ABSTRACT
ABSTRACT: Decreased GABA levels in injury-induced loss of spinal inhibition are still under intense interest and debate. Here, we show that GAD67 haplodeficient mice exhibited a prolonged injury-induced mechanical hypersensitivity in postoperative, inflammatory, and neuropathic pain models. In line with this, we found that loss of 1 copy of the GAD67-encoding gene Gad1 causes a significant decrease in GABA contents in spinal GABAergic neuronal profiles. Consequently, GAD67 haplodeficient males and females were unresponsive to the analgesic effect of diazepam. Remarkably, all these phenotypes were more pronounced in GAD67 haplodeficient females. These mice had significantly much lower amount of spinal GABA content, exhibited an exacerbated pain phenotype during the second phase of the formalin test, developed a longer lasting mechanical hypersensitivity in the chronic constriction injury of the sciatic nerve model, and were unresponsive to the pain relief effect of the GABA-transaminase inhibitor phenylethylidenehydrazine. Our study provides strong evidence for a role of GABA levels in the modulation of injury-induced mechanical pain and suggests a potential role of the GABAergic system in the prevalence of some painful diseases among females.
Subject(s)
Hypersensitivity , Neuralgia , Male , Female , Mice , Animals , Neuralgia/drug therapy , Neuralgia/etiology , Sciatic Nerve/injuries , Pain Management , gamma-Aminobutyric Acid/therapeutic useABSTRACT
Pain, whether acute or persistent, is a serious medical problem worldwide. However, its management remains unsatisfactory, and new analgesic molecules are required. We show here that TAFA4 reverses inflammatory, postoperative, and spared nerve injury (SNI)-induced mechanical hypersensitivity in male and female mice. TAFA4 requires functional low-density lipoprotein receptor-related proteins (LRPs) because their inhibition by RAP (receptor-associated protein) dose-dependently abolishes its antihypersensitive actions. SNI selectively decreases A-type K+ current (IA) in spinal lamina II outer excitatory interneurons (L-IIo ExINs) and induces a concomitant increase in IA and decrease in hyperpolarization-activated current (Ih) in lamina II inner inhibitory interneurons (L-IIi InhINs). Remarkably, SNI-induced ion current alterations in both IN subtypes were rescued by TAFA4 in an LRP-dependent manner. We provide insights into the mechanism by which TAFA4 reverses injury-induced mechanical hypersensitivity by restoring normal spinal neuron activity and highlight the considerable potential of TAFA4 as a treatment for injury-induced mechanical pain.
Subject(s)
Cytokines/metabolism , Hyperalgesia/metabolism , Pain/metabolism , Potassium/metabolism , Receptors, LDL/metabolism , Spinal Cord Dorsal Horn/metabolism , Animals , CHO Cells , Cricetulus , HEK293 Cells , Humans , Mice , RAW 264.7 CellsABSTRACT
Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.