Cyclophilin A Mouse

What is the molecular structure and function of mouse Cyclophilin A?

Mouse Cyclophilin A is a 164 amino acid protein with approximately 17-18 kDa molecular weight that functions as a peptidyl-prolyl cis-trans isomerase. It catalyzes the transition between cis and trans conformations of proline residues, which is critical for proper protein folding . The protein is ubiquitously expressed and has a predicted molecular weight of 17 kDa. Mouse CyPA shares approximately 95% amino acid identity with human CyPA, making it an excellent model for studying human-relevant mechanisms . Beyond its isomerase activity, CyPA functions as a proinflammatory cytokine when secreted in response to inflammatory stimuli .

How does mouse Cyclophilin A compare structurally and functionally to human Cyclophilin A?

Mouse and human CyPA share 95% amino acid identity, indicating high evolutionary conservation of this protein . This high homology extends to functional aspects, with both species' proteins demonstrating similar peptidyl-prolyl isomerase activity, interactions with Cyclosporin A, and roles in inflammatory processes . Both proteins have comparable molecular weights (approximately 17-18 kDa) and can be detected using cross-reactive antibodies . The high degree of conservation makes mouse models particularly valuable for studying CyPA-related human diseases, including cardiovascular disorders, viral infections, and neurodegenerative conditions .

What experimental techniques are available for basic characterization of mouse Cyclophilin A expression?

Several validated methods exist for characterizing mouse CyPA expression:

• Western blot analysis using specific antibodies - detects CyPA at approximately 18 kDa in mouse tissues and cell lines (e.g., C2C12 myoblasts)

• Immunofluorescence staining - allows visualization of CyPA distribution in mouse cells and tissues with appropriate antibodies and controls

• ELISA assays - quantify secreted CyPA in mouse plasma or culture supernatants

• RT-qPCR - measures CyPA mRNA expression levels in mouse tissues

• Recombinant protein expression - full-length mouse CyPA (amino acids 1-164) can be expressed in E. coli with >95% purity for functional studies

When performing these analyses, it's critical to include appropriate controls and standardized protocols to ensure reproducibility.

What genetic mouse models are available for studying Cyclophilin A function?

Several genetic mouse models have been developed to investigate CyPA function:

• Global CyPA knockout mice - complete deletion of CyPA expression across all tissues, useful for studying systemic effects

• Conditional knockout models - tissue-specific CyPA deletion using Cre-loxP systems

• VSMC-specific CyPA overexpressing transgenic mice (VSMC-Tg) - overexpress CyPA specifically in vascular smooth muscle cells to study cardiovascular effects

• Point mutation models - harbor specific mutations affecting CyPA activity or regulation

• Reporter mice - express fluorescent or luminescent proteins under CyPA promoter control

These models have revealed crucial roles of CyPA in various pathological processes, including vascular stenosis, atherosclerosis, and abdominal aortic aneurysm formation . When selecting a model, researchers should consider the specific research question, potential developmental effects, and available facilities for maintaining these specialized mouse lines.

How can researchers effectively distinguish between intracellular and extracellular functions of Cyclophilin A in mouse models?

Distinguishing between intracellular and extracellular CyPA functions requires multiple complementary approaches:

• Cell-specific genetic deletion - Compare phenotypes of tissue-specific knockout models to determine source and target cells

• Exogenous administration - Apply purified recombinant mouse CyPA protein (aa 1-164) to isolate extracellular effects

• Non-cell-permeable inhibitors - Use antibodies or large molecular weight inhibitors that cannot enter cells

• Secretion-deficient mutants - Generate mice expressing CyPA variants that cannot be secreted but retain intracellular function

• Receptor knockout models - Delete putative CyPA receptors like EMMPRIN/CD147 to block extracellular signaling

• Compartmental analysis - Measure CyPA levels in both cellular extracts and culture media/plasma

• Proximity assays - Use techniques like proximity ligation to visualize specific interaction partners in different compartments

These approaches have revealed that intracellular CyPA primarily functions as a chaperone facilitating protein folding, while extracellular CyPA acts as a signaling molecule activating inflammatory and proliferative pathways .

What are optimal protocols for isolating and purifying mouse Cyclophilin A for functional studies?

For high-quality mouse CyPA isolation:

• Recombinant expression system:

  • Clone full-length mouse CyPA (aa 1-164) into a bacterial expression vector with appropriate tag (e.g., His-tag)

  • Express in E. coli with IPTG induction

  • Purify using affinity chromatography (e.g., Ni-NTA for His-tagged proteins)

  • Verify purity (>95%) by SDS-PAGE and confirm identity by mass spectrometry

  • Remove tags if necessary and perform activity assays to confirm functionality

• Native protein isolation:

  • Homogenize mouse tissues or lyse cultured cells in appropriate buffer

  • Perform immunoprecipitation using anti-CyPA antibodies

  • Elute under gentle conditions to preserve activity

  • Confirm identity by Western blot and mass spectrometry

• Quality control measures:

  • Endotoxin testing for recombinant preparations

  • Peptidyl-prolyl isomerase activity assay

  • Binding assays with known partners (e.g., Cyclosporin A)

  • Functional testing in cell culture before in vivo application

These protocols yield functionally active mouse CyPA suitable for mechanistic studies, structural analysis, and functional assays.

What detection methods and antibodies work reliably for mouse Cyclophilin A in different experimental contexts?

Reliable detection methods for mouse CyPA include:

• Western blot analysis:

  • Goat Anti-Human/Mouse/Rat CyPA Antigen Affinity-purified Polyclonal Antibody (e.g., R&D Systems AF3589) at 0.1 μg/mL

  • Detection of specific band at approximately 18 kDa in mouse cell lines (C2C12 myoblasts)

  • Reducing conditions with appropriate immunoblot buffer systems

• Immunofluorescence/Immunohistochemistry:

  • Same antibodies as for Western blot, typically at 10 μg/mL concentration

  • Appropriate secondary antibodies (e.g., Northern-Lights™ 557-conjugated Anti-Goat IgG)

  • Careful optimization of fixation and permeabilization conditions

• Flow cytometry:

  • Cell permeabilization required for intracellular CyPA

  • Surface staining protocols for detecting cell-surface bound CyPA

• ELISA:

  • Commercial kits available specifically for mouse CyPA

  • Development of in-house sandwich ELISA using capture and detection antibodies

When selecting detection methods, researchers should include appropriate positive controls (recombinant CyPA) and negative controls (CyPA-knockout samples) to validate specificity .

What experimental design considerations are critical when studying Cyclophilin A in inflammatory mouse disease models?

Critical experimental design considerations include:

• Control groups:

  • Age and sex-matched wild-type controls

  • Appropriate genetic background controls if using transgenic models

  • Vehicle controls for pharmacological interventions

  • Sham-operated controls for surgical models

• Timing considerations:

  • Acute vs. chronic inflammation models

  • Age-dependent effects (CyPA function changes with aging)

  • Time course experiments to capture dynamic changes

• Cell type-specific analyses:

  • Isolation of relevant cell populations from tissues

  • Single-cell analysis techniques to account for heterogeneity

  • Co-localization studies to identify CyPA-producing and responding cells

• Mechanistic interventions:

  • Pharmacological inhibition using CyPA inhibitors

  • ROS modulation (antioxidants or pro-oxidants) as CyPA secretion is ROS-dependent

  • Receptor blocking (e.g., anti-EMMPRIN antibodies)

• Readouts:

  • Multiple inflammation markers beyond CyPA levels

  • Functional outcomes relevant to the disease model

  • Both tissue and systemic parameters

These considerations help establish causality in CyPA-mediated pathologies rather than just correlative findings.

What are common technical challenges in quantifying mouse Cyclophilin A expression and activity, and how can they be addressed?

Common challenges and solutions include:

• Cross-reactivity issues:

  • Challenge: Antibodies may detect other cyclophilin family members

  • Solution: Validate antibody specificity using CyPA knockout tissues; perform immunodepletion studies

• Post-translational modifications:

  • Challenge: Modified forms of CyPA may affect detection or activity

  • Solution: Use multiple antibodies targeting different epitopes; employ mass spectrometry to identify modifications

• Sample preparation variability:

  • Challenge: CyPA levels can change during sample processing

  • Solution: Standardize collection protocols; include stability controls; process all samples simultaneously

• Activity measurement challenges:

  • Challenge: Environmental factors affect enzymatic activity assays

  • Solution: Include internal standards; perform assays under controlled temperature and pH; use recombinant CyPA standards

• Low signal-to-noise ratio in tissue samples:

  • Challenge: Abundant proteins mask CyPA detection

  • Solution: Perform enrichment steps; use more sensitive detection methods like mass spectrometry

• Secreted vs. intracellular pools:

  • Challenge: Distinguishing origin of CyPA in complex samples

  • Solution: Perform subcellular fractionation; use brefeldin A to block secretion; analyze both cellular and supernatant fractions

Addressing these challenges ensures more reliable and reproducible quantification of CyPA expression and activity in mouse models.

How should researchers interpret apparent data contradictions between different mouse models of Cyclophilin A function?

When faced with contradictory data, researchers should systematically analyze potential sources of discrepancy:

• Genetic background differences:

  • Different mouse strains may have modifier genes affecting CyPA function

  • Solution: Backcross to common background or use multiple backgrounds to test robustness

• Developmental vs. acute effects:

  • Global knockout models may trigger compensatory mechanisms

  • Solution: Use inducible systems to distinguish acute from developmental effects

• Tissue-specific functions:

  • CyPA may have opposing functions in different tissues

  • Solution: Use tissue-specific genetic models; perform tissue-specific analyses

• Disease model differences:

  • CyPA may function differently in acute vs. chronic pathologies

  • Solution: Compare results across multiple disease models; perform time-course studies

• Technical differences:

  • Variations in assay methods, antibodies, or protocols

  • Solution: Standardize techniques; repeat key experiments with identical protocols

• Sex-specific effects:

  • CyPA function may differ between male and female mice

  • Solution: Analyze sexes separately; ensure balanced experimental design

By systematically addressing these factors, apparent contradictions often resolve into a more nuanced understanding of context-dependent CyPA functions in different physiological and pathological settings .

How does Cyclophilin A contribute to cardiovascular pathology in mouse models, and what methodologies best capture these effects?

CyPA plays multiple roles in cardiovascular pathology:

• Mechanisms of action:

  • ROS trigger CyPA secretion from vascular smooth muscle cells (VSMCs)

  • Secreted CyPA activates ERK1/2, Akt, and JAK signaling pathways

  • CyPA stimulates VSMC proliferation and inflammatory cell migration

  • CyPA promotes atherosclerotic plaque formation by stimulating CD34+ progenitor cell differentiation to foam cells

  • CyPA induces matrix metalloproteinase activation via EMMPRIN binding

• Optimal methodologies:

  • Genetic models: Compare CyPA knockout, VSMC-specific overexpression, and wild-type mice

  • Vascular injury models: Wire injury, carotid ligation, or angiotensin II infusion

  • Atherosclerosis models: ApoE-deficient background with high-fat diet

  • Aneurysm models: Angiotensin II infusion in susceptible backgrounds

  • Analytical techniques: Vessel morphometry, en face lesion quantification, immunohistochemistry for inflammatory markers

  • Functional assessments: Vascular reactivity studies, blood pressure measurements, echocardiography

• Key experimental controls:

  • ROS modulation: Antioxidants to block CyPA secretion

  • CyPA neutralization: Antibodies against secreted CyPA

  • Pathway inhibition: Blockers of downstream signaling cascades

These approaches have established CyPA as a critical mediator of vascular stenosis, atherosclerosis, and abdominal aortic aneurysm in mouse models .

What methodological approaches are most effective for studying Cyclophilin A's role in viral infection models using mice?

Effective methodological approaches include:

• Infection models:

  • Viral challenge models with CyPA-dependent viruses (adaptations of HIV-1, HCV)

  • Transgenic mouse models expressing human virus receptors if mouse cells lack susceptibility

  • Humanized mouse models for human-specific viral infections

• Genetic manipulation approaches:

  • CyPA knockout mice to evaluate necessity

  • Knock-in mice with CyPA mutations that disrupt specific viral interactions

  • Cell type-specific CyPA deletion to identify key cellular sources

• Mechanistic analyses:

  • Viral entry assays using labeled viruses and flow cytometry

  • Viral replication measurements (qPCR, plaque assays)

  • Co-immunoprecipitation to assess CyPA-viral protein interactions

  • Subcellular localization studies to track CyPA incorporation into viral particles

  • Pharmacological inhibition using CyPA inhibitors (non-immunosuppressive cyclosporin derivatives)

• Readouts:

  • Viral load quantification in tissues

  • Immune response parameters

  • Tissue pathology and inflammation markers

  • Survival and clinical disease scores

These approaches help delineate CyPA's role in viral assembly, replication, and infectivity for viruses such as HIV-1 and Hepatitis C, where CyPA is known to be incorporated into viral particles .

What experimental approaches best elucidate Cyclophilin A's role in neurodegenerative disease mouse models?

Optimal experimental approaches include:

• Neurodegenerative disease models:

  • Alzheimer's disease: ApoE4 transgenic mice (CyPA-induced blood-brain barrier disruption is implicated)

  • Other models: Parkinson's disease, amyotrophic lateral sclerosis, traumatic brain injury models

• Blood-brain barrier (BBB) assessment techniques:

  • Evans blue extravasation assay

  • Fluorescent tracer penetration studies

  • Immunohistochemistry for tight junction proteins

  • Electron microscopy for ultrastructural BBB analysis

  • Dynamic contrast-enhanced MRI

• Neuroinflammation analyses:

  • Microglia and astrocyte activation markers

  • Inflammatory cytokine profiling

  • Immune cell infiltration quantification

  • Single-cell RNA sequencing of neurovascular unit components

• Genetic and pharmacological interventions:

  • Neuron-specific or astrocyte-specific CyPA knockout models

  • CyPA inhibitors with BBB penetration capability

  • ApoE isoform-dependent effects (comparing E3 vs. E4 backgrounds)

• Functional assessments:

  • Cognitive testing batteries

  • Electrophysiological measurements

  • Neuroimaging (PET, MRI)

  • Behavioral assessments relevant to the specific disease model

These approaches have revealed that CyPA-induced disruption of blood-brain barrier function contributes to the increased risk of developing Alzheimer's disease, particularly in individuals with ApoE4 alleles, with similar mechanisms likely operating in mouse models .

What research design elements are essential when using mouse Cyclophilin A studies to inform human disease understanding?

Essential research design elements include:

• Comparative biology approach:

  • Parallel studies of mouse and human CyPA to confirm conserved functions

  • Assessment of species-specific differences in regulatory pathways

  • Confirmation that mouse phenotypes reflect human disease characteristics

• Validation in human samples:

  • Correlative studies measuring CyPA levels in patient samples

  • Ex vivo functional studies using human tissues

  • Analysis of CyPA polymorphisms in human populations

• Pharmacological validation:

  • Testing CyPA-targeting compounds in both mouse models and human samples

  • Development of biomarkers that work across species

  • Dose-scaling considerations between mouse and human

• Disease-relevant endpoints:

  • Selection of mouse outcomes that match clinical parameters

  • Longitudinal studies mirroring human disease progression

  • Inclusion of comorbidities relevant to human conditions

• Mechanistic depth:

  • Multi-omics approaches to capture system-wide effects

  • Pathway analysis to identify conserved mechanisms

  • Network biology approaches to understand context-dependent functions

These design elements strengthen the translational value of mouse CyPA research for understanding and treating human inflammatory diseases, cardiovascular disorders, viral infections, and neurodegenerative conditions .

How can researchers develop effective pre-clinical therapeutic strategies targeting Cyclophilin A based on mouse model findings?

Developing effective pre-clinical strategies requires:

• Target validation approach:

  • Confirm CyPA's role using genetic models (knockout and overexpression)

  • Validate timing of intervention (preventive vs. therapeutic)

  • Determine tissue-specific requirements for inhibition

  • Establish dose-response relationships

• Therapeutic modality selection:

  • Small molecule CyPA inhibitors (non-immunosuppressive cyclosporin derivatives)

  • Neutralizing antibodies against extracellular CyPA

  • Gene therapy approaches (siRNA, antisense oligonucleotides)

  • Indirect approaches targeting CyPA secretion (antioxidants) or receptors (anti-EMMPRIN)

• Preclinical development considerations:

  • PK/PD studies optimized for the therapeutic modality

  • Multiple disease models to establish broad efficacy

  • Combination approaches with standard-of-care treatments

  • Biomarker development for target engagement

  • Safety assessment focusing on cyclophilin family selectivity

• Translational biomarkers:

  • Circulating CyPA levels

  • Downstream signaling pathway activation

  • Disease-specific functional improvements

  • Imaging approaches for target organs

These strategies have shown promise in preclinical models for various diseases including cardiovascular disorders, where CyPA inhibition reduced vascular inflammation, remodeling, and disease progression .

What proteomics and interactomics approaches are most informative for studying mouse Cyclophilin A functions?

Most informative approaches include:

• Protein-protein interaction analysis:

  • Immunoprecipitation coupled with mass spectrometry

  • Proximity labeling techniques (BioID, APEX) to capture transient interactions

  • Yeast two-hybrid screening followed by co-immunoprecipitation validation

  • Protein arrays to identify novel binding partners

• Post-translational modification mapping:

  • Phosphoproteomics to identify CyPA phosphorylation sites and modified interaction partners

  • Redox proteomics to capture oxidation states relevant to CyPA function

  • Global PTM profiling before and after CyPA manipulation

• Structural proteomics:

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Crosslinking mass spectrometry to capture complex formation

  • Native mass spectrometry to analyze intact complexes

• Dynamic interactomics:

  • Temporal analysis of CyPA interactions during disease progression

  • Stimulus-dependent interaction changes (e.g., after oxidative stress)

  • Cell type-specific interactome mapping

• Computational integration:

  • Network analysis of CyPA-centered protein interactions

  • Pathway enrichment of identified interactors

  • Comparative analysis across disease models

These techniques have revealed CyPA's diverse interaction network, including binding to calcineurin, EMMPRIN, and various components of signaling pathways like MAPK/ERK, providing mechanistic insights into its multifunctional nature .

What transcriptomic approaches best capture the downstream effects of Cyclophilin A in mouse models?

Optimal transcriptomic approaches include:

• Differential gene expression analysis:

  • RNA-seq comparing CyPA knockout vs. wild-type tissues

  • CyPA overexpression models vs. controls

  • Dose-dependent responses to exogenous CyPA treatment

  • Time-course analyses to capture primary vs. secondary effects

• Single-cell transcriptomics:

  • scRNA-seq to identify cell type-specific responses to CyPA

  • Trajectory analysis to capture differentiation changes induced by CyPA

  • Spatial transcriptomics to map responses within tissue architecture

  • Cell type deconvolution in bulk tissue data

• Pathway and network analyses:

  • Gene set enrichment analysis (GSEA) for pathway identification

  • Weighted gene co-expression network analysis (WGCNA)

  • Transcription factor activity inference

  • Causal network analysis to predict upstream regulators

• Integration with other data types:

  • Proteogenomic correlation analysis

  • Epigenomic profiling to identify regulatory mechanisms

  • Metabolomic integration for functional interpretation

• Validation approaches:

  • qRT-PCR confirmation of key targets

  • In situ hybridization for spatial verification

  • Reporter assays for transcriptional regulation studies

These approaches have revealed that CyPA regulates genes involved in inflammation, cell proliferation, extracellular matrix remodeling, and apoptosis, providing insights into its role in various pathological processes .

What emerging technologies and methodologies show promise for advancing mouse Cyclophilin A research?

Promising emerging technologies include:

• Advanced genetic engineering:

  • Base editing and prime editing for precise CyPA modifications

  • Inducible CRISPR systems for temporal control of CyPA editing

  • RNA editing approaches for reversible functional studies

  • Tissue-specific gene editing delivered by AAV vectors

• Single-cell multi-omics:

  • Integrated single-cell transcriptomics, proteomics, and metabolomics

  • Spatial multi-omics to map CyPA function within tissue architecture

  • Lineage tracing combined with functional genomics

• Advanced imaging:

  • Intravital microscopy with genetically encoded CyPA activity sensors

  • Super-resolution microscopy for subcellular localization

  • Whole-body imaging with CyPA-specific probes

  • Functional MRI combined with molecular imaging

• Organoid and microphysiological systems:

  • Organ-specific organoids from CyPA-modified stem cells

  • Multi-organ-on-chip systems to study systemic effects

  • Humanized organoids to enhance translational relevance

• Computational approaches:

  • Machine learning for predicting CyPA-dependent disease progression

  • Systems biology modeling of CyPA regulatory networks

  • Virtual screening for novel CyPA modulators

  • Digital pathology with AI-assisted quantification

These technologies will enable more precise interrogation of CyPA's functions at molecular, cellular, and organismal levels, accelerating both mechanistic understanding and therapeutic development for CyPA-related diseases.

What key knowledge gaps remain in understanding mouse Cyclophilin A biology that require methodological innovation?

Key knowledge gaps requiring methodological innovation include:

• Temporal and spatial dynamics:

  • Real-time tracking of CyPA secretion, diffusion, and signaling

  • Understanding tissue-specific roles in complex disease models

  • Mapping the CyPA interactome with spatiotemporal resolution

• Isoform-specific functions:

  • Differentiating roles of post-translationally modified CyPA forms

  • Understanding potential alternative splicing variants

  • Developing isoform-specific detection and inhibition methods

• Intercellular communication:

  • Identifying all cell types that respond to secreted CyPA

  • Characterizing extracellular vesicle-associated CyPA transport

  • Mapping the complete receptor repertoire for CyPA

• Therapeutic targeting challenges:

  • Achieving tissue-specific CyPA inhibition

  • Differentiating between beneficial and detrimental CyPA functions

  • Developing biomarkers for CyPA activity in vivo

• Systems-level understanding:

  • Integrating CyPA function into larger regulatory networks

  • Understanding compensatory mechanisms after CyPA inhibition

  • Modeling CyPA's role in complex disease progression

Addressing these gaps will require combinatorial approaches integrating genetics, biochemistry, cell biology, and systems biology methodologies to fully understand CyPA's multifaceted roles in health and disease .