In brain disease, microglia can be villains or heroes. How to encourage the latter? In the June 10 Nature Neuroscience, researchers led by Veronique Miron at the University of Edinburgh tried by killing off the troublemakers. In a mouse model of demyelination, the authors found that lesions healed only after inflammatory microglia had died by way of necroptosis. When the researchers blocked this type of programmed cell death, remyelination lagged. “Necroptosis of pro-inflammatory microglia is required to generate pro-regenerative microglia,” Miron told Alzforum. She is investigating whether a similar dynamic plays out in neurodegenerative diseases such as Alzheimer’s, where demyelination also occurs.
- After demyelination, inflammatory microglia die by necroptosis.
- Healthy cells replenish the dead as axons heal.
- Blocking necroptosis holds up remyelination.
“With this work, the researchers solve the long-standing mystery of the fate of microglia that become acutely activated during demyelination,” Constanze Depp and Stefan Berghoff at the Max Planck Institute for Experimental Medicine in Göttingen, Germany, wrote to Alzforum (full comment below). “This work certainly provokes thought about how this natural form of microglial depletion can be boosted experimentally to further stimulate remyelination.”
Miron and colleagues study multiple sclerosis (MS), a disease marked by cycles of demyelination and remyelination. They previously reported that microglia around myelin lesions in mouse brain switch from a pro-inflammatory to an anti-inflammatory state as remyelination begins (Miron et al., 2013). To explore how this happens, first author Amy Lloyd compared microglial gene expression in mouse corpus callosum three and 10 days after inducing demyelination by injecting this region with the toxin lysophosphatidyl choline (LPC). She found differences in 1,020 genes. At three days, when demyelination peaked, microglia around the lesion expressed many cell death and pro-inflammatory genes. At 10 days, when remyelination had revved up, they expressed more regeneration and anti-inflammatory genes.
Did the microglia expressing cell-death genes actually die? Corpus callosum microglial numbers fell by half seven days after the researchers injected the LPC. To confirm that cells were dying, the authors applied LPC to cerebellar explants and used live imaging to watch what happened. Hours after the toxin triggered demyelination, microglia rounded up and burst (see video below). Importantly, LPC had no effect on primary microglia cultures, demonstrating it does not directly induce cell death.
Dying microglia expressed markers of necroptosis, but not apoptosis. Necroptosis is a form of cell suicide that occurs under inflammatory conditions. It triggers different pathways than apoptosis, with receptor-interacting protein kinase 1 (RIPK1) being a key mediator (Jun 2005 news). The authors examined two additional mouse models of demyelination, one induced by cuprizone and the other by an autoimmune reaction to myelin oligodendrocyte glycoprotein. In both cases, RIPK1 levels in microglia peaked around the time remyelination began, suggesting that necroptotic death is a consistent feature of myelin repair.
Annihilated. In the 12 hours after LPC was added to mouse cerebellar explants, microglia expressing green fluorescent protein retract processes and rupture. [Courtesy of Lloyd et al., Nature Neuroscience.]
But is this necessary for myelin to repair itself? Lloyd and colleagues added the RIPK1 inhibitor necrostatin-1 to cerebellar explants at the same time as LPC. A day later, these explants contained twice as many pro-inflammatory microglia as did those treated with LPC alone. Remyelination faltered, with the explants producing only half as much new myelin as control explants one and two weeks later (see image above).
The same thing happened in mice; when microglia around a lesion took up necrostatin-1, that region produced half as much new myelin as normal by day 10. This was true even though microglia containing necrostatin-1 mopped up myelin debris as well as did control microglia. Together, the findings suggested that activated microglia can remove debris, but then need to die off to allow new myelin to form. Why that is, is not clear.
The remyelination 10 days after LPS injection was accompanied by a rebound in the number of microglia. Where do these new cells come from? In remyelinating explants, less than 30 percent of repopulating microglia present during remyelination expressed the precursor cell marker nestin. What about the rest? The researchers traced their lineage by inducing expression of red fluorescent protein in Iba1-positive cells before demyelination. During remyelination, 70 to 80 percent of repopulating microglia expressed this label, indicating that most of these cells arose through expansion of the existing pool, rather than from de novo differentiation. Interferon signaling appeared crucial for this proliferation. Its levels rose during remyelination, and an antibody against the interferon receptor suppressed microglial numbers and delayed myelination.
To see if microglial necroptosis aids remyelination in people, the authors examined lesions in postmortem brain tissue from seven people who had died with relapsing-remitting MS. In active, remyelinating lesions, they found higher levels of necroptotic and proliferating microglia than in inactive lesions that do not heal.
The finding implies that stimulating necroptotic death in inactive lesions might nudge them toward remyelination, the authors claim. They are now trying to identify the signals that trigger necroptosis in hopes of finding a therapeutic target. They will also examine whether subpopulations of microglia are particularly prone or resistant to necroptosis. Human microglia are diverse, with studies identifying up to seven distinct types (Dec 2018 news; Feb 2019 news).
Do the successive death and renewal of microglia play a role in other disorders? Myelin breaks down in Alzheimer’s disease as well, and myelination-related genes are consistently perturbed (May 2019 news). Markers of necroptosis have been found in microglia in the AD brain. In the 5XFAD mouse, they correlate with brain atrophy and cognitive decline, raising the possibility that necroptosis could aid remyelination in AD (Jul 2017 news).
Could microglial replacement encourage healthier, more phagocytic microglia to surround plaques? The answer is unknown, but in mouse models of amyloidosis, depleting microglia slows neurodegeneration, and has been reported to block plaque formation. Intriguingly, remaining microglia still seem to surround plaques (Apr 2014 news; Spangenberg et al., 2016; Mar 2018 news). Miron and colleagues are collaborating with Josef Priller and Tara Spires-Jones at the Edinburgh Dementia Research Institute to investigate whether microglial necroptosis and repopulation can affect AD pathology. Others are studying RIPK1 inhibitors as treatments for inflammatory and neurodegenerative disorders (Harris et al., 2017). Denali Therapeutics, South San Francisco, has begun Phase1 trials of its RIPK1 inhibitor DNL747 in AD (see clinicaltrials.gov) and amyotrophic lateral sclerosis (clinicaltrials.gov) and is targeting MS. GlaxoSmithKline completed Phase 1/2 trials of GSK2982772 for rheumatoid arthritis (clinicaltrials.gov) and psoriasis (clinicaltrials.gov) and is testing in a Phase 2 trial for ulcerative colitis (clinicaltrials.gov).—Madolyn Bowman Rogers
- A New Program for Cell Death: Necroptosis Premiering in a Neuron Near You
- Local Flavor: At Protein Level, Too, Human Microglia Are Diverse
- Single-Cell Profiling Maps Human Microglial Diversity, Flexibility
- When It Comes to Alzheimer’s Disease, Do Human Microglia Even Give a DAM?
- Necroptosis Rampant in the Alzheimer’s Brain?
- Microglial Magic: Drug Wipes Them Out, New Set Appears
- Wiping Out Microglia Prevents Neuritic Plaques
- Miron VE, Boyd A, Zhao JW, Yuen TJ, Ruckh JM, Shadrach JL, van Wijngaarden P, Wagers AJ, Williams A, Franklin RJ, Ffrench-Constant C. M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nat Neurosci. 2013 Sep;16(9):1211-1218. Epub 2013 Jul 21 PubMed.
- Spangenberg EE, Lee RJ, Najafi AR, Rice RA, Elmore MR, Blurton-Jones M, West BL, Green KN. Eliminating microglia in Alzheimer's mice prevents neuronal loss without modulating amyloid-β pathology. Brain. 2016 Apr;139(Pt 4):1265-81. Epub 2016 Feb 26 PubMed.
- Harris PA, Berger SB, Jeong JU, Nagilla R, Bandyopadhyay D, Campobasso N, Capriotti CA, Cox JA, Dare L, Dong X, Eidam PM, Finger JN, Hoffman SJ, Kang J, Kasparcova V, King BW, Lehr R, Lan Y, Leister LK, Lich JD, MacDonald TT, Miller NA, Ouellette MT, Pao CS, Rahman A, Reilly MA, Rendina AR, Rivera EJ, Schaeffer MC, Sehon CA, Singhaus RR, Sun HH, Swift BA, Totoritis RD, Vossenkämper A, Ward P, Wisnoski DD, Zhang D, Marquis RW, Gough PJ, Bertin J. Discovery of a First-in-Class Receptor Interacting Protein 1 (RIP1) Kinase Specific Clinical Candidate (GSK2982772) for the Treatment of Inflammatory Diseases. J Med Chem. 2017 Feb 23;60(4):1247-1261. Epub 2017 Feb 10 PubMed.
- Lloyd AF, Davies CL, Holloway RK, Labrak Y, Ireland G, Carradori D, Dillenburg A, Borger E, Soong D, Richardson JC, Kuhlmann T, Williams A, Pollard JW, des Rieux A, Priller J, Miron VE. Central nervous system regeneration is driven by microglia necroptosis and repopulation. Nat Neurosci. 2019 Jul;22(7):1046-1052. Epub 2019 Jun 10 PubMed.