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3D Brain Tissue-Based Model for Probing Contributions of Microglia and Peripheral Macrophages in Driving Neurodegenerative Disease Mechanisms
- 1. 3D Brain Tissue-Based Model for Probing Contributions of Microglia and Peripheral Macrophages in Driving Neurodegenerative Disease Mechanisms Steven Marinero June 14th, 2016
- 2. Red = microglia stained with anti-Iba1 Green = transfected neurons Microglia appear to surround neurons expressing mutant huntingtin-aggregates
- 3. Red = microglia stained with anti-Iba1 Cyan = mutant Htt aggregates Microglia appear to surround neurons expressing mutant huntingtin-aggregates
- 4. Approach: 1. Model of neurodegenerative disorders in organotypic slice 2. Visualize neurodegeneration 3. How do manipulations of microglia and peripheral macrophages affect neurodegeneration?
- 5. Modeling neurodegenerative disorders using a Gene Gun Neurodegenerative disease relevant DNAs coated onto gold particles Biolistic transfection of DNA-coated gold particles into brain slice explants YFP Htt-nQ
- 6. Gradual degeneration and clearance of medium spiny neurons (MSNs) induced by htt exon-1 transfection 0 10 20 30 40 50 60 3 4 5 p < 0.01 p < 10-7 p < 10-16 No.healthyMSNs/striatum Days after transfection YFP control httN90Q73 0 10 20 30 40 50 60 3 4 5 p < 0.01 p < 10-7 p < 10-16 No.healthyMSNs/striatum Days after transfection YFP control httN90Q73 YFP htt htt htt YFP htt htt htt Green = transfected neurons
- 7. Brain-slice HD assay: typical 8-week screening run ~5,000 brain slices total N=12 for each data point AVR = 1.22 DR = 3.29 #healthyMSNs(%overHtt-pQ) 0 100 200 300 400 500 600 700 0 25 50 75 100 125 150 175 200 225 250 positive control level compound-concentration number No.healthyMSNs(%+SD)
- 8. Day 0 Day 1 Day 3Day 2 Microglial proliferation/activation in brain slice explants Red = microglia stained with anti-Iba1
- 9. Inhibition of IKKβ/NFκB is neuroprotective in brain slice assays YFP htt htt htt YFP htt htt htt = significant by ANOVA followed by Dunnett’s post hoc comparison test at 5% level Green = transfected neurons 0 20 40 60 80 100 YFPcontrol htt-N90Q73 BOC-D W-7810.01 0.03 0.1 0.3 1 3 10 No.healthyneurons+SD 0 10 20 30 40 50 60 70 80 90 YFPControl htt-N90Q73 IKK-NBD0.1 1 No.healthyneurons+SD
- 10. Microglia Depletion screen 1:1000 DMSO BLZ945 30uM GW2580 30uM PLX3397 30uM Liposome Encapsulated Clodronate Clodronate Control (liposome without drug) Red = microglia stained with anti-Iba1 After 4 days in culture
- 11. Depletion of Microglia by PLX3397 in organotypic slice Control PLX3397 0.3µM PLX3397 3µM PLX3397 30µM * Images taken after 4 days exposure to PLX3397 in culture
- 12. 0 20 40 60 80 100 120 YFP mN90Q73 PLX3397 0.3µM PLX3397 3µM PLX3397 30µM No. healthy MSNs + SD Rescue of mHtt transfected neurons by PLX3397 = significant by ANOVA followed by Dunnett’s post hoc comparison test at 5% level * Control PLX3397 0.3µM PLX3397 3µM PLX3397 30µM
- 13. Engraftment of cells into organotypic slice Mouse: Cx3Cr1-eGFP Microglia Human: PBMC Green= Cx3Cr1 mouse microglia engrafted onto rat slice Yellow = reporter transfected neurons Green = human PBMC engrafted onto rat slice Red = microglia stained with anti-IBA1
- 14. Conclusions and Future Directions: Conclusions • Transfection of HttN90Q73 leads to progressive neurodegeneration over 3-4 days • This time course coincides with activation of microglia in slice • Anti-inflammatory compounds are neuroprotective in HttN90Q73- transfected brain slices • Microglia can be selectively targeted and preliminary data suggests PLX3397 is neuroprotective • Transplanted cell types from different species are viable rat brain slices Future Directions • Additional pharmacological and genetic perturbations to probe role of microglia in neurodegeneration • Explore roles of infiltrating of peripheral macrophages in MSN degeneration
- 15. Acknowledgments • Donald Lo • Staci Bilbo • Lo lab • Denise Dunn • Bijal Shah • Linda Kaltenbach • Michael Van Kanegan • Andrew Greenhalgh • Kathleen Grabert • Meeting organizers • Funding • Duke Neurobiology training grant • Lerner Fellowship • Keystone Student Scholarship • Duke Neurobiology conference travel award
- 16. HPBMC integration into slice DIV2. Green HPBMC, red IBA1 imaged on confocal 0-15 µm 15-30 µm 30-45 µm 45-60 µm 60-75 µm
- 17. Explantation of Cx3Cr1 mouse microglia into rat Slice Day 0 Day 1 Day 2 Day 5
- 18. Explantation of human PBMCs into rat slice Day 0 Day 1 Day 2
- 19. YFP only YFP + htt-N90Q73 Microglial migrate towards degenerating, mutant huntingtin-transfected neurons Htt aggregate Green = transfected neurons Red = microglia stained with anti-Iba1 White = mutant Htt aggregates
Editor's Notes
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- Interestingly, during this same time microglia are proliferating and becoming activated Immediately after slicing, ramified. However, after one day in culture, amoeboid and increase in numbers. The correlation between microglial activation and MSN degeneration led us to hypothesize that microglia may play an active role in MSNs degeneration.
- To test this hypothesis, we decided start by to targeting the NFKB, bc neuroinflammation To do this, we used two different small molecule inhibitors and found that they both provided significant neuroprotection to mHtt neurons in a concentration-dependent manner. The pan-caspase inhibitor BOC-D-FMK (BOC-D) was used as positive control Promising, but NFKB pathway is not restricted to neuroinflammation and as the elegant work presented at our keynote by Dr. Glass showed, it certainly is not specific to microglia, so we decided to more specifically target microglia How? Eliminate them (the IKKβ inhibitor W-781 (Wyeth Pharmaceuticals) and the IKK complex inhibitor NEMO binding-domain (IKK-NBD))
- 4 days in culture in the presence of 3 different small molecule inhibitors of the CSF1-R: BLz945 GW2580 (Dr. Easley-Neal) and PLX3397 liposome encapsulated clodronate which becomes selectively toxic to microglia after its phagocytosis (encapsulated do not kill not astrocytes but free does kumamaru 2012) On the left are the controls which illustrate the level of activation and microglia density in the absence of drugs Drug info: CSF1R- family of type III growth factor receptors: PDGFR, c-KIT, FLT3 and c-fms (CSF1R). CSF1R activation has pleiotropic effects, ranging from the control of cell survival, proliferation of chemotaxis, based on the differential binding of adapter proteins and phosphorylation pattern of its ICD GW2580: Gomez-Nicola et al showed that prolonged inhibition with GW2580 leads to blockade of microglial proliferation and shifting of microglial inflammatory profile to anti-inflammatory. Dose does not cause significant change in # of microglia, but prolonged exposure caused reduction in number of microglial cells compared to controls BLZ945: Alters macrophage polarization and blocks glioma progression without depleting TAMS (Joyce lab) decreases TAM turnover rate (Daniel lab) Clodronate: Macrophages metabolize clodronate into an active metabolite that then inhibits mitochondrial oxygen consumption PLX3397: Activity over c-KIT, FLT3 and PDGFRB. Green group says preference for CSF1R. In 3 days of oral tx, 50% reduction in microglia
- We decided to start by testing compound that was recently shown by Green lab to be critical for microglia viability, PLX3397 And here we can see the concentration dependent effect that PLX3397 has on microglia density
- Preliminary data suggest PLX3397 provides significant neuroprotection to mHtt-transfected neurons in this same concentration-range Another question that we are very interested in addressing is the role of infiltrating cells in driving neurodegeneration. As an ex vivo model, organotypic slices give us the opportunity to tightly control the presence and or absence of infiltrating cells
- Transplant cell types from different species into our organotypic slices and they remain viable and even appear to differentiate rat brain slice neurons have transfected and appear in yellow. The green cells are microglia that were isolated. Initially round but I was excited to find that 4 days after engraftment, these microglia are not only still living, but have become more ramified as well Another example of organotypic rat brain slice. microglia labeled anti iBA1 and appear in red. The green cells are peripheral blood mononuclear cells isolated from human blood samples and labeled using a fluorescent dye and explanted into the slices. Encouragingly, these cells appear to differentiate after three days in culture The viability exciting -> will allow us to utilize our model coupled with transgenic mice and even human patient cells to test the effects of specific cell types/genes on neurodegeneration.
- In these images we se an example of microglia-neuron interactions in neurons that have been transfected with a reporter gene (left) and neurons that have been transfected with a mutant huntington’s construct. Qualitatively, it appears that microglia have migrated towards neurons and are more activated and closely associated with neurons. And when we look closer, it is tempting to say in this example highlighted by the box, microglia are in the process of phagocytosing a neuron with an Htt aggregate.