Do Rod-Shaped Microglia Dampen Hyperexcitability in ALS?

Microglia respond to proteinopathies by changing their shapes and expression profiles in different ways. Here’s one that has been somewhat overlooked: rod-shaped microglia. These long, straight cells have been observed in several neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s, but few studies have characterized what they do. In a July 2 preprint on bioRxiv, researchers led by Long-Jun Wu at the University of Texas Health Science Center in Houston shed some light on this, and also reported the first evidence of rod-shaped microglia in the brains of people who died from amyotrophic lateral sclerosis, a TDP-43 proteinopathy.

  • Rod-shaped microglia appear in response to neuronal hyperactivity in TDP-43 mice.
  • The cells cozy up to dendrites and prune excitatory synapses.
  • Without rod-shaped microglia, hyperactivity worsens, and animals die sooner.

In a mouse model, the cells popped up in the motor cortex soon after turning on a TDP-43 transgene. They snuggled up to pyramidal cell dendrites and engulfed excitatory synapses, dampening network activity. Preventing these microglia from forming, on the other hand, amped up neuronal firing, worsened motor abilities, and hastened death. “Our results suggest that rod-shaped microglia play a neuroprotective role by attenuating cortical hyperexcitability,” the authors concluded.

Spare the Rod, Spoil the Brain? In a mouse model of TDP-43 proteinopathy, microglia (green) in the motor cortex changed from a homeostatic ramified shape (left) to a long rod-like appearance (right) associated with neuroprotection. [Courtesy of Xie et al., bioRxiv.]

Adam Bachstetter at the University of Kentucky, Lexington, called the functional findings a major discovery. He also appreciated the study’s identification of potential markers for these cells, including galectin-3, which supports cell-cell signaling among other roles. “Utilizing new, more specific tools to study rod-shaped microglia could significantly advance our understanding of how microglia promote healthy aging and combat disease,” he wrote to Alzforum (comment below).

Ironically, Wu and colleagues did not set out to study rod-shaped microglia. They were interested in how microglia sculpt neuronal circuits through interactions with synapses (Umpierre and Wu, 2021; Zhao et al., 2024). In particular, they wondered if microglia help counteract the hyperexcitability that occurs in the motor cortex early in the course of ALS.

To investigate, they used rNLS8 mice, whose inducible transgene overexpresses human TDP-43 without a nuclear localization signal. When the transgene is switched on via the mice’s diet, the truncated protein accumulates in inclusions in the cytoplasm, mimicking the proteinopathy of ALS. First author Manling Xie turned on this transgene in 2-month-old adult mice while monitoring neuronal activity via probes implanted in the motor cortex. Almost immediately, excitatory pyramidal cells revved up, firing 50 percent more often. This hyperactivity peaked after two to three weeks, dropping back to baseline or even below.

Close Relationship. Rod-shaped microglia (green) sidle up along pyramidal cell dendrites (red) with their processes (left) or cell bodies (middle), or sometimes surround them completely (right). [Courtesy of Xie et al., bioRxiv.]

What did hyperactivity do to brain cells? The authors sacrificed mice at 3 weeks and analyzed gene expression in several regions using spatial and single-cell RNA-Seq. Most expression changes occurred in microglia in the cortex. These cells turned on genes characteristic of disease-associated microglia (DAM), including TREM2, APOE, and galectin-3 (Jun 2017 news).

When the authors immunostained cortical brain sections for activated microglia, they were struck by how many of them had straightened into a rod shape. Before transgene activation, rod-shaped cells were sparse, making up five percent of all microglia. After activation, however, their number swelled, peaking at about one-third of all microglia three weeks later, around the time neuronal hyperactivity peaked. The rod microglia seemed to be a subtype of DAM, expressing genes involved in phagocytosis, cell adhesion, and cell remodeling. Almost two-thirds of them expressed galectin-3, hinting at a potential marker gene.

What did the rod microglia do? Immunostaining showed these long cells aligning themselves along the apical dendrites of pyramidal cells. In some cases, their processes embraced the dendrite; in others, the whole cell body made contact, or even surrounded the dendrite (see image above). Tellingly, these microglia often contained pre- and post-synaptic material engulfed from excitatory synapses (see below). To the authors, the data suggested rod-shaped microglia were pruning synapses to restore homeostasis in the motor cortex.

Synapse Patrol. Rod-shaped microglia (white) contain bits of excitatory presynapse (red) and postsynapse (green). [Courtesy of Xie et al., bioRxiv.]

Xie and colleagues tested this idea by crossing rNLS8 mice with TREM2 knockout mice. TREM2 regulates the transition to DAM (Jul 2018 conference news). As expected, in its absence the rod-shaped microglia did not form. As a result, pyramidal cells fired more wildly. The mice were less coordinated, falling off a rotating rod more quickly. Most dramatically, survival time shrank. Three-fourths of rNLS8 TREM2 knockouts died within a month of TDP-43 transgene activation, compared with about 10 percent of rNLS8 mice that had TREM2. Rod-shaped microglia help protect the brain, the authors concluded.

Blobs to Rods. In healthy control brain (left), microglia are round with thin processes; in ALS brain (right), many have become rod-like. [Courtesy of Xie et al., bioRxiv.]

The authors extended the findings to ALS brain, analyzing postmortem motor cortices from 31 patients and 25 age-matched controls. As in the rNLS8 mice, rod-shaped microglia were abundant in the ALS samples, making up about 15 percent of all microglia, but were nearly nonexistent in control brain (see image to left). In AD, PD, HD, and dementia with Lewy bodies, they make up about 10 percent of all microglia, as well (McGeer et al., 1988; Sapp et al., 2001; Bachstetter et al., 2015). They have also been seen in the APPNL-F mouse model of amyloidosis, and ramp up in APPNL-F and P301S crosses, suggesting tau pathology might energize them further (Dec 2016 conference news).

“Given Long-Jun Wu’s lab expertise in imaging of microglia in vivo, I am incredibly excited to see where this project goes next,” wrote Bachstetter.  One question that needs more work is whether the cells in question are truly microglia or might possibly be infiltrating macrophages, wrote Neta Rosenzweig, Brigham and Women’s Hospital, Boston (comment below).—Madolyn Bowman Rogers

Comments

  1. Rod-shaped microglia fascinated the founders of neuropathology, with pioneers like Nissl, Alzheimer, Cerletti, and Achúcarro describing these cells as Stäbchenzellen. Río-Hortega even dedicated a significant portion of his seminal work on microglia as the “third element” of the brain to analyzing these unique cells (Sierra et al., 2016). Despite early interest, much of what was known about these cells remained descriptive, especially in the context of disease and injury (Giordano et al., 2021).

    Now, Long-Jun Wu’s lab has made another major discovery in microglia biology, demonstrating that microglia wrapping around neuronal processes, particularly apical dendrites, represents a neuroprotective response aimed at limiting neuronal hyperexcitability. This was in a mouse model of TDP-43 neurodegeneration (rNLS8). Using in vivo calcium imaging, spatial transcriptomics, and single-cell transcriptomics, Xie et al. found that rod-shaped microglia are spatially and temporally associated with neuronal hyperexcitability.

    They identified TREM2 as a key regulator in the formation of these microglia. When TREM2 was knocked out, there was a significant reduction in rod-shaped microglia, increased neuronal hyperexcitability, more severe motor deficits, and a shorter lifespan in the mice. These findings suggest that rod-shaped microglia are potentially neuroprotective. However, TREM2 knockout also reduced overall microglia density and size, affecting other microglial phenotypes as well.

    This study adds to the growing body of evidence for the protective effects of reactive microglia. Rod-shaped microglia are observed in aging and Alzheimer’s disease (Bachstetter et al., 2017), potentially indicating a response to reduce neuronal hyperexcitability. Interestingly, not all individuals with Alzheimer’s disease have rod-shaped microglia (Bachstetter et al., 2017), raising critical questions about their role. Are some individuals with Alzheimer’s experiencing hyperexcitable neurons to which microglia fail to respond, worsening brain health? Evidence of rod-shaped microglia in people who age without advanced neurodegenerative pathology supports their protective role. Alternatively, do rod-shaped microglia identify individuals with abnormally high levels of neuronal hyperexcitability?

    Xie et al. identified additional molecular targets, such as Galectin-3, that could be used alongside or as alternatives to TREM2 to manipulate rod-shaped microglia. Utilizing new, more specific tools to study these cells could significantly advance our understanding of how microglia promote healthy aging and combat disease. These advancements may lead to novel therapeutic strategies that enhance microglial protective functions, improving outcomes for those with neurodegenerative diseases.

    Given Long-Jun Wu’s lab expertise in in vivo imaging of microglia, I am incredibly excited to see where this project goes next. It would be fascinating to observe how rod-shaped microglia interact with hyperexcitable neurons in real-time. Manipulating the microglia in real-time and observing the changes in neuronal activity would be a dream experiment. This could provide unprecedented insights into the dynamic role of microglia in regulating neuronal function, and potentially lead to new therapeutic strategies for neurodegenerative diseases.

    References:

    Sierra A, de Castro F, Del Río-Hortega J, Rafael Iglesias-Rozas J, Garrosa M, Kettenmann H. The "Big-Bang" for modern glial biology: Translation and comments on Pío del Río-Hortega 1919 series of papers on microglia. Glia. 2016 Nov;64(11):1801-40. PubMed.

    Giordano KR, Denman CR, Dubisch PS, Akhter M, Lifshitz J. An update on the rod microglia variant in experimental and clinical brain injury and disease. Brain Commun. 2021;3(1):fcaa227. Epub 2021 Jan 4 PubMed.

    Bachstetter AD, Ighodaro ET, Hassoun Y, Aldeiri D, Neltner JH, Patel E, Abner EL, Nelson PT. Rod-shaped microglia morphology is associated with aging in 2 human autopsy series. Neurobiol Aging. 2017 Apr;52:98-105. Epub 2017 Jan 5 PubMed.

    View all comments by Adam Bachstetter

  2. This study entangles an interesting observation related to the detection of “rod-shaped” microglia in ALS pathology. RNA-Seq analysis identified a cluster of disease-associated microglia (DAM) enriched in Lgals3 (Galectin-3) expression in a mouse model of ALS. We, and others, have shown that Lgals3+ microglia play a beneficial role in microglial response to neurodegeneration (MGnD/ aka DAM) (Yin et al., 2023; Yu et al., 2024). Thus, the discovery that TREM2 deletion exacerbates disease progression via the elimination of Lgals3+ “rod-shaped microglia” relevant to the field.

    However, it is still unknown whether the Lgals3+ rod-shaped microglia, detected using immunohistochemistry in this study, truly represent microglia or if they are infiltrating macrophages. Moreover, since global TREM2-KO mice were used in this study, it is possible that the observed effect is due to deletion of TREM2 in peripheral myeloid cells. Future studies will be required to dissect the contribution of microglia and peripheral myeloid cells expressing TREM2 to ALS progression.

    References:

    Yin Z, Rosenzweig N, Kleemann KL, Zhang X, Brandão W, Margeta MA, Schroeder C, Sivanathan KN, Silveira S, Gauthier C, Mallah D, Pitts KM, Durao A, Herron S, Shorey H, Cheng Y, Barry JL, Krishnan RK, Wakelin S, Rhee J, Yung A, Aronchik M, Wang C, Jain N, Bao X, Gerrits E, Brouwer N, Deik A, Tenen DG, Ikezu T, Santander NG, McKinsey GL, Baufeld C, Sheppard D, Krasemann S, Nowarski R, Eggen BJ, Clish C, Tanzi RE, Madore C, Arnold TD, Holtzman DM, Butovsky O. APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFβ-mediated checkpoints. Nat Immunol. 2023 Nov;24(11):1839-1853. Epub 2023 Sep 25 PubMed.

    Yu C, Lad EM, Mathew R, Shiraki N, Littleton S, Chen Y, Hou J, Schlepckow K, Degan S, Chew L, Amason J, Kalnitsky J, Bowes Rickman C, Proia AD, Colonna M, Haass C, Saban DR. Microglia at sites of atrophy restrict the progression of retinal degeneration via galectin-3 and Trem2. J Exp Med. 2024 Mar 4;221(3) Epub 2024 Jan 30 PubMed.

    View all comments by Neta Rosenzweig

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References

News Citations

  1. Hot DAM: Specific Microglia Engulf Plaques
  2. TREM2: Diehard Microglial Supporter, Consequences Be DAMed
  3. Next-Generation Mouse Models: Tau Knock-ins and Human Chimeras

Paper Citations

  1. Umpierre AD, Wu LJ. How microglia sense and regulate neuronal activity. Glia. 2021 Jul;69(7):1637-1653. Epub 2020 Dec 28 PubMed.
  2. Zhao S, Umpierre AD, Wu LJ. Tuning neural circuits and behaviors by microglia in the adult brain. Trends Neurosci. 2024 Mar;47(3):181-194. Epub 2024 Jan 19 PubMed.
  3. McGeer PL, Itagaki S, Boyes BE, McGeer EG. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology. 1988 Aug;38(8):1285-91. PubMed.
  4. Sapp E, Kegel KB, Aronin N, Hashikawa T, Uchiyama Y, Tohyama K, Bhide PG, Vonsattel JP, Difiglia M. Early and progressive accumulation of reactive microglia in the Huntington disease brain. J Neuropathol Exp Neurol. 2001 Feb;60(2):161-72. PubMed.
  5. Bachstetter AD, Van Eldik LJ, Schmitt FA, Neltner JH, Ighodaro ET, Webster SJ, Patel E, Abner EL, Kryscio RJ, Nelson PT. Disease-related microglia heterogeneity in the hippocampus of Alzheimer's disease, dementia with Lewy bodies, and hippocampal sclerosis of aging. Acta Neuropathol Commun. 2015 May 23;3:32. PubMed.

Further Reading

Primary Papers

  1. Xie M, Miller A, Pallegar P, Umpierre A, Liang Y, Wang N, Zhang S, Nagaraj N, Zachary F, Qi F, Ghayal N, Oskarsson B, Zhao S, Zheng J, Nguyen A, Dickson DW, Wu L-J. Rod-shaped microglia interact with neuronal dendrites to regulate cortical excitability in TDP-43 related neurodegeneration. 2024 Jul 02 10.1101/2024.06.30.601396 (version 1) bioRxiv.

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