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Aberrant activation of hippocampal astrocytes causes neuroinflammation and cognitive decline in mice [1]

['Jae-Hong Kim', 'Department Of Pharmacology', 'School Of Medicine', 'Kyungpook National University', 'Daegu', 'Republic Of Korea', 'Brain Science', 'Engineering Institute', 'Brain Korea Four Knu Convergence Educational Program Of Biomedical Sciences For Creative Future Talents', 'Nakamura Michiko']

Date: 2024-07

Reactive astrocytes are associated with neuroinflammation and cognitive decline in diverse neuropathologies; however, the underlying mechanisms are unclear. We used optogenetic and chemogenetic tools to identify the crucial roles of the hippocampal CA1 astrocytes in cognitive decline. Our results showed that repeated optogenetic stimulation of the hippocampal CA1 astrocytes induced cognitive impairment in mice and decreased synaptic long-term potentiation (LTP), which was accompanied by the appearance of inflammatory astrocytes. Mechanistic studies conducted using knockout animal models and hippocampal neuronal cultures showed that lipocalin-2 (LCN2), derived from reactive astrocytes, mediated neuroinflammation and induced cognitive impairment by decreasing the LTP through the reduction of neuronal NMDA receptors. Sustained chemogenetic stimulation of hippocampal astrocytes provided similar results. Conversely, these phenomena were attenuated by a metabolic inhibitor of astrocytes. Fiber photometry using GCaMP revealed a high level of hippocampal astrocyte activation in the neuroinflammation model. Our findings suggest that reactive astrocytes in the hippocampus are sufficient and required to induce cognitive decline through LCN2 release and synaptic modulation. This abnormal glial–neuron interaction may contribute to the pathogenesis of cognitive disturbances in neuroinflammation-associated brain conditions.

To elucidate the disease mechanisms characterized by inappropriate astrocyte reactivity, it is important to understand the relationship between astrocytic dysfunction, particularly prolonged signaling, and neuronal function. To address this issue, we employed repeated optogenetic stimulation and chemogenetic strategies with a combination of electrophysiology and behavioral assessments to better understand the role of the CA1 astrocytes and effects of aberrant activation in the modulation of hippocampal synaptic activity and cognitive decline.

It was determined that astrocytes are integral mediators of cognitive impairment [ 27 , 28 ]. Considering the important role of astrocytes in memory impairment, it is possible that physiologically expressed proinflammatory cytokines are involved in memory formation [ 29 – 33 ], but under pathological conditions, excessive cytokine release may lead to overactivation of astrocytes, thereby resulting in neuronal injury and eventual cognitive decline [ 34 , 35 ]. We have previously identified lipocalin-2 (LCN2) as a mediator of reactive astrocytosis [ 36 – 39 ]. Other studies have also implicated LCN2 in neurodegenerative and cognitive disorders displaying neuronal loss, alterations in astrocytes, neuroinflammatory responses, and synaptic and network dysfunction [ 40 , 41 ]. However, a definitive link between LCN2, sustained neuroinflammation in the hippocampus, and cognitive impairment has not yet been established, and it remains unclear whether hippocampal inflammation persists along with chronic reactive astrocytosis. Moreover, LCN2 has been previously reported to exert both anti-inflammatory [ 42 – 44 ] and proinflammatory [ 45 – 60 ] effector functions in different contexts. Whether LCN2 is neurotoxic or neuroprotective is the subject of controversy.

A number of previous studies have demonstrated that nonneuronal cells, mainly microglia and astrocytes, are involved in the pathogenesis of Alzheimer’s disease (AD) as well as other neurodegenerative conditions and disorders [ 20 – 22 ]. Neuroinflammation is a common feature of diverse nervous system pathologies. In many instances, it begins at the early stage of the disease, which lays the foundations for further exacerbation. The main drivers of neuroinflammation are brain-resident glial cells, such as microglia and astrocytes [ 23 ]. Microglia are the primary responders to any insult to the brain parenchyma, translating the signals into diverse molecules. These microglia-derived molecules regulate the stimuli-dependent reactivity of astrocytes. Once activated, astrocytes can control the microglia phenotype [ 24 ]. Recent evidence indicates that the crosstalk between these glial cells plays an important role in delaying or accelerating neuroinflammation and the overall disease progression [ 24 – 26 ].

Numerous studies have previously shown the importance of astrocytes in memory, demonstrating that the disruption of astrocyte function resulted in memory impairment [ 9 – 12 ] and that memory impairment in genetic models of cognitive deficit can be reduced by correcting the genotype of astrocytes [ 13 , 14 ]. Moreover, a recent study demonstrated that activation of the Gq-coupled pathway in astrocytes enhanced the memory of mice [ 15 ]. In contrast, a more recent study by Li and colleagues demonstrated that the optogenetic stimulation of hippocampal CA1 astrocytes in a transgenic rat model attenuated the contextual fear memory via adenosine A 1 receptors [ 16 ]. Furthermore, other studies have suggested that memory function may be detrimentally affected by certain astrocytic intracellular pathways [ 17 – 19 ]. Interestingly, these seemingly contradictory results provide the true representation in that the effect of astrocytic activity on memory is reflected as an inverted “U-shaped” function in which an optimal level of astrocytic activity is critical to support intact memory, but either a deficit or excess of activity may be detrimental.

The hippocampus has a vital role in the establishment and retention of learning and memory. Processing of brain information is traditionally perceived as a neuronal function. In general, it is believed that astrocytes primarily have supportive functions for neurons in the central nervous system (CNS) [ 1 ], but increasing evidence suggests that astrocytes exert several additional active functions, including signal transmission, information processing, and regulation of neural and synaptic plasticity [ 2 – 4 ]. Recent studies have also linked astrocytes with various behavioral states and brain pathologies in different animal models and presented evidence that cognitive processing, including learning and memory, requires coordinated interplay between astrocytes and different synaptic ensembles [ 5 – 8 ]. Therefore, the coordinated actions of a glial–neuron network may underlie many brain functions, including cognition. However, there is limited information regarding the physiological contribution of astrocytes to cognitive function and underlying mechanisms.

Results

Optogenetic stimulation of CA1 astrocytes induces LCN2 release in the hippocampus To explore whether optogenetic stimulation triggers LCN2 release in the hippocampal CA1 region, we collected interstitial fluid from this region by microdialysis and subjected it to LCN2 ELISA analysis (Fig 3A). Microdialysis samples were collected from the hippocampus during 3 days of photostimulation of AAV-ChR2-eYFP-injected mice. After photostimulation for 20 min, the dialysate collected on day 3 showed significantly increased levels of LCN2 and cytokine (Fig 3B). We determined the cellular localization of LCN2 expression in the hippocampus after optogenetic stimulation by immunostaining for LCN2 and GFAP in the brain sections collected after photostimulation. We detected the colocalization of LCN2 protein with GFAP-positive astrocytes (Fig 3C). These results indicate that stimulated astrocytes release LCN2 and proinflammatory cytokines in the hippocampal CA1 region, concurrent with the induction of neuroinflammation and cognitive impairment. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Optogenetic stimulation of hippocampal astrocytes induces extracellular LCN2 and cytokine release. (A) Experimental timeline. (B) Visual representation of in vivo microdialysis and subsequent ELISA. LCN2, IL-1β, and TNF-α levels in dialysate measured by ELISA. Results are expressed as mean ± SEM (n = 4). *p < 0.05 between the indicated groups; n.s., not significant (one-way ANOVA). (C) Brain tissue samples were subjected to immunofluorescence analysis to localize the expression of LCN2 (red), eYFP (green), and ChR2-eYFP (green) in astrocytes (GFAP, white). The nuclei were stained with DAPI (blue). Arrowheads (yellow) indicate the colocalization of LCN2, ChR2-eYFP, and GFAP. The quantification of colocalization is shown in the adjacent graph. Scale bar: 100 μm. Results are expressed as mean ± SEM (n = 4). *p < 0.05 between the indicated groups (one-way ANOVA). n.d., not detected. Source data can be found in S1 Data. ELISA, enzyme-linked immunosorbent assay. https://doi.org/10.1371/journal.pbio.3002687.g003 Additional in vitro experiments were conducted using cultured astrocytes, which provided further evidence supporting the important role of astrocyte-derived LCN2 in hippocampal neuroinflammation. The production of Lcn2 and proinflammatory mediators in vivo after the optogenetic stimulation of hippocampal CA1 astrocytes was corroborated by the optogenetic experiments conducted using cultured astrocytes (S14A and S14B Fig). Optogenetic stimulation of the cultured astrocytes resulted in significantly elevated mRNA expression of Lcn2, Il1b, and Tnf (S14C Fig). LCN2 protein production was also significantly increased in the astrocyte culture media following optogenetic stimulation (S14D Fig). On the other hand, the 20-min photostimulation of the cultured astrocytes expressing ChR2 had no significant impact on the cell viability (S14E Fig).

Repeated optogenetic stimulation induces neuroinflammation and reactive astrocytes in the hippocampus Neuroinflammation is a common feature of virtually every CNS disease and is being increasingly recognized as a potential mediator of cognitive impairment [85]. After the AAV-mediated delivery, the expression of ChR2-eYFP in GFAP+ astrocytes was confirmed in the hippocampal sections of these animals (S18 Fig). After the optogenetic stimulation of these animals, we evaluated the glial activation and proinflammatory cytokine expression in the hippocampus (Fig 6A). Immunofluorescence analysis of the hippocampal CA1 region revealed a significant increase in the immunoreactivity of GFAP and Iba-1 in the hippocampal CA1 region of the ChR2-eYFP-expressing mice (Fig 6B). An increased intensity of GFAP immunoreactivity in the hippocampal CA1 region after the optogenetic stimulation was accompanied by morphological changes in astrocytes, reminiscent of reactive astrocytes. The repeated optogenetic stimulation increased the length, thickness, and number of branch in hippocampal astrocytes (Fig 6B). Additionally, we observed an increased number of primary processes leaving the soma in astrocytes from the ChR2-eYFP group compared to the eYFP group. Astrocytes in the ChR2-eYFP group exhibited a bushy morphology with many fine terminal processes protruding from the primary cellular processes, and the processes appeared thicker than those in the eYFP group (Fig 6B). Microglia also showed morphological changes from resting to activated states following the optogenetic stimulation of astrocytes (Fig 6B). We next compared the immunoreactivity of GFAP and Iba-1 in the CA1 versus dentate gyrus (DG) areas on day 3 after repeated optogenetic stimulation. Our data showed increased immunoreactivity of GFAP (S19A Fig) and Iba-1 (S19B Fig) in the hippocampal CA1 area, but not in the DG area. As expected, ChR2-eYFP expression was localized to the hippocampal CA1 area. The activation of astrocytes and microglia has been widely acknowledged to contribute to cognitive impairment through the release of proinflammatory mediators, including IL-1β and TNF-α [27,86–88]. To determine whether the optogenetic stimulation of the astrocytes influenced the expression of proinflammatory genes, the hippocampus was harvested from the eYFP or ChR2-eYFP-expressing mice on day 3 after photostimulation. We detected a significantly enhanced expression of Il1b and Tnf mRNA in the hippocampus of ChR2-eYFP-expressing mice compared with that of the eYFP-expressing control mice (Fig 6C). These results indicate that the repeated optogenetic stimulation of CA1 astrocytes induces neuroinflammation and reactive phenotype of astrocytes in the hippocampus. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Lcn2 deficiency attenuates neuroinflammation and cognitive impairment induced by optogenetic stimulation of hippocampal astrocytes. (A) Experimental timeline. (B) Astrocytes and microglia were identified using GFAP and Iba-1 immunolabeling after optogenetic stimulation. The adjacent graph displays the quantification of fluorescence intensity (astrocytes, left; microglia, middle) and percentage of microglia displaying resting and activated morphology (right) in the hippocampal CA1 region of WT and Lcn2-KO mice. In further morphological analysis, the total length and average thickness of astrocyte processes in each group were assessed. The total number of primary, intermediate, and terminal branches for each astrocyte was also measured and compared among experimental groups. Scale bar: 100 μm. Results are expressed as mean ± SEM (n = 5 or 6). *p < 0.05 between the indicated groups; n.s., not significant (one-way ANOVA). (C) Total mRNA was extracted from the hippocampal tissues of each group and subjected to qPCR to determine the expression levels of Il1b and Tnf mRNA. Gapdh was used as an internal control. Results are expressed as mean ± SEM (n = 6). *p < 0.05 between the indicated groups; n.s., not significant (one-way ANOVA). (D–F) Y-maze (n = 10) (D), Barnes maze (n = 5) (E), and passive avoidance (n = 10) (F) cognitive behavior of eYFP or ChR2-eYFP-expressing WT or Lcn2-KO mice after 20-min photostimulation. Results are expressed as mean ± SEM (n = 5 or 10). *p < 0.05 between the indicated groups; n.s., not significant (two-way ANOVA). Source data can be found in S1 Data. LCN2, lipocalin-2. https://doi.org/10.1371/journal.pbio.3002687.g006

Lcn2 deficiency ameliorates neuroinflammation and cognitive impairment after the optogenetic stimulation of hippocampal astrocytes Studies have demonstrated that LCN2 regulates the immune and inflammatory responses in a range of neurological diseases [38,40]. We have previously reported that LCN2 caused glial activation and increases the expression of inflammatory cytokines [38]. In the present study, we determined whether LCN2 was involved in neuroinflammation and cognitive decline after the repeated optogenetic stimulation of astrocytes, for which we first evaluated the hippocampal expression of LCN2 after optogenetic stimulation. Photostimulation for 20 min for 3 consecutive days resulted in a significant increase in the mRNA expression of Lcn2 in the hippocampus (S18C Fig). We also investigated whether Lcn2 deficiency affected glial activation and the subsequent production of proinflammatory cytokines in the hippocampus after photostimulation. As depicted in Fig 6B, the optogenetic stimulation-induced activation of hippocampal astrocytes and microglia was diminished in Lcn2-KO mice. In further morphological analysis of astrocytes, Lcn2 deficiency attenuated the optogenetically enhanced length, thickness, and number of astrocytic branches (primary, intermediate, and terminal processes) (Fig 6B). IMARIS-based 3D morphological analysis showed an enlargement in microglial cell somatic volume, alongside a decrease in the number and length of their processes after repeated optogenetic astrocyte stimulation. These effects were attenuated by Lcn2 deficiency (S20 Fig). In addition, we examined the changes in the Il1b and Tnf mRNA expression in the hippocampus of Lcn2-KO mice. Results of quantitative PCR (qPCR) demonstrated a significant increase in the levels of these proinflammatory cytokines in the hippocampus of wild-type (WT) mice after photostimulation, which was significantly reduced in Lcn2-KO mice (Fig 6C). These findings suggest that LCN2 is an important trigger for neuroinflammation in the hippocampus after the repeated optogenetic stimulation of astrocytes. We compared the photostimulation-induced cognitive impairment between Lcn2-deficient and WT mice. We observed that the cognitive behavior impairment induced by photostimulation was significantly ameliorated in the Lcn2-KO mice, as demonstrated by the Y-maze (Fig 6D), Barnes maze (Fig 6E), and passive avoidance tests (Fig 6F). These data indicate that LCN2 plays a vital role in the development of cognitive impairment after the optogenetic stimulation of hippocampal astrocytes. There was no significant difference in the basal excitatory synaptic transmission, and short- and long-term synaptic plasticity between the WT and Lcn2-KO mice (S21A–S21F Fig). We then examined potential changes in LTP levels in Lcn2-KO mice following 20 min of photostimulation. The degree of TBS-induced LTP alteration showed no significant differences between the eYFP and ChR2-eYFP groups in Lcn2-KO mice, with or without photostimulation (S21G and S21H Fig).

Sustained Gq signaling activation of hippocampal CA1 astrocytes mimics the effect of optogenetic stimulation: LCN2 release, neuroinflammation, and cognitive deficit We used chemogenetic tools to examine whether chronic Gq signaling activation of hippocampal astrocytes mimics the effect of repeated optogenetic stimulation. The muscarinic receptor variant hM3Dq fused to a red fluorescent protein, mCherry, was expressed in the astrocytes within the hippocampal CA1 region using an AAV vector incorporating a GFAP promoter (AAV-GFAP-hM3Dq-mCherry, Fig 7A). To confirm the astrocyte-targeted expression of the hM3Dq-mCherry protein, we performed immunostaining of the hippocampal sections using anti-GFAP antibody and observed that the cells expressing the hM3Dq-mCherry protein were mostly GFAP+ astrocytes (Fig 7B). To verify whether hM3Dq activates astrocytes after treatment with clozapine-N-oxide (CNO), we performed Ca2+ imaging in cultured astrocytes expressing hM3Dq. For this purpose, cultured astrocytes were loaded with Fluo 4-AM and treated with CNO (10 μm) (S22A Fig). We observed that CNO treatment triggered an intracellular Ca2+ increase in hM3Dq-expressing astrocytes (S22B Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 7. Long-term activation of astrocytic hM3Dq in the hippocampal CA1 region induces LCN2 release, neuroinflammation, and cognitive impairments. (A) Experimental timeline. (B) Brain tissue samples were subjected to immunofluorescence analysis to localize the expression of hM3Dq-mCherry (red) in the astrocytes (GFAP, white) and neurons (NeuN, green). The nuclei were stained with DAPI (blue). Arrowheads (yellow) indicate the colocalization of hM3Dq-mCherry and GFAP. The quantification of the colocalization is shown in the adjacent graphs. Scale bar: 200 μm. Results are expressed as mean ± SEM (n = 4). n.d., not detected. (C) Passive avoidance test. Results are expressed as mean ± SEM (n = 5). *p < 0.05 between the indicated groups; n.s., not significant (two-way ANOVA). (D) On day 3 after CNO (1 or 3 mg/kg, i.p.) injection, total mRNA was extracted from the hippocampal tissues of each group and subjected to qPCR to determine the expression levels of Lcn2, Il1b, and Tnf. Gapdh was used as an internal control. The graph displays the quantitative results normalized to Gapdh; results are expressed as mean ± SEM (n = 5). *p < 0.05 between the indicated groups; n.s., not significant (one-way ANOVA). (E) Astrocytes and microglia were identified using GFAP (white) and Iba-1 (eYFP, red; hM3Dq-mCherry, green) immunolabeling after CNO (1 or 3 mg/kg, i.p.) injection. The adjacent graph displays the quantification of the fluorescence intensity (GFAP or Iba-1). In further morphological analysis, the total length and average thickness of astrocyte processes in each group were assessed. Moreover, the total number of primary, intermediate, and terminal branches for each astrocyte was measured. Scale bar: 200 μm. Results are expressed as mean ± SEM (n = 4). *p < 0.05 between the indicated groups; n.s., not significant (one-way ANOVA). Source data can be found in S1 Data. CNO, clozapine-N-oxide; LCN2, lipocalin-2. https://doi.org/10.1371/journal.pbio.3002687.g007 The passive avoidance test was performed on the animals to evaluate the ability of chronic Gq signaling activation of hippocampal CA1 astrocytes to regulate cognitive behavior. hM3Dq-mCherry-expressing mice received CNO (1 or 3 mg/kg, intraperitoneal injection) 3 times a day (8 h interval) for 3 days. After the fourth CNO injection (3 mg/kg), the latency to enter the dark compartment was significantly lower in CNO (3 mg/kg)-injected hM3Dq-expressing mice than in control animals in the passive avoidance test trials performed 24 h after the foot shock, thus suggesting impaired memory due to the chemogenetic stimulation of hippocampal astrocytes (Fig 7C). The hM3Dq-expressing mice exhibited no difference in latency to enter the dark compartment during the conditioning session. No apparent changes in cognitive function were detected in eYFP-expressing control mice. These results indicate that the long-term activation of astrocytic Gq signaling resulted in cognitive function impairment. We explored whether chronic stimulation of astrocytes through hM3Dq triggered LCN2 expression and neuroinflammation. Results from the qPCR analysis revealed the enhanced mRNA expression of Lcn2 and proinflammatory cytokines (Il1b and Tnf) in the hippocampus after chemogenetic stimulation (Fig 7D). Moreover, chemogenetic stimulation induced the activation of both microglia and astrocytes (Fig 7E). In detailed morphological analysis, we observed an increased number of primary processes leaving the soma in astrocytes from the hM3Dq-mCherry group compared to the eYFP group (Fig 7E). Astrocytes in the hM3Dq-mCherry group exhibited a bushy morphology with many fine terminal processes protruding from the primary cellular processes, and the processes appeared thicker than those in the eYFP group (Fig 7E). These effects were only observed after treatment with a high concentration of CNO (3 mg/kg), but not when the concentration was low (1 mg/kg). Overall, these data suggest that the long-term and strong Gq signaling activation of CA1 astrocytes significantly impairs cognitive behavior and increases glial activation and expression of Lcn2 and proinflammatory cytokines.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002687

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