It is critical to share knowledge and harmonize approaches to optimize progress in rare cancers. The International Neuroblastoma Risk Group (INRG) Task Force was formed by the 4 major neuroblastoma cooperative groups in 2004 to achieve this goal. Strategies developed for neuroblastoma are an exemplar for other rare malignancies. Data from an initial cohort of 8800 patients were transferred to the INRG Data Commons, and a data-sharing model was developed. Currently, information on more than 25 000 patients is available to the research community. The INRG staging and risk classification systems have led to harmonized approaches for therapeutic groupings. INRG consensus manuscripts have led to uniform criteria for classifying biological data, evaluating the extent of disease, and defining treatment response. More than 40 INRG research studies have been performed by investigators from around the world, including analyses of rare patients, which would not otherwise be possible. The success of this approach for neuroblastoma has been leveraged to create the Pediatric Cancer Data Commons and the Data for the Common Good. Efforts to enrich the INRG Commons with additional genomic and biomarker data, extracted electronic health records, and digital medical images are ongoing. The international networking model developed by the INRG Task Force has led to new research discoveries and progress in neuroblastoma. The approach has now been applied to 16 other cancers and conditions, including rhabdomyosarcoma, germ cell tumor, Lynch syndrome, and cancer predisposition. This framework of international collaboration and data sharing serves as a model for advancing rare adult malignancies.

Fewer than 10 % of children with diffuse midline glioma (DMG) survive 2 years from diagnosis. Radiation therapy remains the cornerstone of treatment and there are no medicinal products with regulatory approval. Although the biology of DMG is better characterized, this has not yet translated into effective treatments. H3K27-alterations initiate the disease but additional drivers are required for malignant growth. Hence, there is an urgent unmet need to develop new multi-modality therapeutic strategies, including alternative methods of drug delivery. ONC201 (DRD2 antagonist and mitochondrial ClpP agonist) is the most widely evaluated investigational drug. Encouraging early data is emerging for CAR T-cells and oncolytic viruses. GD2, B7-H3 and PI3K signalling are ubiquitous targets across all subtypes and therapeutics directed to these targets would potentially benefit the largest number of children. PI3K, ACVR1, MAPK and PDGFRA pathways should be targeted in rational biological combinations. Drug discovery is a very high priority. New specific and potent epigenetic modifiers (PROTACS e.g. SMARCA4 degraders), with blood-brain penetrance are needed. Cancer neuroscience therapeutics are in early development. Overall survival is the preferred regulatory endpoint. However, the evaluation of this can be influenced by the use of re-irradiation at the time of progression. An efficient clinical trial design fit for regulatory purposes for the evaluation of new therapeutics would aid industry and facilitate more efficient therapy development. Challenges in conducting clinical trials such as the need for comparator data and defining endpoints, could be addressed through an international, first-in-child, randomised, complex innovative design trial. To achieve progress: i) drug discovery; ii) new multi-modality, efficient, collaborative, pre-clinical approaches, possibly including artificial intelligence and, iii) efficient clinical trial designs fit for regulatory purposes are required.

Realising the potentially substantial benefits of chimeric antigen receptor (CAR) T-cell therapy for children with cancer is hindered by non-scientific barriers that are also relevant for other rare diseases. A solely commercial development model will not deliver optimally due to insufficient return on investment for pharmaceutical companies. Access to therapies is restricted for patients who might benefit and advancing innovation in the academic research setting is difficult. Challenges relating to CAR T-cell therapies in paediatric malignancies and how they might be addressed were discussed in a meeting convened by ACCELERATE-an international multistakeholder organisation aiming to advance the timely investigation of new anticancer drugs. New academic and biopharma hybrid development models could benefit rare populations and coordination of early development can promote synergy and avoid duplicative efforts. Following promising first-in-child trials, new models are needed to support pivotal trials, decentralised manufacturing, registration, and reduced costs. The European Medicines Agency and the US Food and Drug Administration encourage academic development and early discussions. A biotech company funded via a pooled investment vehicle could provide access to safe and effective products for children and adolescents with cancer through registration and reimbursement.

High-risk neuroblastoma is a poor prognosis cancer of the sympathetic nervous system that accounts for a disproportionate number of childhood cancer deaths. Many viable biological targets have been identified, and the number of potential combinations is even larger. Several products have attained marketing authorization for treatment of patients with neuroblastoma. Patient outcomes remain poor, with approximately 50% of children with newly diagnosed high-risk neuroblastoma cured of their disease. International, multistakeholder Neuroblastoma Drug Development Strategy (NDDS) meetings were established more than a decade ago. This third NDDS meeting included academia, industry, regulatory, and patient advocacy representatives to prioritize agents and to address key challenges in drug development in this disease. Given the central role that anti-GD2 therapy plays, novel GD2-directed combinations were a key focus, including epigenetic enzymes such as EZH2 and immunologic targets such as IL15 and TIGIT as potential combination partners. GD2-directed chimeric antigen receptor (CAR)-T cells were a top priority, along with emerging CAR-T targets such as B7-H3 and GPC2. Recognizing that combination therapies are likely to be most impactful for patients and for advancing therapies to frontline, another key focus was on high priority combinations of targeted therapies, including Aurora A kinase plus BCL2 or ATR inhibitors. Additional targets and agents were prioritized or deprioritized based upon current data. Access to drugs for clinical trials was viewed as a major barrier to progress. Strategies to overcome this challenge focused on united efforts by the international scientific and advocacy community and early engagement by industry with regulatory authorities.

The twelfth multi-stakeholder Paediatric Strategy focused on cyclin-dependent kinase (CDK) inhibitors. Genetic aberrations in paediatric tumours increase CDK4/6 activity, thus the pathway is a therapeutic target. As a result, CDK4/6 inhibitors have been evaluated in clinical trials for children and young adults with different malignancies, both as single agents and in combination, including molecular enrichment in ESMART. In some instances, trials of different agents within this same class are very similar in design or target population, leading to duplication. Consistent with preclinical data with CDK4/6 inhibitors which mostly demonstrate cytostasis, but not tumour regression, objective responses are rare, although there may be slowing of tumour growth. Similar to adults, no predictive biomarkers have been identified and an integrated comprehensive clinical development strategy of this class of agents is lacking. Patient advocates believe that the many trials of CDK4/6 inhibitors, which have enrolled many children and young people, have not been matched by sufficient gains in knowledge. CDK9, CDK12 and CDK13 inhibitors, based on exploitation of transcriptional vulnerabilities, have shown promise in preclinical models of MYCN-driven neuroblastoma, osteosarcoma and translocation fusion-driven malignancies. The development of these inhibitors should be sequential, iterative and coordinated, including early engagement with regulators. In conclusion, there needs to be prioritisation and coordination of any further development of CDK4/6 inhibitors in paediatric malignancies. Definite tumour regression in preclinical models, not just cytostasis, should be taken as the go/no go decision point to progress such agents to clinical studies. Based on limited single agent activity in preclinical models, trials of new inhibitors should avoid unnecessary exposure of patients to monotherapy and rapidly progress to combination strategies, while retaining designs that allow early evaluation of activity. A framework to support integrated development strategies of new products across sponsors would be very beneficial.

Despite the introduction of anti-GD2 antibody therapy, outcomes for children with high-risk neuroblastoma remain poor, with low cure rates and a high proportion of survivors facing long-term sequelae. In this report, leaders from international cooperative groups and patient advocacy organizations review lessons learnt, identify current challenges, and provide a vision to bring new agents into frontline therapy to increase cure rates and reduce long-term toxicities over the next decade. The implementation of this vision requires improved global collaboration, incorporation of novel biomarkers, and a strengthened interaction with the regulatory landscape.

INTRODUCTION:Phase I/II trials are essential to introduce novel agents for children with cancer. Defining risk factors of early mortality could maximize the efficiency of such trials. METHODS:Patients < 18 years with relapsed/refractory solid tumors in their first phase I/II trial were eligible in retrospect. Mortality at 30 and 90 days on treatment (30-DM, 90-DM) were calculated. Clinical/laboratory parameters and adult prognostic scores (Royal Marsden Hospital -RMH-, MD Anderson Cancer Center -MDACC-) were assessed at baseline and correlated with 90-DM (univariate analysis, logistic regression) to devise a pediatric-specific prognostic score (ITCC). RESULTS:N = 507. Median age 11.6 years (range 0.5-17.9); 45 % females. 30-DM and 90-DM (95 %CI) were 4.7 % (3.1-7.0 %) and 22.9 % (19.3-26.8 %), respectively. RMH (n = 348) and MDACC (n = 345) scores correlated with 90-DM (p < 0.001). Performance status ≤ 80 %, no school attendance and lactate dehydrogenase (LDH) above normal levels strongly correlated with higher 90-DM, constituting the ITCC score (1 point each). The 90-DM with ITCC score (n = 306) of 0, 1, 2 and 3 was 2.7 %, 10.7 %, 36.4 % and 80.0 %, respectively. Odds ratios (95 %CI) for 90-DM with 1, 2 and 3 points were 4.23 (1.28-19.1); 20.0 (6.55-87.4); and 140 (37.4-720), respectively. Among patients with predicted risk of 90-DM ≥ 75 %, those who ultimately died within 90 days represented 1.4 % (RMH, MDACC) versus 7.8 % (ITCC) of the sample; p < 0.001. CONCLUSIONS:The early mortality rates reported here will serve as a reference for future phase I/II trials. Risk scoring based on performance status, school attendance and LDH levels can estimate 90-DM in oncology phase I/II trials.

10052 Background: Outcomes for high-risk neuroblastoma (HR-NBL) following relapse are poor, although there is a paucity of data on overall survival post-relapse (OSPR) in the contemporary era. Prognostic factors associated with OSPR across all neuroblastoma risk groups (INSS stage, MYCN status and time to first relapse) have previously been described. Here we present an analysis focussed specifically on HR-NBL. Methods: We conducted a retrospective analysis using INRG Data Commons including HR-NBL patients diagnosed ≥1985 with relapse/progression ≤2022. HR-NBL was defined as age ≥547days at diagnosis with INRGSS M; or INRGSS L2 MYCN-amplified disease. The final cohort excluded patients without relapse/progression and with death as first event. Primary endpoint was OSPR, with Kaplan-Meier estimates of survival and sub-group comparisons using log-rank tests. Results: Of the 25,245 NBL patients in the INRG Data Commons, 4045 were included in the final analysis. The majority had INRGSS M disease (96%) and were < 5 years at diagnosis (74%), with MYCN amplification in 36% of those with available data. OSPR was 22±0.7% at 2 years and 8±0.4% at 5 years, with median OSPR 0.76 years (95% CI: 0.73-0.82). OSPR improved over time; for example, 2-year OSPR was 16+/-2.8% for patients diagnosed 1985-1989 vs 32%±2.8% for 2015-2019 (p < 0.0001). Across the whole cohort, patients with a diagnosis of NBL (vs nodular ganglioneuroblastoma), greater number of involved metastatic compartments (MSI) or elevated LDH at diagnosis, tumors with MYCN amplification, higher MKI, 1p LOH and presence of ALK mutation had statistically significantly worse OSPR than respective counterparts. OSPR for INRGG M was significantly better than for INRGSS L2. Serum ferritin, tumor grade and ploidy were not associated with OSPR, while 11q LOH and age showed non-proportional hazards, with patients aged ≥5 years at diagnosis having a better early OSPR but poorer long-term outcome than those < 5 years. Conclusions: In this, the largest analysis of patients with relapsed HR-NBL, multiple factors at diagnosis were associated with OSPR, emphasising the importance of tumor biology and disease burden. The finding of improved OSPR for INRG M vs L2 is unexpected but may relate to MYCN amplification. Further analyses will focus on changes in prognostic factors over time, non-proportional hazards and the impact of upfront and relapse treatment paradigms on OSPR and prognostic factors.

Background: In neuroblastoma MYCN amplification is associated with enhanced angiogenesis and poor survival. Mutations in the anaplastic lymphoma kinase (ALK) gene can occur with MYCN amplification, conferring a very poor prognosis. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF)/c-MET signalling are implicated in neuroblastoma progression. Cabozantinib has potent activity against VEGFR2 and MET. Methods: The efficacy of cabozantinib against tumours arising in GEM models of high-risk neuroblastoma was assessed using multiparametric MRI. Tumour-bearing Th-MYCN and Th-ALKF1174L/Th-MYCN mice were imaged prior to, 24 and 48 hrs after treatment with either 30mg/kg/day cabozantinib or vehicle. Treatment-induced changes in tumour volume, native T1, R2* and ADC were evaluated, and histological correlates sought. Additional Th-MYCN mice were treated daily for up to 28 days. Results: Cabozantinib elicited significant 24 and 60 % growth delay 24 and 48 hrs after treatment in tumours in Th-MYCN mice, and a significant 6-8 % reduction in native T1. Tumour R2* was significantly reduced 48 hrs post-treatment. Significantly higher tumour necrosis and apoptosis, and significantly lower Ki67, CD34 and VEGFR2 staining, was determined from the cabozantinib-treated mice. Treatment of Th-ALKF1174L/Th-MYCN mice caused significant 4 % and 21 % tumour growth delay, and a significant 5 % reduction in native T1 at 48 hrs. Daily cabozantinib treatment of Th-MYCN mice elicited significant tumour growth delay over 7 days which translated into significant survival benefit. Conclusion: Cabozantinib exhibits activity against neuroblastomas arising in both Th-MYCN and Th-MYCN/ALKF1174L mice, revealed in situ using MRI. Native T1 is an early, sensitive and clinically translatable imaging biomarker of effective treatment response in neuroblastoma.

Traumatic optic neuropathy (TON), often resulting from repetitive mild traumatic brain injury (r-mTBI), is a major contributor to irreversible visual impairment, with few preventive therapeutic options available. This study evaluates the prophylactic efficacy of ST266, a biologically active secretome derived from amnion epithelial cells, in mitigating TON-related visual and cognitive dysfunction in a murine model of r-mTBI. Mice received five closed-head injuries spaced 48 h apart, preceded by intranasal delivery of ST266 starting 24 h before the first injury and administered twice daily for ten days. Behavioral performance was assessed using the Barnes Maze and optomotor response (OMR). Pathological and biochemical analyses were conducted on the eye and optic nerve tissues. Prophylactic treatment with ST266 preserved retinal ganglion cell (RGC) count, significantly attenuated optic nerve inflammation, and reduced microgliosis, TLR4, and NLRP3 expression in the intracanalicular segment of the optic nerve. ST266 protected spatial learning in the Barnes Maze during the acquisition phase but did not improve memory retention or visual function by OMR. Importantly, this model reflects translationally relevant features of human TON, such as RGC degeneration and visual-cognitive impairments. Our findings identify ST266 as a potential prophylactic therapy for TON, especially in populations at elevated risk (e.g., athletes, military personnel, or individuals prone to falls). Its non-invasive intranasal administration, established safety profile, and anti-inflammatory and neuroprotective effects make it a viable candidate for clinical translation. This is the first study to demonstrate protection against optic nerve injury before trauma onset, addressing a critical unmet need in neuro-ophthalmology.

PURPOSE The aim of the Innovative Therapies for Children with Cancer (ITCC) consortium is to improve access to novel therapies for children and adolescents with cancer. The evolution of the ITCC clinical trial portfolio since 2003 was reviewed. METHODS All ITCC-labeled phase I/II trials opened between January 1, 2003 and February 3, 2018 were analyzed in two periods (2003-2010 and 2011-2018), and data were extracted from the ITCC database, regulatory agencies' registries, and publications. RESULTS Sixty-one trials (62% industry-sponsored) enrolled 3,198 patients. The number of trials in the second period increased by almost 300% (16 v 45). All biomarker-driven trials (n = 14) were conducted in the second period. The use of rolling six and model-based designs increased (1 of 9, 11% v 21 of 31, 68%), and that of 3 + 3 designs decreased (5 of 9, 55% v 5 of 31, 16%; P = .014). The proportion of studies evaluating chemotherapeutics only decreased (5 of 16, 31% v 4 of 45, 9%), the proportion of single-agent targeted therapies did not change (9 of 16, 56.2% v 24 of 45, 53.3%), the proportion of combination targeted therapies trials increased (2 of 16, 12%, v 17 of 45, 38%), the proportion of randomized phase II trials increased (1 of 7, 14% v 8 of 14, 57%). More trials were part of a pediatric investigation plan in the second period (4 of 16, 25% v 21 of 45, 46%). The median time for Ethics Committees' approvals was 1.7 times longer for academic compared with industry-sponsored trials. CONCLUSION This study reports a shift in the paradigm of early drug development for childhood cancers, with more biologically relevant targets evaluated in biomarker-driven trials or in combination with other therapies and with more model-based or randomized designs and a greater focus on fulfilling regulatory requirements. Improvement of trial setup and recruitment could increase the number of patients benefiting from novel agents.

PURPOSE:Outcomes for children with relapsed and refractory high-risk neuroblastoma (RR-HRNB) remain dismal. The BEACON Neuroblastoma trial (EudraCT 2012-000072-42) evaluated three backbone chemotherapy regimens and the addition of the antiangiogenic agent bevacizumab (B). MATERIALS AND METHODS:Patients age 1-21 years with RR-HRNB with adequate organ function and performance status were randomly assigned in a 3 × 2 factorial design to temozolomide (T), irinotecan-temozolomide (IT), or topotecan-temozolomide (TTo) with or without B. The primary end point was best overall response (complete or partial) rate (ORR) during the first six courses, by RECIST or International Neuroblastoma Response Criteria for patients with measurable or evaluable disease, respectively. Safety, progression-free survival (PFS), and overall survival (OS) time were secondary end points. RESULTS:One hundred sixty patients with RR-HRNB were included. For B random assignment (n = 160), the ORR was 26% (95% CI, 17 to 37) with B and 18% (95% CI, 10 to 28) without B (risk ratio [RR], 1.52 [95% CI, 0.83 to 2.77]; P = .17). Adjusted hazard ratio for PFS and OS were 0.89 (95% CI, 0.63 to 1.27) and 1.01 (95% CI, 0.70 to 1.45), respectively. For irinotecan ([I]; n = 121) and topotecan (n = 60) random assignments, RRs for ORR were 0.94 and 1.22, respectively. A potential interaction between I and B was identified. For patients in the bevacizumab-irinotecan-temozolomide (BIT) arm, the ORR was 23% (95% CI, 10 to 42), and the 1-year PFS estimate was 0.67 (95% CI, 0.47 to 0.80). CONCLUSION:The addition of B met protocol-defined success criteria for ORR and appeared to improve PFS. Within this phase II trial, BIT showed signals of antitumor activity with acceptable tolerability. Future trials will confirm these results in the chemoimmunotherapy era.

Importance:In poor-prognosis children's cancers, new therapies may carry fresh hope for patients and parents. However, there is an absolute requirement for any new therapy to be properly evaluated to fulfill scientific, regulatory, and reimbursement requirements. Randomized clinical trials (RCTs) are considered the gold standard, but no consensus exists on how and when they should be deployed to best meet the needs of all stakeholders. Objective:To conduct a multistakeholder meeting to foster a greater shared understanding of perspectives regarding RCTs of new therapies for children with poor-prognosis cancers and develop consensus recommendations on when and how they should be used. Evidence Review:During October 2022 and April 2023, 2 structured workshops were convened, bringing together individuals representing the perspectives of patient advocates and academic clinician-researchers, regulators, and health technology assessment bodies. A premeeting briefing document was prepared and circulated to all attendees. During the workshops, selected attendees presented on behalf of each stakeholder group, focused topic discussions were conducted, and each meeting concluded by agreeing on a consensus set of recommendations. Meeting organizers drafted meeting summary reports that were circulated to all attendees, who commented on and revised them as a group to produce final recommendations from the workshops. Findings:Though the workshops did not reconcile all stakeholder differences, sufficient areas of agreement enabled a set of conclusions to be drawn, resulting in 8 consensus recommendations: (1) drug development strategies for new therapies, including the role of RCTs, should be established at the time of first-in-child studies; (2) engagement with regulators and health technology assessment bodies about RCT design is crucial; (3) involvement of patient advocates is necessary to ensure that an RCT is patient focused; (4) timing of an RCT is critical to preserve clinical equipoise; (5) use of crossover in an RCT can be of benefit, but with important caveats; (6) end point maturity and overall survival in an RCT may be important for regulatory and health technology assessment approvals; (7) in the absence of an RCT, contemporaneous control cohorts are preferred over historical control cohorts; and (8) quality of life should be captured in all prospective RCTs. Conclusions and Relevance:The agreed-upon workshop conclusions provide a basis for key considerations while undertaking future drug development activities for children with poor-prognosis cancers, ensuring that the needs and perspectives of all stakeholders are factored in from the outset.

Phosphatidylinositol 3-kinase (PI3-K) signalling pathway is a crucial path in cancer for cell survival and thus represents an intriguing target for new paediatric anti-cancer drugs. However, the unique clinical toxicities of targeting this pathway (resulting in hyperglycaemia) difficulties combining with chemotherapy, rarity of mutations in childhood tumours and concomitant mutations have resulted in major barriers to clinical translation of these inhibitors in treating both adults and children. Mutations in PIK3CA predict response to PI3-K inhibitors in adult cancers. The same mutations occur in children as in adults, but they are significantly less frequent in paediatrics. In children, high-grade gliomas, especially diffuse midline gliomas (DMG), have the highest incidence of PIK3CA mutations. New mutation-specific PI3-K inhibitors reduce toxicity from on-target PI3-Kα wild-type activity. The mTOR inhibitor everolimus is approved for subependymal giant cell astrocytomas. In paediatric cancers, mTOR inhibitors have been predominantly evaluated by academia, without an overall strategy, in empiric, mutation-agnostic clinical trials with very low response rates to monotherapy. Therefore, future trials of single agent or combination strategies of mTOR inhibitors in childhood cancer should be supported by very strong biological rationale and preclinical data. Further preclinical evaluation of glycogen synthase kinase-3 beta inhibitors is required. Similarly, even where there is an AKT mutation (∼0.1 %), the role of AKT inhibitors in paediatric cancers remains unclear. Patient advocates strongly urged analysing and conserving data from every child participating in a clinical trial. A priority is to evaluate mutation-specific, central nervous system-penetrant PI3-K inhibitors in children with DMG in a rational biological combination. The choice of combination, should be based on the genomic landscape e.g. PTEN loss and resistance mechanisms supported by preclinical data. However, in view of the very rare populations involved, innovative regulatory approaches are needed to generate data for an indication.

Chronic neuroinflammation and microglial activation are key mediators of the secondary injury cascades and cognitive impairment that follow exposure to repetitive mild traumatic brain injury (r-mTBI). Peroxisome proliferator-activated receptor-γ (PPARγ) is expressed on microglia and brain resident myeloid cell types and their signaling plays a major anti-inflammatory role in modulating microglial responses. At chronic timepoints following injury, constitutive PPARγ signaling is thought to be dysregulated, thus releasing the inhibitory brakes on chronically activated microglia. Increasing evidence suggests that thiazolidinediones (TZDs), a class of compounds approved from the treatment of diabetes mellitus, effectively reduce neuroinflammation and chronic microglial activation by activating the peroxisome proliferator-activated receptor-γ (PPARγ). The present study used a closed-head r-mTBI model to investigate the influence of the TZD Pioglitazone on cognitive function and neuroinflammation in the aftermath of r-mTBI exposure. We revealed that Pioglitazone treatment attenuated spatial learning and memory impairments at 6 months post-injury and reduced the expression of reactive microglia and astrocyte markers in the cortex, hippocampus, and corpus callosum. We then examined whether Pioglitazone treatment altered inflammatory signaling mechanisms in isolated microglia and confirmed downregulation of proinflammatory transcription factors and cytokine levels. To further investigate microglial-specific mechanisms underlying PPARγ-mediated neuroprotection, we generated a novel tamoxifen-inducible microglial-specific PPARγ overexpression mouse line and examined its influence on microglial phenotype following injury. Using RNA sequencing, we revealed that PPARγ overexpression ameliorates microglial activation, promotes the activation of pathways associated with wound healing and tissue repair (such as: IL10, IL4 and NGF pathways), and inhibits the adoption of a disease-associated microglia-like (DAM-like) phenotype. This study provides insight into the role of PPARγ as a critical regulator of the neuroinflammatory cascade that follows r-mTBI in mice and demonstrates that the use of PPARγ agonists such as Pioglitazone and newer generation TZDs hold strong therapeutic potential to prevent the chronic neurodegenerative sequelae of r-mTBI.

Repetitive mild traumatic brain injuries (r-mTBI) sustained in the military or contact sports have been associated with the accumulation of extracellular tau in the brain, which may contribute to the pathogenesis of neurodegenerative tauopathies. The expression of the apolipoprotein E4 (apoE4) isoform has been associated with higher levels of tau in the brain, and worse clinical outcomes after r-mTBI, though the influence of apoE genotype on extracellular tau dynamics in the brain is poorly understood. We recently demonstrated that extracellular tau can be eliminated across blood-brain barrier (BBB), which is progressively impaired following r-mTBI. The current studies investigated the influence of repetitive mild TBI (r-mTBI) and apoE genotype on the elimination of extracellular solutes from the brain. Following intracortical injection of biotin-labeled tau into humanized apoETr mice, the levels of exogenous tau residing in the brain of apoE4 mice were elevated compared to other isoforms, indicating reduced tau elimination. Additionally, we found exposure to r-mTBI increased tau residence in apoE2 mice, similar to our observations in E2FAD animals. Each of these findings may be the result of diminished tau efflux via LRP1 at the BBB, as LRP1 inhibition significantly reduced tau uptake in endothelial cells and decreased tau transit across an in vitro model of the BBB (basolateral-to-apical). Notably, we showed that injury and apoE status, (particularly apoE4) resulted in chronic alterations in BBB integrity, pericyte coverage, and AQP4 polarization. These aberrations coincided with an atypical reactive astrocytic gene signature indicative of diminished CSF-ISF exchange. Our work found that CSF movement was reduced in the chronic phase following rmTBI (>18 months post injury) across all apoE genotypes. In summary, we show that apoE genotype strongly influences cerebrovascular homeostasis, which can lead to age-dependent deficiencies in the elimination of toxic proteins from the brain, like tau, particularly in the aftermath of head trauma.

Cerebral blood flow (CBF) is known to increase in response to both dynamic and isometric exercise. However, middle cerebral artery blood velocity (MCAv) kinetics in response to small-muscle isometric exercise, and sex differences in this kinetic response, require further investigation. Previous work suggests that MCAv response times to stressors may be indicative of cerebrovascular function, so the kinetics of this response to isometric exercise would add to the understanding of cerebrovascular health. The objective of this study was to characterize MCAv kinetics in response to isometric handgrip exercise and investigate potential sex differences in the response. We expected that MCAv at baseline would be higher in females than in males. However, we hypothesized that this sex difference would disappear during isometric handgrip exercise. In addition, we hypothesized that the time constant (τ) would be shorter in females compared with males, with a higher maximum response amplitude in females compared with males. Twenty-six healthy older adults with an average age of 56.8 ± 5.0 years (11 male (M) – aged 59.4 ± 4.8 years; 15 female (F) – aged 54.6 ± 4.1 years) completed an isometric handgrip exercise protocol. The exercise protocol consisted of the following: 1) performing a maximum voluntary contraction (MVC) with a handgrip device in the participants’ left hand (on a separate visit); 2) performing a sustained contraction until failure at 40% of the participant’s MVC. MCAv was continuously measured at rest and during handgrip exercise. The time constant was calculated as 63% of the time until steady-state MCAv during handgrip exercise. There was a trend for higher MCAv at baseline (57.8 ± 11.0 cm/s) in females (61.5 ± 11.2 cm/s) compared with males (53.6 ± 9.6 cm/s) that did not reach significance (p=0.08). There were no significant differences between the sexes for max amplitude of MCAv response to isometric handgrip exercise (M: 20.7 ± 11.1 cm/s; F: 15.4 ± 10.0 cm/s; p = 0.24) or time constant (M: 20.5 ± 9.8 sec; F: 18.6 ± 11.3 sec; p = 0.65). These results suggest no significant differences in MCAv between males and females at baseline. There also appear to be no significant sex differences in the kinetic response of MCAv to handgrip exercise. Future work could model MCAv kinetics in response to isometric handgrip exercise. The relationship between age and MCAv kinetics could also be investigated, along with the effect of menopause on MCAv kinetics in females. American Heart Association (19IPLOI34680015; JNB); American Heart Association COVID Supplement (19IPLOI34680015; JNB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Epidemiological studies have unveiled a robust link between exposure to repetitive mild traumatic brain injury (r-mTBI) and elevated susceptibility to develop neurodegenerative disorders, notably chronic traumatic encephalopathy (CTE). The pathogenic lesion in CTE cases is characterized by the accumulation of hyperphosphorylated tau in neurons around small cerebral blood vessels which can be accompanied by astrocytes that contain phosphorylated tau, the latter termed tau astrogliopathy. However, the contribution of tau astrogliopathy to the pathobiology and functional consequences of r-mTBI/CTE or whether it is merely a consequence of aging remains unclear. We addressed these pivotal questions by utilizing a mouse model harboring tau-bearing astrocytes, GFAPP301L mice, subjected to our r-mTBI paradigm. Despite the fact that r-mTBI did not exacerbate tau astrogliopathy or general tauopathy, it increased phosphorylated tau in the area underneath the impact site. Additionally, gene ontology analysis of tau-bearing astrocytes following r-mTBI revealed profound alterations in key biological processes including immunological and mitochondrial bioenergetics. Moreover, gene array analysis of microdissected astrocytes accrued from stage IV CTE human brains revealed an immunosuppressed astroglial phenotype similar to tau-bearing astrocytes in the GFAPP301L model. Additionally, hippocampal reduction of proteins involved in water transport (AQP4) and glutamate homeostasis (GLT1) was found in the mouse model of tau astrogliopathy. Collectively, these findings reveal the importance of understanding tau astrogliopathy and its role in astroglial pathobiology under normal circumstances and following r-mTBI. The identified mechanisms using this GFAPP301L model may suggest targets for therapeutic interventions in r-mTBI pathogenesis in the context of CTE.