TRIM9-Mediated Resolution of Neuroinflammation Confers Neuroprotectionupon Ischemic Stroke in Mice

TRIM9-Mediated Resolution of Neuroinflammation Confers

Neuroprotectionupon Ischemic Stroke in Mice

Jianxiong Zeng

1,9

Yaoming Wang

2,3,9

Zhifei Luo

Lin-Chun Chang

Ji Seung Yoo

Huan

Yan

Younho Choi

Xiaochun Xie

2,3

Benjamin E. Deverman

Viviana Gradinaru

Stephanie L. Gupton

6,7,8

Berislav V. Zlokovic

2,3,*

Zhen Zhao

2,3,*

, and

Jae U. Jung

1,3,10,*

Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of

Southern California, Los Angeles, CA 90033, USA

Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern

California, Los Angeles, CA 90033, USA

Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los

Angeles, CA 90033, USA

Department of Biochemistry and Molecular Medicine, Keck School of Medicine, University of

Southern California, Los Angeles, CA 90033, USA

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA

91125, USA

Neuroscience Center and Curriculum in Neurobiology, University of North Carolina at Chapel

Hill, Chapel Hill, NC 27599, USA

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel

Hill, NC 27599, USA

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel

Hill, NC 27599, USA

These authors contributed equally

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

Author manuscript

Cell Rep

. Author manuscript; available in PMC 2019 April 26.

Published in final edited form as:

Cell Rep

. 2019 April 09; 27(2): 549–560.e6. doi:10.1016/j.celrep.2018.12.055.

Author Manuscript

(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

-transducin repeat-

containing protein (

-TrCP) from the Skp-Cullin-F-box ubiquitin ligase complex, blocking I

degradation and thereby dampening nuclear factor

B (NF-

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-

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.

Zeng et al.

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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

B (NF-

B) signaling pathway in

in vitro

cell culture upon cytokine

stimulation (

Shi et al., 2014

). However, the

in vivo

role of TRIM9 in NF-

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-

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-

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

B kinase (IKK) complex phosphorylates

the amino-terminal serine residues (S32 and S36) of I

, 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-

B p50-p65 complex for transcriptional activation of

targeted genes. NF-

B activation in neurons occurs soon after brain ischemia as evidenced

by I

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-

B-activating IKK have shown that inhibiting NF-

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-

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-

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-

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-

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-

B activation showed that

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

(TNF-

) and IL-1

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

GR1

CD11b

granulocytes and

inflammatory monocytes and CD45

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-

B Signaling

To further examine the role of TRIM9 in brain-specific NF-

B activation, ischemic brains

were harvested 24 hr after MCAO and then subjected to immunoblotting analysis. This

revealed increased I

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-

or IL-1

for 30–120 min.

Immunoblotting assays showed that the degradation of I

was more robust in

Trim9

−/−

primary neurons upon stimulation with OGD (Figures 4A and 4B), TNF-

, or IL-1

(Figures S5B–S5E) than in

Trim9

+/+

primary neurons. Correspondingly, the increase of p65

phosphorylation was also more evident in

Trim9

−/−

primary neurons upon TNF-

or IL-1

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

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-

B activation

via its interaction with

-transducin repeat-containing protein (

-TrCP) (

Shi et al., 2014

), we

further showed the TRIM9 and

-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

A (SA) mutant that

no longer interacts with

-TrCP (

Shi et al., 2014

), followed by stimulation with OGD, TNF-

, or IL-1

. Our data showed that the substitution of TRIM9-WT allowed

Trim9

−/−

primary

neurons to regain their ability to suppress stimulation-induced NF-

B signaling, whereas the

substitution of the TRIM9-SA mutant showed little or no effect on NF-

B signaling (Figures

4F, 4G, and S5H–S5K). When lentivirus-infected primary

Trim9

−/−

neurons were treated

with TNF-

and tested for the

-TrCP interaction with TRIM9 or I

, TRIM9-WT, but

not TRIM9-SA mutant, effectively competed with I

for the

-TrCP interaction (Figure

4H). Consequently,

Trim9

−/−

primary neurons showed a higher I

ubiquitination upon

TNF-

+ MG132 treatment than

Trim9

+/+

primary neurons (Figure 4I). qRT-PCR also

showed higher expression of the inflammatory mediators IL-6, TNF-

, IL-1

, and CCL2 in

Trim9

−/−

primary neurons upon OGD stimulation than in

Trim9

+/+

primary neurons (Figure

4J). These results indicate that TRIM9’s interaction with

-TrCP is critical for fine-tuning

NF-

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

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

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-

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-

pathway. In acute ischemia, NF-

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-

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

(Biolegend, catalog # 575102), TNF-

(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

, 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

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

antibody (Cell Signaling, catalog # 9247, 1:2000),

rabbit monoclonal anti-phospho-I

antibody (Cell Signaling, catalog # 2859, 1:1000),

rabbit monoclonal anti-phospho-p65 antibody (Cell Signaling, catalog # 3033, 1:1000),

mouse monoclonal anti-

-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-

-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

, TNF-

or IL-10 was

determined with a mouse-specific ELISA kit (BD Biosciences, catalog # 555240 for IL-6,

559603 for IL-1

, 555268 for TNF-

, 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

/5%CO

/1%O

. 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

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

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

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

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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