S100b Mouse, His

What are the main S100B mouse models available for neurogenesis research?

S100B transgenic (S100Btg) mice with multiple copies of the murine S100B gene are widely used for studying long-term increased S100B expression. The most documented model contains 12 copies of the murine S100B gene, resulting in nanomolar S100B concentrations in brain tissue, cerebrospinal fluid (CSF), and serum . These levels remain below the toxic micromolar concentrations typically used in cell culture experiments.

In contrast, S100B knockout (S100B KO) mice have been developed to study the effects of S100B deletion. These models show almost non-existent tissue S100B expression compared to wild-type counterparts .

For studying specific mechanisms, researchers should consider:

• S100Btg mice: Optimal for investigating neurotrophic effects at nanomolar concentrations

• S100B KO mice: Valuable for understanding loss-of-function consequences, particularly in neuroinflammatory conditions

How do His-tagged S100B recombinant proteins differ from endogenous mouse S100B?

His-tagged S100B recombinant proteins typically contain a hexahistidine tag (HHHHHH) fused to the S100B protein sequence. This modification facilitates protein purification through affinity chromatography but may influence protein behavior in some experimental settings.

Key differences include:

• Molecular weight: His-tagged mouse S100B recombinant proteins have a predicted molecular weight of approximately 12.8 kDa (112 amino acids), compared to native S100B (approximately 10.5 kDa)

• Formulation: Recombinant His-tagged S100B is typically supplied in buffer solutions containing stabilizers (e.g., 20mM Tris-HCl buffer pH 8.0 with 1mM DTT, 10% glycerol)

• Purity: Commercial preparations are typically >85% pure as assessed by SDS-PAGE

For physiologically relevant experiments, researchers should consider whether the His-tag might interfere with binding to target receptors or downstream signaling pathways.

What are the key parameters to validate when using S100B mouse models?

To ensure experimental rigor with S100B mouse models, researchers should validate:

• S100B expression levels: Confirm elevated S100B in transgenic models or absent expression in knockout models using techniques like western blotting or qPCR

• Age-dependent effects: S100B effects vary significantly across juvenile (28 days), adult (3 months), and senile (one year) mice

• Sex-specific differences: Male and female S100Btg mice show differential effects on ApoE and BDNF expression

• Regional expression patterns: Assess S100B levels in relevant brain regions (hippocampus, frontal cortex)

• Baseline cognitive and behavioral phenotypes: Document any baseline differences in neurological function

Data from transgenic models indicate approximately 50% increased proliferation in juvenile S100Btg mice and 200% increased proliferation in adult S100Btg mice compared to wild-type controls .

How should researchers quantify hippocampal neurogenesis in S100B mouse models?

Quantifying hippocampal neurogenesis in S100B mouse models requires careful methodological consideration:

Method comparison:
Both BrdU incorporation and Ki67 immunoreactivity provide reliable measures of progenitor cell proliferation. Studies have demonstrated a significant correlation between these methods (r = 0.608, p < 0.001) .

Recommended protocol:

• For BrdU labeling: Administer 50 mg/kg BrdU intraperitoneally before tissue collection

• For Ki67 immunohistochemistry: Use standard immunohistochemical procedures on fixed tissue sections

• Quantify proliferating cells specifically in:

  • Subgranular zone (SGZ) - The primary germinative area of the dentate gyrus

  • Granular cell layer (GCL) - The destination area for migrating progenitor cells

• Compare multiple age groups to account for age-dependent decline in neurogenesis

Ki67 immunohistochemistry may be preferable as it avoids the experimental burden of BrdU injections while providing comparable results, aligning with EU Directive 2010/63/EU on reducing animal experimentation burden .

What molecular markers should be assessed alongside S100B to understand its effects on neurogenesis?

To comprehensively evaluate S100B's role in neurogenesis, researchers should assess the following markers:

• Proliferation markers:

  • Ki67: Endogenous marker expressed during all active phases of the cell cycle

  • BrdU: Exogenous thymidine analog that incorporates into newly synthesized DNA

• Associated signaling proteins:

  • GFAP (Glial Fibrillary Acidic Protein): Marker for astrocytes and neural stem cells

  • ApoE (Apolipoprotein E): Associated with lipid metabolism and neuronal repair

  • BDNF (Brain-Derived Neurotrophic Factor): Growth factor that promotes neuronal survival

  • RAGE (Receptor for Advanced Glycation End Products): Principal receptor for S100B

• Neuroinflammatory markers:

  • Iba1: General microglia/macrophage marker

  • iNOS: Marker for pro-inflammatory microglia/macrophages

  • CX3CR1 (Fractalkine receptor): Marker for homeostatic/phagocytic microglia

Studies have shown that long-term increased S100B levels correlate with decreased hippocampal GFAP, suggesting reduced astrogliosis rather than increased reactive astrocytes .

How can researchers effectively analyze His-tagged S100B protein purity and activity?

For His-tagged S100B protein analysis:

Purity assessment:

• SDS-PAGE: Verify a single band at approximately 12.8 kDa (for His-tagged mouse S100B)

• MALDI-TOF: Confirm predicted molecular weight of 12.8 kDa for the 112 amino acid sequence

• Endotoxin testing: Ensure levels are <1 EU per 1μg of protein (using LAL method)

Activity testing:

• Binding assays: Confirm interaction with known S100B receptors (e.g., RAGE)

• Functional assays: Test effects on cell proliferation in neural progenitor cultures

• Downstream signaling: Assess activation of known S100B-dependent pathways

Storage and handling:

• Short-term (1 week): Store at 2-8°C

• Long-term: Aliquot and store at -20°C to -80°C

• Avoid repeated freeze-thaw cycles

How does S100B modulate hippocampal neurogenesis at different concentrations and across different age groups?

S100B demonstrates complex concentration-dependent and age-dependent effects on hippocampal neurogenesis:

Concentration effects:

• Nanomolar (nM) concentrations: Neurotrophic, promoting progenitor cell proliferation

• Micromolar (μM) concentrations: Typically toxic in cell culture models

Age-dependent effects (Ki67+ cells in SGZ):

S100B-induced progenitor cell proliferation in the SGZ increases with age relative to the declining baseline in wild-type mice. Additionally, migration of proliferating cells to the GCL increases with age in S100Btg mice .

The mechanism appears to involve direct S100B effects rather than indirect actions through BDNF, as hippocampal BDNF levels remain unchanged in S100Btg mice despite enhanced neurogenesis .

What are the sex-specific differences in S100B function and how should researchers account for them?

Research reveals important sex-specific differences in S100B function:

Male-specific effects:

• Reduced ApoE levels in both hippocampus and frontal cortex of male S100Btg mice

• No significant changes in BDNF levels in male mice

Female-specific effects:

• Reduced BDNF in frontal cortex of female S100Btg mice

• No significant changes in ApoE levels in female mice

Sex-independent effects:

• Enhanced hippocampal progenitor cell proliferation in both sexes

• Reduced hippocampal GFAP content in both sexes

Methodological recommendations:

• Always include both sexes in experimental designs

• Analyze data separately by sex before pooling

• Consider potential hormonal influences on S100B signaling

• When studying ApoE or BDNF interactions with S100B, sex stratification is crucial

• For translational studies, sex differences may have implications for therapeutic approaches

These findings highlight the complex sex-dependent cross-talk between S100B and other signaling molecules relevant to neurogenesis and neuroinflammation.

How does S100B interact with neuroinflammatory pathways in models of neurological disease?

S100B plays a complex role in neuroinflammatory conditions as demonstrated in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis:

S100B deletion effects in EAE:

• Delayed disease onset

• Reduced paralysis

• Accelerated recovery, particularly in chronic stages

• Reduction in total number and area of demyelinating lesions

Neuroinflammatory marker changes:

• Reduced GFAP expression (astrocyte marker) in S100B KO EAE mice

• Reduced Iba1 expression (microglia/macrophage marker) in S100B KO EAE mice

• Decreased iNOS (pro-inflammatory marker) expression

• Altered CX3CR1 (homeostatic/phagocytic microglia marker) expression

S100B functions as a damage-associated molecular pattern (DAMP) or alarmin, released upon tissue injury and contributing to neuroinflammation. Research suggests therapeutic potential in blocking S100B in neuroinflammatory conditions, as demonstrated by pentamidine treatment in EAE models .

The dual role of S100B (neurotrophic at nanomolar levels, potentially harmful in inflammatory conditions) highlights the importance of context-specific research approaches.

What are the implications of S100B inhibition for potential therapies in neurological disorders?

Research on S100B inhibition has revealed promising therapeutic potential:

In multiple sclerosis models:

• S100B knockout mice show ameliorated clinical symptoms in EAE

• Pharmacological inhibition with pentamidine changed disease course and improved outcome

• Pentamidine treatment prevented lesion formation and oligodendrocyte impairment

• S100B inhibition decreased astrocytic reactivity and promoted non-pro-inflammatory microglia/macrophages

Immunomodulatory effects:

• Enhanced CNS infiltration by regulatory T (Treg) cells

• Reduced T helper (Th)1 and Th17 cell infiltration

• These changes may favor remyelinating processes

These findings suggest repurposing pentamidine or developing other S100B inhibitors as potential therapies for multiple sclerosis and potentially other neuroinflammatory conditions.

How can researchers integrate findings from S100B transgenic and knockout models to understand its therapeutic potential?

The seemingly contradictory findings from S100B transgenic and knockout models can be reconciled to understand therapeutic potential:

Integrative model:

• Context-dependent effects: S100B acts as neurotrophic at nanomolar levels in non-inflammatory contexts, while functioning as a DAMP in inflammatory conditions

• Disease-specific considerations: In traumatic brain injury or developmental contexts, promoting S100B may enhance neurogenesis; in neuroinflammatory conditions, inhibiting S100B may be beneficial

• Concentration-dependent paradigm: Therapeutic approaches should consider whether local S100B concentrations are in the neurotrophic (nM) or potentially harmful (μM) range

Research recommendations:

• Combine transgenic overexpression studies with controlled inhibition experiments

• Monitor both S100B levels and inflammatory markers

• Test therapeutic hypotheses in disease-specific models

• Consider sex as a biological variable, given sex-specific effects

• Evaluate age-dependent effects, as S100B function changes across the lifespan

This integrated approach can help reconcile the dual roles of S100B and guide development of context-appropriate therapeutic strategies.

What experimental approaches can assess the efficacy of His-tagged S100B protein as a potential therapeutic agent?

To evaluate His-tagged S100B as a potential therapeutic agent:

In vitro assessments:

• Dose-response studies (nM vs. μM concentrations) on neural progenitor cultures

• Receptor binding studies (particularly RAGE) with purified His-tagged protein

• Verification that His-tag doesn't interfere with biological activity

• Stability testing under physiological conditions

In vivo approaches:

• Intrathecal or intraperitoneal administration protocols

• Blood-brain barrier penetration studies

• Pharmacokinetic assessment of His-tagged S100B

• Functional outcome measures following administration:

  • Hippocampal-dependent cognitive tasks

  • Histological assessment of neurogenesis (Ki67/BrdU)

  • Evaluation of associated markers (GFAP, ApoE, BDNF)

Prior research has demonstrated that "acute intrathecal and intraperitoneal treatment with S100B in wild-type rodents promoted hippocampal neurogenesis and cognitive function," suggesting potential therapeutic applications in contexts requiring enhanced neurogenesis.