GCDH Human

What is GCDH and what is its primary function in human metabolism?

GCDH (glutaryl-CoA dehydrogenase) is a mitochondrial enzyme belonging to the dehydrogenase/decarboxylase enzyme family that catalyzes a critical step in amino acid metabolism . It is primarily expressed in metabolically active tissues such as the liver, kidneys, and brain . The enzyme's primary function is catalyzing the oxidation of glutaryl-CoA to glutaconyl-CoA, which is then further decarboxylated to crotonyl-CoA . This reaction represents a key step in the catabolism of lysine, hydroxylysine, and tryptophan, essential amino acids whose metabolic byproducts must be efficiently removed to prevent toxic accumulation .

How is GCDH deficiency diagnosed in research settings?

Diagnosis of GCDH deficiency in research contexts typically employs a multi-pronged approach:

• Genetic analysis: Sequencing of the GCDH gene to identify pathogenic variants, with a systematic compilation of variants now available in the Leiden Open Variation Database (LOVD)

• Biochemical evaluation: Measurement of organic acid metabolites (glutaric acid, 3-hydroxyglutaric acid) and glutarylcarnitine (C5DC) in biological samples

• Enzymatic activity assays: Direct measurement of GCDH enzyme activity in patient-derived cells

• Western blot analysis: Protein expression studies using specific antibodies like the GCDH (F2P2M) Rabbit mAb to confirm GCDH deficiency at the protein level

• In silico prediction tools: Computational evidence using tools such as REVEL, SpliceVault, and SpliceAI to evaluate novel variants

Research diagnostic criteria typically require the presence of characteristic metabolite patterns plus identification of pathogenic variants in the GCDH gene.

What cellular and animal models are available for studying GCDH function?

These models recapitulate key aspects of GA1 pathology and serve as platforms for investigating disease mechanisms and testing potential therapeutic approaches . The SH-SY5Y human neuronal cell model was generated using CRISPR-Cas9 technology, with three different gRNAs targeting GCDH exons 3 and 4 . After puromycin selection, individual clones were analyzed for genomic alterations and GCDH expression to confirm successful knockout . For the mouse model, researchers utilize it to study pathogenesis that closely resembles human GA1 pathology, providing insights into the molecular mechanisms of neurodegeneration .

What are the molecular mechanisms of neuronal injury in GCDH deficiency?

Neuronal injury in GCDH deficiency involves multiple interrelated pathways:

• Accumulation of neurotoxic metabolites: The primary pathogenic mechanism involves buildup of glutaric acid (GA) and 3-hydroxyglutaric acid (3-OH-GA) in neural tissues . When GCDH-deficient cells are exposed to high lysine concentrations, they accumulate these neurotoxic metabolites, triggering cellular damage .

• Mitochondrial dysfunction: GCDH deficiency leads to alterations in mitochondrial redox homeostasis . The generated SH-SY5Y GCDH-KO cell model demonstrated that high lysine exposure triggers mitochondrial disturbances, recapitulating the disease phenotype .

• Oxidative stress: The accumulation of toxic metabolites leads to increased reactive oxygen species production and impaired antioxidant defenses.

• Energy metabolism disruption: GCDH deficiency compromises energy supply pathways, particularly affecting highly energy-dependent tissues like the brain .

• Striatal vulnerability: The striatum shows particular susceptibility to damage, possibly due to its high energy requirements and unique metabolic profile.

Research using the SH-SY5Y GCDH-KO cell model has demonstrated that delivering functional GCDH cDNA under the human PGK promoter can restore enzyme activity and prevent cellular damage, suggesting potential therapeutic approaches .

How do GCDH variants correlate with clinical phenotypes in research cohorts?

The genotype-phenotype correlation in GCDH deficiency is complex. The GCDH-LOVD database has systematically compiled 306 different variants from 842 individuals to facilitate such analysis . Key findings include:

• Variant distribution: The variants are distributed throughout the gene without obvious hotspots for specific pathogenicity levels .

• In silico evidence: Approximately 69% (212/306) of GCDH variants have computational evidence of pathogenicity (PP3) at different strength levels . Interestingly, the PP3_Strong criterion was applied more frequently than PP3_Moderate or PP3 criteria (113 variants versus 76 and 23, respectively) .

• Functional evidence: About 18% (54/306) of variants have graded evidence of pathogenicity from functional studies (PS3) .

• Population frequency: Only 18 variants have been reported in ≥10 unrelated affected individuals, suggesting substantial genetic heterogeneity .

• Phase determination: The strength of genotype-related criteria (PM3 and PP4) depends on confirmation of variant phasing, emphasizing the importance of thorough genetic analysis .

Researchers should consider both the specific variant(s) and their combination in compound heterozygotes when designing experiments or interpreting clinical data.

What experimental approaches are used to assess GCDH function in human cell models?

Advanced experimental approaches to assess GCDH function in human cellular models include:

• CRISPR/Cas9 gene editing: Generation of precise GCDH knockout or specific mutations in human cell lines, as demonstrated in the SH-SY5Y neuroblastoma cell model . This approach involves designing multiple guide RNAs (gRNAs) targeting specific exons with minimal off-target effects, followed by puromycin selection and validation through genomic sequencing and protein expression analysis .

• Metabolic flux analysis: Tracing the fate of isotopically labeled lysine, hydroxylysine, or tryptophan to measure flux through GCDH-dependent pathways.

• Mitochondrial function assays: Measurements of oxygen consumption rate, membrane potential, and ATP production to assess the impact of GCDH deficiency on mitochondrial bioenergetics.

• Gene rescue experiments: Introducing wild-type or variant GCDH cDNA into deficient cells to assess functional complementation, as demonstrated by the SH-SY5Y GCDH-GI cell line where GCDH gene insertion under the hPGK promoter restored enzyme activity .

• Metabolite profiling: LC-MS/MS quantification of glutaric acid, 3-hydroxyglutaric acid, and related metabolites in cell culture media and cellular extracts.

• Protein-protein interaction studies: Investigation of GCDH interactions with other mitochondrial proteins to understand its functional network.

• Immunofluorescence techniques: Using specific antibodies like GCDH (F2P2M) Rabbit mAb at dilutions of 1:50-1:200 to visualize subcellular localization and expression patterns of GCDH .

What is the emerging role of GCDH in cancer metabolism research?

Recent studies have uncovered intriguing connections between GCDH and cancer metabolism, with apparent context-dependent effects:

• Melanoma dependency: Studies have shown that melanoma cells can develop a dependency on GCDH . Mechanistically, loss of GCDH results in increased glutarylation of NRF2, a master transcriptional regulator of stress response genes, leading to increased NRF2 stability and activity that ultimately promotes cell death .

• Glioblastoma stem cells: These cells reprogram lysine catabolism, including upregulation of GCDH, which leads to increased histone crotonylation that regulates tumor immunity . This represents a novel epigenetic mechanism linking metabolism to gene regulation in cancer.

• Hepatocellular carcinoma suppression: In contrast to its role in melanoma and glioblastoma, GCDH appears to suppress tumor growth and metastasis in hepatocellular carcinoma (HCC) . This suggests tissue-specific and context-dependent functions of GCDH in cancer biology.

These emerging findings highlight the need for comprehensive research into the metabolic reprogramming involving GCDH in different cancer types, potentially opening new therapeutic avenues.

How can GCDH function be measured in human tissue samples?

Methodological approaches for measuring GCDH function in human tissue samples include:

• Enzyme activity assays: Spectrophotometric or fluorometric assays measuring the conversion of glutaryl-CoA to glutaconyl-CoA in tissue homogenates or isolated mitochondria.

• Immunohistochemistry: Detection of GCDH protein expression in tissue sections using specific antibodies like GCDH (F2P2M) Rabbit mAb at dilutions of 1:100-1:400 . This allows visualization of expression patterns across different cell types within complex tissues.

• Western blotting: Quantitative assessment of GCDH protein levels in tissue lysates using validated antibodies at 1:1000 dilution, with detection of the approximately 48 kDa protein band .

• qRT-PCR: Measurement of GCDH mRNA expression levels to assess transcriptional regulation.

• Metabolite analysis: LC-MS/MS quantification of glutaric acid, 3-hydroxyglutaric acid, and related metabolites in tissue extracts as indirect indicators of GCDH function.

• Isotope tracing: Application of stable isotope-labeled precursors (e.g., 13C-lysine) to tissue slices or explants to trace metabolic flux through GCDH-dependent pathways.

• Single-cell approaches: Newer techniques like single-cell RNA-seq or mass cytometry to assess GCDH expression and function at the single-cell level within heterogeneous tissues.

What gene therapy approaches are being investigated for GCDH deficiency?

Gene therapy research for GCDH deficiency focuses on several complementary strategies:

• Viral vector-mediated gene delivery: Studies using the SH-SY5Y GCDH-KO cell model have demonstrated successful restoration of GCDH function through gene transfer . Researchers used a vector where the GCDH gene was placed under the transcriptional control of the human PGK (hPGK) promoter, with EGFP serving as a reporter for successful integration . FISH analysis confirmed insertion of the transgene in the chromosome 19 GCDH region .

• AAV-based approaches: Adeno-associated virus vectors are being explored due to their neurotropism and safety profile, targeting primarily the liver and/or CNS.

• Lentiviral vectors: Primarily investigated for ex vivo modification of hematopoietic stem cells for subsequent transplantation.

• Genome editing: CRISPR/Cas9-based approaches for direct correction of pathogenic variants in the GCDH gene.

• mRNA therapeutics: Delivery of engineered GCDH mRNA for transient expression of functional enzyme.

Key research challenges include optimizing tissue-specific delivery, achieving sufficient enzyme expression levels, and maintaining long-term expression while minimizing immune responses.

How can bioinformatic approaches aid in GCDH variant classification and research prioritization?

Bioinformatic approaches have become increasingly important in GCDH research:

• Variant databases: The GCDH-LOVD has compiled 306 different variants from 842 individuals, providing a comprehensive resource for variant analysis . This database summarizes known genotypes with clinical and biochemical phenotype information.

• In silico prediction tools: Multiple computational tools are employed to predict variant pathogenicity:

  • Meta-predictor REVEL for missense variant assessment

  • SpliceVault and SpliceAI for splicing effect prediction

  • Population frequency data from gnomAD and DECIPHER

• Constraint metrics: Analysis of missense constraint metrics from population databases helps prioritize potentially pathogenic variants .

• Criteria-based classification: Structured application of evidence criteria (PP3, PS3, PS4, PM3, PP4) at different strength levels enables systematic variant classification . For example, 69% of GCDH variants have computational evidence of pathogenicity (PP3), while 18% have evidence from functional studies (PS3) .

• Structural modeling: Mapping variants onto the 3D protein structure of GCDH to predict functional impacts.

• Machine learning approaches: Development of algorithms that integrate multiple data types to predict variant pathogenicity with higher accuracy.

These approaches facilitate research prioritization by identifying variants most likely to impact GCDH function, guiding experimental designs, and supporting the interpretation of functional studies.

What are the optimal antibodies and detection methods for human GCDH research?

Researchers have several validated options for GCDH detection:

• GCDH (F2P2M) Rabbit mAb: This recombinant monoclonal antibody has been validated for:

  • Western blotting at 1:1000 dilution, detecting the 48 kDa GCDH protein

  • Immunohistochemistry on paraffin-embedded samples at 1:100-1:400 dilution

  • Immunofluorescence at 1:50-1:200 dilution

  • Species reactivity: Human-specific

• Detection considerations:

  • For Western blotting: Use of appropriate positive controls (e.g., human liver lysate) and negative controls (e.g., GCDH knockout cells)

  • For IHC/IF: Proper antigen retrieval methods to expose epitopes without destroying tissue morphology

  • Validation with knockout controls: The SH-SY5Y GCDH-KO cell line serves as an excellent negative control for antibody validation

• Alternative detection methods:

  • Activity-based probes for functional GCDH

  • Mass spectrometry-based approaches for absolute quantification

  • RNA-based detection methods when protein detection is challenging

Researchers should consider the specific experimental context, sample type, and required sensitivity when selecting detection methods.

What are the key considerations for designing CRISPR/Cas9 knockout models of GCDH?

Based on successful generation of SH-SY5Y GCDH-KO cells, key considerations include:

• Guide RNA design:

  • Target early exons (exons 3 and 4 were targeted in the SH-SY5Y model)

  • Design multiple gRNAs (three different guides were used) to increase knockout efficiency

  • Minimize off-target cross-reactivity through careful bioinformatic screening

  • Select guide binding sites 5' to the PAM sequence

• Delivery method:

  • Plasmid-based delivery (pLentiCRISPRv2 containing both Cas9 and guide RNAs was used)

  • Consider cell type-specific transfection optimization

• Selection strategy:

  • Antibiotic selection (puromycin was used for the SH-SY5Y model)

  • Single-cell cloning and expansion for homogeneous populations

• Validation approaches:

  • Genomic DNA analysis to confirm targeted modifications

  • Western blot to verify complete absence of GCDH protein expression

  • Functional assays to confirm loss of GCDH enzymatic activity

  • Phenotypic characterization under stress conditions (e.g., high lysine exposure)

• Rescue experiments:

  • Design of appropriate vectors for gene complementation (e.g., pEGFP/PGK-GCDH)

  • Selection of appropriate promoters (human PGK promoter was effective)

  • Verification of integration and expression (FISH analysis, EGFP reporter)

These considerations ensure generation of valid GCDH knockout models that accurately recapitulate disease phenotypes and can be used for mechanistic studies and therapeutic testing.