASPA) and increased levels of N‐acetylaspartic acid (NAA) in brain and body fluids, severe mental retardation and early death. Gene therapy has been attempted in a number of children with CD. The lack of an animal model has been a limiting factor in developing vectors for the treatment of CD. This paper reports the successful creation of a knock‐out mouse for Canavan disease that can be used for gene transfer.

A Single Intravenous rAAV Injection as Late as P20 Achieves Efficacious and Sustained CNS Gene Therapy in Canavan Mice

Canavans disease (CD) is a fatal pediatric leukodystrophy caused by mutations in aspartoacylase (AspA) gene. Currently, there is no effective treatment for CD; however, gene therapy is an attractive approach to ameliorate the disease. Here, we studied progressive neuropathology and gene therapy in short-lived (≤ 1 month) AspA(-/-) mice, a bona-fide animal model for the severest form of CD. Single intravenous (IV) injections of several primate-derived recombinant adeno-associated viruses (rAAVs) as late as postnatal day 20 (P20) completely rescued their early lethality and alleviated the major disease symptoms, extending survival in P0-injected rAAV9 and rAAVrh8 groups to as long as 2 years thus far. We successfully used microRNA (miRNA)-mediated post-transcriptional detargeting for the first time to restrict therapeutic rAAV expression in the central nervous system (CNS) and minimize potentially deleterious effects of transgene overexpression in peripheral tissues. rAAV treatment globally improved CNS myelination, although some abnormalities persisted in the content and distribution of myelin-specific and -enriched lipids. We demonstrate that systemically delivered and CNS-restricted rAAVs can serve as efficacious and sustained gene therapeutics in a model of a severe neurodegenerative disorder even when administered as late as P20.

Expression of glutamate transporter, GABRA6, serine proteinase inhibitor 2 and low levels of glutamate and GABA in the brain of knock-out mouse for Canavan disease.

Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by spongy degeneration of the brain. The clinical features of CD are hypotonia, megalencephaly, and mental retardation leading to early death. While aspartoacylase (ASPA) activity increases with age in the wild type mouse brain, there is no ASPA activity in the CD mouse brain. So far ASPA deficiency and elevated NAA have been ascribed with the CD. Other factors affecting the brain that result from ASPA deficiency may lead pathophysiology of CD. The NMR spectra and amino acid analysis showed lower levels of glutamate and gamma-aminobutyric acid in the CD mouse brain compared to the wild type. Microarray gene expression on CD mouse brain showed glutamate transporter-EAAT4 and gamma-aminobutyric acid-A receptor, subunit alpha6 (GABRA6) were lower 9.7- and 119.1-fold, respectively. Serine proteinase inhibitor 2 (Spi2) was 29.9-fold higher in the CD mouse brain compared to the wild type. The decrease of GABRA6 and high expression of Spi2 in CD mouse brain were also confirmed by real-time RT-PCR. This first report showing abnormal expression of EAAT4, GABRA6, Spi2 combined with lower levels of glutamate and GABA are likely to be associated with the pathophysiology of CD.

Modification of aspartoacylase for potential use in enzyme replacement therapy for the treatment of Canavan disease

Canavan disease is a fatal neurological disease without any effective treatments to slow the relentless progress of this disorder. Enzyme replacement therapy has been used effectively to treat a number of metabolic disorders, but the presence of the blood-brain-barrier presents an additional challenge in the treatment of neurological disorders. Studies have begun with the aim of establishing a treatment protocol that can effectively replace the defective enzyme in Canavan disease patients. The human enzyme, aspartoacylase, has been cloned, expressed and purified, and the surface lysyl groups modified through PEGylation. Fully active modified enzymes were administered to mice that are defective in this enzyme and that show many of the symptoms of Canavan disease. Statistically significant increases in brain enzyme activity levels have been achieved in this animal model, as well as decreases in the elevated substrate levels that mimic those found in Canavan disease patients. These results demonstrate that the modified enzyme is gaining access to the brain and functions to correct this metabolic defect. The stage is now set for a long term study to optimize this enzyme replacement approach for the development of a treatment protocol.

Neuronal localization of the mitochondrial protein NIPSNAP1 in rat nervous system

The NIPSNAP (4‐nitrophenylphosphatase domain and non‐neuronal SNAP25‐like protein homolog 1) proteins belong to a highly conserved family of proteins of unknown function. We found that NIPSNAP1 binds to the branched‐chain α‐keto acid (BCKA) dehydrogenase enzyme complex, which is disrupted in maple syrup urine disease, a disease of branched‐chain amino acid catabolism that results in neurological dysfunction. Phenylketonuric (PKU) and epileptic mice show altered expression of NIPSNAP1 in the brain. Therefore, the distribution and localization of NIPSNAP1 in rat brain was determined. Results show that NIPSNAP1 is expressed exclusively in neurons including pyramidal neurons in the cerebral cortex, Purkinje neurons in the cerebellum and motor neurons in the spinal cord. Dopaminergic neurons in midbrain and noradrenergic neurons in the brainstem, which are affected in PKU, also express NIPSNAP1. NIPSNAP1 is found to be localized in the mitochondrial matrix and can bind dihydrolipoyl‐transacylase and ‐transacetylase components of the BCKA and pyruvate dehydrogenase complexes in vitro. Our data provide the first experimental evidence for a strictly neuronal expression of this mitochondrial protein in the rat nervous system.

Metabolic changes in the knockout mouse for Canavan's disease: implications for patients with Canavan's disease.

Canavans disease is an autosomal recessive disorder caused by aspartoacylase deficiency, which leads to accumulation of N-acetylaspartic acid in the brain and blood and an elevated level of N-acetylaspartic acid in the urine. The brain of patients with Canavans disease shows spongy degeneration. How the enzyme deficiency and elevated N-acetylaspartic acid cause the pathophysiology observed in Canavans disease is not obvious. The creation of a knockout mouse for Canavans disease is being used as a tool to investigate metabolic pathways in the mouse and correlate them with the patients with Canavans disease. The level of glutamate is lower in the knockout mouse brain than in the wild-type mouse brain, similar to what we have found in children with Canavans disease, and so are the levels of γ-aminobutyric acid (GABA). The level of aspartate is higher in the Canavans disease mouse brain. The activity of aspartate aminotransferase, an enzyme involved in the malate-aspartate shuttle, is lower in the Canavans disease mouse brain. The lower weight of the Canavans disease mouse was in direct proportion to low total-body fat and bone mineral density. These changes might be similar to what is seen in patients with Canavans disease and could have therapeutic implications. (J Child Neurol 2003;18:611—615).