The brain’s resident immune cells, microglia, play a Jekyll-and-Hyde game in Alzheimer’s disease. Researchers at the 12th International Conference on Alzheimer’s and Parkinson’s Diseases, held March 18-22 in Nice, France, outlined their latest efforts to discover what makes these cells good or bad. Some speakers talked about characterizing them in healthy and diseased brains. Others presented new data supporting TREM2’s role in promoting phagocytosis, although controversy continues over whether this receptor even marks microglia or infiltrating macrophages. Other talks pointed to the enzyme arginase-1 as a marker of helpful microglia, or implicated these cells in triggering Parkinson’s disease. As yet, no single clear picture has emerged; instead, accumulating data emphasize the complex and shifting nature of these cells.
Researchers used to divide microglia neatly between an M1 phenotype that promotes inflammation, and a more beneficial M2 phenotype that specializes in phagocytosis. This simple schema is being supplanted by the recognition that microglia can assume numerous states characterized by multiple overlapping markers (see Mar 2013 conference news).
In Nice, Delphine Boche of the University of Southampton, U.K., discussed efforts to more fully characterize healthy versus harmful microglia. She examined multiple microglial markers in postmortem brain slices from 148 dementia patients and 130 age-matched controls. Samples came from the MRC Cognitive Function and Ageing Study (CFAS). She then correlated the findings with cognitive scores on the Mini-Mental State Examination. Microglial expression of CD64 and Iba1 associated with high test performance, whereas CD68 and HLA-DR predominated in people who had scored low. CD64 is involved in activation and Iba1 in motility, while CD68 relates to phagocytosis and HLA-DR to antigen presentation, she noted. She saw high CD68, HLA-DR, and CD64 and low Iba1 in ApoE4 carriers as well, tying this risk factor to inflammation.
How did Alzheimer’s pathology affect the microglia’s phenotype? CD68 was linked to higher tau pathology, while CD64 showed up in brains with more diffuse plaques but less tau, Boche reported. This hints that CD64 might be in play at an earlier and CD68 at a later stage of AD pathology.
Boche saw a difference not only in microglia phenotype but also in overall activation. Brains with Alzheimer’s pathology had more activated phagocytes, but there was a sharp distinction between people with and without dementia. In those with normal cognition, AD pathology was associated with less or no microglial activation, but not more. “Microglia respond differently to Aβ and tau in people with and without dementia,” she noted. “Microglial status may be pivotal in AD pathogenesis.”
The Secret Life of TREM2. TREM2 is cleaved from the surface of microglia by ADAM10, freeing a soluble portion that may interact with ligands on neurons. Disease-associated mutations in TREM2 affect these processes. [Image courtesy of Daniel Sevlever.]
Boche stained for TREM2 in microglia as well, but reported that she saw the marker only on macrophages derived from peripheral cells. The finding fits with recent data from Bruce Lamb at the Cleveland Clinic, Ohio, who argued that TREM2-expressing cells in the brain come from the blood (see Jay et al., 2015). The issue has raised controversy among researchers in the field. “I think the data are convincing that TREM 2 is a protein expressed by infiltrating cells,” David Morgan at the University of South Florida, Tampa, wrote to Alzforum. Others disagree, maintaining that they do see TREM2 on resident microglia (see Dec 2014 conference news). One problem is that the different studies use varying methods. “Further studies using similar protocols, mouse models, and assays will be required to sort out the TREM2 story,” Cynthia Lemere of Brigham and Women’s Hospital, Boston, wrote to Alzforum.
TREM2 has drawn intense interest in AD research ever since geneticists reported that rare variants in this gene confer as much risk as the ApoE4 allele (see Nov 2012 news). Most researchers agree that these variants result in a loss of TREM2 function, implying that the protein helps control AD pathology, although Lamb recently reported fewer plaques in TREM2 knockouts (see Feb 2015 conference news).
In Nice, Christian Haass of Ludwig-Maximilians-Universität, Munich, argued that TREM2 aids phagocytosis. A single-pass transmembrane protein, similar in structure to amyloid precursor protein (APP), TREM2 is cleaved by the α-secretase ADAM10 and its outer portion shed into extracellular space. Haass previously reported that a TREM2 mutation associated with frontotemporal dementia, T66M, inhibited maturation and subsequent shedding of the protein because it got stuck inside endoplasmic reticulum. Both mice and people with T66M and other missense mutations had lowered levels of soluble TREM2 in plasma and cerebrospinal fluid (see Jul 2014 webinar; Kleinberger et al., 2014).
On the other hand, the R47H mutation associated with AD was being shed normally. In ongoing work, Haass is measuring soluble TREM2 in Alzheimer’s disease. At AD/PD, he reported high soluble TREM2 levels in the CSF of people with preclinical AD or mild cognitive impairment due to AD, compared with age-matched peers with normal cognition. In people with dementia, however, the marker returned to nearly normal levels. Soluble TREM2 levels directly correlated with elevated total tau and phospho-tau in the CSF of sporadic AD patients. Haass also saw elevated soluble TREM2 in people with normal cognition who carry a familial AD mutation, and in AD mouse models. It is not yet clear why, but one possibility is that high TREM2 represents an inflammatory defense against disease progression, Haass told Alzforum.
When TREM2 is shed, it leaves the cell surface. Many prior studies reported that TREM2 on microglia promotes phagocytosis (see Feb 2015 news; Hickman and El Khoury, 2014). Microglia without the receptor cannot devour Aβ in vitro, Haass noted. Likewise, microglia with T66M TREM2 poorly phagocytose beads, but when Haass blocked shedding of TREM2 by inhibiting ADAM10, phagocytosis improved. Haass believes faulty phagocytosis underlies disease, but stressed that the problem is not just removal of Aβ. Rather, dysfunctional microglia may fail to clean up cellular debris more broadly, intensifying neuronal stress and precipitating disease, he proposed. Other studies reported no difference in amyloid plaques in mice with little TREM2 (see Jun 2014 news).
Daniel Sevlever of the Mayo Clinic in Jacksonville, Florida, elucidated the effect of various TREM2 mutations. Sevlever reported that a mouse microglial cell line expressing the D87N mutation linked to AD shed less TREM2 into the medium than wild-type, and also had scant cell-surface expression of TREM2. On the other hand, cells with the R47H mutation shed TREM2 normally, agreeing with Haass’ data. The R47H mutation caused other problems, however, leading to reduced expression of TREM2 on the cell surface. Sevlever also looked at what happened to the soluble portion, which can interact with TREM2 ligands on neurons just as membrane-bound TREM2 does. He found that the soluble domain of R47H TREM2 bound poorly to the surface of neuroblastoma cells. Overall, this suggests that the R47H mutation may act through a different mechanism than T66M or D87N, perhaps by reducing binding or cell-surface expression rather than maturation and shedding.
Meanwhile, Karel Otero Gutiérrez of Biogen Idec, Boston, added data that further support TREM2’s role in brain cleanup. He fed mice cuprizone, a chemical that causes oligodendrocytes to die. In this model, the myelin sheath around axons falls apart, activating microglia to mop up the debris. In TREM2 knockout mice, microglia failed to activate or proliferate, and myelin chunks continued to litter the brain. These knockouts had worse motor problems than wild-type mice on cuprizone. Otero saw no signs of a pro-inflammatory effect in their brains, suggesting that defective activation and phagocytosis was the culprit (see Cantoni et al., 2015). “TREM2 may play a general role in controlling the activation of microglia in response to tissue injury,” Otero wrote to Alzforum. He noted that other work suggests TREM2 acts not just by facilitating phagocytosis, but also by supporting microglial survival in diseased and damaged brains.
Bucking the TREM2 tide, Jonathan Cherry, a graduate student with Kerry O’Banion and John Olschowka at the University of Rochester Medical Center, New York, argued that microglia expressing the marker arginase-1 are uniquely in charge of plaque removal. This enzyme was classically associated with M2 microglia, wound healing and suppression of nitric oxide production (see review by Cherry et al., 2014). Examining APPPS1 mice, Cherry found that in areas of chronic inflammation, arginase-1 expression soared while Aβ deposits plummeted. The expression of an anti-inflammatory marker in regions of high inflammation seemed counterintuitive to Cherry, and suggested microglia might be offsetting the inflammatory response. To study this, Cherry induced inflammation in mice. One month later, he found arginase-1 expression on microglia surrounding plaques. These cells had ingested twice the amount of plaque material as microglia expressing the traditional M1 marker iNos. Cherry confirmed that the arginase-1-positive cells were microglia, not macrophages, by using a chimeric mouse with fluorescently labeled bone marrow.
The finding suggested that arginase-1 positive cells might be more efficient phagocytes than their comrades. To confirm this, Cherry induced arginase expression with IL-4 injections; this led to a drop in plaques. Then he knocked out arginase-1 by infusing anti-IL4Rα through a minipump for 28 days. The treatment cut arginase-expressing cells in half and doubled the Aβ load, while not affecting cells positive for iNos. Together, Cherry concluded, his experiments demonstrate that arginase-positive microglia are necessary and sufficient for plaque removal, and validate arginase induction as a therapeutic approach.
Microglia may play a role in Parkinson’s disease, too. Dan Frenkel of Tel Aviv University suggested a mechanism by which the Parkinson’s risk genes DJ-1 and PINK1 could act on microglia to precipitate PD. He knocked down both DJ-1 and PINK1 in cultured microglia, and found the cells became more sensitive to activation, more neurotoxic, and expressed more pro-inflammatory markers (see Trudler et al., 2014). Moreover, when he stimulated the microglia with α-synuclein, he saw they gobbled up only about half as much as did microglia expressing those two genes. Curiously, the defect was specific to α-synuclein, as the cells phagocytosed yeast and inorganic particles normally. Frenkel noted that α-synuclein binds to lipid rafts on the cell membrane, structures that dwindle in cells lacking DJ-1 and PINK1. Deficient cells also had weaker autophagy, suggesting they might degrade α-synuclein poorly as well. Overall, the results hint that mutations in DJ-1 and PINK1 could alter microglia toward a neurotoxic phenotype, triggering disease and allowing α-synuclein deposits to accumulate, Frenkel proposed.—Madolyn Bowman Rogers
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