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

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.

SUBMITTER: Mersmann N

PROVIDER: S-EPMC3098885 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Mutational analysis of aspartoacylase: implications for Canavan disease.

Project description:Mutations that result in near undetectable activity of aspartoacylase, which catalyzes the deacetylation of N-acetyl-l-aspartate, correlate with Canavan Disease, a neurodegenerative disorder usually fatal during childhood. The underlying biochemical mechanisms of how these mutations ablate activity are poorly understood. Therefore, we developed and tested a three-dimensional homology model of aspartoacylase based on zinc dependent carboxypeptidase A. Mutations of the putative zinc-binding residues (H21G, E24D/G, and H116G), the general proton donor (E178A), and mutants designed to switch the order of the zinc-binding residues (H21E/E24H and E24H/H116E) yielded wild-type aspartoacylase protein levels and undetectable ASPA activity. Mutations that affect substrate carboxyl binding (R71N) and transition state stabilization (R63N) also yielded wild-type aspartoacylase protein levels and undetectable aspartoacylase activity. Alanine substitutions of Cys124 and Cys152, residues indicated by homology modeling to be in close proximity and in the proper orientation for disulfide bonding, yielded reduced ASPA protein and activity levels. Finally, expression of several previously tested (E24G, D68A, C152W, E214X, D249V, E285A, and A305E) and untested (H21P, A57T, I143T, P183H, M195R, K213E/G274R, G274R, and F295S) Canavan Disease mutations resulted in undetectable enzyme activity, and only E285A and P183H showed wild-type aspartoacylase protein levels. These results show that aspartoacylase is a member of the caboxypeptidase A family and offer novel explanations for most loss-of-function aspartoacylase mutations associated with Canavan Disease.

| S-EPMC1933483 | biostudies-literature

Brain Nat8l Knockdown Suppresses Spongiform Leukodystrophy in an Aspartoacylase-Deficient Canavan Disease Mouse Model.

Project description:Canavan disease, a leukodystrophy caused by loss-of-function ASPA mutations, is characterized by brain dysmyelination, vacuolation, and astrogliosis ("spongiform leukodystrophy"). ASPA encodes aspartoacylase, an oligodendroglial enzyme that cleaves the abundant brain amino acid N-acetyl-L-aspartate (NAA) to L-aspartate and acetate. Aspartoacylase deficiency results in a 50% or greater elevation in brain NAA concentration ([NAAB]). Prior studies showed that homozygous constitutive knockout of Nat8l, the gene encoding the neuronal NAA synthesizing enzyme N-acetyltransferase 8-like, prevents aspartoacylase-deficient mice from developing spongiform leukodystrophy. We now report that brain Nat8l knockdown elicited by intracerebroventricular/intracisternal administration of an adeno-associated viral vector carrying a short hairpin Nat8l inhibitory RNA to neonatal aspartoacylase-deficient AspaNur7/Nur7 mice lowers [NAAB] and suppresses development of spongiform leukodystrophy.

| S-EPMC5910673 | biostudies-literature

Structure of aspartoacylase, the brain enzyme impaired in Canavan disease.

Project description:Aspartoacylase catalyzes hydrolysis of N-acetyl-l-aspartate to aspartate and acetate in the vertebrate brain. Deficiency in this activity leads to spongiform degeneration of the white matter of the brain and is the established cause of Canavan disease, a fatal progressive leukodystrophy affecting young children. We present crystal structures of recombinant human and rat aspartoacylase refined to 2.8- and 1.8-A resolution, respectively. The structures revealed that the N-terminal domain of aspartoacylase adopts a protein fold similar to that of zinc-dependent hydrolases related to carboxypeptidases A. The catalytic site of aspartoacylase shows close structural similarity to those of carboxypeptidases despite only 10-13% sequence identity between these proteins. About 100 C-terminal residues of aspartoacylase form a globular domain with a two-stranded beta-sheet linker that wraps around the N-terminal domain. The long channel leading to the active site is formed by the interface of the N- and C-terminal domains. The C-terminal domain is positioned in a way that prevents productive binding of polypeptides in the active site. The structures revealed that residues 158-164 may undergo a conformational change that results in opening and partial closing of the channel entrance. We hypothesize that the catalytic mechanism of aspartoacylase is closely analogous to that of carboxypeptidases. We identify residues involved in zinc coordination, and propose which residues may be involved in substrate binding and catalysis. The structures also provide a structural framework necessary for understanding the deleterious effects of many missense mutations of human aspartoacylase.

| S-EPMC1766406 | biostudies-literature

Clinically Distinct Phenotypes of Canavan Disease Correlate with Residual Aspartoacylase Enzyme Activity.

Project description:We describe 14 patients with 12 novel missense mutations in ASPA, the gene causing Canavan disease (CD). We developed a method to study the effect of these 12 variants on the function of aspartoacylase-the hydrolysis of N-acetyl-l-aspartic acid (NAA) to aspartate and acetate. The wild-type ASPA open reading frame (ORF) and the ORFs containing each of the variants were transfected into HEK293 cells. Enzyme activity was determined by incubating cell lysates with NAA and measuring the released aspartic acid by LC-MS/MS. Clinical data were obtained for 11 patients by means of questionnaires. Four patients presented with a non-typical clinical picture or with the milder form of CD, whereas seven presented with severe CD. The mutations found in the mild patients corresponded to the variants with the highest residual enzyme activities, suggesting that this assay can help evaluate unknown variants found in patients with atypical presentation. We have detected a correlation between clinical presentation, enzyme activity, and genotype for CD.

| S-EPMC5412892 | biostudies-literature

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

Project description: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.

| S-EPMC3022971 | biostudies-literature

AMBN mutations causing hypoplastic amelogenesis imperfecta and Ambn knockout-NLS-lacZ knockin mice exhibiting failed amelogenesis and Ambn tissue-specificity.

Project description:BackgroundAmeloblastin (AMBN) is a secreted matrix protein that is critical for the formation of dental enamel and is enamel-specific with respect to its essential functions. Biallelic AMBN defects cause non-syndromic autosomal recessive amelogenesis imperfecta. Homozygous Ambn mutant mice expressing an internally truncated AMBN protein deposit only a soft mineral crust on the surface of dentin.MethodsWe characterized a family with hypoplastic amelogenesis imperfecta caused by AMBN compound heterozygous mutations (c.1061T>C; p.Leu354Pro/ c.1340C>T; p.Pro447Leu). We generated and characterized Ambn knockout/NLS-lacZ (AmbnlacZ/lacZ ) knockin mice.ResultsNo AMBN protein was detected using immunohistochemistry in null mice. ß-galactosidase activity was specific for ameloblasts in incisors and molars, and islands of cells along developing molar roots. AmbnlacZ/lacZ 7-week incisors and unerupted (D14) first molars showed extreme enamel surface roughness. No abnormalities were observed in dentin mineralization or in nondental tissues. Ameloblasts in the AmbnlacZ/lacZ mice were unable to initiate appositional growth and started to degenerate and deposit ectopic mineral. No layer of initial enamel ribbons formed in the AmbnlacZ/lacZ mice, but pockets of amelogenin accumulated on the dentin surface along the ameloblast distal membrane and within the enamel organ epithelia (EOE). NLS-lacZ signal was positive in the epididymis and nasal epithelium, but negative in ovary, oviduct, uterus, prostate, seminal vesicles, testis, submandibular salivary gland, kidney, liver, bladder, and bone, even after 15 hr of incubation with X-gal.ConclusionsAmeloblastin is critical for the initiation of enamel ribbon formation, and its absence results in pathological mineralization within the enamel organ epithelia.

| S-EPMC6732285 | biostudies-literature

Brain ultrasound in Canavan disease.

Project description:Canavan disease (MIM 271900) is a rare autosomal recessive leukodystrophy due to mutations in the ASPA gene (MIM 608034) and characterized by a clinical onset at 3-5 months of life, macrocephaly and poor head control, weak cry and suck, development regression and hypotonia. Here, we report cranial ultrasound findings at birth and at 4 months of age in a patient affected with Canavan disease. The comparison of our sonographic data with few other cases in literature allows us to suggest a characteristic pattern in Canavan disease.

| S-EPMC4142129 | biostudies-literature

Loss of central auditory processing in a mouse model of Canavan disease.

Project description:Canavan Disease (CD) is a leukodystrophy caused by homozygous null mutations in the gene encoding aspartoacylase (ASPA). ASPA-deficiency is characterized by severe psychomotor retardation, and excessive levels of the ASPA substrate N-acetylaspartate (NAA). ASPA is an oligodendrocyte marker and it is believed that CD has a central etiology. However, ASPA is also expressed by Schwann cells and ASPA-deficiency in the periphery might therefore contribute to the complex CD pathology. In this study, we assessed peripheral and central auditory function in the AspalacZ/lacZ rodent model of CD using auditory brainstem response (ABR). Increased ABR thresholds and the virtual loss of waveform peaks 4 and 5 from AspalacZ/lacZ mice, indicated altered central auditory processing in mutant mice compared with Aspawt/wt controls and altered central auditory processing. Analysis of ABR latencies recorded from AspalacZ/lacZ mice revealed that the speed of nerve conduction was unchanged in the peripheral part of the auditory pathway, and impaired in the CNS. Histological analyses confirmed that ASPA was expressed in oligodendrocytes and Schwann cells of the auditory system. In keeping with our physiological results, the cellular organization of the cochlea, including the organ of Corti, was preserved and the spiral ganglion nerve fibres were normal in ASPA-deficient mice. In contrast, we detected substantial hypomyelination in the central auditory system of AspalacZ/lacZ mice. In summary, our data suggest that the lack of ASPA in the CNS is responsible for the observed hearing deficits, while ASPA-deficiency in the cochlear nerve fibres is tolerated both morphologically and functionally.

| S-EPMC4020830 | biostudies-literature

Dual-function AAV gene therapy reverses late-stage Canavan disease pathology in mice.

Project description:The leukodystrophy Canavan disease is a fatal white matter disorder caused by loss-of-function mutations of the aspartoacylase-encoding ASPA gene. There are no effective treatments available and experimental gene therapy trials have failed to provide sufficient amelioration from Canavan disease symptoms. Preclinical studies suggest that Canavan disease-like pathology can be addressed by either ASPA gene replacement therapy or by lowering the expression of the N-acetyl-L-aspartate synthesizing enzyme NAT8L. Both approaches individually prevent or even reverse pathological aspects in Canavan disease mice. Here, we combined both strategies and assessed whether intracranial adeno-associated virus-mediated gene delivery to a Canavan disease mouse model at 12 weeks allows for reversal of existing pathology. This was enabled by a single vector dual-function approach. In vitro and in vivo biopotency assessment revealed significant knockdown of neuronal Nat8l paired with robust ectopic aspartoacylase expression. Following nomination of the most efficient cassette designs, we performed proof-of-concept studies in post-symptomatic Aspa-null mice. Late-stage gene therapy resulted in a decrease of brain vacuoles and long-term reversal of all pathological hallmarks, including loss of body weight, locomotor impairments, elevated N-acetyl-L-aspartate levels, astrogliosis, and demyelination. These data suggest feasibility of a dual-function vector combination therapy, directed at replacing aspartoacylase with concomitantly suppressing N-acetyl-L-aspartate production, which holds potential to permanently alleviate Canavan disease symptoms and expands the therapeutic window towards a treatment option for adult subjects.

| S-EPMC9772617 | biostudies-literature

A disease-associated Aifm1 variant induces severe myopathy in knockin mice.

Project description:OBJECTIVE:Mutations in the AIFM1 gene have been identified in recessive X-linked mitochondrial diseases. Functional and molecular consequences of these pathogenic AIFM1 mutations have been poorly studied in vivo. METHODS/RESULTS:Here we provide evidence that the disease-associated apoptosis-inducing factor (AIF) deletion arginine 201 (R200 in rodents) causes pathology in knockin mice. Within a few months, posttranslational loss of the mutant AIF protein induces severe myopathy associated with a lower number of cytochrome c oxidase-positive muscle fibers. At a later stage, Aifm1 (R200 del) knockin mice manifest peripheral neuropathy, but they do not show neurodegenerative processes in the cerebellum, as observed in age-matched hypomorphic Harlequin (Hq) mutant mice. Quantitative proteomic and biochemical data highlight common molecular signatures of mitochondrial diseases, including aberrant folate-driven one-carbon metabolism and sustained Akt/mTOR signaling. CONCLUSION:Our findings indicate metabolic defects and distinct tissue-specific vulnerability due to a disease-causing AIFM1 mutation, with many pathological hallmarks that resemble those seen in patients.

| S-EPMC6026322 | biostudies-literature

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