Loss-of-function mutations in ISCA2 disrupt 4Fe-4S cluster machinery and cause a fatal leukodystrophy with hyperglycinemia and mtDNA depletion

Abstract

Iron-sulfur (Fe-S) clusters are essential cofactors for proteins that participate in fundamental cellular processes including metabolism, DNA replication and repair, transcriptional regulation, and the mitochondrial electron transport chain (ETC). ISCA2 plays a role in the biogenesis of Fe-S clusters and a recent report described subjects displaying infantile-onset leukodystrophy due to bi-allelic mutation of ISCA2. We present two additional unrelated cases, and provide a more complete clinical description that includes hyperglycinemia, leukodystrophy of the brainstem with longitudinally extensive spinal cord involvement, and mtDNA deficiency. Additionally, we characterize the role of ISCA2 in mitochondrial bioenergetics and Fe-S cluster assembly using subject cells and ISCA2 cellular knockdown models. Loss of ISCA2 diminished mitochondrial membrane potential, the mitochondrial network, basal and maximal respiration, ATP production, and activity of ETC complexes II and IV. We specifically tested the impact of loss of ISCA2 on 2Fe-2S proteins versus 4Fe-4S proteins and observed deficits in the functioning of 4Fe-4S but not 2Fe-2S proteins. Together these data indicate loss of ISCA2 impaired function of 4Fe-4S proteins resulting in a fatal encephalopathy accompanied by a relatively unusual combination of features including mtDNA depletion alongside complex II deficiency and hyperglycinemia that may facilitate diagnosis of ISCA2 deficiency patients.

Keywords: Fe-S clusters; ISCA2; encephalopathy; hyperglycinemia; leukodystrophy; mitochondrial dysfunction.

Figure 1

Pedigrees and cranial MRI for ISCA2 deficiency subjects. A: Pedigree of Subject 1 revealed maternal family history for fatal infantile leukodystrophy (filled symbols indicate affected individual). MRI of Subject 1 identified high T2 signal abnormalities in white matter indicating leukodystrophy which also spanned the entire length of the spinal cord (1–4). B: Subject 2 family history was positive for consanguinity and a history of early infantile leukodystrophy fatality (filled symbols indicate affected individual). Axial T2‐weighted images and Axial T1 images of proband show hyperintensities in the central and periventricular white matter (1–3)

Figure 2

ISCA2 variant alters mitochondrial bioenergetics in subject fibroblasts. A: Mitochondrial membrane potential is significantly diminished in ISCA2 deficiency subject fibroblast cell line. B: Assessment of electron transport chain complex activity of ISCA2 deficiency subject fibroblast revealed mildly elevated activity for complexes I and III, whereas complex II and IV were significantly reduced. C: Protein quantification by immunoblotting shows complex I (NDUFB8) was elevated in patient cells compared with control cells, whereas complex II (SDHA) is diminished. TOMM20 was used a mitochondrial marker and loading control. D: Microscale oxygraphy analysis of live fibroblasts demonstrated a profound respiratory deficiency in ISCA2 subject fibroblasts (black bars), compared with healthy control fibroblast lines (white bars). Error bars for all data indicate standard deviation. ***P < 0.001

Figure 3

ISCA2 subject fibroblast display mitochondrial network disruption, severe mtDNA depletion and lipoylation defects. A and B: Images of control fibroblasts (A) display a ubiquitous mitochondrial distribution and networking by MitoTracker Red staining and a typical distribution of mtDNA nucleoids by PicoGreen staining, whereas Subject 1 cells (B) display blunted mitochondrial networking with an abnormal perinuclear distribution. C: Profound mtDNA depletion was determined in ISCA2 subject fibroblasts relative to controls. D: Quantification of mtDNA copy number in quiescent subject fibroblast lines. ABAT subject fibroblasts show a reduced mtDNA copy number in low serum media (LSM), but mtDNA copy number is rescued with the addition of dNTPs. ISCA2 deficiency subject fibroblasts show reduced mtDNA copy number in LSM media, and this remains unchanged despite the addition of dNTPs. Error bars indicate standard deviation. ***P < 0.001

Figure 4

Characterization of ISCA2 depletion model for mitochondrial bioenergetics and lipoylation defects. A: Immunoblotting of Subject 1 fibroblasts revealed reduced ISCA2 levels. Immunoblotting for lipoic acid bound proteins found severely reduced lipoylated protein in Subject 1 fibroblasts relative to controls. B: qRT‐PCR analysis of T98G glioblastoma cells expressing control non‐targeting shRNA (NT shRNA), ABAT shRNA (shABAT), and two independent ISCA2 shRNAs (shISCA2‐1 and shISCA2‐2). C: Immunoblotting of ABAT across each depletion line displayed a significant reduction in protein for cells expressing ABAT shRNA, but not for cells expressing either ISCA2 shRNA. D: Significantly reduced protein levels were observed in ABAT depleted cells for complex I (NDUFB8) and II (SDHA) proteins, but complex III (UQCRC2) protein levels were significantly elevated. Both ISCA2 depletion cells showed unchanged complex I (NDUFB8), III (UQCRC2), and IV (COXI) protein levels, but significantly reduced complex II protein levels. TOMM20 was used as a mitochondrial marker and loading control. E: Respiratory chain complex biochemical activity was determined in depletion model cells. ABAT depleted cells showed significantly reduced complex I and II levels, but elevated complex III activity. Cells transduced with either ISCA2 shRNA displayed significantly reduced complex II activity, but relatively unchanged activity for complexes I, III, and IV. Citrate synthase (CS) was unchanged across all samples. F: Cells expressing either ISCA2 shRNA displayed significantly reduced ISCA2 protein levels and lipoic acid bound proteins. G: GABA transaminase activity was significantly reduced in cells expressing ABAT shRNA, but not in cells expressing either ISCA2 shRNA. Error bars indicate standard deviation. ***P < 0.001