Microglia, Inflammation, and FTD

FTD Minicourse April, 2009 Microglia, Inflammation, and FTD Li Gan, Ph.D Gladstone Institute of Neurological Disease University of California, San Francisco

Outline Why study inflammation in neurodegeneration? Why study PGRN in neuroinflammation? Ongoing research on PGRN Discussion

Time Magazine, 2004

Effect of NSAIDs on the Relative Risk of Developing AD Relative Risk of Alzheimer s Disease 1.0 0.8 0.6 0.4 0.2 Normalized Risk 200 400 600 1400 Cumulative Days of NSAIDs Use Adapted from In t Veld et al. (2001), New England Journal of Medicine

Microglia Are Resident Immune Cells in the Brain Functionally related to periphery macrophages and other cells of monocytic lineage. Most likely derived from infiltrating hematopoietic or mesodermal cells during early development of CNS Participate in immune surveillance of the CNS

Pathogenic Processes in Neurodegenerative Diseases Aggregates Accumulation Degradation Degradation Microglial Activation Alzheimer s Parkinson s FTD Huntington s ALS + Neurodegeneration

Different Protein Aggregates Accumulate Neurodegenerative Diseases Aggregates Accumulation Amyloid Tau -synuclein Huntingtin TDP-43 Degradation Degradation + Microglial activation Plaques NFT Neurodegeneration

Chronic Inflammation in AD Activated microglia Amyloid Plaques

Activated Microglia Cluster Around Neuritic Plaques in AD Mouse Models Microglia Anti-A Merge

Different Protein Aggregates Accumulate Neurodegenerative Diseases Aggregates Accumulation Amyloid Tau -synuclein Huntingtin TDP-43 Degradation Degradation + Chronic Inflammation Plaques NFT Neurodegeneration

Microglia: Friend or Foe? Microglia as Foe Microglia as Friend Toxic factors Neuronal Damage Aggregates clearance Protection

Outline Why study inflammation in neurodegeneration? Why study PGRN in neuroinflammation in FTD? Ongoing research Discussions

Pathogenic Processes in Neurodegenerative Diseases Aggregates Accumulation TDP-43 Tau Degradation Degradation Chronic Inflammation Alzheimer s Parkinson s FTD Huntington s ALS Neurodegeneration +

Microgliosis and Astrogliosis in FTLD-U with PGRN Mutations TDP-43 Anti-Iba-1 Anti-GFAP

Progranulin Mutations Are Associated With Tau- Negative, Ubiquitin-Positive FTD Baker et al. Nature (2006) Crutis et al. Nature (2006)

What Is Progranulin (PGRN)? Composed of 7.5 tandem repeats of 6-Kda granulin peptide (Granulin A and Granulin B, a.s.k. Epithelin1/2) Involved in wound repair, tumorigenesis, and inflammatory responses

Null Mutations in PGRN Cause Tau-negative FTD

Mutations Are Associated with Reduction in the Levels of PRGN

Expression of Progranulin in Activated Microglia in FTD brains with PRGN Mutations FTD with PRGN Mutation AD

What is the role of PGRN in microglia? Pathogenic? Protective? Bystander?

Outline Why study inflammation in neurodegeneration? Why study PGRN in neuroinflammation in FTD? Ongoing research Discussions

Hypothesis FTD (null mutation) PGRN Microglia activation Neurodegeneration

A Cellular Model of Microglial Toxicity Primary Mimic neuron-glial interaction Cell type-specific genetic manipulation

The Cellular Model: Cortical Neuron-Glia Mixed Cultures MAP2 (neurons) GFAP (astrocytes) Neurons: ~20 30% Astroglia: ~40 50% Microglia: ~10 15% CD11b (microglia) GFAP (astrocytes)

A Toxicity in Cortical Neuronal-Glial Cultures MAP2 (neurons) GFAP (astrocytes) A 1-42 MAP2 (neurons) GFAP (astrocytes)

Microglia Mediate the Toxic Effects of A in Mixed Cortical Cultures NT Chen et al., JBC, 2005 Selective elimination of microglia

A Activates NF- B in Microglia and Astroglia CMV R U5 HIV-1 flap TATA degfp WRE U3 R U5 NT 5X B Enhancer Element NT EGFP expression: NF- B Activation MAP2 A NF- B A Microglia (cd11b) Astroglia (GFAP) MAP2 NF- B Chen et al., JBC, 2005

Inhibition of NF- B Signaling by Overexpressing I B -SR Lenti-I B Super Repressor (I B SR) Adapted from Chen and Greene, J Mol Med., 2003

Targeted Inhibition of NF- B Signaling in Microglia Protects Against A Toxicity A 1-42 A 1-42 Lenti-MCSF-I B -SR MCSF I B -SR Chen et al., JBC, 2005 Adapted from Chen and Greene, J Mol Med., 2003

What is the role of PGRN in microglia? Pathogenic? Protective? Bystander?

Progranulin Is Exclusively Localized in Microglia in Mixed Cortical Cultures PGRN MAP-2 Merge PGRN IBA-1 Merge PGRN GFAP Merge

1. Is PGRN overexpression in microglia protective?

MCSF Promoter Targets Expression in Microglia in Cortical Cultures CMV R U5 HIV-1 flap MCSF EGFP WRE U3 R U5 MCSF (Macrophage Colony Stimulating Factor promoter)

Lenti-MCSF-PRGN Induces Progranulin Expression in Mixed Cortical Cultures CMV R U5 HIV-1 flap MCSF PGRN WRE U3 R U5 Non-Treated MCSF Ctrl Virus MCSF PRGN Virus Ctrl Bf A Ctrl Bf Ctrl Bf A 75kDa PGRN Conditioned Medium

Microglial-derived PRGN Protects Neurons Against Oligomeric A -Induced Toxicity Ctrl A 10 P <0.0005 P <0.0005 Lenti-CTRL Lenti-PRGN MAP2-positive Neurons Survived 7PA2 CM 9 8 7 6 5 4 3 2 1 0 Lenti-MCSF CTRL PGRN CTRL PRGN A + +

Hypothesis FTD (null mutation) PGRN Microglia activation Neurodegeneration

2. Does microglial PGRN suppress NF- B activation?

NF- B-dependent Expression of degfp in Microglial BV2 Cells CMV R U5 HIV-1 flap TATA degfp WRE U3 R U5 5X B Enhancer Element Green:NF- B Activation + LPS 5x B-BV2 Cells Chen et al., JBC, 2005

Transfecting PRGN in BV2 Cells: Electroporation With Amaxa Nucleofactor 1. BV2 microglia in serumcontaining medium 2.Electroporation to transfect mock and PGRN DNA vectors at 1, 2, 4 μg 3. Changed to serum-free medium

Activation of NF- B by Electroporation Is Associated with Downregulation of PRGN Non-transfected Mock (1.0 g) Mock (2.0 g) Mock (4.0 g) Non-transfected Mock Transfection 3 hr 1 day 2 day 3 hr 1 day 2 day PGRN (2.0 g) PGRN (4.0 g) Conditioned Medium

Progranulin Overexpression Inhibits NF- B Signaling Non-transfected Mock (1.0 g) Mock (2.0 g) Mock (4.0 g) PGRN (1.0 g) PGRN (2.0 g) PGRN (4.0 g)

Electroporation-induced PRGN Overexpression Non-transfected Mock Transfection PGRN Transfection 3 hr 1 day 2 day 3 hr 1 day 2 day 3 hr 1 day 2 day PGRN Conditioned Medium

Progranulin Overexpression Inhibits NF- B Signaling in Microglia 1.2 P < 0.0005 Normalized EGFP Expression 1 0.8 0.6 0.4 0.2 0 Mock PGRN N=11

Confirmation of Progranulin Overexpression in LPS-stimulated BV2 Cells Mock PGRN (1.0 g) (2.0 g) (4.0 g) (1.0 g) (2.0 g) (4.0 g) LPS - + - + - + - + - + - + PGRN Conditioned Medium

Recombinant Progranulin Induces Only a Modest Inhibition of LPS-induced NF- B Signaling Ctrl Bf PGRN (20 μg/ml) Non-Treated 10 ng/ml LPS 50 ng/ml LPS

Recombinant Progranulin Induces a Modest Inhibition of LPS-induced NF- B Signaling 12 EGFP Level (Fluorescent Units) 10 8 6 4 2 0 LPS (ng/ml) 0 10 50 * Ctrl Bf PGRN (20 g/ml) (N=3)

3. How is microglial PGRN regulated? Expression (transcription/translation)? Secretion/trafficking? Modifications/interactors?

Mutations Are Associated with Reduction in the Levels of PRGN

Expression of Progranulin in Activated Microglia in FTD brains with PRGN Mutations FTD with PRGN Mutation AD

Electroporation-induced PRGN Overexpression Non-transfected Mock Transfection PGRN Transfection 3 hr 1 day 2 day 3 hr 1 day 2 day 3 hr 1 day 2 day PGRN Conditioned Medium

Viral Infections Reduced Levels of Secreted PGRN CMV R U5 HIV-1 flap MCSF PGRN WRE U3 R U5 Non-Treated MCSF Ctrl Virus MCSF PRGN Virus Ctrl Bf A Ctrl Bf Ctrl Bf A 75kDa PGRN Conditioned Medium

Low Calcium Elevates Secreted PRGN Levels in Microglia [Ca 2+ ] In medium Non-transfected 1.8mM 0.4mM 0mM PGRN Tubulin

Ionomycin Downregulates Levels of Secreted PRGN (Mature Form) in Microglia Non-treated 1 μm Ionomycin 3 μm Ionomycin PGRN Tubulin

Low Calcium Elevates PRGN Levels in Electroporation Transfected PGRN Mock 1.8mM 0.4mM 1.8mM 0.4mM 16 P < 0.01 EGFP Levels (NF- B Activation) 14 12 10 8 6 4 2 Mock PGRN P <0.01 Mock PGRN 0 (N=4) 1.8 mm Ca 2+ 0.4 mm Ca 2+

Hypothesis FTD (null mutation) Environmental Stress PGRN [Ca 2+ ] i Microglia activation Neurodegeneration

Points for Discussion Conceptual Technical Translational

Points for Discussion FTD (null mutation) 3 Stress 1 PGRN Microglial NF- B activation 2 [Ca 2+ ] i Neurodegeneration

Conceptual/Techincal How does calcium homeostasis regulate PRGN? Transcriptional? Post-translational? Trafficking? How is calcium homeostasis regulated by stress? What are the signaling pathways downstream/upstream of the intracellular calcium? Source of calcium matters? Intracellular calcium store? Extracellular calcium influx?

Decrease of Progranulin by Ionomycin is not transcriptionally regulated DMSO 1 μm 3 μm Secreted PGRN Intracellular PGRN 1.5 [121-163 aa] [312-347 aa] 1.25 PGRN / GAPDH 1.0 0.5 PGRN / GAPDH 1.00 0.75 0.50 0.25 0.0 DMSO 1uM 3uM 0.00 Ionomycin / BV2 Ionomycin / BV2 (N=2)

Translational How to promote PGRN levels by modulating calcium homeostasis?