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CRISPR-Cas9-engineered PIGV341E mouse model mirrors human phenotype, shows hippocampal synaptic dysfunctions and suggests a major role of impaired Eph/Abl1 signaling in GPI anchor deficiencies

ABSTRACT: Pathogenic germline mutations in PIGV result in a glycosylphosphatidylinositol biosynthesis deficiency (GPIBD). Individuals with pathogenic biallelic mutations in genes of the GPI pathway show cognitive impairments, a motor delay and often epilepsies. Thus far, the pathophysiology underlying the disease remains unclear and suitable rodent models are lacking that mimic human pathophysiology. We therefore generated a mouse model using CRISPR-Cas9 to introduce the most prevalent hypomorphic missense mutation in European patients, at a site that is also conserved in mice, Pigv:c.1022C>A (p.A341E). Pigv341E mice showed deficits in motor coordination and cognitive impairment with poorer long-term spatial memory. Furthermore, Pigv341E mice showed alterations in sociability and sleep patterns reflecting the human phenotype. Immunohistochemistry showed decreased synaptophysin-immunoreactivity and electrophysiology recordings demonstrated reduced hippocampal synaptic transmission in Pigv341E mice that may explain impaired memory formation. Moreover, we performed single-cell RNA sequencing of hippocampal cells from brains of adult Pigv341E mice that exhibit changes of gene expression, most prominently in a microglia subtype and subicular neurons. A significant reduction of Abl1 transcripts in several cell clusters suggest a link to the signaling pathway of GPI-anchored ephrins. We also identified increased levels of Hdc that might affect histamine metabolism with consequences for circadian rhythm and neuronal excitability. In summary, we present here the first mouse model with a patient-specific hypomorphic mutation that mirrors the human phenotype and will allow us to explore the pathophysiology in GPIBDs in future studies.

ORGANISM(S): Mus musculus

PROVIDER: GSE147722 | GEO | 2020/12/31

REPOSITORIES: GEO

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Project description:Somatic mutation in the X-linked phosphatidylinositol glycan class A (PIG-A) gene causes glycosylphosphatidylinositol (GPI) anchor deficiency in humans with Paroxysmal Nocturnal Hemoglobinuria (PNH). Clinically, patients with PNH have intravascular hemolysis, venous thrombosis and bone marrow failure. We produced a conditional Pig-a knock-out mouse model specifically inactivating the Pig-a gene in hematopoietic cells to study the role of PIG-A deficiency in PNH pathophysiology. We used Affymetrix Mouse Genome 430 2.0 chips to investigate the gene expression pattern in the mouse model of targeted Pig-a deletion. Experiment Overall Design: We performed microarray analysis on 3 pools of sorted GPI-deficient (GPI-) and GPI normal (GPI+) bone marrow cells derived from the same Pig-a knock-out animals, and identified 1275/669 genes of the 45,101 transcripts potentially available for screening using 2-fold change, <10% false discovery rate (FDR) and percentage of present calls as selection criteria. The major representative genes belong to the category of immune response. We tested whether the molecular differences between GPI- and GPI+ bone marrow cells could be preserved when GPI- cells were transplanted in lethally-irradiated wild type mice (C57BL/6). Microarray analysis was performed using sorted bone marrow cells from 4 animals transplanted with GPI-deficient bone marrow cells (T-GPI-) from Pig-a knock-out donors, 3 animals transplanted with GPI-normal bone marrow cells from C57BL/6 donors (T-GPI+), and GPI-normal bone marrow cells from 4 wild-type C57BL/6 mice (WT-GPI+). We found 296 probesets with 2-fold change cutoff, of which T-GPI- cells had 145 up-regulated genes in comparison to WT-GPI+ cells, and had 123 genes differentially expressed when compared to T-GPI+ cells. The gene expression of the GPI-deficient cells was very similar between the two sets of microarray experiments affirming the maintenance of the phenotype before and after bone marrow cell transplantation.

Project description:The decline of cognitive function is a feature of normal human aging and is exacerbated in AlzheimerM-bM-^@M-^Ys disease (AD). DNA repair declines in brain cells during normal aging and even more so in AD. Here we show that experimental reduction in levels of the base excision repair enzyme, DNA polymerase M-NM-2 (Polb) renders neurons vulnerable to age-related dysfunction and degeneration in a mouse model of AD. Whereas 3xTgAD mice exhibit age-related extracellular amyloid b-peptide (Ab) accumulation and cognitive deficits, but no neuronal death, 3xTg/Polb+/- mice accumulates intracellular Ab and neurons die in the hippocampus and cerebral cortex. The DNA repair-deficient 3xTgAD mice exhibited increased DNA strand breaks and apoptotic caspase activation with loss of hippocampal volume, and impaired synaptic plasticity and memory retention. Molecular profiling revealed remarkable similarities in gene expression alterations in brain cells of AD patients and 3xTgAD/Polb+/- mice including multiple abnormalities suggestive of impaired cellular bioenergetics. Our findings demonstrate that a modest decrement in oxidative DNA damage processing is sufficient to render neurons vulnerable to AD-related pathogenic molecular and cellular alterations that result in the dysfunction and death of neurons, and associated cognitive deficits. 4 mouse strains were used in these experiments, the 3xTgAD and Pol M-NM-2 (+/-) mice were bred at the National Institute on Aging (Baltimore, Maryland). The original line 3xTgAD line was generated as described previously (Oddo, et. al 2003) and possess APPswe, PS1M146V, and tauP301L mutations. DNA polymerase beta heterozygous mice, Pol M-NM-2 (+/-), were crossed with the 3xTgAD mice to generate a 3xTgAD/Pol M-NM-2 (+/-) mouse. The Wt strain is C57Bl/6. At 20 months of age these mice were euthanized by cervical dislocation, the brain removed from the skull and dissected into regions of interest, the prefrontal cortex was used for the microarray studies.

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CRISPR-Cas9-engineered PIGV341E mouse model mirrors human phenotype, shows hippocampal synaptic dysfunctions and suggests a major role of impaired Eph/Abl1 signaling in GPI anchor deficiencies

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