MRL/lpr mice develop behavioral deficits and microglial reactivation before the appearance of overt peripheral SLE lesions.
To investigate the CNS disease process in lupus, we first monitored brain and systemic disease development between wildtype (WT) control (MRL/mpj) and lupus-prone MRL/MpJ-Faslpr (hereafter named MRL/lpr) mice (which have an insertion of a retrotransposon in the gene for Fas (CD95) resulting in defective apoptosis of lymphocytes and massive lymphoproliferation30), the best established spontaneous model of SLE and extensively used in lupus-related NP studies31 because NP manifestations appear early in these mice30.
Consistent with previous reports14, we found that serum anti-dsDNA antibody (Ab, the typical serologic indicator of lupus) was evident by 8 weeks in MRL/lpr female mice and continued to increase over time, with lupus nephritis onset at 18-20 weeks (Supplementary Fig. 1a). At 6 weeks of age, anti-dsDNA Ab titers were similar in MRL/lpr and congenic WT mice (Supplementary Fig. 1b), suggesting that no SLE serologic lesion is present in MRL/lpr mice at 6 weeks of age and that the disease onset time is approximately 8 weeks of age, as reported14.
To evaluate neuropathology in lupus, we then carried out a battery of behavioral tests on 16-week-old WT and MRL/lpr mice, the time point when mice began developing mild lupus nephritis (evidenced by increased proteinuria, although the difference was not statistically significant, (Supplementary Fig. 1a). We found that the lupus mice showed anxiety-like phenotypes in the open field test (OFT), a widely used indicator of anxiety-like behavior by evaluating the tendency of mice to remain close to the walls and avoid open spaces (central zone) 32 , and elevated plus maze (EPM) test, which is based on the animals’ natural fear of heights and open spaces, as an additional measurement of anxiety-like behavior (Fig. 1b,c,d and Fig. 1e,f,g), cognitive defects indicated by performance in the novelty Y maze (Supplementary Fig. 1c,d), and increased depression-like phenotypes in the tail suspension test (TST) and forced swim test (FST) (Supplementary Fig. 1e,f). No motor or coordination defects were found in MRL/lpr mice (Supplementary Fig. 1g,h,i). These findings suggest that MRL/lpr mice develop CNS disease and are suitable for NPSLE studies.
In most patients, CNS lupus typically presents at an early phase during SLE2,12, suggesting that NP manifestations may be driven primarily by brain-intrinsic factors rather than as a complication of systemic-autoimmune-activation. To determine whether the brain injury is a preexisting systemic pathology, we further examined the changed behavioral phenotypes observed in 16-week-old lupus mice as early as 6 weeks of age when mice had not yet developed peripheral pathology (Supplementary Fig. 1a,b). As shown in the OFT, 6-week-old MRL/lpr mice were less inclined to explore the central area of the OFT chamber than the peripheral zone (Fig. 1b,c), and likewise, the distance in the center (%) was significantly decreased compared with age-paired control mice (Fig. 1b,d). In the EPM test, MRL/lpr mice showed decreased entries (Fig. 1e,f) and time (Fig. 1e,g) in the open arms of the EPM compared with WT animals. MRL/lpr mice also showed an increased immobility time in the TST but not FST at 6 weeks of age (Supplementary Fig. 1e,f) and no change in cognitive performance in the novelty Y maze (Supplementary Fig. 1c,d). These results indicate that the MRL/lpr mice develop distinct anxiety- and depression- like behaviors that predate peripheral lesions at 6 weeks of age, in contrast to the WT control strain.
Furthermore, a two-way repeated measures (RM) analysis of variance (ANOVA) (age by genotype) analyzing the distance in center revealed a main effect of genotype (F (1,43) =12.28; P = 0.0011, P < 0.01) and no main effect of age (F (1, 43) = 2.026; P = 0.1618, P > 0.05) in the OFT (Fig. 1d). Two-way RM ANOVA analyzing head in the open arm revealed a major effect of genotype (F (1,43) =20.05; P < 0.0001, P < 0.01) and a reduced effect of age (F(1,43) =3.895; P = 0.0549, P > 0.05) in the EPM test (Fig. 1f). Together, these results suggest that anxiety- and depression- like behaviors are major NP changes that occur early in lupus mice, and anxiety-like behaviors are more sensitive. We used this phenomenon as the indicator of NP manifestation in subsequent studies.
Increased anti-NMDAR antibody-mediated excitotoxicity can induce neuronal apoptosis in mice with severe lupus lesions27,33. TUNEL and Nissl’s staining did not detect ongoing apoptosis before 12 weeks of age (Supplementary Fig. 2a) or loss of Nissl’s+ neurons within the circuitry of the prefrontal cortex and hippocampus that mediate emotion and learning in MRL/lpr mice (Supplementary Fig. 2b). These findings suggest that apoptotic neuronal death is unlikely the cause of early behavioral abnormalities in SLE. Although peripheral inflammatory cells have also been reported as participants34, notably, the MRL/lpr strain showed no cellular infiltration in the brain (Supplementary Fig. 2c), suggesting that rather than infiltrating immune cells, CNS-resident nonneuronal cells affect the NPSLE process at this early stage.
Microglia, resident macrophages in brain, respond to local inflammation or CNS damage by becoming reactive, increasing phagocytic activity and inflammatory cytokine production35. To determine whether microglial activation participates in NPSLE development, we assessed the active state of microglia via cell morphology and density in MRL/lpr mouse brains during the early (6-8 weeks) and active (16-20 weeks) stages of SLE. As noted, in addition to increased IBA-1+ cell numbers (Fig. 1i), reactive microglia in MRL/lpr displayed ameboid morphology, as evidenced by increased cellular soma and decreased branching complexity of cytoplasmic processes (Fig. 1h and Supplementary Fig. 1m,o). Additionally, the coimmunostaining of IBA-1 with CD68, a lysosomal-localized indicator of microglial phagocytic activity36, confirmed the reactivation of microglia (Fig. 1i and Supplementary Fig. 1m). We found significantly higher frequencies of reactive microglia in the cortex and hippocampus in MRL/lpr mice at 6 weeks compared with WT mice, which were further exacerbated at 16 weeks (Fig. 1h,i,j and Supplementary Fig. 1n), with similar microglial density in the cerebellum and midbrain (Fig. 1j)
Moreover, GFAP+ astrocytes within the hippocampus displayed no change across strains (Supplementary Fig. 2d), indicating microglia as major players. We also noticed an increase in IBA-1+ intensity in the prefrontal cortex of pristane-induced lupus mice (Supplementary Fig. 1p), as well as increased anxiety-like performance in the OFT, as assessed by distance in the center (Supplementary Fig. 1q). These results were similar to those observed in MRL/lpr lupus-prone mice. Furthermore, relative to the observed rapid and progressive increase in IBA-1+ microglia intensity in the hippocampus beginning from the 6th postnatal week in MRL-lpr mice compared with age-paired WT mice (Fig. 1k,l), complement C3 deposition in the kidney of MRL-lpr mice was significantly increased at 16 weeks compared with age-paired WT mice (Fig. 1k,m).
Thus, the above data together indicate that NP abnormalities occur in the early stage of SLE, prior to peripheral pathology, and are closely related with the specific activation of microglia in brain.
Transcriptional profile reveals molecular changes in the hippocampus of MRL/lpr mice.
To determine the changes in the brain as reactive microglia and NP manifestations develop, we performed whole-transcriptome gene expression analysis of the hippocampus from MRL/lpr and WT mice at 6 weeks, when reactive microglia were detected (Fig. 1a, schematic).
The transcriptome microarray enabled the detection of the differential expression of 1,908 transcripts (cutoff of 1.5-fold change; P < 0.05; Fig. 2a and Supplementary Fig. 3a) and further filtering of 1,019 transcripts (873 upregulated, 146 downregulated in MRL/lpr; adjusted P < 0.05).
In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed signatures of cytokine-cytokine receptor interaction, antigen processing and presentation, activation of innate immune responses, and microglial proteins involved in ligand sensing and protein digestion, as well as of the complement and coagulation cascades. In detail, MRL/lpr mice showed enrichment of immune-activated pathways, including classes of microglial-mediated phagocytosis (KEGG terms ‘Phagosome’ and ‘Protein digestion and absorption’, Fig. 2b and Supplementary Fig. 3b) and the classical complement pathway (KEGG term ‘the complement and coagulation cascades’, Fig. 2c). In the above category, we identified genes associated with microglial-mediated phagocytosis (Cx3cr1, which encodes CX3CR1; Fcgr2b, which encodes FCGR2B; Thbs1, which encodes Thrombospondin1; Itga2, which encodes Integrin α2; Tlr2, which encodes TLR2; and Tap1, which encodes TAP1) and the classical complement pathway (C1qa, which encodes C1QA; C6, which encodes C6; C1ra, which encodes C1R; and Cfp, which encodes properdin), which were validated using quantitative PCR (qPCR) (Fig. 2d,e and Supplementary Fig. 3d). The microglial reactive phagocytosis was further confirmed by a dramatic induction of the immunofluorescence signal for CD68 in IBA-1+ cells in MRL/lpr hippocampus (Fig. 1i and Supplementary Fig. 1m). Although complement components contribute to peripheral tissue damage in SLE37, their expression and function within the brain during lupus have not been well investigated. In murine CNS development and many disorders, C1q and C3 are reported to increase and are required for synaptic pruning38. Immunoblotting confirmed the upregulation of C1q but not C3 protein in both the cortex and hippocampus of MRL/lpr mice (Fig. 2f and Supplementary Fig. 3e). Conversely, MRL/lpr mice expressed lower percentages of genes that regulate neuroactive ligand-receptor interaction, the calcium signaling pathway, long-term depression, axon guidance, and synapse maintenance (Supplementary Fig. 3c and Supplementary information, Table S1), suggesting that synaptic dysfunction may also be involved.
Collectively, these data suggest a link between NP manifestations and increased microglial phagocytosis, as well as complement machinery, in the brain during SLE progression.
The aforementioned specific increase in reactive microglia in lupus mouse brains together with RNA-seq indicated elevation of phagocytic genes encouraged us to study whether microglial phagocytic activation is required for the NPSLE process. For this purpose, we treated MRL/lpr mice with minocycline, a BBB-permeable phagocytic activity inhibitor that can suppress microglial activation initiated at 5 weeks of age (shortly before reactive microglia were detected) (Supplementary Fig. 4a). Then, behavioral and biological analyses were performed at week 8. As expected, minocycline treatment significantly reduced both the IBA1+ microglial number and suppressed its phagocytic activation, as evidenced by reduced IBA1+/CD68+ fluorescence intensity in the hippocampus (Supplementary Fig. 4b,c), without affecting GFAP+ astrocytes and NeuN+ neurons (Supplementary Fig. 4d,e) and C1q intensity (Supplementary Fig. 4f). Notably, minocycline ameliorated the anxiety-like behaviors of MRL/lpr mice in both the OFT (Supplementary Fig. 4g,h) and the EPM test (Supplementary Fig. 4i,j).
To test whether the observed effect of minocycline was due to possible antimicrobial effects rather than suppressed phagocyte activation, we similarly treated a group of MRL/lpr mice with the broad-spectrum antibiotic amoxicillin-clavulanate40 (Supplementary Fig. 5a). However, amoxicillin- clavulanate treatment did not protect lupus mice from brain disease (Supplementary Fig. 5b,c,d). Collectively, these results suggest that increased phagocytosis reactivation of brain microglia is a key mediator of anxiety-like behaviors in MRL/lpr mice.
Synapse loss due to microglial engulfment accounts for early behavioral defects in MRL/lpr mice
Neuron loss can contribute to emotion and cognitive dysfunction4. However, MRL/lpr mice displayed no differences in neuron numbers throughout the hippocampus and prefrontal cortices compared with WT controls at 8 weeks of age (Supplementary Fig. 2b). Sequence analysis detected alterations in synaptic activity-related genes (Supplementary Fig. 3c), which encouraged us to speculate that synaptic defects might be involved, reflecting an earlier subtle change that predates neuronal apoptosis. For confirmation, we quantified synaptic terminals and found that the numbers of synaptic puncta within CA3 (mossy fiber terminals) were decreased at 6 weeks in MRL/lpr mice compared with WT controls (Fig. 3a,b). Respectively, the decrease was traced to a 50% reduction in the number of postsynaptic terminals, with a lower change in the number of presynaptic terminals (Fig. 3a,b and Supplementary Fig. 6a). In diseased MRL/lpr mice, synapse loss occurred predominantly at excitatory (VGLUT1+) presynaptic terminals (Fig. 3c,d), but not at inhibitory presynaptic (VGAT1+) and postsynaptic (Gephyrin+) terminals (Supplementary Fig. 6d,e). Nevertheless, the densities of NeuN (Supplementary Fig. 6b) and phosphorylated neurofilament heavy chain (Supplementary Fig. 6c) within CA3 remained unchanged, indicating that both neurons and axons were preserved, rendering neuro-axonal degeneration an unlikely explanation for the observed loss of synaptic input. We further quantified excitatory synaptic element numbers in the dentate gyrus (DG) granule neurons of Golgi-stained tissue. As observed, dendritic spine numbers were reduced in MRL/lpr mice (Fig. 3h,i), consistent with the immunostaining results.
Synapse pruning by microglia is involved in brain development38 and neurodegenerative processes26,41. We wondered if reactive microglia mediated synapse elimination via a similar process in our model. As noted, we found abundant IBA-1+ phagocytes in close apposition to or even enwrapping neurons in diseased MRL/lpr brains, whereas such phagocytic behavior was relatively rare in the controls (Fig. 3e). Moreover, the juxtaposition of microglia and neurons was associated with a loss of presynaptic SYP+ terminals in MRL/lpr mice but not WT controls (Fig. 3f) in the same cohort of mice. Furthermore, confocal microscopy showed reductions in SYP+ (Fig. 3g), especially excitatory VGLUT+ terminals (Fig. 3c), consistent with the Golgi staining results (Fig. 3h,i). Ultrastructure analysis also revealed displacement of synapses encapsulated by phagocytes (Fig. 3j and Supplementary information Fig, 6f). This evidence collectively indicated active roles of these “phagocytes” in synapse elimination.
To investigate the functional correlates of disturbed synaptic terminals, we performed electrophysiological analysis in acute slices of the hippocampus from MRL/lpr and WT mice. The input/output recordings revealed a significant reduction in basal synaptic activity (Supplementary Fig. 6g) in the lupus group. Long-term potentiation (LTP) indicated that excitatory postsynaptic potentials (EPSPs) were also significantly reduced in MRL/lpr slices compared with WT (Supplementary Fig. 6h), indicating that compromised excitatory synaptic transmission occurred in lupus brain, consistent with the reduced excitatory presynaptic and postsynaptic terminals in the hippocampus.
Together, these results suggest that microglial phagocytosis is a key mediator of the synaptic loss involved in early NP abnormalities in MRL/lpr mice.
Increased complement C1q accumulate at synapses in MRL/lpr mice.
The translatome analyses combined with immunoblotting revealed a much higher abundance of C1q protein in lupus hippocampus. During the NPSLE process, the C1q signal was already prominent in the hippocampus at 6 weeks (Fig. 2f), especially in the DG and CA1/3 regions, in MRL/lpr but not WT mice (Supplementary Fig. 7a). However, unlike microglial activation, there was no significant progressive increase in C1q burden from 6 to 16 weeks of age (Supplementary Fig. 7b). A costaining experiment showed that increased C1q mostly collocated with neurons (Fig.4b) and only slightly with microglia (Fig. 4a) in MRL/lpr hippocampus. In the periphery, classical complement C1q could function as a tag, labeling damaged cells for chemotactic phagocytes42. We thus hypothesized that elevated brain C1q might act as a marker to tag synapses for microglial pruning. Notably, further costaining of C1q and a synaptic marker (synapsin) revealed a significant proportion of C1q immunoreactive puncta located at synapses (Fig. 4c) in lupus brain, as evidenced by a much higher percentage of C1q-labeled synapses in the hippocampal CA3 of MRL/lpr brains (23.6% ± 3.9%) than WT brains (8.2% ± 0.9%) at 6 weeks of age (Fig. 4d).
We also performed Western blotting of C1q in purified PSD fractions versus total lysates in a different cohort of mice at 6 weeks of age and confirmed that C1q was greatly increased (although with interanimal variability) in the PSDs of MRL/lpr hippocampi and barely detectable in the WT PSDs (Fig. 4e,f). These biochemical data suggest that C1q accumulates at synapses in the MRL/lpr brain at or even before overt neurodegeneration has taken place. Importantly, antibodies against synaptophysin+ (SYP+, corresponding to presynaptic terminals) and PSD-95 coimmunoprecipitated C1q from lupus mouse hippocampal lysates at a higher level with PSD-95 (Fig. 4g), suggesting that C1q is predominantly associated with excitatory synapses, especially in the postsynaptic PSD-95 complex. This finding is consistent with observations that the decreased synapses in the MRL/lpr brain were primary excitatory ones (Fig. 3b,c). Consistent with the increased accumulation of C1q in the PSD of MRL/lpr mice, there was a strong positive correlation between the amount of C1q and the decreased center movement in the OFT (Supplementary Fig. 7c).
Although activated microglia have been reported to be a major source of C1q in inflammatory brains38, the inhibition of microglial reactivation by minocycline did not reverse the C1q burden in MRL/lpr mice (Supplementary information, Fig.S4f, 7d), although it reduced the increase in C1q mRNA level in the hippocampus (Supplementary Fig. 7e), indicating that microglia may not be the major source of increased brain C1q protein, rendering the source of C1q a question in the lupus model.
C1q, as a highly abundant serum protein normally produced by many tissues and normally found in serum, is predominantly located in the periphery. During the embryonic period, aging, and neurodegenerative diseases (NDDs), increased brain C1q can be generated by brain-resident cells, such as activated microglia and insulted neurons. However, in autoimmunity conditions, many peripheral activated inflammatory cells can also generate abundant C1q. By immunoblotting C1q in brain, serum, and peripheral tissues of WT and MRL/lpr mice, we found that C1q levels were much higher in the serum and kidney than in the brain (Supplementary Fig. 7f). To further clarify whether increased brain C1q might source from serum, both WT and MRL/lpr mice were intravenously injected with a purified C1q (1 μg/ml). The injected C1q signal in the brain was detected within three hours, as expected due to some permeability of the BBB to C1q, despite intact BBB integrity in MRL/lpr mice at 6 weeks (Supplementary Fig. 7g,h). For additional confirmation, we used a parabiosis mouse model, which has been used to examine circulatory factors that are transferred from one animal to another (Supplementary Fig. 7i)43, to show that connecting WT mice with MRL/lpr or WT littermates changed the C1q amount in the circulation of WT recipients 2 weeks after surgical connection. As expected, the hippocampal homogenate from WT mice in parabiosis with MRL/lpr mice exhibited increased C1q, whereas that from mice in parabiosis with WT controls did not (Supplementary Fig. 7j). Taken together, although the RNA-seq array show that increased C1q transcription in MRL/lpr hippocampus may be due to activated microglia or unfit neurons, however, the observed significant increase in C1q protein in the MRL/lpr mouse brain may originate at least partially from serum.
Although increased brain C1q levels have recently been reported in murine models of viral encephalitis25, frontotemporal dementia36, and Alzheimer’s disease24, they have rarely been reported in lupus. For confirmation, we further observed the PSD level of C1q in the pristane-induced SLE mice (PBS treatment as a control) at 4 months after induction when the mice developed behavioral symptoms without significant renal pathology. The average level of C1q in the PSDs of pristane-treated mice was significantly elevated compared with the controls, although it was lower than in the PSDs of MRL/lpr mice (Fig. 4h). Thus, at least in these mouse models, increased synaptic located C1q seems to function as a common pathologic molecule involved in brain damage during lupus development.
C1q tagging at synapses contribute to microglial synaptic pruning-mediated dendritic spine loss, and C1q-blocking approaches protect synaptic pruning and mitigate anxiety-like behaviors in MRL/lpr mice
Macrophage-mediated phagocytosis in the periphery often requires antibody and complement deposition; however, no differences in the amount of endogenous mouse IgG coating with VGLUT-1- or PSD95-positive staining in CA3 synaptic terminals in 6-week-old MRL/lpr mice were observed compared to the controls (Supplementary Fig. 7k,l). Notably, in addition to increased PSD accumulation, C1q also colocalized within cells with a neuronal morphology and with MAP2-positive neurites (Fig. 5a) in the hippocampus, confirming the idea that C1q rather than IgG may instruct phagocytes to engulf synapses in the early stage of NPSLE. Moreover, C1q protein was more frequently detected in synaptophysin+ (SYP+) perisomatic synaptic boutons surrounded by or adjacent to IBA-1+ cellular processes in MRL/lpr mice, in conjunction with a reactivated IBA-1+ morphology and reduced SYP+ presynaptic terminals (Fig. 5b).
To further examine the relation between C1q, synaptic loss, and microglial activation, we constructed a series of in vitro studies. First, we observed that exogenous C1q colocalized with PSD-95 in neuronal cultures derived from E17 MRL/lpr mice (Fig. 5c). Although C1q has been reported to be involved in the onset of many NDDs and as a classical complement signal, C1q also functions as an initiator to mediate cell death44. However, directly adding C1q to neuronal cultures failed to disturb neuron survival, dendrites, and synapses, even at high concentrations (Supplementary Fig. 8a), suggesting that deposited C1q did not directly affect the integrity and survival of neurons in culture.
A previous study demonstrated that C1q inhibits the proinflammatory effects of HMGB1 on monocytes45. To test whether C1q affected microglial activation, we incubated cultures of primary microglia isolated from WT neonatal mouse brains with or without C1q and assessed the downstream cytokine production, activation markers, and cell morphology. Cultures were performed in serum-free medium to avoid C1q contamination in the serum and to permit accurate control of the concentration of C1q. As anticipated, unlike LPS (a TLR agonist, classic microglia activator) and IFNα (a reported microglia activator in lupus mice), C1q did not alter the transcription of IFN-inducible genes or NF-κB-dependent proinflammatory cytokines at the tested doses (0-50 μg/mL C1q) (Supplementary Fig. 8c) and failed to change the phagocytic activity compared to IFNα and LPS (Supplementary Fig. 8d,e). These observations suggested that C1q alone did not affect the active state of microglia in culture. Thus, both increased C1q and reactivated microglia may be simultaneously required for synaptic elimination.
For confirmation, we next conducted a microglia-neuron coculture system in which primary MRL/lpr mouse hippocampal neurons were cultured. One week later, they were cocultured with latent and LPS prime-induced reactive microglia (3:1, neuron: microglia ratio) for another 3 days (Fig. 5d). Indeed, dendrites of neurons cultured with both exogenous C1q and primed (reactive) but not latent microglia displayed reduced synapse density, as measured by PSD-95 clusters (Fig. 5e,f). Furthermore, when primed microglia were cocultured in a transwell insert, the nondirect-contactable coculture failed to induce C1q-microglia-axis-mediated synapse loss (Supplementary Fig. 8b,f), though the neurite length was slightly shortened. These findings highlighted the activation and close microglial-neuron interactions rather than soluble neurotoxic factors as key factors in microglia’s orchestration of synaptic removal.
Given these findings, we hypothesized that the C1q-dependent guidance of reactive microglia to engulf synapses accounted for the synapse loss in NPSLE. Accordingly, we questioned whether inhibition of C1q could prevent synapse removal by microglia. To test this hypothesis, we used a C1q-blocking antibody, which potently blocked C1q binding to cultured neurons. We first tested whether the anti-C1q antibody could decrease microglial synapse engulfment in vitro. C1q-blocking antibody or isotype control was added concurrently with microglia to the neuron culture (Fig. 5d), and we found that C1q-blocking antibodies, but not the isotype control, prevented the loss of PSD-95 puncta along microglia-proximal dendrites (Fig. 5e,f).
To determine whether C1q-blocking antibodies could prevent synapse engulfment by microglia and rescue synapse loss in vivo, we injected C1q-blocking or isotype control antibodies into the bilateral hippocampus of 5-week-old MRL/lpr mice and analyzed the animals 3 weeks later. Remarkably, we measured a slight but significant rescue of synapsin puncta density in the CA3 region of MRL/lpr mice at 3 weeks after injection of C1q-blocking antibody relative to control IgG by both transmission electron microscopy (TEM) (Fig. 5g,h) and confocal imaging (Fig. 5i). C1q blocking also alleviated the anxiety-like behaviors of MRL/lpr mice (Fig. 5j,k) without affecting IBA-1+ microglia or CD68+ phagocyte intensity in the hippocampus (Supplementary Fig. 8g,h).
The neuronal Nr4a1 defect is an endogenous signal that is critical for the synaptic location of C1q in MRL-lpr mice.
We next investigated the molecular mechanism that guides C1q for synapse tagging. Complement C3 cleaved by C1q into the activated cleavage product C3d has been reported to be involved in phagocyte recruitment in the CNS23,25, and numerous mechanisms have been reported to participate in C1q spinal guidance, including HMGB1 (high mobility group box 1)46, small GTPase-regulating proteins24, and altered neuronal action potential47.
To investigate this phenomenon, we first compared C3 levels. Unexpectedly, no significant changes were found in either total C3 protein or cleavage products in lupus mice at 6 weeks of age or when NPSLE developed (Supplementary Fig. 9a), indicating some other underlying mechanisms. The hippocampus microarray revealed a cluster of reduced genes in the “Nr4a1 related signal transduction” pathway, but not others (Fig. 6a). Nr4a1 belongs to a family of three immediate-early genes that encode three orphan nuclear receptors (Nr4a1, Nr4a2, and Nr4a3)48. In the CNS, Nr4a1/2/3 expression is controlled by NMDARs, CREB, and MEF248, which are key regulators of synaptic function. We confirmed the decrease in Nr4a1 in the prefrontal cortex and hippocampus of 6-week-old MRL/lpr mice compared with WT littermates (Fig. 6b) as well as in both derived primary neurons (Fig. 6c) by q-PCR.
A protein-protein interaction analysis revealed that NR4A1, MEF2D, and TMOD3 act in a highly interconnected network, regulating the cellular response to endogenous stimuli, which in turn controls protein localization (Supplementary Fig. 9b), supporting the idea thatNr4a1 may be related to C1q’s synaptic location. Moreover, Nr4a1 expression is also maintained by synaptic activation48,49, and Nr4a1 loss-of-function causes deficits in L-LTP and long-term memory formation50,51 as well as dendritic spine loss49. Thus, we reasoned that reduced Nr4a1 might be an endogenous signal that reflects weakened neuronal activity and functions as a bridge connecting C1q to defective synapses, as C1q can recognize nonrobust synapses directly linked by local apoptotic-like processes in the synaptic compartment52. We confirmed this phenomenon by incubating neuronal cultures with AP5 (to inactivate NMDARs specifically), which resulted in decreased Nr4a1 expression in a time- and dose-dependent manner (Supplementary Fig. 9c). Then, to examine how depletion of the NR4A1 proteins affects dendritic spines, we further used LV-shRNA-mediated knockdown in cultured hippocampal neurons (Supplementary Fig. 9d) and found that exogenous C1q bound to dendrites (colocalized with PSD-95+ dendrites) of neurons preincubated with shNr4a1 (Fig. 6d) as well as neurons treated with AP5 but not untreated controls (Supplementary Fig. 9e). Moreover, neurons incubated with shNr4a1 showed a 38–46% decrease in dendritic spine density compared with the control, with no effect on dendritic spine density, when cocultured with both primed microglia and exogenous C1q (Fig. 6e,f). Therefore, this study presents a model in which reduced neuronal Nr4a1 activity, an intrinsic signal, serves as a bridge to actively direct C1q and then microglial recruitment to conduct spine pruning.
NR4A1 controls synapse density partially by disturbing the actin cytoskeleton, and dysregulation of the postsynaptic actin network contributes to the loss of dendritic spines in Tau-P301S mice24. Thus, we hypothesized that Nr4a1 knockdown could encourage the recognition of dysregulation in the synaptic cytoskeleton by C1q and investigated whether the pharmacological stabilization of filamentous actin (F-actin) would stabilize and prevent dendritic spine elimination. Neurons that had been treated with shNr4a1 for 3 days in vitro (DIV 7+3) were incubated with fresh shRNAs and the F-actin-stabilizing agent phallacidin for another 2 days. Phallacidin had no effect on dendritic spines in control cultures (data not shown) but partially restored the spine density in neurons treated with shRNAs targeting Nr4a1 (Fig. 6e,f). However, simple disassembly of F-actin seemed insufficient to explain the effect of low Nr4a1 expression because the actin-stabilizing drug phallacidin did not achieve absolute restoration of spines. Indeed, many other mechanisms, either regulated by Nra41 (e.g., BDNF, NMDAR activity) or not regulated by Nr4a1 (astrocyte-derived molecules) have been reported to be involved in microglial synaptic loss regulation53.
To probe the state of synaptic actin polymerization in vivo, we stained F-actin and the postsynaptic marker PSD-95 in the hippocampal CA3 region in WT and MRL/lpr mice. The fluorescence intensity of the F-actin signal that colocalized with PSD-95 clusters was reduced by 36% in MRL/lpr versus WT mice (Fig. 6g,h).
Together, these results highlight that neuronal Nr4a1 is decreased in MRL/lpr mice, which can lead to dysregulation of the synaptic actin cytoskeleton and then guide C1q and microglia, synergistically contributing to the dendritic spine elimination observed in NPSLE.
To determine whether restoring neuronal NR4A1 expression could prevent synapse engulfment by microglia and rescue synapse loss in vivo, we injected an Nr4a1 overexpression lentivirus or vector control into the bilateral hippocampus of 5-week-old MRL/lpr mice and analyzed the animals 3 weeks later (Fig. 7a). The injected Nr4a1-GFP lentivirus was expressed in neurons throughout the hippocampus (Supplementary Fig. 10a). The Nr4a1 lentivirus resulted in a higher protein level than the control injection, confirming the specificity and effectiveness of the Nr4a1 lentivirus (Supplementary Fig. 10b). In brains injected with LV-Nr4a1, the total C1q staining intensity was not affected, but neuron-located C1q was reduced in the CA1 region (Supplementary Fig. 10c,d,e), and a significant reduction was observed in PSD-95 or synapsin puncta within microglial lysosomes compared with LV-Ctl (Fig. 7b,c), suggesting that neuronal Nr4a1 transcription could blunt C1q tagging and consequently synapse engulfment in MRL/lpr brains.
To investigate the role of NRA41 in synaptic plasticity, the input/output curves and LTP were examined in hippocampal slices. Rescued NRA41 expression partially restored the abnormal basal synaptic activity measured by the I/O amplitude in MRL/lpr mice (Fig. 7d). Compared with the control, LV-Nr4a1-treated mice showed enhanced induction of LTP in Schaffer collateral-CA1, although the maintenance (later population spike LTP) was not fully restored at 3 weeks after Nr4a1 construct injection (Fig. 7e). Restored NR4A1 expression also improved the OFT performance of MRL/lpr mice (Fig. 7f,g), though the increase in center movement distance of LV-Nr4a1-injected mice did not reach a significant level compared with the control (Fig. 7h). In summary, our results demonstrate that neuronal NR4A1 rescue can reduce synapse removal by the C1q- microglial axis and lead to a recovery of synapse density and circuit function in vivo (Fig. 8).