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

Comments

  1. Love thy neighbor
    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. Using a wide variety of complementary methods, the authors show that microglia might exchange α-synuclein fibrils with each other, at least in part via tunneling nanotubes, so that those microglia that are overburdened by the protein aggregates can share them with their less-stressed neighbors. In a similar vein, the less-compromised neighboring cells can send mitochondria over to the microglia that have taken up numerous α-synuclein fibrils. In a way, the cells are showing love for their neighbors.

    While researchers have thought for a long time that microglia might play an essential role in clearing α-synuclein aggregates, thereby possibly limiting the prion-like spread of aggregates between neurons, this study sheds new light on how this might take place.

    Very importantly, the most common monogenetic cause of Parkinson’s disease (G2019S LRRK2 mutation) was associated with impairments in the cellular communication between neighboring microglia. For some time, it has been suggested that LRRK2 mutations impact immune cell function and this study pinpoints one cell function that the mutations might affect.

    It would be interesting to know if other gene variants, e.g., related to GBA, also affect the processes described here. Furthermore, it will be interesting to learn more about the molecular mechanisms that control the exchange of protein aggregates and organelles between microglia, so that the traffic moves in the right direction.

  2. This study may have important insights into how microglia deal with extracellular synuclein. While this may be triggered by initial cell death of cells with aggregated synuclein, 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.

    One important question will be to examine how microglial degradation of the aggregates occurs within these cells. This is likely to be phagocytosis pathways that interact with lysosomes and possibly peroxidases. It will also be important to know how these degradative steps change with age, as microglia appear to become less competent, as well as potentially with LRRK2 mutations, as the authors have examined.

    It is also possible that these steps are involved in antigen presentation and lymphocyte activation in Parkinson’s. There is a lot to do on this topic, but this paper is providing a potentially very important insight on how synuclein aggregates are handled in the disease.

  3. Parkinson’s disease is progressive, leading to worsening symptoms over time. This progression in symptoms has been correlated with the appearance of α-synuclein Lewy bodies in more regions through the brain over time. It has been hypothesized that misfolded α-synuclein may be released from neurons and internalized by nearby neurons, where it can propagate its pathological conformation through templated seeding. Studies in animal models have shown that this cell-to-cell transmission of pathological proteins can occur from neuron-to-neuron. The role that other resident brain cells, such as microglia, astrocytes, and oligodendrocytes, may play in this process is less clear. Microglia, with their phagocytic activity, should be poised to help neurons degrade extracellular proteins. However, recent studies have found that even microglia can get fatigued in this line of work, leading to inflammatory and deleterious phenotypes.

    In this remarkable new study, Scheiblich et al. examine what microglia do when challenged with misfolded α-synuclein. Similar to previous studies, Scheiblich and colleagues find that misfolded α-synuclein induces an inflammatory phenotype in cultured microglia that can eventually become toxic. However, microglia are not alone. The authors show that microglia can form a network with other microglia through tunneling nanotubes. When one microglia becomes overloaded with excess amounts of misfolded α-synuclein, it can offload this protein on its neighbor, effectively increasing its degradative capacity. Not only that, but the neighbor can also send some healthy mitochondria back to the overloaded microglia, reducing toxicity associated with handling misfolded proteins. The authors also show that microglia can transfer misfolded α-synuclein in vivo, although the extent of this analysis was limited.

    Several interesting questions arise from this study. 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 pathology out of a high-pathology area into less-burdened areas? Are microglia responsible for the spread of pathogenic proteins from one area to another?

    Recent studies have suggested that microglia, instead of protecting the brain, may aid in spreading α-synuclein across the brain (Guo et al., 2020; Xia et al., 2021). The authors of these studies suggested that microglia-derived exosomes, not direct contacts, are the responsible parties. Future studies will undoubtedly reveal more about the microglial network and how it interacts with the neural network in neurodegeneration.

    References:

    . Microglial exosomes facilitate α-synuclein transmission in Parkinson's disease. Brain. 2020 May 1;143(5):1476-1497. PubMed.

    . Reactive microglia enhance the transmission of exosomal α-synuclein via toll-like receptor 2. Brain. 2021 Aug 17;144(7):2024-2037. PubMed.

  4. This is a very interesting paper that surely will stimulate discussions and further research in the field. The most convincing evidence for the suggested mechanism comes from in  vitro microglia cultures using prelabeled α-synthetic synuclein. The hypothesized mechanism is highly interesting, also in light of our recent findings that abundant microglial α-synuclein inclusions indeed develop in seeded brain slice cultures and endogenously in adult α-synuclein transgenic mice (Barth et al., 2021; Tanriöver et al., 2020).

    In our studies, microglial inclusions developed two to three  weeks later compared to neuronal inclusions and appeared biochemically and structurally different from neuronal α-synuclein inclusions. Because the appearance of the microglial inclusions was always linked to the neuronal inclusions both in magnitude and location, and because anti-α-synuclein antibody treatment blocked both neuronal and microglial inclusions, we hypothesized that the microglial inclusions contain αS of neuronal origins.

    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. 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.

    References:

    . 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.

    . 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.

  5. Decades of research have focused on neuronal abnormalities in Parkinson’s disease (PD), but recently more attention has been given to the glial cells. This elegant work by Michael Heneka and colleagues constitutes an important contribution to the field, highlighting the importance of microglia-to-microglia transfer in the clearance of pathological α-synuclein.

    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. The data from Scheiblich et al. indicate that intercellular exchange of α-synuclein may instead limit the pathology and promote maintenance of a less-inflammatory microglia population.

    Interestingly, Scheiblich et al. describe that microglia promote trafficking of fibrillary α-synuclein, as well as mitochondria, via tunneling nanotubes (TNTs), in a fashion similar to that we have previously reported for astrocytes (Rostami et al., 2017). Moreover, the authors show that microglia carrying the LRRK2 G2019S mutation were unable to support neighboring cells in this fashion, and had difficulties sharing their burden of pathological α-synuclein. It would be interesting to investigate if aged microglia have similar limitations.

    Based on these findings, blocking cell-to-cell spreading of pathological proteins would not be an option for future therapeutic interventions. On the other hand, their study identifies cellular disease mechanisms, including mitochondria donation from healthy microglia to α-synuclein overloaded mitochondria, that may open up other treatment possibilities. 

    Uncovering the relevance of TNTs to the interplay between different cell types, including glial cells and neurons, would be important for better understanding the cellular cross-talk in the PD brain. Importantly, the authors used two-photon microscopy to demonstrate that α-synuclein aggregates also transfer between microglia in the living mouse brain, further supporting the importance of glial cells in PD pathology. A future challenge will be to elucidate whether the in vivo transfer is mainly mediated via TNTs or other cellular structures.

    References:

    . Human Astrocytes Transfer Aggregated Alpha-Synuclein via Tunneling Nanotubes. J Neurosci. 2017 Dec 6;37(49):11835-11853. Epub 2017 Oct 31 PubMed.

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References

Paper Citations

  1. . Microglia clear neuron-released α-synuclein via selective autophagy and prevent neurodegeneration. Nat Commun. 2020 Mar 13;11(1):1386. PubMed.
  2. . 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.
  3. . 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.
  4. . 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.
  5. . 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.
  6. . Neuron-to-neuron transmission of α-synuclein fibrils through axonal transport. Ann Neurol. 2012 Oct;72(4):517-24. PubMed.

Further Reading

Papers

  1. . Alzheimer’s disease alters astrocytic functions related to neuronal support and transcellular internalization of mitochondria. BioRxiv, September 17, 2021

Primary Papers

  1. . 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.