CIAO1 loss of function causes a neuromuscular disorder with compromise of nucleocytoplasmic Fe-S enzymes
Nunziata Maio, … , Tracey A. Rouault, Carsten G. Bönnemann
Nunziata Maio, … , Tracey A. Rouault, Carsten G. Bönnemann
Published June 17, 2024
Citation Information: J Clin Invest. 2024;134(12):e179559. https://doi.org/10.1172/JCI179559.
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Research Article Metabolism Muscle biology

CIAO1 loss of function causes a neuromuscular disorder with compromise of nucleocytoplasmic Fe-S enzymes

Abstract

Cytoplasmic and nuclear iron-sulfur (Fe-S) enzymes that are essential for genome maintenance and replication depend on the cytoplasmic Fe-S assembly (CIA) machinery for cluster acquisition. The core of the CIA machinery consists of a complex of CIAO1, MMS19 and FAM96B. The physiological consequences of loss of function in the components of the CIA pathway have thus far remained uncharacterized. Our study revealed that patients with biallelic loss of function in CIAO1 developed proximal and axial muscle weakness, fluctuating creatine kinase elevation, and respiratory insufficiency. In addition, they presented with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, mild normocytic to macrocytic anemia, and gastrointestinal symptoms. Mutational analysis revealed reduced stability of the variants compared with WT CIAO1. Functional assays demonstrated failure of the variants identified in patients to recruit Fe-S recipient proteins, resulting in compromised activities of DNA helicases, polymerases, and repair enzymes that rely on the CIA complex to acquire their Fe-S cofactors. Lentivirus-mediated restoration of CIAO1 expression reversed all patient-derived cellular abnormalities. Our study identifies CIAO1 as a human disease gene and provides insights into the broader implications of the cytosolic Fe-S assembly pathway in human health and disease.

Authors

Nunziata Maio, Rotem Orbach, Irina T. Zaharieva, Ana Töpf, Sandra Donkervoort, Pinki Munot, Juliane Mueller, Tracey Willis, Sumit Verma, Stojan Peric, Deepa Krishnakumar, Sniya Sudhakar, A. Reghan Foley, Sarah Silverstein, Ganka Douglas, Lynn Pais, Stephanie DiTroia, Christopher Grunseich, Ying Hu, Caroline Sewry, Anna Sarkozy, Volker Straub, Francesco Muntoni, Tracey A. Rouault, Carsten G. Bönnemann

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Figure 7

Mitochondrial dysfunction and compromised biogenesis of Fe-S recipients of the CIA complex in muscle from P1.

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Mitochondrial dysfunction and compromised biogenesis of Fe-S recipients ...
(A) SDS IBs to detect the CIA components (CIAO1, MMS19, FAM96B) and FAM96A in P1 and control (CIAO1+/+) muscle tissue specimens. Levels of cytoplasmic and nuclear Fe-S proteins (RTEL1, POLD1, DPYD, ERCC2), of the IRPs IRP1 and IRP2, and of the IRP-regulated target TFRC were also assessed (TFRC2 designates dimeric TFRC). β-Actin (ACTB) and α-tubulin were included as references for even loading between samples. (B) Top panel illustrates the reaction catalyzed by the cytosolic Fe-S enzyme DPYD. Blot shows DPYD-mediated conversion of [4-14C]-thymine to [4-14C]-dihydrothymine in lysates derived from P1-derived and control-derived (CIAO1+/+; CTRL) muscle tissue specimens, assayed by TLC and autoradiography. The reaction mix containing the substrate of the reaction [4-14C]-T without cell extract was loaded as a negative control (no extract) to visualize the substrate (4-14C-thymine). (C) SDS IBs of lysates from isolated mitochondria to detect subunits of mitochondrial respiratory complex I (NDUFS1 and NDUFS8), complex II (SDHA, SDHB), complex III (UQCRC1, UQCRFS1, MT-CYB), complex IV (MTOC1), and complex V (ATP5A) in P1- and control-derived muscle tissue specimens. Levels of TOM20 and CS were included as a reference for the loading control. (D) In-gel activity assays of mitochondrial respiratory complexes I, -II, and -IV in P1- and control-derived muscle tissue specimens. (E) Native IBs of subunits of complex I (NDUFS1), complex II (SDHA), and complex IV (MTCO1) to assess the overall levels of fully assembled respiratory complexes. (F) SDS IBs of lysates from isolated mitochondria to detect components of the de novo ISC biogenesis pathway proteins HSPA9, NFS1, HSC20, and ISCU, the mitochondrial Fe-S enzymes ACO2 and FECH, and lipoylated PDH and α-KGDH complexes using an anti-lipoate antibody. Lipoylation is a posttranslational modification that depends on the Fe-S enzyme lipoic acid synthase LIAS (A–F, n = 2 biological replicates).