Neuronal Pentraxin 2 Binds Complement Protein, Protects Synapses

In the last five years, neuronal pentraxins have emerged as potential markers of synaptic loss. In the case of NPTX2, researchers may now be able to explain why. In the March 29 Science Translational Medicine, researchers led by Borislav Dejanovic and Morgan Sheng at the Broad Institute of MIT and Harvard, Cambridge, Massachusetts, and Paul Worley of Johns Hopkins University, Baltimore, reported that this synaptic protein binds to complement C1q, a potent instigator of synaptic pruning. In NPTX2 knockout wild-type mice, phagocytic microglia gorged on synapses. Boosting NPTX2 in a mouse model of tauopathy had the opposite effect, preserving synaptic density. The findings highlight a potential means of modulating synapse loss, and they bolster the case that NPTX2 is a marker of synapse health.

Worley previously found that NPTX2 quells hyperexcited neurons by strengthening their connections to inhibitory neurons (Jul 2019 conference news). He and others have also seen NPTX2 fall in the cerebrospinal fluid as Alzheimer’s disease worsens, correlating with synapse loss and waning scores on cognitive tests (Aug 2019 conference news; Aug 2019 conference news). NPTX2 may also be an early marker of neurodegeneration in frontotemporal dementia, dropping in the CSF before symptoms manifest (Mar 2021 conference news).

First author Jiechao Zhou from Johns Hopkins also found that levels of NPTX2 were low in FTD CSF. Twenty-one people who have mutations in GRN or C9ORF72 had less NPTX2 in their CSF than did 40 asymptomatic carriers. All were participants in the Genetic Frontotemporal Dementia Initiative (GENFI) cohort.

In the periphery, pentraxins bind C1q. Pentraxin-3, a long pentraxin distinct from short ones made in the liver, contains a C-reactive protein domain and it can bind C1q to activate the complement cascade. C1q also binds synapses, recruiting microglia to prune them. Indeed, overactive complement signaling has been linked to neurodegeneration in mouse models of amyloidosis and tauopathy (Mar 2015 conference news; Apr 2016 news; Jul 2018 conference news).

Does C1q bind neuronal pentraxins also? These long pentraxins share homology with PTX3. Indeed, Zhou, and another group of researchers, found that purified NPTX2 binds C1q, but not other complement proteins, with nanomolar affinity (Kovács et al., 2020). Now, Zhou reports finding NPTX2-C1q complexes in the FTD CSF samples. Intriguingly, compared to asymptomatic participants symptomatic people had fewer complexes, despite C1q levels being higher than normal.

To examine the relationship between NPTX2 and C1q in vivo, the scientists knocked out NPTX2 in mice. While 2- to 3-month-old knockouts had the same amount of C1q in their cortices as wild-types, the knockouts had more complement C4b, a protein activated downstream of C1q in the complement cascade. NPTX2 knockouts also had as many microglia as wild-types, but more were phagocytic. These cells contained markers of excitatory pre- and post-synapses, Vglut1 and Homer1, respectively, indicating they had pruned these connections. Indeed, NPTX2 knockouts had 15 percent fewer excitatory synapses, despite a normal number of neurons, suggesting that synaptic trimming did not kill the cell3s.

This pruning depended on C1q. When the scientists injected C1q-blocking antibodies into the cortices of NPTX2 knockout mice, the animals mustered fewer phagocytic microglia a week later and had more synapses. Likewise, when the researchers knocked out C1q in the NPTX2 knockouts, the double knockouts retained as many synapses as did wild-type mice (see image below).

No C1q, No Problem. In wild-type mice, microglia (green) prune unwanted synapses, engulfing proteins Homer1 (blue) and Vglut1 (purple). In NPTX2 knockout mice, enlarged microglia gorge on these excitatory synapse markers. Knocking out C1q as well restores normal pruning. [Courtesy of Zhou et al., Science Translational Medicine, 2023.]

Might NPTX2 control synaptic loss in neurodegenerative diseases? Mice carrying the FTD-related P301S tau mutation amass high levels of C1q in their cortices and knocking it out normalizes synaptic density (Dejanovic et al., 2022; Dejanovic et al., 2018).

To see if overexpressing NPTX2 achieves the same results, the researchers injected viruses containing the NPTX2 gene into the hippocampi of 9-month-old P301S mice, then measured complement proteins and microglia activity three weeks later. While levels of C1q stayed steady, the amount of C4b was halved, indicating weakened complement activation. The treated mice overexpressing NPTX2 also had more synapses than their untreated counterparts and fewer phagocytic microglia, which engulfed less Vglut1 and Homer1 than control microglia.

The authors think that boosting NPTX2 expression might be a viable way to prevent synapse loss and neurodegeneration. Scientists are testing a C1q antibody, Annexon’s ANX005, in Phase 2 trials for Huntington’s disease and amyotrophic lateral sclerosis (Jun 2022 news).—Chelsea Weidman Burke

Comments

  1. This is such an interesting finding. NPTX2 concentration appears over and over as clearly reduced in CSF from patients with neurodegenerative diseases but no one has known why. Perhaps this reduction reflects increased complement- and NPTX2-dependent synaptic removal by microglia across neurodegenerative diseases. This would make CSF NPTX-2 a very important biomarker in clinical trials of disease-modifying treatments in the neurodegeneration field as a whole.

    View all comments by Henrik Zetterberg

  2. This study provides an important advance to the field by highlighting a novel complement regulatory function of neuronal pentraxin 2 within the mouse CNS, and by demonstrating its relevance to pathological contexts. In addition, given the broad spectrum of neurodevelopmental and neurodegenerative diseases in which changes in the CSF levels of NPTX2 have been shown to correlate with measures of disease progression, this work provides new mechanistic insight toward understanding NPTX2 as a fluid biomarker.

    Zhou et al. show that NPTX2 can bind C1q and inhibit the classical complement cascade, using a series of in vitro binding and functional assays. In so doing, they recapitulate and expand on the findings of Kovács et al., 2021. Importantly, they demonstrate that, in the absence of NPTX2, adult mice show increased complement pathway activity, as well as elevated microglial engulfment of synaptic proteins and a reduction in the density of different synaptic populations, suggesting that NPTX2 could be eliciting similar complement inhibitory actions in the mouse CNS. Zhou et al. strengthen these findings by demonstrating that some of these phenotypes are mitigated when C1q is genetically ablated or after acute treatment with a C1Q function blocking antibody.

    They also find that that overexpressing NPTX2 in neuronal populations is sufficient to reduce microglial-mediated synapse loss and elicit protection from neuronal death in contexts where expression of complement proteins and activation of the complement pathway are elevated, thus providing evidence that manipulating levels of NPTX2 in pathological/neurodegenerative contexts is sufficient to prevent some of the microglial phenotypes and synapse loss observed in these contexts. Finally, they present compelling evidence that interaction between C1Q and NPTX2 can be observed in the human CNS and, importantly, that reductions in the levels of NPTX2-C1q complexes can be observed in the CSF of symptomatic FTD patients, which they then show correlates with predicted elevations of analytes that reflect downstream complement activation.

    The strengths of the authors’ study lie in the breadth of models, tools, and readouts they have used to characterize NPTX2’s role as a complement inhibitor and its potential to regulate complement activity within the mouse CNS in a variety of contexts. The translational relevance of the complement interactions to human disease is also highlighted by their interrogation of CSF samples from FTD patients, and the implication that a combined measure of complement and NPTX2 could refine the use of these as fluid biomarkers.

    The long-term impact and significance of their strategy to overexpress NPTX2 in the tauopathy model will be important to explore. For example, are the impairments in synaptic function, cognitive performance, and other behavioral phenotypes that have been described to exist in this model, ameliorated or returned to wild-type levels of performance? And are these effects specific to NPTX2, or related to its synaptic localization or neuronal activity-dependent secretion? It also remains to be investigated what upstream pathological mechanism might interrupt NPTX2’s complement inhibitory function, given that levels of NPTX2 itself do not appear to be dramatically reduced in the brains of tauopathy model mice. Is it possible that levels of C1q increase beyond the point at which NPTX2 can restrain its activity, or are other factors at play?

    One caveat to consider is that the phenotypes the authors describe for the NPTX2 KO may not all arise through interactions of NPTX2 with C1Q and a subsequent inhibition of the complement cascade. As the authors themselves raise in both the introduction and discussion, NPTX2 has previously been described to play an activity-dependent role in the strengthening and maturation of GluA4 AMPA-R containing excitatory synapses (Sia et al., 2007), as well as synaptogenesis and the generation of postsynaptic specializations (O’Brien et al., 1999; Lee et al., 2017). As the authors acknowledge, some synaptic phenotypes might arise from these other biological roles of NPTX2, independent of its interactions with complement.

    Future studies to interrogate this will be informative as they will help to address the question of whether, in the healthy adult CNS, the complement pathway is constantly primed and ready to mediate synaptic changes and is being restrained from doing so by the inhibitory actions of NPTX2 and other regulators, or if other factors/mechanisms are required to initiate this process. Further studies to explore what role, if any, NPTX2 plays during brain development, or whether the NPTX2 C1Q interaction shows any regional or temporal specification in the adult, will be informative.

    Given the broad spectrum of neurodevelopmental and neurodegenerative diseases in which changes in CSF levels of NPTX2 have been shown to correlate with measures of disease progression, the translational relevance of this study is particularly high, and the mechanistic insight it provides is valuable (Libiger et al., 2021; Belbin et al., 2020; van der Ende et al., 2020; Xiao et al., 2021). This is particularly true in light of other work that has highlighted changes in CSF complement protein levels in a similarly broad range of disease indications (van der Ende et al., 2022; Zelek et al., 2020; Gracias et al., 2022). It will be interesting to see whether measuring NPTX2-C1Q interactions in human CSF with the PLA assay could offer improvements over measures of C1Q and NPTX2 alone, as either predictive or prognostic biomarkers.

    References:

    Kovács RÁ, Vadászi H, Bulyáki É, Török G, Tóth V, Mátyás D, Kun J, Hunyadi-Gulyás É, Fedor FZ, Csincsi Á, Medzihradszky K, Homolya L, Juhász G, Kékesi KA, Józsi M, Györffy BA, Kardos J. Identification of Neuronal Pentraxins as Synaptic Binding Partners of C1q and the Involvement of NP1 in Synaptic Pruning in Adult Mice. Front Immunol. 2020;11:599771. Epub 2021 Feb 8 PubMed.

    Sia GM, Béïque JC, Rumbaugh G, Cho R, Worley PF, Huganir RL. Interaction of the N-terminal domain of the AMPA receptor GluR4 subunit with the neuronal pentraxin NP1 mediates GluR4 synaptic recruitment. Neuron. 2007 Jul 5;55(1):87-102. PubMed.

    O'Brien RJ, Xu D, Petralia RS, Steward O, Huganir RL, Worley P. Synaptic clustering of AMPA receptors by the extracellular immediate-early gene product Narp. Neuron. 1999 Jun;23(2):309-23. PubMed.

    Lee SJ, Wei M, Zhang C, Maxeiner S, Pak C, Calado Botelho S, Trotter J, Sterky FH, Südhof TC. Presynaptic Neuronal Pentraxin Receptor Organizes Excitatory and Inhibitory Synapses. J Neurosci. 2017 Feb 1;37(5):1062-1080. Epub 2016 Dec 16 PubMed.

    Libiger O, Shaw LM, Watson MH, Nairn AC, Umaña KL, Biarnes MC, Canet-Avilés RM, Jack CR Jr, Breton YA, Cortes L, Chelsky D, Spellman DS, Baker SA, Raghavan N, Potter WZ, Alzheimer's Disease Neuroimaging Initiative (ADNI), Foundation for the National Institutes of Health (FNIH) Biomarkers Consortium, Longitudinal CSF Proteomics Project Team. Longitudinal CSF proteomics identifies NPTX2 as a prognostic biomarker of Alzheimer's disease. Alzheimers Dement. 2021 May 13; PubMed.

    Belbin O, Xiao MF, Xu D, Carmona-Iragui M, Pegueroles J, Benejam B, Videla L, Fernández S, Barroeta I, Nuñez-Llaves R, Montal V, Vilaplana E, Altuna M, Clarimón J, Alcolea D, Blesa R, Lleó A, Worley PF, Fortea J. Cerebrospinal fluid profile of NPTX2 supports role of Alzheimer's disease-related inhibitory circuit dysfunction in adults with Down syndrome. Mol Neurodegener. 2020 Aug 17;15(1):46. PubMed.

    van der Ende EL, Xiao M, Xu D, Poos JM, Panman JL, Jiskoot LC, Meeter LH, Dopper EG, Papma JM, Heller C, Convery R, Moore K, Bocchetta M, Neason M, Peakman G, Cash DM, Teunissen CE, Graff C, Synofzik M, Moreno F, Finger E, Sánchez-Valle R, Vandenberghe R, Laforce R Jr, Masellis M, Tartaglia MC, Rowe JB, Butler CR, Ducharme S, Gerhard A, Danek A, Levin J, Pijnenburg YA, Otto M, Borroni B, Tagliavini F, de Mendonca A, Santana I, Galimberti D, Seelaar H, Rohrer JD, Worley PF, van Swieten JC, Genetic Frontotemporal Dementia Initiative (GENFI). Neuronal pentraxin 2: a synapse-derived CSF biomarker in genetic frontotemporal dementia. J Neurol Neurosurg Psychiatry. 2020 Jun;91(6):612-621. Epub 2020 Apr 9 PubMed.

    Xiao MF, Roh SE, Zhou J, Chien CC, Lucey BP, Craig MT, Hayes LN, Coughlin JM, Leweke FM, Jia M, Xu D, Zhou W, Conover Talbot C Jr, Arnold DB, Staley M, Jiang C, Reti IM, Sawa A, Pelkey KA, McBain CJ, Savonenko A, Worley PF. A biomarker-authenticated model of schizophrenia implicating NPTX2 loss of function. Sci Adv. 2021 Nov 26;7(48):eabf6935. Epub 2021 Nov 24 PubMed.

    van der Ende EL, Heller C, Sogorb-Esteve A, Swift IJ, McFall D, Peakman G, Bouzigues A, Poos JM, Jiskoot LC, Panman JL, Papma JM, Meeter LH, Dopper EG, Bocchetta M, Todd E, Cash D, Graff C, Synofzik M, Moreno F, Finger E, Sanchez-Valle R, Vandenberghe R, Laforce R Jr, Masellis M, Tartaglia MC, Rowe JB, Butler C, Ducharme S, Gerhard A, Danek A, Levin J, Pijnenburg YA, Otto M, Borroni B, Tagliavini F, de Mendonça A, Santana I, Galimberti D, Sorbi S, Zetterberg H, Huang E, van Swieten JC, Rohrer JD, Seelaar H, Genetic Frontotemporal Dementia Initiative (GENFI). Elevated CSF and plasma complement proteins in genetic frontotemporal dementia: results from the GENFI study. J Neuroinflammation. 2022 Sep 5;19(1):217. PubMed.

    Zelek WM, Fathalla D, Morgan A, Touchard S, Loveless S, Tallantyre E, Robertson NP, Morgan BP. Cerebrospinal fluid complement system biomarkers in demyelinating disease. Mult Scler. 2020 Dec;26(14):1929-1937. Epub 2019 Nov 8 PubMed.

    Gracias J, Orhan F, Hörbeck E, Holmén-Larsson J, Khanlarkani N, Malwade S, Goparaju SK, Schwieler L, Demirel İŞ, Fu T, Fatourus-Bergman H, Pelanis A, Goold CP, Goulding A, Annerbrink K, Isgren A, Sparding T, Schalling M, Yañez VA, Göpfert JC, Nilsson J, Brinkmalm A, Blennow K, Zetterberg H, Engberg G, Piehl F, Sheridan SD, Perlis RH, Cervenka S, Erhardt S, Landen M, Sellgren CM. Cerebrospinal fluid concentration of complement component 4A is increased in first episode schizophrenia. Nat Commun. 2022 Nov 3;13(1):6427. PubMed.

    View all comments by Daniel Wilton View all comments by Beth Stevens

Make a Comment

To make a comment you must login or register.

References

News Citations

  1. Do Microglia Finish Off Stressed Neurons Before Their Time?
  2. Synaptic Proteins in CSF: New Markers of Cognitive Decline?
  3. Proteomics Uncovers Potential Markers, Subtypes of Alzheimer’s
  4. FTD Fluid Markers for Degeneration: Check. For Pathology: Not Yet.
  5. Microglia Rely on Mixed Messages to Select Synapses for Destruction
  6. Paper Alert: Microglia Mediate Synaptic Loss in Early Alzheimer’s Disease
  7. Synaptic Tau Clangs the Dinner Bell for Hungry Microglia
  8. C1q Shows Promise as Therapeutic Target to Stop Synapse Loss

Research Models Citations

  1. Tau P301S (Line PS19)

Paper Citations

  1. Kovács RÁ, Vadászi H, Bulyáki É, Török G, Tóth V, Mátyás D, Kun J, Hunyadi-Gulyás É, Fedor FZ, Csincsi Á, Medzihradszky K, Homolya L, Juhász G, Kékesi KA, Józsi M, Györffy BA, Kardos J. Identification of Neuronal Pentraxins as Synaptic Binding Partners of C1q and the Involvement of NP1 in Synaptic Pruning in Adult Mice. Front Immunol. 2020;11:599771. Epub 2021 Feb 8 PubMed.
  2. 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. https://doi.org/10.1038/s43587-022-00281-1 Nature Aging
  3. 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.

External Citations

  1. Huntington’s disease
  2. amyotrophic lateral sclerosis

Further Reading

Primary Papers

  1. Zhou J, Wade SD, Graykowski D, Xiao MF, Zhao B, Giannini LA, Hanson JE, van Swieten JC, Sheng M, Worley PF, Dejanovic B. The neuronal pentraxin Nptx2 regulates complement activity and restrains microglia-mediated synapse loss in neurodegeneration. Sci Transl Med. 2023 Mar 29;15(689):eadf0141. PubMed.

Annotate

To make an annotation you must Login or Register.