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. 2016 Dec;96:323-334.
doi: 10.1016/j.nbd.2016.10.001. Epub 2016 Oct 4.

N-acetylaspartate Supports the Energetic Demands of Developmental Myelination via Oligodendroglial Aspartoacylase

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Free PMC article

N-acetylaspartate Supports the Energetic Demands of Developmental Myelination via Oligodendroglial Aspartoacylase

Jeremy S Francis et al. Neurobiol Dis. .
Free PMC article

Abstract

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.

Keywords: Aspartoacylase; Canavan disease; Energy; Myelination; N-acetylaspartate.

Conflict of interest statement

Potential Conflicts of Interest AAV Oligo patent (TM, SJG) applied for by UNC. RJS is the founder of AskBio and Bamboo Therapeutics. PL is scientific co-founder of Bamboo Therapeutics and scientific advisory Board member of Agilis Biotherapeutics.

Figures

Figure 1
Figure 1
Oligodendroglial tropism of AAV-Olig001. (A) Native GFP fluorescence in corpus callosum of an animal transduced with AAV-Olig001-GFP. (B) Serial sections of AAV-Olig001-GFP-transduced brains were scored for co-labeling with oligodendrocyte (Olig2), neuronal (NeuN), or astrocytic (GFAP) antigens to assess tropism. Mean percentage of GFP-positive cells co-labeling with each antigen presented (n=3, Mean +/− SEM). Over 90% of GFP-positive cells co-label with Olig2. (C) Global transduction of neonatal brains with AAV-Olig001-GFP. Native fluorescence shown in a brain two-weeks post-treatment, highlighting strong labeling of subcortical white matter tracts, motor cortex, and basal ganglia. (D) GFP-positive cells within motor cortex have oligodendroglial morphology and co-label with Olig2 (red).
Figure 2
Figure 2
Rescue of metabolic defects in nur7 brain. (A) Reductions in whole brain NAA in 2 week-old AAVOlig001-ASPA brains to near age-matched wild type levels (***p<0.001/n=5). Representative chromatograms of AAV-Olig001-GFP (B) and AAV-Olig001-ASPA (C) brains. Rescue of whole brain AcCoA (D) and MalCoA (E) levels in the same brains with simultaneous recovery of ATP (F), ATP/AMP ratio (G) and GSH:GSSG ratio (H). *p<0.05, **p<0.01 Mean +/− SEM shown. Significant differences between groups determined using two-tailed Studnet T-test.
Figure 3
Figure 3
Rescue of motor function by AAV/Olig001-ASPA. (A) AAV/Olig001-ASPA animals presented with a highly significant increase in latency to fall on the accelerating rotarod at 12 weeks of age. **p<0.01; n=10. (B) At the conclusion of rotarod testing the same animals were assessed for daily activity levels using automated running wheels for 7 days. Activity levels, measured in wheel turns over the course of the test period, were vastly improved for AAV/Olig001-ASPA animals both in data collected over the entire 7 days and data collected only during the active nocturnal period of the daily cycle. ***p<0.001, n=10. (C) Representative actograms for AAV/Olig001-GFP (green) and AAV/Olig001-ASPA (red) showing increased daily activity levels. Mean +/− SEM presented with significant differences between groups determined by Two-tailed Student T-test.
Figure 4
Figure 4
Rescue of myelination by AAV-Olig001-ASPA. MBP staining in 12 week old AAV-Olig001-ASPA brains (C&D) revealed a drastic improvement in MBP density relative to controls (A&B). Differences in MBP density were especially stark in areas of the brain such as the thalamus (B&D) that are associated with intense vacuolation. MBP staining in AAV-Olig001-ASPA brains was indistinguishable from age-matched wild type reference brains (E&F). MBP fiber density in the motor cortex of AAV-Olig001-ASPA brains was significantly higher than controls (G). Quantification of MBP fiber length density (MBP-LD) by unbiased stereology (H) reveals rescue to wild type levels. MBP-LD expressed in μm/mm3 (volume of area sampled) with the mean for n=5 shown. ***p<0.001, NS= not significant. Significant differences between group means determined using Two-tailed Students T-test.
Figure 5
Figure 5
Rescue of vacuolation by AAV-Olig-001-ASPA. Brains of 12 week old animals were serially processed for H&E staining. The thalamus of AAV-Olig001-ASPA nur7 brains (C) are indistinguishable from wild type reference brains (E), and contrast drastically with the intensely vacuolated AAV/Olig001-GFP thalamus (A). The density of thalamic H&E staining closely mirrors MBP staining (B, D, F). (G) Stereological estimates of thalamus vacuole volume fraction. The percentage of total thalamic volume occupied by vacuoles was calculated using an area fraction probe, revealing a complete rescue in AAV/Olig001-ASPA nur7 animals. Comparison of AAV-Olig001-ASPA nur7 vacuole fraction with age-matched wild type referencecontrols confirms vacuolation was essentially absent. ***p<0.001, n=5. Significant differences between group means (+/− SEM) determined using Two-tailed Student T-test
Figure 6
Figure 6
Rescue of vacuolation in AAV/Olig001-ASPA nur7 brains is associated with increased oligodendrocyte content. Fluorescent immunolabeling of total nuclei (DAPI) and late-stage oligodendrocytes (CC1) in the medial septum (A) and thalamus (B) showing significant increases in CC1-positive cells in AAV/Olig001-ASPA brains relative to GFP controls. Stereological estimates of CC1-positive cell numbers in both the medial septum (MS; C) and thalamus (Thal; D) reveals the restoration of CC1 content to wild type levels. Counts of DAPI-positive nuclei in the severely vacuolated thalamus (E) in the same sections indicates deficiencies in cell numbers can be mainly accounted for by oligodendrocytes. ***p<0.001, **p<0.01. n=5/group, mean +/− sem shown. Significance determined by Two-tailed Student’s t-test.
Figure 7
Figure 7
Long-term ASPA transgene expression in nur7 brain. 12-week old brains from AAV-Olig001-ASPA nur7 (A&C) and AAV-Olig001-GFP nur7 (B&D) stained for ASPA (red) and counterstained with DAPI (blue). White matter tracts of the external capsule (Ext Cap) of AAV-Olig001-ASPA brains (A) label strongly with ASPA-positive fibers, with no staining present in white matter of AAV-Olig001-GFP brains (B). Outer layers of the motor cortex (Mtr Ctx) of AAV-Olig001-ASPA brains contained densely stained multi process-bearing cells (C), while ASPA staining in the motor cortex of AAV-Olig001-GFP was completely absent. Individual ASPA-positive cells in AAV-Olig001-ASPA brains (E) co-labeled with Olig2 (F, G), indicatingpersistent oligodendrocyte transgene expression. (H) HPLC performed on 12-week brains confirmed persistent functionality of transgene expression in the significant reduction in NAA in AAV/Oligo001-ASPA brains relative to controls. *p<0.05, n=4. Significant differences between group means determined using Two-tailed Student t-test.
Figure 8
Figure 8
Chemical shift imaging of NAA and lactate in brains of severely affected (n=3; age 15–24 months) and atypically mild (n=2, ages 5 years and 8 years) Canavan patients. Severely affected patients carried heterozygous compound mutations in the ASPA gene; mild patients were compound heterozygotes for R71H mutation. The same patients were imaged using both single voxel 1H-MRS for NAA and chemical shift imaging (CSI) for lactate quantification, per established protocol. There is a significant difference in mean values of lactate in white matter between the mild and severely affected Canavan patients (p<0.05, Student’s two-sample t-test with 3df) with increasing whole-brain NAA, suggesting that more severe Canavan disease favors anaerobic respiration in white matter and may be associated with metabolic stress.

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