NDUFA5 Human

What is NDUFA5 and what is its role in cellular metabolism?

NDUFA5 (NADH dehydrogenase ubiquinone 1 alpha subcomplex, 5, 13kDa) is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). It functions in the transfer of electrons from NADH to the respiratory chain and belongs to the complex I NDUFA5 subunit family . This protein is located in the peripheral arm of Complex I, although its specific function within the complex remains incompletely characterized . The protein has a calculated and observed molecular weight of 13 kDa and is encoded by the NDUFA5 gene (Gene ID: 4698) . NDUFA5 is also known by several other names including CI-13kD-B, NDUA5, B13, CI-13kB, DKFZp781K1356, FLJ12147, NUFM, and UQOR13 .

Where is NDUFA5 expressed in human and model organisms?

NDUFA5 is ubiquitously expressed across mammalian tissues. Based on antibody detection studies, NDUFA5 protein has been detected in:

The ubiquitous expression pattern is consistent with NDUFA5's fundamental role in mitochondrial respiration, which is essential for most eukaryotic cells .

What are the validated methods for detecting NDUFA5 protein in experimental systems?

Multiple validated methods exist for detecting NDUFA5 in experimental systems, each with specific applications and considerations:

For antigen retrieval in IHC applications, researchers should note that TE buffer pH 9.0 is recommended, although citrate buffer pH 6.0 may be used as an alternative . It is advised that researchers titrate antibodies in each specific testing system to achieve optimal results, as sensitivity may vary between sample types and experimental conditions .

What are the critical considerations for antibody selection in NDUFA5 research?

When selecting antibodies for NDUFA5 research, researchers should consider:

• Host species and clonality: Available antibodies include rabbit polyclonal (e.g., 16640-1-AP) which provides good sensitivity across multiple applications .

• Validated reactivity: Confirm the antibody has been validated in your species of interest. Currently, there are antibodies with demonstrated reactivity for human, mouse, and rat NDUFA5 .

• Application compatibility: Ensure the antibody has been validated for your specific application (WB, IHC, IF/ICC, ELISA) .

• Storage and handling: Most NDUFA5 antibodies require storage at -20°C and are stable for one year after shipment. They are typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Isotype and purification method: NDUFA5 antibodies may be IgG isotype and should be purified by methods such as antigen affinity purification for optimal specificity .

What genetic models are available for studying NDUFA5 function?

Several genetic models have been developed to study NDUFA5 function, with important considerations for researchers:

• Complete knockout models: Homozygous knockout of Ndufa5 in mice (Ndufa5 trap) is embryonic lethal, with embryos not developing beyond day E9. This demonstrates the essential nature of NDUFA5 for mammalian development .

• Conditional knockout models: A conditional knockout mouse model with tissue-specific deletion of Ndufa5 has been created using a Cre-loxP system. This involves:

  • A transgenic mouse expressing NDUFA5 protein under the ROSA26 promoter

  • The transgene contains cDNA encoding NDUFA5 protein flanked by loxP sites

  • Tissue-specific deletion achieved using tissue-specific Cre expression

• CNS-specific knockout model: A neuron-specific CI knockout mouse was created by conditionally deleting the Ndufa5 gene using CaMKIIα-Cre. These mice (Ndufa5 CNS-KO) showed:

  • Normal development until 10-11 months of age

  • Subsequent lethargy and motor coordination deficits

  • Reduced NDUFA5 protein levels in cortex (25% of controls)

  • Reduced fully assembled CI levels and activity (60% of control activity)

These models provide valuable tools for studying both the essential cellular functions of NDUFA5 and its tissue-specific roles, particularly in the nervous system.

What phenotypes are observed in NDUFA5-deficient models, and how are they assessed?

NDUFA5-deficient models display distinct phenotypes that can be assessed using established methodologies:

• CNS-specific knockout phenotypes:

  • Motor function deficits: Observed at 10-11 months of age, including:

    • Lethargy

    • Failure in pole descend test (longer latency to descend)

    • Hind leg clasping when suspended by tail

    • Reduced performance in grid and beam-walking tests

    • Increased percentage of "foot errors" in grid walking

• Biochemical phenotypes:

  • Reduced NDUFA5 protein levels (25% of controls in cortex)

  • Greatly reduced fully assembled Complex I levels

  • Reduced Complex I activity (60% of control activity in homogenates)

• Assessment methods:

  • Motor coordination: Pole descend test, grid walking test, beam-walking test

  • Protein expression: Western blot analysis

  • Complex I assembly: Blue native gel electrophoresis

  • Complex I activity: Enzymatic activity assays in tissue homogenates

Interestingly, despite the biochemical phenotype, no oxidative damage, neuronal death or gliosis were detected in the Ndufa5 CNS-KO mice. Researchers studied this through:

• NeuN-positive cell counting in motor cortex and hippocampus

• Confocal microscopy of cortex, hippocampus, and cerebellum

• GFAP staining for astroglia

• Iba1 staining for microglia

This suggests complex compensatory mechanisms that may protect against neuronal loss despite significant mitochondrial dysfunction.

What is the evidence linking NDUFA5 to autism spectrum disorders?

Several lines of evidence support an association between NDUFA5 and autism spectrum disorders:

• Genetic association studies: A case-control study in a Japanese population (235 patients with autism and 214 controls) examined three single-nucleotide polymorphisms (SNPs) within the NDUFA5 gene and found:

  • Two SNPs (rs12666974 and rs3779262) showed significant association with autism (P=0.00064 and P=0.00046, respectively)

  • A haplotype containing these two SNPs also showed significant association (P-global=0.0013, individual haplotype A-A: P=0.010)

• Transmission disequilibrium test (TDT): Analysis in 148 autistic trios confirmed:

  • Significant associations for both global and A-A haplotype P-values

  • Minor allele and genotype frequencies were decreased in autistic subjects

• SFARI Gene scoring: NDUFA5 has been assigned a SFARI Gene Score of 2, indicating strong evidence for involvement in autism risk .

These findings suggest that variations in NDUFA5 may contribute to autism risk, potentially through effects on mitochondrial function. This association aligns with growing evidence implicating mitochondrial dysfunction in the pathophysiology of autism spectrum disorders.

How do researchers study the role of NDUFA5 in mitochondrial disorders?

Researchers employ several complementary approaches to study NDUFA5's role in mitochondrial disorders:

• Biochemical characterization:

  • Measuring Complex I activity in patient samples or model systems

  • Assessing NADH dehydrogenase activity using spectrophotometric methods

  • Quantifying fully assembled Complex I levels using blue native gel electrophoresis

• Genetic approaches:

  • Sequencing NDUFA5 in patients with suspected mitochondrial disorders

  • Creating genetic models with tissue-specific or conditional deletion of NDUFA5

  • Analyzing SNPs and haplotypes associated with disease phenotypes

• Proteomic analysis:

  • Mass spectrometry to identify NDUFA5 interaction partners

  • Characterization of Complex I assembly in the presence or absence of NDUFA5

  • Comparing mitochondrial proteome changes in NDUFA5-deficient systems

• Functional studies:

  • Respiratory chain enzyme activity measurements

  • Oxygen consumption rate determination

  • Assessment of mitochondrial membrane potential

  • Measurement of reactive oxygen species production

These multidisciplinary approaches allow researchers to understand how NDUFA5 dysfunction contributes to mitochondrial disorders and may inform the development of potential therapeutic strategies.

What are the technical challenges in studying Complex I subunits like NDUFA5?

Researching NDUFA5 and other Complex I subunits presents several technical challenges:

• Complex assembly analysis: The large size and intricate assembly of Complex I (comprising 45 subunits) makes structural and functional studies challenging. Researchers typically employ:

  • Blue native gel electrophoresis

  • Sucrose gradient ultracentrifugation

  • Mass spectrometry for subunit composition analysis

• Functional redundancy: Some Complex I subunits may have partially overlapping functions, making it difficult to attribute specific functions to individual subunits without compensatory effects from other components.

• Tissue-specific effects: As demonstrated by the CNS-specific knockout model, NDUFA5 deficiency may have different consequences in different tissues, requiring tissue-specific approaches .

• Embryonic lethality: Complete knockout of NDUFA5 is embryonic lethal in mice, necessitating conditional knockout strategies to study function in adult tissues .

• Antibody specificity: Ensuring antibody specificity when detecting NDUFA5 in complex biological samples requires careful validation and controls .

• Species differences: While NDUFA5 is highly conserved, there may be species-specific differences in function or regulation that affect the interpretation of results from model organisms.

How can researchers integrate NDUFA5 studies with broader mitochondrial research?

To effectively integrate NDUFA5 research within the broader context of mitochondrial biology:

• Combine biochemical and genetic approaches:

  • Correlate genetic variations with biochemical phenotypes

  • Use both in vitro and in vivo models to validate findings

  • Apply systems biology approaches to understand network effects

• Consider mitochondrial dynamics:

  • Investigate how NDUFA5 deficiency affects mitochondrial morphology

  • Examine potential impacts on mitochondrial biogenesis

  • Assess effects on mitophagy and quality control mechanisms

• Explore metabolic consequences:

  • Measure changes in ATP production

  • Assess alterations in metabolic pathways dependent on mitochondrial function

  • Use metabolomics to identify metabolic signatures of NDUFA5 dysfunction

• Tissue-specific analyses:

  • Compare NDUFA5 function across different tissues

  • Investigate tissue-specific interaction partners

  • Examine how tissue-specific factors influence the consequences of NDUFA5 deficiency

• Translational perspectives:

  • Connect findings from model systems to human patient data

  • Identify potential biomarkers of NDUFA5 dysfunction

  • Explore therapeutic approaches targeting mitochondrial function in NDUFA5-related disorders

By adopting these integrated approaches, researchers can place NDUFA5 findings within the broader context of mitochondrial biology and disease.