Aspartoacylase Research Articles

N-acetylaspartate promotes glycolytic-to-oxidative fiber-type switch and resistance to atrophic stimuli in myotubes

N-acetylaspartate (NAA) is a neuronal metabolite that can be extruded in extracellular fluids and whose blood concentration increases in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Aspartoacylase (ASPA) is the enzyme responsible for NAA breakdown. It is abundantly expressed in skeletal muscle and most other human tissues, but the role of NAA catabolism in the periphery is largely neglected. Here we demonstrate that NAA treatment of differentiated C2C12 muscle cells increases lipid turnover, mitochondrial biogenesis and oxidative metabolism at the expense of glycolysis. These effects were ascribed to NAA catabolism, as CRISPR/Cas9 ASPA KO cells are insensitive to NAA administration. Moreover, the metabolic switch induced by NAA was associated with an augmented resistance to atrophic stimuli. Consistently with in vitro results, SOD1-G93A ALS mice show an increase in ASPA levels in those muscles undergoing the glycolytic to oxidative switch during the disease course. The impact of NAA on the metabolism and resistance capability of myotubes supports a role for this metabolite in the phenotypical adaptations of skeletal muscle in neuromuscular disorders.

Canavan disease: a rare case report

Canavan disease is a very rare, fatal, autosomal recessive neurodegenerative disorder. It usually manifests during infancy. Mutations in the aspartoacylase (ASPA) gene (17p13.3), which code for the aspartoacylase enzyme, cause this disease. It is a leukodystrophy and affects white matter of the brain. This disease is associated with deficiency of an essential enzyme, aspartoacylase. The deficiency leads to loss of white matter in brain, leading to defective transmission of nerve signals. This disorder affects all ethnic groups, but occurs with greater frequency, in individuals of Ashkenazi Jewish descent.

Myelin lesion in the aspartoacylase (Aspa) knockout rat, an animal model for Canavan disease.

Canavan disease (CD) is a fatal hereditary neurological disorder caused by a mutation in the aspartoacylase (ASPA) gene and characterized by neurological signs and vacuolation in the central nervous system (CNS). The mutation inhibits the hydrolysis of N-acetyl-aspartate (NAA) resulting in accumulation of NAA in the CNS. A new Aspa-knockout rat was generated by transcription activator-like effector nuclease (TALEN) technology. Herein we describe the pathological and morphometrical findings in the brain and spinal cords of Aspa-knockout rats. Although Aspa-knockout rats did not show any neurological signs, vacuolation with swollen axons, hypomyelination, and activated swollen astrocytes were observed mainly in the brainstem reticular formation, ascending and descending motor neuron pathway, and in the olfactory tract. Morphometrical analysis revealed no obvious change in the number of neurons. These changes in the CNS are similar to human CD, suggesting that this animal model would be useful for further study of treatment and understanding the pathophysiology of human CD.

NAAG synthetase deficiency has only low influence on pathogenesis in a Canavan disease mouse model.

Canavan disease (CD) is a leukodystrophy caused by mutations in the N-acetylaspartate (NAA) hydrolase aspartoacylase (ASPA). Inability to degrade NAA and its accumulation in the brain results in spongiform myelin degeneration. NAA is mainly synthesized by neurons, where it is also a precursor of the neuropeptide N-acetylaspartylglutamate (NAAG). Hydrolysis of this peptide by glutamate carboxypeptidases is an additional source of extracellular NAA besides the instant neuronal release of NAA. This study examines to what extent NAA released from NAAG contributes to NAA accumulation and pathogenesis in the brain of Aspanur7/nur7 mutant mice, an established model of CD. Towards this aim, Aspanur7/nur7 mice with additional deficiencies in NAAG synthetase genes Rimklb and/or Rimkla were generated. Loss of myelin in Aspanur7/nur7 mice was not significantly affected by Rimkla and Rimklb deficiency and there was also no obvious change in the extent of brain vacuolation. Astrogliosis was slightly reduced in the forebrain of Rimkla and Rimklb double deficient Aspanur7/nur7 mice. However, only minor improvements at the behavioral level were found. The brain NAA accumulation in CD mice was, however, not significantly reduced in the absence of NAAG synthesis. In summary, there was only a weak tendency towards reduced pathogenic symptoms in Aspanur7/nur7 mice deficient in NAAG synthesis. Therefore, we conclude that NAAG metabolism has little influence on NAA accumulation in Aspanur7/nur7 mice and development of pathological symptoms in CD.

Pathological Bergmann glia alterations and disrupted calcium dynamics in ataxic Canavan disease mice.

Canavan disease (CD) is a recessively inherited pediatric leukodystrophy resulting from inactivating mutations to the oligodendroglial enzyme aspartoacylase (ASPA). ASPA is responsible for hydrolyzing the amino acid derivative N-acetyl-L-aspartate (NAA), and without it, brain NAA concentrations increase by 50% or more. Infants and children with CD present with progressive cognitive and motor delays, cytotoxic edema, astroglial vacuolation, and prominent spongiform brain degeneration. ASPA-deficient CD mice (Aspanur7/nur7 ) present similarly with elevated NAA, widespread astroglial dysfunction, ataxia, and Purkinje cell (PC) dendritic atrophy. Bergmann glia (BG), radial astrocytes essential for cerebellar development, are intimately intertwined with PCs, where they regulate synapse stability, functionality, and plasticity. BG damage is common to many neurodegenerative conditions and frequently associated with PC dysfunction and ataxia. Here, we report that, in CD mice, BG exhibit significant morphological alterations, decreased structural associations with PCs, loss of synaptic support proteins, and altered calcium dynamics. We also find that BG dysfunction predates cerebellar vacuolation and PC damage in CD mice. Previously, we developed an antisense oligonucleotide (ASO) therapy targeting Nat8l (N-acetyltransferase-8-like, "Nat8l ASO") that inhibits the production of NAA and reverses ataxia and PC atrophy in CD mice. Here, we show that Nat8l ASO administration in adult CD mice also leads to BG repair. Furthermore, blocking astroglial uptake of NAA is neuroprotective in astroglia-neuron cocultures exposed to elevated NAA. Our findings suggest that restoration of BG structural and functional integrity could be a mechanism for PC regeneration and improved motor function.

Renewal of oligodendrocyte lineage reverses dysmyelination and CNS neurodegeneration through corrected N-acetylaspartate metabolism.

Myelinating oligodendrocytes are essential for neuronal communication and homeostasis of the central nervous system (CNS). One of the most abundant molecules in the mammalian CNS is N-acetylaspartate (NAA), which is catabolized into L-aspartate and acetate by the enzyme aspartoacylase (ASPA) in oligodendrocytes. The resulting acetate moiety is thought to contribute to myelin lipid synthesis. In addition, affected NAA metabolism has been implicated in several neurological disorders, including leukodystrophies and demyelinating diseases such as multiple sclerosis. Genetic disruption of ASPA function causes Canavan disease, which is hallmarked by increased NAA levels, myelin and neuronal loss, large vacuole formation in the CNS, and early death in childhood. Although NAA's direct role in the CNS is inconclusive, in peripheral adipose tissue, NAA-derived acetate has been found to modify histones, a mechanism known to be involved in epigenetic regulation of cell differentiation. We hypothesize that a lack of cellular differentiation in the brain contributes to the disruption of myelination and neurodegeneration in diseases with altered NAA metabolism, such as Canavan disease. Our study demonstrates that loss of functional Aspa in mice disrupts myelination and shifts the transcriptional expression of neuronal and oligodendrocyte markers towards less differentiated stages in a spatiotemporal manner. Upon re-expression of ASPA, these oligodendrocyte and neuronal lineage markers are either improved or normalized, suggesting that NAA breakdown by Aspa plays an essential role in the maturation of neurons and oligodendrocytes. Also, this effect of ASPA re-expression is blunted in old mice, potentially due to limited ability of neuronal, rather than oligodendrocyte, recovery.

366-OR: Regulation of Metabolite Pools Influences Adipocyte Differentiation and Antioxidant Capacity

Obesity and related metabolic disorders, such as insulin resistance and type 2 diabetes (T2D), affect over 40% of U.S. adults and increase the risk of death from cardiovascular disease. Understanding the mechanisms connecting obesity, T2D, and defective adipocyte function bring us closer to developing therapeutic strategies. We discovered expression of the enzyme aspartoacylase (ASPA) in white adipose tissue (WAT) correlates with adipocyte function and insulin sensitivity in mice and people. In the brain, ASPA contributes to the acetate pool necessary for lipid synthesis, and ASPA deficiency causes progressive myelin degeneration and death at a young age. We also observed abundant ASPA expression in WAT at significantly higher levels than whole brain. Aspa is induced during adipocyte differentiation in vitro along with known adipogenic markers. These observations led us to hypothesize that ASPA and its metabolite byproducts regulate adipocyte differentiation and systemic energy balance. When assessing the in vivo effects of Aspa deficiency, Aspa-/- mice accumulated less body mass than Aspa+/+ when fed normal chow diet for 12 weeks. Morphometric tissue analyses indicated Aspa-/- mice maintain smaller adipocytes, corresponding with lower WAT mass at necropsy. Glucose tracing experiments suggested Aspa knockout cells exhibit greater pyruvate carboxylase (PC) anaplerosis that feeds amino acid pools for glutathione synthesis. Mass spectrometry data also indicated higher PC anaplerosis in the adipose tissues of Aspa knockout mice. Persistent and prolonged oxidative stress hinders adipocyte function and insulin sensitivity, while PC activity supports antioxidant capacity. In line with this, Aspa knockout cells showed increased expression of proteins that sustain antioxidant activities. In summary, our studies establish a new role for ASPA outside the brain that contributes to redox activities in adipocytes. Disclosure J.B.Felix: None. P.Saha: None. A.Cox: None. R.A.Sharp: None. P.M.Masschelin: None. S.M.Hartig: None. Funding American Diabetes Association (1-18-IBS-105 to S.M.H.); National Institutes of Health (F31DK130596, R01DK114356)

Adeno-associated virus-mediated gene therapy in a patient with Canavan disease using dual routes of administration and immune modulation

Gene replacement therapy is a rational therapeutic strategy and clinical intervention for neurodegenerative disorders like Canavan disease, a leukodystrophy caused by biallelic mutations in the aspartoacylase (ASPA) gene. We aimed toinvestigate whether simultaneous intravenous (i.v.) and intracerebroventricular (i.c.v.) administration of rAAV9-CB6-ASPA provides a safe and effective therapeutic strategy in an open-label, individual-patient, expanded-access trial for Canavan disease. Immunomodulation was given prophylactically prior to adeno-associated virus (AAV) treatment toprevent an immune response to ASPA or the vector capsid. The patient served as his own control, and change from baseline was assessed by clinical pathology tests, vector genomes in the blood, antibodies against ASPA and AAV capsids, levels of cerebrospinal fluid (CSF) N-acetylaspartate (NAA), brain water content and morphology, clinical status, and motor function tests. Two years post treatment, the patient's white matter myelination had increased, motor function was improved, and he remained free of typical severe epilepsy. NAA level was reduced at 3months and remained stable up to 4 years post treatment. Immunomodulation prior to AAV exposure enables repeat dosing and has prevented an anti-transgene immune response. Dual-route administration of gene therapy may improve treatment outcomes.

Aspartoacylase suppresses prostate cancer progression by blocking LYN activation

BackgroundGlobally, despite prostate cancer (PCa) representing second most prevalent malignancy in male, the precise molecular mechanisms implicated in its pathogenesis remain unclear. Consequently, elucidating the key molecular regulators that govern disease progression could substantially contribute to the establishment of novel therapeutic strategies, ultimately advancing the management of PCa.MethodsA total of 49 PCa tissues and 43 adjacent normal tissues were collected from January 2017 to December 2021 at Zhongnan Hospital of Wuhan University. The advanced transcriptomic methodologies were employed to identify differentially expressed mRNAs in PCa. The expression of aspartoacylase (ASPA) in PCa was thoroughly evaluated using quantitative real-time PCR and Western blotting techniques. To elucidate the inhibitory role of ASPA in PCa cell proliferation and metastasis, a comprehensive set of in vitro and in vivo assays were conducted, including orthotopic and tumor-bearing mouse models (n = 8 for each group). A combination of experimental approaches, such as Western blotting, luciferase assays, immunoprecipitation assays, mass spectrometry, glutathione S-transferase pull-down experiments, and rescue studies, were employed to investigate the underlying molecular mechanisms of ASPA’s action in PCa. The Student’s t-test was employed to assess the statistical significance between two distinct groups, while one-way analysis of variance was utilized for comparisons involving more than two groups. A two-sided P value of less than 0.05 was deemed to indicate statistical significance.ResultsASPA was identified as a novel inhibitor of PCa progression. The expression of ASPA was found to be significantly down-regulated in PCa tissue samples, and its decreased expression was independently associated with patients’ prognosis (HR = 0.60, 95% CI 0.40–0.92, P = 0.018). Our experiments demonstrated that modulation of ASPA activity, either through gain- or loss-of-function, led to the suppression or enhancement of PCa cell proliferation, migration, and invasion, respectively. The inhibitory role of ASPA in PCa was further confirmed using orthotopic and tumor-bearing mouse models. Mechanistically, ASPA was shown to directly interact with the LYN and inhibit the phosphorylation of LYN as well as its downstream targets, JNK1/2 and C-Jun, in both PCa cells and mouse models, in an enzyme-independent manner. Importantly, the inhibition of LYN activation by bafetinib abrogated the promoting effect of ASPA knockdown on PCa progression in both in vitro and in vivo models. Moreover, we observed an inverse relationship between ASPA expression and LYN activity in clinical PCa samples, suggesting a potential regulatory role of ASPA in modulating LYN signaling.ConclusionOur findings provide novel insights into the tumor-suppressive function of ASPA in PCa and highlight its potential as a prognostic biomarker and therapeutic target for the management of this malignancy.

Developing Hypoimmunogenic Human iPSC-Derived Oligodendrocyte Progenitor Cells as an Off-The-Shelf Cell Therapy for Myelin Disorders.

Demyelinating disorders are among the most common and debilitating diseases in neurology. Canavan disease (CD) is a lethal demyelinating disease caused by mutation of the aspartoacylase (ASPA) gene, which leads to the accumulation of its substrate N-acetyl-l-aspartate (NAA), and consequently demyelination and vacuolation in the brain. In this study, hypoimmunogenic human induced pluripotent stem cell (iPSC)-derived oligodendrocyte progenitor cells (OPC) are developed from a healthy donor as an "off-the-shelf" cell therapy. Hypoimmunogenic iPSCs are generated through CRISPR/Cas9 editing of the human leukocyte antigen (HLA) molecules in healthy donor-derived iPSCs and differentiated into OPCs. The OPCs are engrafted into the brains of CD (nur7) mice and exhibit widespread distribution in the brain. The engrafted OPCs mature into oligodendrocytes that express the endogenous wildtype ASPA gene. Consequently, the transplanted mice exhibit elevated human ASPA expression and enzymatic activity and reduced NAA level in the brain. The transplanted OPCs are able to rescue major pathological features of CD, including defective myelination, extensive vacuolation, and motor function deficits. Moreover, the hypoimmunogenic OPCs exhibit low immunogenicity both in vitro and in vivo. The hypoimmunogenic OPCs can be used as "off-the-shelf" universal donor cells to treat various CD patients and many other demyelinating disorders, especially autoimmune demyelinating diseases, such as multiple sclerosis.

Identification of Potential Biomarkers in Stomach Adenocarcinoma using Machine Learning Approaches

Background: Stomach adenocarcinoma (STAD) is a common cancer with poor clinical outcomes globally. Due to a lack of early diagnostic markers of disease, the majority of patients are diagnosed at an advanced stage. Objective: The aim of the present study is to provide some new insights into the available biomarkers for patients with STAD using bioinformatics. Methods: RNA-Sequencing and other relevant data of patients with STAD from The Cancer Genome Atlas (TCGA) database were evaluated to identify differentially expressed genes (DEGs). Then, Machine Learning algorithms were undertaken to predict biomarkers. Additionally, Kaplan–Meier analysis was used to detect prognostic biomarkers. Furthermore, the Gene Ontology and Reactome pathways, protein-protein interactions (PPI), multiple sequence alignment, phylogenetic mapping, and correlation between clinical parameters were evaluated. Results: The results showed 61 DEGs, and the key dysregulated genes associated with STAD are MTHFD1L (Methylenetetrahydrofolate dehydrogenase 1-like), ZWILCH (Zwilch Kinetochore Protein), RCC2 (Regulator of chromosome condensation 2), DPT (Dermatopontin), GCOM1 (GRINL1A complex locus 1), and CLEC3B (C-Type Lectin Domain Family 3 Member B). Moreover, the survival analysis reported ASPA (Aspartoacylase) as a prognostic marker. Conclusion: Our study provides a proof of concept of the potential value of ASPA as a prognostic factor in STAD, requiring further functional investigations to explore the value of emerging markers.

Canavan Disease: A Case Report

Canavan disease is an autosomal recessive leukodystrophy associated with hypotonia, megalencephaly, mental retardation, blindness and spasticity. The biochemical deficiency of aspartoacylase (ASPA) causes CD. Deficiency in ASPA, which hydrolyzes N-acetylaspartate, results in NAA building up to high millimolar amounts in the brain. The hallmarks of the disease are loss of oligodendrocytes and spongy myelin degradation in the CNS. However, it is unclear whether the disease’s pathophysiology is caused by the accumulation of NAA, the absence of NAA-derived acetate, or the absence of any unknown roles of the ASPA enzyme. In this Review, we present an important and timely update on the current and emerging aspects of this neurological disease. Following a brief overview of canavan disease, we discuss current knowledge of neurological findings, pathophysiology, diagnostic approaches, current canavan disease treatment, and gene therapy’s future prospects.

Therapeutic development for Canavan disease using patient iPSCs introduced with the wild-type ASPA gene

SummaryCanavan disease (CD) is a devastating neurological disease that lacks effective therapy. Because CD is caused by mutations of the aspartoacylase (ASPA) gene, we introduced the wild-type (WT) ASPA gene into patient iPSCs through lentiviral transduction or CRISPR/Cas9-mediated gene editing. We then differentiated the WT ASPA-expressing patient iPSCs (ASPA-CD iPSCs) into NPCs and showed that the resultant ASPA-CD NPCs exhibited potent ASPA enzymatic activity. The ASPA-CD NPCs were able to survive in brains of transplanted CD mice. The engrafted ASPA-CD NPCs reconstituted ASPA activity in CD mouse brains, reduced the abnormally elevated level of NAA in both brain tissues and cerebrospinal fluid (CSF), and rescued hallmark pathological phenotypes of the disease, including spongy degeneration, myelination defects, and motor function impairment in transplanted CD mice. These genetically modified patient iPSC-derived NPCs represent a promising cell therapy candidate for CD, a disease that has neither a cure nor a standard treatment.

Increasing Aspartoacylase in the Central Amygdala: The Common Mechanism of Gastroprotective Effects of Monoamine-Based Antidepressants Against Stress.

Monoamine-based antidepressants can prophylactically protect against stress-induced gastric ulcers. Although the central nucleus of amygdala (CeA) has been shown to modulate the severity of stress ulcers, little is known about the molecular mechanisms underlying the gastroprotective effect of this kind of drugs. Here, we first used proton magnetic resonance spectroscopy, a non-invasive tool, to explore the change of neurometabolites of the CeA of rats pretreated with the duloxetine of selective serotonin-norepinephrine reuptake inhibitors during 6 h of water-immersion restraint stress (WIRS). Duloxetine decreased N-acetyl-aspartate/creatine ratio (NAA/creatine) in CeA after WIRS, which was paralleled by the amelioration of gastric lesions. Meanwhile, the gastric ulcer index was negatively correlated with reduced NAA/creatine. Furthermore, the intra-CeA infusion of NAA aggravated WIRS-induced gastric mucosa damage, which suggested the crucial role of reduced NAA. Western blotting was performed to identify the specific enzymes responsible for the change of the contents of NAA at 0.5 h/3 h/6 h after WIRS, considering the preventative gastric protection of duloxetine. The NAA-catabolizing enzyme aspartoacylase (ASPA) was the only enzyme downregulated by 0.5 h WIRS and upregulated by duloxetine. Moreover, overexpressing ASPA in CeA alleviated stress ulcers. Additionally, all of the other three monoamine-based antidepressants, the fluoxetine of selective serotonin reuptake inhibitors, the amitriptyline of tricyclic agents, and the moclobemide of MAOs, increased ASPA expression in CeA. Together, these results indicate that increasing ASPA to hydrolyze NAA in CeA is a common mechanism of gastroprotective effects against stress exerted by monoamine-based antidepressants, and ASPA is a shared target more than monoamine regulation for this kind of drugs.

Mapping the degradation pathway of a disease-linked aspartoacylase variant.

Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we use ASPA C152W as a model to investigate the degradation pathway of a disease-causing protein variant. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution interferes with the de novo folding pathway resulting in increased proteasomal degradation before reaching its stable conformation. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In addition, the disaggregase Hsp104 facilitated refolding of aggregated ASPA C152W, while Cdc48 mediated degradation of insoluble ASPA protein. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. Our findings underscore the use of yeast to determine the protein quality control components involved in the degradation of human pathogenic variants in order to identify potential therapeutic targets.

Feline Spongy Encephalopathy With a Mutation in the ASPA Gene.

Canavan disease is an autosomal recessive leukodystrophy caused by mutations in the gene encoding aspartoacylase (ASPA), which hydrolyses N-acetylaspartate (NAA) to acetate and aspartate. A similar feline neurodegenerative disease associated with a mutation in the ASPA gene is reported herein. Comprehensive clinical, genetic, and pathological analyses were performed on 4 affected cats. Gait disturbance and head tremors initially appeared at 1 to 19 months of age. These cats eventually exhibited dysstasia and seizures and died at 7 to 53 months of age. Magnetic resonance imaging of the brain revealed diffuse symmetrical intensity change of the cerebral cortex, brainstem, and cerebellum. Gas chromatography-mass spectrometry analysis of urine showed significant excretion of NAA. Genetic analysis of the 4 affected cats identified a missense mutation (c.859G>C) in exon 6 of the ASPA gene, which was not detected in 4 neurologically intact cats examined as controls. Postmortem analysis revealed vacuolar changes predominantly distributed in the gray matter of the cerebrum and brain stem as well as in the cerebellar Purkinje cell layer. Immunohistochemically, these vacuoles were surrounded by neurofilaments and sometimes contained MBP- and Olig2-positive cells. Ultrastructurally, a large number of intracytoplasmic vacuoles containing mitochondria and electron-dense granules were detected in the cerebral cortex. All 4 cats were diagnosed as spongy encephalopathy with a mutation in the ASPA gene, a syndrome analogous to human Canavan disease. The histopathological findings suggest that feline ASPA deficiency induces intracytoplasmic edema in neurons and oligodendrocytes, resulting in spongy degeneration of the central nervous system.

Preclinical biodistribution, tropism, and efficacy of oligotropic AAV/Olig001 in a mouse model of congenital white matter disease

Recent advances in adeno-associated viral (AAV) capsid variants with novel oligotropism require validation in models of disease in order to be viable candidates for white matter disease gene therapy. We present here an assessment of the biodistribution, tropism, and efficacy of a novel AAV capsid variant (AAV/ Olig001) in a model of Canavan disease. We first define a combination of dose and route of administration of an AAV/Olig001-GFP reporter conducive to widespread CNS oligodendrocyte transduction in acutely symptomatic animals that model the Canavan brain at time of diagnosis. Administration of AAV/Olig001-GFP resulted in >70% oligotropism in all regions of interest except the cerebellum without the need for lineage-specific expression elements. Intracerebroventricular infusion into the cerebrospinal fluid (CSF) was identified as the most appropriate route of administration and employed for delivery of an AAV/Olig001 vector to reconstitute oligodendroglial aspartoacylase (ASPA) in adult Canavan mice, which resulted in a dose-dependent rescue of ASPA activity, motor function, and a near-total reduction in vacuolation. A head-to-head efficacy comparison with astrogliotropic AAV9 highlighted a significant advantage conferred by oligotropic AAV/Olig001 that was independent of overall transduction efficiency. These results support the continued development of AAV/Olig001 for advancement to clinical application to white matter disease.

Abstract PO-056: Role of the metabolite N-acetylaspartate in breast cancer progression

Abstract Breast cancer (BCa) is a disease that affects over a quarter million women in the US every year. Our laboratory studies reprogrammed energy metabolism in BCa, which is an essential hallmark of the disease. Specifically, we focus on the metabolic rewiring events that occur in BCa, intending to identify cancer-associated metabolic vulnerabilities to treat this aggressive disease. Using a global metabolic profiling approach, we have determined that the metabolite N-acetylaspartate (NAA) is highly accumulated in patients with BCa. Prior studies involving NAA in other cancer types have attributed its accumulation to the upregulation of the enzyme N-acetyl transferase (NAT8L), which synthesizes NAA. However, using matched gene expression data, we found that the accumulation of NAA in these BCa tumors rather coincides with the downregulation of aspartoacylase (ASPA), the enzyme that breaks down NAA. To determine the role of NAA in BCa progression, we generated multiple cell line models containing either NAT8L knockdown, ASPA overexpression, or a combination of the two in the cell line MDA-MB231 in a bid to deplete intracellular NAA. We used these models to test for metastasis by tail vein injection in immunocompromised mice. Consistent with our expectations and other reports, NAT8L knockdown resulted in reduced lung metastasis. Interestingly, however, NAA depletion via ASPA overexpression increased metastasis to the lung in vivo. Furthermore, the combination of ASPA overexpression with the knockdown of NAT8L led to a moderate phenotype between the two independent genetic modifications. Our findings of the metastasis-promoting effect of NAA depletion via ASPA overexpression leads to us believe that it is not just the accumulation of NAA but also its utilization that is important for breast cancer progression. Our future experiments are directed towards determining how NAA utilization causes a pro-tumorigenic phenotype. We would also like to study the current inconsistency between ASPA transcript levels in patient tumors and its observed tumor-promoting function in xenograft experiments. Completion of these studies will potentially define the importance of elevated NAA levels in the context of aggressive BCa. Since NAA is a metabolite that can be detected in serum, our study could also lead to the development of NAA as a non-invasive biomarker for early detection of aggressive BCa, thus providing a bigger window for therapeutic intervention. Citation Format: Akhila Balasubramanian, Weijie Zhang, Franklin Gu, Chandra Ambati, Nagireddy Putluri, Xiang Zhang, Aryan Namboodiri, Stefan Ambs, Arun Sreekumar. Role of the metabolite N-acetylaspartate in breast cancer progression [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-056.

Stem Cell Therapy: Cell‐Based Therapy for Canavan Disease Using Human iPSC‐Derived NPCs and OPCs (Adv. Sci. 23/2020)

Canavan disease (CD) is a leukodystrophy caused by aspartoacylase (ASPA) gene mutation with no cure so far. In this study, a functional ASPA was introduced into patient iPSC-derived neural progenitor cells (iNPCs) and the engineered ASPA iNPCs could rescue major pathological features of CD in a disease mouse model. These patient iPSC-derived cellular products are promising cell therapies for CD patients. [Image designed and prepared by Olivia Sun]

Identification and Characterization of Hypothalamic Alternative Splicing Events and Variants in Ovine Fecundity-Related Genes.

Simple SummaryPrevious studies revealed that alternative splicing (AS) events and gene variants played key roles in reproduction. However, their location and distribution in hypothalamic fecundity-related genes in sheep without the FecB mutation remain largely unknown. In this study, we performed a correlation analysis of transcriptomics and proteomics, and the results suggested several differentially expressed genes (DEGs)/differentially expressed proteins (DEPs), including galectin 3 (LGALS3), aspartoacylase (ASPA) and transthyretin (TTR), could be candidate genes influencing ovine litter size. Further analysis suggested that AS events, single nucleotide polymorphisms (SNPs) and microRNA (miRNA)-binding sites existed in key DEGs/DEPs, such as ASPA and TTR. This study provides a new insight into ovine and even other mammalian reproduction.Previous studies revealed that alternative splicing (AS) events and gene variants played key roles in reproduction; however, their location and distribution in hypothalamic fecundity-related genes in sheep without the FecB mutation remain largely unknown. Therefore, in this study, we described the hypothalamic AS events and variants in differentially expressed genes (DEGs) in Small Tail Han sheep without the FecB mutation at polytocous sheep in the follicular phase vs. monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase vs. monotocous sheep in the luteal phase (PL vs. ML) via an RNA-seq study for the first time. We found 39 DEGs with AS events (AS DEGs) in PF vs. MF, while 42 AS DEGs were identified in PL vs. ML. No DEGs with single nucleotide polymorphisms (SNPs) were observed in PF vs. MF, but five were identified in PL vs. ML. We also performed a correlation analysis of transcriptomics and proteomics, and the results suggested several key DEGs/differentially expressed proteins (DEPs), such as galectin 3 (LGALS3) in PF vs. MF and aspartoacylase (ASPA) and transthyretin (TTR) in PL vs. ML, could be candidate genes influencing ovine litter size. In addition, further analyses suggested that AS events, SNPs and miRNA-binding sites existed in key DEGs/DEPs, such as ASPA and TTR. All in all, this study provides a new insight into ovine and even other mammalian reproduction.