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Page 2 of 9 Klugmann et al. Rare Dis Orphan Drugs J 2023;2:8 https://dx.doi.org/10.20517/rdodj.2023.05

specialized ribonucleic acids (RNAs) and proteins. The importance of this process is highlighted by the
[1]
many diseases resulting from mutations in genes encoding the involved proteins and RNAs . Before
transfer RNAs (tRNAs) can transfer amino acids to the nascent polypeptide chain, they need to be charged
with their cognate amino acid, a reaction catalyzed by specific aminoacyl-tRNA synthetases (ARSs). A
human cell contains 37 cytosolic and mitochondrial synthetase genes, all of which are encoded on nuclear
chromosomes. The correct charging of tRNAs is essential for the fidelity of protein synthesis and there is no
redundancy amongst ARSs. Consequently, dysfunctional tRNA charging is not tolerated. To date,
[2]
pathological mutations that cause neurological diseases have been identified in 24 ARS genes . Intriguingly,
most of the pathological ARS mutations primarily manifest in deficits of the peripheral and central nervous
system, including encephalopathies, Charcot-Marie-Tooth neuropathies, leukodystrophies, or cerebellar
ataxia. This indicates a high susceptibility of neural cells to disturbances in protein synthesis .
[3]

The leukodystrophy Hypomyelination with Brain stem and Spinal cord involvement and Leg spasticity
(HBSL) is caused by recessive missense mutations of the DARS1 gene encoding the cytosolic aspartyl-tRNA
synthetase (AspRS) . For most patients, the disease manifests from 3-36 months of age. The symptoms
[4]
include leg spasticity, inability to walk unsupported or articulate, nystagmus, cognitive impairment,
epilepsy, and premature death. In addition to infantile-onset patients, the disease spectrum has been
[5]
broadened by the description of HBSL cases with late adolescent onset and a milder disease course .
Interestingly, HBSL patients present with similar symptoms as patients afflicted with Leukoencephalopathy
with Brain stem and Spinal cord involvement and elevated Lactate (LBSL), which is caused by missense
mutations of the DARS2 gene encoding mitochondrial AspRS . Although the two conditions might share
[6,7]
a similar underlying pathophysiology, cytosolic and mitochondrial AspRS are not functionally redundant,
as genetic deletion of either of the two genes in mice is embryonically lethal and cannot be compensated for
by the other .
[8,9]

The development of accurate HBSL animal models is crucial to advance the understanding of the disease
mechanism and to develop targeted and effective treatments. However, the lack of redundancy amongst
ARSs and the essential nature of the AspRS enzyme have made disease modelling challenging, as complete
[9]
knockout of the DARS1 gene resulted in early embryonic lethality and the introduction of patient-specific,
disease-causing DARS1 point mutations into the mouse genome has failed to replicate the full HBSL disease
phenotype [10,11] . In the absence of precise animal models, detailed knowledge of the AspRS expression
pattern can provide valuable insight into etiology and help define therapeutic targets. Our recent gene
[12]
[9]
expression studies using mouse and human brain tissue from neurologically healthy individuals
revealed that, despite ubiquitously expressed in all cells, the highest AspRS levels were present in neuronal
lineage cells with comparably little immunoreactivity present in oligodendrocytes, astrocytes, and microglia.
Anatomically, AspRS expression was highly enriched in the cerebellum, a region responsible for motor
control and particularly affected in HBSL patients. Here, we describe in detail the histological and
biochemical methods used to analyze the AspRS expression pattern in human post-mortem brain tissue.
These protocols can readily be adapted to characterize other members of the ARS protein family or, more
broadly, other cytosolic proteins in the human brain.

MATERIALS
Post-mortem brain tissue
Human post-mortem brain tissue was provided by the New South Wales Brain Bank (project no. PID391).
All procedures were approved by the UNSW Sydney Human Research Ethics Advisory Panel D. The tissue
samples were taken from five male subjects aged between 55 and 57 years who died from cardiovascular
disease. Individuals did not suffer from neurological diseases and the brain tissue was free from overt brain