Author response: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The Parkinson's disease (PD)-associated gene leucine-rich repeat kinase 2 (LRRK2) has been studied extensively in the brain. However, several studies have established that mutations in LRRK2 confer susceptibility to mycobacterial infection, suggesting LRRK2 also controls immunity. We demonstrate that loss of LRRK2 in macrophages induces elevated basal levels of type I interferon (IFN) and interferon stimulated genes (ISGs) and causes blunted interferon responses to mycobacterial pathogens and cytosolic nucleic acid agonists. Altered innate immune gene expression in Lrrk2 knockout (KO) macrophages is driven by a combination of mitochondrial stresses, including oxidative stress from low levels of purine metabolites and DRP1-dependent mitochondrial fragmentation. Together, these defects promote mtDNA leakage into the cytosol and chronic cGAS engagement. While Lrrk2 KO mice can control Mycobacterium tuberculosis (Mtb) replication, they have exacerbated inflammation and lower ISG expression in the lungs. These results demonstrate previously unappreciated consequences of LRRK2-dependent mitochondrial defects in controlling innate immune outcomes. eLife digest Parkinson's disease is a progressive nervous system disorder that causes tremors, slow movements, and stiff and inflexible muscles. The symptoms are caused by the loss of cells known as neurons in a specific part of the brain that helps to regulate how the body moves. Researchers have identified mutations in several genes that are associated with an increased risk of developing Parkinson's. The most common of these mutations occur in a gene called LRRK2. This gene produces a protein that has been shown to be important for maintaining cellular compartments known as mitochondria, which play a crucial role in generating energy. It remains unclear how these mutations lead to the death of neurons. Mutations in LRRK2 have also been shown to make individuals more susceptible to bacterial infections, suggesting that the protein that LRRK2 codes for may help our immune system. Weindel, Bell et al. set out to understand how this protein works in immune cells called macrophages, which 'eat' invading bacteria and produce type I interferons, molecules that promote immune responses. Mouse cells were used to measure the ability of normal macrophages and macrophages that lack the mouse equivalent to LRRK2 (referred to as Lrrk2 knockout macrophages) to make type I interferons. The experiments showed that the Lrrk2 knockout macrophages made type I interferons even when they were not infected with bacteria, suggesting they are subject to stress that triggers immune responses. It was possible to correct the behavior of the Lrrk2 knockout macrophages by repairing their mitochondria. When mice missing the gene equivalent to LRRK2 were infected with the bacterium that causes tuberculosis, they experienced more severe disease. The protein encoded by the LRRK2 gene is considered a potential target for therapies to treat Parkinson's disease, and several drugs that inhibit this protein are being tested in clinical trials. The findings of Weindel, Bell et al. suggest that these drugs may have unintended negative effects on a patient's ability to fight infection. This work also indicates that LRRK2 mutations may disrupt immune responses in the brain, where macrophage-like cells called microglia play a crucial role in maintaining healthy neurons. Future studies that examine how mutations in LRRK2 affect microglia may help us understand how Parkinson's disease develops. Introduction Mutations in leucine-rich repeat kinase 2 (LRRK2) are a major cause of familial and sporadic Parkinson's disease (PD), a neurodegenerative disease characterized by selective loss of dopaminergic neurons in the substantia nigra pars compacta region of the midbrain (Cookson, 2017; Kim and Alcalay, 2017; Martin et al., 2014; Schulz et al., 2016). Despite LRRK2 having been implicated in a variety of cellular processes, including cytoskeletal dynamics (Civiero et al., 2018; Kett et al., 2012; Pellegrini et al., 2017), vesicular trafficking (Herbst and Gutierrez, 2019; Sanna et al., 2012; Shi et al., 2017), calcium signaling (Bedford et al., 2016; Calì et al., 2014), and mitochondrial function (Ryan et al., 2015; Singh et al., 2019; Yue et al., 2015), its precise mechanistic contributions to triggering and/or exacerbating PD and other disease pathologies are not known. Of all the cellular pathways affected by LRRK2 mutations, dysregulation of mitochondrial homeostasis has emerged as a centrally important mechanism underlying PD pathogenesis and neuronal loss (Cowan et al., 2019; Panchal and Tiwari, 2019). Indeed, other PD-associated genes, such as PARK2 (Parkin), PINK1, and DJ1, all play crucial roles in mitochondrial quality control via mitophagy. LRRK2 has been implicated in mitophagy directly through interactions with the mitochondrial outer membrane protein MIRO (Hsieh et al., 2016), and several lines of evidence support roles for LRRK2 in controlling mitochondrial network dynamics through interactions with the mitochondrial fission protein DRP1 (Wang et al., 2012). Accordingly, a number of different cell types, including fibroblasts and iPSC-derived neurons from PD patients harboring mutations in LRRK2 exhibit defects in mitochondrial network integrity as well as increased reactive oxygen species (ROS) and oxidative stress (Sison et al., 2018; Smith et al., 2016). In spite of these well-appreciated links, LRRK2's contribution to mitochondrial health in cells outside of the brain remains vastly understudied. There is mounting evidence that mutations in LRRK2, as well as in other genes related to PD including PARK2 and PINK1, contribute to immune outcomes both in the brain and in the periphery. For example, mutations in LRRK2 impair NF-κB signaling pathways in iPSC-derived neurons and render rats prone to progressive neuroinflammation in response to peripheral innate immune triggers (López de Maturana et al., 2016), and chemical inhibition of LRRK2 attenuates inflammatory responses in microglia ex vivo (Moehle et al., 2012). In addition to these strong connections between LRRK2 and inflammatory responses in the brain, numerous genome-wide association studies suggest that LRRK2 is an equally important player in peripheral immune responses. Single nucleotide polymorphisms (SNPs) in LRRK2 are associated with susceptibility to mycobacterial infection, inflammatory colitis (Umeno et al., 2011), and Crohn's disease (Van Limbergen et al., 2009). Consistent with a role for LRRK2 in pathogen defense and autoimmunity, it is abundant in many immune cells (e.g. B cells, dendritic cells, monocytes, macrophages), and expression of LRRK2 is induced in human macrophages treated with IFN-γ (Gardet et al., 2010). Loss of LRRK2 reduces IL-1β secretion in response to Salmonella enterica infection in macrophages (Liu et al., 2017) and enhances expression of pro-inflammatory cytokines in response to Mycobacterium tuberculosis (Mtb) infection at early time points of mouse infection (Härtlova et al., 2018). However, the precise mechanistic contributions of LRRK2 to controlling immune responses in the periphery remain poorly understood. Here, we provide evidence that LRRK2's ability to influence inflammatory gene expression in macrophages is directly linked to its role in maintaining mitochondrial homeostasis. Specifically, we demonstrate that mitochondrial stress and hyper-activation of DRP1 in Lrrk2 KO macrophages leads to the release of mitochondrial DNA (mtDNA), chronic engagement of the cGAS-dependent DNA sensing pathway, and abnormally elevated basal levels of type I IFN and ISGs. These high basal levels of type I IFN appear to completely reprogram Lrrk2 KO macrophages, rendering them refractory to a number of distinct innate immune stimuli, including infection with Mtb. While Mtb-infected Lrrk2 KO mice did not exhibit significant differences in bacterial burdens compared to controls, we did observe exacerbated pathology and lower expression of ISGs in the lungs at early infection timepoints. Collectively, these results demonstrate that LRRK2's role in maintaining mitochondrial homeostasis is critical for proper induction of type I IFN gene expression in macrophages and for downstream inflammatory responses during in vivo infection. Results RNA-seq analysis reveals that LRRK2-deficiency in macrophages results in dysregulation of the type I IFN response during Mtb infection To begin to implicate LRRK2 in the peripheral immune response, we took an unbiased approach and asked how loss of LRRK2 impacts innate immune gene expression during Mtb infection of macrophages ex vivo. Briefly, primary murine bone marrow-derived macrophages (BMDMs) derived from littermate heterozygous (HET) and knockout (KO) Lrrk2 mice were infected with Mtb at MOI of 10. RNA-seq analysis was performed on total RNA collected from uninfected and infected cells 4 hr post-infection (Lrrk2 KO n = 4, Lrrk2 HET n = 3). Previous studies have identified 4 hr as a key innate immune time point during Mtb infection, corresponding to the peak of transcriptional activation downstream of several pattern recognition receptors (PRRs), including the cytosolic DNA sensor cGAS (Manzanillo et al., 2012; Watson et al., 2015; Watson et al., 2012). Following analysis with CLC Genomics Workbench, we first asked whether we could detect gene expression differences in uninfected Lrrk2 HET and KO macrophages. Surprisingly, we identified hundreds of genes whose expression was significantly higher in Lrrk2 KO macrophages (blue genes, Figure 1A). Taking a closer look at the most affected genes (zoom-in, right), we noted that a number of well-characterized ISGs (e.g. Mx1, Ifit1, Irf7, Rsad2, etc.) were expressed several times higher in macrophages lackingLRRK2 (p<0.05). These trends persisted when we compared Lrrk2 WT vs. KO or Lrrk2 HET vs. KO (Figure 1—figure supplement 1B-C). Unbiased canonical pathway analysis confirmed a global upregulation of ISGs, identifying 'Interferon signaling' and 'Activation of IRF by cytosolic PRRs' as the top enriched pathways in uninfected Lrrk2 KO vs. HET BMDMs (Figure 1B). Figure 1 with 1 supplement see all Download asset Open asset Global gene expression analysis reveals that Lrrk2 KO macrophages are deficient at inducing type I IFN expression and have higher basal levels of ISGs. (A) Heatmap depicting significant gene expression differences (Log2 fold-change, p<0.05) between uninfected Lrrk2 KO and HET BMDMs. (B) IPA software analysis showing cellular pathways enriched for differentially expressed genes in uninfected Lrrk2 KO vs. HET BMDMs. (C) Heatmap depicting significant gene expression differences (Log2 fold-change) between Lrrk2 KO and HET BMDMs during infection with Mtb. (D) As in (B) but for pathways enriched for differentially expressed genes in Mtb-infected Lrrk2 KO and HET BMDMs, 4 hr post-infection. (E) RT-qPCR showing expression of Ifnb and IFN stimulated genes in uninfected and Mtb-infected Lrrk2 KO and HET macrophages. Data are shown as ISG/Actb. (F) RT-qPCR of Tnfa in Lrrk2 KO and HET BMDMs. (G) RT-qPCR of Apoe and Ldhb normalized to Actb in uninfected BMDMs. Throughout the manuscript, data are expressed as a mean of three or more biological replicates with error bars depicting SEM. Statistical tests used can be found at the end of the legend. Statistical analysis: *p<0.05, p<0.01, *p<0.005, ****p<0.001 (comparing indicated data points); ##p<0.001 (comparing stimulated to unstimulated of same genotype). In (E–F) a two-way ANOVA Tukey post-test was applied, and in (G) a two-tailed Student's T test. We next looked at gene expression differences in Lrrk2 KO vs. HET BMDMs at 4 hr post-infection with Mtb. Mtb is a potent activator of type I IFN expression, thought to occur mostly through perturbation of the Mtb-containing phagosome and release of bacterial dsDNA into the cytosol, where it is detected by DNA sensors like cGAS, activating the STING/TBK1/IRF3 axis (Collins et al., 2015; Wassermann et al., 2015; Watson et al., 2015; Wiens and Ernst, 2016). Curiously, many of the same ISGs whose expression was statistically higher at baseline in Lrrk2 KO BMDMs failed to induce to the same levels following Mtb infection (e.g. Ifit, Cmpk2, Gbp2, Rsad2; Figure 1C, orange genes, zoom-in, left). This blunted global type I IFN response was also evident via qualitative assessment of genes whose expression was measurably lower in Mtb-infected Lrrk2 KO BMDMs but failed to demonstrate statistical significance (Figure 1—figure supplement 1A). Again, canonical pathway analysis identified an enrichment for immune genes whose expression was impacted by loss of LRRK2 in response to Mtb (Figure 1D). RT-qPCR analysis confirmed higher baseline expression and lower induction during Mtb infection of several ISGs: Rsad2, Gbp2, Cmpk2, Stat2, and Ifit1 in Lrrk2 KO BMDMs (Figure 1E; see 'Statistical analysis' section in Materials and methods for details regarding the statistical analysis of baseline and induced gene expression). We also measured high basal levels of Ifnb and Isg15, although the differences in induction of these genes between Lrrk2 KO and HET macrophages were more modest in this particular experiment (Figure 1E). Increased basal expression and decreased induction of IFN and ISGs was also detected during Mtb infection in the human monocyte cell line U937 (Figure 1—figure supplement 1D) and in RAW 264.7 murine macrophages when Lrrk2 expression was knocked down by shRNA (Lrrk2 KD) (Figure 1—figure supplement 1E). Importantly, blunted expression was not observed for all immune genes; for example, loss of Lrrk2 had no effect on the NFκB gene Tnfa despite the transcript being dramatically induced upon Mtb infection (Figure 1F). Interestingly, expression of several non-ISG, non-immune genes was reduced in uninfected Lrrk2 KO BMDMs, including ApoE, which has been repeatedly linked to inflammatory and neurodegenerative diseases, and Ldhb, a critical metabolic gene involved in post-glycolytic energy production (Figure 1G). Collectively, these transcriptome-focused analyses revealed that Lrrk2 KO macrophages have a high baseline IFN signature but generally fail to induce the type I IFN response to the same level as control cells when infected with Mtb. This phenotype is unusual and suggests that Lrrk2 KO macrophages are somehow fundamentally reprogrammed. Typically, high resting IFN levels potentiate type I IFN responses, leading to a hyperinduction of ISGs following innate immune stimuli (West et al., 2015; Yang et al., 2018). Lrrk2 KO macrophages exhibit blunted type I IFN induction in response to cytosolic nucleic acid agonists We next wanted to define the nature of the innate immune stimuli that would elicit a blunted type I IFN response in Lrrk2 KO macrophages. We began by infecting macrophages with Mycobacterium leprae (Mlep), which shares a virulence-associated ESX-1 secretion system with Mtb and also induces type I IFN through cytosolic nucleic acid sensing (de Toledo-Pinto et al., 2016). We measured a significant defect in ISG expression 8 hr post-infection in Lrrk2 KO BMDMs and Lrrk2 KO RAW 264.7 macrophages compared to control cells (Figure 2A and Figure 2—figure supplement 1A). We next treated primary macrophages and macrophage cell lines with a panel of agonists designed to elicit type I IFN expression downstream of a variety of PRRs. Transfection of immunostimulatory dsDNA (ISD), which is recognized by cGAS and stimulates the STING/TBK1/IRF3 axis, induced blunted Ifnb expression in Lrrk2 KO BMDMs (Figure 2B), Lrrk2 KO peritoneal macrophages (PEM) (significant differences in Ifnb expression were measured between Lrrk2 KO and HET at baseline but induction differences failed to reach statistical significance via 2-way ANOVA Tukey post-hoc testing) (Figure 2C), Lrrk2 KO RAW 264.7 macrophages (Figure 2D), Lrrk2 KO mouse embryonic fibroblasts (MEFs) (Figure 2E), and Lrrk2 KD RAW 264.7 macrophages (Figure 2F). Consistent with the BMDM phenotype from Figure 1, we observed higher basal expression of Ifnb/ISGs and a blunted response to ISD in all the Lrrk2 KO/KD cell lines tested (Figure 2A–E and Figure 2—figure supplement 1C). We also found that Lrrk2 KO BMDMs failed to fully induce Ifnb if we bypassed cGAS and stimulated the DNA sensing adapter STING directly using the agonist DMXAA (Figure 2G). In support of a defect in cytosolic nucleic acid sensing and IFNAR signaling, western blot analysis of IRF3 (phospho-Ser396) and STAT1 (phospho-Tyr701) activation showed a significant defect in the ability of Lrrk2 KO macrophages to signal through IFNAR (phospho-STAT1) and a modest defect in cytosolic DNA sensing (phospho-IRF3) over the course of 6 hr following ISD transfection (Figure 2H, quantitation below, and Figure 2—figure supplement 1B). Collectively, these results suggest that type I IFN-generating pathways are chronically activated in cells lacking LRRK2, but their induction is muted compared to controls when faced with agonists of the cytosolic DNA sensing pathway. Figure 2 with 1 supplement see all Download asset Open asset Lrrk2 KO macrophages exhibit blunted type I IFN expression in response to cytosolic nucleic acid agonists. (A) RT-qPCR of Isg15 expression after 4 and 8 hr of infection with M. leprae (MOI = 50) in Lrrk2 KO BMDMs and HET controls. (B) RT-qPCR of Ifnb in unstimulated Lrrk2 KO and HET BMDMs alongside cells transfected with 1 μg/ml ISD (dsDNA) for 4 hr. (C) As in (B) but with peritoneal macrophages (PEMs) from Lrrk2 KO and HET mice elicited for 4 days with 1 ml 3% Brewer's thioglycolate broth. (D) As in (B) but with RAW 264.7 Lrrk2 KO cells and WT controls. (E) As in (B) but with MEFs from day 14.5 Lrrk2 KO or HET embryos. (F) As in (B) but with RAW 264.7 Lrrk2 KD and scramble (SCR) controls cells. (G) RT-qPCR of Ifnb expression in uninfected Lrrk2 KO or HET BMDMs and in cells treated with 50 ng/ml DMXAA for 2 hr. (H) Western blot analysis and quantification of IRF3 phosphorylation (Ser396) and STAT1 phosphorylation (Tyr701) in BMDMs from HET and Lrrk2 KO mice compared to total IRF3 and STAT1 with tubulin as a loading control following transfection with 1 μg/ml ISD (dsDNA). (I) As in (G) but following transfection with 1 μg/ml poly(I:C), 100 ng/ml LPS, transfection with 10 μM CpG 2395, or stimulation with 1 μM CL097, all for 4 hr. (J) RT-qPCR of Isg15 expression after treatment with 200 IU IFN-β for 4 hr. (K) RT-qPCR of Irf7 gene expression in Lrrk2 HET and KO BMDMs with or without overnight treatment with IFN-β neutralizing antibody (blocking Ab, 1:250). (L) RT-qPCR of Irf7 gene expression in BMDMs from WT, Lrrk2 KO, Ifnar KO, and double knockout (Lrrk2/Ifnar DKO) mice. Statistical analysis: *p<0.05, p<0.01, *p<0.005, ****p<0.001 (comparing indicated data points); ##p<0.001 (comparing stimulated to unstimulated of same genotype). (A–L) two-way ANOVA Tukey post-test. We next tested whether loss of LRRK2 impacted the ability of cells to respond to activators of the type I IFN response outside of the cytosolic DNA sensing cascade. To this end, we treated Lrrk2 KO and HET BMDMs with transfected poly(I:C) (to activate cytosolic RNA sensing), LPS (to stimulate TRIF/IRF3 downstream of TLR4), and CpG and CL097 (to stimulate nucleic acid sensing via TLR9 and TLR7, Interestingly, we observed a defect in Ifnb induction in Lrrk2 KO BMDMs stimulated with poly(I:C), we no in the ability of Lrrk2 KO BMDMs to type I following treatment with LPS, CL097, or CpG (Figure and Figure 2—figure supplement suggesting that responses are in the of LRRK2 but cytosolic DNA and RNA sensing pathways are We observed for and MEFs treated with LPS (Figure 2—figure supplement and poly(I:C) (Figure 2—figure supplement 1E). Lrrk2 KO BMDMs in ISG expression following IFN-β treatment directly with (Figure and Figure 2—figure supplement 1F). Lrrk2 KO BMDMs failed to induce ISG expression following IFN-β we that the elevated basal levels of type I IFN Lrrk2 KO macrophages from inducing a response at the level of To begin to this we wanted to see if IFN-β engagement with IFNAR could this and basal ISGs in Lrrk2 KO macrophages. Indeed, when HET and KO Lrrk2 BMDMs were treated with an IFN-β neutralizing was a in basal levels of Isg15 and Irf7 in Lrrk2 KO cells (Figure and Figure 2—figure supplement 1G). We next tested if loss of IFNAR signaling could the Lrrk2 KO phenotype by Lrrk2 KO mice to Ifnar KO mice. In double KO BMDMs, we also observed a significant in basal ISG levels (Figure and Figure 2—figure supplement These results provide evidence that the type I IFN is chronically in Lrrk2 KO macrophages. Increased basal type I IFN in Lrrk2 KO macrophages is on cytosolic DNA sensing through cGAS both IFN-β and loss of Ifnar normalized basal ISG expression in Lrrk2 KO macrophages, we that Lrrk2 to basal type I IFN expression of cytosolic RNA or DNA nucleic acid sensing pathways that are between and negative et al., To directly the of cGAS in generating elevated resting levels of type I IFN in Lrrk2 KO macrophages, we Lrrk2 KO and KO mice and compared type I IFN transcript levels in double KO BMDMs with of littermate controls. basal Isg15 expression differences between Lrrk2 KO and HET BMDMs were more modest in this loss of cGAS significantly reduced basal ISG expression in Lrrk2 KO BMDMs (Figure and Figure supplement 1A). resting type I IFN double were to respond to innate immune stimuli like which cGAS and stimulates STING directly et al., and poly(I:C) transfection (Figure and Figure supplement 1A). Consistent with the ability of to Lrrk2 KO baseline and induction western blot analysis showed that levels of STAT1 phosphorylation were in double (Figure Together, these results support a where high basal levels of type I IFN and ISGs in Lrrk2 KO macrophages are to chronic engagement of the cGAS-dependent DNA sensing pathway. Figure with 1 supplement see all Download asset Open asset mtDNA basal type I IFN expression in Lrrk2 KO macrophages. (A) Isg15 gene expression in Lrrk2 WT, Lrrk2 KO, KO, and double KO DKO) BMDMs treated with μg/ml DMXAA or transfected with 1 poly(I:C) for 4 hr. (B) Western blot analysis of STAT1 phosphorylation (Tyr701) in BMDMs from WT, Lrrk2 KO, KO, and double knockout mice compared to total STAT1 with tubulin as a loading (C) with antibody to the mitochondrial network of Lrrk2 HET and KO = 10 (D) of total and to (E) As in (D) but cytosolic mitochondrial (F) Western blot of and protein levels in cytosol, and and of Lrrk2 KD and RAW 264.7 cells. (G) Irf7 gene expression normalized to Actb in BMDMs from Lrrk2 WT, Lrrk2 KO, and Lrrk2 HET mice. (H) of normalized to to mtDNA in WT and Lrrk2 KO RAW 264.7 cells treated with 10 μM for 4 (I) RT-qPCR of Ifnb gene expression in WT and Lrrk2 KO RAW 264.7 cells with or without and at 4 hr with 1 μg/ml Statistical analysis: *p<0.05, p<0.01, *p<0.005, ****p<0.001 (comparing indicated data points); ##p<0.001 (comparing stimulated to unstimulated of same genotype). and Tukey and two-tailed Student's T and two-way ANOVA Tukey post-test. sensing of mtDNA to basal type I IFN expression in Lrrk2 KO macrophages We next to the of the chronic DNA has been shown to be a potent activator of type I downstream of cGAS (West et al., 2015), and LRRK2 is known to influence mitochondrial homeostasis et al., through that are not To begin mtDNA in the dysregulation of type I in Lrrk2 KO cells, we first the of the mitochondrial network in Lrrk2 HET and KO As previously for cells or of LRRK2 et al., 2014), Lrrk2 KO MEFs had a more mitochondrial the cell as by (Figure We that this was a of mitochondrial that could mitochondrial including to into the we the cytosolic of control and Lrrk2 KD RAW 264.7 macrophages (Figure and Lrrk2 HET and KO MEFs (Figure supplement and measured cytosolic mtDNA We found that although Lrrk2 KD cells had higher total mtDNA compared to controls (Figure they had more cytosolic mtDNA (Figure phenotype was with Lrrk2 KO MEFs (Figure supplement 1B-C). This in cytosolic mtDNA was not an of cytosolic as an abundant mitochondrial a mitochondrial outer membrane were in the cytosolic by western blot (Figure We that cytosolic mtDNA results in activation of signaling, which the ability of Lrrk2 KO macrophages to respond to cytosolic nucleic acid agonists by canonical IFNAR signaling with a in STAT1 phosphorylation (Figure To the mitochondrial we Lrrk2 KO mice with HET mice. HET mice are deficient in the mitochondrial for maintaining the mtDNA network and have high levels of cytosolic mtDNA et al., et al., in Lrrk2 KO BMDMs to even higher basal ISG expression (Figure suggesting that release of mtDNA into the cytosol in Lrrk2 KO cells to their elevated type I IFN We next to type I IFN defects in Lrrk2 KO macrophages by mtDNA using an of mtDNA and Lrrk2 KO RAW 264.7 cells with reduced mtDNA number (Figure and in basal expression of type I IFN and ISGs in resting Lrrk2 HET and KO cells (Figure and Figure supplement 1D). Importantly, when Lrrk2 KO RAW 264.7 macrophages were stimulated with their ability to induce Ifnb was to that of Lrrk2 KO macrophages induced Ifnb in the of but following treatment WT macrophages induced Ifnb between treatment (Figure and Figure supplement These results demonstrate a critical role for mtDNA in both the high basal levels of type I IFN and the to induce type I IFN expression in Lrrk2 KO macrophages. in type I IFN responses in Lrrk2 KO macrophages are in to increased DRP1 phosphorylation and mitochondrial Previous studies of microglia have shown that LRRK2 to mitochondrial homeostasis through with the mitochondrial fission protein DRP1 et al., 2018). we that the loss of LRRK2 may mitochondrial via of DRP1 leading to and of mtDNA into the To defects in DRP1 in the of LRRK2, we performed and did not observe qualitative to the of DRP1 at the of mitochondrial in Lrrk2 KO although revealed of the peripheral network (Figure supplement 1A). We next asked whether DRP1 was impacted by loss of LRRK2. DRP1 is known to be via phosphorylation at et al., we performed with an antibody specific for DRP1 and observed significantly higher levels of DRP1 in Lrrk2 KD RAW 264.7 cells, Lrrk2 KO BMDMs, and Lrrk2 KO MEFs compared to controls (Figure Western blot analysis of DRP1 confirmed a modest in Lrrk2 KD cells, total DRP1 protein levels (Figure and Figure supplement 1B). of