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The RhoA-ROCK1/ROCK2 Pathway Exacerbates Inflammatory Signaling in Immortalized and Primary Microglia

Elliot J Glotfelty^1,2, Luis B Tovar-Y-Romo³, Shih-Chang Hsueh¹

¹Drug Design & Development Section, Translational Gerontology Branch, Intramural Research Program National Institute on Aging, NIH, Baltimore, MD 21224, USA.

|July 6, 2023

Abstract:

Neuroinflammation is a unifying factor among all acute central nervous system (CNS) injuries and chronic neurodegenerative disorders. Here, we used immortalized microglial (IMG) cells and primary microglia (PMg) to understand the roles of the GTPase Ras homolog gene family member A (RhoA) and its downstream targets Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) in neuroinflammation. We used a pan-kinase inhibitor (Y27632) and a ROCK1- and ROCK2-specific inhibitor (RKI1447) to mitigate a lipopolysaccharide (LPS) challenge. In both the IMG cells and PMg, each drug significantly inhibited pro-inflammatory protein production detected in media (TNF-α, IL-6, KC/GRO, and IL-12p70). In the IMG cells, this resulted from the inhibition of NF-κB nuclear translocation and the blocking of neuroinflammatory gene transcription (iNOS, TNF-α, and IL-6). Additionally, we demonstrated the ability of both compounds to block the dephosphorylation and activation of cofilin. In the IMG cells, RhoA activation with Nogo-P4 or narciclasine (Narc) exacerbated the inflammatory response to the LPS challenge. We utilized a siRNA approach to differentiate ROCK1 and ROCK2 activity during the LPS challenges and showed that the blockade of both proteins may mediate the anti-inflammatory effects of Y27632 and RKI1447. Using previously published data, we show that genes in the RhoA/ROCK signaling cascade are highly upregulated in the neurodegenerative microglia (MGnD) from APP/PS-1 transgenic Alzheimer's disease (AD) mice. In addition to illuminating the specific roles of RhoA/ROCK signaling in neuroinflammation, we demonstrate the utility of using IMG cells as a model for primary microglia in cellular studies.

Insights

Neuroinflammation involves RhoA/ROCK signaling, with inhibitors reducing pro-inflammatory factors in microglial cells. This pathway is implicated in Alzheimer's disease, suggesting potential therapeutic targets.

Area of Science:

Neuroscience
Cell Biology
Immunology

Background:

Neuroinflammation is a common feature of CNS injuries and neurodegenerative diseases.
The RhoA/ROCK signaling pathway plays a critical role in cellular responses.
Microglia are key immune cells in the CNS involved in neuroinflammation.

Purpose of the Study:

To investigate the role of RhoA and its downstream kinases (ROCK1/ROCK2) in microglial neuroinflammation.
To evaluate the therapeutic potential of ROCK inhibitors in mitigating neuroinflammatory responses.
To establish immortalized microglial (IMG) cells as a reliable model for primary microglia (PMg) studies.

Main Methods:

Utilized immortalized microglial (IMG) and primary microglia (PMg) cell models.
Administered lipopolysaccharide (LPS) to induce a neuroinflammatory challenge.
Employed pan-kinase inhibitor Y27632 and ROCK1/ROCK2-specific inhibitor RKI1447.
Assessed pro-inflammatory cytokine production (TNF-α, IL-6, KC/GRO, IL-12p70).
Investigated NF-κB nuclear translocation and inflammatory gene transcription (iNOS, TNF-α, IL-6).
Examined cofilin dephosphorylation and activation.
Used siRNA to differentiate ROCK1 and ROCK2 roles.
Analyzed RhoA activation using Nogo-P4 and narciclasine (Narc).

Main Results:

Both Y27632 and RKI1447 significantly reduced pro-inflammatory protein production in IMG and PMg cells.
Inhibition of NF-κB nuclear translocation and inflammatory gene transcription was observed.
ROCK inhibitors blocked cofilin dephosphorylation and activation.
RhoA activation exacerbated LPS-induced inflammatory responses.
siRNA-mediated blockade of ROCK1 and ROCK2 contributed to anti-inflammatory effects.
RhoA/ROCK pathway genes are upregulated in Alzheimer's disease microglia models.

Conclusions:

RhoA/ROCK signaling is a key mediator of neuroinflammation in microglia.
Inhibiting RhoA/ROCK signaling reduces pro-inflammatory responses and holds therapeutic potential for neurodegenerative diseases.
IMG cells serve as a valid and useful model for studying primary microglia in neuroinflammation research.

Read full analysis

Keywords:

NF-κB ROCK inhibitors ROCK1 ROCK2 RhoA microglia neuroinflammation

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