PEBP1 Mouse

What is PEBP1 and why is it important in mouse model research?

PEBP1 (Phosphatidylethanolamine-binding protein 1) is a multifunctional protein that acts as a pivotal regulator of the mitochondrial component of integrated stress response (ISR). It plays critical roles in amplifying stress signals, particularly those originating from mitochondria. Mouse models are essential for studying PEBP1 as they allow researchers to investigate its role in physiological and pathological conditions in a mammalian system that closely resembles human biology. PEBP1 is abundantly expressed in most mouse tissues, with particularly high expression in the brain, making mouse models valuable for studying its neurological implications . The protein's involvement in the RAF/MEK/ERK pathway, mitochondrial dysfunction responses, and various disease states makes it a significant target for translational research in metabolism, neurodegeneration, and stress response mechanisms.

How is PEBP1 expression distributed across different mouse tissues?

PEBP1 displays a distinct expression pattern across mouse tissues, with spatial transcriptomics data revealing particularly high expression in the brain. The protein is also widely expressed across most, if not all, mouse tissues, suggesting its fundamental role in cellular functions. In particular, hippocampal expression of PEBP1 has been documented, where it is known as the precursor of hippocampal cholinergic neurostimulating peptide . In mouse models of metabolic disorders, such as db/db mice (a model for diabetes), PEBP1 has been found to be highly expressed . The differential expression pattern of PEBP1 across tissues provides valuable insights into tissue-specific functions and potential therapeutic targets. For researchers, understanding this distribution is crucial when designing experiments focused on specific physiological or pathological processes.

What are the key functional differences between total PEBP1 and phosphorylated PEBP1 in mouse models?

Total PEBP1 and its phosphorylated form (particularly at Ser-153) demonstrate distinctly different functional roles in mouse models. When PEBP1 is phosphorylated at Ser-153 by protein kinase C (PKC), it dissociates from Raf-1, which consequently activates the ERK cascade . This phosphorylation-dependent relationship serves as a critical molecular switch in signaling pathways. In animal models of depression, total PEBP1 levels increase while phosphorylated PEBP1 (Ser-153) decreases, suggesting that this balance may play a role in neuropsychiatric conditions . This distinction is important because the unphosphorylated form of PEBP1 inhibits the RAF/MEK/ERK pathway, whereas the phosphorylated form does not. Additionally, recent research indicates that PEBP1 influences the integrated stress response independently of its role in the RAF/MEK/ERK pathway, providing another layer of regulatory control in cellular stress mechanisms .

What are the recommended protocols for genetically manipulating PEBP1 expression in mouse models?

When genetically manipulating PEBP1 expression in mouse models, researchers should consider several methodological approaches based on research objectives:

For PEBP1 knockout studies:

• CRISPR-Cas9 system has proven effective for generating PEBP1 knockout mice, targeting exons to create functional disruption.

• Conditional knockout approaches using Cre-loxP systems are valuable for tissue-specific or inducible PEBP1 deletion, particularly important given PEBP1's widespread expression.

• Validation of knockout efficiency should include both protein expression analysis via Western blotting and functional assessments such as evaluation of downstream pathways (e.g., ERK activation) .

For PEBP1 overexpression:

• Viral vector delivery (particularly AAV) with tissue-specific promoters allows targeted overexpression, though caution is warranted as AAV can cause liver injury in diabetic and obese mouse models .

• Transgenic approaches using tissue-specific promoters (e.g., neuron-specific for brain studies) are preferable for developmental studies.

For studying specific PEBP1 functions:

• Point mutation knockin approaches (particularly at Ser-153) can be used to investigate phosphorylation-dependent functions .

• Protein-protein interaction studies can be conducted using NanoBiT luminescence complementation assays, which have successfully detected PEBP1-eIF2α interactions in vivo .

When reporting results, researchers should thoroughly document genetic background, breeding schemes, and validate genetic manipulations through multiple techniques.

What are the most accurate methods for quantifying PEBP1 expression and activity in mouse tissues?

For rigorous quantification of PEBP1 expression and activity in mouse tissues, researchers should employ multiple complementary approaches:

Protein Expression Quantification:

• Western blotting remains the gold standard for comparing total PEBP1 and phosphorylated PEBP1 levels, with careful consideration of loading controls and phospho-specific antibodies (particularly for Ser-153) .

• Immunohistochemistry/immunofluorescence for spatial distribution analysis within tissues, especially valuable in heterogeneous tissues like brain.

• Mass spectrometry-based approaches, such as MS-CETSA (cellular thermal shift assay), can detect thermal stabilization of PEBP1 during stress responses .

Activity Assessment:

• Functional readouts of PEBP1 activity include measuring:

  • eIF2α phosphorylation levels, which correlate with PEBP1's role in ISR activation

  • Global protein synthesis rates using L-homopropargylglycine (HPG) incorporation (preferred over puromycin assay in energy-starved cells)

  • ERK pathway activation (as PEBP1 inhibits RAF/MEK/ERK signaling)

mRNA Expression Analysis:

• RT-qPCR for PEBP1 transcript levels, with careful primer design to detect potential splice variants

• RNA-seq for comprehensive transcriptome analysis and pathway correlations

• Spatial transcriptomics for tissue-specific expression patterns

For maximum reliability, researchers should triangulate findings using multiple methods and include appropriate positive and negative controls, such as PEBP1 knockout tissues and pharmacological manipulation of PEBP1's downstream pathways.

How can researchers effectively induce and measure mitochondrial stress to study PEBP1 function in mice?

Effective induction and measurement of mitochondrial stress for PEBP1 functional studies in mice requires systematic approaches:

Mitochondrial Stress Induction Methods:

• Pharmacological approaches:

  • ATP synthase inhibitors (oligomycin, bedaquiline) effectively trigger mitochondrial ISR by targeting oxidative phosphorylation

  • Complex I inhibitors (rotenone) disrupt electron transport chain function

  • Uncouplers (BAM15) can dissipate mitochondrial membrane potential for comparative studies

• Genetic approaches:

  • siRNA knockdown of mitochondrial components (e.g., ATP5F1A)

  • Expression of mutant mitochondrial proteins

  • Tissue-specific deletion of essential mitochondrial genes

Measurement Parameters:

• Assessment of mitochondrial morphology:

  • Confocal microscopy to visualize mitochondrial fragmentation

  • Electron microscopy for ultrastructural analysis

• Functional assessments:

  • Oxygen consumption rate (OCR) measurements to evaluate respiratory capacity and ATP production

  • Mitochondrial membrane potential using TMRE staining

  • Extracellular acidification rate (ECAR) to assess glycolytic activity as a compensatory response

• Molecular markers of mitochondrial stress:

  • eIF2α phosphorylation levels (Western blotting)

  • ISR target gene expression (RT-qPCR or RNA-seq)

  • DELE1 cleavage (in DELE1-HA knock-in models)

  • Changes in global protein synthesis rates

When designing these experiments, researchers should include appropriate controls, such as PEBP1 knockout and wild-type comparisons, and assess multiple parameters simultaneously to comprehensively evaluate mitochondrial stress responses mediated by PEBP1.

How does PEBP1 regulate the integrated stress response in mouse models of mitochondrial dysfunction?

PEBP1 serves as a critical amplifier of mitochondrial stress signals in the integrated stress response (ISR) pathway through several mechanistic steps:

• Stress signal detection: Upon mitochondrial dysfunction (induced by inhibitors like oligomycin or genetic perturbations), PEBP1 undergoes thermal stabilization as detected by MS-CETSA, indicating its activation in response to stress conditions .

• Interaction with eIF2α: PEBP1 directly interacts with eIF2α, as demonstrated through NanoBiT luminescence complementation assays. This interaction is disrupted upon eIF2α phosphorylation at Ser-51, suggesting a dynamic regulatory mechanism .

• Amplification of HRI kinase activity: PEBP1 potentiates ISR activation by enhancing the activity of heme-regulated inhibitor (HRI) kinase, the principal eIF2α kinase in the mitochondrial ISR pathway . This amplification is particularly important for mitochondrial stress, which generates relatively weak ISR signals compared to strong ER stressors like tunicamycin.

• Pathway specificity: PEBP1's role in ISR is independent of its canonical function as a RAF kinase inhibitor, as MEK inhibition with trametinib did not rescue the effects of PEBP1 knockout on eIF2α phosphorylation or ISR target gene activation .

• Selective stress response regulation: PEBP1 appears to primarily amplify mitochondrial stress signals rather than all ISR-activating stresses. It has minimal effects on tunicamycin-induced ER stress but significantly influences responses to mitochondrial perturbations and certain viral nucleic acid mimetics (poly(I:C)) .

This regulatory role positions PEBP1 as a critical component that ensures appropriate cellular responses to mitochondrial dysfunction, particularly important in tissues with high metabolic demands.

What is the relationship between PEBP1 and the OMA1-DELE1-HRI mitochondrial stress pathway in mice?

The relationship between PEBP1 and the OMA1-DELE1-HRI mitochondrial stress pathway represents a sophisticated signaling mechanism that ensures appropriate cellular responses to mitochondrial dysfunction:

• Sequential positioning in the pathway: PEBP1 functions downstream of the initial mitochondrial stress sensing by OMA1 and DELE1. Mitochondrial stress activates the mitochondrial protease OMA1, which cleaves and activates DELE1, allowing its translocation to the cytosol where it activates HRI kinase .

• PEBP1 does not affect upstream events: PEBP1 knockout does not impair:

  • Mitochondrial fragmentation induced by oligomycin

  • Mitochondrial hyperpolarization (measured by TMRE staining)

  • DELE1 cleavage by OMA1

• Signal amplification function: PEBP1 appears to act as an amplifier of the relatively weak mitochondrial stress signals. This amplification is particularly important given the abundance differences between pathway components - PEBP1 is approximately 6000 times more abundant than DELE1 in HeLa cells .

• HRI kinase potentiation: PEBP1 overexpression potentiates ISR activation by HRI kinase, suggesting a direct influence on HRI kinase activity or its interaction with eIF2α .

• Interaction with eIF2α: PEBP1 directly interacts with eIF2α as demonstrated by the NanoBiT luminescence complementation assay. This interaction is disrupted by eIF2α phosphorylation at Ser-51, suggesting a dynamic regulatory mechanism that may influence HRI kinase's access to eIF2α .

This relationship demonstrates how PEBP1 serves as a critical link between the initial detection of mitochondrial stress and the resulting ISR, ensuring that authentic mitochondrial dysfunction signals are appropriately amplified while preventing responses to noise or transient fluctuations.

How do different types of cellular stress affect PEBP1 function in mouse models?

Different types of cellular stress elicit distinct effects on PEBP1 function in mouse models, revealing its specialized role in stress response pathways:

• Mitochondrial dysfunction stress:

  • Mitochondrial stressors (oligomycin, bedaquiline, ATP5F1A siRNA, rotenone) lead to thermal stabilization of PEBP1

  • PEBP1 is required for efficient eIF2α phosphorylation and subsequent ISR gene expression in response to these stressors

  • PEBP1 knockout partially rescues protein synthesis rates during oligomycin treatment, indicating its role in translation regulation during mitochondrial stress

• ER stress:

  • PEBP1 shows minimal involvement in tunicamycin-induced ER stress responses, as PEBP1 knockout does not prevent eIF2α phosphorylation or protein synthesis inhibition during tunicamycin treatment

  • This suggests pathway specificity with PEBP1 being more involved in mitochondrial rather than ER stress responses

• Viral-mimetic stress:

  • PEBP1 influences ISR activation by viral nucleic acid mimicking poly(I:C), which activates ISR via PKR kinase

  • This indicates PEBP1's role extends beyond mitochondrial stress to certain inflammatory and innate immune responses

• Metabolic stress:

  • In db/db mice (models of diabetes) and high-fat diet-induced obesity models, PEBP1 is highly expressed

  • Knockdown of PEBP1 alleviates hepatic injury and necroptosis induced by recombinant AAV in mice with diabetes and obesity, suggesting PEBP1 may promote liver damage in metabolic stress conditions

• Psychological stress:

  • Studies in non-human primates suggest altered PEBP1 expression and phosphorylation in depression models, with increased total PEBP1 and decreased phosphorylated PEBP1 (Ser-153)

  • This indicates PEBP1's potential role in stress-related neuropsychiatric conditions

These differential responses to various stressors highlight PEBP1's context-dependent functions and suggest that therapeutic targeting of PEBP1 may need to be tailored to specific stress conditions and tissue types.

What is the significance of PEBP1 in mouse models of depression and neurological disorders?

PEBP1 demonstrates significant relevance in mouse models of depression and neurological disorders through multiple mechanisms:

• Altered expression in depression models: Studies in primates have shown that total PEBP1 increases while phosphorylated PEBP1 (Ser-153) decreases in the hippocampus of spontaneously depressed subjects compared to normal controls . This pattern likely translates to mouse models and suggests PEBP1's involvement in mood regulation.

• Relationship with ERK signaling: The decreased phosphorylation of PEBP1 at Ser-153 observed in depression models indicates reduced activation of ERK signaling . Reduced ERK activation and expression have been detected in the postmortem brains of depressed suicide subjects, and ERK phosphorylation has been hypothesized as an intracellular signaling mechanism mediating antidepressant efficacy .

• Hippocampal function: PEBP1 is particularly abundant in the brain, where it is known as the precursor of hippocampal cholinergic neurostimulating peptide . Given the crucial role of the ISR in the hippocampus for memory formation, PEBP1 may influence cognitive processes through regulation of the integrated stress response.

• Mitochondrial stress response in neurons: PEBP1's role in amplifying mitochondrial stress signals may be particularly important in the brain due to its high metabolic demands and vulnerability to mitochondrial dysfunction . This becomes especially relevant in neurodegenerative disorders characterized by mitochondrial impairment.

• Therapeutic implications: The ISR is thought to play a causative role in a wide array of cognitive and neurodegenerative disorders . PEBP1's function in ISR regulation suggests it could be a potential therapeutic target for these conditions, especially those associated with dysregulated ISR signaling.

These findings position PEBP1 as a potential mediator connecting stress responses, mitochondrial function, and neurological outcomes, with particular relevance to depression, memory formation, and possibly other neurodegenerative conditions.

How does PEBP1 contribute to liver pathology in diabetic and obese mouse models?

PEBP1 plays a significant role in liver pathology in diabetic and obese mouse models through several potential mechanisms:

• Elevated expression in metabolic disorders: PEBP1 is highly expressed in db/db mice, a genetic model of diabetes characterized by leptin receptor deficiency . This elevated expression suggests a potential pathological role in the context of metabolic dysfunction.

• Promotion of hepatic necroptosis: In mice with both diabetes and obesity (but not in mice with either condition alone), knockdown of PEBP1 alleviated hepatic injury and necroptosis induced by recombinant adeno-associated virus (AAV) . This indicates that PEBP1 contributes to programmed necrotic cell death in liver cells under combined metabolic stress conditions.

• AAV-induced liver injury: The combination of high PEBP1 expression in diabetic/obese conditions and AAV exposure appears particularly harmful to the liver, eventually leading to hepatocellular carcinoma in these models . This interaction between PEBP1 and viral vectors has significant implications for gene therapy approaches in patients with metabolic disorders.

• Therapeutic intervention point: Prednisone administration or knockdown of PEBP1 both alleviated hepatic injury and necroptosis induced by recombinant AAV in mice with diabetes and obesity . This suggests that targeting PEBP1 or its downstream pathways may offer therapeutic benefits for liver conditions in metabolic disorders.

• Potential relationship with mitochondrial stress: Given PEBP1's established role in amplifying mitochondrial stress responses , its contribution to liver pathology may involve dysregulated mitochondrial stress signaling, which is particularly relevant in high-energy demanding tissues experiencing metabolic challenge.

These findings highlight PEBP1 as a potential therapeutic target for preventing liver complications in patients with metabolic disorders, particularly when considering gene therapy approaches that utilize AAV vectors.

What are the key metabolic pathways influenced by PEBP1 manipulation in mouse models?

PEBP1 manipulation in mouse models affects several key metabolic pathways, revealing its multifaceted role in cellular metabolism:

The metabolic influence of PEBP1 appears to be context-dependent, with particularly important roles during cellular stress conditions and in tissues experiencing metabolic challenge, such as the liver in diabetic/obese conditions.

How can researchers differentiate between PEBP1's direct effects and its indirect effects through the RAF/MEK/ERK pathway?

Differentiating between PEBP1's direct effects and those mediated through the RAF/MEK/ERK pathway requires sophisticated experimental approaches:

• Pharmacological pathway inhibition:

  • Use specific MEK inhibitors like trametinib to block the RAF/MEK/ERK pathway while studying PEBP1 function

  • If a PEBP1-dependent phenotype persists despite MEK inhibition (as observed with eIF2α phosphorylation), it likely represents a direct effect independent of RAF/MEK/ERK signaling

  • Include proper controls demonstrating effective pathway inhibition (e.g., reduced ERK phosphorylation)

• Genetic complementation strategies:

  • Express mutant PEBP1 variants with selective defects:

    • PEBP1 with mutations in its Raf-binding domain to disrupt RAF/MEK/ERK regulation

    • PEBP1 with mutations affecting its newly identified eIF2α interaction

  • Compare phenotypic rescue capability of these variants in PEBP1 knockout backgrounds

• Temporal analysis:

  • Utilize rapid induction systems (e.g., optogenetic or chemical dimerization) to activate or inhibit PEBP1 function

  • Immediate responses (seconds to minutes) are more likely direct effects, while delayed responses (hours) may involve transcriptional regulation through RAF/MEK/ERK

• Domain-specific targeting:

  • Design domain-specific blocking peptides or nanobodies that selectively interfere with PEBP1's interaction with particular partners

  • This approach can distinguish functions dependent on specific protein-protein interactions

• Pathway cross-validation:

  • Compare PEBP1 knockout phenotypes with those of RAF/MEK/ERK pathway component knockouts

  • Non-overlapping phenotypes suggest independent functions

  • For example, test if ERK pathway activation mimics or opposes the effects of PEBP1 manipulation on ISR activation

• Tissue-specific analysis:

  • Exploit tissues with naturally different levels of RAF/MEK/ERK pathway activity

  • If PEBP1 functions similarly across these tissues despite different baseline ERK activation, this suggests ERK-independent mechanisms

These methodological approaches provide a framework for dissecting the complex functions of PEBP1, distinguishing its canonical role in RAF/MEK/ERK inhibition from its newly discovered functions in stress response pathways.

What are the challenges and solutions for studying tissue-specific functions of PEBP1 in mouse models?

Studying tissue-specific functions of PEBP1 in mouse models presents several challenges with corresponding methodological solutions:

Challenges:

• Ubiquitous expression: PEBP1 is expressed across most mouse tissues, particularly highly in the brain, making it difficult to isolate tissue-specific effects .

• Developmental compensation: Global knockout may trigger compensatory mechanisms during development that mask tissue-specific functions.

• Functional redundancy: Related proteins might compensate for PEBP1 loss in certain tissues but not others.

• Context-dependent effects: PEBP1's function varies with cellular context (e.g., stress conditions, metabolic state), complicating tissue-specific analysis .

• Technical limitations: Tissue-specific manipulation may affect neighboring cells through paracrine signaling.

Solutions:

• Conditional knockout strategies:

  • Employ tissue-specific Cre driver lines with floxed PEBP1 alleles

  • Use tamoxifen-inducible CreERT2 systems for temporal control to minimize developmental compensation

  • Validate tissue specificity through multiple approaches (protein levels, mRNA expression, and downstream functional readouts)

• AAV-mediated manipulation with caution:

  • Utilize serotypes with tissue tropism (e.g., AAV9 for brain, AAV8 for liver)

  • Employ tissue-specific promoters for additional specificity

  • Consider potential toxicity in metabolically compromised tissues, as AAV can cause liver injury in diabetic/obese mice with high PEBP1 expression

• Ex vivo tissue analysis:

  • Isolate primary cells from specific tissues for controlled manipulation

  • Use tissue-specific organoid cultures to maintain physiological context

  • Compare findings between isolated cells and intact tissues to identify cell-autonomous effects

• Tissue-specific rescue experiments:

  • Reintroduce PEBP1 in specific tissues of global knockout mice

  • Use tissue-specific expression of PEBP1 variants to dissect domain-specific functions

• Combinatorial stress conditions:

  • Apply tissue-relevant stressors (e.g., mitochondrial stress in brain, metabolic stress in liver)

  • Compare responses across tissues to identify context-dependent functions

• Multi-omics tissue profiling:

  • Apply spatial transcriptomics to map PEBP1-dependent gene expression across tissue regions

  • Use tissue-specific proteomics to identify tissue-specific PEBP1 interactors

  • Integrate metabolomics to link PEBP1 function to tissue metabolism

These approaches, particularly when used in combination, can help overcome the challenges inherent to studying tissue-specific functions of a widely expressed protein like PEBP1.

What are the current controversies and conflicting data regarding PEBP1 function in mouse models?

Current research on PEBP1 in mouse models reveals several areas of controversy and conflicting data that require careful consideration:

• Dual role in cancer progression:

  • Conflicting evidence exists regarding whether PEBP1 acts primarily as a tumor suppressor or promoter in different contexts

  • While generally considered a metastasis suppressor, studies suggest context-dependent oncogenic roles

  • In liver pathology, PEBP1 appears to promote AAV-induced hepatocellular carcinoma in diabetic and obese mouse models, contradicting its traditional tumor-suppressive role

• Cell-type specific effects on stress responses:

  • The requirement for PEBP1 in amplifying mitochondrial stress signals may vary between cell types

  • Studies indicate that PEBP1's importance in ISR activation might be greater in certain cell types or tissues with high metabolic demands

  • This creates apparent contradictions when comparing research using different cell lineages

• Phosphorylation state interpretation:

  • Discrepancies exist in how to interpret PEBP1 phosphorylation changes

  • In depression models, total PEBP1 increases while phosphorylated PEBP1 (Ser-153) decreases

  • The functional significance of this imbalance remains debated, as does its consistency across different neurological conditions

• Methodological limitations in studying PEBP1-eIF2α interaction:

  • Co-immunoprecipitation assays failed to show interaction between PEBP1 and eIF2α, yet NanoBiT complementation assays detected this interaction

  • This suggests either technical limitations or that the interaction is transient/weak

  • Such methodological discrepancies create controversy about the true nature of PEBP1's direct binding partners

• Tissue-specific mitochondrial ISR activation:

  • The study authors acknowledge limitations in generalizing findings from specific cell lines to in vivo situations

  • Questions remain about whether PEBP1's role in mitochondrial ISR is universal across all tissues or context-dependent

  • The relative importance of PEBP1 versus other ISR regulators may differ based on tissue-specific expression levels

• Therapeutic targeting conflicts:

  • Contradictions exist regarding whether PEBP1 inhibition or activation would be beneficial in different disease contexts

  • In metabolic liver disease, PEBP1 inhibition appears protective

  • In neurological contexts, preserving PEBP1 function might be important for proper stress responses

  • These opposing therapeutic implications create controversy about the optimal direction for drug development

What are the most promising future directions for PEBP1 research in mouse models?

The exploration of PEBP1 in mouse models reveals several promising research directions that could significantly advance our understanding of cellular stress responses and disease mechanisms:

• Therapeutic targeting for age-related disorders: Given PEBP1's role in ISR and its abundance in the brain, investigating its potential as a therapeutic target for cognitive and neurodegenerative disorders represents a compelling research avenue. Small molecules that modulate PEBP1 function could potentially enhance or prolong eIF2α phosphorylation, affecting both healthspan and lifespan .

• Tissue-specific conditional knockout models: Developing sophisticated tissue-specific and inducible PEBP1 knockout mice would allow precise dissection of its functions across different physiological contexts while avoiding developmental compensation issues. This approach would be particularly valuable for understanding PEBP1's role in tissues with high metabolic demands.

• Intersection with metabolic disorders: Further investigation of PEBP1's contribution to liver pathology in metabolic disorders could yield insights relevant to the growing global burden of obesity and diabetes. The finding that PEBP1 knockdown alleviates hepatic injury in diabetic and obese mice warrants deeper mechanistic exploration .

• Mitochondrial stress response amplification: Exploring the precise molecular mechanisms by which PEBP1 amplifies mitochondrial stress signals could reveal new insights into cellular stress adaptation. Understanding how PEBP1 selectively enhances certain stress responses while having minimal effects on others could inform targeted therapeutic approaches .

• Structural biology and protein engineering: Determining the crystal structure of PEBP1 in complex with eIF2α would provide crucial insights for structure-based drug design. Additionally, engineered PEBP1 variants with modified functions could serve as valuable research tools and potential therapeutic agents.

By pursuing these research directions, scientists can build upon the current understanding of PEBP1 biology to develop new therapeutic strategies for disorders ranging from neurodegeneration to metabolic diseases, potentially translating these findings from mouse models to clinical applications.

How can PEBP1 research in mouse models be effectively translated to human health applications?

Translating PEBP1 research from mouse models to human health applications requires strategic approaches that bridge fundamental science with clinical relevance:

• Comparative expression analysis: Systematically compare PEBP1 expression patterns between mouse tissues and corresponding human samples across developmental stages, aging, and disease states. This would validate the relevance of mouse findings to human biology and identify key differences requiring consideration during translation .

• Human iPSC-derived cellular models: Complement mouse studies with human induced pluripotent stem cell (iPSC) models to validate key findings. This approach is particularly valuable for neurological applications, where species differences may be pronounced. Testing PEBP1 manipulation in iPSC-derived neurons, hepatocytes, and other relevant cell types can bridge the species gap.

• Patient-derived xenografts: For cancer and metabolic disease applications, evaluate PEBP1 function in humanized mouse models carrying patient-derived tissues. This would provide insights into how PEBP1 modulation affects human tissues in a physiologically relevant context.

• Biomarker development: Explore correlations between PEBP1 levels/phosphorylation status and disease progression in human patient samples. If consistent relationships are identified, PEBP1 could serve as a biomarker for stress response dysregulation, particularly in neurological and metabolic conditions .

• Pharmacological tool development: Design small molecules that specifically target the interaction between PEBP1 and eIF2α or modulate PEBP1's phosphorylation state. These compounds could be tested initially in mouse models, then advanced to human cell systems and eventually clinical trials if promising .

• Gene therapy considerations: Given the finding that high PEBP1 expression contributes to AAV-induced liver damage in metabolic disorders, incorporate PEBP1 assessment in patient stratification for gene therapy approaches. This could prevent adverse effects in vulnerable populations .

• Multi-species validation: Extend findings from mice to non-human primates before human translation, particularly for neurological applications where primate models may better recapitulate human conditions .