RAPID COMMUNICATION

Transient Focal Cerebral Ischemia UpregulatesImmunoproteasomal Subunits

Lanhai Lu • Hongmin Wang

Received: 14 March 2012 / Accepted: 2 May 2012 / Published online: 22 May 2012

� Springer Science+Business Media, LLC 2012

Abstract This study was designed to determine whether

focal cerebral ischemia alters the expression of the

immunoproteasomal (i-proteasomal) subunits. Transient

cerebral ischemia significantly increased the expression of

the i-proteasomal subunits, 20S b1i (LMP2) and b5i

(LMP7) in the parietal cortex and hippocampus. This

alteration was associated with a remarkable increase in

ubiquitinated proteins. It is likely that the postischemic

induction of the i-proteasome plays an important role in

coping with the damaged proteins and thus may have an

important effect on neuronal survival and death.

Keywords Middle cerebral artery occlusion � Cerebral

ischemia � Neuron � Immunoproteasome � Ubiquitin �Mouse brain

Introduction

Transient cerebral ischemia is associated with an inflam-

matory response and a rapid and excessive production of

various misfolded proteins due to oxidative stress and other

mechanisms (Ge et al. 2007). Overproduction of damaged

proteins following ischemia is reflected in a pronounced

increase of conjugation of targeted proteins with ubiquitin

(Hayashi et al. 1992) in specific brain regions. It remains

unknown how cells, especially neurons, of the brain

respond to the dramatic increase of ubiquitinated proteins.

The proteasome is the major protein degradation

machinery present in both the cytoplasm and nucleus. The

functional proteasome is a 26S large protein complex

composed of multiple subunits. It contains the multicata-

lytic 20S core complex and two 19S regulatory complexes

that regulate substrate binding, unfolding, and entry into

the 20S subunits (Dahlmann 2005). The 20S core particle

consists of four-stacked heptameric ring structures that are

themselves composed of two different types of subunits, asubunits (structural in nature) and b subunits (predomi-

nantly catalytic) (Zwickl et al. 1999). In response to viral,

bacterial or other types of stress, the three constitutive

catalytic subunits, b1, b2, and b5, can be replaced by the

three inducible subunits, low molecular weight protein 2

(LMP2 or b1i), multicatalytic endopeptidase complex like

1 (MECL1 or b2i), and b5i (LMP7), respectively (Loukissa

et al., 2000). When this occurs, the proteasomes that con-

tain the three catalytic immunosubunits are usually referred

to as the immunoproteasome (i-proteasome). In this report,

we show that transient focal cerebral ischemia can signif-

icantly upregulate the protein levels of i-proteasome sub-

units LMP2 and LMP7.

Materials and Methods

Animals and Focal Cerebral Ischemia

Adult male C57BL/6 mice (10–12 weeks old, 20–30 g)

were used in this study. All experiments were approved by

the Institutional Animal Care and Use Committee of the

University of South Dakota. Before experiment, mice were

fasted with free access to water overnight. Transient focal

cerebral ischemia was induced by unilateral occlusion

of the left middle cerebral artery (MCA). Mice were

L. Lu � H. Wang (&)

Division of Basic Biological Sciences, Sanford School of

Medicine, University of South Dakota, Vermillion, SD 57069,

USA

e-mail: hongmin.wang@usd.edu

123

Cell Mol Neurobiol (2012) 32:965–970

DOI 10.1007/s10571-012-9854-y

anesthetized in a chamber filled with 5 % isoflurane/100 %

oxygen using a respirator (E–Z systems). The inspired

isoflurane concentration was then reduced to 1.5–2% until

the surgery was complete. Focal cerebral ischemia was

induced by inserting an intraluminal filament into the left

MCA according to methods described previously (Moisse

et al. 2008). In brief, a midline incision was made on the

ventral surface of the neck, and the common carotid artery

and the internal and external carotid artery were isolated

from the vagus nerve and its sheath under a stereomicro-

scope (Labomed). The left external carotid artery was then

ligated with a 6.0 silk suture and the internal carotid artery

was clipped using a microvascular clip before an arteriot-

omy was performed between the proximal ligated suture

and the vessel clip on the common carotid artery (CCA)

with microscissors. The heat-blunted 5-0 nylon suture was

then introduced via the arteriotomy and slowly advanced

through the left internal carotid artery to block the left

MCA. After 15 or 30 min of occlusion of the MCA, the

occluding filament was gently removed to allow reperfu-

sion for 3 or 24 h. After the surgery, the mouse body

temperature was maintained at 37 �C using a heating pad

(Gaymar T/Pump Heat Therapy System). When the mice

regained complete consciousness, their neurological deficit

was evaluated using a five-point scale as previously

described (Atochin et al. 2003). The animals that were

scored between 2 and 3 were included in the experiments.

TTC Staining

Twenty-four hours after reperfusion, the animals were

sacrificed and their brains were immediately removed. Four

coronal sections (2 mm thick) were promptly made from

the olfactory bulb to the cerebellum using a brain slicer.

The sections were stained in 2 % 2,3,5-triphenyltetrazoli-

um chloride (TTC) (Sigma, St. Louis, MO) dissolved in

normal phosphate-buffered saline (PBS) and stained for

20 min at room temperature in the dark. The sections were

then washed twice with PBS and fixed with 10 % formalin

(Sigma, St. Louis, MO) for 30 min at room temperature.

The caudal face of the TTC-stained brain sections derived

from the ischemic animals that were scored only between 2

and 3 was photographed.

Western blot Analysis

Samples used for Western blot were isolated from the

ischemic (ipsilateral) and control (contralateral) hemi-

spheres from the frontal and parietal cortex and the hip-

pocampus. The brain tissues were lysed with tissue lysis

buffer (50 mM Tris–HCl, pH 6.8, 150 mM NaCl, 20 mM

EDTA, 1 mM EGTA, 0.5 % SDS, 0.5 % NP40, 0.5 %

sarkosyl, and protease inhibitor cocktail) and centrifuged at

14,0009g for 20 min at 4 �C. The supernatant was used for

Western blot analysis. Protein concentration in the super-

natant was determined using the BCA assay kit (Pierce)

and the procedure for the Western blot was described

previously (Dong et al. 2012). The primary antibodies used

in the studies include the antibodies against proteasomal

20S b1i (LMP2) (Enzo, 1:2,000), 20S b2i (MECL1) (Enzo,

1:2,000), 20S b5i (LMP7) (Enzo, 1:2,000), ubiquitin

(Enzo, 1:2,000), and b-tubulin (Cell Signaling, 1:5,000).

All horse radish peroxidase-conjugated secondary anti-

bodies (used at 1:5,000 dilution) were purchased from

Santa Cruz Biotechnology. Western blot results were

quantified with film scanning software (UN-SCAN-IT

gel6.1).

Statistical Analysis

Data are presented as mean ± SD. Statistical comparisons

between two groups were evaluated using two-tailed

student’s t test. P \ 0.05 was regarded as statistically

significant.

Results and Discussion

Transient cerebral ischemia is associated with a rapid and

excessive production of various misfolded proteins due to

oxidative stress (Ge et al. 2007) and a pronounced increase

of conjugation of targeted proteins with ubiquitin (Hayashi

et al. 1992) in specific brain regions. However, it remains

unknown how cells, especially neurons, of the brain

respond to this. To examine whether transient focal cere-

bral ischemia alters expression of the i-proteasomal sub-

units, we performed middle cerebral artery occlusion for 15

or 30 min. After 24 h of reperfusion, brain injury levels

were determined by triphenyltetrazolium chloride (TTC)

staining (Fig. 1a) and the i-proteasomal subunits were

examined by Western blot analysis. As shown in Fig. 1b,

after 15 min of MCA occlusion and 24 h reperfusion,

LMP2 increased nearly threefold in the cortex and 3.6-fold

in hippocampus of the ischemic hemisphere compared to in

the control hemisphere, whereas LMP7 increased 1.4-fold

in the cortex and 1.8-fold in the hippocampus of the

ischemic hemisphere (Fig. 1b). The elevated i-proteasomal

subunit expression corresponded with an increase of

ubiquitinated proteins, more evident in the hippocampus of

the ischemic hemisphere as showed as a smear (Fig. 1b)

suggesting that numerous proteins with ubiquitin are

present following ischemia/reperfusion.

The expression pattern of the two i-proteasomal subunits

induced by 30 min MCA occlusion (Fig. 1c) is similar to

that induced by 15 min MCA occlusion. Both LMP2 and

LMP7 expression showed a significant rise in both the cortex

966 Cell Mol Neurobiol (2012) 32:965–970

123

and hippocampus of the ischemic hemisphere (Fig. 1b).

However, the expression of MECL1 (20S b2i) did not show

any significant change in the examined areas of the ischemic

animals (data not shown). These data indicate that the

transient focal cerebral ischemia upregulates LMP2 and

LMP7 subunits in the brain regions examined.

To further determine the temporal relationship between

transient cerebral ischemia and proteasome induction, we

also examined the two i-proteasomal subunit levels after

0.5, 1, and 3 h reperfusion following 30 min MCA

occlusion. Thirty minutes ischemia followed by \1 h

reperfusion did not induce LMP7 overexpression and only

slightly upregulated LMP2 expression (data not shown).

However, following 30 min MCA occlusion and 3 h of

reperfusion, both LMP2 and LMP7 proteins showed

remarkable upregulation in the cortex of the ischemic

hemisphere (Fig. 1d). Meanwhile, a constitutive protea-

some subunit of the 20S proteasome, 7a, did not show a

significant change in level in the same brain region

examined under the same conditions (Fig. 1d).

Fig. 1 Effect of transient MCA occlusion on LMP2 (b1i) and LMP7

(b5i) expression. Representative photographs a showing cerebral

infarction in coronal forebrain sections (2 mm thick) stained with 2 %

TTC following 15 min (upper panel) or 30 min (lower panel) MCA

occlusion and 24 h reperfusion in age-matched mice. The unstained

regions of the brain sections indicate ischemic infarct damage due to

occlusion of the middle cerebral artery (MCA). Alternatively,

following the ischemic stroke and reperfusion, brains samples were

taken from the cortex and hippocampus of the ischemic (ischemia)

and nonischemic (control) hemispheres and used for protein analysis

by Western blot. *40 lg of total protein was loaded on each lane and

the expression levels of LMP2 and LMP7 were evaluated by Western

blot analysis. Ischemia-induced changes in LMP2 and LMP7 were

evaluated after 15 min MCA occlusion and 24 h of reperfusion (b),

after 30 min MCA occlusion and 24 h of reperfusion (c), and after

30 min MCA occlusion and 3 h of reperfusion (d). Protein band

intensities of LMP2 or LMP7 were measured and normalized against

b-tubulin level. A constitutive proteasome subunit 7a of the 20S

proteasome was also immunoblotted (d), although ischemia did not

upregulate its expression. Data are presented as means ± SD n = 3.

*P \ 0.05; **P \ 0.01

Cell Mol Neurobiol (2012) 32:965–970 967

123

Next, we performed immunohistochemical staining and

fluorescence microscopy to determine whether increased

i-proteasomal subunits are found in neurons or in glia

following MCA occlusion and reperfusion. Images were

captured from the border regions of the MCA territory

where cells showed strong immunoreactivity after ischemia

(data not shown). After 15 min occlusion and 24 h reper-

fusion, both LMP2 and LMP7 showed weak expression and

even distribution of immunoreactivity in the sections of the

non-ischemic hemispheres (Fig. 2a, b, upper panels). In

Fig. 1 continued

968 Cell Mol Neurobiol (2012) 32:965–970

123

contrast, ischemia induced a remarkable increase in

expression of LMP2 and LMP7 in the same area of the

ischemic hemisphere (Fig. 2a, b, upper panels). Notably,

ischemic induction of the proteins was not evenly

distributed. Immunostaining using neuron-specific marker

NeuN revealed that both LMP2 and LMP7 immunoreac-

tivity was significantly higher in neurons than in non-

neuronal cells (Fig. 2a, b, lower panels). The upregulated

Fig. 2 Immunohistochemical analysis of the distribution of LMP2

(b1i) (a) and LMP7 (b5i) (b) expression in the contralateral control

hemisphere (control) and the ipsilateral ischemic hemisphere (ische-

mia) of animals subjected to 15 min MCA occlusion and 24 h of

reperfusion. Images were captured from the border regions of the

MCA territory of the parietal cortex. LMP2 and LMP7 are in redcolor and neurons were identified using a NeuN antibody (greencolor, arrows). Nuclei were stained with Hoechst 33342. The arrows-

pointed cells are shown at higher magnification in the insets and the

scale bars represent 10 lm (Color figure online)

Cell Mol Neurobiol (2012) 32:965–970 969

123

LMP2 and LMP7 proteins were seen in both the cytoplasm

and nucleus (Fig. 2a, b, insets of the lower panels). These

results indicate that the induction of the two i-proteasomal

subunits mainly occurred in neurons following transient

cerebral ischemia.

Recent data have shown that compared to the non-

demented elderly, both the Alzheimer’s disease-(Mishto

et al. 2006) and Huntington’s disease-affected brains

(Diaz-Hernandez et al. 2003) show a higher expression of

i-proteasomal subunits. Interestingly, inhibition of i-prote-

asomal induction decreases neuronal survival in a rat

model of amyotrophic lateral sclerosis (Ahtoniemi et al.

2007), suggesting that normal i-proteasomal activity is

required for neuronal survival. In this report, we demon-

strate that transient cerebral ischemia upregulates the

expression of the two i-proteasomal subunits, LMP2 and

LMP7 and this occurs selectively in neurons. Previous

observations reveal that following a brief transient ische-

mia, the activity of the 26S proteasomes rapidly decreases

(Kamikubo and Hayashi 1996). Since the i-proteasome has

the enhanced proteolytic activity (van Deventer and

Neefjes 2010), this upregulation of i-proteasomal subunits

may contribute to recovery of the proteasomal activity

following a transient cerebral ischemic stroke, leading to

efficient clearance of the unwanted proteins and thus pos-

sibly conferring neurons with increased tolerance to tran-

sient ischemic stroke. If this is proven to be true, it could

open up a new avenue for therapeutic intervention targeting

cerebral ischemia-caused neuronal injury.

Acknowledgments We would like to thank Dr. Robin Miskimins

for critical reading of the manuscript, Drs. Joyce Keifer and Fran Day

at the South Dakota Imaging Core Facility for help in fluorescence

microscopy. This work was supported by Start-up Funds from the

University of South Dakota (HW) and an Inside TRACK Award of

the University of South Dakota (HW).

Conflict of interest The authors have declared no conflict of

interest.

References

Ahtoniemi T, Goldsteins G, Keksa-Goldsteine V, Malm T, Kanninen

K, Salminen A, Koistinaho J (2007) Pyrrolidine dithiocarbamate

inhibits induction of immunoproteasome and decreases survival

in a rat model of amyotrophic lateral sclerosis. Mol Pharmacol

71:30–37

Atochin DN, Clark J, Demchenko IT, Moskowitz MA, Huang PL

(2003) Rapid cerebral ischemic preconditioning in mice deficient

in endothelial and neuronal nitric oxide synthases. Stroke 34:

1299–1303

Dahlmann B (2005) Proteasomes. Essays Biochem 41:31–48

Diaz-Hernandez M, Hernandez F, Martin-Aparicio E, Gomez-Ramos

P, Moran MA, Castano JG, Ferrer I, Avila J, Lucas JJ (2003)

Neuronal induction of the immunoproteasome in Huntington’s

disease. J Neurosci 23:11653–11661

Dong G, Callegari EA, Gloeckner CJ, Ueffing M, Wang H (2012)

Prothymosin-alpha interacts with mutant huntingtin and sup-

presses its cytotoxicity in cell culture. J Biol Chem 287:1279–

1289

Ge P, Luo Y, Liu CL, Hu B (2007) Protein aggregation and

proteasome dysfunction after brain ischemia. Stroke 38:3230–

3236

Hayashi T, Takada K, Matsuda M (1992) Post-transient ischemia

increase in ubiquitin conjugates in the early reperfusion.

NeuroReport 3:519–520

Kamikubo T, Hayashi T (1996) Changes in proteasome activity

following transient ischemia. Neurochem Int 28:209–212

Loukissa A, Cardozo C, Altschuller-Felberg C, Nelson JE (2000)

Control of LMP7 expression in human endothelial cells by

cytokines regulating cellular and humoral immunity. Cytokine

12:1326–1330

Mishto M, Bellavista E, Santoro A, Stolzing A, Ligorio C, Nacmias

B, Spazzafumo L, Chiappelli M, Licastro F, Sorbi S et al (2006)

Immunoproteasome and LMP2 polymorphism in aged and

Alzheimer’s disease brains. Neurobiol Aging 27:54–66

Moisse K, Welch I, Hill T, Volkening K, Strong MJ (2008) Transient

middle cerebral artery occlusion induces microglial priming in

the lumbar spinal cord: a novel model of neuroinflammation.

J Neuroinflammation 5:29

van Deventer S, Neefjes J (2010) The immunoproteasome cleans up

after inflammation. Cell 142:517–518

Zwickl P, Ng D, Woo KM, Klenk HP, Goldberg AL (1999) An

archaebacterial ATPase, homologous to ATPases in the eukary-

otic 26 S proteasome, activates protein breakdown by 20 S

proteasomes. J Biol Chem 274:26008–26014

970 Cell Mol Neurobiol (2012) 32:965–970

123