Generation and phenotyping of NAD(P)HX repair deficient zebrafish and iPSC-derived microglia-like cells

PEBEL (progressive early onset encephalopathy with brain edema and/or leukoencephalopathy) disorder
is a severe infantile neurometabolic disorder that falls within the broader spectrum of rare inherited
diseases known as Inborn Errors of Metabolism. The underlying cause of PEBEL is a genetic deficiency in
the NAD(P)HX metabolite repair system. This system is responsible for correcting hydration damage of
the central cofactors NADH and NADPH, which leads to the formation of non-canonical and inactive
metabolites called NAD(P)HX.
The NAD(P)HX repair system comprises two key enzymes: S-NAD(P)HX dehydratase, also referred to as
NAXD, and NAD(P)HX epimerase, known as NAXE. Mutations in either NAXD or NAXE result in the onset
of PEBEL during the early years of life, and in most documented cases, it has so far ledto premature death.
The appearance of disease symptoms is typically triggered by febrile incidents and/or viral/bacterial
infections. In a few recently published trials, the treatment of PEBEL patients with high doses of Vitamin
B3, a precursor of NAD+, was very efficient in treating the severe skin lesions and exerted a stabilizing
effect of the neurological condition of patients. However, the exact molecular mechanisms underlying the
disorder remain unclear. It remains yet to be establishedwhether the depletion of normal cofactors, the
accumulation of non-canonical metabolites, or a combination of both factors or even additional
perturbations play most critical roles in disease development. Similarly, the reason for the immunerelated trigger-dependent onset of the disease symptoms remains fully unclear.
To shed light on these aspects, in this study, we aimed to establish zebrafish models of NAXD and NAXE
deficiency using the CRISPR/Cas9 technology. Through gross and molecular phenotyping of these
zebrafish models, we observed that naxe mutants exhibit a mild immune deficiency that does not
apparently impact their behavior or survival. On the other hand, naxd mutants display a severe
phenotype, with readily detectable differences in locomotion behavior and a significant decrease in
survival rate.
To complement the zebrafish model work and study specifically the impact of NAXD deficiency on the
immune system, we used patient-derived and genetically modified induced pluripotent stem cells (iPSCs)
to generate microglia-like cells(iMGLs) lacking functional NAXD. Patient-derived microglia-like cells with
NAXD deficiency demonstrated main hallmarks of this cell type showing that the methodology can be
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used to generate a suitable disease model. NADHX accumulation, the main molecular characteristic of
NAXD disease, could also be shown to occur in the NAXD deficient iMGLs. Significant differences between
NAXD mutant and isogenic control iMGLs could be measured for several microglia specific functions such
as phagocytic activity, ramification morphology, and expression of inflammation responsive genes. For
some of these parameters, conflicting results were obtained in NAXD patient-derived and knockout
iMGLs, which calls for repeating this type of phenotyping in a larger collection of cell lines with different
genetic backgrounds, to identify the traits specifically affected by mutations in the NAXD gene.
The original findings in this thesis provide initial insights into the effect of NAXD and NAXE deficiency in
developmental processes and their connection with the immune system in the context of PEBEL disorder.
Longitudinal studies in our cell and whole-organism models, guided by our findings here, will be required
to understand the causal chain of events leading from a failure to repair damaged NAD(P)H to an impaired
immune function and an eventually fatal neurological decline.