Aging microglia and maintenance of neuronal activity and synapses

As the “constant gardener” of synapses, microglia in recent studies have been found to constitutively regulate synaptic structure and neuronal activity. The constant dynamic behavior of microglial processes (Davalos et al., 2005; Nimmerjahn et al., 2005) appears not to be a random, cell-autonomous feature but are instead have their overall rate, extent, and direction regulated and guided by neuronal activity (Fontainhas et al., 2011; Li et al., 2012). These dynamic processes are observed to make repeated contact with synaptic structures and influence synaptic stability and pruning in an activity-dependent manner (Wake et al., 2009; Tremblay et al., 2010; Paolicelli et al., 2011; Schafer et al., 2012), potentially altering overall activity patterns in neuronal networks (Pascual et al., 2012). Microglia are capable of modulating the activity of neurons by directly contacting neuronal somata with their processes, down-regulating the activity of contacted neurons (Li et al., 2012). Taken together, these forms of regulation seem to constitute a homeostatic mechanism regulating overall levels of activity in the CNS; neurons with greater levels of activity release the attractant ATP in an activity-dependent manner via probenecid-sensitive pannexin-1 channels (Fontainhas et al., 2011; Li et al., 2012), inducing microglia to migrate and polarize their processes toward them, and which are then reduced in their activity following microglial contact.

Is this “gardening” function of microglia influenced by aging? In our work, we have observed that aged microglia interestingly decline in their dynamic motility of their processes and in their migration rates through neural parenchyma (Damani et al., 2011). These decrements may translate into less frequent and extensive synaptic and neuronal contacts and prolonged microglia response times. These may potentially be related to (1) age-related changes in the structure and electrotonic properties of dendrites and dendritic spines (Duan et al., 2003; Kabaso et al., 2009; Morrison and Baxter, 2012) and (2) a decreased homeostatic regulation of neuronal activity. Also, we had found that microglia migration to sites of neural injury is slower to initiate in aged animals—but once accomplished, microglial aggregation tends to be more sustained and less reversible. Dysregulated fluxes of microglia can therefore result in more sustained imbalances in activity homeostasis, and possibly contribute to differential responses to excitotoxic damage observed in young versus aged brains (Campuzano et al., 2008).

Anti-inflammatory and anti-oxidant approaches

The pro-inflammatory and primed characteristics of the aging microglia phenotype prompt the use of an anti-inflammatory approach to alleviate the consequences of microglial aging. Minocycline, a highly bioavailable tetracycline-derived antibiotic with anti-inflammatory properties (Yrjanheikki et al., 1998, 1999; Tikka et al., 2001), has been investigated as an inhibitor of microglial activation in numerous animal models of neuroinflammation and CNS injury (Kim and Suh, 2009). Specific to its effect on aging microglia, minocycline has been shown to ameliorate pro-inflammatory cytokine production and sickness behavior following LPS-administration in aged mice (Henry et al., 2008) and to reduce aging-exacerbated deficits in long-term potentiation (LTP) in the aged rat hippocampus (Griffin et al., 2006; Liu et al., 2012). Clinical studies involving the use of minocycline for neurodegenerative diseases are ongoing; results of completed studies have produced both positive and negative results in separate disease contexts, revealing the need for further investigations (Plane et al., 2010). In contrast to minocycline, which has broad and somewhat varied anti-inflammatory actions, specific and targeted therapeutic approaches have also been considered. The central role of IL1β in mediating age-related pro-inflammatory effects of microglia has led to investigations of the IL1 receptor antagonist, IL1RA, as a potential therapy. Administration of IL1RA was found to ameliorate deficits in long term memory (Frank et al., 2006), impairments in theta-burst late-phase LTP (Chapman et al., 2010), and sickness behavior (Abraham and Johnson, 2009a) following immune challenges by E. coli or LPS administration. These findings may prompt future clinical investigations involving approved inhibitors of IL-1 signaling (anakinra, rilonacept, canakinumab) for the treatment of neurodegenerative disease (Dinarello et al., 2012).

As the generation of ROS and increased oxidative stress has been associated with the activation of pro-inflammatory programs in primed microglia (Verri et al., 2012), anti-oxidant approaches involving the use of dietary compounds have been examined in multiple studies, with the goal of reducing microglial activation in the course of reducing oxidative stress (McGahon et al., 1999; Gemma et al., 2002; Abraham and Johnson, 2009b). Flavonoids, a class of plant phenolics found widely in a variety of fruits, vegetables, and grains, have been investigated in the forms of luteolin, quercetin, genistein, hesperetin, among others, and have been found to decrease markers of neuroinflammation (Jang and Johnson, 2010; Izzi et al., 2012). Dietary administration of luteolin, one of the more potent agents in this regard (Comalada et al., 2006), decreased inflammatory markers and improved spatial working memory in aged mice (Jang et al., 2008, 2010). Dietary components of the retinoid/carotenoid class (which include astaxanthin, crocin, crocetin, retinoic acid, lutein, zeaxanthin, and their synthetic derivatives) have also been found to decrease microglia and macrophage activation in numerous in vitro studies (Dheen et al., 2005; Xu and Drew, 2006; Kim et al., 2008, 2010; Nam et al., 2010; Bian et al., 2012). Anti-oxidant supplements such as alpha-tocopherol (Vitamin E) (Murray and Lynch, 1998; Berg et al., 2005) and vitamin D3 (Moore et al., 2005) have been found to exert anti-inflammatory benefits. While the combination of in vitro, animal model, and epidemiological studies indicates the promise of dietary supplementation, verification of benefit in large scale controlled clinical studies is a necessary next step. Currently, there are few therapies of this nature that have been proven and approved for age-related neurodegenerative disease. One exception is AMD, a disease in which activated microglia in the aged retina feature (Gupta et al., 2003; Ma et al., 2013), for which anti-oxidant supplementation with vitamin A, C, E, and zinc, constitutes the current standard of care in reducing risk for disease progression (Age-Related Eye Disease Study Research, 2001). Results for a large scale trial involving supplementation with the carotenoids, lutein and zeaxanthin, and omega-3 fatty acids, for preventing AMD progression are expected in mid-2013 (AREDS2 Research Group et al., 2012).

Preventing or reversing microglial “run-down” in aging

The concept that aging microglia accumulate progressive physiological changes (build-up of lipofuscin, increasing number of mtDNA mutations) that drive the emergence of senescent phenotypes suggest the feasibility of strategies that aim to ameliorate or reverse these changes. Measures targeted at lipofuscin accumulation in microglia may help alleviate lysosomal dysfunction, increase autophagy, decrease ROS production, and in general, ameliorate the detrimental phenotypes of aging microglia (Nakanishi and Wu, 2009). In the retina, lipofuscin accumulates in microglia of aged and AMD retinas (Ma et al., 2013) and is compositionally akin to that accumulating in nearby RPE cells (Xu et al., 2008). Current clinical studies of visual cycle modulators (ACU-4429, fenretinide) (Kubota et al., 2012; Mata et al., 2012) that aim to decrease lipofuscin accumulation in RPE cells may also be helpful in decreasing accumulation in retinal microglia. Alternatively, measures that stimulate autophagy, a process slowed by lipofuscin accumulation, may also be helpful. Anti-lipolytic drugs and rapamycin, which are agents found increase autophagy (Donati, 2006; Cai et al., 2012), have also been associated with reductions in age-related oxidative damage (Donati et al., 2006; Yang and Ming, 2012) and microglial activation (Dello Russo et al., 2012), and may therefore be useful in the therapy of neurodegenerative disease (Chong et al., 2012). Early stage clinical trials have been conducted with rapamycin for AMD and multiple sclerosis (NCT00095329 and 00766649) but results are not currently available.

With respect to the age-dependent accumulation of mtDNA mutations in aged microglia, a nucleus-encoded protein called transcription factor A, mitochondrial (TFAM) has been found to promote mtDNA transcription and maintain its architectural structure (Kanki et al., 2004). TFAM overexpression can reduce ROS production and NF-κB activation in vitro and decrease age-related mtDNA damage and IL1β expression in hippocampal microglia in vivo (Hayashi et al., 2008). As a result, compounds that can stimulate TFAM expression or activity, such as resveratrol (Lagouge et al., 2006; Vetterli et al., 2011) and brimonidine (Lee et al., 2012a) may be useful in this strategy.

Modulation of the aging microglial milieu

The increased basal inflammatory state and priming in aged microglia that result from an age-dependent shift in balance between “On” and “Off” signals may be another target for microglial “rejuvenation.” During aging, the expression levels of “Off” ligands CX3CL1 and CD200 decrease concurrently with increases in microglial activation status; the causal influence of these changes are demonstrated by the ability of exogenous CX3CL1 or a CD200 fusion protein to ameliorate phenotypes associated with microglial aging (Lyons et al., 2007, 2009a,b; Cox et al., 2012; Vukovic et al., 2012). These results present these signaling pathways as promising molecular targets for intervention. For the CX3CL1-CX3CR1 signaling axis, despite increasing attention in different disease areas, suitable modulatory agents have yet to be investigated in clinical studies (D'Haese et al., 2012). For the CD200-CD200R axis, measures that increase IL4 (phospholipid microparticles-incorporating phosphatidylserine, atorvastatin), which up-regulates CD200 expression, can improve age-related decreases in LTP attributed to microglial aging (Nolan et al., 2005; Lyons et al., 2007, 2009b, 2011; Clarke et al., 2008). Also, a neural cell adhesion molecule (NCAM)-derived peptide, FGL, which can increase CD200 expression in vitro and in vivo, has been found to abrogate age-related increases in glial activation and synaptic changes (Downer et al., 2009, 2010; Ojo et al., 2012). Discovery of other “Off” signaling pathways can augment this strategy of modulating the milieu of aging microglia in ways that promote microglial quiescence.

Exercise

Physical exercise has been associated with a number of salubrious effects on CNS in animal studies, including increasing adult neurogenesis, improving performance on cognitive tasks, and ameliorating various declining neurological parameters observed with aging (Van Praag et al., 1999, 2005; Kim et al., 2004; Blackmore et al., 2009). Also, exercise has been implicated in improving aspects of systemic immune function (Woods, 2005). Recent studies have connected these two classes of effects in showing that exercise may exert its effects on the CNS, at least in part, through the modulation of microglia activity. The exaggerated neuroinflammatory response following a peripheral immune challenge in aged animals, which is associated with increased cytokine production, reduced BDNF expression, and increased cognitive deficits, was found to be completely reversed by voluntary exercise (Barrientos et al., 2011). This effect of exercise appears to be mediated by the “de-priming” of aged microglia as microglia from isolated the brains from exercising mice similarly demonstrated a more modest pro-inflammatory response to LPS administered in vitro relative to those from non-exercising mice. In a separate study, exercise was found to promote adult neurogenesis by stimulating greater NPC proliferation (Vukovic et al., 2012). These effects again appear to be mediated via microglia as prior depletion of microglia completely eliminates them. In this study, exercise seems to influence microglia through increased expression of CX3CL1, which confers on microglia the ability to up-regulate NPC proliferation. In other studies, exercise in animals was found to decrease age-related basal microglial proliferation (Kohman et al., 2012), and to lower microglial activation in models of hypoxia (Lin et al., 2012), high-fat diet (Yi et al., 2012), tau overexpression (Leem et al., 2011), and closed-head injury (Chen et al., 2012), further supporting the notion that exercise can play a role in deactivating and de-priming microglia. The molecular mechanisms underlying how exercise mediates these effects will be a fascinating area for future study, and may discover agents that can elicit exercise-induced effects without the need for actual exercise.

Replacement of microglia as therapy?

Although microglia are long-lived residents of the CNS and are not readily turned-over in the uninjured CNS, it remains plausible that aged microglia, instead of being modulated, may actually be wholly replaced or reinforced by exogenously introduced cells capable of carrying out the functions of young, non-senescent microglia. Studies in organotypic hippocampal slice cultures have shown that brain slices deprived of microglia can be again replenished by exogenously introduced cultured microglia which are able to colonize the brain parenchyma, acquire a ramified morphology, and confer neuroprotection to excitotoxic injury (Vinet et al., 2012). Animal studies have demonstrated that lethal irradiation followed by bone marrow transplantation allows the elimination of endogenous microglia and their replacement by bone marrow-derived cells which acquire features of microglia within the CNS parenchyma (Simard and Rivest, 2004). This technique of “microglial replacement” has enabled microglia expressing a mutant version of a particular gene in a transgenic mouse to be replaced by wild type microglia expressing a functional version of the same gene. These microglial “replacements” have been successful in alleviating the neurological and systemic symptoms in MECP2 mutant mice, a model for Rett syndrome (Derecki et al., 2012), in correcting abnormal grooming behavior in Hoxb8 mutant mice, a possible model for obsessive-compulsive disorder (Chen et al., 2010), and in increasing survival in SOD1G93A transgenic mice, a model for amyotrophic lateral sclerosis (ALS) (Lee et al., 2012b). Conversely, replacement of wild type microglia with transgenic microglia deficient in MyD88 signaling was found to increase disease susceptibility in a mouse ALS model (Kang and Rivest, 2007). Even in the absence of bone-marrow transplantation, the depletion of brain microglia has been found to induce an influx of peripheral monocytes into the CNS that repopulates the microglia-depleted areas (Varvel et al., 2012). These “replacement” cells intriguingly resemble endogenous microglia in distribution, morphology, and physiology, suggesting the notion that worn-out, aged brain microglia can perhaps be replaced by fresher and more able recruits from the periphery. The possibility that we can harness our intrinsic capability to “self-rejuvenate” our CNS microglia using our peripheral immune system is a radical but tantalizing concept.

The ability of microglia to be replenished in the CNS by peripherally derived cells that can take on features and functions of microglia present the possibility that these approaches may be in the future be translated into cell-based therapies to rejuvenating the population of senescent microglia in aged brains. Further studies still remain to be done regarding (1) possible sources of replacement cells (including stem cells sources) (Selvaraj et al., 2012), (2) the physiological functioning of newly integrated cells in an aged brain milieu, and (3) the use of brain conditioning regimens to optimize the spatial and temporal extents of microglial replacement (Capotondo et al., 2012) in order to increase and to realize the feasibility of such strategies.

Research in our laboratory is supported by the National Eye Institute Intramural Research Program. Acknowledgment is made for grant support to the donors of Macular Degeneration Research (MDR), a program of the American Health Assistance Foundation (AHAF, now the BrightFocus Foundation).