Highlights d Piezo cell autonomously inhibits axon regeneration in Drosophila sensory neurons d Piezo channels are activated after axon injury or during axon regeneration d Piezo inhibits axon regeneration through calcium signaling, Nos, and PKG d Piezo1 also inhibits axon regeneration in mammals in vitro and in vivo

Highlights d The dynamic assembly shows varying tunnel width, helical rise, and domain orientation d The N-terminal domain and C-terminal tail are not required for CtHsp104 hexamerization d CtHsp104 rescues TDP-43, polyGlu and a-Syn toxicity in yeast and is well tolerated d CtHsp104 confers thermotolerance to yeast suggesting collaboration with Hsp70/Hsp40

Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate the DNA damage checkpoint by phosphorylating Chek1, which inhibits Cdc25, causing cell cycle arrest. This pathway has not been implicated in neuroregeneration. We show that in Drosophila sensory neurons removing Atr or Chek1, or overexpressing Cdc25 promotes regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown impedes regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes regeneration and improves synapse/behavioral recovery after CNS injury. Independent of DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the mechanosensitive ion channel Piezo and its downstream NO signaling. Sensory neuron-specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in mammalian neurons in vitro and in flies in vivo enhances regeneration. Our findings reveal the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for regeneration, and identify potential therapeutic targets for treating nervous system trauma.

Cells sense and respond to mechanical stimuli by converting those stimuli into biological signals, a process known as mechanotransduction. Mechanotransduction is essential in diverse cellular functions, including tissue development, touch sensitivity, pain, and neuronal pathfinding. In the search for key players of mechanotransduction, several families of ion channels were identified as being mechanosensitive and were demonstrated to be activated directly by mechanical forces in both the membrane bilayer and the cytoskeleton. More recently, Piezo ion channels were discovered as a bona fide mechanosensitive ion channel, and its characterization led to a cascade of research that revealed the diverse functions of Piezo proteins and, in particular, their involvement in neuronal repair.

26Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate 27 the DNA damage checkpoint by phosphorylating Chek1, which inhibits Cdc25, causing cell 28 cycle arrest. This pathway has not been implicated in neuroregeneration. We show that in 29 Drosophila sensory neurons, removing Atr or Chek1, or overexpressing Cdc25 promotes 30 regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown impedes 31 regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes 32 regeneration and improves synapse/behavioral recovery after CNS injury. Independent of 33 DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the 34 mechanosensitive ion channel Piezo and its downstream NO signaling. Sensory neuron-35 specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in 36 mammalian neurons in vitro and in flies in vivo enhance regeneration. Our findings reveal 37the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for 38 regeneration, and identify potential therapeutic targets for treating nervous system 39 trauma. 40 41 DNA damage response (DDR). We found that mediators of the DNA single-strand break (SSB) 63 response specifically inhibit axon regeneration. 64SSBs are known to activate Atr (ataxia telangiectasia and Rad3 related), a 65 serine/threonine kinase that directly phosphorylates Chek1 (checkpoint kinase-1). Chek1 in turn 66 phosphorylates and inhibits the phosphatase Cdc25C (cell division cycle 25C) or Cdc25A, which 67 would prevent Cdk1(cyclin-dependent kinase 1)/CycB (cyclin B) from being dephosphorylated 68 and therefore cause a cell cycle arrest in G2/M or S-phase, respectively 13, 14 . A multistep model 69 4 has been proposed for Atr checkpoint activation in response to DNA damage 15 , which involves 70 DNA damage sensing, signal transduction and execution. DNA damage generates ssDNA 71 (single-stranded DNA), which is recognized and coated by RPA (Replication protein A). The 72 primed ssDNA recruits Atr-Atrip (Atr interacting protein) and facilitates the loading of 9-1-1 73 (Rad9-Hus1-Rad1) by the Rad17 complex. The 9-1-1 complex may then stimulate the kinase 74 activity of Atr-Atrip, leading to phosphorylation of its substrates including Rad17 and Rad9. 75Phosphorylated Rad17 and Rad9 may facilitate the recruitment of downstream signaling proteins 76Claspin and TopBP1 (topoisomerase (DNA) II binding protein 1), allowing them to be efficiently 77 phosphorylated by Atr. Phosphorylated TopBP1 may further stimulate the kinase activity of Atr, 78whereas phosphorylation of Claspin may promote the phosphorylation and activation of Chek1. 79Atr can also be activated by mechanical force. It has been reported that Atr can respond 80 to mechanical stimuli, such as osmotic stress, in mediating chromosome dynamics, which is 81 independent of DNA damage 16 . However, the underlying mechanoreceptor remains unknown. 82We have recently demonstrated that the mechanosensitive (MS) i...