PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice

Abstract

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3' splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

Keywords: CRISPR/Cas; autophagy; humanized mouse model; phenylketonuria; splice variant.

Graphical Abstract

Figure 1

Functional analysis of the c.1066-11G>A PAH variant. (A) Schematic representation of minigene construction in the pcDNA3.1 vector, RT-PCR after transfection in Hep3B cells with wild-type (WT) and mutant minigenes and sanger sequencing. (B) RT-PCR analysis in HepG2 WT and mutant cell lines. Identity of the amplified bands was confirmed by sanger sequencing. (C) Western blot for immunodetection of PAH from HepG2 WT and mutant cell lines. COS cells were used as negative control. Tubulin was used as loading control.

Figure 2

Characterization of Pah c.1066-11A (PKU) mice. (A) Schematics (not drawn to scale) of the edited humanized mouse Pah gene B) PKU mice survival. (C) Body weight distribution by genotype during 12 weeks. (D) Hypopigmentation (12 weeks-old mice) and microphthalmia (20 weeks-old mice) in homozygous PKU mice compared with wild-type (WT). (E) Western blot for immunodetection of PAH from WT, heterozygous (Het) and PKU mice livers. GAPDH was used as loading control. (F) Overview of PAH specific activity in liver lysates. Statistical analysis performed by one-way ANOVA (^****P < 0.0001). All plots show mean values ± SEM.

Figure 3

Brain neurotransmitters and related metabolites levels in Pah c.1066-11A (PKU) mice. Measures of neurotransmitters dopamine, 3,4-Dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin and 5-hydroxyindole acetic acid (5-HIAA) in brain lysates by HPLC. Statistical analysis performed by unpaired t test (^***P < 0.001, ^****P < 0.0001). Data are presented as mean ± SEM.

Figure 4

Behavioural performance of Pah c.1066-11A (PKU) mice. (A) Distance travelled and velocity in open field test. (B) Latency to fall off the rotarod. (C) Latency to fall off the inverted grid test. (D) Hind-limb clasping. (E) Time spent interacting with empty cage and unfamiliar mouse in social approach test. Statistical analysis performed by unpaired t test (^**P < 0.01, ^****P < 0.0001). Data are presented as mean ± SEM.

Figure 5

Analysis of different types of neuronal markers. Immunohistochemistry brain image of Iba1 (A) and GFAP (B) in WT and PKU mice. Statistical analysis performed by unpaired t test (^*P < 0.05, ^**P < 0.01, ns, not significant). Data are presented as mean ± SEM.

Figure 6

Myelinization defects. (A) Immunohistochemistry brain images of Olig2 in WT and PKU mice. Statistical analysis performed by unpaired t test (^*P < 0.05). Data are presented as mean ± SEM. (B) Transmission electron microscopy (TEM) images of lumbar transverse sections of the spinal cord of two different WT and Pah c.1066-11A (PKU) mice.

Figure 7

Evaluation of the effect of the c.1066-11G>A variant on PAH folding and stability in liver. (A and B) Western blot analysis of PAH, ubiquitin and DNAJC12 and HSP70 co-chaperones and autophagy markers p62, LC3BII and LAMP1 in liver samples from 12-week-old mice. β-actin was used as loading control. (C) Results of protein quantification performed by laser densitometry. Statistical analysis performed by unpaired t test (^*P < 0.05, ^**P < 0.01, (^***P < 0.001, ^****P < 0.0001, ns, not significant). Data are presented as mean ± SEM.

Figure 8

Evaluation of the effect of the c.1066-11G>A variant on PAH folding and stability in HepG2 cells. (A and B) Representative western blot analysis of PAH and DNAJC12 and HSP70 co-chaperones and autophagy markers p62, LC3BII and LAMP1 in HepG2 cells. β-actin was used as loading control. (C) Results of protein quantification performed by laser densitometry. Data are presented as mean ± SD of two independent experiment. (D) PAH levels detected by western blot in HepG2 cells 72 h after transfection with 2 μg of the plasmid coding for DNAJC12. GAPDH was used as loading controls.