Katrin Färber

Purinergic receptors on microglial cells: functional expression in acute brain slices and modulation of microglial activation in vitro

Microglial cells are the pathologic sensors in the brain. ATP released from damaged cells is a candidate for signalling neural injury to microglia. Moreover, ATP is an extracellular messenger for propagating astrocyte activity in the form of Ca2+ waves. To test for the functional expression of purinoreceptors in microglial cells we employed the patch‐clamp technique in acute slices of adult mouse brain. ATP triggered a nonselective cationic and a K+ current. Pharmacological screening with purinergic ligands indicated the presence of P2Y1 and P2Y2/4 receptors linked to the activation of a K+ current and P2X receptors, including P2X7, linked to the activation of a nonselective cationic current. These findings suggest that microglial cells in situ express different purinergic receptors with distinct sensitivity and functional coupling. To test for the involvement of purinoreceptors in microglial activation, we stimulated cultured microglial cells with lipopolysaccharide and measured the release of tumour necrosis factor α, interleukin‐6, interleukin‐12 and macrophage inflammatory protein 1α, induction of K+ outward currents and nitric oxide release. All these parameters were reduced in the presence of purinergic ligands, indicating that purinergic receptor activation attenuated indicators of microglial activation.

Physiology of microglial cells

Microglial cells in culture and in situ express a defined pattern of K(+) channels, which is distinct from that of other glial cells and neurons. This pattern undergoes defined changes with microglial activation. As expected for a cell with immunological properties, microglia express a variety of cytokine and chemokine receptors, which are linked to the mobilization of Ca(2+) (cytosolic free calcium) from internal stores. Microglial cells also have the capacity to respond to neuronal activity: they express receptors for the major excitatory receptor glutamate and the main inhibitory receptor GABA (gamma-amino butyric acid). By expressing purinergic receptors, microglia can sense astrocyte activity in the form of Ca(2+) waves. Activation of transmitter receptors can affect cytokine release which is a potential means as to how brain activity can affect immune function.

Purinergic signaling and microglia.

Microglial cells are considered as the pathologic sensors of the brain. In this paper, we review mechanisms of purinergic signaling in microglia. As ATP is not only considered as a physiological signaling substance but is also elevated in pathology, it is not surprising that microglia express a variety of P2X, P2Y and adenosin receptors. As a rapid physiological event, ATP triggers a cationic conductance, increases the potassium conductance and also elicits a calcium response. As a long-term effect, purinergic receptor activation is linked to the movement of microglial processes and, in the context of pathology, to chemotaxis. The purinoreceptors also modulate the release of substances from microglia, such as cytokines, nitric oxide, or superoxide, which are important in the context of a pathologic response.

Dopamine and noradrenaline control distinct functions in rodent microglial cells

Microglial cells are the immune-competent elements of the brain. They not only express receptors for chemokines and cytokines but also for neurotransmitters such as GABA [Charles et al., Mol. Cell Neurosci. 24 (2003) 214], glutamate [Noda et al., J. Neurosci. 20 (2000) 251], and adrenaline [Mori et al., Neuropharmacology 43 (2002) 1026]. Here we report the functional expression of dopamine receptors in mouse and rat microglia, in culture and brain slices. Using the patch clamp technique as the functional assay we identified D1- and D2-like dopamine receptors using subtype-specific ligands. They triggered the inhibition of the constitutive potassium inward rectifier and activated potassium outward currents in a subpopulation of microglia. Chronic dopamine receptor stimulation enhanced migratory activity and attenuated the lipopolysaccharide (LPS)-induced nitric oxide (NO) release similar as by stimulation of adrenergic receptors. While, however, noradrenaline attenuated the LPS-induced release of TNF-alpha and IL-6, dopamine was ineffective in modulating this response. We conclude that microglia express dopamine receptors which are distinct in function from adrenergic receptors.

Functional role of calcium signals for microglial function

In this review we summarize mechanisms of Ca2+ signaling in microglial cells and the impact of Ca2+ signaling and Ca2+ levels on microglial function. So far, Ca2+ signaling has been only characterized in cultured microglia and thus these data refer rather to activated microglia as observed in pathology when compared with the resting form found under physiological conditions. Purinergic receptors are the most prominently expressed ligand‐gated Ca2+‐permeable channels in microglia and control several microglial functions such as cytokine release in a Ca2+‐dependent fashion. A large variety of metabotropic receptors are linked to Ca2+ release from intracellular stores. Depletion of these intracellular stores triggers a capacitative Ca2+ entry. While microglia are already in an activated state in culture, they can be further activated, for example, by exposure to bacterial endotoxin. This activation leads to a chronic increase of [Ca2+]i and this Ca2+ increase is a prerequisite for the release of nitric oxide and cytokines. Moreover, several factors (TNFα, IL‐1β, and IFN‐γ) regulate resting [Ca2+]i levels.

Microglia express GABAB receptors to modulate interleukin release

gamma-Aminobutyric acid (GABA) can act as a neuroprotective agent besides its well-established role as the main inhibitory neurotransmitter in the CNS. Here we report that microglial cells express GABA(B) receptors indicating that these prominent immunocompetent cells in the brain are a target for GABA. Agonists of GABA(B) receptors triggered the induction of K(+) conductance in microglial cells from acute brain slices and in culture. Both subunits of GABA(B) receptors were identified in cultured microglia by Western blot analysis and immunocytochemistry, and were detected on a subpopulation of microglia in situ by immunohistochemistry. In response to facial nerve axotomy, we observed an increase in GABA(B) receptor expressing microglial cells in the facial nucleus. We activated microglial cells in culture with lipopolysaccharide (LPS) to induce the release of interleukin-6 and interleukin-12p40. This release activity was attenuated by simultaneous activation of the GABA(B) receptors indicating that GABA can modulate the microglial immune response.

Bradykinin-Induced Microglial Migration Mediated by B1-Bradykinin Receptors Depends on Ca2+ Influx via Reverse-Mode Activity of the Na+/Ca2+ Exchanger

Bradykinin (BK) is produced and acts at the site of injury and inflammation. In the CNS, migration of microglia toward the lesion site plays an important role pathologically. In the present study, we investigated the effect of BK on microglial migration. Increased motility of cultured microglia was mimicked by B1 receptor agonists and markedly inhibited by a B1 antagonist, but not by a B2 receptor antagonist. BK induced chemotaxis in microglia isolated from wild-type and B2-knock-out mice but not from B1-knock-out mice. BK-induced motility was not blocked by pertussis toxin but was blocked by chelating intracellular Ca2+ or by low extracellular Ca2+, implying that Ca2+ influx is prerequisite. Blocking the reverse mode of Na+/Ca2+ exchanger (NCX) completely inhibited BK-induced migration. The involvement of NCX was further confirmed by using NCX+/− mice; B1-agonist-induced motility and chemotaxis was decreased compared with that in NCX+/+ mice. Activation of NCX seemed to be dependent on protein kinase C and phosphoinositide 3-kinase, and resultant activation of intermediate-conductance (IK-type) Ca2+-dependent K+ currents (IK(Ca)) was activated. Despite these effects, BK did not activate microglia, as judged from OX6 staining. Using in vivo lesion models and pharmacological injection to the brain, it was shown that microglial accumulation around the lesion was also dependent on B1 receptors and IK(Ca). These observations support the view that BK functions as a chemoattractant by using the distinct signal pathways in the brain and, thus, attracts microglia to the lesion site in vivo.

WNT signaling in activated microglia is proinflammatory.

Microglia activation is central to the neuroinflammation associated with neurological and neurodegenerative diseases, particularly because activated microglia are often a source of proinflammatory cytokines. Despite decade‐long research, the molecular cascade of proinflammatory transformation of microglia in vivo remains largely elusive. Here, we report increased β‐catenin expression, a central intracellular component of WNT signaling, in microglia undergoing a proinflammatory morphogenic transformation under pathogenic conditions associated with neuroinflammation such as Alzheimers disease. We substantiate disease‐associated β‐catenin signaling in microglia in vivo by showing age‐dependent β‐catenin accumulation in mice with Alzheimers‐like pathology (APdE9). In cultured mouse microglia expressing the WNT receptors Frizzled FZD4,5,7,8 and LDL receptor‐related protein 5/6 (LRP5/6), we find that WNT‐3A can stabilize β‐catenin. WNT‐3A dose dependently induces LRP6 phosphorylation with downstream activation of disheveled, β‐catenin stabilization, and nuclear import. Gene‐expression profiling reveals that WNT‐3A stimulation specifically increases the expression of proinflammatory immune response genes in microglia and exacerbates the release of de novo IL‐6, IL‐12, and tumor necrosis factor α. In summary, our data suggest that the WNT family of lipoglycoproteins can instruct proinflammatory microglia transformation and emphasize the pathogenic significance of β‐catenin‐signaling networks in this cell type.

The ectonucleotidase cd39/ENTPDase1 modulates purinergic‐mediated microglial migration

Microglia is activated by brain injury. They migrate in response to ATP and although adenosine alone has no effect on wild type microglial migration, we show that inhibition of adenosine receptors impedes ATP triggered migration. CD39 is the dominant cellular ectonucleotidase that degrades nucleotides to nucleosides, including adenosine. Importantly, ATP fails to stimulate P2 receptor mediated migration in cd39−/− microglia. However, the effects of ATP on migration in cd39−/− microglia can be restored by co‐stimulation with adenosine or by addition of a soluble ectonucleotidase. We also tested the impact of cd39‐deletion in a model of ischemia, in an entorhinal cortex lesion and in the facial nucleus after facial nerve lesion. The accumulation of microglia at the pathological sites was markedly decreased in cd39−/− animals. We conclude that the co‐stimulation of purinergic and adenosine receptors is a requirement for microglial migration and that the expression of cd39 controls the ATP/adenosine balance.

The potassium channels Kv1.5 and Kv1.3 modulate distinct functions of microglia

Activation of microglia by LPS leads to an induction of cytokine and NO release, reduced proliferation and increased outward K(+) conductance, the latter involving the activation of Kv1.5 and Kv1.3 channels. We studied the role of these channels for microglial function using two strategies to interfere with channel expression, a Kv1.5 knockout (Kv1.5(-/-)) mouse and an antisense oligonucleotide (AO) approach. The LPS-induced NO release was reduced by AO Kv1.5 and completely absent in the Kv1.5(-/-) animal; the AO Kv1.3 had no effect. In contrast, proliferation was augmented with both, loss of Kv1.3 or Kv1.5 channel expression. After facial nerve lesion, proliferation rate was higher in Kv1.5(-/-) animals as compared to wild type. Patch clamp experiments confirmed the reduction of the LPS-induced outward current amplitude in Kv1.5(-/-) microglia as well as in Kv1.5- or Kv1.3 AO-treated cells. Our study indicates that induction of K(+) channel expression is a prerequisite for the full functional spectrum of microglial activation.