Temporal mapping and pharmacological manipulation of signaling architecture of Tyrosine kinases in Traumatic Brain Injury (TBI)

Abstract

Traumatic Brain Injury (TBI) is a highly complicated pathology involving multiple events occurring simultaneously including, but not limited to, inflammation, blood brain barrier disruption, apoptosis and alteration in neuronal signaling. Due to limited understanding of complex protein interactions and therapeutic exploration so far in TBI, the focus of this thesis. was on mapping large scale signaling architecture originating from the key regulators of multiple signaling events (such as cell proliferation, differentiation, apoptosis); Receptor Tyrosine Kinases (RTKs) at therapeutically effective time point. We opted for an advanced unbiased approach to explore the temporal dynamics of 14 different RTK families post TBI and developed an R software-based analysis pipeline for analyzing protein array data, available on open-access GitHub repository PROTEAS (PROTein array Expression AnalysiS; github.com/Rida-Rehman/PROTEAS. Our initial investigation of temporal activation revealed that most signaling events initiate as early as 3 hours post TBI, thereby limiting our time window for acute intervention. After establishing the temporal significance, we performed a large-scale in-depth analysis of 223 tyrosine and serine/theronine kinase proteins and identified distinct signaling modules at 3h after trauma. We selected three distinct RTKs and NRTKs from the screening; Met, VEGFR1, Btk and FGFR family to exploit the therapeutic potential. Spatial relevance of these RTKs reveal increase in phosphorylation levels primarily in microglia indicating critical role of these resident immune cells. Proteomics analysis at 3h, 3d and 7d post injury also revealed changes in protein expression over time with interesting immune fingerprint over time. Inhibitor treatment with single dose pre-trauma, against these receptors, revealed positive regulation of immune response and subsequent neuroprotection. Acute and Prolonged treatment (starting 2 hours pre-TBI until 7 days, one dose/day) of Met inhibitor revealed significant behavioral improvement until treatment withdrawal. We concluded that long term pathological outcomes can be mitigated and regulated as early as 1 day post TBI by signal specific RTK treatment during therapeutically effective time window and microglia are drivers of most, if not all, critical immune specific events.