Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Nov 27.
Published before final editing as: J Immunol. 2025 Sep 4:vkaf215. doi: 10.1093/jimmun/vkaf215

LRRK2 kinase activity restricts NRF2-dependent mitochondrial protection in microglia

Chi G Weindel 1,2,*, Aja K Coleman 1,#, Lily M Ellzey 3,#, Sandeep Kumar 2, Sara L Chaisson 2, Jacob R Davis 1, Kristin L Patrick 1,3, Robert O Watson 1,4,*
PMCID: PMC12412900  NIHMSID: NIHMS2098502  PMID: 40906893

Abstract

Mounting evidence supports a critical role for central nervous system (CNS) glial cells in neuroinflammation and neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s Disease (PD), Multiple Sclerosis (MS), as well as neurovascular ischemic stroke. Previously, we found that loss of the PD-associated gene leucine-rich repeat kinase 2 (Lrrk2) in macrophages, peripheral innate immune cells, induced mitochondrial stress and elevated basal expression of type I interferon (IFN) stimulated genes (ISGs) due to chronic mitochondrial DNA engagement with the cGAS/STING DNA sensing pathway. Here, we report that loss of LRRK2 results in a paradoxical response in microglial cells, a CNS-specific macrophage population. In primary murine microglia and microglial cell lines, loss of Lrrk2 reduces tonic IFN signaling leading to a reduction in ISG expression. Consistent with reduced type I IFN, mitochondria from Lrrk2 KO microglia are protected from stress and have elevated metabolism. These protective phenotypes involve upregulation of NRF2, an important transcription factor in the response to oxidative stress and are restricted by LRRK2 kinase activity. Collectively, these findings illustrate a dichotomous role for LRRK2 within different immune cell populations and give insight into the fundamental differences between immune regulation in the CNS and the periphery.

INTRODUCTION

Microglia are slow dividing long-lived resident macrophage populations of the CNS (1, 2), that develop from yolk sac myeloid hematopoietic precursors and travel to the brain prior to closure of the blood brain barrier (3–6). As an innate immune population, microglia act as the first line of defense against invading pathogens and cellular damage through multiple processes including phagocytosis, neuron pruning, cytokine release, and direct cell-cell communication (7–12). The tight regulation of these processes is necessary to prevent neuroinflammation, a hallmark of neurodegenerative diseases of the CNS (13–16).

The type I IFN response has gained recent interest for its role in both maintaining neuron health as well as promoting inflammation. Signaling through the IFNα/β receptor (IFNAR) has been shown to be critical for the development of healthy neurons, as cell-type specific loss of IFNAR signaling results in Lewy body accumulation, α-synuclein aggregation, and a PD-like phenotype due to a blockade of neuronal autophagy (17). Microglial type I IFN responses also play crucial roles in neurodevelopment, where IFNAR signaling facilitates phagocytosis and clearance of damaged neurons (18). Although type I IFN is critical for the healthy development of the CNS, IFNAR signaling can act as a double-edged sword to promote neurodegeneration. For example, in the 5xFAD model of amyloid β-induced Alzheimer’s, blocking IFNAR in CNS cell populations was protective against memory loss and synaptic damage (19, 20). In naturally aging brains, type I IFN signatures in microglial cells are enhanced (21) and are associated with low level inflammation, bystander cell activation, and microgliosis (22). Given what we know, increased IFN signatures could be a product of elevated microglial phagocytic activity and protection, or aberrant inflammation, bystander cell activation, and CNS damage. Thus, there is a critical need to better understand the regulatory nodes that govern protective vs. pathogenic type I IFN responses in the brain, and how this regulation drives the maintenance of healthy glia and neurons while restricting neuroinflammation.

In the peripheral immune system, the tempering of type I IFN responses is a critical means to restrict inflammation and prevent interferonopathies and autoimmunity. One well-known restrictive pathway is the NRF2-mediated redox response. Classically, NRF2 is a transcription factor that upregulates antioxidant proteins to protect against oxidative stress. NRF2 has also been shown to regulate immune signaling; it is a negative regulator of type IFN during viral infection (23, 24) and can restrict IFNβ activation and inflammation following LPS stimulation or sepsis (25–27). NRF2 restricts the type I IFN response at several nodes, including inhibiting dimerization of the transcription factor IRF3 (28–30), and reducing STING expression and mRNA stability in human cells (31). Proteins upregulated by NRF2 during oxidative stress such as HMOX1 also have regulatory effects on the type I IFN response through degradation of transcription factors IRF3/IRF7 via autophagy (32), suggesting a complex interplay between the two pathways. Less is known about the connection between NRF2 and type I IFN responses in the brain. It has been shown that mice lacking NRF2 have neuroinflammation with astrogliosis (33), and NRF2 modulation impacts neuroinflammation in several PD models (34, 35). Despite the intriguing connections between NRF2 and brain health, the role of NRF2 in glial cell inflammation remains under studied.

LRRK2 is a multifunctional PD-associated kinase expressed in neurons and immune cell populations, including monocytes and macrophages (36). Because LRRK2 has been implicated in both genetic and sporadic forms of PD, cells lacking LRRK2 or carrying mutant forms of LRRK2 are excellent models to investigate overall mechanisms of PD (37–39). In addition to PD, LRRK2 has been implicated in increased risk for stroke, traumatic brain injury, neurocognitive disorders, and depression (40–44)While LRRK2 has been well studied in the context of neurons, less is known about the function of LRRK2 in immune cell populations. Previously, we found that loss of LRRK2 in peripheral macrophages including BMDMs, peritoneal macrophages, and macrophage cell lines, results in significantly elevated basal levels of type I IFN/ISGs and an inability to induce interferon responses after infection (45). We linked these elevated basal I IFN responses to mitochondrial stress, namely mitochondrial fragmentation and oxidative stress, that causes mitochondrial DNA leakage into the cytosol and chronic engagement of the cGAS/STING signaling pathway (45).

Here, motivated by our Lrrk2 KO macrophage findings, we investigated how loss of LRRK2 impacts CNS resident microglia cells. Surprisingly, we found that microglial cells lacking LRRK2 had a reduction in type I IFN transcripts compared to controls. Differential gene expression analysis uncovered that the NRF2 redox pathway was upregulated in Lrrk2 KO microglia. Consistent with enhanced protection, Lrrk2 KO microglial cells were better at maintaining a healthy mitochondrial membrane potential and had a higher capacity for OXPHOS, which was dependent on NRF2. Inhibition of LRRK2 kinase activity was sufficient to upregulate NRF2 and reduce ISGs in microglial cells. Surprisingly, LRRK2 kinase inhibition had the opposite effect on peripheral macrophages and reduced NRF2 protein expression. Likewise, macrophages harboring a point mutation that increases LRRK2 kinase activity (Lrrk2 G2019S) had elevated nuclear NRF2 expression, whereas Lrrk2 G2019S microglia had reduced NRF2 expression and elevated ISGs. Taken together these data show LRRK2 kinase function plays differential and active roles in regulating both the type I IFN response and NRF2 activity in microglial cells and macrophages. This work gives insight into the functional requirements of different macrophage populations and how anti- and pro- inflammatory processes are regulated in various tissues.

MATERIALS AND METHODS

Mice

Lrrk2 KO mice (C57BL/6-Lrrk2tm1.1Mjff /J) stock #016121, and Lrrk2 G2019S KI mice (B6.Cg-Lrrk2tm1.1Hlme/J) stock #030961 were purchased from The Jackson Laboratories (Bar Harbor, ME). All mice used in experiments were compared to age- and sex- matched controls by pooling equal males and females between genotypes. To ensure littermate controls were used in all experiments Lrrk2 KO crosses were made with (KO) Lrrk2−/− x (HET) Lrrk2+/− mice. Mice used to make glial cultures were between P1 and P1.5 days old. All animals were housed, bred, and studied at Texas A&M Health Science Center under approved Institutional Care and Use Committee guidelines.

Primary cells

Mixed glial cultures were differentiated from the brains of neonatal mice as described (46). Briefly, glial cells were isolated from the cortexes of neonate mice at P1-P1.5. Disaggregation media was used to liberate glial cells. Glial cells were centrifuged twice 400 rcf, 5 min and washed in complete media (DMEM, 10% FBS, 1 mM sodium pyruvate, 10% MCSF conditioned media), and grown in 10 mL of media 10 cm TC-treated dishes, one dish per brain at 37 °C 5% CO2. Complete media was replaced on day 1 following gentle aspiration. Cells were allowed to differentiate in complete media feeding 5 mL on day 5 and then replacing 5 mL of media on every other day afterward. Following 10 days of culture, microglial cells were isolated from glial cultures by washing briefly with cold 1x PBS + EDTA to detach the microglial layer. Cells were then counted, plated on non-tissue culture treated plates, and washed after 4 hrs with 1x PBS to remove contaminating cell populations. Bone marrow derived macrophages (BMDMs) were differentiated from BM cells isolated by washing mouse femurs with 10 mL DMEM 1 mM sodium pyruvate. Cells were then centrifuged for 5 min at 400 rcf and resuspended in BMDM media (DMEM, 20% FBS (Millipore), 1 mM sodium pyruvate (Lonza), 10% MCSF conditioned media (Watson lab)). BM cells were counted and plated at 5×106 cells per 15 cm non-TC treated dishes in 30 mL complete BMDM media. Cells were fed with an additional 15 mL of BMDM media on day 3. Cells were harvested on day 7 with 1X PBS EDTA (Lonza).

Cell lines

SIMA9 cells (ATCC® SC-6004™), and RAW 264.7 cells (ATCC® TIB-71™) were obtained from the ATCC. BV2 cells were gifted by Dr. Jianrong Li, Texas A&M. For BV2 and SIMA9 cells stably expressing scramble knockdown (KD) and Lrrk2 KD, Lenti-X cells were transfected with a pSICOR scramble non-targeting shRNA construct and pSICOR Lrrk2 targeting constructs using Polyjet (SignaGen Laboratories). Virus was collected 24 and 48 h post transfection. Microglia were transduced using Lipofectamine 2000 (Thermo Fisher). After 48 h, the media was supplemented with puromycin (Invitrogen) to select cells containing the shRNA plasmid.

Flow cytometry

To confirm purity, isolated astrocytes were gated by SSC/FSC and identified as GFAP+. Microglial cells isolated from glial cultures were gated on SSC/FSC followed by CD45+ and defined as CD45+ (eBiosciences) CD11b+ (eBiosciences). Activation markers IAb (eBioceiences), and CD86 (eBiosciences) were analyzed on this population. To assess mitochondrial membrane potential, cells were released from culture plates with 1x PBS + EDTA. Single cell suspensions were made in 1x PBS 2% FBS. For TMRE assays, cells were stained for 20 min at 37 °C in 25 nM TMRE dye and analyzed on an LSR Fortessa X20 (BD Biosciences). Fluorescence was measured under PE (585/15). To assess mitochondrial membrane potential under stress, cells were treated for 15 min with 50 μM FCCP. For JC-1 assays, JC-1 dye was sonicated for 5 min with 30 sec intervals. Cells were stained for 20 min at 37 °C in 1 μM JC-1 dye and analyzed on an LSR Fortessa X20 (BD Biosciences). Aggregates were measured under Texas Red (610/20 600 LP) and monomers under FITC (525/50 505 LP). To assess mitochondrial membrane potential under stress, cells were treated for 3 hrs. with 2.5 μM rotenone prior to being lifted of the culture plates. 5 μM ATP was then added for 5, 15, or 30 min, or 50 μM FCCP was added for 15 min.

Western blot

Cells were lysed in 1x RIPA buffer with protease and phosphatase inhibitors (Pierce). DNA was degraded using 1 U/mL benzonase (EMD Millipore). Proteins were separated by SDS- PAGE and transferred to nitrocellulose membranes. Membranes were blocked overnight in either 5% BSA or non-fat milk, and incubated overnight at 4 °C with the following antibodies: IBA1 (Wako Chemical 019–19741) 1:2000; RSAD2 (Proteintech) 1:1000; STAT1 (Cell Signaling) 1:1000 pRAB10 T73 (Abcam) 1:1000; IFIT1 (Proteintech) 1:1000; IFIT3 (Proteintech) 1:1000; NRF2 (Cell Signaling), 1:1000; HMOX1 (Proteintech); ACTB (Abcam), 1:5000; and TUBB (Abcam), 1:5000. Membranes were incubated with appropriate secondary antibody (Licor) for 2 hrs at RT prior to imaging on Odyssey Fc Dual-Mode Imaging System (Licor).

Immunofluorescence microscopy

Microglia or macrophages were seeded at 2.5×105 cells/well on glass coverslips in 24-well dishes. Cells were fixed in 4% paraformaldehyde for 10 min at RT and then washed three times with PBS. Coverslips were incubated in diluted primary antibody 1:200 ACTIN (Abcam), 1:200 NRF2 (1:200), in 1x PBS + 5% non-fat milk + 0.3% Triton-X (PBS-MT) for 3 hrs. Cells were then washed three times in 1x PBS and incubated in secondary antibodies at 1:500 for 1 hrs and then DAPI diluted in PBS-MT for 5 min. Coverslips were washed twice with 1x PBS and twice with deionized water and mounted on glass slides using Prolong Gold Antifade Reagent (Invitrogen)

Seahorse metabolic assays

Seahorse XF Mito Stress test kits and cartridges (Agilent) were prepared per manufacturer’s protocol and as previously described (47). Microglia were seeded at 5×104 cells/well and analyzed the following day on a Seahorse XF 96well Analyzer (Agilent). For treatments cells were stimulated overnight with 10 ng/ml LPS (Invivogen), or 100 IU IFN-β (PBL), or for 4 hrs with 5 μM ML385 (Selleckchem) or 5 μM Brusatol (Selleckchem) for NRF2 inhibition.

mRNA sequencing

Microglial cell library preparation was carried out by the Baylor College of Medicine Genomic and RNA Profiling Core (GARP) in biological triplicate. RNA sequencing (150 bp paired- end reads) was performed on an Illumina NovaSeq 6000 with S4 flow cell. Data was analyzed by ROSALIND® (https://rosalind.bio/), with a HyperScale architecture developed by ROSALIND, Inc. (San Diego, CA). Quality scores were assessed using FastQC. Reads were aligned to the Mus musculus genome build GRCm39 using Agilent software. Differentially expressed genes were selected as those with p-value threshold <0.05 in the heatmaps represented. Transcriptome analysis was performed using IPA analysis to generate GO term, disease pathway lists, and to compare loss of LRRK2 between microglia and macrophages. Heatmaps were generated using GraphPad Prism software (GraphPad, San Diego, CA). Rosalind and IPA were used for pathways analysis and to generate volcano plots and Venn diagrams.

qRT-PCR

RNA was isolated using Directzol RNAeasy kits (Zymogen). cDNA was made with iScript Direct Synthesis kits (BioRad) per manufacturer’s protocol. qRT-PCR was performed in triplicate using Sybr Green Power up (ThermoFisher). Data was analyzed on a ViiA 7 Real-Time PCR System (Applied Biosystems) or a BioRad CFX96 analyzer (BioRad).

Statistical analysis

All data are representative of 3 or more independent experiments with an n=3 or more. Graphs were generated using Prism (GraphPad). Image analysis was performed using either Image J or cellpose. Significance for assays were determined using a student’s two-tailed t-test, or a one-way ANOVA followed by a Sidak’s multiple comparisons test for more than two variables, unless otherwise noted.

RESULTS

The type I IFN signature is reduced in microglial cells upon loss of LRRK2.

To understand how loss of LRRK2 impacts microglial cells, we first developed a process to generate and isolate pure populations of non-activated primary microglial cells from the cerebral cortices of P1.0-P1.5 neonates using sex and age matched littermate controls (Lrrk2 KO vs. Lrrk2 HET). Key to this approach is the ability to separate microglial cells from astrocytes, another abundant glial cell population in the brain. We achieved this by dissecting out cerebral cortices followed by a high trypsin digestion (2.5%) to disrupt cells of the meninges followed by 6 days of culture for astrocytes, or 10 days of culture for microglial cells in complete media containing MCSF. Microglial cells were gently washed off the astrocyte layer with PBS/EDTA. We then verified microglial vs. astrocyte cell populations measuring GFAP mRNA expression (an abundant astrocyte transcript) and IBA1 protein (a key surface marker of microglia) (Fig 1A.). Astrocyte purity was also assessed by measuring GFAP+ cells (>90%) by flow cytometry (Fig S1A). Microglia, defined as CD45+ CD11b+, were measured to be >85% pure by flow cytometry (Fig S1B). To identify the major LRRK2-dependent differences in transcript abundance, we performed bulk RNA-SEQ analysis from RNA collected from resting microglia and generated sequencing libraries. Using the Rosalind RNA-seq platform, we compared transcripts between HET and Lrrk2 KO microglia and identified 96 significantly differentially expressed genes (Adj. p-value < 0.05), with 77 downregulated and 19 upregulated genes (Fig 1B, Fig S1B, Table S1). Notably, many of the most significantly down regulated genes, aside from Lrrk2, were ISGs including Rsad2, Ifit2, Mx1, Ifit3, Cxcl10, Cmpk2, among others (Fig 1B, 1D purple). Consistent with the type I IFN pathway being impacted, pathway analysis through NCATS BioPlanet identified the most significant pathway impacted as IFN α/β signaling (Fig 1C). Only a handful of genes were significantly upregulated in Lrrk2 KO microglia. These genes (Id1, Id3, Dmpk, Cd34, Serpine2 and Dhfr) have been associated with neurogenesis and microglial cell division (48–52) (Fig 1E). qRT-PCR confirmed downregulation of multiple ISG transcripts including but not limited to Rsad2, Ifit1, Gbp2, Isg15, and Irf7 (Fig 1F). Importantly, downregulation of ISGs was also observed at the protein level (Fig 1G, Fig S1D). By generating lentiviral shRNA knockdowns (KDs) of Lrrk2 alongside a scramble (SCR) control, we observed the same reduced type I IFN signature in the spontaneously immortalized microglial cell line SIMA9 (Fig 1H), supporting a role for LRRK2 in promoting ISG expression in microglial cells.

Figure 1: Loss of LRRK2 reduces tonic IFN signaling in microglial cells.

Figure 1:

Open in a new tab

(A.) Transcript levels of Gfap in Lrrk2 KO and Lrrk2 HET (control) astrocytes and microglia measured by qRT-PCR (upper graph). Protein levels of IBA1 relative to ACTIN in Lrrk2 KO and HET astrocytes and microglia measured by western blot (lower graph). (B.) Volcano plot of genes differentially expressed between Lrrk2 KO and HET microglia (left, purple) down in KO, (right, orange) up in KO. (C.) Ingenuity pathway analysis of major transcriptional pathways differentially expressed between Lrrk2 KO and HET microglia (D.) Heatmap of significant genes downregulated in Lrrk2 KO microglia compared to HET controls. (E.) Heatmap of significant genes upregulated in Lrrk2 KO microglia compared to HET controls. (F.) Transcript levels of ISGs Rsad2, Ifit1, Gbp2, Isg15, and Irf7 in Lrrk2 KO and HET microglia measured by qRT-PCR. (G.) Protein levels of RSAD2, compared to TUBB in Lrrk2 KO and HET microglia measured by western blot; n=3. Quantification on right. (H.) Transcript levels Lrrk2 in Lrrk2 KD and SCR SIMA9 microglia measured by qRT-PCR. (I.) The same as in (H), but ISGs Rsad2, Ifit1, and Irf7. Two-tailed Student’s t-test was used to determine statistical significance. *p<0.05, **p<0.01, ***p<0.005.

To better understand potential drivers of this phenotype, we looked toward known regulators of the type I IFN response in macrophages (53, 54). We saw no major differences in activation (I-Ab) or costimulatory marker (CD86) expression, indicating that loss of LRRK2 did not impact major states of cellular activation (Fig S1E). Likewise, we saw no difference in the expression of negative regulators of type I IFN gene expression like Sosc1, Smad2/3, and Pias4 (Fig S1F) (55). We also confirmed that genes associated with M1-like vs M2-like macrophage states were not differentially regulated in Lrrk2 KO microglia (the type I IFN response has previously been linked to M1 polarization through Irf7 (56) (Fig S1G). Taken together, these data argue that LRRK2 is required to maintain tonic levels of ISGs in microglial cells through a previously undescribed mechanism.

Loss of LRRK2 differentially impacts microglia and peripheral macrophages.

Given the surprising phenotype of Lrrk2 KO microglia, we decided to compare the transcriptional profile differences of Lrrk2 KO microglia to Lrrk2 KO peripheral macrophages. We observed an overlap of 26 genes differentially expressed genes (DEGs) in both Lrrk2 KO microglia and macrophages, with 67 and 352 DEGs distinct for microglia and macrophages respectively (Fig 2A, Table S1, S2). Within the group of 26 shared DEGs, many genes were oppositely impacted by loss of LRRK2 in macrophages and microglial cells, with 19 genes increased in macrophages but reduced in microglia (Fig 2B). Half of these “conflicting” DEGs, including Mx1, Ifit1–3, Oasl2, and Gbp2 etc., are associated with the type I IFN response and Ingenuity Pathway Analysis identified “IFNα/β Signaling” as the most significantly impacted pathway shared between Lrrk2 KO macrophages and microglia (Fig 2D).

Figure 2: The type I IFN response and NRF2 pathways are opposing in microglial cells and macrophages.

Figure 2:

Open in a new tab

(A.) Venn diagram depicting genes differentially expressed in Lrrk2 KO microglial cells (left), both Lrrk2 KO microglia and macrophages (center) or only Lrrk2 KO macrophages (right). (B.) Heatmap of genes differentially expressed in Lrrk2 KO microglia and macrophages. (C.) Transcript levels of ISGs Rsad2, Gbp2, Irf7, Isg15, in Lrrk2 KO and HET astrocytes measured by qRT-PCR. (D.) IPA pathway plot of shared pathways impacted by a loss of LRRK2 in microglia and macrophages. (E.) Pathway analysis of genes upregulated in Lrrk2 KO microglia only (upper) and Lrrk2 KO macrophages only (lower). (F.) Hierarchal clustered heatmap depicting z-scores and pathway differences between microglial cells and macrophages. (G.) Transcript levels of Nfe2l2 (NRF2) in Lrrk2 KO and HET microglia measured by qRT-PCR (H.) The same as in (G) but NRF2 proteins levels relative to TUBB. Two-tailed Student’s t-test was used to determine statistical significance. *p<0.05, **p<0.01, ***p<0.005.

We initially hypothesized that the type I IFN phenotype in Lrrk2 KO microglia could have something to do with the CNS residency of these cells. To test if other CNS glial cells also downregulated type I IFN in the absence of LRRK2, we performed qRT-PCR analysis on Lrrk2 KO and HET astrocytes, purified as described in (Fig 1A, S1A). Unexpectedly, we found that ISGs were upregulated in Lrrk2 KO astrocytes compared to HET controls (Fig 2C). We also noted that transcripts associated with astrocyte maturation and activation, e.g. Gfap, S100b, Icam1, and Ccl5, were also elevated in Lrrk2 KO astrocytes (57) (Fig S2). These data suggest that Lrrk2 KO astrocytes display a more macrophage-like type I IFN phenotype and that the downregulation of ISGs in Lrrk2 KO microglia is not shared by other glial populations.

To better understand why loss of LRRK2 differentially impacts ISG expression in astrocytes/macrophages vs. microglia, and perhaps identify the driver of the phenotype, we performed pathway analysis of non-type I IFN genes in Lrrk2 KO microglia and macrophages. We found that non-ISG DEGs in Lrrk2 KO microglial cells were enriched in pathways related to cell cycle, metabolism, and cancer cells (Fig 2E upper graph), whereas non-ISG DEGs in Lrrk2 KO macrophages were enriched in pathways related to in immune mediated processes, including antigen presentation, neutrophil degranulation, cytokine signaling, and NRF2-mediated antioxidant protection (Fig 2E lower graph). To begin to understand what differentially regulated pathways might be contributing to the inverse phenotype of Lrrk2 KO macrophages vs. microglia, we performed hierarchical clustering of the most significant differentially regulated pathways. (Fig 2F). Two major clusters were noted. One cluster contained upregulated inflammatory signaling pathways (Fig 2F, orange box); the other contained several pathways downregulated only in Lrrk2 KO macrophages, including sphingolipid metabolism, NRF2 stress response, and pathogenesis of coronavirus (Fig 2F, purple box). We chose to focus on NRF2 due to its previous association with downregulating type I IFN responses (23, 25, 32).

To establish that NRF2 expression could be impacted by loss of LRRK2, we performed qRT-PCR (Fig 2G), and western blot analysis (Fig 2H) of NRF2 in Lrrk2 KO and HET microglia. We found that NRF2 expression was increased at the protein but not mRNA levels, indicating NRF2 redox sensing is upregulated in the absence of LRRK2. Taken together, these data suggest that compared to peripheral macrophages, microglial cells employ additional regulatory nodes to restrict type I IFN responses that rely on the redox regulator NRF2.

Lrrk2 KO microglia are protected from stressors and have enhanced mitochondrial metabolism during activation.

Because we previously found that the increase in LRRK2-dependent basal type I IFN in macrophages was linked to mitochondrial dysfunction, including reduced mitochondrial membrane potential and decreased OXPHOS (45), we hypothesized that Lrrk2 KO microglial mitochondria would have the inverse phenotype. To test this, we performed mitochondrial membrane potential assays using JC-1 and TMRE on Lrrk2 KO and HET microglia. JC-1 is a carbocyanine dye that accumulates within healthy mitochondria with normal membrane potential to form red fluorescent aggregates. Upon loss of mitochondrial membrane potential, JC-1 diffuses to the cytosol as a monomer where it emits a green fluorescence, providing a facile tool to determine mitochondrial health. In line with our hypothesis, loss of LRRK2 resulted in increased mitochondrial membrane potential (more red aggregates) in resting microglia and microglia exposed to rotenone/ATP (Fig 3A). We further confirmed this by using the mitochondrial membrane potential dye TMRE in resting Lrrk2 KO and HET microglia (Fig S3A, Fig 3B), and microglia treated with the uncoupling agent FCCP (Fig 3B). Consistent with an inverse correlation between type I IFN upregulation and mitochondrial health, we saw the opposite response in astrocytes (Fig S3C). Protection of mitochondrial membrane potential was also observed in Lrrk2 KD SIMA9 microglial cells compared to SCR control (Fig S3B). Given that Lrrk2 KO microglial mitochondria had increased membrane potential even under high stress conditions, we next used the Agilent Seahorse Metabolic Analyzer to further investigate the impact of stress on the mitochondrial metabolic state. In the Seahorse assay, oxidative phosphorylation (OXPHOS) and glycolysis are assayed by oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively. We found that OCR in Lrrk2 KO microglia was enhanced in terms of basal, spare and maximal capacity (Fig. 3C, lower quantification), indicating that Lrrk2 KO mitochondria were not only more active at rest, but they had a greater capacity for maintaining high levels of OXPHOS during stress, indictive of a protected state. This protection was maintained following activation with type I IFN, which can enhance OCR in macrophages (58) (Fig 3D, lower quantification), as well as LPS, which has been shown to reduce OCR in macrophages (59) (Fig 3E, lower quantification). While oxidative phosphorylation relied heavily on LRRK2, glycolysis, as measured by ECAR, was not impacted under any of these conditions (Fig S3C–E). These data are consistent with the mitochondria of Lrrk2 KO microglia having enhanced protection at the level of mitochondrial homeostasis and suggest that LRRK2 negatively regulates NRF2 to restrict OXPHOS capacity in microglia.

Figure 3: Loss of LRRK2 promotes mitochondrial protection in microglial cells.

Figure 3:

Open in a new tab

(A.) JC-1 staining of mitochondrial membrane potential in Lrrk2 KO and HET microglia measured by flow cytometry. Cells were treated with 2.5 μM rotenone for 3 hrs. followed by 5 μM ATP for 5 and 30 min. (B.) TMRE staining of mitochondrial membrane potential in Lrrk2 KO and HET microglia measured by flow cytometry. Cells were treated with vehicle control (untreated) or 50 μM FCCP for 30 min. (C.) Oxygen consumption rate (OCR) of resting Lrrk2 KO and HET microglia measured by the seahorse analyzer mito-stress test. Arrows and numbers indicate reads between injections times. Quantification of major OCR readouts below. (D.) The same as in (C) but cells were treated for 16 hrs with 100 IU IFN-β. (E.) The same as in (C) and (D) but cells were treated for 16hrs with 10 ng/mL LPS. Two-tailed Student’s t-test or One way ANOVA with Sidak’s multiple comparisons was used to determine statistical significance. *p<0.05, **p<0.01, ***p<0.005.

Upregulation of NRF2 drives the protection of Lrrk2 KO microglial cells

In addition to downregulating the type I IFN response directly, NRF2 has been also shown to protect the mitochondria through the upregulation of reactive oxygen scavengers, induction of autophagy, and other protective metabolic programs (60–63). We therefore choose to further explore the divergence between NRF2 in microglial cells and macrophages focusing on NRF2 protective capacity and the mitochondria. To begin to understand how NRF2 might be differentially regulated in Lrrk2 KO microglial cells compared to controls, we examined both NRF2 expression and localization. Cytoplasmic NRF2 is bound to the KEAP1 complex where it is ubiquitinated and constitutively degraded by the proteosome (64). During cellular stress, KEAP1 releases NRF2, allowing it to rapidly translocate to the nucleus and turn on protective gene expression programs. Consistent with a more activated (protective) state in Lrrk2 KO microglia, we observed elevated levels of nuclear NRF2 (green) colocalizing with DAPI (blue) (Fig 4A) and an increase in the overall expression of NRF2. Consistent with enhanced protection we also saw greater induction of nuclear NRF2 in Lrrk2 KO microglia treated with rotenone for 4h to induce mitochondrial stress (Fig 4B right graph). Because Lrrk2 KO microglia showed enhanced mitochondrial OXPHOS at baseline and during stress with greater OXPHOS reserves, we next wanted to ask if this protection was dependent on NRF2. We therefore performed the Seahorse Mito Stress test on Lrrk2 KO and HET microglia in the presence or absence of NRF2 inhibitors ML385 and brusatol, which inhibit NRF2’s DNA binding ability and expression, respectively (65, 66). Compared to untreated Lrrk2 KO microglia (Fig 4C, left panel), we found that Lrrk2 KO microglia treated with ML385 (Fig 4C, central panel) or brusatol (Fig 4C right panel), lost their protective metabolic state and exhibited reduced basal, spare and maximal respiration potential compared to controls (Fig 4D). Taken together, these data indicate that NRF2 is elevated and activated in Lrrk2 KO microglia compared to HETs, and this enhanced activity provides protection to the mitochondria.

Figure 4: Upregulation of NRF2 drives mitochondrial protection in LRRK2 KO microglial cells.

Figure 4:

Open in a new tab

(A.) Protein levels and localization of NRF2 in resting Lrrk2 KO and HET microglia measured by Immunofluorescence microscopy NRF2 (green), DAPI (blue). Left graph measures NRF2 expression based on mean fluorescence intensity, right graph measures the number of NRF2hi nuclei per field of vision (B.) The same as in (A) but cells were treated with 200 ng/mL rotenone for either 0 or 4 hrs. to induce mitochondrial stress. (C.) Oxygen consumption rate (OCR), a proxy for oxidative phosphorylation, of Lrrk2 KO and HET microglia measured by the seahorse bioanalyzer mito-stress test. Cells were either treated with vehicle control (untreated) (left panel) or treated with NRF2 inhibitors for 4 hrs., ML385 5 μM (middle panel), Brustol 5 μM (right panel). (D.) Quantification of the major OCR readouts from the above line graphs. Two-tailed Student’s t-test or One way ANOVA with Sidak’s multiple comparisons was used to determine statistical significance. *p<0.05, **p<0.01, ***p<0.005.

LRRK2 kinase activity promotes tonic ISG expression and tempers NRF2 activity

Because increased LRRK2 kinase activity is associated with pathophysiology of both familial and sporadic PD (38, 67, 68), inhibition of LRRK2 kinase activity has been proposed as a possible route of PD intervention. Currently multiple LRRK2 kinase inhibitors are being tested in clinical trials for PD therapeutics (69–71). Given the importance of the type I IFN response in multiple neurodegenerative disorders, we wanted to determine if LRRK2 kinase activity was necessary for controlling ISG expression and restricting NRF2 activity. To this end, we treated SIMA9 and BV2 cells with the blood brain barrier (BBB) penetrant small molecule inhibitor of LRRK2, GNE9605. We found that as early as 24h post-treatment with GNE9605, the ISG transcript Rsad2 was depleted in both microglial cell lines (Fig 5A) and RSAD2 protein levels were reduced in GNE9605-treated SIMA9 cells (Fig 5B). The decrease in RSAD2 expression caused by LRRK2 kinase inhibition was dependent on NRF2 activity and expression, as cotreatment with ML385 partially prevented the reduction in RSAD2 (Fig S4A). LRRK2 kinase activity also restricted the NRF2 antioxidant response, as SIMA9 microglia treated with GNE9605 had elevated nuclear expression of NRF2 (Fig 5C) and increased expression of the NRF2 induced antioxidant target protein HMOX1 (Fig 5D). To better understand the differences between the LRRK2 kinase dependent control of NRF2 activity in microglia vs. macrophages, we performed a side-by-side comparison of NRF2 expression in SIMA9 microglial cells compared to the macrophage cell line RAW264.7 in the presence of LRRK2 kinase inhibition. We found that LRRK2 kinase inhibition by GNE9605 significantly increased the expression of NRF2 measured by western blot in SIMA9 cells (Fig 5E), but reduced expression of NRF2 in RAW 264.7 cells (Fig 5D). In both cell types, phosphorylation of RAB10 at T73, a known LRRK2 target protein, was used as a control to measure LRRK2 kinase inhibition (Fig 5E, F). Together, these data further support cell-type specific roles for LRRK2 kinase activity in modulating NRF2.

Figure 5: LRRK2 kinase activity regulates the type I IFN response and NRF2 activity in microglial cells.

Figure 5:

Open in a new tab

(A.) Transcript levels of Rsad2, in SIMA9 and BV2 microglia measured by qRT-PCR. Cells were treated for 0, 24, 48, and 72 hrs. with the small molecule LRRK2 inhibitor 10 μM GNE9605. (B.) Protein levels of RSAD2, compared to TUBB in SIMA9 microglia measured by western blot. Cells were treated for 24 and 48 hrs. with 10 μM GNE9605 (C.) Protein levels and localization of NRF2 in SIMA9 microglia treated with or without 10 μM GNE9605 for 72 hrs. measured by immunofluorescence microscopy NRF2 (green), DAPI (blue). (D.) The same as in (C) but looking at HMOX1 (red). (E.) Protein levels as measured by western blot for NRF2 and pRAB10 T73 in SIMA9 cells treated for 72 hrs with 10 μM GNE9605 or vehicle control (untreated). Protein normalized to tubulin (TUBB). Quantification on right. (F.) The same as in (E) but using RAW 264.7 cells. Two-tailed Student’s t-test or One way ANOVA with Sidak’s multiple comparisons was used to determine statistical significance. *p<0.05, **p<0.01, ***p<0.005.

The PD-associated Lrrk2 G2019S mutation divergently regulates NRF2 expression in peripheral macrophages and microglial cells

Given the inverse response LRRK2 kinase inhibition had on NRF2 expression between microglia and macrophages, we lastly wanted to explore the impact of increased LRRK2 kinase activity in peripheral macrophages compared to microglial cells. The PD-associated missense mutation LRRK2 G2019S has been shown to enhance the kinase activity of LRRK2 (72, 73). We previously reported that LRRK2 mutant macrophages (Lrrk2 G2019S) were prone to proinflammatory cell death and fragmented mitochondria (74). However, Lrrk2 G2019S macrophages did not have an elevated tonic ISG signature like Lrrk2 KO macrophages, which was surprising given the state of their mitochondrial network (74). We hypothesized that the increased kinase activity of LRRK2 increased NRF2 levels, contributing to antioxidant protection and dampened type I IFN responses.

In contrast to BMDMs, when we looked at NRF2 protein levels in Lrrk2 G2019S primary microglial cells compared to WT controls we observed reduced NRF2 protein expression and increased ISG proteins including RSAD2 and IFIT1 (Fig 6B). Elevated ISGs were also visible at the mRNA level and were dependent on LRRK2 kinase activity as treatment with GNE6905 for 24 hrs was sufficient to reduce expression of ISGs Rsad2 and Ifit3 (Fig 6C). Transcript expression of Lrrk2 was not impacted by the G2019S mutation nor was expression of Nfe2l2, the gene that encodes NRF2 (Fig 6D). Taken together, these data demonstrate that microglial cells rely on a novel LRRK2-NRF2 circuit to control type I IFN expression that is LRRK2 kinase-dependent. Peripheral macrophages, on the other hand, employ an opposing regulon, providing an example of how different macrophage populations evolve regulatory mechanisms to meet the requirements of their tissue environment.

Figure 6: The G2019S mutation in LRRK2 has opposing effects on NRF2 expression in macrophages and microglia.

Figure 6:

Open in a new tab

(A.) Protein levels and localization of NRF2 in resting Lrrk2 G2019S and WT BMDMs measured by immunofluorescence microscopy NRF2 (green), DAPI (blue) ACTIN (red). Graph measures NRF2 expression within the nuclei based on mean signal intensity. (B.) Protein levels of RSAD2, IFIT1 and NRF2 compared to GAPDH in resting primary microglia from Lrrk2 G2019S and WT mice measured by western blot. (C.) Transcript levels of Rsad2, and Ifit3 measured by qRT-PCR in Lrrk2 G2019S and WT microglia treated for 24 hrs with 10 μM GNE9605 or vehicle control. (D.) The same as in (C) but looking at Lrrk2 and Nfe2l2 expression. Two-tailed Student’s t-test or One way ANOVA with Sidak’s multiple comparisons was used to determine statistical significance. *p<0.05, **p<0.01, ***p<0.005.

DISCUSION

Despite being most famously associated with antiviral immunity, the type I IFN response is also necessary for the development of healthy neurons, neuron survival, and neurite outgrowth (17). Consistent with playing a role in neuronal function, type I IFN is gaining traction as a key mediator of neurodegenerative diseases including Alzheimer’s and PD, in addition to neuroinflammatory conditions like ischemic stroke. It follows then, that resident CNS glial immune cells need ways to regulate expression of type I IFN and ISGs, both in the healthy brain and in response to pathogens or inflammatory triggers. Because little is known about type I IFN regulation in glia, we sought to gain understanding of how PD-related protein, LRRK2, influences immune responses in microglia compared to peripheral macrophages. We found that loss of Lrrk2 resulted in significantly reduced tonic type I IFN signaling compared to WT or heterozygous (Lrrk2 HET) controls in both primary microglia and microglial cell lines (Fig 1). We linked this decrease in basal type I IFN to upregulation of the transcription factor nuclear factor erythroid 2–related factor 2 (NRF2), which promotes cellular resistance to oxidative stress (Fig 4B, C). Conversely, loss of Lrrk2 in astrocytes or peripheral macrophages, resulted in an increase in type I IFN, reduced NRF2 expression, and a failure to protect cells from oxidative stress (Fig 2C, S2C). Our findings suggest fundamental differences in LRRK2-dependent immune regulation between microglia, other CNS resident immune cells, and the periphery.

The current type I IFN paradigm in macrophages is that cells are primed via cytosolic sensing of mitochondrial DNA through a cGAS/STING dependent axis to maintain tonic type I IFN signaling, allowing for rapid upregulation of IFNβ and ISGs upon viral infection (75). The capacity of mitochondrial DNA to prime through cGAS/STING has previously been shown in microglia from aged mice and Lrrk2 KO peripheral macrophages (22). This study suggests that additional regulation of type I IFN is present in microglia from neonatal mice where a loss of LRRK2 is sufficient to downregulate ISGs and protect mitochondria through upregulation of the redox associated transcription factor NRF2. This disparity indicates that in microglial cells, type I IFN priming is either unnecessary or detrimental directly after birth, at time of intense neural development (76, 77). These combined findings of microglial type I IFN regulation highlight new and interesting avenues for further research. Future studies investigating the role of LRRK2 in microglia across a broad age range of mice will likely yield exciting insights, considering PD and other neurodegenerative diseases often develop later in life. Given the importance of type I IFN in neuron development, it is understandable that additional regulatory nodes would be necessary to prevent runaway neuroinflammation, which is seen in young mice lacking NRF2 (35, 78–81). The idea that cGAS/STING primes old but not young microglia is especially intriguing and suggests that NRF2 protection is lost over time, possibly to enhance antiviral immunity in the CNS. This same loss of NRF2 protection could also act as a major factor in the pathobiology of diseases like Alzheimer’s and PD, which have a strong type I IFN component to disease progression.

Previous work investigating PD pathology highlights the importance of NRF2 mediated protection. SNPs in NFE2L2 that decrease antioxidant function are associated with early onset PD and susceptibility to PD (82, 83). Additionally, several drug studies have shown NRF2 activators protect against PD pathology and neurodegeneration in mouse models (84–87). In addition to being protective in neurodegenerative disease, activation of NRF2 also ameliorates damage caused from ischemic stroke where it protects the BBB, improves edema, and mitigates neurological defects by reducing oxidative stress (79, 88). One mechanism of action is NRF2 restriction of hyperactive type I IFN in microglia which occurs during the acute phase of stroke (89). Upregulation of NRF2 could also protect mitochondria by preventing mitochondrial DNA release and subsequent activation of the type I IFN response. LRRK2 kinase inhibitors are also prime targets for use as disease modifying agents in PD, stroke, and traumatic brain injury (40, 41, 90). One mechanism of protection following LRRK2 kinase inhibition is the upregulation of the AMPK/p38 pathway, that heavily regulates NRF2 (91, 92). This could provide a mechanism through which inhibition of LRRK2 upregulates NRF2 for protective effects, increasing protection against mitochondrial stress, reducing deleterious type I IFN, acting in a similar function as other NRF2 activators. By defining novel links between LRRK2, NRF2, and mitochondrial homeostasis, our findings can now be further expanded upon in the context of disease-associated Lrrk2 mutations.

Supplementary Material

1

ACKNOWLEDGEMENTS

We would like to acknowledge the members of the Patrick and Watson labs for their helpful discussions and feedback. We would particularly like to thank Haley Scott for help with microglial cell isolation practices and written protocol development. We’d like to thank the Li lab for their help with the culture of primary microglial cells and gift of BV2 cells. We’d also like to thank A. Phillip West and the West lab for their help with mitochondrial experiments.

FUNDING

The National Institutes of Health (NIH) grant R01 AI155621 (R.O.W.), NIH grant R01 AI145287 (R.O.W.), and the Parkinson’s Foundation Launch Award PF-Launch-938138 (C.G.W.).

Footnotes

CONFLICTS OF INTEREST

The authors declare that the research described herein was conducted in the absence of any commercial or financial relationships that could be considered a conflict of interest.

REFERENCES

  • 1.Askew K, Li K, Olmos-Alonso A, Garcia-Moreno F, Liang Y, Richardson P, Tipton T, Chapman MA, Riecken K, Beccari S, Sierra A, Molnár Z, Cragg MS, Garaschuk O, Perry VH, and Gomez-Nicola D. 2017. Coupled Proliferation and Apoptosis Maintain the Rapid Turnover of Microglia in the Adult Brain. Cell Rep 18. [Google Scholar]
  • 2.Kierdorf K, Erny D, Goldmann T, Sander V, Schulz C, Perdiguero EG, Wieghofer P, Heinrich A, Riemke P, Hölscher C, Müller DN, Luckow B, Brocker T, Debowski K, Fritz G, Opdenakker G, Diefenbach A, Biber K, Heikenwalder M, Geissmann F, Rosenbauer F, and Prinz M. 2013. Microglia emerge from erythromyeloid precursors via Pu.1-and Irf8-dependent pathways. Nat Neurosci 16. [Google Scholar]
  • 3.Ginhoux F, Lim S, Hoeffel G, Low D, and Huber T. 2013. Origin and differentiation of microglia. Front Cell Neurosci . [Google Scholar]
  • 4.Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF, Conway SJ, Ng LG, Stanley ER, Samokhvalov IM, and Merad M. 2010. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science (1979) 330. [Google Scholar]
  • 5.Alliot F, Godin I, and Pessac B. 1999. Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain. Developmental Brain Research 117. [Google Scholar]
  • 6.Gomez Perdiguero E, Klapproth K, Schulz C, Busch K, Azzoni E, Crozet L, Garner H, Trouillet C, De Bruijn MF, Geissmann F, and Rodewald HR. 2015. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature 518. [Google Scholar]
  • 7.Paolicelli RC, Bolasco G, Pagani F, Maggi L, Scianni M, Panzanelli P, Giustetto M, Ferreira TA, Guiducci E, Dumas L, Ragozzino D, and Gross CT. 2011. Synaptic pruning by microglia is necessary for normal brain development. Science (1979) 333. [Google Scholar]
  • 8.Zengeler KE, and Lukens JR. 2021. Innate immunity at the crossroads of healthy brain maturation and neurodevelopmental disorders. Nat Rev Immunol 21. [Google Scholar]
  • 9.Salter MW, and Stevens B. 2017. Microglia emerge as central players in brain disease. Nat Med 23. [Google Scholar]
  • 10.Chien C-H, Lee M-J, Liou H-C, Liou H-H, and Fu W-M. 2016. Microglia-Derived Cytokines/Chemokines Are Involved in the Enhancement of LPS-Induced Loss of Nigrostriatal Dopaminergic Neurons in DJ-1 Knockout Mice. PLoS One 11: e0151569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, Dustin ML, and Gan WB. 2005. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 8. [Google Scholar]
  • 12.Cowan MN, Sethi I, and Harris TH. 2022. Microglia in CNS infections: insights from Toxoplasma gondii and other pathogens. Trends Parasitol 38. [Google Scholar]
  • 13.McGeer PL, Itagaki S, Akiyama H, and McGeer EG. 1988. Rate of cell death in parkinsonism indicates active neuropathological process. Ann Neurol 24: 574–576. [DOI] [PubMed] [Google Scholar]
  • 14.McGeer PL, Itagaki S, Boyes BE, and McGeer EG. 1988. Reactive microglia are positive for HLA-DR in the: Substantia nigra of Parkinson’s and Alzheimer’s disease brains. Neurology 38. [Google Scholar]
  • 15.Bartels T, De Schepper S, and Hong S. 2020. Microglia modulate neurodegeneration in Alzheimer’s and Parkinson’s diseases. Science (1979) 370. [Google Scholar]
  • 16.Muzio L, Viotti A, and Martino G. 2021. Microglia in Neuroinflammation and Neurodegeneration: From Understanding to Therapy. Front Neurosci 15. [Google Scholar]
  • 17.Ejlerskov P, Hultberg JG, Wang JY, Carlsson R, Ambjørn M, Kuss M, Liu Y, Porcu G, Kolkova K, Friis Rundsten C, Ruscher K, Pakkenberg B, Goldmann T, Loreth D, Prinz M, Rubinsztein DC, and Issazadeh-Navikas S. 2015. Lack of Neuronal IFN-β-IFNAR Causes Lewy Body- and Parkinson’s Disease-like Dementia. Cell 163. [Google Scholar]
  • 18.Escoubas CC, Dorman LC, Nguyen PT, Lagares-Linares C, Nakajo H, Anderson SR, Barron JJ, Wade SD, Cuevas B, Vainchtein ID, Silva NJ, Guajardo R, Xiao Y, Lidsky PV, Wang EY, Rivera BM, Taloma SE, Kim DK, Kaminskaya E, Nakao-Inoue H, Schwer B, Arnold TD, Molofsky AB, Condello C, Andino R, Nowakowski TJ, and Molofsky AV. 2024. Type-I-interferon-responsive microglia shape cortical development and behavior. Cell 187. [Google Scholar]
  • 19.Roy ER, Wang B, Wan YW, Chiu G, Cole A, Yin Z, Propson NE, Xu Y, Jankowsky JL, Liu Z, Lee VMY, Trojanowski JQ, Ginsberg SD, Butovsky O, Zheng H, and Cao W. 2020. Type I interferon response drives neuroinflammation and synapse loss in Alzheimer disease. Journal of Clinical Investigation 130. [Google Scholar]
  • 20.Roy ER, Chiu G, Li S, Propson NE, Kanchi R, Wang B, Coarfa C, Zheng H, and Cao W. 2022. Concerted type I interferon signaling in microglia and neural cells promotes memory impairment associated with amyloid β plaques. Immunity 55. [Google Scholar]
  • 21.Hammond TR, Dufort C, Dissing-Olesen L, Giera S, Young A, Wysoker A, Walker AJ, Gergits F, Segel M, Nemesh J, Marsh SE, Saunders A, Macosko E, Ginhoux F, Chen J, Franklin RJM, Piao X, McCarroll SA, and Stevens B. 2019. Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes. Immunity 50. [Google Scholar]
  • 22.Gulen MF, Samson N, Keller A, Schwabenland M, Liu C, Glück S, Thacker VV, Favre L, Mangeat B, Kroese LJ, Krimpenfort P, Prinz M, and Ablasser A. 2023. cGAS–STING drives ageing-related inflammation and neurodegeneration. Nature 620. [Google Scholar]
  • 23.Gunderstofte C, Iversen MB, Peri S, Thielke A, Balachandran S, Holm CK, and Olagnier D. 2019. Nrf2 Negatively Regulates Type I Interferon Responses and Increases Susceptibility to Herpes Genital Infection in Mice. Front Immunol 10. [Google Scholar]
  • 24.Wyler E, Franke V, Menegatti J, Kocks C, Boltengagen A, Praktiknjo S, Walch-Rückheim B, Bosse J, Rajewsky N, Grässer F, Akalin A, and Landthaler M. 2019. Single-cell RNA-sequencing of herpes simplex virus 1-infected cells connects NRF2 activation to an antiviral program. Nat Commun 10. [Google Scholar]
  • 25.Ryan DG, Knatko EV, Casey AM, Hukelmann JL, Dayalan Naidu S, Brenes AJ, Ekkunagul T, Baker C, Higgins M, Tronci L, Nikitopolou E, Honda T, Hartley RC, O’Neill LAJ, Frezza C, Lamond AI, Abramov AY, Arthur JSC, Cantrell DA, Murphy MP, and Dinkova-Kostova AT. 2022. Nrf2 activation reprograms macrophage intermediary metabolism and suppresses the type I interferon response. iScience 25. [Google Scholar]
  • 26.Thimmulappa RK, Lee H, Rangasamy T, Reddy SP, Yamamoto M, Kensler TW, and Biswal S. 2006. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. Journal of Clinical Investigation 116. [Google Scholar]
  • 27.Kobayashi EH, Suzuki T, Funayama R, Nagashima T, Hayashi M, Sekine H, Tanaka N, Moriguchi T, Motohashi H, Nakayama K, and Yamamoto M. 2016. Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription. Nat Commun 7. [Google Scholar]
  • 28.Olagnier D, Peri S, Steel C, van Montfoort N, Chiang C, Beljanski V, Slifker M, He Z, Nichols CN, Lin R, Balachandran S, and Hiscott J. 2014. Cellular Oxidative Stress Response Controls the Antiviral and Apoptotic Programs in Dengue Virus-Infected Dendritic Cells. PLoS Pathog 10. [Google Scholar]
  • 29.Olagnier D, Lababidi RR, Hadj SB, Sze A, Liu Y, Naidu SD, Ferrari M, Jiang Y, Chiang C, Beljanski V, Goulet ML, Knatko EV, Dinkova-Kostova AT, Hiscott J, and Lin R. 2017. Activation of Nrf2 Signaling Augments Vesicular Stomatitis Virus Oncolysis via Autophagy-Driven Suppression of Antiviral Immunity. Molecular Therapy 25. [Google Scholar]
  • 30.Olagnier D, Farahani E, Thyrsted J, Blay-Cadanet J, Herengt A, Idorn M, Hait A, Hernaez B, Knudsen A, Iversen MB, Schilling M, Jørgensen SE, Thomsen M, Reinert LS, Lappe M, Hoang HD, Gilchrist VH, Hansen AL, Ottosen R, Nielsen CG, Møller C, van der Horst D, Peri S, Balachandran S, Huang J, Jakobsen M, Svenningsen EB, Poulsen TB, Bartsch L, Thielke AL, Luo Y, Alain T, Rehwinkel J, Alcamí A, Hiscott J, Mogensen T, Paludan SR, and Holm CK. 2020. SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate. Nat Commun 11. [Google Scholar]
  • 31.Olagnier D, Brandtoft AM, Gunderstofte C, Villadsen NL, Krapp C, Thielke AL, Laustsen A, Peri S, Hansen AL, Bonefeld L, Thyrsted J, Bruun V, Iversen MB, Lin L, Artegoitia VM, Su C, Yang L, Lin R, Balachandran S, Luo Y, Nyegaard M, Marrero B, Goldbach-Mansky R, Motwani M, Ryan DG, Fitzgerald KA, O’Neill LA, Hollensen AK, Damgaard CK, de Paoli F. v., Bertram HC, Jakobsen MR, Poulsen TB, and Holm CK. 2018. Nrf2 negatively regulates STING indicating a link between antiviral sensing and metabolic reprogramming. Nat Commun 9. [Google Scholar]
  • 32.Wu M, Fan Y, Li L, and Yuan J. 2024. Bi-directional regulation of type I interferon signaling by heme oxygenase-1. iScience 27. [Google Scholar]
  • 33.Hubbs AF, Benkovic SA, Miller DB, O’Callaghan JP, Battelli L, Schwegler-Berry D, and Qiang M. 2007. Vacuolar leukoencephalopathy with widespread astrogliosis in mice lacking transcription factor Nrf2. American Journal of Pathology 170. [Google Scholar]
  • 34.Jazwa A, Rojo AI, Innamorato NG, Hesse M, Fernández-Ruiz J, and Cuadrado A. 2011. Pharmacological targeting of the transcription factor NRf2 at the basal ganglia provides disease modifying therapy for experimental parkinsonism. Antioxid Redox Signal 14. [Google Scholar]
  • 35.Rojo AI, Innamorato NG, Martín-Moreno AM, De Ceballos ML, Yamamoto M, and Cuadrado A. 2010. Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson’s disease. Glia 58. [Google Scholar]
  • 36.Giesert F, Hofmann A, Bürger A, Zerle J, Kloos K, Hafen U, Ernst L, Zhang J, Vogt-Weisenhorn DM, and Wurst W. 2013. Expression Analysis of Lrrk1, Lrrk2 and Lrrk2 Splice Variants in Mice. PLoS One 8: e63778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Rocha EM, Keeney MT, Di Maio R, De Miranda BR, and Greenamyre JT. 2022. LRRK2 and idiopathic Parkinson’s disease. Trends Neurosci 45: 224–236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kluss JH, Mamais A, and Cookson MR. 2019. LRRK2 links genetic and sporadic Parkinson’s disease. Biochem. Soc. Trans. 47: 651–661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Smith WW, Pei Z, Jiang H, Dawson VL, Dawson TM, and Ross CA. 2006. Kinase activity of mutant LRRK2 mediates neuronal toxicity. Nat Neurosci 9: 1231–1233. [DOI] [PubMed] [Google Scholar]
  • 40.Rui Q, Ni H, Gao F, Dang B, Li D, Gao R, and Chen G. 2018. LRRK2 contributes to secondary brain injury through a p38/drosha signaling pathway after traumatic brain injury in rats. Front Cell Neurosci 12. [Google Scholar]
  • 41.Hwang JA, Choi SK, Kim SH, and Kim DW. 2024. Pharmacological Inhibition of LRRK2 Exhibits Neuroprotective Activity in Mouse Photothrombotic Stroke Model. Exp Neurobiol 33: 36–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Cao J, Zhuang Y, Zhang J, Zhang Z, Yuan S, Zhang P, Li H, Li X, Shen H, Wang Z, and Chen G. 2018. Leucine-rich repeat kinase 2 aggravates secondary brain injury induced by intracerebral hemorrhage in rats by regulating the P38 MAPK/Drosha pathway. Neurobiol Dis 119: 53–64. [DOI] [PubMed] [Google Scholar]
  • 43.Puccini JM, Marker DF, Fitzgerald T, Barbieri J, Kim CS, Miller-Rhodes P, Lu SM, Dewhurst S, and Gelbard HA. 2015. Leucine-rich repeat kinase 2 modulates neuroinflammation and neurotoxicity in models of human immunodeficiency virus 1-associated neurocognitive disorders. Journal of Neuroscience 35: 5271–5283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.LRRK2 Inhibition by PF06447475 Antagonist Modulates Early Neuronal Damage after Spinal Cord Trauma _ Enhanced Reader. .
  • 45.Weindel CG, Bell SL, Vail KJ, West KO, Patrick KL, and Watson RO. 2020. LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis. Elife 9. [Google Scholar]
  • 46.Lian H, Roy E, and Zheng H. 2016. Protocol for Primary Microglial Culture Preparation. Bio Protoc 6. [Google Scholar]
  • 47.Van den Bossche J, Baardman J, and de Winther MPJ. 2015. Metabolic characterization of polarized M1 and M2 bone marrow-derived macrophages using real-time extracellular flux analysis. Journal of Visualized Experiments 2015. [Google Scholar]
  • 48.Quan Y, Xu J, Xu Q, Guo Z, Ou R, Shang H, and Wei Q. 2023. Association between the risk and severity of Parkinson’s disease and plasma homocysteine, vitamin B12 and folate levels: a systematic review and meta-analysis. Front Aging Neurosci 15. [Google Scholar]
  • 49.Krawczyk MC, Godoy M, Vander P, Zhang AJ, and Zhang Y. 2023. Loss of Serpin E2 alters antimicrobial gene expression by microglia but not astrocytes. Neurosci Lett 811. [Google Scholar]
  • 50.Asheuer M, Pflumio F, Benhamida S, Dubart-Kupperschmitt A, Fouquet F, Imai Y, Aubourg P, and Cartier N. 2004. Human CD34+ cells differentiate into microglia and express recombinant therapeutic protein. Proc Natl Acad Sci U S A 101. [Google Scholar]
  • 51.Caillet-Boudin ML, Fernandez-Gomez FJ, Tran H, Dhaenens CM, Buee L, and Sergeant N. 2014. Brain pathology in myotonic dystrophy: When tauopathy meets spliceopathy and RNAopathy. Front Mol Neurosci 6. [Google Scholar]
  • 52.Tzeng SF, and De Vellis J. 1998. Id1, Id2, and Id3 gene expression in neural cells during development. Glia 24. [Google Scholar]
  • 53.Lee AJ, and Ashkar AA. 2018. The dual nature of type I and type II interferons. Front Immunol 9. [Google Scholar]
  • 54.Ivashkiv LB, and Donlin LT. 2014. Regulation of type i interferon responses. Nat Rev Immunol 14: 36–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Arimoto KI, Miyauchi S, Stoner SA, Fan JB, and Zhang DE. 2018. Negative regulation of type I IFN signaling. J Leukoc Biol 103. [Google Scholar]
  • 56.Tanaka T, Murakami K, Bando Y, and Yoshida S. 2015. Interferon regulatory factor 7 participates in the M1-like microglial polarization switch. Glia 63. [Google Scholar]
  • 57.Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, Bennett ML, Münch AE, Chung W-S, Peterson TC, Wilton DK, Frouin A, Napier BA, Panicker N, Kumar M, Buckwalter MS, Rowitch DH, Dawson VL, Dawson TM, Stevens B, and Barres BA. 2017. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541: 481–487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Wu D, Sanin DE, Everts B, Chen Q, Qiu J, Buck MD, Patterson A, Smith AM, Chang CH, Liu Z, Artyomov MN, Pearce EL, Cella M, and Pearce EJ. 2016. Type 1 Interferons Induce Changes in Core Metabolism that Are Critical for Immune Function. Immunity 44. [Google Scholar]
  • 59.Kelly B, and O’Neill LA. 2015. Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res 25: 771–784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Abdalkader M, Lampinen R, Kanninen KM, Malm TM, and Liddell JR. 2018. Targeting Nrf2 to suppress ferroptosis and mitochondrial dysfunction in neurodegeneration. Front Neurosci 12. [Google Scholar]
  • 61.Gumeni S, Papanagnou ED, Manola MS, and Trougakos IP. 2021. Nrf2 activation induces mitophagy and reverses Parkin/Pink1 knock down-mediated neuronal and muscle degeneration phenotypes. Cell Death Dis 12. [Google Scholar]
  • 62.Bento-Pereira C, and Dinkova-Kostova AT. 2021. Activation of transcription factor Nrf2 to counteract mitochondrial dysfunction in Parkinson’s disease. Med Res Rev 41. [Google Scholar]
  • 63.Cvetko F, Caldwell ST, Higgins M, Suzuki T, Yamamoto M, Prag HA, Hartley RC, Dinkova-Kostova AT, and Murphy MP. 2021. Nrf2 is activated by disruption of mitochondrial thiol homeostasis but not by enhanced mitochondrial superoxide production. Journal of Biological Chemistry 296. [Google Scholar]
  • 64.Baird L, and Yamamoto M. 2020. The Molecular Mechanisms Regulating the KEAP1-NRF2 Pathway. Mol Cell Biol 40. [Google Scholar]
  • 65.Singh A, Venkannagari S, Oh KH, Zhang YQ, Rohde JM, Liu L, Nimmagadda S, Sudini K, Brimacombe KR, Gajghate S, Ma J, Wang A, Xu X, Shahane SA, Xia M, Woo J, Mensah GA, Wang Z, Ferrer M, Gabrielson E, Li Z, Rastinejad F, Shen M, Boxer MB, and Biswal S. 2016. Small molecule inhibitor of NRF2 selectively intervenes therapeutic resistance in KEAP1-deficient NSCLC tumors. ACS Chem Biol 11. [Google Scholar]
  • 66.Olayanju A, Copple IM, Bryan HK, Edge GT, Sison RL, Wong MW, Lai ZQ, Lin ZX, Dunn K, Sanderson CM, Alghanem AF, Cross MJ, Ellis EC, Ingelman-Sundberg M, Malik HZ, Kitteringham NR, Goldring CE, and Park BK. 2015. Brusatol provokes a rapid and transient inhibition of Nrf2 signaling and sensitizes mammalian cells to chemical toxicity - Implications for therapeutic targeting of Nrf2. Free Radic Biol Med 78. [Google Scholar]
  • 67.Ilieva NM, Hoffman EK, Ghalib MA, Greenamyre JT, and De Miranda BR. 2024. LRRK2 kinase inhibition protects against Parkinson’s disease-associated environmental toxicants. Neurobiol Dis 196. [Google Scholar]
  • 68.Lubben N, Brynildsen JK, Webb CM, Li HL, Leyns CEG, Changolkar L, Zhang B, Meymand ES, O’Reilly M, Madaj Z, DeWeerd D, Fell MJ, Lee VMY, Bassett DS, and Henderson MX. 2024. LRRK2 kinase inhibition reverses G2019S mutation-dependent effects on tau pathology progression. Transl Neurodegener 13. [Google Scholar]
  • 69.West AB 2017. Achieving neuroprotection with LRRK2 kinase inhibitors in Parkinson disease. Exp Neurol 298. [Google Scholar]
  • 70.Wojewska DN, and Kortholt A. 2021. Lrrk2 targeting strategies as potential treatment of parkinson’s disease. Biomolecules 11. [Google Scholar]
  • 71.Taymans JM, Fell M, Greenamyre T, Hirst WD, Mamais A, Padmanabhan S, Peter I, Rideout H, and Thaler A. 2023. Perspective on the current state of the LRRK2 field. NPJ Parkinsons Dis 9. [Google Scholar]
  • 72.Kelly K, Wang S, Boddu R, Liu Z, Moukha-Chafiq O, Augelli-Szafran C, and West AB. 2018. The G2019S mutation in LRRK2 imparts resiliency to kinase inhibition. Exp Neurol 309: 1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Ozelius LJ, Senthil G, Saunders-Pullman R, Ohmann E, Deligtisch A, Tagliati M, Hunt AL, Klein C, Henick B, Hailpern SM, Lipton RB, Soto-Valencia J, Risch N, and Bressman SB. 2006. LRRK2 G2019S as a Cause of Parkinson’s Disease in Ashkenazi Jews . New England Journal of Medicine 354: 424–425. [DOI] [PubMed] [Google Scholar]
  • 74.Weindel CG, Zhao X, Martinez E, Bell SL, Vail KJ, Coleman AK, Vanportfliet JJ, Zhao B, Mabry CJ, Li P, West AP, Karpac J, Patrick KL, and Watson RO. Mitochondrial dysfunction promotes alternative gasdermin D-mediated inflammatory cell death and sus-ceptibility to infection. .
  • 75.West AP, Khoury-Hanold W, Staron M, Tal MC, Pineda CM, Lang SM, Bestwick M, Duguay BA, Raimundo N, Macduff DA, Kaech SM, Smiley JR, Means RE, Iwasaki A, and Shadel GS. Mitochondrial DNA stress primes the antiviral innate immune response. [Google Scholar]
  • 76.Yin W, Li T, Wu Z, Hung SC, Hu D, Gui Y, Cho S, Sun Y, Woodburn MA, Wang L, Li G, Piven J, Elison JT, Wu CW, Zhu H, Cohen JR, Lin W, Zhang H, Yap PT, Smith K, Styner M, Wolff J, Hazlett HC, Yacoub E, Elison J, Ugurbil K, Shen D, Gilmore J, and Piven J. 2025. Charting brain functional development from birth to 6 years of age. Nat Hum Behav . [Google Scholar]
  • 77.Ben-Yehuda H, Matcovitch-Natan O, Kertser A, Spinrad A, Prinz M, Amit I, and Schwartz M. 2020. Maternal Type-I interferon signaling adversely affects the microglia and the behavior of the offspring accompanied by increased sensitivity to stress. Mol Psychiatry 25: 1050–1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 78.Shah ZA, Li RC, Thimmulappa RK, Kensler TW, Yamamoto M, Biswal S, and Doré S. 2007. Role of reactive oxygen species in modulation of Nrf2 following ischemic reperfusion injury. Neuroscience 147. [Google Scholar]
  • 79.Shih AY, Li P, and Murphy TH. 2005. A small-molecule-inducible Nrf2-mediated antioxidant response provides effective prophylaxis against cerebral ischemia in vivo. Journal of Neuroscience 25. [Google Scholar]
  • 80.Chen PC, Vargas MR, Pani AK, Smeyne RJ, Johnson DA, Kan YW, and Johnson JA. 2009. Nrf2-mediated neuroprotection in the MPTP mouse model of Parkinson’s disease: Critical role for the astrocyte. Proc Natl Acad Sci U S A 106. [Google Scholar]
  • 81.Branca C, Ferreira E, Nguyen TV, Doyle K, Caccamo A, and Oddo S. 2017. Genetic reduction of Nrf2 exacerbates cognitive deficits in a mouse model of Alzheimer’s disease. Hum Mol Genet 26. [Google Scholar]
  • 82.Comprehensive Assessment of Genetic Sequence Variants in the Antioxidant ‘Master Regulator’ Nrf2 in Idiopathic Parkinson’s Disease _ Enhanced Reader.
  • 83.Gui Y, Zhang L, Lv W, Zhang W, Zhao J, and Hu X. 2016. NFE2L2 variations reduce antioxidant response in patients with Parkinson disease,.
  • 84.Peng S, Chen Y, Wang R, and Zhang J. 2024. Z-ligustilide provides a neuroprotective effect by regulating the phenotypic polarization of microglia via activating Nrf2-TrxR axis in the Parkinson’s disease mouse model. Redox Biol 76. [Google Scholar]
  • 85.Zhang N, Shu HY, Huang T, Zhang QL, Li D, Zhang GQ, Peng XY, Liu CF, Luo WF, and Hu LF. 2014. Nrf2 signaling contributes to the neuroprotective effects of urate against 6-OHDA toxicity. PLoS One 9. [Google Scholar]
  • 86.Nakano-Kobayashi A, Fukumoto A, Morizane A, Nguyen DT, Le TM, Hashida K, Hosoya T, Takahashi R, Takahashi J, Hori O, and Hagiwara M. 2020. Therapeutics potentiating microglial p21-Nrf2 axis can rescue neurodegeneration caused by neuroinflammation,.
  • 87.Sun W, Shen Y, Xiao H, and Li H. 2021. Resveratrol attenuates rotenone-induced inflammation and oxidative stress via STAT1 and Nrf2/Keap1/SLC7A11 pathway in a microglia cell line. Pathol Res Pract 225. [Google Scholar]
  • 88.Fan W, Chen H, Li M, Fan X, Jiang F, Xu C, Wang Y, Wei W, Song J, Zhong D, and Li G. 2024. NRF2 activation ameliorates blood–brain barrier injury after cerebral ischemic stroke by regulating ferroptosis and inflammation. Sci Rep 14. [Google Scholar]
  • 89.Cui P, Song B, Xia Z, and Xu Y. 2024. Type I Interferon Signalling and Ischemic Stroke: Mechanisms and Therapeutic Potentials. Transl Stroke Res . [Google Scholar]
  • 90.West AB 2017. Achieving neuroprotection with LRRK2 kinase inhibitors in Parkinson disease. Exp Neurol 298: 236–245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 91.Naidu S, Vijayan V, Santoso S, Kietzmann T, and Immenschuh S. 2009. Inhibition and Genetic Deficiency of p38 MAPK Up-Regulates Heme Oxygenase-1 Gene Expression via Nrf2. The Journal of Immunology 182: 7048–7057. [DOI] [PubMed] [Google Scholar]
  • 92.Hourihan JM, Moronetti Mazzeo LE, Fernández-Cárdenas LP, and Blackwell TK. 2016. Cysteine Sulfenylation Directs IRE-1 to Activate the SKN-1/Nrf2 Antioxidant Response. Mol Cell 63: 553–566. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2098502-supplement-1.pdf (1.5MB, pdf)

RESOURCES