Repulsive Guidance Molecule-a Is Involved in Th17- Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis

Article
Repulsive Guidance Molecule-a Is Involved in Th17Cell-Induced Neurodegeneration in Autoimmune
Encephalomyelitis
Graphical Abstract
Authors
Shogo Tanabe, Toshihide Yamashita
Correspondence
[email protected]
In Brief
The mechanism of neurodegeneration
under inflammation in the CNS remains
largely unknown. Tanabe and Yamashita
demonstrate that RGMa is highly expressed in Th17 cells and induces
dephosphorylation of Akt, leading to
death of neurons. A neutralizing antibody
to RGMa attenuates axonal degeneration
and severity of Th17-cell-mediated autoimmune encephalomyelitis.
Highlights
RGMa binding to neogenin induces dephosphorylation of Akt in
neurons
RGMa expressed in Th17 cells induces neurodegeneration
Depletion of RGMa rescues axonal degeneration in EAE
Neutralizing antibody to RGMa attenuates severity of Th17 cellmediated EAE
Tanabe & Yamashita, 2014, Cell Reports 9, 1–12
November 20, 2014 ª2014 The Authors
http://dx.doi.org/10.1016/j.celrep.2014.10.038
Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in
Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038
Cell Reports
Article
Repulsive Guidance Molecule-a Is Involved
in Th17-Cell-Induced Neurodegeneration
in Autoimmune Encephalomyelitis
Shogo Tanabe1,2 and Toshihide Yamashita1,2,*
1Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
2Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 5 Sanbancho, Chiyoda-ku,
Tokyo 102-0075, Japan
*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.celrep.2014.10.038
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
SUMMARY
Multiple sclerosis (MS) is a chronic autoimmune disease characterized by inflammation, demyelination,
and neurodegeneration in the CNS. Although it is
important to prevent neurodegeneration for alleviating neurological disability, the molecular mechanism of neurodegeneration remains largely unknown.
Here, we report that repulsive guidance molecule-a
(RGMa), known to regulate axonal growth, is associated with neurodegeneration in experimental autoimmune encephalomyelitis (EAE), a mouse model of
MS. RGMa is highly expressed in interleukin-17-producing CD4+ T cells (Th17 cells). We induced EAE by
adoptive transfer of myelin oligodendrocyte glycoprotein (MOG)-specific Th17 cells and then inhibited
RGMa with a neutralizing antibody. Inhibition of
RGMa improves EAE scores and reduces neuronal
degeneration without altering immune or glial responses. Th17 cells induce cultured cortical neuron
death through RGMa-neogenin and Akt dephosphorylation. Our results demonstrate that RGMa is
involved in Th17-cell-mediated neurodegeneration
and that RGMa-specific antibody may have a therapeutic effect in MS.
INTRODUCTION
In multiple sclerosis (MS), immune cells, such as T cells and
monocytes, infiltrate the CNS and induce inflammation, demyelination, and neurodegeneration (Trapp et al., 1999). MS
pathogenesis is attributed to acquired autoimmunity to CNS
components, including myelin sheaths (Stinissen et al., 1998).
Previous studies have demonstrated that CD4+ T cells play critical roles in inducing CNS inflammation (Goverman, 2009). T
helper type 1 cells (Th1 cells), characterized by their secretion
of interferon-gamma (IFN-g), are abundant in the cerebrospinal
fluid (CSF) of MS patients and are an essential initiator of
encephalomyelitis (Ando et al., 1989; Rotteveel et al., 1990). A
more recent study identified T helper type 17 cells (Th17 cell),
which produce interleukin-17 (IL-17), as critical drivers for autoimmune diseases, including MS (Bettelli et al., 2006). Th17 cells
are differentiated by IL-6 and transforming growth factor-b1
(TGF-b1), and stimulation of Th17 cells with IL-23 enhances their
pathogenic activity via proinflammatory cytokine production
(Dong, 2008; Lee et al., 2012). Indeed, IL-17-, IL-17 receptor-,
or IL-23 receptor-deficient mice are resistant to experimental
autoimmune encephalomyelitis (EAE; Hu et al., 2010; Komiyama
et al., 2006; McGeachy et al., 2009). IL-17 also exerts cytotoxic
effects on NG2 glial cells (Kang et al., 2013), and an in vivo imaging study revealed that Th17 cells directly contact neurons in
EAE and induce neuronal dysfunction (Siffrin et al., 2010). The
existing evidence suggests that Th17 cells are important mediators of inflammation in EAE and MS.
As mentioned above, the mechanism of inflammation in
EAE and MS has been extensively investigated. However, it
is currently accepted that neurodegeneration, which is often
accompanied by demyelination, is the major cause of permanent
neurological disability in MS (Trapp et al., 1999; Trapp and Nave,
2008), although this phenomenon is rather poorly understood
and its mechanism remains largely unknown. Neurodegeneration is induced in the regions of inflammation and begins at disease onset of MS, and it becomes irreversibly progressive in a
secondary progressive stage of MS. Indeed, neurodegeneration
is a major pathological hallmark in both the acute and chronic
et al., 2011;
phases of MS and EAE (Franklin et al., 2012; Nikic
Soulika et al., 2009). Therefore, elucidation of the molecular
mechanism of neurodegeneration may provide efficient neuroprotective therapy to treat progressive MS.
Repulsive guidance molecule-a (RGMa) is a glycosylphosphatidylinositol-anchored membrane protein that plays an important
role in axon guidance in the visual system (Severyn et al., 2009;
Stahl et al., 1990). RGMa is expressed in oligodendrocytes and
contributes to axonal growth inhibition after CNS injury (Hata
et al., 2006; Tao et al., 2013). RGMa is also involved in apoptosis
in Xenopus and chick embryos, but the precise function of
RGMa in cell death remains controversial (Fujita et al., 2008;
Matsunaga et al., 2004; Shin and Wilson, 2008). We previously
reported that RGMa is expressed in dendritic cells and promotes T cell activation in EAE (Muramatsu et al., 2011).
We immunized mice with myelin oligodendrocyte glycoprotein
Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors 1
Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in
Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038
Figure 1. RGMa Is Expressed in Th17 Cells
(A) Intracellular labeling and subsequent flow cytometry analysis of IFN-g, IL-17, and Foxp3 in CD4+ T cells differentiated to Th0, Th1, Th17, or Treg cells.
(B) Quantitative RT-PCR analysis of RGMa in CD4+ T cells differentiated to Th0, Th1, Th17, or Treg cells. The graph shows the relative expression level to that of
GAPDH mRNA (n = 3).
(C) Representative western blots showing the expression levels of RGMa (32 kDa) and b-actin (45 kDa) in CD4+ T cells polarized to Th0, Th1, Th17, or Treg cells.
The level of RGMa was normalized to that of b-actin.
(D) Quantitative RT-PCR analysis of RGMa in CD4+ T cells differentiated with cytokine combinations of TGF-b1, IL-6 and IL-23, or no cytokines (n = 3).
Statistical analysis was performed by one-way ANOVA, followed by Tukey-Kramer tests for (B) and (D) (***p < 0.001, *p < 0.05). NS, not significant. Error bars
represent mean ± SEM.
(MOG)35–55 or proteolipid protein (PLP)139–151 and treated these
mice with an RGMa-neutralizing antibody before neurological
symptoms of EAE appeared to determine if interfering with
RGMa-signaling could ameliorate EAE. The observed preventive
effect fits well with the observation that RGMa facilitates T cell
activation by dendritic cells. Thus, RGMa plays an important
role in initiation of inflammation in EAE. However, the therapeutic
effect of this antibody on progressive EAE remained to be determined. This issue is important from a clinical point of view, but
information regarding the roles of RGMa in EAE or MS progression is scarce.
The present study sought to elucidate the function of RGMa
in progression of EAE. Our results demonstrate that RGMa is
involved in Th17-cell-induced neurodegeneration in EAE. We
found high RGMa expression in Th17 cells, and RGMa depletion
with the neutralizing antibody attenuated the severity of EAE
induced by the adoptive transfer of Th17 cells. Moreover,
RGMa depletion reduced acute axonal degeneration in inflammatory lesions without altering immune or glial responses.
Indeed, coculture of Th17 cells and cortical neurons revealed
that Th17 cells induced neuronal cell death in an RGMa-dependent manner. Collectively, we identified the molecular mechanism of Th17-induced neurodegeneration, and our findings
demonstrate that RGMa in Th17 cells is a major inducer of neurodegeneration in EAE.
2 Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors
RESULTS
RGMa Is Expressed in Th17 Cells
We investigated RGMa expression in each type of CD4+ T cell
that plays critical roles in the pathologies of EAE and MS. Naive
CD4+ T cells were isolated from wild-type (WT) mouse spleen
and differentiated in vitro into Th0 (IL-2), Th1 (IL-2 + IL-12),
Th17 (IL-6 + TGF-b1 + IL-23), and regulatory T cells (Treg; IL2 + TGF-b1). The differentiation status was confirmed by intracellular staining and flow cytometry analysis (Figure 1A). RGMa
expression levels were determined in T cells by quantitative
RT-PCR and western blot analysis. While Th0, Th1, and Treg
cells expressed low levels of RGMa, abundant mRNA and protein levels of RGMa were detected in Th17 cells (Figures 1B
and 1C). Th17 cells have crucial roles in promoting inflammation
and tissue injury to facilitate the development of autoimmune
diseases. It has been reported that Th17 cells require IL-23 stimulation to maintain and enhance these activities (Dong, 2008; Lee
et al., 2012). To examine whether IL-23 was required to express
RGMa, Th17 cells were differentiated by IL-6 and TGF-b1 with
or without IL-23. Although Th17 cells differentiated by only
TGF-b1 + IL-6 express low levels of RGMa, IL-23 stimulation
enhanced RGMa expression in Th17 cells (Figure 1D). These
observations suggest that RGMa may be associated with
Th17-cell-induced disease progression.
Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in
Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038
Th17-EAE as assessed by EAE scores in the late stage of disease progression (Figure 2A). Although the day of onset was
not altered by RGMa antibody treatment, the accumulated
EAE scores and maximum scores were significantly decreased
by RGMa antibody treatment (Figures 2B–2D). In this model,
58.8% of Th17-EAE mice died within 30 days after transfer.
However, RGMa antibody treatment reduced the mortality
rate to 21.4% (Figure 2E). These data demonstrate that
RGMa inhibition reduced the severity and mortality of Th17induced EAE.
Figure 2. RGMa Inhibition Ameliorates EAE Severity Induced by
2D2-Th17 Cells
(A) Th17 cells from 2D2 MOG35–55 TCR transgenic mice were adoptively
transferred to wild-type irradiated recipient mice (2.0 3 106). The graph shows
EAE scores in mice treated with control IgG (n = 17) or anti-RGMa antibody
(n = 14) after 2D2-Th17 cell transfer.
(B–D) The average data of onset day (B), maximum scores (C), and accumulated scores (D) after 2D2-Th17 cell transfer.
(E) The rate of disease mortality within 30 days after adoptive transfer.
Statistical analysis was performed by two-way ANOVA followed by Bonferroni
tests for (A), Student’s t test for (B)(D), *p < 0.05, **p < 0.01. Error bars
represent mean ± SEM.
RGMa Inhibition Attenuates Th17-Induced EAE Severity
To examine the role of RGMa in Th17 cells, we employed a
mouse model of Th17-induced EAE. We adoptively transferred
in vitro differentiated Th17 cells from 2D2 MOG35-55-specific
T cell receptor (TCR) transgenic mice to irradiated WT recipient
mice (Th17-EAE; Bettelli et al., 2003; Ja¨ger et al., 2009; Rothhammer et al., 2011). This model system allowed us to
estimate the effect of RGMa in the EAE progression phase
apart from the function of RGMa in dendritic-cell-induced
T cell responses. An RGMa-specific neutralizing antibody
was administered intraperitoneally to Th17-EAE mice every
7 days. RGMa antibody treatment attenuated the severity of
RGMa-Specific Antibody Does Not Affect Immune
Responses in Th17-EAE
As RGMa modulates T cell responses and promotes inflammation, we examined whether treatment with an RGMa antibody
suppressed immune cell activation or infiltration. In EAE, inflammatory lesions are formed by accumulated immune cells;
therefore, we quantified the number of inflammatory lesions
by assessing hematoxylin and eosin (H&E)-stained spinal
cord of Th17-EAE mice 30 days after transfer. We did not
observe any alteration in the number of inflammatory lesions
in mice treated with an RGMa-specific antibody compared
with mice treated with control immunoglobulin G (IgG) (Figure 3A). Moreover, we assessed the extent of demyelination
with fluoromyelin staining. The demyelinated area was not
altered by RGMa-specific antibody treatment (Figure 3B).
These histological analyses suggest that RGMa antibody
treatment did not affect inflammatory lesion formation or
demyelination processes.
We next examined whether RGMa-specific antibody affects
infiltration of Th17 cells to the CNS. T cells from 2D2 mice could
be distinguished by their expression of TCR Va 3.2, which is
only expressed in 2D2 T cells (Bettelli et al., 2003; Ja¨ger et al.,
2009). Thirty days after transfer, mononuclear cells were isolated from the brain and spinal cord, and the number of exogenous T cells (CD4+, TCR Va 3.2+ cells) was quantified by flow
cytometry analysis. The number of infiltrated exogenous
T cells was not different in either the brain or spinal cord between the control IgG and RGMa-specific antibody treatment
groups (Figure 3C). We confirmed that these exogenous
T cells were Th17 cells by performing intracellular cytokine
staining (Figure 3D). Furthermore, to determine whether the
RGMa antibody suppressed T cell activation, we stimulated
T cells isolated from Th17-EAE mouse spleen with MOG35–55
peptide and measured proliferation activity with bromodeoxyuridine (BrdU) incorporation. We did not observe any differences
in proliferation activity between control IgG and RGMa-specific
antibody-treated mice (Figure 3E). In EAE, astrocyte and microglia activation contributes to disease exacerbation (Colombo
et al., 2012; Ding et al., 2014). Thus, we examined whether
RGMa-specific antibody treatment reduced astrocyte and
microglia activation by performing immunohistochemistry with
glial fibrillary acidic protein (GFAP) and Iba1 antibodies. We
determined that RGMa-specific antibody treatment did not alter
the activation state of astrocyte or microglia (Figure 3F). Collectively, these results indicate that RGMa-specific antibody treatment did not affect immune responses, demyelination, or gliosis
in Th17-EAE mice.
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Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in
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Figure 3. RGMa Is Not Involved in Immune Responses in Th17-EAE Mice
(A) H&E staining of cervical spinal cord sections of control IgG- and anti-RGMa-treated Th17-EAE mice 30 days after transfer. Arrowheads indicate EAE lesions.
The graph shows the number of EAE lesions per section (control IgG, n = 3; anti-RGMa, n = 4). Scale bar represents 100 mm.
(legend continued on next page)
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Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in
Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038
RGMa-Specific Antibody Reduces Neuronal Damage in
Th17-EAE
To address the role of RGMa in Th17-EAE, we performed
immunohistochemistry to analyze the expression of neogenin,
which is a receptor of RGMa, in the spinal cord of Th17-EAE
mice. Although neogenin was not expressed in noninflammatory lesions, neogenin signals were detected in inflammatory
lesions and colocalized with axons immunostained with an
axonal neurofilament (SMI-312) antibody (Figure 4A). Based
on this result, we hypothesized that RGMa contributes to neurodegeneration in Th17-EAE. To assess the extent of neuronal
damage, we stained the sections for amyloid precursor protein
(APP), which is a marker of axonal damage, and found that the
number of APP signals in lesions was significantly decreased
in Th17-EAE mice treated with the RGMa-specific antibody
(Figure 4B). In addition, we stained the sections with SMI-312
antibody to assess whether RGMa-specific antibody preserves
axons in inflammatory lesions. SMI-312 staining revealed that
RGMa antibody treatment significantly preserved axons in the
lesions of dorsal column (Figure 4C). These results suggest
that RGMa is involved in acute axonal degeneration in Th17EAE mice.
RGMa in Th17 Cells Induces Neuronal Cell Death
A recent study demonstrated that Th17 cells induce neurodegeneration via cell-cell contacts both in vitro and in vivo (Siffrin
et al., 2010). Furthermore, RGMa is a membrane protein and
is involved in apoptosis (Shin and Wilson, 2008). These lines
of evidence and our in vivo results prompted us to investigate whether RGMa expressed in Th17 cells can directly
induce neurodegeneration. Cortical neurons and Th17 cells
were cocultured with or without RGMa-specific antibody,
and neuronal cell death was detected by TUNEL staining. Th17
cells, but not Th0 cells or conditioned medium from Th17
cells, induced neuronal apoptosis. Moreover, this effect was inhibited by the RGMa-specific antibody treatment (Figures 5A
and 5B). As we demonstrated that IL-23 stimulation was required
to express RGMa in Th17 cells, cortical neurons were cocultured
with Th17 cells differentiated by only TGF-b1 and IL-6. Coculturing with unstimulated Th17 cells resulted in fewer TUNELpositive cells than incubation with IL-23-stimulated Th17 cells
(Figure 5C). These results demonstrate that Th17 cells directly
induce neuronal cell death through RGMa and that IL-23 exposure is required to exert this effect.
Neogenin is an RGMa receptor and has various physiological
functions, including roles in neurite outgrowth and cell death (De
Vries and Cooper, 2008). We next examined whether neogenin
inhibition reduced Th17-cell-induced neuronal death. Knock-
down of neogenin expression in cortical neurons was carried
out by small interfering RNA (siRNA) transfection, and the knockdown efficiency was confirmed by assessing mRNA and protein
levels (Figures 5D and 5E). Although Th17 cells increased the
number of TUNEL-positive neurons that were transfected with
control siRNA, this effect was attenuated by neogenin knockdown in neurons (Figure 5F). Neogenin knockdown by itself or
coculturing with Th0 cells did not affect the number of TUNELpositive neurons. Therefore, Th17-cell-induced neuronal death
is dependent on RGMa expressed in Th17 cells and neogenin
in neurons.
RGMa Is Not Involved in T Cell-Neuron Adhesion
As we demonstrated that contact between Th17 cells and neurons was required to induce neuronal death, we next examined
whether RGMa is involved in T cell-neuron adhesion. Th0 or
Th17 cells were labeled with calcein-AM and plated on cortical
neurons for 6 hr. Nonadherent cells were removed by several
washes and the fluorescent intensity was measured. After a
6 hr incubation, although Th17 cells adhered to neurons significantly more than Th0 cells, RGMa antibody treatment had no effect on the adherent activity of either Th0 or Th17 cells (Figures
6A and 6B). This result excludes the possibility that RGMa in
Th17 cells is involved in adhesion to neurons.
RGMa in Th17 Cells Mediates Akt Dephosphorylation in
Neurons
To determine which signaling pathways Th17 cells affect to
induce neuronal death, we investigated the expression of cellsurvival-related molecules in neurons cocultured with Th17 cells.
We examined the phosphorylation of Akt and extracellular
signal-related kinase (ERK), which are antiapoptotic factors
that are dephosphorylated by RGMa-neogenin signaling (Endo
and Yamashita, 2009 and Manning and Cantley, 2007). We
isolated neurons from the T cell-neuron coculture system with
anti-CD4 magnetic beads and confirmed that CD4+ T cells
were not contaminated with neurons by flow cytometry (Figure 7A). Akt in neurons cocultured with Th17 cells was dephosphorylated compared to neurons cocultured with Th0 cells,
and RGMa-specific antibody significantly inhibited Th17 cellinduced Akt dephosphorylation (Figures 7B and 7C). In contrast,
while ERK1 phosphorylation was not altered in neurons cocultured with Th17 cells, ERK2 was dephosphorylated in neurons
cocultured with either Th0 or Th17 cells (Figures 7D–7F). As
both Th0 and Th17 cells dephosphorylated ERK2 to a similar
extent in neurons and RGMa-specific antibody did not modulate
ERK2 phosphorylation, we concluded that ERK dephosphorylation is not involved in Th17-induced neuronal death. These
(B) Fluoromyelin staining of cervical spinal cord sections in both groups. The graph shows the percentages of demyelinated area in sections (control IgG, n = 3;
anti-RGMa, n = 3). Scale bar represents 50 mm.
(C) The number of infiltrated CD4+, TCR Va 3.2+ cells in the brain and spinal cord of control IgG- and anti-RGMa-treated Th17-EAE mice were identified by flow
cytometry analyses (control IgG, n = 4; anti-RGMa, n = 4).
(D) The percentages of IL-17+ cells in infiltrated CD4+, TCR Va 3.2+ cells in the brain and spinal cord.
(E) Splenocytes were isolated from control IgG- and anti-RGMa-treated Th17-EAE mice 30 days after transfer and restimulated with MOG35–55 peptide for 3 days.
Proliferation was measured by BrdU incorporation (control IgG, n = 3; anti-RGMa, n = 4).
(F) Immunohistochemical staining for Iba1 (green) and GFAP (red). Scale bar represents 60 mm.
Statistical analysis was performed by Student’s t test for (A)–(C) and one-way ANOVA for (E) (*p < 0.05). Error bars represent mean ± SEM.
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Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in
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Figure 4. RGMa Antibody Suppresses
Axonal Damage in EAE Lesions
(A) Immunohistochemical staining for axons (SMI312, green) and neogenin (red) in EAE lesions and
normal sites of spinal cord. Scale bar represents
30 mm.
(B) Immunohistochemical staining for APP to
detect axonal damage in EAE lesions of control
IgG- and RGMa antibody-treated Th17-EAE mice.
The graph shows the number of APP signals in
EAE lesions per mm2 (control IgG, n = 3; antiRGMa, n = 3). Scale bar represents 30 mm.
(C) Axonal neurofilament (SMI-312) staining in the
spinal cord of intact and Th17-EAE mice treated
with control IgG or anti-RGMa. Representative
images show the EAE lesion sites (dorsal column)
of Th17-EAE mice and the same regions in intact
mice. The graph shows the percentage of preserved axons in EAE lesions or the corresponding
regions in intact mice (intact, n = 3; control IgG, n =
4; anti-RGMa, n = 4). Scale bar represents 20 mm.
Statistical analysis was performed by Student’s
t test for (B), and one-way ANOVA followed by
Tukey Kramer test for (C). *p < 0.05. Error bars
represent mean ± SEM.
DISCUSSION
results strongly suggest that dephosphorylation of Akt in neurons by RGMa expressed in Th17 cells plays a critical role in
neuronal death.
6 Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors
In the present study, we elucidated a
mechanism of neurodegeneration in
Th17-cell-induced EAE. In MS and EAE,
activated immune cells infiltrate the CNS
and induce demyelination and neurodegeneration. These phenomena aggravate
the disease condition and limit spontaneous functional recovery. Because neurodegeneration is a major pathological
hallmark in both the acute and chronic
phases of MS and EAE (Franklin et al.,
et al., 2011; Soulika et al.,
2012; Nikic
2009), identifying ways to inhibit neurodegeneration is an important therapeutic
strategy for the effective treatment of MS.
RGMa was first identified as an axon
repulsive guidance molecule in the retina
(Severyn et al., 2009; Monnier et al.,
2002). Subsequent studies revealed
that RGMa has other functions, such as
growth cone collapse and neural tube
closure (Conrad et al., 2007; Niederkofler
et al., 2004). RGMa is expressed in glial
cells, including oligodendrocytes, and
potently inhibits axonal growth in the
CNS (Hata et al., 2006). Indeed, RGMa inhibition promotes drastic axonal growth
and recovery after spinal cord injury
(Hata et al., 2006; Kitayama et al., 2011). On the other hand,
RGMa regulates inflammation through T cell activation, and an
RGMa polymorphism is associated with the expression of
Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in
Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038
Figure 5. RGMa in Th17 Cells Induces
Neuronal Cell Death In Vitro
(A) Representative figures show dual staining with
TUNEL (red) and b-tubulin III (green) in cortical
neurons. Cortical neurons were cocultured with
CD4+ T cells polarized to Th0 or Th17 cells with or
without anti-RGMa antibody (10 mg/ml). Scale bar
represents 50 mm.
(B) The rate of TUNEL+ apoptotic neurons in
the coculture system (n = 3; CM, conditioned
medium).
(C) The rate of TUNEL+ apoptotic neurons cocultured with IL-23-stimulated or unstimulated Th17
cells (n = 3).
(D) The graph shows the relative expression level
of neogenin mRNA quantified by quantitative
RT-PCR (n = 3).
(E) Neogenin expression was detected by western
blot analysis and normalized to a-tubulin.
(F) The rate of TUNEL+ neurons transfected with
control or neogenin siRNA and cocultured with
Th0 or Th17 cells (n = 3).
Statistical analysis was performed by one-way
ANOVA followed by Tukey Kramer tests for (B),
Student’s t test for (C) and (D), and two-way
ANOVA followed by Bonferroni test for (F). Error
bars represent mean ± SEM.
IFN-g and tumor necrosis factor-a (TNF-a) in the CSF of MS patients (Muramatsu et al., 2011; Nohra et al., 2010). The present results demonstrate a function of RGMa wherein its expression by
Th17 cells induces neuronal damage. Several reports have suggested the involvement of RGMa in cell death. Neogenin overexpression induced cell death in both chick embryos and human
embryonic kidney 293 (HEK293) cells, but this effect was inhibited by RGMa (Matsunaga et al., 2004; Fujita et al., 2008). In
contrast, RGMa overexpression induced cell death via neogenin
signaling in Xenopus (Shin and Wilson,
2008). Thus, the bidirectional effect of
RGMa in cell death may be cell context
dependent. Our in vitro results indicated
that RGMa in Th17 cells induced neuronal
death. However, recombinant RGMa by
itself did not induce neuronal death (data
not shown), suggesting that additional
molecules or a specific inflammatory
environment may be required for RGMa
to facilitate neuronal death. In fact, the
proinflammatory cytokine TNF-a induces
neogenin upregulation in a nuclear factor-kappa B (NF-kB)-dependent manner
(Mirakaj et al., 2012). Indeed, our histological analysis of Th17-EAE mice indicated
that axons expressed neogenin only in inflammatory lesions. Accordingly, in EAE
lesions, inflammation leads to neogenin
upregulation in neurons; therefore RGMa
in Th17 cells may be able to drive degenerative signaling in neurons.
We found that inhibition of RGMa attenuated the severity of
EAE induced by 2D2-Th17 cells. To elucidate the underlying
mechanism, we examined the possibility that the RGMa antibody could suppress inflammation. However, treatment with
the RGMa-neutralizing antibody did not alter the number of
lesions, Th17 cell infiltration activity, T cell activation, or gliosis.
These results suggest that RGMa is not involved in inflammation and contributes to alternative functions of Th17 cells
in EAE. While Th17 cells induce inflammation by secreting
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Figure 6. RGMa Is Not Involved in T CellNeuron Adhesion
(A) Representative figures demonstrate adherent
calcein AM-labeled Th0 or Th17 cells to cortical
neurons. T cells and neurons were cultured
for 6 hr.
(B) Quantitative analysis of (A). Fluorescent intensity was measured after 6 hr of coculture (n = 5).
Statistical analysis was performed by a Student’s t
test (*p < 0.05). Error bars represent mean ± SEM.
proinflammatory cytokines, such as IL-17, IL-21, and IL-22, it has
been reported that Th17 cells are involved in processes other
than inflammation (Dong, 2008). For example, Th17 cells express
receptor activator of NF-kB ligand (RANKL) on their surface,
which allows them to stimulate osteoclasts and promote bone
destruction (Sato et al., 2006). It is well known that CNS neurodegeneration is a pathological hallmark of MS, and a previous
in vivo imaging study revealed that Th17 cells adhere to axons
and induce axonal degeneration in brain stem lesions (Siffrin
et al., 2010). However, the mechanisms underlying Th17 cellmediated neurodegeneration in MS were largely unknown. Our
in vivo findings demonstrate that RGMa inhibition is neuroprotective in Th17-EAE, and our in vitro results indicate that RGMa
expressed by Th17 cells is required for Th17-cell-mediated
neuronal death. In addition, RGMa in Th17 cells induced dephosphorylation of Akt, which is an antiapoptotic factor (Dudek et al.,
1997). Notably, dephosphorylation of Akt is involved in both
apoptosis and axonal degeneration (Wakatsuki et al., 2011).
Hence, it is possible to speculate that Akt dephosphorylation
mediates axonal degeneration in Th17-EAE lesions.
We performed intraperitoneal administration of the RGMa
antibody to Th17-EAE mice. This administration route is considered to allow the antibody to contact majority of the circulating
Th17 cells. Histological analysis showed that the RGMa antibody
treatment did not affect immune responses or demyelination.
Therefore, RGMa is not involved in immune responses, except
T cell activation induced by dendritic cells, or demyelination
process. Considering the mechanism of action of the RGMa
antibody in the model, intrathecal administration of the RGMa
antibody is expected to show more effective neuroprotective
activity than systemic administration. However, it should be
noted that systemic administration of the RGMa antibody may
be feasible and useful in treating MS.
We also showed that IL-23 stimulation led to RGMa upregulation in Th17 cells. IL-23 is known to promote inflammation and
enhance Th17 cell pathogenic activities (Dong, 2008). In fact,
our in vitro experiments indicated that IL-23-unstimulated Th17
cells exert lower neurotoxicity than IL-23-stimulated Th17 cells.
There are two possible reasons for this result. First, RGMa
expression in Th17 cells was not sufficient to induce neuronal
death. Second, neogenin expression in neurons was inadequate
because of low levels of proinflammatory cytokines from Th17
cells. It was reported that IL-23 is secreted from macrophages
8 Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors
and contributes to inflammation following
brain ischemia (Shichita et al., 2009). IL23 is also an essential factor for the development of EAE (McGeachy et al., 2009 and Langrish et al., 2005).
The present study revealed that IL-23 contributes to both inflammation and neuronal damage via Th17 cells.
As RGMa has also an adherent function (Lah and Key, 2012),
it was suggested that the RGMa antibody could reduce neuronal
death by inhibiting T cell-neuron adhesion. However, the RGMa
antibody did not affect adhesion between T cells and neurons in
the present study. Interestingly, Th17 cells adhered to neurons
more efficiently than Th0 cells. T cells express various types of
adhesion molecules, such as very late antigen 4 (VLA-4) and
lymphocyte function-associated antigen 1 (LFA-1; Rothhammer
et al., 2011; Tian et al., 2000). Th17 cells may express high levels
of adhesion molecules that facilitate interactions with neurons.
Indeed, treatment with the RGMa antibody did not completely
inhibit Th17-cell-induced neuronal death. Thus, the neurotoxic
effects of T cells may depend partially on the strength of adhesion between T cells and neurons.
The proposed mechanisms that contribute to neuronal
damage in MS are antibodies, complements, mitochondrial
dysfunction caused by aberrant ion channels, and proinflammatory mediators from macrophages, microglia, and astrocytes
(Davalos et al., 2012; Friese et al., 2007; Mathey et al., 2007;
Mead et al., 2002, 2004; Schattling et al., 2012; Storch et al.,
2002; Wang et al., 2005). However, effective treatments for
neurodegeneration in patients with MS have not been developed until now. Here, we report a molecule involved in both
inflammation and neurodegeneration in MS. It should also be
noted that degeneration of the optic nerve is also induced in
Th17-EAE mice (Herges et al., 2012). Therefore, the RGMa antibody may be useful for protecting neurons from the attack of
Th17 cells in the optic nerve as well as the spinal cord of MS
patients. Our present study builds on our previous results (Muramatsu et al., 2011) and reveals a mechanism of Th17 cellinduced neurodegeneration in EAE. Collectively, our findings
indicate that RGMa inhibition could be an effective treatment
to ameliorate both inflammation and subsequent neurodegeneration in MS.
EXPERIMENTAL PROCEDURES
Animals
C57BL/6J WT mice were purchased from SLC Japan. 2D2 MOG35–55 TCR
transgenic mice were a kind gift from Dr. Masaaki Murakami (Osaka University,
Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in
Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038
Figure 7. RGMa in Th17 Cells Induces Akt
Dephosphorylation in Neurons
(A) Schema (left) for T cell removal from the
T cell-neuron coculture system. T cell removal
was confirmed by flow cytometry analysis of CD4
labeling (right).
(B) Phospho-Akt levels in neurons were detected
by western blot.
(C) The quantification of phospho-Akt obtained
from (B) (n = 4), normalized to total Akt levels.
(D) Phospho-ERK expression in neurons was
detected.
(E) The quantification of ERK1 phosphorylation
obtained from (D) (n = 4), normalized to total ERK
levels.
(F) The quantification of ERK2 phosphorylation
levels obtained from (D) (n = 4).
Statistical analysis was performed by oneway ANOVA followed by Tukey-Kramer tests
(*p < 0.05, **p < 0.01). Error bars represent
mean ± SEM.
145-2C11, eBioscience) and anti-CD28 (2.5 mg/ml,
37.51, eBioscience) antibodies for 4 days in the
presence of appropriate cytokines and antibodies.
For the differentiation of each type of T cell, the
following cytokines and antibodies were used.
Th0: IL-2 (10 ng/ml, R&D Systems); Th1: IL-2
(10 ng/ml), IL-12 (10 ng/ml, R&D Systems) and
anti-IL-4 antibody (25% culture supernatant of
hybridoma, HB-188); Th17: IL-6 (30 ng/ml, R&D
Systems), TGF-b1 (3 ng/ml, R&D Systems), IFN-g
antibody (2% culture supernatant of hybridoma,
HB-170), and presence or absence of IL-23
(20 ng/ml, R&D Systems); and Treg: IL-2
(10 ng/ml) and TGF-b1 (5 ng/ml). After 4 days,
T cell differentiation efficiency was examined by
flow cytometry analyses.
Intracellular Cytokine Staining
Differentiated T cells were collected and stimulated with phorbol 12-myristate 13-acetate (PMA:
100 ng/ml, Sigma-Aldrich), ionomycin (750 ng/ml,
Calbiochem), and brefeldin A (1 mg/ml, SigmaAldrich) for 4 hr at 37 C. Cells were fixed in fixation
buffer (eBioscience), permeablized in permeabilization buffer (eBioscience), and stained with fluorescent-labeled IFN-g (1:250, BioLegend), IL-17A
(1:100, BioLegend), CD4 (1:100, BioLegend), and
Foxp3 (1:100, BioLegend) antibodies diluted in
permeabilization buffer for 30 min at 4 C. Data
were collected with a FACS Canto II (BD Biosciences) and analyzed with FlowJo software (Tree
Star).
Osaka, Japan). All experiments adhered to the guidelines for the care and use
of laboratory animals of Osaka University.
In Vitro T Cell Differentiation
CD4+ T cells were purified from spleen with a CD4+ T cell isolation kit II (Miltenyi
Biotech). Isolated T cells were stimulated with plate-bound anti-CD3ε (5 mg/ml,
Reverse Transcription and qRT-PCR
Total RNA was extracted from neurons and each
type of T cell with an RNeasy Mini Kit (QIAGEN),
and reverse transcription was performed using a PrimeScript RT Master
Mix (Takara), both according to the manufacturers’ protocols. Quantitative
RT-PCR was performed with SYBR green real-time PCR master mix (Applied
Biosystems) with oligonucleotide primer sets corresponding to the
cDNA sequences of glyceraldehyde phosphate dehydrogenase (GAPDH),
RGMa, and neogenin as follows: mouse GAPDH, 50 -TGTGTCCGTCGTGGA
Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors 9
Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in
Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038
TCTGA-30 , 50 - TTGCTGTTGAAGTCGCAGGAG-30 ; mouse RGMa, 50 -TTGGAG
GATGGCAAGATGCT-30 , 50 -CCCGAGTCCTTTCAAACAGATG-30 ; and mouse
neogenin, 50 -TCCAAACACAATAAGCCTGACG-30 , 50 - ATGGGACCAAATCTG
CATTAACT-30 (sense and antisense, respectively). PCR reactions and analyses were carried out using a 7300 real-time PCR system (Applied Biosystems). The relative intensity versus GAPDH and the fold change relative
to control were calculated.
Induction of EAE and Disease Analysis
Induction of Th17-EAE using 2D2 mice was performed as previously
described (Ja¨ger et al., 2009). CD4+ T cells were isolated from spleen of
8- to 12-weeks-old 2D2 MOG35-55 TCR-specific transgenic mice by using
a CD4+ T cell isolation kit II. CD4+ T cells were stimulated with anti-CD3ε
antibody (2.5 mg/ml) in the presence of irradiated splenocytes (3,000 rad)
under Th17-polarizing conditions, including IL-23. After 5 days, cells were
collected and re-stimulated with plate-bound anti-CD3ε (5 mg/ml) and antiCD28 (2.5 mg/ml) antibodies for 2 days. The differentiation status was
checked on day 5 by intracellular cytokine staining, and 2.0 3 106 cytokine-producing cells were intravenously injected into 8-weeks-old female
mice. The recipient mice were irradiated with X-ray before injection
(500 rad). Three hundred micrograms of rabbit isotype control IgG (I-5006,
Sigma-Aldrich) or rabbit anti-RGMa antibody (28045, Immuno-Biological
Laboratories [IBL]) was administered intraperitoneally every 7 days. We
monitored and assessed the EAE scores daily according to the following
criteria: 0, no abnormalities noted; 1, loss of tail reflex; 2, partial hind limb
paralysis; 3, complete hind limb paralysis; 4, front and hind limb paralysis;
and 5, moribund state. EAE scores were assessed by investigator who
was blinded to the treatments.
Histological Analysis
Thirty days after the transfer of 2D2 Th17 cells, mice were anesthetized with a
cocktail of domitor (0.3 mg/kg), dormicum (4 mg/kg), and butorphanol (5 mg/
kg) and transcardially perfused ice-cold PBS followed by 4% PFA (Merck).
The cervical spinal cords were dissected and postfixed with 4% PFA overnight at 4 C, then immersed in phosphate-buffered 20% sucrose (pH 7.2)
overnight at 4 C. The spinal cords were then embedded in optimal cutting
temperature compound (Tissue-Tek), and cross-sections were cut at
40 mm thickness on a cryostat. For immunohistochemistry, sections were
permeabilized in PBS containing 0.1% Triton X-100 and blocked with 3%
normal goat serum (Sigma-Aldrich) for 1 hr at room temperature. Sections
were incubated with primary antibodies overnight at 4 C and then incubated
with fluorescent-labeled secondary antibodies for 1 hr at room temperature.
The primary antibodies were used as follows: rabbit anti-Iba1 (1:3,000, 01919741, Wako), mouse anti-GFAP (1:1,000, G-A-5, Sigma-Aldrich), rabbit antiAPP (1:200, Sigma-Aldrich), rabbit anti-neogenin (1:500, NBP1-89651, Novus
Biologicals), and mouse anti-axonal neurofilament (1:1,000, SMI-312, Covance). For secondary antibodies, Alexa 488- or Alexa Fluor 568-conjugated
goat anti-rabbit IgG or mouse IgG (1:500, Molecular Probes) were used. To
assess demyelination in the spinal cord, we stained myelin with green fluorescent lipophilic dye (FluoroMyelin, 1:500, Invitrogen) for 30 min at room
temperature. The labeled sections were imaged under confocal microscopy
(FV1200, Olympus). To assess the numbers of inflammatory lesions, we performed H&E staining (Muto Kagaku). Inflammatory lesions were defined as
accumulation of more than ten cells. The numbers of inflammatory lesions
were counted from five to eight sections per mouse. For APP signal quantification, ten inflammatory lesions per mouse were imaged under confocal
microscopy, and the number of APP signals was quantified by ImageJ software (US NIH). Counted APP signals were normalized to the area of lesions
per mm2. For quantification of the axon preservation, the spinal cord sections
were stained with SMI-312 antibody, and the number of SMI-312+ axons in
inflammatory lesions of dorsal column was quantified by ImageJ software.
The average of axon number in intact mice was defined as 100%, and we
calculated the percentage of preserved axons from EAE mice treated
with control IgG or anti-RGMa. For quantification of demyelinated areas, fluoromyelin-positive areas in white matter were quantified by ImageJ, and
the percentages of demyelinated areas in the total white matter areas were
calculated.
10 Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors
Flow Cytometry Analysis of CNS-Infiltrating Immune Cells
At 30 days after 2D2 T cell transfer, mice were anesthetized and transcardially
perfused with ice-cold PBS. The brain and spinal cord were dissected out and
minced with a scalpel. The minced tissues were digested with 0.1% collagenase D (Roche Applied Science) containing 2.5 mM calcium chloride at 37 C
for 30 min. We prepared single-cell suspensions by trituration, and the
cells were resuspended in 30% Percoll (GE Healthcare). Next, 70% Percoll
was layered underneath and centrifugation was performed at 2,000 rpm for
20 min at room temperature. We isolated infiltrated immune cells from the interfaces of 30%/70% Percoll gradients. For flow cytometry analysis, cells were
treated with anti-CD16/32 antibody (1:100, eBioscience) for 10 min on ice to
block Fc receptors and then stained with fluorescent-labeled antibodies for
TCR Va 3.2 TCR Va 3.2 (1:250, eBioscience), CD11b (1:100, BioLegend),
CD4 (1:100, BioLegend), and/or CD45 (1:100, BioLegend). For intracellular
cytokine staining, cells were stimulated with PMA (100 ng/ml), ionomycin
(750 ng/ml), and brefeldin A (1 mg/ml) at 37 C for 4 hr. After fixation and
permeabilization, cells were stained with antibodies for TCR Va 3.2 (1:250,
eBioscience), IL-17A (1:100, BioLegend), and/or CD4 (1:100, BioLegend).
Proliferation of T Cells
T cell proliferation was measured with Cell Proliferation ELISA, BrdU (colorimetric) (Roche Diagnostics). Splenocytes were isolated from Th17-EAE mice
at 30 days after transfer and stimulated with MOG35–55 peptide (0, 1, 5, or
10 mg/ml, CS Bio) in 96-well plates (12.5 3 105/ml). After 48 hr, BrdU was
added, and the cells were cultured for an additional 24 hr. BrdU incorporation
was measured according to the manufacturer’s protocol.
Coculture of T Cells and Neurons
Cortical neurons were collected from the cerebral cortices of embryonic
day 17 to 18 mice embryos. Harvested cortex was minced and digested in
0.25% trypsin for 15 min at 37 C. Cells were triturated and passed through
70 mm nylon cell strainer. The resultant cell suspension was diluted with Neurobasal medium (Life Technologies) supplemented with B-27 (Life Technologies), GlutaMAX (Life Technologies), and penicillin/streptomycin (Gibco) and
plated on culture slides coated with poly-D-lysine (Sigma-Aldrich) at a density
5.0 3 105 cells per ml. Three days after plating, differentiated Th0 or Th17 cells
were added at 1:1 ratio to the neurons with or without anti-RGMa antibody
(10 mg/ml, IBL).
TUNEL Labeling and Immunocytochemistry
Cell death was detected by TUNEL staining with the In Situ Cell Death Detection
Kit, TMR red (Roche Applied Science) according to the manufacturer’s protocol.
Briefly, cells were fixed with 4% PFA and permeabilized with 1% sodium citrate
containing 0.1% Triton X-100 for 5 min. Then, cells were reacted with TMR-dUTP
in the presence of terminal deoxynucleotidyl-transferase enzyme for 1 hr at
37 C. For immunocytochemistry, cells were blocked with 3% normal goat
serum in PBS containing 0.1% Triton X-100 for 1 hr and incubated with anti-b
Tubulin III antibody (1:2,000, TUJ1, Covance) overnight at 4 C. Cells were
washed three times and incubated with Alexa 488-conjugated goat anti-mouse
IgG (1:500, Molecular Probes) and DAPI (1 mg/ml, Santa Cruz Biotechnology) for
1 hr. After three washes, slides were mounted with fluorescent mounting medium (Dako). Neuronal apoptosis was quantified using fluorescence microscopy
(BZ-9000, Keyence). To count the total cells, DAPI+ cells were counted and then
TUNEL+ cells were counted from ten random fields. The rate of TUNEL-positive
cells was calculated as the number of TUNEL/DAPI 3 100 (%).
Adhesion Assay for Neurons and T Cells
Differentiated T cells were labeled with 5 mg/ml 30 ,60 -Di(O-acetyl)-40 ,50 -bis
[N,N-bis(carboxymethyl)aminomethyl] fluorescein, tetraacetoxymethyl ester
(calcein-AM, Dojindo) for 30 min at 37 C. After three washes with RPMI1640 medium, 2.0 3 105 T cells were added to cortical neurons (2.5 3 105)
that were cultured for 3 days and incubated at 37 C with rabbit IgG
(10 mg/ml, Sigma-Aldrich) or RGMa antibody (10 mg/ml). After 6 hr incubation,
nonadherent cells were removed by washing with RPMI medium containing
0.5% bovine serum albumin four times. The fluorescent intensity was
measured with a SpectraMAX (Molecular Devices) at excitation and emission
wavelengths of 485 and 538 nm, respectively.
Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in
Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038
siRNA Transfection
Cortical neurons (5.0 3 106 cells) were suspended in 100 ml Nucleofector solution (Amaxa Biosystems) containing 500 pmol control (MISSION siRNA Universal negative control, Sigma-Genosys) or neogenin siRNA (Sigma-Genosys).
Electroporation was performed by program O-005 as described by the manufacturer’s protocol (Amaxa Biosystems). After 3 days, neogenin expression
was confirmed by quantitative RT-PCR and western blot, and Th0 or Th17
cells were added to siRNA-electroporated neurons. The neogenin siRNA sequences were 50 -CAAUUCCAUGGAUAGCAAU-30 and 50 -AUUGCUAUCCA
UGGAAUUG-30 .
Western Blot
T cells were lysed in lysis buffer containing 150 mM NaCl, 1% Triton X-100,
20 mM HEPES (pH 7.4), 10% glycerol, 5 mM EDTA, and complete protease
inhibitor cocktail (Roche Applied Science). Neurons and T cells were cocultured for 6 hr. Neurons were separated from the coculture system with antiCD4 microbeads (Miltenyi Biotech), and unlabeled fractions were regarded
as neurons. Neurons were lysed in radioimmunoprecipitation assay (RIPA)
buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM Tris-HCl (pH 7.5), complete protease inhibitor
cocktail, and phosSTOP phosphatase inhibitor (Roche Applied Science).
After incubation on ice for 30 min, lysates were centrifuged at
15,000 rpm for 30 min at 4 C, and the supernatants were collected. Protein
concentrations were determined with a bicinchoninic acid assay (BCA) kit
(Pierce). Thirty micrograms of each sample were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene
fluoride (PVDF) membranes (Millipore). Membranes were blocked with
5% bovine serum albumin in PBS containing 0.05% Tween-20 for 1 hr
and incubated with rat anti-RGMa (2 mg/ml, MAB2458, R&D Systems), rabbit anti-phospho Akt (1:1,000, 9271, Cell Signaling Technology), rabbit antitotal Akt (1:1,000, 9272, Cell Signaling Technology), rabbit anti-phospho
ERK (1:1,000, 4370, Cell Signaling Technology), rabbit anti-total ERK
(1:1,000, 9102, Cell Signaling Technology), rabbit anti-neogenin (1:1,000,
H-175, Santa Cruz), rabbit anti-b-actin (1:1,000, 4970, Cell Signaling Technology), or mouse anti-a-tubulin (1:250, sc-5286, Santa Cruz) in blocking
solution overnight at 4 C. After washing, membranes were incubated with
horseradish peroxidase-conjugated secondary antibody to rat, rabbit, or
mouse IgG (1:3,000, Cell Signaling Technology) for 1 hr. Detection was performed by Pierce Western Blotting Substrate Plus (Pierce) and RAS-3000
(Fuji Film).
Statistics
Student’s t tests, one-way ANOVA followed by Tukey-Kramer tests, and twoway ANOVA followed by Bonferroni tests were performed with GraphPad
Prism 6 software. p < 0.05 was considered significant.
AUTHOR CONTRIBUTIONS
S.T. designed and performed the experiments and analyzed the data. T.Y.
coordinated and directed the project. T.Y. and S.T. conceived the project
and wrote the manuscript.
ACKNOWLEDGMENTS
We thank M. Murakami and D. Kamimura (Osaka University) for their kind gift of
2D2 TCR transgenic mice, H. Ishii (Columbia University) for technical advice,
and R. Muramatsu (Osaka University) for helpful suggestions. This work was
supported by a grant for Core Research for Evolutional Science and Technology from the Japan Science and Technology Agency to T.Y. and by a Grant-inAid for Scientific Research (S) from the Japan Society for the Promotion of
Sciences (25221309) to T.Y.
Received: July 12, 2014
Revised: September 22, 2014
Accepted: October 14, 2014
Published: November 13, 2014
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