NADHX repair deficiency Zebrafish and IPSC-derived microglia like cells

The central NAD(P)H cofactors are prone to hydration in vivo, producing damaged hydroxylated
derivatives (NAD(P)HX). The NAD(P)HX metabolite repair system converts the redox inactive forms
back to the active cofactors. The repair system consists of two partner enzymes, an ATP-dependent
NAD(P)HX dehydratase (NAXD) and an NAD(P)HX epimerase (NAXE). Loss-of-function mutations, in
either one of those genes, cause a progressive encephalopathy with brain edema and/or
leukoencephalopathy (PEBEL). PEBEL is a fever or inflammation-induced neurodegenerative disorder
in children characterized by ataxia, muscular hypotonia, respiratory insufficiency and/or respiratory
failure and skin manifestations followed by premature death. The observation that symptoms seem
to manifest only after an inflammatory trigger and include pancytopenia as well as recurrent viral
infections indicate that the immune system plays a pivotal role in disease development. In this study
we aim to understand how the accumulation of NAD(P)HX and/or the depletion of NAD(P)H affect
the physiological function of microglia, the immune cells of the brain, in connection to the observed
neurodegeneration. For this purpose, we have generated naxd and naxe mutant zebrafish lines using
CRISPR/Cas9 technology. Preliminary results show that Naxe deficient zebrafish larvae display a
stronger inflammatory response after LPS treatment and a higher number of microglia cells x
compared to control larvae. Naxd mutant zebrafish larvae show decreased viability and further
phenotypic analyses are currently ongoing. To complement this work, we have also derived
microglial cells from NAXD and NAXE knockout human iPSCs. These NAD(P)HX repair deficient
microglial cells exhibit NADHX accumulation, decreased cell viability, increased phagocytic activity
and, in agreement with our zebrafish model, higher expression of inflammatory genes. Taken
together our results indicate that NAXD and NAXE function play an important role in microglial health
and that accumulation of NAD(P)HX over time might lead to a heightened inflammatory response,
potentially contributing to the etiology of the associated neurodegenerative disease.
Keywords: rare diseases, metabolite repair, microglia,ipsc,zebrafish

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