A Pvr–AP-1–Mmp1 signaling pathway is activated in astrocytes upon traumatic brain injury

Reviewer #1 (Public Review):

Li et al report that upon traumatic brain injury (TBI), Pvr signalling in astrocytes activates the JNK pathway and up-regulates the expression of the well-known JNK target MMP1. The FACS sort astrocytes, and carry out RNAseq analysis, which identifies pvr as well as genes of the JNK pathway as particularly up-regulated after TBI. They use conventional genetics loss of function, gain of function and epistasis analysis with and without TBI to verify the involvement of the Pvr-JNK-MMP1 signalling pathway.

The strengths are that multiple experiments are used to demonstrate that TBI in their hands damaged the BBB, induced apoptosis and increased MMP1 levels. The RNAseq analysis on FACS sorted astrocytes is nice and will be valuable to scientists beyond the confines of this paper. The functional genetic analysis is conventional, yet sound, and supports claims of JNK and MMP1 functioning downstream of Pvr in the TBI context.

However, the weaknesses are that novelty and insight are both rather limited, some data are incomplete and other data do not support some claims. Some approaches used lacked resolution and some experiments lacked rigour. The authors may wish to improve some of their data as this would make their case more convincing. Alternatively, they should remove unsupported claims.

Novelty and insight:
Others had previously published that both JNK signalling and MMP1 were activated upon injury, in multiple contexts (as well as the articles cited by the authors, they should also see Losada-Perez et al 2021). That Pvr can regulate JNK signalling was also known (Ishimaru et al 2004). And it was also known that astrocytes can respond to injury by proliferating, both in larval ventral nerve cords and adult brains (Kato et al 2011; Losada-Perez et al 2016; Harrison et al 2021; Simoes et al 2022). The authors argue that the novelty of the work is the investigation of the response of astrocytes to TBI. However, this is of somewhat limited scope. The authors mention that Mmp1 regulates tissue remodelling, the inflammatory process and cancer. Exploring these functions further would have been an interesting addition, but the authors do not investigate what consequences the up-regulation of Mmp1 after injury has in repair or regeneration processes.

Incomplete or unconvincing data:
The authors failed to detect PCNA-GFP and pH3 in brains after TBI and conclude that that TBI does not induce astrocyte proliferation. However, this is a surprising claim, as it would be rather different from all previous prevalent observations of cell proliferation induced by injury. Cell proliferation can be notoriously difficult to detect (ie due to timing and sample size), thus instead this raises doubts on the experimental protocol or execution.
Others have previously reported: cells in S- phase using PCNA-GFP and other reporters (eg BrdU, EdU, FUCCI) in the intact adult brain (Kato et al 2009; Foo et al 2017; Li et al 2020; Fernandez-Hernandez et al 2013; Simoes et al 2022); that injury to the adult brain and VNC induces cell proliferation that can be detected with cell proliferation markers like BrdU, Myc, FUCCI and the mitotic marker pH3 (Kato et al 2009; Fernandez-Hernandez et al 2013; Losada-Perez et al 2021; Simoes et al 2022); and that injury to the brain and CNS induces glial proliferation in adult and larval brains/CNS, specifically of astrocytes (Kato et al 2011; Losada-Perez et al 2016; Fernandez-Hernandez et al 2013; Simoes et al 2022). Thus, the fact that they did not observe PCNAGFP+ cells in control, intact adult brains nor after TBI could suggest that they had technical, experimental difficulties. Detecting mitotic cells with anti-pH3 is difficult because M phase is very brief, but others have succeeded (Simoes et al 2022). Given that in all previous reports mentioned above cells were seen to proliferate after injury in the CNS, it would be rather surprising if no cell proliferation occurred after TBI. Resolving this conflicting result is important, as it could imply that TBI induces very different cellular responses from various other lesions or injury types. It is conceivably not impossible, but the most parsimonious start point would be that multiple injury types could cause equivalent responses in cells. Thus, the authors ought to consider whether technical or experimental design problems affected their experimental outcome instead.

Other claims not supported by data:
(1) astrocyte hypertrophy, as the tools used do not have the resolution to support this claim;

(2) localisation of anti-Pvr to specific cells, as the images show uniform signal or background instead;

(3) astrocytes do not engulf cell debris after TBI, as the tools and images do not have the resolution to make this claim.

The authors could improve these data with alternative experiments to maintain the claims; alternatively, these unsupported claims should be removed.

Statistical analysis:
The statistical analysis needs revising as it is wrong in multiple places. Revising the statistics will also require revision of the validity of the claims and adjusting interpretations accordingly.

Altogether, this is an interesting and valuable addition to the repertoire of articles investigating neuron-glia communication and glial responses to injury in the Drosophila central nervous system (CNS). It is good and important to see this research area in Drosophila grow. This community together is building a compelling case for using Drosophila and its unparalleled powerful genetics to investigate nervous system injury, regeneration and repair, with important implications. Thus, this paper will be of interest to scientists investigating injury responses in the CNS using Drosophila, other model organisms (eg mice, fish) and humans.

Reviewer #2 (Public Review):

In this study, Li et al. examined the involvement of astrocyte-like glia in responding to traumatic brain injury in Drosophila. Using a previously-established method that induces high-impact, whole-body trauma to flies (HIT device), the authors observed increased blood-brain-barrier permeabilization, neuronal cell death, and hypertrophy of astrocyte-like cells in the fly brain following injury. The authors provide compelling evidence implicating a signaling pathway involving the PDGF/VEGF-like Pvr receptor tyrosine kinase, the AP-1 transcription factor, and the matrix metalloprotease Mmp1 in the astrocytic cell response to TBI. The authors' data was generally high-quality data and combined multiple experimental approaches (microscopy, RNA sequencing, and transgenic), increasing the rigor of the study. The identification of injury-induced gene expression changes in astrocytic cells helps increase our limited understanding of roles this glial subtype plays in the adult fly brain. Limitations of the study include a reliance on RNAi-mediated gene silencing without validation via genetic mutants and a limited examination of how astrocyte-like and ensheathing glia could interact following TBI, especially given that several genes identified in this study are known to mediate ensheathing glial responses to axotomy. The conclusions are generally well-supported by the presented data, however some further clarification of quantitative methods and analyses will help to strengthen the findings:

1. The significance and quantification method for the astrocytic cell body sizes in Fig. 2C, D and appearance of GFP+ accumulations in Fig. 2F should be better defined - how were cell bodies and GFP+ puncta identified relative to other astrocytic cell structures, are they homogeneous in size/intensity in different brain regions following injury, and what could the GFP+ puncta represent?
2. The relative contributions of astrocyte-like and ensheathing glia in the brain's response to TBI is unclear. RNA sequencing identified Mmp1 and Draper as genes upregulated following TBI, however, these genes have previously been implicated in ensheathing glial (and not astrocytic) responses to acute nerve injury. The authors provide convincing evidence that their transcriptomic data is devoid of neuronal genes, but what about the possibility of ensheathing glial contaminants? Figures 2I-Q suggest that the majority of Mmp1 protein co-localizes with ensheathing rather than astrocytic glial membranes following TBI. Does knockdown of Pvr, Jra, or kay in ensheathing glia affect Mmp1 upregulation following injury? A closer examination of how these two glial subtypes contribute to and interact-and what proportion of Mmp1 is cell autonomous to astrocytes-during injury responses would be valuable.
3. The authors rely on RNAi and overexpression methods to manipulate expression of candidate genes in Figures 4, 5, and 7. In most cases, only a single RNAi line is used to reduce expression of a candidate gene, increasing the possibilities of off-target effects or insufficient gene knockdown. These data could be strengthened by using multiple RNAi lines as well as mutants to validate findings for Pvr, Jra, and kay knockdown in Figures 4 and 5, and perhaps confirmation of knockdown efficiency, particularly in Fig. 7.
4. Single channel images should be included in Fig. 1L and M to help strengthen the conclusion that Dcp-1+ puncta are elav+ and repo-.
5. Sample sizes and a description of power analysis should be included in figure legends/methods. Based on the graphs, some sample sizes appear low (e.g., Fig. 1H+K and 2D+Q).

Reviewer #3 (Public Review):

In this study, authors used the Drosophila model to characterize molecular details underlying traumatic brain injury (TBI). The authors used the transcriptomic analysis of astrocytes collected by FACS sorting of cells derived from Drosophila heads following brain injury and identified upregulation of multiple genes, such as Pvr receptor, Jun, Fos, and MMP1. Additional studies identified that Pvr positively activates AP-1 transciption factor (TF) complex consisting of Jun and Fos, of which activation leads to the induction of MMP1. Finally, authors found that disruption of endocytosis and endocytotic trafficking facilitates Pvr signaling and subsequently leads to induction of AP-1 and MMP1.

Overall, this study provides important clues to understanding molecular mechanisms underlying TBI. The identified molecules linked to TBI in astrocytes could be potential targets for developing effective therapeutics. The obtained data from transcriptional profiling of astrocytes will be useful for future follow-up studies. The manuscript is well-organized and easy to read. However, I would like to request the authors to address the following issue to improve the quality of their study.

It is unclear why the authors did not explore the involvement of the JNK pathway in their study. While they described the potential involvement of the JNK pathway based on previous literature, they did not include any evidence on the JNK pathway in their own study.

It is important to note that the mechanism by which JNK activates AP-1 is primarily through phosphorylation, not the quantitative control of amounts, as much as I know. This raises questions about the authors' proposed hierarchical relationship between Pvr and AP-1 and the potential involvement of the JNK pathway in mediating this relationship.

Given the significance of the mechanistic link between Pvr and AP-1 in solidifying the authors' conclusion, it would have been beneficial for them to explore the involvement of the JNK pathway in their study, even if only minimally. The lack of such exploration may weaken the overall strength of their findings and the potential implications for understanding TBI.