FOLH1 Mouse

What is FOLH1 and what is its primary function in mice?

FOLH1, also known as glutamate carboxypeptidase II (GCPII) or PSMA, is a type II transmembrane glycoprotein expressed in various mouse tissues. It functions primarily as an enzyme that cleaves specific substrates including N-acetylaspartylglutamate (NAAG). In mice, FOLH1 is encoded by the Folh1 gene (MGI:1858193) .

The protein exhibits significant glutamate carboxypeptidase enzymatic activity, which can be measured using specific substrates such as Ac-Asp-Glu. Under standard conditions, recombinant mouse FOLH1 shows activity exceeding 350 pmol/min/μg . Importantly, FOLH1 plays a critical role in inflammatory processes, particularly in intestinal mucosa, where its activity is significantly elevated during inflammation .

How are FOLH1 knockout mice generated and what are their key phenotypic characteristics?

FOLH1 knockout (FOLH1-/-) mice are generated using standard gene targeting techniques where the Folh1 gene is disrupted. The most notable phenotypic characteristic of these mice is their significant resistance to experimentally induced colitis. When challenged with dextran sodium sulfate (DSS), FOLH1-/- mice consistently demonstrate:

• Lower disease activity index (DAI) scores compared to wild-type controls

• Significantly longer colon length, indicating reduced inflammation

• Healthier colonic mucosa with noticeably less neutrophil infiltration

• Better preserved crypts and goblet cells

These observations strongly suggest that FOLH1 contributes to inflammatory pathology in the intestinal tract, making these knockout mice valuable models for studying protective mechanisms in inflammatory bowel disease.

What are the commonly used mouse models for studying FOLH1 function?

Several established mouse models are instrumental in FOLH1 research:

These complementary models allow researchers to study both the consequences of FOLH1 absence and its response to different inflammatory stimuli .

What is the relationship between FOLH1 and inflammatory bowel disease in mouse models?

Research demonstrates a significant relationship between FOLH1 and inflammatory bowel disease (IBD) in mouse models:

• Elevated Enzymatic Activity:

  • DSS-induced colitis increases FOLH1 activity 1.5-fold in the colon

  • IL-10-/- spontaneous colitis model shows 3.9-fold increased activity in colonic mucosa

• Protective Effect of FOLH1 Deficiency:

  • FOLH1-/- mice show significantly reduced disease severity when challenged with DSS

  • Histologically, these mice maintain better intestinal architecture and reduced inflammation

• Therapeutic Potential:

  • The FOLH1 inhibitor 2-PMPA (IC50 = 300 pM) significantly reduces disease activity

  • 2-PMPA treatment inhibits colonic FOLH1 activity by >90% and substantially reduces disease severity

These findings position FOLH1 as both a biomarker for IBD and a potential therapeutic target.

How is FOLH1 expression and activity measured in mouse tissues?

Multiple methodological approaches are used to assess FOLH1 in mouse tissues:

The enzymatic activity assay is particularly valuable as it directly measures functional FOLH1, which correlates with disease activity in IBD models .

What experimental controls are essential when studying FOLH1 in mice?

Rigorous experimental controls are critical for valid FOLH1 research:

• Genotype Controls:

  • Wild-type littermates as positive controls

  • FOLH1-/- mice as negative controls for enzymatic activity

• Treatment Controls:

  • Vehicle-treated mice for drug studies (e.g., when testing 2-PMPA)

  • Substrate blanks (no enzyme activity control) for enzymatic assays

• Tissue Controls:

  • Uninvolved tissue from the same animal

  • Multiple tissue types to assess tissue-specific effects

• Assay Quality Controls:

  • Recombinant mouse FOLH1 protein (Catalog # 4946-ZN) as a positive control

  • Defined buffers: 50 mM HEPES, 0.1 M NaCl, pH 7.5 for activity assays

• Drug Level Monitoring:

  • Measuring inhibitor concentrations (e.g., 2-PMPA levels in plasma and tissue) to confirm target engagement

How do FOLH1 inhibitors like 2-PMPA affect experimental colitis in mouse models?

The FOLH1/GCPII inhibitor 2-PMPA (2-phosphonomethyl pentanedioic acid) demonstrates significant therapeutic effects in colitis models:

These findings establish that pharmacological inhibition of FOLH1/GCPII effectively ameliorates experimental colitis, validating FOLH1 as a therapeutic target .

What methodological approaches are used to measure FOLH1/GCPII enzymatic activity in mouse tissue samples?

The following standardized protocol is used for measuring FOLH1/GCPII enzymatic activity:

Materials Required:

• Assay Buffer: 50 mM HEPES, 0.1 M NaCl, pH 7.5

• o-PA Buffer: 0.2 M Sodium Hydroxide containing 0.1% mercaptoethanol (v/v)

• Substrate: Ac-Asp-Glu (10 mM stock in 40 mM Sodium Hydroxide)

• o-phthaldialdehyde (o-PA): 50 mg/mL (0.373 M) stock in DMSO

• F16 Black Maxisorp Plate for fluorescence detection

• Fluorescent Plate Reader (e.g., SpectraMax Gemini EM)

Procedure:

• Dilute tissue samples or recombinant FOLH1 to 0.2 μg/mL in Assay Buffer

• Dilute Substrate to 40 μM with Assay Buffer

• Mix equal volumes (125 μL) of enzyme sample and substrate

• Prepare substrate blank as negative control

• Incubate under appropriate conditions

• Add o-PA reagent for detection

• Measure fluorescence

• Calculate specific activity (pmol/min/μg)

This methodology enables quantitative assessment of FOLH1 activity in various experimental conditions, with recombinant mouse FOLH1 showing activity >350 pmol/min/μg under standard conditions .

How do results from FOLH1 mouse models translate to human IBD pathology?

The translational relevance of FOLH1 mouse models to human IBD is supported by several parallel findings:

• Comparable Enzymatic Upregulation:

  • Human IBD: 2.8-41-fold increase in affected intestinal mucosa

  • Mouse models: 1.5-3.9-fold increase in experimental colitis

• Cross-Species Methodological Validation:

  • The same enzymatic activity assays applied to both human and mouse samples

  • Consistent upregulation pattern observed across species

• Gene Expression Correlations:

  • FOLH1 identified as a "hub gene" with significant correlations to multiple known IBD biomarkers in human samples

  • Immunohistochemical validation confirms elevated protein expression in affected tissues

• Therapeutic Implications:

  • Protection in FOLH1-/- mice suggests potential benefit of FOLH1 inhibition in humans

  • Efficacy of 2-PMPA provides preclinical evidence for pursuing FOLH1 inhibitors as IBD therapeutics

These findings suggest strong translational relevance of FOLH1 mouse models to human IBD, though differences in magnitude and specific mechanisms require careful consideration when extrapolating to human disease.

What are the molecular mechanisms behind FOLH1-mediated effects in experimental colitis?

While the complete molecular mechanisms remain under investigation, several key pathways have been identified:

• Enzymatic Function:

  • FOLH1/GCPII functions as a glutamate carboxypeptidase

  • Cleaves substrates like N-acetylaspartylglutamate (NAAG) to release glutamate

  • This activity increases dramatically in inflamed tissues

• Mechanistic Pathways:

  • Glutamate Signaling: FOLH1 inhibition may reduce glutamate levels, affecting inflammatory signaling

  • Epithelial Barrier Function: FOLH1-/- mice show preserved goblet cells and crypt architecture

  • Inflammatory Cell Recruitment: FOLH1 deficiency reduces neutrophil infiltration

• Evidence for Causality:

  • Protection in FOLH1-/- mice against DSS-induced colitis

  • Dose-dependent therapeutic effect of 2-PMPA

  • Target engagement confirmed by >90% reduction in enzymatic activity with treatment

The integration of FOLH1 with multiple IBD-related pathways suggests it may serve as a central regulatory node in intestinal inflammation, though further research is needed to fully elucidate the molecular interactions involved.