SURF1 knockout cloned pigs: Early onset of a severe lethal phenotype

Abstract

Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an early onset, fatal mitochondrial encephalopathy, leading to multiple neurological failure and eventually death, usually in the first decade of life. Mutations in SURF1, a nuclear gene encoding a mitochondrial protein involved in COX assembly, are among the most common causes of LS. LSSURF1 patients display severe, isolated COX deficiency in all tissues, including cultured fibroblasts and skeletal muscle. Recombinant, constitutive SURF1−/− mice show diffuse COX deficiency, but fail to recapitulate the severity of the human clinical phenotype. Pigs are an attractive alternative model for human diseases, because of their size, as well as metabolic, physiological and genetic similarity to humans. Here, we determined the complete sequence of the swine SURF1 gene, disrupted it in pig primary fibroblast cell lines using both TALENs and CRISPR/Cas9 genome editing systems, before finally generating SURF1−/− and SURF1−/+ pigs by Somatic Cell Nuclear Transfer (SCNT). SURF1−/− pigs were characterized by failure to thrive, muscle weakness and highly reduced life span with elevated perinatal mortality, compared to heterozygous SURF1−/+ and wild type littermates. Surprisingly, no obvious COX deficiency was detected in SURF1−/− tissues, although histochemical analysis revealed the presence of COX deficiency in jejunum villi and total mRNA sequencing (RNAseq) showed that several COX subunit-encoding genes were significantly down-regulated in SURF1−/− skeletal muscles. In addition, neuropathological findings, indicated a delay in central nervous system development of newborn SURF1−/− piglets. Our results suggest a broader role of sSURF1 in mitochondrial bioenergetics.

Therapeutic Approaches to Treat Mitochondrial Diseases: “One-Size-Fits-All” and “Precision Medicine” Strategies

“…Shreds of evidence suggest that SURF1 mutations lead to metabolic impairments in neural progenitor cells (NPCs), which cannot switch from glycolytic to OXPHOS metabolism, with subsequent aberrant proliferation and insufficient support for neuronal morphogenesis [ 416 ]. A similar neuronal impairment was reported in a SURF1 ko pig model [ 417 ]. Cerebral organoids from LS patients carrying MT-ATP6 mutations also showed defective corticogenesis and suggest pre-natal impairment [ 418 ].…”

SURF1 mutations causative of Leigh syndrome impair human neurogenesis

“…Microcephaly has often been reported in LS patients 2 . SURF1 knock-out piglets, while not fully recapitulating the clinical phenotype of LS SURF1 , also exhibited developmental alterations and reduced cortical thickness 18 . Moreover, a mutation in the mtDNA gene MT-ATP6 that is associated with LS 39 also caused abnormal NPC functionality 31 .…”

“…However, the mechanistic and pathophysiological understanding of LS SURF1 is hindered by the lack of effective model systems. SURF1 knock-out has been carried out in several animals (flies, mice, zebrafish, and piglets) but all these efforts failed to fully recapitulate the neurological phenotypes of human LS SURF1 [14][15][16][17][18] . Here, we report the generation of a human mechanistic model of LS SURF1 based on patient-derived 2D neural cultures and 3D brain organoids.…”

“…SURF1 knock-down in zebrafish caused reduced COX activity and developmental defects in endodermal tissue with a neurological impairment mainly occurring at the level of motoneurons and the peripheral nervous system 17 . SURF1 knock-out in piglets resulted in a severe lethal phenotype but the neurological defects were mild, mainly comprising a slight delay of CNS development without lactic acidosis and without apparent COX deficiency 18 .…”

The m.9143T>C Variant: Recurrent Infections and Immunodeficiency as an Extension of the Phenotypic Spectrum in MT-ATP6 Mutations?

“…Appropriately it was reported that in a cohort of 221 paediatric patients with mitochondrial disease, the global mortality rate was 14%, with sepsis (55%) and pneumonia (29%) being the two most common causes of death [ 21 ]. Furthermore, there are some animal models, underlying the connection of immunological dysfunction and mitochondrial diseases [ 22 , 23 ]. The presented index patient had a history of recurrent lactic acidosis and ketoacidosis; an immunodeficiency with T cell lymphopenia and hypogammaglobulinemia was suspected.…”