Homer1 Scaffold Proteins Govern Ca2+ Dynamics in Normal and Reactive Astrocytes

Abstract

In astrocytes, the intracellular calcium (Ca2+) signaling mediated by activation of metabotropic glutamate receptor 5 (mGlu5) is crucially involved in the modulation of many aspects of brain physiology, including gliotransmission. Here, we find that the mGlu5-mediated Ca2+ signaling leading to release of glutamate is governed by mGlu5 interaction with Homer1 scaffolding proteins. We show that the long splice variants Homer1b/c are expressed in astrocytic processes, where they cluster with mGlu5 at sites displaying intense local Ca2+ activity. We show that the structural and functional significance of the Homer1b/c-mGlu5 interaction is to relocate endoplasmic reticulum (ER) to the proximity of the plasma membrane and to optimize Ca2+ signaling and glutamate release. We also show that in reactive astrocytes the short dominant-negative splice variant Homer1a is upregulated. Homer1a, by precluding the mGlu5-ER interaction decreases the intensity of Ca2+ signaling thus limiting the intensity and the duration of glutamate release by astrocytes. Hindering upregulation of Homer1a with a local injection of short interfering RNA in vivo restores mGlu5-mediated Ca2+ signaling and glutamate release and sensitizes astrocytes to apoptosis. We propose that Homer1a may represent one of the cellular mechanisms by which inflammatory astrocytic reactions are beneficial for limiting brain injury.

Keywords: Homer; astrocytes; intracellular Ca2+; mGlu5; neuroinflammation.

Figure 1.

Homer1b/c are expressed in astrocytes and their immunoreactivity overlaps with mGlu1/5 immunoreactivity. (A, B) Homer1b/c are expressed in cortical astrocytes across species. (A) P5 rats, a diffuse staining for Homer1b/c (red) is present within cell bodies and processes of cortical S100β-positive cells (green). (B) P30 GFAP-EGFP mice, cortical astrocytes (green) largely express Homer1b/c (red) in their cell bodies and processes. (C) High magnification of an astrocyte in B. (D) Double labeling of P10 GFAP-EGFP astrocytes. The punctate staining for Homer1b/c (red) observed in juvenile mice partially colocalized with mGlu5 (green) staining in the large astrocytic processes; left panel shows a single plane of EGFP fluorescence expressed under the GFAP promoter. Boxed region marks large astrocytic process shown in E–F. (E, F) High magnification of the Homer1b/c and the mGlu1/5 puncta on the large astrocytic process (box in D) shows an overlap of the 2 signals (F, arrowheads).

Figure 2.

Functional role of Homer1 isoforms in regulation of Ca²⁺ microdomains in cultured astrocytes. (A, B) TIRF images of mGlu1/5 and ER distributions in astrocytes not transfected or transfected with Homer1a-CFP or Homer1b-CFP. (A) Overexpression of Homer1b or Homer1a induces clustering and declustering of mGlu1/5, respectively. Cultured astrocytes are transfected with Homer1b-CFP or Homer1a-CFP and subsequently immunostained for mGlu5. TIRF illumination reveals a sub-plasmalemmal distribution of mGlu5. Larger clusters of the receptor are present in Homer1b-transfected cells compared with nontransfected cells (middle and left, respectively). The distribution of the receptor is more diffuse when cells are transfected with Homer1a (right). Scale bars: 20 μm (B) Overexpression of Homer1b or Homer1a induce association and dissociation of ER tubules with the plasma membrane. Cultured astrocytes are transfected with only ER-GFP (left) or co-transfected with ER-GFP and Homer1b-CFP or Homer1a-CFP (middle or left, respectively). Under TIRF illumination, Homer1b cotransfected cells exhibit an increase in GFP fluorescence (middle), while the GFP florescence is decreased in Homer1a cotransfected cells. Scale bars: 20 μm (C–E) Global and sub-membrane intracellular Ca²⁺ concentration [Ca²⁺]_i induced by stimulation with 3,5-dihydroxyphenylglycine (DHPG). (C) Cultured astrocytes transfected with Homer1b-CFP (top panel) or Homer1a-CFP are incubated for 20 min with 5 μM Fluo4-AM and illuminated with epifluorescence (EPI, middle panel) for global Ca²⁺ imaging or with TIRF illumination (lower panel) for sub-membrane, local Ca²⁺ imaging. Scale bars: 20 μm (D) Traces corresponding to Ca²⁺-EPI or Ca²⁺-TIRF in CFP-expressing or nontransfected (Control) cells before and after local application of DHPG (100 μM, 2 s) were plotted as background-subtracted ΔF/F₀. Note faster response of sub-membrane [Ca²⁺]_i of Homer1b-transfected cells (middle). *P < 0.05, one-way ANOVA. (E) The amplitude of the global [Ca²⁺]_i response to DHPG (Ca²⁺ EPI, left) is significantly larger in Homer1b/c and decreased in Homer1a-overexpressing cells in comparison with control nontransfected cells (CTRL). The amplitudes of the sub-membrane [Ca²⁺]_i responses to DHPG (Ca²⁺ TIRF, right) are significantly decreased in Homer1a-transfected cells while remain unchanged in Homer1b-transfected cells. **P < 0.01, one-way ANOVA. Group sample sizes, n = 8. (F–I) Properties of localized Ca²⁺ events. (F) High magnification image of a sub-membrane Ca²⁺ event, with a typical ΔF/F₀ trace. Scale bar: 2 μm (G) Three representative example traces of localized Ca²⁺ events illustrate what is summarized in (H). In Homer1a-overexpressing cells there is a cessation of spontaneous Ca²⁺ spiking activity compared with control or Homer1b-overexpressing cells. Group I mGlu activation only marginally increases the frequency of events in Homer1a-overexpressing cells, while a large increase in spiking frequency is observed in controls and Homer1b-overexpressing cells. Also, Homer1a-overexpressing cells exhibit reduction in amplitude. (H) Frequency (left) and amplitude (right) of localized Ca²⁺ events, grouped into three 1-second bins, one before (-1-0) and 2 after the DHPG application. **P < 0.01,*P < 0.05, one-way ANOVA (I) Before and after the local application of DHPG, the sub-membrane Ca²⁺ events occurring in 100 ms-long frames are counted and plotted (mean ± SD for each histogram bar) to obtain their temporal distribution (n = 5 cells for each of the groups). The overexpression of Homer1a results in a dramatic reduction of the number of localized events of Ca²⁺.

Figure 3.

Reactive astrocytes upregulate Homer1a. (A–C) Reactive astrogliosis and Homer1a expression in a rat model of neonatal HI. (A) In HI animals, Homer1a immunohistochemistry staining is present in the cortical areas peripheral to the lesion (LP, lesion periphery) at 1 day after the insult. At 7 days after HI the staining is stronger in the area (LB, lesion border) adjacent to the lesion core (LC). (B) In ischemic animals, a mild astrocytic reaction (identified by GFAP immunohistochemistry staining) is present in the lesion periphery at 1 day after the insult. At 7 days after HI there is a gradient of reactive astrocytosis that culminates in an astrocytic scar in the lesion border. (C) The double immunofluorescence staining for S100β (green) and Homer1a (red) shows that at 1 day after HI, Homer1a expression is stronger in neurons (white arrowheads) than in astrocytes, whereas at 7 days postinsult Homer1a strongly overlaps with S100β-positive cells on the astrocytic scar. (D, E) Homer1a is expressed in reactive astrocytes of human HIE newborns. Left panels show representative images of GFAP immunohistochemistry staining on postmortem brain sections of human newborn controls (D) and HIE (E) cases. In the right panels, images of GFAP (green) and Homer1a (red) immunofluorescence costaining in consecutive brain sections of the same control (D) or HIE (E) case illustrate that Homer1a expression is largely increased and concurrent with reactive astrocytes in HIE newborns. Insets (bottom-left side of immunofluorescence images) show higher magnification of selected astrocytes (white boxes). Fluorescence images are z-stack maximal projections. Scale bars: (A, B) 100 μm, (C) 50 μm, (D, E) 200 μm (immunohistochemistry), 50 μm (immunofluorescence).

Figure 4.

Global Ca²⁺ response evoked by mGlu1/5 stimulation is impaired in reactive astrocytes from ischemic tissue. (A) At 7 days after induction of HI, acute brain slices (250 μm, coronal) are incubated 60 min at 34°C in aCSF with the Ca²⁺ indicator Fluo-4 AM (10 μM) and the specific astrocytic marker sulforhodamine 101 (SR101, 2 μM) and subsequently washed for 20 min. The left panels show the SR101 (red) stained astrocytes. The series of sequential images in pseudo color illustrate typical examples of the evolution of Fluo-4 fluorescence intensity induced by application of the group I mGluR agonist DHPG (100 μM) on tissue from sham-operated (normal astrocytes, top) or HI (reactive astrocytes, bottom) animals. (B) To analyze the global Ca²⁺ response of astrocytes, ROIs outlining the cell bodies and the large processes of SR101-positive cells are defined and their Fluo-4 intensity changes are monitored and expressed as ΔF/F₀. (C) Representative traces of the [Ca²⁺]_i increases after application of DHPG and ionomycin (IONO, 10 μM), which is used as positive control, on slices from sham-operated and HI animals (left and right, respectively) as shown in (B). (D) The time-course of the average % of ΔF/F₀ normalized to the maximal ionomycin response shows a decrease in the maximal amplitude of the response to DHPG in reactive astrocytes (n = 5 animals, 7 slices, approximately 250 cells for sham; n = 5 animals, 10 slices, approximately 250 cells for HI, mean ± SEM per slice). (E) The amplitude of the response to DHPG in reactive astrocytes is significantly reduced (P = 0.015, unpaired Student's t-test) while no discernable change in the number of responding cells is observed (P = 0.187, Mann–Whitney U-test). (F) The immunostaining of GFAP (green) and mGlu5 (red) in sham animals (left panels) and in HI animals (right panels) shows the increased and extensive expression of mGlu5 at 7 days after the ischemic insult in reactive astrocytes from the area bordering the lesion (z-stack maximum projection). Scale bars: 50 μm. Asterisks indicate significant differences between groups (*P < 0.05, unpaired Student's t-test).

Figure 5.

Local Ca²⁺ response evoked by mGlu1/5 stimulation is impaired in reactive astrocytes from ischemic tissue. (A) SR101-positive astrocytes responsive to DHPG are selected (left) from slices of healthy rats and ROI (1 µm²) are placed on the cell body and along the main process (right). Dashed box (left) indicates the location of the insets (right). The ROIs are placed starting on the soma and numbered according to their distance from the initial point. Ca²⁺ signals (grayscale images) in each ROI underwent measurable rises and falls as shown in the traces in (B); dots underneath represent each detected Ca²⁺ spike. Two localized Ca²⁺ signals seen in (A) are highlighted with arrowheads in the respective ROI traces. DHPG application causes an elevation in global Ca²⁺ signal as seen in each trace as a positive deviation from the background level, and changes of some parameters of localized Ca²⁺ spikes (quantified in G and F). Scale bars: 800 nm (C, D) Analysis of reactive astrocytes is shown as in (A, B), with fewer and smaller localized Ca²⁺ responses (D) compared with their healthy counterparts. Mean traces with standard errors (B, D bottom) represent the sum of ROIs and show a pattern of global [Ca²⁺]_i change (see Fig. 4D for comparison). Localized Ca²⁺ events of normal (E) and reactive (F) astrocytes are pooled together and are shown grouped based on their location (soma/process) and stimulation (basal/DHPG). Black bold traces in each of the aggregates represent the mean localized events ± SD. Frequency (G) and amplitude (H) of identified localized events are measured under basal conditions and after DHPG challenge, and are split into events occurring in the somata and processes of normal and reactive astrocytes. Asterisks indicate significant differences between groups [*P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student t-test (normal vs. reactive) or one-way ANOVA with Bonferroni post-test (within normal or reactive)].

Figure 6.

Homer1a reduces exocytotic glutamate release from astrocytes. (A) Differential interference contrast (DIC) image (top left) of a solitary astrocyte coexpressing RCaMP1h (top center, red) and Homer1a-YFP (top right, green). The lower panels show NADH fluorescence images, reporting on extracellular glutamate surrounding somata of solitary astrocytes, taken at different time points (black arrowheads in B) before and after mechanical stimulation (indicated by an arrow in B). The pseudo color scale is a linear representation of the fluorescence intensity ranging from 125 to 200 intensity units. (B) Average kinetics of extracellular NADH fluorescence (means ± SEM) in solitary astrocytes expressing either RCaMP1h alone (control, black squares, n = 15 cells) or along with Homer1a-YFP (open triangles, n = 15 cells) in response to mechanical stimulation (arrow). Changes in NADH fluorescence are shown as % ΔF/F₀ after background subtraction and correction for bleaching. SEMs are shown in single direction for clarity. (C) Normalized peak of glutamate (Glut) release; dashes represent medians with interquartile range and asterisks indicate significant changes (**P < 0.01, Mann–Whitney U-test). Scale bar: 20 μm. (D) Temporal distribution of fusion events of VGLUT1-pHluorin expressing vesicles evoked by DHPG application (2 s, 100 μM) in control cells and in cells overexpressing Homer1a-CFP. Each individual histogram represents the number (mean ± SEM) of fusion evens detected in a 50-ms-long frame (n = 8 cells). (E) Frequency histograms of VGLUT1-pHluorin events in control cells and in cells transfected with Homer1a-CFP. Note that overexpression of Homer1a significantly decreases the frequency of fusion events. (**P < 0.01, one-way ANOVA).

Figure 7.

Effects of Homer1a knockdown on reactive astrocytes. (A) Intracerebroventricularly delivered nontargeting control siRNA and anti-Homer1a siRNA2 (red) are present in GFAP-positive reactive astrocytes (green) 2 days after the injection, as seen in single plane confocal images (high magnification shown in right panels) (B) Representative ΔF/F₀, normalized to the maximal ionomycin response, time-course traces of DHPG-stimulated normal and reactive astrocytes in acute slices from animals injected with nontargeting control siRNA or anti-Homer1a siRNA2. (C) The amplitude of DHPG-evoked intracellular Ca²⁺ transients is significantly smaller in control siRNA-treated reactive astrocytes than in control siRNA-treated normal astrocytes and is restored by the knockdown of Homer1a with siRNA2 (mean ± SEM, n = 3 animals, 3 slices per condition). (D) Acute slices from siRNA-injected sham- or HI-operated rats are incubated with bafilomycin A1 and stimulated with DHPG to induce the release of extracellular glutamate from astrocytes. In slices from ischemic animals injected with control siRNA (n = 6) the release of extracellular glutamate is significantly smaller than in slices from sham animals (n = 5). The knockdown of Homer1a with siRNA2 on slices from ischemic animals (n = 3) significantly amplified the extracellular glutamate release from astrocytes (mean ± SEM). (E) Immunofluorescence analysis of tissue from HI animals injected with control siRNA or anti-Homer1a siRNA2 reveals the presence of GFAP-positive (red) reactive astrocytes that immunopositive for the active form of caspase-3 (green). (F) Quantification of active caspase-3 positive reactive astrocytes shows that the knockdown of Homer1a with siRNA2 increases the number of active caspase-3 positive reactive astrocytes (mean ± SD, n = 3 animals). (*P < 0.05, ***P < 0.001, unpaired Student's t-test and one-way ANOVA with Bonferroni's post-test). Scale bars: 50 μm (low magnification) and 20 μm (higher magnification).

Figure 8.

Working model. Under physiological conditions (left), astrocytes transduce an extracellular signal (glutamate, blue dots) through the membrane-bound mGlu1/5 receptor into the cell. In the cytoplasm, the mGlu1/5 receptor is physically linked to Homer1b/c oligomers and, through them, to the ER. This tether enhances the vicinity of the Ca²⁺-containing ER and together with the Ca²⁺-sensitive exocytotic machinery forms a functionally relevant structural microdomain. Under pathological conditions (right) Homer1b/c is replaced by its nonoligomerizing splice variant Homer1a, which lacks the coiled-coil domain and thus prevents the formation of mGlu1/5-Homer1-ER clusters. As a consequence of this and together with the fact that astrocytes under ischemic condition undergo hypertrophy, the transduction is impaired and very little Ca²⁺ is immediately available in these functional domains on both local and global levels to fuel processes such as gliotransmission.