Author ORCID Identifier

Defense Date


Document Type


Degree Name

Master of Science


Anatomy & Neurobiology

First Advisor

Andrew Ottens

Second Advisor

Carmen Sato-Bigbee

Third Advisor

Dong Sun


The research of this thesis investigated the mechanism of action for the therapeutic compound CLP290, which our laboratory has previously demonstrated to reverse degradation of the protein potassium-chloride co-transporter 2 (KCC2) and restore neuronal function following modeled traumatic brain injury (TBI) in the rat. The loss of membranous KCC2 from the plasma membrane after TBI worsens the excitation/inhibition imbalance common in neurotrauma. Addressed here were the specific aims to: 1) assess that CLP290 treatment restores the functional oligomeric form of KCC2 to the neuronal plasma membrane as necessary to provide chloride homeostasis; 2) investigate the signaling mechanism by which CLP290 treatment inhibits TBI-induced KCC2 degradation; 3) determine the neuroprotective capacity of CLP290 treatment two-weeks after TBI and any laminar specificity within the perilesional cortical mantle. Studies here used brain tissues collected at one, two or fourteen days following controlled cortical impact injury in the rat, with animals administered a 50 mg/kg effective dose of CLP290 at one day following injury. Outcomes were assessed with a series of immunochemical assays to probing molecular and cellular outcomes with selective antibodies and histological stains. Results demonstrated that CLP290 was effective in preventing the loss of oligomeric KCC2 from the membrane, supporting a capacity to maintain chloride homeostasis within the perilesional neocortical tissue. We further established that the induced loss of KCC2 starting two days after TBI was preceded by a significant decrease in KCC2 phosphorylation at serine 940 (pS940), a key regulator of stability within the membrane. Moreover, we were able to connect this loss with TBI altered activation of protein kinase C delta isoform (PKCδ). Within 24-h of injury, we observed a significant decline in PKCδ at the membrane, a known regulator of KCC2-pS940. Moreover, we determined that this was partially compensated for early on by an endothelin-1 mediated increase in PKCδ phosphorylation at threonine 505 (T505). However, this transient state was reversed by two days after injury, at which point we observed an alternative PKCδ activation state through increased phosphorylation at tyrosine 311 (Y311). This modification is known to shift PKCδ localization and activity away from the membrane, which promoted a greater decline in KCC2-pS940 and significant destabilization of oligomeric KCC2. Importantly, administering CLP290 one day after TBI inhibit PKCδ-pY311 modification and restored PKCδ to the membrane along with normal (sham) levels of KCC2-pS940 and oligomeric KCC2. Findings here thus track CLP290’s mechanism of action back to its effect on PKCδ-pY311. However, this PKCδ regulatory site has been shown in other neuronal stress conditions to promote apoptotic cell death. Results here demonstrate that day-one administration of CLP290 was neuroprotective out to two-weeks after injury, preventing significant neuronal loss across all layers of perilesional neocortex. Ultimately, studies here significantly expanded our understanding of CLP290’s mechanism of action in preventing significant loss of KCC2 following TBI, maintaining chloride homeostasis and providing neuroprotection, supporting its therapeutic potential in neurotrauma.


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Available for download on Wednesday, July 30, 2025


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