PTEN inhibition dedifferentiates long-distance axon-regenerating intrinsically photosensitive retinal ganglion cells and upregulates mitochondria-associated DYNLT1A and LARS2

The central nervous system (CNS) projection neurons fail to spontaneously regenerate injured axons. Targeting the developmentally regulated genes in order to reactivate embryonic intrinsic axon growth capacity, or targeting tumor suppressor genes such as Pten, promote axon regeneration in a subset of injured retinal ganglion cells (RGCs). The subset of RGCs that regenerate axons in response to inhibition of Pten was narrowed-down to the Opn4+ intrinsically photosensitive (ip) and a subtypes of RGCs. Here, we used single cell RNA-sequencing (scRNA-seq) to investigate why only a subset of injured RGCs regenerate axons in response to Pten knockdown (KD), and to characterize the relationship between axon regeneration promoted by targeting Pten and the developmental decline in intrinsic axon growth capacity. We found that, only the most similar to embryonic state ipRGC subtypes C33 and C40 regenerated axons in response to Pten KD, which dedifferentiated them even further towards an embryonic state. We also found that genes downstream of Pten KD, specifically in the RGCs that regenerated axons, include mitochondria-associated developmentally regulated and non-developmentally regulated Dynlt1a and Lars2, respectively, which promoted axon regeneration on their own. Overall, we show that injury itself reverts transcriptome towards an embryonic state only in some neuronal subtypes and not sufficiently close to embryonic state to confer response to Pten KD, whereas certain mature neuronal subtypes are similar to embryonic state even without injury, which primes them to regenerate axons in response to Pten KD, that dedifferentiates them further towards an embryonic state and upregulates mitochondria-associated Dynlt1a and Lars2. Overall design: Pten was KD in the adult RGCs using an intravitreally injected adeno-associated virus serotype 2 (AAV2) vector. The regenerating RGCs were FACS-isolated from the retinal single cell suspension 2 weeks after ONC injury, based on the expression of an mCherry reporter (identifying the transduced cells) and a signal of Alexa Fluor 488 conjugated to the Cholera toxin subunit B (CTB). After sorting, cells were processed by plate-based droplet scRNA-seq. The transcriptomes of 101 regenerating RGCs passed quality control.