Constitutive regulation of the glutamate/aspartate transporter EAAT1 by Calcium-Calmodulin-Dependent Protein Kinase II.

Glutamate clearance by astrocytes is an essential part of normal 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 in human astrocytes are EAAT1 and EAAT2 (GLAST and GLT-1, respectively, in rodents). While the inhibition of calcium/calmodulin-dependent kinase (CaMKII), a ubiquitously expressed serine/threonine protein kinase, results in diminished glutamate uptake in cultured primary rodent astrocytes (Ashpole et al. 2013), the molecular mechanism underlying this regulation is unknown. Here, we use a heterologous expression model to explore CaMKII regulation of EAAT1 and EAAT2. In transiently transfected HEK293T cells, pharmacological inhibition of CaMKII (using KN-93 or tat-CN21) reduces [3 H]-glutamate uptake in EAAT1 without altering EAAT2-mediated glutamate uptake. While over-expressing the Thr287Asp mutant to enhance autonomous CaMKII activity had no effect on either EAAT1 or EAAT2-mediated glutamate uptake, over-expressing a dominant-negative version of CaMKII (Asp136Asn) diminished EAAT1 glutamate uptake. SPOTS peptide arrays and recombinant glutathione S-transferase-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) at Thr37 diminished EAAT1-mediated glutamate uptake, suggesting that the phosphorylation state of this residue is important for constitutive EAAT1 function. Our study is the first to identify a glutamate transporter as a direct CaMKII substrate and suggests that CaMKII signaling is a critical driver of constitutive glutamate uptake by EAAT1.

Loss of calcium/calmodulin-dependent protein kinase II activity in cortical astrocytes decreases glutamate uptake and induces neurotoxic release of ATP.

The extent of calcium/calmodulin-dependent protein kinase II (CaMKII) inactivation in the brain after ischemia correlates with the extent of damage. We have previously shown that a loss of CaMKII activity in neurons is detrimental to neuronal viability by inducing excitotoxic glutamate release. In the current study we extend these findings to show that the ability of astrocytes to buffer extracellular glutamate is reduced when CaMKII is inhibited. Furthermore, CaMKII inhibition in astrocytes is associated with the rapid onset of intracellular calcium oscillations. Surprisingly, this rapid calcium influx is blocked by the N-type calcium channel antagonist, ω-conotoxin. Although the function of N-type calcium channels within astrocytes is controversial, these voltage-gated calcium channels have been linked to calcium-dependent vesicular gliotransmitter release. When extracellular glutamate and ATP levels are measured after CaMKII inhibition within our enriched astrocyte cultures, no alterations in glutamate levels are observed, whereas ATP levels in the extracellular environment significantly increase. Extracellular ATP accumulation associated with CaMKII inhibition contributes both to calcium oscillations within astrocytes and ultimately cortical neuron toxicity. Thus, a loss of CaMKII signaling within astrocytes dysregulates glutamate uptake and supports ATP release, two processes that would compromise neuronal survival after ischemic/excitotoxic insults.

Community signalling between Streptococcus gordonii and Porphyromonas gingivalis is controlled by the transcriptional regulator CdhR.

Interspecies signalling between Porphyromonas gingivalis and Streptococcus gordonii serves to constrain development of dual species communities. Contact with S. gordonii propagates a tyrosine phosphorylation-dependent signal within P. gingivalis that culminates in reduced transcription of adhesin and signalling genes. Here we demonstrate the involvement of the P. gingivalis orphan LuxR family transcription factor PGN_1373, which we designate CdhR, in this control pathway. Expression of cdhR is elevated following contact with S. gordonii; however, regulation of cdhR did not occur in a mutant lacking the tyrosine phosphatase Ltp1, indicating that CdhR and Ltp1 are components of the same regulon. Contact between S. gordonii and a CdhR mutant resulted in increased transcription of mfa, encoding the subunit of the short fimbriae, along with higher levels of Mfa protein. Expression of luxS, encoding AI-2 synthase, was also increased in the cdhR mutant after contact with S. gordonii. The Mfa adhesive function and AI-2-dependent signalling participate in the formation and development of dual species communities, and consistent with this the cdhR mutant displayed elevated accumulation on a substratum of S. gordonii. Recombinant CdhR protein bound to upstream regulatory regions of both mfa and luxS, indicating that CdhR has a direct effect on gene expression. LuxS was also found to participate in a positive feedback loop that suppresses CdhR expression. Interaction of Mfa fimbriae with S. gordonii is necessary to initiate signalling through CdhR. These results reveal CdhR to be an effector molecule in a negative regulatory network that controls P. gingivalis-S. gordonii heterotypic communities.

Disruption of nNOS-NOS1AP protein-protein interactions suppresses neuropathic pain in mice.

Elevated N-methyl-D-aspartate receptor (NMDAR) activity is linked to central sensitization and chronic pain. However, NMDAR antagonists display limited therapeutic potential because of their adverse side effects. Novel approaches targeting the NR2B-PSD95-nNOS complex to disrupt signaling pathways downstream of NMDARs show efficacy in preclinical pain models. Here, we evaluated the involvement of interactions between neuronal nitric oxide synthase (nNOS) and the nitric oxide synthase 1 adaptor protein (NOS1AP) in pronociceptive signaling and neuropathic pain. TAT-GESV, a peptide inhibitor of the nNOS-NOS1AP complex, disrupted the in vitro binding between nNOS and its downstream protein partner NOS1AP but not its upstream protein partner postsynaptic density 95 kDa (PSD95). Putative inactive peptides (TAT-cp4GESV and TAT-GESVΔ1) failed to do so. Only the active peptide protected primary cortical neurons from glutamate/glycine-induced excitotoxicity. TAT-GESV, administered intrathecally (i.t.), suppressed mechanical and cold allodynia induced by either the chemotherapeutic agent paclitaxel or a traumatic nerve injury induced by partial sciatic nerve ligation. TAT-GESV also blocked the paclitaxel-induced phosphorylation at Ser15 of p53, a substrate of p38 MAPK. Finally, TAT-GESV (i.t.) did not induce NMDAR-mediated motor ataxia in the rotarod test and did not alter basal nociceptive thresholds in the radiant heat tail-flick test. These observations support the hypothesis that antiallodynic efficacy of an nNOS-NOS1AP disruptor may result, at least in part, from blockade of p38 MAPK-mediated downstream effects. Our studies demonstrate, for the first time, that disrupting nNOS-NOS1AP protein-protein interactions attenuates mechanistically distinct forms of neuropathic pain without unwanted motor ataxic effects of NMDAR antagonists.

Proteome-wide lysine acetylation in cortical astrocytes and alterations that occur during infection with brain parasite Toxoplasma gondii.

Lysine acetylation is a reversible post-translational modification (PTM) that has been detected on thousands of proteins in nearly all cellular compartments. The role of this widespread PTM has yet to be fully elucidated, but can impact protein localization, interactions, activity, and stability. Here we present the first proteome-wide survey of lysine acetylation in cortical astrocytes, a subtype of glia that is a component of the blood-brain barrier and a key regulator of neuronal function and plasticity. We identified 529 lysine acetylation sites across 304 proteins found in multiple cellular compartments that largely function in RNA processing/transcription, metabolism, chromatin biology, and translation. Two hundred and seventy-seven of the acetylated lysines we identified on 186 proteins have not been reported previously in any other cell type. We also mapped an acetylome of astrocytes infected with the brain parasite, Toxoplasma gondii. It has been shown that infection with T. gondii modulates host cell gene expression, including several lysine acetyltransferase (KAT) and deacetylase (KDAC) genes, suggesting that the host acetylome may also be altered during infection. In the T. gondii-infected astrocytes, we identified 34 proteins exhibiting a level of acetylation >2-fold and 24 with a level of acetylation <2-fold relative to uninfected astrocytes. Our study documents the first acetylome map for cortical astrocytes, uncovers novel lysine acetylation sites, and demonstrates that T. gondii infection produces an altered acetylome.

Characterization of Antibodies that Detect Human GFAP after Traumatic Brain Injury.

After traumatic brain injury (TBI), glial fibrillary acidic protein (GFAP) and other brain-derived proteins and their breakdown products are released into biofluids such as CSF and blood. Recently, a sandwich ELISA was constructed that measured GFAP concentrations in CSF or serum from human mild-moderate TBI patients. The goals of the present study were to characterize the same two antibodies used in this ELISA, and to determine which GFAP bands are detected by this antibody combination. Here, both antibodies recognized GFAP specifically in human brain and post-TBI CSF in a cluster of bands ranging from 50-38 kDa, that resembled bands from calpain-cleaved GFAP. By immunoprecipitation, the anti-GFAP Capture antibody recovered full length GFAP and its breakdown products from human brain lysate and post-TBI CSF. These findings demonstrate that the anti-GFAP ELISA antibodies non-preferentially detect intact GFAP and GFAP breakdown products, underscoring their utility for detecting brain injury in human patients.