IF a dual-vector adeno-associated virus (AAV9) system — (i) a tyrosine hydroxylase (TH)-promoter-driven phospho-mimetic DRP1^S616E/S637A transgene (constitutively fission-active; ~1×10¹³ vg/kg, intracranial stereotaxic or CSF delivery) combined with (ii) a synapsin-promoter-driven BNIP3L/NIX overexpression cassette — is administered bilaterally to 20-month-old male and female C57BL/6J mice (a standard aging background with documented nigral mtDNA deletion accumulation),

THEN single-neuron droplet digital PCR (ddPCR) quantification of Δ4977bp heteroplasmy in substantia nigra pars compacta (SNpc) dopaminergic neurons at 6 and 12 months post-injection will reveal a ≥30% reduction in mean per-cell deletion heteroplasmy relative to AAV-GFP vehicle controls, accompanied by restoration of mitochondrial Complex I/IV enzyme activity, preservation of TH⁺ neuronal counts, and improved open-field locomotor performance,

BECAUSE the following causal chain operates in sequence:

1. In aged post-mitotic neurons, a decline in endogenous DRP1 GTPase activity and an age-associated shift toward DRP1-pS637 (the inhibitory, PKA-phosphorylated state) promotes mitochondrial hyperfusion, creating large, interconnected networks in which wild-type mtDNA gene products functionally complement the bioenergetic deficit caused by Δ4977bp molecules — a phenomenon termed "phenotypic masking" — that shields the deleted genomes from the ΔΨm threshold required to trigger PINK1 stabilization and Parkin recruitment (Burman et al., Nature Cell Biology, 2023; Yang et al., Cell Death & Disease, 2022). [The hyperfused state is the primary failure mode of endogenous quality control]

2. The AAV9-TH::DRP1^S616E/S637A transgene replaces the function of endogenous DRP1 specifically in dopaminergic neurons, delivering a constitutively fission-active isoform that mimics CDK1/CDK5-mediated S616 phosphorylation while ablating the PKA-mediated S637 inhibitory brake. This forces asymmetric fission events that physically partition the mitochondrial network into polarized (ΔΨm-high, WT-mtDNA-enriched) and depolarized (ΔΨm-low, Δ4977bp-enriched) daughter organelles — a segregation geometry documented by Kleele et al. as the mechanistic prerequisite for selective mitophagy (Kleele et al., Nature, 2021). [Forced asymmetric fission disrupts phenotypic masking by isolating deleted molecules in daughter organelles below the ΔΨm threshold]

3. In aged neurons, even when depolarized daughter mitochondria are correctly isolated, downstream mitophagy is rate-limited by insufficient expression of the receptor-mediated mitophagy adaptors BNIP3L/NIX and FUNDC1, which are required to tether the depolarized organelle to the autophagosome membrane independently of ubiquitin-dependent PINK1/Parkin flux (Burman et al., Nature Cell Biology, 2023). The AAV9-Synapsin::BNIP3L cassette provides pan-neuronal overexpression of NIX, ensuring that every depolarized, Δ4977bp-enriched daughter mitochondrion generated b...

SENS category: LysoSENS