Recombinant Human ATPase family AAA domain-containing protein 3A (ATAD3A)

What is the structure and localization of ATAD3A?

ATAD3A is a member of the AAA+ (ATPases Associated with diverse cellular Activities) superfamily, characterized by a conserved ATPase domain responsible for ATP hydrolysis. The protein has three main structural components:

• An N-terminal domain that anchors the protein to the inner mitochondrial membrane

• A coiled-coil domain critical for protein-protein interactions and oligomerization

• A C-terminal AAA-ATPase domain located in the mitochondrial matrix, containing the Walker motifs necessary for ATPase activity

Regarding localization, ATAD3A is primarily enriched in mitochondrial-associated membranes (MAMs), which are contact sites between mitochondria and the endoplasmic reticulum (ER). It spans from the outer mitochondrial membrane to the inner membrane, with its ATPase domain extending into the mitochondrial matrix. This unique topology allows ATAD3A to interact with proteins in different mitochondrial compartments and the ER, facilitating its diverse functions .

How does ATAD3A regulate mitochondrial DNA maintenance?

ATAD3A plays a crucial role in maintaining mitochondrial DNA (mtDNA) through several mechanisms:

• Nucleoid binding and organization: ATAD3A binds to the displacement loop (D-loop) of mtDNA to regulate the distribution of mtDNA across mitochondrial membranes. When ATAD3A is depleted through antisense RNA treatment, visible nucleoids disappear without affecting mtDNA copy number, suggesting that ATAD3A is essential for the formation of nucleoid structures .

• Nucleoid trafficking: Live imaging experiments demonstrate that ATAD3A mediates the movement of nucleoids within mitochondria. In ATAD3A knockdown cells, nucleoid trafficking is severely impaired, with most nucleoids moving very slowly compared to control cells. This trafficking function requires both the ATPase domain and the coiled-coil domain of ATAD3A .

• Interaction with nucleoid proteins: ATAD3A interacts with mitochondrial transcription factor A (TFAM), a key protein involved in mtDNA packaging and transcription. This interaction is maintained in the presence and absence of ATP or ADP, suggesting that ATAD3A regulates nucleoid structure through protein-protein interactions .

• Nucleoid clustering regulation: In cells deficient for mitochondrial fission factor dynamin-related protein 1 (Drp1), ATAD3A is required for nucleoid clustering. When both ATAD3A and Drp1 are depleted, nucleoid enlargement is prevented, resulting in many small, dispersed nucleoids throughout elongated mitochondria .

These findings collectively indicate that ATAD3A functions as a key regulator of mtDNA organization, distribution, and dynamics within mitochondria.

What experimental models are commonly used to study ATAD3A?

Researchers employ multiple experimental models to investigate ATAD3A functions:

• Cell culture models:

  • Immortalized neuronal cell lines (HT-22 hippocampal neurons, Neuro2a neuroblastoma cells)

  • Primary cortical neurons

  • Stable APP wild-type and APP Swedish mutant-expressing cell lines

  • ATAD3A knockout or knockdown cell lines created through RNAi or CRISPR-Cas9

• Animal models:

  • ATAD3A knockout mice (both complete and heterozygous models)

  • 5XFAD mice (a model of Alzheimer's disease)

  • Tissue-specific ATAD3A knockout models

• Human samples:

  • Post-mortem brain tissue from Alzheimer's disease patients

  • Patient-derived cells with ATAD3A mutations

• Biochemical and imaging approaches:

  • Protein oligomerization analysis using non-reducing conditions and chemical cross-linkers

  • High-speed live imaging of nucleoid movement using spinning-disk confocal microscopy

  • Mitochondrial fractionation to isolate MAMs

  • ATPase activity assays

  • Immunoprecipitation to study protein-protein interactions

When designing experiments using these models, researchers should consider the species-specific expression of ATAD3 paralogs. While humans and other primates express three paralogs (ATAD3A, ATAD3B, and ATAD3C), mice and rats express only ATAD3A, which may impact the interpretation of results across different model systems .

How does ATAD3A oligomerization affect mitochondrial function and disease pathology?

ATAD3A oligomerization represents a critical regulatory mechanism that influences multiple aspects of mitochondrial function and contributes to disease pathogenesis:

• Regulation of nucleoid dynamics:

  • ATAD3A oligomerization is essential for nucleoid trafficking within mitochondria

  • The AAA-ATPase domain and coiled-coil domains are both required for proper oligomerization and nucleoid movement

  • Mutations in the conserved Walker motif B (E412Q) of the ATPase domain impair nucleoid trafficking without affecting TFAM interaction

• Role in neurodegenerative diseases:

  • ATAD3A oligomerization increases in various Alzheimer's disease models, including:

    • Neurons exposed to oligomeric Aβ peptides

    • APP wild-type and Swedish mutant-expressing cells

    • 5XFAD mouse model (specifically in the cortex, hippocampus, and thalamus)

    • Post-mortem hippocampal tissues from AD patients

• Impact on MAM integrity and cholesterol metabolism:

  • Aberrant ATAD3A oligomerization induces hyperconnectivity of MAMs

  • Oligomerized ATAD3A inhibits CYP46A1 gene expression, impairing neuronal cholesterol turnover

  • This promotes APP processing and synaptic loss, contributing to AD pathology

  • Suppression of ATAD3A oligomerization (through heterozygous knockout or pharmacological inhibition) enhances MAM integrity and cholesterol metabolism in AD mice

• Therapeutic implications:

  • Targeting ATAD3A oligomerization with compounds like DA1 reduces AD neuropathology and cognitive deficits in animal models

  • This approach represents a potential therapeutic strategy for slowing AD progression

These findings position ATAD3A oligomerization as a molecular switch linking metabolic dysfunction to neurodegenerative disease, suggesting new avenues for therapeutic intervention.

What is the relationship between ATAD3A and mitochondrial dynamics?

ATAD3A plays a multifaceted role in regulating mitochondrial dynamics through several mechanisms:

• Interaction with fission and fusion machinery:

  • ATAD3A interacts with multiple proteins involved in mitochondrial fission and fusion, including:

    • Dynamin-related protein 1 (DRP1), a key mitochondrial fission protein

    • Mitofusins 1 and 2 (MFN1, MFN2), which mediate mitochondrial fusion

    • Coiled-coil-domain-containing protein 56 (CCDC56), a positive regulator of mitochondrial fission

• Regulation of nucleoid distribution during fission/fusion events:

  • In cells deficient for Drp1, nucleoids cluster together in elongated mitochondria, forming enlarged "mito-bulbs"

  • ATAD3A is required for this nucleoid clustering, as ATAD3A depletion in Drp1-deficient cells prevents nucleoid enlargement

  • Notably, ATAD3A affects nucleoid trafficking without affecting mitochondrial membrane dynamics, suggesting a specialized role in coordinating nucleoid behavior during fusion/fission events

• Influence on mitophagy and quality control:

  • ATAD3A coordinates with TOM40 and TIM23 to regulate the import of PINK1, a key mitophagy initiator

  • ATAD3A deletion in mouse hematopoietic cells induces increased mitophagy

  • ATAD3A forms a complex with AMBRA1 and PINK1 at the outer mitochondrial membrane to regulate mitophagy

  • Binding of AMBRA1 to ATAD3A prevents PINK1 degradation, affecting mitophagy initiation

• Impact on mitochondrial structure:

  • ATAD3A plays roles in cristae formation and the assembly of respiratory complexes

  • Mutations in ATAD3A lead to alterations in mitochondrial number, size, and cristae morphology

These functions position ATAD3A as a key regulator at the interface of mitochondrial structure, dynamics, and quality control, coordinating these processes to maintain mitochondrial homeostasis.

How do ATAD3A mutations affect mitochondrial function in disease contexts?

ATAD3A mutations have been identified in patients with various neurological disorders and impact mitochondrial functions through several mechanisms:

• Effects on protein oligomerization and function:

  • Nearly all documented disease-associated mutations in ATAD3A exhibit either loss-of-function or dominant-negative effects

  • Many mutations disrupt the dimerization/oligomerization of ATAD3A, which is vital for its various functions

  • Despite affecting different domains of the protein, mutations commonly lead to similar mitochondrial dysfunction

• Impact on mitochondrial ultrastructure:

  • ATAD3A mutations alter mitochondrial number, size, and cristae morphology

  • These structural changes compromise mitochondrial function and contribute to disease pathology

• Effects on mitochondrial respiration:

  • Mutations in ATAD3A lead to diminished activity of mitochondrial respiratory chain complexes I, IV, and V

  • This respiratory dysfunction contributes to energy deficits in affected tissues, particularly in energy-demanding organs like the brain

• Disruption of quality control mechanisms:

  • ATAD3A mutations affect autophagy and mitophagy processes

  • These disruptions impair the clearance of damaged mitochondria, leading to the accumulation of dysfunctional organelles

• Tissue-specific effects:

  • The consequences of ATAD3A mutations may vary across different tissues

  • In the context of Alzheimer's disease, ATAD3A oligomerization shows region-specific increases in the brain, correlating with areas of Aβ aggregation

Understanding the specific mechanisms by which ATAD3A mutations affect mitochondrial function is critical for developing therapeutic strategies for ATAD3A-associated diseases.

What techniques are optimal for studying ATAD3A oligomerization?

Investigating ATAD3A oligomerization requires specialized techniques to preserve and detect oligomeric states:

• Non-reducing SDS-PAGE analysis:

  • Prepare protein samples in buffer lacking reducing agents like β-mercaptoethanol

  • This preserves disulfide bonds that may stabilize oligomeric structures

  • Western blotting with ATAD3A-specific antibodies can detect oligomeric bands at higher molecular weights

  • This technique has successfully identified increased ATAD3A oligomerization in AD models and patient samples

• Chemical cross-linking approaches:

  • Treat cells or isolated mitochondria with cross-linking agents such as bismaleimidohexane (BMH)

  • Cross-linkers stabilize protein-protein interactions, allowing detection of transient complexes

  • This method has been used to confirm ATAD3A oligomerization in APP wild-type and Swedish mutant-expressing cells

• Blue native PAGE (BN-PAGE):

  • This technique preserves native protein complexes and can be used to study ATAD3A oligomers

  • Combine with second-dimension SDS-PAGE to identify components of ATAD3A-containing complexes

• Fluorescence resonance energy transfer (FRET):

  • Tag ATAD3A with fluorescent proteins (e.g., CFP/YFP pairs)

  • Measure FRET efficiency to detect protein-protein interactions in living cells

  • This allows real-time monitoring of oligomerization dynamics

• Bimolecular fluorescence complementation (BiFC):

  • Split fluorescent protein approach to visualize protein interactions

  • Can be used to study ATAD3A oligomerization in different mitochondrial compartments

• Structure-function analysis:

  • Generate point mutations in key domains (ATPase domain, coiled-coil region)

  • Assess the impact on oligomerization and function

  • The E412Q mutation in the Walker B motif has been used to study the role of ATPase activity in ATAD3A function

• Small molecule modulators:

  • Compounds like DA1 that inhibit ATAD3A oligomerization can be valuable tools

  • These can help establish causality between oligomerization and downstream effects

When designing experiments to study ATAD3A oligomerization, researchers should consider that different extraction conditions and sample preparation methods may affect the detection of oligomeric states.

How can researchers effectively investigate ATAD3A's role in nucleoid trafficking?

To study ATAD3A's function in nucleoid trafficking, researchers can employ several complementary approaches:

• Live-cell imaging of nucleoid dynamics:

  • Use high-speed spinning-disk confocal microscopy to track nucleoid movement in real-time

  • Label nucleoids with DNA-binding dyes (e.g., PicoGreen) or fluorescently tagged nucleoid proteins (e.g., TFAM-GFP)

  • Measure the speed and trajectory of nucleoid movement under different conditions

  • This technique has revealed that ATAD3A knockdown significantly reduces nucleoid mobility

• Genetic manipulation strategies:

  • Generate ATAD3A knockdown or knockout cells using RNAi or CRISPR-Cas9

  • Create rescue experiments with wild-type or mutant ATAD3A constructs

  • Combine with Drp1 knockdown to study nucleoid clustering phenotypes

  • Expression of ATAD3A variants with mutations in specific domains (e.g., E412Q in the ATPase domain) can dissect domain-specific functions

• Photoactivation experiments:

  • Use photoactivatable fluorescent proteins to track mitochondrial content diffusion

  • This can distinguish between general effects on mitochondrial dynamics versus specific effects on nucleoid movement

  • Previous studies have shown that ATAD3A specifically affects nucleoid trafficking without altering mitochondrial membrane dynamics

• Protein-protein interaction studies:

  • Investigate interactions between ATAD3A and nucleoid proteins like TFAM

  • Purify recombinant ATAD3A domains and perform in vitro binding assays with nucleoid components

  • Analyze the effect of nucleotides (ATP, ADP) on these interactions

• Structural analysis:

  • Examine how ATAD3A mutations affect mitochondrial morphology and nucleoid distribution

  • Quantify nucleoid size, number, and clustering under different conditions

  • Correlate nucleoid behavior with mitochondrial function

• Combined approaches:

  • Integrate live imaging with electron microscopy to correlate nucleoid dynamics with ultrastructural changes

  • Combine nucleoid tracking with functional assays (e.g., respiratory measurements) to link trafficking defects to mitochondrial dysfunction

These methodologies provide complementary information about ATAD3A's role in nucleoid trafficking and can help elucidate the mechanisms by which ATAD3A coordinates nucleoid behavior with mitochondrial dynamics and function.

What approaches can be used to study ATAD3A's influence on cholesterol metabolism in neurodegenerative diseases?

Investigating ATAD3A's role in cholesterol metabolism, particularly in the context of neurodegenerative diseases, requires multidisciplinary approaches:

• Analysis of cholesterol levels and distribution:

  • Measure total cholesterol content in brain tissue or cultured neurons using enzymatic assays or mass spectrometry

  • Visualize cholesterol distribution using filipin staining or fluorescent cholesterol analogs

  • Fractionate cells to analyze cholesterol content in different compartments (ER, mitochondria, MAMs)

  • Compare cholesterol profiles between wild-type and ATAD3A-modified models (knockdown, overexpression, or mutant expression)

• Gene expression analysis of cholesterol metabolism enzymes:

  • Quantify expression of CYP46A1, the key enzyme for brain cholesterol clearance, using qRT-PCR, Western blotting, or immunohistochemistry

  • Investigate how ATAD3A oligomerization affects CYP46A1 expression

  • Analyze expression of other cholesterol metabolism genes (e.g., HMGCR, SREBP) to assess broader metabolic effects

  • Chromatin immunoprecipitation (ChIP) to identify potential transcriptional mechanisms

• MAM isolation and characterization:

  • Isolate MAM fractions using differential centrifugation and density gradient techniques

  • Quantify ATAD3A oligomerization in MAM fractions under different conditions

  • Measure cholesterol content and distribution within MAMs

  • Analyze protein composition of MAMs to identify ATAD3A interaction partners

• Functional studies in neurodegenerative disease models:

  • Use 5XFAD mouse model or other AD models to study ATAD3A oligomerization and cholesterol metabolism

  • Measure cognitive function in relation to ATAD3A status using behavioral tests

  • Analyze Aβ production and APP processing in relation to ATAD3A-mediated cholesterol changes

  • Assess synaptic markers to correlate cholesterol alterations with synaptic integrity

• Pharmacological approaches:

  • Use ATAD3A oligomerization inhibitors (e.g., DA1) to normalize cholesterol metabolism

  • Employ CYP46A1 activators to bypass ATAD3A-mediated inhibition

  • Manipulate cholesterol levels using statins or cyclodextrins to determine if cholesterol alterations mediate ATAD3A effects

• Human tissue studies:

  • Analyze post-mortem brain samples from AD patients for ATAD3A oligomerization and CYP46A1 expression

  • Correlate findings with cholesterol levels and disease severity

  • Implement spatial transcriptomics or proteomics to map regional variations

These approaches can help elucidate the molecular mechanisms by which ATAD3A affects cholesterol metabolism and contributes to neurodegenerative disease pathology, potentially identifying new therapeutic targets.

What are the major challenges in studying ATAD3A function and therapeutic targeting?

Researchers face several significant challenges when investigating ATAD3A:

• Structural complexity and membrane association:

  • ATAD3A's membrane-spanning topology makes it difficult to purify and crystallize for structural studies

  • The protein's association with multiple membrane compartments complicates the interpretation of localization studies

  • These challenges have limited our understanding of ATAD3A's precise molecular mechanism

• Functional redundancy and paralog interactions:

  • In humans and primates, three paralogs (ATAD3A, ATAD3B, ATAD3C) exist, with ATAD3B acting as a negative regulator of ATAD3A

  • This redundancy complicates genetic studies and may mask phenotypes in single knockout models

  • Species differences in paralog expression (mice and rats express only ATAD3A) create translational challenges

• Multifunctional nature:

  • ATAD3A participates in diverse processes including mtDNA maintenance, mitochondrial dynamics, cholesterol metabolism, and MAM regulation

  • This functional diversity makes it difficult to isolate specific pathways for targeted intervention

  • Perturbation of ATAD3A can have pleiotropic effects, complicating the interpretation of experimental results

• Therapeutic targeting challenges:

  • ATAD3A's essential functions in mitochondria make complete inhibition potentially toxic

  • Selective targeting of pathological oligomerization while preserving normal function requires sophisticated drug design

  • Mitochondrial drug delivery presents additional challenges for therapeutic development

• Tissue-specific effects:

  • ATAD3A pathology shows tissue and region specificity (e.g., in AD, changes are prominent in cortex and hippocampus but not other brain regions)

  • Understanding the basis for this selectivity is important for developing targeted therapeutics

Future research directions should focus on:

• Developing conditional and tissue-specific knockout models to overcome lethality issues

• Creating small molecules that specifically target pathological ATAD3A oligomerization

• Establishing high-resolution structures of ATAD3A in different functional states

• Identifying biomarkers for ATAD3A dysfunction that could guide therapeutic development

How does ATAD3A integrate into broader mitochondrial quality control networks?

ATAD3A functions within complex mitochondrial quality control networks, interacting with multiple pathways:

• Coordination with mitochondrial fission/fusion machinery:

  • ATAD3A interacts with key fission (DRP1) and fusion (MFN1/2) proteins

  • These interactions may help coordinate nucleoid distribution during mitochondrial dynamics

  • ATAD3A likely serves as a link between nucleoid behavior and mitochondrial structural changes

• Role in mitophagy regulation:

  • ATAD3A regulates PINK1 import and processing, a critical step in mitophagy initiation

  • It forms a complex with AMBRA1 and PINK1 at the outer mitochondrial membrane

  • ATAD3A deletion induces increased mitophagy in mouse hematopoietic cells

  • These findings position ATAD3A as a checkpoint in the decision between mitochondrial maintenance and elimination

• Interaction with ER-mitochondria contact sites:

  • ATAD3A is enriched at MAMs and regulates ER-mitochondria connectivity

  • This localization allows ATAD3A to influence calcium signaling, lipid transfer, and organelle communication

  • In neurodegenerative disease models, aberrant ATAD3A oligomerization disrupts MAM integrity

• Regulation of metabolic adaptation:

  • ATAD3A influences cholesterol metabolism through CYP46A1 expression

  • It also regulates mTOR, SREBP-1c, and cyclin D1 signaling for cell proliferation

  • ATAD3A phosphorylation is regulated by insulin and serum

  • These functions suggest ATAD3A serves as a metabolic sensor linking environmental cues to mitochondrial adaptation

• Integration with respiratory complex assembly:

  • ATAD3A mutations impair the activity of mitochondrial respiratory chain complexes I, IV, and V

  • This suggests a role in respiratory complex assembly or stability

  • Such integration allows ATAD3A to coordinate mitochondrial bioenergetics with other quality control mechanisms

Future research should focus on mapping the temporal dynamics of these interactions to understand how ATAD3A prioritizes different quality control pathways under various stress conditions. Developing technologies to monitor ATAD3A activity in real-time could provide insights into its decision-making role in mitochondrial quality control.