The High‐Mobility Group Box 1 Cytokine Induces Transporter‐Mediated Release of Glutamate from Glial Subcellular Particles (Gliosomes) Prepared from in Situ‐Matured Astrocytes

The multifunctional protein high‐mobility group box 1 (HMGB1) is expressed in restricted areas of adult brain where it can act as a proinflammatory cytokine. We report here that HMGB1 affects CNS transmission by inducing glutamatergic release from glial (gliosomes) but not neuronal (synaptosomes) resealed subcellular particles isolated from mouse cerebellum and hippocampus. Confocal microscopy showed that gliosomes are enriched with glia‐specific proteins such as GFAP and S‐100, but not with neuronal proteins such as PSD‐95, MAP‐2, and β‐tubulin III. Furthermore, gliosomes exhibit labeling neither for integrin‐αM nor for myelin basic protein, specific for microglia and oligodendrocytes, respectively. The gliosomal fraction contains proteins of the exocytotic machinery coexisting with GFAP. Consistent with ultrastructural analysis, several ∼30‐nm nonclustered vesicles are present in the gliosome cytoplasm. Finally, gliosomes represent functional organelles that actively export glutamate when subjected to releasing stimuli, such as ionomycin or ATP, by mechanisms involving extracellular Ca²⁺ and Ca²⁺ release from intracellular stores. HMGB1‐induced release of the stable glutamate analogue [³H]d‐aspartate and endogenous glutamate form gliosomes, whereas nerve terminals were insensitive to the protein. The HMGB1‐evoked release of glutamate was independent on modifications of cytosolic Ca²⁺ concentration, but it was blocked by dl‐threo‐β‐benzyloxyaspartate, suggesting the involvement of transporter‐mediated release mechanisms. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 does not block the HMGB1 effect, indicating a role for the glial glutamate–aspartate transporter (GLAST) subtype in this response. HMGB1 bind to gliosomes but not to synaptosomes and can physically interact with GLAST and receptor for advanced glycation end products (RAGE). Taken together, these results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammalian brain.

Introduction

In the last decade, exciting results have led to dramatic conceptual changes about the role of glial cells in the brain. For a long time, it was believed that glial cells provide structural, metabolic, and trophic support, while the task of information processing was attributed exclusively to neurons. An increasing number of papers suggest that glia shares at least some of the features typical of neurons, particularly those concerned with excitatory neurotransmission (reviewed in Haydon, 2001). In fact, glial cells are endowed with transporters able to remove glutamate from the extracellular space; a process even more efficient than that actuated by neurons (Bergles 1997, Mennerick 1994). Astrocytes, due to their intimate spatial relationship with neuronal synaptic contacts, can directly respond to synaptically released messengers and, in turn, communicate via signaling substances with neurons. The disclosure of this active role of glia has led to the model of the tripartite synapse (Araque et al., 1999), which affirms that a pivotal role in regulating synaptic function, strength, and plasticity is played by glial cells tightly surrounding pre‐ and postsynaptic neuronal elements.

Numerous stimuli have been reported to increase the intracellular Ca²⁺ concentration in astrocytes. As one of the foremost players, glutamate released from neurons can induce Ca²⁺ mobilization from intracellular stores by activating ionotropic and metabotropic receptors located on astroglial cells, which is associated with astrocytic glutamate release, through a process resembling neuronal exocytosis (reviewed in Montana et al., 2006). Interestingly, exocytotic‐like intracellular Ca²⁺‐driven glutamate release from astrocytes can also be elicited by substances involved in the inflammatory and immune pathways, including bradykinin (Parpura et al., 1994), prostaglandins (Bezzi et al., 1998), and chemokines (Bezzi et al., 2001).

A recently discovered member of the cytokine family is the high‐mobility group box 1 (HMGB1) protein that is widely expressed in developing nervous system and transformed cells of nerve origin, including neuroblastoma and glioma (Guazzi 2003, Sajithlal 2002, Taguchi 2000) and that can be actively exported by several cell types (Müller et al., 2004).

In this chapter, we summarize the results obtained studying the glutamate releasing properties of HMGB1, as well as the mechanisms underlining the extracellular export of the excitatory transmitter from astrocytes by exploiting the characteristics of gliosomes, a preparation of astrocyte subcellular particles acutely isolated from the brain of adult animals. This preparation appears well suited to investigate the functional neurochemistry of mature astrocytes. In particular, we show here that release of previously taken up [³H]d‐aspartate ([³H]d‐Asp) and of endogenous glutamate can be evoked from gliosomes by HMGB1 throughout reversal of the glial glutamate–aspartate transporter (GLAST).