Microglia-Neuron Interaction in Chronic Pain Long-Jun Wu

Microglia-neuron interaction in chronic pain

Long-Jun Wu, PhD Professor and Consultant Department of Neurology Department of Neuroscience Mayo Clinic Microglia: principal immune cells in the brain

Miller, Science, 2005

Microglia are highly dynamic

Microglia basal motility, 10 min

Microglia: CX3CR1-GFP

Microglia process chemotaxis, 30 min

ATP

Wu et al., Glia, 2007 Two-photon imaging microglia-neuron interaction in vivo

Neuron: Thy1-YFP Microglia: CX3CR1-GFP http://wulab.cbn.rutgers.edu/ http://neuroimmunelab.mayo.edu/ Overview

• How microglia sense neuronal activity?

• What is the function of microglia in chronic pain?

- 63mV - 62mV Microglial process extension to hyperactiv to extension process Microglial

Glutamate + TTX+ Glutamate Glutamate

30 mV 0.5 s 0.5 e neurons Microglial process extension to hyperactive neurons

Px1

Eyo et al., J Neurosci, 2014

Neuronal firing triggers microglial process convergence

Microglia: GFP Neuron: Alexa 594

Alexa 594 Neuron Microglia sense ATP from hyperactive neurons

Microglia

P2Y12 Hv1/TREM2? Neuron

Neuronal activities induce microglial process Microglia dampen neuronal extension/convergence/phagocytosis activities

Eyo et al., J Neurosci, 2014 Eyo et al., Cell Rep, 2018 Eyo et al., J Neurosci, 2015 Mo et al., J Neurosci, 2019 Dr. Ukpong Eyo Tian et al., J Neurosci, 2017 Eyo and Wu, Prog Neurobiol, 2019 Now at U Virginia A critical question in the microglia field:

Most microglia in vivo imaging was done under anesthesia.

How about microglia dynamics in awake mice? Neuronal activity in awake and anesthetized mice Microglial process dynamics in awake and anesthetized mice Increased microglial process dynamics after anesthesia What is the molecular mechanisms?

P2Y12-/- mice

Norepinephrine controls microglial process dynamics The functional significance?

CX3CR1-GFP:Thy1-YFP Increased contact with neuronal dendrites after anesthesia Neuronal activity inhibits microglial process dynamics via norepinephrine signaling in awake mice

Awake Anesthetized

Dr. Yong U. Liu Now at South China U Liu et al., Nat Neurosci, 2019

Summary

Biphasic neuronal activity increases microglial process dynamics

Anesthesia Seizures NE ATP

β2R P2Y12R

Awake

hypoactive hyperactive Microglial process dynamics

Neuronal activity Microglial Ca2+ imaging in vivo

1 2 3

CX3CR1-CreER:ROSA-CAG-GCaMP6s mice Microglial Ca2+ in response to biphasic neuronal activity

Umpierre et al., eLife, 2020 Biphasic neuronal activity increases microglial Ca2+ activity

Anesthetized Seizures ?? ?? ?? ??

Awake

hypoactive hyperactive Microglial Ca2+ Ca2+ Microglial activity

Neuronal Dr. Anthony activity Umpierre

Umpierre et al, eLife, 2020 Overview

• How microglia sense neuronal activity?

• What is the function of microglia in chronic pain?

Microgliosis and neuropathic pain

a b Day 7 after SNT

Contralateral Ipsilateral L4 spinal nerve transection (SNT) Microglia/monocyte ablation

CX3CR1CreER/+:R26iDTR/+ mice a DT SNT Tamoxifen Immunostaining Behaviors

Day-10 Day-4 -1 0 1 3 5 7 9 11 14 Days after SNT (POD) Microglia/monocyte ablation attenuated neuropathic pain

CX3CR1CreER/+:R26iDTR/+ mice a DT SNT Tamoxifen Immunostaining Behaviors

Day-10 Day-4 -1 0 1 3 5 7 9 11 14 Days after SNT (POD)

e f 2.0 Control Ablation 4.0 Control Ablation Ipsi Ipsi Ipsi Ipsi 1.6 Contra Contra 3.5 Contra Contra

1.2 3.0

0.8 2.5 * ** ** *** *** *** 0.4 2.0 *** ***

*** *** *** *** *** *** (s) latency Withdrawal 0.0 1.5 50% withdrawal 50% threshold (g) 0 1 3 5 7 9 11 14 0 1 3 5 7 9 11 14 POD (days) POD (days) How about only microglia ablation?

a CX3CR1CreER/+:R26iDTR/+ mice DT Tamoxifen SNT Immunostaining Behavioral test

Day-27 Day-21 -1 0 1 3 5 7 9 11 14 POD

b Dorsal horn (POD3) c L4 DRG (POD3) Contralateral Ipsilateral Contralateral Ipsilateral

d 1600 e Control MG ablation Control 1.6 Ipsi Ipsi

2 MG ablation 1200 Contra Contra 1.2 *** 800

cells/mm 0.8 + *** Iba1 400 *** 0.4 *** * 0 0.0 Contra Ipsi Contra Ipsi (g) threshold withdrawal 50% 0 1 2 3 5 7 9 11 14 Dorsal horn L4 DRG POD (days)

Resident microglia are required for chronic pain initiation In vivo imaging of monocytes in the spinal cord No monocyte infiltration using CCR2RFP / CX3CR1GFP mice

a b c Spinal Cord (POD 14) DH VH

CCR2RFP / CX3CR1GFP a’ CCR2RFP b‘ CCR2RFP a’ mice CX3CR1GFP CX3CR1GFP GFP/+ GFP/+ :CX3CR1 b’ RFP/+ CCR2

Merged Merged Microglial proliferation dominates microgliosis

a b + DH (POD 7) Iba1 + + IBA1 / Iba1 / BrdU 700 Iba1 BrdU Brdu

2 600

500

400

300

200 Number of Number cells/mm 100

0 Sham POD3 POD7 c Mitotic microglia / total microglia cells % Microglia / total mitotic cells % POD 3 POD 7 POD 3 POD 7 15.4% 88.7% 89.4% 13.9%

11.3% 10.6% 86.1% 84.6%

IBA-1+/BrdU+BrdU+Iba1+ IBA-1+BrdU-Iba1+ IBA-1+/BrdU+BrdU+Iba1+ BrdU+BrdU+Iba1- Microglial proliferation peaks at day 3 after SNT

a b + + 100 Ki-67 Iba1 + - Ki-67 80 Ki-67 Iba1

POD 3 POD 7 Numberof cells/DH 0

0 2 4 6 8 10 12 14 POD (days) Microglial proliferation is correlated with neuropathic pain

a b Sham SNT SNT+Ara C ## POD 7 500 ### *** 2

400

300

200

100 Number of microglia/mm of Number 0 Iba1 Sham SNT

SNT+Ara C c d Control Ara C Control Ara C Sham

4.0 2 600 2.0 Ipsi Ipsi Ipsi Ipsi SNT Contra Contra Contra Contra 500 SNT+Ara C 1.6 3.5 3.0 400 1.2 R2 = 0.92 300 2.5 *** 0.8 ** ** *** ** *** *** *** *** 200 ** *** 2.0 0.4 100 1.5 0.0 Microglia number/mm 0 50% threshold withdrawal (g)

0 1 2 3 4 5 6 7 Thermal (s)latency withdrawal 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 POD (days) POD (days) Index of AUC Microglia and monocytes synergistically promote pain

Dr. Jiyun Peng Peng et al., Nat Commun, 2016; Gu et al., Cell Rep, 2016 Now at Nanchang U

Question: chronic pain model without monocyte activation?

If so,

(1) What is microglial function?

(2) What is microglial signaling involved?

HFS induces spinal LTP and chronic pain a Sciatic nerve sti, in vivo spinal recordings 500 b Sham 10V HFS Sham 1V HFS 10V HFS 20V HFS 400 1V HFS 20V HFS i 300 Before 200

ii 100 After HFS C-fiber response C-fiber (%) 0i ii -60 0 60 120 180 240 300 360 Time (mins)

c 2.0 d 4.0 Sham 10V HFS Sham 10V HFS 1V HFS 20V HFS 3.5 1V HFS 20V HFS 1.5 3.0 1.0 2.5 PWL(s)

PWT(g) 2.0 .5 1.5 0.0 1.0 -1 1 3 5 7 10 14 21 -1 1 3 5 7 10 14 21 Time (days) Time (days)

In collaboration with Dr. Xianguo Liu (Sen Yat-sen University) HFS increases long-term microglial activation

a 10V HFS Iba1 b Sham 5h 3d 7d 21d Iba1

β-actin

Sham

* * * * * * * * *

HFS 3d

HFS 7d HFS HFS-induced chronic pain is a unique model

(1) Short-term stimulation but long-lasting pain

(2) No nerve injury

(3) Minimal peripheral inflammation

(4) Dramatic microglial activation

What is the role of microglia in HFS-induced chronic pain? What is the role of microglia?

TM Immunostaining DT HFS Pain Beha. EP

-30 -24 -3 -1 0 1 3 5 7 DT 3dDT HFS CX3CR1CreER/+:R26iDTR/+ mice Days after HFS

Iba1

Control

DT 3d

DT 21d What is the role of microglia?

TM Immunostaining DT HFS Pain Beha. EP

-30 -24 -3 -1 0 1 3 5 7 DT 3dDT HFS CX3CR1CreER/+:R26iDTR/+ mice Days after HFS b a Control MG abl + HFS Sham DT 3d DT 21 Control MG ablation + HFS Sham DT 3d DT 21d 400 1.5

300 * * * * * 1.0 * * * * * 200 .5 100 PTW(g) 0.0 0 C-fiber response C-fiber (%) -1 1 3 5 7 -60 0 60 120 180 240 300 Time (days) Time (mins)

What is microglial mechanism underlying acute to chronic transition? HFS increases the nerve sprouting

a b CGRP I&IIo IIi II&IVo

** ** *** * ** *** * **

Sham HFS 7d I I IIo IIo IIi IIi

III III

IV IV HFS HFS HFS

V V

Microglial BDNF involved? Microglial BDNF in HFS-induced nerve spouting and pain

a CGRP

BDNF+/+ HFS BDNF-/- sham BDNF-/- HFS

CX3CR1CreER/+:BDNFflox/flox mice BDNF+/+ BDNF-/- b 1.6 c HFS Sham * * * HFS * * * * 1.2

### # ### .8 PTW(g) .4 * * * * * * * * * * * *

0.0 I&IIo IIi II&IVo -1 1 3 5 7 Time (days) How microglia get activated after HFS?

a CSF1R CSF1R/Iba1 b Control Anti-CSF1

Sham HFS Sham HFS

CSF1R

Iba1

β-actin WT Sham

** * ### ### WT HFS 3d

CSFR1 Iba1

HFS activates microglia via CSF1 signaling Microglial CSF1R and BDNF?

+/+ -/- b BDNF BDNF a PBS PBS 1.6 CSF1 CSF1 Anti-CSF1

Sham HFS Sham HFS 1.2

BDNF .8 * * * CGRP PWT (g) PWT * * * * * * * * * β-actin .4

0.0 -1 0 1 2 3 Time (days)

CSF1 signaling is coupled to BDNF in HFS-induced pain

Dr. Lijun Zhou Zhou et al., Cell Rep, 2019 Now at Sun Yat-sen U How can we directly manipulate microglia to study their function in chronic pain?

Optogenetics and chemogenetics

Red-activated channelrhodopsinNMO (ReaChR)- in microglia IgG T10

CX3CR1creER/+: R26LSL-ReaChR/+ mice Activation of microglial ReaChR NMOtriggers- chronic pain IgG T10 Microglial ReaChR induces chronicNMO pain- via IL1β signaling IgG T10

Dr. Min-Hee Yi

Yi et al., BioRxiv, 2020; Yi et al., Plos Biol, under revision Summary

• How microglia sense neuronal activity? Microglia sense neuronal hyperactivity via ATP-P2Y12 signaling Microglia sense neuronal hypoactivity via NE-β2 signaling U shape microglial responses to neuronal activity

• What is the function of microglia in chronic pain? Microglial proliferation induces microgliosis and neuropathic pain after nerve injury HFS-induced chronic pain requires microglial CSF1R signaling Optogenetic activation of spinal microglia induces chronic pain via IL1β signaling Acknowledgements Current lab: Postdocs • Tingjun Chen • Dale Bosco • Min-Hee Yi • Tony Umpierre • Yujia Wei • Yanlu Ying • Lei Li • Huijie Huang • Aastha Dheer • Koichi Haruwaka PhD students • Fiona Zheng • Manling Xie • Lingxiao Wang

Supported by: • NIH, R01NS088627 Collaborators: • NIH, R01NS112144 • Vanda Lennon, Greg Worrell, Guojun Bu (Mayo Clinic) • NIH, R01NS110949 • Xianguo Liu (Sun Yat-sen University) • NIH, R01NS110825 • Mark Mattson (National Institute on Aging, NIH) • NIH, R21AG064159 • Junfang Wu (University of Maryland) • CMSAN at Mayo Clinic • Seog Bae Oh (Seoul National University)