Mitochondrial complex II in intestinal epithelial cells regulates T cell-mediated immunopathology.

Intestinal epithelial cell (IEC) damage by T cells contributes to graft-versus-host disease, inflammatory bowel disease and immune checkpoint blockade-mediated colitis. But little is known about the target cell-intrinsic features that affect disease severity. Here we identified disruption of oxidative phosphorylation and an increase in succinate levels in the IECs from several distinct in vivo models of T cell-mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses, identified disruption of IEC-intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations. The relevance of IEC-intrinsic SDHA in mediating disease severity was confirmed by complementary chemical and genetic experimental approaches and validated in human clinical samples. These data identify a critical role for the alteration of the IEC-specific mitochondrial complex II component SDHA in the regulation of the severity of T cell-mediated intestinal diseases.

Ambient oxygen levels regulate intestinal dysbiosis and GVHD severity after allogeneic stem cell transplantation.

The severity of T cell-mediated gastrointestinal (GI) diseases such as graft-versus-host disease (GVHD) and inflammatory bowel diseases correlates with a decrease in the diversity of the host gut microbiome composition characterized by loss of obligate anaerobic commensals. The mechanisms underpinning these changes in the microbial structure remain unknown. Here, we show in multiple specific pathogen-free (SPF), gnotobiotic, and germ-free murine models of GI GVHD that the initiation of the intestinal damage by the pathogenic T cells altered ambient oxygen levels in the GI tract and caused dysbiosis. The change in oxygen levels contributed to the severity of intestinal pathology in a host intestinal HIF-1α- and a microbiome-dependent manner. Regulation of intestinal ambient oxygen levels with oral iron chelation mitigated dysbiosis and reduced the severity of the GI GVHD. Thus, targeting ambient intestinal oxygen levels may represent a novel, non-immunosuppressive strategy to mitigate T cell-driven intestinal diseases.

Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease.

The effect of alterations in intestinal microbiota on microbial metabolites and on disease processes such as graft-versus-host disease (GVHD) is not known. Here we carried out an unbiased analysis to identify previously unidentified alterations in gastrointestinal microbiota-derived short-chain fatty acids (SCFAs) after allogeneic bone marrow transplant (allo-BMT). Alterations in the amount of only one SCFA, butyrate, were observed only in the intestinal tissue. The reduced butyrate in CD326(+) intestinal epithelial cells (IECs) after allo-BMT resulted in decreased histone acetylation, which was restored after local administration of exogenous butyrate. Butyrate restoration improved IEC junctional integrity, decreased apoptosis and mitigated GVHD. Furthermore, alteration of the indigenous microbiota with 17 rationally selected strains of high butyrate-producing Clostridia also decreased GVHD. These data demonstrate a heretofore unrecognized role of microbial metabolites and suggest that local and specific alteration of microbial metabolites has direct salutary effects on GVHD target tissues and can mitigate disease severity.

C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation.

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.

Acute downregulation of emerin alters actomyosin cytoskeleton connectivity and function.

Fetal lung fibroblasts contribute dynamic infrastructure for the developing lung. These cells undergo dynamic mechanical transitions, including cyclic stretch and spreading, which are integral to lung growth in utero. We investigated the role of the nuclear envelope protein emerin in cellular responses to these dynamic mechanical transitions. In contrast to control cells, which briskly realigned their nuclei, actin cytoskeleton, and extracellular matrices in response to cyclic stretch, fibroblasts that were acutely downregulated for emerin showed incomplete reorientation of both nuclei and actin cytoskeleton. Emerin-downregulated fibroblasts were also aberrantly circular in contrast to the spindle-shaped controls and exhibited an altered pattern of filamentous actin organization that was disconnected from the nucleus. Emerin knockdown was also associated with reduced myosin light chain phosphorylation during cell spreading. Interestingly, emerin-downregulated fibroblasts also demonstrated reduced fibronectin fibrillogenesis and production. These findings indicate that nuclear-cytoskeletal coupling serves a role in the dynamic regulation of cytoskeletal structure and function and may also impact the transmission of traction force to the extracellular matrix microenvironment.

Fertility Desire and Associations with Condomless Sex, Antiretroviral Adherence, and Transmission Potential in a Cohort of Kenyan Women Living with HIV in Sero-discordant Relationships: A Mixed Methods Study.

For women living with HIV (WLH) in serodiscordant partnerships, decisions about childbearing can challenge condom use and antiretroviral adherence. In a prospective cohort of 148 WLH in serodiscordant partnerships, 58 (39%) wanted more children in the future but were not currently trying to conceive (fertility desire), and 32 (22%) were currently trying to become pregnant (fertility intent). Detection of prostate specific antigen (PSA) in vaginal secretions, a marker for recent condomless sex, was lowest in women with fertility desire and highest in women with fertility intent. Detectable viral load followed a similar pattern. Risk of HIV transmission, when condomless sex and PSA detection occurred concurrently, was three to fourfold higher at visits with fertility intent compared to visits with fertility desire. Qualitative interviews underscored the importance women place on childbearing and suggested that they had limited information about the role of antiretroviral therapy in reducing sexual HIV transmission.

The nucleoskeleton as a genome-associated dynamic 'network of networks'.

In the cytosol, actin polymers, intermediate filaments and microtubules can anchor to cell surface adhesions and interlink to form intricate networks. This cytoskeleton is anchored to the nucleus through LINC (links the nucleoskeleton and cytoskeleton) complexes that span the nuclear envelope and in turn anchor to networks of filaments in the nucleus. The metazoan nucleoskeleton includes nuclear pore-linked filaments, A-type and B-type lamin intermediate filaments, nuclear mitotic apparatus (NuMA) networks, spectrins, titin, 'unconventional' polymers of actin and at least ten different myosin and kinesin motors. These elements constitute a poorly understood 'network of networks' that dynamically reorganizes during mitosis and is responsible for genome organization and integrity.

Global Mental Health: Where We Are and Where We Are Going.

PURPOSE OF REVIEW: To summarize recent findings in global mental health along several domains including socioeconomic determinants, inequities, funding, and inclusion in global mental health research and practice. RECENT FINDINGS: Mental illness continues to disproportionately impact vulnerable populations and treatment coverage continues to be low globally. Advances in integrating mental health care and adopting task-shifting are accompanied by implementation challenges. The mental health impact of recent global events such as the COVID-19 pandemic, geo-political events, and environmental change is likely to persist and require coordinated care approaches for those in need of psychosocial support. Inequities also exist in funding for global mental health and there has been gradual progress in terms of building local capacity for mental health care programs and research. Lastly, there is an increasing effort to include people with lived experiences of mental health in research and policy shaping efforts. The field of global mental health will likely continue to be informed by evidence and perspectives originating increasingly from low- and middle-income countries along with ongoing global events and centering of relevant stakeholders.

Youth intentions to provide social support to a peer with a concussion.

OBJECTIVES: 1) To describe demographic factors, concussion knowledge, attitudes, subjective norms, self-efficacy and intentions to provide social support to a peer with a concussion and 2) to examine if demographic factors and concussion knowledge are associated with components of the Theory of Planned Behavior. METHODS: The survey was completed between October 2018 and February 2019 by 200 youth (M = 15.30 years, SD = 1.52). Questions were designed for athletes and non-athletes and inquired about various types of social support. Data analysis included descriptive statistics, Wilcoxon Rank Sum Tests and Spearman's Rank-Order Correlation Coefficients. RESULTS: More favorable attitudes and intentions to provide social support were observed among females (W = 2576, p ≤ 0.001; W = 2411, p ≤ 0.001), older youth (rho = 0.32, p ≤ 0.001; rho = 0.41, p ≤ 0.001) and those with higher concussion knowledge (rho = 0.29, p ≤ 0.001; rho = 0.22; p ≤ 0.001). Participating in sports with a high-risk of concussion was associated with lower attitudes and intentions to provide social support (W = 6677; p ≤ 0.001; W = 6721; p ≤ 0.001). Self-reported concussion history or knowing someone with a concussion history was not significantly associated with social support intentions. CONCLUSION: This study identified characteristics of youth who had positive intentions to provide social support. These findings identify individuals who may model providing social support to a peer, as well as opportunities for future concussion education.

Redox and Nucleophilic Reactions of Naphthoquinones with Small Thiols and Their Effects on Oxidization of H2S to Inorganic and Organic Hydropolysulfides and Thiosulfate.

Naphthoquinone (1,4-NQ) and its derivatives (NQs, juglone, plumbagin, 2-methoxy-1,4-NQ, and menadione) have a variety of therapeutic applications, many of which are attributed to redox cycling and the production of reactive oxygen species (ROS). We previously demonstrated that NQs also oxidize hydrogen sulfide (H2S) to reactive sulfur species (RSS), potentially conveying identical benefits. Here we use RSS-specific fluorophores, mass spectroscopy, EPR and UV-Vis spectrometry, and oxygen-sensitive optodes to examine the effects of thiols and thiol-NQ adducts on H2S-NQ reactions. In the presence of glutathione (GSH) and cysteine (Cys), 1,4-NQ oxidizes H2S to both inorganic and organic hydroper-/hydropolysulfides (R2Sn, R=H, Cys, GSH; n = 2-4) and organic sulfoxides (GSnOH, n = 1, 2). These reactions reduce NQs and consume oxygen via a semiquinone intermediate. NQs are also reduced as they form adducts with GSH, Cys, protein thiols, and amines. Thiol, but not amine, adducts may increase or decrease H2S oxidation in reactions that are both NQ- and thiol-specific. Amine adducts also inhibit the formation of thiol adducts. These results suggest that NQs may react with endogenous thiols, including GSH, Cys, and protein Cys, and that these adducts may affect both thiol reactions as well as RSS production from H2S.

Potassium penicillin and gentamicin pharmacokinetics in healthy conscious and anesthetized horses.

OBJECTIVE: To determine the effects of general anesthesia on the safety and efficacy of co-administered potassium penicillin G (PEN) and gentamicin (GENT) in horses. STUDY DESIGN: Nonrandomized crossover. ANIMALS: Six adult, Thoroughbred horses. METHODS: Horses were administered PEN (22 000 IU/kg IV) and GENT (6.6 mg/kg IV). Plasma samples were collected over a 6 h period and synovial fluid was collected at 30 min and 6 h respectively. Drug administration and sample collection protocols were repeated after at least a 48 hour washout period and induction of anesthesia using xylazine/ketamine and maintenance with isoflurane gas. Drug concentrations were determined using ultrapressure liquid chromatography with mass spectrometry. A 2-compartment model was used to determine pharmacokinetics and differences were determined between conscious and anesthetized horses using paired t-tests (significance P < .05). RESULTS: Potassium penicillin g and GENT had higher minimum plasma concentrations (PEN 0.44 vs. 0.11 µg/mL, P = .002; GENT 3.0 vs. 1.9 µg/mL, P = .009), longer half lives (PEN 71 vs. 59 min, P = .018; GENT 149 vs. 109 min, P = .038), and slower clearances (PEN 3.41 vs. 5.1 mL/kg/min, P = .005; GENT 1.18 vs. 1.48 mL/kg/min, P = .028) in anesthetized horses vs. conscious horses. The PEN concentrations remained above the breakpoint minimum inhibitory concentration (MIC, 0.5 µg/mL) for 332 min in anesthetized vs. 199 min in conscious horses. The GENT concentrations reached 10 times higher than the breakpoint MIC (2 µg/mL) in all horses and were maintained for 58 vs. 59 min in anesthetized and conscious states, respectively. Synovial fluid concentrations were higher in conscious horses vs. anesthetized horses at 30 min for PEN (7.0 vs. 0.93 µg/mL, P < .001) and 30 (5.3 µg/mL vs. 0.79 µg/mL, P < .001) and 360 min (3.4 vs. 1.82 µg/mL, P < .003) for GENT. CONCLUSION: General anesthesia resulted in lower intrasynovial concentrations and delayed clearance of PEN/GENT in horses. CLINICAL SIGNIFICANCE: Redosing healthy anesthetized horses with PEN prior to 4-5 h is not necessary. When administered to anesthetized horses, intravenous PEN/GENT may not reach adequate intrasynovial concentrations to treat or prevent common pathogens. The doses or dosing intervals of antimicrobials administered to horses undergoing anesthesia may need to be adjusted to ensure maintenance of safe and effective plasma concentrations.

Development of a sensitive trial-ready poly(GP) CSF biomarker assay for C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis.

OBJECTIVE: A GGGGCC repeat expansion in the C9orf72 gene is the most common cause of genetic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). As potential therapies targeting the repeat expansion are now entering clinical trials, sensitive biomarker assays of target engagement are urgently required. Our objective was to develop such an assay. METHODS: We used the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by the C9orf72 repeat expansion in cerebrospinal fluid (CSF) of people with C9orf72-associated FTD/ALS. RESULTS AND CONCLUSIONS: We show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intraplate and interplate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze-thaw and no haemoglobin interference. We used this assay to measure poly(GP) in CSF samples collected through the Genetic FTD Initiative (N=40 C9orf72 and 15 controls). We found it had 100% specificity and 100% sensitivity and a large window for detecting target engagement, as the C9orf72 CSF sample with the lowest poly(GP) signal had eightfold higher signal than controls and on average values from C9orf72 samples were 38-fold higher than controls, which all fell below the lower limit of quantification of the assay. These data indicate that a Simoa-based poly(GP) DPR assay is suitable for use in clinical trials to determine target engagement of therapeutics aimed at reducing C9orf72 repeat-containing transcripts.

Grouper source levels and aggregation dynamics inferred from passive acoustic localization at a multispecies spawning site.

Four species of grouper (family Epinephlidae), Red Hind (Epinephelus guttatus), Nassau (Epinephelus striatus), Black (Mycteroperca bonaci), and Yellowfin Grouper (Mycteroperca venenosa) share an aggregation site in Little Cayman, Cayman Islands and produce sounds while aggregating. Continuous observation of these aggregations is challenging because traditional diver or ship-based methods are limited in time and space. Passive acoustic localization can overcome this challenge for sound-producing species, allowing observations over long durations and at fine spatial scales. A hydrophone array was deployed in February 2017 over a 9-day period that included Nassau Grouper spawning. Passive acoustic localization was used to find positions of the grouper-produced calls recorded during this time, which enabled the measurement of call source levels and evaluation of spatiotemporal aspects of calling. Yellowfin Grouper had the lowest mean peak-to-peak (PP) call source level, and Nassau Grouper had the highest mean PP call source level (143.7 and 155.2 dB re: 1 µPa at 1 m for 70-170 Hz, respectively). During the days that Nassau Grouper spawned, calling peaked after sunset. Similarly, when Red Hind calls were abundant, calls were highest in the afternoon and evening. The measured source levels can be used to estimate communication and detection ranges and implement passive acoustic density estimation for these fishes.

Current process and outcomes of the surgical management of LUTS due to benign prostatic enlargement: how consistent are we? - results from the multi-institutional audit of surgical management of BPE (AuSuM BPE) in the United Kingdom.

OBJECTIVE: In view of changing landscape of surgical treatment for LUTS secondary to BPE, this audit was undertaken to assess key aspects of the processes and outcomes of the current interventional treatments for BPE, across different units in the UK. MATERIALS AND METHOD: A multi-institutional snapshot audit was conducted for patients undergoing interventions for LUTS/BPE over 8-week period. Using Delphi process two-part proforma was designed to capture data. RESULTS: 529 patients were included across 20 NHS trusts in England and Wales. Median age was 73 years. Indications for surgery were acute retention (47%) and LUTS (45%). 80% of patients had prior medical therapy. TURP formed the commonest procedure. 27% patients had <23 hour hospital stay. Immediate (21%) and delayed (18%) complications were Clavien-Dindo <2 category. High proportion of patients reported residual symptoms. Type and indication of surgery were significant predictor of complications, length of stay and failure of TWOC outcomes, on multivariate analyses. There were variations in departmental processes, 50% centres used PROMs. CONCLUSION: Monopolar TURP still remains the commonest intervention for BPE. Most departments are adopting newer technologies. The audit identified opportunities for development of consistent, effective and patient centric practices as well as need for large-scale focused studies.

Sensitivity and Specificity of a Multimodal Approach for Concussion Assessment in Youth Athletes.

CONTEXT: Current international consensus endorses a multimodal approach to concussion assessment. However, the psychometric evaluation of clinical measures used to identify postconcussion performance deficits once an athlete is asymptomatic remains limited, particularly in the pediatric population. OBJECTIVE: To describe and compare the sensitivity and specificity of a multimodal assessment battery (balance, cognition, and upper and lower body strength) versus individual clinical measures at discriminating between concussed youth athletes and noninjured controls when asymptomatic. DESIGN: Prospective cohort study. SETTING: Hospital laboratory setting. PARTICIPANTS: A total of 32 youth athletes with a concussion and 32 matched (age and sex) noninjured control participants aged 10-18 years. INTERVENTION(S): Participants were administered preinjury (baseline) assessments of cognition (Immediate Post-Concussion Assessment and Cognitive Testing [ImPACT]), balance (BioSway), and upper and lower body strength (grip strength and standing long jump). Assessments were readministered when concussed participants reported symptom resolution (asymptomatic time point). Noninjured control participants were reassessed using the same time interval as their concussion matched pair. Sensitivity and specificity were calculated using standardized regression-based methods and receiver operating characteristic curves. MAIN OUTCOME MEASURES: Outcome measures included baseline and postinjury ImPACT, BioSway, grip strength, and standing long jump scores. RESULTS: When asymptomatic, declines in performance on each individual clinical measure were seen in 3% to 22% of the concussion group (sensitivity = 3%-22%) compared with 3% to 13% of the noninjured control group (specificity = 87%-97%) (90% confidence interval). The multimodal battery of all combined clinical measures yielded a sensitivity of 41% and a specificity of 77% (90% confidence interval). Based on discriminative analyses, the multimodal approach was statistically superior compared with an individual measures approach for balance and upper and lower body strength, but not for cognition. CONCLUSIONS: Results provide a foundation for understanding which domains of assessment (cognition, balance, and strength) may be sensitive and specific to deficits once symptoms resolve in youth athletes. More work is needed prior to clinical implementation of a preinjury (baseline) to postinjury multimodal approach to assessment following concussion in youth athletes.

Glycans modify mesenchymal stem cell differentiation to impact on the function of resulting osteoblasts.

Glycans are inherently heterogeneous, yet glycosylation is essential in eukaryotes, and glycans show characteristic cell type-dependent distributions. By using an immortalized human mesenchymal stromal cell (MSC) line model, we show that both N- and O-glycan processing in the Golgi functionally modulates early steps of osteogenic differentiation. We found that inhibiting O-glycan processing in the Golgi prior to the start of osteogenesis inhibited the mineralization capacity of the formed osteoblasts 3 weeks later. In contrast, inhibition of N-glycan processing in MSCs altered differentiation to enhance the mineralization capacity of the osteoblasts. The effect of N-glycans on MSC differentiation was mediated by the phosphoinositide-3-kinase (PI3K)/Akt pathway owing to reduced Akt phosphorylation. Interestingly, by inhibiting PI3K during the first 2 days of osteogenesis, we were able to phenocopy the effect of inhibiting N-glycan processing. Thus, glycan processing provides another layer of regulation that can modulate the functional outcome of differentiation. Glycan processing can thereby offer a novel set of targets for many therapeutically attractive processes.

Proteome-Wide Analysis of Cysteine Reactivity during Effector-Triggered Immunity.

A surge in the accumulation of oxidants generates shifts in the cellular redox potential during early stages of plant infection with pathogens and activation of effector-triggered immunity (ETI). The redoxome, defined as the proteome-wide oxidative modifications of proteins caused by oxidants, has a well-known impact on stress responses in metazoans. However, the identity of proteins and the residues sensitive to oxidation during the plant immune response remain largely unknown. Previous studies of the thimet oligopeptidases TOP1 and TOP2 placed them in the salicylic acid dependent branch of ETI, with a current model wherein TOPs sustain interconnected organellar and cytosolic pathways that modulate the oxidative burst and development of cell death. Herein, we characterized the ETI redoxomes in Arabidopsis (Arabidopsis thaliana) wild-type Col-0 and top1top2 mutant plants using a differential alkylation-based enrichment technique coupled with label-free mass spectrometry-based quantification. We identified cysteines sensitive to oxidation in a wide range of protein families at multiple time points after pathogen infection. Differences were detected between Col-0 and top1top2 redoxomes regarding the identity and number of oxidized cysteines, and the amplitude of time-dependent fluctuations in protein oxidation. Our results support a determining role for TOPs in maintaining the proper level and dynamics of proteome oxidation during ETI. This study significantly expands the repertoire of oxidation-sensitive plant proteins and can guide future mechanistic studies.

Mitochondrial Deacetylase SIRT3 Plays an Important Role in Donor T Cell Responses after Experimental Allogeneic Hematopoietic Transplantation.

Allogeneic hematopoietic cell transplantation (allo-HCT) through its graft-versus-tumor (GVT) effects is a curative therapy against many hematological malignancies. However, GVT is linked to harmful graft-versus-host disease (GVHD) after allo-HCT. Both GVT and GVHD require allogeneic T cell responses, which is an energetically costly process that causes oxidative stress. Sirtuin 3 (SIRT3), a mitochondrial histone deacetylase (HDAC), plays an important role in cellular processes through inhibition of reactive oxygen species (ROS). Nonmitochondrial class of HDACs regulate T cell responses, but the role of mitochondrial HDACs, specifically SIRT3, on donor T cell responses after allo-HCT remains unknown. In this study, we report that SIRT3-deficient (SIRT3-/-) donor T cells cause reduced GVHD severity in multiple clinically relevant murine models. The GVHD protective effect of allogeneic SIRT3-/- T cells was associated with a reduction in their activation, reduced CXCR3 expression, and no significant impact on cytokine secretion or cytotoxic functions. Intriguingly, the GVHD protective effect of SIRT3-/- T cells was associated with a reduction in ROS production, which is contrary to the effect of SIRT3 deficiency on ROS production in other cells/tissues and likely a consequence of their deficient activation. Notably, the reduction in GVHD in the gastrointestinal tract was not associated with a substantial reduction in the GVT effect. Collectively, these data reveal that SIRT3 activity promotes allogeneic donor T cell responses and ROS production without altering T cell cytokine or cytolytic functions and identify SIRT3 as a novel target on donor T cells to improve outcomes after allo-HCT.

The youth concussion awareness network (You-CAN) - a school-based peer-led intervention to improve concussion reporting and social support: the protocol for a cluster randomized trial.

BACKGROUND: Concussion prevalence is increasing in the pediatric population, and is a matter of public health concern. Concussion symptoms can be physical, cognitive, emotional and behavioural, and last longer in high school aged youth than adults. Concussions are underreported in youth due to their lack of knowledge, social environment, perceived outcomes of reporting, norms, and self-efficacy. The Youth Concussion Awareness Network (You-CAN) is a school-based peer-led program designed to increase high school students' intent to report a concussion, and provide social support to a peer. This study aims to investigate whether participation in You-CAN, a program grounded in service learning principles, impacts concussion knowledge, attitudes, intent to report a suspected concussion to an adult, and intent to provide social support to a peer. Secondary aims include assessing the implementation fidelity and acceptability of the intervention. METHODS: This longitudinal study will use a cluster randomized trial design. Three high schools from six randomly selected Canadian school boards will participate and be randomized to three study arms: (1) You-CAN led by school staff; (2) You-CAN led by school staff and research team; and (3) untreated comparison group. Intervention arms 1 and 2 will deliver the You-CAN program and create a Concussion Council at their school. The Concussion Council will deliver a concussion awareness campaign and participate in an online showcase with other participating schools. In addition, arm 2 will have monthly video-calls with the research team. A survey based on the Theory of Planned Behaviour will be administered school-wide with all arms (1, 2, 3) at two time points (beginning {T0} and end {T1} of the school year). Exit interviews will be completed with the Concussion Councils and participating school staff. DISCUSSION: This study will provide evidence of the effectiveness of a school-based peer-led concussion program on increasing concussion knowledge, attitudes, subjective norms, perceived behavioural control, intent to report a concussion to an adult, and intent to provide social support to a peer amongst Canadian high school students. It will also provide important information about the implementation and acceptability of the You-CAN program for high school students and staff. TRIAL REGISTRATION: This trial is registered with the ISRCTN registry (ISRCTN64944275, 14/01/2020, retrospectively registered).