microglia roles in neuronal spatial patterning and synaptic wiring in the adult CNS

Posted comment on ´Microglia function in central nervous system development and plasticity` by D.P. Schafer and B. Stevens published in Cold Spring Harbour Perspectives in Biology Advance July 17th 2015, doi: 10.1101/cshperspect.a020545

SUMMARY

Schafer and Steven`s article is a review of the spatial patterning and synaptic wiring functions of microglia in the adult CNS which can lead to a change in behavior. Early research linked the microglia to responses to injury and disease, but as Schafer and Steven`s describe they are also part of normal healthy brain functioning. It has been found that the processes of ´resting` microglia are highly motile in healthy, intact brain and can reach the whole brain parenchyma within hours.

The authors begin by describing two roles for microglia in spatial patterning in the developing and mature: one in cell death and the other in cell proliferation. In its role in programmed cell death, microglia exhibit different phagocytotic signaling pathways such as TNF, TREM2. There is induced apoptosis after microglial mediated phagocytosis of debris or in the case of injury/disease (termed ´phagoptosis`) microglia actively engulf live cells and this is linked to inflammation. Microglia can also promote survival of neural precursor cells (NPCs) in the developing CNS linked to the insulin-like growth factor -1 which binds to receptors and promotes survival of these cells. Other species demonstrate different mechanisms, eg the Draper/dCED-6-dependent signaling pathway in Drosophila and Caenorhabditis elegans and a v0-ATPase a1 subunit mechanism in developing zebrafish. In the neonatal hippocampus, the mechanism involves the cell surface receptor integrin and the transmembrane signal transduction adapter molecule DAP12 as seen using knockout mice. These molecules are expressed by peripheral immune cells including macrophages and in normal brain later on exclusively by macrophages where they carry out effector functions such as cell adhesion and phagocytosis. CD 11b and DAP12 also regulate production of superoxide ions by microglia in the cerebellum, however further research is required to find out whether they are part of the general mechanism or just involved in these specific regions.

The authors also describe the other microglial function in adult spatial patterning, that of the promotion of cell survival and cell proliferation. Some research has shown that a lack of microglia leads to enhanced NPC proliferation, but other reports show a reduction. The differences in results are attributed to experimental conditions, eg culture, age of mice, region-specific effects. The effect appears in vivo, but only in development between P3 and p5 where the microglia stabilizes the number of apopteric neurons particularly in layer V. The number of layer V cells was also found to be decreased in mice deficient in the fractalkine receptor (CX3CR1), but these mice also have a reduction in free IGF-1 protein although demonstrate an increase in IGF-1 binding proteins suggesting that IGF-1 may be factor downstream from CX3CR1 that promotes neuron survival. Schafer and Stevens conclude that the research is too non-specific to make definitive conclusions.

Although the authors describe the various ways in which microglia can affect spatial patterning in the developing and adult cells, they ask the question how both such disparate functions (ie. cell death and cell proliferation) can occur in the same time frame. Explanations were suggested in the form that the microglia may in fact be a heterogenous cell population; that they overlap certain functions with astrocytes which can also phagocytose and have been shown to perform a similar role as microglia in synaptic pruning; that pharmacological strategies are non-specific; and that the knockout mice used in the experiments may express the relevant neural factors by different means.

Schafer and Stevens continue in their article with a description of the other attributed function of microglia in adult CNS that of a role in synaptic wiring. They give evidence for the microglial role in synaptic pruning, eg. that phagocytic microglia were enriched in brain regions undergoing active synaptic remodeling, including the cerebellum, hippocampus, and visual system; that  microglia processes dynamically interact with developing synapses and can detect local changes in neuronal activity, ie. reduced processes when neuronal activity was blocked by tetradotoxin (TTX), enucleation of an eye, or lowering the temperature; and also described imaging of the V1 showing that in time-dependent increased structural and functional synaptic plasticity that microglia appeared to contact synaptic elements and spines, in particular, appeared to change size upon microglia contact. In dark-adapted mice, imaging revealed that microglia changed their motility and interactions with spines as compared with light-reared control animals. The microglia under these conditions appeared more frequently with contact synaptic elements and had more phagocytic inclusions, which was speculated to be synaptic elements in response to experience dependent remodeling of the synapse.

The authors describe synaptic pruning in postnatal development and gave the example of mouse retinogeniculate pathway where postsynaptic thalamic relay neurons within the dorsal lateral geniculate nucleus (dLGN) are initially innervated by multiple weak retinal ganglion cell inputs originating from the retina. A subset of these presynaptic inputs is later eliminated, while the remaining inputs are maintained and strengthened. Microglial processes were observed closely situated to presynaptic inputs undergoing remodeling within the dLGN and that during the height of the presynaptic input remodeling (P5 in the mouse), the microglia contained presynaptic inputs within their processes and within lysosomal compartments. Furthermore, imaging analysis revealed engulfed retinal ganglion cell axonal terminals throughout the dLGN. The engulfed synapses were believed to be less active synapses existing during the activity-dependent synaptic competition.

One mechanism mediating the microglia-synapse interaction and synaptic pruning was suggested as the classic complement cascade in particular involving the cascade proteins, C1q and C3. Evidence for the involvement was given as the observations that: the microglia were localized in synaptic compartments; and that in the innate immune system C1q and/or C3 bind cellular material, inducing its removal by several mechanisms, including phagocytic pathways. It was suggested that complement C1q and C3 target synapses for elimination by the microglia and it was found that CR3 is expressed by microglia at particularly high levels during the peak of microglia-mediated synaptic engulfment (eg. in the P5). However, the identities of specific receptors for the complement at the synapse remain unclear although an in vitro study suggests that terminal sugar residues in the extracellular matrix of glycoproteins surrounding neurons mediate C1q binding to the neurites. However, Schafer and Stevens report that it is not clear if the complement proteins are localized to specific (eg.less active/weaker) synapses and whether changes in neuronal activity impact this process.

The other mechanism proposed by Schafer and Stevens comes from recent data showing a hippocampus CX3CR1-dependent mechanism. Research showed that mice deficient in CX3CR1 (CX3CR1 knockouts) exhibited a transient increase in spine density in postnatal weeks 2 and 3 in the hippocampus. A concomitant increase in postsynaptic density protein 95 (PSD95)-positive immunoreactivity within the microglia was also found. The results suggest that fractalkine signaling regulates microglia number and/or recruitment to synaptic sites in the early postnatal brain and it has also been found that there is a link between fractalkine signaling and learning, spatial memory, and conditioning in the adult.

Schafer and Stevens report in their article that microglia also regulate synapse maturation and long-range synaptic connectivity in the synapses that remain. It was found that in ostnatal CX3CR1 knockout mice there is a diminished spontaneous postsynaptic current (EPSC), a transient increase in spine density and enhanced hippocampal long-term depression, with a reduced susceptibility to drug-induced seizures. Some effects were found to be transient, eg. microglia and spine numbers in the hippocampus returned to control levels in young adult mice, but the sEPSC/mEPSC ratio remained low in CX3CR1 knockout mice as compared with the controls. This was thought to be because CA3 axons in knockout mice fail to form multi-synapse boutons (i.e. axons forming synapses on more than one dendritic spine) in the CA1 and there is a reduction in functional connectivity between the hippocampus and the prefrontal cortex.

Another mechanism for the role of microglia in cell survival was also suggested and that involves the effect on ion channels and receptors. This was observed in the maturation of hippocampal synapses which was altered in mice harboring a mutation in the KARAP/DAP12 gene coding for a transmembrane immune receptor expressed by developing microglia from birth. The receptors had a subunit composition of NMDA receptors and calcium permeability status of AMPA receptors that are both characteristic of immature synapses and the cells appeared to have enhanced LTP. Microglia are known to express an array of ion channels and receptors that can be stimulated by neurotransmitters and activity-dependent signals, such as ATP and glutamate. Thus, in addition to responding to neuronal activity, microglia also have the potential to modulate basal glutamatergic and GABAergic synaptic transmission in the healthy CNS.

The authors proceed in their article to explain the link between behavior and microglia. In neuropsychiatric disorders such as autism spectrum disorder, obsessive–compulsive disorder, and schizophrenia, abnormal microglial cell activation exists along with deficits in synaptic connectivity. This is also observed in animal models and there is particular interest with rodent models in cases of maternal immune activation and later changes in microglial activity. Mice subjected to early-life infection in utero develop several behavioral phenotypes associated with schizophrenia (eg. decreases in exploratory behavior and social interactions) and/or memory and learning loss, particularly if combined with a later-life stress or challenges to the immune system (eg. peripheral LPS administration). Changes to the microglia include the activation state and numbers in a brain region–specific manner. These results provide the evidence that microglia may be “primed” prenatally during early-life infection and that a subsequent challenge can significantly enhance their effects on later-life behavior. This view has been further substantiated by showing that microglial-derived interleukin-1β is a primary mechanism by which deficits in hippocampal-dependent learning and memory are induced following an in utero immune challenge and later-life stress. Furthermore, a similar cytokine-mediated “priming” mechanism was suggested as also contributing to age-dependent memory loss associated with aging and neurodegenerative disease.

Evidence is also presented in this article that microglia can affect the baseline behavior in the absence of disease. For example, increased numbers of activated microglia were observed in the preoptic nucleus of healthy male rats as compared to females. Administration of minocycline to males caused a decrease in activated microglia to a level more closely resembling female microglia, and a coincident display of feminine behaviors and vice versa. This observation suggested that microglia could be playing key roles in regulating sexual behavior. Another example relates to CX3CR1-deficient mice which have deficits in LTP accompanied by significant deficits in contextual fear learning and performance in the Morris water maze. The results are not considered conclusive since another study found the opposite  with very little effect on behavioral performance. However, more evidence comes from the manipulation of gene expression specifically in the microglia. This system takes advantage of the relatively low turnover rate of microglia versus other peripheral CX3CR1+ immune cells, which turn over regularly (within a week) during hematopoiesis. After tamoxifen treatment, peripheral cells that undergo recombination turned over within 1 week, resulting in microglia-specific, Cre-mediated expression of genes. Using these mice, the microglia were depleted using a diphtheria toxin strategy, or brain-derived neurotrophic factor (BDNF) was specifically knocked out in microglia. In either case, the result was a significant deficit in memory performance, as well as in a fear-conditioning situation. Furthermore, these effects were accompanied by a decrease in dendritic spine formation, specifically in the motor cortex.

Although the mechanisms are not clear at this time, Schafer and Stevens show in their article that microglia play important roles in neuronal functioning in adult CNS relating to cell death and cell survival and proliferation and hence demonstrate critical functions in regulating behavior, particularly if these cells are manipulated during development (postnatal through to juvenile stages).

COMMENT

What makes this article interesting is that it brings to the foreground a particular cell type in the adult brain that has in the past stayed in the shadows of research and public attention. Studies of the brain in injury and disease have highlighted that cell types other than neurons also play important roles in neuronal structure and function and Schafer and Stevens in their article describe one such cell type, that of microglia. Their meta-analysis of research findings shows what is known and points to what is not known about the role and mechanisms of this type of glial cell in the adult brain. It appoints them two major roles in neuronal physiology that of cell destruction and aiding cell proliferation.

The role of the microglia in the clearing away of non-functioning cells designated for destruction as well as debris is known and the relevant mechanism of phagocytosis moderately well researched. What is less clear however is how the neuronal cells signal that destruction and how microglia recognize that signal. It is obvious that the signals must be associated with the membrane in some way and Schafer and Stevens suggest mechanisms found in other species that support this, eg. inflammation signals, Draper/dCED-6-dependent signaling pathway components and the v0-ATPase a1 subunit. They also indicate the role of the fractalkine receptor (CX3CR1) produced exclusively by microglia in the adult brain. This appears to be linked to IGF-1 perhaps release since CX3CR1 knockout mice have decreased levels of free IGF-1 protein and an increase in the IGF-1 binding proteins. Schafer and Stevens also describe the situation with the human hippocampus and cerebellum. The hippocampal system appears to have a requirement for the cell surface receptor integrin and transmembrane signal transduction adapter molecule DAP12 responsible for cell adhesion and phagocytosis. CD 11b and DAP12 also regulate production of superoxide ions by microglia also in the cerebellum. However, the evidence that apoptotic neurons exist in the absence of microglial or NPC contact and that phagocytosis can still proceed suggests that this may not be a microglial dependent process. The observation that in the adult mouse hippocampal dentate gyrus microglia appear to infiltrate small cytoplasmic openings of the cells before cell death and that the microglial processes closely approach the nuclei provides another aspect to the microglial cell destruction method.

This function of the microglia of clearing away of non-functioning-designated- for-destruction cells and debris also appears to play a role in cell survival and cell proliferation as well and Schafer and Stevens describe in their article how this type of glial cells aids synaptic wiring. Perhaps this other role is just the other side of the cell-destruction ´coin`. Maybe it is just a simple case of clearing weeds in the garden around plants promoting growth of those plants since weeds also use valuable natural resources and may produce by-products that hinder growth. This may be also the case in the  synapse, however, it is also possible that heterogenous cell populations, appropriate and concise functional timing and overlapping astrocyte functions, the explanations put forward by the authors of this article may also be valid.

The role of microglia in advantageous synaptic wiring appears to be particularly appropriate to those areas of the adult brain that undergo high levels of neuronal plasticity such as the cerebellum, hippocampus and the visual system. In the case of the hippocampus, this function may play a role in brain memory formation where normally there is a high level of activity dependent neurogenesis and memory formation is linked solely to newly formed cells. These cells exhibit activity dependent long-term potentiation (LTP). A possible version of what happens in this area during stimulation could be that the CA3 cells representing the informational input exhibit LTP and hence increased PSD95 is observed representing increased synaptic firing capacity. The observed increased firing of the CA3 cells incites increased firing of the CA1 cells with formation of multi-axonic boutons representing information from multiple cells bound together. Connectivity of the CA1 cells with relevant cells of the enterorhinal cortex transmits the complexity and context of the experience to this area which then through its connectivity to the dentate gyrus, prefrontal cortex and other areas transfers the information to where appropriate. The firing cells of the CA1 and CA3 cells representing the stimulation could then possibly be reduced to the long-term depression (LTD) state with time and microglia will ´cull` these less active cells so that the first-response relay function of the hippocampus is maintained. The microglia can also remove any neural precursor cells formed as part of the activity dependent neurogenesis that are not part of the experience representation. Therefore, under normal conditions there is a balance between cell proliferation and cell death so that the hippocampus function in brain memory formation is maintained.

However, this balance can be upset as is shown in the case of Alzheimer`s disease where there are reports of hyper-excitability of hippocampal cells and limited success of anti-inflammatory drugs in reducing the disease symptoms. A possible explanation of the mechanism involved here could be that in the beginning of the disease there is a lack of relevancy of incoming information as observed with age and this may over-stimulate the hippocampal cells causing excessive neurogenesis and the observed hyper-excitability. An increase in PSD95 and hence increased synaptic function would result as in the normal situation, but microglial removal of NPCs not part of the experience would be low since more information would be inputted than normal.  The CA3 cells would exhibit LTP and appropriate CA1 cells as a consequence would also be fired, but not as multi-axonal boutons representing bound information, but more single cells representing single characteristics. This higher level of information, but lower level of related useful information would be transmitted by the connectivity of the CA1 to the enterorhinal cortex, but because the information is not connected single characteristics are further transmitted and not complex, bound information. This change in firing could mean that the level of beta amyloid involved in the endocytosis mechanisms in place with firing cells in the enterorhinal cortex is increased and binding and blocking of endocytic vesicle formation occurs. Hence, the cells are signaled for destruction by the microglia with the result that the activity of the microglia in the area is greater and connectivity to the CA3 area is reduced. Therefore, CA1 cell number reduction has a negative effect on upstream cells and on informational input, which is observed in Alzheimer`s disease. Since cell number reduction implies an imbalance of the cell death and cell survival ratio microglial number and function is also affected and the over-activity observed manifests itself as neuroinflammation which is also considered a factor in Alzheimer`s disease. However, this version of events at the synapse is speculation and much more research is needed to determine the correct direct or indirect role of microglia in Alzheimer`s disease.

Therefore, the topic of microglia and its role in the neuronal mechanism in the adult brain is an interesting topic that is far from being solved. The link with Alzheimer`s disease with its elements of neurodegeneration and neuroinflammation and the evidence of involvement in other neuropsychiatric disorders makes it an important ´player` to study.  The question is whether microglia is an instigator of the diseases or just the result and if we can manipulate them to bring us a therapeutic advantage.

Since we`re talking about the topic…………….

…….if we use an animal model of Alzheimer disease and look at CX3CR1 in the hippocampus and enterorhinal cortex can we see changes in activity consistent with the pathology of the disease.

….can we assume that conditioning experiments are faster in CX3CR1 knock out mice since there should be hyper-excitability in the hippocampal area and increased stimulation? If the amygdala is lesioned, is there any effect on microglial function?

…..fluorescent binding of the CX3CR1 and appropriate imaging might lead to the observations of membrane microdomains formed in the same way as endocytosis and clathrin. Should microglial processes binding within the synapse and within the cell itself also be observed and can we chart the progress of the receptor during phagocytosis?

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