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

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.

Dietary triheptanoin rescues oligodendrocyte loss, dysmyelination and motor function in the nur7 mouse model of Canavan disease.

The inherited pediatric leukodystrophy Canavan disease is characterized by dysmyelination and severe spongiform degeneration, and is currently refractory to treatment. A definitive understanding of core disease mechanisms is lacking, but pathology is believed to result at least in part compromised fatty acid synthesis during myelination. Recent evidence generated in an animal model suggests that the breakdown of N-acetylaspartate metabolism in CD results in a heightened coupling of fatty acid synthesis to oligodendrocyte oxidative metabolism during the early stages of myelination, thereby causing acute oxidative stress. We present here the results of a dietary intervention designed to support oxidative integrity during developmental myelination in the nur7 mouse model of Canavan disease. Provision of the odd carbon triglyceride triheptanoin to neonatal nur7 mice reduced oxidative stress, promoted long-term oligodendrocyte survival, and increased myelin in the brain. Improvements in oligodendrocyte survival and myelination were associated with a highly significant reduction in spongiform degeneration and improved motor function in triheptanoin treated mice. Initiation of triheptanoin treatment in older animals resulted in markedly more modest effects on these same pathological indices, indicating a window of therapeutic intervention that corresponds with developmental myelination. These results support the targeting of oxidative integrity at early stages of Canavan disease, and provide a foundation for the clinical development of a non-invasive dietary triheptanoin treatment regimen.

Canavan disease: clinical features and recent advances in research.

Canavan disease (CD) is a genetic neurodegenerative leukodystrophy that results in the spongy degeneration of white matter in the brain. CD is characterized by mutations in the gene encoding aspartoacylase (ASPA), the substrate enzyme that hydrolyzes N-acetylaspartic acid (NAA) to acetate and aspartate. Elevated NAA and subsequent deficiency in acetate associated with this disease cause progressive neurological symptoms, such as macrocephaly, visuocognitive dysfunction, and psychomotor delay. The prevalence of CD is higher among Ashkenazi Jewish people, and several types of mutations have been reported in the gene coding ASPA. Highly elevated NAA is more specific to CD than other leukodystrophies, and an examination of urinary NAA concentration is useful for diagnosing CD. Many researchers are now examining the mechanisms responsible for white matter degeneration or dysmyelination in CD using mouse models, and several persuasive hypotheses have been suggested for the pathophysiology of CD. One is that NAA serves as a water pump; consequently, a disorder in NAA catabolism leads to astrocytic edema. Another hypothesis is that the hydrolyzation of NAA in oligodendrocytes is essential for myelin synthesis through the supply of acetate. Although there is currently no curative therapy for CD, dietary supplements are candidates that may retard the progression of the symptoms associated with CD. Furthermore, gene therapies using viral vectors have been investigated using rat models. These therapies have been found to be tolerable with no severe long-term adverse effects, reduce the elevated NAA in the brain, and may be applied to humans in the future.

Microvascular damage is involved in the pathogenesis of heroin induced spongiform leukoencephalopathy.

OBJECTIVE: To investigate whether microvascular damage is involved in the pathogenesis of heroin induced spongiform leukoencephalopathy (HSLE). METHODS: The brain tissues were collected from 4 HSLE patients and 5 controls and then fixed in 4% paraformaldehyde. The frontal lobe, corpus callosum and cerebellum were separated. The expressions of myelin base protein (MBP) and CD34 were detected by immunohistochemistry. TUNEL staining was applied to detect cell apoptosis. The correlation between microvascular changes and pathological vacuoles was evaluated. RESULTS: No obvious abnormalities were found in the brain of controls. Immunohistochemistry for MBP showed the collapse and fracture of myelin sheath and vacuole formation in the subcortical white matter, corpus callosum, and cerebellar white matter of HSLE patients. TUNEL staining showed the number of apoptotic cells in the cerebellar white matter and corpus callosum of HSLE patients was significantly higher than that in controls (F = 389.451, P < 0.001). Masson's trichrome staining revealed vacuolar degeneration in the cerebral white matter of HSLE patients, and the vacuoles were distributed around the microvessels. Immunohistochemistry revealed CD34 positive cells were seldom found besides the vessels in the cerebellar white matter and corpus callosum of HSLE patients, but a variety of CD34 positive cells was found in the vascular wall of controls (F = 838.500, P < 0.001). CONCLUSION: Apoptosis of oligodendrocytes may be related to the HSLE. Cerebral vascular injury and microcirculation dysfunction are involved in the pathogenesis of HSLE. The interrelation between apoptosis of oligodendrocytes and the microvascular damage are required to be studied in future investigations.

N-acetylaspartic acid impairs enzymatic antioxidant defenses and enhances hydrogen peroxide concentration in rat brain.

N-Acetylaspartic acid accumulates in Canavan Disease, a severe inherited neurometabolic disease clinically characterized by severe mental retardation, hypotonia, macrocephaly and generalized tonic and clonic type seizures. Considering that the mechanisms of brain damage in this disease remain poorly understood, in the present study we investigated the in vitro and in vivo effects of N-acetylaspartic acid on the activities of catalase, superoxide dismutase and glutathione peroxidase, as well as on hydrogen peroxide concentration in cerebral cortex of 14-day-old rats. Catalase and glutathione peroxidase activities were significantly inhibited, while hydrogen peroxide concentration was significantly enhanced by N-acetylaspartic acid both in vitro and in vivo. In contrast, superoxide dismutase activity was not altered by N-acetylaspartic acid. Our results clearly show that N-acetylaspartic acid impairs the enzymatic antioxidant defenses in rat brain. This could be involved in the pathophysiological mechanisms responsible for the brain damage observed in patients affected by Canavan Disease.

Lack of aspartoacylase activity disrupts survival and differentiation of neural progenitors and oligodendrocytes in a mouse model of Canavan disease.

Loss of the oligodendrocyte (OL)-specific enzyme aspartoacylase (ASPA) from gene mutation results in the sponginess and loss of white matter (WM) in Canavan disease (CD). This study addresses the fate of OLs during the pathophysiology of CD in an adult ASPA knockout (KO) mouse strain. Massive arrays of neural stem/progenitor cells, immunopositive for PSA-NCAM, nestin, vimentin, and NG2, were observed within the severely affected spongy WM of the KO mouse brain. In these mice, G1-->S cell cycle progression was confirmed by an increase in cdk2-kinase activity, a reduction in mitotic inhibitors p21(Cip1) and p27(Kip1), and an increase in bromodeoxyuridine (BrdU) incorporation. Highly acetylated nuclear histones H2B and H3 were detected in adult KO mouse WM, suggesting the existence of noncompact chromatin as seen during early development. Costaining for BrdU- or Ki67-positive cells with markers for neural progenitors confirmed a continuous generation of OL lineage cells in KO WM. We observed a severe reduction in 21.5- and 18.5-kDa myelin basic protein and PLP/DM20 proteolipid proteins combined with a decrease in myelinated fibers and a perinuclear retention of myelin protein staining, indicating impairment in protein trafficking. Death of OLs, neurons, and astrocytes was identified in every region of the KO brain. Immature OLs constituted the largest population of dying cells, particularly in WM. We also report an early expression of full-length ASPA mRNA in normal mouse brain at embryonic day 12.5, when OL progenitors first appear during development. These findings support involvement of ASPA in CNS development and function.

Are astrocytes the missing link between lack of brain aspartoacylase activity and the spongiform leukodystrophy in Canavan disease?

Canavan disease (CD) is a genetic degenerative brain disorder associated with mutations of the gene encoding aspartoacylase (ASPA). In humans, the CD syndrome is marked by early onset, hydrocephalus, macroencephaly, psychomotor retardation, and spongiform myelin sheath vacuolization with progressive leukodystrophy. Metabolic hallmarks of the disease include elevated N-acetylaspartate (NAA) levels in brain, plasma and CSF, along with daily excretion of large amounts of NAA and its anabolic metabolite, N-acetylaspartylglutamate (NAAG). Of the observed neuropathies, the most important appears to be the extensive demyelination that interferes with normal neuronal signaling. However, finding the links between the lacks of ASPA activity in oligodendrocytes, the buildup of NAA in white matter (WM) and the mechanisms underlying the edematous spongiform leukodystrophy have remained elusive. In this analytical review we consider what those links might be and propose that in CD, the pathological buildup of NAA in limited WM extracellular fluid (ECF) is responsible for increased ECF osmotic-hydrostatic pressure and initiation of the demyelination process. We also hypothesize that NAA is not directly liberated by neurons in WM as it is in gray matter, and that its source in WM ECF is solely as a product of the catabolism of axon-released NAAG at nodes of Ranvier by astrocyte NAAG peptidase after it has docked with the astrocyte surface metabotropic glutamate receptor 3. This hypothesis ascribes for the first time a possible key role played by astrocytes in CD, linking the lack of ASPA activity in myelinating oligodendrocytes, the pathological buildup of NAA in WM ECF, and the spongiform demyelination process. It also offers new perspectives on the cause of the leukodystrophy in CD, and on possible treatment strategies for this inherited metabolic disease.

[Long term clinical course of Canavan disease--a rare Japanese case].

Canavan disease (CD), which is a rare disease in Japan, is an autosomal-recessive neurodegenerative disorder caused by mutations in aspartoacylase, an enzyme that deacetylates N-acetylaspartate to generate free acetate in the brain. CD affected children usually die by the age of 10 years. Here we report a long term clinical course of a 21-year-old Japanese woman who was diagnosed as CD at the age 4. This patient is the only reported case of CD in Japan that has been biochemically confirmed. Although this patient is currently bed-ridden with spastic quadriplegia and severe mental retardation, her general condition is quite stable. This patient showed a milder clinical course compared to the majority of CD patients. Because this is the only reported case of CD in Japan, we hypothesize that there might be an ethnic phenotypic polymorphism in CD.

A Missense Variant in KCNJ10 in Belgian Shepherd Dogs Affected by Spongy Degeneration with Cerebellar Ataxia (SDCA1).

Spongy degeneration with cerebellar ataxia (SDCA) is a severe neurodegenerative disease with monogenic autosomal recessive inheritance in Malinois dogs, one of the four varieties of the Belgian Shepherd breed. We performed a genetic investigation in six families and seven isolated cases of Malinois dogs with signs of cerebellar dysfunction. Linkage analysis revealed an unexpected genetic heterogeneity within the studied cases. The affected dogs from four families and one isolated case shared a ∼1.4 Mb common homozygous haplotype segment on chromosome 38. Whole genome sequence analysis of three affected and 140 control dogs revealed a missense variant in the KCNJ10 gene encoding a potassium channel (c.986T>C; p.Leu329Pro). Pathogenic variants in KCNJ10 were reported previously in humans, mice, and dogs with neurological phenotypes. Therefore, we consider KCNJ10:c.986T>C the most likely candidate causative variant for one subtype of SDCA in Malinois dogs, which we propose to term spongy degeneration with cerebellar ataxia 1 (SDCA1). However, our study also comprised samples from 12 Malinois dogs with cerebellar dysfunction which were not homozygous for this variant, suggesting a different genetic basis in these dogs. A retrospective detailed clinical and histopathological analysis revealed subtle neuropathological differences with respect to SDCA1-affected dogs. Thus, our study highlights the genetic and phenotypic complexity underlying cerebellar dysfunction in Malinois dogs and provides the basis for a genetic test to eradicate one specific neurodegenerative disease from the breeding population. These dogs represent an animal model for the human EAST syndrome.

Non-genetic therapeutic approaches to Canavan disease.

Canavan disease (CD) is a rare leukodystrophy characterized by diffuse spongiform white matter degeneration, dysmyelination and intramyelinic oedema with consequent impairment of psychomotor development and early death. The molecular cause of CD has been identified as being mutations of the gene encoding the enzyme aspartoacylase (ASPA) leading to its functional deficiency. The physiological role of ASPA is to hydrolyse N-acetyl-l-aspartic acid (NAA), producing l-aspartic acid and acetate; as a result, its deficiency leads to abnormally high central nervous system NAA levels. The aim of this article is to review what is currently known regarding the aetiopathogenesis and treatment of CD, with emphasis on the non-genetic therapeutic strategies, both at an experimental and a clinical level, by highlighting: (a) major related hypotheses, (b) the results of the available experimental simulatory approaches, as well as (c) the relevance of the so far examined markers of CD neuropathology. The potential and the limitations of the current non-genetic neuroprotective approaches to the treatment of CD are particularly discussed in the current article, in a context that could be used to direct future experimental and (eventually) clinical work in the field.

A case of Canavan disease with microcephaly.

BACKGROUND: Canavan disease is an autosomal recessive disorder with spongy degeneration of white matter of the brain. It presents with developmental delay, visual problems and macrocephaly. PATIENT DESCRIPTION: We report a ten-month old boy with Canavan disease who presented with global developmental delay, seizures, abnormal eye movements and microcephaly. RESULTS: MRI brain revealed diffuse involvement of the supra tentorial white matter, globus pallidi, thalami, dentate nuclei and brainstem with sparing of the corpus callosum. The genetic testing revealed homozygous mutation of aspartoacylase gene [c.859 G>A (p.Ala287Thr)] in Exon 6. CONCLUSION: Possibility of Canavan disease should be considered even in the presence of microcephaly.

Aspartoacylase gene knockout in the mouse: impact on reproduction.

Canavan disease (CD) is an autosomal recessive disorder caused by aspartoacylase (ASPA) gene mutations resulting enzyme deficiency. The homozygous knockout mouse for CD showed symptoms similar observed in patients with CD. Canavan disease leads to early death. Therefore, a role of ASPA in reproduction was investigated using the mouse model for CD. Homozygous (KO/KO) pups, produced by mating female heterozygous (KO/+) mouse with KO/+ males had approximately 12% death incidence rates in the first 2 months of life. KO/KO mothers mated with KO/+ males showed fetal death. KO/KO mothers produced fewer offspring compared to KO/+ mothers. These data suggest that ASPA is necessary for normal reproduction and postnatal survival.

An atypical case of Canavan disease with stroke-like presentation.

BACKGROUND: Canavan disease is an autosomal recessive leukodystrophy caused by a deficiency of aspartoacylase. The disease has a severe course, with death occurring in the first few years of life. Atypical patients with mild courses have been reported, but acute presentations similar to stroke have not been well described. PATIENT DESCRIPTION: We present a boy who presented at 4 months of age with seizures after an episode of cardiopulmonary arrest is discussed. RESULTS: He was initially thought to have an ischemic watershed stroke based on his initial clinical presentation and magnetic resonance imaging. However, biochemical and follow-up radiologic evaluation were consistent with mild Canavan disease. DNA sequencing of the ASPA gene indicated one known mutation (A305E) and a novel mutation, L30V. Follow-up magnetic resonance imaging did not reveal the atrophy which would have been expected with watershed ischemia. Magnetic resonance spectroscopy demonstrated elevated N-acetyl aspartate to creatinine and N-acetyl aspartate to choline ratios. At 4 years of age, he was normocephalic, with mild clumsiness, speech delay, and seizures. CONCLUSIONS: This child's unusual acute presentation, along with his prolonged mild course, raises questions about the relationship between biochemical signs of abnormal aspartoacylase function and clinical findings. This patient highlights the need for long-term clinical follow-up of children with mild Canavan disease to clarify the significance of these biochemical abnormalities.

Spongy degeneration of the brain, Canavan disease: biochemical and molecular findings.

Canavan disease is a severe progressive leukodystrophy characterized by swelling and spongy degeneration of the white matter of the brain. It is an autosomal recessive disease found more frequently among Ashkenazi Jews. The clinical features are those of severe mental retardation with inability to gain developmental milestones. Hypotonia, head lag and macrocephaly are characteristic of Canavan disease and become apparent after 5-6 months of age. Massive excretion in the urine of N-acetylaspartic acid is the biochemical marker for Canavan disease, which is caused by deficiency of the enzyme aspartoacylase. This discovery allowed for accurate diagnosis of Canavan disease, while prior to that, a brain biopsy was needed. The gene for aspartoacylase has been cloned and two mutations predominate among Ashkenazi Jewish individuals with Canavan disease and account for more than 98% of the Ashkenazi Jewish patients. The mutations among other ethnic groups are more diverse. The carrier frequency for the two common mutations among Ashkenazi Jews was found to be surprisingly high, 1:37. Screening for carriers is now common practice for this population. A knock-out mouse for Canavan disease is being genetically engineered in our laboratory. The mouse model will allow for development of strategies for gene therapy.

Brain N-acetylaspartate as a molecular water pump and its role in the etiology of Canavan disease: a mechanistic explanation.

N-acetyl-L-aspartate (NAA), an abundant amino acid present in the vertebrate brain, is synthesized and stored primarily in neurons. Its metabolism is also dynamic, with NAA turning over more than once each day by its regulated efflux into extracellular fluid (ECF), cycling between an anabolic L-aspartate acetylating compartment in neurons and a catabolic NAA deacetylating compartment in oligodendrocytes. An inborn error in NAA metabolism results in Canavan disease (CD), a rare and usually fatal early-onset autosomal recessive human central nervous system (CNS) disease, caused by failure of the catabolic metabolism of NAA resulting from a lack of sufficient amidohydrolase II activity in oligodendrocytes. Various hypotheses regarding the metabolism of NAA and its role have been considered, and although NAA may perform several functions in the CNS, an important role of NAA appears to be osmoregulatory. Based on this role, an osmotic-hydrostatic mechanism for the etiology of the CD phenotype is proposed. In CD, the daily addition of 13375 Pascals (0.132 atmospheres or 1.94 lbs per square inch) of hydrostatic pressure to brain ECF, on the brain cell side of brain-barrier epithelial membranes, resulting from the continuous synthesis and efflux of NAA, is considered to be responsible for the syndrome.

Aspartoacylase is restricted primarily to myelin synthesizing cells in the CNS: therapeutic implications for Canavan disease.

Canavan disease is a devastating neurodegenerative childhood disease caused by mutations in aspartoacylase, an enzyme that deacetylates N-acetylaspartate to generate free acetate in the brain. Localization of aspartoacylase in different cell types in the rat brain was examined in an attempt to understand the pathogenesis of Canavan disease. In situ hybridization histochemistry with a riboprobe based on murine aspartoacylase cDNA was used in this study. The hybridization signal was detectable primarily in the myelin-synthesizing cells, namely oligodendroglia. These findings provide strong additional support for insufficient myelin synthesis as the pathogenic basis of Canavan disease and make a compelling case for acetate supplementation as a simple and noninvasive therapy for this fatal disease with no treatment.

Global CNS gene transfer for a childhood neurogenetic enzyme deficiency: Canavan disease.

The neurogenetic prototypic disease on which we chose to test our gene therapy strategy is Canavan disease (CD). CD is an autosomal recessive leukodystrophy associated with spongiform degeneration of the brain. At present the disease is uniformly fatal in affected probands. CD is characterized by mutations in the aspartoacylase (ASPA) gene, resulting in loss of enzyme activity. In this review, recent evidence is summarized on the etiology and possible treatments for CD. In particular, we discuss two gene delivery systems representing recent advances in both viral and liposome technology: a novel cationic liposome-polymer-DNA (LPD) complex, DCChol/DOPE-protamine, as well as recombinant adeno-associated virus (AAV) vectors.

Selected leukodystrophies.

The clinicopathologic profiles of the major leukodystrophies (adreno-leukodystrophy, metachromatic leukodystrophy, globoid cell leukodystrophy or Krabbe's disease, Pelizaeus-Merzbacher disease, and spongy degeneration of infancy or Canavan's disease) are reviewed. Particular attention is paid to distinctive imaging characteristics, molecular advances, pathogeneses, and potential therapies.

Protracted course of N-acetylaspartic aciduria in two non-Jewish siblings: identical clinical and magnetic resonance imaging findings.

Canavan disease (CD) or N-acetylaspartic aciduria (NAA) is a severe, progressive, autosomal recessive leukodystrophy, occurring mainly among Ashkenazi Jewish individuals. We report clinical and MRI findings in two, non-Jewish, Greek siblings, 7 and 5 years, respectively, with a protracted form of NAA. The constellation of identical clinical course and identical MRI findings with involvement of the basal ganglia, the brainstem, the dentate nucleus and the subcortical white matter in both siblings, as well as the absence of the three commonest mutations found in both Jewish and non-Jewish CD patients, give support to the existence of a protracted form of NAA with a milder clinical course, presumably genetically determined.