A neuroprotective tetrapeptide for treatment of acute traumatic brain injury
Aman P Mann et al. EMBO Mol Med. 2025 Nov.
Abstract
Traumatic brain injury (TBI) is a major clinical problem because of the high incidence and the severity of the subsequent sequelae. Despite extensive efforts, there are no therapeutic drugs clinically approved for treating acute TBI patients. To address this unmet need, we assessed the activity of the tetrapeptide, CAQK, in mice. When administered intravenously shortly after moderate or severe TBI, CAQK accumulates in the injured brain in mice and pigs. CAQK binds to an extracellular matrix glycoprotein complex that is upregulated in injured brain. Treatment of TBI mice with CAQK resulted in reduction in the size of the injury compared to control mice. There was reduced upregulation of the glycoprotein complex, less apoptosis, and lower expression of inflammatory markers in the injured area, indicating that CAQK alleviates neuroinflammation and the ensuing secondary injury. CAQK treatment also improved functional deficit in TBI mice, with no overt toxicity. Our findings suggest that CAQK may have therapeutic applications in TBI.
Keywords: Brain Injury; Neurotherapeutic; Peptide; Traumatic Brain Injury.
© 2025. The Author(s).
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Conflict of interest statement
Disclosure and competing interests statement. APM, SH, PS, and ER are inventors on the patents. APM, SH and ER have ownership interest in AivoCode and are founders and officers of the company. The remaining authors declare no competing financial interests.
Figures Figure 1. CAQK binds to TnC in the ECM.
(A) FAM-CAQK binding to purified CSPG complex isolated from chicken brain was analyzed by fluorescence polarization of FAM label on the peptide. FAM-CAQK (20 nM) was incubated with CSPG complex for 60 min at 37 °C. Binding was analyzed by measuring the increase in millipolarization units (mP). FAM-AAQK was used as the control peptide. *P = 0.02617, **P = 0.03684 (Fisher’s exact test). Data expressed as mean ± SEM from three independent experiments. (B) Fluorescence polarization of FAM was analyzed to determine FAM-CAQK (20 nM in PBS) binding to purified human TnC after incubation at indicated concentrations for 60 min at 37 °C. Data expressed as mean ± SEM and analyzed using an unpaired t-test. *P = 0.049, **P = 0.0071, ***P = 0.0037. n = 3. (C) CAQK binding to two recombinant TnC fragments was analyzed by fluorescence polarization of FAM on the peptide. FAM-CAQK (20 nM in PBS) was incubated with fragments representing the EGF domains or FN(III) domains of TnC at the indicated concentrations (μM) for 1 h at 37 °C. Data expressed as mean ± SEM and analyzed using an unpaired t-test. **P < 0.01, ***P < 0.005. n = 3. (D) To localize the CAQK-binding site more accurately, FAM-CAQK (20 nM in PBS) was incubated with a recombinant fragment containing the TnC FN(III) 5–6 domains at indicated concentrations (μM) for 1 h at 37 °C and fluorescence polarization of FAM was analyzed. Cold = excess unlabeled peptide (1 μM) added to TnC fragment. Data expressed as mean ± SEM. Differences were analyzed using an unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.005. n = 3. (E) Brightfield and fluorescence images of gross mouse brain with CCI and injected with FAM-CAQK at 24 h after injury. Coronal. Left panel shows the intrinsic fluorescence of FAM-CAQK seen macroscopically using an Illumatool bright light system in the green channel. Higher magnification regions (injured and uninjured) are shown stained with anti-TnC antibody (red), FAM (CAQK; green), and nuclei (DAPI, blue). Scale bar, 40 μm. Quantification of positive staining (% area) was performed using Image J. Data were expressed as mean ± SEM. Differences were analyzed using Welch’s t-test (**P < 0.01, **P = 0.0059, **P = 0.0052). n = 3. (F) Immunohistochemical staining for TnC expression in cortical brain sections from frozen human brain tissue obtained from two patients with head trauma (Trauma 1 and 2) compared to cortical sections of the brain in a patient without TBI (control). Data expressed as median of five regions of interest. Differences were analyzed using an unpaired t-test (**P = 0.0233). n = 1. Figure 2. Pharmacokinetic analysis of CAQK.
(A) Mice with CCI received i.v. bolus of CAQK (12.5 mg/kg) at 4 h after CCI. Blood was collected at different time points, and plasma was analyzed by LC/MS. Plasma concentration of CAQK was plotted using WinNonlin. N = 3 per time point. (B) Plasma clearance data were used to calculate pharmacokinetic parameters using a two-compartment analysis. (C) CAQK accumulation was analyzed in brain homogenates from injured (ipsilateral) and uninjured (contralateral) sides at different time points after i.v. injection and plotted as ng/g of brain tissue. N = 3/group. Data were expressed as mean ± SEM. Differences were analyzed using an unpaired t-test. ***P = 0.0216 vs contralateral side. Figure 3. CAQK targets brain injury in pig CCI.
(A) FAM-CAQK (2.5 mg/kg) was injected i.v. into pigs with CCI at 1 h post-injury. The peptide was allowed to circulate for 60 min. The pigs were perfused, and brains were isolated and imaged using an Illumatool Bright Light System in the green channel. Intrinsic fluorescence of FAM-CAQK is seen macroscopically. Bottom panel shows immunofluorescence staining of FAM signal (green) in the uninjured area (lower left panel) and injured area (lower right panel). N = 2. (B) Fluorescence imaging on cortical brain sections from CCI pig injected with FAM-CAQK. Sections were immunostained with anti-TnC antibody (red), FAM (CAQK; green) and counterstained for nuclei with DAPI (blue). Scale bar, 50 μm. (C) Plasma concentration (Cp) of FAM-CAQK plotted over time. FAM-CAQK (2.5 mg/kg) was injected i.v. into a pig with CCI at 1 h post-injury. Plasma concentration was detected by measuring fluorescence from the FAM label in the peptide at different time points. Figure 4. CAQK effect on TBI in mice.
(A) Mouse brains were isolated at 7 days post-injury after i.v. treatment with either CAQK peptide (200 nmol (5 mg/kg) per injection) or vehicle (saline) at 6 h after injury and once daily for 7 days. (B) Stereological analysis of coronal brain sections (cresyl violet staining) after treatment with CAQK at post-injury day 7 shows significantly reduced tissue loss (n = 6/group). (C) Quantification of cortical lesion volumes, including the anterior and posterior margins of the contusion, from the stereological analysis in (B). The results indicate a significant difference in lesion areas between the saline-treated and CAQK-treated mice (n = 6/group). Data were expressed as mean ± SEM. Significance was calculated using Fisher’s LSD test (*P = 0.0166). (D) Effect of CAQK treatment on apoptotic cell death examined by TUNEL staining (brown) in an area surrounding commissural fibers of the corpus callosum. Scale bar, 100 μm. Quantification of TUNEL staining (% area) was performed using Image J. Data were expressed as mean ± SEM. Differences were analyzed using Fisher’s LSD test (**P = 0.001). n = 6/group. (E) Effect of treatment on astrogliosis and microglial activation in injured brain. Coronal sections showing cortex and hippocampus from TBI mice treated with CAQK or vehicle were immunostained for GFAP (red) and Iba1 (green). DAPI staining of nuclei is blue. Left panel shows lower magnification. Scale bar, 100 μm. Insets were magnified and shown on the right panel. Scale bar, 50 μm. Quantification of positive staining (% area) was performed using Image J. Data were expressed as mean ± SEM. Differences were analyzed using Fisher’s LSD test (*P = 0.003, **P = 0.001). n = 6/group (F). Serum levels of GFAP in TBI mice at 24 h and 14 days after brain injury are plotted in a box and whiskers plot with whiskers down to the minimum, and up to the maximum, values. Data compared to naïve mice with no injury and no treatment. Plasma concentration shown (pg/ml; n = 5/group). Data were expressed as mean ± SEM. Differences were analyzed using an unpaired t-test (*P = 0.0294). Figure 5. CAQK treatment reduces expression of neuroinflammatory genes in TBI.
(A) Volcano plots showing −log10(p value) and log 2-fold change in genes that were expressed differentially at a significant level (fold increase >1.2 or fold decrease <1.2 and P < 0.05) in vehicle-treated and naïve mice (left panel) and CAQK-treated and naïve mice (right panel) using the neuroinflammation panel (n = 3). The generalized linear model (GLM) developed by NanoString is used for calculating differential expression for count data. (B) Number of differentially expressed genes (DEGs) in injured brains at day 14 after injury compared to 12-week-old naïve brains from uninjured mice. Genes were filtered based on fold change >1.20, P < 0.05 as a threshold. (C) Nanostring-based heatmaps showing differential gene patterns in CAQK-treated and vehicle-treated groups using the neuroinflammation panel (left panel) and neuropathology panels (right panel). Color coding was based on z-score scaling. (D) Pathway analysis of the genes that differed most in the neuroinflammation panel between CAQK-treated and vehicle-treated mice after CCI. Differentially expressed genes based on fold change >1.20, P < 0.05 as threshold. Gene set analysis (GSA) module from NanoString® used for statistical analysis. (E) Analysis of key inflammatory genes implicated in TBI arranged in order of fold change in the vehicle-treated group. LCN2 (lipocalin 2), Tgfb1 (transforming growth factor-beta 1), C3 (complement factor 3), CXCL10 (C-X-C Motif Chemokine Ligand 10), TNF (tumor necrosis factor), S100b (S100 Calcium Binding Protein B), and TnC (tenascin-C). Figure 6. CAQK treatment improves behavioral outcome in TBI mice.
(A) Rotarod test performed on TBI and naive mice at day 15–18 after injury. Results expressed as mean rotational velocity in rpm at the time of falling off the rod in three successive tests. n = 10 per group. *P = 0.0246, CAQK treated vs. vehicle control (ANOVA). (B) Hanging wire test performed in mice on days 22–23 after injury. The latency from the beginning of the test until the time the mouse fell off was timed up to a maximum of 30 s, and each animal got three trials separated by 30 s. n = 8 per group. Data were expressed as mean ± SEM. *P = 0.0393. Fisher’s LSD test for CAQK-treated vs. vehicle control. (C) Novel object recognition test in mice on days 25–29 after injury. n = 10 per group. Data were expressed as mean ± SEM. *P = 0.0246, one-way ANOVA with Bonferroni test. Figure EV1. CAQK binding to CSPG components.
(A) Fluorescence polarization (FP) measurement of CAQK binding to CSPG. FAM-CAQK (20 nM) was incubated with CSPG (200 nM) for 60 min at 37 °C. Binding with CSPG pretreated with chondroitinase ABC (chABC) at two concentrations low (5mU) and high (15mU) was compared to untreated CSPG. (B) FP assay to assess the binding of CAQK to brain ECM from different sources. FAM-labeled CAQK (20 nM) was incubated for 1 h at 37 °C with purified CSPG isolated from chicken brain (1 μM) or supernatant collected from cultured U251 human glioblastoma cell line. (C) Top hits from proteomic analysis of CSPG complex isolated from chicken brains and U251 conditioned media. (D) FP assay of binding of different peptides to TnC. FAM-labeled peptides (20 nM) were incubated with TnC (1 μM) for 1 h at 37 °C. (E) FP assay to assess the effect of the thiol group in cysteine of CAQK on binding to TnC. FAM-labeled CAQK (20 nM) was incubated with TnC (1 μM) for 1 h at 37 °C in the presence of GSH and Iodoacetamide. n = 3. Figure EV2. CAQK plasma clearance in rats.
(A) CAQK was injected daily i.v. in healthy adult Sprague-Dawley rats at a 10 mg/kg dose for 7 days. Blood was collected at different time points at Day 1 and Day 7, and plasma was analyzed by LC/MS to analyze CAQK. Plasma concentration of CAQK was plotted. N = 3 per time point. Plasma clearance data were also used to calculate pharmacokinetic parameters in (B). (C) FP measurement of CAQK binding to BSA. FAM-CAQK (20 nM) was incubated with BSA at the indicated concentration for 1 h at 37 °C. n = 3. Figure EV3. CAQK targeting in the pig model of TBI.
(A) TnC expression is upregulated in pig CCI. Immunohistochemical staining for TnC on cortical pig brain sections shows elevated tenascin-C expression in the cortex surrounding CCI brain injury compared to the cortex of a sham-injured animal. Scale bar—300 μm. (B) Control peptide does not accumulate in the brain of a pig with CCI. Fluorescence imaging on cortical brain sections from CCI pig injected with FAM-AAQK. Sections were immunostained with anti-FAM (AAQK; green) and nuclei (blue). Scale bar—100 μm. (C) CAQK accumulation in different organs in pig CCI. A male Yorkshire pig with CCI was injected with FAM-CAQK at 6 h post-injury. IHC staining of FAM label on fixed paraffin-embedded tissue sections from different organs collected 1 h after peptide injection. Scale bar, 200 μm. IVS interventricular septum.
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