IF a dual-AAV2/7 system co-delivering (i) a phospho-site-deficient ASCL1 variant (ASCL1-6SA; serine/threonine-to-alanine substitutions at CDK5/GSK3β consensus sites, under gfaABC1D promoter, 1×10¹² vg/ml) together with (ii) CasRx-mediated dual knockdown of NFIA and NFIB (the NFI-family glial identity maintainers, under gfaABC1D promoter, 2×10¹² vg/ml) and (iii) a third AAV2/7 encoding LMX1A–T2A–NURR1 under the same promoter (1×10¹² vg/ml; total 4 μl co-injected at AP −3.1 mm, ML ±1.2 mm, DV −4.6 mm from bregma) is delivered stereotaxically to the substantia nigra pars compacta (SNc) of 8–10-week-old male Aldh1l1-CreERT2;Rosa26-tdTomato C57BL/6 mice at 1 week post-6-OHDA lesion (tamoxifen induction 2 weeks pre-lesion),

THEN at 16 weeks post-injection, ≥15% of tdTomato⁺ astrocyte-lineage cells in the SNc will be tdTomato⁺/TH⁺/NeuN⁺ triple-positive, striatal dopamine content (HPLC-ECD) will be restored to ≥40% of the intact contralateral hemisphere, and amphetamine-induced rotational asymmetry will be reduced by ≥50% relative to lesion-only vehicle controls,

BECAUSE three mechanistically independent and sequentially necessary barriers to astrocyte-to-dopaminergic-neuron conversion are jointly dismantled:

1. Wild-type ASCL1 pioneer activity is suppressed by CDK5/GSK3β phosphorylation in mature astrocytes, preventing chromatin remodeling at neuronal loci. The phospho-site-deficient ASCL1-6SA variant retains full bHLH DNA binding while escaping kinase-mediated inhibition, enabling it to open silent neuronal chromatin in post-mitotic astrocytes in a manner unachievable by wild-type ASCL1 overexpression alone. (Phospho-site-deficient Ascl1 redirects fate toward fast-spiking interneurons in cortical astroglia with far greater efficiency than wild-type)[https://doi.org/10.1101/2023.11.03.565289]

2. NFI transcription factors (NFIA, NFIB) maintain a transcriptional lock on astrocyte identity by occupying astrocyte-specific loci and actively repressing neuronal gene modules. In the adult SNc, these factors constitute a persistent epigenetic barrier that ASCL1 overexpression alone cannot overcome. RBPJ (Notch effector) directly binds and promotes NFI factor expression; integrated single-cell transcriptomic and CUT&Tag data demonstrate that inhibiting both RBPJ-driven Notch effectors and NFI factors is necessary and sufficient to unlock glia-to-neuron conversion in a lineage-traced CNS glia population. (RBPJ directly drives NFI expression, and dual inhibition of Notch effectors plus NFI factors achieves robust glia-to-neuron reprogramming confirmed by fate-mapping)[https://doi.org/10.1101/2023.10.29.560483]

3. [SPECULATIVE] PTBP1 knockdown contributes a post-transcriptional derepression layer — releasing neuronal mRNAs (e.g., Ptbpn1, Snap25, Syt1) from suppression — but this is insufficient alone because it does not address epigenetic or transcriptional locks. When PTBP1 knockdown is layered onto an already-initiated neuro...

SENS category: RepleniSENS

Key references: • doi.org/10.1101/2023.11.03.565289] • doi.org/10.1101/2023.10.29.560483] • doi.org/10.1101/2023.11.03.565289 • doi.org/10.1101/2023.10.29.560483