TRIM9-Mediated Resolution of Neuroinflammation Confers
Neuroprotectionupon Ischemic Stroke in Mice
Jianxiong Zeng
1,9
,
Yaoming Wang
2,3,9
,
Zhifei Luo
4
,
Lin-Chun Chang
1
,
Ji Seung Yoo
1
,
Huan
Yan
1
,
Younho Choi
1
,
Xiaochun Xie
2,3
,
Benjamin E. Deverman
5
,
Viviana Gradinaru
5
,
Stephanie L. Gupton
6,7,8
,
Berislav V. Zlokovic
2,3,*
,
Zhen Zhao
2,3,*
, and
Jae U. Jung
1,3,10,*
1
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of
Southern California, Los Angeles, CA 90033, USA
2
Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern
California, Los Angeles, CA 90033, USA
3
Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los
Angeles, CA 90033, USA
4
Department of Biochemistry and Molecular Medicine, Keck School of Medicine, University of
Southern California, Los Angeles, CA 90033, USA
5
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA
91125, USA
6
Neuroscience Center and Curriculum in Neurobiology, University of North Carolina at Chapel
Hill, Chapel Hill, NC 27599, USA
7
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel
Hill, NC 27599, USA
8
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel
Hill, NC 27599, USA
9
These authors contributed equally
10
Lead Contact
SUMMARY
Excessive and unresolved neuroinflammation is a key component of the pathological cascade in
brain injuries such as ischemic stroke. Here, we report that TRIM9, a brain-specific tripartite motif
This is an open access article under the CC BY-NC-ND license (
http://creativecommons.org/licenses/BY-NC-ND/4.0/
).
*
Correspondence: zlokovic@usc.edu (B.V.Z.), zzhao@usc.edu (Z.Z.), jaeujung@med.usc.edu (J.U.J.).
AUTHOR CONTRIBUTIONS
J.Z., Z.Z., and J.U.J. designed all experiments, analyzed data, and wrote the paper; J.Z., Y.W., and Z.L. performed experiments and
analyzed data, and L.-C.C., J.S.Y., H.Y., Y.C., and X.X. performed experiments. B.E.D., V.G., S.L.G., and B.V.Z. contributed key
materials, provided guidance for some experiments, and edited the paper.
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at
https://doi.org/10.1016/j.celrep.2018.12.055
.
DECLARATION OF INTERESTS
The authors declare no competing interests.
HHS Public Access
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Published in final edited form as:
Cell Rep
. 2019 April 09; 27(2): 549–560.e6. doi:10.1016/j.celrep.2018.12.055.
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(TRIM) protein, was highly expressed in the peri-infarct areas shortly after ischemic insults in
mice, but expression was decreased in aged mice, which are known to have increased
neuroinflammation after stroke. Mechanistically, TRIM9 sequestered
β
-transducin repeat-
containing protein (
β
-TrCP) from the Skp-Cullin-F-box ubiquitin ligase complex, blocking I
κ
B
α
degradation and thereby dampening nuclear factor
κ
B (NF-
κ
B)-dependent proinflammatory
mediator production and immune cell infiltration to limit neuroinflammation. Consequently,
Trim9
-deficient mice were highly vulnerable to ischemia, manifesting uncontrolled
neuroinflammation and exacerbated neuropathological outcomes. Systemic administration of a
recombinant TRIM9 adeno-associated virus that drove brain-wide TRIM9 expression effectively
resolved neuroinflammation and alleviated neuronal death, especially in aged mice. These findings
reveal that TRIM9 is essential for resolving NF-
κ
B-dependent neuroinflammation to promote
recovery and repair after brain injury and may represent an attractive therapeutic target.
Graphical Abstract
In Brief
Neuroinflammation drives pathology during brain injury. Zeng et al. show that TRIM9 is induced
after ischemic insults in young mice, but not old mice, and promotes resolution of
neuroinflammation. AAV-mediated TRIM9 therapy in aged mice restricts neuroinflammation and
alleviates stroke damage, representing a potential therapeutic target for brain injury.
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INTRODUCTION
Tripartite motif containing 9 (TRIM9), a brain-specific ubiquitin (Ub) ligase, is primarily
expressed in neurons and regulates netrin-dependent axon guidance and morphogenesis
through the interaction with vasodilator-stimulated phosphoprotein (VASP), a mechanism
conserved between different species (
Hao et al., 2010
;
Menon et al., 2015
;
Plooster et al.,
2017
;
Winkle et al., 2016a
,
2016c
). We have shown that TRIM9 is a potent inhibitor of
nuclear factor
κ
B (NF-
κ
B) signaling pathway in
in vitro
cell culture upon cytokine
stimulation (
Shi et al., 2014
). However, the
in vivo
role of TRIM9 in NF-
κ
B-mediated
neuroinflammation remains elusive.
Ischemic stroke remains a leading cause of mortality and disabilities in the elderly
(
Benjamin et al., 2017
). Excitotoxicity, oxidative and nitrosative stress, necrosis, and
inflammation are key pathogenic events that contribute to neuronal injury and cell death
after ischemic stroke (
Chamorro et al., 2016
;
Lo et al., 2005
). NF-
κ
B is a master regulator of
hypoxia-induced inflammation (
Eltzschig and Carmeliet, 2011
) and plays important roles in
neuronal plasticity, aging, and degeneration in CNS diseases (
Gabuzda and Yankner, 2013
;
Mattson and Camandola, 2001
;
Salminen et al., 2008
;
Zhang et al., 2013
). NF-
κ
B is a
dimeric transcription factor consisting members of the Rel family, including Rel-A (p65), c-
Rel, Rel-B, p50, and p52, and is often held in the cytoplasm by inhibitor IkB proteins as its
latent form (
Chen, 2005
). Upon stimulation, the I
κ
B kinase (IKK) complex phosphorylates
the amino-terminal serine residues (S32 and S36) of I
κ
B
α
, triggering its ubiquitination and
degradation by the Skp-Cullin-F-box (SCF) Ub ligase complex and the 26S-proteasome
pathway, respectively (
Chen, 2005
;
Frescas and Pagano, 2008
). Consequently, this allows
the nuclear translocation of NF-
κ
B p50-p65 complex for transcriptional activation of
targeted genes. NF-
κ
B activation in neurons occurs soon after brain ischemia as evidenced
by I
κ
B
α
degradation and p65 phosphorylation (
Stephenson et al., 2000
), which drives the
neuronal expression of inflammatory mediators such as inter-leukin 6 (IL-6) (
Ohtaki et al.,
2006
) and chemokine C-C motif ligand 2 (CCL2) (
Stowe et al., 2012
). Genetic and
pharmacological studies targeting NF-
κ
B-activating IKK have shown that inhibiting NF-
κ
B
is generally beneficial for stroke recovery (
Herr-mann et al., 2005
;
Iadecola and Anrather,
2011
). However, this has been challenged by stroke studies in mouse models with p50 or
cRel deficiency (
Harari and Liao, 2010
), as well as in systemic injury models (
Elsharkawy
and Mann, 2007
), suggesting that NF-
κ
B-mediated acute inflammatory response is not just
deleterious. In fact, the acute inflammation responses triggered by CNS injuries usually
resolve within a short period of time, which set up tissue boundaries for subsequent repair
process (
Buckley et al., 2013
;
Iadecola and Anrather, 2011
;
Jin et al., 2010
). However, brain-
specific factors that govern inflammation resolution have not been well defined (
Iadecola
and Anrather, 2011
). Hence, understanding the brain’s regulatory mechanisms that ensure
the timely activation and subsequent inactivation of NF-
κ
B-mediated neuroinflammation is
essential to develop a therapeutic strategy for the recovery and repair after ischemic brain
injury.
Stroke mostly occurs in elderly people, and outcomes of stroke patients are highly
influenced by age, indicating that aging is an inherent risk factor for stroke (
Markus et al.,
2005
;
Popa-Wagner et al., 2011
). Compared to the young brain, the aged brain displays a
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compromised ability to resolve stroke-mediated inflammation, causing high susceptibility to
ischemia and poor functional recovery (
Chen et al., 2010
). Indeed, upon middle cerebral
artery occlusion (MCAO), aged mice exhibit elevated proinflammatory mediators, large
infarction volumes, severe behavioral impairment, and high mortality rates compared to
young mice (
DiNapoli et al., 2008
;
Jin et al., 2004
;
Liu et al., 2009
;
Rosen et al., 2005
;
Shapira et al., 2002
), reflecting the effects seen in elderly patients who often experience
severe functional disabilities following an ischemia (
Hankey et al., 2002
). However, little is
known about brain-specific mechanisms that regulate the resolution of neuroinflammation
that are potentially undermined during aging. Hence, investigating the timely regulation of
NF-
κ
B-mediated neuroinflammation is key to a better understanding of pathophysiology
and repair after acute brain ischemia.
Here, we report that TRIM9 provides an innate mechanism to resolve ischemic-stroke-
induced neuroinflammation via fine-tuning of
in vivo
NF-
κ
B signaling activity in a mouse
model, and thus, targeting TRIM9 and its related neuroinflammatory pathway may offer a
target for immunomodulatory therapy for stroke.
RESULTS
TRIM9 Upregulation in the Peri-infarct Brain Region after Ischemic Stroke in Mice
Transient MCAO, which mimics ischemic stroke and reperfusion in rodents, is widely used
to investigate post-ischemic inflammatory responses and resolution (
Liesz et al., 2009
;
Shichita et al., 2009
,
2012
,
2017
). Immunoblotting analysis of NF-
κ
B activation showed that
I
κ
B
α
degradation and p65 phosphorylation were evidently induced between 1 and 12 hr
after 30-min MCAO and returned close to a baseline level after 48 and 72 hr in C57BL/6J
(wild-type [WT]) mice (Figures 1A and 1B). This was consistent with the self-limiting
characteristics of post-ischemic inflammation (
Liu et al., 2015
) that preceded the repair
processes (
Iadecola and Anrather, 2011
). We also observed the highly upregulated levels of
p-p65 in NeuN-positive neurons of the ischemic brains at 12 hr after 30-min MCAO
compared to those in the sham-operated group (Figures 1C and 1D). When ischemic brain
tissues isolated 12 hr after surgical procedures were subjected to RNA sequencing (RNA-
seq) analysis, the expression of numerous genes was altered in MCAO-treated tissues
compared to those in sham-operated tissues (Figure 1E). Remarkably, among those top 20
upregulated genes, only
Trim9
exhibited a brain-specific expression pattern (
Berti et al.,
2002
) (
https://www.ncbi.nlm.nih.gov/gene/94090
) (Figure S1A; Table S1). Gene ontology
analysis showed that
Trim9
was in the neurological disease and inflammation pathway in
which genes experienced the most significant alteration of expressions (adjusted p value <
0.01) (Figure 1F; Table S2). Immunoblotting assays showed the upregulation of TRIM9 in
the ischemic brain hemisphere, when compared with the unaffected contralateral hemisphere
(Figures S1B and S1C). In addition, high resolution
in situ
hybridization analysis with
RNAscope gene-specific probes (
Wang et al., 2012a
) demonstrated that the upregulation of
Trim9
was primarily detected at the periphery of the ischemic areas in the ipsilateral
hemisphere, as compared to a basal level of the unaffected contralateral hemisphere (Figure
1G). In addition, no significant difference of stroke-induced
Trim9
expression between 12-
week-old male and female mice was observed (Figure S1D). Taken together, these findings
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indicate that
Trim9
is highly upregulated in the peri-infarct cortical areas of mouse brain
upon ischemic stroke.
Exacerbated Ischemic Brain Injury in
Trim9
-Deficiency Mice
Besides aberrant axonal branching in the corpus callosum (
Winkle et al., 2014
) and
excessive dendritic arborization of den-tate granule cells in the hippocampus (
Winkle et al.,
2016b
),
Trim9
-deficient (
Trim9
−/−
) mice exhibit no gross anatomical defects or impairment
in motor functions (
Winkle et al., 2016b
). We found that the numbers of NeuN
+
neurons,
SMI-312
+
neuritic density, Oligo2
+
oligodendrocytes, NG2
+
Oligo2
+
oligodendrocyte
precursor cells, GFAP
+
astrocytes, and Iba1
+
microglia in both the somatosensory cortex and
hippocampus were nearly identical between
Trim9
−/−
mice and WT
Trim9
+/+
littermates
(Figures S2A–S2F). In addition, consistent with the lack of expression in endothelial cells
(
Zhang et al., 2014
),
Trim9
−/−
mice showed no change of the middle cerebral artery (MCA)
territory (Figures S2G and S2H) or alteration of cerebral blood flow (CBF) before, after, and
during 30-min MCAO (Figures 2A and S2I) compared to WT littermates, indicating that the
cerebrovascular system was not affected by
Trim9
deficiency. By contrast,
Trim9
−/−
mice
were more prone to ischemic injury than WT litter-mates, exhibiting a 2-fold increase in
infarct volume (Figures 2B and 2C) and a 2.3-fold enhancement in edema volume (Figure
2C) at 24 hr after MCAO. Behavioral analysis using a 6-point motor neurological score
method (
Wang et al., 2005
) showed an ~2-fold decline of neurological outcomes in
Trim9
−/−
mice relative to WT littermates at 24 hr after MCAO (Figure 2C). Cytokines and
chemokines are important mediators of neuroinflammation in stroke (
Chamorro et al., 2012
;
Eltzschig and Carmeliet, 2011
;
Iadecola and Anrather, 2011
). At 24 hr after 30-min MCAO,
Trim9
−/−
mice showed a dramatic increase in a panel of inflammatory cytokines, including
IL-6 and CCL2/5 but no alteration of IL-10 compared to WT littermates (Figures 2D and
S3A). ELISA showed that tumor necrosis factor
α
(TNF-
α
) and IL-1
β
were marginally
increased in Trim9
−/−
mice at 24 hr after 30-min MCAO but became more evident at 36 hr
(
Liesz et al., 2013
) (Figure S3B). These data suggest TRIM9 functions in dampening
neuronal expression of inflammation mediators of IL-6 and CCL2 that are upregulated
within the first day of stroke (
Ohtaki et al., 2006
;
Stowe et al., 2012
). A terminal
deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and
immunohistological analysis also indicated that the ischemic ipsilateral brain regions of
Trim9
−/−
mice showed ~2-fold increases in apoptotic cells and neuronal death compared to
those of WT lit termates (Figures 2E and 2F).
Trim9
Deficiency Causes the Elevation of Peripheral Immune Cell Infiltration in Mouse
Brain
CCL2 (also known as monocyte chemoattractant protein-1) attracts inflammatory immune
cells such as monocytes to infiltrate into brain parenchyma under pathological conditions,
which triggers further neuronal dysfunction and damage (
Mennicken et al., 1999
). Flow
cytometry analysis showed that compared to MCAO-treated WT littermates, MCAO-treated
Trim9
−/−
mice had significant increases of CD45
hi
GR1
+
CD11b
+
granulocytes and
inflammatory monocytes and CD45
hi
CD11b
−
CD3e
+
T cells (Figures 3). These results
indicate that TRIM9 plays a critical role in dampening the stroke-induced production and
recruitment of inflammation mediators and immune cells, respectively.
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TRIM9 Suppresses Neuroinflammation by Inhibiting NF-
κ
B Signaling
To further examine the role of TRIM9 in brain-specific NF-
κ
B activation, ischemic brains
were harvested 24 hr after MCAO and then subjected to immunoblotting analysis. This
revealed increased I
κ
B
α
degradation and p65 phosphorylation (p-p65) in the ischemic
brains of
Trim9
−/−
mice compared to those of
Trim9
+/+
mice (Figures S4A and S4B).
Consistently, immunostaining also showed increases of p-p65-positive neurons (NeuN) in
the peri-infarct area of
Trim9
−/−
mice (Figures S4C and S4D). To further test this, primary
cortical neurons isolated from
Trim9
+/+
and
Trim9
−/−
embryos (Figure S5A) were
challenged either by 30- or 60-min oxygen-glucose deprivation (OGD) with reoxygenation
for 24 hr (R24h) or by the pro-inflammatory cytokine TNF-
α
or IL-1
β
for 30–120 min.
Immunoblotting assays showed that the degradation of I
κ
B
α
was more robust in
Trim9
−/−
primary neurons upon stimulation with OGD (Figures 4A and 4B), TNF-
α
, or IL-1
β
(Figures S5B–S5E) than in
Trim9
+/+
primary neurons. Correspondingly, the increase of p65
phosphorylation was also more evident in
Trim9
−/−
primary neurons upon TNF-
α
or IL-1
β
stimulation than in
Trim9
+/+
primary neurons (Figures S5B–S5E). Finally, human neural
progenitor cell (hNPC)-derived neurons (
Liang et al., 2016
;
Wang et al., 2016
) were infected
with scramble- or
TRIM9
-specific short hairpin RNA (shRNA) lentivirus for 24 hr, followed
by OGD stimulation for 30- or 60-min shRNA-mediated knockdown of
TRIM9
expression
in hNPC-derived neurons led to robust I
κ
B
α
degradation and increased p65 phosphorylation
upon OGD stimulation (Figures S5F and S5G). Furthermore,
TRIM9
-specific shRNA-
treated hNPC-derived neurons were more vulnerable to OGD-induced cell death than
scramble shRNA-treated neurons (Figures 4C and 4D). These results indicate that TRIM9 is
required for neuronal survival under ischemia and inflammation.
As our previous study demonstrates the TRIM9-mediated suppression of NF-
κ
B activation
via its interaction with
β
-transducin repeat-containing protein (
β
-TrCP) (
Shi et al., 2014
), we
further showed the TRIM9 and
β
-TrCP interaction in brain tissue of MCAO-treated or sham-
operated mice (Figure 4E). In addition, primary neurons isolated from
Trim9
−/−
embryos
were infected with lentivirus containing vector, TRIM9-WT, or S
76
AS
80
A (SA) mutant that
no longer interacts with
β
-TrCP (
Shi et al., 2014
), followed by stimulation with OGD, TNF-
α
, or IL-1
β
. Our data showed that the substitution of TRIM9-WT allowed
Trim9
−/−
primary
neurons to regain their ability to suppress stimulation-induced NF-
κ
B signaling, whereas the
substitution of the TRIM9-SA mutant showed little or no effect on NF-
κ
B signaling (Figures
4F, 4G, and S5H–S5K). When lentivirus-infected primary
Trim9
−/−
neurons were treated
with TNF-
α
and tested for the
β
-TrCP interaction with TRIM9 or I
κ
B
α
, TRIM9-WT, but
not TRIM9-SA mutant, effectively competed with I
κ
B
α
for the
β
-TrCP interaction (Figure
4H). Consequently,
Trim9
−/−
primary neurons showed a higher I
κ
B
α
ubiquitination upon
TNF-
α
+ MG132 treatment than
Trim9
+/+
primary neurons (Figure 4I). qRT-PCR also
showed higher expression of the inflammatory mediators IL-6, TNF-
α
, IL-1
β
, and CCL2 in
Trim9
−/−
primary neurons upon OGD stimulation than in
Trim9
+/+
primary neurons (Figure
4J). These results indicate that TRIM9’s interaction with
β
-TrCP is critical for fine-tuning
NF-
κ
B signaling and inflamma-tory responses in neurons.
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AAV-PHP.B-Mediated Brain-wide TRIM9 Expression Ameliorates Ischemic Injury in Trim9
−/−
Mice
To explore whether
in vivo
brain-wide expression of
Trim9
could alleviate ischemia-induced
neuroinflammation and brain injury in mice, we utilized an engineered variant of adeno-
associated virus (AAV) serotype 9 (AAV-PHP.B) that can efficiently transfer genes
throughout the CNS following systemic delivery (
Deverman et al., 2016
;
Morabito et al.,
2017
). AAV-PHP.B-mediated expression of the green fluorescent protein (GFP), the Flag-
tagged murine TRIM9-WT or the SA mutant was readily detected in
Trim9
−/−
mouse
embryonic fibroblasts (MEFs) and primary neurons (Figure S6A; see also STAR Methods).
Subsequently, AAV-PHP.B:CAG-GFP (PHP.B-GFP), AAV-PHP.B:CAG-FLAG-TRIM9
(PHP.B-TRIM9), and AAV-PHP.B:CAG-FLAG-TRIM9-SA (PHP.B-SA) were administrated
twice to 12-week-old
Trim9
−/−
mice following a retro-orbital injection protocol (
Deverman
et al., 2016
) (Figure 5A). GFP and FLAG-tagged TRIM9-WT or SA mutant expression were
tested 21 days post-injection. Efficient expression of GFP, FLAG-tagged TRIM9-WT, or SA
mutant was observed in neurons throughout the brain, including the cortex and hippocampus
(Figures 5B and S6B). When compared with PHP.B-GFP- or PHP.B-SA-infected
Trim9
−/−
mice 24 hr after 30-min MCAO, PHP.B-TRIM9-infected
Trim9
−/−
mice showed
substantially reduced infarct volume (Figures 5C and 5D) and neurological impairments, as
indicated by neurological scores (Figure 5E). An immunoblotting assay of tissue extracts
from MCAO-induced ischemic brain hemispheres showed the reduced I
κ
B
α
degradation in
PHP.B-TRIM9-infected
Trim9
−/−
mice compared to PHP.B-GFP- or PHP.B-SA-infected
Trim9
−/−
mice (Figures S6C and S6D). Finally, when primary neurons isolated from
Trim9
−/−
mice were infected with PHP.B-GFP, PHP.B-TRIM9, or PHP.B-SA and then
subjected to OGD conditions, I
κ
B
α
degradation was considerably lower in PHP.B-TRIM9-
infected
Trim9
−/−
neurons than in PHP.B-GFP- or PHP.B-SA-infected
Trim9
−/−
neurons
(Figures S6E and S6F). These results collectively indicate that AAV-PHP.B-mediated brain-
wide TRIM9 expression effectively resolves inflammatory responses in neurons, providing
neuroprotection against ischemic stroke.
AAV-PHP.B-Mediated Brain-wide TRIM9 Expression Alleviates Ischemic Injury in Middle-
Aged Mice
A previous genome-wide analysis reports that the stroke-induced expression of a specific
group of genes, including
Trim9
, is considerably lower in the ipsilateral cortex of aged rats
than in that of young rats (
Buga et al., 2012
). A similar level of TRIM9 expression in the
brain was observed between 12-week-old young mice and 70-week-old middle-aged mice
under normal conditions. In contrast, the stroke-induced upregulation of TRIM9 expression
was detectably lower in aged mouse brains than in young mouse brains (Figures 6A and 6B).
The stroke-induced expression of two key inflammatory mediators, IL-6 (Figures 6C and
6D) and CCL2 (Figures 6E and 6F), was induced much higher in aged mouse brains than in
young mouse brains.
To examine
in vivo
expression of
Trim9
to alleviate ischemia-induced neuroinflammation in
aged mice, 70-week-old C57BL/6J mice were retro-orbitally injected with PHP.B-GFP or
PHP.B-TRIM9 and subjected to 30-min MCAO, followed by neurological tests at 24 hr.
When compared with PHP.B-GFP-infected aged mice, PHP.B-TRIM9-infected aged mice
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showed substantially reduced infarct areas (Figures 6G and 6H) and neurological
impairments, as indicated by neurological scores (Figure 6I). Confocal microscopy showed
that compared to PHP.B-GFP-infected aged mice, PHP.B-TRIM9-infected aged mice had
reduced levels of p-p65-positive neurons (NeuN) in the peri-infarct cortex (Figures 6J and
6K) and neuronal cell death, as indicated by TUNEL
+
NeuN
+
double-positive cells in the
infarct cortex (Figures 6L and 6M). The strong correlation between the p-p65-positive
neuron population level and the motor neurological behavior score was observed in PHP.B-
GFP- or PHP.B-TRIM9-infected aged mice (Figure 6N). These results collectively
demonstrate that AAV-PHP.B-mediated brain-wide TRIM9 expression effectively alleviates
neuroinflammation and ameliorates neuropathological and neurological outcomes after
ischemic stroke.
DISCUSSION
The acute inflammatory response and its resolution are indispensable for body’s
physiological repair process after injuries, while unresolved inflammation is implicated in a
body of human diseases, including CNS injuries (
Schwartz and Baruch, 2014
).
Inflammation resolution in the CNS is a highly organized active process that requires
complex crosstalk between injured neurons, glial cells, and infiltrating inflammatory cells
(
Schwartz and Baruch, 2014
). However, brain factors that govern the resolution of
neuroinflammation are yet to be defined (
Iadecola and Anrather, 2011
). By revisiting the
brain’s endogenous genes within the inflammatory response network that were activated
upon ischemic injury, we identified TRIM9 as a brain-specific modulator of NF-
κ
B-
dependent neuroinflammation (Figure 1). It governs the resolution of post-ischemic
inflammation and consequently improves neuropathological and neurological outcomes in
the mouse model of acute ischemic stroke (Figure 2). Accordingly,
Trim9
deficiency is
accompanied by elevated pro-inflammatory cytokine production, inflammatory cell
infiltration, subsequent neuronal death, and exacerbated brain injury after MCAO (Figures 2
and 3).
Pro-inflammation, anti-inflammation, and pro-resolution signals converge on the NF-
κ
B
pathway. In acute ischemia, NF-
κ
B activation in injured neurons initiates strong
inflammatory responses via production of proinflammatory mediators. TRIM9 expression is
strongly increased in the peri-infarct area in a mouse stroke model (Figure 1G). This delayed
surge of
Trim9
expression ensures timely antagonization of NF-
κ
B activity and tissue
resolution of inflammation, as we showed that the brain-wide expression of TRIM9
effectively promoted resolution of neuroinflammation and alleviated neuronal death in
TRIM9-deficient mice (Figure 5). Our findings indicated that the balance between these
pathways could be adjusted temporally by TRIM9 after ischemic stroke. AAV-PHP.B-
mediated TRIM9 expression ameliorates the ischemic neuropathology and neurological
outcomes in young
Trim9
−/−
mice (Figure 5) and 70-week-old WT mice (Figure 6). This
suggests that targeting TRIM9 for better resolution of neuroinflammation may offer a
potential target for immunomodulatory therapy for acute ischemia. Previous studies have
shown that the infiltration pattern of peripheral immune cells and timing of immune
activation differ in stroke (
Chamorro et al., 2012
;
Iadecola and Anrather, 2011
;
Jin et al.,
2010
), suggesting that the infarct or behavior could possibly change over time. Investigation
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of stroke-related neuropathology on later time-point than 24 hr could provide more useful
information about the resolution of inflammation, especially in aged mice.
Older women have a much higher incidence of stroke than men as well as a worse prognosis,
which is recapitulated in rodent model; for instance, aged female mice display a larger
infarct volume upon stroke than aged male mice (
Liu et al., 2009
). In the current study, aged
female mice were specifically chosen to evaluate the therapeutic potential of AAV-based
gene therapy, since TRIM9 expression was similar between young male and female mice
(Figure S1D). As a huge body of literature on stroke and inflammation has used young male
mice as MACO animal models (
Herrmann et al., 2005
;
Kawano et al., 2006
;
Liesz et al.,
2009
,
2013
;
Reischl et al., 2014
;
Sarabi et al., 2008
;
Shichita et al., 2009
,
2012
,
2017
;
Strecker et al., 2011
;
Wang et al., 2016
), and we also utilized young male mice to maintain
consistency with previous studies. It is well known that the aged population is not only
highly susceptible to ischemic stroke but also shows slow recovery from ischemic stroke.
However, the basis of this high susceptibility and slow recovery of the aged population is
very complex, with numerous contributing factors. We found that the upregulation of
TRIM9 expression appeared to be critical to resolve inflammation in the peri-infarct region
of brain and minimize the injury size. However, this stroke-induced upregulation of TRIM9
expression was compromised in aged mice, causing sustained neuroinflammation and
enhanced ischemic damage. Induction of
Trim9
expression in the peri-infarct areas of
ischemic brain occurred during the early stage. Additional studies are necessary to
understand the molecular mechanism of Trim9 expression under normal conditions versus
ischemic stroke conditions.
The MCAO model we used here is an acute stroke mouse model, which does not allow a
sufficient time window for target gene delivery for gene therapy. For example, while AAV
mutant AAV-PHP.B enabled brain-wide expression of TRIM9 gene with high efficiency
(
Deverman et al., 2016
), TRIM9 gene delivery still required days (or even more than a
week) after AAV injection. Alternative methods to quickly upregulate endogenous TRIM9
expression may provide its systemic expression for post-stroke treatment. Lastly, the pre-
stroke strategies may be utilized as prevention approaches, which would represent a
potential therapeutic method for the aging population, especially in at-risk older women.
Further study is needed to evaluate a number of methods to upregulate brain-specific TRIM9
expression.
Parkinson’s disease (PD), the second most common age-associated neurodegenerative
disorder, is characterized by the loss of dopaminergic neurons (
Obeso et al., 2010
), as well
as chronic neuroinflammation (
McGeer and McGeer, 2004
). Specifically, post-mortem
analyses of human PD patients and experimental animal studies shows that the chronic
increase of proinflammatory cytokines and the infiltration and accumulation of immune cells
from the periphery are also implicated in the pathogenesis of PD (
Barcia et al., 2003
;
Hirsch
and Hunot, 2009
;
Hirsch et al., 2012
). Intriguingly, a previous study showed that Trim9
expression was significantly downregulated in the brains of PD and Lewy body dementia
(
Tanji et al., 2010
). We showed that AAV-PHP.B-mediated Trim9 expression markedly
reduced neuroinflammation in young Trim9
−/−
mice (Figure 5) and middle-aged WT mice
(Figure 6), leading to improvement of the ischemic neuropathology and neurological
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outcomes. This suggests that manipulating Trim9 expression for better resolution of
neuroinflammation may offer prospect to immunomodulatory therapy for PD, ischemic
stroke, and even other neurodegenerative diseases. A better understanding of the role of
TRIM9-mediated regulation of inflammation would provide insight into the
neuropathological processes and help to establish effective therapeutic strategies.
STAR
★
METHODS
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and request for resources and reagents should be directed to and will be
fulfilled by Lead Contact Jae U. Jung (jaeujung@med.usc.edu).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell culture—
HEK293 cells were purchased from ATCC (catalog # CRL-11268; the sex is
female) and maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and
100 U ml
−1
penicillin-streptomycin. IL-1
β
(Biolegend, catalog # 575102), TNF-
α
(Biolegend, catalog # 575202), and proteasome inhibitor MG132 (R&D Systems, catalog #
1748) were used for stimulation on mouse primary neurons.
Mouse primary neuron culture—
Cerebral cortices from E18 mouse embryos (the sex is
unavailable) were dissected, carefully stripped of their meninges, digested with TrypLE
Express Enzyme (Thermo Fisher Scientific, catalog # 12604013) with DNaseI (0.5 mg/ml)
(Sigma, catalog # AMPD1) for 20 min at 37°C, and dispersed to single-cell level by passing
through a cell strainer (70 μm). The cell suspension was then cultured with Neurobasal
medium supplemented with B27 Supplement (Thermo Fisher Scientific, catalog #
17504044) at 37°C in humiliated 5% CO
2
, 95% air on poly-D-Lysine (Millipore, catalog #
A-003-E) and laminin (Thermo Fisher Scientific, catalog # 23017015) pre-coated coverslips
or in 12-well culture plates. Medium was replaced at 50% every other day.
Human neural progenitor cell (hNPC)-derived neurons—
Human neural progenitor
cell (hNPC) from fetal origin (the sex is unavailable) (
Guo et al., 2013
;
Wang et al., 2010
)
were maintained as neurospheres in DMEM/F12-N2 supplemented with 20 ng/ml (1.4 nM)
bFGF (Sigma-Aldrich, St. Louis, MO) in low-attachment T-25 flasks (Corning, Acton, MA)
(
Wang et al., 2016
). For differentiation, hNPC were plated on poly-L-Orthithine (10 μg/ml)
and laminin (10 μg/ml) coated round cover glasses in 4-well plates at a density of 4 × 10
5
per well in STEMdiff Neuron Maturation medium (StemCell Technologies, Inc.) and
incubated for 10 days
in vitro
to allow for differentiation.
Mouse embryo fibroblasts (MEFs)—
MEFs from
Trim9
−/−
mice (the sex is unavailable)
was isolated and cultured, as previously described (
Durkin et al., 2013
). Briefly, E13
embryos were harvested and digested by 0.25% trypin-EDTA. Cell suspension was cultured
with MEF culture medium containing DMEM supplemented with 10% FBS and 100 U ml
−1
penicillin-streptomycin in T75 flasks.
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Mice—
The Institutional Animal Care and Use Committee at the University of Southern
California approved all procedures per the National Institutes of Health guidelines. Mice
were housed in plastic cages on a 12h light cycle, with
ad libitum
access to water, with
standard laboratory diet, and in a specific pathogen-free facility.
Trim9
+/−
heterozygous mice
(
Winkle et al., 2014
) were bred to generate age-matched
Trim9
−/−
and littermates
Trim9
+/+
mice. Male (
Liesz et al., 2009
;
O’Collins et al., 2006
;
Shichita et al., 2009
,
2012
)
Trim9
+/+
and
Trim9
−/−
mice with 12-week-old were used. C57BL/6J mice were purchased from the
Jackson Laboratory (JAX stock 000664). Because there is a significant
sex
effect on the
pathology of brain ischemia (
Liu et al., 2009
;
Wendeln et al., 2018
), where both male and
female mice were compared, only female aged C57BL/6J mice with 70-week-old were used
in the present study. All the animals that survived surgical procedures were included in the
study. All animals were randomized for all stroke studies and procedures. All experiments
were blinded; the operators responsible for experimental procedure and data analysis were
blinded and unaware of group allocation throughout the experiments.
METHOD DETAILS
Transient middle cerebral artery occlusion (MCAO)—
Using a 27-½ gauge needle,
mice were anesthetized with 100 mg/kg Ketamine intraperitoneally (IP) and 10 mg/kg IP
xylazine. Rectal temperature was maintained at 37°C using a feedback-controlled heating
system. MCA was occluded for 30 minutes using a silicon-coated nylon monofilament
(DOCCOL, CO) as we previously described (
Wang et al., 2012b
). Cerebral blood flow was
monitored by laser Doppler flowmetry (Transonic Systems). Mice with an adequacy of
MCAO as evidenced by R 80% drop in the cerebral blood flow were included in the study.
Motor neurological examination was determined after 24 hours, using the following criteria:
no neurological deficit, 0; failure to extend left forepaw fully, 1; turning to left, 2; circling to
left, 3; unable to walk spontaneously, 4; and stroke-related death, 5. All mice were
euthanized 0, 1, 4, 12, 24, 36, 48, or 72 hours after the MCAO for indicated analysis. All
animals that were survived from surgical procedures were included in the study.
Immunoblotting—
Brain tissue or cell lysates were collected in 1% NP40 buffer with the
protease inhibitor cocktail (Roche, catalog # 4693159001) and phosphatase inhibitor
PhosSTOP (Roche, catalog # 4906845001), and protein amounts were quantified by BCA
protein assay kit (Thermo Fisher Scientific, catalog # 23227). Proteins were separated by
SDS-PAGE and transferred to PVDF membrane (Bio-Rad, catalog # 1620177)) by semi-dry
transfer at 25V for 30 min. All membranes were blocked in 5% milk in PBST for 1 h and
probed overnight with indicated primary antibodies in 5% BSA at 4°C. Primary antibodies
included: mouse monoclonal anti-I
κ
B
α
antibody (Cell Signaling, catalog # 9247, 1:2000),
rabbit monoclonal anti-phospho-I
κ
B
α
antibody (Cell Signaling, catalog # 2859, 1:1000),
rabbit monoclonal anti-phospho-p65 antibody (Cell Signaling, catalog # 3033, 1:1000),
mouse monoclonal anti-
β
-actin antibody (Santa Cruz, catalog # sc-47778, 1:2000), rabbit
polyclonal anti-TRIM9 antibody (
Winkle et al., 2014
) (1:2000, generated using murine
TRIM9 recombinant protein aa 158–271; reacting with three isoform a/b/c), mouse
monoclonal anti-ubiquitin antibody (Santa Cruz, catalog # sc-8017, 1:1000), rabbit
monoclonal anti-
β
-TrCP antibody (Cell Signaling, catalog # 4394, 1:1000), mouse
monoclonal anti-HA antibody (Santa Cruz, catalog # sc-57594, 1:2000), mouse monoclonal
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anti-GFP antibody (Santa Cruz, catalog # sc-101525, 1:2000), and mouse monoclonal anti-
Flag antibody (Sigma, catalog # F1804, 1:2000). Appropriate HRP-conjugated secondary
antibodies were incubated on membranes in 5% milk and bands were developed with
ChemiDoc Touch imaging system (Bio-Rad) and analyzed in Image Lab software.
Tissue staining—
At endpoint, mice were anesthetized and transcardially perfused with
PBS and fixed with 4% PFA. Mouse brains were post-fixed overnight in 4% PFA at 4°C. For
cryosectioning, fixed tissues were cryoprotected in 30% sucrose in PBS overnight at 4°C
and embedded in Tissue-Tek OCT compound (VWR, catalog # 25608–930). Cryostat
sections were cut at 20 μm thickness. Mouse brain sections were permeabilized in PBS-T
(PBS containing 0.2% Triton X-100) for 10 min, blocked with 5% normal donkey serum
(Jackson ImmunoResearch, catalog # 017-000-121) for 60 min and incubated in primary
antibody diluted in the blocking solution overnight at 4°C. Primary antibodies used in this
study include mouse monoclonal anti–glial fibrillary acidic protein (GFAP) (cell signaling,
catalog # 3670), Rabbit monoclonal anti-Iba1 (Abcam, catalog # ab178846), Rabbit
polyclonal anti-NeuN (Millipore, catalog # ABN78), Mouse monoclonal anti-NeuN
(Millipore, catalog # MAB377), Mouse monoclonal anti-SMI-312 (Abcam, catalog #
ab24574), Rabbit polyclonal anti-NG2 (Abcam, catalog # ab83178), Mouse monoclonal
anti-Olig2 (Abcam, catalog # MABN50), Mouse monoclonal anti-Flag (Sigma, catalog #
F1804), Rabbit monoclonal anti-phospho-p65 (Cell Signaling, catalog # 3033), Rat
monoclonal anti-IL6 (Thermo Fisher Scientific, catalog # AMC0864), and Rabbit polyclonal
anti-CCL2 (Abcam, catalog # 9779). After three washes with PBS, sections were incubated
with the secondary antibodies for 1 h, including Alexa 488-conjugated donkey anti-mouse
(Thermo Fisher Scientific, catalog # A21202), Alexa 488-conjugated donkey anti-rabbit
(Thermo Fisher Scientific, catalog # A-21206), Alexa 568-conjugated donkey anti-mouse
(Thermo Fisher Scientific, catalog # A10037), Alexa 568-conjugated donkey anti-rabbit
(Thermo Fisher Scientific, catalog # A10042), Alexa 647-conjugated donkey anti-rabbit
(Thermo Fisher Scientific, catalog # A-31573), Alexa 488-conjugated donkey anti-rat
(Thermo Fisher Scientific, catalog # A-21208), and Alexa 647-conjugated donkey anti-rat
(Jackson ImmunoResearch Laboratories, catalog # 712-605-153). All images were taken
with the Zeiss 510 confocal microscopy or using the BZ 9000 all-in-one Fluorescence
Microscope from Keyence (Osaka, Japan), and analyzed using NIH ImageJ software.
RNA sequencing—
RNA was prepared using TRIzol Reagent (Thermo Fisher Scientific,
catalog # 15596018) followed by RNeasy Mini Kit (QIAGEN, catalog # 74104). The
libraries were made using KAPA stranded mRNA-seq kits (KAPA Biosystems, catalog #
kk8421) according to manufacturer’s protocol. The cDNA libraries were sent to the
Technology Center for Genomics & Bioinformatics at UCLA for sequencing on HiSeq 3000
with single end read (~30 million reads per sample). RNA-seq data was inspected by FastQC
(
Simon, 2010
). Using default settings, STAR version 2.5.2b (
Dobin et al., 2013
) were used
to map reads to mouse genome GRCm38. Alignment results were processed for gene
quantification using featureCounts version 1.5.1 (
Liao et al., 2014
) with the second strand-
specific option. Differentially expressed genes were determined using DESeq2 version
1.14.1 (
Love et al., 2014
). The significantly changed genes were analyzed using the
Ingenuity Pathway Analysis tool for functional analysis. The comparison of expression
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levels of the upregulated genes was performed by RPKM value of each gene from existing
RNaseq data in NCBI.
RNAscope
in situ
hybridization—
Trim9
mRNA transcripts were detected using murine
gene-specific probe (Advanced Cell Diagnotics, catalog # 479071) and visualized using the
RNAscope 2.5 HD Reagent Kit RED (Advanced Cell Diagnotics, catalog # 322360) on 4%
paraformaldehyde (PFA) fixed frozen mouse brain tissue sections, according the
manufacturer’s instructions, followed by counterstaining with hematoxylin (Vector
Laboratories, catalog # H3401). Sections were imaged using the BZ 9000 all-in-one
Fluorescence Microscope from Keyence (Osaka, Japan), and analyzed using NIH ImageJ
software.
Visualization of the anastomotic line between the middle cerebral artery
(MCA) and anterior cerebral artery (ACA)—
We performed an assay with the
procedures as previously described (
Wang et al., 2005
). Briefly, latex mixed with carbon
black was injected through the cannulated aorta of mouse. Anastomotic lines between the
MCA and the ACA territories were determined by tracing peripheral branches on dorsal
brain surfaces of mice to the points at which vessels were connected (dotted red line).
Cresyl violet staining and neuropathological analysis—
Mouse brain sections from
five equidistant rostrocaudal brain levels, at −1.6 mm, 0.8 mm, 0 mm, 0.8 mm and 1.6 mm
from bregma, were fixed by methanol and stained with the Cresyl Echt Violet staining − kit
(American − MasterTech, catalog # AHC0443). Sections were digitized and transformed
into gray model, and the border between infarct and non-infarct tissue was outlined using an
image analysis system (ImageJ). On these sections, infarct volume and brain swelling were
quantified. The infarct volume was calculated by subtracting the volume of the non-lesioned
area in the ipsilateral hemisphere from the volume of the whole area in the contralateral
hemisphere (
Wang et al., 2013
). The edema volume was calculated by subtracting the
volume of the contralateral hemisphere from the volume of the ipsilateral hemisphere (
Wang
et al., 2005
).
RT-qPCR—
Total RNAs were extracted from mouse brain tissues or mouse primary neurons
using TRIzol Reagent (Thermo Fisher Scientific, catalog # 15596018) followed by RNeasy
Mini Kit (QIAGEN, catalog # 74104) according to the manufacturer’s instructions. The
purified RNA was reversely transcripted to cDNA using iScript cDNA Synthesis Kit (Bio-
Rad, catalog # 1708891). All gene transcripts were quantified by quantitative PCR using iQ
SYBR Green supermix (Bio-Rad, catalog # 1708880) on CFX96 real-time PCR system
(Bio-Rad). Primer sequences are listed in Table S3.
ELISA—
Mouse brain tissues were isolated and homogenized mechanically. The resulting
supernatants were collected and their concentration of IL-6, IL-1
β
, TNF-
α
or IL-10 was
determined with a mouse-specific ELISA kit (BD Biosciences, catalog # 555240 for IL-6,
559603 for IL-1
β
, 555268 for TNF-
α
, 555252 for IL-10), and this was followed by analysis
with the FilterMax F5 multi-mode microplate reader (Molecular Devices).
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TUNEL staining—
Terminal deoxynucleotidyl transferase-mediated dUTP nick
endlabeling (TUNEL) assay was employed to demonstrate apoptotic cells. Using the
In Situ
Cell Death Detection Kit, Fluorescein (Roche, catalog # 11684795910) or
In Situ
Cell Death
Detection Kit, TMR red (Roche, catalog # 12156792910), brain sections were treated
following the procedure specified by the manufacturer.
Analyzing infiltrating immune cells—
The mice were transcardially perfused with PBS
containing 0.05 M EDTA extensively to remove blood cells in the circulation. The forebrain
(bregma from −3 to 3) of the hemisphere was removed and dissociated using neural tissue
dissociation kit (MACS Miltenyl Biotec, catalog # 130093231). Ipsilateral (ischemic)
hemisphere of the forebrain was used while contralateral hemisphere of the fore-brain was
used as sham control. Debris was removed by Debris Removal Solution (MACS Miltenyl
Biotec, catalog # 130109398) followed by removal of red blood cells (Biolegend, catalog #
420301). Cell suspensions were stained with propidium iodide (Invitrogen, catalog # P3566)
and fluorochrome-conjugated antibodies including CD45 (APC/Cy7, 1:100, catalog #
103116), CD11b (eFluor450, 1:100, catalog # 48-0112-82), CD11c (PE/Cy7, 1:100, catalog
# 117318), CD3e (PerCP/Cy5.5, 1:100, catalog # 100328), or GR1 (APC, 1:100, catalog #
108412). Subsequently, the stained suspensions were analyzed with BD FACSCanto II flow
cytometer and analyzed with FlowJo software. Each cell type was indicated by percentage of
CD45 positive cells. All antibodies were purchased from eBioscience or BD PharMingen.
Propidium iodide staining was used to gate live cells.
Immunocytochemistry—
Cultured mouse primary neurons were fixed with 4% PFA at
room temperature for 20 min, permeabilized, and stained with primary antibodies, including
rabbit polyclonal anti-TRIM9(
Winkle et al., 2014
) and mouse monoclonal anti-MAP2
(Abcam, catalog # ab11267), followed by incubation of secondary antibodies including
Alexa 488-conjugated donkey anti-mouse (Thermo Fisher Scientific, catalog # A21202) and
Alexa 568-conjugated donkey anti-rabbit (Thermo Fisher Scientific, catalog # A10042). All
images were taken with the BZ 9000 all-in-one Fluorescence Microscope from Keyence
(Osaka, Japan) and analyzed using NIH ImageJ software.
Oxygen and Glucose Deprivation (OGD)—
Mouse primary cortical neurons or hNPC-
derived neurons were treated with OGD by replacing culture medium with a glucose-free
Neurobasal medium (Thermo Fisher Scientific, catalog # 21103049), and they were
immediately placed in a hypoxic incubator chamber (STEMCELL, catalog # 27310) flushed
with a gas mixture of 94%N
2
/5%CO
2
/1%O
2
. After OGD for the indicated time, medium
was replaced by standard medium, and cells were cultured under normal condition for
reoxygenation of 24 h.
Lentivirus infection—
Lentiviral vector-mediated
in vitro
gene delivery was performed as
previously described (
Shi et al., 2014
). Briefly, hNPC-derived neurons or mouse primary
neurons were cultured with medium containing 5 μg/ml polybrene (Sigma, catalog # H9268)
and 5 × 10
5
infectious units of lentiviruses containing the following: scrambled shRNA,
TRIM9
-specific shRNAs (target sequence for human: 5-
CGATGCCCTCAACAGAAGAAA-3), pCDH lentivirus expressing mouse HA-TRIM9, or
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pCDH lentivirus expressing HA-SA mutant of TRIM9. At 48 h post infection, cells were
further treated with OGD or other conditions as indicated.
Live-Dead Cell assay—
hNPC-derived neurons were washed with PBS. LIVE-DEAD
Viability/Cytotoxicity Kit (Thermo Fisher Scientific, catalog # L3224) was used to quickly
discriminate live from dead cells by simultaneously staining with green-fluorescent calcein-
AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to
indicate loss of plasma membrane integrity. The samples were immediately imaged by the
BZ 9000 all-in-one Fluorescence Microscope from Keyence (Osaka, Japan) and analyzed
using ImageJ software.
Immunoprecipitation—
For co-IPs, brain tissues and cells were lysed with RIPA
minimum lysis buffer (Millipore, catalog # 20–188)). After clarification and pre-clearing,
protein amounts were quantified by BCA protein assay kit (Thermo Fisher Scientific,
catalog # 23227). 1/10 of cell lysates were heated as whole cell lysate in immunoblotting,
and the remained 9/10 lysates were incubated for 16 h with indicated antibodies, followed
by additional incubation with Pierce Protein A/G Agarose (Thermo Fisher Scientific, catalog
# 20422) for 2h. Immune complexes were washed with lysis buffer and subjected to
immunoblotting analysis. For ubiquitination, cells were initially lysed with RIPA buffer
containing 1% SDS, then cell extracts were diluted with RIPA buffer to 0.1% SDS
concentration. Finally 1/9 of the diluted extracts were heated as whole cell lysate in
immunoblotting and the remained 9/10 lysates were subjected to IP and IB.
Adenovirus associated virus (AAV) mediated
in vivo
delivery of Trim9—
For
in
vivo
delivery of
Tirm9
to the central nervous system, we intravenously injected 12-week-old
Trim9
−/−
mice or 70-week-old C57BL/6J mice at
retro-orbital
site using a AAV-PHP.B
vector, a method that allows widespread gene transfer to the adult brain (
Deverman et al.,
2016
), either expressing mouse
Trim9
(NM_001110203.1) under the control of a CAG
promoter (AAVPHP.B:CAG-TRIM9), TRIM9-SA mutant (AAV-PHP.B:CAG-TRIM9-SA),
or a control vector encoding the green fluorescent protein (AAV-PHP.B:CAG-GFP). Retro-
orbital injections of 1.5 × 10
12
genome copies per mouse was performed twice at 21 and 18
days before MCAO, to allow sufficient re-expression of TRIM9 in the CNS.
QUANTIFICATION AND STATISTICAL ANALYSIS
The sample size chosen for our animal experiments in this study was estimated based on our
prior experience performing similar experiments. For all the bar graphs, data was expressed
as mean ± s.d. The various types of statistical analysis were performed using GraphPad
Prism and by an investigator blinded to the experimental conditions, including two-way
ANOVA test, Mann-Whitney
U
test, Wilcoxon matched pair test, one-way ANOVA followed
by Tukey’s post hoc analysis, one-way ANOVA followed by Bonferroni’s post hoc analysis,
and two-tailed Student’s t test was performed using GraphPad Prism.
P value
< 0.05 was
considered statistically significant.
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DATA AVAILABILITY
The RNA-seq data has been deposited to the NCBI GEO database under the accession
number GEO: GSE114652.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
ACKNOWLEDGMENTS
This research was supported in part by the NIH (grants CA200422, CA180779, DE023926, DE027888, DE28521,
AI073099, AI116585, AI129496, AI140718, and AI140705), the Hastings Foundation, and the Fletcher Jones
Foundation (J.U.J.); NIH grant 9R01NS090904-16 (B.V.Z.); the Alzheimer’s Association (grant NIRG-15-363387)
and Whittier Foundation (Z.Z.); the Cure for Alzheimer’s Fund (B.V.Z. and Z.Z.), NS090904, and Foundation
Leducq Translatlantic Network of Excellence for the Study of Perivascular Spaces in Small Vessel Disease
(reference 16 CVD 05) (B.V.Z.); and GM108970 (S.L.G.). We acknowledge funding from the Beckman Institute at
Caltech (to V.G. and B.E.D.) through the Resource Center for CLARITY, Optogenetics, and Vector Engineering.
V.G. is a Heritage Principal Investigator supported in this work by an NIH Director’s New Innovator Award
(DP20D017782 to V.G.).
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