GPCR-mediated regulation of glial TNF production

Reviewer #1 (Public review):

In this manuscript, the authors explore whether GPCR signaling in astrocytes affects the production of TNF by astrocytes and, to a lesser extent, microglia. Unfortunately, the method used by the authors to acquire astrocyte-enriched cultures is known to result in meaningful rates of contamination by myeloid cells (microglia and others), oligodendrocyte-lineage cells, and neurons. Alternative methods of generating highly enriched astrocyte cultures, as well as purifying astrocytes with little to no neuronal or myeloid contamination across age and brain regions, have shown no evidence of TNF expression by astrocytes (Zhang et al., J Neurosci, 2014; Zhang et al., Neuron, 2016; Clarke et al., PNAS, 2018). In fact, the paper cited by the authors as demonstrating differences between human and rodent astrocytes found no evidence of TNF expression in immature or mature human astrocytes (Zhang et al., Neuron, 2016). The idea that the majority of the observed TNF transcriptomic signal, at least in culture, comes from myeloid or neuronal contamination also aligns with the authors' observation that myeloid-enriched cultures act identically to astrocyte-enriched cultures.

The authors also use a GFAP virus to drive GPCR signaling in astrocytes and neuronal progenitor cells in their cultures, but, given that these cultures are known to have meaningful contamination by other cell types, such signaling could be due to astrocyte → microglia/neuron signaling or other multicellular pathways that cannot be excluded. Similar concerns mean that we cannot assume the effect of DREADD activation of astrocytes in vivo (Figure 6) reflects a bulk change in TNF expression driven by astrocyte-specific changes rather than by multicellular signaling.

The most compelling evidence for their claim of astrocyte TNF expression comes from the human-induced astrocytes. However, their antibody staining is not sufficient to claim these cells are truly astrocyte-like. Antibody staining is highly prone to non-specificity, as highlighted by the fact that their ALDH1L1 antibody staining appears perfectly nuclear despite ALDH1L1 being a cytoplasmic protein.

To address both the purity concerns of the astrocyte-enriched cultures and the concerns about the astrocyte identity of the induced astrocytes, the authors should perform RNA sequencing. By profiling gene expression in these cultures at the genome-wide level, readers can truly assess the degree of contamination and thus the likelihood of the proposed mechanism (i.e., astrocyte-specific TNF production). Importantly, previous studies have suggested that very little neuronal and myeloid contamination is required to dramatically change cellular responses (Foo et al., Neuron, 2011; Liddelow et al., Nature, 2017).

Reviewer #2 (Public review):

Summary:

Abbasi et al. examine how signaling through the major G-protein pathways (Gs, Gq, and Gi) influences tumor necrosis factor expression in astrocytes and microglia. Using a combination of pharmacological receptor activation, chemogenetic manipulation, primary rodent glial cultures, human induced pluripotent stem cell-derived astrocytes, and an in vivo astrocyte-targeted Gi manipulation, the authors report a broadly consistent pattern in which Gs- and Gq-associated signaling reduces tumor necrosis factor expression, whereas Gi signaling increases it. The study's cross-species and cross-preparation design, spanning astrocytes and microglia as well as in vitro and in vivo systems, provides a potentially valuable framework for understanding how neuromodulatory pathways may regulate glial inflammatory signaling.

Strengths:

A major strength of the study is the breadth of experimental systems used, which includes primary rat glia, human induced pluripotent stem cell-derived astrocytes, and an in vivo manipulation, allowing for comparison across species and levels of biological complexity. The use of chemogenetic receptors in astrocytes provides relatively direct control over Gq and Gi signaling, and these experiments yield consistent effects on both tumor necrosis factor messenger RNA and protein, strengthening the internal validity of the astrocyte findings. The observation that similar directional effects are seen in human-derived astrocytes and in microglial cultures further supports the idea that aspects of this regulatory relationship may be conserved across glial cell types. More broadly, the study addresses an important and timely question about how neuromodulatory signaling pathways interface with glial inflammatory outputs, and it generates a coherent set of observations that could serve as a foundation for more mechanistic work.

Weaknesses:

The central claim that Gs, Gq, and Gi signaling broadly and directly constitute a general regulatory code for tumor necrosis factor expression is more expansive than the current evidence fully supports. In particular, the evidence for Gs-dependent effects is indirect, relying on beta-adrenergic receptor activation and forskolin-mediated adenylyl cyclase stimulation rather than direct manipulation of Gs itself, leaving uncertainty about pathway specificity. More generally, the use of different endogenous receptors to represent each G-protein class in microglia complicates interpretation, since individual receptors may engage additional signaling pathways beyond their canonical G-protein coupling, limiting the extent to which the results can be attributed to G-protein class alone.

The in vivo experiment also does not definitively establish the cellular source of the observed increase in tumor necrosis factor, as measurements are taken from bulk cortical tissue following astrocyte-targeted Gi activation. This leaves open the possibility that the observed changes arise indirectly from other cell types, particularly microglia, which are shown elsewhere in the study to be strongly responsive to Gi-related manipulations. In addition, the specificity of chemogenetic expression in vivo is not quantitatively demonstrated, further limiting cell-type attribution.

There are also important issues related to experimental design and statistical interpretation. Across several experiments, it is unclear whether reported sample sizes reflect independent biological replicates, technical replicates, or imaging fields, which is especially consequential for the human induced pluripotent stem cell-derived astrocyte experiments where donor-level independence is not clearly established. The in vivo design also appears to treat hemispheres as independent observations despite their paired nature, which may inflate statistical independence given the small sample size.

Finally, several conclusions would benefit from more cautious framing. The data support differential regulation of tumor necrosis factor relative to interleukin-1 rather than strict cytokine specificity, and measurements based solely on messenger RNA should not be interpreted as direct evidence of cytokine production. The comparison between glial signaling effects and neuronal excitation or inhibition also juxtaposes fundamentally different biological readouts and should not be interpreted as a direct functional opposition. Overall, while the study provides interesting and potentially important observations, the broader pathway-level and cell-type-specific conclusions are not yet fully established by the current experimental evidence.