Altered motility of plaque-associated microglia in a model of Alzheimer’s disease

doi:10.1016/j.neuroscience.2016.05.061

Neuroscience

Volume 330, 25 August 2016, Pages 410-420

https://doi.org/10.1016/j.neuroscience.2016.05.061 Get rights and content

Highlights

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Plaques-associated microglia show a hyperactive motility in slices from 5xFAD mice.
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Microglia in 5xFAD mice do not respond appropriately to tissue damage.
•
An adenosine A2A receptor antagonist did not enhance microglial response to damage.
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A2A receptor antagonism reduced the hypermotility of plaque-associated microglia.

Abstract

Alzheimer’s disease (AD), the most common form of dementia in the elderly, is characterized by the presence of extracellular plaques composed of amyloid β (Aβ) peptides and intracellular tau aggregates. The plaques are surrounded by microglia, the brain’s resident immune cells, which likely participate in the clearance of Aβ by phagocytosis. The microglia that are associated with plaques display an abnormal ameboid morphology and do not respond to tissue damage, in contrast to microglia in healthy brains. Here, we used time lapse confocal microscopy to perform a detailed real-time examination of microglial motility in acute hippocampal brain slices from the 5xFAD mouse model of AD, which was crossed to Cx3cr1^GFP/GFP mice to achieve microglia-specific GFP expression for visualization. During baseline conditions, microglia around plaques appeared hypermotile, moving the processes that were pointing away from plaques at higher speed than microglia not associated with plaques. Yet, neither plaque-associated, nor plaque-free microglia were able to extend processes toward sites of modest mechanical damage. Application of the selective adenosine A2A receptor antagonist preladenant, which restores microglial response to cellular damage in a mouse model of Parkinson’s disease, reduced the hypermotility of plaque-associated microglia, but did not restore motility toward damaged cells in slices from 5xFAD mice. Our results suggest that process hypermotility and resistance to A2A antagonism during response to tissue damage may represent unique functional phenotypes of plaque-associated microglia that impair their ability to function properly in the AD brain.

Introduction

Alzheimer’s disease (AD) is the most common neurodegenerative disease and the leading cause of dementia in the elderly. Patients affected by AD display cognitive deficits such as impaired memory and difficulties with daily functions. Some of the pathological features associated with the disease are brain shrinkage and the presence of two types of abnormal protein aggregates: tau protein aggregated in intraneuronal neurofibrillary tangles and amyloid β (Aβ) peptides aggregated in extracellular plaques (Spires-Jones and Hyman, 2014).

Another prominent feature of AD pathology is the presence of an inflammatory response, including increased levels of inflammatory mediators and activated microglia (Akiyama et al., 2000, Heneka et al., 2010), the brain’s resident immune cells. Interestingly, the greatest inflammatory reaction is observed around Aβ plaques, which are often completely surrounded by microglia with activated morphology in both humans (Perlmutter et al., 1992, Itagaki et al., 1994, Sheng et al., 1997) and mouse models of the disease (Frautschy et al., 1998).

Despite being considered immune cells, the two most prominent features of microglia in the healthy brain are their highly ramified morphology and motility. Microglia have small cell bodies that give rise to several primary processes that branch to generate multiple secondary and tertiary processes. These processes constantly move back-and-forth, appearing to sample the brain parenchyma and interacting with synapses (Davalos et al., 2005, Nimmerjahn et al., 2005, Wake et al., 2009). When cell damage occurs, microglia extend their processes to surround the damaged area (Davalos et al., 2005). This response is mediated by ATP release at sites of damage and activation of P2Y12 receptors on microglia (Haynes et al., 2006). In the context of AD, microglia exposed to Aβ oligomers may release ATP to enhance migration to Aβ plaques (Kim et al., 2012). However, when microglia become activated by toll-like receptor activation or Aβ peptides, they downregulate their P2Y12 receptors and upregulate adenosine A2A receptors at the mRNA level in vitro (Orr et al., 2009). A2A receptor activation by the ATP breakdown product adenosine leads to process retraction by activated microglia in vitro (Orr et al., 2009), and impairs microglial responses to tissue damage in models of systemic inflammation and Parkinson’s disease (PD) (Gyoneva et al., 2014a, Gyoneva et al., 2014b).

A2A receptors may also play a role in AD pathogenesis. Consumption of caffeine, a nonselective adenosine receptor antagonist, has been linked to a lower risk for developing AD (Eskelinen and Kivipelto, 2010, Santos et al., 2010), as well as reduced neuronal loss, lowered Aβ levels and improved cognition in animal models of AD (Arendash et al., 2009, Canas et al., 2009, Cao et al., 2009). Caffeine also blocks the actions of adenosine on microglial motility (Gyoneva et al., 2014a), raising the possibility that this contributes to its role in AD. Microglia in a mouse model of AD show a reduced response to tissue damage by laser ablation (Krabbe et al., 2013), although the involvement of A2A receptors in modulating microglial motility has not been evaluated in models of AD.

We hypothesized that adenosine A2A receptors will modulate the motility of microglia and microglial response to tissue damage in AD. In this study, we used an ex vivo preparation of acute brain slices to evaluate the motility of microglia and response to damage in the 5xFAD mouse model of AD. Moreover, we examined the contribution of A2A receptors to this response by perfusing the slices with the A2A receptor-selective antagonist preladenant. We found that plaque-associated microglia have a unique motility response, consistent with a functional difference between plaque-associated and plaque-free microglia. These data suggest that microglial function is altered in AD, raising the possibility that cell surface receptors that restore microglial function could serve as a therapeutic target.

Section snippets

Animals and acute slice preparation

All procedures involving the use of animals were reviewed and approved by the Emory University Institutional Animal Care and Use Committee. Two mouse stains were used here: 5xFAD mice [obtained from Dr. Doug Feinstein (University of Illinois, Chicago)] and Cx3cr1^GFP/GFP mice (purchased from The Jackson Laboratories); both strains are on the C57Bl/6 background. 5xFAD mice carry five mutations associated with familial AD [Swedish (K670N, M671L), Florida (I716V) and London (V717I) mutations in

Imaging microglial motion in acute brain slices from the 5xFAD mouse model of AD

We prepared acute 200-μm-thick brain slices from 5xFAD:mg-GFP and non-transgenic (non-Tg) littermate controls, both of which contain Cx3cr1-driven GFP expression in microglia (see Methods for description of strains). To visualize plaques in tissues, we injected the mice with the congophilic dye Methoxy-X04 (Klunk et al., 2002) one day before slice preparation (Hefendehl et al., 2011). In our system, we were able to detect multiple plaques in the hippocampus and cortex as early as 2 month of age (

Discussion

In the present study, we examined microglial motility at baseline and following antagonism of adenosine A2A receptors in acute brain slices of an animal model of AD. We crossed 5xFAD mice, which carry five total familial mutations in APP and PS1 (Oakley et al., 2006), to Cx3cr1^GFP/GFP mice that exhibit microglial GFP expression (Jung et al., 2000) and monitored microglial motility with confocal microscopy (Fig. 1). Our data allow us to draw three main conclusions. First, microglia that are

Conclusion

We studied the motility of microglia in the 5xFAD model of AD. We show that microglia that are physically associated with plaques show hypermotile behavior for the processes pointing away from plaques and confirm that microglia in tissues with AD-like pathology show a delayed response to tissue damage. While the adenosine A2A receptor antagonist preladenant was able to ameliorate the hyperactivity of plaque-associated microglia, it did not restore the response to tissue damage. The exact roles

Author contributions

S.G., S.A.S., S.F.T. and D.W. planned the experiments. S.G., S.A.S. and J.Z. performed the research. S.G. and S.A.S. analyzed the data. S.G., S.A.S., D.W. and S.F.T. discussed the results. All authors wrote the manuscript.

Disclosure

The authors have no conflict of interest to declare.

Acknowledgements

Funding was provided by NINDS NRSA (F31NS076215, S.G.), NIH Pharmacological Sciences Institutional Training grant (T32GM008602, S.G.), and a pilot grant from the Emory University Alzheimer’s Disease Research Center (NIHP50 AG025688, S.F.T.). We thank Dr. Ethel Garnier-Amblard for synthesizing preladenant.

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^†: Current address: Biogen, 225 Binney Street, Cambridge, MA 02142, USA.

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Altered motility of plaque-associated microglia in a model of Alzheimer’s disease

Highlights

Abstract

Introduction

Section snippets

Animals and acute slice preparation

Imaging microglial motion in acute brain slices from the 5xFAD mouse model of AD

Discussion

Conclusion

Author contributions

Disclosure

Acknowledgements

Neurobiol Aging

Neurobiol Dis

J Biol Chem

Brain Res

Neuron

Up-regulation of adenosine receptors in the frontal cortex in Alzheimer’s disease

Brain Pathol

A1 adenosine receptors accumulate in neurodegenerative structures in Alzheimer’s disease and mediate both amyloid precursor protein processing and tau phosphorylation and translocation

Brain Pathol

Caffeine reverses cognitive impairment and decreases brain amyloid-β levels in aged Alzheimer’s disease mice

J Alzheimer Dis

Status epilepticus induces a particular microglial activation state characterized by enhanced purinergic signaling

J Neurosci

Accelerated microglial pathology is associated with Aβ plaques in mouse models of Alzheimer’s disease

Aging Cell

Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance

J Neurosci

Adenosine A2A receptor blockade prevents synaptotoxicity and memory dysfunction caused by β-amyloid peptides via p38 mitogen-activated protein kinase pathway

J Neurosci

Caffeine suppresses amyloid-β levels in plasma and brain of Alzheimer’s disease transgenic mice

J Alzheimer Dis

ATP mediates rapid microglial response to local brain injury in vivo

Nat Neurosci

Caffeine as a protective factor in dementia and Alzheimer’s disease

J Alzheimer Dis

Microglial response to amyloid plaques in APPsw transgenic mice

Am J Pathol

Systemic inflammation regulates microglial responses to tissue damage in vivo

Glia