Variant in NHLRC2 leads to increased hnRNP C2 in developing neurons and the hippocampus of a mouse model of FINCA disease

Abstract

Background FINCA disease is a pediatric cerebropulmonary disease caused by variants in the NHL repeat-containing 2 (NHLRC2) gene. Neurological symptoms are among the first manifestations of FINCA disease, but the consequences of NHLRC2 deficiency in the central nervous system are currently unexplored. Methods The orthologous mouse gene is essential for development, and its complete loss leads to early embryonic lethality. In the current study, we used CRISPR/Cas9 to generate an Nhlrc2 knockin (KI) mouse line, harboring the FINCA patient missense mutation (c.442G > T, p.Asp148Tyr). A FINCA mouse model, resembling the compound heterozygote genotype of FINCA patients, was obtained by crossing the KI and Nhlrc2 knockout mouse lines. To reveal NHLRC2-interacting proteins in developing neurons, we compared cortical neuronal precursor cells of E13.5 FINCA and wild-type mouse embryos by two-dimensional difference gel electrophoresis. Results Despite the significant decrease in NHLRC2, the mice did not develop severe early onset multiorgan disease in either sex. We discovered 19 altered proteins in FINCA neuronal precursor cells; several of which are involved in vesicular transport pathways and actin dynamics which have been previously reported in other cell types including human to have an association with dysfunctional NHLRC2. Interestingly, isoform C2 of hnRNP C1/C2 was significantly increased in both developing neurons and the hippocampus of adult female FINCA mice, connecting NHLRC2 dysfunction with accumulation of RNA binding protein. Conclusions We describe here the first NHLRC2-deficient mouse model to overcome embryonic lethality, enabling further studies on predisposing and causative mechanisms behind FINCA disease. Our novel findings suggest that disrupted RNA metabolism may contribute to the neurodegeneration observed in FINCA patients.

Expanding the phenotypic spectrum of FINCA (fibrosis, neurodegeneration, and cerebral angiomatosis) syndrome beyond infancy

“…The hypothesis that NDD is the key clinical finding of FINCA phenotype was strengthened by the FINCA mouse model (Nhlrc2 D148Y/− ), mimicking the genotype described in the Finish probands (P7‐P9,Table 1). 11 Contrary to the human FINCA phenotype, those mice did not develop a severe, early onset multiorgan disease. An altered protein expression in murine neuronal precursor cells (NPCs) compared to wildtype was noticed.…”

“…An altered protein expression in murine neuronal precursor cells (NPCs) compared to wildtype was noticed. The authors assume that an impaired autophagy of mutated Nhlrc2 RNA and vesicular trafficking might result in NDD 11 …”

Nhlrc2 is crucial during mouse gastrulation

“…Phire Hot Start II Polymerase (Thermo Fisher Scientific, Waltham, MA) and a Piko Thermal Cycler (Thermo Fisher Scientific, Vantaa, Finland) were used according to the manufacturer's instructions. Sanger sequencing was performed to validate the PCR product as described previously (Hiltunen et al, 2020). The PCR product from E6.5-8.5 embryos was precipitated with NaCl (4 M, 1:10) and ethanol before digestion overnight at À20 C or for 30 min at À70 C. The PCR product was digested using the SacI restriction enzyme (Thermo Scientific, Vilnius, Lithuania) according to the manufacturer's instructions and run on 1.5% agarose gel (BioNordika, Helsinki, Finland).…”

“…The protein isolation and immunoblotting protocol used has been described in detail previously (Hiltunen et al, 2020). In brief, ES cell pellets were solubilized with 1.5% dodecyl β-d-maltopyranoside…”

“…4.9 | Histology Decidua were dissected on E7.5-8.5 and processed as described previously (Hiltunen et al, 2020). The decidua were fixed with 10% neutral buffered formalin (FF Chemicals, Haukipudas, Finland) for 24 hr at room temperature under agitation.…”