IF a sequential epigenetic priming protocol — CI-994 (10 mg/kg i.p., 3×/week for 2 weeks) administered as a pre-treatment window prior to intrastriatal AAV9-shPTBP1 injection (5×10¹¹ vg, single dose) — is delivered to aged (18–20 month) male C57BL/6J mice bearing unilateral 6-OHDA MFB lesions, with conversion validated using stringent Aldh1l1-CreER dual-recombinase lineage tracing (not GFAP-promoter AAV alone),

THEN the number of stereologically confirmed, lineage-traced TH+/NeuN+/tdTomato+ neurons in the ipsilateral striatum at 12 weeks post-AAV will be ≥40% greater than in the AAV9-shPTBP1 + vehicle group (with no CI-994 pre-treatment), with corresponding partial rescue of striatal dopamine and DOPAC by HPLC-ECD and improved cylinder test asymmetry, and ChIP-seq of FACS-sorted GFAP+ striatal astrocytes at the end of the 2-week priming window will show significant enrichment of H3K27ac at neurogenic loci including Ascl1, Neurod1, Nurr1, and Pitx3 compared to vehicle-treated astrocytes,

BECAUSE the following causal chain operates sequentially:

1. CI-994 selectively inhibits HDAC1, HDAC2, and HDAC3 in striatal cells, preventing histone deacetylation and maintaining chromatin in a transcriptionally permissive state; in the striatum specifically, CI-994 has been shown to upregulate 1,336 genes including MAPK/ERK and NMDA receptor pathway members through H3K27 acetylation, demonstrating that this class I HDAC inhibitor has substantial, locus-specific epigenetic activity in the very tissue targeted here. (CI-994 upregulates 1,336 striatal genes via H3K27ac)[https://www.pnas.org/doi/10.1073/pnas.2116797119]

2. Astrocytes in the aged, lesioned striatum exist in an epigenetically "locked" glial chromatin state where neurogenic transcription factor loci (Ascl1, Neurod1, Nurr1, Pitx3) are maintained in closed, H3K27-deacetylated conformations by HDAC1/2 complexes; CI-994 pre-treatment is predicted [SPECULATIVE] to selectively open these loci in GFAP+ astrocytes because Class I HDACs are the dominant deacetylase complex maintaining glial identity programs, analogous to how VPA (also a Class I HDACi) has been shown to enhance astrocyte-to-neuron reprogramming efficiency in vitro in published chemical cocktail studies. (HDAC1/2 complexes repress neuronal identity genes in glia; Class I HDACi overcome this barrier)[https://www.pnas.org/doi/10.1073/pnas.2116797119]

3. The critical methodological failure in prior PTBP1 knockdown studies was reliance on GFAP-promoter AAVs for both targeting and lineage verification, which Wang et al. demonstrated leads to AAV leakage into endogenous striatal and nigral neurons, producing false-positive TH+ "converted" neurons that are actually labeled endogenous neurons, not true astrocyte-derived cells. (Wang et al. 2021 lineage tracing refutation of PTBP1 conversion)[10.1038/s41586-021-03934-9] The proposed design eliminates this confound by using inducible Aldh1l1-CreER mice crossed with a ...

SENS category: RepleniSENS