Jeremy S. Francis

Immune responses to AAV in a phase I study for Canavan disease

Canavan disease is a rare leukodystrophy with no current treatment. rAAV‐ASPA has been developed for gene delivery to the central nervous system (CNS) for Canavan disease. This study represents the first use of a viral vector in an attempt to ameliorate a neurodegenerative disorder.

Long-Term Follow-Up After Gene Therapy for Canavan Disease

Gene therapy for Canavan disease results in a decrease in pathologically elevated N-acetyl-aspartate concentrations in the brain and long-term clinical stabilization. Gene Therapy for Canavan Disease Canavan disease is a fatal childhood neurodegenerative disorder for which there is no effective treatment. It is caused by a defect in a single gene (ASPA) that results in a deleterious buildup of N-acetyl-aspartate in the brain. This process starts at birth and is accompanied by a failure to form and maintain myelin, the protective sheath surrounding nerves. As a brain-specific disorder with simple Mendelian inheritance, Canavan disease represents an excellent target for enzyme replacement using gene therapy. Leone et al. now report the long-term results of gene therapy in 13 Canavan disease patients using adeno-associated viral vector delivery of the ASPA gene. The investigators found that gene therapy was safe and led to a decrease in N-acetyl-aspartate in the brain, together with decreased seizure frequency and clinical stabilization. Clinical stabilization was greatest in the youngest patients. Early detection and treatment with gene therapy–mediated enzyme replacement in the neonatal period may offer the best opportunity for a reduction in symptoms and long-term stabilization in patients with Canavan disease. Canavan disease is a hereditary leukodystrophy caused by mutations in the aspartoacylase gene (ASPA), leading to loss of enzyme activity and increased concentrations of the substrate N-acetyl-aspartate (NAA) in the brain. Accumulation of NAA results in spongiform degeneration of white matter and severe impairment of psychomotor development. The goal of this prospective cohort study was to assess long-term safety and preliminary efficacy measures after gene therapy with an adeno-associated viral vector carrying the ASPA gene (AAV2-ASPA). Using noninvasive magnetic resonance imaging and standardized clinical rating scales, we observed Canavan disease in 28 patients, with a subset of 13 patients being treated with AAV2-ASPA. Each patient received 9 × 1011 vector genomes via intraparenchymal delivery at six brain infusion sites. Safety data collected over a minimum 5-year follow-up period showed a lack of long-term adverse events related to the AAV2 vector. Posttreatment effects were analyzed using a generalized linear mixed model, which showed changes in predefined surrogate markers of disease progression and clinical assessment subscores. AAV2-ASPA gene therapy resulted in a decrease in elevated NAA in the brain and slowed progression of brain atrophy, with some improvement in seizure frequency and with stabilization of overall clinical status.

Gata6 regulates aspartoacylase expression in resident peritoneal macrophages and controls their survival

Gata6 regulates differentiation, metabolism and survival of peritoneal macrophages.

Aspartoacylase supports oxidative energy metabolism during myelination.

The inherited leukodystrophy Canavan disease arises due to a loss of the ability to catabolize N-acetylaspartic acid (NAA) in the brain and constitutes a major point of focus for efforts to define NAA function. Accumulation of noncatabolized NAA is diagnostic for Canavan disease, but contrasts with the abnormally low NAA associated with compromised neuronal integrity in a broad spectrum of other clinical conditions. Experimental evidence for NAA function supports a role in white matter lipid synthesis, but does not explain how both elevated and lowered NAA can be associated with pathology in the brain. We have undertaken a systematic analysis of postnatal development in a mouse model of Canavan disease that delineates development and pathology by identifying markers of oxidative stress preceding oligodendrocyte loss and dysmyelination. These data suggest a role for NAA in the maintenance of metabolic integrity in oligodendrocytes that may be of relevance to the strong association between NAA and neuronal viability. N-acetylaspartic acid is proposed here to support lipid synthesis and energy metabolism via the provision of substrate for both cellular processes during early postnatal development.

Novel role for aspartoacylase in regulation of BDNF and timing of postnatal oligodendrogenesis.

Neuronal growth factors are thought to exert a significant degree of control over postnatal oligodendrogenesis, but mechanisms by which these factors coordinateoligodendrocyte development with the maturation of neural networks are poorly characterized. We present here a developmental analysis of aspartoacylase (Aspa)‐null tremor rats and show a potential role for this hydrolytic enzyme in the regulation of a postnatal neurotrophic stimulus that impacts on early stages of oligodendrocyte differentiation. Abnormally high levels of brain‐derived neurotrophic factor (BDNF) expression in the Aspa‐null Tremor brain are associated with dysregulated oligodendrogenesis at a stage in development normally characterized by high levels of Aspa expression. BDNF promotes the survival of proliferating cells during the early stages of oligodendrocyte maturation in vitro, but seems to compromise the ability of these cells to populate the cortex in vivo. Aspartoacylase activity in oligodendrocytes is shown to provide for the negative regulation of BDNF in neurons, thereby determining the availability of a developmental stimulus via a mechanism that links oligodendroglial differentiation with neuronal maturation.

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.

GFP-transgenic Lewis rats as a cell source for oligodendrocyte replacement

We have investigated the gliogenic potential of cells isolated from a recently described GFP-transgenic rat [Inoue, H., Ohsawa, I., Murakami, T., Kimura, A., Hakamata, Y., Sato, Y., Kaneko, T., Takahashi, M., Okada, T., Ozawa, K., Francis, J., Leone, P., Kobayashi, E., 2005. Development of new inbred transgenic strains of rats with LacZ or GFP. Biochem Biophys Res Commun 329 288-295.] for application to oligodendrocyte replacement in models of white matter insult and disease. These transgenic rats present native GFP fluorescence in oligodendrocytes of the CNS, with no detectable fluorescence in astrocytes or mature neurons. By targeting a highly gliogenic period of postnatal development, we show that sphere-forming cultures of proliferating cells generated from the GFP-transgenic brain give rise to significant numbers of differentiated oligodendrocytes in vitro. Postnatal source tissue was significantly more gliogenic than embryonic source tissue, with greater than 50% of postnatally derived cells differentiating into GFP-positive oligodendrocytes. Differentiated oligodendrocytes exhibited an increased intensity of GFP fluorescence concomitant with the acquisition of mature oligodendrocyte-specific markers in both isolated cultures and in co-culture with primary neurons. Transplantation of postnatally derived GFP-positive sphere-forming cells into ethidium bromide lesioned Kyoto-Wistar rats resulted in the engraftment and survival of GFP-positive oligodendrocytes for at least 6 weeks in the host white matter and cerebral cortex. Our results show that sphere-forming cultures of cells isolated from the early postnatal GFP-Lewis rat brain are a useful tool for oligodendrocyte replacement studies.

Endogenous aspartoacylase expression is responsive to glutamatergic activity in vitro and in vivo.

Aspartoacylase (ASPA) is an enzyme that functions to catabolize the neuronal amino acid derivative N‐acetyl‐L‐aspartic acid (NAA). Loss of this function results in the failure of developmental myelination. NAA synthesis and catabolism are tightly compartmentalized within neurons and oligodendrocytes, respectively, and there is evidence to suggest the developmental regulation of ASPA activity is transcriptional. NAA has no known direct physiological mode of action, and the identification of a transcriptional regulator of aspa would provide an opportunity to link NAA to relatively more characterized physiological contexts with a view to definitive functional classification. Using transcriptional and immunohistochemical endpoints, we define a window of postnatal development punctuated by increases in aspa within a pre‐existing population of oligodendrocytes in the rat brain. Ontological mining of expression data generated in aspa‐null rats during this developmental window identifies both neuronal and oligodendroglial transcriptional abnormalities that suggest an association between glutamatergic synaptic activity and ASPA. Glutamate, but not NAA, is shown to activate endogenous aspa expression in vitro, and endogenous aspa expression is upregulated in the brains of animals over expressing vesicular glutamate transporter type‐I (vglut1). These results define a discrete period of postnatal development within which glutamate provides a means by which the tightly compartmentalized NAA metabolic cycle can function in sync with normal development and may be a factor in pathological models of dysregulated NAA metabolism.

Transcriptional Regulation of N-acetylaspartate Metabolism in the 5xFAD Model of Alzheimer's Disease: Evidence for Neuron-Glia Communication During Energetic Crisis

N-acetylaspartate (NAA) provides a non-invasive clinical index of neuronal metabolic integrity across the entire neurodegenerative spectrum. While NAA function is not comprehensively defined, reductions in the brain are associated with compromised mitochondrial metabolism and are tightly linked to ATP. We have undertaken an analysis of abnormalities in NAA during early stage pathology in the 5xFAD mouse model of familial Alzheimers disease and show here that dysregulated expression of the gene encoding for the rate-limiting NAA synthetic enzyme (Nat8L) is associated with deficits in mitochondrial oxidative phosphorylation in this model system. Downreguation of Nat8L is particularly pronounced in the 5xFAD hippocampus, and is preceded by a significant upregulation of oligodendrocytic aspartoacylase (aspa), which encodes for the sole known NAA-catabolizing enzyme in the brain. Reductions in 5xFAD NAA and Nat8L cannot be accounted for by discrepancies in either neuron content or activity of the substrate-providing malate-aspartate shuttle, thereby implicating transcriptional regulation in a coordinated response to pathological energetic crisis. A central role for ASPA in this response is supported by a parallel developmental analysis showing highly significant increases in Nat8L expression in an ASPA-null mouse model during a period of early postnatal development normally punctuated by the transcriptional upregulation of aspa. These results provide preliminary evidence of a signaling mechanism in Alzheimers disease that involves cross talk between neurons and oligodendrocytes, and suggest that ASPA acts to negatively regulate Nat8L expression. This mechanism is proposed to be a fundamental means by which the brain conserves available substrate during energy crises.