Recombinant Mouse Fibroblast growth factor 17 (Fgf17) (Active)

What is the molecular structure of recombinant mouse FGF-17 and how does it compare to native proteins?

Recombinant mouse FGF-17 protein typically consists of amino acids Thr23-Thr216 with an N-terminal methionine, derived from E. coli expression systems . The mouse or human FGF-17 cDNA encodes a cleavable 22 amino acid signal sequence and a 194 amino acid secreted mature protein . In terms of sequence homology, mature mouse FGF-17 shares remarkable conservation across species: 100% amino acid identity with rat, 99% with human and porcine, and 97% with canine and equine FGF-17 . The FGF domain of FGF-17 shares 75% amino acid identity with FGF-8 and 64% with FGF-18, forming a distinct subfamily with overlapping expression patterns and functions . This high degree of conservation suggests evolutionary importance and functional significance across mammalian systems.

How should FGF-17 be properly stored and reconstituted to maintain optimal bioactivity for experiments?

For optimal bioactivity, lyophilized recombinant mouse FGF-17 should be reconstituted in sterile buffer solutions containing a carrier protein (such as BSA) to prevent adhesion to tubes and loss of activity. The typical ED50 for biological effects is 150-750 ng/mL in the presence of 10 μg/mL heparin, which is essential for proper FGF receptor binding and activation . Researchers should note that heparin co-administration is critical for experimental protocols, as FGFs require heparan sulfate proteoglycans as cofactors for receptor binding. Once reconstituted, working aliquots should be stored at -20°C to -80°C to prevent freeze-thaw cycles, and solutions should be prepared fresh for critical experiments. Biological activity should be validated using established assays such as cell proliferation in responsive cell lines before proceeding to complex experiments.

What are the optimal experimental conditions for using recombinant FGF-17 in neural progenitor cell differentiation protocols?

When using recombinant FGF-17 for neural progenitor differentiation, particularly for ventral midbrain dopaminergic (VM DA) progenitor patterning, optimal conditions include:

• Concentration: Recent studies show that FGF-17 induces higher expression of critical VM DA progenitor markers FOXA2 and LMX1A compared to the traditionally used FGF-8 .

• Timing: Application during early neural patterning stages is critical, as FGF-17's expression is developmentally regulated, appearing slightly later than FGF-8 during embryogenesis but still essential for proper midbrain-hindbrain boundary (MHB) development .

• Co-factors: Include 10 μg/mL heparin in the medium, as FGF-17 requires heparan sulfate for proper receptor binding and signaling .

• Culture conditions: For dopaminergic differentiation protocols, FGF-17 treatment should be combined with appropriate basal medium and supplements that support neural development, followed by maturation factors for terminal differentiation .

• Validation: Verify patterning efficiency through immunostaining for FOXA2, LMX1A, OTX2, and EN1 markers, which define the VM DA progenitor identity .

How can FGF-17 activity be quantitatively measured in experimental settings?

Quantitative measurement of FGF-17 activity can be accomplished through several complementary approaches:

• Receptor Activation Assays: Measure phosphorylation of downstream signaling molecules (particularly MAPK/ERK pathway components) in cells expressing FGF receptors (especially FGFR1c-3c and FGFR4) following FGF-17 treatment .

• Proliferation Assays: Assess cell proliferation in responsive cell populations, particularly oligodendrocyte progenitor cells (OPCs), where FGF-17 has been shown to induce significant proliferation. Quantification can be performed using BrdU incorporation, Ki67 staining, or cell counting methods .

• Gene Expression Analysis: Measure the upregulation of SRF (Serum Response Factor) target genes, as FGF-17 has been demonstrated to activate SRF signaling through actin modulation .

• Functional Bioassays: For OPC differentiation, quantify differentiation markers by immunofluorescence, flow cytometry, or qRT-PCR for markers like FOXA2 and LMX1A in neural progenitor models .

• In Vivo Activity Measurements: For in vivo studies, assess OPC proliferation in specific brain regions (e.g., hippocampus) following FGF-17 infusion, using BrdU labeling or other cell proliferation markers .

What are the most reliable methods for analyzing behavioral abnormalities in FGF-17 knockout mice?

When analyzing behavioral abnormalities in FGF-17 knockout mice, researchers should employ a comprehensive battery of tests focusing on the specific domains affected by FGF-17 deficiency:

• Social Behavior Assessment: As FGF-17-/- mice exhibit abnormal social behaviors, use the three-chamber social approach test, social recognition tests, and reciprocal social interaction paradigms. These should be conducted under controlled lighting conditions (approximately 350 lux) and experimenters should remain blind to genotype .

• Olfactory Testing: Since social deficits may relate to sensory processing, implement olfactory recognition tests using cotton swabs with different odorants (e.g., cineole, limonene, isoamyl acetate) and mineral oil as vehicle control. Record exploration time and frequency across multiple trials to assess habituation and dishabituation .

• Cognitive Assessment: Employ Y-maze, novel object recognition, and fear conditioning paradigms to assess various cognitive domains. For memory testing, use validated protocols like novelty-suppressed feeding and contextual fear conditioning .

• Motor Function: As FGF-17-/- mice may display cerebellar abnormalities and ataxia, include rotarod testing and open field activity monitoring to quantify motor deficits .

• Data Analysis: Compare results across genotypes (Fgf17+/+, Fgf17+/-, Fgf17-/-) with appropriate statistical methods, controlling for sex differences as these may influence behavioral outcomes .

What molecular and anatomical phenotypes should be examined in FGF-17 deficient models?

When examining FGF-17 deficient models, researchers should focus on the following molecular and anatomical phenotypes:

• Brain Regional Development:

  • Dorsal frontal cortex morphology and layer formation

  • Midbrain-hindbrain boundary (MHB) patterning

  • Cerebellar development, particularly foliation patterns and cellular organization

• Cellular Composition Analysis:

  • Oligodendrocyte progenitor cell (OPC) density and distribution across brain regions

  • Myelination patterns using myelin-specific markers

  • Neuronal subtypes, particularly in regions with high FGF-17 expression during development

• Molecular Marker Expression:

  • SRF (Serum Response Factor) activation and downstream target expression

  • Regional expression of developmental transcription factors including FOXA2, LMX1A, OTX2, and EN1

  • Actin cytoskeleton organization, as FGF-17 regulates actin dynamics

• Circuit-level Analysis:

  • Synaptic density and organization in frontal cortex

  • Electrophysiological properties of neurons in affected regions

  • Functional connectivity between brain regions involved in social behavior

• Age-dependent Changes:

  • Comparative analysis across developmental stages and in aging to assess progressive phenotypes

  • Cognitive performance correlation with cellular and molecular changes

How does FGF-17 signaling interact with other morphogens in establishing the midbrain-hindbrain boundary during development?

FGF-17 operates within a complex morphogen network at the midbrain-hindbrain boundary (MHB), with precise spatiotemporal interactions:

• Temporal Sequence: FGF-17 expression follows FGF-8 at the MHB, suggesting a sequential activation mechanism where initial patterning by FGF-8 is refined and maintained by FGF-17 . This temporal regulation is critical, as premature or delayed expression disrupts proper boundary formation.

• Receptor Specificity: FGF-17 preferentially signals through FGFR1c-3c and FGFR4, but with different binding affinities than FGF-8 and FGF-18, creating distinct downstream signaling outcomes despite utilizing the same receptors . This differential receptor activation contributes to unique developmental outcomes.

• Wnt1-FGF Feedback Loops: FGF-17 participates in reciprocal regulatory interactions with Wnt1, which is expressed at the MHB. This cross-regulation establishes a molecular boundary and maintains progenitor domains. Experimental disruption of either pathway affects the other, indicating their interdependence.

• Regulatory Relationships with Otx2/Gbx2: FGF-17 signaling reinforces the expression boundary between Otx2 (midbrain) and Gbx2 (hindbrain), which initially establish the MHB. FGF-17's role appears more prominent in maintaining rather than establishing this boundary, as evidenced by the viable phenotype of FGF-17-/- mice compared to lethal FGF-8 knockouts .

• Sonic Hedgehog (Shh) Interactions: FGF-17 signaling intersects with ventral Shh gradients to establish proper dorsal-ventral patterning in the developing midbrain, particularly for dopaminergic neuron specification. This is evidenced by the successful patterning of ventral midbrain dopaminergic progenitors using FGF-17 in experimental protocols .

What are the molecular mechanisms explaining FGF-17's effects on oligodendrocyte progenitor cell proliferation and differentiation in the context of aging?

The molecular mechanisms underlying FGF-17's effects on oligodendrocyte progenitor cells (OPCs) in aging involve several interconnected pathways:

• SRF-Mediated Transcriptional Regulation: FGF-17 robustly activates Serum Response Factor (SRF) signaling through actin modulation, enhancing or inhibiting actin polymerization. This activation is dose-dependent and stronger than other CSF factors, providing a mechanistic explanation for FGF-17's potent effects on OPC biology .

• Actin Cytoskeleton Dynamics: FGF-17 modulates actin polymerization, which is critical for OPC process extension, migration, and differentiation. This effect can be experimentally manipulated using jasplakinolide (enhances polymerization) or latrunculin A (inhibits polymerization), directly affecting SRF activation outcomes .

• Age-Dependent Expression Patterns: FGF-17 levels decrease with age in human plasma, CSF, and mouse neurons, correlating with reduced oligodendrogenesis and myelination in the aging brain. This suggests that the decline in FGF-17 may be a causal factor in age-related oligodendrocyte dysfunction .

• Receptor Signaling Specificity: FGF-17 likely activates specific FGFR subtypes on OPCs, triggering MAPK/ERK, PI3K/AKT, and potentially JAK/STAT pathways that regulate both proliferation and differentiation programs. The balance between these signaling outcomes determines whether OPCs proliferate or differentiate into mature oligodendrocytes .

• Interaction with Age-Related Inflammation: FGF-17 may counteract age-related inflammatory signals that inhibit OPC function, providing a permissive environment for remyelination in the aged brain. This represents a potential mechanism for the cognitive improvements observed following FGF-17 administration to aged mice .

What are the optimal delivery methods and dosing regimens for FGF-17 in preclinical models of age-related cognitive decline?

Based on current research, the following delivery methods and dosing parameters appear most effective for FGF-17 administration in preclinical models:

How do the effects of FGF-17 treatment compare with other approaches for addressing age-related oligodendrocyte dysfunction?

FGF-17 offers distinct advantages and mechanisms compared to other approaches for treating age-related oligodendrocyte dysfunction:

• Comparative Effectiveness:

  • Young CSF Infusion: FGF-17 treatment produces comparable effects to young CSF infusion but offers the advantage of being a defined molecular entity rather than a complex biological fluid with variable composition .

  • Growth Factor Therapies: Unlike PDGF-AA (which primarily promotes OPC proliferation) or IGF-1 (which mainly supports survival), FGF-17 appears to effectively promote both proliferation and differentiation of OPCs, potentially offering more complete remyelination .

  • Anti-inflammatory Approaches: While anti-inflammatory treatments may create a permissive environment for remyelination, FGF-17 directly drives the cellular processes needed for new myelin formation through SRF activation .

• Mechanistic Advantages:

  • Targeted Cell Population: FGF-17 specifically acts on OPCs through defined receptor systems, potentially minimizing off-target effects compared to broader interventions .

  • Physiological Relevance: As an endogenous factor that decreases with age, FGF-17 replacement represents a more physiologically relevant approach than introducing exogenous compounds .

  • Multiple Mechanisms: FGF-17 affects both proliferation and differentiation stages of oligodendrogenesis, potentially addressing multiple aspects of age-related dysfunction .

• Practical Considerations:

  • Production and Stability: As a recombinant protein with established production methods, FGF-17 offers practical advantages over cell-based therapies or complex biological fluids .

  • Delivery Challenges: Like other protein therapeutics, FGF-17 faces delivery challenges across the blood-brain barrier, requiring direct CNS administration or development of novel delivery systems for clinical translation .

How does FGF-17 signaling differ from other members of the FGF8 subfamily, and what are the functional consequences of these differences?

FGF-17 exhibits subtle but significant differences from its subfamily members (FGF-8 and FGF-18) with important functional implications:

• Receptor Binding Profiles:

  • While FGF-17, FGF-8, and FGF-18 all bind FGFR1c-3c and FGFR4, they do so with different affinities and kinetics

  • These differences in binding characteristics translate to distinct downstream signaling intensities and durations, particularly evident in the stronger activation of FOXA2 and LMX1A expression by FGF-17 compared to FGF-8 in ventral midbrain patterning

• Developmental Expression Patterns:

  • FGF-17 is expressed slightly later than FGF-8 during embryogenesis at the midbrain-hindbrain boundary, suggesting sequential roles in development

  • FGF-17 shows more restricted expression patterns in adult tissues compared to FGF-18, being primarily found in ovarian follicles and prostate, while FGF-18 has broader expression

• Knockout Phenotype Severity:

  • FGF-17 deletion produces viable mice with specific brain abnormalities affecting the dorsal frontal cortex, midbrain, and cerebellum

  • In contrast, FGF-8 and FGF-18 knockouts are lethal, indicating more fundamental developmental roles

  • This suggests FGF-17 has more specialized functions that cannot be fully compensated by other family members

• Signaling Pathway Activation:

  • FGF-17 demonstrates particularly strong activation of SRF signaling through actin modulation, which is critical for its effects on oligodendrocyte progenitor cells

  • This potent SRF activation may explain why FGF-17 was identified as a key component from young CSF responsible for cognitive improvements in aged mice

What experimental approaches can determine the specific contribution of FGF-17 to neural circuit function in regions affected by its expression?

Advanced experimental approaches to delineate FGF-17's specific contributions to neural circuit function include:

• Spatiotemporally Controlled Manipulation:

  • Conditional genetic systems using inducible Cre/loxP to delete or overexpress FGF-17 in specific brain regions and developmental stages

  • Viral vector-mediated expression of FGF-17 or dominant-negative receptors in adult brain circuits to distinguish developmental from maintenance functions

  • Optogenetic or chemogenetic activation of FGF-17-expressing neurons to determine their circuit-level contributions to behavior

• High-Resolution Anatomical Analysis:

  • Single-cell RNA sequencing to identify cell populations responsive to FGF-17 signaling

  • Spatial transcriptomics to map FGF-17 signaling effects across brain regions

  • Volume electron microscopy to assess ultrastructural changes in myelination and synaptic organization in FGF-17 deficient models

• Functional Circuit Assessment:

  • In vivo calcium imaging in FGF-17-deficient animals during social and cognitive tasks to identify altered circuit dynamics

  • Electrophysiological recording in brain slices from regions affected by FGF-17 deficiency to determine synaptic and intrinsic neuronal properties

  • Chemogenetic silencing of specific pathways to determine which circuits mediate behavioral abnormalities in FGF-17 knockout models

• Molecular Pathway Dissection:

  • Phospho-proteomics to map the complete signaling cascade activated by FGF-17 in different neural cell types

  • CRISPR screening to identify essential mediators of FGF-17's effects on oligodendrocyte biology

  • Chromatin immunoprecipitation sequencing (ChIP-seq) to identify SRF binding sites regulated by FGF-17 signaling

• Translational Validation:

  • Correlation of FGF-17 levels in human CSF with cognitive performance and white matter integrity

  • Development of FGF-17 mimetics or small molecule activators of downstream pathways as potential therapeutic agents

  • PET imaging with tracers for oligodendrocyte activity to monitor effects of FGF-17-based interventions in vivo