It takes a village—of microglia, that is—to vanquish the scourge of α-synuclein aggregates. That’s the upshot of a paper published September 21 in Cell. Led by Michael Heneka of the German Center for Neurodegenerative Diseases in Bonn, scientists found that when microglia become overloaded with α-synuclein aggregates, they form an on-demand network of contacts with neighboring, healthy microglia, through which they pass excess aggregates. The conduits? Tunneling nanotubes.
- Microglia hand off α-synuclein aggregates to naïve neighbors.
- The aggregates pass via tunneling nanotubes and gap junctions.
- Acceptor microglia pass healthy mitochondria to their overwhelmed peers, calming cell stress.
In turn, those neighbors promptly dispose of the refuse, and pass back fresh mitochondria to rejuvenate their overwhelmed peers. This exchange essentially quiets pro-inflammatory responses and relieves cell stress. Notably, microglia bearing the G2019S mutation in the LRRK2 kinase that causes Parkinson’s disease fumbled these hand-offs. In all, the findings suggest that microglia share the burden of α-synuclein aggregates, and that disease may worsen when this communal effort falls short.
“This is a truly fascinating study suggesting a new, and somewhat unexpected, set of cellular mechanisms that microglia might use to combat accumulation of pathogenic α-synuclein aggregates,” commented Patrik Brundin of the Van Andel Institute in Grand Rapids, Michigan. “In a way, the cells are showing love for their neighbors.”
“It is likely to be an important piece of the jigsaw puzzle of how pathogenesis is perpetuated in synucleinopathies, and particularly how relatively few aggregates could lead to widespread changes in many microglial and astrocytic cells, a central step in CNS inflammation,” commented David Sulzer of Columbia University in New York.
Lending a Tube. Microglia overloaded with α-synuclein (top left) crank out pro-inflammatory cytokines and reactive oxygen species, careening toward death. Healthy microglia (blue) connect via nanotubes and gap junctions to relieve struggling neighbors of their α-synuclein aggregates (top); they even gift them fresh mitochondria (bottom). [Courtesy of Scheiblich et al., Cell, 2020.]
A characteristic feature of all synucleinopathies, including Parkinson’s disease, is the accumulation of α-synuclein aggregates within cells. Aggregated forms of this synaptic protein can pass between neurons, propagating the pathology through the brain. Microglia have been reported to limit α-synuclein spread by taking up aggregates and digesting them via autophagy (Choi et al., 2020). Recent studies have spotted α-synuclein aggregates lurking within microglia (Tanriöver et al., 2020; Barth et al., 2021). How microglia acquire and deal with α-synuclein aggregates, and how the cells influence the progression of pathology, are crucial questions in the field.
First author Hannah Scheiblich and colleagues investigated how microglia deal with α-synuclein aggregates in cell culture. When exposed to recombinant human α-synuclein fibrils, primary mouse microglia readily phagocytosed, yet stopped short of fully digesting them. Gene-expression analysis revealed that cells overstuffed in this way revved up expression of pro-inflammatory genes, including TNF-α and interferon-stimulated genes, as well as genes involved in cell stress and programmed cell death. The overloaded cells also pumped out reactive oxygen species, and their mitochondria appeared to deteriorate.
What the researchers found next surprised them, Heneka said. When exposed to α-synuclein fibrils, microglia sprouted membrane projections of various sizes. Using electron microscopy and immunocytochemistry to zero in on these projections, the researchers found that they connected directly to other microglia, implying some form of communication. Bolstered by actin filaments, these processes contained α-synuclein fibrils, various vesicles, and cytoplasmic organelles such as mitochondria and endoplasmic reticulum.
To see if these contents passed between connected cells, the authors used time-lapse immunocytochemistry. They found that smaller α-synuclein aggregates cruised inside the long, thin tubes to pass from one microglia to another in about three minutes. Larger aggregates lumbered through shorter, thicker passageways, taking 40 to 60 minutes to move from one cell to another.
By analyzing a barrage of co-culture experiments with α-synuclein-laden “donor” and naïve “acceptor” cells, the researchers found that α-synuclein preferentially passes from overloaded microglia to unburdened cells via tunneling nanotubes and gap junctions. The acceptor microglia then make quick work of the incoming cargo. “It’s as if the acceptor microglia are waiting for the α-synuclein to arrive,” Heneka told Alzforum. “Once it enters the acceptor cytosol, it rapidly disappears,” he said (see movie below).
Mutual Aid. An α-synuclein-loaded microglial cell (upper) passes α-synuclein aggregates (black clumps) to a less-burdened cell (bottom). Once aggregates enter the cytoplasm of the acceptor, they vanish. [Courtesy of Scheiblich et al., Cell, 2021.]
Exactly how the acceptor microglia degrade the incoming α-synuclein is the subject of ongoing experiments, Heneka said. Previous studies have demonstrated the transfer of α-synuclein aggregates via tunneling nanotubes in neurons, astrocytes, and pericytes, but not microglia (Abounit et al., 2016; Dieriks et al., 2017; Freundt et al., 2012).
With their α-synuclein load lightened, the donor microglia reverted to a calmer state, turning down expression of pro-inflammatory and cell death genes, and squelching their release of reactive oxygen species. On the other hand, the α-synuclein transfer had no substantial influence on genes expressed in the acceptor microglia. Within 300 minutes of co-culture, the transcriptomes of the donor and acceptor microglia had roughly equalized, suggesting an overall restoration of homeostasis.
Disposal of α-synuclein was not the only service acceptor microglia provided. Intact mitochondria also traversed these intercellular corridors. These organelles predominantly moved from α-synuclein-naïve, healthy microglia to α-synuclein-loaded cells. The researchers believe that the infusion of fresh mitochondria explains the recovery of mitochondrial function, and reduction of oxidative stress, in the α-synuclein donor cells.
Caring Connections. A donor microglia loaded with α-synuclein (red) hooks up with a healthy, α-synuclein-free acceptor via a tunneling nanotube. Both α-synuclein and mitochondria (green) migrate through the corridor (bottom right). [Courtesy of Scheiblich et al., Cell, 2021.]
Do microglia in this way form an α-synuclein disposal network in the brain? The researchers explored this question using two-photon microscopy in mice. Indeed, injected α-synuclein fibrils wound up within microglia, and many microglia then sprouted α-synuclein-containing projections that connected them to other microglia. In postmortem brain samples from people with dementia with Lewy bodies (DLB) and multisystem atrophy (MSA), the researchers spotted α-synuclein-laden microglia that appeared to be connected to other microglia.
In all, the findings support the existence of an on-demand microglial network that forms in response to α-synuclein overload, Heneka believes. For now, the response appears somewhat specific for α-synuclein, because the researchers reported that neither Aβ nor tau aggregates spurred microglia to join forces to the same extent. Still, how this mechanism might unfold in the context of age-related neurodegenerative disease, which progresses over decades, remains to be seen, Heneka said. He suggested that an eventual failure of the microglial network—due to aging and/or pathogenic mutations—might herald exacerbation of disease.
The scientists found that primary microglia from G2019S-LRRK2 mutant mice were less adept at transferring α-synuclein from donors to acceptors. Even when they did, mutant acceptors did not take it in their stride, but revved up production of reactive oxygen species. While mitochondria passed between LRRK2-mutant cells, only mitochondria from wild-type α-synuclein cells were able to dampen the release of ROS from α-synuclein-loaded microglia carrying the LRRK2 mutation. In other words, the LRRK2 mutation quashed the exchange and degradation of α-synuclein, and neutralized mitochondria coming from α-synuclein-free cells.
For Michael Henderson of the Van Andel Institute, the study raised many questions. “How do microglia signal who needs help? Can neurons similarly signal to other neurons or microglia when their degradative capacity is compromised? In the event of neurodegeneration, can microglia form a bucket brigade of sorts that moves synuclein out of a high-pathology area into less-burdened areas? Are microglia responsible for the spread of pathogenic proteins from one area to another?”
To Heneka's mind, chief among these questions is whether microglia can relieve neurons or other cell types of excess α-synuclein; his lab is investigating this.
Gaye Tanriöever and Mathias Jucker of the German Center for Neurodegenerative Diseases in Tubingen hypothesize that microglia in the brain probably contain α-synuclein that came from neurons. “Thus, it will be important to have a fresh look at the network dynamics and relationship between neuronal versus microglial α-synuclein inclusions with a focus on the suggested nanotube and cell-to-cell connections,” they wrote to Alzforum. “The hypothesized and exciting cooperative strategy of pathogenic protein degradation of microglia will surely inspire the field to re-evaluate the contribution of microglia in disease progression when the degradation network fails.”
The findings call into question the idea that α-synuclein transfer between cells is always bad. “To date, intercellular propagation of α-synuclein aggregates has mainly been considered a negative process, constituting a possible mechanism for spreading of PD pathology throughout the brain tissue,” commented Anna Erlandsson of Uppsala University, Sweden. “The data from this study indicate that intercellular exchange of α-synuclein may instead limit the pathology and promote maintenance of a less-inflammatory microglia population.”—Jessica Shugart
- Choi I, Zhang Y, Seegobin SP, Pruvost M, Wang Q, Purtell K, Zhang B, Yue Z. Microglia clear neuron-released α-synuclein via selective autophagy and prevent neurodegeneration. Nat Commun. 2020 Mar 13;11(1):1386. PubMed.
- Tanriöver G, Bacioglu M, Schweighauser M, Mahler J, Wegenast-Braun BM, Skodras A, Obermüller U, Barth M, Kronenberg-Versteeg D, Nilsson KP, Shimshek DR, Kahle PJ, Eisele YS, Jucker M. Prominent microglial inclusions in transgenic mouse models of α-synucleinopathy that are distinct from neuronal lesions. Acta Neuropathol Commun. 2020 Aug 12;8(1):133. PubMed.
- Barth M, Bacioglu M, Schwarz N, Novotny R, Brandes J, Welzer M, Mazzitelli S, Häsler LM, Schweighauser M, Wuttke TV, Kronenberg-Versteeg D, Fog K, Ambjørn M, Alik A, Melki R, Kahle PJ, Shimshek DR, Koch H, Jucker M, Tanriöver G. Microglial inclusions and neurofilament light chain release follow neuronal α-synuclein lesions in long-term brain slice cultures. Mol Neurodegener. 2021 Aug 11;16(1):54. PubMed.
- Abounit S, Bousset L, Loria F, Zhu S, de Chaumont F, Pieri L, Olivo-Marin JC, Melki R, Zurzolo C. Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes. EMBO J. 2016 Oct 4;35(19):2120-2138. Epub 2016 Aug 22 PubMed.
- Dieriks BV, Park TI, Fourie C, Faull RL, Dragunow M, Curtis MA. α-synuclein transfer through tunneling nanotubes occurs in SH-SY5Y cells and primary brain pericytes from Parkinson's disease patients. Sci Rep. 2017 Feb 23;7:42984. PubMed.
- Freundt EC, Maynard N, Clancy EK, Roy S, Bousset L, Sourigues Y, Covert M, Melki R, Kirkegaard K, Brahic M. Neuron-to-neuron transmission of α-synuclein fibrils through axonal transport. Ann Neurol. 2012 Oct;72(4):517-24. PubMed.
- Lampinen R, Belaya I, Saveleva L, Liddell JR, Rait D, Huuskonen MT, Giniatullina R, Sorvari A, Soppela L, Mikhailov N, Boccuni I, Giniatullin R, Cruz-Haces M, Konovalova J, Koskuvi M, Rauramaa T, Domanskyi A, Hämäläinen RH, Goldsteins G, Koistinaho J, Malm T, Chew S, Rilla K, White AR, Marsh-Armstrong N, Kanninen KM. Alzheimer’s disease alters astrocytic functions related to neuronal support and transcellular internalization of mitochondria. BioRxiv, September 17, 2021
- Scheiblich H, Dansokho C, Mercan D, Schmidt SV, Bousset L, Wischhof L, Eikens F, Odainic A, Spitzer J, Griep A, Schwartz S, Bano D, Latz E, Melki R, Heneka MT. Microglia jointly degrade fibrillar alpha-synuclein cargo by distribution through tunneling nanotubes. Cell. 2021 Sep 30;184(20):5089-5106.e21. Epub 2021 Sep 22 PubMed.