Ataxin-1 poly(Q)-induced proteotoxic stress and apoptosis are attenuated in neural cells by docosahexaenoic acid-derived neuroprotectin D1.

Neurodegenerative diseases share two common features: enhanced oxidative stress and cellular inability to scavenge structurally damaged abnormal proteins. Pathogenesis of polyglutamine (poly(Q)) diseases involves increased protein misfolding, along with ubiquitin and chaperon protein-containing nuclear aggregates. In spinocerebellar ataxia, the brain and retina undergo degeneration. Neuroprotectin D1 (NPD1) is made on-demand in the nervous system and retinal pigment epithelial (RPE) cells in response to oxidative stress, which activates prosurvival signaling via regulation of gene expression and other processes. We hypothesized that protein misfolding-induced proteotoxic stress triggers NPD1 synthesis. We used ARPE-19 cells as a cellular model to assess stress due to ataxin-1 82Q protein expression and determine whether NPD1 prevents apoptosis. Ectopic ataxin-1 expression induced RPE cell apoptosis, which was abrogated by 100 nm docosahexaenoic acid, 10 ng/ml pigment epithelium-derived factor, or NPD1. Similarly, NPD1 was protective in neurons and primary human RPE cells. Furthermore, when ataxin-1 82Q was expressed in 15-lipoxygenase-1-deficient cells, apoptosis was greatly enhanced, and only NPD1 (50 nm) rescued cells from death. NPD1 reduced misfolded ataxin-1-induced accumulation of proapoptotic Bax in the cytoplasm, suggesting that NPD1 acts by preventing proapoptotic signaling pathways from occurring. Finally, NPD1 signaling interfered with ataxin-1/capicua repression of gene expression and decreased phosphorylated ataxin-1 in an Akt-independent manner, suggesting that NPD1 signaling modulates formation or stabilization of ataxin-1 complexes. These data suggest that 1) NPD1 synthesis is an early response induced by proteotoxic stress due to abnormally folded ataxin-1, and 2) NPD1 promotes cell survival through modulating stabilization of ataxin-1 functional complexes and pro-/antiapoptotic and inflammatory pathways.

Astrogliosis involves activation of retinoic acid-inducible gene-like signaling in the innate immune response after spinal cord injury.

Spinal cord injury (SCI) induces a glial response in which astrocytes become activated and produce inflammatory mediators. The molecular basis for regulation of glial-innate immune responses remains poorly understood. Here, we examined the activation of retinoic acid-inducible gene (RIG)-like receptors (RLRs) and their involvement in regulating inflammation after SCI. We show that astrocytes express two intracellular RLRs: RIG-I and melanoma differentiation-associated gene 5. SCI and stretch injury of cultured astrocytes stimulated RLR signaling as determined by phosphorylation of interferon regulatory factor 3 (IRF3) leading to production of type I interferons (IFNs). RLR signaling stimulation with synthetic ribonucleic acid resulted in RLR activation, phosphorylation of IRF3, and increased expression of glial fibrillary acidic protein (GFAP) and vimentin, two hallmarks of reactive astrocytes. Moreover, mitochondrial E3 ubiquitin protein ligase 1, an RLR inhibitor, decreased production of GFAP and vimentin after RIG-I signaling stimulation. Our findings identify a role for RLR signaling and type I IFN in regulating astrocyte innate immune responses after SCI.

Inflammasome Proteins as Inflammatory Biomarkers of Age-Related Macular Degeneration.

Purpose: Age-related macular degeneration (AMD) can result in severe vision loss and blurriness in the older population. The early and intermediate stages of AMD typically start without noticeable symptoms and can only be detected with a comprehensive eye exam. Because of the quiet onset of the disease, it is necessary to identify potential biomarkers to aid in the diagnosis, staging, and association with disease onset. Inflammasome signaling proteins are prominent biomarkers in the central nervous system, and the inflammasome has been shown to play a role in the innate inflammatory response in aging and AMD. Methods: Serum from healthy controls and AMD patients were analyzed for the protein levels of Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), interleukin (IL)-18 and C-reactive protein (CRP) to determine cutoff points, positive and negative predictive values, and receiver operator characteristic curves, as well as univariate and multivariate linear and logistic regression models. Results: ASC, IL-18, and CRP were elevated in the serum of AMD patients when compared to healthy controls. The area under the curve (AUC) for ASC was 0.98 with a cutoff point of 365.6 pg/mL, whereas IL-18 had an AUC of 0.73 and a cutoff point of 242.4 pg/mL, and the AUC for CRP was 0.67 with a cutoff point of 8,684,152 pg/mL. Levels of IL-18 had a statistically significant linear correlation with that of ASC with an adjusted R2 of 0.1906, indicating that 19% of IL-18 could be explained by ASC protein levels in serum. Moreover, a logistic regression model for the diagnosis of AMD consists of ASC and having a diagnosis of hypertension, indicating that these two factors (elevated levels of ASC and a diagnosis of hypertension [HTN]) are associated with the diagnosis of AMD. Conclusions: ASC, IL-18, and CRP are elevated in patients with AMD, and the protein levels of IL-18 are partially the result of ASC protein expression. Moreover, elevated protein levels of ASC in serum and a diagnosis of HTN increase the odds of patients having a diagnosis of AMD. Translational Relevance: Biomarkers of AMD may be used to monitor disease risk, response to treatment and disease progression.

The Inflammasome in Times of COVID-19.

Coronaviruses (CoVs) are members of the genus Betacoronavirus and the Coronaviridiae family responsible for infections such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and more recently, coronavirus disease-2019 (COVID-19). CoV infections present mainly as respiratory infections that lead to acute respiratory distress syndrome (ARDS). However, CoVs, such as COVID-19, also present as a hyperactivation of the inflammatory response that results in increased production of inflammatory cytokines such as interleukin (IL)-1ß and its downstream molecule IL-6. The inflammasome is a multiprotein complex involved in the activation of caspase-1 that leads to the activation of IL-1ß in a variety of diseases and infections such as CoV infection and in different tissues such as lungs, brain, intestines and kidneys, all of which have been shown to be affected in COVID-19 patients. Here we review the literature regarding the mechanism of inflammasome activation by CoV infection, the role of the inflammasome in ARDS, ventilator-induced lung injury (VILI), and Disseminated Intravascular Coagulation (DIC) as well as the potential mechanism by which the inflammasome may contribute to the damaging effects of inflammation in the cardiac, renal, digestive, and nervous systems in COVID-19 patients.

Pronounced cell death in the absence of NMDA receptors in the developing somatosensory thalamus.

Genetic deletion of NMDA glutamate receptors disrupts development of whisker-related neuronal patterns in the somatosensory system. Independent studies have shown that NMDA receptor antagonists increase cell death among developing neurons. Here, we report that a dramatic feature of the developing somatosensory system in newborn NMDA receptor 1 (NMDAR1) knock-out mice is increased cell death in the ventrobasal nucleus (VB) of the thalamus. Sections were subject to terminal deoxynucleotidyl transferase dUTP nick end labeling staining for apoptotic DNA fragmentation, thionine staining for pyknotic nuclei, silver staining for degenerating cells, and immunostaining for caspase-3. All four methods demonstrated that deletion of NMDAR1 causes a large (on the order of threefold to fivefold) increase in cell death in the VB. The NMDA receptor antagonists dizocilpine maleate (MK-801) and phencyclidine also increase cell death in this structure. The onset of increased cell death in the VB in the absence of NMDA receptor function is approximately the time of birth, overlaps with naturally occurring cell death and synaptogenesis, and displays some anatomical specificity. For example, there was no increase in cell death in the hippocampus or neocortex of NMDAR1 knock-out mice at any of the time points examined: embryonic day 15.5 (E15.5), E17.5, and postnatal day 0. We also report a significant reduction in the size of the VB that is evident starting at E17.5. The results indicate that NMDA receptors play a major role in cell survival during naturally occurring cell death in the VB and demonstrate that cell death is a consideration in NMDA receptor knock-out studies.

RIG-I contributes to the innate immune response after cerebral ischemia.

BACKGROUND: Focal cerebral ischemia induces an inflammatory response that when exacerbated contributes to deleterious outcomes. The molecular basis regarding the regulation of the innate immune response after focal cerebral ischemia remains poorly understood. METHODS: In this study we examined the expression of retinoic acid-inducible gene (RIG)-like receptor-I (RIG-I) and its involvement in regulating inflammation after ischemia in the brain of rats subjected to middle cerebral artery occlusion (MCAO). In addition, we studied the regulation of RIG-I after oxygen glucose deprivation (OGD) in astrocytes in culture. RESULTS: In this study we show that in the hippocampus of rats, RIG-I and IFN-α are elevated after MCAO. Consistent with these results was an increased in RIG-I and IFN-α after OGD in astrocytes in culture. These data are consistent with immunohistochemical analysis of hippocampal sections, indicating that in GFAP-positive cells there was an increase in RIG-I after MCAO. In addition, in this study we have identified n-propyl gallate as an inhibitor of IFN-α signaling in astrocytes. CONCLUSION: Our findings suggest a role for RIG-I in contributing to the innate immune response after focal cerebral ischemia.

Mincle signaling in the innate immune response after traumatic brain injury.

The innate immune response contributes to the inflammatory activity after traumatic brain injury (TBI). In the present study we identify macrophage-inducible C-type lectin (mincle) as a pattern recognition receptor that contributes to innate immunity in neurons after TBI. Here we report that mincle is activated by SAP130 in cortical neurons in culture, resulting in production of the inflammatory cytokine TNF. In addition, mincle and SAP130 are elevated in the brain and cerebrospinal fluid of humans after TBI and the brain of rodents after fluid percussion brain injury. Thus, these findings suggest the involvement of mincle to the pathology of TBI. Importantly, blocking mincle with a neutralizing antibody against mincle in cortical neurons in culture treated with SAP130 resulted in inhibition of mincle signaling and decreased TNF production. Therefore, our findings identify mincle as a contributor to the inflammatory response after TBI.

Gap junctions are required for NMDA receptor dependent cell death in developing neurons.

A number of studies have indicated an important role for N-methyl-D-aspartate (NMDA) receptors in cell survival versus cell death decisions during neuronal development, trauma, and ischemia. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all three of these conditions. However, whether neuronal gap junctions contribute to NMDA receptor-regulated cell death is not known. Here we address the role of neuronal gap junction coupling in NMDA receptor-regulated cell death in developing neurons. We report that inactivation or hyperactivation of NMDA receptors induces neuronal cell death in primary hypothalamic cultures, specifically during the peak of developmental gap junction coupling. In contrast, increasing or decreasing NMDA receptor function when gap junction coupling is low has no or greatly reduced impact on cell survival. Pharmacological inactivation of gap junctions or knockout of neuronal connexin 36 prevents the cell death caused by NMDA receptor hypofunction or hyperfunction. The results indicate the critical role of neuronal gap junctions in cell death caused by increased or decreased NMDA receptor function in developing neurons. Based on these data, we propose the novel hypothesis that NMDA receptors and gap junctions work in concert to regulate neuronal survival.

Photoreceptor outer segment phagocytosis attenuates oxidative stress-induced apoptosis with concomitant neuroprotectin D1 synthesis.

Photoreceptor cell (rods and cones) renewal is accompanied by intermittent shedding of the distal tips of the outer segment followed by their phagocytosis in the retinal pigment epithelial (RPE) cells. This renewal is essential for vision, and it is thought that it fosters survival of photoreceptors and of RPE cells. However, no specific survival messenger/mediators have as yet been identified. We show here that photoreceptor outer segment (POS) phagocytosis markedly attenuates oxidative stress-induced apoptosis in ARPE-19 cells in culture. This phenomenon does not seem to be a generalized outcome of phagocytosis because nonbiological (polystyrene microsphere) phagocytosis did not elicit protection. The free docosahexaenoic acid (DHA) pool size and neuroprotectin D1 (NPD1) content increased during POS phagocytosis but not during microspheres phagocytosis. We have also explored other lipid mediators [lipoxin A4 and 15(S)- and 12(S)-hydroxyeicosatetraenoic acids] under these conditions and found them unchanged upon POS phagocytosis. Moreover, oxidative stress challenge to RPE cells undergoing POS phagocytosis further increased DHA and NPD1 content. Under these conditions, NPD1 was found within the RPE cells as well as in the culture medium, suggesting autocrine and paracrine bioactivity. Furthermore, using deuterium-labeled DHA, we show that as the availability of free DHA increases during oxidative stress, NPD1 synthesis is augmented in ARPE-19 cells. Our data suggest a distinct signaling that promotes survival of photoreceptor and RPE cells by enhancing the synthesis of NPD1 during phagocytosis. Taken together, NPD1 may be a mediator that promotes homeostatic regulation of cell integrity during photoreceptor cell renewal.

NMDA receptors promote survival in somatosensory relay nuclei by inhibiting Bax-dependent developmental cell death.

Naturally occurring cell death is a universal feature of developing nervous systems that plays an essential role in determining adult brain function. Yet little is known about the decisions that select a subset of CNS neurons for survival and cause others to die. We report that postnatal day 0 NMDA receptor subunit 1 (NMDAR1) knockout mice display an approximately 2-fold increase in cell death in the brainstem trigeminal complex (BSTC), including all four nuclei that receive somatosensory inputs from the face (principalis, oralis, interpolaris, and caudalis). Treatment with the NMDA receptor antagonist dizocilpine maleate (MK-801) for 24 h before birth also caused an increase in cell death that reached statistical significance in two of the four nuclei (oralis and interpolaris). The neonatal sensitivity to NMDA receptor hypofunction in the BSTC, and in its main thalamic target, the ventrobasal nucleus (VB), coincides with the peak of naturally occurring cell death and trigeminothalamic synaptogenesis. At embryonic day 17.5, before the onset of these events, NMDAR1 knockout does not affect cell survival in either the BSTC or the VB. Immunostaining for active caspase-3 and the neuronal marker Hu specifically confirms the presence of dying neurons in the BSTC and the VB of NMDAR1 knockout neonates. Finally, genetic deletion of Bax rescues these structures from the requirement for NMDA receptors to limit naturally occurring cell death. Taken together, the results indicate that NMDA receptors play a survival role for somatosensory relay neurons during synaptogenesis by inhibiting Bax-dependent developmental cell death.