Neurotoxic reactive astrocytes are induced by activated
microglia
Shane A Liddelow
1,2
, Kevin A Guttenplan
1
, Laura E Clarke
1
, Frederick C Bennett
1,3
,
Christopher J Bohlen
2
, Lucas Schirmer
4,5
, Mariko L Bennett
1
, Alexandra E Münch
1
, Won-
Suk Chung
6
, Todd C Peterson
7
, Daniel K Wilton
8
, Arnaud Frouin
8
, Brooke A Napier
9
, Nikhil
Panicker
10,11,12
, Manoj Kumar
10,11,12
, Marion S Buckwalter
7
, David H Rowitch
16,17
, Valina L
Dawson
10,11,12,13,14
, Ted M Dawson
10,11,12,14,15
, Beth Stevens
8
, and Ben A Barres
1
1
Department of Neurobiology, Stanford University, School of Medicine, Stanford, CA 94305, USA
2
Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, Victoria
3010, AUSTRALIA
3
Department of Psychiatry and Behavioral Sciences, Stanford University, School of Medicine,
Stanford, CA 94305, USA
4
Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of
California San Francisco, San Francisco, CA, 94143, USA
5
Department of Neurology, Klinikum rechts der Isar, Technical University of Munich, Munich,
81675, GERMANY
6
Department of Biological Sciences, Korea Advanced Institute of Science and Technology
(KAIST), Daejeon 34141, South Korea
7
Department of Neurology & Neurological Sciences, Stanford University, School of Medicine,
Stanford, CA 94305, USA
8
Department of Neurology, F. M. Kirby Neurobiology Center, Boston Children’s Hospital, Boston,
MA 02115, USA
9
Department of Microbiology and Immunology, Stanford University, School of Medicine, Stanford,
CA 94305, USA
10
Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins
University School of Medicine, Baltimore, MD 21205, USA
11
Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205,
USA
12
Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA 70130-2685, USA
Reprints and permissions information is available at www.nature.com/reprints.
Corresponding Author: Liddelow, Shane A (liddelow@stanford.edu).
BAB is a co-founder of Annexon Biosciences, Inc., a company working to make new drugs for treatment of neurological diseases.
Author Contributions
See Supplementary Notes for author contributions.
Data Availability:
The data that support the findings of this study are available from the corresponding author upon reasonable request.
HHS Public Access
Author manuscript
Nature
. Author manuscript; available in PMC 2017 July 26.
Published in final edited form as:
Nature
. 2017 January 26; 541(7638): 481–487. doi:10.1038/nature21029.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
13
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205,
USA
14
Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of
Medicine, Baltimore, MD 21205, USA
15
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of
Medicine, Baltimore, MD 21205, USA
16
Departments of Pediatrics and Neurosurgery, University of California San Francisco, San
Francisco, CA 94143, USA
17
Department of Paediatrics, University of Cambridge, Cambridge, CB2 0AH, UK
Summary
Reactive astrocytes are strongly induced by central nervous system (CNS) injury and disease but
their role is poorly understood. Here we show that A1 reactive astrocytes are induced by
classically-activated neuroinflammatory microglia. We show that activated microglia induce A1s
by secreting Il-1α, TNFα, and C1q, and that these cytokines together are necessary and sufficient
to induce A1s. A1s lose the ability to promote neuronal survival, outgrowth, synaptogenesis and
phagocytosis, and induce death of neurons and oligodendrocytes. Death of axotomized CNS
neurons
in vivo
is prevented when A1 formation is blocked. Finally, we show that A1s are highly
present in human neurodegenerative diseases including Alzheimer’s, Huntington’s, Parkinson’s,
ALS, and Multiple Sclerosis. Taken together these findings explain why CNS neurons die after
axotomy, strongly suggest that A1s help to drive death of neurons and oligodendrocytes in
neurodegenerative disorders, and point the way forward for developing new treatments of these
diseases.
Introduction
Astrocytes are abundant cells in the central nervous system (CNS) that provide trophic
support for neurons, promote formation and function of synapses, and prune synapses by
phagocytosis, in addition to fulfilling a range of other homeostatic maintenance functions
1–4
.
Astrocytes undergo a dramatic transformation called “reactive astrocytosis” after brain
injury and disease and up-regulate many genes
5,6
and form a glial scar after acute CNS
trauma
1,6,7
. Functions of reactive astrocytes have been a subject of some debate, with
previous studies showing they both hinder and support CNS recovery
1,6–9
. It has not been
clear under what contexts they may be helpful or harmful and many questions remain about
their functions.
We previously purified and gene profiled reactive astrocytes from mice treated either with a
systemic injection of lipopolysaccharide (LPS), or received middle cerebral artery occlusion
to induce ischemia
5
. We found neuroinflammation and ischemia induced two different types
of reactive astrocytes that we termed “A1” and “A2” respectively (in analogy to the “M1”/
“M2” macrophage nomenclature, a nomenclature under current refinement because
macrophages clearly can display more than two polarization states
8,9
). A1s highly up-
regulate many classical complement cascade genes previously shown to be destructive to
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synapses, so we postulated that A1s might be harmful. In contrast, A2s up-regulated many
neurotrophic factors and we thus postulated that A2s are protective. Consistent with this
latter possibility, previous studies have provided evidence that reactive astrocytes induced by
ischemia promote CNS recovery and repair
1,10,11
.
Here we show that A1 reactive astrocytes are induced by activated microglia. A1s lose most
normal astrocyte functions but gain a new neurotoxic function, rapidly killing neurons and
mature differentiated oligodendrocytes. We show A1s rapidly form
in vivo
after CNS injury
and are highly present in many human neurodegenerative diseases. Lastly we show that
inhibition of A1 reactive astrocyte formation after acute CNS injury, prevents death of
axotomized neurons. Thus A1 reactive astrocytes are harmful, contributing to neuron death
after acute CNS injury. Understanding the multidimensional roles of reactive astrocytes has
great potential to contribute to development of new treatment strategies to reduce CNS cell
loss and neurological impairment after acute CNS injury as well as in neurodegenerative
diseases.
1. Screen for cellular and molecular inducers of the A1 phenotype
We first investigated whether microglia induce A1 reactive astrocytes because LPS is a
strong inducer of A1s
1
and is an activator of TLR4 signaling, a receptor expressed
specifically by microglial in the rodent CNS
12–15
. We took advantage of
Csf1r
−/−
knock-out
mice that lack microglia
16
(Extended Data Fig. 1) to ask whether A1s can be produced
without microglia. To assess astrocyte reactivity, we used a microfluidic qPCR screen to
determine gene expression changes in astrocytes purified by immunopanning from saline-
and LPS-treated wild type control or
Csf1r
−/−
mice. As expected, wild-type littermate
controls had a normal response to LPS injection
5,17
, with robust induction of an A1 response
(Fig. 1a), however astrocytes from
Csf1r
−/−
mice failed to activate A1s. These findings show
reactive microglia are required to induce A1 reactive astrocytes
in vivo
.
To determine what microglia-secreted signals induce A1s, we next performed a screen to
individually test various candidate molecules. We used immunopanning
18
to prepare highly
pure populations of resting (non-reactive) astrocytes (Extended Data Fig. 2a,b). We cultured
purified astrocytes in serum-free conditions and tested effects of various molecules on gene
expression using our microfluidic assay. As a control, we first investigated if astrocytes in
culture can respond to LPS and found they do not (Extended Data Fig. 2). This was expected
as rodent astrocytes lack receptors and downstream signaling components required for LPS-
activation (TLR4 and MYD88)
12–14
. We found however, that several cytokines could induce
some, but not all, A1 reactive genes. Our best inducers of a partial A1 phenotype were
interleukin 1 alpha (Il-1α), tumor necrosis factor alpha (TNFα), and complement
component 1, q subcomponent (C1q,). When purified astrocytes were cultured with all three
cytokines, astrocytes exhibited an A1 phenotype nearly identical to the A1 phenotype
induced by LPS
in vivo
(Fig. 1a). All three of these cytokines are highly expressed
specifically by microglia
13,15
, again suggesting a critical role for microglia in inducing A1
reactive astrocytes.
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2. Reactive microglia induce A1 reactive astrocytes by secreting Il-1α,
TNF
α and C1q
To further confirm that microglia induce A1 reactive astrocytes, we purified microglia by
immunopanning and cultured astrocytes in control microglia conditioned medium (MCM) or
MCM from microglia that had first been made reactive with LPS. LPS-activated MCM, but
not resting MCM, strongly induced A1 reactive astrocytes (Fig. 1a). The level to which these
transcripts were induced was comparable to that seen
in vivo
following systemic LPS
injection
5
(Extended Data Fig. 3).
To verify which cytokines microglia use to signal A1 induction, we purified microglia by
immunopanning and determined which cytokines are secreted by resting and LPS-activated
microglia. Levels of Il-1α, TNFα and C1q were all significantly elevated after microglial
activation (Fig. 1b, c). Il-1β secretion also increased in LPS-activated MCM, but was unable
to induce expression of A1 transcripts (Fig. 1a). We also tested a range of other microglia-
secreted cytokines that were unable to induce A1s (Extended Data Fig. 2). The combination
of Il-1α, TNFα and C1q however, closely mimicked that of LPS-reactive MCM (Fig. 1a).
To ensure no other factors secreted by LPS-activated microglia could also make A1s, we
collected LPS-activated MCM and pre-treated it with neutralizing antibodies to Il-1α,
TNFα, and C1q. This pre-treated MCM was unable to induce reactive astrocyte genes (Fig.
1a, Extended Data Fig. 3e). Thus Il-1α, TNFα and C1q together are sufficient to induce the
A1 phenotype, and are necessary for LPS-reactive microglia to induce A1s
in vitro
.
Does cessation of Il-1α, TNFα, and C1q signaling enable A1 reactive astrocytes
in vitro
to
revert back to resting astrocytes or is the A1 phenotype stable? To find out, we removed all
three cytokines from A1 cultures, and added neutralizing antibodies to all three to make sure
they were fully inhibited. After 7 days, we assessed levels of A1 transcripts and found the
A1 phenotype remained. As a proof of principal, we also investigated if additional molecules
could revert A1s to a non-reactive phenotype. We tested the anti-inflammatory cytokine
TGFβ and FGF (as it has been previously shown that astrocyte activation is suppressed in
the injured brain by FGF signaling
19
). We grew A1s in culture, then treated with TGFβ or
FGF and found both significantly decreased reactive astrocyte transcript levels (Fig. 1d,
Extended Data Fig. 3). Whether or not there are additional signaling processes that can
revert A1s
in vivo
is an important question for future studies.
We next investigated if genetic deficiency of Il-1α, TNFα, or C1q would be sufficient to
prevent A1 astrocyte reactivity
in vivo
. First we checked if single knock mice (Il-1α
−/−
,
TNFα
−/−
, or C1q
−/−
) were still able to produce neuroinflammatory reactive microglia
following systemic LPS injection. Using qPCR we saw microglia from these animals still
had many reactive transcripts
15
highly upregulated 24 h following LPS injection (Extended
Data Fig. 4). We next used astrocytes purified from these same mice and used our
microfluidic qPCR screen to determine whether they were reactive. Each knock-out mouse
had significantly decreased A1 astrocyte reactivity (Fig. 1e). Additionally, we looked at
double (
Il-1α
−/−
TNFα
−/−
) and triple knock-out mice (
Il-1α
−/−
TNFα
−/−
C1q
−/−
) and saw
decreases in A1 reactivity, with triple knock-out animals having no response following
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systemic LPS injection (Fig. 1e). Microglia from these same knock-out mice still upregulate
inflammatory mediators in response to LPS injection, but simply fail to release A1 initiators
(Extended Data Fig. 4). Taken together our data show that microglia-derived Il-1α, TNFα,
and C1q work together to mediate A1 reactive astrocytes.
3. A1 reactive astrocytes lose many normal astrocyte functions
A1 reactive astrocytes have decreased synaptic functions
Can A1 reactive astrocytes induce formation of functional synapses
in vitro?
We cultured
purified retinal ganglion cells (RGCs) with resting or A1 reactive astrocytes and quantified
synapse number by double immunostaining for pre- and post-synaptic proteins (Fig. 2a,
Extended Data Fig. 5). RGCs cultured with A1s had 50% less synapses compared to those
grown with control astrocytes (Fig. 2b). When RGCs were cultured with control astrocytes
to induce synapse formation and then cultured with A1s, synapse number significantly
decreased by about 40%, suggesting that A1s are either unable to maintain these synapses or
actively disassemble them.
Astrocytes induce formation of excitatory synapses by secreting GPCG4/6
20
, SPARCL1
21
,
and thrombospondins (THBS1/2)
22
, so we next investigated whether reactive astrocytes still
produce these factors. Quantitative PCR showed decreased
Gpc6
and
Sparcl1
, while
simultaneously showing increased expression of
Thbs1/2
(Fig. 2c). This increase in
thrombospondins (which should increase synaptic number) suggests the decreased synapse
number may reflect an A1-induced toxicity to synapses (see below). To determine effects of
A1 reactive astrocytes on synapse function we used whole-cell patch clamp recording on
RGCs cultured with resting astrocytes or A1s. RGCs cultured with A1s had significantly
decreased frequency and amplitude of miniature excitatory postsynaptic currents when
compared to RGCs cultured with resting astrocytes (Fig. 2d–g). Taken together these results
show A1 reactive astrocytes induce formation of fewer synapses, and the few synapses they
do induce are significantly weaker when compared to those produced by healthy resting
astrocytes.
A1 reactive astrocytes have decreased phagocytic capacity
To compare phagocytic ability of normal and A1 astrocytes, we measured engulfment of
purified synaptosomes. A1s engulfed 50–75% fewer synaptosomes than control astrocytes
(Fig. 3a,b). Similarly, we found control astrocytes are able to robustly phagocytose myelin
debris, but upon conversion to an A1 reactive phenotype almost completely lose this
capacity (Fig. 3a,c). This phagocytic deficit corresponded with a 90% decrease in
Mertk
and
60% decrease in
Megf10
mRNA, phagocytic receptors we have previously found mediate
synaptic phagocytosis
3
(Fig. 3f). To determine whether A1s also display decreased
phagocytic ability
in vivo
, we used
Aldh1l1
-eGFP transgenic mice and LPS injection
(Extended Data Fig. 6) to visualize phagocytosis of Alexa594-conjugated cholera toxin-β,
CTB-594, labelled synapses by control and A1 astrocytes. Confocal microscopy was used to
visualize engulfed CTB-labelled synapses inside
Aldh1l1
-eGFP fluorescent astrocytes as we
previously reported
3
. We found A1 reactive astrocytes in the LGN
in vivo
show the same
significant loss of synaptic engulfment ability (around 50% compared to astrocytes in saline-
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