Activation of microglia in the spinal cord dorsal horn following peripheral nerve injury contributes to the development of pain hypersensitivity. How activated microglia selectively enhance the activity of spinal nociceptive circuits is not well understood. We discovered that following peripheral nerve injury, microglia degrade extracellular matrix structures, perineuronal nets (PNNs), in lamina I of the spinal cord dorsal horn. Lamina I PNNs selectively enwrap spinoparabrachial projection neurons, which integrate nociceptive information in the spinal cord and convey it to supraspinal brain regions to induce pain sensation. Degradation of PNNs by microglia enhances the activity of projection neurons and induces pain-related behaviors. Thus, nerve injury-induced degradation of PNNs is a mechanism by which microglia selectively augment the output of spinal nociceptive circuits and cause pain hypersensitivity.
BACKGROUND. Regeneration of adult tissues requires the activity of rare, mitotically quiescent somatic stem cells. These features are illustrated by the muscle stem cell (MuSC), also known as the satellite cell for its satellite position underneath the basal lamina of the myofiber.Isolation of MuSCs results in their rapid activation of the myogenic program and their subsequent culture ex vivo leads to loss of stem cell regenerative capacity. These shortcomings make MuSCs difficult to study, manipulate and prevent cell based therapies. We have previously shown that muscle stem cells (MuSCs) require tightly regulated protein synthesis through the phosphorylation of eIF2α. Sal003, an analog of salubrinal that blocks eIF2α dephosphorylation, promotes ex vivo expansion of MuSCs retaining regenerative capacity after engraftment into the Dmd mdx mouse model of Duchenne muscular dystrophy.METHODS. Since micromolar concentrations of sal003 (10μM) are required to expand MuSCs ex vivo, we undertook a structure relations study to identify novel sal003 analogs with efficacy at lower concentrations. We demonstrate ex vivo expansion of MuSCs isolated from wild-type and mdx mice using new compounds, and use CRISPR/Cas9 genome editing tools to restore dystrophin expression in cultured MuSCs.RESULTS. Here, we have synthesized and screened chemical analogs of sal003 to identify a novel compound promoting the ex vivo expansion of MuSCs. The novel compound expands wild-type and mdx MuSCs more efficiently than sal003 and also prolongs culture of primary myoblasts from isolated MuSCs.CONCLUSIONS. We identify a novel sal003 analog, C10, with increased potency at lower concentrations. Culture conditions including sal003 or C10 can extend culture of primary myoblasts from isolated MuSCs, which we predict will enable their further study, genetic manipulation and cell based therapies.
Unique Organization of actin cytoskeleton in magnocellular vasopressin neurons in normal conditions and in response to salt-loading.
Translational control of gene expression is an important regulator of adult stem cell quiescence, activation and self-renewal. In skeletal muscle, quiescent satellite cells maintain low levels of protein synthesis, mediated in part through the phosphorylation of eIF2α (P-eIF2α). Pharmacological inhibition of the eIF2α phosphatase with the small molecule sal003 maintains P-eIF2α and permits the expansion of satellite cells ex vivo. Paradoxically, P-eIF2α also increases the translation of specific mRNAs, which is mediated by P-eIF2α-dependent read-through of inhibitory upstream open reading frames (uORFs). Here, we ask whether P-eIF2α-dependent mRNA translation enables expansion of satellite cells. Using transcriptomic and proteomic analyses, we show a number of genes associated with the assembly of the spindle pole to be upregulated at the level of protein, without corresponding change in mRNA levels, in satellite cells expanded in the presence of sal003. We show that uORFs in the 5′ UTR of mRNA for the mitotic spindle stability gene Tacc3 direct P-eIF2α-dependent translation. Satellite cells deficient for TACC3 exhibit defects in expansion, self-renewal and regeneration of skeletal muscle.
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