Anniina E Hiltunen1,2, Salla M Kangas3,4, Steffen Ohlmeier5, Ilkka Pietilä3,6, Jori Hiltunen3, Heikki Tanila7, Colin McKerlie8,9, Subashika Govindan10, Hannu Tuominen11,12, Riitta Kaarteenaho13,14, Mikko Hallman3, Johanna Uusimaa3,15, Reetta Hinttala3,4. 1. Medical Research Center Oulu and PEDEGO Research Unit, University of Oulu and Oulu University Hospital, PO Box 5000, 90014, Oulu, Finland. anniina.hiltunen@oulu.fi. 2. Biocenter Oulu, University of Oulu, Oulu, Finland. anniina.hiltunen@oulu.fi. 3. Medical Research Center Oulu and PEDEGO Research Unit, University of Oulu and Oulu University Hospital, PO Box 5000, 90014, Oulu, Finland. 4. Biocenter Oulu, University of Oulu, Oulu, Finland. 5. Proteomics Core Facility, Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5400, Oulu, 90014, Finland. 6. Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Rudbeck Laboratory, Uppsala, Sweden. 7. A.I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland. 8. The Hospital for Sick Children, Toronto, Canada. 9. Faculty of Medicine, University of Toronto, Toronto, Canada. 10. Tissue Engineering Laboratory, Hepia/HES-SO, University of Applied Sciences Western Switzerland, Geneva, Switzerland. 11. Department of Pathology, Cancer and Translational Medicine Research Unit, University of Oulu, Oulu, Finland. 12. Department of Pathology, Oulu University Hospital, Oulu, Finland. 13. Research Unit of Internal Medicine, Respiratory Research, University of Oulu, Oulu, Finland. 14. Medical Research Center Oulu and Unit of Internal Medicine and Respiratory Medicine, Oulu University Hospital, Oulu, Finland. 15. Clinic for Children and Adolescents, Paediatric Neurology Unit, Oulu University Hospital, Oulu, Finland.
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.
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 NHLRC2deficiency 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 Nhlrc2knockin (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.
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