Aspartoacylase Research Articles

Cell-Based Therapy for Canavan Disease Using Human iPSC-Derived NPCs and OPCs.

Canavan disease (CD) is a fatal leukodystrophy caused by mutation of the aspartoacylase (ASPA) gene, which leads to deficiency in ASPA activity, accumulation of the substrate N‐acetyl‐L‐aspartate (NAA), demyelination, and spongy degeneration of the brain. There is neither a cure nor a standard treatment for this disease. In this study, human induced pluripotent stem cell (iPSC)‐based cell therapy is developed for CD. A functional ASPA gene is introduced into patient iPSC‐derived neural progenitor cells (iNPCs) or oligodendrocyte progenitor cells (iOPCs) via lentiviral transduction or TALEN‐mediated genetic engineering to generate ASPA iNPC or ASPA iOPC. After stereotactic transplantation into a CD (Nur7) mouse model, the engrafted cells are able to rescue major pathological features of CD, including deficient ASPA activity, elevated NAA levels, extensive vacuolation, defective myelination, and motor function deficits, in a robust and sustainable manner. Moreover, the transplanted mice exhibit much prolonged survival. These genetically engineered patient iPSC‐derived cellular products are promising cell therapies for CD. This study has the potential to bring effective cell therapies, for the first time, to Canavan disease children who have no treatment options. The approach established in this study can also benefit many other children who have deadly genetic diseases that have no cure.

Monitoring prostate cancer (PCa) with appraise the gene expression of PRUNE2, NCAPD3 and ASPA and their connection with age, family history and tumor stage

IntroductionA major cause of cancer mortality among men is prostate cancer (PCa). In this survey we tried to find a new way to screening and monitoring PCa patients by specific biomarkers like protein prune homolog 2 (PRUNE2), non-SMC condensing complex subunit D3 (NCAPD3), and aspartoacylase (ASPA). Material and methodsBiopsy-proven PCa of 40 Iranian men were collected from the Shahid Akbar Abadi Hospital during 2018 year. Following a screening to determine their family history of prostate cancer encounter, smoking habit and stages and grades of their PCa by tumor, node, and metastasis staging system (TNM) were done by telephone and check the patient file. In the following, their biomarkers are detected by real-time PCR method. ResultsPursuant to our outcome, there is no correlation between neither smoking habits nor age average of PCa and control groups, significantly (P-value > 0.05). There is a significant decreasing in NCAPD3 and ASPA expression in PCa group, yet we can see an opposite result about PRUNE2 expression (P-value < 0.05). Our investigation showed that 5.7% frequency among all PCa samples, which all of them are both family history and smoking positive, presented T4N1M1. ConclusionsUp and down in expression of biomarkers in patients with prostate cancer, give us a clear perspective of “what will happen next” these patients, but if we want to use these biomarkers instead PSA test, we need more studies and information of PRUNE2, NCAPD3, ASPA and other genes connection to PCa.

Bioinformatics Analysis of Stromal Molecular Signatures Associated with Breast and Prostate Cancer.

This study aimed to identify stromal molecular signatures associated with breast and prostate cancer. The microarray data GSE26910 was downloaded from Gene Expression Omnibus database, including six invasive breast tumor stroma, six matched normal controls, six invasive prostate tumor stroma, and six matched controls. The differentially expressed genes (DEGs) in invasive breast and prostate tumors stroma were, respectively, identified. Then common stromal genes (B_P.DEGs) were further screened. Protein-protein interaction (PPI) network was constructed and Gene Ontology analysis was performed. Besides, gene-chemical interactions were mapped in Comparative Toxicogenomics Database to screen the chemicals related to feature genes. The results showed that, in total, 16 B_P.DEGs were identified. Thereinto, only seven B_P.DEGs were mapped into PPI, and only four functional modules (adenylate cyclase activating polypeptide 1 (pituitary) receptor type I (ADCYAP1R1) module, aspartoacylase (ASPA) module, glutathione S-transferase mu 5 (GSTM5) module, and periplakin (PPL) module) were involved in important biological processes associated with cancer progression. In addition, the chemicals, such as dihydrotestosterone, apocarotenal, testosterone, and progesterone, were screened for the roles of feature genes in the progression of breast and prostate cancer. In conclusion, ADCYAP1R1, GSTM5, and PPL were stromal molecular signatures and might play a key role in the progression of breast and prostate cancer.

Downregulation of aspartoacylase during the progression of myelin breakdown in the dmy mutant rat with mitochondrial magnesium channel MRS2 defect

ObjectiveThe dmy rat is an autosomal recessive mutant that exhibits severe rapid myelin breakdown throughout the central nervous system at 7–8 weeks of age. The dmy rat has a point mutation in Mrs2 gene, which encodes an essential component of the major electrophoretic Mg2+ influx system in the mitochondria. However, it remains unknown how mitochondrial dysfunction leads to the myelin breakdown. MethodsWe focused on the aspartoacylase (ASPA) and mitochondrion-related metabolites to clarify the mechanism of myelin pathology in dmy rats. Aspa mRNA was significantly decreased in both the gray matter and the ventral white matter of spinal cord in the dmy rats from 4 to 8 weeks of age. Very faint immunohistochemical expression for ASPA was noted in the gray and white matter of the affected dmy rats at 8 weeks. Liquid chromatography mass spectrometry revealed no different amount of N-acetylaspartate (NAA), which is synthesized from aspartate and acetyl-coenzyme A (CoA) in neurons, in the brain and spinal cord between the dmy and control rats. ConclusionOur results indicated that the pyruvate dehydrogenase activity might be reduced due to the loss of Mg2+ transport activity in the mitochondria of the dmy rats, suggesting acetyl CoA production might be reduced. The number of oligodendrocytes was well preserved until 7 weeks. It is intriguing that prior to the myelin destruction at 7–8 weeks, disrupted expression of Aspa mRNA and ASPA protein undergoes from early stage of myelinogenesis. These data indicate that ASPA expression would be a useful index to evaluate a function of oligodendrocyte in the dmy rat.

Next generation sequencing of RNA reveals novel targets of resveratrol with possible implications for Canavan disease

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.

Comparative computational assessment of the pathogenicity of mutations in the Aspartoacylase enzyme.

Aspartoacylase (ASPA) is a zinc-dependent abundant enzyme in the brain, which catalyzes the conversion of N-acetyl aspartate (NAA) into acetate and aspartate. Mutations in the ASPA gene are associated with the development of Canavan disease (CD), leading to the deficiency of ASPA activity. Patients with CD were characterized by degeneration of the white matter of the brain. We reported earlier on two patients with severe form of CD that both had two novel missense mutations in the ASPA: c.427 A > G; p. I143V and c.557 T > A; p. V186D (Zaki et al. 2017a), patient 1 harbored both mutations (p.I143V and p.V186D) in a heterozygous form together with four other mutations, and patient 2 had both mutations in homozygous form. Wijayasinghe et al. (2014) crystallized the 3D structures of four different ASPA mutants (p.K213E, p.Y231C, p.E285A, and p.F295S). In this study, we used in silico prediction methods and molecular dynamics simulation (MDS) to understand the structural impact of all these mutations. Moreover, we used molecular docking (MD) to investigate the binding patterns of the NAA substrate to the native and mutant proteins. Among the mutations, p.E285A (crystallized mutant) was predicted to be the most deleterious for the protein function and the least deleteriousness mutant was the p.I143V (novel mutant). Among the novel mutations, p.V186D was observed to be disruptive for both the zinc binding and NAA binding than the p.I143V. This study provides practical insights on the effect of these mutations on the ASPA function and might serve as a platform for drug design for CD treatment.

Increasing N-acetylaspartate in the Brain during Postnatal Myelination Does Not Cause the CNS Pathologies of Canavan Disease.

Canavan disease is caused by mutations in the gene encoding aspartoacylase (ASPA), a deacetylase that catabolizes N-acetylaspartate (NAA). The precise involvement of elevated NAA in the pathogenesis of Canavan disease is an ongoing debate. In the present study, we tested the effects of elevated NAA in the brain during postnatal development. Mice were administered high doses of the hydrophobic methyl ester of NAA (M-NAA) twice daily starting on day 7 after birth. This treatment increased NAA levels in the brain to those observed in the brains of Nur7 mice, an established model of Canavan disease. We evaluated various serological parameters, oxidative stress, inflammatory and neurodegeneration markers and the results showed that there were no pathological alterations in any measure with increased brain NAA levels. We examined oxidative stress markers, malondialdehyde content (indicator of lipid peroxidation), expression of NADPH oxidase and nuclear translocation of the stress-responsive transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF-2) in brain. We also examined additional pathological markers by immunohistochemistry and the expression of activated caspase-3 and interleukin-6 by Western blot. None of the markers were increased in the brains of M-NAA treated mice, and no vacuoles were observed in any brain region. These results show that ASPA expression prevents the pathologies associated with excessive NAA concentrations in the brain during postnatal myelination. We hypothesize that the pathogenesis of Canavan disease involves not only disrupted NAA metabolism, but also excessive NAA related signaling processes in oligodendrocytes that have not been fully determined and we discuss some of the potential mechanisms.

In search of druggable targets for GBM amino acid metabolism

BackgroundAmino acid (AA) pathways may contain druggable targets for glioblastoma (GBM). Literature reviews and GBM database (http://r2.amc.nl) analyses were carried out to screen for such targets among 95 AA related enzymes.MethodsFirst, we identified the genes that were differentially expressed in GBMs (3 datasets) compared to non-GBM brain tissues (5 datasets), or were associated with survival differences. Further, protein expression for these enzymes was also analyzed in high grade gliomas (HGGs) (proteinatlas.org). Finally, AA enzyme and gene expression were compared among the 4 TCGA (The Cancer Genome Atlas) subtypes of GBMs.ResultsWe detected differences in enzymes involved in glutamate and urea cycle metabolism in GBM. For example, expression levels of BCAT1 (branched chain amino acid transferase 1) and ASL (argininosuccinate lyase) were high, but ASS1 (argininosuccinate synthase 1) was low in GBM. Proneural and neural TCGA subtypes had low expression of all three. High expression of all three correlated with worse outcome. ASL and ASS1 protein levels were mostly undetected in high grade gliomas, whereas BCAT1 was high. GSS (glutathione synthetase) was not differentially expressed, but higher levels were linked to poor progression free survival. ASPA (aspartoacylase) and GOT1 (glutamic-oxaloacetic transaminase 1) had lower expression in GBM (associated with poor outcomes). All three GABA related genes -- glutamate decarboxylase 1 (GAD1) and 2 (GAD2) and 4-aminobutyrate aminotransferase (ABAT) -- were lower in mesenchymal tumors, which in contrast showed higher IDO1 (indoleamine 2, 3-dioxygenase 1) and TDO2 (tryptophan 2, 3-diaxygenase). Expression of PRODH (proline dehydrogenase), a putative tumor suppressor, was lower in GBM. Higher levels predicted poor survival.ConclusionsSeveral AA-metabolizing enzymes that are higher in GBM, are also linked to poor outcome (such as BCAT1), which makes them potential targets for therapeutic inhibition. Moreover, existing drugs that deplete asparagine and arginine may be effective against brain tumors, and should be studied in conjunction with chemotherapy. Last, AA metabolism is heterogeneous in TCGA subtypes of GBM (as well as medulloblastomas and other pediatric tumors), which may translate to variable responses to AA targeted therapies.

Redirecting N-acetylaspartate metabolism in the central nervous system normalizes myelination and rescues Canavan disease.

Canavan disease (CD) is a debilitating and lethal leukodystrophy caused by mutations in the aspartoacylase (ASPA) gene and the resulting defect in N-acetylaspartate (NAA) metabolism in the CNS and peripheral tissues. Recombinant adeno-associated virus (rAAV) has the ability to cross the blood-brain barrier and widely transduce the CNS. We developed a rAAV-based and optimized gene replacement therapy, which achieves early, complete, and sustained rescue of the lethal disease phenotype in CD mice. Our treatment results in a super-mouse phenotype, increasing motor performance of treated CD mice beyond that of WT control mice. We demonstrate that this rescue is oligodendrocyte independent, and that gene correction in astrocytes is sufficient, suggesting that the establishment of an astrocyte-based alternative metabolic sink for NAA is a key mechanism for efficacious disease rescue and the super-mouse phenotype. Importantly, the use of clinically translatable high-field imaging tools enables the noninvasive monitoring and prediction of therapeutic outcomes for CD and might enable further investigation of NAA-related cognitive function.

Suppressing N-Acetyl-l-Aspartate Synthesis Prevents Loss of Neurons in a Murine Model of Canavan Leukodystrophy.

This is the first demonstration of cortical and cerebellar neuron depletion and progressive cerebral cortical thinning in an animal model of Canavan disease. Genetic suppression of N-acetyl-l-aspartate (NAA) synthesis, previously shown to block brain vacuolation in aspartoacylase-deficient mice, also prevents neuron loss and cerebral cortical atrophy in these mice. These results suggest that lowering the concentration of NAA in the brains of children with Canavan disease would prevent or slow progression of neurological deficits.

The neuronal metabolite NAA regulates histone H3 methylation in oligodendrocytes and myelin lipid composition.

The neuronal mitochondrial metabolite N-acetylaspartate (NAA) is decreased in the multiple sclerosis (MS) brain. NAA is synthesized in neurons by the enzyme N-acetyltransferase-8-like (NAT8L) and broken down in oligodendrocytes by aspartoacylase (ASPA) into acetate and aspartate. We have hypothesized that NAA links the metabolism of axons with oligodendrocytes to support myelination. To test this hypothesis, we performed lipidomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance thin-layer chromatography (HPTLC) to identify changes in myelin lipid composition in postmortem MS brains and in NAT8L knockout (NAT8L-/-) mice which do not synthesize NAA. We found reduced levels of sphingomyelin in MS normal appearing white matter that mirrored decreased levels of NAA. We also discovered decreases in the amounts of sphingomyelin and sulfatide lipids in the brains of NAT8L-/- mice compared to controls. Metabolomic analysis of primary cultures of oligodendrocytes treated with NAA revealed increased levels of α-ketoglutarate, which has been reported to regulate histone demethylase activity. Consistent with this, NAA treatment resulted in alterations in the levels of histone H3 methylation, including H3K4me3, H3K9me2, and H3K9me3. The H3K4me3 histone mark regulates cellular energetics, metabolism, and growth, while H3K9me3 has been linked to alterations in transcriptional repression in developing oligodendrocytes. We also noted the NAA treatment was associated with increases in the expression of genes involved in sulfatide and sphingomyelin synthesis in cultured oligodendrocytes. This is the first report demonstrating that neuronal-derived NAA can signal to the oligodendrocyte nucleus. These data suggest that neuronal-derived NAA signals through epigenetic mechanisms in oligodendrocytes to support or maintain myelination.

351. Efficacious Non-Oligodendrocyte Gene Therapy Suggests a New Dogma About CNS Compartmentalization of NAA Metabolism and Supports a Metabolic Sink Theory

N-acetylaspartate (NAA) is the second most abundant amino acid derivative in the mammalian central nervous system (CNS). Although its physiologic function remains elusive, many CNS disorders have been associated with changes in NAA levels, e.g. Alzheimer's disease, bipolar disorder. The disease that is known to have direct connection to altered NAA metabolism is Canavan disease (CD); a leukodystrophy caused by mutations in the aspartoacylase (ASPA) gene. The current understanding is that, physiologically, the ASPA enzyme hydrolyzes NAA into L-aspartate and acetate in oligodendrocytes. Consequently, it was postulated that Canavan gene therapy has to restore ASPA expression in oligodendrocytes. However, we hypothesized that NAA can move freely and its cell-type independent break-down ameliorates Canavan disease. We constructed several tissue/cell-specific expression cassettes limiting hASPA expression to either astrocytes, neurons, oligodendrocytes, liver, heart, or muscle. We opted for the Canavan disease knock-out (CD KO) mouse model because it shows early lethality at around post-natal day 28 and the severest disease phenotype of all Canavan mouse models available. To our surprise, mice expressing hASPA restricted to peripheral organs showed extended survival and normalization of the growth curve at later time points, suggesting a contribution of peripheral organs to the disease pathomechanism. Moreover, astrocyte restricted hASPA expression produced the strongest disease recovery matching the performance of wild-type (WT) mice. Thus, our data seem to support our oligodendrocytes independent NAA metabolic sink theory. In an attempt to verify this theory, we used lower dose rAAVhASPA for localized brain injections to demonstrate that localized T2 hyperintensity signal clearance on MRI was well correlated with reduction of NAA levels by MRS. In other words, the further away from the injection site, the higher the NAA levels, which supports the idea of drainage and hydrolytic activity of NAA towards the injection side. Currently, we are investigating this metabolic sink theory in more detail by creating a functional map of therapeutic gene transfer in the brain by mass spectrometry quantification of NAA, and vector genome and ASPA transcripts analyses in different anatomic regions. Overall, our data present evidence that ASPA expression does not have to be restored in oligodendrocytes in order to rescue lethality and Canavan disease phenotype, which is in congruence with the metabolic sink theory. Furthermore, this calls into question a common hypothesis that NAA breakdown in oligodendrocytes is the mainstay for myelination and myelin defects in Canavan disease and might even support the idea that other metabolic disorders could benefit from treatment under the premise of the metabolic sink theory.

349. The Cure of Canavan Disease: Is It a Scientific Fiction or Clinical Reality?

Canavan Disease (CD) is a rare and lethal inherited pediatric CNS disorder with recessive mutations in the aspartoacylase (ASPA) gene. Traditionally, its variable disease phenotypes were descripted with the congenital sub-form showing neonatal onset and the severest phenotype with early death. The two other sub-forms, infantile, and juvenile, manifest with delayed onset and milder symptoms. Some CD patients are now 20 years of age and older. To date, there is no effective treatment available. Thus, gene replacement therapy is an attractive approach for treating this devastating disease. Previously, we have shown that a single intravenous (i.v.) injection of recombinant adeno-associated virus (rAAV) expressing human ASPA (hASPA) rescues early lethality and partially restores motor function (1st generation gene therapy) in the CD knock-out (CD KO) mouse, which resembles the congenital sub-form of CD and displays the severest phenotype of all available CD mouse models, with early death at around post-natal day (p) 28. After this remarkable improvement of symptoms with our 1st generation treatment, we further optimized our gene therapy. Now, in its 2nd and 3rd generation, our gene therapy cures the disease in the CD KO mouse. Interestingly, our 3rd generation gene therapy turns CD KO mice into “supermice”, outperforming WT mice on rotarod motor function test. This rescue is persistent and currently mice at 1.5 years of age still show no signs of disease reoccurrence. CNS pathology and magnet resonance imaging (MRI) at p25 and p365 show complete normalization. To further support the efficacy of our 3rd generation gene therapy, we performed neurometabolome profiling with over 400 characterized metabolites that showed reversal of the Canavan disease related metabolic changes including myelin associated lipids. To further evaluate the potency of our 3rd generation gene therapy, we tested different doses and routes of administration. Of note, 200-fold lower doses intraventricularly (ICV) administered still rescues lethality, while mice treated ICV with 20-fold reduced dose draw even with WT mice on motor function testing. Next, we moved to the Nur7 mouse model (resembles infantile and juvenile sub-form) that displays a similar disease pattern as the CD KO mouse with respect to growth curve and neurologic symptoms but eventually re-gains weight and shows survival similar to wild-type mice. Again, we treated mice i.v. with a single dose of rAAVhASPA at p1 as our gold standard and subsequent groups at 6 and 12 weeks of age to determine the therapeutic window. Of note, mice treated at 6 weeks of age recovered within 4 weeks post-treatment. Mice treated later than 6 weeks require more time to recover but still showed significant improvement over Nur7 mutants. This recovery was also correlated by CNS pathology and MRI. Currently, we are evaluating mice that were treated at 24 weeks of age to determine if there is a time point of no return. Overall, our data show clear evidence for the cure of the disease at early and late stages of the disease in two different mouse models. In addition, this is confirmed on different levels of cellular complexity by MRI, fMRI, CNS pathology, and neurometabolic profiling.

Modeling the Complete Catalytic Cycle of Aspartoacylase.

The complete catalytic cycle of aspartoacylase (ASPA), a zinc-dependent enzyme responsible for cleavage of N-acetyl-l-aspartate, is characterized by the methods of molecular modeling. The reaction energy profile connecting the enzyme-substrate (ES) and the enzyme-product (EP) complexes is constructed by the quantum mechanics/molecular mechanics (QM/MM) method assisted by the molecular dynamics (MD) simulations with the QM/MM potentials. Starting from the crystal structure of ASPA complexed with the intermediate analogue, the minimum-energy geometry configurations and the corresponding transition states are located. The stages of substrate binding to the enzyme active site and release of the products are modeled by MD calculations with the replica-exchange umbrella sampling technique. It is shown that the first reaction steps, nucleophilic attack of a zinc-bound nucleophilic water molecule at the carbonyl carbon and the amide bond cleavage, are consistent with the glutamate-assisted mechanism hypothesized for the zinc-dependent hydrolases. The stages of formation of the products, acetate and l-aspartate, and regeneration of the enzyme are characterized for the first time. The constructed free energy diagram from the reactants to the products suggests that the enzyme regeneration, but not the nucleophilic attack of the catalytic water molecule, corresponds to the rate-determining stage of the full catalytic cycle of ASPA.

PSA-NCAM positive neural progenitors stably expressing BDNF promote functional recovery in a mouse model of spinal cord injury.

BackgroundNeural stem cells for the treatment of spinal cord injury (SCI) are of particular interest for future therapeutic use. However, until now, stem cell therapies are often limited due to the inhibitory environment following the injury. Therefore, in this study, we aimed at testing a combinatorial approach with BDNF (brain-derived neurotrophic factor) overexpressing early neural progenitors derived from mouse embryonic stem cells. BDNF is a neurotrophin, which both facilitates neural differentiation of stem cells and favors regeneration of damaged axons.MethodsMouse embryonic stem cells, modified to stably express BDNF-GFP, were differentiated into PSA-NCAM positive progenitors, which were enriched, and SSEA1 depleted by a sequential procedure of magnetic-activated and fluorescence-activated cell sorting. Purified cells were injected into the lesion core seven days after contusion injury of the spinal cord in mice, and the Basso mouse scale (BMS) test to evaluate motor function was performed for 5 weeks after transplantation. To analyze axonal regeneration the anterograde tracer biotinylated dextran amine was injected into the sensorimotor cortex two weeks prior to tissue analysis. Cellular differentiation was analyzed by immunohistochemistry of spinal cord sections.ResultsMotor function was significantly improved in animals obtaining transplanted BDNF-GFP-overexpressing cells as compared to GFP-expressing cells and vehicle controls. Stem cell differentiation in vivo revealed an increase of neuronal and oligodendrocytic lineage differentiation by BDNF as evaluated by immunohistochemistry of the neuronal marker MAP2 (microtubule associated protein 2) and the oligodendrocytic markers ASPA (aspartoacylase) and Olig2 (oligodendrocyte transcription factor 2). Furthermore, axonal tracing showed a significant increase of biotin dextran amine positive corticospinal tract fibers in BDNF-GFP-cell transplanted animals caudally to the lesion site.ConclusionsThe combinatorial therapy approach by transplanting BDNF-overexpressing neural progenitors improved motor function in a mouse contusion model of SCI. Histologically, we observed enhanced neuronal and oligodendrocytic differentiation of progenitors as well as enhanced axonal regeneration.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0268-x) contains supplementary material, which is available to authorized users.

Atypical clinical and radiological course of a patient with Canavan disease

Canavan disease (CD) is a rare metabolic disorder caused by aspartoacylase (ASPA) deficiency. It leads to severe neurological degeneration with spongiform brain degeneration. Accumulation of N-acetylaspartate (NAA) in brain and urine is specific to the disease and guides diagnosis. Magnetic resonance imaging (MRI) usually shows diffuse white matter abnormalities with involvement of the basal ganglia. Mild forms of the disease with a more favorable clinical course and radiological involvement of the basal ganglia without white matter abnormalities have also been reported. Here we report an atypical case of a girl aged nine years with CD. The disease started at the classical age of five months. Classical elevation of NAA in brain and urine was present and genetic analysis identified mutations in the ASPA gene. However, clinical evolution was milder than typical CD, with partial motor impairment and relatively well-preserved cognitive skills. MRI was also atypical with low white matter involvement and unusual topography and evolution of abnormalities in the basal ganglia.

N-Acetylaspartate Synthase Deficiency Corrects the Myelin Phenotype in a Canavan Disease Mouse Model But Does Not Affect Survival Time.

Canavan disease (CD) is a severe, lethal leukodystrophy caused by deficiency in aspartoacylase (ASPA), which hydrolyzes N-acetylaspartate (NAA). In the brains of CD patients, NAA accumulates to high millimolar concentrations. The pathology of the disease is characterized by loss of oligodendrocytes and spongy myelin degeneration in the CNS. Whether accumulating NAA, absence of NAA-derived acetate, or absence of any unknown functions of the ASPA enzyme is responsible for the pathology of the disease is not fully understood. We generated ASPA-deficient (Aspa(nur7/nur7)) mice that are also deficient for NAA synthase Nat8L (Nat8L(-/-)/Aspa(nur7/nur7)). These mice have no detectable NAA. Nevertheless, they exhibited normal myelin content, myelin sphingolipid composition, and full reversal of spongy myelin and axonal degeneration. Surprisingly, although pathology was fully reversed, the survival time of the mice was not prolonged. In contrast, Aspa(nur7/nur7) mice with only one intact Nat8L allele accumulated less NAA, developed a less severe pathology, phenotypic improvements, and, importantly, an almost normal survival time. Therefore, inhibition of NAA synthase is a promising therapeutic option for CD. The reduced survival rate of Nat8L(-/-)/Aspa(nur7/nur7) mice, however, indicates that complete inhibition of NAA synthase may bear unforeseeable risks for the patient. Furthermore, we demonstrate that acetate derived from NAA is not essential for myelin lipid synthesis and that loss of NAA-derived acetate does not cause the myelin phenotype of Aspa(nur7/nur7) mice. Our data clearly support the hypothesis that NAA accumulation is the major factor in the development of CD.

Canavan disease: A first case from Jordan

Canavan disease (CD) is a fatal, hereditary disorder of central nervous system development that has been linked to mutations in the gene for the enzyme aspartoacylase (ASPA) which leads to accumulation of N-acetyl aspartic acid (NAA) in the brain with resultant leukodystrophy. CD is most prevalent in people of Ashkenazi Jewish descent but has been observed in other ethnic groups. Patients have severe mental retardation, poor head control, macrocephaly, and seizures. We report a 3 months old male infant, the sixth child of healthy consanguineous second cousins parents, presenting with poor visual fixation poor response to sounds, axial and peripheral hypotonia, severe head lag, and exaggerated deep tendon reflexes in the upper and lower limbs. Family history revealed that the proband had four brothers and one sister with severe global delay, poor visual fixation, poor response to sounds, spasticity, poor head control and large heads. Urinary organic acids showed high levels of NAA, and ASPA gene mutation analysis identified a rare mutation in exon 1 (G27 R). This is the first case of CD to be reported from Jordan. Organic acid screening in urine for all infants with global delay and macrocephaly could lead to the diagnosis of more cases of CD in Jordan with determination of the most prevalent gene mutation and the designation of the relevant screening test.

Canavan disease: Can two wrongs make a right?

Double-knockout mice that lack both aspartoacylase (ASPA) and NAA synthase (Nat8L) show improved myelination and behavioral recovery, partially circumventing the effects of Canavan disease.

Acetate supplementation as a means of inducing glioblastoma stem-like cell growth arrest.

Glioblastoma (GBM), the most common primary adult malignant brain tumor, is associated with a poor prognosis due, in part, to tumor recurrence mediated by chemotherapy and radiation resistant glioma stem-like cells (GSCs). The metabolic and epigenetic state of GSCs differs from their non-GSC counterparts, with GSCs exhibiting greater glycolytic metabolism and global hypoacetylation. However, little attention has been focused on the potential use of acetate supplementation as a therapeutic approach. N-acetyl-l-aspartate (NAA), the primary storage form of brain acetate, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis, are significantly reduced in GBM tumors. We recently demonstrated that NAA supplementation is not an appropriate therapeutic approach since it increases GSC proliferation and pursued an alternative acetate source. The FDA approved food additive Triacetin (glyceryl triacetate, GTA) has been safely used for acetate supplementation therapy in Canavan disease, a leukodystrophy due to ASPA mutation. This study characterized the effects of GTA on the proliferation and differentiation of six primary GBM-derived GSCs relative to established U87 and U251 GBM cell lines, normal human cerebral cortical astrocytes, and murine neural stem cells. GTA reduced proliferation of GSCs greater than established GBM lines. Moreover, GTA reduced growth of the more aggressive mesenchymal GSCs greater than proneural GSCs. Although sodium acetate induced a dose-dependent reduction of GSC growth, it also reduced cell viability. GTA-mediated growth inhibition was not associated with differentiation, but increased protein acetylation. These data suggest that GTA-mediated acetate supplementation is a novel therapeutic strategy to inhibit GSC growth.