Recombinant Danio rerio Mitochondrial inner membrane protease subunit 2 (immp2l)

What is immp2l and what is its primary function in Danio rerio?

Mitochondrial inner membrane protease subunit 2 (immp2l) is a critical enzyme that catalyzes the removal of transit peptides required for targeting proteins from the mitochondrial matrix across the inner membrane into the inter-membrane space. In Danio rerio (zebrafish), as in other organisms, immp2l is involved in processing the signal peptide sequences of mitochondrial proteins, particularly cytochrome c1 (Cyc1) . The protein is encoded by the immp2l gene, also known by its ORF name zgc:100888, and has the UniProt accession number Q6AZD4 . Its primary function is to ensure proper mitochondrial protein localization and subsequent appropriate mitochondrial function, which is crucial for cellular energy production and homeostasis .

How is recombinant Danio rerio immp2l typically produced for research purposes?

Recombinant Danio rerio immp2l is typically produced by cloning the full-length protein sequence (amino acids 1-183) into expression vectors suitable for prokaryotic or eukaryotic expression systems . The recombinant protein is often tagged to facilitate purification, though the specific tag type may vary depending on the production process requirements . Following expression, the protein is purified and typically stored in a Tris-based buffer with 50% glycerol at -20°C for short-term use or -80°C for extended storage . For research applications, aliquoting is recommended to avoid repeated freeze-thaw cycles, which can compromise protein integrity, and working aliquots can be stored at 4°C for up to one week .

What are the observed phenotypic effects of immp2l mutations in zebrafish models?

Mutations in the immp2l gene produce distinct behavioral and physiological phenotypes in zebrafish. At the behavioral level, immp2l-/- mutant zebrafish exhibit enhanced cohesion states and increased shoaling behavior, forming tighter aggregations compared to wild-type fish . This alteration in collective behavior patterns is quantifiable through computer vision and unsupervised machine learning analysis of fish movements, which revealed specific changes in the ethogram states of mutant fish .

At the physiological level, immp2l mutations impair the signal peptide sequence processing of mitochondrial proteins, particularly cytochrome c1, leading to elevated reactive oxygen species (ROS) production . This increased oxidative stress affects multiple organ systems and can accelerate aging-associated phenotypes . The specific behavioral phenotype of enhanced shoaling in zebrafish appears to be a distinctive effect that could potentially be linked to the neurological impacts of altered mitochondrial function .

How does immp2l deficiency impact mitochondrial function and oxidative stress in research models?

Immp2l deficiency significantly disrupts mitochondrial function through several documented mechanisms:

• Impaired processing of target proteins: Deficiency in immp2l leads to incomplete cleavage of mitochondrial proteins, particularly cytochrome c1 (Cyc1) .

• Altered mitochondrial membrane potential: Homozygous mutation of Immp2l (Immp2l-/-) elevates mitochondrial membrane potential, creating bioenergetic imbalances .

• Increased ROS production: Both homozygous and heterozygous immp2l mutations significantly increase superoxide (- O₂⁻) production in various tissues, including the brain .

• Compromised respiratory function: Mitochondrial respiratory rate, total respiratory capacity, and respiratory complex activities are decreased in Immp2l+/- mice compared to wild-type, particularly under stress conditions such as ischemia-reperfusion .

These mitochondrial dysfunctions collectively contribute to increased oxidative stress, which damages cellular components and accelerates aging-associated pathologies . The oxidative stress effects are substantial enough that even heterozygous mutations (Immp2l+/-) exacerbate ischemic brain damage through enhanced superoxide production and mitochondrial functional impairment .

What is the relationship between immp2l mutations and age-associated disorders?

Research indicates a significant relationship between immp2l mutations and accelerated onset of age-associated disorders. Experimental evidence shows that Immp2l mutants exhibit multiple aging-associated phenotypes, including:

• Wasting and sarcopenia (muscle loss)

• Loss of subcutaneous fat

• Kyphosis (abnormal spine curvature)

• Ataxia (impaired coordination)

• Increased oxidative stress in multiple organs including brain and kidney

Notably, female mutants demonstrate earlier onset and more severe age-associated disorders compared to their male counterparts, suggesting sex-specific effects of immp2l mutation . The underlying mechanism appears to be related to mitochondrial ROS acting as a driving force for accelerated aging processes. Additionally, research suggests that ROS damage to adult stem cells could be one of the mechanisms for these age-associated disorders, as adipose-derived stromal cells (ADSC) from mutant mice show impaired proliferation capability and form significantly fewer and smaller colonies, despite retaining their adipogenic differentiation capability in vitro .

What are the optimal storage and handling conditions for recombinant Danio rerio immp2l protein?

Optimal storage and handling of recombinant Danio rerio immp2l protein requires careful attention to temperature conditions and buffer composition to maintain structural integrity and enzymatic activity:

The protein should be stored in a Tris-based buffer with 50% glycerol, which has been optimized for this specific protein . Repeated freezing and thawing cycles should be strictly avoided as they can lead to protein denaturation and activity loss . When designing experiments, researchers should prepare appropriately sized working aliquots at 4°C to minimize freeze-thaw cycles while ensuring experiment reproducibility.

For handling during experiments, maintain the protein on ice when removed from storage, and centrifuge briefly before opening tubes to collect any condensation. If dilution is necessary for experimental procedures, use the same buffer composition to maintain protein stability .

What experimental approaches can be used to assess the functional consequences of immp2l mutations in zebrafish?

Multiple experimental approaches can be employed to comprehensively assess the functional consequences of immp2l mutations in zebrafish:

• Behavioral Analysis:

  • Computer vision and unsupervised machine learning techniques to quantify collective behaviors such as shoaling patterns

  • Analysis of swimming patterns, velocity, and group cohesion through automated tracking systems

  • Categorization of behavioral states through ethogram generation

• Mitochondrial Function Assessment:

  • Measurement of mitochondrial membrane potential using fluorescent dyes (e.g., TMRM, JC-1)

  • Oxygen consumption rate (OCR) analysis to evaluate respiratory capacity

  • Assessment of mitochondrial complex activities through enzymatic assays

• Oxidative Stress Evaluation:

  • Quantification of superoxide (- O₂⁻) production using dihydroethidium (DHE) or MitoSOX

  • Measurement of lipid peroxidation (e.g., malondialdehyde assay)

  • Assessment of DNA oxidative damage through 8-OHdG quantification

  • Analysis of antioxidant enzyme expression and activity (e.g., SOD, catalase)

• Molecular Characterization:

  • Protein processing analysis to confirm improper cleavage of target proteins like cytochrome c1

  • Western blotting to assess protein expression levels and post-translational modifications

  • RNA sequencing to identify transcriptional changes resulting from immp2l mutation

• Stress Response Testing:

  • Hypoxia challenge to assess mitochondrial resilience

  • Age-dependent phenotypic characterization to evaluate premature aging markers

These methodological approaches allow for comprehensive assessment of the phenotypic spectrum resulting from immp2l mutations, from molecular alterations to behavioral consequences.

How can CRISPR-Cas9 be optimized for generating immp2l knockout zebrafish models?

Optimizing CRISPR-Cas9 for generating immp2l knockout zebrafish models requires attention to several critical factors:

• Guide RNA (gRNA) Design:

  • Target conserved functional domains within the immp2l gene, particularly the catalytic region

  • Use multiple prediction algorithms to select gRNAs with high on-target efficiency and minimal off-target effects

  • Design gRNAs targeting early exons to ensure complete loss of function

  • Consider including gRNAs targeting different regions to allow for comparison of phenotypes

• Delivery Method Optimization:

  • For maximum efficiency, inject 1-cell stage embryos with a ribonucleoprotein (RNP) complex of Cas9 protein and synthesized gRNA

  • Empirically determine optimal concentrations of Cas9 protein (typically 300-500 ng/μL) and gRNA (typically 50-100 ng/μL)

  • Include phenol red in the injection mix (0.05%) to visualize successful injections

• Mutation Validation:

  • Employ T7 endonuclease I assay or heteroduplex mobility assay for initial screening

  • Confirm mutations by direct sequencing of PCR amplicons from the targeted region

  • Use quantitative PCR and Western blotting to confirm reduced/absent expression

• Founder Selection and Breeding Strategy:

  • Screen F0 mosaic fish for germline transmission by outcrossing to wild-type

  • Select lines with frameshift mutations that result in premature stop codons

  • Establish heterozygous lines (immp2l+/-) before generating homozygous knockouts

• Control Development:

  • Generate control lines using non-targeting gRNAs to account for off-target effects

  • Maintain wild-type siblings from the same breeding pairs as appropriate controls

  • Consider rescue experiments by co-injecting wild-type immp2l mRNA to confirm specificity

This optimized approach ensures the generation of reliable immp2l knockout models for investigating the protein's functions in zebrafish development, behavior, and physiology .

How does research on immp2l in zebrafish inform our understanding of human neurological disorders?

Research on immp2l in zebrafish has provided valuable insights into human neurological disorders through several key mechanisms:

Zebrafish immp2l studies have revealed that mutations in this gene lead to distinctive behavioral changes, particularly enhanced cohesion and increased shoaling behavior . This is particularly relevant as the human IMMP2L gene has been associated with neurological conditions including Gilles de la Tourette Syndrome and Tic Disorder . The behavioral phenotypes observed in zebrafish models may represent analogous manifestations of the neural circuit disruptions underlying these human conditions.

The mechanistic relationship appears to center on mitochondrial dysfunction. Immp2l mutations impair the processing of mitochondrial proteins, leading to increased ROS production and oxidative stress in neural tissues . This mitochondrial dysregulation is increasingly recognized as a pathogenic mechanism in various neurodevelopmental and neurodegenerative disorders in humans.

Importantly, zebrafish models allow for high-throughput behavioral analysis that would be challenging in mammalian models. The application of computer vision and unsupervised machine learning to quantify collective behaviors provides a sophisticated approach to phenotyping that can detect subtle behavioral alterations resulting from genetic mutations . This approach enables researchers to identify potential therapeutic targets and test compounds that might mitigate the neurological manifestations of immp2l dysfunction.

What are the implications of immp2l research for understanding aging and age-related pathologies?

Research on immp2l has significant implications for understanding the molecular mechanisms of aging and age-related pathologies:

Studies have demonstrated that immp2l mutation increases oxidative stress in multiple organs and leads to premature onset of aging-associated phenotypes including wasting, sarcopenia, loss of subcutaneous fat, kyphosis, and ataxia . This direct evidence supports the mitochondrial theory of aging, which posits that accumulated mitochondrial ROS damage drives the aging process.

Particularly notable is the finding that female mutants show earlier onset and more severe age-associated disorders than male mutants, suggesting important sex-specific differences in mitochondrial biology and aging processes . This sexual dimorphism mirrors observations in human aging and age-related diseases, where prevalence and progression often differ between sexes.

The research also highlights the potential role of stem cell dysfunction in aging. Adipose-derived stromal cells from immp2l mutant mice showed impaired proliferation capability and formed significantly fewer and smaller colonies, despite retaining their differentiation capability . This suggests that mitochondrial ROS damage to adult stem cells could be one mechanism through which immp2l dysfunction accelerates aging, potentially explaining the systemic effects observed.

These findings provide a mechanistic framework linking mitochondrial proteostasis, ROS production, and stem cell maintenance to the progression of aging, offering potential targets for interventions aimed at extending healthy lifespan.

How can immp2l be targeted for potential therapeutic interventions in mitochondrial diseases?

Based on current research, several approaches for targeting immp2l in therapeutic interventions for mitochondrial diseases can be considered:

• Antioxidant Therapies:

  • Mitochondria-targeted antioxidants could potentially mitigate the increased ROS production resulting from immp2l dysfunction

  • Compounds such as MitoQ, SkQ1, or SS-31 that specifically target mitochondria may prove more effective than general antioxidants

• Gene Therapy Approaches:

  • AAV-mediated delivery of functional immp2l could restore proper mitochondrial protein processing in affected tissues

  • CRISPR-based approaches could potentially correct immp2l mutations in specific tissues

• Small Molecule Modulators:

  • High-throughput screening could identify compounds that enhance residual immp2l activity or compensate for its loss

  • Chemical chaperones might improve the folding/function of mutated immp2l protein

• Mitochondrial Transplantation:

  • Emerging techniques for mitochondrial transplantation could potentially restore function in tissues severely affected by immp2l deficiency

• Metabolic Bypassing Strategies:

  • Alternative metabolic pathways could be enhanced to compensate for compromised mitochondrial function

  • Ketogenic diets or metabolic intermediates might provide alternative energy sources for affected tissues

The zebrafish model provides an excellent platform for screening potential therapeutic compounds due to its amenability to high-throughput analysis, transparency during development, and conservation of mitochondrial biology with humans . Initial testing could evaluate compounds' ability to rescue the increased superoxide production and behavioral alterations characteristic of immp2l mutants before advancing to mammalian models.

What are the key methodological challenges in studying immp2l function in zebrafish models?

Researchers face several significant methodological challenges when studying immp2l function in zebrafish models:

• Phenotypic Variability:

  • Immp2l deficiency can produce variable phenotypes depending on genetic background and environmental conditions

  • Standardizing housing conditions, diet, and experimental timing is crucial for reproducible results

  • Larger sample sizes may be needed to account for inherent biological variability

• Developmental Compensation:

  • Zebrafish possess remarkable compensatory mechanisms that may mask the effects of immp2l knockout

  • Distinguishing between true negative results and compensatory adaptations requires careful experimental design

  • Analysis of related pathways and potential compensatory genes is advisable

• Mitochondrial Assay Limitations:

  • Measuring mitochondrial function in vivo in zebrafish presents technical challenges

  • Tissue-specific differences in mitochondrial density and function require targeted approaches

  • Integration of multiple complementary assays (membrane potential, ROS production, respiratory capacity) provides more comprehensive assessment

• Behavioral Analysis Complexities:

  • Collective behaviors require sophisticated tracking and analysis systems

  • Environmental factors (light, temperature, tank size) can significantly impact behavioral outcomes

  • Standardization of behavioral testing conditions is essential for reproducibility

• Translating Findings Across Species:

  • While immp2l function is conserved, differences in tissue-specific expression and redundancy may exist between zebrafish and mammals

  • Validation of key findings in mammalian models may be necessary for translational relevance

Addressing these challenges requires rigorous experimental design, appropriate controls, and integration of multiple methodological approaches to provide robust insights into immp2l function.

How can contradictory findings in immp2l research be reconciled and interpreted?

Contradictory findings in immp2l research can be systematically reconciled through several analytical approaches:

• Context-Dependent Effects Analysis:

  • Compare experimental conditions between studies (age of animals, genetic background, environmental factors)

  • Analyze tissue-specific differences in immp2l expression and function

  • Consider that heterozygous (immp2l+/-) and homozygous (immp2l-/-) mutations may produce distinct phenotypes

• Methodological Variation Assessment:

  • Evaluate differences in knockout strategies (e.g., complete gene deletion vs. point mutations)

  • Compare protein detection methods (antibodies, detection limits, etc.)

  • Assess differences in functional assays (e.g., variations in ROS detection methods)

• Phenotypic Spectrum Mapping:

  • Create a comprehensive phenotypic map across multiple studies

  • Identify consistent core phenotypes versus variable secondary effects

  • Consider that seemingly contradictory findings may represent different aspects of a complex phenotypic spectrum

• Sex-Specific Analysis:

  • Separate analysis of male and female subjects, as research has shown significant sex differences in immp2l mutation effects

  • Evaluate hormonal influences on mitochondrial function and oxidative stress responses

• Temporal Dynamics Consideration:

  • Age-dependent effects may explain apparent contradictions

  • Acute versus chronic responses to immp2l deficiency may differ substantially

  • Developmental timing of immp2l function may influence phenotypic outcomes

• Compensatory Mechanism Identification:

  • Investigate potential compensatory pathways that may be differentially activated across studies

  • Evaluate expression of related mitochondrial proteases like immp1l that might partially compensate for immp2l loss

This systematic approach allows researchers to integrate seemingly contradictory findings into a more comprehensive understanding of immp2l biology, recognizing that variations may reflect biological complexity rather than experimental error.

What are the most promising future directions for immp2l research in zebrafish models?

Future immp2l research in zebrafish models holds significant promise in several key directions:

• Tissue-Specific Conditional Knockouts: Developing tissue-specific and temporally controlled immp2l knockout systems would help dissect the differential roles of this protein across various organs and developmental stages. This approach could clarify whether the diverse phenotypes observed result from primary effects in specific tissues or secondary consequences of systemic mitochondrial dysfunction .

• Integration with Omics Approaches: Combining immp2l manipulation with transcriptomics, proteomics, and metabolomics analyses would provide comprehensive insights into the molecular networks affected by immp2l dysfunction. This systems biology approach could identify novel therapeutic targets and biomarkers for related human conditions .

• Expanded Behavioral Phenotyping: Further refinement of the computer vision and machine learning approaches to behavioral analysis could reveal subtle phenotypes that connect mitochondrial dysfunction to specific neural circuit alterations. This could establish zebrafish as a premier model for high-throughput screening of compounds targeting neurological manifestations of mitochondrial disorders .

• Aging Research Applications: Given the clear connection between immp2l dysfunction and accelerated aging phenotypes, longitudinal studies of immp2l-deficient zebrafish could provide valuable insights into the progression of age-related changes. This could establish immp2l mutants as a useful model for testing interventions aimed at extending healthy lifespan through mitochondrial targeting .

• Therapeutic Screening Platforms: Developing standardized assays in immp2l-deficient zebrafish for high-throughput screening of compounds that mitigate mitochondrial dysfunction, reduce ROS production, or rescue behavioral phenotypes would accelerate the discovery of potential therapeutics for related human disorders .