Uncoupling N-acetylaspartate from brain pathology: implications for Canavan disease gene therapy

Abstract

N-Acetylaspartate (NAA) is the second most abundant organic metabolite in the brain, but its physiological significance remains enigmatic. Toxic NAA accumulation appears to be the key factor for neurological decline in Canavan disease—a fatal neurometabolic disorder caused by deficiency in the NAA-degrading enzyme aspartoacylase. To date clinical outcome of gene replacement therapy for this spongiform leukodystrophy has not met expectations. To identify the target tissue and cells for maximum anticipated treatment benefit, we employed comprehensive phenotyping of novel mouse models to assess cell type-specific consequences of NAA depletion or elevation. We show that NAA-deficiency causes neurological deficits affecting unconscious defensive reactions aimed at protecting the body from external threat. This finding suggests, while NAA reduction is pivotal to treat Canavan disease, abrogating NAA synthesis should be avoided. At the other end of the spectrum, while predicting pathological severity in Canavan disease mice, increased brain NAA levels are not neurotoxic per se. In fact, in transgenic mice overexpressing the NAA synthesising enzyme Nat8l in neurons, supra-physiological NAA levels were uncoupled from neurological deficits. In contrast, elimination of aspartoacylase expression exclusively in oligodendrocytes elicited Canavan disease like pathology. Although conditional aspartoacylase deletion in oligodendrocytes abolished expression in the entire CNS, the remaining aspartoacylase in peripheral organs was sufficient to lower NAA levels, delay disease onset and ameliorate histopathology. However, comparable endpoints of the conditional and complete aspartoacylase knockout indicate that optimal Canavan disease gene replacement therapies should restore aspartoacylase expression in oligodendrocytes. On the basis of these findings we executed an ASPA gene replacement therapy targeting oligodendrocytes in Canavan disease mice resulting in reversal of pre-existing CNS pathology and lasting neurological benefits. This finding signifies the first successful post-symptomatic treatment of a white matter disorder using an adeno-associated virus vector tailored towards oligodendroglial-restricted transgene expression.Electronic supplementary materialThe online version of this article (10.1007/s00401-017-1784-9) contains supplementary material, which is available to authorized users.

Antisense Oligonucleotide Reverses Leukodystrophy in Canavan Disease Mice

“…How might elevated [NAA B ] elicit vacuolation and Purkinje cell dendropathy? Raising [NAA B ] in aspartoacylase‐expressing mice, for example by engineering neuronal transgenic Nat8l overexpression, does not alter brain histology . However, several human and murine mutations that perturb expression of astroglial ion channel–associated proteins cause vacuolar leukodystrophies .…”

“…Raising [NAA B ] in aspartoacylase-expressing mice, for example by engineering neuronal transgenic Nat8l overexpression, does not alter brain histology. 18 However, several human and murine mutations that perturb expression of astroglial ion channel-associated proteins cause vacuolar leukodystrophies. 19 Because astroglia express a sodium-coupled plasma membrane dicarboxylic acid transporter with high affinity for NAA, NaDC3 (encoded by Slc13a3), 20 vacuolation in aspartoacylase-deficient brains may be attributable to the osmotic effects of astroglial NAA overaccumulation, 3,5 and the rapid therapeutic response to Nat8l gapmer may be attributable to restoration of normal astroglial NAA content and osmolar homeostasis.…”

Adeno‐associated virus‐based Alzheimer's disease mouse models and potential new therapeutic avenues

“…The popularity of AAV as a gene delivery system, particularly for CNS applications, can be explained by their non‐pathogenic nature, their ability to transduce neurons and convey long‐term transgene expression as well as mild immune responses. Moreover, the simplistic capsid surface can be easily manipulated to engineer the vectors with targeted cellular tropism and enhanced transduction efficiency (von Jonquieres et al, ). Wild‐type AAV is a member of the parvovirus family of non‐enveloped single‐stranded DNA viruses.…”

“…Interestingly, the cytomegalovirus (CMV) promoter or the CMV/chicken β‐actin hybrid promoter have been reported to exert activity in neurons. However, swapping CMV‐based promoters for glia‐specific promoters achieved cell lineage‐selective green fluorescence protein (GFP) expression in astrocytes and oligodendrocytes in mice (Georgiou et al, ; von Jonquieres et al, ; von Jonquieres et al, ; von Jonquieres et al, ; Watakabe et al, ). Similar promoter selectivity has been confirmed in mouse models of neurodegenerative diseases (Georgiou et al, ; von Jonquieres et al, ) and also in rat and NHP (Bassil et al, ; Lawlor et al, ; Mudannayake, Mouravlev, Fong, & Young, ).…”

“…However, swapping CMV-based promoters for glia-specific promoters achieved cell lineage-selective green fluorescence protein (GFP) expression in astrocytes and oligodendrocytes in mice (Georgiou et al, 2017;von Jonquieres et al, 2013;von Jonquieres et al, 2016;von Jonquieres et al, 2018;Watakabe et al, 2015).…”