Research PaperNeuronal ablation of mt-AspRS in mice induces immune pathway activation prior to severe and progressive cortical and behavioral disruption
Introduction
Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare, autosomal recessive disorder characterized by slowly progressive spasticity, ataxia, proprioceptive deficits, and in some cases, cognitive decline. Most patients harbor compound heterozygous mutations in the DARS2 gene (Tzoulis et al., 2012) which encodes mitochondrial aspartyl-tRNA synthetase (mt-AspRS), a ubiquitously expressed enzyme which charges tRNA molecules with cognate amino acids essential for mitochondrial protein translation. Diagnosis of LBSL includes identification of pyramidal, cerebellar, and dorsal column abnormalities on MRI, often presenting with increased lactate detected by proton-magnetic resonance spectroscopy (Scheper et al., 2007; van Berge et al., 2013). Age of onset and degree of disability vary widely with genotypic variation complicating a genotype-phenotype correlation (van Berge et al., 2014). With this said, more severe early infantile onset cases with seizures, microcephaly and global delay have also been reported (Sauter et al., 2015; Steenweg et al., 2012). Since the first descriptions of LBSL, human diseases have now been associated with each of the 19 mitochondrial tRNA synthetases, all presenting with diverse clinical symptoms (Sissler et al., 2017; Theisen et al., 2017).
Recapitulating DARS2 deficiency and pathology in mouse or cell systems has proven difficult. Previous efforts to develop model animals have revealed that Dars2 is essential for embryonic development, as a complete knock-out results in lethality around embryonic day 8.5, most likely due to the absence of proteins necessary for mitochondrial DNA maintenance (Dogan et al., 2014; Tyynismaa and Suomalainen, 2009) and attempts to recreate human mutations in the mouse have so far been unsuccessful. LBSL patient deficits are limited to the central nervous system (CNS) and thus CNS specific deletions of DARS2 may best expose the roles of DARS2 in the brain. Inducing DARS2 mutations in various human cell types in vitro revealed neuronal cells types to show the greatest deficit (van Berge et al., 2012) and further, selective deletion of Dars2 from CamKIIα-expressing neurons in the mouse results in severe neurodegeneration and microglial activation, while selective oligodendrocyte Dars2 deletion produces a more mild phenotype (Aradjanski et al., 2017). In this study we conducted long-term detailed characterization of a similar mouse model describing their motor-behavioral profile in conjunction with in vivo Magnetic Resonance Imaging followed by post-mortem histological studies and gene expression studies to elucidate the underlying disease pathophysiology.
Section snippets
Mouse breeding and maintenance
Tissue-specific Dars2 knock-out mice were generated using the Cre-LoxP system. LoxP-flanked Dars2 mice (as described in Dogan et al., 2014) were mated with mice expressing cre-recombinase under the regulation of the CamKIIα promotor (The Jackson Laboratory stock no. 005359) to selectively disrupt Dars2 expression in CamKIIα-expressing neurons (mutant mice). All experimental mice were bred in house and genotyped for presence of homozygous flox sites and cre by Transnetyx (Cordova, TN). All
CamKII-Dars2 mutant mice show increased activity and brain atrophy by 6 months of age
Analysis of open field activity revealed a significant increase of overall activity in CamKII-Dars2 mutant mice compared to littermate controls (Fig. 1A; main effect: genotype, F1,212 = 207.35, p < .0001; age, F15,212 = 8.52, p < .0001). Sidak's correction for multiple comparisons demonstrated significant differences between control and mutant total distance traveled beginning at 22 weeks (p < .05) and continuing throughout the duration of testing. Rearing behavior was also increased in CamKII-
Discussion
In mouse, the selective loss of Dars2 in CamKIIα neurons results in a severe and progressive phenotype which includes hyperactivity, concurrent atrophy of cortical tissues, and increased presence and activation of microglial cells. Previous work in a similar mouse model (models differ in generation of CamKIIα-Cre line; see Xu et al., 2000) show cortical and hippocampal morphological changes occur as early as 20 weeks of age (Aradjanski et al., 2017), with atrophy emanating from the second and
Conclusions
To date, studies of LBSL describe case reports of brain pathology and corresponding symptomology, or patient-derived cell line function (DARS2 production or splice efficiency). Mutation heterozygosity and overall lack of post-mortem tissue blur our ability to decipher variant contributions or effects on mixed cell populations. Further, as these reports have demonstrated, patient cells produce decreased but sufficient amounts of DARS2 protein and mutations most commonly affect residues conserved
Acknowledgements
Research reported in the manuscript was funded by A Cure for Ellie (acureforellie.org) and by the National Institutes of Health (NINDS 5R01NS097511; NICHD U54HD079123; and the Office of the NIH Director S10OD016374). The authors thank Lauren LeBon, Katherine Martin, Carmela Sidrauski, and Margaret Roy at Calico Life Sciences LLC for RNA-seq data, analysis, and consultation. Additional thanks to Karen Smith-Connor and Mary E Blue for histology consultation, Joseph Mertz for IPA consultation, and
Declaration of Competing Interest
AF is a paid drug safety monitoring board member for Bluebird Bio, Stealth Biotherapeutics, and a paid consultant to Calico Labs.
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