Massive Solubility Changes in Neuronal Proteins upon Simulated Traumatic Brain Injury Reveal the Role of Shockwaves in Irreversible Damage

Traumatic brain injury (TBI) is a significant global health concern, affecting millions annually. Often underreported, TBI carries substantial individual and societal burdens, including a notable link to dementia. Current treatments for moderate to severe TBI remain challenging, with cytotoxic brain tissue edema a major predictor of outcome. While the molecular mechanisms of TBI are partially understood, involving primary and secondary injury stages, the primary molecular events immediately following impact remain poorly understood. Research has focused on secondary injury processes, leading to limited therapeutic success. This study hypothesizes that external impact causes protein unfolding, triggering subsequent TBI phenomena. Previous simulations showed dynamic pressure pulses impacting laminin aggregation. This research employed a novel apparatus and PISA assay to analyze proteome-wide effects of simulated dynamic impacts on human brain-derived cells, aiming to identify primary molecular mechanisms and potential therapeutic targets.

The literature extensively documents the prevalence and consequences of TBI, highlighting its association with dementia and the challenges in treating severe cases. Existing research has detailed the secondary injury mechanisms, including Wallerian degeneration, mitochondrial dysfunction, excitotoxicity, neuroinflammation, oxidative stress, autophagy impairment, and apoptosis. Numerous potential therapeutic agents targeting these secondary processes have failed in clinical trials, suggesting the need to focus on the less-understood primary injury mechanisms. This lack of understanding of primary injury, particularly the immediate molecular events, has hindered the development of effective TBI treatments. The researchers highlight the need for a shift in focus towards the primary, potentially reversible, events immediately following the initial impact.

Human SH-SY5Y neuroblastoma cells were cultured and subjected to simulated TBI using a 5.1 kg dummy head dropped from varying heights (55-110 cm) onto a static bottom plate, with and without an energy-absorbing mat. An accelerometer measured g-force. Cell viability was assessed using CellTiter-Blue and Trypan Blue assays. The Proteome Integral Solubility Alteration (PISA) assay, a high-throughput version of Thermal Proteome Profiling (TPP), quantified changes in protein solubility across the proteome immediately after impact. LC-MS/MS was used for protein quantification. Principal component analysis (PCA) analyzed the PISA data. Pathway analysis (STRING DB) identified enriched pathways among significantly altered proteins. Centrifugation experiments compared the effects of g-force alone to the combined effects of g-force and shockwave. Statistical analysis included two-tailed unpaired Student's t-tests.

Dynamic impacts reduced cell viability in a height-dependent manner, with greater reduction after 48 h compared to 24 h, indicating persistent cell death. The energy-absorbing mat significantly reduced cell death at lower impact heights but had less effect at higher heights. PISA analysis revealed massive solubility changes in approximately 25% of the proteome (1382 proteins) after hard impacts, compared to only 1.5% (86 proteins) after soft impacts. PCA analysis identified the first principal component (PC1) as a 'shock meter' quantitatively assessing impact severity. Proteins involved in cell adhesion, collagen, laminin, and stress response were affected, along with proteins in immune response, complement, and coagulation cascades. Proteins involved in neurodegenerative disorders (APP, APLP2, MAPT, SNCA) also showed solubility changes. Comparison of dynamic impact with centrifugation revealed that the cellular effects were primarily due to shockwaves, as cell viability partially recovered after centrifugation. Pathway analysis indicated that the top 50 most affected proteins are involved in pathways such as cell adhesion, the immune system and cellular anatomical features.

This study demonstrates a novel methodology for simulating TBI using cultured neuronal cells. The findings suggest that shockwaves, rather than g-force acceleration alone, are primarily responsible for irreversible cell damage in TBI. The massive solubility changes observed in the proteome, including proteins implicated in neurodegenerative diseases, highlight a potential link between TBI and these disorders. The PC1 component in PCA provides a quantitative measure ('shock meter') of impact severity. The study suggests that disruption of protein complexes and organelles may be the most significant damaging effect, while protein unfolding exacerbates this damage. Increased protein solubility after impact may cause cellular swelling due to increased hydration shell volume. The identification of proteins involved in complement and coagulation cascades as potential early responders to mechanical trauma highlights their potential as biomarkers for TBI severity. The researchers suggest that this proteome-based shock meter could be used to design protective measures and assess the protective effect at the molecular level.

This study provides a new approach to TBI simulation and analysis using a proteome-based shock meter, demonstrating the crucial role of shockwaves in irreversible cellular damage. The identification of numerous affected proteins, including those involved in neurodegenerative diseases, and the development of the 'shock meter' advance the understanding of TBI pathophysiology and provide potential targets for intervention and prevention. Future research could focus on investigating the long-term consequences of these solubility changes and developing targeted therapies based on identified protein biomarkers.

The study used an in vitro model of TBI, which may not fully replicate the complexity of in vivo injury. The 'shock meter' requires further validation and standardization. While the study used the PISA assay to provide a global view of protein solubility changes, focusing on specific interactions or post-translational modifications could give further insights.