Aspartoacylase-LacZ Knockin Mice: An Engineered Model of Canavan Disease

Nadine Mersmann,Wiebke Möbius,Wolfgang Weber-Fahr,Dmitri Tkachev,Torben Ruhwedel,Sabine Rühle,Philipp Thomas Röth,Ruth Jelinek,Alexander Sartorius,Matthias Klugmann

doi:10.1371/journal.pone.0020336

Abstract

Canavan Disease (CD) is a recessive leukodystrophy caused by loss of function mutations in the gene encoding aspartoacylase (ASPA), an oligodendrocyte-enriched enzyme that hydrolyses N-acetylaspartate (NAA) to acetate and aspartate. The neurological phenotypes of different rodent models of CD vary considerably. Here we report on a novel targeted aspa mouse mutant expressing the bacterial β-Galactosidase (lacZ) gene under the control of the aspa regulatory elements. X-Gal staining in known ASPA expression domains confirms the integrity of the modified locus in heterozygous aspa lacZ-knockin (aspalacZ/+) mice. In addition, abundant ASPA expression was detected in Schwann cells. Homozygous (aspalacZ/lacZ) mutants are ASPA-deficient, show CD-like histopathology and moderate neurological impairment with behavioural deficits that are more pronounced in aspalacZ/lacZ males than females. Non-invasive ultrahigh field proton magnetic resonance spectroscopy revealed increased levels of NAA, myo-inositol and taurine in the aspalacZ/lacZ brain. Spongy degeneration was prominent in hippocampus, thalamus, brain stem, and cerebellum, whereas white matter of optic nerve and corpus callosum was spared. Intracellular vacuolisation in astrocytes coincides with axonal swellings in cerebellum and brain stem of aspalacZ/lacZ mutants indicating that astroglia may act as an osmolyte buffer in the aspa-deficient CNS. In summary, the aspalacZ mouse is an accurate model of CD and an important tool to identify novel aspects of its complex pathology.

Highlights

Aspartoacylase (ASPA) deacetylates N-acetyl-aspartate (NAA) to produce acetate and L-aspartate
Disruption of ASPA expression in aspa-lacZ knockin mice Based on a knockout-first strategy that produces a knockout at the RNA processing level [11], targeting of the aspa locus in C57bl/6 embryonic stem (ES) cell was achieved by inserting the bgeo cassette flanked by frt sites into intron 1 of the intact aspa gene
Using a TaqMan probe binding to a downstream sequence, aspa mRNA levels were undetectable in aspalacZ/lacZ mice, while we observed a 60.464.3% (p = 0.0001) reduction of mRNA expression in aspalacZ/+ brains compared to controls (Fig. 1D)

Summary

Introduction

Aspartoacylase (ASPA) deacetylates N-acetyl-aspartate (NAA) to produce acetate and L-aspartate This enzyme is a marker of mature oligodendrocytes [1,2,3] and mutations of the aspa gene cause the fatal recessive leukodystrophy Canavan disease (CD) [4,5]. The monogenic nature of CD, and the lack of an effective treatment have provided the rationale for in vivo gene transfer into the CNS of patients and ASPA-deficient animals [7]. While these animal models generally reprise the pathological hallmarks observed in CD, they show substantial differences in disease severity and longevity [8,9,10]. The identification of all ASPA expression domains is essential to gain a comprehensive picture of the complex CD pathology and design effective therapies

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Results

Conclusion