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RIPK3 coordinates RHIM domain-dependent inflammatory transcription in neurons

Sigal B. Kofman, Lan H. Chu, Joshua M. Ames, Suny Dayane Chavarria, Katrina Lichauco, Brian P. Daniels, Andrew Oberst

Posted on: 8 April 2024 , updated on: 9 April 2024

Preprint posted on 2 March 2024

Neurons repurpose death complex components to induce an anti-viral response during Zika virus infection.

Selected by Zoie Magri

Background

Programmed cell death is essential to maintain homeostasis and defend against pathogens. Cell death pathways are triggered by pathogens to induce an inflammatory response and eliminate the replicative niche of the pathogen. To ensure this pro-death response remains intact, there are many pathways that can be activated, including “back up” methods that can be triggered even after the pathogen has attempted to inhibit cell death. The RHIM (RIP Homotypic Interaction Motifs) domain-containing proteins play an important role in this. These proteins have been found to bind and induce cell death or inflammation depending on the stimulus. RIPK1, TRIF, and ZBP1 form a pro-death complex that can induce cell death. If this complex is inhibited, signaling diverts to a RIPK3-dependent inflammatory necroptotic pathway.

While programmed cell death is crucial for controlling viral pathogens, neurons are incapable of triggering cell death. Neurons are non-regenerative cells that are extremely resistant to programmed cell death – mostly to preserve synapses. However, this leaves neurons vulnerable to viral pathogens, as they cannot eliminate the replicative niche by undergoing cell death. Previous work from the authors of the preprint highlighted here described a unique function for the pro-death kinase RIPK3 in death-resistant neurons. During Zika infection, the neurons were predictably resistant to necroptosis, but exhibited a RIPK3-dependent pro-inflammatory response. The mechanism of RIPK3, a typically necroptotic kinase, inducing an anti-viral response in neurons is investigated for the first time in this preprint.

Key Findings

RIPK3 mediates an anti-viral response in neurons.

Mouse embryonic fibroblasts (MEFs), typically quite responsive to inflammatory stimuli, were compared to neurons in this study. While Zika virus did induce a small inflammatory response in MEFs, this response was not dependent on RIPK3. In contrast, neurons showed a RIPK3-dependent inflammatory response. The small inflammatory response in MEFs subsided quickly, while neurons sustained this response even as long as 24 hours post Zika exposure. Interestingly, the RIPK3-dependent response observed in neurons majorly favored an anti-viral profile, showing reduced NFκB and Type I interferon activation in Zika-stimulated Ripk3-/- neurons. This suggests an intriguing role for RIPK3 in neuronal viral defense – while neurons cannot die by RIPK3 activation to control the infection, they can at least create an environment that is hostile to the virus.

Neurons are intrinsically resistant to Zika-induced death.

The authors then studied what makes neurons resistant to Zika-induced necroptosis. A simple explanation for this phenomenon would be low expression of MLKL, the necroptotic protein that is activated by RIPK3 and physically creates deadly pores in the cell membrane. While neurons did express little to no MLKL, the authors quickly found there is more to the story. Using adeno-associated viral particles, murine MLKL was successfully exogenously expressed in neurons. Despite strong expression of exogenous MLKL, activation of RIPK3 was unable to induce death in neurons. Therefore, there must be additional mechanisms that are neuron-specific that protect these cells from necroptotic death, regardless of MLKL availability.

A RHIM protein complex drives inflammation when cell death is blocked.

In utilizing the MEFs, the authors were able to provide some clarity regarding the inflammatory activity of RIPK3. While MEFs normally had an underwhelming inflammatory response to RIPK3 activation, expression of inflammatory markers increased when cell death was blocked. In other words, the MEFs mimicked the neuronal anti-viral phenotype when cell death was inhibited. The most intriguing candidates for cooperation with RIPK3 are other RHIM-containing proteins, such as TRIF, ZBP1, and RIPK1. Indeed, the MEFs neuron-like antiviral response was inhibited when any of these three RHIM domain proteins were knocked down. Immunoprecipitation further confirmed physical binding of RIPK3, RIPK1, and ZBP1 only after activation of RIPK3 in MEFs.

Excitingly, the model in which an RHIM protein complex drives inflammation when cell death is prevented could be confirmed in neurons. Once again, the inflammatory response was dependent on all RHIM-containing proteins. In support of direct RHIM involvement, neurons expressing a RHIM-less RIPK1 mutant were not able to induce the inflammatory response.

In summary, when RIPK3 is activated by a virus or other stimulus, and cell death is prevented, RIPK3 utilizes other RHIM-containing proteins to promote an anti-viral response.

Why I think this preprint is important

This preprint is important because it shows a novel mechanism in which neurons participate in an immune response. As they are incapable of removing a viral niche via cell death, one may consider them a vulnerable cell population. However, this work demonstrates that neurons are able to co-opt existing pro-death proteins, that are activated by a virus, to induce an antiviral response. While the neurons cannot eliminate themselves as a replicative niche, RIPK3 allows them to create an anti-viral environment via cytokines. Additionally, this work validates the potential targeting of RIPK3 and other RHIM-containing proteins in preventing or reducing neuroinflammation in viral or pathological contexts.

Tags: cell death, necroptosis, neurons, zika

doi: https://doi.org/10.1242/prelights.37058

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A discussion with the authors

Andrew Oberst shared

1. Many viral infections, in mouse models and humans, induce neuroinflammation. Is it possible that a RIPK3- or even TRIF-knockout mouse would be protected from neuroinflammation in an in vivo Zika model?

Our past work has shown that mice lacking RIPK3 or ZBP1 are more sensitive to neuroinvasive infection with West Nile and Zika virus (see, for example Daniels et al. Cell 2017.) We actually attribute this to a failure of neuroinflammation; that is, in the absence of the “necroptotic” pathway, you don’t get the ingress of leukocytes to the infected brain that’s required to control infection. However, you’re correct to assert that while this response is protective during acute infection, it can drive long-term pathologies, including the persistent motor and learning deficits experienced by many patients who survive neuroinvasive WNV infection. I’d point anyone interested to the great work of Robyn Klein’s lab on this topic. If/whether modulating RIPK signaling could improve some of these sequelae of infection is something we’re now working on. But I think these findings also raise some concerns about using RIPK inhibitors that target the brain, as has been proposed for ALS and other neurodegenerative diseases; you’d want to be careful that you’re not sensitizing to viral infection.

2. MLKL is not able to kill neurons, even when it is exogenously expressed. In the MLKL-expressing neurons, is MLKL phosphorylated upon RIPK3 activation? It would be interesting to know what stage of death signaling is interrupted in neurons.

How MLKL works (or doesn’t work) in neurons is a great question and something we’re trying to understand. MLKL has been shown to be regulated at multiple levels including by interaction with inositol phosphates (PMID: 29883610) and via ESCRT membrane repair (PMID: 28388412). We’re working to understand if these or other mechanisms shield neurons from MLKL-induced lethality.

3. Does the inhibition of cell death effectors lead to RIPK3-dependent inflammation in other cell types, besides MEFs?

Yes, for sure. RIPK3 can act as a platform for transcription in any cell with other RHIM components, as far as we know. MEFs were just used as a handy and commonly employed “normal cell”.

4. Other cell types, such as cancerous cells, are resistant to cell death. Do they perhaps mimic the neuronal phenotype, in that they can utilize the activation of RIPK3 not for death but for inflammation?

We generally see that RIPK3 itself is lost in many cancer cells (PMID: 25952668) and we and others have found that re-activating RIPK signaling (but not cell death) in tumors activates anti-tumor immunity (Snyder et al Sci. Imm 2019). Philipp Jost’s lab has some really spectacular data suggesting that loss of RIPK3 is a component of tumor immune evasion in both mouse models and human patients. So we’d suggest that the type of inflammation driven by this pathway is likely to promote cytotoxic type-I responses that are anti-tumor. I think a slightly different question that we find interesting is, how to other long-lived, non-regenerative cells (besides neurons) respond to activation of this pathway? And is re-directing “necroptotic” signaling away from cell death a general feature of such cells?

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