Proper mechanical stimulation can improve rotator cuff enthesis injury repair. However, the underlying mechanism of mechanical stimulation promoting injury repair is still unknown. In this study, we found that Prrx1⁺ cell was essential for murine rotator cuff enthesis development identified by single-cell RNA sequence and involved in the injury repair. Proper mechanical stimulation could promote the migration of Prrx1⁺ cells to enhance enthesis injury repair. Meantime, TGF-β signaling and primary cilia played an essential role in mediating mechanical stimulation signaling transmission. Proper mechanical stimulation enhanced the release of active TGF-β1 to promote migration of Prrx1⁺ cells. Inhibition of TGF-β signaling eliminated the stimulatory effect of mechanical stimulation on Prrx1⁺ cell migration and enthesis injury repair. In addition, knockdown of Pallidin to inhibit TGF-βR2 translocation to the primary cilia or deletion of Ift88 in Prrx1⁺ cells also restrained the mechanics-induced Prrx1⁺ cells migration. These findings suggested that mechanical stimulation could increase the release of active TGF-β1 and enhance the mobilization of Prrx1⁺ cells to promote enthesis injury repair via ciliary TGF-β signaling.

Lineage reprograming of resident glial cells to dopaminergic neurons (DAns) is an attractive prospect of the cell-replacement therapy for Parkinson's disease (PD). However, it is unclear whether repressing polypyrimidine tract binding protein 1 (PTBP1) could efficiently convert astrocyte to DAns in the substantia nigra and striatum. Although reporter-positive DAns were observed in both groups after delivering the adeno-associated virus (AAV) expressing a reporter with shRNA or CRISPR-CasRx to repress astroglial PTBP1, the possibility of AAV leaking into endogenous DAns could not be excluded without using a reliable lineage-tracing method. By adopting stringent lineage-tracing strategy, two other studies showed that either knockdown or genetic deletion of quiescent astroglial PTBP1 fails to obtain induced DAns under physiological condition. However, the role of reactive astrocytes might be underestimated because upon brain injury, reactive astrocyte can acquire certain stem cell hallmarks that may facilitate the lineage conversion process. Therefore, whether reactive astrocytes could be genuinely converted to DAns after PTBP1 repression in a PD model needs further validation. In this study, we used Aldh1l1-CreERT2-mediated specific astrocyte-lineage-tracing method to investigate whether reactive astrocytes can be converted to DAns in a 6-hydroxydopamine (6-OHDA) mouse model of PD. However, we found that no astrocyte-originated DAn was generated after effective and persistent knockdown of astroglial PTBP1 either in the substantia nigra or in striatum, while AAV 'leakage' to nearby neurons was easily observed. Our results confirmed that repressing PTBP1 does not convert astrocytes to DAns, regardless of physiological or PD-related pathological conditions.