Cypin: A novel target for traumatic brain injury.

Cytosolic PSD-95 interactor (cypin), the primary guanine deaminase in the brain, plays key roles in shaping neuronal circuits and regulating neuronal survival. Despite this pervasive role in neuronal function, the ability for cypin activity to affect recovery from acute brain injury is unknown. A key barrier in identifying the role of cypin in neurological recovery is the absence of pharmacological tools to manipulate cypin activity in vivo. Here, we use a small molecule screen to identify two activators and one inhibitor of cypin's guanine deaminase activity. The primary screen identified compounds that change the initial rate of guanine deamination using a colorimetric assay, and secondary screens included the ability of the compounds to protect neurons from NMDA-induced injury and NMDA-induced decreases in frequency and amplitude of miniature excitatory postsynaptic currents. Hippocampal neurons pretreated with activators preserved electrophysiological function and survival after NMDA-induced injury in vitro, while pretreatment with the inhibitor did not. The effects of the activators were abolished when cypin was knocked down. Administering either cypin activator directly into the brain one hour after traumatic brain injury significantly reduced fear conditioning deficits 5 days after injury, while delivering the cypin inhibitor did not improve outcome after TBI. Together, these data demonstrate that cypin activation is a novel approach for improving outcome after TBI and may provide a new pathway for reducing the deficits associated with TBI in patients.

Multi-Dimensional Mapping of Brain-Derived Extracellular Vesicle MicroRNA Biomarker for Traumatic Brain Injury Diagnostics.

The diagnosis and prognosis of traumatic brain injury (TBI) is complicated by variability in the type and severity of injuries and the multiple endophenotypes that describe each patient's response and recovery to the injury. It has been challenging to capture the multiple dimensions that describe an injury and its recovery to provide clinically useful information. To address this challenge, we have performed an open-ended search for panels of microRNA (miRNA) biomarkers, packaged inside of brain-derived extracellular vesicles (EVs), that can be combined algorithmically to accurately classify various states of injury. We mapped GluR2+ EV miRNA across a variety of injury types, injury intensities, history of injuries, and time elapsed after injury, and sham controls in a pre-clinical murine model (n = 116), as well as in clinical samples (n = 36). We combined next-generation sequencing with a technology recently developed by our lab, Track Etched Magnetic Nanopore (TENPO) sorting, to enrich for GluR2+ EVs and profile their miRNA. By mapping and comparing brain-derived EV miRNA between various injuries, we have identified signaling pathways in the packaged miRNA that connect these biomarkers to underlying mechanisms of TBI. Many of these pathways are shared between the pre-clinical model and the clinical samples, and present distinct signatures across different injury models and times elapsed after injury. Using this map of EV miRNA, we applied machine learning to define a panel of biomarkers to successfully classify specific states of injury, paving the way for a prognostic blood test for TBI. We generated a panel of eight miRNAs (miR-150-5p, miR-669c-5p, miR-488-3p, miR-22-5p, miR-9-5p, miR-6236, miR-219a.2-3p, miR-351-3p) for injured mice versus sham mice and four miRNAs (miR-203b-5p, miR-203a-3p, miR-206, miR-185-5p) for TBI patients versus healthy controls.

A Role for Postsynaptic Density 95 and Its Binding Partners in Models of Traumatic Brain Injury.

Postsynaptic density 95 (PSD-95), the major scaffold protein at excitatory synapses, plays a major role in mediating intracellular signaling by synaptic N-methyl-d-aspartate (NMDA) type glutamate receptors. Despite the fact that much is known about the role of PSD-95 in NMDA-mediated toxicity, less is known about its role in mechanical injury, and more specifically, in traumatic brain injury (TBI). Given that neural circuitry is disrupted after TBI and that PSD-95 and its interactors end-binding protein 3 (EB3) and adenomatous polyposis coli (APC) shape dendrites, we examined whether changes to these proteins and their interactions occur after brain trauma. Here, we report that total levels of PSD-95 and the interaction of PSD-95 with EB3 increase at 1 and 7 days after moderate controlled cortical impact (CCI), but these changes do not occur after mild injury. Because changes occur to PSD-95 following brain trauma in vivo, we next considered the functional consequences of PSD-95 alterations in vitro. Rapid deformation of cortical neurons leads to neuronal death 72 h after injury, but this outcome is not dependent on PSD-95 expression. However, disruptions in dendritic arborization following stretch injury in vitro require PSD-95 expression, and these changes in arborization can be mimicked with expression of PSD-95 mutants lacking the second PDZ domain. Thus, PSD-95 and its interactors may serve as therapeutic targets for repairing dendrites after TBI.