myelin-basic-protein and Canavan-Disease

Canavan disease (CD) is an autosomal-recessive neurodegenerative disorder caused by inactivation of the enzyme aspartoacylase (ASPA, EC 3.5.1.15) due to mutations. ASPA releases acetate by deacetylation of N-acetylaspartate (NAA), a highly abundant amino acid derivative in the central nervous system. CD results in spongiform degeneration of the brain and severe psychomotor retardation, and the affected children usually die by the age of 10. The pathogenesis of CD remains a matter of inquiry. Our hypothesis is that ASPA actively participates in myelin synthesis by providing NAA-derived acetate for acetyl CoA synthesis, which in turn is used for synthesis of the lipid portion of myelin. Consequently, CD results from defective myelin synthesis due to a deficiency in the supply of the NAA-derived acetate. The demonstration of the selective localization of ASPA in oligodendrocytes in the central nervous system (CNS) is consistent with the acetate deficiency hypothesis of CD. We have tested this hypothesis by determining acetate levels and studying myelin lipid synthesis in the ASPA gene knockout model of CD, and the results provided the first direct evidence in support of this hypothesis. Acetate supplementation therapy is proposed as a simple and inexpensive therapeutic approach to this fatal disease, and progress in our preclinical efforts toward this goal is presented.

Topics: Aging; Animals; Aspartic Acid; Canavan Disease; Central Nervous System; Humans; Mice; Mice, Knockout; Myelin Basic Protein

Resveratrol (RSV) is a small compound first identified as an activator of sirtuin 1 (SIRT1), a key factor in mediating the effects of caloric restriction. Since then, RSV received great attention for its widespread beneficial effects on health and in connection to many diseases. RSV improves the metabolism and the mitochondrial function, and more recently it was shown to restore fatty acid β-oxidation (FAO) capacities in patient fibroblasts harboring mutations with residual enzyme activity. Many of RSV's beneficial effects are mediated by the transcriptional coactivator PGC-1α, a direct target of SIRT1 and a master regulator of the mitochondrial fatty acid oxidation. Despite numerous studies RSV's mechanism of action is still not completely elucidated. Our aim was to investigate the effects of RSV on gene regulation on a wide scale, possibly to detect novel genes whose up-regulation by RSV may be of interest with respect to disease treatment. We performed Next Generation Sequencing of RNA on normal fibroblasts treated with RSV. To investigate whether the effects of RSV are mediated through SIRT1 we expanded the analysis to include SIRT1-knockdown fibroblasts. We identified the aspartoacylase (ASPA) gene, mutated in Canavan disease, to be strongly up-regulated by RSV in several cell lines, including Canavan disease fibroblasts. We further link RSV to the up-regulation of other genes involved in myelination including the glial specific transcription factors POU3F1, POU3F2, and myelin basic protein (MBP). We also observe a strong up-regulation by RSV of the riboflavin transporter gene SLC52a1. Mutations in SLC52a1 cause transient multiple acyl-CoA dehydrogenase deficiency (MADD). Our analysis of alternative splicing identified novel metabolically important genes affected by RSV, among which is particularly interesting the α subunit of the stimulatory G protein (Gsα), which regulates the cellular levels of cAMP through adenylyl cyclase. We conclude that in fibroblasts RSV stimulates the PGC-1α and p53 pathways, and up-regulates genes affecting the glucose metabolism, mitochondrial β-oxidation, and mitochondrial biogenesis. We further confirm that RSV might be a relevant treatment in the correction of FAO deficiencies and we suggest that treatment in other metabolic disorders including Canavan disease and MADD might be also beneficial.

Topics: Amidohydrolases; Canavan Disease; Cell Line; Cells, Cultured; Fibroblasts; Gene Expression Regulation; Genes, p53; Glucose; High-Throughput Nucleotide Sequencing; Humans; Lipid Metabolism; Molecular Targeted Therapy; Myelin Basic Protein; Oxidation-Reduction; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Receptors, G-Protein-Coupled; Resveratrol; Sequence Analysis, RNA; Sirtuin 1; Transcription Factors; Up-Regulation

Breakdown of neuro-glial N-acetyl-aspartate (NAA) metabolism results in the failure of developmental myelination, manifest in the congenital pediatric leukodystrophy Canavan disease caused by mutations to the sole NAA catabolizing enzyme aspartoacylase. Canavan disease is a major point of focus for efforts to define NAA function, with available evidence suggesting NAA serves as an acetyl donor for fatty acid synthesis during myelination. Elevated NAA is a diagnostic hallmark of Canavan disease, which contrasts with a broad spectrum of alternative neurodegenerative contexts in which levels of NAA are inversely proportional to pathological progression. Recently generated data in the nur7 mouse model of Canavan disease suggests loss of aspartoacylase function results in compromised energetic integrity prior to oligodendrocyte death, abnormalities in myelin content, spongiform degeneration, and motor deficit. The present study utilized a next-generation "oligotropic" adeno-associated virus vector (AAV-Olig001) to quantitatively assess the impact of aspartoacylase reconstitution on developmental myelination. AAV-Olig001-aspartoacylase promoted normalization of NAA, increased bioavailable acetyl-CoA, and restored energetic balance within a window of postnatal development preceding gross histopathology and deteriorating motor function. Long-term effects included increased oligodendrocyte numbers, a global increase in myelination, reversal of vacuolation, and rescue of motor function. Effects on brain energy observed following AAV-Olig001-aspartoacylase gene therapy are shown to be consistent with a metabolic profile observed in mild cases of Canavan disease, implicating NAA in the maintenance of energetic integrity during myelination via oligodendroglial aspartoacylase.

Topics: Amidohydrolases; Animals; Aspartic Acid; Autophagy-Related Proteins; Basic Helix-Loop-Helix Transcription Factors; Brain; Canavan Disease; Child; Child, Preschool; Dependovirus; Disease Progression; Energy Metabolism; Female; Gene Expression Regulation; Green Fluorescent Proteins; HEK293 Cells; Humans; Infant; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Transgenic; Movement Disorders; Myelin Basic Protein; Myelin Sheath; Neurodegenerative Diseases; Oligodendroglia

To investigate whether microvascular damage is involved in the pathogenesis of heroin induced spongiform leukoencephalopathy (HSLE).. 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.. 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).. 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.

Topics: Adult; Antigens, CD34; Apoptosis; Canavan Disease; Cerebellum; Cerebrovascular Trauma; Corpus Callosum; Female; Frontal Lobe; Heroin; Heroin Dependence; Humans; Male; Microvessels; Middle Aged; Myelin Basic Protein; Oligodendroglia

Recombinant adeno-associated virus (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in adult rodents have shown that the use of cell type-specific promoters is sufficient to target AAV-mediated transgene expression to glia. However, neurological disorders caused by glial pathology usually have an early onset. Therefore, modelling and treatment of these conditions require expanding the concept of targeted glial transgene expression by promoter selectivity for gene delivery to the immature CNS. Here, we have investigated the AAV-mediated green fluorescent protein (GFP) expression driven by the myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoters in the developing mouse brain. Generally, the extent of transgene expression after infusion at immature stages was widespread and higher than in adults. The GFAP promoter-driven GFP expression was found to be highly specific for astrocytes following vector infusion to the brain of neonates and adults. In contrast, the selectivity of the MBP promoter for oligodendrocytes was poor following neonatal AAV delivery, but excellent after vector injection at postnatal day 10. To extend these findings obtained in naïve mice to a disease model, we performed P10 infusions of AAV-MBP-GFP in aspartoacylase (ASPA)-deficient mouse mutants presenting with early onset oligodendrocyte pathology. Spread of GFP expression and selectivity for oligodendrocytes in ASPA-mutants was comparable with our observations in normal animals. Our data suggest that direct AAV infusion to the developing postnatal brain, utilising cellular promoters, results in targeted and long-term transgene expression in glia. This approach will be relevant for disease modelling and gene therapy for the treatment of glial pathology.

Topics: Age Factors; Animals; Animals, Newborn; Astrocytes; Brain; Canavan Disease; Cells, Cultured; Dependovirus; Gene Expression; Genetic Therapy; Genetic Vectors; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Mice, Inbred C57BL; Myelin Basic Protein; Oligodendroglia; Organ Specificity; Promoter Regions, Genetic; Transgenes

To investigate the role of oligodendrocyte apoptosis under the regulation of the bcl-2/bax protein expression in brain white matter in the pathogenesis of heroin-induced spongiform leucoencephalopathy (HSLE).. Samples of frontal lobe, cerebellum, and corpus callosum were obtained from the brains during autopsy of 4 HSLE cases and 5 normal controls and underwent light microscopy and electron microscopy. Immunocytochemistry was used to detect the expression of myelin basic protein (MBP), caspase-3, bcl-2 protein, and bax protein.. Widespread demyelination was seen in the white matter of the frontal lobe, cerebellum and corpus callosum of the HSLE cases, most severely in the cerebellum. The levels of caspase-3 and bax expression of the HSLE group were significantly higher than those of the control group (both P <0.05) , however, the bcl-2 level of the HSLE group was no significantly different from that of the control group (P > 0.05).. Widespread demyelination in the white matter is a prevailed pathological change of HSLE. Oligodendrocyte apoptosis under induced by the decrease of bcl-2/bax ratio may contribute to the pathogenesis.

Topics: Adult; Aged; Apoptosis; Autopsy; bcl-2-Associated X Protein; Canavan Disease; Female; Heroin; Heroin Dependence; Humans; Immunohistochemistry; Male; Microscopy, Electron; Middle Aged; Myelin Basic Protein; Oligodendroglia; Proto-Oncogene Proteins c-bcl-2