Pharmacologic inhibition of reactive gliosis blocks TNF-α-mediated neuronal apoptosis.

Reactive gliosis is an early pathological feature common to most neurodegenerative diseases, yet its regulation and impact remain poorly understood. Normally astrocytes maintain a critical homeostatic balance. After stress or injury they undergo rapid parainflammatory activation, characterized by hypertrophy, and increased polymerization of type III intermediate filaments (IFs), particularly glial fibrillary acidic protein and vimentin. However, the consequences of IF dynamics in the adult CNS remains unclear, and no pharmacologic tools have been available to target this mechanism in vivo. The mammalian retina is an accessible model to study the regulation of astrocyte stress responses, and their influence on retinal neuronal homeostasis. In particular, our work and others have implicated p38 mitogen-activated protein kinase (MAPK) signaling as a key regulator of glutamate recycling, antioxidant activity and cytokine secretion by astrocytes and related Müller glia, with potent influences on neighboring neurons. Here we report experiments with the small molecule inhibitor, withaferin A (WFA), to specifically block type III IF dynamics in vivo. WFA was administered in a model of metabolic retinal injury induced by kainic acid, and in combination with a recent model of debridement-induced astrocyte reactivity. We show that WFA specifically targets IFs and reduces astrocyte and Müller glial reactivity in vivo. Inhibition of glial IF polymerization blocked p38 MAPK-dependent secretion of TNF-α, resulting in markedly reduced neuronal apoptosis. To our knowledge this is the first study to demonstrate that pharmacologic inhibition of IF dynamics in reactive glia protects neurons in vivo

FGF signal interpretation is directed by sprouty and spred proteins during mesoderm formation

SummaryVertebrate gastrulation requires coordination of mesoderm specification with morphogenetic movements. While both of these processes require FGF signaling, it is not known how mesoderm specification and cell movements are coordinated during gastrulation. The related Sprouty and Spred protein families are recently discovered regulators of receptor tyrosine kinase signaling. We identified two genes for each family in Xenopus tropicalis: Xtsprouty1, Xtsprouty2, Xtspred1, and Xtspred2. In gain- and loss-of-function experiments we show that XtSprouty and XtSpred proteins modulate different signaling pathways downstream of the FGF receptor (FGFR), and consequently different developmental processes. Notably, XtSproutys inhibit morphogenesis and Ca2+ and PKCδ signaling, leaving MAPK activation and mesoderm specification intact. In contrast, XtSpreds inhibit MAPK activation and mesoderm specification, with little effect on Ca2+ or PKCδ signaling. These differences, combined with the timing of their developmental expression, suggest a mechanism to switch FGFR signal interpretation to coordinate mesoderm formation and cell movements during gastrulation

ROS detoxification and proinflammatory cytokines are linked by p38 MAPK signaling in a model of mature astrocyte activation.

Astrocytes are the most abundant glial cell in the retinal nerve fiber layer (NFL) and optic nerve head (ONH), and perform essential roles in maintaining retinal ganglion cell (RGC) detoxification and homeostasis. Mature astrocytes are relatively quiescent, but rapidly undergo a phenotypic switch in response to insult, characterized by upregulation of intermediate filament proteins, loss of glutamate buffering, secretion of pro-inflammatory cytokines, and increased antioxidant production. These changes result in both positive and negative influences on RGCs. However, the mechanism regulating these responses is still unclear, and pharmacologic strategies to modulate select aspects of this switch have not been thoroughly explored. Here we describe a system for rapid culture of mature astrocytes from the adult rat retina that remain relatively quiescent, but respond robustly when challenged with oxidative damage, a key pathogenic stress associated with inner retinal injury. When primary astrocytes were exposed to reactive oxygen species (ROS) we consistently observed characteristic changes in activation markers, along with increased expression of detoxifying genes, and secretion of proinflammatory cytokines. This in vitro model was then used for a pilot chemical screen to target specific aspects of this switch. Increased activity of p38α and β Mitogen Activated Protein Kinases (MAPKs) were identified as a necessary signal regulating expression of MnSOD, and heme oxygenase 1 (HO-1), with consequent changes in ROS-mediated injury. Additionally, multiplex cytokine profiling detected p38 MAPK-dependent secretion of IL-6, MCP-1, and MIP-2α, which are proinflammatory signals recently implicated in damage to the inner retina. These data provide a mechanism to link increased oxidative stress to proinflammatory signaling by astrocytes, and establish this assay as a useful model to further dissect factors regulating the reactive switch

Additional file 1 of Lipoxins A4 and B4 inhibit glial cell activation via CXCR3 signaling in acute retinal neuroinflammation

Additional file 1: Table S1. Antibodies Used. Figure S1. High magnification images of cell marker staining. A) Representative retinal staining at 1 day following intravitreal LPS challenge, shown at higher magnification to better demonstrate cell morphology of GFAP, F4-80, and GR-1. B) Representative retinal staining for ramified or amoeboid microglial morphology following staining for Iba1 or CD68. (scale bars represent 20 μm). Figure S2. LPS challenge did not induce an apparent T-cell response. Representative negative staining for CD4- and CD3-positive T cells in retinal sections at three days following intravitreal LPS treatment showed no signal in the retina. A parallel stained section from a retina three days after exposure to the potent oxidative stressor paraquat (PQ) is presented as a positive control (arrows, scale bars represent 100 μm). Figure S3. LPS treatment results in significant RGC loss after three weeks. A) Brn3a staining in retinas over a time course showed no clear reduction in RGC density compared to control until a 21 day time point (d21) after LPS injection (scale bars represent 100 μm). B) Corresponding quantification after 21 days reveals a small, but significant loss of RGCs at three weeks following LPS-induced retinal inflammation (*p <0.05, bars represent SE, GCL; ganglion cell layer). Figure S4. LXA4, LXB4 or Amg487 treatment alone do not induce retinal inflammation markers. Retinal staining for A)GFAP, B) Iba1, or C) F4-80, do not show any difference at two days following treatment, compared to vehicle alone (scale bars represent 100 μm)

Evidence of an Annexin A4 mediated plasma membrane repair response to biomechanical strain associated with glaucoma pathogenesis

Glaucoma is a common neurodegenerative blinding disease that is closely associated with chronic biomechanical strain at the optic nerve head (ONH). Yet, the cellular injury and mechanosensing mechanisms underlying the resulting damage have remained critically unclear. We previously identified Annexin A4 (ANXA4) from a proteomic analyses of human ONH astrocytes undergoing pathological biomechanical strain that mimics glaucomatous conditions. Annexins are a family of calcium-dependent phospholipid binding proteins with key functions in plasma membrane repair (PMR); an active mechanism to limit and mend cellular injury that involves membrane and cytoskeletal reorganizations. However, a role for direct membrane damage and PMR has not been well studied in the context of biomechanical strain, such as that associated with glaucoma. Here we report that this moderate strain surprisingly damages cell membranes to increase permeability in a calcium-dependent manner, and induces rapid aggregation of ANXA4 at injury sites. ANXA4 loss-of-function increases permeability, while exogenous ANXA4 reduces it. Furthermore, ANXA4 aggregation is associated with F-actin dynamics in vitro, and remarkably this interaction and aggregation signature is also observed in the glaucomatous ONH in patient samples. Together these studies link moderate biomechanical strain with direct membrane damage and actin dynamics, and identify an active PMR role for ANXA4 in new model of cell injury associated with glaucoma pathogenesis

p38 MAPK regulates secretion of specific proinflammatory cytokines.

<p>Conditioned media were collected from control primary astrocytes, and from cells treated with 1+SB for 24 hours. Significant increases were observed for <b>A)</b> IL-6, <b>B)</b> MCP-1, and <b>C)</b> MIP-2, which were blocked by p38 MAPK inhibition (n = 6, *p<0.05 compared to control, **p<0.05 compared to PQ alone, bars represent S.E.).</p

ROS exposure in primary mature astrocytes induces robust dose and time-dependent cell death and detoxifying responses.

<p><b>A)</b> Exposure of primary astrocytes to increasing mM concentrations of PQ results in dose and time dependent ROS (DCF fluorescence) over 24 and 48 hours, as measured by flow cytometry. This result can be compared to acute exposure to 0.5 mM H₂O₂, which generates a sharp spike in peroxide formation by one hour (n = 3, bars represent S.E., *p<0.05). <b>B)</b> Dose and time dependent increases in cell death (PI signal), at 24 and 48 hours, following exposure to increasing mM concentrations of PQ. ETOH was used as a positive control for cell death after 1 hour (n = 3, bars represent S.E.). <b>C–F)</b> Time course of RT-qPCR results from cells exposed to 1 mM PQ shows significant increases in expression of MnSOD, catalase, HO-1, and GPX1 by eight hours (n = 4, *p<0.05 at peak/trough, bars represent S.E.).</p

Oxidative stress mediates robust induction of activation and stress markers in mature retinal astrocytes.

<p><b>A)</b> Representative western blots from cells exposed to 0.3 and 1.0 mM PQ, and control, for 24 hours. Dose dependent increases were observed for GFAP, HSP70, PGC-1α, and there was a dramatic decrease in GS, compared to a loading control GAPDH. <b>B)</b> Corresponding quantification of repeated experiments as in (A) showing consistent responses for each protein probed from fresh cultures (n≥6 independent cultures, *p<0.05, bars represent S.E.).</p

Relative protein concentrations in conditioned media.

<p>BLQ; below quantification,</p>*<p>p<0.05 compared to control,</p>**<p>p<0.05 compared to PQ (n = 6).</p