Weight-drop model of mild brain injury caused motor and sensory cortex dysfunction in CCL5 knockout (CCL5-KO) mice

Both wild-type (WT, C57BL/6) and CCL5-KO mice were given mild brain injury using a weight-drop (WD) system at about two months of age as in our previous study [22]; the weight drop induced head injury as shown in Fig. 1A. The redlined circled impact area includes the mouse motor and sensory cortex. The expression level of CCL5 in WT mouse cortical tissues was about 40 pg/mg in the cortex, which rapidly increased to 60–160 pg/mg after 1-, 4-, and 7-day post-injury (dpi) by ELISA assay (Fig. 1B). The mNSS was used to evaluate neurological injury after 1-, 7-, 14-, and 28 days after injury. The mNSS score was lower than 4 in all four groups at all time-points consistent with a mild injury in mice. The mNSS score slightly increased at 1 dpi and was reduced to 1 at 7 dpi in the WT-mTBI group, indicating a functional recovery in WT mice. In contrast, the mNSS score was not reduced in CCL5-KO mice even one month after brain injury (Fig. 1C). Motor cortex function and sensory function were evaluated by Rotarod, beam walking, and limb adhesive removal tests (ART). The coordination and balance of mice on the Rotarod (time falling from Rotarod) after mTBI was significantly reduced in both WT and KO mice at 1 dpi (Fig. 1D). As with mNSS, the balance performance on Rotarod improved in the WT mTBI group of mice but not in CCL-KO mice after 7 dpi (Fig. 1D). The number of foot faults on beam walking was slightly increased in WT mice but markedly increased in CCL5-KO at 1-dpi (Fig. 1E). This parameter improved after 7–14 dpi in both groups of mice (Fig. 1E). The walking time over the beam was 10.25 ± 0.716 s in WT sham and 14.67 ± 0.99 s in WT mTBI groups at 1 dpi (p = 0.002); in KO mice, the walking time at 1-dpi was 12.65 ± 1.36 s in the sham group and 24.62 ± 2.73 s in the TBI group (p = 0.002, compared to sham; p = 0.0082 compared to WT TBI group). The time to remove adhesive stickers on the paws also increased in both WT and KO mice after mTBI, which improved more slowly in CCL5-KO mice after 7–14 dpi compared to 1–7 dpi in WT mice (Fig. 1F). Taken together, the delayed recovery suggests expression of CCL5 is important for neural functional recovery after mild TBI.

The recovery of axonal injury and synapse reformation was impaired in the CCL5-KO cortex after mild TBI

Golgi staining was used to show the neurite structures in cortical neurons adjacent to the injured region (Fig. 2A, F). The number of intersections from soma and total intersections of neurites were evaluated by Sholl analysis. The number of intersections, total intersections, and spine density were reduced, and the number of swollen spines was increased in WT mice after 14 days of injury (Fig. 2B–E) when cortical function performance had already recovered. Those parameters were improved after 28 days as enhanced numbers of intersections, total intersections, and spine density (Fig. 2B–D); the swollen spines were also reduced after 28 days (Fig. 2E). In contrast, the number of intersections, total intersections and the spine density continued to be reduced in CCL5-KO mouse cortex after mTBI over 14 to 28 dpi (Fig. 2G–I), and the number of swollen spines also continually increased (Fig. 2J). The level of synaptic proteins—PSD95 reduced slightly and synaptophysin levels reduced and were maintained in CCL5-KO mouse cortex tissue over 14 to 28 dpi (Fig. 2L); this was not seen in the WT cortex after injury (Fig. 2K).

Fig. 2

The recovery of axonal injury was impaired in the CCL5-KO cortex after mild TBI. Golgi staining revealed the axon and spine structures in WT and CCL5-KO mouse cortex with sham treatment and mild TBI – 14 and − 28 days of injury (dpi). A, F The representative images of neurites and spine structures in WT and CCL5-KO mouse cortex; boxed regions were enlarged on the right. Black arrowheads point to the normal dendritic spines, and white arrows point to swollen neurites and spines. Scale bar = 1 mm and 20 μm. B, G The number of intersections (WT sham vs. 14 dpi, p < 0.0001; WT 14 dpi vs. 28 dpi, p < 0.0001; KO sham vs. 14 dpi, p < 0.0001; KO 14 dpi vs. 28 dpi, p < 0.0001. Data were analyzed by two-way ANOVA and presented as mean ± SEM), C, H total intersections (WT sham vs. 14 dpi, p = 0.0018; WT 14 dpi vs. 28 dpi, p = 0.0493; KO sham vs. 14 dpi, p < 0.0001; KO 14 dpi vs. 28 dpi, p = 0.0004.), D, I spine density (WT sham vs. 14 dpi, p < 0.0001; WT 14 dpi vs. 28 dpi, p < 0.0001; WT sham vs. 28 dpi, no significant difference, NS; KO sham vs. 14 dpi, p < 0.0001; KO 14dpi vs. 28 dpi, p = 0.0399; KO sham vs. 28 dpi, p < 0.0001.), and E, J swollen spines (WT sham vs. 14 dpi, p < 0.0001; WT 14 dpi vs. 28 dpi, p < 0.0001; WT sham vs. 28 dpi, p = 0.0231; KO sham vs. 14 dpi, p = 0.0004; KO 14dpi vs. 28dpi, p = 0.0229; KO sham vs. 28 dpi, p < 0.0001.) were quantified in different groups of mice (n = 10 in each group). Data in C–E and H–J were analyzed by unpaired t-test and presented as mean ± SEM. K, L The expression of synaptic proteins – PSD95 and synaptophysin in different groups of WT and CCL5-KO mouse cortex, including sham, 4, 7, 14, and 28 dpi, was analyzed by western blot. Quantification of results from 3 independent mouse samples in each group is listed above the images of protein blots in 2 K–L. (KO PSD95: sham vs. 4 dpi, p = 0.0015; sham vs. 7 dpi, p = 0.0038; sham vs. 14 dpi, p = 0.0147; sham vs. 28 dpi, p = 0.0025. KO Synaptophysin: sham vs. 4 dpi, p = 0.0126; sham vs. 7 dpi, p = 0.0019; sham vs. 14 dpi, p = 0.0089; sham vs. 28 dpi, p = 0.0186. Data were presented as mean ± SEM and analyzed by t-test following Mann-Whitney test)

Taken together, these findings suggest that impairment of cortical motor and sensory function in CCL5-KO mice might result from the loss of neurites and spines after mTBI. The repair of neurites and regrowth of spines was manifested around post-injury 14–28 days according to Golgi staining in WT, and this repair process may be compromised by lack of CCL5 in the KO animals.

Reduced activation of axonogenesis, synaptogenesis, and myelination signaling molecules in CCL5-KO cortex after mTBI

To identify the protein profile changes in the damaged brain and its correlation with axon injury in CCL5-KO mice after mild TBI, tissues from the CCL5-KO sham and injured mouse cortex were harvested and then analyzed by LC-MS/MS. Gene Ontology (GO) enrichment analysis of the entire list of 932 proteins was carried out and identified 183 proteins, including 84 up-regulated and 99 down-regulated proteins (adjusted p-value < 0.05) (Fig. 3A and Supplementary Fig. 1A, also see Supplementary Data file 1); identified GO terms for each category are shown in Fig. 3B and Supplementary Data File 2. Synapse and intermediate filament-related proteins were identified in the cellular component category (Fig. 3B, blue characters). Ingenuity Pathway Analysis (IPA) revealed that the major affected disease and function moieties were associated with cell morphology, organization, and development; organ degeneration, neurodegeneration, and necrosis were increased in the Organ injury category (Fig. 3C, Supplementary Fig. 1B). Nervous system development and function were also the major affected disease and function categories; an outgrowth of neurites, axonogenesis, and myelination were reduced in the Nerve system category (Fig. 3D, Supplementary Fig. 1B). Canonical pathways analysis of nervous system subcategories identified signaling pathways primarily associated with axonogenesis, axon guidance, and neuritogenesis (Supplementary Fig. 1C). These were EIF2, eIf4, and p70S6K, Rho family GTPase, axonal guidance, CDK5, semaphorin, mTOR, Ephrin, and ERK/MAPK signaling (Fig. 3E, F). Neuregulin and ERBB signaling are myelination-related signaling molecules (Fig. 3G). Proteins identified in each pathway are listed in the Supplementary Data file 3. We detected the activation of axon guidance-related signaling molecules—Semaphorin 3A, EphinA5, EphA4, and eIF2a, which were significantly lower in the CCL5-KO groups of mice (Fig. 3H, H’). Downstream signaling molecules—phosphorylation of mTOR (S2448), which is also involved in synaptogenesis, was increased in the WT-TBI group but reduced in the CCL5-KO TBI group by western blot analysis (Fig. 3I, I’). This suggests that mTOR-related function in axonogenesis and synaptogenesis was impaired in CCL5-KO after injury.

Fig. 3

LC-MS/MS analysis identified a reduction of axonogenesis and myelination signaling pathways in CCL5-KO mice with mild brain injury. A Venn diagram comparing DEPs (differentially expressed proteins) and volcano plot of significant DEPs between sham and mild TBI CCL5-KO mouse cortex. DEPs: p-value < 0.05 in comparison to sham control, respectively. Colored points represent log2 ratio > 0 upregulated protein (red) and log2 ratio < 0 downregulated protein (blue). Selected axonogenesis and myelination pathway-related proteins are highlighted as indicated (Red: myelinations, Green: axonogenesis and synaptogenesis, Yellow: overlapping). Results from Gene Ontology (GO) enrichment analysis of 183 identified proteins (84 up regulated, 99 down regulated) against a background list of all known mouse protein symbols. B Identified GO terms from the three GO groups are shown (Green bar: biological process, Red bar: cellular component, Blue bar: molecular function. Blue character: neuron function related). The strength of enrichment of each GO term was indicated by the Log10 p-value (X-axis). C, D IPA analysis identified affected diseases and function in organ injury (C) and nervous system (D) categories. Z-score values indicated that functions are predicted to be activated (red) or inhibited (blue). Selected IPA canonical pathways in the nervous system identified significant DEPs related to axon (E), synapse (F), and myelination signaling pathways (G). Protein blots analyzed the expression of identified proteins in sham and mTBI groups of WT and CCL5-KO mouse cortical tissue, including H, H' axon guidance-related Sema3a (WT sham vs. KO sham, p < 0.0001; WT TBI vs. KO TBI, p = 0.0067), EphrinA5 (WT sham vs. KO sham, p = 0.0015; WT TBI vs. KO TBI, p = 0.0223), EphA4 (WT TBI vs. KO TBI, p = 0.0162), and p-EIF2/EIF2; I, I' synapse-related p-mTOR/mTOR (WT sham vs. TBI, p = 0.0319; WT TBI vs. KO TBI, p = 0.0002), and J, J' myelination-related: NRG-1 (WT sham vs. KO sham, p = 0.0045; KO sham vs. KO TBI, p = 0.05; WT TBI vs. KO TBI, p = 0.0006), p-Erk/Erk (WT sham vs. KO sham, p = 0.014; KO sham vs. KO TBI, p = 0.0146), and SMI32 (WT sham vs. KO sham, p = 0.0328; WT TBI vs. KO TBI, p = 0.030). K, The immunostaining of unmyelinated neuritis with SMI32 antibody (green) and oligodendrocyte by Oligo2 antibody (red) in different groups of mouse cortex. DAPI, blue, for nucleus. Scale = 100 μm. The quantification of L SMI-32 and M Oligo-2 in sham and mTBI groups of WT and KO mice (SMI32: WT sham vs. KO sham, p = 0.0003; WT sham vs. TBI, p = 0.0002; KO sham vs. TBI, p < 0.0009; WT TBI vs. KO TBI, p = 0.0002) (Oligo2: WT sham vs. KO sham, p = 0.0003; WT TBI vs. KO TBI, p = 0.0007). Data were analyzed by unpaired t-test

Neuregulin, ErbB2/3, and Erk signaling are essential in promoting Schwann cell growth and survival, migration, extending axons, and myelination. The expression of Neuregulin and phosphorylated-Erk were reduced in the cortex tissue of the CCL5-KO TBI group of mice (Fig. 3J, J’); in contrast, SMI-32 labeled non-phosphorylated neurofilaments, which indicates damaged axons, was significantly increased in the injured cortex of CCL5-KO mice after 1 month (Fig. 3J–L, J’). Oligo2-labeled oligodendrocytes were raised in the injured cortex of WT mice after mTBI but not in the KO mice (Fig. 3K, M). Together, these data support our finding of axonal injury and synapse loss shown in Golgi staining (Fig. 2) and cortical neuron dysfunction (Fig. 1) after brain injury in KO mice.

CCL5 is an important chemokine in microglia activation, but the impact of inflammatory responses was not the major affected pathway (Supplementary Fig. 1B, green); the inflammatory response – macrophage activation and phagocytosis pathways were reduced in CCL5-KO mice after TBI (Supplementary Fig. 1E).

Intranasal delivery of recombinant CCL5 after injury improves both motor and sensory function

As shown in Fig. 1B, the level of CCL5 protein increased to 60 ~ 160 pg/mg in cortex tissue after mild TBI; a low dose of CCL5 was thus used here in the following rescue experiments. To confirm the effect of CCL5 in neurite repair and synapse regrowth after brain injury, we studied intranasally (in) delivered recombinant CCL5 (rCCL5) into the mouse brain, as we previously used [22]. First, a single dosage of rCCL5 (300 pg/g) was administered into the CCL5-KO mouse brain just before inducing mTBI (Fig. 4A-1). However, a therapeutic treatment after an injury is also necessary for the clinical application; thus, we also administered a lower dosage series of rCCL5 (30 pg/g) into the CCL5-KO mouse brain 3 days after inducing mTBI every two days until 28 dpi (Fig. 4A-2). PBS treatment was used as a CCL5 TBI control. The rCCL5 was first conjugated with Alex FluroTM-594 and the distribution of rCCL5- Alex FluroTM-594 in the brain was detected by Alex FluroTM-594 (red) as well as a CCL5-specific antibody in mouse cortex (Fig. 4B). The boxed region indicates the injured cortex region; B’ is part of an enlarged image from the boxed region (Fig. 4B). The tissue level of CCL5 was detected by CCL5 specific ELISA assay to confirm penetration efficiency. 40 pg/mg of CCL5 was detected in mouse prefrontal cortex (CTX) tissues after i.n. CCL5 for 6 h and was gradually reduced to 20 ~ 30 pg/mg of CCL5 after 24 h (Supplementary Fig. 2A), which is similar to the endogenous level of CCL5 in WT mouse cortex (Fig. 1B). The localization of rCCL5 in mouse cortex was co-labeled with the neuron marker-NeuN (Supplementary Fig. 2B), the astrocyte marker-GFAP (Supplementary Fig. 2C) and the microglia marker-Ibal (Supplementary Fig. 2D); the results showed that rCCL5 colocalized mostly with NeuN-positive neurons (~ 70%, Supplementary Fig. 2B, E).

Fig. 4

Cortical neuron dysfunctions were improved by intranasally delivering CCL5 into CCL5-KO mice. A An illustration of intranasal delivery (i.n.) of recombinant CCL5 into mice. Recombinant CCL5 was administered into mice either (1) 30 min before injury with a single dosage of 300 pg/g or (2) 3 days after injury with 30 pg/g every 2 days until 28 dpi. B Recombinant CCL5 conjugated with Alexa Fluor™ 594 was detected by Alexa Fluor™ 594 (red) and CCL5 specific antibody (green) in mouse cortex. Images of CCL5 at the injury site in the cortex were enlarged on the right B' (Scale bar = 1 mm in B and 100 µm in B’). DAPI labeled the nucleus. C-J A Black dashed line points to the time of brain injury. The Purple dashed line indicates the treatment with CCL5 before the weight drop impact in C-F; the green dashed line and green area indicate the post-treatment with CCL5 from 3 dpi until 28 dpi in G-J. C, G The mNSS score of the CCL5-KO sham group and mice treated with PBS (control) and CCL5 (300 pg/g, single dose) before mTBI or PBS (control) and CCL5 (30 pg/g, every two days) (G: PBS vs Post-L5, p = 0.0418) after mTBI. Motor function of CCL5-KO mice with i.n. PBS or CCL5 was analyzed by Rotarod (D, H) (D: sham vs PBS, p < 0.0001; sham vs Pre-L5, p = 0.0010; PBS vs Pre-L5, p < 0.0001. H: PBS vs Post-L5, p=0.0009), and beam walking (E, I), which was improved in both i.n. CCL5 treated groups. (E: sham vs PBS, p < 0.0001; sham vs Pre-L5, p = 0.0247; PBS vs Pre-L5, p = 0.0038. I: PBS vs Post-L5, p = 0.0006). F, J Sensory function was analyzed by sicker removal test (F: sham vs PBS, p = 0.0028; sham vs Pre-L5, NS; PBS vs Pre-L5, p = 0.0013. J: PBS vs Post-L5 at 4 dpi, p = 0.015, by t-test). The time to remove stickers in CCL5-KO mice was reduced after being treated with CCL5. (n = 4 ~ 5 in C-F; n = 6 in G-J). Data in D-I was analyzed by two-way ANOVA between groups and presented as mean ± SEM

In the pretreatment with CCL5 (PreL5) study, the neurological mNSS score was lower than 4 in all three groups of mice, including CCL5-KO sham, TBI + i.n. PBS, and TBI + i.n. CCL5 (PreL5) after 1-, 3- and 7-dpi (Fig. 4C). Motor function, assessed by Rotarod, improved in CCL5-treated CCL5-KO mice after 3 dpi (Fig. 4D); the foot faults also improved faster in mice receiving recombinant CCL5 compared to the PBS group (Fig. 4E). In ART - the time to remove paw stickers in CCL5-KO TBI mice receiving recombinant CCL5 was the same as in the CCL5-KO sham control animals (Fig. 4F). In contrast, the CCL5-KO TBI i.n. PBS group had a much slower time for sticker removal at dpi 1 and 4 (Fig. 4F). Thus, the cortical function parameters were improved within seven days in the PreL5 group of mice, which suggests early CCL5 intervention is beneficial for cortex neuron function recovery.

In the post-treatment of CCL5 (PostL5) study, the neurological mNSS score was also lower than 4 in TBI + i.n. PBS, and TBI + i.n. CCL5 (PostL5) at -3, -7, and − 14 dpi; the mNSS score was reduced faster in mice treated with CCL5 than in mice treated with PBS (Fig. 4G). The falling times from the Rotarod were reduced in both PBS and PostL5 groups of mice after mTBI, but which improved immediately when animals received rCCL5 (PostL5) but not PBS (Fig. 4H). The number of foot faults from the balance beam also improved after rCCL5 treatment but not PBS treatment (Fig. 4I). In ART - the time to remove paw stickers was also reduced quickly right after rCCL5 treatment (Fig. 4J).

These findings strongly support an important function of CCL5 in cortical neuronal recovery after mild brain injury. Both early intervention and subsequent treatment with CCL5 improved mouse motor and sensory function.

CCL5 treatment enhanced axonogenesis, synaptogenesis, and myelination signaling in mouse CCL5-KO cortex after mTBI

To further confirm the rescue effect of CCL5 in cortical neuron function, cortical tissues from CCL5-KO mice with TBI, TBI + PreL5, and TBI-PostL5 were harvested and analyzed by LC-MS/MS. GO enrichment evaluated the entire list of 1531 proteins and identified 134 proteins from PreL5 group vs. TBI, 275 proteins from PostL5 vs. TBI, and 54 proteins from PreL5 overlapping with PostL5 vs. TBI (45 up-regulation, 8 down-regulations. adjusted p-value < 0.05) (Fig. 5A–C and Supplementary Figs. 3A, 4 A-B, 5A-B also see Supplementary Data file 1). Identified GO terms for each category in PreL5 or PostL5 treatment are shown in Supplementary Fig. 4C and 5C and Supplementary Data file 6, 7. The overlapping portions of PreL5 and PostL5 were analyzed by DAVID GO analysis and IPA (Fig. 5C). GO analysis identified that CCL5 treatment affected transport, synapse, and synaptosome-related pathways in the cellular component category (Fig. 5D, Blue character—Supplementary Data File 4. CCL5 treatment reduced abdominal lesion inflammation, necrosis neurodegeneration, the organismal death, organ degeneration, and degeneration of brain categories in IPA analysis (Supplementary Fig. 3B). The major affected diseases and functions are cell death and survival, organismal injury and abnormality, especially Nervous system development, and Function and cellular assembly and organization (Supplementary Fig. 3C). In the nervous system category, the quantity of neuroglia, brain formation, and neuritogenesis were increased in the TBI + CCL5 treatment groups (Fig. 5E). The reduced axonogenesis, neuritogenesis, and myelination-related signaling pathways in KO TBI tissue, such as eIF2, mTOR, synaptogenesis, Neuregulin, and myelination signaling were all increased after CCL5 treatments (Fig. 5F–I, and Supplementary Fig. 3D). IPA analysis showed up-regulation in the axon regeneration signaling pathway (Fig. 5F), synaptogenesis signaling pathway (Fig. 5G), and myelination signaling pathway (Fig. 5I) in both PreL5 and PostL5 CCL5 treatment. Interestingly, CCL5 treatment also enhanced neuron development-related signaling, such as CNTF, VEGF, Huntington’s disease, and Reelin (Fig. 5H). (The gradient of yellow to red indicates the Z-score value in Fig. 5F–I.). Proteins identified in each pathway are listed in the Supplementary Data File 5, and their connections in different pathways are in Supplementary Fig. 3F, G.

Fig. 5

Both pretreatment and post-treatment with CCL5 enhanced neurite and synapse growth and myelination-related signaling pathways in injured cortical tissue. A Volcano plot of significant DEPs between mTBI and TBI + CCL5 pretreatment (PreL5) CCL5-KO mouse cortex. B Volcano plot of significant DEPs between mTBI and TBI + CCL5 post-treatment (PostL5) CCL5-KO mouse cortex. DEPs: p-value < 0.05 in comparison to TBI, respectively. Colored points represent: log2 ratio > 0 upregulated protein (red) and log2 ratio < 0 downregulated protein (blue). Selected axonogenesis, neuritogenesis, synaptogenesis, and myelination pathway-related proteins are highlighted as indicated (Red: myelinations, Green: axonogenesis, neuritogenesis, and synaptogenesis, Yellow: overlapping). C Venn diagram comparing DEPs between the TBI group, TBI + CCL5 pretreatment (PreL5), and TBI + CCL5 post-treatment (PostL5) groups. 54 identified proteins (46 up regulated, 8 down regulated) were affected by both treatments. D Identified GO terms from each of the three GO groups (Green bar: biological process, Red bar: cellular component, Blue bar: molecular function. Blue character: neuron function-related) were shown. The strength of enrichment of each GO term was indicated by the Log10 p-value (X-axis). E IPA analysis identified affected diseases and functions in the nervous system category (Z-score value indicated that functions are predicted to be activated (red). Selected IPA canonical pathways in the nervous system identified significant DEPs related to axon (F), synapse (G), neuron development (H), and myelination (I) related signaling pathways in both PreL5 and PostL5 treatments. The Z-score in different identified pathways was shown as a gradient of yellow to red. See also Supplementary Figs. 3, 4, and 5

The upstream analysis identified several proteins correlated with neuron activation (PI3 kinase, PKA, mTOR pathway-related, such as OTUD3, FUNDC2, PPP1R1B, HTR6), neuronal migration (HTR6, DCLK1), retrograde transport (DCLK1), dendrite development and synaptic vesicle transport release (LRP8, SV2C), as well as transcription factors increasing neurogenesis (TCF7L2, HDAC7) or cortical complexity formation (LGALS3BP) (green labeled in Supplementary Fig. 3E). ACSBG1 promotes long-chain fatty acid metabolism and myelinogenesis (red marked in Supplementary Fig. 3E). These proteins and signaling pathways may facilitate the restoration of cortical neuron regrowth after trauma.

Cortical neuron axonogenesis, synaptogenesis, and myelination were enhanced by CCL5 treatment

We further validated these findings from proteomic analysis anatomically. Synapse and spine structure in sham, TBI, and TBI with pretreatment of CCL5 (TBI + PreL5) or TBI with post-treatment of CCL5 (TBI + PostL5) cortex were examined using Golgi staining. The reduced spine density in the TBI group of KO mice was restored in the TBI + PreL5 and TBI + PostL5 groups of mice (Fig. 6A-B); the increased number of swollen spines in the TBI group was reduced in both TBI + PreL5 and TBI + PostL5 groups of mice (Fig. 6A, C). Synaptic proteins - PSD95 , synaptophysin and GAP43 in the injured mouse cortex also increased after CCL5 treatment (Fig. 6D, D’).

Fig. 6

CCL5 treatment enhanced synaptogenesis and myelination by activating the mTOR signaling pathway and the NGR-ERBB signaling pathway after mTBI. Golgi staining of cortical neurons in sham, TBI, TBI with CCL5 pretreatment (PreL5), and TBI with CCL5 post-treatment (PostL5) groups of mice. A The representative images of neurites and dendritic spines in different groups of mice. Black arrows point to swollen spines. Scale bar = 10 µm. The spine density (B) and the number of swollen spines (C) were quantified in different groups of mice (n = 10 in each group). (B: sham vs TBI, p < 0.0001; TBI vs Pre-L5, p = 0.0447; TBI vs Post-L5, p < 0.0001). (C: sham vs TBI, p < 0.0001; TBI vs Pre-L5, p < 0.0001; TBI vs Post-L5, p < 0.0001) D The expression of synaptic proteins – PSD95 and synaptophysin in KO mice cortex after mTBI with/without CCL5 administration. (D’: PSD95: TBI vs Pre-L5, p = 0.0169; TBI vs Post-L5, p = 0.0008; Synaptophysin: sham vs TBI, p = 0.004; TBI vs Pre-L5, p = 0.0032; TBI vs Post-L5, p = 0.0013; GAP43: TBI vs Pre-L5, p = 0.0079; TBI vs Post-L5, p = 0.0177). Western blot analyzed the expression of E axon-related signaling proteins - Sema3, EIF2, and mTOR phosphorylation. (E’: SEMA3a: TBI vs Pre-L5, p = 0.0193; TBI vs Post-L5, p = 0.0483; p-p70S6T421: TBI vs Pre-L5, p = 0.0121; TBI vs Post-L5, p = 0.0238; p-mTOR: TBI vs Pre-L5, p = 0.0121). F myelination-related proteins - Neuregulin, Erk, and SMI32 (F’: NRG-1: TBI vs Pre-L5, p = 0.0420; TBI vs Post-L5, p = 0.0127; p-ERK1/2: TBI vs Pre-L5, p = 0.0317; TBI vs Post-L5, p = 0.0303); and G FGF signaling - FAK phosphorylation (G’: p-FAK: sham vs TBI, p = 0.0476; TBI vs Pre-L5, p = 0.0476; TBI vs Post-L5, p = 0.0238) in injured cortex in different groups. (n = 4 ~ 5 in each group) H The immunostaining of unmyelinated axon - SMI-32 and oligo-2 in 4 groups of CCL5-KO mouse cortex; arrows point to the Oligo-2 positive cells under the pial surface around the injured cortex. The quantification results were in (J) SMI-32 and (K) oligodendrocytes. (J: sham vs TBI, p < 0.0001; TBI vs Pre-L5, p < 0.0001; TBI vs Post-L5, p < 0.0001). (K: TBI vs Pre-L5, p = 0.0004; TBI vs Post-L5, p = 0.0008). I The immunostaining of Reelin (red, arrows) and CXCR4 (Cyan) in mouse cortex in different groups of mice; the q-PCR quantitative result of CXCR4 in mouse cortex L (L: sham vs TBI, p = 0.0075; TBI vs Pre-L5, p = 0.0047; TBI vs Post-L5, p < 0.0001). Data were analyzed by unpaired t-test. See also Supplementary Fig. 6

Those reduced axonal and spine formation-related signaling proteins in the TBI group of CCL5-KO mice, including Sema3A, phosphor-p70S6, and phosphor-mTOR, were increased, in both TBI + preCCL5 and TBI + PostCCL5 mouse cortex (Fig. 6E, E’). Myelination-related signaling pathways proteins—NRG-1, phosphor-Erk (Fig. 6F, F’), and phosphor-FAK-increased (Fig. 6G, G’). Unmyelinated neurofilament labeled by SMI-32 was reduced in those mice receiving CCL5 treatment (Fig. 6H, J) with increased oligodendrocytes (Arrowheads pointed, Fig. 6H, K). In addition, receptor CXCR4, identified by LC-MS/MS, was also increased in the CCL5-administered cortex by both immunostaining and q-PCR analysis (Fig. 6I–L, and Supplementary Fig. 6). Interestingly, Reelin signal was increased in the post-CCL5 treatment group which guide axon extension/migration in the cortex region (Fig. 6I, and Supplementary Fig. 6).

Although neuronal function in WT mice recovered from mTBI quickly (7–14 dpi), the recovery of spine and axon was seen only after 14 dpi. Therefore, similar CCL5 administration was also performed in WT mice. The mNSS, Rotarod, beam walking and ART scores changed significantly in WT mice receiving CCL5 after TBI, especially in the PostL5 group (Supplementary Fig. 7A–C). The spine density of cortical neurons was significantly increased in both pre- and post-CCL5 treatment groups (Supplementary Fig. 7E, F) and the percent of swollen spines was reduced by CCL5 administration at 14 dpi (Supplementary Fig. 7E, G) by Golgi staining. The fluorescent intensity of SMI-32 immunostaining was reduced in both pre- and post-CCL5 treatment groups of mice (Supplementary Fig. 7H, I); the number of Oligo2 positive cells was increased in the Post-CCL5 group (Supplementary Fig. 7H, J).

CCL5 administration significantly increased the protein expression of synaptophysin and GAP43 in the injured mouse cortex in both pre- and post-CCL5 treated groups (Supplementary Fig. 7K, K’), but the protein level of PSD95 was only also slightly increased after CCL5 treatment (Supplementary Fig. 7K, K’). With the axon guidance and mTOR related signaling molecules, Post-CCL5 administration significantly increased Reelin expression as well as p70S6K and mTOR phosphorylation (Supplementary Fig. 7L, L’), but not Sema3a (Supplementary Fig. 7L, L’). Both pre- and post-CCL5 treatment increased the NRG-1 downstream molecule – Erk1/2 phosphorylation (Supplementary Fig. 7M, M’) but not NRG-1. Interestingly, post-CCL5 treatment increased FAK protein expression but not the phosphorylation (Supplementary Fig. 7N, N’). Although CCL5 administration showed some different activations of signaling molecules between WT and CCL5-KO, CCL5 makes a significant contribution to in vivo axonogenesis, synaptogenesis, and remyelination processes after brain injury in both WT and CCL-KO mice.

CCL5 promoted growth cone formation and sprouting through receptor CCR5 and mTOR and ERK signaling pathway

CCL5 promotes synaptogenesis in hippocampal neurons through the PI3K-Akt-GSK3β pathway shown in our previous study [1], but the growth cone regrowth is the critical step of axonogenesis after injury. Herein, this study identified several signaling pathways activated by CCL5 in mouse brains, such as the mTOR, Rho, and FGF pathways; we further validated those signaling pathways in in vitro cortical neuron cultures. Cortical neurons were cultured from WT and CCK5-KO mice and treated with different doses of CCL5 (0, 250, 500, and 1000 pg/ml of CCL5). FGF treatment was taking as a positive control. Phalloidin was used to label the growth cones, and Tuj-1 labeled the whole neuron structure. The signal intensity of Phalloidin was significantly lower in the CCL5-KO neurons around growth cones compared to WT neurons, which increased with CCL5 treatment in KO neurons but slightly in WT neurons (Fig. 7A, B). The number of branching was lower in CCL5-KO neurons, and increased with CCL5 treatment in both WT and KO neurons (Fig. 7A, C). The phosphorylation of mTOR/p70s6k and Erk signaling was also increased after CCL5 treatment (Fig. 7D–H).

Fig. 7

CCL5 increased growth cone formation through activating the mTOR and FAK pathway. A Phalloidin (red) labeled the filopodia in axon growth cones of WT or CCL5-KO neurons after treating with CCL5 (0, 100, 250, 500, 1000 pg/ml). Tuj-1 labeled neurites, and DAPI labeled the nucleus. Phalloidin labeled growth cones in different groups were enlarged in the boxed regions: scale bar = 50 μm and 5 μm. The fluorence intensity of Phalloidin and neurite branching in different groups was quantified in B, C. (B: KO neuron treated with CCL5, p = 0.027; WT vs. KO neuron, p < 0.0001). (C: WT neuron treated with CCL5, p = 0.0089; KO neuron treated with CCL5, p < 0.0001; WT contol vs. KO contol, p = 0.0009 by t-test). D The activation of p70s6k/mTOR, NRG-1, FAK, and Erk signaling proteins in neurons after treatment with CCL5 (0, 100, 250, 500, 1000 pg/ml) in westen blot analysis. FGF was taken as positive control. Quantification data were in E-H. E The activation of p70s6k by CCL5 p = 0.0182; the activation of p70s6k by FGF p = 0.0079. F The activation of mTOR by CCL5 p = 0.0217; the activation of mTOR by FGF p = 0.0169. G The activation of ERK1/2 by CCL5 p = 0.0437; the activation of ERK1/2 by FGF p = 0.0025. Data was analyzed by one-way ANOVA in same group and two-way ANOVA between groups. Data between control and FGF treatment was analyzed by t-test

Blocking CCL5 receptor - CCR5 with a FAD proved inhibitor - Maraviroc (5 nM) reduced phalloidin signals intensity (Fig. 8A, B) but not branching (Fig. 8A, C); the activation of FAK/Erk1/2, mTOR/p70s6k signaling by CCL5 was also reduced by Maraviroc (Fig. 8D–H). Rapamycin, the inhibitor of mTOR signaling, inhibits the activation of mTOR and caused inactivation of p70s6K (Fig. 8E) which also abolished CCL5’s effect on growth cones (Fig. 8A, B). The Rho kinase inhibitor - Y27632 (50 µM, ROCK inhibitor) blocked CCL5’s effect on growth cone (Fig. 8A, B) but increased the branching number (Fig. 8C) which modulated the activation of Erk/FAK singlaing upon CCL5 administration. Thus, the CCL5/CCR5 axis promotes cortical neuron axon formation through mTOR and Erk signaling pathways.

Fig. 8

Inhibiting CCR5 receptor and mTOR signaling pathway reduced CCL5’s effect on growth cone formation. A Phalloidin (red) labeled the filopodia in axon growth cones of WT neurons treating with CCL5 (250 pg/ml), CCR5 inhibitor-Maraviroc (5 nM), mTOR inhibitor-Rapamycin (1 µM) and ROCK inhibitor-Y27632 (50 µM). Tuj-1 labeled neurites, and DAPI labeled the nucleus. Phalloidin and growth cones in different groups were enlarged in the boxed regions: scale bar = 50 μm and 5 μm. The fluorence intensity of Phalloidin and number of branching in different groups was quantified in B, C. (B: control vs. Maraviroc, p = 0.0451; control vs. Rapamycin (Rapa), p = 0.0047; control vs. Y-27632, p = 0.02. CCL5 vs. CCL5 + Maraviroc, p = 0.0173; CCL5 vs. CCL5 + Rapamycin, p = 0.0079; CCL5 vs. CCL5 + Y-27632, p = 0.0159.). (C: control vs. Y-27632, p = 0.0033, by t-test). D The activation of p70s6k/mTOR, NRG-1, FAK, and Erk signaling proteins in neurons after cotreatment with CCL5 (250 pg/ml) and CCR5 inhibitor-Maraviroc, Y27632 or Rapamycin (1 µM) in western blot analysis. FGF was taken as positive control. Quantification data were in E-H. E The quantification of p70s6k activation. (Control vs. Maraviroc, p = 0.0002; control vs. CCL5 + Maraviroc, p = 0.0002; control vs. Y-27632, p = 0.0005; control vs. CCL5 + Y-27632, p = 0.0002. ) F The quantification of mTOR activation. (Control vs. Maraviroc, p < 0.0001; control vs. CCL5 + Maraviroc, p < 0.0001; control vs. Rapa, p < 0.0001; control vs. CCL5 + Rapa, p < 0.0001; control vs. Y-27632, p < 0.0001; control vs. CCL5 + Y-27632, p < 0.0001.). G The quantification of ERK1/2 activation. (Control vs. Maraviroc, p = 0.0013; control vs. Rapa, p = 0.0013; control vs. Y-27632, p = 0.0030.) H The quantification of FAK activation. (Control vs. Maraviroc, p < 0.0109; control vs. CCL5 + Maraviroc, p = 0.0109; control vs. Rapa, p = 0.0191; control vs. CCL5 + Rapa, p < 0.0001; control vs. CCL5 + Y-27632, p = 0.0361.). Data was analyzed by unpaired t-test. NS: no significant difference