Lall D, Lorenzini I, Mota TA, Bell S, Mahan TE, Ulrich JD, Davtyan H, Rexach JE, Muhammad AK, Shelest O, Landeros J, Vazquez M, Kim J, Ghaffari L, O'Rourke JG, Geschwind DH, Blurton-Jones M, Holtzman DM, Sattler R, Baloh RH. C9orf72 deficiency promotes microglial-mediated synaptic loss in aging and amyloid accumulation. Neuron. 2021 Jul 21;109(14):2275-2291.e8. Epub 2021 Jun 15 PubMed.

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  1. C9ORF72 repeat expansions associated with neurodegeneration are thought to be detrimental through both accumulation of toxic, gain-of-function protein products and loss of functional C9ORF72 mRNA and protein (LOF). Previous studies from Baloh’s group revealed that C9ORF72 suppresses STING-mediated inflammation in peripheral myeloid cells, which underlies the inflammatory phenotype manifested in C9ORF72-null mice (O'Rourke et al., 2016; McCauley et al., 2020). However, the direct relevance of such LOF phenomenon to CNS neurodegenerative processes was not clear.

    Here, Lall et al. focused on the effects of C9ORF72 deficiency in microglia and uncovered enhanced basal inflammation, in particular type I IFN signaling as well as antigen presentation. Single-cell RNA-Seq analysis pinpointed an elevated transcriptional module associated with interferon response microglia (IRM), coupled with diminished activated response microglia (ARM) module. These findings are original and consistent with the crucial function of STING in promoting interferon response and inflammation (Li et al., 2018), and further highlights that microglial activation and subset functionality is critically shaped by innate immune signaling activation.

    Using a variety of genetic approaches in vitro and in vivo, Lall et al. revealed an intriguing microglia-intrinsic effect of C9ORF72 deficiency. C9ORF72 -/- microglia, with their heightened IFN signaling, influence aspects of brain homeostasis, such as synaptic density and neuronal morphology, culminating in behavioral deficits.

    By analyzing 5XFAD mice crossed to C9ORF72 -/-, the authors found that the deletion severely exacerbated the pathological activities of microglia in the context of AD pathology. These findings corroborate our previous work showing that IFN-activated microglia are intimately involved in synapse removal, both in wild-type animals and in models of Aβ plaque pathology (Roy et al., 2020). 

    It was interesting to see the increased number of C9ORF72 -/- microglia clustered around amyloid plaques; however, it remains to be determined if and how the balance between IRMs and ARMs is skewed in 5XFAD by the lack of C9ORF72, relative to that observed in wild-type brains. Better understanding of the cross-regulation of different microglial activation states will offer more insights on their functional involvements in pathophysiological processes.

    References:

    O'Rourke JG, Bogdanik L, Yáñez A, Lall D, Wolf AJ, Muhammad AK, Ho R, Carmona S, Vit JP, Zarrow J, Kim KJ, Bell S, Harms MB, Miller TM, Dangler CA, Underhill DM, Goodridge HS, Lutz CM, Baloh RH. C9orf72 is required for proper macrophage and microglial function in mice. Science. 2016 Mar 18;351(6279):1324-9. PubMed.

    McCauley ME, O'Rourke JG, Yáñez A, Markman JL, Ho R, Wang X, Chen S, Lall D, Jin M, Muhammad AK, Bell S, Landeros J, Valencia V, Harms M, Arditi M, Jefferies C, Baloh RH. C9orf72 in myeloid cells suppresses STING-induced inflammation. Nature. 2020 Sep;585(7823):96-101. Epub 2020 Aug 19 PubMed.

    Li T, Chen ZJ. The cGAS-cGAMP-STING pathway connects DNA damage to inflammation, senescence, and cancer. J Exp Med. 2018 May 7;215(5):1287-1299. Epub 2018 Apr 5 PubMed.

    Roy ER, Wang B, Wan YW, Chiu G, Cole A, Yin Z, Propson NE, Xu Y, Jankowsky JL, Liu Z, Lee VM, Trojanowski JQ, Ginsberg SD, Butovsky O, Zheng H, Cao W. Type I interferon response drives neuroinflammation and synapse loss in Alzheimer disease. J Clin Invest. 2020 Apr 1;130(4):1912-1930. PubMed.

    View all comments by Wei Cao

  2. This paper from Lall et al. in Neuron is very interesting and consistent with some of our work. C9ORF72 loss of function (due to expanded repeats in human diseases such as ALS/FTD and AD) appears to induce an upregulation of type I Interferon pathways that results in complement deposition and microglia-mediated elimination of complement-tagged synapses. This, in turn, leads to neuronal dysfunction and spatial learning and memory impairment.

    Interestingly, C9ORF72 deficiency in 5XFAD mice enhanced IFN response microglia that phagocytosed more Aβ, as well as synapses, leading to lower plaque load, fewer synapses, and enhanced cognitive deficits. Roy and colleagues had demonstrated that the type I Interferon response is upstream of complement signaling and synapse loss (Roy et al., 2020). They found that IFN blockade rescues synapse loss in 5XFAD mice but had no effect on Aβ plaque burden. In our own work, we found that lifelong complement C3-deficiency protected against age-related hippocampal synapse loss and cognitive decline in both WT and APP/PS1 mice (Shi et al., 2015; Shi et al., 2017). However, C3-deficiency also led to increased plaque load in 16-month-old APP/PS1 mice. Interestingly, the microglia in our mice and the amyloid mice with IFN blockade in the Roy paper seemed less activated. This is consistent with the idea that IFN signaling drives a pro-inflammatory signaling cascade that increases complement deposition and synaptic elimination by microglia.

    Lall et al. suggest that C9ORF72 may act as a negative regulator of type I Interferon. Thus, when there is C9ORF72 loss of function (due to its expanded repeat), the brakes are released on IFN, which then allows upregulation of complement and its downstream effects. Thus, one would expect less microglial phagocytosis of both synapses and Aβ. Conversely (and consistent with Lall’s results), we observed less microglial phagocytosis of synapses and Aβ, which resulted in sparing of cognitive decline, despite more and larger plaques.

    Also, it is very interesting that the loss of C9ORF72 induced anti-amyloid antibodies in the 5XFAD mice. This, too, is likely to involve complement receptor-mediated microglial phagocytosis of amyloid. Lastly, we have now generated global and microglia- or astrocyte-specific C3 inducible conditional knockout mice and are crossing them to APP knock-in mice. It would not be surprising to find cell-autonomous effects of C3 lowering in the brain—time will tell.

    References:

    Roy ER, Wang B, Wan YW, Chiu G, Cole A, Yin Z, Propson NE, Xu Y, Jankowsky JL, Liu Z, Lee VM, Trojanowski JQ, Ginsberg SD, Butovsky O, Zheng H, Cao W. Type I interferon response drives neuroinflammation and synapse loss in Alzheimer disease. J Clin Invest. 2020 Apr 1;130(4):1912-1930. PubMed.

    Shi Q, Colodner KJ, Matousek SB, Merry K, Hong S, Kenison JE, Frost JL, Le KX, Li S, Dodart JC, Caldarone BJ, Stevens B, Lemere CA. Complement C3-Deficient Mice Fail to Display Age-Related Hippocampal Decline. J Neurosci. 2015 Sep 23;35(38):13029-42. PubMed.

    Shi Q, Chowdhury S, Ma R, Le KX, Hong S, Caldarone BJ, Stevens B, Lemere CA. Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP/PS1 mice. Sci Transl Med. 2017 May 31;9(392) PubMed.

    View all comments by Cynthia Lemere

  3. The article by Lall and colleagues reported important findings on C9ORF72 disease mechanism with significant value for other neurodegenerative disorders. It demonstrated that knockout C9ORF72 mice showed abnormal neuronal morphology, enhanced cortical synaptic loss, and memory deficits. This study revealed the connection between altered microglial function, neurodegeneration, and aging, which has several parallels with our human-based studies.

    For instance, we and others demonstrated that increased DNA methylation (DNAm) at the C9ORF72 locus (in response to a repeat-expansion) leads to a downregulation of C9ORF72 expression and correlates with disease onset and duration, indicating that the loss of C9ORF72 could play a role in disease mechanism (Xi et al., 2013; Xi et al., 2015; Gijselinck et al., 2016; McGoldrick et al., 2018). Likely RNA foci and DPRs alone are insufficient to drive neurodegeneration and require additional stressors (e.g., downregulation of C9ORF72). Notably, the highest C9ORF72 levels among CNS tissues was detected in the cerebellum, a region with high abundance of DPRs and RNA foci in C9ORF72 cases but no neurodegeneration (McGoldrick et al., 2018). 

    The important aspect of Lall et al. is showing the key role of C9ORF72 in antigen presentation and inflammation. Specifically, the loss of C9ORF72 in mice was linked with a significant upregulation of Trem2 and Ctss, encoding cathepsin S that is involved in antigen presentation. Recently, we implicated a 16 Kb region at the 1q21.3 locus as a modifier of ALS age of onset in both C9ORF72 carriers and noncarriers. Importantly, the risk allele is linked with the increased expression of CTSS in cerebellum (Zhang et al., 2021). The role of antigen presenting processes in modulating disease age of onset is also supported by another study, in which we reported that age of onset in a C9ORF72 cohort and general FTD cohort was associated with variants within a 125 Kb region at 6p21.32 locus (Zhang et al., 2018). The risk allele was linked with increased frontal cortex expression of HLA-DRB1 (the major histocompatibility complex class II protein HLA-DR). Notably, HLA-DRB1 is implicated in neurodegenerative diseases as a marker of activated microglia, and important in initiating immune responses by presenting peptides derived from exogenous and endogenous proteins.

    The study by Lall et al. also points to the critical connection between neurodegeneration and aging. For instance, it demonstrated that C9ORF72-depleted microglia trigger age-dependent neuronal defects. This brings up another parallel with our human-based studies of ALS and other neurodegenerative diseases. DNA methylation (DNAm) at certain CpG dinucleotides is closely associated with aging (Bergsma and Rogaeva, 2020). The cumulative assessment of DNAm levels at age-related CpGs allow determining DNAm-age reflecting the biological age. DNAm-age acceleration is associated with disease age of onset, duration of survival in C9ORF72-carriers (Zhang et al., 2017), and general cohort of ALS patients (Zhang et al., 2020). 

    Hence, more studies are needed to understand the role of DNAm-age and immune pathways in neurodegenerative diseases.

    References:

    Xi Z, Zinman L, Moreno D, Schymick J, Liang Y, Sato C, Zheng Y, Ghani M, Dib S, Keith J, Robertson J, Rogaeva E. Hypermethylation of the CpG Island Near the G4C2 Repeat in ALS with a C9orf72 Expansion. Am J Hum Genet. 2013 May 22; PubMed.

    Xi Z, Zhang M, Bruni AC, Maletta RG, Colao R, Fratta P, Polke JM, Sweeney MG, Mudanohwo E, Nacmias B, Sorbi S, Tartaglia MC, Rainero I, Rubino E, Pinessi L, Galimberti D, Surace EI, McGoldrick P, McKeever P, Moreno D, Sato C, Liang Y, Keith J, Zinman L, Robertson J, Rogaeva E. The C9orf72 repeat expansion itself is methylated in ALS and FTLD patients. Acta Neuropathol. 2015 May;129(5):715-27. Epub 2015 Feb 26 PubMed.

    Gijselinck I, Van Mossevelde S, van der Zee J, Sieben A, Engelborghs S, De Bleecker J, Ivanoiu A, Deryck O, Edbauer D, Zhang M, Heeman B, Bäumer V, Van den Broeck M, Mattheijssens M, Peeters K, Rogaeva E, De Jonghe P, Cras P, Martin JJ, de Deyn PP, Cruts M, Van Broeckhoven C. The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter. Mol Psychiatry. 2016 Aug;21(8):1112-24. Epub 2015 Oct 20 PubMed.

    McGoldrick P, Zhang M, van Blitterswijk M, Sato C, Moreno D, Xiao S, Zhang AB, McKeever PM, Weichert A, Schneider R, Keith J, Petrucelli L, Rademakers R, Zinman L, Robertson J, Rogaeva E. Unaffected mosaic C9orf72 case: RNA foci, dipeptide proteins, but upregulated C9orf72 expression. Neurology. 2018 Jan 23;90(4):e323-e331. Epub 2017 Dec 27 PubMed.

    Zhang M, Xi Z, Saez-Atienzar S, Chia R, Moreno D, Sato C, Montazer Haghighi M, Traynor BJ, Zinman L, Rogaeva E. Combined epigenetic/genetic study identified an ALS age of onset modifier. Acta Neuropathol Commun. 2021 Apr 23;9(1):75. PubMed.

    Zhang M, Ferrari R, Tartaglia MC, Keith J, Surace EI, Wolf U, Sato C, Grinberg M, Liang Y, Xi Z, Dupont K, McGoldrick P, Weichert A, McKeever PM, Schneider R, McCorkindale MD, Manzoni C, Rademakers R, Graff-Radford NR, Dickson DW, Parisi JE, Boeve BF, Petersen RC, Miller BL, Seeley WW, van Swieten JC, van Rooij J, Pijnenburg Y, van der Zee J, Van Broeckhoven C, Le Ber I, Van Deerlin V, Suh E, Rohrer JD, Mead S, Graff C, Öijerstedt L, Pickering-Brown S, Rollinson S, Rossi G, Tagliavini F, Brooks WS, Dobson-Stone C, Halliday GM, Hodges JR, Piguet O, Binetti G, Benussi L, Ghidoni R, Nacmias B, Sorbi S, Bruni AC, Galimberti D, Scarpini E, Rainero I, Rubino E, Clarimon J, Lleó A, Ruiz A, Hernández I, Pastor P, Diez-Fairen M, Borroni B, Pasquier F, Deramecourt V, Lebouvier T, Perneczky R, Diehl-Schmid J, Grafman J, Huey ED, Mayeux R, Nalls MA, Hernandez D, Singleton A, Momeni P, Zeng Z, Hardy J, Robertson J, Zinman L, Rogaeva E, International FTD-Genomics Consortium (IFGC). A C6orf10/LOC101929163 locus is associated with age of onset in C9orf72 carriers. Brain. 2018 Oct 1;141(10):2895-2907. PubMed.

    Bergsma T, Rogaeva E. DNA Methylation Clocks and Their Predictive Capacity for Aging Phenotypes and Healthspan. Neurosci Insights. 2020;15:2633105520942221. Epub 2020 Jul 21 PubMed.

    Zhang M, Tartaglia MC, Moreno D, Sato C, McKeever P, Weichert A, Keith J, Robertson J, Zinman L, Rogaeva E. DNA methylation age-acceleration is associated with disease duration and age at onset in C9orf72 patients. Acta Neuropathol. 2017 Aug;134(2):271-279. Epub 2017 Apr 24 PubMed.

    Zhang M, McKeever PM, Xi Z, Moreno D, Sato C, Bergsma T, McGoldrick P, Keith J, Robertson J, Zinman L, Rogaeva E. DNA methylation age acceleration is associated with ALS age of onset and survival. Acta Neuropathol. 2020 May;139(5):943-946. Epub 2020 Mar 7 PubMed.

    View all comments by Ekaterina Rogaeva

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  1. Sans C9ORF72, Microglia Devour More Aβ Plaques. Synapses, Too.