- Email: [email protected]
Calming Neurons with a Microglial Touch
TINS 1578 No. of Pages 3
Trends in Neurosciences
Spotlight
Calming Neurons with a Microglial Touch
Insight into the functional significance of microglia–neuronal soma interactions first came from zebrafish studies in which microglial contacts with hyperactive 1,2 Kaushik Sharma, neuronal somata served to reduce neuroLong-Jun Wu,3,4,5 and nal hyperactivity [4]. Similarly, mouse Ukpong B. Eyo 1,2,* microglial P2Y12Rs have been suggested to play a role in modulating neuronal hyIn vivo two-photon imaging of peractivity in a chemoconvulsive seizure microglia in the intact brain has context [5]. Results from combined imagrevealed that microglia constantly ing and electrophysiological studies in survey neuronal soma. Research mouse cortical brain slices further supover the past decade and a recent ported this hypothesis because microglial paper by Cserép et al. published contact with neurons prevented repetitive in Science are now uncovering depolarization-induced neurotoxicity [6]. the nature, mechanisms, and con- Moreover, in ischemia, the presence of misequences of these interactions in croglia correlated with reduced neuronal hyperactivity [7]. Thus, the picture emerghealth and injury. ing over the past decade of microglial imaging research suggests that microglia Advances in in vivo two-photon micros- target neuronal somata and dendrites to copy and microglia-specific mouse re- dampen neuronal hyperactivity. A recent porter lines enabled the first detailed elaborate study by Cserép et al. beautifully in vivo studies of microglia in 2005 [1,2]. confirms this hypothesis and elucidates These studies indicated that microglia several novel features of the nature are highly dynamic in surveying their and significance of microglia–neuronal microenvironment through their many soma contacts, as well as their underlying ramified processes. Later studies further mechanisms [8]. showed that microglia survey specific neuronal elements such as synapses Cserép et al. [8] first document anatomi(e.g., [3]) and neuronal cell bodies cally extensive physical interactions be(e.g., [4]). However, at that time, the tween microglial processes and neuronal mechanisms underlying such interac- cell bodies in both mouse and human tions and their functional significance brains. Through real-time in vivo analyses were mostly unknown. A 2014 paper raised in mice, the authors showed that only a mithe possibility of NMDA receptor-dependent nority (~10%) of synapses are contacted coupling to pannexin-independent ATP by microglial processes at any given time, release as a predominant mechanism for whereas ~90% of somata are contacted microglia–neuron physical interactions by microglia. The average duration of con[5]. In this study, the microglia-specific tact was more than threefold longer for P2Y12 purinergic receptor (P2Y12R) somata (~25 minutes) than for dendrites was shown to be crucial for several (~7.5 minutes). Together, these results unforms of physical interactions between derline the importance of looking into the microglia and neuronal somata and den- issue of contacts between microglial prodrites. This and other studies highlighted cesses and neuronal soma, in addition to that microglia play an active role in neuro- the often-studied contacts with dendrites.
previously determined. Among their findings, Cserép et al. identified clusters of Kv2.1 voltage-gated potassium channels on neuronal somata as sites of microglial contact in both mouse and human brains, pointing to a possible mechanism of microglia–neuronal soma interactions. The localization of microglial processes adjacent to neuronal mitochondria is especially noteworthy, as well as the fact that vesicular release of purinergic signals is required for chemoattraction of microglial processes. Of interest, given the known role of P2Y12Rs in regulating microglia–neuron interactions [5], the authors examined and confirmed the expression of P2Y12Rs at sites of microglia–neuronal soma contacts. In addition, pharmacological blockade of P2Y12Rs reduced the duration of these contacts. Consistent with this, when neurons were chemogenetically activated they were able to recruit microglial processes in wild-type mice, but not in P2Y12R-deficient mice, a feature that is reminiscent of previous observations following chemoconvulsive seizures [5]. Taken together, the findings by Cserép et al. indicate a mechanism for microglia– neuronal soma interactions that involves mitochondrial activity in the neuronal cell body, leading to purine release through somatic vesicles at Kv2.1 sites that attracts microglial processes through P2Y12R activation. This mechanism further appears to be required for adequate and prolonged stabilization of these contacts.
The authors next asked whether the communication sites between microglia and neuronal somata, that are abundant in healthy brain, are altered in response to brain injury. Specifically, the authors conducted ischemia studies in mice and examined post-mortem tissue from stroke patients. Following ischemia in both mice and humans, there was increased nal surveillance even in homeostatic concoverage of neuronal soma by microglial ditions, underscoring the broader roles of Despite known contacts between microglial processes. Further, in mice, pharmacomicroglia beyond those in response to processes and neuronal somata, the logical blockade with a P2Y12R inhibitor precise sites of contact had not been decreased these interactions, and this disease and injury. Trends in Neurosciences, Month 2020, Vol. xx, No. xx
1
Trends in Neurosciences
previous observations about the neuro- interesting therapeutic targets in patholoprotective role of contacts between gies with hyperactive neurons. microglia and neuronal somata in seizure studies [5]. The recent study by Cserép et al. [8] raises several questions. First, although the study The ability to visualize the activity of highlights the role of microglial P2Y12Rs microglial processes in vivo, and the eluci- in neuronal hyperactivity, the relative contridation of their interactions with neuronal butions of these receptors in regulating somata, have led to considerable under- broader physiological interactions between standing of the possible beneficial func- microglial processes and neuronal somata tions of microglial P2Y12Rs in monitoring is still not fully understood. Microglial neuronal health in physiology, and poten- P2Y12Rs clearly play at least some role tial approaches for restoring them in hy- in regulating circuits and behavior, as demperactive pathology. Given the selective onstrated for instance by recent evidence expression of P2Y12R by brain microglia showing that conditional targeting of [9], microglial P2Y12Rs could become microglial P2Y12Rs can modulate innate fear responses [10]. However, whether and to what extent this (and other) physiological behavior(s) are specifically regulated by physical interactions between microglia and neurons remains to be clarified. Second, mechanistic questions remain, for instance with regard to NMDA receptors which were previously implicated in regulating microglia–neuronal soma interactions [5]. It is also not fully clear at this point precisely how microglial processes reduce neuronal hyperactivity upon contact. Do they reduce glutamate spillover, regulate receptor expression, 'plug' membrane leaks to prevent calcium influx and overload, or deploy other mechanisms? The interpretation of some of the findings by Cserép et al., for instance the involvement of a vesicular pathway for purine release to facilitate contacts with microglial processes, is complicated by the use of an L-type calcium channel blocker because L-type channels are required for both calcium influx and vesicle release. Because Kv2.1 channel Microglia calcium influx through NMDA receptors is Hyperactivity P2Y12R required for microglia–neuron physical Normal activity ATP Resting neuron Hyperactive Dying neuron interactions [5], future work using more neuron selective vesicular release inhibitors will be Trends in Neurosciences necessary to clarify this issue. Lastly, Figure 1. Microglia–Neuron Interactions during Hyperactivity States. Purinergic P2Y12 receptors it should be noted that many of the mech(P2Y12Rs) on microglia mediate the attraction and subsequent interaction of microglia processes with anistic studies on microglial P2Y12Rs neuronal somata in response to ATP released by hyperactive neurons during conditions such as ischemia and have relied on loss-of-function genetic museizure. Microglial contacts with these neurons correlate with dampened neuronal activity. Inhibiting the function of these contacts (as in the case of P2Y12R knockout mice or using pharmacological P2Y12R tations or pharmacological agents against P2Y12Rs to examine their detrimental inhibitors) can result in excitotoxic cell death of the hyperactive neurons.
correlated with worsened ischemic infarct. At the cellular level, P2Y12R inhibition in ischemia resulted in significantly elevated cytosolic calcium in neurons in comparison with conditions where P2Y12R activity was maintained. Together, these results show that microglial P2Y12R activity is neuroprotective during ischemic injury at the level of both tissue and cellular activity (Figure 1). The analyses by Cserép et al., which use a functional approach (Ca2+ imaging) and focus on neuronal somata, complement and extend a body of earlier work that described microglia interactions with neuronal synapses following ischemia (e.g., [3]), and are also somewhat akin to
2
Trends in Neurosciences, Month 2020, Vol. xx, No. xx
Trends in Neurosciences
effects in acute pathologies. However, from a therapeutic perspective, and to be more clinically relevant, the reverse gain-of-function experiments are eagerly awaited. In particular, it would be of interest to test pharmacological approaches to stimulate microglial P2Y12Rs in models of acute pathologies, with the hope that they would yield beneficial effects. Acknowledgments We thank members of the laboratory of U.B.E. for their contributions in discussion of this manuscript. Our work is supported by grants from the National Institutes of Health (K22NS104392 to U.B.E. and R01NS088627 to L-J.W.).
1
Center for Brain Immunology and Glia (BIG), University of Virginia, Charlottesville, VA 22908, USA Department of Neuroscience, University of Virginia, Charlottesville, VA 22908, USA
2
3
Department of Neurology, Mayo Clinic, Rochester, MN 55905, USA Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA 5 Department of Immunology, Mayo Clinic, Rochester, MN 55905, USA 4
*Correspondence: [email protected] (U.B. Eyo). https://doi.org/10.1016/j.tins.2020.01.008 Published by Elsevier Ltd.
References 1. Davalos, D. et al. (2005) ATP mediates rapid microglial response to local brain injury in vivo. Nat. Neurosci. 8, 752–758 2. Nimmerjahn, A. et al. (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314–1318 3. Wake, H. et al. (2009) Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. J. Neurosci. 29, 3974–3980 4. Li, Y. et al. (2012) Reciprocal regulation between resting microglial dynamics and neuronal activity in vivo. Dev. Cell 23, 1189–1202 5. Eyo, U.B. et al. (2014) Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and
microglial P2Y12 receptors after status epilepticus. J. Neurosci. 34, 10528–10540 6. Kato, G. et al. (2016) Microglial contact prevents excess depolarization and rescues neurons from excitotoxicity. eNeuro 3, e0004-16.2016 7. Szalay, G. et al. (2016) Microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke. Nat. Commun. 7, 11499 8. Cserép, C. et al. (2019) Microglia monitor and protect neuronal function via specialized somatic purinergic junctions. Science Published online December 12, 2019. https://doi.org/10.1126/science.aax6752 9. Hickman, S.E. et al. (2013) The microglial sensome revealed by direct RNA sequencing. Nat. Neurosci. 16, 1896–1905 10. Peng, J. et al. (2019) Microglial P2Y12 receptor regulates ventral hippocampal CA1 neuronal excitability and innate fear in mice. Mol. Brain 12, 71
Trends in Neurosciences, Month 2020, Vol. xx, No. xx
3
Trends in Neurosciences
Spotlight
Calming Neurons with a Microglial Touch
Insight into the functional significance of microglia–neuronal soma interactions first came from zebrafish studies in which microglial contacts with hyperactive 1,2 Kaushik Sharma, neuronal somata served to reduce neuroLong-Jun Wu,3,4,5 and nal hyperactivity [4]. Similarly, mouse Ukpong B. Eyo 1,2,* microglial P2Y12Rs have been suggested to play a role in modulating neuronal hyIn vivo two-photon imaging of peractivity in a chemoconvulsive seizure microglia in the intact brain has context [5]. Results from combined imagrevealed that microglia constantly ing and electrophysiological studies in survey neuronal soma. Research mouse cortical brain slices further supover the past decade and a recent ported this hypothesis because microglial paper by Cserép et al. published contact with neurons prevented repetitive in Science are now uncovering depolarization-induced neurotoxicity [6]. the nature, mechanisms, and con- Moreover, in ischemia, the presence of misequences of these interactions in croglia correlated with reduced neuronal hyperactivity [7]. Thus, the picture emerghealth and injury. ing over the past decade of microglial imaging research suggests that microglia Advances in in vivo two-photon micros- target neuronal somata and dendrites to copy and microglia-specific mouse re- dampen neuronal hyperactivity. A recent porter lines enabled the first detailed elaborate study by Cserép et al. beautifully in vivo studies of microglia in 2005 [1,2]. confirms this hypothesis and elucidates These studies indicated that microglia several novel features of the nature are highly dynamic in surveying their and significance of microglia–neuronal microenvironment through their many soma contacts, as well as their underlying ramified processes. Later studies further mechanisms [8]. showed that microglia survey specific neuronal elements such as synapses Cserép et al. [8] first document anatomi(e.g., [3]) and neuronal cell bodies cally extensive physical interactions be(e.g., [4]). However, at that time, the tween microglial processes and neuronal mechanisms underlying such interac- cell bodies in both mouse and human tions and their functional significance brains. Through real-time in vivo analyses were mostly unknown. A 2014 paper raised in mice, the authors showed that only a mithe possibility of NMDA receptor-dependent nority (~10%) of synapses are contacted coupling to pannexin-independent ATP by microglial processes at any given time, release as a predominant mechanism for whereas ~90% of somata are contacted microglia–neuron physical interactions by microglia. The average duration of con[5]. In this study, the microglia-specific tact was more than threefold longer for P2Y12 purinergic receptor (P2Y12R) somata (~25 minutes) than for dendrites was shown to be crucial for several (~7.5 minutes). Together, these results unforms of physical interactions between derline the importance of looking into the microglia and neuronal somata and den- issue of contacts between microglial prodrites. This and other studies highlighted cesses and neuronal soma, in addition to that microglia play an active role in neuro- the often-studied contacts with dendrites.
previously determined. Among their findings, Cserép et al. identified clusters of Kv2.1 voltage-gated potassium channels on neuronal somata as sites of microglial contact in both mouse and human brains, pointing to a possible mechanism of microglia–neuronal soma interactions. The localization of microglial processes adjacent to neuronal mitochondria is especially noteworthy, as well as the fact that vesicular release of purinergic signals is required for chemoattraction of microglial processes. Of interest, given the known role of P2Y12Rs in regulating microglia–neuron interactions [5], the authors examined and confirmed the expression of P2Y12Rs at sites of microglia–neuronal soma contacts. In addition, pharmacological blockade of P2Y12Rs reduced the duration of these contacts. Consistent with this, when neurons were chemogenetically activated they were able to recruit microglial processes in wild-type mice, but not in P2Y12R-deficient mice, a feature that is reminiscent of previous observations following chemoconvulsive seizures [5]. Taken together, the findings by Cserép et al. indicate a mechanism for microglia– neuronal soma interactions that involves mitochondrial activity in the neuronal cell body, leading to purine release through somatic vesicles at Kv2.1 sites that attracts microglial processes through P2Y12R activation. This mechanism further appears to be required for adequate and prolonged stabilization of these contacts.
The authors next asked whether the communication sites between microglia and neuronal somata, that are abundant in healthy brain, are altered in response to brain injury. Specifically, the authors conducted ischemia studies in mice and examined post-mortem tissue from stroke patients. Following ischemia in both mice and humans, there was increased nal surveillance even in homeostatic concoverage of neuronal soma by microglial ditions, underscoring the broader roles of Despite known contacts between microglial processes. Further, in mice, pharmacomicroglia beyond those in response to processes and neuronal somata, the logical blockade with a P2Y12R inhibitor precise sites of contact had not been decreased these interactions, and this disease and injury. Trends in Neurosciences, Month 2020, Vol. xx, No. xx
1
Trends in Neurosciences
previous observations about the neuro- interesting therapeutic targets in patholoprotective role of contacts between gies with hyperactive neurons. microglia and neuronal somata in seizure studies [5]. The recent study by Cserép et al. [8] raises several questions. First, although the study The ability to visualize the activity of highlights the role of microglial P2Y12Rs microglial processes in vivo, and the eluci- in neuronal hyperactivity, the relative contridation of their interactions with neuronal butions of these receptors in regulating somata, have led to considerable under- broader physiological interactions between standing of the possible beneficial func- microglial processes and neuronal somata tions of microglial P2Y12Rs in monitoring is still not fully understood. Microglial neuronal health in physiology, and poten- P2Y12Rs clearly play at least some role tial approaches for restoring them in hy- in regulating circuits and behavior, as demperactive pathology. Given the selective onstrated for instance by recent evidence expression of P2Y12R by brain microglia showing that conditional targeting of [9], microglial P2Y12Rs could become microglial P2Y12Rs can modulate innate fear responses [10]. However, whether and to what extent this (and other) physiological behavior(s) are specifically regulated by physical interactions between microglia and neurons remains to be clarified. Second, mechanistic questions remain, for instance with regard to NMDA receptors which were previously implicated in regulating microglia–neuronal soma interactions [5]. It is also not fully clear at this point precisely how microglial processes reduce neuronal hyperactivity upon contact. Do they reduce glutamate spillover, regulate receptor expression, 'plug' membrane leaks to prevent calcium influx and overload, or deploy other mechanisms? The interpretation of some of the findings by Cserép et al., for instance the involvement of a vesicular pathway for purine release to facilitate contacts with microglial processes, is complicated by the use of an L-type calcium channel blocker because L-type channels are required for both calcium influx and vesicle release. Because Kv2.1 channel Microglia calcium influx through NMDA receptors is Hyperactivity P2Y12R required for microglia–neuron physical Normal activity ATP Resting neuron Hyperactive Dying neuron interactions [5], future work using more neuron selective vesicular release inhibitors will be Trends in Neurosciences necessary to clarify this issue. Lastly, Figure 1. Microglia–Neuron Interactions during Hyperactivity States. Purinergic P2Y12 receptors it should be noted that many of the mech(P2Y12Rs) on microglia mediate the attraction and subsequent interaction of microglia processes with anistic studies on microglial P2Y12Rs neuronal somata in response to ATP released by hyperactive neurons during conditions such as ischemia and have relied on loss-of-function genetic museizure. Microglial contacts with these neurons correlate with dampened neuronal activity. Inhibiting the function of these contacts (as in the case of P2Y12R knockout mice or using pharmacological P2Y12R tations or pharmacological agents against P2Y12Rs to examine their detrimental inhibitors) can result in excitotoxic cell death of the hyperactive neurons.
correlated with worsened ischemic infarct. At the cellular level, P2Y12R inhibition in ischemia resulted in significantly elevated cytosolic calcium in neurons in comparison with conditions where P2Y12R activity was maintained. Together, these results show that microglial P2Y12R activity is neuroprotective during ischemic injury at the level of both tissue and cellular activity (Figure 1). The analyses by Cserép et al., which use a functional approach (Ca2+ imaging) and focus on neuronal somata, complement and extend a body of earlier work that described microglia interactions with neuronal synapses following ischemia (e.g., [3]), and are also somewhat akin to
2
Trends in Neurosciences, Month 2020, Vol. xx, No. xx
Trends in Neurosciences
effects in acute pathologies. However, from a therapeutic perspective, and to be more clinically relevant, the reverse gain-of-function experiments are eagerly awaited. In particular, it would be of interest to test pharmacological approaches to stimulate microglial P2Y12Rs in models of acute pathologies, with the hope that they would yield beneficial effects. Acknowledgments We thank members of the laboratory of U.B.E. for their contributions in discussion of this manuscript. Our work is supported by grants from the National Institutes of Health (K22NS104392 to U.B.E. and R01NS088627 to L-J.W.).
1
Center for Brain Immunology and Glia (BIG), University of Virginia, Charlottesville, VA 22908, USA Department of Neuroscience, University of Virginia, Charlottesville, VA 22908, USA
2
3
Department of Neurology, Mayo Clinic, Rochester, MN 55905, USA Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA 5 Department of Immunology, Mayo Clinic, Rochester, MN 55905, USA 4
*Correspondence: [email protected] (U.B. Eyo). https://doi.org/10.1016/j.tins.2020.01.008 Published by Elsevier Ltd.
References 1. Davalos, D. et al. (2005) ATP mediates rapid microglial response to local brain injury in vivo. Nat. Neurosci. 8, 752–758 2. Nimmerjahn, A. et al. (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314–1318 3. Wake, H. et al. (2009) Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. J. Neurosci. 29, 3974–3980 4. Li, Y. et al. (2012) Reciprocal regulation between resting microglial dynamics and neuronal activity in vivo. Dev. Cell 23, 1189–1202 5. Eyo, U.B. et al. (2014) Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and
microglial P2Y12 receptors after status epilepticus. J. Neurosci. 34, 10528–10540 6. Kato, G. et al. (2016) Microglial contact prevents excess depolarization and rescues neurons from excitotoxicity. eNeuro 3, e0004-16.2016 7. Szalay, G. et al. (2016) Microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke. Nat. Commun. 7, 11499 8. Cserép, C. et al. (2019) Microglia monitor and protect neuronal function via specialized somatic purinergic junctions. Science Published online December 12, 2019. https://doi.org/10.1126/science.aax6752 9. Hickman, S.E. et al. (2013) The microglial sensome revealed by direct RNA sequencing. Nat. Neurosci. 16, 1896–1905 10. Peng, J. et al. (2019) Microglial P2Y12 receptor regulates ventral hippocampal CA1 neuronal excitability and innate fear in mice. Mol. Brain 12, 71
Trends in Neurosciences, Month 2020, Vol. xx, No. xx
3