Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair

Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB) repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ), single-strand annealing (SSA), and homology directed repair (HDR/GC). Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI–induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy

Blocking microglial pannexin-1 channels alleviates morphine withdrawal in rodents

Opiates are essential for treating pain, but termination of opiate therapy can cause a debilitating withdrawal syndrome in chronic users. To alleviate or avoid the aversive symptoms of withdrawal, many of these individuals continue to use opiates. Withdrawal is therefore a key determinant of opiate use in dependent individuals, yet its underlying mechanisms are poorly understood and effective therapies are lacking. Here, we identify the pannexin-1 (Panx1) channel as a therapeutic target in opiate withdrawal. We show that withdrawal from morphine induces long-term synaptic facilitation in lamina I and II neurons within the rodent spinal dorsal horn, a principal site of action for opiate analgesia. Genetic ablation of Panx1 in microglia abolished the spinal synaptic facilitation and ameliorated the sequelae of morphine withdrawal. Panx1 is unique in its permeability to molecules up to 1 kDa in size and its release of ATP. We show that Panx1 activation drives ATP release from microglia during morphine withdrawal and that degrading endogenous spinal ATP by administering apyrase produces a reduction in withdrawal behaviors. Conversely, we found that pharmacological inhibition of ATP breakdown exacerbates withdrawal. Treatment with a Panx1-blocking peptide (10panx) or the clinically used broad-spectrum Panx1 blockers, mefloquine or probenecid, suppressed ATP release and reduced withdrawal severity. Our results demonstrate that Panx1-mediated ATP release from microglia is required for morphine withdrawal in rodents and that blocking Panx1 alleviates the severity of withdrawal without affecting opiate analgesia

Macrophages Regulate Schwann Cell Maturation after Nerve Injury

Summary: Pro-regenerative macrophages are well known for their role in promoting tissue repair; however, their specific roles in promoting regeneration of the injured nerve are not well defined. Specifically, how macrophages interact with Schwann cells following injury during remyelination has been largely unexplored. We demonstrate that after injury, including in humans, macrophages function to clear debris and persist within the nerve microenvironment. Macrophage ablation immediately preceding remyelination results in an increase in immature Schwann cell density, a reduction in remyelination, and long-term deficits in conduction velocity. Targeted RNA-seq of macrophages from injured nerve identified Gas6 as one of several candidate factors involved in regulating Schwann cell dynamics. Functional studies show that the absence of Gas6 within monocyte lineage cells impairs Schwann cell remyelination within the injured nerve. These results demonstrate a role for macrophages in regulating Schwann cell function during nerve regeneration and highlight a molecular mechanism by which this occurs. : Stratton et al. demonstrate that macrophages persist in the injured rodent and human nerve and regulate Schwann cells. Macrophages have a unique transcriptional profile, including the expression of Gas6, that functions to regulate Schwann cell remyelination. Keywords: nerve injury, macrophage, Schwann cell, regeneration, remyelination, population-based RNA-se

A dynamic role for dopamine receptors in the control of mammalian spinal networks

Authors acknowledge studentships from the Natural Sciences and Engineering Research Council of Canada (NSERC-PGS-D: SAS), the Canadian Institute for Health Research (CIHR: NEB), Alberta Innovates—Health Solutions (AIHS: SAS, NEB), Hotchkiss Brain Institute (SAS, NEB) and the Faculty of Veterinary Medicine (CHTK). This research was supported by grants from NSERC (Discovery grant: PJW), CIHR (PJW, YZ) and the VP Research Office and the Faculty of Medicine & Dentistry at the University of Alberta (GBB).Dopamine is well known to regulate movement through the differential control of direct and indirect pathways in the striatum that express D1 and D2 receptors respectively. The spinal cord also expresses all dopamine receptors; however, how the specific receptors regulate spinal network output in mammals is poorly understood. We explore the receptor-specific mechanisms that underlie dopaminergic control of spinal network output of neonatal mice during changes in spinal network excitability. During spontaneous activity, which is a characteristic of developing spinal networks operating in a low excitability state, we found that dopamine is primarily inhibitory. We uncover an excitatory D1-mediated effect of dopamine on motoneurons and network output that also involves co-activation with D2 receptors. Critically, these excitatory actions require higher concentrations of dopamine; however, analysis of dopamine concentrations of neonates indicates that endogenous levels of spinal dopamine are low. Because endogenous levels of spinal dopamine are low, this excitatory dopaminergic pathway is likely physiologically-silent at this stage in development. In contrast, the inhibitory effect of dopamine, at low physiological concentrations is mediated by parallel activation of D2, D3, D4 and α2 receptors which is reproduced when endogenous dopamine levels are increased by blocking dopamine reuptake and metabolism. We provide evidence in support of dedicated spinal network components that are controlled by excitatory D1 and inhibitory D2 receptors that is reminiscent of the classic dopaminergic indirect and direct pathway within the striatum. These results indicate that network state is an important factor that dictates receptor-specific and therefore dose-dependent control of neuromodulators on spinal network output and advances our understanding of how neuromodulators regulate neural networks under dynamically changing excitability.Publisher PDFPeer reviewe

Erratum: Blocking microglial pannexin-1 channels alleviates morphine withdrawal in rodents.

Opiates are essential for treating pain, but termination of opiate therapy can cause a debilitating withdrawal syndrome in chronic users. To alleviate or avoid the aversive symptoms of withdrawal, many of these individuals continue to use opiates. Withdrawal is therefore a key determinant of opiate use in dependent individuals, yet its underlying mechanisms are poorly understood and effective therapies are lacking. Here, we identify the pannexin-1 (Panx1) channel as a therapeutic target in opiate withdrawal. We show that withdrawal from morphine induces long-term synaptic facilitation in lamina I and II neurons within the rodent spinal dorsal horn, a principal site of action for opiate analgesia. Genetic ablation of Panx1 in microglia abolished the spinal synaptic facilitation and ameliorated the sequelae of morphine withdrawal. Panx1 is unique in its permeability to molecules up to 1 kDa in size and its release of ATP. We show that Panx1 activation drives ATP release from microglia during morphine withdrawal and that degrading endogenous spinal ATP by administering apyrase produces a reduction in withdrawal behaviors. Conversely, we found that pharmacological inhibition of ATP breakdown exacerbates withdrawal. Treatment with a Panx1-blocking peptide (10panx) or the clinically used broad-spectrum Panx1 blockers, mefloquine or probenecid, suppressed ATP release and reduced withdrawal severity. Our results demonstrate that Panx1-mediated ATP release from microglia is required for morphine withdrawal in rodents and that blocking Panx1 alleviates the severity of withdrawal without affecting opiate analgesia