Inhibition of heat shock protein family A member 8 attenuates spinal cord ischemia–reperfusion injury via astrocyte NF-κB/NLRP3 inflammasome pathway

Background Astrocyte over-activation and extensive neuron loss are the main characteristic pathological features of spinal cord ischemia–reperfusion injury (SCII). Prior studies have placed substantial emphasis on the role of heat shock protein family A member 8 (HSPA8) on postischemic myocardial inflammation and cardiac dysfunction. However, it has never been determined whether HSPA8 participates in astrocyte activation and thus mediated neuroinflammation associated with SCII. Methods The left renal artery ligation-induced SCII rat models and oxygen–glucose deprivation and reoxygenation (OGD/R)-induced rat primary cultured astrocytes were established. The lentiviral vector encoding short hairpin RNA targeting HSPA8 was delivered to the spinal cord by intrathecal administration or to culture astrocytes. Then, the spinal neuron survival, gliosis, and nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome and its related pro-inflammatory cytokines were analyzed. Results SCII significantly enhanced the GFAP and HSPA8 expression in the spinal cord, resulting in blood–brain barrier breakdown and the dramatical loss of spinal neuron and motor function. Moreover, injury also increased spinal nuclear factor-kappa B (NF-κB) p65 phosphorylation, NLRP3 inflammasome-mediated caspase-1 activation, and subsequent interleukin (IL)-1β as well as IL-18 secretion. Silencing the HSPA8 expression efficiently ameliorated the spinal cord tissue damage and promoted motor function recovery after SCII, through blockade of the astrocyte activation and levels of phosphorylated NF-κB, NLRP3, caspase-1, IL-1β, and IL-18. Further in vitro studies confirmed that HSPA8 knockdown protected astrocytes from OGD/R-induced injury via the blockade of NF-κB and NLRP3 inflammasome activation. Conclusion Our findings indicate that knockdown of HSPA8 inhibits spinal astrocytic damage after SCII, which may provide a promising therapeutic strategy for SCII treatment.

Conserved genetic signatures parcellate cardinal spinal neuron classes into local and projection subsets

Motor and sensory functions of the spinal cord are mediated by populations of cardinal neurons arising from separate progenitor lineages. However, each cardinal class is composed of multiple neuronal types with distinct molecular, anatomical, and physiological features, and there is not a unifying logic that systematically accounts for this diversity. We reasoned that the expansion of new neuronal types occurred in a stepwise manner analogous to animal speciation, and we explored this by defining transcriptomic relationships using a top-down approach. We uncovered orderly genetic tiers that sequentially divide groups of neurons by their motor-sensory, local-long range, and excitatory-inhibitory features. The genetic signatures defining neuronal projections were tied to neuronal birth date and conserved across cardinal classes. Thus, the intersection of cardinal class with projection markers provides a unifying taxonomic solution for systematically identifying distinct functional subsets.

Inhibition of lncRNA H19/miR-370-3p pathway mitigates neuronal apoptosis in an in vitro model of spinal cord injury (SCI)

Background Spinal cord injury (SCI) is the most serious complication of spinal injury, often leading to severe dysfunction of the limbs below the injured segment. Conventional therapy approaches are becoming less and less effective, and gene therapy is a new research direction by now. Methods The Sprague-Dawley rats were haphazardly assigned to two groups, namely sham group and SCI model group, and lncRNA H19 and miR-370-3p levels were investigated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Correlation between lncRNA H19 and miR-370-3p was ascertained by luciferase report assay and RT-qPCR. After transfection with si-H19, miR-370-3p inhibitor, negative controls (NC), or both, primary spinal neurons were subjected to the simulation of lipopolysaccharide (LPS) for inducing in vitro model of SCI. Cell viability, apoptotic rate, caspase-3 activity, Bax and Bcl-2 protein, ROS generation, TNF-α, IL-1β, and IL-6 protein, as well as IκBα and p65 phosphorylation ratio were evaluated adopting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), apoptosis, caspase-3 activity, ROS generation, and western blot assays, thereby searching for the specific action mechanism on LPS-induced spinal never injury. Results SCI resulted in lncRNA H19 higher expression and miR-370-3p lower expression. LPS simulation raised a series of cellular biological changes, such as decreased viability, promoted apoptosis, generated ROS, and released inflammatory factors. lncRNA H19 inhibition reversed above LPS-induced changes. Besides, as the downstream target of lncRNA H19, miR-370-3p was oppositely regulated by lncRNA H19. The above biological changes induced by lncRNA H19 inhibition were reversed by miR-370-3p upregulation. Moreover, lncRNA H19 inhibition could block NF-κB pathway through miR-370-3p upregulation. Conclusion Inhibition of lncRNA H19/miR-370-3p mitigated spinal neuron apoptosis in an in vitro model of SCI. This provided the possibility for clinical use of gene therapy.

Evolution of lbx spinal cord expression and function

Ladybird homeobox (Lbx) transcription factors have crucial functions in muscle and nervous system development in many different animals. Amniotes have two Lbx genes, Lbx1 and Lbx2, but only Lbx1 is expressed in the spinal cord. In contrast, teleosts have three lbx genes, lbx1a, lbx1b and lbx2. In this study, we characterize the spinal cord expression of zebrafish lbx1a, lbx1b and lbx2 and show that each of these genes is expressed by distinct cell types. Our data suggest that lbx1a is expressed by dI4, dI5 and dI6 spinal interneurons, whereas lbx1b and lbx2 are primarily expressed in different spinal cord progenitor domains. We investigated the evolution of Lbx spinal cord expression patterns by examining Lbx1 and Lbx2 expression in the lesser spotted dogfish, Scyliorhinus canicula and Lbx1 expression in the tetrapod, Xenopus tropicalis. Our results suggest that zebrafish lbx1a spinal cord expression is conserved with that of Lbx1 in other vertebrates, whereas lbx1b spinal cord expression probably evolved in teleosts after the duplication of lbx1 into lbx1a and lbx1b. lbx2 spinal expression was probably acquired somewhere in the ray-finned lineage, as this gene is not expressed in the spinal cords of either amniotes or S. canicula. Consistent with its conserved spinal cord expression pattern, we also show that the spinal cord function of zebrafish lbx1a is conserved with mouse Lbx1. In zebrafish lbx1a mutants, there is a reduction of inhibitory spinal neurons and an increase in excitatory neurons, similar to the phenotype of mouse Lbx1 mutants. Interestingly, we also see a reduction of inhibitory spinal neurons in lbx1b mutants, although in this case there is not a corresponding increase in the number of excitatory neurons and lbx1a;lbx1b double mutants do not have a more severe spinal cord phenotype than lbx1a single mutants, suggesting that lbx1a and lbx1b do not act redundantly in spinal neuron development. This suggests that lbx1b and lbx1a may be required in succession for correct specification of inhibitory dI4 and dI6 interneurons, although only lbx1a is required for suppression of excitatory fates in these cells.

Spinal neuron-glia-immune interaction in cross-organ sensitization

Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), historically considered as regional gastrointestinal disorders with heightened colonic sensitivity, are increasingly recognized to have concurrent dysfunction of other visceral and somatic organs, such as urinary bladder hyperactivity, leg pain, and skin hypersensitivity. The interorgan sensory cross talk is, at large, termed “cross-organ sensitization.” These organs, anatomically distant from one another, physiologically interlock through projecting their sensory information into dorsal root ganglia (DRG) and then the spinal cord for integrative processing. The fundamental question of how sensitization of colonic afferent neurons conveys nociceptive information to activate primary afferents that innervate distant organs remains ambiguous. In DRG, primary afferent neurons are surrounded by satellite glial cells (SGCs) and macrophage accumulation in response to signals of injury to form a neuron-glia-macrophage triad. Astrocytes and microglia are major resident nonneuronal cells in the spinal cord to interact, physically and chemically, with sensory synapses. Cumulative evidence gathered so far indicate the indispensable roles of paracrine/autocrine interactions among neurons, glial cells, and immune cells in sensory cross-activation. Dichotomizing afferents, sensory convergency in the spinal cord, spinal nerve comingling, and extensive sprouting of central axons of primary afferents each has significant roles in the process of cross-organ sensitization; however, more results are required to explain their functional contributions. DRG that are located outside the blood-brain barrier and reside upstream in the cascade of sensory flow from one organ to the other in cross-organ sensitization could be safer therapeutic targets to produce less central adverse effects.

MiR-34a Inhibits Spinal Cord Injury and Blocks Spinal Cord Neuron Apoptosis by Activating Phatidylinositol 3-kinase (PI3K)/AKT Pathway Through Targeting CD47

Objective: Dysregulation of miR-34a has been reported for its implication in neuronal development. This study aims to explore the effect and possible mechanism of miR-34a on neuron apoptosis induced by Spinal Cord Injury (SCI). Materials and Methods: SCI model was established using Allen's weight-drop method and rats in the sham group were performed with laminectomy without weight-drop injury. Basso Bcattie Bresnahan (BBB) rating scale was applied to evaluate the locomotor function of rats. Pathological changes of spinal cord tissues in SCI rats were observed after hematoxylin and eosin (HE) staining. Rats were separately injected with miR-34a agomir, miR-34a agomir NC, si-CD47 and si- CD47 NC before their spinal cord tissues were collected for terminal-deoxynucleoitidyl Transferase Mediated nick end labeling (TUNEL) staining. Expressions of miR-34a, si-CD47, apoptosis related proteins and AKT pathway related proteins were measured by quantitative reverse transcription- polymerase chain reaction (qRT-PCR) and western blot. Results: SCI rat models were successfully established evidenced by decreased BBB scores and HE staining. Injection of miR-34a agomir and/or si-CD47 could suppress neuron cell apoptosis, with deceased apoptotic index (AI) and pro-apoptotic protein (cleaved caspase-3 and Bax) levels, and increased expressions of anti-apoptotic proteins (Bcl-2 and Mcl-1). Phosphorylated levels of phatidylinositol 3-kinase (PI3K) and AKT were further increased in rats injected with miR-34a agomir and si-CD47, compared with miR-34a agomir or si-CD47 injection alone. Conclusion: MiR-34a can downregulate CD47 expression to activate PI3K/AKT signal pathway, and thus inhibit SCI induced spinal neuron apoptosis.

Developmentally regulated KCC2 phosphorylation is essential for dynamic GABA-mediated inhibition and survival

Despite its importance for γ-aminobutyric acid (GABA) inhibition and involvement in neurodevelopmental disease, the regulatory mechanisms of the K+/Cl− cotransporter KCC2 (encoded by SLC12A5) during maturation of the central nervous system (CNS) are not entirely understood. Here, we applied quantitative phosphoproteomics to systematically map sites of KCC2 phosphorylation during CNS development in the mouse. KCC2 phosphorylation at Thr906 and Thr1007, which inhibits KCC2 activity, underwent dephosphorylation in parallel with the GABA excitatory-inhibitory sequence in vivo. Knockin mice expressing the homozygous phosphomimetic KCC2 mutations T906E/T1007E (Kcc2E/E), which prevented the normal developmentally regulated dephosphorylation of these sites, exhibited early postnatal death from respiratory arrest and a marked absence of cervical spinal neuron respiratory discharges. Kcc2E/E mice also displayed disrupted lumbar spinal neuron locomotor rhythmogenesis and touch-evoked status epilepticus associated with markedly impaired KCC2-dependent Cl− extrusion. These data identify a previously unknown phosphorylation-dependent KCC2 regulatory mechanism during CNS development that is essential for dynamic GABA-mediated inhibition and survival.