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Queuine ameliorates impaired mitochondrial function caused by mt-tRNAAsn variants

Journal of Translational Medicine volume 22, Article number: 780 (2024) Cite this article

Abstract

Background

Mitochondrial tRNA (mt-tRNA) variants have been found to cause disease. Post-transcriptional queuosine (Q) modifications of mt-tRNA can promote efficient mitochondrial mRNA translation. Q modifications of mt-tRNAAsn have recently been identified. Here, the therapeutic effectiveness of queuine was investigated in cells from patients with mt-tRNAAsn variants.

Methods

Six patients (from four families) carrying mt-tRNAAsn variants were included in the study. Queuine levels were quantified by mass spectrometry. Clinical, genetic, histochemical, biochemical, and molecular analysis was performed on muscle tissues and lymphoblastoid cell lines (LCLs) from patients to investigate the pathogenicity of the novel m.5708 C > T variant. The use of queuine in mitigating mitochondrial dysfunction resulting from the mt-tRNAAsn variants was evaluated.

Results

The variants included the m.5701 delA, m.5708 C > T, m.5709 C > T, and m.5698 G > A variants in mt-tRNAAsn. The pathogenicity of the novel m.5708 C > T variant was confirmed, as demonstrated by a decreased steady-state level of mt-tRNAAsn, mtDNA-encoded protein levels, oxygen consumption rate (OCR), and the respiratory complex activity. Notably, the serum queuine level was significantly reduced in these patients and in vitro queuine supplementation was found to restore the reductions in mitochondrial protein activities, mitochondrial membrane potential, OCR, and increases in reactive oxygen species.

Conclusions

The study not only confirmed the pathogenicity of the m.5708 C > T variant but also explored the therapeutic potential of queuine in individuals with mt-tRNAAsn variants. The recognition of the novel m.5708 C > T variant’s pathogenic nature contributes to our comprehension of mitochondrial disorders. Furthermore, the results emphasize queuine supplementation as a promising approach to enhance the stability of mt-tRNAAsn and rescue mitochondrial dysfunction caused by mt-tRNAAsn variants, indicating potential implications for the development of targeted therapies for patients with mt-tRNAAsn variants.

Introduction

Mitochondrial diseases (MDs) are caused by variants in both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) [1,2,3]. In the mtDNA category, mitochondrial tRNA (mt-tRNA) genes, although constituting only a small fraction (5–10%), have been identified as crucial sites for pathogenic point variants [4,5,6]. The molecular pathomechanisms associated with these variants may be related to faulty mt-tRNA folding, reduced stability, aminoacylation, and post-transcriptional modifications [7, 8]. Post-transcriptional modifications at the anticodon wobble position of mt-tRNA, such as the 5-taurinomethyluridine (τm5U) [9, 10], 5-formylcytidine (f5C) [11], N6-threonylcarbamoyladenosine (t6A) [12], and queuosine (Q) [13] modifications, are essential for the accurate transcription of the genetic code and maintaining the stability of the mt-tRNA molecules [14]. Rencently, the mt-tRNALeu(UUR) and mt-tRNATrp variants were associated with τm5U at the wobble position, and supplementation of taurine was found to mitigate the affected mitochondrial function and reduce the likelihood of stroke-like manifestations in individuals with m.3243 A > G and m.5541 C > T variants [15,16,17].

The Q modification is a conserved post-transcriptional modification in tRNA that is essential for the accurate decoding of specific codons related to asparagine (Asn), tyrosine (Tyr), histidine (His), and aspartic acid (Asp) [18, 19]. This modification is closely involved with maintaining the efficiency and fidelity of protein translation by facilitating precise codon recognition [20]. Queuine is the mammalian substrate for the Q modification in tRNA. Queuine is a 7-deazaguanine derivative that is obtained from the diet and the gut microflora and is able to traverse the gut epithelium. It is then integrated into the wobble position (nucleoside 34) on tRNAs, both mitochondrial and cytoplasmic, specifically those with the G34U35N36 anticodon sequence. Recent investigations have shown that queuine at position 34 (Q34) in mt-tRNA regulates the elongation rates of mitochondria in a similar manner to cytoplasmic tRNAs [21]. Lack of the Q modification is associated with reduced translation of mRNAs associated with mitochondrial function [22]. However, the specific mechanisms through which the Q modification influences MDs, as well as the potential therapeutic applications of queuine in addressing mt-tRNAAsn variants, require further investigation.

Here, the clinical, pathological, and genetic characteristics of four MD pedigrees carrying mt-tRNAAsn variants were comprehensively analyzed. Mitochondrial function was assessed in cells from patients harboring the novel m.5708 C > T variant, thus exploring the potential of queuine for treating individuals with mt-tRNAAsn variants.

Materials and methods

Subjects

A comprehensive examination of our database on MDs identified six patients from four unrelated families. All possessed variants in mt-tRNAAsn, include m.5701 delA, m.5708 C > T, m.5709 C > T, and m.5698 G > A. The subjects underwent thorough clinical assessments, encompassing blood tests, brain magnetic resonance imaging (MRI), and muscle biopsies, to characterize their clinical phenotypes. Written informed consent was obtained from all subjects, and the study protocol received approval from the Research Ethics Board of Qilu Hospital, under registration number KYLL-2021(KS)-079.

Genomic analysis

Total DNA was harvested from various samples (blood, urine sediment, oral mucosa, and muscle biopsy samples) using the TIANamp Genomic DNA Kit (Tiangen, China), as described [23]. Complete mtDNA sequences were obtained from muscle biopsy samples by next-generation sequencing, while Sanger sequencing was used for analyzing mtDNA sequences from other tissue samples of the probands (P1, P2, P4 and P6) and family members. For Sanger sequencing, the region encompassing the mt-tRNAAsn variants was amplified using the forward primer (5′- CTA ACC GGC TTT TTG CCC-3′) and reverse primer (5′- ACC TAG AAG GTT GCC TGG CT-3′).

Queuine quantification by mass spectrometry

Serum queuine levels were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The samples underwent solid-phase extraction and subsequent derivatization to improve detection. The samples were analyzed on a C18 column with gradient elution using water and acetonitrile containing 0.1% formic acid. The positive electrospray ionization mode was used for MS analysis with multiple reaction monitoring to enhance specificity. Calibration curves were established to ensure precise quantitation, and internal standards were used to ensure the reliability of the results.

Histopathological analysis of muscle biopsy specimens

Muscle biopsy samples were collected from the left biceps brachii. Serial cryosections (10 μm) were prepared and stained [24], using cytochrome C oxidase (COX), succinate dehydrogenase (SDH) stains as well as the SDH-COX combination (S/C). Single fibers were isolated using a tungsten needle and used to extract single-cell DNA from COX-positive or deficient samples, followed by single-fiber PCR analysis, as reported previously [25].

Lymphoblastoid cell lines generation

Lymphoblastoid cell lines (LCLs) were generated using Epstein-Barr Virus (EBV) following a previously established method [26]. LCLs harboring the m.5708 C > T variant was cultivated from a freshly obtained blood sample from Patient 2, in conjunction with LCLs derived from healthy controls of similar age and sex. All LCLs were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin&streptomycin(P&S).

Cell culture and queuine supplementation protocols

LCLs were grown for 15 days, either in conventional culture medium or medium supplemented with 1 µM queuine (+ Q). The cells were then used for subsequent experiments. We selected the concentration and time course based on preliminary experiments and established literature references [20] (Fig.S1).

Western blotting

To evaluate the levels of mitochondrial-related proteins, protein samples (30–50 µg) obtained from muscle sample homogenates and LCLs were subjected to SDS-PAGE separation and transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% skim milk for 1 h and then incubated overnight at 4 °C with the primary antibodies, namely, an OXPHOS cocktail (ab110413, Abcam, UK), anti-ATP6 (55313-1-Ap, Proteintech, USA), anti-CYB (55090-1-Ap, Proteintech), anti-ND4 (A9941, ABclonal, USA), anti-CO4 (11242-1-AP, Proteintech), anti-VDAC1 (ab15895, Abcam), GAPDH (60004-1-Ig, Proteintech), and anti-β-actin (ab8226, Abcam). HRP-conjugated goat anti-mouse or anti-rabbit IgG (Jackson, USA) were then used, and the proteins were visualized using an enhanced chemiluminescence (ECL) system.

Northern blotting

Total RNA was harvested from LCLs using TRIzol (Life Technologies, USA), as described [27]. Twenty micrograms of the RNA were denatured in TBE buffer followed by transfer to nylon membranes (RPN303B, GE Healthcare, USA) for hybridization with digoxigenin-labeled oligodeoxynucleotide probes specific to mt-tRNAHis, mt-tRNAAsn, mt-tRNALeu(UUR), mt-tRNATrp, mt-tRNAGly, mt-tRNAVal, and 5 S rRNA. The internal control was 5 S rRNA. Detailed probe sequences are provided in Table S1.

Oxygen consumption rate

A Seahorse XFe24 analyzer (Agilent, USA) was used for measuring the oxygen consumption rate (OCR) [28]. LCLs (20 × 105) were seeded in Seahorse XFe24 plates and subjected to a mitochondrial stress test using successive additions of 1 µM oligomycin, 1 µM FCCP, 0.5 µM rotenone, and 0.5 µM antimycin A. The basal, maximal, and ATP-linked OCR values were calculated accordingly.

Measurement of mitochondrial reactive oxygen species

The reactive oxygen species (ROS) levels in the mitochondria were assessed using MitoSOX Red dye (M36008, Molecular Probes, USA). Cells (1 × 106 cells/mL in PBS) were incubated in 5 µM MitoSOX Red for 20 min at 37 °C and 5% CO2. After washing with PBS, the MitoSOX Red fluorescence (Ex/Em: 510/580 nm) was measured by flow cytometry, with 10 000 cells recorded per sample.

Mitochondrial membrane potential analysis

The mitochondrial membrane potential (MMP) was evaluated by JC-1 staining (C2003S, Beyotime, China). Cells (1 × 106/mL) were treated with 10 µg/mL JC-1 dye (20 min, 37 °C, 5% CO2). After rinsing with PBS, the JC-1 monomers were measured using flow cytometry (excitation/emission: 488/530 nm) and aggregates (excitation/emission: 525/590 nm), recording 10 000 cells per sample.

Mitochondrial harvesting and measurement of enzymatic activity

Mitochondria were isolated from LCL for the assessment of respiratory chain enzyme activities. Briefly, harvested cells were rinsed with ice-cold PBS and resuspended in ice-cold isolation buffer (210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EDTA, and 0.5% BSA, pH 7.4). The cells were homogenized with 30‒40 strokes of a Dounce homogenizer and centrifuged (800 × g, 10 min, 4 °C) to pellet the cell debris. The supernatants were re-centrifuged (10 000 × g, 15 min, 4 °C) to pellet the mitochondria which were then washed with isolation buffer and resuspended in mitochondrial resuspension buffer (250 mM mannitol, 5 mM HEPES, pH 7.4) for enzyme activity assays. Respiratory chain enzyme activity levels were determined spectrophotometrically, following standard protocols [29].

Statistics

Data were analyzed by GraphPad Prism (v5.04, GraphPad Software, USA) and SPSS (v23.0, IBM Corporation., USA). Quantitative data are represented as the mean ± SEM based on three or more independent experiments. Independent samples t-tests were used for comparing the mutant (MUT) and wild-type (WT) systems. P-values < 0.05 were considered statistically significant.

Results

Identification and characterization of mt-tRNAAsn variants

From a total of 643 patients diagnosed with MD, six individuals from four unrelated families that harbored mt-tRNAAsn variants were identified. These variants included m.5701 delA, m.5708 C > T, m.5709 C > T, and m.5698 G > A. The presence of these variants was observed in various tissues with remarkable heterogenicity (Fig. 1A). The Mitomap database [30] classified the variants as either reported or novel, and computational analysis showed that the variant sites involved evolutionarily conserved nucleotide residues (Fig. 1B).

Fig. 1
figure 1

Characterization of mt-tRNAAsn variants in four MD families. (A)Pedigrees of four families affected by MDs with variations in mt-tRNAAsn are presented, encompassing a total of 6 patients. The mutation load in various tissues, namely peripheral blood (B), oral mucosa (O), urinary sediment (U), and muscle (M), is displayed. (B) Conservative analysis of the mt-tRNAAsn variant sites identified in this article. The nucleotide sequences observed at these sites across a range of mammalian species demonstrate a notable level of conservation. (C) Secondary structures of mt-tRNAAsn molecules, with these variants being highlighted

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All the patients presented with muscle weakness and other associated symptoms. Five patients displayed elevated basal lactate levels. None of the patients had any infections, trauma, or other stressors as triggers prior to the onset of their symptoms. Muscle biopsies conducted on five of the patients indicated a significant deficiency of COX-positive fibers, as indicated by the presence of blue staining in the SDH/COX double-stained sections. In some patients, the presence of strongly SDH-reactive blood vessels (SSVs) was observed on SDH staining, further highlighting the mitochondrial pathology (Fig. 2A). Furthermore, serum queuine levels were quantified by mass spectrometry in two patients (P2, P6), showing significantly reduced levels relative to those seen in healthy controls (P2: 5.48 nM, P6: 1.71 nM, P < 0.01) (Fig. 2B and Fig. S2). The clinical characteristics of the six patients with mt-tRNAAsn variants are detailed in Table 1.

Fig. 2
figure 2

Results of muscle biopsies and serum queuine levels of the patients. (A) Histological analysis of muscle biopsies from 5 patients (P1, P2, P3, P4, P6) with mt-tRNAAsn variants. COX deficient fibers on COX staining, ragged blue fibers (RBFs) on SDH staining, and blue staining fibers in the SDH/COX double-stained sections are indicated by black arrows. (B) Quantitative analysis of serum queuine levels in three patients (P2, P6) with mt-tRNAAsn variants, revealing a statistically decrease compared with controls. The data are presented as the means ± SEM.**P < 0.01 as determined by Mann-Whitney test

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Table 1 Clinical, pathological, and genetical characteristics of patients with mt-tRNAAsn variants
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The m.5708 C > T variant reduces mt-tRNAAsn and mitochondrial protein levels

To explore the effects of the m.5708 C > T variant on the stability of mt-tRNAAsn, Northern blotting was performed on total RNA extracted from patient-derived LCLs.

The average mt-tRNAAsn levels in the MUT cells were found to be reduced to 57.4, 47.6, 53.2, 47.5, 36.9, and 55.2% after normalization to mt-tRNALeu(UUR), mt-tRNAGly, mt-tRNAHis, mt-tRNATrp, mt-tRNAVal, and 5 S rRNA, in comparison to the WT cells. This suggests that the m.5708 C > T variant led to a significant reduction in steady-state mitochondrial tRNAAsn levels (Fig. 3A).

Fig. 3
figure 3

Molecular and bioenergetic impact of the novel m.5708 C > T variant in mt-tRNAAsn. (A) Northern blot analysis demonstrates mt-tRNA levels in WT and MUT LCLs, utilizing DIG-labeled oligodeoxynucleotide probes specific to several mt-tRNAs including mt-tRNAAsn. Quantitative comparison, mt-tRNAAsn normalized to 5 S rRNA and other mt-tRNAs, is shown. (B) Western blot analysis evaluates the subunits of mitochondrial respiratory chain complexes I-V in control (C1, C2) and patient-derived (P2 F2 III-1, P3 F2 II-2) muscle samples, with GAPDH and VDAC1 as loading controls. (C) Western blot analysis of nDNA encoded and mtDNA encoded respiratory complex subunits in WT and MUT LCLs. (D) Flow cytometric analysis of mitochondrial ROS production in WT and MUT LCLs, indicated by the shift in fluorescence intensity, with quantification on the right. (E) Fluorescence microscopy using the JC-1 dye to assess MMP in WT and MUT LCLs, with a quantified shift from red to green fluorescence indicating membrane depolarization. (F) Single-fiber PCR analysis of P2 shows a higher variant load in COX-deficient muscle fibers (89.4%, n = 8) compared to COX-positive fibers (31.1%, n = 11). (G) Seahorse XF Analyzer-derived OCR in WT and MUT LCLs, quantifies basal, ATP-linked and maximal respiration, with various mitochondrial inhibitors applied at indicated time points. (H) Enzymatic activity assays of mitochondrial complexes I, II, IV, and citrate synthase in WT and MUT LCLs, with bar graphs showing relative activities. The data are presented as the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by Student’s t-test

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Western blotting was used to explore how the m.5708 C > T variant affected the expression of nDNA- and mtDNA-encoded respiratory chain complex subunits in mutant systems. The average levels of several nDNA-encoded proteins, such as UQCRC2, SDHB and NDUFB8, as well as the mtDNA-encoded proteins CO1, CYB, and ND4, were significantly decreased in muscle samples and LCLs from the patient compared to the control samples (Fig. 3B and C).

The m.5708 C > T variant leads to mitochondrial dysfunction

MitoSOX staining was used to evaluate the impact of the m.5708 C > T variant on mitochondrial ROS levels. The results demonstrated a substantial increase in ROS in MUT LCL cells, reaching 138.7% (P < 0.01) above the control levels (Fig. 3D). This finding suggested the presence of oxidative stress in the MUT cells. Additionally, measurement of MMPs revealed a significant decrease in MUT cells, indicating compromised MMP integrity (Fig. 3E). The results of the single-fiber PCR analysis indicate significant increases in the variant load of COX-deficient muscle fibers (89.4%, n = 8) compared to COX-positive muscle fibers (31.1%, n = 11) (P < 0.001) (Fig. 3F).

To assess the effect of the m.5708 C > T variant on mitochondrial respiratory function, we quantified the OCR of LCLs with MUT and WT cells. This showed significantly reduced OCR across all measured parameters in the MUT cells. The MUT cells showed lower OCRs with markedly decreased basal (35.4%, P < 0.001), ATP-linked (51.6%, P < 0.001), and maximal (18.7%, P < 0.001) OCR values, relative to the means in WT cells (Fig. 3G). These findings indicate significant impairment in the ability of MUT cells to produce energy through mitochondrial processes, further confirming the involvement of the variant in mitochondrial dysfunction. Furthermore, assays of mitochondrial enzyme activities showed a 46.7% reduction in complex I activity and a 61.3% reduction in that of complex IV in MUT LCLs compared to the WT (Fig. 3H).

Queuine ameliorates reductions in mitochondrial protein levels resulting from the m.5708 C > T variant

To explore the therapeutic possibilities of queuine on mitochondrial dysfunction caused by mt-tRNAAsn variants, patient-devrived cells with the m.5708 C > T variant were investigated. LCLs with both the MUT and WT genotypes were cultured in standard medium or medium containing additional queuine (+ Q) for 15 days. Western blotting showed that queuine supplementation significantly increased the expression of the nDNA-encoded proteins UQCRC2 (149.1%, P < 0.05) and SDHB (182.8%, P < 0.01), as well as the mtDNA-encoded proteins ATP6 (151.2%, P < 0.05), CO1 (211.6%, P < 0.01), CYB (158.7%, P < 0.01), and ND4 (150.8%, P < 0.01) in MUT LCLs. Additionally, in WT LCLs, queuine supplementation led to increased expression of UQCRC2 (220.2%, P < 0.05), ATP6 (137.3%, P < 0.05), CYB (168.6%, P < 0.05), and ND4 (179.3%, P < 0.01) compared to the standard medium (Fig. 4A and B).

Fig. 4
figure 4

Queuine ameliorates mitochondrial protein levels and OCR impaired by m.5708 C > T variant. (A) Western blot analysis of nDNA encoded subunits of mitochondrial respiratory complexes in WT and MUT LCLs, with queuine (+ Q) and standard medium. Relative protein levels of ATP5A, CO4, UQCRC2 and SDHB normalized to ACTIN are quantified in the adjacent graphs. (B) Western blot evaluation of mtDNA encoded subunits in WT and MUT cells, treated and untreated with queuine. Densitometry quantification for ATP6, CO1, CYB, and ND4 is presented, with VDAC1 as the loading control. (C) Seahorse XF Analyzer measurements of OCR delineating the bioenergetic profiles of WT and MUT cells under the influence of queuine. The graphs on the right detail the effects on basal respiration, ATP production, and maximal respiration. The data are presented as the means ± SEM. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 as determined by Student’s t-test

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Queuine mitigates impairment of mitochondrial function induced by the m.5708 C > T variant

The assessment of mitochondrial OXPHOS activities was conducted by measuring the OCR. The MUT LCLs exhibited increases in basal (265.5%, P < 0.001), ATP-linked (198.4%, P < 0.01), and maximal (384.9%, P < 0.01) OCR values, suggesting an enhancement in mitochondrial respiration as a result of queuine treatment. Notably, this effect was also observed in WT cells, with increases in both basal (150.1%, P < 0.05) and maximal (139.9%, P < 0.05) OCRs observed after queuine supplementation (Fig. 4C).

Flow cytometry indicated significant increases in MMP in both MUT and WT LCLs following queuine treatment. This effect was particularly pronounced in the MUT group, which exhibited a substantial elevation in MMP levels (550.2%, P < 0.001) compared to standard medium (Fig. 5A). Additionally, queuine treatment resulted in a marked decrease in ROS generation in MUT cells (36.1%, P < 0.001) compared to standard medium, indicating a potential protective effect against oxidative stress (Fig. 5B).

Fig. 5
figure 5

Queuine alleviates of ROS levels and MMP caused by the m.5708 C > T variant and the mechanistic overview. (A) Fluorescent JC-1 assay depicting the MMP in WT and MUT cells, comparing the effects of queuine treatment. Quantification of the red/green fluorescence ratio is shown to reflect membrane potential changes. (B) Flow cytometric analysis of mitochondrial superoxide production using MitoSOX Red staining in WT and MUT cells. The right panel quantifies MitoSOX fluorescence intensity, indicating relative ROS levels. A significant decrease in ROS levels following treatment, particularly in MUT cells. (C) Schematic illustrations provide a detailed representation of the pathogenic process induced by the mt-tRNAAsn variants, as well as the mitigating effect of queuine treatment. The data are presented as the means ± SEM. *P < 0.05, ***P < 0.001 as determined by Student’s t-test

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Discussion

Clinical phenotypes associated with mt-tRNA variants in MD are diverse and lack effective therapeutic measures [31, 32]. Emerging research indicates that addressing post-transcriptional modification abnormalities in mt-tRNA could potentially ameliorate clinical symptoms [33,34,35,36]. In this study, we presented an analysis of the clinical characteristics associated with four rare variants identified in mt-tRNAAsn and conducted a comprehensive investigation into the pathogenicity of the novel m.5708 C > T variant. Interestingly, individuals harboring these variants displayed markedly reduced levels of queuine in their serum, and the in vitro investigations further verified the efficacy of queuine supplementation in ameliorating mitochondrial dysfunction in cells carrying variants in mt-tRNAAsn. Therefore, it is postulated that the addition of queuine could potentially ameliorate mitochondrial dysfunction resulting from mt-tRNAAsn variants.

It appears that mt-tRNAAsn variants lead to significant muscle involvement in patients with MD. Currently, MITOMAP has documented only six disease-associated variants in mt-tRNAAsn(Fig. S3 and Table S2). Most of these variants (5/6) are associated with myopathy phenotypes, including chronic progressive external ophthalmoplegia (CPEO) and mitochondrial myopathy (MM), while one patient also showed multi-organ involvement. Similarly, all the patients in our cohort presented with muscle weakness and most had elevated basal lactate levels.

Among the six patients, patient 1 (P1) manifested the neuropathy ataxia with retinitis pigmentosa (NARP) phenotype, while P2 and P3 showed the MM phenotype, P4 and P5 presented with muscle weakness and seizures, and P6 exhibited muscle weakness, ophthalmoplegia, and peripheral neuropathy. Considering the rarity of mt-tRNAAsn variants in MDs and the previously unreported variant observed at our center, a thorough evaluation was undertaken. The pathogenicity of the novel m.5708 C > T variant was confirmed, as demonstrated by reduced steady-state levels of mt-tRNAAsn, mtDNA-encoded protein levels, OCR values, and respiratory complex activity.

Interestingly, the patients with mt-tRNAAsn variants all displayed markedly reduced serum queuine levels, which underscores the potential therapeutic advantages of queuine supplementation. In vitro studies further supported the beneficial effects of queuine supplementation in rectifying mitochondrial dysfunctions in cells harboring mt-tRNAAsn variants. Specifically, the administration of queuine effectively alleviated instability and abnormalities in the translational apparatus linked to the m.5708 C > T variants, successfully restoring the respiratory functions of complexes I and IV.

Queuine concentrations in human serum typically fall within the range of 1‒10 nM [37]. It is noteworthy that these levels do not fluctuate with age in healthy individuals, although they tend to be higher in women than men [38]. The molecular mechanisms underlying the lower serum levels of queuine and the therapeutic effects of queuine supplementation in individuals with mt-tRNAAsn variants remain unclear. The hypothesis suggests that the mt-tRNAAsn variant may induce structural abnormalities in mt-tRNAAsn, rendering them more vulnerable to ribonuclease cleavage. This phenomenon could result in an excessive presence of aberrant tRNA-derived stress-induced fragments (tRFs), which might impede the efficiency of mt-DNA transcription [39, 40]. Evidence suggests that supplementation with queuine can counteract abnormal tRNA cleavage, thus improving transcriptional efficacy. Additionally, a recent study has highlighted the importance of the queuine salvage protein QNG1 (C9ORF64) in queuine salvage and recycling [41]. However, it remains uncertain whether the reduced queuine levels in these patients can be attributed to deficiencies in the recycling and reutilization mechanisms.

Queuine holds significant importance in both medical and biomedical fields. In cellular studies and mouse models, queuine depletion disrupts protein translation, leading to accumulations of misfolded proteins and triggering the unfolded protein response (UPR) [20]. In human HepG2 cells, the absence of queuine impairs phenylalanine conversion to tyrosine, causing phenylketonuria [42]. Additionally, queuine deficiency in Hela cells increased Warburg metabolism, which promotes glycolysis and cancer cell survival [43]. Furthermore, recent studies showed that queuine supplementation can achieve full remission in a mouse model of multiple sclerosis [44]. Studies also suggested that queuine deficiency may contribute to neurodegenerative diseases such as Parkinson’s and Alzheimer’s, and ensuring adequate queuine levels could potentially slow the progression of these diseases. Proper queuine supplementation could enhance the accuracy and efficiency of mitochondrial protein synthesis, thereby improving the assembly and activity of the OXPHOS complexes. Additionally, queuine’s influence on cellular metabolism, including the enhancement of glycolysis and modulation of the Warburg effect, can indirectly affect mitochondrial function. By optimizing cellular energy production pathways, queuine helps maintain a balance between glycolysis and oxidative phosphorylation, ensuring adequate ATP production and overall cellular energy homeostasis [38]. These findings underscore the critical role of queuine in maintaining cellular homeostasis and highlight its therapeutic potential across various diseases.

The limitations of this study include the limited sample size and the absence of in vivo experiments, which are particularly challenging due to the rarity of mutations in mt-tRNAAsn. Furthermore, the development of mouse models harboring mt-tRNA mutations has been fraught with difficulties, and currently, there are no available mouse models with mt-tRNAAsn mutations. Additionally, experiments on other mt-tRNA mutations, including tRNAHis, tRNAAsp, and tRNATyr, are necessary to gather more evidence.

In summary, we provides a comprehensive analysis of the clinical and pathological characteristics in MD patients harboring mt-tRNAAsn variants, and also identified molecular pathomechanisms associated with the novel m.5708 C > T variant. The effectiveness of queuine supplemental was verified in vitro, suggesting a viable therapeutic strategy for individuals harboring mt-tRNAAsn variants.

Data availability

All data relevant to the study are included in the article and in and its supplementary materials. Any reagents used within this study can be obtained from the corresponding author upon a reasonable request.

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Acknowledgements

The authors express their gratitude to the study participants and their families for generously contributing samples and providing invaluable clinical information.

Funding

This study was supported by the National Natural Science Foundation of China (No. 82301590, 82171394 and 82371410), Grants from the National Key R&D Program of China (No.2021YFC2700904), Key R&D Program of Shandong Province (2022ZLGX03), China Postdoctoral Science Foundation (2023M742116), Natural Science Foundation of Shandong Province (ZR2023QH106), Shandong Provincial Postdoctoral Innovation Talent Support Program (SDBX2022061).

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Author notes

  1. Yan Lin, Jiayin Wang, Chuanzhu Yan and Kunqian Ji contributed equally to this work.

Authors and Affiliations

  1. Department of Neurology, Shandong Key Laboratory of Mitochondrial Medicine and Rare Diseases, Research Institute of Neuromuscular and Neurodegenerative Diseases, Qilu Hospital of Shandong University, No. 107 West Wenhua Road Jinan, Jinan, Shandong, 250012, China

    Yan Lin, Jiayin Wang, Xingyu Zhuang, Ying Zhao, Wei Wang, Dongdong Wang, Yuying Zhao, Chuanzhu Yan & Kunqian Ji

  2. Qingdao Key Laboratory of Mitochondrial Medicine and Rare Disease, Qilu Hospital (Qingdao), Shandong University, Qingdao, Shandong, 266035, China

    Chuanzhu Yan

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  1. Yan Lin
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Contributions

YL conceived the study. YL and JYW designed and performed most of the experiments; XYZ, YZ and WW helped with some of the experiments; DDW, FCL and YYZ supervised the study; YL and JYW wrote the paper with critical edits from CZY and KQJ. The authors read and approved the final manuscript.

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Correspondence to Chuanzhu Yan or Kunqian Ji.

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Supplementary Material 1: Table S1: The detailed sequences for tRNAs and 5 S rRNA

Supplementary Material 2: Table S2: Clinical manifestations of mutation in the mitochondrial tRNAAsn

12967_2024_5574_MOESM3_ESM.tif

Supplementary Material 3: Fig S1: A preliminary experiment to determine the concentration and time of administration of queuine. The data are presented as the means ± SEM. ns P > 0.05, *P < 0.05, ***P < 0.001 as determined by One-way ANOVA and Tukey’s multiple comparisons test

12967_2024_5574_MOESM4_ESM.tif

Supplementary Material 4: Fig S2: Original data of mass spectrometry. A, B. Chromatogram for the real sample (P2 and P6). C, D.Chromatogram of the standard and reference standards

12967_2024_5574_MOESM5_ESM.tif

Supplementary Material 5: Fig S3: Reported and our mt-tRNAAsn variants, along with their respective positions on the cloverleaf structure of tRNAAsn

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Lin, Y., Wang, J., Zhuang, X. et al. Queuine ameliorates impaired mitochondrial function caused by mt-tRNAAsn variants. J Transl Med 22, 780 (2024). https://doi.org/10.1186/s12967-024-05574-0

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Journal of Translational Medicine

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