Norepinephrine acting through α- and β-adrenergic receptors reduces TNF mRNA levels in astrocytes.

(A) Schematic representation of the experimental design, with Norepinephrine (NE) binding to β-adrenergic and α1- adrenergic receptors on astrocytes to influence TNF production. (B) Astrocyte cultures treated with 50μM NE for 4h had significantly decreased TNF mRNA levels as measured by qPCR (Mann-Whitney, P=0.0286, n=4). The -ΔΔCt values are plotted on the left axis, while fold change is plotted on the right axis. (C) NE treatment did not increase the rate of TNF mRNA degradation. Astrocytes were treated with NE, followed by the RNA synthesis inhibitor actinomycin (5 µg/mL). NE treatment did not alter the rate of TNF RNA degradation (2-way repeated measure ANOVA; effect of treatment: F(1, 16) = 1.708, P = 0.2097; effect of time: F(3, 16) = 34.14, P < 0.0001; interaction: F(3, 16) = 0.734, P = 0.5468). (D) The α1-adrenergic receptor agonist Phenylephrine (PE; 50μM, 4h), significantly decreased TNF mRNA levels in astrocytes (Mann-Whitney, P=0.0079, n=5). (E) Astrocyte cultures treated with the β-adrenergic receptor agonist Isoprenaline (ISO; 50μM, 4h) also had a similar decrease in TNF mRNA levels (Mann-Whitney, P=0.0286, n=4). (F) Astrocyte cultures were treated with the adenylyl cyclase activator Forskolin (FSK; 20μM, 4h) had significantly reduced TNF mRNA levels (Mann-Whitney, P=0.0286, n=4). (G) TNF protein was also decreased following 4h FSK treatment. Micrographs of astrocyte cultures immunostained for TNF (green) and DAPI (blue) from non-treated cultures and cultures treated with 20μM FSK. (H) Mean intensity was significantly decreased by FSK treatment (Mann-Whitney, P= 0.0002, n=8).

Gq-receptor activation via DREADDs decreased TNF mRNA levels in astrocytes.

(A) Schematic representation of Gq-DREADD activation in astrocytes using clozapine-N-oxide (CNO) on Gq-DREADD expressing astrocytes, leading to the modulation of TNF production. (B) Micrograph showing that the majority of astrocytes were expressing mCherry (red; counterstained with DAPI in blue) six days after being infected with AAV8-gfaABC1D-hM3Gq-mCherry virus. (C) Activation of Gq-DREADDS on astrocytes with 10μM CNO for 6h significantly reduced TNF mRNA expression compared with non-treated (NT) DREADD expressing astrocytes (Mann-Whitney, P= 0.0043, n=6). (D) Micrographs of TNF protein expression in Gq-DREADD-expressing astrocytes comparing non-treated (NT) and CNO-treated cultures. (E) Quantification of TNF protein fluorescent intensity, showing that CNO treatment significantly reduced TNF protein in astrocytes expressing Gq-DREADDs (Mann-Whitney, P=0.0085, n=16).

Gi receptor activation using DREADDs increases TNF mRNA levels in astrocytes.

(A) Schematic representation of Gi-DREADD activation in astrocyte using CNO to activate Gi-DREADDs expressed on astrocytes. (B) Micrograph of astrocyte cultures infected with AAV2/5-gfaABC1D-hM4D(Gi)-mCherry virus. Six days post-infection, the majority of astrocytes expressed mCherry (red; counterstained with DAPI in blue). (C) Gi-DREADD receptor activation by CNO significantly increased TNF mRNA expression, compared with non-treated DREADD expressing astrocytes (Mann-Whitney, P=0.0079, n=5). Cultures were first treated with 100μM Glutamate for 24h, before being treated with 10μM CNO for 6h. (D) Micrographs of TNF protein expression in Gi DREADD-expressing astrocytes comparing non-treated (NT) and CNO-treated cultures. (E) Quantification of TNF protein fluorescent intensity, showing that CNO treatment significantly increased TNF protein in astrocytes expressing Gi-DREADDs (Mann-Whitney, P<0.0001, n=10).

Human iPSC-derived astrocytes have similar responses to GPCRs as rat astrocytes.

(A) Micrographs showing immunocytochemistry demonstrating that the iPSC-derived astrocytes were positive for the astrocytic markers GFAP, S100β and ALDH1L1 but negative for the NPC marker SOX2. Scale bar (50μm). (B) Group data showing that expression of TNF mRNA in human astrocytes decreased following forskolin treatment to activate the Gs pathway (FSK; 20μM, 4h), compared with non-treated sister cultures (Mann-Whitney, P=0.0286, n=4). (C) Group data showing that activation of Gq-DREADDs by CNO (10μM, 6h) reduced the expression of TNF mRNA in human astrocytes compared with non-treated sister cultures (Mann-Whitney, P=0.0286, n=5). (D) Group data showing that activation of Gi-DREADDs by CNO (10μM, 6h) increased the expression of TNF mRNA in human astrocytes compared with non-treated sister cultures (Mann-Whitney, P=0.0286, n=4).

Microglia have an identical pattern of GPCR-regulation of TNF production as astrocytes.

(A) Schematic representation of the pathways for GPCR activation in microglia by various ligands to determine the impact on TNF mRNA expression. (B) Treating enriched microglia cultures with 50μM NE for 4h significantly decreased TNF mRNA levels (Mann-Whitney, P=0.0286, n=4). (C) Treating microglia cultures with the β adrenergic receptor agonist Isoprenaline (ISO; 50μM, 4h) also significantly reduced TNF mRNA levels (Mann-Whitney, P=0.0079, n=5). (D) Microglia cultures treated with adenylyl cyclase activator Forskolin (FSK; 20μM, 4h) had a similar reduction in TNF mRNA levels (Mann-Whitney, P=0.0079, n=5). (E) To test the Gq pathway, microglia cultures were treated with 15 µM DHPG, an agonist of group I mGluRs, for 4h. DHPG treatment significantly decreased TNF mRNA levels in microglia (Mann-Whitney, P=0.0286, n=4). (F) To test the Gi pathway, microglia cultures were treated with 50 µM Baclofen, a GABAB receptor agonist, for 4h. Baclofen treatment significantly reduced TNF levels in microglia (Mann-Whitney, P=0.0286, n=4).

Astrocytic Gi-DREADD activation increases TNF mRNA in vivo.

(A) Schematic of the injection and treatment protocol for in vivo expression of Gi-DREADDs in V1 of one hemisphere and a control virus in the contralateral hemisphere. The micrograph shows the areas dissected for analysis. (B) Micrographs showing the expression of mCherry bilaterally in V1, with a higher magnification showing expression in astrocytes. (C) Following DCZ treatment (3 µg/kg, IP, 3 h), the Gi-DREADD expressing V1 had significantly more TNF mRNA expression than the control V1 (Mann-Whitney, P=0.0286, n=4).

Summary of GPCR activation on TNF expression in astrocytes and microglia.

Activation of Gs and Gq pathways in both astrocytes and microglia results in a decrease in TNF levels, while activation of Gi pathways increases TNF levels. This response is the opposite of what occurs in neurons, where Gq and Gs activation typically leads to neuronal activation, and Gi activation results in inhibition.

Other pro-inflammatory cytokines were not co-regulated with TNF by GPCRs in astrocytes.

(A) IL-1α mRNA expression was increased by forskolin treatment (FSK; 20μM, 4h) in cultured astrocyte cultures (Mann–Whitney, P=0.0286, n=4). (B) Activation of Gq-DREADD receptors on astrocytes with CNO treatment (10 μM, 6h) did not significantly change IL-1α mRNA (Mann–Whitney, P>0.9999, n=4). (C) Activation of Gi-DREADD receptors on astrocytes with CNO did not lead to significant changes in IL-1α mRNA (Mann–Whitney, P=0.7000, n=3). (D) FSK treatment (20 μM; 4h) increased IL1β mRNA levels in astrocytes (Mann–Whitney, P=0.0227, n=7), as it did with IL-1α. (E) Activation of Gq-DREADD receptors in astrocytes with CNO did not lead to significant changes in IL-1β (Mann–Whitney, P=0.1801, n=7). (F) Activation of Gi-DREADD receptors in astrocytes with CNO did not lead to significant changes in IL-1β (Mann–Whitney, P=0.1462, n=7).

Assessment of other cytokines and activation markers in microglia.

(A) Microglia cultures were treated with 20μM Forskolin (FSK) for 4h, followed by qPCR to measure IL1a mRNA level. Forskolin led to a significant decrease in IL-1α (Mann–Whitney, P=0.0286, n=4) (B) Microglia cultures were treated with 15μM DHPG for 4h, followed by qPCR to measure IL1a mRNA level. No specific pattern of response was observed following Gq activation with DHPG (Mann–Whitney, P>0.9999 n=4). (C) Microglia cultures were treated with 50μM Baclofen for 4h, followed by qPCR to measure IL1a mRNA level. No specific pattern was observed following Gi activation with Baclofen (Mann–Whitney, P>0.9999, n=4). (D) Microglia cultures were treated with 20μM Forskolin (FSK) for 4h, followed by qPCR to measure IL1b mRNA level. There was a non-significant trend for an increase in IL-1β (Mann–Whitney, P=0.3143, n=4). (E) Microglia cultures were treated with 15μM DHPG for 4h, followed by qPCR to measure IL1β mRNA level. No specific pattern was observed following Gq activation with DHPG (Mann–Whitney, P>0.9999, n=4). (F) Microglia cultures were treated with 50μM Baclofen for 4h, followed by qPCR to measure IL1β mRNA level. No specific pattern was observed following Gi activation with Baclofen (Mann–Whitney, P=0.3143, n=4). (G) Microglia cultures were treated with 20μM Forskolin (FSK) for 4h, followed by qPCR to measure Iba1 mRNA level as a microglia activation marker. Forskolin led to a non-significant increase in Iba1 mRNA level (Mann–Whitney, P=0.3143, n=4). (H) Microglia cultures were treated with 15μM DHPG for 4h, followed by qPCR to measure Iba1 mRNA level. DHPG led to a significant increase in Iba1 mRNA level (Mann–Whitney, P=0.0286, n=4). (I) Microglia cultures were treated with 50μM Baclofen for 4h, followed by qPCR to measure Iba1 mRNA level. Baclofen also led to a significant increase in Iba1 mRNA level (Mann–Whitney, P=0.0286, n=4).

(A) iPSC quality control for colonies reprogrammed from fibroblasts. All three iPSC lines exhibited typical round morphology and expressed pluripotency markers including Sox2 and SSEA4. (B) IPSCs also expressed pluripotency markers Oct4 and TRA-1-60. (C) NPC quality control for neural progenitor cells differentiated from iPSC colonies. All three lines expressed the NPC markers PAX6 and SOX1. (D) The NPCs also expressed Nestin and SOX2.