STRC AC1/CREB Alternative Hypothesis
Replacement for the NFAT-based sonogenetic approach, after critical review found that Ca2+ from MET channels does not reach the OHC soma where NFAT/calcineurin reside.
Why NFAT Fails
- Ca2+ from MET channels is trapped in stereocilia by millimolar buffers, PMCA2 pumps, mitochondrial barrier, and 4-6 mM oncomodulin
- NFATc4 nuclear translocation in OHCs observed ONLY during damage (noise, ototoxins)
- NFATc4 downstream targets in OHC: TNF, Caspase-8, Caspase-3 (apoptosis)
- NFATc4 knockout mice have normal hearing
The AC1/CREB Cascade
Sound → MET channels → Ca2+ (in stereocilia)
→ Calmodulin (in stereocilia)
→ AC1/ADCY1 (IN stereocilia — confirmed by immunolocalization)
→ cAMP (small molecule, diffuses freely past cuticular plate)
→ PKA (in soma)
→ CREB phosphorylation (Ser133)
→ CRE-dependent promoter → mini-STRC expression
Why AC1/CREB is Better
| Problem with NFAT | Solution in AC1/CREB |
|---|---|
| Ca2+ doesn’t reach soma | AC1 is IN stereocilia, converts Ca2+ to cAMP locally |
| Ca2+ is heavily buffered | cAMP is not buffered, diffuses freely |
| NFAT activation = apoptosis | CREB = neuroplasticity, no death link |
| NFATc4 KO = normal hearing | ADCY1 mutations = deafness (essential gene) |
Key Evidence
- ADCY1 in stereocilia: Protein localized to cochlear hair cell stereocilia and nuclei (PubMed 24482543)
- ADCY1 causes deafness: Recessive hearing impairment in humans and zebrafish from ADCY1 mutations
- Ca2+-calmodulin activates AC1: Well-characterized biochemistry. AC1 is one of two CaM-activated adenylyl cyclases (with AC8)
- CRE promoter: Standard responsive element (TGACGTCA), used in viral vectors in vivo, well-characterized
What Remains Unknown
- Does AC1 produce enough cAMP during normal hearing (not just loud sound)?
- Does cAMP from stereocilia reach the nucleus at sufficient concentration?
- Would a CRE-responsive promoter fire in OHCs? Never tested.
- Specificity: cAMP/CREB responds to many signals beyond sound. May have unwanted basal activity.
- All ODE parameters need re-derivation for AC1/cAMP/PKA/CREB kinetics.
Construct Design (Preliminary)
5' ITR (145 bp) + CRE-responsive promoter (~200 bp) + mini-STRC CDS (3,546 bp) + bGH polyA (250 bp) + 3' ITR (145 bp)
= ~4,286 bp (414 bp margin in AAV)
CRE promoter options:
- 6xCRE + minimal promoter (standard, ~200 bp)
- Hybrid CRE-SRE (as in Pan et al. 2018, higher specificity)
Speculative: DFNB16-Specific Calcium Leak
In Strc-/- mice, stereocilia lose horizontal top connectors by P15, bundle stiffness drops 60-74%. Possible that disrupted structure allows Ca2+ to leak into soma, making NFAT viable ONLY in diseased cells. If true: disease activates promoter, cure silences it. Perfect self-dosing.
No one has measured somatic Ca2+ in Strc-/- OHCs. Testable by calcium imaging in cochlear explants.
Next Steps
- Build ODE model for AC1/cAMP/PKA/CREB pathway with cochlear parameters
- Ask Dr. Holt: “Does MET-channel Ca2+ reach the soma? Would AC1/CRE be more realistic?”
- Search for ADCY1 expression levels in OHC transcriptomes (Liu et al. 2014, Sdata 2018)
- Design 6xCRE promoter construct
- Consider calcium imaging experiment in Strc-/- explants
Connections
- Sonogenetic STRC Computational Proof — NFAT pathway has critical biological gaps
- STRC Sonogenetics Bifurcation Analysis — math is correct but biology is wrong
[about]Misha[see-also]Alternative STRC Delivery Hypotheses