Glutamate, the predominant excitatory neurotransmitter in the central nervous system, is involved in almost all aspects of neurological function including cognition, motor function, memory, learning, decision making, and neuronal plasticity. For normal neurological function, glutamate signaling must be properly regulated. Disrupted glutamate regulation plays a pivotal role in the acute pathophysiology of traumatic brain injury (TBI), disrupting neuronal signaling, initiating secondary injury cascades, and producing excitotoxicity. Increases in extracellular glutamate have been correlated with unfavorable outcomes in TBI survivors, emphasizing the importance of glutamate regulation. The aim of this thesis was to examine disruptions in the regulation of extracellular glutamate after experimental TBI. In these studies, we used glutamate-sensitive microelectrode arrays (MEAs) to examine the regulation of extracellular glutamate two days after diffuse brain injury. First, we examined which brain regions were vulnerable to post-traumatic increases in extracellular glutamate. We detected significant increases in extracellular glutamate in the dentate gyrus and striatum, which correlated to the severity of brain injury. Second, we examined the regulation of extracellular glutamate by neurons and glia to determine the mechanisms responsible for post-traumatic increases in extracellular glutamate. In the striatum of brain-injured rats, we detected significant disruptions in release of glutamate by neurons and significant decreases in the removal of glutamate from the extracellular space by glia. Third, we examined if a novel therapeutic strategy, a viral-vector mediated gene delivery approach, could improve the regulation of extracellular glutamate. Infusion of an adeno-associated virus expressing a glutamate transporter into the rat striatum produced significant improvements in glutamate clearance, identifying a novel strategy to reduce excitotoxicity. Lastly, we examined the translational potential of MEAs as novel neuromonitoring device for clinical TBI research. Overall, these studies have demonstrated the translational potential of MEAs to aid in the diagnosis and treatment of TBI survivors.

Indiana University-Purdue University Indianapolis (IUPUI) / Glutamate clearance by astrocytes is an essential part of physiological excitatory neurotransmission. Failure to adapt or maintain low levels of glutamate in the central nervous system is associated with multiple acute and chronic neurodegenerative diseases. The primary excitatory amino acid transporters (EAATs) in human astrocytes are EAAT1 and EAAT2 (GLAST and GLT-1 respectively in rodents). While the inhibition of a ubiquitously-expressed serine/threonine protein kinase, the calcium/calmodulindependent kinase (CaMKII) results in diminished glutamate uptake in cultured primary rodent astrocytes, the molecular mechanism underlying this regulation is unknown. In order to delineate this mechanism, we use a heterologous expression model to explore CaMKII regulation of EAAT1 and EAAT2. In transiently transfected HEK293T cells, pharmacological inhibition of CaMKII and overexpression of a dominant-negative version of CaMKII (Asp136Asn) reduces [3H]-glutamate uptake by EAAT1, without altering EAAT2 mediated glutamate uptake. Surprisingly, overexpression of a constitutively active autophosphorylation mutant (Thr287Asp) to increase autonomous CaMKII activity and a mutant incapable of autophosphorylation (Thr287Val) had no effect on either EAAT1 or EAAT2 mediated glutamate uptake. Pulldown of FLAGtagged glutamate transporters suggests CaMKII does not interact with EAAT1 or EAAT2. SPOTS peptide arrays and recombinant GST-fusion proteins of the intracellular N- and C-termini of EAAT1 identified two potential phosphorylation sites at residues Thr26 and Thr37 in the N-terminus. Introducing an Ala (a non-phospho mimetic) but not an Asp (phosphomimetic) at Thr37 diminished EAAT1-mediated glutamate uptake, suggesting that the phosphorylation state of this residue is important for constitutive EAAT1 function. In sum, this is the first report of a glutamate transporter being identified as a direct CaMKII substrate. These findings indicate that CaMKII signaling is a critical driver of homeostatic glutamate uptake by EAAT1. Aberrations in basal CaMKII activity disrupt glutamate uptake, which can perpetuate glutamate-mediated excitotoxicity and result in cellular death.