Nlrx1 regulates neuronal cell death
© Imbeault et al.; licensee BioMed Central. 2014
Received: 29 September 2014
Accepted: 14 December 2014
Published: 24 December 2014
Regulation of cell death during neurodegeneration is one of the key factors that play a role in the speed at which a disease progresses. Out of several cellular pathways responsible for this progression, necrosis and apoptosis are situated on the opposite spectrum of cell death regulation. Necrosis produces an environment that promotes inflammation and cytotoxicity and apoptosis is a highly organized process that maintains tissue homeostasis. A recently discovered protein, Nlrx1, regulates inflammatory and cell death responses during infection.
Using transfections of N2A cell line, we demonstrate that Nlrx1 redirects cells away from necrosis and towards an apoptotic pathway following rotenone treatments. In addition, Nlrx1 promotes DRP1 phosphorylation and increases mitochondrial fission.
Our results suggest a novel molecular pathway for regulating mitochondrial dynamics and neuronal death. Nlrx1 may play an important role in neurodegenerative diseases, where necrosis is a prominent factor.
KeywordsNlrx1 Cells death Necrosis Apoptosis
Neuronal cell death is a fundamental process that governs development and homeostasis of the central nervous system (CNS) . During development many neurons die off in the process of pruning, which leaves only those neurons that have meaningful connections. Throughout adult life, neurons have to survive under constant environmental stress such as toxins, infections, and inflammatory mediators. Inability to cope with these stimuli results in neuronal cell death and neurodegeneration that lead to neurological dysfunction . There are three major types of cell death: necrosis, apoptosis, and autophagy. Necrotic cell death is the least controlled process that triggers cellular pathways, which leads to bursting of cells and leakage of the internal materials (such as HMGB1) in the extracellular environment. This leakage is highly cytotoxic and induces robust pro-inflammatory responses. Apoptosis is an organized step-like process that initiates with nuclear condensation, membrane blebbing, and leads to formation of apoptotic bodies that are phagocytized by microglia and astrocytes. Finally, autophagy may be considered a cell survival pathway as it mobilizes cell resources in response to many stress events including inflammation, starvation, hypoxia, etc. Driven to extreme, autophagy may lead to cell death . Remarkably, mitochondria is situated at the crossroads of all three pathways, and thus regulate the balance between the three types of cell death . Mitochondria are well known for their ability to induce apoptosis by releasing cytochrome c and by activating downstream caspases. In addition, mitochondrial fusion and fission are critical to the survival of neurons. Interestingly, mitochondrial fission was shown to be protective during ischemia and during Huntington’s disease .
Inflammation is an integral part of the tissue response to any kind of cell death. This response may become cytotoxic and even damaging to surrounding cells depending on the milieu. For example, during infection or tissue damage, microglia and astrocytes are activated by pathogen-associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs). Once activated, these cells release cytokines and chemokines that attract more inflammatory cells. In addition, they release reactive oxygen and nitrogen species thus, increasing the cytotoxicity of the environment and leading to excessive neuronal cell death. The concentrations and compositions of PAMPs and DAMPs are monitored by sensors and receptors including Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I helicases (RLRs), etc. ,. Multiple proteins from the NLR family regulate intestinal homeostasis, regulating susceptibility to inflammatory bowel diseases and cancer .
Of these receptors, Nlrx1, belongs to the NLR family of intracellular sensors that regulate major cellular pathways including cell death and inflammation. Previous research implicated Nlrx1 in the regulation of autophagy and reactive oxygen species production during viral infection . In addition, most recent publications implicated Nlrx1 in the regulation of cell death, gastritis, and colon cancer –. Moreover, we demonstrate that Nlrx1 modulates neuronal apoptosis by regulating mitochondrial fission.
Materials and methods
BRD, Mdivi, staurosporine, and rotenone were purchased from Sigma-Aldrich. Z-VAD FMK was purchased from R&D systems. MitoTracker Mitochondrion-Selective Probes were purchased from Invitrogen. Trizol was purchased from Life Technologies. M-MLV Reverse Transcriptase and RNasin Ribonuclease Inhibitor were purchased from Promega. Oligo(dT) primer was purchased from Fermentas Life Sciences and PCR Nucleotide Mix was purchased from GE Healthcare. Brilliant III Ultra-Fast SYBR Green QPCR Master Mix was purchased from Agilent Technologies. α/β-Tubulin rabbit, cleaved caspase-3 rabbit, DRP1 rabbit, phospho-DRP1 (Ser616) rabbit, HSP90 Rabbit, HMGB1 Rabbit, COX IV Rabbit, and anti-rabbit IgG HRP-linked antibodies were purchased from Cell Signaling Technology. Nlrx1 polyclonal antibody was purchased from Proteintech.
Cell culture and cell lines
All cell lines were generated on the basis of mouse neuroblastoma (N2A) cells. Nlrx1 stable Knock-In N2A cells were generated using Origene TrueORF cDNA Clone Nlrx1 vector system with TurboFectin. Nlrx1 stable Knock-Down cells were generated using Origene ShRNA plasmid against Nlrx1. The vector contains a tGFP gene, which expresses tGFP constitutively in mammalian cells. Transfection stable control was generated using a GFP Scrambled ShRNA from Origene. Nlrx1 Knock-In GFP positive cells were selected with neomycin and Nlrx1 GFP positive Knock-Down and Scrambled were selected with puromycin. Real time Quantitative PCR and RT-PCR was used to verify expression of Nlrx1. Primers sequences Nlrx1 F: 5′-CCT CTG CTC TTC AAC TTG CTC-3′, Nlrx1 R: 5′-CCC ATC TGA TCC AGA ACA TCG-3′, 18S F: 5′-CGG CTA CCA CAT CCA AGG AA-3′, 18S R: 5′-GCT GGA ATT ACC GCG GCT-3′ were purchased from IDT.
Membranes were incubated with primary antibody (1:1000) overnight at 4°C and secondary (1:2000) for 2 hours at room temperature.
Cell death assay
Cell death was detected by LDH release with a microtiter plate based colorimetric absorbance assay that was developed based on a protocol from Chan and al, 2013.
Mitochondrial mass was evaluated using Mitotracker Mitochondrion-Selective Probes. 2×105 cells were resuspended in 200 μl of media containing 100 nm of Mitotracker and were incubated at 37°C for 20 minutes. Samples were analyzed by flow cytometry using a FACS Calibur. Data were analyzed using FlowJo software.
Cells were fixed using standard protocol by glutaraldehyde in sodium cacodylate followed by osmic acid and Epon 3 impregnation. Images were collected using Hitachi H-7500.
Mean values were compared using Two-way ANOVA followed by Tukes’ test for comparison; significance was accepted at p < 0.05.
The field of NLR biology is young and the majority of research has been directed towards the role of NLRs in the host-pathogen interaction. Nlrx1 is one of the few NLRs that, in addition to mediating the immune response, regulates cell death in multiple cell types. In this report, we provide evidence that Nlrx1 controls cell death by regulating the mitochondrial homeostasis. In particular, we found that Nlrx1 augments mitochondrial fission that protects cells from the rotenone toxicity. We found that Nlrx1 protects N2A cells during necrosis-like cell death but not against reagents like staurosporine that potentially induce apoptotic cell death. While in absence of Nlrx1, cells are more protected against apoptosis-inducing stimuli, they are more sensitive to necrosis. In deciphering Nlrx1’s molecular pathway, we found that Nlrx1 associates with DRP1, which augments mitochondrial fission and thus saves cells from necrosis. Indeed, the inhibition of DRP1 resulted in loss of Nlrx1-mediated protection during rotenone-induced cell death.
Overexpression of Nlrx1 in N2A cells significantly reduced rotenone-mediated cell death, while reduction of Nlrx1 made cells more vulnerable to rotenone toxicity. Previous research suggested that Nlrx1 may mediate ROS production ,. We used BRD treatment that enhances non-toxic ROS production. Although this treatment increased rotenone-dependent cell death, the effect in the different cell lines was similar, which suggests that mechanisms of Nlrx1 neuroprotection are not ROS dependent. Rotenone can induce mitochondrial dysfunction, increase in ROS production, and an increase in caspases-dependent apoptosis. At the same time, cytotoxic events within cells initiate necrosis –. Our results suggest that Nlrx1 inhibits both rotenone-induced necrosis and apoptosis. Indeed, after rotenone treatment, we observed reduced presence of HSP90 and HMGB1 in the supernatants from KI cells compared to KD cells. In KD cells, low levels of Nlrx1 allowed cell to shift towards necrosis, which was most notable when apoptosis was inhibited by Z-VAD.
Our observations are also confirmed by another study from Girardin group who found that Nlrx1 accelerates intrinsic apoptotic pathway . In that paper, Nlrx1 augmented intrinsic apoptotic pathway while inhibiting TNF cyclohexamide-sensitive cell death. In a different report, authors demonstrated that TNF may induce necrotic programed cell death mediated through TNFRI RIP2 TRAF2 . Allen et al. demonstrated that viral infection induces Nlrx1-mediated autophagy in cells. Interestingly, another recent report found that Nlrx1 protects macrophages by blocking the function of the viral proteins that induce apoptosis. Another group demonstrated that Nlrx1 is mediating virally-induced autophagy, but they did not report an effect of Nlrx1 on cell death . Our work suggests that in the absence of viral infection, Nlrx1 redirects cellular stress towards apoptosis thus, protecting cells from necrosis-like cell death. We did not notice any physiological or biochemical differences between Nlrx1 KI and Nlrx1 KD cells at basal level suggesting that Nlrx1 functions are triggered only during stressful conditions. These results are collaborated by multiple studies with Nlrx1 KO mice. Although, Nlrx1 has been implicated in many cellular pathways, Nlrx1 KO mice are viable and fertile and do not show any deviations from WT mice at the basal conditions ,–.
Several groups have shown Nlrx1 to localize to mitochondria, although the exact distribution of Nlrx1 within the inner and outer mitochondrial membrane is still under debate.
Electron microscopy studies enforced by flow cytometry experiments suggest an increased number of mitochondria in Nlrx1 KI cells. We demonstrated that increase in Nlrx1 expression resulted in augmented mitochondrial fission with an upsurge in phosphorylated levels of DRP1. Several reports suggest that Nlrx1 may bind and regulate functions of mitochondria-localized proteins including MAVS and UQCRC2 ,. The exact molecular pathway that phosphorylates DRP1 is still under investigation. Overexpressing Nlrx1 resulted in the increased number of mitochondria, but these mitochondria had a reduced number of cristae of which all were swollen, which suggests that excessive fission induced mitochondrial stress. In our opinion, those mitochondria are more sensitive to cytotoxic events, which explains why Nlrx1 KI cells were more sensitive to some of the apoptosis inducing reagents. This observation is collaborated by several studies, which evaluate mitochondrial function in cell death. A dysfunction of DRP1 and altered mitochondrial fission led to a switch from apoptotic to necrotic cell death . In addition, an increase in mitochondrial fission has been implicated in the etiology of neuronal cell death in Huntington’s disease .
In conclusion, to our knowledge this work describes for the first time the involvement of Nlrx1 in mitochondrial dynamics during neuronal death. We would like to note that these experiments were conducted in N2A transformed cells lines and that these cells possess neuronal-like properties. Future studies will confirm this observation in primary neuronal cultures and in transgenic mice.
Central nervous system
Scrambled Sh RNA transfected cells
Nlrx1 Sh RNA transfected cells
Nlrx1-GFP transfected cells
Reactive oxygen species
We thank National Science and Engineering Research Counsil and Fonds de recherche du Québec - Santé for financial support.
We would like to extent our regards to Daniel Serrano for his valuable help with confocal microscopy. We would also like to acknowledge Anne Vézina from Plateforme d’Histologie et de Microscopie Électronique of University of Sherbrooke who helped with electron microscopy procedure. Finally, we want to thank Leonid Volkov for his helpful insights with Flow cytometry. Quebec-Bavaria relation program grant.
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