Abstract
Parkinson’s disease (PD) is a multifactorial disease caused by irreversible progressive loss of dopaminergic neurons (DANs). Recent studies have reported successful conversion of astrocytes into DANs by repressing polypyrimidine tract binding protein 1 (PTBP1), which led to the rescue of motor symptoms in a chemically-induced mouse model of PD. However, several follow-up studies have questioned the validity of this astrocyte to DAN conversion model. In this study, we devised an adenine base editing strategy to downregulate PTBP1 in astrocytes and neurons in a chemically-induced PD mouse model. While PTBP1 downregulation in astrocytes had no effect, we observed that PTBP1 downregulation in neurons of the substantia nigra pars compacta and striatum resulted in the expression of the DAN marker tyrosine hydroxylase (TH) in non-dividing neurons, which was associated with an increase in striatal dopamine concentrations and a rescue of forelimb akinesia and spontaneous rotations. Phenotypic analysis using multiplexed iterative immunofluorescence imaging further revealed that most of the TH-positive cells in the striatum co-expressed the dopaminergic marker DAT and the pan-neuronal marker NEUN, with the majority of these triple-positive cells being classified as mature GABAergic neurons. Additional research is needed to fully elucidate the molecular mechanisms underlying the expression of the observed markers and understand how the formation of these cells contributes to the rescue of spontaneous motor behaviors. Nevertheless, our findings support a model where neuronal, but not astrocytic, downregulation of PTBP1 can mitigate symptoms in PD mice.
Competing Interest Statement
G.S. is an advisor to Prime Medicine.
Footnotes
Key improvements include: i)We confirm functionality of intein-split AAV-ABE8e vectors by quantifying editing efficiencies at the Ptbp1 locus in vitro in neuronal and astrocytic cell lines. ii)We demonstrate proof of editing in the striatum and SNc of treated PD mice, confirming successful base editor delivery in vivo. Additionally, we show downregulation of Ptbp1 in the targeted brain regions at the RNA and protein level. iii)We performed sgRNA-dependent off-target analysis using GUIDE-seq and detected low base editor activity at off-target sites. RT-qPCR analysis revealed, however, no changes at the RNA level of these genes, strengthening our conclusion that the observed effects are attributed to Ptbp1 downregulation. iv)We further characterized TH+ neurons in vivo using multiplexed iterative immunofluorescence imaging and found that TH+ neurons either express markers of medium spiny neurons or various types of interneurons. v)We provide better-quality representative images in all figures to support the quantitative data and conclusions. vi)We provide a more detailed discussion on the potential mechanistic relationship between Ptbp1 downregulation and the partial phenotypic rescue, and highlight limitations of our study and remaining questions for future studies.
