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:
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
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
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
This work was supported by Grants from the US NIH (1R01NS097679 and RF1AG056320) and U.S. Alzheimer’s
The authors declare no competing financial interests.
Correspondence should be addressed to Xinglong Wang at [email protected]
Copyright © 2020 the authors
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
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
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,
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
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
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:
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.
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.
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/
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
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.
Harland et al. • Mitochondria for Neuroinflammation J. Neurosci., February 19, 2020 • 40(8):1756 –1765 • 1759