Leigh Syndrome Research Articles

Generation of a human induced pluripotent stem cell line NTUHi004-A from a patient with Leigh syndrome harboring a homozygous missense mutation c.836 T > G (p.Met279Arg) in NDUFAF5 gene

Leigh syndrome is a rare autosomal recessive disorder showcasing a diverse range of neurological symptoms. Classical Leigh syndrome is associated with mitochondrial complex I deficiency, primarily resulting from biallelic mutations in the NDUFAF5 gene, encoding the NADH:ubiquinone oxidoreductase complex assembly factor 5. Using the Sendai virus delivery system, we generated an induced pluripotent stem cell line from peripheral blood mononuclear cells of a 47-years-old female patient who carried a homozygous NDUFAF5 c.836 T > G (p.Met279Arg) mutation. This cellular model serves as a tool for investigating the underlying pathogenic mechanisms and for the development of potential treatments for Leigh syndrome.

Identification of a novel MT-ND3 variant and restoring mitochondrial function by allotopic expression of MT-ND3 gene

Mitochondrial diseases are caused by nuclear, or mitochondrial DNA (mtDNA) variants and related co-factors. Here, we report a novel m.10197G > C variant in MT-ND3 in a patient, and two other patients with m.10191 T > C. MT-ND3 variants are known to cause Leigh syndrome or mitochondrial complex I deficiency. We performed the functional analyses of the novel m.10197G > C variant that significantly lowered MT-ND3 protein levels, causing complex I assembly and activity deficiency, and reduction of ATP synthesis. We adapted a previously described re-engineering technique of delivering mitochondrial genes into mitochondria through codon optimization for nuclear expression and translation by cytoplasmic ribosomes to rescue defects arising from the MT-ND3 variants. We constructed mitochondrial targeting sequences along with the codon-optimized MT-ND3 and imported them into the mitochondria. To achieve the goal, we imported codon-optimized MT-ND3 into mitochondria in three patients with m.10197G > C and m.10191 T > C missense variants in the MT-ND3. Nuclear expression of the MT-ND3 gene partially restored protein levels, complex I deficiency, and significant improvement of ATP production indicating a functional rescue of the mutant phenotype. The codon-optimized nuclear expression of mitochondrial protein and import inside the mitochondria can supplement the requirements for ATP in energy-deficient mitochondrial disease patients.

Lack of mitochondrial complex I assembly factor NDUFAF2 results in a distinctive infantile-onset brainstem neurodegenerative disease with early lethality

BackgroundCongenital disorders of the mitochondrial respiratory chain are a heterogeneous group of inborn errors of metabolism. Among them, NADH:ubiquinone oxidoreductase (complex I, CI) deficiency is the most common. Biallelic pathogenic variants in NDUFAF2, encoding the nuclear assembly CI factor NDUFAF2, were initially reported to cause progressive encephalopathy beginning in infancy. Since the initial report in 2005, less than a dozen patients with NDUFAF2-related disease have been reported.MethodsClinical, biochemical, and neuroradiological features of four new patients residing in Northern Israel were collected during 2016–2022 at Emek Medical Center. Enzymatic activities of the five respiratory-chain complexes were determined in isolated fibroblast mitochondria by spectrophotometric methods. Western blot analyses were conducted with anti-human NDUFAF2 antibody; antibody against the mitochondrial marker VDAC1 was used as a loading control. Genetic studies were performed by chromosome microarray analysis using Affymetrix CytoScan 750 K arrays.ResultsAll four patients presented with infantile-onset growth retardation, ophthalmological impairments with nystagmus, strabismus (starting between 5 and 9 months), and further progressed to life-threatening episodes of apnea usually triggered by trivial febrile illnesses (between 10 and 18 months) with gradual loss of acquired developmental milestones (3 of 4 patients). Serial magnetic-resonance imaging studies in two of the four patients showed a progressive pattern of abnormal T2-weighted hyperintense signals involving primarily the brainstem, the upper cervical cord, and later, the basal ganglia and thalami. Magnetic-resonance spectroscopy in one patient showed an increased lactate peak. Disease progression was marked by ventilatory dependency and early lethality. 3 of the 4 patients tested, harbored a homozygous 142-kb partial interstitial deletion that omits exons 2–4 of NDUFAF2. Mitochondrial CI activity was significantly decreased in the only patient tested. Western blot analysis disclosed the absence of NDUFAF2 protein compared to normal controls. In addition, we reviewed all 10 previously reported NDUFAF2-deficient cases to better characterize the disease.ConclusionsBiallelic loss-of-function mutations in NDUFAF2 result in a distinctive phenotype in the spectrum of Leigh syndrome with clinical and neuroradiological features that are primarily attributed to progressive brainstem damage.

Molecular and in silico investigation of a novel ECHS1 gene mutation in a consanguine family with short-chain enoyl-CoA hydratase deficiency and Mt-DNA depletion: effect on trimer assembly and catalytic activity.

Short-chain enoyl-CoA hydratase deficiency (ECHS1D) is a rare congenital metabolic disorder that follows an autosomal recessive inheritance pattern. It is caused by mutations in the ECHS1 gene, which encodes a mitochondrial enzyme involved in the second step of mitochondrial β-oxidation of fatty acids. The main characteristics of the disease are severe developmental delay, regression, seizures, neurodegeneration, high blood lactate, and a brain MRI pattern consistent with Leigh syndrome. Here, we report three patients belonging to a consanguineous family who presented with mitochondrial encephalomyopathy. Whole-exome sequencing revealed a new homozygous mutation c.619G > A (p.Gly207Ser) at the last nucleotide position in exon 5 of the ECHS1 gene. Experimental analysis showed that normal ECHS1 pre-mRNA splicing occurred in all patients compared to controls.Furthermore, three-dimensional models of wild-type and mutant echs1 proteins revealed changes in catalytic site interactions, conformational changes, and intramolecular interactions, potentially disrupting echs1 protein trimerization and affecting its function. Additionally, the quantification of mtDNA copy number variation in blood leukocytes showed severe mtDNA depletion in all probands.

Impact of dietary ketosis on volatile anesthesia toxicity in a model of Leigh syndrome.

Genetic mitochondrial diseases impact over 1 in 4000 individuals, most often presenting in infancy or early childhood. Seizures are major clinical sequelae in some mitochondrial diseases including Leigh syndrome, the most common pediatric presentation of mitochondrial disease. Dietary ketosis has been used to manage seizures in mitochondrial disease patients. Mitochondrial disease patients often require surgical interventions, leading to anesthetic exposures. Anesthetics have been shown to be toxic in the setting of mitochondrial disease, but the impact of a ketogenic diet on anesthetic toxicities in this setting has not been studied. Our aim in this study was to determine whether dietary ketosis impacts volatile anesthetic toxicities in the setting of genetic mitochondrial disease. The impact of dietary ketosis on toxicities of volatile anesthetic exposure in mitochondrial disease was studied by exposing young Ndufs4(-/-) mice fed ketogenic or control diet to isoflurane anesthesia. Blood metabolites were measured before and at the end of exposures, and survival and weight were monitored. Compared to a regular diet, the ketogenic diet exacerbated hyperlactatemia resulting from isoflurane exposure (control vs. ketogenic diet in anesthesia mean difference 1.96 mM, Tukey's multiple comparison adjusted p = .0271) and was associated with a significant increase in mortality during and immediately after exposures (27% vs. 87.5% mortality in the control and ketogenic diet groups, respectively, during the exposure period, Fisher's exact test p = .0121). Our data indicate that dietary ketosis and volatile anesthesia interact negatively in the setting of mitochondrial disease. Our findings suggest that extra caution should be taken in the anesthetic management of mitochondrial disease patients in dietary ketosis.

NDUFS7 variant in dogs with Leigh syndrome and its functional validation in a Drosophila melanogaster model

Two Jack-Russell Terrier × Chihuahua mixed-breed littermates with Leigh syndrome were investigated. The dogs presented with progressive ataxia, dystonia, and increased lactate levels. Brain MRI showed characteristic bilateral symmetrical T2 hyperintense lesions, histologically representing encephalomalacia. Muscle histopathology revealed accumulation of mitochondria. Whole genome sequencing identified a missense variant in a gene associated with human Leigh syndrome, NDUFS7:c.535G > A or p.(Val179Met). The genotypes at the variant co-segregated with the phenotype in the investigated litter as expected for a monogenic autosomal recessive mode of inheritance. We investigated the functional consequences of the missense variant in a Drosophila melanogaster model by expressing recombinant wildtype or mutant canine NDUFS7 in a ubiquitous knockdown model of the fly ortholog ND-20. Neither of the investigated overexpression lines completely rescued the lethality upon knockdown of the endogenous ND-20. However, a partial rescue was found upon overexpression of wildtype NDUFS7, where pupal lethality was moved to later developmental stages, which was not seen upon canine mutant overexpression, thus providing additional evidence for the pathogenicity of the identified variant. Our results show the potential of the fruit fly as a model for canine disease allele validation and establish NDUFS7:p.(Val179Met) as causative variant for the investigated canine Leigh syndrome.

ETHICALLY MANAGING LEIGH’S SYNDROME: A CLINICAL CASE STUDY

Leigh syndrome, a rare inherited neurometabolic disorder described as subacute necrotizing encephalomyelopathy, affects the central nervous system and falls within a group of mitochondrial cytopathies involving disordered oxidative phosphorylation in the affected neural cells leading to disrupted enzyme of mitochondrial aberrations are attributed to variously inherited nDNA or mtDNA mutations The present study strives to demonstrate the efficacy, and therefore the need and ethical justification of therapeutic intervention at-least in the speech-language and swallowing realm; despite poor prognosis and the high risk of early death, in such infants through a representative case study. The case “x”, a three years old girl suffered classical cognitive, neuromotor, and linguistic regression following suspected pneumonia at 18 months (probable trigger), before which she had 10-15 words vocabulary, able to recognize body parts, follow simple verbal command. The child presented severe dystonia, hypotonia, moderate dysphagia with persistent of sucking reflexes, oromotor inadequacies, absence of spoken words, absence of receptive language except to name-calling. Intensive physiotherapeutic and speech-language intervention were initiated. Orofacial exercises, dysphagia management protocols including the compensatory approaches and dietary modifications as well as direct language therapy for increase in vocabulary were taken up. After 2 months of therapy, there were significant improvements especially in swallowing rate and strength, integration of infantile reflexes and increase in linguistic receptivity, which clearly warrants continued therapy and consideration of AAC. Keywords: Neurological disorder, Speech disorder, Leigh’s syndrome, Speech therapy.

A rare pediatric neurometabolic disorder: Leigh syndrome

Subacute necrotizing encephalomyelopathy called Leigh Disease is a rare inherited pediatric neurological disorder generally involving infants. The condition primarily involves the central nervous system and the most involved structures are basal ganglia, thalamus, and brainstem. In this disorder, there is a progressive loss of mental and movement abilities associated with abnormal muscle tones, weakness, visual loss, respiratory failure, and strikingly, we got raised lactate levels in the blood, cerebral spinal fluid, and other body fluids. The prognosis of this condition is bad as there is no effective treatment and finally, the patient succumbs to death mostly due to respiratory failure. Here, we present a case of Leigh disease in an 8-month-old boy having chief complaints of delayed motor (weakness), and cognitive development with a previous history of recurrent respiratory infection. Upon recent radiological imaging, gross neurological abnormalities were seen which suggested the possibility of Leigh disease which was further confirmed by raised body fluid lactate levels.

Interoperability of Leigh Syndrome Patient Registry Data with Regulatory Submission Standards

Introduction: Leigh syndrome (LS) is a rare, severe neurometabolic disorder and a type of primary mitochondrial disease. The Cure Mito Foundation is a nonprofit foundation founded in 2018 by parents of affected children. The organization's mission is to unite the global LS community to accelerate patient-centered research, treatments, and cures. Cure Mito has launched a global patient registry for Leigh syndrome in September 2021 to better understand the disease, facilitate clinical trials recruitment, and build a strong global community. Objectives: Patient data is collected in registries for getting a real-world view of patient reported outcomes, and to improve the understanding of the disease. Establishment of interoperability of therapies. The project described here has been undertaken to establish such interoperability for Leigh syndrome patient registry data.Methods: Establishing interoperability consisted of assessment of all data elements and collected data, alignment of data elements and variables with CDASH domains and variables, terminology alignment, data transformation, and compliance assessment. Results: Data assessment has been done, domain map has been developed, and data has been converted to CDASH standard.Conclusion: Cure Mito Foundation and Sumptuous Data Sciences, LLC completed a project focused on interoperability of Leigh syndrome patient registry data with CDISC standards, converting the data to CDASH standards, with SDTM conversion in progress. This project is a key milestone for all stakeholders who collect real world data in rare diseases.

Direct neuronal reprogramming of NDUFS4 patient cells identifies the unfolded protein response as a novel general reprogramming hurdle

Mitochondria account for essential cellular pathways, from ATP production to nucleotide metabolism, and their deficits lead to neurological disorders and contribute to the onset of age-related diseases. Direct neuronal reprogramming aims at replacing neurons lost in such conditions, but very little is known about the impact of mitochondrial dysfunction on the direct reprogramming of human cells. Here, we explore the effects of mitochondrial dysfunction on the neuronal reprogramming of induced pluripotent stem cell (iPSC)-derived astrocytes carrying mutations in the NDUFS4 gene, important for Complex I and associated with Leigh syndrome. This led to the identification of the unfolded protein response as a major hurdle in the direct neuronal conversion of not only astrocytes and fibroblasts from patients but also control human astrocytes and fibroblasts. Its transient inhibition potently improves reprogramming by influencing the mitochondria-endoplasmic-reticulum-stress-mediated pathways. Taken together, disease modeling using patient cells unraveled novel general hurdles and ways to overcome these in human astrocyte-to-neuron reprogramming.

ChemRAP uncovers specific mRNA translation regulation via RNA 5′ phospho-methylation

5′-end modifications play key roles in determining RNA fates. Phospho-methylation is a noncanonical cap occurring on either 5′-PPP or 5′-P ends. We used ChemRAP, in which affinity purification of cellular proteins with chemically synthesized modified RNAs is coupled to quantitative proteomics, to identify 5′-Pme “readers”. We show that 5′-Pme is directly recognized by EPRS, the central subunit of the multisynthetase complex (MSC), through its linker domain, which has previously been involved in key noncanonical EPRS and MSC functions. We further determine that the 5′-Pme writer BCDIN3D regulates the binding of EPRS to specific mRNAs, either at coding regions rich in MSC codons, or around start codons. In the case of LRPPRC (leucine-rich pentatricopeptide repeat containing), a nuclear-encoded mitochondrial protein associated with the French Canadian Leigh syndrome, BCDIN3D deficiency abolishes binding of EPRS around its mRNA start codon, increases its translation but ultimately results in LRPPRC mislocalization. Overall, our results suggest that BCDIN3D may regulate the translation of specific mRNA via RNA-5′-Pme.

Expanding the genetic spectrum of mitochondrial diseases in Tunisia: novel variants revealed by whole-exome sequencing.

Introduction: Inherited mitochondrial diseases are the most common group of metabolic disorders caused by a defect in oxidative phosphorylation. They are characterized by a wide clinical and genetic spectrum and can manifest at any age. In this study, we established novel phenotype-genotype correlations between the clinical and molecular features of a cohort of Tunisian patients with mitochondrial diseases. Materials and methods: Whole-exome sequencing was performed on five Tunisian patients with suspected mitochondrial diseases. Then, a combination of filtering and bioinformatics prediction tools was utilized to assess the pathogenicity of genetic variations. Sanger sequencing was subsequently performed to confirm the presence of potential deleterious variants in the patients and verify their segregation within families. Structural modeling was conducted to study the effect of novel variants on the protein structure. Results: We identified two novel homozygous variants in NDUFAF5 (c.827G>C; p.Arg276Pro) and FASTKD2 (c.496_497del; p.Leu166GlufsTer2) associated with a severe clinical form of Leigh and Leigh-like syndromes, respectively. Our results further disclosed two variants unreported in North Africa, in GFM2 (c.569G>A; p.Arg190Gln) and FOXRED1 (c.1261G>A; p.Val421Met) genes, and we described the first case of fumaric aciduria in a Tunisian patient harboring the c.1358T>C; p.Leu453Pro FH variant. Conclusion: Our study expands the mutational and phenotypic spectrum of mitochondrial diseases in Tunisia and highlights the importance of next-generation sequencing to decipher the pathomolecular mechanisms responsible for these disorders in an admixed population.

Interleukin-6-elicited chronic neuroinflammation may decrease survival but is not sufficient to drive disease progression in a mouse model of Leigh syndrome

BackgroundMitochondrial diseases (MDs) are genetic disorders characterized by dysfunctions in mitochondria. Clinical data suggest that additional factors, beyond genetics, contribute to the onset and progression of this group of diseases, but these influencing factors remain largely unknown. Mounting evidence indicates that immune dysregulation or distress could play a role. Clinical observations have described the co-incidence of infection and the onset of the disease as well as the worsening of symptoms following infection. These findings highlight the complex interactions between MDs and immunity and underscore the need to better understand their underlying relationships.ResultsWe used Ndufs4 KO mice, a well-established mouse model of Leigh syndrome (one of the most relevant MDs), to test whether chronic induction of a neuroinflammatory state in the central nervous system before the development of neurological symptoms would affect both the onset and progression of the disease in Ndufs4 KO mice. To this aim, we took advantage of the GFAP-IL6 mouse, which overexpresses interleukin-6 (IL-6) in astrocytes and produces chronic glial reactivity, by generating a mouse line with IL-6 overexpression and NDUFS4 deficiency. IL-6 overexpression aggravated the mortality of female Ndufs4 KO mice but did not alter the main motor and respiratory phenotypes measured in any sex. Interestingly, an abnormal region-dependent microglial response to IL-6 overexpression was observed in Ndufs4 KO mice compared to controls.ConclusionOverall, our data indicate that chronic neuroinflammation may worsen the disease in Ndufs4 KO female mice, but not in males, and uncovers an abnormal microglial response due to OXPHOS dysfunction, which may have implications for our understanding of the effect of OXPHOS dysfunction in microglia.

Structural insights into respiratory complex I deficiency and assembly from the mitochondrial disease-related ndufs4−/− mouse

Respiratory complex I (NADH:ubiquinone oxidoreductase) is essential for cellular energy production and NAD+ homeostasis. Complex I mutations cause neuromuscular, mitochondrial diseases, such as Leigh Syndrome, but their molecular-level consequences remain poorly understood. Here, we use a popular complex I-linked mitochondrial disease model, the ndufs4−/− mouse, to define the structural, biochemical, and functional consequences of the absence of subunit NDUFS4. Cryo-EM analyses of the complex I from ndufs4−/− mouse hearts revealed a loose association of the NADH-dehydrogenase module, and discrete classes containing either assembly factor NDUFAF2 or subunit NDUFS6. Subunit NDUFA12, which replaces its paralogue NDUFAF2 in mature complex I, is absent from all classes, compounding the deletion of NDUFS4 and preventing maturation of an NDUFS4-free enzyme. We propose that NDUFAF2 recruits the NADH-dehydrogenase module during assembly of the complex. Taken together, the findings provide new molecular-level understanding of the ndufs4−/− mouse model and complex I-linked mitochondrial disease.

Leigh syndrome: a must-know bilateral symmetrical basal ganglia affection

Leigh's syndrome also known as subacute necrotizing encephalomyopathy is a rare progressive neurological disease with a reserved prognosis affecting the brainstem and the basal nuclei, the symptomatology of which is revealed in the majority of cases during the first two years of life. In this article we report the case of an 4 years old child referred to our center following a consultation for delayed psychomotor acquisitions with axial hypotonia and elevated lactate levels. Brain MRI showed a characteristic appearance of this syndrome. This clinical case reports the MRI aspect of Leigh's syndrome, a rare and serious pathology responsible for bilateral and symmetrical lesions of the basal ganglia.

Chapter 4 - Primary mitochondrial diseases

Primary mitochondrial diseases (PMDs) are a heterogeneous group of hereditary disorders characterized by an impairment of the mitochondrial respiratory chain. They are the most common group of genetic metabolic disorders, with a prevalence of 1 in 4,300 people. The presence of leukoencephalopathy is recognized as an important feature in many PMDs and can be a manifestation of mutations in both mitochondrial DNA (classic syndromes such as mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; myoclonic epilepsy with ragged-red fibers [RRFs]; Leigh syndrome; and Kearns-Sayre syndrome) and nuclear DNA (mutations in maintenance genes such as POLG, MPV17, and TYMP; Leigh syndrome; and mitochondrial aminoacyl-tRNA synthetase disorders). In this chapter, PMDs associated with white matter involvement are outlined, including details of clinical presentations, brain MRI features, and elements of differential diagnoses. The current approach to the diagnosis of PMDs and management strategies are also discussed. A PMD diagnosis in a subject with leukoencephalopathy should be considered in the presence of specific brain MRI features (for example, cyst-like lesions, bilateral basal ganglia lesions, and involvement of both cerebral hemispheres and cerebellum), in addition to a complex neurologic or multisystem disorder. Establishing a genetic diagnosis is crucial to ensure appropriate genetic counseling, multidisciplinary team input, and eligibility for clinical trials.

A Method for Producing Induced Pluripotent Stem Cell-Derived Cardiomyocytes from Leigh Syndrome Patients for Its Application in Disease Modeling and Drug Validation.

Leigh syndrome (LS), a complex multisystemic disorder, poses significant challenges in genetic medicine due to its intricate pathogenesis and wide-ranging clinical manifestations. Notably, these arise from mutations in either nuclear genetic DNA or mitochondrial DNA, affecting ATP production and resulting in diverse clinical outcomes. The unpredictable trajectory of this disease, ranging from severe developmental delays to early mortality, underscores the need for improved therapeutic solutions. This research pivots toward the novel use of induced pluripotent stem cells (iPSCs) as a promising platform for understanding disease mechanisms and spearheading patient-specific drug discoveries. Given the past successes of iPSCs in delineating organ-specific disorders and the recent endorsement of human iPSC-derived cardiomyocytes (CMs) by the FDA for drug evaluation, our work seeks to bridge this innovation to Leigh syndrome research. We detail a methodological approach to generate iPSCs from LS patients and differentiate them into iPSCs-CMs. Using multi-electrode array (MEA) analyses, we evaluate the field potential of these cells, spotlighting the potential of hiPSC-CM in drug validation and disease modeling. This pioneering approach offers a glimpse into the future of patient-centric therapeutic interventions for Leigh/Leigh-like syndrome.

Sedation Considerations in Leigh Syndrome

Sedation Considerations in Leigh Syndrome

Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA

DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by>99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.

Stress Pathways Induced by Volatile Anesthetics and Failure of Preconditioning in a Mitochondrial Complex I Mutant.

Carriers of mutations in the mitochondrial electron transport chain are at increased risk of anesthetic-induced neurotoxicity. To investigate the neurotoxicity mechanism and to test preconditioning as a protective strategy, this study used a Drosophila melanogaster model of Leigh syndrome. Model flies carried a mutation in ND23 (ND2360114) that encodes a mitochondrial electron transport chain complex I subunit. This study investigated why ND2360114 mutants become susceptible to lethal, oxygen-modulated neurotoxicity within 24 h of exposure to isoflurane but not sevoflurane. This study used transcriptomics and quantitative real-time reverse transcription polymerase chain reaction to identify genes that are differentially expressed in ND2360114 but not wild-type fly heads at 30 min after exposure to high- versus low-toxicity conditions. This study also subjected ND2360114 flies to diverse stressors before isoflurane exposure to test whether isoflurane toxicity could be diminished by preconditioning. The ND2360114 mutation had a greater effect on isoflurane- than sevoflurane-mediated changes in gene expression. Isoflurane and sevoflurane did not affect expression of heat shock protein (Hsp) genes (Hsp22, Hsp27, and Hsp68) in wild-type flies, but isoflurane substantially increased expression of these genes in ND2360114 mutant flies. Furthermore, isoflurane and sevoflurane induced expression of oxidative (GstD1 and GstD2) and xenobiotic (Cyp6a8 and Cyp6a14) stress genes to a similar extent in wild-type flies, but the effect of isoflurane was largely reduced in ND2360114 flies. In addition, activating stress response pathways by pre-exposure to anesthetics, heat shock, hyperoxia, hypoxia, or oxidative stress did not suppress isoflurane-induced toxicity in ND2360114 mutant flies. Mutation of a mitochondrial electron transport chain complex I subunit generates differential effects of isoflurane and sevoflurane on gene expression that may underlie their differential effects on neurotoxicity. Additionally, the mutation produces resistance to preconditioning by stresses that protect the brain in other contexts. Therefore, complex I activity modifies molecular and physiologic effects of anesthetics in an anesthetic-specific manner.