Project Summary
Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare,
autosomal recessive neurological disorder caused by mutations in the gene DARS2, which encodes the
mitochondrial aspartyl-tRNA synthetase. LBSL patients are compound heterozygote, and experience slow and
progressive dorsal column dysfunction, resulting in the need for mobility assistance by the early teenage years,
or in severe cases, death. DARS2 has a well-defined role in mitochondrial protein translation, however, this
canonical function appears unaltered in patients with LBSL. Furthermore, mutations within the family of
mitochondrial tRNA synthetases (mt-ARS) each produces a unique pattern of dysfunction, suggesting unique
mechanisms independent of translation. Ubiquitous expression of DARS2 and selective CNS dysfunction
within LBSL patients underscores the need to study disease mechanism in relevant cell types. Induced
pluripotent stem cells (iPSCs) allow us to differentiate patient peripheral cells into mature and functional motor
neurons (Aim 1). Within this proposal we plan to culture LBSL patient and isogenic control motor neurons (Aim
1.1) to establish phenotypes in LBSL patient lines (Aim 1.2). Preliminary data reveals deficits in LBSL neurons
and also shows feasibility of cell phenotyping experiments. Characterization of CNS cell types in LBSL is novel
and will inform the field on how patient variant combinations affect cell function. Preliminary evaluation of the
mitochondrial proteome in LBSL revealed no errors in translation, and only diffuse effects overall, thus we
propose to expand our search into disease mechanism by probing DARS2 interaction partners (Aim 2).
Cytosolic tRNA synthetases are reported to participate in protein signaling and even to behave as RNA-binding
proteins influencing gene expression, translation, and self-regulation, we therefore suspect that DARS2 as a
mitochondrial tRNA synthetase may have similar non-translational functions. Evaluation of RNA and protein
interaction through cross-linking immunoprecipitation (CLIP-seq; Aim 2.1) and cross-linking co-
immunoprecipitation (XL-MS; Aim 2.2) will reveal RNA and proteins partners pertinent to normal DARS2
activity. We plan to assess interactors in both control and LBSL patient iPSC-derived motor neurons, with the
understanding that patient mutations may affect technical aspects of these experiments. Novel functional data
collected from healthy patient samples alone stand to change our understanding of mt-ARS in the cell and may
dictate therapeutic development for mt-ARS disorders. As LBSL pathology is unique from other synthetase
disorders, it is possible that alternative functions relate to disease mechanism, and identifying these functions
would provide targets for intervention. Patient iPSCs and iPSC-derived motor neurons are essential to
understand endogenous DARS2 behavior and LBSL pathophysiology. Our overall goal is to use patient iPSC-
derived neurons to understand LBSL disease mechanism and to use this information to guide therapeutic
development.