Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer's disease model methods
Aim. Evidence-backed execution summary for Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer's disease model methods from Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer's disease model.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Development of a CSF1R inhibitor for microglial elimination
reagent used in the protocol.
- Use
- To explore the roles of microglia in the initial development of plaque pathology, we required a method that allowed for the extended and specific elimination of microglia throughout the plaque forming period, using non-invasive/non-stressful approaches. As we previously demonstrated, microglia are critically depende...
Discussion
reagent used in the protocol.
- Use
- In this study, we sought to investigate the role of microglia throughout the onset and development of AD pathology in 5xFAD mice via the sustained depletion of microglia from the adult mouse brain for a period of ~6 months. While various methods of microglial ablation are available, the extent/duration of microglial...
Discussion
reagent used in the protocol.
- Use
- In conclusion, we have designed and created a specific CSF1R inhibitor, PLX5622, that allows for the sustained and specific elimination of microglia. This novel method of microglial depletion provided us with the means to eliminate microglia for the duration of AD pathogenesis. Ultimately, these data demonstrate tha...
RNA Sequencing
Reads were mapped to the reference mouse genome (mm10) using STAR aligner and quantified with the featureCounts function of the Rsubread package in R. After filtering out low-count genes, count distributions were scaled using the calcNormFactors function of the edgeR package. Transgene/human alignments were not fil...
- Use
- Reads were mapped to the reference mouse genome (mm10) using STAR aligner and quantified with the featureCounts function of the Rsubread package in R. After filtering out low-count genes, count distributions were scaled using the calcNormFactors function of the edgeR package. Transgene/human alignments were not fil...
Statistics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Every reported n is the number of biologically independent replicates. No statistical methods were used to predetermine sample sizes; however, our sample sizes are similar to those reported in recently published similar studies,. Behavioral, biochemical, and immunohistological data were analyzed using either two-t...
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Development of a CSF1R inhibitor for microglial elimination
To explore the roles of microglia in the initial development of plaque pathology, we required a method that allowed for the extended and specific elimination of microglia throughout the plaque forming period, using non-invasive/non-stressful approaches. As we previously demonstrated, microglia are critically dependent on CSF1R signaling for their survival, and we set out to develop specific CSF1R inhibitors that were orally bioavailable, brain-penetrant, and able to achieve robust brain-wide microglia elimination for extended periods of time. Using a structure-guided drug design strategy based on the existing CSF1R/KIT/FLT3 inhibitor PLX3397,, combined with in vivo screening for optimal PK, brain penetrance, and microglial depletion, we created PLX5622 (Fig. ), with crystallography revealing the interactions between PLX5622 and the CSF1R (Fig. ). The synthesis schematic...
Discussion
In this study, we sought to investigate the role of microglia throughout the onset and development of AD pathology in 5xFAD mice via the sustained depletion of microglia from the adult mouse brain for a period of ~6 months. While various methods of microglial ablation are available, the extent/duration of microglial depletion and the technical requirements necessary to achieve sustained microglial elimination prohibit the use of most depletion paradigms. For example, the CX3CR1cre ER xDTR ff mouse model relies on administration of diphtheria toxin, which not only induces a cytokine storm, but also limits microglial elimination to 5 days,. The CD11b-HSVTK model requires intracerebroventricular infusion of ganciclovir in order to produce substantial microglial depletion, which in turn induces BBB damage and myelotoxicity, resulting in increased mortality following 4 weeks of ganciclov...
Discussion
In conclusion, we have designed and created a specific CSF1R inhibitor, PLX5622, that allows for the sustained and specific elimination of microglia. This novel method of microglial depletion provided us with the means to eliminate microglia for the duration of AD pathogenesis. Ultimately, these data demonstrate that microglial elimination is associated with the prevention of plaque formation and the downregulation of hippocampal neuronal genes that occur in a preclinical model of AD progression. These results indicate that microglia appear to contribute to multiple facets of AD etiology - microglia appear crucial to the initial appearance and structure of plaques, and following plaque formation, promote a chronic inflammatory state modulating neuronal gene expression changes in response to Aβ/AD pathology.
Measurement outputs
What raw and processed outputs should exist?
To explore the roles of microglia in the initial development of plaque pathology, we required a method that allowed for the extended and specific elimination of microglia throug...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Investigations into oligomeric species of Aβ (via A11 immunoblotting) found no significant changes in protein levels with microglia elimination in 5xFAD mice (Fig. )....
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
In this study, we sought to investigate the role of microglia throughout the onset and development of AD pathology in 5xFAD mice via the sustained depletion of microglia from th...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
In conclusion, we have designed and created a specific CSF1R inhibitor, PLX5622, that allows for the sustained and specific elimination of microglia. This novel method of microg...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
To explore the roles of microglia in the initial development of plaque pathology, we required a method that allowed for the extended and specific elimination of microglia throughout the plaque forming period, using non-invasive/non-stressful approaches.
from paperScoring or quantification
Quantify the primary readouts for this experiment: To explore the roles of microglia in the initial development of plaque pathology, we required a method that allowed for the extended and specific elimination of microglia throug...; Investigations into oligomeric species of Aβ (via A11 immunoblotting) found no significant changes in protein levels with microglia elimination in 5xFAD mice (Fig. )....; In this study, we sought to investigate the role of microglia throughout the onset and development of AD pathology in 5xFAD mice via the sustained depletion of microglia from th...; In conclusion, we have designed and created a specific CSF1R inhibitor, PLX5622, that allows for the sustained and specific elimination of microglia. This novel method of microg....
from paperStatistical comparison
To explore the roles of microglia in the initial development of plaque pathology, we required a method that allowed for the extended and specific elimination of microglia throug...; Investigations into oligomeric species of Aβ (via A11 immunoblotting) found no significant changes in protein levels with microglia elimination in 5xFAD mice (Fig. )....; Every reported n is the number of biologically independent replicates. No statistical methods were used to predetermine sample sizes; however, our sample sizes are similar to th...
from paperReporting output
Report representative outputs alongside summary comparisons for To explore the roles of microglia in the initial development of plaque pathology, we required a method that allowed for the extended and specific elimination of microglia throug..., Investigations into oligomeric species of Aβ (via A11 immunoblotting) found no significant changes in protein levels with microglia elimination in 5xFAD mice (Fig. )...., In this study, we sought to investigate the role of microglia throughout the onset and development of AD pathology in 5xFAD mice via the sustained depletion of microglia from th..., In conclusion, we have designed and created a specific CSF1R inhibitor, PLX5622, that allows for the sustained and specific elimination of microglia. This novel method of microg....
inferred from protocolStructured statistical methods
To explore the roles of microglia in the initial development of plaque pathology, we required a method that allowed for the extended and specific elimination of microglia throug...; Investigations into oligomeric species of Aβ (via A11 immunoblotting) found no significant changes in protein levels with microglia elimination in 5xFAD mice (Fig. )....; Every reported n is the number of biologically independent replicates. No statistical methods were used to predetermine sample sizes; however, our sample sizes are similar to th...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (3)
To explore the roles of microglia in the initial development of plaque pathology, we required a method that allowed for the extended and specific elimination of microglia throughout the plaque forming period, using non-invasive/non-stressful approaches. As we previously demonstrated, microglia are critically dependent on CSF1R signaling for their survival, and we set out to develop specific CSF1R inhibitors that were orally bioavailable, brain-penetrant, and able to achieve robust brain-wide microglia elimination for extended periods of time. Using a structure-guided drug design strategy based on the existing CSF1R/KIT/FLT3 inhibitor PLX3397,, combined with in vivo screening for optimal PK, brain penetrance, and microglial depletion, we created PLX5622 (Fig. ), with crystallography revealing the interactions between PLX5622 and the CSF1R (Fig. ). The synthesis schematic for the synthesis of PLX5622 and the formation of the optimized PLX5622-fumaric acid salt is shown (Fig. ). Cell free kinase inhibitor profiling revealed that PLX5622 is highly specific for the CSF1R, showing > 20-fold selectivity over KIT and FLT3, the two most homologous recepto...
In this study, we sought to investigate the role of microglia throughout the onset and development of AD pathology in 5xFAD mice via the sustained depletion of microglia from the adult mouse brain for a period of ~6 months. While various methods of microglial ablation are available, the extent/duration of microglial depletion and the technical requirements necessary to achieve sustained microglial elimination prohibit the use of most depletion paradigms. For example, the CX3CR1cre ER xDTR ff mouse model relies on administration of diphtheria toxin, which not only induces a cytokine storm, but also limits microglial elimination to 5 days,. The CD11b-HSVTK model requires intracerebroventricular infusion of ganciclovir in order to produce substantial microglial depletion, which in turn induces BBB damage and myelotoxicity, resulting in increased mortality following 4 weeks of ganciclovir treatment. Additionally, clodronate liposomes can be administered to deplete microglia; however, this effect is short-lived and requires intrahippocampal infusion, as clodronate liposomes are incapable of crossing the BBB. Previously, we discovered that microglia are critically dependent upon C...
In conclusion, we have designed and created a specific CSF1R inhibitor, PLX5622, that allows for the sustained and specific elimination of microglia. This novel method of microglial depletion provided us with the means to eliminate microglia for the duration of AD pathogenesis. Ultimately, these data demonstrate that microglial elimination is associated with the prevention of plaque formation and the downregulation of hippocampal neuronal genes that occur in a preclinical model of AD progression. These results indicate that microglia appear to contribute to multiple facets of AD etiology - microglia appear crucial to the initial appearance and structure of plaques, and following plaque formation, promote a chronic inflammatory state modulating neuronal gene expression changes in response to Aβ/AD pathology.
Machine-readable layer
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