Citation
- Authors: Taylor MJ. et al.
- Year: 2022
- Journal: Sci Adv 8 eabq2611
- Applications: in vitro / in vivo / shRNA plasmid / in vivo-jetPEI
Method
To perform an initial dose response study to knock down Chk2 in vivo after DC injury by shRNA, 1 to 4 µg of plasmid DNA for shNull, shChk1, and shChk2 (all from Dharmacon) were complexed in in vivo JetPEI (catalog no. 101000040, Polyplus, New York, USA) and injected intra-DRG as described by us previously (20). Shamtreated animals (partial laminectomy but no DC injury) were also included as additional controls. At 4 weeks after DC injury and treatment, ipsilateral L4/L5 DRG pairs were harvested, total RNA was extracted using TRIzol reagent as described above, and Chk1 and Chk2 mRNA were knocked down using quantitative qRT-PCR, as described above.
Abstract
DNA double-strand breaks occur in many acute and long-term neurological conditions, including neurodegeneration, neurotrauma, and stroke. Nonrepaired breaks chronically activate the DNA damage response in neurons, leading to neural dysfunction and apoptosis. Here, we show that targeting of the central ATM-Chk2 pathway regulating the response to double-strand breaks slows neural decline in Drosophila models of chronic neurodegeneration. Inhibitors of ATM-Chk2, but not the parallel ATR-Chk1 pathway, also promote marked, functional recovery after acute central nervous system injury in rats, suggesting that inhibiting nonhomologous end-joining rather than homologous recombination is crucial for neuroprotection. We demonstrate that the Chk2 inhibitor, prexasertib, which has been evaluated in phase 2 clinical trials for cancer, has potent neuroprotective effects and represents a new treatment option to promote functional recovery after spinal cord or optic nerve injury.