In vivocharacterization of the aspartyl-tRNA synthetase DARS: Homing in on the leukodystrophy HBSL.
The recently diagnosed leukodystrophy Hypomyelination with Brain stem and Spinal cord involvement and Leg spasticity (HBSL) is caused by mutations of the cytoplasmic aspartyl-tRNA synthetase geneDARS. The physiological role of DARS in translation is to accurately pair aspartate with its cognate tRNA. Clinically, HBSL subjects show a distinct pattern of hypomyelination and develop progressive leg spasticity, variable cognitive impairment and epilepsy. To elucidate the underlying pathomechanism, we comprehensively assessed endogenous DARS expression in mice. Additionally, aiming at creating the first mammalian HBSL model, we genetically engineered and phenotyped mutant mice with a targetedDarslocus.



DARS, although expressed in all organs, shows a distinct expression pattern in the adult brain with little immunoreactivity in macroglia but enrichment in neuronal subpopulations of the hippocampus, cerebellum, and cortex. Within neurons, DARS is mainly located in the cell soma where it co-localizes with other components of the translation machinery. Intriguingly, DARS is also present along neurites and at synapses, where it potentially contributes to local protein synthesis.Dars-null mice are not viable and die before embryonic day 11. Heterozygous mice with only one functionalDarsallele display substantially reduced DARS levels in the brain; yet these mutants show no gross abnormalities, including unchanged motor performance. However, we detected reduced pre-pulse inhibition of the acoustic startle response indicating dysfunction of attentional processing inDars+/-mice.



Our results, for the first time, show an in-depth characterization of the DARS tissue distribution in mice, revealing surprisingly little uniformity across brain regions or between the major neural cell types. The complete loss of DARS function is not tolerated in mice suggesting that the identified HBSL mutations in humans retain some residual enzyme activity. The mild phenotype of heterozygousDars-null carriers indicates that even partial restoration of DARS levels would be therapeutically relevant. Despite the fact that they do not resemble the full spectrum of clinical symptoms, the robust pre-pulse inhibition phenotype ofDars+/-mice will be instrumental for future preclinical therapeutic efficacy studies. In summary, our data is an important contribution to a better understanding of DARS function and HBSL pathology.
Translational Neuroscience Facility & Department of Physiology, School of Medical Sciences, UNSW Sydney, NSW 2052, Australia. Electronic address: d.frohlich@unsw.edu.au.
Dominik
D
Fröhlich
Neurodegenerative and Repair Unit, School of Medical Science, UNSW Sydney, NSW 2052, Australia. Electronic address: a.suchowerska@unsw.edu.au.
Alexandra K
AK
Suchowerska
Translational Neuroscience Facility & Department of Physiology, School of Medical Sciences, UNSW Sydney, NSW 2052, Australia. Electronic address: ziggy@unsw.edu.au.
Ziggy H T
ZH
Spencer
Translational Neuroscience Facility & Department of Physiology, School of Medical Sciences, UNSW Sydney, NSW 2052, Australia. Electronic address: g.jonquieres@unsw.edu.au.
Georg
G
von Jonquieres
Translational Neuroscience Facility & Department of Physiology, School of Medical Sciences, UNSW Sydney, NSW 2052, Australia. Electronic address: c.klugmann@unsw.edu.au.
Claudia B
CB
Klugmann
Biomedical Resources Imaging Laboratory, UNSW Sydney, NSW 2052, Australia. Electronic address: a.bongers@unsw.edu.au.
Andre
A
Bongers
Transgenic Animal Unit, Mark Wainwright Analytical Centre, UNSW Sydney, NSW 2052, Australia. Electronic address: fabien.delerue@unsw.edu.au.
Fabien
F
Delerue
Neurodegenerative and Repair Unit, School of Medical Science, UNSW Sydney, NSW 2052, Australia. Electronic address: h.stefen@unsw.edu.au.
Holly
H
Stefen
Transgenic Animal Unit, Mark Wainwright Analytical Centre, UNSW Sydney, NSW 2052, Australia; Dementia Research Unit, School of Medical Science, UNSW Sydney, NSW 2052, Australia; Neuroscience Research Australia, Sydney, NSW 2031, Australia. Electronic address: l.ittner@unsw.edu.au.
Lars M
LM
Ittner
Neurodegenerative and Repair Unit, School of Medical Science, UNSW Sydney, NSW 2052, Australia. Electronic address: t.fath@unsw.edu.au.
Thomas
T
Fath
Translational Neuroscience Facility & Department of Physiology, School of Medical Sciences, UNSW Sydney, NSW 2052, Australia. Electronic address: g.housley@unsw.edu.au.
Gary D
GD
Housley
Translational Neuroscience Facility & Department of Physiology, School of Medical Sciences, UNSW Sydney, NSW 2052, Australia. Electronic address: m.klugmann@unsw.edu.au.
Matthias
M
Klugmann
D000818:Animals; D001226:Aspartate-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D001253:Astrocytes / Q000201:enzymology / Q000473:pathology; D001288:Attention / Q000502:physiology; D001921:Brain / Q000201:enzymology / Q000254:growth & development / Q000473:pathology; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005106:Exploratory Behavior / Q000502:physiology; D020279:Hereditary Central Nervous System Demyelinating Diseases / Q000201:enzymology* / Q000473:pathology; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009043:Motor Activity / Q000502:physiology; D009474:Neurons / Q000201:enzymology / Q000473:pathology; D009836:Oligodendroglia / Q000201:enzymology / Q000473:pathology; D010641:Phenotype; D065808:Prepulse Inhibition / Q000502:physiology; D013216:Reflex, Startle / Q000502:physiology; D013116:Spinal Cord / Q000201:enzymology / Q000254:growth & development / Q000473:pathology; D013574:Synaptosomes / Q000201:enzymology; D020931:ran GTP-Binding Protein / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; C000605781:RAN-1 protein, C elegans; D020931:ran GTP-Binding Protein; D001226:Aspartate-tRNA Ligase
Aminoacyl-tRNA synthetase; Aspartyl-tRNA synthetase; DARS; HBSL; Leukodystrophy; Mouse model