DDHD2 Antibody

What is DDHD2 and what are its primary cellular functions?

DDHD2 (DDHD domain containing 2), also known as KIAA0725p, belongs to the intracellular phospholipase A1 (PLA1) protein family. These enzymes hydrolyze the sn-1 ester bond of phospholipids . The protein has a calculated molecular weight of 81 kDa (711 amino acids), though it is typically observed at 80-90 kDa in experimental contexts . DDHD2 preferentially targets phosphatidic acid as a substrate and plays a crucial role in membrane trafficking from the Golgi apparatus to the plasma membrane . Recent research has revealed that DDHD2 is particularly enriched in synaptic terminals of neurons, where it colocalizes with synapsin (Pearson's coefficient 0.62 ± 0.016 in hippocampal hilus and 0.60 ± 0.048 in cultured hippocampal neurons) . The protein serves essential functions in neuronal metabolism and fatty acid processing, as evidenced by mitochondrial dysfunction in DDHD2 knockout neurons, which display significantly reduced basal respiration, maximal respiration, and ATP levels (20.3% decrease) .

What applications can DDHD2 antibodies be validated for?

DDHD2 antibodies have been successfully validated for multiple experimental applications:

The antibody has demonstrated reactivity with human, mouse, and rat samples, making it suitable for comparative studies across these species . For optimal results, researchers should titrate the antibody concentration for each specific testing system and sample type, as sensitivity can be sample-dependent .

What is the recommended protocol for DDHD2 antibody in immunohistochemistry of brain tissue?

For successful immunohistochemical detection of DDHD2 in brain tissue, the following optimized protocol is recommended:

• Tissue preparation: Dissect adult mouse brain (28-30 days) and fix in 4% paraformaldehyde (PFA) with 15% sucrose overnight at 4°C.

• Sectioning: Mount brain on cryostat base with sucrose and prepare 30 μm thick coronal sections.

• Washing: Wash sections 3 times (5 minutes each) with PBS to remove cryo-protectant.

• Antigen retrieval: Heat sections at 95°C in citric acid buffer (10 mM citrate, 0.05% tween-20, pH 6) for 20 minutes. Alternatively, antigen retrieval with TE buffer pH 9.0 has also shown effective results .

• Blocking: After washing with PBST (0.5% tween-20, 0.1% triton X-100 in PBS) 3 times (10 minutes each), block sections in buffer containing 0.1% BSA, 2% NDS, 0.1% Triton X-100, 0.05% tween-20, and 50 mM glycine in PBS for 1 hour at room temperature.

• Primary antibody: Apply DDHD2 antibody at 1:200 dilution in antibody signal enhancer solution (10 mM glycine, 0.1% of 30% H₂O₂, 0.05% tween-20, 0.1% Triton X-100 in PBS) and incubate overnight at 4°C in a humidified chamber .

• Secondary antibody: After washing with PBST, incubate with appropriately labeled secondary antibody for 1 hour at room temperature.

• Mounting: After final washing, mount with refractive index-matched mounting media and coverslip .

This protocol has been demonstrated to produce specific staining throughout the hippocampus, with particularly strong signal in areas enriched with large synaptic terminals such as the hilus .

How can researchers validate the specificity of DDHD2 antibody?

Validating antibody specificity is crucial for ensuring reliable experimental results. For DDHD2 antibody, multiple validation approaches have been documented:

• RNA interference: Specificity of the DDHD2 antibody has been confirmed through shRNA-mediated knockdown, where neurons with DDHD2 ablation showed significantly reduced immunostaining compared to control neurons . This genetic validation provides strong evidence for antibody specificity.

• Western blot validation: The antibody detects a protein band at the expected molecular weight range (80-90 kDa) in rat brain tissue lysates, corresponding to the calculated 81 kDa size of DDHD2 .

• Cross-species reactivity: Consistent detection across human, mouse, and rat samples supports conservation of the recognized epitope and antibody specificity .

• Knockout validation: Studies using DDHD2-/- neurons demonstrate the absence of signal with the antibody, providing definitive validation of specificity .

• Co-localization studies: The strong co-localization of antibody staining with known synaptic markers (e.g., synapsin) in brain tissue and cultured neurons (Pearson's coefficient 0.60-0.62) provides functional validation of the antibody's specificity for DDHD2 in its native cellular context .

What is the subcellular localization pattern of DDHD2 in neurons?

DDHD2 exhibits a distinctive subcellular localization pattern in neurons that provides insights into its functional role:

• Synaptic terminal enrichment: Immunocytochemistry studies reveal that DDHD2 is distributed throughout neurons but is highly enriched in nerve terminals within axons. Quantitative analysis demonstrates that DDHD2 accumulates to the same extent as synapsin in nerve terminals .

• Hippocampal distribution: Throughout the hippocampus, DDHD2 is present with particularly strong expression in areas enriched with large synaptic terminals, such as the hilus. The fluorescence signal of DDHD2 and synapsin in the hippocampal hilus overlaps with a Pearson's colocalization coefficient of 0.62 ± 0.016 .

• Co-expression studies: GFP-DDHD2 and mRuby-synapsin co-expression experiments in hippocampal neurons showed strong co-localization with a Pearson's coefficient of 0.60 ± 0.048, further confirming the synaptic localization of DDHD2 .

• Membrane association: Consistent with its role in membrane trafficking from the Golgi apparatus to the plasma membrane, DDHD2 associates with membrane structures within cells .

The enrichment of DDHD2 in synaptic terminals suggests it plays a critical role in synaptic function, potentially through its involvement in lipid metabolism and membrane trafficking at these sites.

How does DDHD2 contribute to neuronal metabolism and mitochondrial function?

DDHD2 plays a critical role in neuronal metabolism and mitochondrial function through its involvement in fatty acid processing:

• Mitochondrial respiration: Oxygen consumption rate (OCR) measurements reveal that DDHD2-/- neurons exhibit significantly reduced mitochondrial respiration compared to control neurons. This includes decreased basal respiration, maximal respiration, ATP production (20.3% decrease), and non-mitochondrial oxygen consumption .

• Fatty acid metabolism: As an intracellular phospholipase A1, DDHD2 likely contributes to the release of fatty acids from phospholipids, particularly phosphatidic acid, providing essential substrates for energy metabolism in neurons .

• Metabolic adaptation: Proteomic analysis by label-free mass spectrometry identified 2,511 proteins in cultured control and DDHD2-/- neurons, revealing a global proteomic shift in DDHD2-/- neurons that indicates widespread metabolic adaptations in response to DDHD2 loss .

• Myristic acid supplementation effects: Treatment of DDHD2-/- neurons with Myr-CoA (myristic acid) resulted in a distinct proteomic profile compared to untreated knockout neurons, suggesting that fatty acid supplementation can partially rescue metabolic defects caused by DDHD2 deficiency .

These findings collectively indicate that DDHD2 provides a critical flux of saturated fatty acids to support neuronal energy metabolism, with its absence leading to significant mitochondrial dysfunction.

What is the role of DDHD2 in lipid droplet regulation at synapses?

DDHD2 serves a crucial function in regulating lipid droplet (LD) dynamics at synapses:

• LD accumulation in DDHD2 deficiency: Genetic deletion of DDHD2 leads to massive accumulation of LDs in mouse neurons, indicating that DDHD2 normally prevents excessive LD formation by facilitating lipid mobilization .

• Activity-dependent regulation: DDHD2 appears necessary for activity-driven fatty acid fueling of nerve terminals, with inhibition of DDHD2 activity causing accumulation of LDs at synapses . This suggests that DDHD2 is activated during neuronal activity to mobilize fatty acids from stored lipids.

• Phospholipase activity: As a phospholipase A1 that preferentially targets phosphatidic acid, DDHD2 likely generates lyso-phosphatidic acid and free fatty acids at synaptic terminals, providing substrates for membrane remodeling and energy production .

• Synaptic enrichment: The strong colocalization of DDHD2 with synaptic markers indicates that its lipid metabolic functions are particularly important at synapses, potentially supporting the high energy demands and membrane dynamics of these structures .

• Membrane trafficking: Beyond direct lipid metabolism, DDHD2's role in efficient membrane trafficking from the Golgi apparatus to the plasma membrane suggests it may coordinate lipid supply with membrane dynamics at synapses .

The collective evidence indicates that DDHD2 functions as a key regulator of lipid homeostasis at synapses, with particular importance during periods of elevated neuronal activity.

How does DDHD2 knockout affect secretory pathway membrane trafficking?

Recent research has revealed important insights into how DDHD2 deficiency impacts the secretory pathway:

• Perturbation of membrane trafficking: Loss of DDHD2 leads to disruption in secretory pathway membrane trafficking, consistent with its known role in facilitating efficient membrane movement from the Golgi apparatus to the plasma membrane .

• Proteomic changes: Global proteomic analysis in DDHD2-/- neurons revealed alterations in protein expression patterns that cluster distinctly from control neurons, indicating widespread cellular adaptations to compensate for disrupted membrane trafficking .

• Hierarchical clustering: Principal component and hierarchical clustering analyses of proteomic data confirm consistent and significant changes in protein expression patterns in DDHD2-/- neurons compared to control neurons, with these patterns partially rescued by Myr-CoA supplementation .

• Lipid composition effects: As a phospholipase that targets phosphatidic acid, DDHD2 deficiency likely alters membrane lipid composition, potentially affecting membrane curvature, fluidity, and protein recruitment essential for normal trafficking .

• Structural consequences: The accumulation of lipid droplets observed in DDHD2-deficient neurons may physically impede normal membrane trafficking processes, creating a secondary effect on secretory pathway function .

These findings highlight the critical role of DDHD2-mediated lipid metabolism in maintaining proper membrane trafficking through the secretory pathway, with implications for neuronal function and viability.

What are the optimal experimental approaches to study DDHD2 function in primary neuronal cultures?

Based on published methodologies, the following experimental approaches are recommended for investigating DDHD2 function in primary neuronal cultures:

• Neuronal culture preparation:

  • For dissociated hippocampal neurons, sparsely plate primary rat hippocampal neurons to allow clear visualization of axonal structures .

  • For mitochondrial studies, establish cultures from control C57BL6/J and DDHD2-/- mice .

• Loss-of-function studies:

  • shRNA-mediated knockdown has been successfully used to ablate DDHD2 expression in neurons, with validated reduction in protein levels .

  • DDHD2-/- knockout cultures provide a complete loss-of-function model for comprehensive analysis .

• Functional assays:

  • Mitochondrial function: Use Cell Mito Stress assay to measure oxygen consumption rate (OCR) as a direct readout of mitochondrial respiration activity .

  • Lipid droplet visualization: Apply appropriate lipid staining techniques to quantify LD accumulation at synapses .

  • Membrane trafficking: Monitor protein transport through the secretory pathway using tagged reporter proteins.

• Rescue experiments:

  • Myr-CoA supplementation has been shown to partially rescue phenotypes in DDHD2-/- neurons, providing a tool to distinguish direct from indirect effects .

  • Re-expression of wild-type or mutant DDHD2 (e.g., GFP-DDHD2) can confirm specificity of observed phenotypes .

• Proteomic analysis:

  • Label-free mass spectrometry has successfully identified over 2,500 proteins in neuronal cultures, enabling comprehensive analysis of proteome changes in DDHD2-deficient neurons .

These approaches provide a comprehensive toolkit for investigating the multifaceted functions of DDHD2 in neuronal lipid metabolism, membrane trafficking, and synaptic function.

How do different fixation and antigen retrieval methods affect DDHD2 antibody performance?

Optimizing fixation and antigen retrieval methods is critical for successful DDHD2 immunodetection:

• Fixation methods:

  • For tissue sections: 4% paraformaldehyde (PFA) with 15% sucrose solution overnight at 4°C has proven effective for preserving DDHD2 antigenicity in brain tissue .

  • For cultured cells: -20°C ethanol fixation has been successfully used for immunofluorescent analysis of HEK-293 cells .

• Antigen retrieval options:

  • Heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) is suggested as the primary method for paraffin-embedded tissue sections .

  • Alternatively, citric acid buffer (10 mM citrate, 0.05% tween-20, pH 6.0) heated at 95°C for 20 minutes has been successfully employed for frozen brain sections .

  • The choice between these methods may depend on the specific tissue preparation and embedding method.

• Buffer composition considerations:

  • The addition of detergents (0.5% tween-20, 0.1% triton X-100) to PBS washing buffer enhances antibody penetration in tissue sections .

  • Antibody signal enhancer solution containing glycine, H₂O₂, and detergents can improve signal-to-noise ratio .

• Section thickness optimization:

  • 30 μm thick coronal sections have been successfully used for brain tissue immunostaining, balancing structural integrity with antibody penetration .

• Blocking conditions:

  • Effective blocking has been achieved using 0.1% BSA, 2% NDS, 0.1% Triton X-100, 0.05% tween-20, and 50 mM glycine in PBS for 1 hour at room temperature .

Researchers should consider these parameters when optimizing DDHD2 detection protocols for their specific experimental systems, as the effectiveness of antigen retrieval methods can vary depending on tissue type, fixation duration, and embedding method.

What are common challenges in DDHD2 antibody applications and how can they be addressed?

Researchers may encounter several challenges when working with DDHD2 antibodies, with the following solutions recommended:

• Variability in signal intensity:

  • Carefully titrate antibody concentration for each experimental system; recommended dilution ranges are 1:1000-1:4000 for WB, 1:50-1:500 for IHC, and 1:200-1:800 for IF/ICC .

  • Sample-dependent variations may require optimization beyond the standard recommended dilutions.

• Background staining:

  • Implement thorough blocking with 0.1% BSA, 2% normal donkey serum, 0.1% Triton X-100, 0.05% tween-20, and 50 mM glycine in PBS .

  • Consider antibody signal enhancer solution containing 10 mM glycine, 0.1% of 30% H₂O₂, 0.05% tween-20, and 0.1% Triton X-100 in PBS for primary antibody incubation .

• Antigen masking in fixed tissues:

  • Test both recommended antigen retrieval methods: Tris-EDTA buffer (pH 9.0) and citric acid buffer (10 mM citrate, 0.05% tween-20, pH 6.0) .

  • Heat-mediated antigen retrieval at 95°C for 20 minutes is critical for exposing the DDHD2 epitope in fixed tissues .

• Specificity concerns:

  • Include appropriate negative controls, such as shRNA-mediated knockdown or DDHD2-/- tissues/cells .

  • Confirm expected molecular weight (80-90 kDa) in Western blot applications .

• Storage and stability issues:

  • Store antibody at -20°C, where it remains stable for one year after shipment .

  • For long-term storage, aliquoting is unnecessary but may be preferred to avoid freeze-thaw cycles.

By addressing these common challenges through careful optimization, researchers can achieve reliable and specific detection of DDHD2 across multiple experimental applications.

How can researchers distinguish between DDHD2 and related phospholipase family members?

Distinguishing DDHD2 from other phospholipase family members requires careful experimental design:

• Antibody selection criteria:

  • Use antibodies raised against unique epitopes in DDHD2 that are not conserved in related family members.

  • The DDHD2 antibody (25203-1-AP) is generated against a specific DDHD2 fusion protein (Ag18373), enhancing specificity .

• Western blot validation:

  • DDHD2 appears at 80-90 kDa on western blots, which may differ from the molecular weights of related phospholipases .

  • Run appropriate controls including recombinant proteins of related family members to confirm antibody specificity.

• Expression pattern analysis:

  • DDHD2 shows characteristic enrichment in synaptic terminals with strong colocalization with synapsin (Pearson's coefficient 0.60-0.62) .

  • Compare localization patterns with other family members, which may show distinct subcellular distributions.

• Functional assays:

  • DDHD2 preferentially targets phosphatidic acid as a substrate, which may differ from the substrate preferences of related phospholipases .

  • Design substrate-specific activity assays to differentiate between family members.

• Genetic approaches:

  • Use specific shRNA sequences or CRISPR-Cas9 targeting that are unique to DDHD2.

  • Validate knockdown/knockout specificity by checking expression of related family members to ensure they remain unaffected.

These approaches, used in combination, can provide high confidence in the specific detection and functional analysis of DDHD2 distinct from related phospholipase family members.

How can DDHD2 antibodies be applied in neurodegenerative disease research?

DDHD2 antibodies offer valuable research tools for investigating neurodegenerative conditions:

• SPG54 hereditary spastic paraplegia:

  • DDHD2 is identified as the SPG54 gene, with mutations causing a form of hereditary spastic paraplegia .

  • Antibodies enable characterization of DDHD2 expression, localization, and processing in patient-derived samples or disease models.

• Lipid droplet pathology:

  • The massive accumulation of lipid droplets in neurons following DDHD2 deletion suggests a role in neurodegenerative conditions featuring abnormal lipid storage .

  • Antibodies can help quantify DDHD2 levels and correlate with lipid droplet accumulation in disease states.

• Mitochondrial dysfunction:

  • DDHD2 knockout causes significant mitochondrial impairment, including reduced respiration and ATP production .

  • Antibodies can be used to examine DDHD2 expression in diseases characterized by mitochondrial dysfunction.

• Membrane trafficking defects:

  • DDHD2's role in secretory pathway membrane trafficking suggests potential involvement in neurodegenerative conditions featuring trafficking deficits .

  • Immunolocalization studies can reveal altered DDHD2 distribution in disease models.

• Therapeutic development:

  • Antibodies can track DDHD2 expression and localization in response to experimental therapeutics targeting lipid metabolism or membrane trafficking.

  • They provide essential tools for validating target engagement in drug development pipelines.

These applications highlight the value of DDHD2 antibodies in understanding the pathological mechanisms of neurodegenerative conditions and developing potential therapeutic strategies.

What methodological considerations are important when using DDHD2 antibody in multi-protein colocalization studies?

When designing multi-protein colocalization studies involving DDHD2, researchers should consider the following methodological factors:

• Antibody compatibility:

  • Select primary antibodies raised in different host species (e.g., rabbit anti-DDHD2 with mouse anti-synapsin) to enable simultaneous detection with species-specific secondary antibodies .

  • If using multiple rabbit antibodies, consider sequential staining with direct labeling of the first primary antibody.

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap to reduce bleed-through artifacts.

  • For DDHD2 colocalization with synaptic markers, successful combinations include GFP-DDHD2 with mRuby-synapsin for exogenous expression .

• Imaging parameters:

  • Use confocal microscopy with appropriate channel separation and sequential scanning to minimize crosstalk.

  • Collect z-stacks to capture the full three-dimensional distribution of proteins, especially important in complex neuronal structures.

• Quantification methods:

  • Calculate Pearson's colocalization coefficient as demonstrated in successful DDHD2 studies (yielding values of 0.60-0.62 for DDHD2 and synapsin) .

  • Consider additional metrics such as Manders' overlap coefficient or intensity correlation analysis for more nuanced colocalization assessment.

• Controls and validation:

  • Include single-label controls to set imaging parameters and establish bleed-through levels.

  • Use DDHD2 knockdown/knockout samples as negative controls to validate specificity of colocalization patterns .

• Sample preparation consistency:

  • Maintain consistent fixation and antigen retrieval methods across all samples to avoid artificial differences in colocalization patterns.

  • For brain sections, 30 μm thickness with heat-mediated antigen retrieval has proven effective .

These methodological considerations will help ensure reliable and interpretable results when studying DDHD2 colocalization with other proteins of interest.

What emerging technologies could enhance DDHD2 detection and functional analysis?

Several cutting-edge technologies hold promise for advancing DDHD2 research:

• Proximity labeling methods:

  • BioID or TurboID fused to DDHD2 could identify proximal interacting proteins at synaptic terminals, revealing the protein's immediate functional network.

  • APEX2-DDHD2 fusion could enable electron microscopy visualization of DDHD2's precise ultrastructural localization relative to synaptic vesicles and active zones.

• Super-resolution microscopy:

  • STORM or PALM imaging could resolve DDHD2 distribution at the nanoscale level, potentially revealing subsynaptic domains of enrichment not visible with conventional microscopy.

  • Two-color super-resolution could precisely map DDHD2 relative to lipid droplets and synaptic structures with unprecedented detail.

• Live-cell imaging approaches:

  • Split-fluorescent protein complementation could visualize dynamic DDHD2 interactions with suspected binding partners in living neurons.

  • FRET-based activity sensors could detect DDHD2 enzymatic activity in real-time during neuronal stimulation.

• Single-cell analysis:

  • Spatial transcriptomics combined with DDHD2 immunostaining could correlate protein levels with gene expression patterns at the single-cell level.

  • Mass cytometry could quantify DDHD2 alongside dozens of other proteins across large neuronal populations.

• Cryo-electron tomography:

  • Direct visualization of DDHD2 in its native cellular environment could reveal structural details of its association with membranes and lipid droplets.

  • Immunogold labeling combined with cryo-ET could precisely locate DDHD2 within the complex architecture of synaptic terminals.

These emerging technologies hold tremendous potential for advancing our understanding of DDHD2's precise localization, dynamics, and functional interactions in neuronal systems.

What are the key unanswered questions regarding DDHD2 function in neuronal metabolism?

Despite significant progress in DDHD2 research, several important questions remain unanswered:

• Activity-dependent regulation:

  • How is DDHD2 enzymatic activity regulated during periods of increased neuronal activity?

  • What post-translational modifications control DDHD2 function in response to synaptic signaling?

• Substrate specificity in vivo:

  • While DDHD2 preferentially targets phosphatidic acid in vitro, what are its physiological substrates within neuronal membranes?

  • How does substrate availability change under different metabolic conditions in neurons?

• Interaction with lipid droplet machinery:

  • What molecular mechanisms link DDHD2 activity to lipid droplet formation or mobilization at synapses?

  • Does DDHD2 interact directly with lipid droplet proteins or indirectly influence droplet dynamics through its enzymatic products?

• Mitochondrial connections:

  • How does DDHD2-mediated lipid metabolism integrate with mitochondrial function to support neuronal energy demands?

  • What is the molecular pathway connecting DDHD2 deficiency to the observed reductions in mitochondrial respiration and ATP production?

• Therapeutic potential:

  • Can targeted manipulation of DDHD2 activity or expression protect against neurodegeneration in models of SPG54 or related disorders?

  • Would bypassing DDHD2 through direct fatty acid supplementation provide therapeutic benefit in DDHD2-deficient conditions?

Addressing these questions will require integrative approaches combining DDHD2 antibody-based detection methods with advanced functional assays, genetic manipulations, and metabolomic analyses to fully elucidate the complex roles of this protein in neuronal metabolism and function.