Recombinant Human Fibroblast growth factor 12 (FGF12) (Active)

Basic Research Questions

• What is FGF12 and what is its role in neuronal function?

  FGF12 belongs to a subfamily of FGF proteins called FGF homologous factors (FHFs), which were traditionally considered non-signaling intracellular proteins until recent discoveries revealed their extracellular activities . FGF12 functions primarily by interacting with the C-terminal domain of voltage-gated sodium channels (VGSCs) 1.2, 1.5, and 1.6, where it promotes neuronal excitability by delaying fast inactivation of these channels . This interaction is critical for proper neural development and function, explaining why FGF12 variants are associated with developmental and epileptic encephalopathy (DEE) . When investigating FGF12 function, researchers should consider both its intracellular regulatory roles with sodium channels and its emerging extracellular signaling capabilities, particularly under stress conditions such as elevated temperatures (42°C) or serum starvation .

• What are the key structural and functional differences between FGF12 isoforms?

  FGF12 exists in at least two major isoforms: the long "a" isoform and the short "b" isoform, which display distinct cellular behaviors and functional properties . The "a" isoform contains an extended N-terminal region that is crucial for its secretion, despite lacking a conventional signal peptide. This isoform binds efficiently to the A1 subunit of Na+/K+ ATPase (ATP1A1) and phosphatidylserine, enabling its secretion pathway . In contrast, the "b" isoform binds ATP1A1 and phosphatidylserine less efficiently and consequently is not secreted from cells . For experimental work, researchers should carefully select the appropriate isoform based on their research question, as these structural differences translate to significant functional distinctions. When expressing recombinant FGF12, specifying the isoform is essential, as is verifying protein identity through western blotting with specific antibodies against tags such as GFP or SBP that may be incorporated into expression constructs .

• How can researchers effectively express and purify recombinant human FGF12?

  An efficient method for producing bioactive recombinant human FGF12 (rhFGF12) involves using an Escherichia coli expression system with the following protocol: First, clone the hFGF12 gene and ligate it into an expression vector such as pET-3a to construct a recombinant plasmid (e.g., pET-3a-hFGF12) . After transformation, screen single colonies to identify high-expression engineering strains. Optimize fermentation conditions to maximize protein yield while maintaining proper folding. For purification, a combination of chromatographic techniques typically yields the best results . Verify protein purity using SDS-PAGE and confirm biological activity through functional assays such as MTT proliferation assays with NIH3T3 or PC12 cell lines . If working with specific isoforms, ensure your expression construct contains the complete sequence for either the "a" or "b" isoform based on your research needs. Testing for proper folding and biological activity is crucial, as improperly folded FGF12 may lack the ability to interact with its binding partners such as voltage-gated sodium channels .

• What experimental models are recommended for studying FGF12 function?

  Several experimental models have proven valuable for investigating FGF12 function:

  • Cell culture models: PC12 and NIH3T3 cell lines are effective for studying proliferative effects and neuroprotective properties of rhFGF12 . U2OS cells expressing FGF12-GFP-myc constructs have been used to study secretion mechanisms .

  • Neuronal models: Primary neuronal cultures or neuronal-like cells (ND7/23) co-expressing FGF12 with sodium channels NaV1.2 or NaV1.6 (including their beta-1 and beta-2 subunits) can be used to study channel modulation .

  • In vivo models: Transgenic zebrafish with GFP fluorescence in motor neurons provide an excellent system for studying central nervous system injury and FGF12 neuroprotection . Drosophila models have been employed for in vivo functional analysis of FGF12 variants .

  • Disease models: H₂O₂-induced oxidative injury in PC12 cells serves as an in vitro neurotoxicity model, while mycophenolate mofetil (MMF)-induced central injury in zebrafish creates an in vivo injury model . Both are valuable for testing the neuroprotective effects of rhFGF12.

  When selecting a model system, researchers should consider the specific aspect of FGF12 biology they wish to investigate, whether it's channel modulation, secretion mechanisms, or neuroprotective functions.

• What detection methods are most suitable for analyzing FGF12 expression and localization?

  Several complementary methods can be employed for comprehensive analysis of FGF12:

  • Western blotting: Effective for quantifying FGF12 expression levels in cell lysates and for detecting secreted FGF12 in media supernatants after immunoprecipitation with antibodies against tags like myc .

  • Proximity Ligation Assay (PLA): Useful for detecting protein-protein interactions in situ, such as between FGF12 and ATP1A1. This technique involves fixing cells with 4% paraformaldehyde, permeabilizing with 0.1% Triton X-100, and using antibodies against the proteins of interest in combination with PLA probes .

  • Confocal microscopy: High-content screening systems like Opera Phenix enable visualization of FGF12 cellular localization using appropriate fluorescent markers and cellular stains .

  • Real-time quantitative PCR: For sensitive detection of FGF12 gene expression at the mRNA level, particularly important when analyzing expression in patient-derived cells like lymphoblastoid cells .

  • Long-read sequencing: For detecting structural variations in the FGF12 gene that may be missed by conventional sequencing methods .

  Selection of detection methods should be based on the specific research question, bearing in mind that combination approaches often provide the most comprehensive insights.

Advanced Research Questions

• How do biallelic structural variations in FGF12 contribute to developmental and epileptic encephalopathy (DEE)?

  Biallelic structural variations (SVs) in FGF12 represent a novel disease mechanism for developmental and epileptic encephalopathy that differs from previously described gain-of-function mechanisms. While heterozygous missense variants or duplications of FGF12 cause epilepsy through gain-of-function effects, biallelic SVs appear to operate through loss-of-function mechanisms . These biallelic SVs, often involving intronic, GC-rich, or repetitive regions, can be missed by conventional exome sequencing but are detectable through long-read whole genome sequencing .

  To investigate these mechanisms, researchers should employ a multi-pronged approach:

  1. Perform gene expression analyses using patient-derived lymphoblastoid cells to quantify FGF12 transcript levels

  2. Conduct structural analysis of mutant proteins to predict functional consequences

  3. Develop in vivo functional assays, such as Drosophila models, to validate loss-of-function effects

  These investigations reveal that biallelic SVs/SNVs disrupt FGF12's normal interaction with sodium channels, leading to altered neuronal excitability through mechanisms distinct from the gain-of-function variants previously described. This dual disease mechanism (gain-of-function for heterozygous variants versus loss-of-function for biallelic variants) highlights the complex role of FGF12 in neuronal function and epileptogenesis .

• What molecular mechanisms underlie FGF12 secretion despite its lack of a conventional signal peptide?

  FGF12 secretion, particularly of the long "a" isoform, occurs through an unconventional pathway that has been recently characterized. This process involves:

  1. Protein-protein interactions: The A1 subunit of Na⁺/K⁺ ATPase (ATP1A1) serves as a critical interaction partner for FGF12a, facilitating its secretion. This interaction can be confirmed through pull-down assays using streptavidin magnetic beads with FGF12-SBP constructs and detection by western blotting .

  2. Kinase involvement: Tec kinase plays an essential role in FGF12 secretion, as demonstrated by inhibition studies using LFM-A13 (275 μM) .

  3. Lipid interactions: Phosphatidylinositol and phosphatidylserine are crucial lipid components that facilitate FGF12a secretion. The short "b" isoform binds phosphatidylserine less efficiently, explaining its lack of secretion .

  4. Phase separation dynamics: Liquid-liquid phase separation appears to be involved in FGF12 secretion, which can be investigated through droplet formation assays. In these assays, recombinant FGF12a and FGF12b (5 μM) are incubated with 10% PEG-8000 and heparin (100 nM) on ice for 30 minutes, then visualized using differential interference contrast microscopy .

  5. Stress conditions: Environmental factors such as elevated temperature (42°C) or serum starvation enhance FGF12 secretion, suggesting a stress-response component to this mechanism .

  This unconventional secretion pathway represents a paradigm shift in understanding FHF biology, as these proteins were previously thought to function exclusively intracellularly.

• What signaling pathways are activated by recombinant human FGF12, and how do they contribute to neuroprotection?

  Recombinant human FGF12 (rhFGF12) exerts its neuroprotective effects through multiple signaling pathways:

  1. ERK/MAPK pathway: rhFGF12 stimulates proliferation in PC12 cells through activation of the ERK/MAPK signaling cascade . This pathway can be assessed through western blotting for phosphorylated ERK following rhFGF12 treatment.

  2. Anti-apoptotic mechanisms: rhFGF12 protects against H₂O₂-induced oxidative injury in PC12 cells by reducing apoptosis . This protection can be quantified through apoptosis assays such as Annexin V/PI staining and flow cytometry.

  3. Proliferative signaling: Cell proliferation stimulated by rhFGF12 can be measured using MTT assays in appropriate cell lines such as NIH3T3 and PC12 .

  The neuroprotective effects of rhFGF12 have been demonstrated in multiple models, including H₂O₂-induced oxidative injury in PC12 cells and mycophenolate mofetil (MMF)-induced central injury in transgenic zebrafish . These findings suggest that rhFGF12 may offer therapeutic potential for nerve injury through its dual action of promoting cellular proliferation and inhibiting apoptosis. Researchers investigating these mechanisms should incorporate pathway inhibitors in their experimental design to confirm the specific contribution of each signaling cascade to the observed neuroprotective effects.

• How do FGF12 variants modulate NaV1.2 and NaV1.6 channels, and what are the implications for neuronal excitability?

  FGF12 variants exert differential effects on voltage-gated sodium channels NaV1.2 and NaV1.6, which are critical determinants of neuronal excitability. To investigate these effects, researchers can use co-expression systems in neuronal-like cells (such as ND7/23) where FGF12 (wild-type or mutant) is expressed alongside NaV1.2 or NaV1.6, including their beta-1 and beta-2 sodium channel subunits (SCN1B and SCN2B) .

  Key experimental approaches include:

  1. Electrophysiological recordings: Patch-clamp techniques can directly measure changes in sodium channel kinetics, including activation, inactivation, and recovery from inactivation.

  2. Co-immunoprecipitation assays: These can quantify the binding affinity between FGF12 variants and sodium channel alpha subunits.

  3. Computational modeling: Neuronal excitability models can predict how changes in channel properties affect action potential generation and propagation.

  Wild-type FGF12 typically delays fast inactivation of voltage-gated sodium channels, thereby promoting neuronal excitability . Different variants can either enhance this effect (gain-of-function) or disrupt it (loss-of-function), leading to dysregulated neuronal activity that manifests as developmental and epileptic encephalopathy . The specific nature of the variant (missense, copy number, or structural) determines its functional impact on channel modulation, underscoring the importance of comprehensive functional characterization of FGF12 variants identified in patients with neurological disorders.

• What advanced technical approaches can detect structural variations in FGF12 that are missed by conventional sequencing?

  Detecting structural variations (SVs) in FGF12, particularly those in intronic, GC-rich, or repetitive regions, requires specialized approaches beyond standard exome sequencing:

  1. Long-read whole genome sequencing (LRS): This technology enables the generation of reads that span entire or partial SVs throughout the genome, allowing direct characterization of complex SVs . Oxford Nanopore or PacBio platforms are commonly used for LRS.

  2. Bioinformatic analysis pipelines: Specialized algorithms are needed to detect SVs from long-read data, including tools that can identify insertions, deletions, inversions, and complex rearrangements.

  3. Validation strategies: After identifying candidate SVs, validation through orthogonal methods is essential:

     • PCR amplification across breakpoints

     • Sanger sequencing of junction fragments

     • Comparative genomic hybridization arrays

     • Droplet digital PCR for copy number analysis

  4. Expression analysis: For intronic variants that may affect splicing, RNA sequencing of patient-derived cells (such as lymphoblastoid cell lines) can confirm aberrant splicing patterns .

  5. Functional genomics approaches: CRISPR-Cas9 genome editing can be used to recreate patient-specific SVs in cellular or animal models to assess their functional impact.

  These comprehensive approaches have successfully identified biallelic SVs in FGF12 that were previously missed by exome sequencing, highlighting the importance of considering SVs in the molecular diagnosis of patients with developmental and epileptic encephalopathy .

• How can liquid-liquid phase separation of FGF12 be investigated experimentally?

  Liquid-liquid phase separation (LLPS) has emerged as an important mechanism in FGF12 biology, particularly in relation to its unconventional secretion pathway . Researchers can investigate LLPS of FGF12 using several experimental approaches:

  1. Droplet formation assays: Recombinant FGF12a or FGF12b (5 μM) can be incubated with molecular crowding agents such as 10% PEG-8000 and heparin (100 nM) on ice for 30 minutes. The resulting droplets can be visualized using differential interference contrast (DIC) microscopy with a 40×/0.6 objective . Comparative analysis between FGF12a, FGF12b, and other FGFs (such as FGF2) can reveal isoform-specific differences in phase separation properties.

  2. Fluorescence recovery after photobleaching (FRAP): This technique assesses the dynamics of molecules within phase-separated droplets. By tagging FGF12 with fluorescent proteins, researchers can photobleach a region within a droplet and measure the rate of fluorescence recovery, providing insights into molecular mobility and droplet material properties.

  3. Mutagenesis studies: Systematic mutation of specific residues or domains in FGF12 can identify regions critical for phase separation. This approach has already revealed that the N-terminal fragment of FGF12a is crucial for its secretion, likely due to its role in phase separation .

  4. Modulation of cellular conditions: Since FGF12 secretion increases under stress conditions such as elevated temperature (42°C) or serum starvation , researchers can systematically vary these parameters to determine their effects on phase separation.

  5. Colocalization with phase separation markers: Immunofluorescence studies can assess whether FGF12 colocalizes with known markers of phase-separated compartments under various cellular conditions.

  These approaches collectively provide a comprehensive toolkit for investigating the role of LLPS in FGF12 biology, potentially revealing new insights into its unconventional secretion mechanism and function.

• What are the most effective in vitro and in vivo models for evaluating the neuroprotective effects of rhFGF12?

  To evaluate the neuroprotective effects of recombinant human FGF12 (rhFGF12), researchers can utilize the following complementary models:

  In vitro models:

  1. H₂O₂-induced oxidative injury in PC12 cells: This model involves treating PC12 cells with H₂O₂ to induce oxidative stress, then assessing the protective effects of rhFGF12 through:

     • MTT assays to measure cell viability

     • Apoptosis assays (Annexin V/PI staining)

     • Western blotting for apoptotic markers

  2. Glutamate excitotoxicity in primary neurons: Primary cortical or hippocampal neurons can be exposed to glutamate to induce excitotoxic injury, with rhFGF12 added as a potential neuroprotectant.

  3. Oxygen-glucose deprivation (OGD) in neuronal cultures: This model mimics ischemic conditions and can be used to assess whether rhFGF12 protects against ischemic neuronal damage.

  In vivo models:

  1. Transgenic zebrafish central injury model: Transgenic zebrafish with GFP-labeled motor neurons in the hindbrain can be treated with mycophenolate mofetil (MMF) to induce central injury, then treated with rhFGF12 to assess neuroprotection .

  2. Rodent models of neuronal injury: Various rodent models can be employed, including:

     • Traumatic brain injury models

     • Spinal cord injury models

     • Stroke models (middle cerebral artery occlusion)

     • Seizure models (kainic acid or pilocarpine-induced)

  3. Drosophila models: These have been successfully used for in vivo functional analysis of FGF12 variants and could be adapted to test rhFGF12 neuroprotective effects.

  For all these models, appropriate outcome measures include behavioral assessments, histological analysis of neuronal survival, electrophysiological recordings, and molecular markers of neuronal health and function. The combination of in vitro and in vivo approaches provides a comprehensive evaluation of rhFGF12's neuroprotective potential and underlying mechanisms.

• How do different FGF12 variants identified in patients with epilepsy correlate with distinct clinical phenotypes?

  FGF12 variants exhibit genotype-phenotype correlations that provide insights into the molecular basis of associated neurological disorders:

  1. Heterozygous gain-of-function variants: These include recurrent missense variants and entire duplications of FGF12, which enhance its ability to delay sodium channel inactivation, resulting in increased neuronal excitability and epilepsy phenotypes .

  2. Biallelic loss-of-function variants: These include homozygous single-nucleotide variants (SNVs) and biallelic structural variations (SVs), which impair FGF12's normal modulatory function on sodium channels . These variants are associated with developmental and epileptic encephalopathy (DEE).

  To establish genotype-phenotype correlations, researchers should:

  • Collect comprehensive clinical data from patients with FGF12 variants, including seizure types, age of onset, developmental milestones, neuroimaging findings, and treatment responses

  • Perform functional characterization of variants through electrophysiological studies of sodium channel modulation in expression systems

  • Use patient-derived cells (such as induced pluripotent stem cells differentiated into neurons) to study the impact of specific variants on neuronal function

  • Develop animal models harboring specific FGF12 variants to assess behavioral and electrophysiological phenotypes

  By correlating clinical features with functional data, researchers can develop a more nuanced understanding of how different types of FGF12 dysfunction lead to specific epilepsy syndromes, potentially guiding the development of precision medicine approaches for affected individuals .