Cong Q, Soteros BM, Wollet M, Kim JH, Sia GM.
The endogenous neuronal complement inhibitor SRPX2 protects against complement-mediated synapse elimination during development.
Nat Neurosci. 2020 Jul 13;
PubMed.
This paper describes a novel complement inhibitor, SRPX2, expressed by neurons, that directly binds C1q and suppresses complement activation, C3 deposition on synapses, and subsequent C3-mediated synaptic elimination. They used CRISPR technology to develop an SRPX2 KO mouse model and examined complement, dendritic spines, and synapses in two brain regions: the retinogeniculate pathway in dorsal lateral geniculate nucleus (dLGN) and Layer 4 of the somatosensory cortex (L4 SS) up to age postnatal day P90. Interestingly, they found both spatial- and temporal-dependent effects of SPRX2 deletion. In dLGN, C3 levels were elevated at P4 and P10 but returned to WT levels by P30. In L4 SS, C3 levels were similar to WT at P30 but elevated at P60 and returned to WT levels by P90. C3 levels were unaffected in Layers 2 and 4 of SS. Microglial engulfment was increased and synapse number decreased at peak C3 elevation (i.e., P4 and P10 in dLGN and P60 in L4 SS). Although the C3 levels returned to normal by P90 in L4 SS, the synapse number remained low, suggesting a possible long-term effect of over-pruning during this apparent critical window during brain development. The authors also double-crossed the SPRX2 KO with C3 KO mice and showed that C3 KO dominated, suggesting that SRPX2 is upstream of C3. Synaptic pruning increased in SPRX2 KO mice and reduced in C3 KO and SPRX2-C3 double KOs.
This new data is very consistent with published reports, including our own, showing regional differences in the effects of constitutive complement deletion. We and others have shown that C3 depletion protects against age- and AD-associated synapse loss and cognitive decline in WT and amyloid Tg mouse models. Similar results have been shown in complement-deficient tau models.
What’s exciting is that this novel complement inhibitor, which clearly plays an important role during brain development, may also play a role later in life. It will be very interesting to determine whether SPRX2 levels are reduced with aging and/or neurodegeneration in human and animal model brains, thereby allowing classical complement activation leading to more C3 deposition on synapses and binding to CR3 on microglia to induce synaptic elimination. If so, elevating SPRX2 levels therapeutically may suppress complement-induced microglia-mediated synaptic pruning that occurs in aging (Shi et al., 2015) and/or neurological diseases, including Alzheimer’s, Parkinson’s, and schizophrenia. However, the authors point out that they do not yet know whether SPRX2 inhibits other molecules or pathways. More research is needed, but this work suggests a new potential therapeutic target (or pathway).
Recently, Drs. Wei Cao and Hui Zheng reported that Type 1 Interferon drives C3-mediated microglial synaptic elimination in Alzheimer’s disease (Roy et al., 2020). They demonstrate the presence of nucleic acid (NA), likely derived from neurons, in a subset of amyloid plaques in both human AD brain and amyloid mouse models, that are surrounded by reactive microglia of the neurodegenerative phenotype (MGnD). They hypothesize that the NA+ plaques stimulate an IFN-induced innate immune response that promotes the conversion of microglia to the MGnD phenotype, which then phagocytose local synapses via IFN-C3-mediated synaptic elimination. Interestingly, hippocampal injection of RNA-containing amyloid caused a significant increase in complement gene expression, including C3, and a downregulation of negative regulators of complement (Prelp and Cd55). They convincingly demonstrate that Type 1 IFNβ signaling, like SPRX2, is upstream of C3.
Taken together, it is clear that innate immunity plays a large role in synaptic health during brain development, aging, and neurological disorders. These papers help to identify drivers and repressors of these signaling pathways that ultimately end with complement-mediated synaptic engulfment. Further understanding of signaling, molecules including cell specificity, brain region and timing of effect, will hopefully help to inform drug development for treatment of developmental and neurodegenerative diseases.
References:
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.
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.
This unique paper deserves a broad readership because of the clever strategies the authors used in determining the role of inhibition of the complement system (via SRPX2) in regulating C1q activity, C3 levels and, in turn, preventing synaptic elimination via microglia. This is truly a well-done study. It involves SRPX2, C3, and double-knockout mouse lines coupled with excellent tools/techniques (e.g., electrophysiology, retinogeniculate synapse elimination assay in the dLGN in vivo, etc.) that very well support the authors’ conclusions on the SRPX2-C1q-C3 pathway in selective synapse elimination at specific time points during development and in specific brain regions.
While the determination of C3 levels supports their conclusions, it would be important to determine the levels of both C3a/C3b (in addition to total C3 levels) in SRPX2 KO mice, which would further prove the increased activity of C1q in SRPX2 KO mice.
A note regarding Figure 4: While the evidence presented strongly argues for differences in the number of functional synapses in SRPX2 KO cells, there are a couple of explanations that need further exploration. First, the reduction in AMPAR currents without a change in NMDA current may suggest the presence of more silent synapses, rather than a reduction in the total number of synapses. However, the non-significant finding for NMDARs is potentially due to a small number of neurons that had very large NMDAR currents, while the majority did show diminished amplitudes. A complementary method that could be used would be to assess the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) for both NMDA and AMPA. A reduction in frequency of mEPSCs for both receptor types, without a corresponding change in paired pulse ratio between wild-type and SRPX2 KO neurons, would provide further supporting evidence for the hypothesized reduction in the total number of synapses via increased complement-mediated phagocytosis.
The authors nicely discuss the potential relevance of SRPX2 and other complement inhibitors’ role in neurological/psychiatric conditions. To complement what is observed, it is important to test if SRPX2 overexpression would do the opposite and reduce C3 levels at P4/P10 and prevent elimination of synapse. Also, it is important to determine which complement inhibitor (if any) may play a regulatory role in synapse elimination at P30 and in adult dLGN.
Perhaps extending this study to hippocampal synaptic plasticity, the role of microglial C3Rs and any regulators of microglial complement receptors, would be important to investigate in the context of AD/ADRD.
Comments
Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School
This paper describes a novel complement inhibitor, SRPX2, expressed by neurons, that directly binds C1q and suppresses complement activation, C3 deposition on synapses, and subsequent C3-mediated synaptic elimination. They used CRISPR technology to develop an SRPX2 KO mouse model and examined complement, dendritic spines, and synapses in two brain regions: the retinogeniculate pathway in dorsal lateral geniculate nucleus (dLGN) and Layer 4 of the somatosensory cortex (L4 SS) up to age postnatal day P90. Interestingly, they found both spatial- and temporal-dependent effects of SPRX2 deletion. In dLGN, C3 levels were elevated at P4 and P10 but returned to WT levels by P30. In L4 SS, C3 levels were similar to WT at P30 but elevated at P60 and returned to WT levels by P90. C3 levels were unaffected in Layers 2 and 4 of SS. Microglial engulfment was increased and synapse number decreased at peak C3 elevation (i.e., P4 and P10 in dLGN and P60 in L4 SS). Although the C3 levels returned to normal by P90 in L4 SS, the synapse number remained low, suggesting a possible long-term effect of over-pruning during this apparent critical window during brain development. The authors also double-crossed the SPRX2 KO with C3 KO mice and showed that C3 KO dominated, suggesting that SRPX2 is upstream of C3. Synaptic pruning increased in SPRX2 KO mice and reduced in C3 KO and SPRX2-C3 double KOs.
This new data is very consistent with published reports, including our own, showing regional differences in the effects of constitutive complement deletion. We and others have shown that C3 depletion protects against age- and AD-associated synapse loss and cognitive decline in WT and amyloid Tg mouse models. Similar results have been shown in complement-deficient tau models.
What’s exciting is that this novel complement inhibitor, which clearly plays an important role during brain development, may also play a role later in life. It will be very interesting to determine whether SPRX2 levels are reduced with aging and/or neurodegeneration in human and animal model brains, thereby allowing classical complement activation leading to more C3 deposition on synapses and binding to CR3 on microglia to induce synaptic elimination. If so, elevating SPRX2 levels therapeutically may suppress complement-induced microglia-mediated synaptic pruning that occurs in aging (Shi et al., 2015) and/or neurological diseases, including Alzheimer’s, Parkinson’s, and schizophrenia. However, the authors point out that they do not yet know whether SPRX2 inhibits other molecules or pathways. More research is needed, but this work suggests a new potential therapeutic target (or pathway).
Recently, Drs. Wei Cao and Hui Zheng reported that Type 1 Interferon drives C3-mediated microglial synaptic elimination in Alzheimer’s disease (Roy et al., 2020). They demonstrate the presence of nucleic acid (NA), likely derived from neurons, in a subset of amyloid plaques in both human AD brain and amyloid mouse models, that are surrounded by reactive microglia of the neurodegenerative phenotype (MGnD). They hypothesize that the NA+ plaques stimulate an IFN-induced innate immune response that promotes the conversion of microglia to the MGnD phenotype, which then phagocytose local synapses via IFN-C3-mediated synaptic elimination. Interestingly, hippocampal injection of RNA-containing amyloid caused a significant increase in complement gene expression, including C3, and a downregulation of negative regulators of complement (Prelp and Cd55). They convincingly demonstrate that Type 1 IFNβ signaling, like SPRX2, is upstream of C3.
Taken together, it is clear that innate immunity plays a large role in synaptic health during brain development, aging, and neurological disorders. These papers help to identify drivers and repressors of these signaling pathways that ultimately end with complement-mediated synaptic engulfment. Further understanding of signaling, molecules including cell specificity, brain region and timing of effect, will hopefully help to inform drug development for treatment of developmental and neurodegenerative diseases.
References:
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.
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 Cynthia LemereUniversity of New Mexico
University of New Mexico
This unique paper deserves a broad readership because of the clever strategies the authors used in determining the role of inhibition of the complement system (via SRPX2) in regulating C1q activity, C3 levels and, in turn, preventing synaptic elimination via microglia. This is truly a well-done study. It involves SRPX2, C3, and double-knockout mouse lines coupled with excellent tools/techniques (e.g., electrophysiology, retinogeniculate synapse elimination assay in the dLGN in vivo, etc.) that very well support the authors’ conclusions on the SRPX2-C1q-C3 pathway in selective synapse elimination at specific time points during development and in specific brain regions.
While the determination of C3 levels supports their conclusions, it would be important to determine the levels of both C3a/C3b (in addition to total C3 levels) in SRPX2 KO mice, which would further prove the increased activity of C1q in SRPX2 KO mice.
A note regarding Figure 4: While the evidence presented strongly argues for differences in the number of functional synapses in SRPX2 KO cells, there are a couple of explanations that need further exploration. First, the reduction in AMPAR currents without a change in NMDA current may suggest the presence of more silent synapses, rather than a reduction in the total number of synapses. However, the non-significant finding for NMDARs is potentially due to a small number of neurons that had very large NMDAR currents, while the majority did show diminished amplitudes. A complementary method that could be used would be to assess the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) for both NMDA and AMPA. A reduction in frequency of mEPSCs for both receptor types, without a corresponding change in paired pulse ratio between wild-type and SRPX2 KO neurons, would provide further supporting evidence for the hypothesized reduction in the total number of synapses via increased complement-mediated phagocytosis.
The authors nicely discuss the potential relevance of SRPX2 and other complement inhibitors’ role in neurological/psychiatric conditions. To complement what is observed, it is important to test if SRPX2 overexpression would do the opposite and reduce C3 levels at P4/P10 and prevent elimination of synapse. Also, it is important to determine which complement inhibitor (if any) may play a regulatory role in synapse elimination at P30 and in adult dLGN.
Perhaps extending this study to hippocampal synaptic plasticity, the role of microglial C3Rs and any regulators of microglial complement receptors, would be important to investigate in the context of AD/ADRD.
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