1) What is LPS (not just what does it stand for)? Why is it used as a model for neuroinflammation?

2) Describe microglia: where are they found, what role do they play, why can’t that role be carried out the same way it is in the rest of the body?

3) Mitofusin2 (Mfn2) is a mitochondrial protein. What is its apparent role? Can you think of a reason why overexpression could be protective against a stress? Is it reasonable that overexpression of this gene could also cause problems (if so, how)?

4) How did the authors arrange that Mfn2 was only upregulated in the brain and spinal cord of TMFN mice, and not in other tissues of the mice? How do they demonstrate this?

5) Briefly describe the roles of these molecules in immunity/inflammation:
 

IL-1β
IL-6
IL-10
TNFα
 

They all belong to a class of molecules; what is that class called?

6) What evidence do the authors provide that microglia are not “activated” in response to LPS challenge in TMFN mice? What is the difference (in this paper, in terms of what they represent) between the roles of Iba1 and GFAP?

7) Do mitochondria of TMFN mice respond differently to LPS insult than those of nontransgenic mice? How do the authors demonstrate this? Name a way this method could be subject to bias on the part of the experimenter, and how one might avoid such bias.

8) What does CX3CL1 do, normally? How is its expression affected (in terms of RNA and protein quantity) by inflammation in normal mice and in TMFN mice?

9) How do the authors investigate the role of CX3CL1 in microglial inflammation and neuron damage? What method(s) do they use to achieve this? 

10) Given that the transgene used is only upregulated in the brain and spinal cord of TMFN mice, what might account for the TMFN mice having less severe cardiac dysfunction in response to LPS challenge?

Neurobiology of Disease

Neuronal Mitochondria Modulation of LPS-Induced
Neuroinflammation

Micah Harland,1 Sandy Torres,1 Jingyi Liu,1 and Xinglong Wang1,2
1Department of Pathology, and 2Center for Mitochondrial Diseases, Case Western Reserve University, Cleveland, Ohio 44106

Neuronal mitochondria dysfunction and neuroinflammation are two prominent pathological features increasingly realized as important
pathogenic mechanisms for neurodegenerative diseases. However, little attempt has been taken to investigate the likely interactions
between them. Mitofusin2 (Mfn2) is a mitochondrial outer membrane protein regulating mitochondrial fusion, a dynamic process
essential for mitochondrial function. To explore the significance of neuronal mitochondria in the regulation of neuroinflammation, male
and female transgenic mice with forced overexpression of Mfn2 specifically in neurons were intraperitoneally injected with lipopolysac-
charide (LPS), a widely used approach to model neurodegeneration-associated neuroinflammation. Remarkably, LPS-induced lethality
was almost completely abrogated in neuronal Mfn2 overexpression mice. Compared with nontransgenic wild-type mice, mice with
neuronal Mfn2 overexpression also exhibited alleviated bodyweight loss, behavioral sickness, and myocardial dysfunction. LPS-induced
release of IL-1� but not TNF-� was further found greatly inhibited in the CNS of mice with neuronal Mfn2 overexpression, whereas
peripheral inflammatory responses in the blood, heart, lung, and spleen remained unchanged. At the cellular and molecular levels, neuronal
Mfn2 suppressed the activation of microglia, prevented LPS-induced mitochondrial fragmentation in neurons, and importantly, upregulated
the expression of CX3CL1, a unique chemokine constitutively produced by neurons to suppress microglial activation. Together, these results
reveal an unrecognized possible role of neuronal mitochondria in the regulation of microglial activation, and propose neuronal Mfn2 as a likely
mechanistic linker between neuronal mitochondria dysfunction and neuroinflammation in neurodegeneration.

Key words: LPS; Mfn2; mitochondrial dynamics; neuroinflammation; sepsis; septic myocardial dysfunction

Introduction
Progressive loss or dysfunction of neurons in the CNS or periph-
eral nervous system (PNS) is a characteristic feature of a wide
range of neurodegenerative disorders including Alzheimer’s
disease (AD), Parkinson’s disease, Huntington’s disease, amyo-
trophic lateral sclerosis (ALS), and frontotemporal lobar degen-
eration. Although the cause that drives the progression of each of
these neurodegenerative diseases remains elusive, it has been well
recognized that these devastating diseases are multifactorial and

involve many pathogenic mechanisms such as glutamate excito-
toxicity, oxidative stress, neuroinflammation, and mitochondrial
dysfunction in addition to the widely studied accumulation of
misfolded or aggregated proteins. Among them, mitochondrial
dysfunction and neuroinflammation have been extensively stud-
ied in the past decade. As prominent pathological features, both
mitochondrial dysfunction and neuroinflammation are closely
associated with pathological hallmarks (Heneka et al., 2015; Gao
et al., 2017), and have been implicated as interdependent pathological
lesions in neurodegenerative diseases (Wilkins and Swerdlow, 2016).
However, despite intensive effort devoted to understanding the un-
derlying cause(s) of these two prominent pathological features for
neurodegenerative diseases, a clear mechanistic linker(s) between
them has yet to be identified.

Mitofusin2 (Mfn2) is a conserved dynamin-like GTPase pro-
tein predominantly localized in the mitochondrial outer mem-
brane regulating mitochondrial fusion (Chen et al., 2003), a
process reported to be essential for various aspects of mitochon-

Received Sept. 27, 2019; revised Dec. 4, 2019; accepted Jan. 1, 2020.
Author contributions: X.W. designed research; M.H., S.T., and J.L. performed research; M.H. and X.W. wrote the

paper.
This work was supported by Grants from the US NIH (1R01NS097679 and RF1AG056320) and U.S. Alzheimer’s

Association (AARG-17-499682).
The authors declare no competing financial interests.
Correspondence should be addressed to Xinglong Wang at xinglong.wang@case.edu.
https://doi.org/10.1523/JNEUROSCI.2324-19.2020

Copyright © 2020 the authors

Significance Statement

Our study suggests that Mfn2 in neurons contributes to the regulation of neuroinflammation. Based on the remarkable suppres-
sion of LPS-induced neuroinflammation and neurodegeneration-associated mitochondrial dysfunction and dynamic abnormal-
ities by neuronal Mfn2, this study centered on Mfn2-mediated neuroinflammation reveals novel molecular mechanisms that are
involved in both mitochondrial dysfunction and neuroinflammation in neurodegenerative diseases. The pharmacological target-
ing of Mfn2 may present a novel treatment for neuroinflammation-associated diseases.

1756 • The Journal of Neuroscience, February 19, 2020 • 40(8):1756 –1765

mailto:xinglong.wang@case.edu

drial function including respiratory complex assembly (Cogliati
et al., 2013), ATP production (Benard et al., 2007), Ca 2� homeo-
stasis (Frieden et al., 2004; Szabadkai et al., 2004), and reactive
oxygen species production (Yu et al., 2006). Mfn2 has also been
reported to be present in the endoplasmic reticulum (ER) or
mitochondria-associated membranes to regulate ER and mito-
chondria tethering (de Brito and Scorrano, 2008; Sebastián et al.,
2012; Sugiura et al., 2013), autophagosome formation (Hailey et
al., 2010), autophagosome-lysosome fusion (Zhao et al., 2012),
mitophagy (McLelland et al., 2018), and axonal transport of cal-
pastatin, an endogenous specific inhibitor of the calpain sys-
tem, to maintain neuromuscular synapses based on our most
recent study (Wang et al., 2018). Altered mitochondrial dy-
namics (Kandimalla et al., 2016), distribution (Kopeikina et
al., 2011), function (David et al., 2005), transport (Rodríguez-
Martín et al., 2016), ER/mitochondria association (Perreault
et al., 2009), autophagy (Schaeffer et al., 2012), and calpain
(Reinecke et al., 2011), all Mfn2-related pathways, have been con-
sistently observed in experimental models for neurodegenerative
diseases.

Sepsis is a multi-symptomatic, life-threatening immune reaction
to an infection. Although sepsis and neurodegenerative diseases are
generally thought to be unrelated, both share similar neuroinflam-
mation and neurologic symptoms (Sankowski et al., 2015).
Additionally, a growing body of evidence supports that neuroin-
flammation and neurodegeneration can be initiated by robust in-
flammatory events in the periphery, including single dose
lipopolysaccharide (LPS; Sheng et al., 2003; Kitazawa et al., 2005;
Qin et al., 2007; Sy et al., 2011; Ifuku et al., 2012; Okuyama et al.,
2013; Jin et al., 2014). Despite previous studies reporting altered
mitochondrial dynamics in heart, lung, liver, kidney, and skeletal
muscle tissue in sepsis animal models (Hansen et al., 2015; Liu et al.,
2015; Yu et al., 2016; Park et al., 2018; Haileselassie et al., 2019; Tan et
al., 2019), their role in the CNS during sepsis remains largely un-
known. To examine this, we generated transgenic mice overexpress-
ing Mfn2 specifically in CNS neurons under the control of Thy1.2
promotor, i.e., TMFN mice (Wang et al., 2015, 2018) and investi-
gated the role of neuronal Mfn2 in neuroinflammation by intraperi-
toneally injecting TMFN and age-matched nontransgenic (NTg)
mice with LPS, one of the most widely used approaches for periph-
erally induced neuroinflammation (Catorce and Gevorkian, 2016),
and explored the potential pathways by which neuronal mitochon-
dria regulate neuroinflammation via Mfn2.

Materials and Methods
Transgenic mice. All mouse procedures were performed in accordance

with NIH guidelines and the Institutional Animal Care and Use Com-
mittee (IACUC) at Case Western Reserve University (CWRU). Mfn2
transgenic (TMFN) mice were created via pronuclear injection of the
murine Thy1.2 genomic expression cassette (gift from Dr. Philip C.
Wong, Johns Hopkins University) expressing human Mfn2 into C57BL/6
fertilized eggs. C57BL/6 Mfn2fl/fl mice were obtained from Dr. David
Chan (California Institute of Technology). All experiments used 3-month-
old male and female littermates raised in specific pathogen-free facilities.
The animal number needed to reach statistical significance was calcu-
lated and reviewed by CWRU IACUC before the experiments.

LPS injection and survival. Three-month-old littermate mice were intra-
peritoneally injected with 10 mg/kg bodyweight of 0.22 �m Millex filtered
O111:B4 LPS (L2630, Sigma Aldrich) in PBS (in mM: 137 NaCl, 2.7 KCl, 10
Na2HPO4, 17.6 KH2PO4, pH 7.4) or an equal volume of PBS ipsilaterally. Sur-
vival was assessed daily up to 14 d postinjection (dpi), at which point remaining
mice were killed in accordance with CWRU IACUC protocol.

Intrahippocampal injection. Three-month-old mice were intrahip-
pocampally injected with 1 �l of AAV1-shCX3CL1 and AAV1-

Scrambled control (TMFN and NTg mice) or AAV1-Cre-EGFP and
AAV1-EGFP control (Mfn2fl/fl mice; all from Vector Biolabs) on the left
and right sides of the mice, respectively. Briefly, mice were anesthetized
with 2% isoflurane inhalation and maintained at 2% via nosecone. A
stereotactic platform with heating pad support was used to position the
heads of the mice. Eye ointment was administered and the necks/heads of
the mice were shaved before sterilization with betadine/alcohol. Bupiva-
caine/lidocaine (1:1 v/v) was injected subcutaneously at the base of the
neck. The skull was exposed with a scalpel midline incision from the
frontal cranial bones to the parietal cranial bones. Small holes were
drilled in the skull and AAV1 was injected (2.1 mm anteroposterior, �2.0
mm mediolateral, and �1.5 mm dorsoventral relative to bregma) with a
30-gauge needle at a rate of 0.2 �l/min followed by 5 additional minutes
for absorption. The incision was closed with nylon suture, carprofen was
intraperitoneally administered, and mice were returned to a clean cage
with warming pad for recovery. At 15 dpi, TMFN and NTg mice were
intraperitoneally injected with 10 mg/kg LPS and killed 2 d later (17 dpi
of AAV1). Mfn2fl/fl mice were killed at 21 dpi.

Microglia depletion. Three-month-old TMFN and NTg littermate mice
were fed Purina Prolab RMH 3000 chow (5P75, Lab Diets) with or with-
out 290 mg/kg PLX3397 (206178, MedKoo Biosciences) prepared by Lab
Diets ad libitum for 4 weeks. Mice were intraperitoneally injected with 10
mg/kg LPS or PBS control and remained on PLX3397 or control diet
during the experiments.

Open-field tests. Open-field tests were conducted at 2 dpi during sim-
ilar daytimes. A multiple unit open-field maze consisting of four cham-
bers (50 cm length � 50 cm width � 38 cm height) was used for all tests.
Each chamber was wiped with 10% ethanol before use and before subse-
quent tests to remove scent cues left by the previous mouse. Four lamps
were placed at the outside corners of the test unit to allow for ample
visibility within the chambers. Mice were acclimated in the chamber for
3 min before each trial and video recorded for 10 min. Anymaze 6.13
software (Stoelting) was used to evaluate mouse movement/position
(immobility settings at 65% and 2 s). Inner zones were defined as a 40 cm
length � 40 cm width square in the center of each chamber. Total trav-
eled distance, immobile time, average speed, body rotations, and track
plots were calculated using Anymaze 6.13.

Tissue collection. The majority of tissues and whole blood were col-
lected at 2 dpi unless specified otherwise in the text. Briefly, mice were
anesthetized with 2% isoflurane inhalation and killed via cervical dislo-
cation for collection of non-perfused tissues. Perfused tissues, used
where specified, were collected from mice anesthetized with Avertin (tri-
bromoethanol, 2.5% in PBS) and transcardially perfused with 4°C PBS
for 6 min. Tissues for lysates were snap frozen on dry ice and stored at
�80°C before processing. Tissues for embedding were fixed in 10% for-
malin, dehydrated in increasing ethanol concentrations (70, 95, and
100%), cleared in 100% xylene, and paraffinized in blocks.

Electrocardiogram. Electrocardiogram (ECG) recordings of mouse
cardiac function was performed using the PowerLab 4/35 data acquisi-
tion system, FE136 Animal Bio Amp, and 29-G needle electrodes (AD-
Instruments). Mice were anesthetized with 2% isoflurane inhalation and
maintained at 2% via nosecone. Sterile electrodes were inserted �1 cm
subcutaneously proximal to each side of the thorax near the upper limbs and
a reference grounding electrode was inserted subcutaneously near the left
lower limb. ECGs were recorded with LabChart 8.1.13 software (ADInstru-
ments) for 5 min with a 4000/s sampling rate. A low-pass filter with a cutoff
frequency of 50 Hz was applied to improve signal-to-noise ratio. ECGs were
block averaged to produce a single representative ECG and LabChart 8.1.13
software was used to automatically identify wave positions and amplitudes.
Parameters were qualitatively validated against published mouse ECGs (Ho
et al., 2011; Boukens et al., 2014) to ensure proper denotation of waves.
Amplitude was measured as the voltage difference between 0 mV baseline to
the peak of each wave. Time intervals were measured as the time difference
from the peak of one wave to another.

Hematology. Mouse tail-vein blood samples were collected in Mi-
crovette 200 lithium heparin capillary tubes (Sarsedt). White blood cell
differentials were assessed with HEMAVET 950 hematology system
(Drew Scientific). Plasma was prepared by centrifugation (1000 � g, 4°C)
of tail vein blood for 10 min followed by collection of the clear top layer.

Harland et al. • Mitochondria for Neuroinflammation J. Neurosci., February 19, 2020 • 40(8):1756 –1765 • 1757

Immunoblotting. Mouse tissues were lysed in 1� lysis buffer (9801,
Cell Signaling Technology) with 1 mM phenylmethyl sulfonyl fluoride
(10837091001, MilliporeSigma), Protease Inhibitor Cocktail (P8340,
MilliporeSigma), and Phosphatase Inhibitor (4906845001, Roche). Pro-
tein concentrations were resolved using Pierce BCA Protein Assay
(23227, ThermoFisher Scientific). Equal protein concentrations and vol-
umes were run on 10% SDS-PAGE gels and blotted on Immobilon-P
(IPVH00010, MilliporeSigma). Blots were blocked in 10% nonfat milk in
tris-buffered saline Tween (TBST; 50 mM Tris and 150 mM NaCl, 0.1%
Tween20, pH 7.6) before probing with primary and detection anti-
bodies in 1% milk in TBST. Blots were developed with Immobilon
Western chemiluminescent horse radish peroxidase (HRP) substrate
(WBKLS0500, MilliporeSigma) or ImmunoCruz Western Blotting
Luminol Reagent (sc-2048, Santa Cruz Biotechnology) and imaged
with ChemiDoc MP Imaging System (Bio-Rad).

Primary antibodies (all diluted 1:3000 in TBST from stock concentra-
tions): mouse monoclonal anti-Drp1 (611112, BD Biosciences), rabbit
monoclonal anti-GAPDH (2118, Cell Signaling Technology), mouse
monoclonal anti- glial fibrillary acidic protein (GFAP; 14-9892-82, In-
vitrogen), mouse monoclonal anti-HIF-1� (610958, BD Biosciences),
rabbit monoclonal anti-Iba1 (013-27691, Wako Chemical), rabbit poly-
clonal anti-MFF (12741, Abcam), mouse monoclonal anti-Mfn1 (sc-
166644, Santa Cruz Biotechnology), mouse monoclonal anti-Mfn2
(sc-100560, Santa Cruz Biotechnology), rabbit monoclonal anti-Mfn2
(11925, Cell Signaling Technology), mouse monoclonal anti-Opa1
(612606, BD Biosciences), rabbit polyclonal anti-MAP2 (AB5622, Milli-
poreSigma), rabbit monoclonal anti-Synaptophysin (5461S, Cell Signal-
ing Technology), and mouse monoclonal anti- voltage-dependent anion
channel 1 (VDAC1; ab14734, Abcam). Detection antibodies (diluted
1:10,000 in TBST from stock concentrations): anti-rabbit IgG, HRP-
linked (7074S, Cell Signaling Technology), and anti-mouse IgG, HRP-
linked (7076S, Cell Signaling Technology).

Immunohistochemistry (IHC). Formaldehyde-fixed paraffin embedded
mouse tissue was used to prepare serial adjacent sections. Tissue sections
were de-paraffinized with 100% xylene twice and rehydrated with de-
creasing ethanol concentrations (100, 95, 70, and 50%) before incuba-
tion in tris-buffered saline (TBS; 50 mM Tris and 150 mM NaCl, pH 7.6).
Tissue antigen retrieval was conducted with 1� immunoDNA retriever
with citrate (BSB 0021, Bio SB) in a TintoRetriever pressure cooker (BSB
7008, Bio SB). Slides were rinsed with deionized (DI) water and incu-
bated with TBS. Individual sections were circled with hydrophobic
marker and blocked with 10% normal goat serum (NGS; 50062Z, Ther-
moFisher Scientific) in TBS for 30 min at room temperature. Sections
were rinsed with 1% NGS in TBS and excess liquid was removed with a
paper towel. Sections were incubated with primary antibodies diluted in
1% NGS in TBS overnight at 4°C, then rinsed and incubated with 1%
followed by 10% NGS in TBS. Species-specific secondary antibodies were
then placed on the tissue sections for 30 min at room temperature. Sec-
tions were rinsed and incubated in 1 and 10% NGS an additional time
before incubation with species-specific peroxidase-anti-peroxidase for
1 h at room temperature. Slides were developed using a DAB chromogen
kit (DB801L, Biocare Medical; or ENZ-ACC105, Enzo Life Sciences).
Tris buffer (50 mM Tris, pH 7.6) was used to stop the reaction and slides
were rinsed with DI water and dehydrated with 70, 95, 100% ethanol, and
xylene. Coverslips were mounted with Permount (SP15-500, Fisher Sci-
entific). Slides were imaged using a Zeiss Axio Imager.A2 equipped with
an AxioCam 503 using Zeiss EC Plan-Neofluar 10� and 20� objectives.

For hematoxylin and eosin staining, tissue sections were rehydrated in
decreasing ethanol concentrations as above and then placed in DI water.
Slides were incubated in hematoxylin for 3 min, rinsed in DI water, and
excess stain removed with acid alcohol. Slides were thoroughly rinsed with
DI water and incubated in eosin for 30 s. Sections were dehydrated in 95 and
100% ethanol followed by xylene. Coverslips were mounted and slides were
imaged as described for immunohistochemistry.

Primary antibodies (diluted in 1% NGS in TBS from stock concentra-
tions): mouse monoclonal anti-GFAP (1:250; 14-9892-82, Invitrogen), rab-
bit monoclonal anti-Iba1 (1:250; 013-27691, Wako Chemical), rabbit
monoclonal anti-Ki-67 (1:100; 9129S, Cell Signaling Technology), rabbit
polyclonal anti-MAP2 (1:500; AB5622, MilliporeSigma), mouse mono-

clonal anti-NeuN (1:1000; MAB377, MilliporeSigma), rabbit monoclo-
nal anti-synaptophysin (1:100; 5461S, Cell Signaling Technology).
Secondary and detection antibodies: goat anti-mouse IgG (1:50; AP124,
MilliporeSigma), goat ant-rabbit IgG (1:50; AP132, MilliporeSigma),
mouse peroxidase-anti-peroxidase (1:250; 223-005-024, Jackson Immu-
noResearch), rabbit peroxidase-anti-peroxidase (1:250; 323-005-024,
Jackson ImmunoResearch).

Immunofluorescent microscopy. Formaldehyde-fixed paraffin embed-
ded mouse tissue was sectioned, de-paraffinized, rehydrated, and antigen
retrieved. Slides were rinsed with DI water and incubated with PBS.
Sections were circled with a hydrophobic marker and blocked with 10%
NGS in PBS for 30 min. Sections were rinsed with 1% NGS in PBS and
incubated with individual primary antibodies at 4°C overnight. Sections
were then rinsed with 1% NGS, blocked in 10% NGS for 10 min, and
rinsed with 1% NGS. Sections were incubated with species-specific Alex-
aFluor 488- or 568-conjugated Abs diluted 1:300 in PBS for 2 h at room
temperature in the dark. Sections were rinsed 3� with PBS, incubated
with DAPI diluted 1:1000 in PBS for 15 min, and rinsed 3� with PBS.
Excess liquid was removed and slides were coverslipped using
Fluoromount-G mounting medium (0100-01, SouthernBiotech). Slides
were imaged using a Zeiss Celldiscoverer 7 equipped with an AxioCam
512 and Hamamatsu Orca Flash 4.0 V3 using Zeiss Plan-Apochromat
20� and 50� autocorr objectives with 0.5�, 1�, and 2� magnification
changers.

Primary antibodies (diluted in 1% NGS in PBS from stock concentra-
tions): rabbit monoclonal anti-Iba1 (1:250; 013-27691, Wako Chemical),
mouse monoclonal anti-VDAC1 (1:3000; ab14734, Abcam). Detection an-
tibodies: anti-mouse IgG (H�L) AlexaFluor 568 (1:300; A-11031, Invitro-
gen), anti-rabbit IgG (H�L) AlexaFluor 488 (1:300; A-11034, Invitrogen).

Enzyme-linked immunosorbent assay (ELISA). Optical 96-well plates
were coated with polyclonal antigen capture antibody in ELISA Coating
Buffer (421701, BioLegend) or, for CX3CL1 direct ELISA, 20 �g/ml
mouse brain extract in PBS overnight at 4°C. Wells were aspirated and
blocked with 1% bovine serum albumin (BSA) in PBS for 1.5 h at room
temperature. Wells were aspirated, rinsed 3� with PBS, and aspirated.
Recombinant protein standards and samples diluted in PBS were incu-
bated in individual wells overnight at 4°C. Wells were aspirated, rinsed
with PBS 3�, and aspirated. Primary antibody targeting the antigen of
interest was diluted in PBS and added to each well. The plate was incu-
bated at 4°C overnight. After aspirating, rinsing 3�, and aspirating again,
each well was incubated in diluted species-specific HRP-conjugate detec-
tion antibody for 2 h at room temperature. Wells were aspirated, rinsed
4�, aspirated, and then developed with TMB substrate (N301, Thermo-
Fisher Scientific) at 37°C for 30 min. 450 nm stop solution (ab171529,
Abcam) was added each well. The OD450 of each well was read with a
BioTek 800TS microplate reader. Standard curves and protein concen-
trations were calculated from triplicate well averages in Microsoft Excel.

Capture antibodies (diluted in PBS): rabbit polyclonal anti-IL-1�
(1 �g/ml; ab9722, Abcam), rabbit polyclonal anti-TNF� (0.5 �g/ml;
ab6671, Abcam). Primary antibodies: rabbit polyclonal anti-CX3CL1
(1 �g/ml; PA1–29026, Invitrogen), mouse monoclonal anti-IL-1�
(1 �g/ml; MAA563Mu21, Cloud-Clone), mouse monoclonal anti-
TNF� (1 �g/ml; MAA133Mu21, Cloud-Clone). Detection antibodies
(diluted 1:1000 in 1% BSA in PBS from stock concentrations): anti-
rabbit IgG, HRP-linked (7074S, Cell Signaling Technology), anti-mouse
IgG, HRP-linked (7076S, Cell Signaling Technology). Protein standards:
recombinant mouse IL-1� (ab78839, Abcam), recombinant mouse
TNF� (ab9740, Abcam).

Real-time PCR. Total RNA was isolated from fresh perfused mouse
whole brain tissue using RNeasy Mini kit (74104, Qiagen) according to
the manufacturer’s specifications. Complimentary DNA was synthesized
using High-Capacity cDNA Reverse Transcription kit (4368814, Applied
Biosystems) and Simpliamp Thermal Cycler (ThermoFisher Scientific)
according to the manufacturer’s cycling parameters. Real-time PCR for
target mRNAs was assayed using Power SYBR Green Master Mix
(4367660, Applied Biosystems) in a StepOne Real-Time PCR System
(Life Technologies) according to the manufacturer’s cycling parameters.
StepOne 2.3 software (Life Technologies) was used to measure CT values

1758 • J. Neurosci., February 19, 2020 • 40(8):1756 –1765 Harland et al. • Mitochondria for Neuroinflammation

and fold-change standardized to GAPDH mRNA was calculated in
Microsoft Excel.

The following primer pairs were used: CX3CL1 F: 5�-CGCGTTCTTC
CATTTGTGTA-3� and R: 5�-CTGTGTCGTCTCCAGGACAA-3�, CX3
CR1 F: 5�-CAGCATCGACCGGTACCTT-3� and R: 5�-GCTGCACT
GTCCGGTTGTT-3�, GAPDH F: 5�-ATGTTCCAGTATGACTCCAC
TCACGG-3� and R: 5�-GAAGACACCAGTAGACTCCACGACA-3�,
IL-1� F: 5�-AACCTGCTGGTGTGTGACGTTC-3� and R: 5�-CAGCAC
GAGGCTTTTTTGTT GT-3�, IL-6 F: 5�-ACAACCACGGCCTTCCCT
ACTT-3� and R: 5�-CACGATTTCCCAGAGAACATGTG-3�, IL-10 F:
5�-ATAACTGCACCCACTTCCCA-3� and R: 5�-GGGCATCACTTC
TACCA GGT-3�, TNF� F: 5�-CTCCAGGCGGTGCCTATGT-3� and R:
5�-GAAGAGCGTGGTGGCCC-3�.

Image analysis. Immunoblot band densities were quantified with
open-source WCIF ImageJ (developed by W. Rasband at the National
Institutes of Health) and Image Lab 6.0.1 (Bio-Rad). IHC staining and
immunofluorescent intensities were quantified with ZEN 2.3 (Carl Zeiss
Microscopy). Immunofluorescent VDAC1 (AF568; green) and DAPI
(blue) Z-stacks were processed for background subtraction and con-
strained iterative deconvolution before 3D projection in Zen software.
Wide-field VDAC1 Z-stack images were similarly processed and mito-
chondrial length were calculated from single plane images using Zen 2.3
automated image analysis and Microsoft Excel. Mitochondrial length
was defined as the Feret maximum ( F).

Experimental design and statistical analysis. Statistical analyses were
conducted with Prism 8.0 (GraphPad). Data are mean � SEM. Data were
compared by unpaired two-tailed t tests for two samples or one-way
ANOVA followed by Tukey’s multiple comparison post hoc test for �3
samples. Sample size (n) was defined as the number of cells counted in
imaging experiments, or the number of mice per experimental group.
The null hypothesis was rejected at the 0.05 level. p values 0.05 were
considered statistically significant. The statistical test, sample size (n),
and the p values are all described in the figure legends.

Results
TMFN mice are resistant to
LPS-induced endotoxic shock
Mfn2 was only upregulated in the brain
and spinal cord of TMFN mice and no
transgene expression was noted in the
peripheral immune system and other tis-
sues, including the heart, lung, gastro-
cnemius muscle, liver and kidney (Fig.
1-1 A, B, available at https://doi.org/10.
1523/JNEUROSCI.2324-19.2020.f1-1).
Three-month-old TMFN and NTg litter-
mates were intraperitoneally injected with
a single lethal dose of LPS or PBS. Survival
was monitored for up to 14 d, whereas all
other assays were conducted at 2 dpi (Fig.
1A). After intraperitoneal injection of le-
thal dose LPS, 3-month-old NTg mice
showed body weight loss and died largely
within 1 week (Fig. 1B and Fig. 1-1C,
available at https://doi.org/10.1523/
JNEUROSCI.2324-19.2020.f1-1). In striking
contrast, TMFN mice demonstrated signifi-
cantly alleviated body weight loss and most
survived after lethal dose LPS challenge (Fig.
1-1C, available at https://doi.org/10.1523/
JNEUROSCI.2324-19.2020.f1-1), even
though they also exhibited symptoms of
acute illness. LPS-induced splenomegaly,
a prominent feature reported in experi-
mental sepsis models (Altamura et al.,
2001), was similar in NTg and TMFN lit-

termates (Fig. 1-1 D, available at https://doi.org/10.1523/
JNEUROSCI.2324-19.2020.f1-1). Weights of other tissues or
organs were also identical, and showed similar pathological dam-
age between NTg and TMFN littermates (Fig. 1-1 D, E, available
at https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1),
suggesting that peripheral inflammatory responses and gross or-
gan constitution are likely analogous in TMFN and NTg mice
with LPS injection. Three independent lines of TMFN mice with
similar transgene expression were tested and there was no phe-
notypic difference between them. Nonspecific psychological and
behavioral symptoms, usually referred to as “sickness behavior”,
virtually accompany all acute inflammatory illnesses (Dantzer et
al., 2008). To further investigate whether LPS-induced sickness be-
havior was improved in TMFN mice, we assessed the performance of
mice in the open field test at 2 dpi. Compared with mice with PBS
injection, NTg mice with LPS injection showed greatly reduced trav-
eling distance and stayed largely immobile in proximity to the walls
of the maze (Fig. 1C,D), indicative of both locomotor impairment
and anxiety-related behavior. TMFN mice with LPS injection exhib-
ited greater overall movement throughout the test with augmented
distance traveled, reduced time spent immobile, and less wall-
hugging behavior (Fig. 1C,D). Together, these data demonstrate that
forced expression of Mfn2 in neurons is sufficient to greatly alleviate
LPS-induced endotoxic shock and associated behavioral sickness.

LPS-induced neuroinflammation is attenuated in TMFN mice
Peripheral LPS challenge causes widespread immune activation,
including lasting neuroinflammation and neuronal dysfunction
in mice (Catorce and Gevorkian, 2016). However, TMFN and
NTg mice with the same injection type showed no differences in
leukopenia and levels of plasma proinflammatory cytokines

Figure 1. Forced Mfn2 expression in neurons attenuates LPS-induced mortality and behavioral sickness. A, Experimental design
for the intraperitoneal LPS injection experiment. Three-month-old TMFN and NTg littermates were intraperitoneally injected with
a single dose of LPS (10 mg/kg bodyweight) or PBS and survival was assessed up to 14 dpi, whereas all other assays were conducted
at 2 dpi. B, Kaplan–Meier survival curve of TMFN and NTg mice following LPS injection (n
10 mice/group). C, D, Representative
track plots (C) and quantifications (D) of average distance traveled, immobility duration, average speed, and body rotations of
TMFN and NTg mice at 2 dpi in open-field tests (n
10 mice/group). Error bars represent mean � SEM, representative of triplicate
experiments. Student’s t test or one-way ANOVA followed by Tukey’s multiple-comparison test. **p 0.01, ***p 0.001,
****p 0.0001. ns, Nonsignificant. For further details on Mfn2 expression and pathology in TMFN mice see Figure 1-1, available
at https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1. For cardiac dysfunction see Figure 1-2, available at https://doi.
org/10.1523/JNEUROSCI.2324-19.2020.f1-2.

Harland et al. • Mitochondria for Neuroinflammation J. Neurosci., February 19, 2020 • 40(8):1756 –1765 • 1759

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-2

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-2

IL-1� and TNF� (Fig. 2-1 A–C, available at https://doi.org/
10.1523/JNEUROSCI.2324-19.2020.f2-1), further supporting
that peripheral inflammation is unaltered in TMFN mice. Since
plasma is specific to the circulatory system and does not consti-
tute the entire immune environment, we then examined IL-1�
and TNF� in multiple tissues from TMFN and NTg mice at 2 dpi,
including the brain, spinal cord, heart, lung, and spleen. Consis-
tent with their elevated plasma levels, both IL-1� and TNF� were
greatly increased in these tissues of TMFN and NTg mice with
LPS challenge (Fig. 2 A, B). Surprisingly, although TNF� was sim-
ilarly high in all surveyed tissues of LPS-treated TMFN and NTg
mice, LPS-induced IL-1� increase was remarkably suppressed
specifically in the brain and spinal cord of TMFN mice (Fig.
2 A, B, and Fig. 2-1 D, available at https://doi.org/10.1523/
JNEUROSCI.2324-19.2020.f2-1). Subsequent gene expression
analyses validated the specific inhibition of IL-1� but not IL-6,
IL-10, or TNF� at the mRNA level in the brains of TMFN mice
with LPS injection compared with NTg mice with LPS (Fig. 2C).
Of note, these cytokines were similar at basal levels in TMFN and
NTg mice (Fig. 2A–C). These results indicate that forced Mfn2
expression in neurons attenuates peripherally induced neuroin-
flammation, likely through specific diminished induction of pro-
inflammatory IL-1�.

LPS-induced microglia activation is suppressed in
TMFN mice
IL-1� is the main inflammatory mediator of the CNS and, when
augmented, is commonly accompanied by microglia activation
(Liu and Quan, 2018). Because TMFN mice displayed reduced
IL-1� in the CNS following LPS injection, we next sought to
determine whether microglia were differentially activated in
TMFN mice. Brain sections from TMFN and NTg mice at 2 dpi
were stained for ionized calcium binding adaptor molecule 1
(Iba1), a well established marker of microglia activation
(Hoogland et al., 2015). Microglia of NTg mice with LPS injec-
tion exhibited fewer processes and increased Iba1 staining, indi-
cating microglia activation in the brain (Fig. 3 A, B). In contrast,

TMFN microglia maintained ramified, inactive morphology with
weak Iba1 staining after peripheral LPS challenge (Fig. 3 A, B).
Staining for GFAP, a marker of astrocyte activation, was in-
creased to similar levels in the brains and spinal cords of
TMFN and NTg mice with LPS injection (Fig. 3-1 A, B, available
at https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-1). To
further quantify whether microglia were inactive in TMFN mice
following LPS injection, we conducted immunofluorescent
staining for Iba1 in the brains of TMFN and NTg mice at 2 dpi.
Microglia in NTg mice with LPS had rounded morphology with
fewer processes that were shorter on average (Fig. 3C,D, and Fig.
3-1C, available at https://doi.org/10.1523/JNEUROSCI.2324-
19.2020.f3-1). Contrastingly, microglia morphology, process
number, and Iba1 intensity were unaltered by LPS injection in
TMFN mice with average process length only decreasing slightly
compared with PBS injected control mice (Fig. 3C,D). To con-
firm that microglia were explicitly inactive in TMFN mice, we
immunoblotted for Iba1 and GFAP in brain extracts from TMFN
and NTg mice at 2 dpi. Iba1 levels were greatly increased in NTg
mice with LPS injection, but only marginally increased in TMFN
mice with LPS (Fig. 3-1 D, available at https://doi.org/10.1523/
JNEUROSCI.2324-19.2020.f3-1). GFAP was increased equally
in brains of TMFN and NTg mice with LPS injection (Fig. 3-1 D,
available at https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-1),
suggesting that microglia quiescence is likely driving reduced neuroin-
flammation in TMFN mouse brains.

LPS-induced mitochondrial fragmentation in neurons is
inhibited in TMFN mice
To determine whether LPS could induce mitochondrial fragmen-
tation in neurons of mice and whether forced Mfn2 expression
could prevent this, mitochondrial morphology was assessed
by immunofluorescent staining for mitochondria marker
VDAC1 in brain sections from TMFN and NTg mice at 2 dpi.
Compared with neurons of NTg mice, mitochondria in TMFN
mouse neurons became elongated as evidenced by the increase in
mitochondrial length; Fig. 4 A, B, and Fig. 4-1 A, B, available at

Figure 2. TMFN mice are protected from peripheral LPS-induced neuroinflammation. IL-1� (A) and TNF� (B) concentrations in the indicated tissues of TMFN and NTg mice at 2 dpi. Concentra-
tions are per milligram of organ tissue (n
3 mice/group). C, Quantification of IL-1�, IL-6, IL-10, and TNF� mRNA levels in brains of TMFN and NTg mice at 2 dpi. Quantifications are normalized to
GAPDH mRNA (n
3 mice/group). Error bars represent mean � SEM, representative of triplicate experiments. Student’s t test or one-way ANOVA followed by Tukey’s multiple-comparison test.
*p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. ns, Nonsignificant. For further details on peripheral inflammation see Figure 2-1, available at https://doi.org/10.1523/JNEUROSCI.2324-
19.2020.f2-1. For the necessity of Mfn2 for neuroinflammation see Figure 2-2, available at https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f2-2.

1760 • J. Neurosci., February 19, 2020 • 40(8):1756 –1765 Harland et al. • Mitochondria for Neuroinflammation

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f2-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f2-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f2-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f2-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f2-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f2-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f2-2

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-1, which
was expected. In systemic LPS models, mitochondrial fragmen-
tation has been demonstrated in multiple peripheral tissues
(Gonzalez et al., 2014; Hansen et al., 2015; Haileselassie et al.,
2019). Consistently, the significant reduction in mitochondrial
length could also be noted in neurons of NTg mice with LPS
injection (Fig. 4 A, B). Mitochondria in TMFN neurons, however,
still remained elongated after LPS challenge (Fig. 4 A, B), demon-
strating that peripheral LPS-induced mitochondrial fragmenta-
tion in neurons could be prevented by forced Mfn2 expression.
We also investigated the expression of mitochondrial fission and
fusion key regulators in brain and spinal cord tissues from TMFN
and NTg mice with and without LPS injection. Immunoblot
analysis confirmed significantly higher Mfn2 levels in TMFN
mice. However, no significant changes in Mfn2 or other fusion
regulators Mfn1 and Opa1 were noted in LPS versus PBS-injected
NTg or TMFN mice (Fig. 4-1C,D, available at https://doi.org/
10.1523/JNEUROSCI.2324-19.2020.f4-1). Fission related pro-
teins Drp1 and MFF were also unaltered (Fig. 4-1C,D, available at
https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-1), sug-
gesting that mitochondrial fragmentation is unlikely because of
the altered expression of fission and fusion regulators. Of note,
overall mitochondrial contents were similar among all samples as
evidenced by the constant expression of mitochondrial marker
VDAC1 (Fig. 4-1C,D, available at https://doi.org/10.1523/
JNEUROSCI.2324-19.2020.f4-1). These data collectively impli-
cate that peripheral inflammation can induce mitochondrial
fragmentation in neurons and that increased fusion protein Mfn2
can alleviate fragmented mitochondrial morphology caused by
LPS.

Microglia activation inhibitor CX3CL1 is an indispensable
factor for Mfn2-mediated microglia inhibition
In the brain, CX3CL1 is produced by neurons and acts as the sole
ligand of microglia expressed chemokine receptor CX3CR1 to
mediate microglia activation in response to inflammatory stimuli
such as LPS, IL-1�, and TNF� (Zujovic et al., 2000, 2001; Mizuno
et al., 2003; Biber et al., 2007). Although quantitative PCR
(qPCR) analyses found the greatly increased level of CX3CL1
mRNA in the TMFN brains, CX3CR1 mRNA was found to be
unchanged (Fig. 5A), implying a likely specific effect of neuronal
Mfn2 on CX3CL1 transcription. Notably, LPS injection induced
strong downregulation of CX3CL1 mRNA in the brains of NTg,
which was not observed in TMFN mouse brains, whereas
CX3CR1 mRNA expression was similar between genotypes and
injection groups (Fig. 5A). Consistently, the protein level of
CX3CL1 was greatly augmented in TMFN brains and remained at
similarly high levels after LPS injection, even though NTg mice
with LPS injection only exhibited a trend, but not statistically
significant, toward reduced expression of CX3CL1 at the protein
level in brains (Fig. 5B). We next virally knocked down CX3CL1
in the hippocampi of TMFN and NTg mice and injected periph-
eral LPS to determine whether CX3CL1 was critical for prevent-
ing microglia activation in TMFN mice and whether its
knockdown could exacerbate neuroinflammation in LPS-
injected NTg mice. Astonishingly, CX3CL1 knockdown restored
microglia activation in the hippocampi of TMFN mice with LPS
and augmented microglia activation in LPS-injected NTg mice as
evidenced by increased Iba1/IL-1� staining and increased num-
ber of Iba1� microglia at the injection site compared with
scrambled-control injected side (Fig. 5C,D, and Fig. 5-1, available

Figure 3. Microglia in TMFN mice are quiescent during peripheral LPS challenge. A, Representative IHC staining for Iba1 (microglia) in hippocampi of TMFN and NTg mice at 2 dpi. Enlarged images
from boxed regions show individual microglia morphology (n
5 mice/group). B, Quantification of Iba1 intensity in frontal cortices of TMFN and NTg mice at 2 dpi from Iba1 IHC staining. AU,
arbitrary units (n
5 mice/group). C, Representative brain CA1 region immunofluorescent staining for Iba1 (AF568; green) and nuclei (DAPI; blue) of TMFN and NTg mice at 2 dpi of LPS. Enlarged
images from boxed regions show individual microglia morphology (n
4 mice/group). D, Quantification of average microglia process length and process number from Iba1 (AF568) immunoflu-
orescent staining of brains from TMFN and NTg mice at 2 dpi. The longest process on each microglia and number of processes per microglia were averaged from images of the hippocampus and frontal
cortex for each mouse. Data points represent the average process lengths or process numbers for individual mice (n
4 mice/group). Error bars represent mean � SEM, representative of triplicate
experiments. Student’s t test or one-way ANOVA followed by Tukey’s multiple-comparison test. *p 0.05, **p 0.01, ****p 0.0001. ns, Nonsignificant. For further details on microglia and
astrocyte activation see Figure 3-1, available at https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-1. For the effect of microglia depletion see Figure 3-2, available at https://doi.org/10.1523/
JNEUROSCI.2324-19.2020.f3-2.

Harland et al. • Mitochondria for Neuroinflammation J. Neurosci., February 19, 2020 • 40(8):1756 –1765 • 1761

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-2

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-2

at https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f5-1),
supporting the necessity of CX3CL1 for Mfn2-mediated micro-
glia inhibition. While further mechanistic insight is needed to
identify how mitochondrial dynamics or Mfn2 expression regu-
late CX3CL1 expression in neurons, these data suggest that
forced Mfn2 expression in neurons drives expression of microglia
inhibitory CX3CL1, potentially blocking peripheral-induced
neuroinflammation.

Discussion
In this study we demonstrate for the first time that genetic up-
regulation of Mfn2 in neurons is sufficient to remarkably prevent
sepsis lethality and sickness behavior induced by peripheral in-
jection of lethal dose LPS. Interestingly, neuronal Mfn2 overex-
pression resulted in the specific inhibition of microglia but not
astrocytes. Likewise, LPS-induced IL-1� but not TNF�, IL-6, or
IL-10 increase was also specifically suppressed by neuronal Mfn2.
Because microglial inhibitor CX3CL1 produced by neurons was
found greatly upregulated by neuronal Mfn2, Mfn2 likely acts as
an unrecognized neuroinflammatory modulator in neurons.
Neuronal Mfn2 ablation alone induces IL-1� release (Fig.
2-2 A–C, available at https://doi.org/10.1523/JNEUROSCI.2324-

19.2020.f2-2), implying its necessity for neuroinflammation. No-
tably, Mfn2 is reduced in patients with AD or ALS (Wang et al.,
2009, 2018). Therefore, neuronal Mfn2 may be a point of conver-
gence for neuronal mitochondria dysfunction and neuroinflam-
mation in neurodegeneration, worthy of further detailed
investigation.

One of the most important and surprising findings of this
study was that upregulation of Mfn2 in neurons was associated
with almost completely abolished mortality in mice injected with
a lethal dose of LPS. Mfn2 is only upregulated in the brain and
spinal cord of TMFN mice. Even though the Thy1 promoter can
drive transgene expression in enteric neurons (Goto et al., 2013),
TMFN mouse peripheral tissues did not show increased Mfn2
expression (Fig. 1-1 A, B, available at https://doi.org/10.1523/
JNEUROSCI.2324-19.2020.f1-1). On top of these, TMFN mice
exhibited LPS-induced peripheral inflammatory responses
similar to NTg mice, together excluding the involvement of pe-
ripheral immune system in the resistance of TMFN mice to LPS-
induced lethality. Cardiac dysfunction is a prominent feature of
septic shock in response to infection and has been implicated as
an important component contributing to its high mortality (Ru-

Figure 4. Mfn2 overexpression alleviates peripheral LPS-induced mitochondrial fragmentation in neurons. A, Representative single plane (top) and 3D z-stack illustrations (middle/bottom) of
immunofluorescent staining for VDAC1 (AF488; green) and nuclei (DAPI; blue) in CA1 neurons of TMFN and NTg mice at 2 dpi. Red boxes indicate region enlarged in the bottom panels (n
4
mice/group). B, Quantification of mitochondrial length in wide-field VDAC1 (AF488) immunofluorescent images of CA1 neurons of TMFN and NTg mice at 2 dpi. Measurements were averaged for
individual CA1 neuronal images from each mouse. Data points represent the average mitochondrial length for individual mice (PBS injection: n
6 mice/group; LPS injection: n
5 mice/group).
Error bars represent mean � SEM, representative of triplicate experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. ***p 0.001. ns, Nonsignificant. For further details on
mitochondrial dynamics see Figure 4-1, available at https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-1. For neuronal viability see Figure 4-2, available at https://doi.org/10.1523/
JNEUROSCI.2324-19.2020.f4-2.

1762 • J. Neurosci., February 19, 2020 • 40(8):1756 –1765 Harland et al. • Mitochondria for Neuroinflammation

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f5-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f2-2

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f2-2

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-2

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-2

diger and Singer, 2007). Although inflammatory cytokine release
and Mfn2 expression were similar in NTg and TMFN heart tis-
sues, there was clear absence of ischemic QT depression and a
decrease in myocardial ischemia and infarction indicators
HIF-1� and Ki-67 in TMFN mice (Fig. 1-2 A–F, available at
https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-2), indi-
cating that neuronal Mfn2-regulated cardiac function, poten-
tially through autonomic regulation, is likely an important
mechanism for the suppression of lethality associated with septic
shock in TMFN mice. In support of this notion, previous studies
have identified autonomic control of myocardial dysfunction in
LPS rodent models, particularly through cholinergic neurons of
the sympathovagal system (Plaschke et al., 2018; Sallam et al.,
2018; Ndongson-Dongmo et al., 2019). Nevertheless, additional
work is still needed to clarify whether autonomic neurotransmis-
sion is altered by Mfn2 overexpression in neurons. LPS has been
reported to severely affect neural activity and dopamine release in
reward-related brain regions in association with reduced motiva-
tion and anhedonia (Felger et al., 2016). Accordingly, despite the
absence of neuronal loss or altered expression of neuronal
markers NeuN, MAP2, and synaptophysin in our results (Fig.
4-2 A–D, available at https://doi.org/10.1523/JNEUROSCI.2324-
19.2020.f4-2), mitigated LPS-induced sickness behavior was
noted in TMFN mice. Therefore, Mfn2 may regulate neural ac-
tivity and dopamine release in preventing the development of

cognitive and behavioral complications, worthy of further explo-
ration. In addition to heart injury, systematic ischemic organ
damage is present in murine LPS models (Cecconi et al., 2018).
Because TMFN animals challenged with LPS displayed signifi-
cantly alleviated bodyweight loss, future studies will also be inter-
esting to investigate the possible Mfn2-mediated autonomic
protection of other peripheral organs and tissues, which are
known to be tightly controlled by the CNS and PNS.

Another interesting finding of the present study was the spe-
cific inhibition of LPS-induced IL-1� but not IL-6, IL-10, or
TNF� increase in the CNS by forced overexpression of neuronal
Mfn2. IL-1� is a proinflammatory cytokine that is upregulated in
AD and many neurodegenerative diseases, and believed to be the
key player driving the neuroinflammatory process (Shaftel et al.,
2008). By binding to its cognate receptor, IL-1� initiates a signal-
ing cascade through the type I IL-1 receptor to activate NF-�B-
dependent transcription of inflammatory gene expression
(Weber et al., 2010). The majority of IL-1� research has been
conducted on peripheral cells, but it appears that similar signal-
ing mechanisms could be involved in IL-1�-mediated neuroin-
flammation in CNS cells. IL-1� and other members of the IL-1
family can be constitutively synthesized by neural cells in the
brain and have long been implied as neuromodulators in addi-
tion to proinflammatory factors (Vitkovic et al., 2000). Although
the specific cellular source and the regulation mechanisms for

Figure 5. CX3CL1 is upregulated and required for microglia inhibition in TMFN mice. A, CX3CL1 (top) and CX3CR1 (bottom) mRNA levels in brains of TMFN and NTg mice at 2 dpi were determined
by qPCR. Fold changes were standardized to GAPDH mRNA for each mouse (n
3 mice/group). B, CX3CL1 protein levels in brains of TMFN and NTg mice at 2 dpi were assessed by ELISA. AU, Arbitrary
units (n
4 mice/group). C, Representative whole brain and hippocampus IHC staining for Iba1 and IL-1� in LPS-injected TMFN mice with intrahippocampal AAV1-shCX3CL1 (right) and
AAV1-Scrambled (left, control). Brains were isolated from TMFN and NTg mice 17 dpi of AAV1 and 2 dpi LPS. Arrowheads show hippocampal injection sites with increased Iba1� cell number and
IL-1� staining in AAV1-shCX3CL1-injected mice (n
4 mice/group). D, Quantifications from IHC of Iba1� cell count (left), Iba1 intensity (middle), and IL-1� intensity (right) in hippocampal
injection sites of TMFN and NTg mice from C (n
4 mice/group). Error bars represent mean � SEM, representative of triplicate experiments. Student’s t test and one-way ANOVA followed by Tukey’s
multiple-comparison test. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. ns, Nonsignificant. For further details on CX3CL1 ablation see Figure 5-1, available at https://doi.org/
10.1523/JNEUROSCI.2324-19.2020.f5-1.

Harland et al. • Mitochondria for Neuroinflammation J. Neurosci., February 19, 2020 • 40(8):1756 –1765 • 1763

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f1-2

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-2

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f4-2

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f5-1

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f5-1

expression, cleavage, and release remain largely elusive, activated
microglia are generally believed the to be the primary source of IL-1�
in the brain (Rothwell and Luheshi, 2000). In support of this notion,
microglia depletion was found to suppress LPS-induced loco-
motive deficits (Fig. 3-2 A–D, available at https://doi.org/10.1523/
JNEUROSCI.2324-19.2020.f3-2). Indeed, TMFN mice demon-
strated greatly abrogated basal microglia activation. Furthermore,
despite the fact that astrocytes also play a critical role in regulating
neuroinflammation and that IL-1� alone is capable of eliciting as-
trocyte and microglia activation in the rodent brain (Rothwell and
Luheshi, 2000; Felger et al., 2016), neuronal Mfn2 appears to have no
effect on basal or LPS-induced astrocyte activation. Therefore, our
results indicate that neuronal Mfn2 may control IL-1� expression or
release by specifically participating in microglia activation.

CX3CR1 and its sole ligand CX3CL1 are critical mediators
between neurons and microglia in neurodegenerative diseases
(Finneran and Nash, 2019). Despite the dependence of neuro-
protective or neurotoxic functions of the CX3CL1/CX3CR1 sig-
naling pathway on microglial activation stimuli or pathological
condition, a large body of evidence has implicated CX3CL1 as a
potent inhibitor of microglial activation (Biber et al., 2007). Ex-
citingly, we found significantly increased expression of CX3CL1
but not CX3CR1 in the CNS of TMFN mice. Knockdown of
CX3CL1 was also sufficient to restore microglia activation and
IL-1� release in hippocampi of LPS-injected TMFN mice. There-
fore, a plausible mechanistic explanation for the specific inhibi-
tory effects of neuronal Mfn2 on microglial activation and related
cytokine release is that Mfn2 in neurons might contribute to the
regulation of CX3CL1/CX3CR1 signaling by modulating the ex-
pression of CX3CL1. As a predominant mitochondrial outer
membrane protein, Mfn2 unlikely regulates CX3CL1 expression
or transcription directly. Since LPS-induced mitochondrial frag-
mentation was alleviated in TMFN mice, it is highly possible that
neuronal Mfn2 may regulate CX3CL1 expression via mitochon-
drial dynamics-dependent metabolism or the transport of neu-
ronal factors required for CX3CL1 expression. However,
considering the functional role of Mfn2 in ER and mitochondria
tethering, autophagy, and calpastatin transport, we could not
exclude the possibility that Mfn2 acts via other pathways to reg-
ulate neuroinflammation via CX3CL1. Future detailed investiga-
tion of the dependence of Mfn2-mediated CX3CL1 expression on
mitochondrial fission and fusion dynamics and the sufficiency of
mitochondrial fission and fusion dynamics for CX3CL1 regula-
tion will provide a clear answer.

Together, these data suggest that Mfn2 in neurons contributes
to the regulation of neuroinflammation. Although mitochon-
drial dysfunction and neuroinflammation are two prominent
pathological features of neurodegenerative diseases, whether and
how neuronal mitochondria and neuroinflammation are interre-
lated is largely unknown. Based on the remarkable suppression
of LPS-induced neuroinflammation and neurodegeneration-
associated mitochondrial dysfunction and dynamic abnormal-
ities by neuronal Mfn2 (Wang et al., 2011, 2012, 2018), this
study centered on Mfn2-mediated neuroinflammation reveals
novel molecular mechanisms that are involved in both mito-
chondrial dysfunction and neuroinflammation in neurodegenera-
tive diseases. The pharmacological targeting of Mfn2 may present a
novel treatment for neuroinflammation-associated diseases.

References
Altamura M, Caradonna L, Amati L, Pellegrino NM, Urgesi G, Miniello S

(2001) Splenectomy and sepsis: the role of the spleen in the immune-
mediated bacterial clearance. Immunopharmacol Immunotoxicol 23:
153–161.

Benard G, Bellance N, James D, Parrone P, Fernandez H, Letellier T, Ros-
signol R (2007) Mitochondrial bioenergetics and structural network or-
ganization. J Cell Sci 120:838 – 848.

Biber K, Neumann H, Inoue K, Boddeke HW (2007) Neuronal “on” and
“off” signals control microglia. Trends Neurosci 30:596 – 602.

Boukens BJ, Rivaud MR, Rentschler S, Coronel R (2014) Misinterpretation
of the mouse ECG: “musing the waves of Mus musculus”. J Physiol
592:4613– 4626.

Catorce MN, Gevorkian G (2016) LPS-induced murine neuroinflammation
model: main features and suitability for pre-clinical assessment of nutra-
ceuticals. Curr Neuropharmacol 14:155–164.

Cecconi M, Evans L, Levy M, Rhodes A (2018) Sepsis and septic shock.
Lancet 392:75– 87.

Chen H, Detmer SA, Ewald AJ, Griffin EE, Fraser SE, Chan DC (2003) Mito-
fusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and
are essential for embryonic development. J Cell Biol 160:189 –200.

Cogliati S, Frezza C, Soriano ME, Varanita T, Quintana-Cabrera R, Corrado
M, Cipolat S, Costa V, Casarin A, Gomes LC, Perales-Clemente E, Salviati
L, Fernandez-Silva P, Enriquez JA, Scorrano L (2013) Mitochondrial
cristae shape determines respiratory chain supercomplexes assembly and
respiratory efficiency. Cell 155:160 –171.

Dantzer R, O’Connor JC, Freund GG, Johnson RW, Kelley KW (2008)
From inflammation to sickness and depression: when the immune system
subjugates the brain. Nat Rev Neurosci 9:46 –56.

David DC, Hauptmann S, Scherping I, Schuessel K, Keil U, Rizzu P, Ravid R,
Drose S, Brandt U, Müller WE, Eckert A, Götz J (2005) Proteomic and
functional analyses reveal a mitochondrial dysfunction in P301L tau
transgenic mice. J Biol Chem 280:23802–23814.

de Brito OM, Scorrano L (2008) Mitofusin 2 tethers endoplasmic reticulum
to mitochondria. Nature 456:605– 610.

Felger JC, Li Z, Haroon E, Woolwine BJ, Jung MY, Hu X, Miller AH (2016)
Inflammation is associated with decreased functional connectivity within
corticostriatal reward circuitry in depression. Mol Psychiatry 21:1358 –
1365.

Finneran DJ, Nash KR (2019) Neuroinflammation and fractalkine signaling
in Alzheimer’s disease. J Neuroinflammation 16:30.

Frieden M, James D, Castelbou C, Danckaert A, Martinou JC, Demaurex N
(2004) Ca 2� homeostasis during mitochondrial fragmentation and pe-
rinuclear clustering induced by hFis1. J Biol Chem 279:22704 –22714.

Gao J, Wang L, Liu J, Xie F, Su B, Wang X (2017) Abnormalities of mito-
chondrial dynamics in neurodegenerative diseases. Antioxidants 6:E25.

Gonzalez AS, Elguero ME, Finocchietto P, Holod S, Romorini L, Miriuka SG,
Peralta JG, Poderoso JJ, Carreras MC (2014) Abnormal mitochondrial
fusion–fission balance contributes to the progression of experimental
sepsis. Free Radic Res 48:769 –783.

Goto K, Kato G, Kawahara I, Luo Y, Obata K, Misawa H, Ishikawa T, Kuni-
yasu H, Nabekura J, Takaki M (2013) In vivo imaging of enteric neuro-
genesis in the deep tissue of mouse small intestine. PLoS One 8:e54814.

Haileselassie B, Mukherjee R, Joshi AU, Napier BA, Massis LM, Ostberg NP,
Queliconi BB, Monack D, Bernstein D, Mochly-Rosen D (2019) Drp1/
Fis1 interaction mediates mitochondrial dysfunction in septic cardiomy-
opathy. J Mol Cell Cardiol 130:160 –169.

Hailey DW, Rambold AS, Satpute-Krishnan P, Mitra K, Sougrat R, Kim PK,
Lippincott-Schwartz J (2010) Mitochondria supply membranes for au-
tophagosome biogenesis during starvation. Cell 141:656 – 667.

Hansen ME, Simmons KJ, Tippetts TS, Thatcher MO, Saito RR, Hubbard ST,
Trumbull AM, Parker BA, Taylor OJ, Bikman BT (2015) Lipopolysac-
charide disrupts mitochondrial physiology in skeletal muscle via disparate
effects on sphingolipid metabolism. Shock 44:585–592.

Heneka MT, Carson MJ, El Khoury J, Landreth GE, Brosseron F, Feinstein
DL, Jacobs AH, Wyss-Coray T, Vitorica J, Ransohoff RM, Herrup K,
Frautschy SA, Finsen B, Brown GC, Verkhratsky A, Yamanaka K, Koisti-
naho J, Latz E, Halle A, Petzold GC, et al. (2015) Neuroinflammation in
Alzheimer’s disease. Lancet Neurol 14:388 – 405.

Ho D, Zhao X, Gao S, Hong C, Vatner DE, Vatner SF (2011) Heart rate and
electrocardiography monitoring in mice. Curr Protoc Mouse Biol 1:123–
139.

Hoogland IC, Houbolt C, van Westerloo DJ, van Gool WA, van de Beek D
(2015) Systemic inflammation and microglial activation: systematic re-
view of animal experiments. J Neuroinflammation 12:114.

Ifuku M, Katafuchi T, Mawatari S, Noda M, Miake K, Sugiyama M, Fujino T
(2012) Anti-inflammatory/anti-amyloidogenic effects of plasmalogens

1764 • J. Neurosci., February 19, 2020 • 40(8):1756 –1765 Harland et al. • Mitochondria for Neuroinflammation

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-2

https://doi.org/10.1523/JNEUROSCI.2324-19.2020.f3-2

in lipopolysaccharide-induced neuroinflammation in adult mice. J Neu-
roinflammation 9:197.

Jin M, Jang E, Suk K (2014) Lipocalin-2 acts as a neuroinflammatogen in
lipopolysaccharide-injected mice. Exp Neurobiol 23:155–162.

Kandimalla R, Manczak M, Fry D, Suneetha Y, Sesaki H, Reddy PH (2016)
Reduced dynamin-related protein 1 protects against phosphorylated tau-
induced mitochondrial dysfunction and synaptic damage in Alzheimer’s
disease. Hum Mol Genet 25:4881– 4897.

Kitazawa M, Oddo S, Yamasaki TR, Green KN, LaFerla FM (2005)
Lipopolysaccharide-induced inflammation exacerbates tau pathology by
a cyclin-dependent kinase 5-mediated pathway in a transgenic model of
Alzheimer’s disease. J Neurosci 25:8843– 8853.

Kopeikina KJ, Carlson GA, Pitstick R, Ludvigson AE, Peters A, Luebke JI,
Koffie RM, Frosch MP, Hyman BT, Spires-Jones TL (2011) Tau accu-
mulation causes mitochondrial distribution deficits in neurons in a
mouse model of tauopathy and in human Alzheimer’s disease brain. Am J
Pathol 179:2071–2082.

Liu L, Song Y, Zhao M, Yi Z, Zeng Q (2015) Protective effects of edaravone,
a free radical scavenger, on lipopolysaccharide-induced acute kidney in-
jury in a rat model of sepsis. Int Urol Nephrol 47:1745–1752.

Liu X, Quan N (2018) Microglia and CNS interleukin-1: beyond immuno-
logical concepts. Front Neurol 9:8.

McLelland GL, Goiran T, Yi W, Dorval G, Chen CX, Lauinger ND, Krahn AI,
Valimehr S, Rakovic A, Rouiller I, Durcan TM, Trempe JF, Fon EA
(2018) Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent
release of ER from mitochondria to drive mitophagy. eLife 7:e32866.

Mizuno T, Kawanokuchi J, Numata K, Suzumura A (2003) Production and
neuroprotective functions of fractalkine in the central nervous system.
Brain Res 979:65–70.

Ndongson-Dongmo B, Lang GP, Mece O, Hechaichi N, Lajqi T, Hoyer D,
Brodhun M, Heller R, Wetzker R, Franz M, Levy FO, Bauer R (2019)
Reduced ambient temperature exacerbates SIRS-induced cardiac auto-
nomic dysregulation and myocardial dysfunction in mice. Basic Res Car-
diol 114:26.

Okuyama S, Makihata N, Yoshimura M, Amakura Y, Yoshida T, Nakajima M,
Furukawa Y (2013) Oenothein B suppresses lipopolysaccharide (LPS)-
induced inflammation in the mouse brain. Int J Mol Sci 14:9767–9778.

Park JS, Choi HS, Yim SY, Lee SM (2018) Heme oxygenase-1 protects the
liver from septic injury by modulating TLR4-mediated mitochondrial
quality control in mice. Shock 50:209 –218.

Perreault S, Bousquet O, Lauzon M, Paiement J, Leclerc N (2009) Increased
association between rough endoplasmic reticulum membranes and mito-
chondria in transgenic mice that express P301 L tau. J Neuropath Exp
Neurol 68:503–514.

Plaschke K, Do TQM, Uhle F, Brenner T, Weigand MA, Kopitz J (2018)
Ablation of the right cardiac vagus nerve reduces acetylcholine content
without changing the inflammatory response during endotoxemia. Int J
Mol Sci 19:E442.

Qin L, Wu X, Block ML, Liu Y, Breese GR, Hong JS, Knapp DJ, Crews FT
(2007) Systemic LPS causes chronic neuroinflammation and progressive
neurodegeneration. Glia 55:453– 462.

Reinecke JB, DeVos SL, McGrath JP, Shepard AM, Goncharoff DK, Tait DN,
Fleming SR, Vincent MP, Steinhilb ML (2011) Implicating calpain in
tau-mediated toxicity in vivo. Plos One 6:e23865.

Rodríguez-Martín T, Pooler AM, Lau DHW, Morotz GM, De Vos KJ, Gilley J,
Coleman MP, Hanger DP (2016) Reduced number of axonal mitochon-
dria and tau hypophosphorylation in mouse P301L tau knockin neurons.
Neurobiol Dis 85:1–10.

Rothwell NJ, Luheshi GN (2000) Interleukin 1 in the brain: biology, pathol-
ogy and therapeutic target. Trends Neurosci 23:618 – 625.

Rudiger A, Singer M (2007) Mechanisms of sepsis-induced cardiac dysfunc-
tion. Crit Care Med 35:1599 –1608.

Sallam MY, El-Gowilly SM, El-Gowelli HM, El-Lakany MA, El-Mas MM
(2018) Additive counteraction by �7 and �4�2-nAChRs of the hypoten-
sion and cardiac sympathovagal imbalance evoked by endotoxemia in
male rats. Eur J Pharmacol 834:36 – 44.

Sankowski R, Mader S, Valdés-Ferrer SI (2015) Systemic inflammation and
the brain: novel roles of genetic, molecular, and environmental cues as
drivers of neurodegeneration. Front Cell Neurosci 9:28.

Schaeffer V, Lavenir I, Ozcelik S, Tolnay M, Winkler DT, Goedert M (2012)
Stimulation of autophagy reduces neurodegeneration in a mouse model
of human tauopathy. Brain 135:2169 –2177.

Sebastián D, Hernández-Alvarez MI, Segalés J, Sorianello E, Muñoz JP, Sala
D, Waget A, Liesa M, Paz JC, Gopalacharyulu P, Orešič M, Pich S, Burce-
lin R, Palacín M, Zorzano A (2012) Mitofusin 2 (Mfn2) links mitochon-
drial and endoplasmic reticulum function with insulin signaling and is
essential for normal glucose homeostasis. Proc Natl Acad Sci U S A
109:5523–5528.

Shaftel SS, Griffin WS, O’Banion MK (2008) The role of interleukin-1 in
neuroinflammation and alzheimer disease: an evolving perspective.
J Neuroinflammation 5:7.

Sheng JG, Bora SH, Xu G, Borchelt DR, Price DL, Koliatsos VE (2003)
Lipopolysaccharide-induced-neuroinflammation increases intracellular
accumulation of amyloid precursor protein and amyloid beta peptide in
APPswe transgenic mice. Neurobiol Dis 14:133–145.

Sugiura A, Nagashima S, Tokuyama T, Amo T, Matsuki Y, Ishido S, Kudo Y,
McBride HM, Fukuda T, Matsushita N, Inatome R, Yanagi S (2013)
MITOL regulates endoplasmic reticulum-mitochondria contacts via
Mitofusin2. Mol Cell 51:20 –34.

Sy M, Kitazawa M, Medeiros R, Whitman L, Cheng D, Lane TE, Laferla FM
(2011) Inflammation induced by infection potentiates tau pathological
features in transgenic mice. Am J Pathol 178:2811–2822.

Szabadkai G, Simoni AM, Chami M, Wieckowski MR, Youle RJ, Rizzuto R
(2004) Drp-1-dependent division of the mitochondrial network blocks
intraorganellar Ca 2� waves and protects against Ca 2�-mediated apopto-
sis. Mol Cell 16:59 – 68.

Tan Y, Ouyang H, Xiao X, Zhong J, Dong M (2019) Irisin ameliorates septic
cardiomyopathy via inhibiting DRP1-related mitochondrial fission and
normalizing the JNK-LATS2 signaling pathway. Cell Stress and Chaper-
ones 24:595– 608.

Vitkovic L, Bockaert J, Jacque C (2000) “Inflammatory” cytokines: neuro-
modulators in normal brain? J Neurochem 74:457– 471.

Wang L, Gao J, Liu J, Siedlak SL, Torres S, Fujioka H, Huntley ML, Jiang Y, Ji
H, Yan T, Harland M, Termsarasab P, Zeng S, Jiang Z, Liang J, Perry G,
Hoppel C, Zhang C, Li H, Wang X (2018) Mitofusin 2 regulates axonal
transport of calpastatin to prevent neuromuscular synaptic elimination in
skeletal muscles. Cell Metab 28:400 – 414.e8.

Wang W, Zhang F, Li L, Tang F, Siedlak SL, Fujioka H, Liu Y, Su B, Pi Y, Wang
X (2015) MFN2 couples glutamate excitotoxicity and mitochondrial
dysfunction in motor neurons. J Biol Chem 290:168 –182.

Wang X, Su B, Lee HG, Li X, Perry G, Smith MA, Zhu X (2009) Impaired
balance of mitochondrial fission and fusion in Alzheimer’s disease. J Neu-
rosci 29:9090 –9103.

Wang X, Su B, Liu W, He X, Gao Y, Castellani RJ, Perry G, Smith MA,
Zhu X (2011) DLP1-dependent mitochondrial fragmentation mediates
1-methyl-4-phenylpyridinium toxicity in neurons: implications for Par-
kinson’s disease. Aging Cell 10:807– 823.

Wang X, Petrie TG, Liu Y, Liu J, Fujioka H, Zhu X (2012) Parkinson’s
disease-associated DJ-1 mutations impair mitochondrial dynamics and
cause mitochondrial dysfunction. J Neurochem 121:830 – 839.

Weber A, Wasiliew P, Kracht M (2010) Interleukin-1 (IL-1) pathway. Sci
Signal 3:cm1.

Wilkins HM, Swerdlow RH (2016) Relationships between mitochondria
and neuroinflammation: implications for Alzheimer’s disease. Curr Top
Med Chem 16:849 – 857.

Yu J, Shi J, Wang D, Dong S, Zhang Y, Wang M, Gong L, Fu Q, Liu D (2016)
Heme oxygenase-1/carbon monoxide-regulated mitochondrial dynamic
equilibrium contributes to the attenuation of endotoxin-induced acute
lung injury in rats and in lipopolysaccharide-activated macrophages. An-
esthesiology 125:1190 –1201.

Yu T, Robotham JL, Yoon Y (2006) Increased production of reactive
oxygen species in hyperglycemic conditions requires dynamic change
of mitochondrial morphology. Proc Natl Acad Sci U S A 103:2653–
2658.

Zhao T, Huang X, Han L, Wang X, Cheng H, Zhao Y, Chen Q, Chen J, Cheng H,
Xiao R, Zheng M (2012) Central role of mitofusin 2 in autophagosome-
lysosome fusion in cardiomyocytes. J Biol Chem 287:23615–23625.

Zujovic V, Benavides J, Vigé X, Carter C, Taupin V (2000) Fractalkine mod-
ulates TNF-alpha secretion and neurotoxicity induced by microglial acti-
vation. Glia 29:305–315.

Zujovic V, Schussler N, Jourdain D, Duverger D, Taupin V (2001) In vivo
neutralization of endogenous brain fractalkine increases hippocampal
TNFalpha and 8-isoprostane production induced by intracerebroventric-
ular injection of LPS. J Neuroimmunol 115:135–143.

Harland et al. • Mitochondria for Neuroinflammation J. Neurosci., February 19, 2020 • 40(8):1756 –1765 • 1765

• Neuronal Mitochondria Modulation of LPS-Induced Neuroinflammation

Introduction
Materials and Methods
Results
Discussion
References