Periodic Reporting for period 4 - REMIND (Targeting pathological synaptic pruning by microglia in neurodegeneration)
Periodo di rendicontazione: 2023-07-01 al 2024-12-31
Synapse loss is the major correlate of cognitive impairment in neurodegenerative diseases. Recent studies suggest that microglia, which mediate activity-dependent synaptic pruning during development, can also be responsible for synapse loss in neurodegeneration. Although the underlying mechanisms are not well understood, evidence indicates that impaired synaptic activity, along with dysfunction in microglia, might be at the origin of the pathology. GWAS studies reveal that the majority of genes associated with neurodegeneration are highly expressed in microglia. However, little is known about the causal mechanisms that link risk variants to microglial function.
Understanding the cellular and molecular mechanisms underlying pathological synapse pruning by microglia will help design selective therapeutic targeting approaches, to prevent cognitive dysfunction at the early stages of neurodegeneration.
This research will have high relevance for the society, as it will contribute to the development of novel therapeutic strategies to target microglial in brain diseases.
We have focused our studies on genetic factors associated with the risk of neurodegeneration, such as INPP5D and TDP-43. Using conditional knockout mouse models, we have been able to study the specific roles of those genes as modulators of microglial function, and their implication in brain diseases. Our findings showed that dysfunctional microglia significantly contribute to the onset and progression of neurodegeneration, an led to the identification of important microglia-dependent underlying mechanisms.
The project REMIND aims to investigate the role of microglia in the refinement of synaptic connections and to understand the contribution of this mechanism to the pathogenesis of neurodegeneration.
Understanding the cellular and molecular mechanisms underlying pathological synapse pruning by microglia will help design selective therapeutic targeting approaches, to prevent cognitive dysfunction at the early stages of neurodegeneration.
This research will have high relevance for the society, as it will contribute to the development of novel therapeutic strategies to target microglial in brain diseases.
We have focused our studies on genetic factors associated with the risk of neurodegeneration, such as INPP5D and TDP-43. Using conditional knockout mouse models, we have been able to study the specific roles of those genes as modulators of microglial function, and their implication in brain diseases. Our findings showed that dysfunctional microglia significantly contribute to the onset and progression of neurodegeneration, an led to the identification of important microglia-dependent underlying mechanisms.
The project REMIND aims to investigate the role of microglia in the refinement of synaptic connections and to understand the contribution of this mechanism to the pathogenesis of neurodegeneration.
The project REMIND investigated the role of microglia in the pathogenesis of neurodegeneration, with a focus on activity-dependent refinement of synapses, and relevance of risk variants associated to neurodegeneration. The main research objectives achieved under this project can be summarized as follows:
• The role of the AD risk gene INPP5D as a modulator of microglia in early brain development:
We showed that SHIP1 is enriched in early stages of healthy brain development. Combining in vivo loss-of-function approaches and proteomics, we discovered that mice lacking microglial SHIP1 displayed increased complement and synapse loss in the early postnatal brain. SHIP1-deficient microglia showed altered signatures and abnormal synaptic pruning, dependent on the complement system. Mice exhibited cognitive defects in adulthood only when microglial SHIP1 was depleted early postnatally, but not at later stages. iPSC-derived microglia lacking SHIP1 also showed increased engulfment of synaptic structures. These findings suggest that SHIP1 is essential for proper microglia-mediated synapse remodeling in the healthy developing brain. This work has been recently published in Immunity Cell Press (Matera, Compagnion et al., 2025) and presented at several international conferences.
In addition, we have expanded our studies on SHIP1 investigating potential pharmacological approaches. In collaboration with the group of Prof. Kerr, SUNY University USA, we have developed and characterized a novel small molecule SHIP1 agonist. Our studies in microglia describe a positive effect on intracellular degradation of lipid laden cargos, in association with reduced inflammatory response. These findings have been published (Pedicone, Fernandes, Matera et al., 2022) and further studies are ongoing to test the beneficial effects of this approach in neurodegenerative contexts.
• The role of TDP-43 in modulating microglial function:
TDP-43 proteinopathy is a hallmark of neurodegenerative disorders. Mislocalization of TDP-43 has been observed in neurons and glial cells. However, the role of TDP-43 in microglia and the consequences of its loss-of-function remain unexplored. In this study, we utilized magnetic resonance imaging, confocal, and electron microscopy and uncovered early structural changes and myelin abnormalities in mice lacking microglial TDP-43. Spatial transcriptomics further revealed an enriched interferon-response signature associated with oligodendrocyte dysfunction. Early microglial TDP-43 depletion resulted in motor behavior deficits in adult mice. Mechanistically, knocking out TDP-43 impaired microglial ability to engulf and degrade myelin. It also led to cryptic exon inclusion in the Tyrobp mRNA, resulting in a C-terminally truncated protein causing defective TREM2 signaling. Our findings reveal a novel role for TDP-43 in regulating the TREM2-TYROBP axis in mice, highlighting a previously unrecognized mechanism by which microglial dysfunction contributes to neurodegeneration. This work is under revision and findings have been presented at several international conferences.
• Microglia and synaptic profiling at early stages in the ArcAbeta AD mouse model.
Using lipidomics, confocal live imaging and functional assays, we showed that synaptosomes isolated from juvenile AD mice are more prone to be eliminated by microglia than controls. Increased synapse engulfment was also confirmed in the hippocampi of P15 AD mice compared to controls. This work, currently in preparation (Ginggen et al.), indicates that synaptic and microglia alterations occur already at early developmental stages in a mouse model of AD long before the onset of symptoms. These findings have been presented at multiple international conferences.
• Finally, we have established a novel in vivo model to induce hindlimb paralysis based on chemical denervation, to assess the implication of microglia in potential circuit refinement. We have characterized the model by behavioral tests and histology and found that the synaptic markers VGAT and PSD95 are reduced and the microglial density and morphology altered. In addition, we use microglia depletion approaches to test the effects on functional recovery. These findings are included in a manuscript in preparation.
Finally, we have developed several international collaborations, focused on microglia-mediated synapse refinement in diverse contexts.
• The role of the AD risk gene INPP5D as a modulator of microglia in early brain development:
We showed that SHIP1 is enriched in early stages of healthy brain development. Combining in vivo loss-of-function approaches and proteomics, we discovered that mice lacking microglial SHIP1 displayed increased complement and synapse loss in the early postnatal brain. SHIP1-deficient microglia showed altered signatures and abnormal synaptic pruning, dependent on the complement system. Mice exhibited cognitive defects in adulthood only when microglial SHIP1 was depleted early postnatally, but not at later stages. iPSC-derived microglia lacking SHIP1 also showed increased engulfment of synaptic structures. These findings suggest that SHIP1 is essential for proper microglia-mediated synapse remodeling in the healthy developing brain. This work has been recently published in Immunity Cell Press (Matera, Compagnion et al., 2025) and presented at several international conferences.
In addition, we have expanded our studies on SHIP1 investigating potential pharmacological approaches. In collaboration with the group of Prof. Kerr, SUNY University USA, we have developed and characterized a novel small molecule SHIP1 agonist. Our studies in microglia describe a positive effect on intracellular degradation of lipid laden cargos, in association with reduced inflammatory response. These findings have been published (Pedicone, Fernandes, Matera et al., 2022) and further studies are ongoing to test the beneficial effects of this approach in neurodegenerative contexts.
• The role of TDP-43 in modulating microglial function:
TDP-43 proteinopathy is a hallmark of neurodegenerative disorders. Mislocalization of TDP-43 has been observed in neurons and glial cells. However, the role of TDP-43 in microglia and the consequences of its loss-of-function remain unexplored. In this study, we utilized magnetic resonance imaging, confocal, and electron microscopy and uncovered early structural changes and myelin abnormalities in mice lacking microglial TDP-43. Spatial transcriptomics further revealed an enriched interferon-response signature associated with oligodendrocyte dysfunction. Early microglial TDP-43 depletion resulted in motor behavior deficits in adult mice. Mechanistically, knocking out TDP-43 impaired microglial ability to engulf and degrade myelin. It also led to cryptic exon inclusion in the Tyrobp mRNA, resulting in a C-terminally truncated protein causing defective TREM2 signaling. Our findings reveal a novel role for TDP-43 in regulating the TREM2-TYROBP axis in mice, highlighting a previously unrecognized mechanism by which microglial dysfunction contributes to neurodegeneration. This work is under revision and findings have been presented at several international conferences.
• Microglia and synaptic profiling at early stages in the ArcAbeta AD mouse model.
Using lipidomics, confocal live imaging and functional assays, we showed that synaptosomes isolated from juvenile AD mice are more prone to be eliminated by microglia than controls. Increased synapse engulfment was also confirmed in the hippocampi of P15 AD mice compared to controls. This work, currently in preparation (Ginggen et al.), indicates that synaptic and microglia alterations occur already at early developmental stages in a mouse model of AD long before the onset of symptoms. These findings have been presented at multiple international conferences.
• Finally, we have established a novel in vivo model to induce hindlimb paralysis based on chemical denervation, to assess the implication of microglia in potential circuit refinement. We have characterized the model by behavioral tests and histology and found that the synaptic markers VGAT and PSD95 are reduced and the microglial density and morphology altered. In addition, we use microglia depletion approaches to test the effects on functional recovery. These findings are included in a manuscript in preparation.
Finally, we have developed several international collaborations, focused on microglia-mediated synapse refinement in diverse contexts.
REMIND has led to the identification of novel mechanisms underlying microglia-mediated synapse remodeling, as detailed above.
We believe that several progresses beyond the state of the art have been achieved during the execution of the project, including the advances of knowledge in the role of microglial SHIP1 in refining synapses in a complement-dependent manner; the development and characterization of a novel SHIP1 small molecule agonist, currently under testing in vivo, in animal models of neurodegeneration; the identification of a novel regulation of the TREM2/DAP12 axis dependent on TDP-43 in microglia.
Altogether these discoveries have set the basis for novel drug development projects, aimed at specific microglial targeting in brain diseases.
We believe that several progresses beyond the state of the art have been achieved during the execution of the project, including the advances of knowledge in the role of microglial SHIP1 in refining synapses in a complement-dependent manner; the development and characterization of a novel SHIP1 small molecule agonist, currently under testing in vivo, in animal models of neurodegeneration; the identification of a novel regulation of the TREM2/DAP12 axis dependent on TDP-43 in microglia.
Altogether these discoveries have set the basis for novel drug development projects, aimed at specific microglial targeting in brain diseases.