Neuronal ablation of mt-AspRS in mice induces immune pathway activation prior to severe and progressive cortical and behavioral disruption

Highlights

• Neuronal loss of Dars2 in mice results in a slow and progressive neurodegeneration.

• Dars2 induced degeneration is preceded by activation of several immune pathways.

• Neuronal Dars2 mutant mice may be useful for the testing of therapeutics targeting the pediatric leukodystrophy, LBSL.

Abstract

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare, slowly progressive white matter disease caused by mutations in the mitochondrial aspartyl-tRNA synthetase (mt-AspRS, or DARS2). While patients show characteristic MRI T2 signal abnormalities throughout the cerebral white matter, brainstem, and spinal cord, the phenotypic spectrum is broad and a multitude of gene variants have been associated with the disease. Here, Dars2 disruption in CamKIIα-expressing cortical and hippocampal neurons results in slowly progressive increases in behavioral activity at five months, and culminating by nine months as severe brain atrophy, behavioral dysfunction, reduced corpus callosum thickness, and microglial morphology indicative of neuroinflammation. Interestingly, RNAseq based gene expression studies performed prior to the presentation of this severe phenotype reveal the upregulation of several pathways involved in immune activation, cytokine production and signaling, and defense response regulation. RNA transcript analysis demonstrates that activation of immune and cell stress pathways are initiated in advance of a behavioral phenotype and cerebral deficits. An understanding of these pathways and their contribution to significant neuronal loss in CamKII-Dars2 deficient mice may aid in deciphering mechanisms of LBSL pathology.

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.