Abstract
Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1,2,3,4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.
Main
Mitochondrial dysfunction is an important contributor to pathogenesis of neurodegenerative diseases, with a considerable range of manifestations from severe epilepsy to various forms of peripheral or central nervous system degeneration7. MIRAS, which is caused by genetic mutation(s) in the nuclear-encoded catalytic α-subunit of POLG1, is unusually variable in age of onset and clinical manifestations8. Disease symptoms in patients with MIRAS carrying identical homozygous founder mutations may even manifest differently—in early adolescence, early adulthood or middle age. The clinical spectrum varies from treatment-resistant epilepsy and valproate hepatotoxicity to ataxia–polyneuropathy with or without epilepsy, or polyneuropathy–parkinsonism without epilepsy5,8,9,10,11,12. The underlying POLG1 variant (c.2243G>C, p.W748S; coinciding with the neutral p.E1143G cis-variant; hereafter the MIRAS allele) is common in populations of European descent with a carrier frequency of 1:84 and 1:100 in Finnish and Norwegian populations, respectively5,13. The allele originates from a single ancestral founder individual, dated back to Viking times5,13. The p.W748S change affects the intrinsic processivity region of POLG1 that is involved in replisome contacts and mtDNA processivity, without altering the polymerase catalytic functions14. Out of the variable MIRAS phenotypes, the most severe is the acute-status epilepticus in a previously healthy teenager, manifesting a few weeks after a minor viral infection5,15 and closely mimicking viral encephalitis16,17. These observations suggest that a viral infection could trigger the symptomatic MIRAS disease.
Abundant lines of research implicate mitochondria as key immune modulators in mouse models and human materials. Stress-induced mtDNA or mtRNA release to cytoplasm triggers a IFN-I response that confers resistance to viral infection18,19,20,21. However, these reports suggest that chronic activation of mitochondrial-induced immune responses could contribute to degenerative disease, including neurodegeneration. The variable manifestations of MIRAS and the POLG1 mutation affecting mtDNA replication make MIRAS an excellent candidate for a disease involving a viral trigger.
Immunity defects in MIRAS carriers
We first queried FinnGen, a Finnish population genome database with links to medical history data6, for diagnoses that are enriched in individuals carrying the MIRAS-associated POLG1 variant (rs113994097). Immunodeficiencies stood out as the most significant diagnosis (a sample of 309,154 Finnish individuals, P = 2.01 × 10−7; Fig. 1a). No similar enrichment of immunodeficient traits existed in a set of other mitochondrial and related disease gene variants (Extended Data Fig. 1a). The finding prompted us to examine the role of POLG1 and, particularly, the MIRAS allele in innate immune signalling.
Decreased IFN-I and mtDNA/mtRNA release
The primary fibroblasts from patients with MIRAS (characteristics are shown in Extended Data Fig. 1b,c and Supplementary Table 1) showed decreased stability of POLG1 protein, with around a 50% reduction in the protein amount compared with the matched controls (Fig. 1b). No discernible changes were found in POLG1 transcripts, POLG2 (the accessory β-subunit of POLG replisome), mitochondrial transcription factor A (TFAM), respiratory chain enzyme protein or transcript levels, or in the mtRNA or mtDNA abundance at the baseline (Extended Data Fig. 1d,e).
To examine the immune responses of these patients’ cells, we challenged the fibroblasts with synthetic double-stranded DNA (dsDNA) or dsRNA (polyinosinic:polycytidylic acid, poly(I:C)). They mimic the pathogen-associated molecular patterns (PAMPs) of viruses, which are either released into the cytosol during host cell entry or produced during viral replication. They activate host cytosolic pattern recognition receptors (PRRs), including RNA receptors such as retinoic-acid-inducible gene I (RIG-I) and melanoma-differentiation-associated protein 5 (MDA5) and DNA receptors such as cyclic GMP-AMP synthase (cGAS) and RNA polymerase III, which can convert DNA into RNA intermediates, activating RIG-I. The activation triggers an immune cascade and converges on the production of IFN-I and pro-inflammatory cytokines leading to downstream auto/paracrine antiviral defence22,23,24 (a schematic of the response is shown in Fig. 1c). Although the basal immune and cytokine gene expression levels were comparable between control and MIRAS cells, the latter showed a delayed and dampened initial IFNβ response to dsRNA or dsDNA challenges compared with the controls (Fig. 1d): MIRAS cells induced around a twofold decrease in IFNB1 (encoding IFNβ) expression after 7 h of dsRNA treatment, and at 7 and 24 h after dsDNA treatment. Under these conditions, MIRAS cells also expressed reduced levels of IFN-inducible RIGI and IFN-stimulated genes (ISGs), including ISG15 and IFN-induced protein with tetratricopeptide repeats 3 (IFIT3), while inflammatory cytokine genes (tumour necrosis factor (TNF), interleukin-6 (IL6) and IL1B) displayed variable induction dynamics to the two viral PAMP mimetic treatments (Fig. 1d and Extended Data Fig. 2a). The amounts of PRRs (RIG-I and MDA5), IFN-induced proteins (IFIT3 and IFIT2) and signal transducer and activator of transcription 2 (STAT2) were low after 24 h of PAMP mimetic treatment in MIRAS cells (Fig. 1e and Extended Data Fig. 2b,c; no difference in STING protein). We next tested the ability of viral-PAMP-mimetic-treated MIRAS and control cells to induce paracrine immune activation in naive cells (Extended Data Fig. 2d). Medium transferred from MIRAS cells resulted in a lower activation of the IFNβ pathway, supporting attenuated IFN-I cytokine release and paracrine immune response in MIRAS cells (Fig. 1f and Extended Data Fig. 2e). Co-expression of constitutively active RIG-I and mitochondrial antiviral-signalling protein (MAVS) proteins in MIRAS cells enhanced IFNβ pathway activation in response to dsRNA treatment (Extended Data Fig. 3a–d). These results demonstrate that cells of patients with MIRAS mount a compromised early IFN-I response to viral PAMP mimetics.
mtDNA and mtRNA release into the cytoplasm has been reported to activate cGAS18, RIG-I21,25,26 and MDA519, and the IFN pathway. We investigated the ability of MIRAS and control fibroblasts to present mtDNA and/or mtRNA in the cytosol after exposure to viral PAMP mimetic. Both mtDNA and mtRNA amounts were decreased in the MIRAS cytosol compared with the total mtDNA or mtRNA pools (Fig. 1g and Extended Data Fig. 3e,f). These data support the conclusion that dampened mtDNA/mtRNA release from mitochondria contributes to lowered innate immunity activation in MIRAS fibroblasts.
Overactivated pro-inflammatory response
Delayed and/or dampened early IFN-I response during viral infection can elicit a secondary aberrant activation of pro-inflammatory responses, particularly NF-κB signalling27. We investigated whether a prolonged viral PAMP mimetic exposure would trigger such a pro-inflammatory response in MIRAS fibroblasts. We found an increased amount of NF-κB transcription factor component (p65) and its Ser536-phosphorylated form that activates NF-κB signalling during viral infection28 in MIRAS cells after 32 h of viral PAMP mimetic exposure (Fig. 1h and Extended Data Fig. 3g). This was accompanied by an increase amount of TNF—a pro-inflammatory cytokine that is associated with NF-κB activation. Neither IRF-3 transcription factor (which upregulates IFN-I cytokine expression), nor its activating kinase TBK-1 were induced under this treatment condition. IFNB1 expression was modestly decreased in MIRAS cells at this prolonged treatment duration, pointing to a time-dependent cellular activation of IFN-I and inflammatory responses (Extended Data Fig. 3h). TNF-mediated pro-inflammatory signalling can activate necroptotic cell death through MLKL phosphorylation29,30. The phosphorylated MLKL (p-MLKL) signal was increased in MIRAS cells compared with in controls after 32 h of dsRNA and after 72 h of dsDNA treatment, before any gross changes in cell morphology (Fig. 1i and Extended Data Fig. 3g,i). Overall, MIRAS cells show a slow activation of the early IFN-I response, followed by overactivated pro-inflammatory NF-κB signalling and increased necroptotic sensitivity when challenged by viral PAMP mimetics.
Aberrant responses to neurotropic viruses
Next, we tested the responses of MIRAS cells to bona fide viral infections. As the teenage-onset MIRAS manifestation resembles viral encephalitis, we included two neurotropic viruses: HSV-1, a dsDNA virus, and tick-borne encephalitis virus (TBEV), a positive-strand RNA flavivirus. The neuroinvasive SARS-CoV-2 virus, a positive-strand RNA virus underlying the COVID-19 pandemic, was also studied. All of these viruses share the characteristic of causing mild infections to most individuals, but severe delayed complications to a minority. The encephalitis caused by neurotropic HSV-131 or TBEV32 are proposed to be a consequence of an overactivated innate immune response and/or a cytokine storm33. In HSV-1-infected MIRAS cells, the intermediate–early regulatory protein of HSV-1, ICP27, showed around 1.6-fold higher expression compared with that in the similarly infected control cells at 24 and 48 h after infection, indicating decreased cellular restriction of viral replication in MIRAS (Fig. 2a–d and Extended Data Fig. 4a–d). HSV-1 infection decreased the POLG1 protein and mtDNA levels, especially in MIRAS, the latter being 40% less than in controls at 48 h after infection (Fig. 2b,d,e and Extended Data Fig. 4e). HSV-1 has evolved extensive strategies to evade and/or downregulate the host innate immune response. These include inhibiting IFN-I signalling and inducing host shut off (both are known functions of HSV-1 ICP-27) to facilitate viral gene expression and replication34,35. Accordingly, the host cell chaperone HSP60 showed progressive decline and IFN-I signalling protein levels in MIRAS and control cells changed after HSV-1 infection (Fig. 2b and Extended Data Fig. 4a–c). However, the infection activated pro-inflammatory NF-κB (Fig. 2b,c). At 24 h after HSV-1 infection, MIRAS cells showed an increase in NF-κB-p65 and the Ser536 phosphorylated form (Fig. 2d and Extended Data Fig. 4b,d). This is in accordance with previous reports of HSV-1-induced persistent activation of NF-κB for efficient virus replication36,37. Consistent with the response induced by prolonged treatment with the PAMP mimetic, MIRAS cells also induced p-MLKL at 24 and 48 h of HSV-1 infection compared with the controls, but did not affect cellular viability at 48 h after infection (Fig. 2b–d and Extended Data Fig. 4e,f). These results corroborate the findings of viral PAMP exposure in MIRAS: a dampened early IFN response favours viral replication, resulting in overactivation of the pro-inflammatory response during prolonged infection and increased susceptibility to infection-induced necroptosis. CRISPR-correction of MIRAS POLG1 p.W748S successfully restored POLG1 stability in induced patient fibroblasts (Extended Data Fig. 5a,b). After 48 h of HSV-1 infection, the corrected cells showed less NF-κB-p65, p-NF-κB-p65 and p-MLKL compared with the patient cells (Extended Data Fig. 5c). Furthermore, mtDNA depletion induced by HSV-1 in the corrected MIRAS mutant lines was similar to the infected controls, indicating the causal role of POLG1 p.W748S (Extended Data Fig. 5d).
