Mild Repetitive Injury-induced Chronic Traumatic Encephalopathy Phenotype in Human Triculture 3D in vitro Brain Tissue Model.

Introduction: Chronic traumatic encephalopathy (CTE) is characterized as a progressive, neurodegenerative disease associated with mild repetitive head trauma. Current diagnosis of CTE is limited to postmortem identification of p-tau aggregates in neurons, astrocytes, and around microvasculature. Due to this limitation, nothing is known about the onset and the progression of the disease; thus, no treatment or diagnostic criteria are currently available for mild traumatic brain injury (TBI) patients. Thus, developing a system that would allow the scientific community to study the molecular mechanisms and biomarkers associated with the CTE disease timeline is of utmost importance. Using an earlier developed human 3D in vitro triculture model, we determined the criteria for a mild injury that ensures neuronal network stability and allows for repetitive impacts. Next, we narrowed down the required injury quantity and time interval in between that leads to CTE-like p-tau phenotype. We further demonstrated the involvement of mitochondria dysfunction in the p-tau diseased samples.

Materials and

Methods: Human neurons, primary astrocytes, and a microglial cell line were seeded in silk protein scaffolds (d=6mm) with a collagen gel central window (d=2mm) at 2:0.5:0.1 million cells, respectively. The injury was inflicted using a controlled cortical impactor (CCI) with different conditions – tip size: 1mm, 3mm, speed: 1m/s, 3m/s, and 6m/s, number of hits: 1hit, 2hits, and 3hits, time interval: 0D, 1D, 3D, 7D, and 14D, time point: 24H, 7D, 14D, 4W. Western blot, immunofluorescence, and seahorse assays were performed following the manufacturer's protocol.

Results, Conclusions, and Discussions: Our study revealed that 1mm and 3mm tips with a 1m/s injury maintained neuronal network integrity, enabling us to conduct repetitive injuries. We also found that 7D and 14D intervals between injuries led to a significant increase in p-tau aggregates within 7D, 14D, and 4W after the last injury. Moreover, we observed substantial changes in energy production mechanisms in response to injury. For instance, mild repetitive injury triggered an increase in ECAR rate (glycolysis) within the first week of acute injury, followed by a potential glycolytic depression four weeks after the injury. Additionally, the immediate injury caused a marked decrease in the cellular capacity of oxygen consumption, with a shift to overconsumption of oxygen within 72 hours after injury and stabilization of its OXPHOS rate by the first week.
Our findings underscore the potential of our model in advancing the study of mild repetitive injury-induced neurodegenerative disease. While our current analysis focused on p-tau and beta-amyloid (Aß) as neurodegenerative markers, future work will incorporate alpha-synuclein and phosphorylated TDP-43 (pTDP-43). Building on our preliminary data, we will also explore the vascular contribution to the disease progression. These insights could facilitate the development of new diagnostic tools and therapeutic strategies for CTE and other neurodegenerative diseases.

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