Coenzyme Q headgroup intermediates can ameliorate a mitochondrial encephalopathy

Shi, Guangbin; Miller, Claire; Kuno, Sota; Rey Hipolito, Alejandro G.; El Nagar, Salsabiel; Riboldi, Giulietta M.; Korn, Megan; Tran, Wyatt C.; Wang, Zixuan; Ficaro, Lia; Lin, Tao; Spillier, Quentin; Gamallo-Lana, Begoña; Jones, Drew R.; Snuderl, Matija; Song, Soomin C.; Mar, Adam C.; Joyner, Alexandra L.; Sillitoe, Roy V.; Banh, Robert S.; Pacold, Michael E.

doi:10.1038/s41586-025-09246-x

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Published: 09 July 2025

Coenzyme Q headgroup intermediates can ameliorate a mitochondrial encephalopathy

Nature volume 645, pages 466–474 (2025)Cite this article

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Abstract

Decreased brain levels of coenzyme Q₁₀ (CoQ₁₀), an endogenously synthesized lipophilic antioxidant^1,2, underpin encephalopathy in primary CoQ₁₀ deficiencies^3,4 and are associated with common neurodegenerative diseases and the ageing process^5,6. CoQ₁₀ supplementation does not increase CoQ₁₀ pools in the brain or in other tissues. The recent discovery of the mammalian CoQ₁₀ headgroup synthesis pathway, in which 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL) makes 4-hydroxymandelate (4-HMA) to synthesize the CoQ₁₀ headgroup precursor 4-hydroxybenzoate (4-HB)⁷, offers an opportunity to pharmacologically restore CoQ₁₀ synthesis and mechanistically treat CoQ₁₀ deficiencies. To test whether 4-HMA or 4-HB supplementation promotes CoQ₁₀ headgroup synthesis in vivo, here we administered 4-HMA and 4-HB to Hpdl^−/− mice, which model an ultra-rare, lethal mitochondrial encephalopathy in humans. Both 4-HMA and 4-HB were incorporated into CoQ₉ and CoQ₁₀ in the brains of Hpdl^−/− mice. Oral treatment of Hpdl^−/− pups with 4-HMA or 4-HB enabled 90–100% of Hpdl^−/− mice to live to adulthood. Furthermore, 4-HB treatment stabilized and improved the neurological symptoms of a patient with progressive spasticity due to biallelic HPDL variants. Our work shows that 4-HMA and 4-HB can modify the course of mitochondrial encephalopathy driven by HPDL variants and demonstrates that CoQ₁₀ headgroup intermediates can restore CoQ₁₀ synthesis in vivo.

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Main

Many common diseases are associated with decreased tissue CoQ₁₀ levels⁸. Furthermore, some pharmaceuticals, such as statins, reduce CoQ₁₀ levels, resulting in statin-associated muscle symptoms⁹. The perceived role of decreased tissue CoQ₁₀ in diseases has fuelled a long-standing desire to replete tissue CoQ₁₀ pools¹⁰. However, CoQ₁₀ pools are refractory to pharmacological augmentation^11,12,13, particularly in the central nervous system, owing to the poor bioavailability^11,14 of CoQ₁₀. New therapeutic approaches are needed to address the limitations of CoQ₁₀ supplementation or to promote endogenous CoQ₁₀ synthesis.

A potential avenue of enabling CoQ₁₀ synthesis in vivo is our recent discovery of the mammalian CoQ₁₀ headgroup synthesis pathway⁷, in which HPDL catalyses the synthesis of 4-HMA, which generates 4-HB, the immediate precursor of the CoQ₁₀ headgroup^5,6,15 (Fig. 1a). Both 4-HMA and 4-HB are small molecules (<200 Da) that are readily taken up by cells and are efficiently incorporated into CoQ₁₀. Our work, and recent research showing that high doses of 4-HB rescue the survival of mice with hypomorphic Coq2 (Coq2^A252V)¹⁶, indicates that 4-HMA and 4-HB might enable mammalian CoQ₁₀ synthesis in patients with deficits in the CoQ₁₀ headgroup synthesis pathway, providing a therapeutic strategy for patients with CoQ₁₀ deficiencies.

**Fig. 1: CoQ headgroup intermediates improve *Hpdl*^−/− mouse survival and are incorporated into brain CoQ.**

To investigate this hypothesis, we tested whether 4-HMA and 4-HB could be used to treat Hpdl^−/− mice, which robustly model neurodevelopmental disorder with progressive spasticity and brain white matter abnormalities (NEDSWMA)^{17,18,19,20,21,22,23}, an ultra-rare lethal primary CoQ₁₀ deficiency caused by HPDL variants that presents with severe neurodevelopmental symptoms. Hpdl^−/− mice develop movements consistent with spastic paresis and epileptic seizures and die by postnatal day 15 (P15)²¹. Here we show that treatment with 4-HMA or 4-HB restores CoQ biosynthesis in the brains of Hpdl^−/− mice and demonstrate rescue of Hpdl^−/− mice by supplementation with CoQ₁₀ headgroup intermediates. We also show that 4-HB treatment improves neurological symptoms and modifies the clinical course of a patient with HPDL variants. Restoration of brain CoQ₁₀ synthesis with CoQ₁₀ headgroup intermediates might alleviate the neurological symptoms of primary CoQ₁₀ deficiencies and diseases associated with low tissue CoQ₁₀ pools.

4-HMA and 4-HB can treat Hpdl ^−/− pups

As previously described²¹, Hpdl^−/− pups do not gain weight as quickly as do Hpdl^+/+ pups after birth, develop seizures (Extended Data Fig. 1a,b and Supplementary Video 1) and die by P15 (Fig. 1b). Hpdl^−/− pups have lower plasma 4-HMA concentrations than do Hpdl^+/+ or Hpdl^+/− pups (Fig. 1c) and a higher plasma lactate:pyruvate ratio (Extended Data Fig. 1c).

We hypothesized that treatment of Hpdl^−/− pups with the CoQ₁₀ headgroup intermediates 4-HMA or 4-HB would improve the neurological symptoms of Hpdl^−/− mice. 4-HMA and 4-HB are orally bioavailable with half-lives of 2.8 h and 0.35 h, respectively (Extended Data Fig. 1d and Supplementary Table 1), and neither compound induces inflammation or necrosis in the brain, heart, lungs, liver, kidneys or spleen of mice at single doses of 500 mg kg⁻¹ or at a dose of 500 mg kg⁻¹ per day for 3 months (Extended Data Fig. 1e). 4-HMA was detected in the plasma of 4-HMA-treated Hpdl^−/− pups on P10 (Extended Data Fig. 1f).

Hpdl^−/− pups treated orally with 4-HMA or 4-HB at a minimum of 10 mg kg⁻¹ once daily starting on P3 or P7 gained weight and had improved mobility (Extended Data Fig. 1a,g, Supplementary Table 2 and Supplementary Video 2). Hpdl^−/− pups treated on P10 did not survive (Extended Data Fig. 1h). After weaning, we treated Hpdl^−/− pups with 10 mg kg⁻¹ 4-HMA or 4-HB per day in their drinking water and discontinued 4-HMA or 4-HB treatment on P30. Most Hpdl^−/− pups treated with 4-HMA or 4-HB lived for 18 months or more (Fig. 1d). By contrast, Hpdl^−/− pups treated orally with CoQ₉, CoQ₁₀, the CoQ analogue idebenone or the mitochondrially targeted antioxidant MitoTEMPO^24,25,26 did not survive (Fig. 1d). We conclude that 4-HMA or 4-HB treatment extends the lifespan of Hpdl^−/− pups to the average lifespan of a mouse.

4-HMA and 4-HB restore CoQ synthesis

To test whether 4-HMA and 4-HB crossed the blood–brain barrier and were incorporated into CoQ₉, the predominant coenzyme Q species in mice, and CoQ_10, which is also found in mouse brain, we treated Hpdl^+/+, Hpdl^+/− and Hpdl^−/− pups with ¹³C₆-4-HMA or ¹³C₆-4-HB, in which the ring carbons are labelled with ¹³C, at 10 mg kg⁻¹ per day from P3 to P20. We expected that incorporation of all ring carbons from ¹³C₆-4-HMA or ¹³C₆-4-HB into CoQ₉ and CoQ₁₀ would result in ¹³C₆-CoQ₉ and ¹³C₆-CoQ₁₀ (m6-CoQ₉ and m6-CoQ₁₀, respectively; Fig. 1e). In Hpdl^−/− pups, at least 50% of CoQ₉ and CoQ₁₀ came from ¹³C₆-4-HMA and ¹³C₆-4-HB (Fig. 1f). Treatment with ¹³C₆-4-HMA and ¹³C₆-4-HB also increased brain CoQ₉ and CoQ₁₀ pools in Hpdl^−/− pups (Extended Data Fig. 2a), albeit to levels lower than those of Hpdl^+/+ and Hpdl^+/− mice²⁷, and these lower CoQ levels persisted after 4-HMA treatment was stopped (Extended Data Fig. 2b). 4-HMA did not increase CoQ₉ and CoQ₁₀ levels in the brains of P90 wild-type mice (Extended Data Fig. 2c). These data show that Hpdl loss results in a primary CoQ deficiency in the brains of mice and that orally administered ¹³C₆-4-HMA and ¹³C₆-4-HB enable CoQ₉ and CoQ₁₀ synthesis in the brain.

4-HMA improves histology of Hpdl ^−/− pup brains

CoQ₁₀ is essential for electron transport chain activity and maintenance of mitochondrial membrane potential²⁸. Coq8a^−/− mice develop cerebellar ataxia and have abnormal cerebellar mitochondrial morphology²⁹. Moreover, primary CoQ₁₀ deficiencies are a cause of cerebellar ataxia in patients³⁰. Therefore, we examined cerebellar mitochondrial morphology and cerebellar morphology in Hpdl^−/− pups. Electron microscopy revealed that mitochondria in the cerebella of P11 Hpdl^−/− pups were smaller than those of Hpdl^+/+ or Hpdl^+/− control pups and that mitochondria in the cerebella of Hpdl^+/− pups were smaller than those of Hpdl^+/+ controls (Fig. 2a,b). 4-HMA increased cerebellar mitochondrial size in P11 Hpdl^−/− pups, although not to the same size as those of Hpdl^+/+ pups (Fig. 2a,b). P11 Hpdl^−/− pups had small cerebella, with a thinner external granular layer, vacuolization and intracellular oedema in cerebellar Purkinje cells (PCs) and depletion of the internal granular layer (Fig. 2c). Cerebellum size in Hpdl^−/− pups was greatly reduced on P6 (Extended Data Fig. 3a–c), when these animals responded to 4-HMA (Extended Data Fig. 1h). Cerebellar histology in P6 and P11 Hpdl^−/− pups and in P56–P57 mice was restored by 4-HMA supplementation (Fig. 2c and Extended Data Fig. 3a,h). Immunohistochemical staining for calbindin showed underdeveloped PC dendritic arbors in Hpdl^−/− pups on P11 (Extended Data Fig. 3e,f). 4-HMA treatment largely restored PC dendritic arborization (Extended Data Fig. 3e,f and Extended Data Fig. 4a). Granule cell precursors in P11 Hpdl^−/− pups had lower Ki-67 staining than did those in Hpdl^+/+ pups (Extended Data Fig. 3e). Ki-67 staining was restored by 4-HMA treatment (Extended Data Fig. 3e). Similarly, the external granular layer thickness was reduced in Hpdl^−/− pups on P11 compared with Hpdl^+/+ and Hpdl^+/− pups and was partially restored with 4-HMA treatment (Extended Data Fig. 3e,g). To determine whether these changes were initiated before P11, we analysed cerebellar morphology on P6. PAX6 staining revealed that granule cells were depleted in the external and internal granular layers of P6 Hpdl^−/− pups relative to Hpdl^+/+ pups and was improved with 4-HMA treatment (Extended Data Fig. 3b). Calbindin staining of the PCs in P6 Hpdl^−/− pups revealed a multilayer without dendrites. 4-HMA treatment resulted in a single layer of PCs with stunted dendrites (Extended Data Fig. 3b). The cerebella of P6 Hpdl^−/− pups had higher TUNEL staining indicating cell death, which decreased with 4-HMA treatment of Hpdl^−/− pups (Extended Data Fig. 3b,d). 4-HMA increased cerebellar size in P6 Hpdl^−/− pups (Extended Data Fig. 3a,c).

**Fig. 2: 4-HMA supplementation improves cerebellar histology in *Hpdl*^−/− mice.**

The cerebral cortex of P11 Hpdl^−/− pups showed no major changes in mitochondrial morphology (Extended Data Fig. 4b) but subtle loss of healthy cortical architecture (Extended Data Fig. 4c). The hippocampus was largely unremarkable (Extended Data Fig. 4d). However, TUNEL staining was higher in the cortex, striatum and thalamus of P6 Hpdl^−/− pups and treatment with 4-HMA significantly decreased TUNEL staining in the striatum (Extended Data Fig. 5a).

These findings indicate that part of the phenotype of Hpdl^−/− mice involves cerebellar cell death associated with a delay in cerebellar maturation and increased cell death in the cortex, striatum and thalamus. Metabolite replacement therapy with 4-HMA, the metabolic product of HPDL, largely reverted these abnormalities. Our data demonstrate that 4-HMA-dependent CoQ synthesis is important for brain development and indicate that treatment with 4-HMA or 4-HB during infancy or childhood could improve the outcomes of patients with HPDL variants.

4-HMA improves Hpdl ^−/− Purkinje cell function

To test whether Hpdl loss affects mitochondrial electron transport chain activity, we measured electron flow through complexes I, II and IV (Extended Data Fig. 6a) in mitochondria isolated from the brains of P9 Hpdl^+/+, Hpdl^+/− and Hpdl^−/− pups and P9 Hpdl^−/− pups treated with 4-HMA. Consistent with loss of CoQ, electron flow through complexes I and II was reduced in mitochondria from Hpdl^−/− pups and was improved by 4-HMA treatment (Fig. 3a). There was a non-statistically significant decrease in electron flow, rescued by 4-HMA, through complex IV (Fig. 3a). We observed similar decreases in electron flow through complexes I and II in mitochondria from the cortices of Hpdl^−/− pups (Extended Data Fig. 6b). Mitochondria isolated from the cortices and cerebella of P90 Hpdl^−/− mice treated with 4-HMA from P3 to P30 had electron flow through complexes I, II and IV comparable to that of P90 Hpdl^+/+ and Hpdl^+/− mice (Extended Data Fig. 6c). 4-HMA treatment did not increase electron flow through complexes I, II or IV in mitochondria isolated from the cerebella of P12 Hpdl^+/+ pups (Extended Data Fig. 6d). These data demonstrate that loss of Hpdl attenuates electron flow through isolated cortex and cerebellar mitochondria in Hpdl^−/− pups, presumably because of decreased CoQ levels. 4-HMA treatment from P3 to P30 restores electron flow through complexes I and II to near-wild-type levels even on P90. These data indicate that there is a CoQ threshold above which there is a durable rescue of mitochondrial electron flow, and that HPDL maintains CoQ pools above that threshold during development.

**Fig. 3: 4-HMA improves PC function and behavioural phenotypes of *Hpdl*^−/− mice.**

PCs are the sole output of the cerebellar cortex and depend on high metabolic activity to sustain rapid, chronic firing. To determine whether Hpdl loss affected the function of PCs, we manually patch-clamped PCs in brain slices from Hpdl^+/+ and Hpdl^+/− mice, as well as untreated and 4-HMA-treated Hpdl^−/− mice, in three age brackets: P8–10 pups (during cerebellar development), P17–19 pups (after cerebellar maturation was mostly completed)³¹ and P56–57 pups (the age at which we did our behavioural studies). The P17–19 and P56–57 groups did not have untreated Hpdl^−/− pups as these animals die by P15. In the P8–10 group, PCs from Hpdl^−/− mice had increased input resistance (R_i) compared with PCs from Hpdl^+/+ and Hpdl^+/− mice (Fig. 3b and Table 1), indicative of fewer membrane ion channels. There was a significant reduction in membrane capacitance (C_m) in Hpdl^−/− PCs relative to Hpdl^+/− PCs^32,33, indicative of smaller or less developed cells (Fig. 3b). These electrophysiological deficits were partially restored by 4-HMA treatment such that R_i was comparable to that of PCs of Hpdl^+/− mice and lower than the R_i of PCs from Hpdl^+/+ mice (Fig. 3b). Moreover, the C_m of PCs from 4-HMA-treated Hpdl^−/− mice significantly increased relative to that of PCs from Hpdl^−/− mice and was higher than that of PCs from Hpdl^+/+ and Hpdl^+/− mice (Fig. 3b). By P17–19 or P56–57, no differences were observed in the R_i and C_m of PCs from Hpdl^+/+, Hpdl^+/− and Hpdl^−/− pups treated with 4-HMA (Fig. 3c, Table 1 and Extended Data Fig. 7h). These data are consistent with 4-HMA treatment correcting the aberrant passive electrophysiological characteristics of cerebellar PCs from Hpdl^−/− pups.

Table 1 Passive electrophysiological characteristics of cerebellar PCs

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We next tested whether Hpdl loss affected the current–voltage (I–V) curves of PCs in mouse pups. Cerebellar PCs from P8–10 Hpdl^−/− pups had a tighter grouping of currents at hyperpolarized membrane potentials (−100 to −60 mV) relative to that of Hpdl^+/+ PCs (Extended Data Fig. 7a, insets), resulting in a flatter I–V curve for Hpdl^−/− PCs at hyperpolarized membrane potentials (Extended Data Fig. 7a). Interestingly, 4-HMA treatment seemed to overcompensate for this, resulting in a steeper I–V curve of PCs in Hpdl^−/− pups relative to those of the other groups (Extended Data Fig. 7a), closer in slope and magnitude to the I–V curves of P17–19 PCs (Extended Data Fig. 7b). The I–V curves of PCs from P17–19 and P56–57 Hpdl^+/+, Hpdl^+/− and 4-HMA-treated Hpdl^−/− pups were comparable (Extended Data Fig. 7b,c). These data are consistent with PCs from Hpdl^−/− pups having differential sensitivity to inputs at hyperpolarized membrane potentials than do Hpdl^+/+ and Hpdl^+/− PCs and partial reversion of these electrophysiological properties by 4-HMA.

The peak frequency of PCs from P8–10 Hpdl^−/− pups was significantly lower than those of Hpdl^+/+ and Hpdl^+/− PCs but was increased by 4-HMA treatment to a level higher than that of Hpdl^+/+ pups (Extended Data Fig. 7d). These differences resolved by P17–19 (Extended Data Fig. 7e). These findings are consistent with immaturity of Hpdl^−/− PCs relative to PCs of Hpdl^+/+ and Hpdl^+/− pups and accelerated maturation of 4-HMA-treated Hpdl^−/− PCs. Hpdl loss and 4-HMA rescue did not affect the firing rate–current curves of PCs from P7–10 or 17–19 pups (Extended Data Fig. 7f,g). However, PCs from P56–57 Hpdl^+/+ mice had a higher firing rate at higher input currents than PCs from Hpdl^+/− and 4-HMA-treated Hpdl^−/− mice (Extended Data Fig. 7i). Likewise, action-potential parameters of PCs from Hpdl^+/+, Hpdl^+/− and Hpdl^−/− pups were mostly similar (Extended Data Fig. 8a). However, spike thresholds of PCs from Hpdl^−/− pups treated with 4-HMA were more hyperpolarized than those of PCs from Hpdl^+/+, Hpdl^+/− and untreated Hpdl^−/− pups on P8–10 (Extended Data Fig. 8a). The action half potential and spike half-width of PCs from Hpdl^−/− pups treated with 4-HMA were slightly less than those of PCs from Hpdl^+/+ pups (Extended Data Fig. 8a). The differences in action half potential between PCs from Hpdl^+/+ or untreated Hpdl^−/− pups and those from Hpdl^−/− pups treated with 4-HMA persisted on P17–19 (Extended Data Fig. 8b). No significant differences in peak frequency or action-potential characteristics in PCs from P56–57 (8-week-old) Hpdl^+/+, Hpdl^+/− and 4-HMA-treated Hpdl^−/− mice were observed (Extended Data Fig. 8c).

These results demonstrate that homozygous Hpdl loss delays the functional maturation of cerebellar PCs. 4-HMA treatment accelerates and ultimately restores the functional maturity of cerebellar PCs as measured by electrophysiological parameters.

4-HMA treats Hpdl ^−/− motor symptoms in pups

Patients with HPDL variants typically present with ataxia, spastic tetraplegia and other deficits in motor function. To test whether 4-HMA improved the metabolic physiology and neurological symptoms associated with Hpdl variants, we carried out metabolic home-cage measurements and tests of motor function on P58 (approximately 8-week-old) Hpdl^−/− mice treated with 4-HMA and compared them with Hpdl^+/+ and Hpdl^+/− mice. 4-HMA reduced seizures in Hpdl^−/− mice (Extended Data Fig. 1b). At the start of testing, male, but not female, Hpdl^−/− mice weighed less than their Hpdl^+/+ and Hpdl^+/− counterparts (Extended Data Fig. 9c) but had no significant differences in food or water consumption, activity, maximal oxygen consumption, carbon dioxide production or energy expenditure (Extended Data Fig. 9a). Hpdl^−/− male, but not female, mice treated with 4-HMA had a significantly elevated respiratory exchange ratio (RER) compared with Hpdl^+/− and Hpdl^+/+ mice (sex × genotype interaction: F_2,17 = 9.913, P < 0.01), suggestive of altered energy substrate use (Extended Data Fig. 9b,d). This effect seemed to be gene-dosage dependent, as Hpdl^+/− male mice had statistically intermediate RER values (male Hpdl^+/− versus Hpdl^+/+, P < 0.05; male Hpdl^+/− versus Hpdl^−/−, P < 0.05; Extended Data Fig. 9b,d). Hpdl^−/− mice treated with 4-HMA had a mild, significant reduction in the regularity of their step sequence (F_2,17 = 3.826, P < 0.05; Fig. 3d). The first-order effect of the gait analysis was mouse survival that enabled ambulation. However, 4-HMA-treated Hpdl^−/− mice showed no significant impairments in any other gait parameter or in the open-field, rotarod, pole and transverse-beam tests, which assess motor learning, coordination and balance (Fig. 3e,f, Extended Data Figs. 9e,f and 10a and Supplementary Videos 3–5). A gene-dosage effect was observed in forelimb grip strength, with significant reductions found in 4-HMA-treated Hpdl^−/− mice relative to Hpdl^+/− and Hpdl^+/+ mice (F_2,17 = 38.908, P < 0.001), and with Hpdl^+/− mice having significantly lower forelimb grip strength than Hpdl^+/+ mice (Fig. 3g). This pattern was similar but less pronounced in measures of grip strength for all four limbs. Only Hpdl^−/− mice treated with 4-HMA had significant reductions in grip strength relative to Hpdl^+/+ mice (F_2,17 = 4.358, P < 0.05; Fig 3g). These results are potentially consistent with the firing rate–current curves of PCs from P56–57 Hpdl^+/− mice and 4-HMA-treated Hpdl^−/− mice, which have lower firing rates at higher input currents than do PCs from P56–57 Hpdl^+/+ mice (Extended Data Fig. 7i).

The gene-dosage effects on RER in male mice and on mouse grip strength suggest that there are neurological and metabolic features affected by Hpdl loss that are partially restored by our current 4-HMA treatment regimen, indicating that treatment may need optimization or that some neurological features are refractory to 4-HMA treatment. However, the improvement of motor phenotypes in 4-HMA-treated Hpdl^−/− mice suggests that 4-HMA could improve the outcomes of humans with neurological symptoms due to HPDL variants.

4-HB can treat a patient with HPDL encephalopathy

An eight-year-old child with biallelic HPDL variants (c.3G>C (p.Met1Ile), c.789delG (p.P264Lfs*51)) had a healthy birth and healthy development until eight years of age, when he presented with rapidly progressing spasticity over 2–3 months, although he probably had subtle symptoms before then. The child was able to run and participate in typical sports in August 2023, developed bilateral ankle clonus in September 2023 and could no longer run or participate in typical sports in October 2023. He was started on systemic CoQ₁₀ supplementation in mid-October 2023. In early November 2023, medical records note that he could stand without support but had difficulty maintaining an upright posture with feet together. By late November 2023, he fell frequently and his ambulation was impaired to the degree that physical therapists advised the family to obtain a wheelchair. The patient’s family history included two younger siblings who had died in infancy (Fig. 4a), and who both developed rapidly progressing and catastrophic encephalopathy and seizures. The second sibling received genetic testing, revealing biallelic HPDL variants (c.3G>C (p.Met1Ile), c.789delG (p.P264Lfs*51)). After the onset of symptoms in the eight-year-old child, genetic testing confirmed the same biallelic variants. Both variants are reported in ClinVar³⁴ as pathogenic (VCV001344855.6 and VCV002221909.2) and the association of the c.3G>C (p.Met1Ile) variant with NEDSWMA has previously been reported¹⁹. HPDL variants are ultra-rare based on calculated allele frequencies (Table 2).

**Fig. 4: Treatment of a single patient with *HPDL* encephalopathy with the CoQ₁₀ headgroup intermediate 4-HB.**

Table 2 Allele frequency data for HPDL variants

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Given the rapid and apparently unrelenting progression of the patient’s symptoms (including 1.5 months while the patient was taking CoQ₁₀), the history of a sibling with NEDSWMA and the availability of 4-HB in highly pure form, we proposed treatment with 4-HB. The administration of 4-HB was approved by the US Food and Drug Administration (single patient investigational new drug (IND) 170119) and the treatment protocol was approved by the NYU institutional review board (IRB; protocol 23-01512). The parents provided informed consent and the child agreed to treatment with 4-HB; data are shared with permission. Initial assessments included a brain MRI, with scans showing patchy T2 hyperintensity of the corticospinal tract of unclear relevance and no major cerebellar abnormalities (Extended Data Fig. 10b).

The patient started treatment with 30 mg of 4-HB in a 5 mg ml⁻¹ solution (6 ml) in water on day 1. On the basis of preclinical data, the dose was rapidly increased over the first 4 days to 100 mg kg⁻¹ (600 ml of solution); that dose was maintained until day 30 of treatment. The dose was then decreased to 50 mg kg⁻¹ (300 ml of solution) for an additional 95 days and then decreased to the current dose of 25 mg kg⁻¹. The child generally tolerated the treatment well; the solution was palatable and was reported to have a sour taste. During the first 30 days of the study, on several occasions, the child had emesis shortly after drinking the 600 ml solution, attributed to the large volume of solution ingested over a short period of time (15–20 min). No further emesis occurred during the 30 days with the lower dose volume of 300 ml. Safety monitoring, including electrocardiograms and bloodwork, was reassuring; there was no substantial change in these values for the duration of treatment. The data presented include 250 days of treatment. CoQ₁₀ was discontinued after several months of treatment with 4-HB.

Outcome measures (Table 3) demonstrate improvement of spasticity according to the Modified Ashworth Scale³⁵ with the sum total of scores decreasing from 10 to 6 (Fig. 4b). The Spastic Paraplegia Rating Scale (SPRS)³⁶ score, a measure of spasticity and its effects on quality of life, improved from 13 to 7 (Fig. 4c). Scores for the nine-hole peg test³⁷, a measure of hand–finger dexterity, are consistent with stabilization or subtle improvement of the child’s hand movement to times typical for children his age (Table 3). The 10-m walk test³⁸ results are varied, in part because the child compensated for profound imbalance with increased walking speed at the beginning of the study. The patient’s brain MRI did not change after 30 days of treatment (Extended Data Fig. 10b). Subjective reports, some of which are components of the SPRS, include improvement in endurance (the patient initially struggled to walk across a hospital lobby and by 30 days walked for 20 min), improvement in balance (the patient initially was unable to stand in tandem and was able to do so about 2 weeks after treatment initiation; after 8 months of treatment he was able to step laterally to catch a ball), decreased fall frequency from multiple times daily to rarely and restoration of running by approximately 1.5 months of treatment (Table 4).

Table 3 Clinical outcomes for a patient with HPDL encephalopathy treated with 4-HB

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Table 4 Patient-reported outcomes for a patient with HPDL encephalopathy treated with 4-HB

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These data and observations demonstrate the safety and tolerance of 4-HB treatment in a child at a dose of 100 mg kg⁻¹ for 27 treatments, followed by 50 mg kg⁻¹ for 95 treatments and subsequent treatment with a maintenance dose of 25 mg kg⁻¹. We show stabilization over the course of a rapidly progressing spasticity, followed by objective and subjective improvement in the child’s motor function, providing evidence of improvement in neurological symptoms in a patient with HPDL encephalopathy^{17,18,19,20,21,22,23,39,40}.

Discussion

The central role of CoQ₁₀ in mitochondrial health has long been understood. Attempts to improve CoQ₁₀ synthesis have been difficult in the periphery⁴¹ and impossible in the central nervous system. Here we demonstrate that the CoQ₁₀ headgroup intermediates 4-HMA and 4-HB efficiently rescue the neurological phenotypes of Hpdl^−/− mice, which model an ultra-rare primary CoQ₁₀ deficiency. 4-HB modified the course of an HPDL-variant mitochondrial disease in a patient with no toxicity during eight months of treatment. Our results demonstrate that the mammalian CoQ₁₀ synthesis pathway is amenable to pharmacological repletion with CoQ₁₀ headgroup intermediates. We demonstrate that metabolite replacement therapy improves the neurological symptoms of a primary CoQ₁₀ deficiency in a patient.

Although 4-HMA treatment improves most neurological deficits and improves the survival of Hpdl^−/− mice when administered by day 6 or 7 postnatally, initiation of treatment after P10 did not improve Hpdl^−/− pup survival. These data indicate that there is a critical window in neural development during which the effects of HPDL encephalopathy will be partially reversible in patients. Treatment with CoQ₁₀ headgroup intermediates during the critical window restores cellular function and development and improves neurological symptoms, even though our current 4-HMA and 4-HB treatment regimen only partially restores CoQ pools in the brains of Hpdl^−/− mice. Patients, such as the person we treated, may have a longer critical window if they retain any residual HPDL activity. Once brain development is complete⁴², CoQ₁₀ headgroup synthesis might be dispensable. In support of this hypothesis, 4-HMA-treated Hpdl^−/− mice survive without supplementation of CoQ₁₀ headgroup intermediates after P30.

The durable rescue of mitochondrial function in Hpdl^−/− mice suggests a CoQ threshold effect as well as differential requirements for CoQ synthesis during development and in adulthood. CoQ headgroup synthesis may not be required for the survival of adult mice living under controlled conditions, particularly if there are alternative sources of CoQ or CoQ headgroups. Conditional deletion of Hpdl will enable us to address the tissue-specific and temporal requirements of HPDL activity.

Loss of CoQ headgroup synthesis is associated with increased TUNEL staining, consistent with apoptosis, in the developing cerebellum and in the cortex, striatum and thalamus (Extended Data Figs. 3b,d and 5a). This may be caused by loss of mitochondrial function or increased mitochondrial or cellular reactive oxygen species due to decreased CoQ. Increased apoptosis in these brain regions may contribute to the clinical symptoms of patients with HPDL variants. Determining the mechanisms underlying increased sensitivity of specific brain regions to loss of HPDL and CoQ headgroup synthesis will require further research.

Treatment with ¹³C₆-4-HMA or ¹³C₆-4-HB restores CoQ₉ and CoQ₁₀ pools in Hpdl^−/− mice to about half that of wild-type mice, and a substantial fraction of CoQ₉ and CoQ₁₀ remains unlabelled. These results are consistent with a parallel CoQ headgroup synthesis pathway or the uptake of CoQ headgroup from maternal milk, diet or another source. This alternative pathway is not sufficient for development but enables the survival of mice once development is complete. An alternative source of CoQ₁₀ headgroup intermediates (or CoQ₁₀ itself) may account for some of the variability in the presentation of HPDL encephalopathy and other primary CoQ₁₀ deficiencies. Surprisingly, lower CoQ₉ and CoQ₁₀ levels restore mitochondrial electron flow and improve the survival of Hpdl^−/− mice, although lower CoQ levels may be responsible for the reduced grip strength of 4-HMA-treated Hpdl^−/− mice. Additional studies will be needed to test whether prenatal treatment, higher doses and improved formulations of 4-HMA and 4-HB increase brain CoQ₉ and CoQ₁₀ pools to wild-type levels. Our data are consistent with a role of HPDL in controlling the amount of CoQ₉ and CoQ₁₀ in the brain.

Oral administration of 4-HB at high doses rescues perinatal lethality in mice with hypomorphic Coq2, which conjugates 4-HB to its isoprenoid tail during CoQ₁₀ synthesis¹⁶. This previous work indicates that CoQ₁₀ headgroup intermediates could treat other primary CoQ₁₀ deficiencies. The authors of this previous work suggested that the treatment of HPDL pathogenic variants with 4-HB would require further investigation. Here we demonstrate that both 4-HMA and 4-HB restore the growth of mice lacking Hpdl and that 4-HB can improve the clinical outcomes of a child with a neurodevelopmental disorder driven by HPDL variants. We anticipate that early treatment with 4-HMA or 4-HB, enabled by newborn screening tests to detect inborn errors in CoQ₁₀ headgroup synthesis or conjugation, will maximize the therapeutic benefits of treatment with CoQ₁₀ headgroup intermediates. Larger trials of CoQ₁₀ headgroup replacement therapy in patients with HPDL variants will be required to determine the outcomes of treatment with CoQ₁₀ headgroup intermediates.

Note added in proof: We note the recent publication of two genotype–phenotype correlation studies characterizing HPDL variants^43,44.

Methods

Detailed methods are included in the Supplementary Methods.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Metabolomics data are available at Zenodo (https://doi.org/10.5281/zenodo.15361205)⁴⁶. Additional patient data that cannot be shared due to privacy reasons are located in controlled access storage at NYU Langone Health. Source data are provided with this paper.

Code availability

Code used to analyse Patch Clamp data is available at GitHub (https://github.com/songs05/IonLab/tree/Ephys-Analysis).

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Acknowledgements

We thank the staff of the DART Microscopy Laboratory, A. Liang, J. Sall and J. Liang, for their consultation and assistance with TEM work; I. Diaz for help and statistical discussions in presenting the single-patient IND data; D. N. Stephen for sectioning early postnatal mouse brains; D. M. Sabatini, D. E. Biancur, G. Francis Jr, J. Rodencal, S. Feske and W. Coetzee for critical reading and comments on the paper; K. Astudillo for the assistance in implementing the IRB protocol; the NYU Metabolomics Core Resource Laboratory (RRID: SCR_017935), Rodent Behavioral Laboratory (RRID: SCR_017942), IonLab (RRID: SCR_021754), Experimental Pathology Laboratory (RRID: SCR_017928) and DART Microscopy Laboratory (RRID: SCR_017934) for their assistance in obtaining the data presented here. These cores are partially supported by the Cancer Center Support Grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center. We are particularly grateful to the patient and his family and to the members of the Division of Neurology I, US Food and Drug Administration and the NYU Institutional Review Board. M.E.P. is a Damon Runyon-Rachleff Innovation awardee supported in part by the Damon Runyon Cancer Research Foundation (DRR 63-20) and is the recipient of a Pershing Square Sohn Prize for Young Investigators in Cancer Research and the MIND Prize from the Pershing Square Foundation, the Tara Miller Melanoma Foundation – MRA Young Investigator Award (668365), a Basic Research Grant from the Harry J. Lloyd Charitable Trust, an American Cancer Society Research Scholar Grant (RSG-21-115-01-MM), an NIGMS R35 MIRA from the NIH (1R35GM147119), an Irma T. Hirschl Career Scientist Award, a Concern Foundation Conquer Cancer Now Grant, a Harrington Scholar–Innovator Award from the Harrington Discovery Institute and an Oxford-Harrington Rare Disease Scholar Award, and research funding from NYU Langone Health Technology Opportunities and Ventures. R.S.B. is a Damon Runyon Dale F. Frey awardee supported by the Damon Runyon Cancer Research Foundation (DRG-50-22) and the NCI (R37CA289040). A.L.J. is supported by grants from the NIH (R37MH085726 and R01NS092096) and by an NCI Cancer Center Support Grant (CCSG, P30 CA08748) and Cycle for Survival. S.E.N. was supported by a François Wallace Monahan Fellowship. R.V.S. was supported by R01NS119301, R01NS127435 and P50HD103555 (Cell and Tissue Pathogenesis Core of the BCM IDDRC). S.K. is supported by a fellowship of the Takeda Science Foundation, a fellowship of the Astellas Foundation for Research on Metabolic Disorders and a fellowship of the Mochida Memorial Foundation for Medical and Pharmaceutical Research. The clinical portion of this research is supported in part by the NYU-H + H Clinical and Translational Science Institute (CTSI), which is supported by the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, through grant award number UL1TR001445. The NYU Metabolomics Core Resource Laboratory (RRID: SCR_017935) is partially supported by Cancer Center Support Grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center. Support for NYU Langone Health’s Ion Laboratory (RRID: SCR_021754) is partially provided through the Office of Scientific Research, NIH R01 AI097302, NIH subaward D009105901, Sophion Industries and Nanion Technologies. Behavioural and continuous metabolic home-cage experiments were performed in the NYULMC Rodent Behavior Laboratory (RRID: SCR_017942) supported by the NIH BRAIN Initiative U19NS1076 (A.C.M.).

Author information

These authors contributed equally: Guangbin Shi, Claire Miller

Authors and Affiliations

Department of Radiation Oncology, NYU Grossman School of Medicine, New York, NY, USA
Guangbin Shi, Sota Kuno, Zixuan Wang, Quentin Spillier, Drew R. Jones & Michael E. Pacold
Laura and Isaac Perlmutter Cancer Center, NYU Langone Health, New York, NY, USA
Guangbin Shi, Sota Kuno, Megan Korn, Wyatt C. Tran, Zixuan Wang, Quentin Spillier, Robert S. Banh & Michael E. Pacold
Department of Neurology, The Marlene and Paolo Fresco Institute for Parkinsons and Movement Disorders, NYU Grossman School of Medicine, New York, NY, USA
Claire Miller & Giulietta M. Riboldi
Department of Pathology and Immunology, Baylor College of Medicine, Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX, USA
Alejandro G. Rey Hipolito, Tao Lin & Roy V. Sillitoe
Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Salsabiel El Nagar & Alexandra L. Joyner
Department of Biochemistry, NYU Grossman School of Medicine, New York, NY, USA
Megan Korn, Wyatt C. Tran, Drew R. Jones & Robert S. Banh
Metabolomics Core Research Laboratory, NYU Grossman School of Medicine, New York, NY, USA
Lia Ficaro & Drew R. Jones
Department of Neuroscience and Physiology Neuroscience Institute, NYU Grossman School of Medicine, New York, NY, USA
Begoña Gamallo-Lana & Adam C. Mar
Department of Pathology, NYU Grossman School of Medicine, New York, NY, USA
Matija Snuderl
IonLab, Department of Pathology, NYU Langone Health, New York, NY, USA
Soomin C. Song

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Guangbin Shi
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Sota Kuno
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Contributions

G.S., R.S.B. and M.E.P. conceived and planned the research. G.S., Q.S., M.K., S.K., Z.W., W.C.T., S.C.S., A.G.R.H., T.L. and S.E.N. designed and carried out experiments. A.G.R.H., T.L., R.V.S., S.E.N., A.L.J. and M.S. interpreted histology. M.K., L.F. and D.R.J. performed metabolomic analysis. B.G.L. and A.C.M. designed, conducted, analysed and interpreted the behavioural assays. S.C.S. carried out and S.C.S., A.G.R.H. and R.V.S. interpreted electrophysiological measurements. C.M. and G.M.R. wrote the single-patient IND and IRB protocol for treatment of the patient. C.M. and G.M.R. wrote the clinical-trial protocol, treated the patient, collected and analysed all single-patient data without input from the other authors. C.M. treated the patient and collected all single-patient data without input from the other authors. M.E.P. wrote and A.G.R.H., A.L.J., R.V.S., R.S.B. and M.E.P. edited the paper, except for the section on the treatment of the single patient, which C.M. and G.M.R. wrote and edited independently. All authors reviewed and approved the paper.

Corresponding author

Correspondence to Michael E. Pacold.

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Competing interests

G.S., Q.S., R.S.B. and M.E.P. are co-inventors on patents related to the use of 4-HMA, 4-HB and analogues in the diagnosis and treatment of neurodevelopmental and other diseases assigned to New York University. M.E.P. directly supervised the preclinical research. The treatment was conducted and supervised by C.M. and G.M.R. under an institutional conflict-of-interest management plan designed and implemented by NYU Langone Health in accordance with its policies. M.E.P. consulted on the clinical protocol and C.M. and G.M.R. directed the course of treatment in accordance with the institutional plan. Financial support for the clinical treatment was provided by the Pershing Square Foundation. The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Related to Fig. 1: mammalian CoQ headgroup intermediates improve the survival of Hpdl^−/− mice and are incorporated into mouse brain CoQ₉ and CoQ₁₀ in vivo.

a, Hpdl^−/− pups (n = 5) exhibit significant weight loss by post-natal day 10 (P10) relative to Hpdl^+/+ (n = 8) and Hpdl^+/− (n = 6) pups. Treatment of Hpdl^−/− pups with 4-HMA (n = 4), the product of HPDL, and 4-HB (n = 5), the immediate precursor of the CoQ headgroup, results in weight gain similar to Hpdl^+/− pups after P9. Error bars represent median ± interquartile range. b, Seizure frequencies in pups. All Hpdl^−/− pups (n = 6) have observed seizures. No Hpdl^+/+ (n = 6) or Hpdl^+/− (n = 8) pups had observed seizures. Treatment with 4-HMA reduced the number of Hpdl^−/− pups with seizures to 6 out of a total of 93 treated Hpdl^−/− pups. c, Lactate/pyruvate ratio in plasma from Hpdl^+/+ (n = 8), Hpdl^+/− (n = 8), and Hpdl^−/− (n = 5) pups. Hpdl^−/− pups have a higher lactate/pyruvate ratio than Hpdl^+/+ and Hpdl^+/− pups. d, Pharmacokinetic data for 4-HMA and 4-HB at 500 mg kg⁻¹ orally and 1 mg kg⁻¹ intravenously. Error bars represent mean ± standard deviation for data from 3 mice. e, Long-term toxicity histology for 4-HMA and 4-HB administered orally for 3 months at 500 mg kg⁻¹ per day. No inflammation or necrosis were observed in the brain, heart, kidneys, liver, lungs, or spleen from untreated, 4-HMA, or 4-HB-treated mice. Sections are representative of n = 4 mice in each group. f, Plasma concentrations of 4-HMA in P10 Hpdl^−/− pups treated with 10 mg kg⁻¹ per day, orally administered 4-HMA starting at P3. Plasma 4-HMA concentrations are higher in treated Hpdl^−/− pups than in Hpdl^+/+, Hpdl^+/−, and untreated Hpdl^−/− pups. Hpdl^+/+, Hpdl^+/−, and Hpdl^−/− pup data were obtained for Fig. 1c and are presented in a faded shade for ease of interpretation. 4-HMA-treated Hpdl^−/− pup n = 7. g, Hpdl^−/− pups can stand 3 days after supplementation with 4-HMA (10 mg/kg/day, oral administration). h, Survival of Hpdl^−/− pups treated on P7 or P10. Hpdl^−/− pups treated by postnatal day 7 (n = 32) survive into adulthood. Treatment of Hpdl^−/− pups at 10 days after birth (n = 4) does not improve their survival. Significance was tested by two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test.

Source data

Extended Data Fig. 2 Related to Fig. 1: CoQ concentrations in the brains of pups and P90 mice following 4-HMA and 4-HB treatment.

a, CoQ₉ and CoQ₁₀ concentrations from mouse brains following treatment with ¹³C₆-4-HMA or ¹³C₆-4-HB starting at postnatal day 3. ¹³C₆-4-HMA and ¹³C₆-4-HB are incorporated into CoQ₉ and CoQ₁₀ in Hpdl^−/− mice, and increase brain CoQ₉ and CoQ₁₀ pools relative to untreated Hpdl^−/− mice. ¹³C₆-4-HMA and ¹³C₆-4-HB are minimally incorporated into CoQ₉ and CoQ₁₀ in Hpdl^+/+ mice and are incorporated into CoQ₉ and CoQ₁₀ at low fractions in Hpdl^+/− mice. ¹³C₆-4-HMA: Hpdl^+/+ n = 3, Hpdl^+/− n = 6, and Hpdl^−/− treated n = 5. ¹³C₆-4-HB: Hpdl^+/+ n = 3, Hpdl^+/− n = 4, and Hpdl^−/− treated n = 3. Hpdl^−/− untreated n = 3. CoQ₉ and CoQ₁₀ data from Hpdl^−/− untreated mice were obtained in the 4-HMA experiment and are presented in a faded shade of color in the 4-HB experiment for ease of interpretation. b, CoQ₉ and CoQ₁₀ concentrations from the cortex and cerebellum of mice treated with ¹³C₆-4-HMA from P2-P30 and sacrificed at P90. The concentrations of CoQ₉ and CoQ₁₀ in the brains of treated Hpdl^−/− mice are ~50% of the concentrations of these molecules in the brains of Hpdl^+/+ mice. Only a minor fraction of CoQ₉ and CoQ₁₀ is labeled with ¹³C. Hpdl^+/+ n = 3, Hpdl^+/− n = 3, and Hpdl^−/− treated n = 3. c, CoQ₉ and CoQ₁₀ concentrations from the brains of P90 wild-type mice that were not treated with 4-HMA, or treated with 4-HMA from P2-P30. 4-HMA does not increase the brain concentrations of CoQ₉ and CoQ₁₀ in wild-type mice. Significance was tested by two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test.

Source data

Extended Data Fig. 3 Related to Fig. 2: 4-HMA supplementation improves the histologic appearances of the brains of Hpdl^−/− mice.

a, Hematoxylin and eosin staining of cerebellar samples from P6 Hpdl^+/+, Hpdl^−/−, and Hpdl^−/− mice treated with 4-HMA (Hpdl^−/− + 4-HMA). Three Hpdl^+/+ (A, D and G), Hpdl^−/− (B, E and H) and Hpdl^−/− + 4-HMA (C, F and I) samples were analyzed to determine the reproducibility of the phenotype. Hpdl^−/− mouse cerebella have pronounced atrophy that is partially rescued after 4-HMA treatment. b, Immunostaining for PAX6 (green) and Calbindin (red) on mid-sagittal sections of P6 cerebella from Hpdl^+/+, Hpdl^−/−, and Hpdl^−/− mice treated with 4-HMA from P2-3 (Hpdl^−/− + 4-HMA) (Panels A-C). Nuclei were counterstained with Hoechst (blue). Panels D-I are higher-magnification views of the boxed area in each image above with single-channel PAX6 (green) and Calbindin (red) staining. PAX6 stanning of granule cells shows that the EGL and IGL are depleted in the Hpdl^−/− cerebella (Panel F compared to D, H). Calbindin staining of Purkinje cells shows the cells form a multilayer in Hpdl^−/− cerebella (panel G). Purkinje cells form a single layer following 4-HMA treatment, but dendrites are stunted (panel I). Panels J-L contain lobule 3 from mid-sagittal cerebellar sections immunostained for Hoechst (blue) and TUNEL (yellow), showing an increase in cell death in Hpdl^−/− cerebella (panel K) compared to control cerebella (panel J). 4-HMA treatment decreases cell death (Panel L). The white dashed line delineates the external granular layer of lobule 3. c, Quantitation of cerebellar area in midline sections from P6 pups in Extended Data Fig. 3b. Hpdl^−/− pups have smaller cerebella than Hpdl^+/+ pups. Cerebellar size in Hpdl^−/− pups is partially rescued by 4-HMA treatment. n = 4 for all groups. d, Quantitation of TUNEL staining, a measure of apoptosis, in the cerebella of P6 pups, from Extended Data Fig. 3b. Hpdl^−/− pups have higher TUNEL staining in their cerebella than Hpdl^+/+ pups. TUNEL staining in Hpdl^−/− pups is partially decreased by 4-HMA treatment, consistent with decreased levels of apoptosis when CoQ headgroup synthesis is restored. n = 4 for all groups. e, P11 Hpdl^−/− pups show reduced Purkinje cell dendrite height (panels G-J) and EGL thickness (panels T-V) as revealed by calbindin antibody and Ki-67 antibody staining, respectively. P11 Hpdl^−/− pups also demonstrate reduced cerebellar size (panel G) and ectopic Purkinje cells (panel H), indicative of abnormal Purkinje cell development. These abnormalities appear restored by 4-HMA treatment from P2-3 (panels K-M, W-Y). f, Quantitation of dendrite height shown in the calbindin stains from Extended Data Fig. 2e, which confirms that P11 Hpdl^−/− pups have significantly reduced dendrite height that was rescued in the 4-HMA treated group. n = 3 animals per group. g, Quantitation of External Granular Layer (EGL) thickness shown in the Ki-67 stains from Extended Data Fig. 2e, which confirms that P11 Hpdl^−/− pups have significantly reduced EGL thickness that was rescued in the 4-HMA treated group. n = 3 animals per group. h, Hematoxylin and eosin staining of cerebellar samples from P56-57 Hpdl^+/+, Hpdl^+/−, and Hpdl^−/− mice treated with 4-HMA. The cerebellar histology is grossly normal in all three groups of animals. For panels c, d, f, and g, significance was tested by two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test.

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Extended Data Fig. 4 Related to Fig. 2: additional histological characterization of untreated and 4-HMA-treated brains from Hpdl^−/− mice.

a, Representative calbindin staining of cerebellar sections from P56-57 Hpdl^+/+, Hpdl^+/−, and 4-HMA-treated Hpdl^−/− mice and quantitation of dendrite height. The dendrite height is similar in all three groups of animals. b, Representative electron micrographs of the cortex in P11 Hpdl^+/+, Hpdl^+/−, and Hpdl^−/− pups, as well as Hpdl^−/− pups treated with 4-HMA. We observed minimal changes in cortical mitochondrial ultrastructure in Hpdl^−/− pups (panels C and G) compared to Hpdl^+/+ pups (panels A and E) and Hpdl^+/− pups (panels B and F). c, P11 Hpdl^−/− pups exhibit subtle loss of normal cortical layers of the cerebral cortex (panels C, G, K), as well as edema, shrinking of the cortical neurons, and red neurons. These histological findings are partially restored by supplementation with 4-HMA (panels D, H, L). d, P11 Hpdl^−/− pups do not exhibit major changes in hippocampal histology in comparison to Hpdl^+/+ pups, Hpdl^+/− pups, and Hpdl^−/− pups treated with 4-HMA. n = 4 for all groups. For panel a, significance was tested by two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test.

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Extended Data Fig. 5 Related to Fig. 2: TUNEL staining in the brains of untreated and 4-HMA-treated Hpdl^−/− mice.

a, Staining for Hoechst (blue) and TUNEL (yellow) showing the forebrain of Hpdl^+/+, Hpdl^−/− and Hpdl^−/− + 4-HMA mice at P6 (panels A-C). The white dashed lines delineate the approximate areas of the cortex, striatum, thalamus and hippocampus that were quantified. Magnified view of the red-boxed areas are on the right (panels D-O). n = 3 slides per mouse and 3 mice/genotype. The graphs represent the density of TUNEL+ particles per mm squared in the cortex (panel P), striatum (panel Q), thalamus (panel R) and hippocampus (panel S) of the Hpdl^+/+, Hpdl^−/− and Hpdl^−/− + 4-HMA mice at P6. For panels P-S, significance was tested by two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test.

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Extended Data Fig. 6 Related to Fig. 3: electron flow measurements in cerebellum and cortex from Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− mice.

a, Electron transport chain substrates and inhibitors used for the electron flow assay. I, Complex I. II, Complex II. III, Complex III. IV, Complex IV. Q, CoQ. b, Electron flow measurements of mitochondria isolated from the cortex of P9 Hpdl^+/+, Hpdl^+/−, and Hpdl^−/− pups, as well as Hpdl^−/− pups treated with 4-HMA. Electron flow through complex I and complex II is reduced in Hpdl^−/− pups, and is improved by treatment with 4-HMA. c, Electron flow measurements of mitochondria isolated from the cortex and cerebellum of P90 (3-month-old) Hpdl^+/+ and Hpdl^+/− pups, as well as Hpdl^−/− pups treated with 4-HMA from P3-30. Electron flow through complexes I, II, and IV are similar in all groups. d, Electron flow measurements of mitochondria isolated from P12 Hpdl^+/+ mice treated with 4-HMA starting on P3. 4-HMA treatment does not increase electron flow through complex I, II, or IV in wild-type pups. Mitochondria were isolated from n = 5 animals for all groups. Significance was tested by two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test.

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Extended Data Fig. 7 Related to Fig. 3: electrophysiologic characterization of Purkinje cells from Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− mice.

a, The I/V curves of Purkinje cells from P8-10 Hpdl^−/− pups and 4-HMA-treated Hpdl^−/− pups are significantly different from each other and from the I/V curves of Hpdl^+/+ and Hpdl^+/− pups, which are similar. P-values indicating statistical significance are listed. The Hpdl^−/− pups have a flatter I/V curve at hyperpolarized membrane potentials relative to the other curves due to a tighter grouping of currents at hyperpolarized membrane potentials (-100 to -60 mV) than the Hpdl^+/+ and Hpdl^+/− pups (see insert). The 4-HMA-treated Hpdl^−/− pups have a steeper I/V curve similar to more mature Purkinje cells. b, The I/V curves of Purkinje cells from P17-19 Hpdl^+/+, Hpdl^+/−, and 4-HMA-treated Hpdl^−/− pups are similar. The inset depicts an example Hpdl^+/+ Purkinje cell current response to the voltage step commands, which is similar to the Hpdl^+/− and 4-HMA-treated Hpdl^−/− pup Purkinje cells responses. c, The I/V curves of Purkinje cells from P56-57 Hpdl^+/+, Hpdl^+/−, and 4-HMA-supplemented Hpdl^−/− mice are comparable. d, Peak frequency of Purkinje cells from P8-10 Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− pups. The 4-HMA treated Hpdl^−/− group has higher peak frequency than any of the other groups. Differences in frequency were identified by two-way ANOVA followed by Dunnett’s multiple comparison test. e, Peak frequency of Purkinje cells from P17-19 Hpdl^+/+, Hpdl^+/−, and 4-HMA-treated Hpdl^−/− pups. The peak frequencies in all three groups are comparable. f, F/I curves of Purkinje cells from P8-10 Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− pups. There were no significant differences in the linear portions of each curve between each group. The insert shows the simple firing pattern of an immature Purkinje cell. g, F/I curves of Purkinje cells from P17-19 Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− pups. There were no significant differences in the linear portions of each curve between each group. The insert shows the simple firing pattern of a more mature Purkinje cell. h, Input resistance and membrane capacitance in Purkinje cells from P56-57 (8-week-old; age comparable to behavioral assays) Hpdl^+/+ and 4-HMA-supplemented Hpdl^−/− mice are comparable, with a nonsignificant decrease in input resistance and a nonsignificant increase in membrane capacitance in Hpdl^+/− mice. i, The F/I curve of Purkinje cells from P56-57 Hpdl^+/+ mice exhibits a higher firing rate at higher input currents than Purkinje cells from Hpdl^+/− and 4-HMA-treated Hpdl^−/− mice. All electrophysiological data are presented as the averages of at least 10 Purkinje cells (Supplementary Table 4). Significance was tested by two-way ANOVA followed by Tukey’s post-hoc test.

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Extended Data Fig. 8 Related to Fig. 3: action potential characteristics of Purkinje cells from Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− mice.

a, Action potential characteristics of Purkinje cells from P8-10 Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− pups. An average action potential is shown in panel A and the phase plane diagram is shown in panel B. Significant action potential shape differences can be seen between Purkinje cells from Hpdl^+/+ and 4-HMA-treated Hpdl^−/− pups. Purkinje cells from 4-HMA-treated Hpdl^−/− pups had a significantly lower spike threshold than the Hpdl^+/+ (p = 0.0076) and Hpdl^−/− pups (p = 0.0207; panel C). The spike threshold for Purkinje cells from the Hpdl^+/− and 4-HMA-treated Hpdl^−/− pups trended to difference but was not significant (p = 0.0506), consistent with 4-HMA treatment rescuing Hpdl^−/− pup grip strength to the level of Hpdl^+/− mice (Fig. 3g). The spike half-width of Purkinje cells from the 4-HMA-treated Hpdl^−/− pups was significantly narrower than all three treatments (Hpdl^+/+: p < 0.0001; Hpdl^+/−: p = 0.0002; Hpdl^−/−: p < 0.0001; panel E). Finally, Purkinje cells from 4-HMA-treated Hpdl^−/− pups had a shallower action half-potential (AHP) amplitude compared to Hpdl^+/+and Hpdl^−/− pups (WT: p = 0.0008; KO: p = 0.0461; panel F). The 4-HMA-treated Hpdl^−/− pup statistics resemble those of Hpdl^+/+ pups in the P17-19 group (Extended Data Fig. 8b). b, Action potential characteristics of P17-19 Hpdl^+/+, Hpdl^+/−, and 4-HMA-treated Hpdl^−/− pups. An average action potential is shown in panel A and the phase plane diagram is shown in panel B. The spike threshold, spike amplitude, and spike half-width are comparable in all three groups. The AHP for Purkinje cells from 4-HMA-treated Hpdl^−/− pups was lower than for Hpdl^+/+pups (p = 0.0268). c, Peak frequency and action potential characteristics of Purkinje cells from P56-57 Hpdl^+/+, Hpdl^+/−, and 4-HMA-treated Hpdl^−/− mice. The peak frequency, spike amplitude, spike half-width, spike threshold, and AHP amplitude are comparable in all three groups. All electrophysiological data are presented as the averages of at least 10 Purkinje cells (Supplementary Table 4). Significance was tested by two-way ANOVA followed by Dunnett’s multiple comparison test.

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Extended Data Fig. 9 Related to Fig. 3: additional metabolic, behavioral, and neurological tests of Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− mice.

a, Metabolic phenotypes such as food and water intake, activity, O₂ consumption, CO₂ production, and energy expenditure are similar in 4-6-week-old Hpdl^+/+, Hpdl^+/−, and 4-HMA-treated Hpdl^−/− mice, as measured in metabolic cages. There were no significant changes in any of these parameters between Hpdl^+/+, Hpdl^+/−, and 4-HMA-treated Hpdl^−/− mice. b, Respiratory exchange ratio (vO₂/vCO₂) in Hpdl^+/+, Hpdl^+/−, and 4-HMA-treated Hpdl^−/− mice. c, 4-HMA treatment partially restores the weight of male Hpdl^−/− mice and rescues the weight of Hpdl^−/− female mice. Hpdl^+/− mice have comparable weights to Hpdl^+/+ mice. The combined data demonstrate that 4-HMA partially restores the weight of Hpdl^−/− mice relative to Hpdl^+/− mice. d, Respiratory exchange ratio summary in Hpdl^+/+, Hpdl^+/−, and 4-HMA-treated Hpdl^−/− mice. Male Hpdl^−/− mice treated with 4-HMA had a significantly increased respiratory exchange ratio (RER) compared to both Hpdl^+/+ male mice and Hpdl^+/− and 4-HMA-treated Hpdl^−/− female mice. e, Rotarod performance is similar in Hpdl^+/+, Hpdl^+/−, and 4-HMA-treated Hpdl^−/− male and female mice. f, Open field data from 6–8-week-old Hpdl^+/+, Hpdl^+/−, and 4-HMA-treated Hpdl^−/− mice revealed no significant differences in distance moved or time that mice spent in the center of the open field. For behavioral tests, data from male mice (n: Hpdl^+/+= 4, Hpdl^+/−= 5, 4-HMA-treated Hpdl^−/−=4) are plotted as triangles. Data from female mice (n: Hpdl^+/+= 4, Hpdl^+/−=2, 4-HMA-treated Hpdl^−/−=5) are plotted as circles. Behavioral data do not contain comparisons to untreated Hpdl^−/− mice, which do not survive to the age at which these behavioral assays are carried out. Data presented are mean ± standard error of the mean, as well as raw data. Behavioral data are presented as box and whisker plots, where the center is the median, the box bounds are the 25^th to 75^th percentiles, and whiskers indicate minimum to maximum. Significance was tested by two-way ANOVA followed by Tukey’s post-hoc test.

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Extended Data Fig. 10 Related to Figs. 3 and 4: gait characterization of Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− mice, and brain MRI from a patient with biallelic HPDL variants receiving the CoQ₁₀ headgroup intermediate 4-HB.

a, Additional catwalk data from 6–8-week-old Hpdl^+/+, Hpdl^+/−, and 4-HMA-treated Hpdl^−/− mice reveal no significant differences in gait parameters between the groups of mice. A, stride length. B, swing phase duration. C, stance phase duration. D, cadence. E, base of support. F, maximum contact intensity. G, symmetry index for stride length. b, T1- and T2-weighted brain MRI images from a patient with biallelic HPDL variants before treatment with 4-HB (day 0; panels A and B) and 30 days after treatment with 4-HB (panels C and D). For behavioral tests, data from male mice (n: Hpdl^+/+= 4, Hpdl^+/−= 5, 4-HMA-treated Hpdl^−/−=4) are plotted as triangles. Data from female mice (n: Hpdl^+/+= 4, Hpdl^+/−=2, 4-HMA-treated Hpdl^−/−=5) are plotted as circles. Behavioral data do not contain comparisons to untreated Hpdl^−/− mice, which do not survive to the age at which these behavioral assays are carried out. Data presented are mean ± standard error of the mean, as well as raw data. Behavioral data are presented as box and whisker plots, where the center is the median, the box bounds are the 25^th to 75^th percentiles, and whiskers indicate minimum to maximum. Significance was tested by two-way ANOVA followed by Tukey’s post-hoc test.

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Supplementary Tables 1–4

Supplementary Table 1. Maximum tolerated dose and pharmacokinetics of 4-HMA and 4-HB. Maximum tolerated dose of 4-HMA and 4-HB and half-life of 4-HMA and half-life of 4-HB dosed at treatment dose (10 mg kg⁻¹ orally), 500 mg kg⁻¹ orally and 1 mg kg⁻¹ intravenously. Data are mean ± s.d. Supplementary Table 2. Dose response of 4-HMA treatment in improving the survival of Hpdl^−/− pups. 4-HMA doses of 10 mg kg⁻¹ per day or 20 mg kg⁻¹ per day improves the 30-day survival of 90% of pups at 30 days after birth. Doses of 0.1 mg kg⁻¹ per day or 1 mg kg⁻¹ per day did not improve the overall survival of Hpdl^−/− pups. Supplementary Table 3. Genotyping primers for Hpdl^−/− mice. Supplementary Table 4. Number of Purkinje cells that were used for patch-clamp for electrophysiological studies.

Supplementary Video 1

Phenotypes of Hpdl^−/− pups. P12 Hpdl^−/− pups (left) have spasticity, posturing and seizures, whereas Hpdl^+/+ and Hpdl^+/− littermates are healthy in appearance.

Supplementary Video 2

Effects of 4-HMA treatment on an Hpdl^−/− pup. Two Hpdl^−/− pups are on the left and two Hpdl^+/− littermates are on the right. The Hpdl^−/− pup second from the left was treated with 4-HMA at 10 mg per kg per day orally and showed improvement in growth and motor function. The untreated Hpdl^−/− pup died by P15.

Supplementary Video 3

Gait of an Hpdl^+/+ mouse on the catwalk system. Example video of an 8-week-old Hpdl^+/+ mouse during gait evaluation.

Supplementary Video 4

Gait of an Hpdl^+/− mouse on the catwalk system. Example video of an 8-week-old Hpdl^+/− mouse during gait evaluation.

Supplementary Video 5

Gait of an Hpdl^−/− mouse treated with 4-HMA on the catwalk system. Example video of an 8-week-old Hpdl^−/− mouse previously treated with 4-HMA during gait evaluation.

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Shi, G., Miller, C., Kuno, S. et al. Coenzyme Q headgroup intermediates can ameliorate a mitochondrial encephalopathy. Nature 645, 466–474 (2025). https://doi.org/10.1038/s41586-025-09246-x

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Received: 30 April 2024
Accepted: 05 June 2025
Published: 09 July 2025
Issue date: 11 September 2025
DOI: https://doi.org/10.1038/s41586-025-09246-x

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4-HMA and 4-HB can treat Hpdl −/− pups

4-HMA and 4-HB restore CoQ synthesis

4-HMA improves histology of Hpdl −/− pup brains

4-HMA improves Hpdl −/− Purkinje cell function

4-HMA treats Hpdl −/− motor symptoms in pups

4-HB can treat a patient with HPDL encephalopathy

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Extended Data Fig. 1 Related to Fig. 1: mammalian CoQ headgroup intermediates improve the survival of Hpdl−/− mice and are incorporated into mouse brain CoQ9 and CoQ10 in vivo.

Extended Data Fig. 2 Related to Fig. 1: CoQ concentrations in the brains of pups and P90 mice following 4-HMA and 4-HB treatment.

Extended Data Fig. 3 Related to Fig. 2: 4-HMA supplementation improves the histologic appearances of the brains of Hpdl−/− mice.

Extended Data Fig. 4 Related to Fig. 2: additional histological characterization of untreated and 4-HMA-treated brains from Hpdl−/− mice.

Extended Data Fig. 5 Related to Fig. 2: TUNEL staining in the brains of untreated and 4-HMA-treated Hpdl−/− mice.

Extended Data Fig. 6 Related to Fig. 3: electron flow measurements in cerebellum and cortex from Hpdl+/+, Hpdl+/−, Hpdl−/− and 4-HMA-treated Hpdl−/− mice.

Extended Data Fig. 7 Related to Fig. 3: electrophysiologic characterization of Purkinje cells from Hpdl+/+, Hpdl+/−, Hpdl−/− and 4-HMA-treated Hpdl−/− mice.

Extended Data Fig. 8 Related to Fig. 3: action potential characteristics of Purkinje cells from Hpdl+/+, Hpdl+/−, Hpdl−/− and 4-HMA-treated Hpdl−/− mice.

Extended Data Fig. 9 Related to Fig. 3: additional metabolic, behavioral, and neurological tests of Hpdl+/+, Hpdl+/−, Hpdl−/− and 4-HMA-treated Hpdl−/− mice.

Extended Data Fig. 10 Related to Figs. 3 and 4: gait characterization of Hpdl+/+, Hpdl+/−, Hpdl−/− and 4-HMA-treated Hpdl−/− mice, and brain MRI from a patient with biallelic HPDL variants receiving the CoQ10 headgroup intermediate 4-HB.

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4-HMA and 4-HB can treat Hpdl ^−/− pups

4-HMA improves histology of Hpdl ^−/− pup brains

4-HMA improves Hpdl ^−/− Purkinje cell function

4-HMA treats Hpdl ^−/− motor symptoms in pups

Extended Data Fig. 1 Related to Fig. 1: mammalian CoQ headgroup intermediates improve the survival of Hpdl^−/− mice and are incorporated into mouse brain CoQ₉ and CoQ₁₀ in vivo.

Extended Data Fig. 3 Related to Fig. 2: 4-HMA supplementation improves the histologic appearances of the brains of Hpdl^−/− mice.

Extended Data Fig. 4 Related to Fig. 2: additional histological characterization of untreated and 4-HMA-treated brains from Hpdl^−/− mice.

Extended Data Fig. 5 Related to Fig. 2: TUNEL staining in the brains of untreated and 4-HMA-treated Hpdl^−/− mice.

Extended Data Fig. 6 Related to Fig. 3: electron flow measurements in cerebellum and cortex from Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− mice.

Extended Data Fig. 7 Related to Fig. 3: electrophysiologic characterization of Purkinje cells from Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− mice.

Extended Data Fig. 8 Related to Fig. 3: action potential characteristics of Purkinje cells from Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− mice.

Extended Data Fig. 9 Related to Fig. 3: additional metabolic, behavioral, and neurological tests of Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− mice.

Extended Data Fig. 10 Related to Figs. 3 and 4: gait characterization of Hpdl^+/+, Hpdl^+/−, Hpdl^−/− and 4-HMA-treated Hpdl^−/− mice, and brain MRI from a patient with biallelic HPDL variants receiving the CoQ₁₀ headgroup intermediate 4-HB.