In TDP-43 Mouse, Hesitant Microglia Eventually Swoop to the Rescue

Despite probing and prodding microglia every way they can think of, scientists are still unsure whether these cells help or hinder neurodegeneration. Now, mice whose neurons express a truncated form of the ALS-related protein TDP-43 reveal a microglial state that helps neurons heal. Researchers led by Virginia Lee, University of Pennsylvania, Philadelphia, report that microglia appear surprisingly unreactive as TDP-43 accumulates in the animals’ spinal cords. However, turning off TDP-43 expression seemed to awaken the cells. They proliferated, gobbled up TDP-43 aggregates, and motor function improved. In their activated state, the microglia expressed a unique set of genes, distinct from the set expressed during disease progression. The findings are reported in the February 20 Nature Neuroscience.

  • An hTDP-43 conditional mouse model unmasks distinct microglial states associated with neurodegeneration versus repair.
  • When TDP-43 is switched off, microglia divide, switch gene expression, and clear TDP-43.
  • When these reactive microglia are deleted, the recovery stalls.

“This is a very elegant model,” said Stanley Appel, Houston Methodist Neurological Institute, Texas. Oleg Butovsky, Brigham and Women's Hospital in Boston, said the study was extremely important, given the dearth of information on ALS disease mechanisms, but cautioned it has limitations.

Studies have long implicated microglial activation in amyotrophic lateral sclerosis (ALS) pathology (e.g., Engelhardt and Appel, 1990; Turner et al., 2004; Corcia et al., 2012), as well as in the superoxide dismutase 1 (mSOD1) mouse model of familial ALS (Alexianu et al., 2001; Hall et al., 1998). Blocking microglial proliferation or function slows disease progression and reduces motor neuron loss in mSOD1 mice (Martínez-Muriana et al., 2016; Frakes et al., 2014). However, microglia have also been reported to be neuroprotective in the early stages of disease (Liao et al., 2012; Hooten et al., 2015). 

Microglia to the Rescue. Three days after shutting down hTDP-43 (left column), microglia (red) begin to proliferate (middle row). Within a week, TDP-43 aggregates (green) have begun to clear (middle column). Eight weeks later (right column) they are all but gone, and microglia have quieted down. Motor neurons (blue) are preserved. [Courtesy of Spiller et al., Nature Neuroscience, 2018.]

Hoping to clarify the different roles microglia can play, first author Krista Spiller used a conditional TDP-43 model created in Lee’s lab (Nov 2015 news). The rNLS8 mouse expresses human TDP-43 in neurons but without its nuclear localization signal. The model mimics pathology seen in more than 90 percent of ALS patients and in some patients with frontotemporal degeneration, namely, the accumulation of TDP-43 aggregates in the cytoplasm. A neurofilament heavy chain promoter, which can be blocked with doxycycline, drives expression of the transgene. Lee explained that this mouse model enables her to study neurodegeneration separately from recovery; the former happens while the hTDP-43 gene is turned on, the latter after the transgene is turned off and pathology subsides. “It has been really hard to separate favorable microglia effects from unfavorable ones. Here we can at least separate the damage stage from the healing stage,” said Spiller.

Raised on a doxycycline-laced diet, the rNLS8 mice led healthy lives until they were switched to doxycycline-free chow, usually at three months of age. Consistent with the group’s previous findings, two weeks after the switch hTDP-43 aggregates began showing up in the lumbar spinal cord. Also at this time motor symptoms began to surface: The mice clasped their hind limbs abnormally when suspended by their tails, trembled, developed an arched back, and scored poorly on grip strength, motor coordination, and balance. Surprisingly, Spiller found no change in microglial number, shape, or in the expression of the microglial activation marker CD68 compared with controls that remained on doxycycline. Even six weeks after turning on the transgene, when about 30 percent of lumbar motor neurons had died off, Spiller saw slightly more microglia, but still no change in their shape or CD68 expression. “I was really surprised,” she said. “I thought maybe it was just one or two weird animals.” However, the findings held across five animals each at zero, two, and six weeks after inducing hTDP-43 expression.

To test what happened after the hTDP-43 spigot closed again, the scientists added doxycycline back to the chow when the mice were about 20 weeks old and had been expressing hTDP-43 for six weeks. Within a week, microglia in the spinal cord doubled in number (image above), with many adopting an amoeboid shape typical of reactive microglia. The researchers saw a similar transformation when TDP-43 had been on for only two weeks and motor neuron loss was minimal, suggesting the response is independent of cell death. Also, the fraction of microglia that had taken up hTDP-43, presumably by phagocytosis, shot up from about 10 percent to nearly half after a weeklong hTDP-43 holiday. Interestingly, only rarely did the researchers spot other neuron-specific proteins, such as the neuronal nuclear antigen NeuN and choline acetyl transferase, within microglia, suggesting the cells selectively cleared hTDP-43.

Curiously, the authors detected no TDP-43 in the extracellular space in the spinal cord. “We joked that the microglia might be sucking it out from neurons with a straw,” said Spiller. Three days after hTPD-43 suppression, microglia were seen sidling up to neurons. Appel suggested that aggregated TDP-43 released by neurons would be phagocytized by microglia, and “if the microglia are kissing motor neuron membranes, you may not see much extracellular TDP-43.”

To probe molecular differences between the microglial phenotypes, the authors analyzed transcriptional profiles two and six weeks after turning on hTDP-43, as well as one week after switching it off, a time the researchers call “recovery.” In the late stage of TDP-43 pathology, i.e., after six weeks of hTDP-43 expression, many of the upregulated genes matched those in a microglial neurodegenerative phenotype (MGnD) and in disease-associated microglia (DAMs), as reported previously (Sep 2017 news; Jun 2017 news). Interestingly, these genes remained highly expressed during recovery. A separate group of genes were specific to recovery, including genes coding for cell adhesion molecules and phagosome proteins (image below).

Recovery Signature. Expression of a unique subset of microglial-specific genes ramped up after hTDP-43 was shut off in rNLS8 mice. [Courtesy of Spiller et al., Nature Neuroscience, 2018.]

To test the effect of removing microglia during the recovery stage, the researchers fed mice PLX3397, a compound that blocks the kinase activity of colony-stimulating factor 1 receptor and is known to selectively kill microglia. Spiller started giving PLX3397 a week before turning off hTDP-43 and continued for another two weeks after. Whereas PLX3397-free mice were essentially symptom-free at the end of the intervention, PLX3397-treated mice still clasped their hind limbs and some remained hunched over. Muscle action potentials, motor neuron numbers, muscle denervation, and hTDP-43 levels all correlated with the extent of microglial depletion, with lower numbers of microglia linked to poorer signs of recovery.

Why do microglia swing into action only after hTDP-43 is switched off? “That’s the million-dollar question,” said Spiller. At first she thought hTDP-43 expression was making the microglia sick, but when she challenged them with lipopolysaccharide or injured a nerve, the microglia responded normally. “Maybe they are lacking a signal,” said Spiller, who is now testing candidate regulators in vivo.

Johnathan Cooper-Knock, University of Sheffield in England, was impressed by the model, but cautioned that the microglial phenotype associated with recovery depends entirely on switching off hTDP-43, which “has no parallel in the human disease.”

Butovsky said understanding the rNLS8 model better was an important next step. He and Cooper-Knock were both puzzled by the lack of microgliosis during hTDP-43 expression. “Microglia are the sensors of the brain and usually respond to very small changes. How can they not be responding to this massive neuronal loss? This is completely unexpected,” Butovsky said. He also found it odd that no astrocytosis accompanied the recovery microgliosis, and that these microglia continued to express homeostatic genes. For his part, Appel wondered if other immune cells, such as regulatory T lymphocytes and astrocytes, collaborate with microglia in this model during both the disease and recovery stages (Henkel et al., 2013).—Marina Chicurel

Comments

  1. This paper from Virginia Lee's group features further characterization of mice with doxycycline-suppressible expression of human TDP-43, including a defective nuclear localization signal (Walker et al., 2015). Importantly, the expression of the human TDP-43 is almost entirely limited to neuronal cells by placing it under control of a neurofilament heavy chain promoter. The groundbreaking aspect of this model is that while the mice develop TDP-43 pathology and neuronal toxicity when the human gene is expressed, when expression is turned off the pathology is cleared and motor neuron loss ceases.

    In this paper Spiller et al. describe analysis of the role of microglia in this process. The authors did not observe significant microgliosis during the period in which the mice were losing motor neurons, but in the recovery phase there was an increase in reactive microglia which were observed to clear human TDP-43 through contact with diseased neurons. This effect was not a byproduct of doxycycline, and was necessary for motor recovery—when microglia were suppressed recovery did not occur.

    The immediate inference of this work is that microglia are necessary for recovery of motor neurons in human patients with ALS where TDP-43 pathology is almost universal. The authors suggest that modulation of microglial activity may be an attractive therapeutic target; indeed, certain microglial phenotypes have previously been linked to clinical severity in ALS patients (e.g., Cooper-Knock et al., 2017). Some caution should be reserved, however, because aspects of the model are not obviously physiological: The authors point out that it is unclear why significant microglial activation did not occur in the period when the human gene was being actively expressed. The microglia themselves do not express the human gene, so a direct effect of defective TDP-43 protein is not a possible answer. What is clear is that the change in microglial activity is entirely dependent on the change in expression of the human gene; this event has no parallel in the human disease. Understanding the basis of this observation is crucial to understanding this model and I suspect will offer further insights into human ALS as well.

    References:

    Walker AK, Spiller KJ, Ge G, Zheng A, Xu Y, Zhou M, Tripathy K, Kwong LK, Trojanowski JQ, Lee VM. Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43. Acta Neuropathol. 2015 Nov;130(5):643-60. Epub 2015 Jul 22 PubMed.

    Cooper-Knock J, Green C, Altschuler G, Wei W, Bury JJ, Heath PR, Wyles M, Gelsthorpe C, Highley JR, Lorente-Pons A, Beck T, Doyle K, Otero K, Traynor B, Kirby J, Shaw PJ, Hide W. A data-driven approach links microglia to pathology and prognosis in amyotrophic lateral sclerosis. Acta Neuropathol Commun. 2017 Mar 16;5(1):23. PubMed.

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  2. Disease-associated microglia (DAM) emerged last year as a shared hallmark of brain aging, AD-like pathology in 5xFAD and PS2APP and APPPS1 models, ALS in mSOD1 model, and other neurodegenerative conditions (Keren-Shaul et al., 2017; Krasemann et al., 2017). The transcriptional profile of DAM cells, compared to that of homeostatic microglia, involves changes in expression of genes identified as risk factors in neurodegeneration (Lambert et al., 2013), suggesting that DAM are protective cells that clear apoptotic bodies, myelin debris, and Aβ, and create a barrier around bigger amyloid plaques to limit their toxicity (Krasemann et al., 2017; Poliani et al., 2015; Song et al., 2017; Song et al., 2018; Yuan et al., 2016). 

    Mislocalization and aggregation of TDP-43 is a common feature of FTD and ALS. Using a doxocyclin-controlled human TDP-43–mediated ALS model, Spiller et al. illuminate the role of microglia in TDP-43 pathology. They find that TDP-43 acts as a negative regulator of microglial differentiation into cells displaying markers of DAM (e.g., expression of Apoe, Itgax, Spp1, Lpl, Ctsb and Ctsd). A very important finding was reported here: In the late disease and early recovery phase (after hTDP-43 expression was suppressed), microglia differentiated dramatically to DAM (Supplementary Fig. 10) and cleared neuronal TDP-43 (Fig. 7). Blocking microglia in the early recovery phase using PLX3397 suppressed the ability to recover motor function (Fig.8), demonstrating the protective role of activated microglia in this context.

    Besides this great scientific advancement in our understanding of the role of TDP-43 and microglia in ALS pathology, this paper should set the direction for the future work in the field in several ways:

    1. We should use mouse models that more accurately reflect human conditions. Here the authors used a model of human TDP-43 pathology, which is present in >90 percent of ALS patients. In contrast, the most popular model of ALS, the mSOD1 mice, corresponds to only 3 percent of human ALS cases associated with SOD1 mutation.
    2. We should incorporate more molecular data from human samples to our mouse-based research. Spiller et al. find confirmation of their initial findings from mouse models in human spinal cord specimens of patients with different mutations corresponding to animal models used in the mouse study.
    3. The combination of advanced genetic and molecular tools needs to be incorporated to elucidate the cross-talk and dynamics of the pathology.
    4. We need to better understand the mechanism of how TDP-43 suppresses microglial ability to differentiate into DAM.
    5. We need to test if suppression of DAM activity is a common mechanism in other neurodegeneration conditions, and what are the suppressive factors.

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References

News Citations

  1. ALS Model Mice Roar Back When Human Transgene Silenced
  2. ApoE and Trem2 Flip a Microglial Switch in Neurodegenerative Disease
  3. Hot DAM: Specific Microglia Engulf Plaques

Research Models Citations

  1. NEFH-tTA x hTDP-43ΔNLS

Paper Citations

  1. Engelhardt JI, Appel SH. IgG reactivity in the spinal cord and motor cortex in amyotrophic lateral sclerosis. Arch Neurol. 1990 Nov;47(11):1210-6. PubMed.
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Further Reading

No Available Further Reading

Primary Papers

  1. Spiller KJ, Restrepo CR, Khan T, Dominique MA, Fang TC, Canter RG, Roberts CJ, Miller KR, Ransohoff RM, Trojanowski JQ, Lee VM. Microglia-mediated recovery from ALS-relevant motor neuron degeneration in a mouse model of TDP-43 proteinopathy. Nat Neurosci. 2018 Mar;21(3):329-340. Epub 2018 Feb 20 PubMed.

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