Scott-Hewitt N, Mahoney M, Huang Y, Korte N, Yvanka de Soysa T, Wilton DK, Knorr E, Mastro K, Chang A, Zhang A, Melville D, Schenone M, Hartigan C, Stevens B. Microglial-derived C1q integrates into neuronal ribonucleoprotein complexes and impacts protein homeostasis in the aging brain. Cell. 2024 Aug 8;187(16):4193-4212.e24. Epub 2024 Jun 27 PubMed.
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University of California, Irvine
This is an incredibly well-done and compelling study that convincingly demonstrates that a microglial-derived protein is taken up by neurons and modulates their function. We know that C1q has a diverse array of functions, particularly in the extracellular space, and this suggests that it also now has critical intraneuronal functions as well. Given the large upregulation of C1q in disease and aging, it suggests that this novel mechanism may play roles in these processes. I am sure the authors are exploring these possibilities already and look forward to their findings!
View all comments by Kim GreenVigil Neuroscience, Cambridge, Massachusetts
The study unveils the fascinating and unexpected discovery that C1q regulates protein homeostasis in the aging brain independently of its role in the complement pathway.
Our research, along with that of others, including Beth Stevens’ lab, has demonstrated that increased expression of C1q and downstream complement components drives synapse loss and neuronal damage in models of tauopathy and amyloidosis (Dejanovic et al., 2018; Dejanovic et al., 2020).
Interestingly, we previously described that C1q deficiency in mice results in a reduction in brain size. Deletion of C3, a central component of the complement system, on the other hand, had no effect on brain volume (Dejanovic et al., 2020). This observation could potentially be explained if C1q plays a role in regulating protein translation in the brain in a complement-independent manner.
C1q expression is relatively high in a healthy brain and increases significantly in the AD brain. It’s compelling to hypothesize that elevated levels of C1q in AD might lead to complement-dependent neuronal damage, while also contributing to neuronal dysfunction through the aberrant regulation of neuronal protein homeostasis. If this hypothesis is confirmed, it could provide a fresh perspective on the role of C1q in the brain and its implications for neurodegenerative diseases like AD.
References:
Dejanovic B, Huntley MA, De Mazière A, Meilandt WJ, Wu T, Srinivasan K, Jiang Z, Gandham V, Friedman BA, Ngu H, Foreman O, Carano RA, Chih B, Klumperman J, Bakalarski C, Hanson JE, Sheng M. Changes in the Synaptic Proteome in Tauopathy and Rescue of Tau-Induced Synapse Loss by C1q Antibodies. Neuron. 2018 Dec 19;100(6):1322-1336.e7. Epub 2018 Nov 1 PubMed.
Dejanovic B, Wu T, Tsai MC, Graykowski D, Gandham VD, Rose CM, Bakalarski CE, Ngu H, Wang Y, Pandey S, Rezzonico MG, Friedman BA, Edmonds R, De Mazière A, Rakosi-Schmidt R, Singh T, Klumperman J, Foreman O, Chang MC, Xie L, Sheng M, Hanson JE. Complement C1q-dependent excitatory and inhibitory synapse elimination by astrocytes and microglia in Alzheimer's disease mouse models. Nat Aging. 2022 Sep;2(9):837-850. Epub 2022 Sep 20 PubMed.
View all comments by Borislav DejanovicVIB-UAntwerp
VIB-Center for Molecular Neurology
This exciting and thought-provoking work by Scott-Hewitt et al. uncovers a new intraneuronal function for the microglial secreted protein C1q. The authors performed C1q co-immunoprecipitation from synaptosomes isolated from developing, young adult, and adult brain tissue, and identified ribosomal proteins and RNA-binding proteins as C1q interactors by unbiased proteomics. Following these findings, they show that C1q is internalized by neurons by endocytosis, and that it interacts with ribonucleoprotein complexes (RNP) in situ. Purified C1q protein was also confirmed to undergo liquid-liquid phase separation (LLPS) in vitro; this was RNA-dependent, as was the in vivo interaction with RNP. C1q knockout mice were reported to have age-dependent alterations in neuronal protein synthesis in vivo and impaired fear memory extinction.
This is an intriguing story that highlights a noncanonical role for the complement protein C1q in the aged brain. It does not come as a surprise that C1q can exert noncanonical roles that are independent from classical complement activation, as it was found to mediate a wide array of functions across different fields ranging from cancer (Bulla et al., 2016), to atherosclerosis (Maffia et al., 2024) and embryonic development (Agostinis et al., 2017). It is very exciting to see such a parallelism in the brain, where C1q has been mostly described as a microglial secreted protein mediating synapting pruning via classical complement pathway during development and disease. This novel, complement-independent, physiological role of C1q could impact neuronal homeostasis and plasticity in disease contexts or developmental stages in ways that are yet to be understood, but might be critical to explore in the future. For example, in models of Alzheimer’s disease, where complement-dependent functions of C1q are already known to mediate early synapse loss (Hong et al., 2016), the impact of non-classical C1q pathways is unexplored.
As does all thought-provoking work, this article raises important questions, including, what is the exact mechanism by which microglial-secreted C1q is internalized by neurons? Do one or more of the C1q receptors that have been described mediate the endocytosis process? And how does the internalized C1q traffic from endosomes to RNP complexes within neurons?
Finally, there is an important technical aspect worth highlighting. The optimization of tissue preparation for immunostaining, including a fixation protocol and blocking conditions that allowed for the visualization of sensitive RNA-dependent structures, is very elegant and could be potentially of great value for the field.
References:
Bulla R, Tripodo C, Rami D, Ling GS, Agostinis C, Guarnotta C, Zorzet S, Durigutto P, Botto M, Tedesco F. C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation. Nat Commun. 2016 Feb 1;7:10346. PubMed.
Maffia P, Mauro C, Case A, Kemper C. Canonical and non-canonical roles of complement in atherosclerosis. Nat Rev Cardiol. 2024 Apr 10; PubMed.
Agostinis C, Tedesco F, Bulla R. Alternative functions of the complement protein C1q at embryo implantation site. J Reprod Immunol. 2017 Feb;119:74-80. Epub 2016 Sep 17 PubMed.
Hong S, Beja-Glasser VF, Nfonoyim BM, Frouin A, Li S, Ramakrishnan S, Merry KM, Shi Q, Rosenthal A, Barres BA, Lemere CA, Selkoe DJ, Stevens B. Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science. 2016 May 6;352(6286):712-6. Epub 2016 Mar 31 PubMed.
View all comments by Renzo MancusoUniversity of California, Irvine
Overall, this manuscript provides provocative novel observations and hypotheses that expand our knowledge of the potential roles of C1q, a recognition component of the innate immune system and of the classical complement pathway.
C1q has been shown to be involved in activation of the classical complement pathway, in synaptic pruning, and in multiple other roles in the CNS and in the periphery C1q (Benoit et al., 2013; Parker et al., 2022; Wang et al., 2020; Benavente et al., 2020; Peterson et al., 2015; Yu et al., 2023). It increases with age and disease/injury in the brain. However, what C1q binds to, and the functional consequences thereof, are unresolved. This manuscript investigates the association of C1q with RNA-containing complexes inside neurons. Importantly, the authors determined that C1q derived from microglia gained access to neurons via endocytosis, and once inside the cells it associated with RNA/ribonucleoproteins (RNPs).
Mass spectroscopic analysis of material that immunoprecipitated with an anti-C1q antibody found an enrichment of proteins associated with ribosomes and RNA-binding proteins. C1q is highly cationic with exposed patches of positively charged amino acids—e.g., arginine ladders in both the collagen-like domain and globular heads. These have the propensity to bind arrays of negatively charged nucleic acids and phospholipids, such as phosphatidyl serine, which can be exposed on the cell surface by energy depletion (Scott-Hewitt et al., 2020). C1q also binds cardiolipin, a lipid in the inner mitochondrial membrane that is exposed during tissue/cell damage (Peitsch et al., 1988). Obviously, the functional consequences of the RNA/RNP association reported here may demonstrate yet another novel role for C1q, an evolutionarily ancient protein.
Whether the RNA/RNP association has a positive or negative role in cognitive decline in disorders such as Alzheimer’s disease remains to be determined. While the authors report that microglia-specific depletion of C1q in 6- to 8-week-old animals resulted in selective impairment of fear memory extinction (perhaps consistent with lack of forgetting as demonstrated in a different behavioral paradigm (Wang et al., 2020)), it will be relevant to assess behavioral phenotypes of this deletion at older ages and in models of neurodegenerative disorders, where the levels of C1q in the brain, and associated with RNA, would be much higher than in the younger mice.
The authors also report differential changes in brain proteins between 1-year-old wild-type and C1q knockouts, although as they write, the relevance of these changes to the C1q-RNA associations remain to be discerned, since lifelong deficiency of C1q could manifest in multiple ways and not necessarily via the RNA/RNP interactions, though the possibility of brain-specific consequences is indeed quite intriguing. In addition, since a number of C1q-binding proteins/C1q cellular receptors have been identified (Benavente et al., 2020), these can be explored to determine how C1q gains access to the neuron-specific intracellular space—a complement to the unbiased approaches suggested by the authors.
Importantly, this paper has a message for therapeutic targeting of C1q in age-related CNS diseases. It will be critical to discern the balance between beneficial and detrimental processes influenced by C1q (Benoit et al., 2013; Parker et al., 2022; Wang et al., 2020; Benavente et al., 2020; Peterson et al., 2015; Yu et al., 2023) to determine therapeutic efficacy/window of treatment.
Finally, this paper is important for reporting technicalities that impact the investigation of C1q interactions, such as duration of tissue fixation, and the impact of heat-resistant enzymes (RNases here) in solutions containing serum. These details will accelerate investigations into these novel interactions.
References:
Scott-Hewitt N, Mahoney M, Huang Y, Korte N, Yvanka de Soysa T, Wilton DK, Knorr E, Mastro K, Chang A, Zhang A, Melville D, Schenone M, Hartigan C, Stevens B. Microglial-derived C1q integrates into neuronal ribonucleoprotein complexes and impacts protein homeostasis in the aging brain. Cell. 2024 Aug 8;187(16):4193-4212.e24. Epub 2024 Jun 27 PubMed.
Benoit ME, Hernandez MX, Dinh ML, Benavente F, Vasquez O, Tenner AJ. C1q-induced LRP1B and GPR6 Proteins Expressed Early in Alzheimer Disease Mouse Models, Are Essential for the C1q-mediated Protection against Amyloid-β Neurotoxicity. J Biol Chem. 2013 Jan 4;288(1):654-65. PubMed.
Parker SE, Bellingham MC, Woodruff TM. Complement drives circuit modulation in the adult brain. Prog Neurobiol. 2022 Jul;214:102282. Epub 2022 May 6 PubMed.
Wang C, Yue H, Hu Z, Shen Y, Ma J, Li J, Wang XD, Wang L, Sun B, Shi P, Wang L, Gu Y. Microglia mediate forgetting via complement-dependent synaptic elimination. Science. 2020 Feb 7;367(6478):688-694. PubMed.
Benavente F, Piltti KM, Hooshmand MJ, Nava AA, Lakatos A, Feld BG, Creasman D, Gershon PD, Anderson A. Novel C1q receptor-mediated signaling controls neural stem cell behavior and neurorepair. Elife. 2020 Sep 7;9 PubMed.
Peterson SL, Nguyen HX, Mendez OA, Anderson AJ. Complement protein C1q modulates neurite outgrowth in vitro and spinal cord axon regeneration in vivo. J Neurosci. 2015 Mar 11;35(10):4332-49. PubMed.
Yu Q, Zhang N, Guan T, Guo Y, Marzban H, Lindsey B, Kong J. C1q is essential for myelination in the central nervous system (CNS). iScience. 2023 Dec 15;26(12):108518. Epub 2023 Nov 23 PubMed.
Scott-Hewitt N, Perrucci F, Morini R, Erreni M, Mahoney M, Witkowska A, Carey A, Faggiani E, Schuetz LT, Mason S, Tamborini M, Bizzotto M, Passoni L, Filipello F, Jahn R, Stevens B, Matteoli M. Local externalization of phosphatidylserine mediates developmental synaptic pruning by microglia. EMBO J. 2020 Aug 17;39(16):e105380. Epub 2020 Jul 13 PubMed.
Peitsch MC, Tschopp J, Kress A, Isliker H. Antibody-independent activation of the complement system by mitochondria is mediated by cardiolipin. Biochem J. 1988 Jan 15;249(2):495-500. PubMed.
View all comments by Andrea TennerUniversity of Modena and Reggio Emilia
In Alzheimer's disease, microglia are activated when they recognize the Aβ peptide, secreting a variety of inflammatory factors and neurotoxins, leading to neuronal damage/death. One of these factors, the complement C1q, is thought to modulate phagocytosis by microglia that respond to amyloid plaques. In this article, the Stevens lab demonstrates that C1q can be internalized by neurons, where it escapes the endosomal compartment and interacts with neuronal ribonucleoproteins (RNPs), undergoing RNA-mediated phase separation and changing neuronal protein synthesis. This unravels a new function for microglia in Alzheimer's disease: Not only do they initiate an immune reaction, leading to neuronal cell death, but they secrete a factor that can enter neurons and hinder their "normal function."
There are several intriguing similarities between C1q and viral proteins that also escape lysosome degradation and induce stress granules that modify translation. Stress granules, a type of RNP, have been repeatedly associated with protein aggregation and neurodegeneration. Important questions that arise from this study include, does C1q preferentially interact with TDP-43-containing RNPs? Does it contribute to TDP-43 aggregation? Of note, deletion of the C1qa gene reduced TDP-43 proteinopathy and neurodegeneration (Zhang et al., 2020). In addition, although the authors report pan-neuronal C1q staining, studies should address if C1q is preferentially taken up by specific neuronal populations, and to what extent this may contribute to disease spreading and evolution.
References:
Zhang J, Velmeshev D, Hashimoto K, Huang YH, Hofmann JW, Shi X, Chen J, Leidal AM, Dishart JG, Cahill MK, Kelley KW, Liddelow SA, Seeley WW, Miller BL, Walther TC, Farese RV Jr, Taylor JP, Ullian EM, Huang B, Debnath J, Wittmann T, Kriegstein AR, Huang EJ. Neurotoxic microglia promote TDP-43 proteinopathy in progranulin deficiency. Nature. 2020 Dec;588(7838):459-465. Epub 2020 Aug 31 PubMed.
View all comments by Serena CarraStanford University
Stanford University
This exciting paper beautifully complements previous work showing the role of microglial C1q in synaptic pruning during neurodevelopment and neurodegeneration alike (Stevens et al., 2007; Hong et al., 2016; Wilton et al., 2023). This paper elegantly reveals a novel mechanism, in which C1q interacts with neuronal ribonucleoprotein complexes and impacts neuronal protein synthesis and learning and memory over the course of normal aging.
This study also demonstrates that typical aldehyde fixation and serum blocking procedures used in immunostaining drastically blunt RNase-sensitive C1q signal. This discovery underscores the critical need to optimize these methods when investigating RNA-dependent structures, a factor that may have been disregarded in previous research efforts
Of particular interest is the finding that C1q and RNA can form liquid-liquid phase-separated droplets in vitro. These sorts of RNA-containing droplets are abundant in neurons and, at synapses, are thought to be critical for sequestering RNAs until they undergo local translation, a dynamic process that often needs to be suddenly “flipped on” in conjunction with synaptic activity (Bauer et al., 2023).
Phase separation involving RNA in neurons has also gained attention in the context of the numerous RNA-binding proteins implicated in ALS and frontotemporal dementia, such as TDP-43 and FUS (Ito et al., 2017). TDP-43 and FUS are predominantly nuclear proteins, but under pathological conditions, they can be found in RNA-containing droplets in the neuronal cytoplasm. While it’s still unclear how exactly these droplets are deleterious, if at all, it’s plausible that TDP-43 and FUS might be inappropriately sequestering their RNA targets, impairing neuronal function.
C1q could be doing something similar, given that in this study, knocking out C1q increased protein synthesis in neurons. The authors used a mass spectrometry approach to identify actively translated proteins and found that septins and mitochondrial proteins are among the proteins whose translation is most impacted by C1q knockout. Further investigation could identify specific RNAs that interact with C1q in droplets and provide potential targets whose translation could be modulated to boost neuronal health and cognitive performance. It would be interesting to investigate the extent to which the overall impacts of C1q during aging result from alterations in neuronal protein homeostasis, compared to the influences of synaptic pruning or even the induction of reactive astrocytes (Liddelow et al., 2017).
References:
Stevens B, Allen NJ, Vazquez LE, Howell GR, Christopherson KS, Nouri N, Micheva KD, Mehalow AK, Huberman AD, Stafford B, Sher A, Litke AM, Lambris JD, Smith SJ, John SW, Barres BA. The classical complement cascade mediates CNS synapse elimination. Cell. 2007 Dec 14;131(6):1164-78. PubMed.
Hong S, Beja-Glasser VF, Nfonoyim BM, Frouin A, Li S, Ramakrishnan S, Merry KM, Shi Q, Rosenthal A, Barres BA, Lemere CA, Selkoe DJ, Stevens B. Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science. 2016 May 6;352(6286):712-6. Epub 2016 Mar 31 PubMed.
Wilton DK, Mastro K, Heller MD, Gergits FW, Willing CR, Fahey JB, Frouin A, Daggett A, Gu X, Kim YA, Faull RL, Jayadev S, Yednock T, Yang XW, Stevens B. Microglia and complement mediate early corticostriatal synapse loss and cognitive dysfunction in Huntington's disease. Nat Med. 2023 Nov;29(11):2866-2884. Epub 2023 Oct 9 PubMed. Correction.
Bauer KE, de Queiroz BR, Kiebler MA, Besse F. RNA granules in neuronal plasticity and disease. Trends Neurosci. 2023 Jul;46(7):525-538. Epub 2023 May 16 PubMed.
Ito D, Hatano M, Suzuki N. RNA binding proteins and the pathological cascade in ALS/FTD neurodegeneration. Sci Transl Med. 2017 Nov 8;9(415) PubMed.
Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, Bennett ML, Münch AE, Chung WS, Peterson TC, Wilton DK, Frouin A, Napier BA, Panicker N, Kumar M, Buckwalter MS, Rowitch DH, Dawson VL, Dawson TM, Stevens B, Barres BA. Neurotoxic reactive astrocytes are induced by activated microglia. Nature. 2017 Jan 26;541(7638):481-487. Epub 2017 Jan 18 PubMed.
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