Accepted Manuscript

Spinal cord-specific deletion of the glutamate transporter GLT1 causes motor neurondeath in mice

Kaori Sugiyama, Kohichi Tanaka

PII: S0006-291X(18)30363-2

DOI: 10.1016/j.bbrc.2018.02.132

Reference: YBBRC 39497

To appear in: Biochemical and Biophysical Research Communications

Received Date: 2 February 2018

Accepted Date: 15 February 2018

Please cite this article as: K. Sugiyama, K. Tanaka, Spinal cord-specific deletion of the glutamatetransporter GLT1 causes motor neuron death in mice, Biochemical and Biophysical ResearchCommunications (2018), doi: 10.1016/j.bbrc.2018.02.132.

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Title:

Spinal cord-specific deletion of the glutamate transporter GLT1 causes motor

neuron death in mice

Kaori Sugiyama1 and Kohichi Tanaka1,2*

1Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo

Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510,

Japan.

2Center for Brain Integration Research (CBIR), TMDU, 1-5-45 Yushima, Bunkyo-ku,

Tokyo 113-8510, Japan.

*Corresponding author

Kohichi Tanaka, M.D., Ph.D.

Laboratory of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and

Dental University. 1-5-45 Yushima, Bunkyo, Tokyo 113-8510, Japan. TEL/FAX:

+81-3-5803-5846/5843.

E-mail: tanaka.aud@mri.tmd.ac.jp

Conflicts of interest: none

Abstract:

Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disorder

characterized by the selective loss of motor neurons. The precise mechanisms that cause

the selective death of motor neurons remain unclear, but a growing body of evidence

suggests that glutamate-mediated excitotoxicity has been considered to play an

important role in the mechanisms of motor neuron degeneration in ALS. Reductions in

glutamate transporter GLT1 have been reported in animal models of ALS and the motor

cortex and spinal cord of ALS patients. However, it remains unknown whether the

reduction in GLT1 has a primary role in the induction of motor neuron degeneration in

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ALS. Here, we generated conditional knockout mice that lacked GLT1 specifically in

the spinal cord by crossing floxed-GLT1 mice and Hoxb8-Cre mice.

Hoxb8-Cre/GLT1flox/flox mice showed motor deficits and motor neuron loss. Thus, loss

of the glial glutamate transporter GLT1 is sufficient to cause motor neuron death in

mice.

Keywords:

glutamate transporter, GLT1, motor neuron, amyotrophic lateral sclerosis, excitotoxicity

1. Introduction

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that

is characterized by the selective loss of motor neurons [1,2]. ALS can be classified into

two major types: familial ALS (FALS), which accounts for approximately 10% of cases,

and sporadic ALS (SALS), which comprises the remaining 90%. Among the several

potential pathogenic mechanisms of ALS, glutamate-mediated excitotoxicity has been

considered to play an important role in the mechanisms of motor neuron death [1-4].

The link between excitotoxicity and ALS was supported by early studies indicating that

ALS patients exhibited large increases in the glutamate levels in cerebrospinal fluid

[5,6], a finding that has since been confirmed in 40% of SALS patients [7]. Furthermore,

ALS patients show reduced glutamate uptake in the motor cortex and spinal cord [8],

which results from a reduction of the astrocytic glutamate transporter GLT1 (also

known as EAAT2) [9]. Together with the finding that the loss of GLT1 was observed in

ALS rodent models with copper/zinc superoxide dismutase (SOD1) [10] or TAR

DNA-binding protein 43 (TDP-43) mutations [11], it seems most likely that deficiencies

in the expression and function of the glial glutamate transporter GLT1 may result in

elevated levels of extracellular glutamate, which could lead to neuronal death through

excitotoxicity in both SALS and FALS. However, GLT1 impairment could be a

consequence of motor neuron loss, because the expression of GLT1 is critically

dependent on the presence of neurons [12]. Furthermore, previous studies showed that

the chronic in vivo administration of glutamate transport inhibitors elevated

extracellular glutamate but did not result in motor neuron death, gliosis or motor deficits

[13,14].

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Here we tested whether a primary deficit in GLT1 was sufficient to induce

spinal motor neuron death and motor deficits in vivo. Because GLT1 null mice die

prematurely with seizures [15], we generated GLT1 conditional knockout mice that

lacked GLT1 specifically in the spinal cord.

2. Materials and methods

2.1. Mice

All animal procedures were conducted according to the animal experiment

plan approved by the Tokyo Medical and Dental University Animal Care and Use

Committee. Three to five mice were housed per cage on 12-hour light/dark cycle at 23-

25 °C. Food and water were available ad libitum. Floxed-GLT1 [16-18] mice,

Hoxb8-Cre transgenic [19] mice, and ROSAtdTomato reporter [20] mice were previously

described. Male mice were used except for GLT1 immunohistochemistry. Age and

number of mice used for all experiments are described in the figure legends.

2.2. Behavioral tests

Behavioral analyses were conducted as described previously [18]. For the

hanging wire test, the wire netting was turned over and held for 60 sec after placing

mice on it. The time until the hindlimbs disengaged from the wire netting was recorded

as the latency to fall. For the hindlimb reflex score, mice were suspended from their

tails for 14 sec and we evaluated the hindlimb reflex score using an established

definition (Fig. 2D). 0, normal; 1, failure to stretch their hindlimbs; 2, hindlimb

clasping; 3, hindlimb paralysis.

2.3. Histology

Histological analysis was performed as previously described [16-18]. Mice

deeply anesthetized with pentobarbital (100 mg/kg, i.p.) were fixed by perfusion with

4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). After further fixation

of brains and spinal cords with 4% PFA overnight, tissues were transferred to 30%

sucrose/PBS for cryoprotection and embedded in OCT compound (Sakura, Tokyo,

Japan). Cryosections were prepared at a 20 µm thickness for spinal cords, and a 50 µm

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thickness for sagittal sections and mounted on MAS-coated slides (Matunami, Osaka,

Japan) (Leica CM3050s). Cryosections were washed, permeabilized and blocked with

0.3% Triton X-100, 1% BSA and 10% normal goat serum in PBS, and incubated with

primary antibodies overnight at 4 °C. For ChAT immunofluorescence, sections were

incubated in 10mM citrate buffer (pH 6.0) at 100 °C for 20 min. After air-cooling,

sections were permeabilized and blocked with 0.3% Triton X-100, 1% BSA and 10%

normal horse serum in PBS. The following antibodies were used: polyclonal anti-GLT1

(1:5,000, a gift from M. Watanabe, Hokkaido University) [21], polyclonal anti-ChAT

(1:200, MAB144P, Merck Millipore), polyclonal anti-glial fibrillary acidic protein

(GFAP, 1:1,000, Z0334, Dako), polyclonal anti-CD68 (1:100, MCA1957, AbD

Serotec), and polyclonal anti-MAP2 (1:200, MAB3418, Millipore) antibodies. Sections

were washed, incubated with secondary antibodies conjugated with Alexa Fluor 488,

594, or 633, and DAPI as nuclear marker (Life Technologies) for 2 hours, and mounted

with Fluoromount (Diagnostic BioSystems). For ChAT immunofluorescence, sections

were incubated with biotinylated donkey anti-goat IgG (1:200, Vector Lab) for 2 h,

washed and incubated with streptavidin conjugated with Alexa Fluor 488 (1:200,

Invitrogen) for 2 h. For GLT1 immunohistochemistry, sections were incubated with

biotinylated goat anti-rabbit IgG (1:1,000, Vector Lab.) for 1 h, immersed in 1%

H2O2/PBS, and incubated with AB complex (1:500, Vector Lab) for 1 h, and then

visualized with a DAB substrate kit (Vector Lab). Images were acquired using an

LSM710 laser scanning confocal microscope (Carl Zeiss), a BX-700 fluorescent

microscopy (KEYENCE) for GLT1 immunohistochemistry, or an Olympus SZX16

stereoscopic microscope (Olympus).

2.4. Cell counts

Cell counts were conducted as described previously [18]. The number of

ChAT positive cells in the bilateral ventral horns of L3-L5 in 6-9 serial sections was

counted using Image J software with the cell counter plug-in (NIH, Bethesda, MD)

under the conditions blind to genotype.

2.5. Statistical analysis

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Results are presented as the mean ± SEM. Student’s t-test was used to

compare differences between any two groups. For longitudinal observations,

repeated-measure ANOVA with post hoc unpaired two-tailed t-test was performed. To

analyze the significant differences in survival rate, Kaplan-Meier and logrank test were

used. All statistical analyses were performed using Statcel3 software. A p-value of less

than 0.05 was considered statistically significant.

3. Results

3.1. Generation of spinal cord-specific GLT1 knockout mice

Because GLT1 knockout animals die prematurely with seizures [15], we have

generated GLT1 conditional mutant mice that lacked GLT1 specifically in the spinal

cord to examine the effects of GLT1 loss on spinal cord motor neuron survival. We

crossed mice carrying a loxP-flanked GLT1 allele (GLT1flox/flox, denoted as control)

[16-18] with Hoxb8-Cre mice [20] to obtain spinal cord-specific GLT1 knockout mice

(Hoxb8-Cre/GLT1flox/flox mice, Fig. 1A). The Hoxb8-Cre line expresses Cre

recombinase in the spinal cord caudal to cervical level C5 but express very little Cre in

the brain [19]. To assess Cre expression, we crossed the Hoxb8-Cre mice to the

ROSAtdTomato reporter mice. Consistent with previous report [19], the strong tdTomato

signal was observed caudal to cervical spinal cord of Hoxb8-Cre/ROSAtdTomato mice

(Fig. 1B). Thus, using this Cre mouse line enabled us to delete GLT1 in the spinal cord.

Immunohistochemistry of GLT1 showed that the amount of GLT1 protein in

the spinal cord caudal to cervical level C5 in Hoxb8-Cre/GLT1flox/flox mice was much

lower than age-matched GLT1flox/flox mice but that GLT1 expression in other brain

regions was normal (Fig.1C). There was no compensatory upregulation of other

subtypes of glutamate transporters, GLAST and EAAC1, in Hoxb8-Cre/GLT1flox/flox

mice [18].

3.2. Hoxb8-Cre/GLT1flox/flox mice develop motor dysfunction

In contrast to GLT1 null mice, Hoxb8-Cre/GLT1flox/flox mice did not show

lethal seizures and survived to adulthood (Fig. 2A). Starting at 4 months of age,

Hoxb8-Cre/GLT1flox/flox mice had slightly lower body weight than did the controls and

showed both grip strength reduction and hindlimb reflex impairment (Fig. 2B, C, E).

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These motor deficits stabilized after 6 months of age and Hoxb8-Cre/GLT1flox/flox mice

exhibited no overt paralysis until at least 22 months of age (data not shown). These

results indicate that GLT1 deletion in spinal cord causes mild motor deficits in mice.

3.3. Hoxb8-Cre/GLT1flox/flox mice show motor neuron loss and gliosis

We next examined whether GLT1 deletion caused motor neuron loss and

gliosis. Immunohistochemical analysis using an anti-ChAT antibody showed a loss of

spinal motor neurons in the lumbar ventral horn of Hoxb8-Cre/GLT1flox/flox mice at 5

months of age (symptomatic stage) (Fig. 3A, B). In contrast, there was no difference in

the number of ChAT-positive cells between control and Hoxb8-Cre/GLT1flox/flox mice at

2 months of age (presymptomatic stage) (Fig. 3A, B). In addition, immunoreactivity for

GFAP, a marker of astrocyte, and CD68, a marker of activated microglia, was increased

in the ventral horn of the lumbar spinal cord of Hoxb8-Cre/GLT1flox/flox mice at 5

months of age (Fig. 3C). Taken together, these results indicate that GLT1 deletion in

spinal cord is sufficient to induce motor neuron loss and gliosis in vivo.

4. Discussion

Although several lines of evidence suggest the possibility that glutamate

transport dysfunction may play an important role in the mechanisms of motor neuron

degeneration in ALS [5-9], a few reports do not support glutamate-transport deficits as a

primary cause of neuronal death in ALS. Spinal infusion of glutamate transporter

inhibitors in rats failed to cause motor neuron death or motor deficits [13,14].

Furthermore, adeno-associated virus-based GLT1 overexpression failed to prevent loss

of phrenic nerve motor neurons or rescue respiratory function in SOD1(G93A) mice,

a widely studied ALS model [22]. Thus, currently the evidence for glutamate transporter

dysfunction as a primary cause of motor neuron death is inconclusive. In this study, we

found that conditional knockout mice that lacked GLT1 specifically in the spinal cord

developed motor dysfunction and spinal motor neuron loss. These data demonstrate that

dysfunction of GLT1 is sufficient to cause motor neuron death in vivo.

Although Hoxb8-Cre/GLT1flox/flox mice showed motor neuron death, motor

dysfunction and the loss of motor neurons in Hoxb8-Cre/GLT1flox/flox mice were very

mild, which suggested that Hoxb8-Cre/GLT1flox/flox mice could not faithfully

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recapitulate the rapidly progressive clinical phenotype observed in ALS patients. In a

recent paper, we demonstrated that a concurrent reduction in the GLAST (another glial

glutamate transporter) expression levels from Hoxb8-Cre/GLT1flox/flox mice accelerated

motor deficits and resulted in earlier motor neuron loss and a reduction in survival [18],

thus recapitulating the clinical phenotypes of ALS as accurately as SOD1 and TDP-43

mutants [10,11]. Since GLAST knockout mice did not show grip strength reduction,

hindlimb reflex impairment, and spinal motor neuron loss [23], these results suggest

that loss of GLAST is a contributor to, but not the cause of, spinal motor neuron loss.

Taken together, our studies suggest that glial glutamate transporter

GLT1 plays an important role in the survival of spinal motor neurons and loss of GLT1

in spinal cord directly contributes to spinal motor neuron death and motor deficits.

.

Acknowledgements

This work was supported by the Strategic Research Program for Brain Sciences

(SRPBS) from the Ministry of Education, Culture, Sports, Science and Technology of

Japan (MEXT, to KT), JSPS KAKENHI Grant Number 17H02216 (to KT) and a

Grant-in-Aid for JSPS Fellows (to KS).

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Figure Legends

Figure 1. Generation of spinal cord-specific GLT1 knockout mice

(A) Schematic diagram showing the generation of spinal cord-specific GLT1 knockout

(Hoxb8-Cre/GLT1flox/flox) mice.

(B) Characterization of the brain-sparing Hoxb8-Cre line. Red fluorescence is observed

in the spinal cord caudal to cervical level C5 in Hoxb8-Cre mice harboring the reporter

R26tdTomato (ROSAtdTomato) at postnatal day 0.

(C) GLT1 immunohistochemistry on brain and spinal cord from control (GLT1flox/flox)

and Hoxb8-Cre/GLT1flox/flox mice at 4 weeks of age.

Scale bars, (A and C) 2 mm.

Figure 2. Mild motor deficits in spinal cord-specific GLT1 knockout mice.

(A) Percent survival of control (n = 14) and Hoxb8-Cre/GLT1flox/flox (n = 17) mice

calculated using the Kaplan-Meier method (p = 0.36, logrank test). n. s. = not

significant.

(B) Body weight changes, (C) hanging wire test, and (E) hindlimb reflex score of

control (n = 12) and Hoxb8-Cre/GLT1flox/flox animals (n = 9). *p < 0.05, **p < 0.01,

***p < 0.001 (post hoc unpaired two-tailed t-test at corresponding time-point after

two-way repeated measures ANOVA).

(D) The scoring criteria for the hindlimb reflex score. Mice were suspended by the tail

approximately 30 cm above a tabletop for 14 sec. The posture adopted was scored

according to the following criteria: score of 0, normal; score of 1, failure to stretch

hindlimbs; score of 2, hindlimb clasping; score of 3, hindlimb paralysis.

Figure 3. Motor neuronal loss and gliosis in spinal cord-specific GLT1 knockout

mice.

(A) ChAT immunofluorescence of the lumbar ventral horn from control and

Hoxb8-Cre/GLT1flox/flox mice at 2 and 5 months of age.

(B) Quantification of ChAT-positive motor neurons from 2 and 5 months of age of

control (P2M, n = 5; P5M, n = 3) and Hoxb8-Cre/GLT1flox/flox (P2M, n = 5; P5M, n = 4)

mice. **p < 0.01, n.s. = not significant (unpaired two-tailed t-test).

(C) GFAP and CD68 immunofluorescence in the lumbar ventral horn from control and

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Hoxb8-Cre/GLT1flox/flox mice at 5 months of age. Mice were triple labeled with GFAP

(red), CD68 (cyan) and MAP2 (green) antibodies.

Scale bars, (A and C) 50 µm.

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Highlights:

GLT1 deletion in spinal cord causes motor deficits.

GLT1 deletion in spinal cord induces spinal motor neuron loss.

GLT1 deletion in spinal cord induces gliosis in the ventral horn.