Glutamate, Inflammation, Depression and the Brain.



Program#/Poster#: 51.16/U1
Presentation Title: Glutamate stimulates astrocyte release of ATP: A potential mechanism for riluzole’s antidepressant action
Location: WCC Hall A-C
Presentation time: Saturday, Nov 15, 2014, 1:00 PM - 5:00 PM
Presenter at Poster: Sat, Nov. 15, 2014, 4:00 PM - 5:00 PM
Topic: ++C.15.d. Animal models
1Neurophychiatry, Tottori Univ., Tottori, Japan; 2Mol. Psychiatry, Yale Univ., New Haven, CT
Abstract: Stress decreases neurogenesis and synaptogenesis in the adult hippocampus, leading to depressive-like behavior; however the mechanism by which stress causes neuronal damage is unknown. We previously demonstrated that stress increases ATP in the hippocampus, which stimulates the release of interleukin-1β (IL-1β) and causes decreased neurogenesis and depressive behavior. Those changes are ameliorated by the purinergic P2X7 receptor (P2X7R) antagonist (A-804598), indicating that ATP is critical for stress-induced depression. Here, we investigated the source of ATP induced by stress. Glutamate, an excitatory neurotransmitter, is another molecule that we have previously confirmed to be increased by stress. The released glutamate is taken up by the surrounding astrocytes and transformed into glutamine to maintain homeostasis of the synapse. Thus, we hypothesized that stress increases glutamate that is sensed by astrocytes, which in turn release ATP as a gliotransmitter. To test this hypothesis, we used rat astrocyte primary cell culture to examine ATP release. We found that glutamate (2 to 10 µM) releases ATP in astrocyte cell culture; thus the excess glutamate is a potential stimulus for stress-induced changes in ATP and inflammatory responses in the brain. We next investigated whether inhibition of excess glutamate can ameliorate the stress response induced by immobilization stress. Cortisol is a well-known stress marker, and we first confirmed that P2X7R antagonist (A-804598) suppresses the increase of cortisol, indicating that ATP regulates the increase of cortisol. Cortisol is easy to measure by peripheral blood, so we employed it as an indicator of stress reaction. Riluzole is a drug used for the treatment of amyotrophic lateral sclerosis, and it is thought to prevent glutamate release from presynaptic terminals and stimulate glutamate uptake in the synapse. We thus employed riluzole to reduce glutamate and then measured cortisol levels. Riluzole was administered intraperitoneally one hour prior to 40 minutes of immobilization stress. The concentration of cortisol in serum was measured by ELISA. Riluzole decreased the increase of cortisol caused by immobilization stress. Together, the results support the hypothesis that stress increases glutamate, which is sensed by astrocytes and induces ATP release, which in turn induces pro-inflammatory responses, including up-regulation of cortisol. Riluzole is thought to be a potential drug for depression, and these mechanisms may contribute to its antidepressant effects.
Disclosures: T. Yamanashi: None. M. Kusunose: None. T. Yamauchi: None. K.T. Ota: None. M. Iwata:None. R.S. Duman: None. K. Kaneko: None.
Keyword (s): ATP
Support: Grant-in-Aid for Young Scientists B


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Wow Emma - that brings together a whole bunch of stuff.

Together, the results support the hypothesis that stress increases glutamate, which is sensed by astrocytes and induces ATP release, which in turn induces pro-inflammatory responses.

Check this out on P2X7 - a critical player in this process - from Follow ME in Denmark. It's involved in pain, microglial activation, perhaps ME/CFS and a variety of neurological diseases

P2X7 in ME/CFS and other diseases

I have previously referred to articles about TRPA1 involvement in inflammation, vasodilation, central sensitization and hypothesized that TRPA1 has a role in ME/CFS/POTS/MCS/pain and more.

TRPA1 is of course only a small part of what is affected in the human body during these diseases. Many other receptors must also be involved. The latest article from Light et al mentions higher expression of P2RX7, so let us take a closer look at the P2X7 receptor.

The P2X7 receptor (P2X7R) is a nonselective cation channel that is activated by extracellular ATP and triggers the secretion of several proinflammatory substances, such as IL-1β, IL-18, TNF-α, and nitric oxide.

Quotes from P2X7 articles:

Following the activation of P2X7R through high concentrations of and/or prolonged exposure to ATP, a large conductance channel is elicited, which leads to dynamic changes in the membrane potential that include an intracellular potassium efflux. This cationic efflux induces inflammasome complex assembly and subsequent pro-caspase-1 maturation into caspase-1 through the NOD-like receptor protein (NLRP3). Caspase-1 plays a key role in the cleavage of pro-IL-1β to form mature IL-1β, which plays a principal role in nitric oxide synthase (NOS), cyclooxygenase-2, and tumor necrosis factor-alpha (TNF-α) activities
Reference: Physiological Roles and Potential Therapeutic Applications of the P2X7 Receptor in Inflammation and Pain

The expression of the purinergic, P2X7 receptor (P2X7R), is known to be enhanced in many brain pathologies where presence of activated microglia is a concurrent feature. This review focuses on the links between P2X7R expression and microglial activation and proliferation. The P2X7R is identified as a key player in the process of microgliosis, where by driving microglial activation, it can potentially lead to a deleterious cycle of neuroinflammation and neurodegeneration.
Reference: Microglia: Proliferation and activation driven by the P2X7 receptor

P2X7 is involved in central sensitization. Purinergic signaling, involving P2X4, P2X7 and P2Y12 receptors, plays a central role in the recruitment and activation of microglia, which have emerged as key regulators of central sensitization.
Reference: Pain hypersensitivity mechanisms at a glance
Reference: Central sensitization of nociceptive neurons in rat medullary dorsal horn involves purinergic P2X7 receptors
Reference: P2X(7) inhibition in stellate ganglia prevents the increased sympathoexcitatory reflex via sensory-sympathetic coupling induced by myocardial ischemic injury

Lipid rafts are important in the control of nociceptor excitability – P2X7 is associated with lipid rafts

Lipid rafts are microdomains of the plasma membrane highly enriched in cholesterol and sphingolipids. Lipid rafts tune the spatial and temporal organisation of proteins and lipids on the plasma membrane. They are thought to act as platforms on the membrane where proteins and lipids can be trafficked, compartmentalised and functionally clustered.
Reference: Association between tetrodotoxin resistant channels and lipid rafts regulates sensory neuron excitability

Reference: Palmitoylation of the P2X7 receptor, an ATP-gated channel, controls its expression and association with lipid rafts
(Palmitoylation is the covalent attachment of fatty acids, such as palmitic acid, to cysteine and less frequently to serine and threonine residues of proteins, which are typically membrane proteins.)

Caveolae are a special type of lipid raft

It has recently been shown in epithelial cells that the ATP-gated ion channel P2X7R is in part, associated with caveolae and colocalized with caveolin-1. In the present study of the mouse heart, we show for the first time, using immunohistochemistry and cryoimmunoelectron microscopy, that P2X7R is expressed in atrial cardiomyocytes and in cardiac microvascular endothelial cells, but not in the ventricle cardiomyocytes. In cardiac tissue homogenates derived from caveolin-1 deficient mice (cav-1-/-), an increase of the P2Xrx7 mRNA and P2X7R protein (80 kDa) was found, particularly in atrial samples.
Reference: Increased P2X7R expression in atrial cardiomyocytes of caveolin-1 deficient mice

Some guessing:
Some ME patients have POTS. POTS patients may have autoimmunoreactive IgGs against proteins associated with caveolae structure. Cavelolin-1 is a protein in the caveolae. Increased P2X7R expression is found in atrial cardiomyocytes of caveolin-1 deficient mice. ME patients show increased P2RX7 expression.

P2X7 and motor neruron death/cell death:

Here we found that P2X(7) receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death
Reference: Extracellular ATP and the P2X7 receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis

P2X7-induced motor neuron death was dependent on neuronal nitric oxide synthase-mediated production of peroxynitrite, p38 activation, and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well-established peroxynitrite/FAS death pathway in motor neurons.
Reference: P2X7 receptor-induced death of motor neurons by a peroxynitrite/FAS-dependent pathway

A cytofluorometric assay demonstrated that P2X7 activation induced ROS formation in EOC13 cells, via a mechanism independent of Ca2+ influx and K+ efflux. Cytofluorometric measurements of Annexin-V binding and 7AAD uptake demonstrated that P2X7 activation induced EOC13 cell death. The ROS scavenger N-acetyl-L-cysteine impaired both P2X7-induced EOC13 ROS formation and cell death, suggesting that ROS mediate P2X7-induced EOC13 death. In conclusion, P2X7 activation induces the uptake of organic cations, ROS formation, and death in EOC13 microglia.
Reference: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

Let us keep an eye on P2X7 and ME/CFS research to find out what the higher expression of P2RX7 actually means in ME/CFS.

Surface expression of P2X7R on PBMC in patients with pSS was significantly higher than controls, suggesting P2X7R may contribute to the complex pathogenesis of pSS and also anxiety and/or depression.
Reference: The expression of P2X7 receptors on peripheral blood mononuclear cells in patients with primary Sjögren's syndrome and its correlation with anxiety and depression

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