Science tells us that a lot of good things happen in our brains while we sleep – learning and memories are consolidated and waste is removed, among other things.
ew research shows for the first time that important immune cells called microglia – which play an important role in reorganizing the connections between nerve cells, fighting infections, and repairing damage – are also primarily active while we sleep.
The findings, which were conducted in mice and appear in the journal Nature Neuroscience, have implications for brain plasticity, diseases like autism spectrum disorders, schizophrenia, and dementia, which arise when the brain’s networks are not maintained properly, and the ability of the brain to fight off infection and repair the damage following a stroke or other traumatic injury.
“It has largely been assumed that the dynamic movement of microglial processes is not sensitive to the behavioral state of the animal,” said Ania Majewska, Ph.D., a professor in the University of Rochester Medical Center’s (URMC) Del Monte Institute for Neuroscience and lead author of the study.
“This research shows that the signals in our brain that modulate the sleep and awake state also act as a switch that turns the immune system off and on.”
Microglia serve as the brain’s first responders, patrolling the brain and spinal cord and springing into action to stamp out infections or gobble up debris from dead cell tissue.
It is only recently that Majewska and others have shown that these cells also play an important role in plasticity, the ongoing process by which the complex networks and connections between neurons are wired and rewired during development and to support learning, memory, cognition, and motor function.
In previous studies, Majewska’s lab has shown how microglia interact with synapses, the juncture where the axons of one neuron connects and communicates with its neighbors.
The microglia help maintain the health and function of the synapses and prune connections between nerve cells when they are no longer necessary for brain function.
The current study points to the role of norepinephrine, a neurotransmitter that signals arousal and stress in the central nervous system.
This chemical is present in low levels in the brain while we sleep, but when production ramps up it arouses our nerve cells, causing us to wake up and become alert.
The study showed that norepinephrine also acts on a specific receptor, the beta2 adrenergic receptor, which is expressed at high levels in microglia.
When this chemical is present in the brain, the microglia slip into a sort of hibernation.
The study, which employed an advanced imaging technology that allows researchers to observe activity in the living brain, showed that when mice were exposed to high levels of norepinephrine, the microglia became inactive and were unable to respond to local injuries and pulled back from their role in rewiring brain networks.
The current study points to the role of norepinephrine, a neurotransmitter that signals arousal and stress in the central nervous system.
“This work suggests that the enhanced remodeling of neural circuits and repair of lesions during sleep may be mediated in part by the ability of microglia to dynamically interact with the brain,” said Rianne Stowell, Ph.D. a postdoctoral associate at URMC and first author of the paper.
“Altogether, this research also shows that microglia are exquisitely sensitive to signals that modulate brain function and that microglial dynamics and functions are modulated by the behavioral state of the animal.”
The research reinforces to the important relationship between sleep and brain health and could help explain the established relationship between sleep disturbances and the onset of neurodegenerative conditions like Alzheimer’s and Parkinson’s.
Additional co-authors on the study include Ryan Dawes, Hanna Batchelor, Katheryn Lordy, Brandan Whitelaw, Mark Stoessel, Jean Bidlack, and Edward Brown with URMC, and Grayson Sipe and Mriganka Sur with the Massachusetts Institute of Technology.
Funding: The research was supported with funding from the National Eye Institute, the National Institute of Neurological Disorders and Stroke, the National Institute of Alcohol Abuse and Alcoholism, the National Science Foundation, the Schmitt Program on Integrative Brain Research, the University of Rochester Bilski-Mayer Fellowship, and the URMC Summer Scholars Fellowship.
Microglia are highly specialized and dynamic cellular components of the central nervous system (CNS) originating from embryonic precursors in the yolk sac, comprising approximately 10% of the total glial cell number in the adult brain (Ginhoux et al., 2010; Labzin, Heneka, & Latz, 2017; Li & Barres, 2017).
Microglia have traditionally been considered to be in a resting and quiescent state in physiological conditions.
With the advent of elegant two/multiple photon microscopy image techniques, genetic and molecular targeting tools, we now appreciate that in normal conditions microglia have a ramified morphology, are maintained by diverse signals from neurons and can continuously move their dendrites, which allows for constant active screening of the surrounding microenvironment (Kierdorf & Prinz, 2017; Nimmerjahn, Kirchhoff, & Helmchen, 2005).
Microglia are long‐lived cells with a relatively low turnover. By genetically labeling microglia in pathogen‐free mice it was recently determined that microglia can survive during the whole lifespan of an animal, and can thus exert crucial long‐lasting influences on neurodegenerative disorders (Fuger et al., 2017).
However, it is well documented that microglia can be self‐regulated without contribution from peripheral myeloid cells and their turnover is tightly controlled by the coupling of apoptosis, with approximately 1% murine microglia dying in 1 day and the whole population of cells renewing several times throughout life (Askew et al., 2017; Tay et al., 2017).
Although significant species differences in microglial biology such as microglial density were noted, this finding also concords with observations in humans, a recent study highlighting that more than 96% of human microglia can be slowly renewed throughout life (Reu et al., 2017).
Microglial cells are believed to play multifunctional roles in both inflammatory and physiological contexts (Grabert et al., 2016; Thompson & Tsirka, 2017). In the healthy brain microglia efficiently monitor CNS homeostasis and have a marked impact on neural development. In order to actively survey the CNS they have recently been demonstrated to require the proper activity of tandem‐pore domain halothane‐inhibited K+ channel 1, which is the main K+ channel expressed in microglial cells (Madry et al., 2017).
In several pathological conditions such as epilepsy, single‐cell RNA sequencing of hippocampal microglia indicated that microglia undergo dramatically transcriptomic alterations (more than 2,000 differentially expressed genes) and immunological activation during early time points, particularly regarding mitochondrial activity and metabolic pathways (Bosco et al., 2018).
As such they play an indispensable role in the inflammatory cascade. Some studies based on comprehensive single cell RNA sequencing experiments have reported that microglia do not vary considerably in the whole brain (Keren‐Shaul et al., 2017; Matcovitch‐Natan et al., 2016).
However, a recent study provides further novel evidence that CD11b+ microglia in the circumventricular regions are actually maintained in the activated state even during physiological conditions (Takagi, Furube, Nakano, Morita, & Miyata, 2017). Microglia in this specific region not only display the amoeboid morphology rather than the ramified form, but also express high protein levels of activation markers in the healthy mouse brain (Takagi et al., 2017).
This recent report is consistent with the view that while microglia are uniformly distributed throughout the CNS they appear to perform characteristic functions in specific regions (De Biase et al., 2017; Marshall, Deleyrolle, Reynolds, Steindler, & Laywell, 2014). Indeed, genome‐wide transcriptional studies have reported that the bio‐energetic and immunoregulatory functions of microglia varied considerably in different anatomical regions, evidenced by cerebellar and cortical microglia displaying distinct gene expression profiles under steady‐state conditions (Grabert et al., 2016).
More specifically, a recent study provides convincing evidence of an epigenetic mechanism involved in the clearance activity of microglia that differs regionally in the adult brain (Ayata et al., 2018).
Variations in microglial profiles may also depend on the specific diseases states (Mastroeni et al., 2017), significantly altered transcripts having been reported in the hippocampus of Alzheimer’s disease (AD) and in the substantia nigra of Parkinson’s disease (PD), respectively (Mastroeni et al., 2017).
It is now well accepted that alterations in microglial activity and dysregulated microglial‐induced neuroinflammation have dual effects on many neurological diseases (Du et al., 2017; Salter & Stevens, 2017). Although the microglial field is intensively researched at present, less is still known about how microglia can be precisely targeted for optimal therapeutic efficacy.
The concept of glial replacement therapy using progenitors has recently been proposed (Cartier, Lewis, Zhang, & Rossi, 2014; Shen, Li, Bao, & Wang, 2017; Srivastava, Bulte, Walczak, & Janowski, 2017). In this review we will introduce and discuss a new experimental paradigm to specifically control the excessive activation of microglia in vivo using fully differentiated and pre‐activated cells, and provide a rationale for its translation into clinical practice.
MULTI‐TASKING MICROGLIA: A FRIEND FOR BRAIN HOMEOSTASIS
Microglia can perform diverse functions to maintain overall tissue integrity during steady‐state conditions (Colonna & Butovsky, 2017; Kabba et al., 2017; Mosser, Baptista, Arnoux, & Audinat, 2017; Prinz, Erny, & Hagemeyer, 2017). Indeed, a growing body of evidence convincingly demonstrates that microglia are recognized for acting as “busy bees” and maintain an expanding array of functions during both early brain development and adult homeostasis (Figure (Figure1).1).
In particular, microglia can secrete a broad range of protective neurotrophic substances such as brain‐derived neurotrophic factor (BDNF), vascular endothelial growth factor, neuronal growth factor (NGF), insulin‐like growth factor‐1 (IGF‐1), platelet‐derived growth factors and transforming growth factor‐β (TGF‐β) (Butovsky et al., 2014; Parkhurst et al., 2013; Shibata & Suzuki, 2017; Wlodarczyk et al., 2017), thus ensuring appropriate neuronal network development and maintenance as well as enhancing memory and learning (Molteni & Rossetti, 2017; Parkhurst et al., 2013).
There is a widespread consensus that microglia are also in active intimate contact with neighboring neuronal and non‐neuronal cells, thereby regulating neuronal proliferation, migration and differentiation and refining the neural circuits (Frost & Schafer, 2016; Mosser et al., 2017).
In order to perform these above‐mentioned functions a diverse array of receptors including TAM receptors, glutamate receptors, and purinergic receptors are used by microglia to efficiently communicate with other cells (Fourgeaud et al., 2016; York, Bernier, & MacVicar, 2017). Among these complex systems the CX3CL1/CX3CR1 and CD200‐CD200R axes play key roles in microglia–neuron contact (Eyo & Wu, 2013; Kierdorf & Prinz, 2017; Limatola & Ransohoff, 2014; Mecca, Giambanco, Donato, & Arcuri, 2018). Indeed, the CX3CR1−/− mouse exhibited profound alterations in both morphology and connectivity of the mature newborn hippocampal granule neurons (Bolos et al., 2017). The CD200−/− mouse exhibits an activated microglial phenotype accompanied by high expression of CD11b and CD45 (Hoek et al., 2000). Concurrently, microglia are critically involved in neural repair through phagocytic scavenging, such as clearing dead tissues, ingesting plaques, and apoptotic cells (Michell‐Robinson et al., 2015).
Furthermore, microglia participate in synaptogenesis by producing neurotrophic substances, such as BDNF, as well as eliminating excessive presynaptic and postsynaptic elements through synaptic pruning via the activation of the complement pathway (Masuda & Prinz, 2016; Sominsky, De Luca, & Spencer, 2017; Stevens et al., 2007; Um, 2017). In addition to the complement‐mediated mechanism, astrocyte‐derived interleukin (IL)‐33 is also physiologically required to maintain synapse homeostasis by modulating microglial synapse engulfment (Vainchtein et al., 2018). Microglia are involved in regulating and shaping both excitatory and inhibitory synapses, such as γ‐aminobutyric acid‐expressing and glycinergic synapses (Cantaut‐Belarif et al., 2017; Um, 2017).
Conversely, early evidence obtained from CX3CR1 gene‐deleted mice indicated that reduced numbers of microglial cells during brain development could impair the processes of synaptic pruning, resulting in a significantly higher density of dendritic spines and immature synapses (Paolicelli et al., 2011; Shibata & Suzuki, 2017). During adult neurogenesis, microglia actively contribute to regulate the dynamics, maintenance, and functions of synapses of adult‐born neurons (Reshef et al., 2017).
In addition to their well‐described immunological roles, newly emerging neurobiological functions of microglia are currently being recognized and studied. It has been recently determined that a subpopulation of transiently activated microglia, identified in early postnatal white matter region, can directly contribute to the maintenance of oligodendrocyte progenitor numbers and subsequent myelinogenesis in the mouse, since a decreased oligodendrocyte progenitor number was noted following injection of the selective colony‐stimulating factor 1 receptor (CSF‐1R) inhibitor BLZ945 that effectively depletes microglia (Hagemeyer et al., 2017). Other recent observations provide evidence that microglia actively regulate neurovascular homeostasis, such as forming new blood vessels and the vascular branching of the retina and hindbrain (Arcuri, Mecca, Bianchi, Giambanco, & Donato, 2017; Brandenburg et al., 2016; Dudvarski Stankovic, Teodorczyk, Ploen, Zipp, & Schmidt, 2016; Zhao, Eyo, Murugan, & Wu, 2018). In a historical perspective remarkable progress has been made in deciphering many aspects of microglial biology. Further in vivo studies are still warranted to characterize and explore the versatile features of microglia.
MICROGLIAL REPOPULATION RESOLVES NEUROINFLAMMATION: NEW CELLS, NEW FRIENDS
Basic and clinical research demonstrates that suppressing the immune response by depleting autoreactive immune cells may re‐establish the immune balance (Wraith, 2017). Taking MS for example, monoclonal antibodies (including Rituximab and Ocrelizumab) that selectively target and deplete CD20+ B cells have been approved for the treatment of MS (Greenfield & Hauser, 2017; Hauser et al., 2017; Sabatino, Zamvil, & Hauser, 2018; Salzer et al., 2016).
Our colleagues have demonstrated that relapsing–remitting MS patients with Rituximab as the initial treatment have better relapse control and tolerability in comparison with other disease‐modifying drugs (Granqvist et al., 2018; Spelman, Frisell, Piehl, & Hillert, 2017). Available MS treatments mainly resolve the peripheral inflammation but further specific and effective cell‐depletion therapies still represent a highly unmet medical need, especially in chronic disease states.
Due to the self‐renewal ability of microglia, following microglial depletion by pharmacological therapies or genetic targeting the empty CNS niche can be entirely repopulated within a relatively short period without serious side‐effects (Elmore et al., 2015; Han et al., 2017; Varvel et al., 2012).
In a previous pioneering study, Elmore and colleagues indicated that rapid repopulation of microglial cells can be noted after administration of the selective CSF‐1R inhibitor PLX3397 (Elmore et al., 2014). They claimed that following cessation of PLX3397 treatment the newly repopulated cells did not arise from bone marrow‐derived cells, but instead from local nestin+ microglial progenitor cells in the brain parenchyma (Figure (Figure2b)2b) (Elmore et al., 2014). However, Bruttger et al demonstrated that following partial depletion (80%) in the CX3CR1CreER iDTR mouse, microglia proliferate by themselves rather than from nestin+ microglial progenitor cells to finally refill the niche
(Figure (Figure2d)2d) (Bruttger et al., 2015; Jakel & Dimou, 2017). This mechanism has received further support (Askew et al., 2017) including using elegant fate‐mapping approaches, single‐cell RNA sequencing and parabiosis (Huang, Xu, Xiong, Sun, et al., 2018).
The recruitment of circulating precursors does not contribute to the resident microglial pool in the healthy CNS (Ajami, Bennett, Krieger, McNagny, & Rossi, 2011; Mildner et al., 2007). However, contradictory findings have been reported by using the CD11b‐HSVTK transgenic mouse in which circulating monocytes have the ability to potentially replace the adult CNS myeloid niche after microglial depletion (Figure (Figure2c)2c) (Varvel et al., 2012) and the newly engrafted peripheral cells have a unique functional phenotype compared with resident microglia (Cronk et al., 2018).
By contrast, two repopulating origins following microglial depletion in the retina have been described (Huang, Xu, Xiong, Qin, et al., 2018).
One is the resident central‐emerging microglia in the optic nerve and the other is the extra‐retinal periphery‐emerging microglia from the ciliary body/iris (Huang, Xu, Xiong, Qin, et al., 2018).
Two distinct resources including peripheral macrophages could also contribute to robust microglial regeneration independently of irradiation (Cronk et al., 2018).
However, intrinsic regulatory mechanisms that mediate the replacement of microglia‐like cells after selective depletion are not yet fully understood.
One recent study provided novel evidence that CX3CR1 signaling may actively regulate microglial compensation in the retina since CX3CR1−/− deficient mice had significantly lower numbers of repopulated microglial during early recovery when compared to CX3CR1 signaling‐sufficient mice (Zhang et al., 2018).
Furthermore, we have recently demonstrated that CNS repopulated monocytes required TGF‐β signaling to colonize the functional microglial niche following microglia depletion in the CX3CR1CreER DTA transgenic mouse model (Figure (Figure2a)2a) (Lund et al., 2018). Specific TGF‐β signaling deficiency on the new microglia‐like cells led to development of progressive motor disease similar to ALS‐like symptoms (Lund et al., 2018).
In this context, the next critical question is whether the newly engrafted microglia‐like cells including local hyperproliferation and/or bone marrow‐derived cells could completely adapt the embryonically seeded microglial phenotypes and functions.
Even though they are numerically and morphologically different from embryonically seeded microglia, the newly repopulated cells can still perform the same general functions as resident microglia including constantly surveying the microenvironment and appropriately responding to the acute events (Varvel et al., 2012; Zhang et al., 2018).
Additionally, embryonically seeded microglia and the newly repopulated cells may respond differently to environmental stimuli, as evidenced by two distinct cell types showing differential motilities in response to laser burn injury in vivo (Cronk et al., 2018).
Using RNA sequencing it was determined that the gene profiles of fully repopulated microglia had been little influenced by administration of PLX5622 because no inflammatory related genes were up‐regulated or down‐regulated during depletion and repopulation processes (Huang, Xu, Xiong, Qin, et al., 2018). Consistent with this idea, Elmore and colleagues determined that repopulated microglia have relatively larger cell bodies than do resident microglia.
These two types of microglial cells shared the same level of mRNA gene expression as well as similar responses to lipopolysaccharide stimulation (Elmore et al., 2015). Furthermore, mice with repopulated microglia did not exhibit any cognitive or behavioral abnormalities (Elmore et al., 2015).
More interestingly, the same research group further reported that repopulated microglia have the ability to largely resolve the pro‐inflammatory response and promote functional recovery after brain damage by replacing the active and highly swollen microglia with normal ramified microglia, downregulating the expression of reactive microglial markers and reducing the levels of inflammatory‐related genes (Rice et al., 2017).
Furthermore, peripherally derived microglia‐like cells remained transcriptionally and functionally distinct from microglia arising through local proliferation (Cronk et al., 2018). Further evaluations are needed to confirm if overall brain function can be affected by mixed engrafted microglia‐like cells during diverse neurological disease models.
ENFORCED MICROGLIAL REPOPULATION: A NEW PROSPECT FOR FIXING A DYSFUNCTIONAL NICHE
So microglial depletion can lead to natural repopulation of the empty niche through either (a) hyperproliferation of remaining microglia, (b) stimulation of microglial precursors, or (c) infiltrating of monocytes from the circulation. An interesting aspect of myeloid cell repopulation with peripheral monocytes is that transcriptomic analyses reveal different outcomes in different tissues (Guilliams & Scott, 2017).
Monocytes can thus replace Kupffer cells in the liver (Scott et al., 2016) and alveolar macrophages in the lung (van de Laar et al., 2016) with almost identical cellular phenotypes, while in the CNS bone marrow monocyte‐derived microglia only become microglia‐like cells, retaining more than 2,000 differentially expressed genes compared to resident microglia (Cronk et al., 2018).
It is currently unknown why the CNS should differ in this respect to other tissues, but indicates that environmental cues must be tissue‐specific and of varied instructional consequence in different tissues (Bennett et al., 2018).
It is also apparent that the repopulation process is tightly regulated, cells only occupying available tissue niches and the repopulation process (through either surviving microglia proliferation or monocyte infiltration) being halted through as yet undetermined mechanisms once the tissue is full.
In certain studies an “overshoot” of repopulating cell numbers appears to be adjusted through selective loss of cells so that a homeostatic numerical occupancy is achieved.
The important concept herein is that myeloid cell niches can be both efficiently depleted of resident cells and that repopulating cells can occupy the available niche with partial or total restoration of normal homeostatic functionality.
Microglial repopulation can arise from either bone marrow‐derived elements or local self‐renewal proliferation to replenish the empty niche in the CNS, which can be therapeutically targeted (Varvel et al., 2012; Waisman et al., 2015).
It is also important to note that the therapeutic outcomes of the newly derived microglia can be fundamentally different during these two processes, as evidenced by recent data indicating that bone marrow‐derived microglia are functionally distinct from yolk sac‐derived microglia (Cronk et al., 2018).
It follows that there might be specific advantages depending on the disease state. For example, if microglia are genetically dysfunctional, then self‐proliferation following depletion will not help. If the dysfunction extends to all myeloid cells (e.g. TREM2 in Nasu‐Hakola disease, ALS, and X‐linked adrenoleukodystrophy) then even repopulation by infiltrating monocytes will also not be beneficial.
As monocyte‐derived repopulating cells would have a potentially different response to systemic stimuli compared to normal repopulating microglia, this is also an issue to contemplate. In contrast, it has been shown that bone marrow‐derived cells are much more efficient in clearing amyloid beta deposits compared to their endogenous counterparts (Kawanishi et al., 2018; Simard, Soulet, Gowing, Julien, & Rivest, 2006) and so monocyte repopulation would potentially be more efficient in AD.
A final scenario is enforced repopulation of pre‐defined myeloid cell through adoptive transfer. Natural microglia are excluded from this scenario, but blood‐derived monocytes or bone marrow‐derived macrophages, either unactivated, pre‐activated or genetically modified, have potential, as do stem cell‐derived myeloid cells. In order to successfully generate a sufficient source of renewable microglia‐like cells, several different protocols using cultured human inducible pluripotent stem cells (hiPSCs) have been recently established (Abud et al., 2017; Pocock & Piers, 2018). Specifically, hiPSCs are cultured with neuroglia differentiation media by supplement of CSF1 and IL‐34 to differentiate into Tmem119+/P2RY12+ microglia‐like cells that perform phagocytic functions (Muffat et al., 2016).
Another well‐characterized method has been used to differentiate human and murine hiPSCs into microglia‐like cells through a hematopoietic progenitor‐like intermediate stage by adding defined factors and then co‐culturing with astrocytes (Pandya et al., 2017). A recently developed protocol for the derivation of microglia‐like cells from human monocytes could be the adoptive cell of most practical and functional relevance (Sellgren et al., 2017).
Considering that we have demonstrated the beneficial action of adoptively transferred immunomodulatory macrophages to prevent pathogenesis in settings of both Type 1 Diabetes (Parsa et al., 2012) and EAE (Zhang, Lund, Mia, Parsa, & Harris, 2014) and that other researchers have consequently successfully therapeutically employed our protocol in settings of spinal cord injury (Ma et al., 2015) and in wound healing (Riabov et al., 2017) then conceptually an initial transfer of immunomodulatory macrophages (either stimulated or gene‐modified) to halt the neuroinflammatory process could then be followed by transfer of microglia‐like cells.
These approaches may provide a potential novel therapeutic angle for a wide array of neurological disorders and we currently actively explore this potential.
We thus propose that an immunotherapy protocol comprising total microglial ablation followed by the immediate enforced repopulation of the available niche through the adoptive transfer of myeloid cells could be considered as a means of replacing dysfunctional microglia in neurodegenerative states such as ALS and AD (Figure (Figure33).
University of Rochester Medical Center
Mark Michaud – University of Rochester Medical Center
The image is in the public domain.
Original Research: Closed access
“Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex”. Rianne D. Stowell, Grayson O. Sipe, Ryan P. Dawes, Hanna N. Batchelor, Katheryn A. Lordy, Brendan S. Whitelaw, Mark B. Stoessel, Jean M. Bidlack, Edward Brown, Mriganka Sur & Ania K. Majewska.
Nature Neuroscience doi:10.1038/s41593-019-0514-0.