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. 2011;6(5):e20336.
doi: 10.1371/journal.pone.0020336. Epub 2011 May 20.

Aspartoacylase-lacZ knockin mice: an engineered model of Canavan disease

Nadine Mersmann  1 , Dmitri Tkachev, Ruth Jelinek, Philipp Thomas Röth, Wiebke Möbius, Torben Ruhwedel, Sabine Rühle, Wolfgang Weber-Fahr, Alexander Sartorius, Matthias Klugmann
Affiliations

Aspartoacylase-lacZ knockin mice: an engineered model of Canavan disease

Nadine Mersmann et al. PLoS One. 2011.

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 (aspa(lacZ/+)) mice. In addition, abundant ASPA expression was detected in Schwann cells. Homozygous (aspa(lacZ/lacZ)) mutants are ASPA-deficient, show CD-like histopathology and moderate neurological impairment with behavioural deficits that are more pronounced in aspa(lacZ/lacZ) males than females. Non-invasive ultrahigh field proton magnetic resonance spectroscopy revealed increased levels of NAA, myo-inositol and taurine in the aspa(lacZ/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 aspa(lacZ/lacZ) mutants indicating that astroglia may act as an osmolyte buffer in the aspa-deficient CNS. In summary, the aspa(lacZ) mouse is an accurate model of CD and an important tool to identify novel aspects of its complex pathology.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of aspalacZ mice by homologous recombination.
A, Genomic structure of the murine aspa locus, which spans 6 exons (black boxes). Homologous recombination of the targeting vector inserts the βgeo cassette (blue box), encoding β-Galactosidase (lacZ), into intron 1 of the intact aspa gene. This cassette is flanked by frt-sites (green circles). Additional loxP sites flank exon 2 (red triangles). The vector includes a DTA cassette for negative selection. In the targeted allele, exon 1 is spliced to the splice acceptor site preceding βgeo, and transcription is terminated at the introduced pA site. The conditional aspaflox allele is produced by breeding aspalacZ mutants to FLP-deleter mice for recombination of the βgeo cassette in vivo. Note that frt and loxP sites are not drawn to scale. βgeo, β-galactosidase-neomycin resistance cassette; SA, splice acceptor; pA, polyadenylation site, DTA, Diphteria toxin gene. B, For Southern blot analysis genomic DNA was digested with SmaI/ApaI. The neo probe (white box in A) detected the expected 7.5 kb band in heterozygotes and homozygotes. C, Genomic PCR of littermates with primers 1 & 3 produces the expected amplicons in aspa+/+ (336 bp), aspalacZ/+ (387 bp and 336 bp) and aspalacZ/lacZ (387 bp) animals. The upstream loxP site was detected by PCR with primers 1 & 2 yielding a 307 bp band. D, Q-RT-PCR using a TaqMan probe for quantification of aspa mRNA levels in brains of aspa+/+, aspalacZ/+, and aspalacZ/lacZ mice (n = 3) confirms the attenuation of transcription downstream of exon 2 in the targeted allele. E, Representative Western blot of whole brain lysates of aspa +/+, aspalacZ/+ and aspalacZ/lacZ mice (aged 4 months, n = 3). The 37 kD protein ASPA was detected in aspa +/+ and aspalacZ/+ mice but not in aspalacZ/lacZ mutants. β-Galactosidase is expressed in aspalacZ/+ and aspalacZ/lacZ brain but not controls. α-Tubulin was used as loading control. F. Representative picture of a male aspalacZ/lacZ mutant (asterisk) and an aspa+/+ littermate at P70.
Figure 2
Figure 2. Analysis of aspa expression in CNS and periphery.
A–D, Representative pictures of β-Galactosidase staining in adult aspalacZ/+ mouse tissues show activity of the aspa gene in oligodendrocytes of white and grey matter throughout the brain. Abundant staining was detected in white matter tracts in cerebellum and corpus callosum. While in the hippocampus and cortex X-Gal staining was found in oligodendrocyte cell bodies (A), lacZ-positive fibres were detected in the thalamus (B). In the cerebellum, proximal processes and somata of white matter oligodendrocytes were stained (C, D). E–F, Outside the CNS, the cortex of the kidney (E), small intestine (F) and sciatic nerve fibres (G) show intense X-Gal staining. H–J, Confocal detection of ASPA immunoreactivity (H) in cerebellar white matter of an adult Plp-DsRed-1 transgenic mouse expressing the red fluorescent reporter protein (I) in oligodendrocytes . ASPA is expressed in somata and processes of oligodendrocytes. The pattern of ASPA-immunopositive cells matches the DsRed-expressing oligodendrocytes (J). K–M, Confocal detection of ASPA immunoreactivity in cytosolic domains of wildtype Schwann cells. ASPA is enriched at the paranode (asterisk) and bands of Cajal (arrow heads) and segregates from MBP. N, Q-PCR analysis of aspa mRNA levels in different tissues of adult aspa+/+ mice (n = 6). hc, hippocampus; ctx, cortex; med, medulla; tha, thalamus; wm, white matter. Bars: 500 µm in A,C,E; 200 µm in B, D; 400 µm in F,G; 20 µm in H–J; 10 µm in K–M.
Figure 3
Figure 3. CNS vacuolisation in aspalacZ/lacZ mutants.
Representative pictures of Nissl stained brain sections of aspa+/+ (A,C) and aspalacZ/lacZ (B,D) mice (4 months). Vacuoles are abundant in forebrain grey matter of cortex and thalamus while white matter of the corpus callosum is spared (B). In the hippocampus, the histopathology is exclusively seen in the pyramidal but not the dentate granule cell layer. Magnifications of thalamic areas show substantial spongy degeneration in the mutant (arrows in D) but not in the control (C). hc, hippocampus; cc, corpus callosum; ctx, cortex; dcl, granule cell layer; pcl, pyramidal cell layer; tha, thalamus. Bars: 400 µm in A,B; 200 µm in C,D.
Figure 4
Figure 4. Histopathology of the brain stem and cerebellum in aspalacZ/lacZ mice.
Caudal brain stem of controls (A, C, F, G) and aspalacZ/lacZ mice (B, D, E, H): Vacuolization is abundant in the dorsal region aspalacZ/lacZ mice (solitary tract nu, commissural, SolC) as shown in semithin sections (B) and electron microscopy (H). In the pyramidal tract of mutant mice myelin appears normal, but axonal swellings and degeneration are present (D, asterisks). Axonal swellings as well as hypomyelination were found in the dorsal region with abundant vacuolization (F, asterisk). Cerebellum of controls (I, K, M, O) and aspalacZ/lacZ mice (J, L, N, P): In semithin sections of mutant cerebellum, vacuoles are detectable in the white matter (WM), granule cell layer (GL) and in the Purkinje cell layer (J, L). Parallel fibre to Purkinje cell dendrite synapses appear normal (M, N), but in aspalacZ/lacZ mice the Bergman glia (BG) is swollen and shows accumulation of electron dense particles (N, P). BG, Bergman glia; CC, central canal; GL, granule cell layer; ML, molecular layer; PC, Purkinje cell; PF, parallel fibre; SolC, solitary tract nu, commissural; WM, white matter. Scale bars: 100 µm (A, B, I, J), 5 µm (K, L), 2 µm (G, H), 1 µm (C, D, O, P), 500 nm (M, N).
Figure 5
Figure 5. Myelin protein levels are reduced in aspalacZ/lacZ mutants.
Western blot analysis of PLP, DM20, CNP and MBP levels in whole brain lysates of aspa+/+, aspalacZ/+ and aspalacZ/lacZ mice (P60, n = 3). Quantification of immunoblots shows statistically significant decreases of all proteins tested in homozygous mutants while the presence of one functional aspa allele is sufficient to preserve protein quantities at control levels. Note: PLP, DM20, and the MBP21.5 kD and MBP18.5/17 kD isoforms were analysed separately.
Figure 6
Figure 6. Reactive gliosis in aspalacZ/lacZ mice.
Immunohistochemical staining in the thalamus of aspa+/+ controls and aspalacZ/lacZ mice showed substantially increased numbers of GFAP-positive astrocytes and Iba-1-positive microglia in the mutants (at 3 months of age). Note that ASPA immunoreactivity is present in the control but not in the mutant. Bars: 25 µm.
Figure 7
Figure 7. 1H-MRS analysis.
Spectra were acquired from aspa+/+ and aspalacZ/lacZ littermates using a 2×2×3 mm3 single-voxel PRESS Sequence in the thalamus. Representativ metabolites are marked in the spectra (Cr: Creatine and Phosphocreatine, NAA: NAA+NAAG, Cho: Phosphocholine and Glycerophosphorylcholine, Glu: Glutamate, Gln: Glutamine, mI: Myo-Inositol, Tau: Taurine, Glx: Glu+Gln). For metabolite concentrations see Table 1.
Figure 8
Figure 8. Special behavioural deficits in male but not female aspalacZ/lacZ mutants.
A. Analysis of motor coordination in the rotarod test shows impairment of both female and male aspalacZ/lacZ mice compared with their aspa+/+ controls at P90. B. Spontaneous exploratory activity and aspects of anxiety-related behaviour of both sexes and genotypes were tested in an open field for 30 min (see Methods section). Male mutants (n = 8) travelled less and spent more time in the centre than male controls (n = 8). This behaviour is suggestive of impaired exploratory activity and anxiolysis. The behaviour of female mutants (n = 6) did not differ from female controls (n = 7).
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