Browsing by Subject "EAAT2"

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    CaMKII regulation of astrocytic glutamate uptake
    (2016-05-19) Chawla, Aarti R.; Hudmon, Andy; Cummins, Theodore; Oxford, Gerry S.; Chen, Jinhui; Hoang, Quyen
    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.
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    Effects of ceftriaxone on ethanol, nicotine or sucrose intake by alcohol-preferring (P) rats and its association with GLT-1 expression
    (Elsevier, 2016-06-21) Sari, Youssef; Toalston, Jamie E.; Rao, P.S.S.; Bell, Richard L.; Psychiatry, School of Medicine
    Increased glutamatergic neurotransmission appears to mediate the reinforcing properties of drugs of abuse, including ethanol (EtOH). We have shown that administration of ceftriaxone (CEF), a β-lactam antibiotic, reduced EtOH intake and increased glutamate transporter 1 (GLT-1) expression in mesocorticolimbic regions of male and female alcohol-preferring (P) rats. In the present study, we tested whether CEF administration would reduce nicotine (NIC) and/or EtOH intake by adult female P rats. P rats were randomly assigned to 4 groups: (a) 5% sucrose (SUC) or 10% SUC [SUC], (b) 5% SUC+0.07mg/ml NIC and 10% SUC+0.14mg/ml NIC [NIC-SUC], 15% EtOH and 30% EtOH [EtOH] and (d) 15% EtOH+0.07mg/ml NIC and 30% EtOH+0.14mg/ml NIC [NIC-EtOH]. After achieving stable intakes (4weeks), the rats were administered 7 consecutive, daily i.p. injections of either saline or 200mg/kg CEF. The effects of CEF on intake were significant but differed across the reinforcers; such that ml/kg/day SUC was reduced by ∼30%, mg/kg/day NIC was reduced by ∼70% in the NIC-SUC group and ∼40% in the EtOH-NIC group, whereas g/kg/day EtOH was reduced by ∼40% in both the EtOH and EtOH-NIC group. The effects of CEF on GLT-1 expression were also studied. We found that CEF significantly increased GLT-1 expression in the prefrontal cortex and the nucleus accumbens of the NIC and NIC-EtOH rats as compared to NIC and NIC-EtOH saline-treated rats. These findings provide further support for GLT-1-associated mechanisms in EtOH and/or NIC abuse. The present results along with previous reports of CEF's efficacy in reducing cocaine self-administration in rats suggest that modulation of GLT-1 expression and/or activity is an important pharmacological target for treating polysubstance abuse and dependence.