Decreased Astrocytic CCL5 by MiR-324-5p Ameliorates Ischemic Stroke Injury via the CCR5/ERK/CREB Pathway

Reviewer #1 (Public review):

Summary:

Here, the authors attempt to show that CCL5 is increased after stroke, possibly due to decreased miR-324, and that this is a modifiable system to decrease stroke damage. By bidirectionally manipulating CCL5 levels through direct injection of CCL5; a CCL5 blocking antibody; miR324; miR324 antagomir; or CCR5-blocking Maraviroc, they broadly show improvement with lower CCL5 levels. This includes infarct size, behavioral analysis, and immunohistochemical analysis of astrocytes, microglia, and neurons. They further try to mechanistically tie miR324 and CCL5 in astrocytes specifically to stroke-induced changes using a neuronal/astrocytic coculture system. They argue that decreasing CCL5 leads to increased ERK and CREB phosphorylation as a potential neuroprotective mechanism. CCL5 is one potential ligand for CCR5, and recent work identified CCR5 as a targetable mechanism by clinically-approved drug Maraviroc to enhance stroke recovery. Particularly given the high level of interest in CCR5 in stroke recovery, the focus on CCL5 - one of CCR5's potential ligands - and its miR regulation is an exciting expansion of this area of stroke biology.

Strengths:

The authors' findings that decreasing CCL5 acutely after stroke shows behavioral improvement appear robust. This broadly replicates work from other groups, although the finding that miR324 manipulation can phenocopy direct CCL5 manipulation is novel and intriguing. However, many of their other claims are difficult to evaluate based on a combination of missing methodological information, inappropriate statistical testing, and a flawed culture system.

Weaknesses:

Broadly speaking, the manuscript takes a zoomed-out view of what is fundamentally highly localized biology.

(1) miRNA-based regulation, by definition, has to include miR and mRNA in the same cell type; as the authors note, CCL5 is expressed in many cells. It is therefore impossible to propose any interaction on the basis of the tissue-level changes described; any evidence of in vivo cell-type specificity would dramatically improve the claims.

(2) The authors treat an extensive area of ipsilesional cortex uniformly as "IP". Astrocytic and microglial responses to localized injuries such as stroke are highly location-dependent and undoubtedly change dramatically within this area. The presented data cannot be interpreted without confirmation that these were taken at identical distances from the injury, and what that distance was. These do not appear to be adjacent to the injury, where the responses would presumably be the most informative. Similarly, it is difficult to interpret the neuronal Sholl and spine data without more information on where within the large IP region these neurons were found.

The authors attempt to narrow in on cell-type specificity via culture. However, astrocytes are notoriously prone to a dramatic change in culture and require careful methods (immunopanning; see eg doi: 10.1016/j.neuron.2011.07.022) to maintain much resemblance to their in vivo counterpart. It is difficult to conclude much about the role of astrocytes in the CCL5 pathway based on the use of this shaking-based culture system, particularly in the absence of cell-type specific validation in vivo.

There is missing methodological information, including infarct size measurements, TUNEL staining, and statistical testing. The TTC figures look very odd, like a collection of overlapping stars have been placed on the images rather than the natural relatively smooth infarct edges one would expect. It is unclear if the infarct volume measurements accounted for edema, as is standard; there is no description of the protocol used for quantification. It is also unclear if the infarct volume measurement comparisons were also done with t-tests vs ANOVA, as the statistical test used is not listed in the figure legends. In numerous cases where statistical testing is listed, repeated t-tests between subgroups are used vs the more appropriate ANOVA (assuming normality; nonparametric testing as appropriate), making it difficult to have confidence in the results.

Reviewer #2 (Public review):

The authors presented evidence from various in vivo and in vitro experiments demonstrating the mutual interaction between CCL5 and astrocytic miR-342-5p in the ipsilateral core of cerebral ischemia. However, miR-342-5p was downregulated only late after MCAO (D3-7). Additionally, this downregulation was observed not only in the ipsilateral core but also in the ipsilateral penumbra and contralateral sides. Therefore, it is not convincing that the upregulation of CCL5 in the ipsilateral core at later time points (D3 and D7) is attributable to the decreased expression of miR-342-5p. In particular, infarct injury was already evident within a short time period (say 24 h) following MCAO.

(1) The temporal and spatial expression patterns of miR-324-5p do not match those of CCL-5, especially at D1 and D3 (see Figure 1C, 1D). Despite the inverse relationship between miR-324-5p and CCL-5 expression at D7 after MCAO, what was the purpose of administering miR-324-5p agomir (or antagomir) at D1 post-MCAO? If the connection cannot be clearly established, the conclusion reached at the end will be difficult to accept.

(2) Would administering miR-342-5p or anti-CCL5 at later time points (e.g., after D3) reduce infarct size or improve functional recovery? If this is not the case, the effect of CCL5 on neuronal cell damage (infarct size formation) must occur within a very short time after MCAO. Additionally, if the increased CCL5 expression is due to the downregulation of miR-342-5p, its impact would likely be less significant.

(3) While the study offers valuable insights into the roles of CCL5 and its connection with the regulation of miR-342-5p (though this connection is somewhat weak), it is recommended that the authors explore potential translational applications of these findings.

Overall, given the experimental designs and results, it is difficult to support the conclusions drawn in the manuscript.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

(1) miRNA-based regulation, by definition, requires the miRNA and its target mRNA to be present in the same cell type. CCL5 is expressed in many cell types, making it impossible to propose any miRNA–mRNA interaction based solely on tissue-level expression changes. In vivo cell-type specificity data would substantially strengthen the claims.

We fully agree with this critique, and we consider this the most critical point to address. To directly examine cell-type-specific expression in vivo, we performed FISH combined with immunofluorescence for GFAP (astrocytes) or IBA1 (microglia) in the peri-infarct region of MCAO mice at D3 (Figure 1E–G). Our results show that Ccl5 mRNA is expressed in both GFAP-positive astrocytes and IBA1-positive microglia, with comparable co-localization rates across both cell populations. In contrast, miR-324-5p showed a significantly higher positive rate in astrocytes than in microglia (Figure 1G). Given that miR-324-5p is more abundantly expressed in astrocytes, the regulatory capacity of the miR-324-5p/CCL5 axis is predicted to be more pronounced in this cell type. These findings provide direct in vivo evidence supporting an astrocyte-predominant miR-324-5p/CCL5 regulatory interaction in the peri-infarct region. We acknowledge that the current data do not constitute cell-type-specific in vivo manipulation, and we discuss this limitation and propose future directions (including viral or transgenic approaches) in the revised Discussion.

(2) The authors treat an extensive area of ipsilesional cortex uniformly as "IP." Astrocytic and microglial responses to localized injuries such as stroke are highly location-dependent and undoubtedly change dramatically within this area. Data cannot be interpreted without confirmation that samples were collected at identical, defined distances from the injury. Similarly, it is difficult to interpret the Sholl and spine data without knowing where within the large IP region these neurons were found.

We thank the reviewer for identifying this important concern. Upon review, we recognized that part of the tissue samples used for qPCR, ELISA and Western blot in the original submission were collected from an excessively broad region of the ipsilateral cortex, which likely introduced heterogeneity into the data. We have re-collected these samples specifically from the peri-infarct zone, defined as the 1–2 mm cortical rim immediately surrounding the visibly pale infarct core, beginning from the second and third coronal slices from the most rostral aspect of the cerebral cortex. The qPCR, ELISA and Western blot data have been updated accordingly (Figures 1C–D, 2A, 3A, 6A), and the sampling definition has been specified in the Methods. We also confirmed that all immunofluorescence and Golgi staining analyses were performed within this same peri-infarct zone, where cells retain intact morphology and show the most informative between-group differences. This sampling region has now been defined consistently across all in vivo analyses in the revised manuscript.

(3) Astrocytes are notoriously prone to dramatic change in serum-containing culture. The shaking-based culture system makes it difficult to conclude much about the role of astrocytes in the CCL5 pathway, particularly without cell-type-specific validation in vivo.

We acknowledge this limitation. We attempted to implement the immunopanning protocol described by Barres et al. (doi: 10.1016/j.neuron.2011.07.022) to obtain a more purified astrocyte culture. However, several essential reagents — including sodium selenite, putrescine, and N-acetyl-L-cysteine — could not be procured due to import and purchasing restrictions in our region. The use of a commercially available O4 antibody substitute (clone O4, R&D, MAB1326) in place of O4 hybridoma supernatant, combined with the absence of these chemicals, likely contributed to the very low astrocyte yields and poor cell viability observed across multiple independent attempts. We therefore retained the shaking-based isolation and purification method for the present study.

To characterize the composition of our primary cortical astrocyte cultures, we have included immunofluorescence data from co-labeling of GFAP with Tuj1, Olig2, and IBA1 at P0 and P1 in Supplementary Figure S4, confirming that GFAP-positive cells comprised approximately 88% of total cells at P1. We have also added a paragraph to the Discussion acknowledging that more refined culture systems, as well as cell-type-specific in vivo manipulation of CCL5 and miR-324-5p via viral or transgenic approaches, would further consolidate the conclusions of the present study.

(4) Missing methodological information, including infarct size measurements, TUNEL staining, and statistical testing.

We apologize for these omissions. Detailed descriptions of infarct volume quantification (including the edema-correction formula), TUNEL staining procedures, NeuN/TUNEL co-labeling, and all statistical tests have been added to the Methods section.

(5) The TTC figures appear unusual, with infarct edges resembling overlapping stars rather than natural smooth boundaries. It is unclear whether infarct volume measurements accounted for edema, and no quantification protocol is described.

We apologize for the confusion. The unusual appearance of the infarct edges in the original TTC figures resulted from dotted-line annotations we had added to highlight the infarct boundaries; these have now been removed to present the unmodified TTC images (Figure 2B, 3B). Infarct volume was corrected for edema-induced hemispheric swelling using the formula: [(contralateral hemisphere volume − ipsilateral non-infarcted volume) / contralateral hemisphere volume] × 100%. This formula and the complete quantification protocol have been added to the Methods section.

(6) Repeated t-tests between subgroups are used instead of the more appropriate ANOVA, making it difficult to have confidence in the results.

We agree. We identified that t-tests had been applied inappropriately in the original qPCR and ELISA analyses. All qPCR and ELISA data have been re-analyzed using two-way ANOVA with Tukey's post-hoc test, as appropriate for datasets with multiple groups and time points. We have also reviewed all other figures and corrected any inappropriate use of t-tests.

Reviewer #2 (Public review):

(1) The temporal and spatial expression patterns of miR-324-5p do not match those of CCL5, especially at D1 and D3. Despite the inverse relationship between miR-324-5p and CCL5 being apparent only at D7 after MCAO, what was the purpose of administering miR-324-5p agomir (or antagomir) at D1 post-MCAO? If the connection cannot be clearly established, the conclusion reached at the end will be difficult to accept.

We thank the reviewer for identifying this critical issue. Upon re-examination, we recognized that the original qPCR and ELISA samples had been collected from an excessively broad region of the ipsilateral cortex, which likely introduced heterogeneity into the expression data and obscured the true temporal dynamics in the viable peri-infarct tissue. We have re-collected samples specifically from the peri-infarct zone — defined as the 1–2 mm cortical rim immediately surrounding the infarct core — and updated the data accordingly (Figures 1C–D, 2A, 3A).

The updated data reveal a clearer and more consistent temporal pattern. Ccl5 mRNA levels in the IP region are significantly elevated as early as D1 compared with sham controls, and continue to increase progressively through D7. Regarding miR-324-5p, although IP region levels at D1 do not yet differ significantly from sham controls, they are already significantly lower than in the contralateral CP region at this early time point. This ipsilateral-versus-contralateral difference at D1 indicates that miR-324-5p downregulation begins in the acute phase following stroke, even before it reaches statistical significance relative to the sham baseline. From D3 onwards, miR-324-5p levels in the IP region are significantly reduced relative to both sham and CP groups, coinciding with the period of sustained and progressive CCL5 upregulation. Taken together, these updated findings support an early and progressive inverse relationship between miR-324-5p and CCL5 in the peri-infarct cortex following MCAO, consistent with our previously published finding that miR-324-5p suppresses astrocytic CCL5 expression (Sun et al., Cell Death Dis., 2019), and functionally validated by the ELISA data showing that miR-324-5p agomir injection significantly reduces CCL5 protein concentrations in the IP region at D3 and D7 (Figure 3A).

Regarding the rationale for administering miR-324-5p agomir at D1: this timing was chosen to model a clinically realistic therapeutic scenario targeting the early post-stroke period. MicroRNA agomir/antagomir interventions typically require 2–10 days to achieve peak target gene modulation in the mouse brain; administration at D1 therefore ensures that meaningful miR-324-5p-mediated suppression of CCL5 is achieved during the critical acute-to-subacute transition period, as confirmed by the ELISA results at D3 (Figure 3A).

(2) Would administering miR-342-5p or anti-CCL5 at later time points (e.g., after D3) reduce infarct size or improve functional recovery? If this is not the case, the effect of CCL5 on neuronal cell damage must occur within a very short time after MCAO. Additionally, if the increased CCL5 expression is due to the downregulation of miR-342-5p, its impact would likely be less significant.

We acknowledge that the current study did not include experimental groups with delayed administration, and we recognize this as a limitation.

However, several points inform our interpretation. First, as described in our response to Comment 1 above, the updated data demonstrate that miR-324-5p downregulation in the IP region is already detectable relative to the contralateral CP region at D1 and progresses further through D3 and D7, indicating that the miR-324-5p/CCL5 regulatory axis is engaged from the acute phase of stroke, providing a biological basis for early intervention. Second, in experimental stroke models, the infarct core is largely established within the first 24–72 h following vessel occlusion, with the majority of ischemic neuronal death occurring during this window. CCL5, as a pro-inflammatory mediator, is expected to amplify immune cell recruitment and inflammatory cascades most consequentially during this early period, making D1 administration mechanistically rational for limiting neuronal loss. Third, the superior early behavioral outcomes in the CCL5 antibody group relative to the miR-324-5p agomir group (Figures 2D–E, 3D–E) support the value of early CCL5 suppression. As the antibody acts immediately while the agomir requires time for post-transcriptional regulation, this difference highlights that timely agomir delivery is essential to achieve effective CCL5 suppression during the critical early window.

(3) The study would benefit from the exploration of potential translational applications.

We thank the reviewer for this constructive suggestion. We are planning to investigate whether astrocyte-derived extracellular vesicles engineered to overexpress miR-324-5p can enhance neurological recovery after stroke. Extracellular vesicles offer several translational advantages: they can traverse the blood-brain barrier, provide a stable and biocompatible vehicle for miRNA delivery, and may be less immunogenic than viral approaches. This would leverage the neuroprotective regulatory mechanism identified in the present study while offering a clinically viable delivery strategy.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) In vivo cell-type specificity data for CCL5/miR-324-5p would substantially strengthen the manuscript.

We have performed FISH combined with GFAP and IBA1 immunofluorescence in the peri-infarct region of MCAO mice at D3 to characterize the cell-type-specific in situ expression of Ccl5 mRNA and miR-324-5p (Figure 1E–G).

(2) Cell-type-specific in vivo manipulation of CCL5/miR-324-5p (e.g., virally or transgenically) would further substantiate the conclusions. A more compelling astrocytic culture model is needed.

We fully agree that cell-type-specific in vivo manipulation would represent a major advance. As described in our response to Comment 3 above, we were unable to successfully implement immunopanning in the current study. The FISH data provide in vivo evidence supporting the astrocyte-enriched expression of miR-324-5p in the peri-infarct region. We have added a Discussion paragraph explicitly identifying viral or transgenic astrocyte-specific manipulation of CCL5 and miR-324-5p in vivo as a critical next step to validate and extend the conclusions of this study.

(3) The measurement shown in Figure 4D is unclear. A more informative measure might be TUNEL/DAPI, with additional cell-type-specific markers to identify what cells are dying in what proportions.

We agree with this suggestion and have revised the quantification accordingly. In the updated manuscript, apoptotic cell death is reported as the proportion of TUNEL-positive cells among total DAPI-positive nuclei (TUNEL/DAPI). As a complementary cell-type-specific measure, we quantified the proportion of NeuN-positive neurons among total DAPI-positive nuclei (NeuN/DAPI) to specifically assess neuronal survival within the co-culture system. These two measures together provide a clear and interpretable readout of both overall cell death and neuronal viability under each experimental condition. The revised quantification is presented in Figure 4C–E and Figure 5B–D.

(4) qPCR and ELISA data should be normalized to internal controls, sham values should be presented, and ANOVA (or appropriate non-parametric tests) should be used.

All qPCR data are now normalized to Gapdh (for mRNA) or U6 snRNA (for miRNA). Sham group values are presented in all relevant figures. Two-way ANOVA with Tukey's post-hoc test is now used for all qPCR and ELISA comparisons. The updated statistical approach is summarized in the Statistical Analysis section of the Methods.

(5) The description "within 24 hrs" for the timing of CCL5 in vivo manipulation is ambiguous.

We have revised the description to "at 24 h post-MCAO" throughout the Methods and Results sections to specify the precise time point of intervention.

(6) Only some statistical comparisons are shown in Figure 2E, which inaccurately implies that the other groups are not different.

We have updated Figure 2E and Figure 3E to include all statistically significant pairwise comparisons, ensuring that the significance markers accurately represent the complete set of statistical relationships among all groups.

(7) "Activation" is not the appropriate term for astrocytes in pathological contexts; A1/A2 terminology should be removed.

We thank the reviewer for this important correction. In line with the consensus recommendations by Escartin et al. (Nat Neurosci, 2021), we have replaced all instances of "astrocyte activation" in pathological contexts with "astrocyte reactivity" or "reactive astrogliosis" throughout the manuscript, including the Abstract, Results, and Discussion. All references to A1 and A2 subtypes have been removed from the Discussion.

(8) There are typographical errors, and the repeated use of "Besides" is awkward.

We have carefully proofread the entire manuscript to correct typographical errors. All instances of "Besides" used as a sentence-opening connector have been replaced with contextually appropriate alternatives, such as "Furthermore," "Moreover," "In addition," or "Additionally."