Neuronal nuclear calcium signaling suppression of microglial reactivity is mediated by osteoprotegerin after traumatic brain injury

Buffering nuclear calcium in neurons enhances the early accumulation of microglia upon TBI

In order to demonstrate the effective target engagement of PV.NLS expressed in neurons, we assessed the levels of phospho-CREB (pCREB) 3 h after TBI (since NC is critical for the phosphorylation of CREB; [8, 30, 40].

AAV9 encoding for hSyn::PV.NLS-mCherry (or an empty vector for control) was injected into the somatosensory cortex of adult mice, generating > 90% infection efficiency (30 days after injection > 90% of NeuN + cells were mCherry + (Additional file 1: Fig. S1A, B). Thirty days after AAV9 injection, mice were randomized to undergo either mild TBI (all animals scored 0 at the NSS test; scores of individual animals are reported in Additional file 6: Table S1) or sham surgery. There were a total of four experimental groups: 1. empty vector (control) sham (CS); 2. control TBI (CT); 3. PV.NLS sham (PS); 4. PV.NLS TBI (PT), all of which were killed 3 h after treatment. CT samples displayed a significant increase in neuronal pCREB in the site of injury compared to CS (as previously reported; [14], but the expression of PV.NLS largely blunted it (Fig. 1A, B). At this time point no neuronal loss was detected, irrespective of PV.NLS expression (Fig. 1C. Notably, a large number of small, elongated pCREB + nuclei were found in PT, but not in CT images; co-immunostaining of pCREB with GFAP and IBA1 revealed that almost all the pCREB + , small, elongated nuclei were detected in IBA1 + cells and therefore identified as microglia (> 98%; (Additional file 1: Fig. S1C, D)).

Fig. 1figure 1

Buffering nuclear calcium in neurons enhances the accumulation of microglia 3 h after TBI. A, B Significant increase of neuronal pCREB intensity 3 h post-TBI compared to Sham (CS vs CT; 1.00 ± 0.01 vs 2.11 ± 0.44). Buffering of nuclear Calcium significantly decreases neuronal pCREB intensity 3 h post-TBI (CT vs PT; 2.11 ± 0.44 vs 1.37 ± 0.35). Red datapoints indicate average per animal (N = 4, used for statistics); smaller datapoints depict individual neurons. Mean ± SD. C Neuronal density at the injury site 3 h post-TBI; no difference across groups. N = 4. D, E Unchanged microglial pCREB intensity 3 h post-TBI compared to sham. Buffering of nuclear calcium does not significantly increase microglial pCREB intensity 3 h post-TBI. N = 4. F Buffering of nuclear Calcium significantly increased microglia density upon TBI (CT vs PT; 7.65 ± 3.65 vs 15.66 ± 3.81). No significant difference in microglia density in animals injected with control virus (CS vs CT). Data are shown as mean ± SD. N = 4 mice. Scale bar: 25 µm. *p < 0.05; ***p < 0.001

In an independent set of experiments, we explored the effect of neuronal NC blunting on microglia upon TBI. In agreement with the spatially heterogeneous nature of TBI lesions at this time point [14, 15], we considered two regions of interest: one located at the center of the lesion (“core”) and the other located at a fixed lateral distance from the core (“perilesional area”, Additional file 2: Fig. S2A). At 3 h post-TBI, CT samples displayed only a small increase in IBA1 + cells compared to CS, whereas PT samples showed a massive increase in the number of IBA1 + cells in the site of injury (Fig. 1D, F) as well as in the perilesional area (Additional file 1: Fig. S1E, F). pCREB levels in microglia were not significantly altered across experimental groups (Fig. 1D, E). Thus, buffering of neuronal NC in the acute phases of TBI unexpectedly resulted in the massive increase in local microglia.

Buffering of neuronal nuclear calcium induces a disease-associated microglia (DAM)-like phenotype upon TBI

We further characterized the IBA1 + population expanded in PT by immunostaining with the microglia marker TMEM119 and the disease-associated microglia (DAM)-like markers CD11c and CST7 [36, 57]. We also determined the expression of CD169, a marker of pathogenic phagocytes [11, 56].

Across the four experimental groups, over > 95% of IBA1 + cells were also TMEM119 + at 3 h post-injury (Fig. 2A, C), indicating that the contribution of infiltrating peripheral cells was comparatively minimal at this time point. Interestingly, PT samples showed the highest density of TMEM119 + cells (Fig. 2B).

Fig. 2figure 2

Buffering neuronal nuclear calcium induces a disease-associated microglia (DAM)-like phenotype upon TBI. A, B Nuclear calcium buffering significantly increases the density of IBA1 + /TMEM119 + , IBA1 + /CD11c + and IBA1 + /CST7 + cells 3 h post-TBI (CT vs PT; IBA1 + /TMEM119 + : 6.68 ± 3.09 vs 12.52 ± 3.79; IBA1 + /CD11c + : 4.96 ± 1.99 vs 14.80 ± 5.33; IBA1 + /CST7 + : 2.15 ± 1.84 vs 7.53 ± 4.14). Significant increase in IBA1 + /CD169 + (PS vs PT; 1.638 ± 2.446 vs 10.630 ± 3.989). N = 4–5. C Nuclear calcium buffering significantly increase the fraction of IBA1 + /CD11c (vs total IBA1 + cells) 3 h post-TBI compared to PV.NLS-Sham (CS vs CT; 14.18 ± 14.37% vs 76.19 ± 29.49%), but not to TBI alone (CT vs PT; 76.19 ± 29.49% vs 98.13 ± 3.64%). The fraction of IBA1 + /CD169 double-positive cells was increased upon TBI by nuclear calcium buffering cells (CT vs PT; 16.80 ± 14.86% vs 82.90 ± 20.20%). N = 4–5. Scale bar 50 µm (overview) and 25 µm (inserts). Arrows indicate double-positive cells. Data are shown as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001

The density of IBA1 + CD11c + cells was massively increased in PT samples compared to either CT or PS (Fig. 2A, B); however, their portion compared to overall IBA1 + population was unchanged (Fig. 2C). Likewise, the density of IBA1 + /CST7 + cells was significantly increased in PT samples compared to CS, CT or PS but the presence of total IBA1 + cells was comparable in CT and PT. On the other hand, only very few IBA1 + cells co-expressed CD169 in CS, CT or PT samples, but the number of IBA1 + /CD169 + cells was massively increased in PT brains. Most notably, the population of IBA1 + /CD169 + cells was also substantially increased in PT, implying that the strong expression of CD169 + in microglia corresponded to a reactive phenotype following the NC blockade as well as TBI (Fig. 2A, C). Thus, the blockade of NC results, upon TBI, in the appearance of a large microglial population with a DAM-like and unique phenotype characterized by high CD169 expression.

Blockade of neuronal nuclear calcium/calmodulin signaling recapitulates the enhanced recruitment of microglia after TBI

Since Ca2+/CaM-dependent kinases play a significant role in regulating signaling downstream of NC [62], we hypothesized that sequestering nuclear Ca2+/CaM would recapitulate the effect of blunting NC with PV.NLS. For this purpose, we expressed a CaMBP4.mCherry construct, designed to bind and sequester nuclear Ca2+/CaM [73] or an empty vector for control in neurons of the somatosensory cortex via AAV injection. We considered four experimental groups: 1. control sham (CS), 2. control TBI (CT); 3. CaMBP4 sham (CaS); and 4. CaMBP4 TBI (CaT). Observing the core area following TBI, the expression of CaMBP4 significantly reduced the upregulation of pCREB (Fig. 3A, B), but did not affect the vulnerability of neurons 3 h after TBI (Fig. 3C). Likewise, the expression of neuronal CaMBP4 caused a massive increase in microglial density upon TBI (Fig. 3D, F), but was ineffective in sham-treated animals. Finally, we found that, compared to CS, the levels of pCREB were substantially increased in microglial cells when CaMBP4 was expressed in neurons (Fig. 3D, E). Thus, the inhibition of CaM-dependent NC signaling exerted by CaMBP4 in neurons largely recapitulates the effects of NC buffering by PV.NLS. This data suggests that NC-dependent action on CREB phosphorylation and microglial accumulation is specific and mediated by a CaM-dependent pathway.

Fig. 3figure 3

Blockade of neuronal nuclear calcium/calmodulin pathway recapitulates the enhanced recruitment of microglia after trauma. A, B Buffering of CaM by CAMBP4 significantly decreases neuronal pCREB intensity 3 h post-TBI (CT vs CaT; 2.33 ± 0.50 vs 1.62 ± 0.29). N = 4. C No significant difference of neuronal density at the injury site 3 h post-TBI. Mean ± SD. N = 4. DF Buffering of CamK activation does not significantly increase microglial pCREB intensity 3 h post-TBI. E CT vs CaT; 1.33 ± 0.41 vs 1.72 ± 0.62; N = 4) but significantly increased microglia density post-TBI F CT vs CaT; 4.58 ± 0.53 vs 11.44 ± 2.66). Individual neurons are depicted as small datapoints; average per mouse (N = 4, used for statistics) depicted as red datapoints. Arrows indicate IBA1 + cells expressing pCREB. Data are shown as mean ± SD. Scale bar: 25 µm. N = 4. *p < 0.05; ****p < 0.0001

Buffering of neuronal nuclear calcium enhances subacute microgliosis and synapse loss in TBI

We then explored the impact of NC buffering in TBI observed at later stages such as 24 h post-injury (1dpi) and 7d post-injury.

At 24 h post-injury, CT brains displayed an increased density of IBA1 + cells compared to sham mice (CT vs CS). However, in PT samples there was no evidence for a larger expansion of the IBA1 + population compared to CT samples, whereby cells showed a distinct ameboid morphology (Fig. 4A, B). The abundant IBA1 + population was associated with a significantly higher density (cells/area unit) of IBA1 + /TMEM119 + cells in the core and perilesional areas (Fig. 4A, B and Additional file 2: Fig. S2B-C). In addition, virtually all IBA1 + cells were TMEM119 + , indicating a limited contribution of peripheral immune cells within and around the lesion (Fig. 4C). The IBA1 + /CD11c + subpopulation was substantially larger in PT samples (Fig. 4A, B), yet maintaining a comparable component of total IBA1 + cells in PT and CT brains (Fig. 4C), suggesting an overall expansion of the microglial population in PT. Notably, the density of IBA1 + /Cst7 + as well as of IBA1 + /CD169 + cells and their relative representation was significantly increased at 24 h in PT samples (Fig. 4A-C). Thus, NC blunting resulted, upon TBI, in a substantially larger microglial population with a distinctive over-representation of CST7 + and CD169 + cells.

Fig. 4figure 4

Buffering of neuronal nuclear calcium enhances subacute microgliosis and synapse loss 24 h post-trauma. AC At 24 h after injury, neuronal nuclear Calcium buffering drives the significant increase of IBA1 + , IBA1 + /TMEM119 + , IBA1 + /CD11c + , IBA1 + /CST7 + and IBA1 + /CD169 + compared to TBI in mice injected with control AAV (B CT vs PT; for IBA1 + cells 7.92 ± 2.44 vs 15.74 ± 2.24; for IBA1 + /TMEM119 + cells 7.89 ± 2.40 vs 15.57 ± 2.30; for IBA1 + /CD11c + cells 7.88 ± 2.41 vs 14.50 ± 2.40; CT vs PT for IBA1 + /CST7 + cells; 2.44 ± 1.06 vs 8.79 ± 4.64; CT vs PT for IBA1 + /CD169 + cells; 3.48 ± 0.52 vs 7.95 ± 1.89). Buffering of nuclear calcium signaling in TBI did not alter the fraction of IBA1 + /CD11c + cells (C: CT vs PT; 97.06 ± 4.48% vs 91.80 ± 2.72%), but increased the fraction of CST7 + and CD169 +  + (CT vs PT for CST7; 29.72 ± 5.31% vs 63.82 ± 18.50%; CT vs PT vor CD169; 47.13 ± 17.14% vs 61.36 ± 7.11%). Data are shown as mean ± SD. N = 4. Scale bar 100 µm (overview) and 20 µm (insert). DE TBI decreases excitatory (Shank2/3+) synaptic density in the core (CS vs CT; 843.10 ± 84.62 vs 545.32 ± 138.21) but not in the perilesional area of the injury. Buffering of nuclear calcium signaling did not worsen the core synaptic density upon TBI but significantly decreased synaptic density in the perilesional area (CT vs PT; 769.9 ± 152.8 vs 471.3 ± 80.9). N = 4. F Significant decrease in neuronal density in the injury core 24 h post-TBI (CS vs CT; 33.82 ± 3.32 vs 18.50 ± 3.44). Buffering nuclear calcium signaling did not worsen neuronal density in the core compared to TBI alone (CT vs PT; 18.50 ± 3.44 vs 20.09 ± 3.41). Significant decrease in neuronal density in the perilesional area post-TBI to sham and nuclear calcium buffering (CS vs CT; 34.2 ± 1.55 vs 22.25 ± 4.67; CT vs PT; 22.25 ± 4.67 vs 29.88 ± 4.52). Data are shown as mean ± SD. N = 4. Scale bar 100 µm (overview) and 5 µm (insert). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

To assess the relationship between microglia infiltration and the extent of synapse loss, we quantified the integrity of the cortical architecture by measuring the density of excitatory synapses (number of pan-Shank + puncta per area unit), inhibitory synapses (number of Gephyrin + puncta per area unit) and the number of surviving neurons in the core and perilesional brain areas.

At 24 h after injury CT animals showed a significant loss of excitatory synapses was observed in the core of the lesion, whereas in the perilesional area the synaptic density was only slightly reduced compared to CS brains (Fig. 4D, E). In contrast, we detected a robust loss of excitatory synapses in PT samples particularly in the perilesional area, indicating that the area of synaptic involvement is much larger than expected, as clearly manifested in the low-magnification pictures (Fig. 4D). On the other hand, Gephyrin puncta (inhibitory synapses) were reduced to a similar extent in the core of the lesion as well as in the perilesional area at 24 h (Additional file 2: Fig. S2D–F).

Together, these experiments suggest that NC blockade resulted in a more extensive microgliosis and an exacerbated loss of excitatory synapses in the cortical area affected by TBI at 24 h post injury. Neuronal density was substantially decreased in the core of both CT and PT groups (Fig. 4F). Interestingly, an increased preservation of neuronal density was found in the perilesional area of PT brains despite the increased microgliosis and excitatory synaptic loss.

At 7dpi, CT brains still displayed a slight accumulation of microglia, however their density remained substantially higher in PT sections both in the core and perilesional areas (Fig. 5A, B). Although there was little difference in neuronal loss between CT and PT both in the core and in the perilesional area (Fig. 5A, C), PT brains still displayed substantially reduced counts of synapses in the perilesional area (Fig. 5D-F), indicating the persistent larger area of synaptic loss which was already established at 24 h post-injury.

Fig. 5figure 5

Buffering of neuronal nuclear calcium enhances subacute microgliosis and synapse loss 7 days post-trauma. AC Buffering of nuclear calcium signaling in TBI significantly increased the density of IBA1 + cells compared to TBI alone in the core and perilesional areas (B CT vs PT; Core 7.20 ± 2.87 vs 14.90 ± 4.89; perilesional area 5.68 ± 1.37 vs 10.06 ± 2.72). Buffering of nuclear calcium signaling in TBI reduced the neuronal loss in the core (C CT vs PT; 10.42 ± 3.55 vs 18.32 ± 0.81), but not in the perilesional area (CT vs PT; 24.06 ± 5.78 vs 25.05 ± 1.44). Data are shown as mean ± SD. N = 4. Scale bar: 100 µm. DF Significant decrease of excitatory synapses 7d post-TBI observed in animals injected with control AAV (CS vs CT; 819.12 ± 61.44 vs 607.12 ± 134.12) or nuclear calcium buffer (CT vs PT; 607.11 ± 134.52 vs 423.32 ± 60.11) in the core of the injury. Buffering of nuclear calcium signaling also resulted in a significant decrease in synaptic density in the perilesional area (CT vs PT; 845.2 ± 53.7 vs 599.2 ± 103.7). Data are shown as mean ± SD. N = 4.*p < 0.05; **p < 0.01; ***p < 0.001

Taken together, these findings demonstrate that enhanced microgliosis resulting from NC buffering following TBI is not transient, but is maintained for up to 7 days and is associated with a larger area of synaptic loss, thus linking the robust cellular inflammatory response with synaptic damage.

Blunting neuronal nuclear calcium worsens acute motor disturbances upon TBI

Next we explored the functional impact of the expanded, reactive microgliosis due to NC buffering after TBI. Since the somatosensory area of the brain provides strong excitatory drive to the primary and secondary whisker motor area [16, 43, 76], and based on evidence that silencing of the somatosensory area results in decreased whisking [63], we hypothesized that the disruption of synaptic networks in the somatosensory cortex might affect the whisking activity even in absence of a direct lesion of the motor area.

In fact, high-speed recordings of the spontaneous whisking activity of whiskers contralateral to the injury site assessed as number of whisking events (Fig. 6A) revealed that CT mice did show a decrease in spontaneous whisking already at 1 dpi which further deteriorated at 3dpi before showing a trend towards recovery at 7 dpi (Fig. 6B, C, F). While the whisking activity of PS mice were comparable to CS mice, PT mice displayed a significantly larger decline in whisking at 1 dpi compared to CT mice (Fig. 6C, E, F), indicating a more severe sensorimotor dysfunction in this group. However, the activity of PT mice converged with the CT counterparts by 3 dpi and with a similar recovery at 7 dpi. The kinetic parameters of single whisking events (Additional file 3: Fig. S3A–F) were comparable in the four groups, underscoring the sparing of the motor cortices; likewise, the activity of ipsilateral whiskers, controlled by the contralateral, uninjured side, was also comparable across the four groups (Fig. 6G). Of note, mice could not be tested at time points earlier than 1d because the stress of the TBI procedure made them characteristically uncooperative.

Fig. 6figure 6

Blunting neuronal nuclear calcium worsens acute motor disturbances upon TBI. A Representative images of analyzed mouse whiskers, showing the affected (red) and unaffected (blue) whiskers. BE Representative traces depicting the angular position of the affected whiskers (red) compared to the unaffected (blue) whiskers over time. Decreased activity is seen at D1 post-injury in PV.NLS TBI mice, but not in the other treatment groups. F Significant decrease of whisker activity after nuclear calcium buffering in TBI compared to TBI alone 1d post-TBI (CT vs PT; 0.82 ± 0.08 vs 0.47 ± 0.09). The difference is no longer detectable at later timepoints. G No significant changes to the whisking activity of the unaffected whisker was observed. N = 8 per group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Thus, buffering of neuronal NC leads to a more severe acute disruption of the affected network.

Targeted transcriptome analysis reveals new neuronal nuclear calcium-regulated mediators of neuro–glia crosstalk after TBI

In order to gain mechanistic insights into the processes triggering enhanced microgliosis and phagocytic phenotype occurring when neuronal NC is inhibited in TBI, we obtained a targeted nanostring transcriptome analysis of genes involved in neuroinflammatory cascades of the injected/injured area from CS, CT, PS and PT groups at 3 h post-injury. After pre-processing, quality control and normalization, the principal component analysis (PCA) distinctly defined the four groups (Fig. 7A), indicating significant differences in their transcriptome. Furthermore, the biological replications belonging to the four groups clustered together, segregated by treatment, in the unsupervised hierarchical clustering (not shown). The comparison of the transcriptome of CT vs PT samples (Fig. 7B, C) identified 74 upregulated and 72 downregulated genes (full list in Additional file 7: Table S2). Specifically, PT samples displayed a significant elevation in the expression of genes associated with phagocytic activity (CD68, Lamp1, Lamp2, Itgam, Ctss, Tlr2, Ifi30), antibody-mediated phagocytosis (Fcgr1, Fcgr2b, Fcgr3, Fcer1g) and in particular with DAM (TREM2, Clec7a, Cst7, complement C4a, ApoE, Cx3cr1, Csf1r, Spp1, Tyrobp, Grn). Furthermore, it showed a distinct elevation in the transcription of several other complement factors of the classical pathway (C1qa, C1qb, C1qc, C3), interferon-response genes (Irf7, Irf8, STAT1), chemokines and other migration factors (Ccl3, Ccl5, Cxcl9, Cxcl10). Thus, PT samples displayed a transcriptome compatible with the histological evidence of increased microglial recruitment as early as 3 h after injury, with a DAM and phagocytic phenotype.

Fig. 7figure 7

Targeted transcriptome analysis reveals new neuronal nuclear calcium-regulated mediators of neuro–glia crosstalk after TBI. A Principal component analysis (PCA) plot showing distinct clustering of each treatment. B Volcano plot of PV.NLS TBI vs control TBI shows upregulation of genes related to disease-associated microglia (green) or complement system (blue) and a down regulation of genes related to synaptic function (red) and transcriptional regulation (purple) 3 h after TBI. C, D A subset of the 74 genes upregulated and 72 genes downregulated in the PV.NLS TBI vs control (C) and of the 65 genes upregulated and 81 genes downregulated in the PV.NLS TBI vs PV.NLS sham (D) is depicted. A complete list of significantly differentially expressed genes can be found in Additional file 7: Table S2

On the other hand, a number of genes associated with synaptic proteins (Shank3, Grin2a, Grm2, Grm3) or involved in transcriptional and epigenetic regulation (Kdm4b and c, Kdm5d, Dnmt3a, Hdac6, CamKIV and Creb1) were downregulated.

The comparison of the PT and PS transcriptomes revealed 65 upregulated and 81 downregulated genes (Fig. 7D). The list of upregulated and downregulated genes was remarkably similar to those that were revealed by the comparison of PT and CT transcriptomes, with a selective upregulation of complement-associated and DAM-associated genes (among others, Clec7a, Cst7, C4a, Spp1, ApoE as well as C5ar1, C1qb, C1qa, C3ar1), antibody-dependent phagocytosis and interferon-response genes. Among the downregulated genes, synaptic proteins and epigenetic regulators were also prominently represented. Taken together these findings suggest that the induction of DAM-like transcriptional signatures and downregulation of synaptic genes is not a consequence of NC blockade alone but a fingerprint of TBI occurring in the context of NC blockade.

Neuronal expression of osteoprotegerin is upregulated by nuclear calcium signaling and neuronal activity in TBI

We interrogated the transcriptome dataset for soluble mediators, which may be potentially involved in neuron–microglia crosstalk, among the genes downregulated by PV.NLS upon TBI, under the hypothesis that the downregulation of one or more anti-inflammatory mediators may account for the upregulation of reactive microglia. We focused on TNFRSF11b, also known as osteoprotegerin (OPG), because (i) it is known to be released in the extracellular space and (ii) it reduces the activity of phagocytic cells in bone and connective tissue [26].

In fact, OPG is a soluble decoy receptor for RANKL, normally released in the extracellular space and readily detectable in biological fluids [26]; by antagonizing RANKL, OPG decreases osteoclast-dependent bone resorption. In the bone of OPG−/− mice osteoclasts are overactive and OPG overexpression suppresses their phagocytic activity [13]. Interestingly, OPG also restricts microglial reactivity to bacterial infection [37].

In this context, we used single molecule in situ mRNA hybridization to confirm the modulation of OPG expression and pinpoint the cellular source of OPG. OPG mRNA colocalized with NeuN and VGLUT2 in all four treatment groups, indicating a neuronal source (Fig. 8A), further confirmed by the colocalization of 75% of OPG mRNA molecules with NeuN immunoreactivity (Additional file 4: Fig. S4B). Notably, the number of OPG mRNA molecules increased in neurons 3 h after TBI (Fig. 8A, C, D), but this upregulation was abolished in neurons expressing PV.NLS.mCherry (Fig. 8A, C). We further considered the expression level of OPG mRNA 24 h and 7 days after injury; at the former timepoint, a significant upregulation of OPG was detected in CT animals but not in PT animals, whereas at the latter only a non-significant trend was seen (Additional file 4: Fig. S4C–F). Thus, OPG upregulation is sustained between 3 and 24 h but goes back to baseline by 7 dpi.

Fig. 8figure 8

Neuronal expression of osteoprotegerin is upregulated by nuclear calcium signaling and neuronal activity in TBI. A, B Significant increase of OPG (TNFRSF11b) mRNA density 3 h post-TBI compared to sham (CS vs CT; 8.78 ± 1.95 vs 18.03 ± 2.08). Buffering of nuclear calcium suppresses the upregulation OPG mRNA upon TBI (CT vs PT; 18.03 ± 2.09 vs 11.06 ± 2.80). Small datapoints depict individual neurons, red datapoints depict average per animal (N = 4, used for statistics). C, D Chemogenetic inhibition of PV interneurons in TBI significantly increases OPG mRNA density compared to TBI alone (Sal-T vs PSEM-T; 22.24 ± 4.43 vs 31.77 ± 4.10). Note that all animals express the PSAM chemogenetic construct (green) but only the PSEM + groups receive the agonist. Small datapoints depict individual neurons, red datapoints depict average per animal (N = 3, used for statistics).Data are shown as mean ± SD *p < 0.05; **p < 0.01

We then wondered whether OPG expression could be enhanced by increasing neuronal firing through chemogenetic approaches. To achieve this goal, we injected an AAV encoding the inhibitory PSAM(Gly)-GFP construct into PV-Cre mice. PSAM(Gly)-GFP was successfully expressed in PV + interneurons (Fig. 8B). Upon PSEM administration, PSAM(Gly) decreased the excitability of PV interneurons, thereby reducing perisomatic inhibition of neighboring excitatory neurons and resulting in their increased firing [14]. Reduced PV firing (PSAM(Gly) + PSEM) resulted in the upregulation of OPG in sham mice and even more robustly in TBI mice, compared to control mice (PSAM(Gly) + vehicle; Fig. 8B, D)).

So it can be concluded that OPG is upregulated in neurons upon TBI through a process involving neuronal firing and NC signals.

Re-expression of OPG in neurons with nuclear calcium buffering reduces microgliosis and prevents synaptic degradation after TBI

To establish a mechanistic link between neuronal OPG and TBI-induced microgliosis, we re-expressed OPG in neurons during NC blockade. The AAV expressing OPG under the hSyn promoter was sufficient to produce a massive upregulation of OPG expression irrespective of PV.NLS expression (Additional file 5: Fig. S5A, B). Mice were injected with a mix of AAV encoding either PV.NLS alone (PT) or PV.NLS and OPG (POT) 30 days before being subject to TBI. Two more groups were considered, both injected with AAV encoding an empty control vector, and subject to sham surgery (CS) or TBI (CT).

Re-expression of OPG substantially decreased the extent of microgliosis as determined by the density of IBA1 + and IBA1 + /TMEM119 + cells (Fig. 9A, B) and IBA1 + /CD11c + cells after TBI in POT mice compared to PT mice (Fig. 9A, B). Furthermore, OPG re-expression substantially decreased the proportion of cells expressing CST7 (Fig. 9A, C) as well as of CD169 + microglia (Fig. A, C), demonstrating that OPG re-expression is sufficient to normalize the recruitment and phenotype of microglia upon TBI.

Fig. 9figure 9

Re-expression of OPG in neurons with nuclear calcium buffering reduces microgliosis and prevents synaptic degradation after TBI. AC Buffering of nuclear calcium signaling significantly increased the density of IBA1 + /TMEM119 + , IBA1 + /CD11c + , IBA1 + /CST7 + and IBA1 + /CD169 + cells 24 h after TBI compared to TBI alone (CT vs PT; for IBA1 + /TMEM119 + 7.56 ± 0.48 vs 14.37 ± 1.74; for IBA1 + /CD11c + 6.74 ± 0.42 vs 11.70 ± 1.33; for IBA1 + /CST7 + 3.17 ± 0.29 vs 6.44 ± 1.05; for IBA1 + /CD169 + 1.38 ± 0.329 vs 9.56 ± 0.934). Re-expression of OPG decreased IBA1 + /TMEM119 + , IBA1 + /CD11c + , IBA1 + /CST7 + ; IBA1 + /CD169 cells compared to PV.NLS TBI (PT vs POT; for IBA1 + /TMEM119 + cells 14.37 ± 1.74 vs 8.02 ± 0.40; for IBA1 + /CD11c + cells 11.7 ± 1.33 vs 7.52 ± 0.21; for IBA1 + /CST7 + cells 6.44 ± 1.05 vs 2.20 ± 0.54; for IBA1 + /CD169 + 9.56 ± 0.93 vs 1.44 ± 1.38). Likewise, re-expression of OPG normalized the fraction of IBA1 + /CST7 + and IBA1 + /CD169 + compared to PV.NLS TBI (PT vs POT; for CST7 + 52.14 ± 4.91% vs 28.98 ± 6.44%; for CD169 + 68.01 ± 15.06% vs 20.67 ± 14.95%). N = 3. Mean ± SD Scale bar: 40 µm (overview) and 20 µm (insert). D, E Re-expression of OPG together with buffering of nuclear calcium signaling did not alter core synaptic density compared to PV.NLS-TBI but significantly increased the synaptic density in the perilesional area compared to PV.NLS-TBI (PT vs POT; 298.3 ± 78.1 vs 576.3 ± 66.8). N = 4. F OPG re-expression together with nuclear calcium buffering did not alter core neuronal density compared to TBI alone but significantly increased neuronal density in the perilesional areas (CT vs POT 25.13 ± 2.26 vs 32.01 ± 5.38). Yellow inserts show analyzed core and perilesional areas. Scale bar: 100 µm (overview) and 5 µm (insert). Data are shown as mean ± SD. N = 4. *p < 0.05; **p < 0.01; ***p &

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