National Institute for Basic Biology

Neurobiology

DIVISION OF MOLECULAR NEUROBIOLOGYProfessor

NODA, Masaharu

Associate Professor

SHINTANI, Takafumi

Assistant Professor:

SAKUTA, Hiraki
HIYAMA, Takeshi
Technical Staff:
TAKEUCHI, Yasushi
NIBB Research Fellow:
NOMURA, Kengo
Postdoctoral Fellow:
FUJIKAWA, Akihiro
SUZUKI, Ryoko
KUBOYAMA, Kazuya
LIN, Chia-Hao
SOKENDAI Graduate Student:
MATSUDA, Takashi
YU, Yang
TANGA, Naomi
HIGASHI, Satoru
Technical Assistant:
MIURA, Seiko
NAKANISHI, Norie
DOKYO, Yumi
WADA, Kotoe
KONISHI, Mie
ISOSHIMA, Yoshiko
HASHIMOTO, Terumi
Secretary:KODAMA, Akiko
We have been studying the molecular and cellular mechanisms underlying the development of the vertebrate central
nervous system (CNS), mainly using mice. This researchcovers many developmental events including the patterningof the retina, neuronal terminal differentiation, axonal navigation, branching and targeting, synapse formation, refinement and plasticity. The scope of our interests also encompasses the mechanisms for various functions of the mature
brain, including body-fluid regulation, behavior control,
learning, and memory.

I. Mechanisms for neural circuit formation

Adenomatous polyposis coli 2 (APC2) is preferentiallyexpressed in the nervous system from early developmental
stages through to adulthood. APC2 is distributed along actin
fibers as well as microtubules in neurons. Our investigationsuggests that APC2 is involved in the signaling pathway
from membrane receptors for extracellular guidance factors
to the intracellular migration machinery.
Sotos syndrome (OMIM #117550) is characterized byintellectual disability and a combination of typical facial
features and large head circumference. Sotos syndrome has
been known to be caused by haploinsufficiency in the NSD1gene. Our knockdown experiments revealed that the expression of APC2 in the nervous system was under the controlof NSD1. Moreover, Apc2-knockout (KO) mice also showed
Sotos syndrome-like abnormalities. We are now investigating
the relationship between NSD1 and APC2 in more detail byexamining Nsd1-KO mice.

II. Physiological roles of receptor-like protein
tyrosine phosphatases

various biological events such as cellular proliferation, differentiation, survival, migration, and metabolism. Cellulartyrosine phosphorylation levels are governed by the opposing
activities of protein tyrosine kinases (PTKs) and protein
tyrosine phosphatases (PTPs). The physiological functions
and regulatory mechanisms of receptor-like PTPs (RPTPs)
are not fully elucidated. We have been making efforts toreveal the functional roles of RPTPs, especially of the R3and R5 subfamilies.

2-1 R3 RPTP subfamily
The R3 RPTP subfamily, which is comprised of PTPRB,PTPRH, PTPRJ, and PTPRO, reportedly plays pivotal
roles in the development of several tissues including thevascular and nervous systems. We demonstrated, for thefirst time, that PTPRJ is a physiological enzyme attenuating insulin signaling in vivo. Ptprj-KO mice show lower
weight gain associated with lesser food intake comparedwith wild-type mice. Thus we are now investigating a roleof PTPRJ in the regulation of energy homeostasis.
2-2 R5 RPTP subfamily

PTPRZ is the most abundant RPTP in oligodendrocyte
precursor cells (OPCs), which are the principal source ofmyelinating oligodendrocytes. Three PTPRZ isoforms aregenerated by alternative splicing from a single PTPRZ gene:two transmembrane isoforms, PTPRZ-A and PTPRZ-B, andone secretory isoform, PTPRZ-S, all of which expressedin the CNS are heavily modified with chondroitin sulfate(CS) chains. The CS moiety on their extracellular domain ofPTPRZ is essential for achieving high-affinity binding sites
for the endogenous ligands; pleiotrophin (PTN), midkine,
and interleukin-34. However, its functional significance andmolecular mechanism in regulating PTPRZ activity remainsobscure.
We have shown that PTPRZ receptors have functions tomaintain the immature status of OPCs by dephosphorylating
various substrate molecules including p190 RhoGAP. Thisyear, we revealed that protein expression of CS-modified
PTPRZ-A began earlier, peaking at postnatal days 5–10 (P5–P10), and then that of PTN peaked at P10 at the developmental stage corresponding to myelination onset in the mousebrain. Ptn-KO mice consistently showed a later onset of theexpression of myelin basic protein, a major component of
the myelin sheath, than wild-type mice. Upon ligand application, PTPRZ-A/B in cultured oligodendrocyte precursor cells
exhibited punctate localization on the cell surface instead ofdiffuse distribution, causing the inactivation of PTPRZ andoligodendrocyte differentiation. Notably, the same effectwas observed with the removal of CS chains with chondroitinase ABC (chABC), but not polyclonal antibodies against
the extracellular domain of PTPRZ (Figure 1). These data
indicate that PTN-PTPRZ-A signaling controls the timingof oligodendrocyte precursor cell differentiation in vivo, inwhich the negatively charged CS moiety maintains PTPRZin a monomeric active state, and that the positively-charged
PTN induces receptor clustering, potentially by neutralizing
electrostatic repulsion between CS chain.

Protein tyrosine phosphorylation plays crucial roles in35

National Institute for Basic Biology

Neurobiology

subfornical organ (SFO) and organum vasculosum lamina
terminalis (OVLT), and is involved in [Na+] sensing for thecontrol of salt-appetite.

CS modification

α-PTPRZ-S

chABC
PTN

3-1 Thirst control in the brain by Nax and TRPV4cell membrane
Transmembrane

Inhibition

No inhibition

Repulsive fieldPTN

α-PTPRZ-SPTPRZFigure 1. Schematic representation of the role of the CS chains in regulating PTPRZ activity. The CS modification in PTPRZ was essential for
maintaining the monomeric active form, possibly by electrostatic repulsion, and, at the same time, provided ligand binding sites. The binding ofPTN appears to neutralize the negative charges of the CS chains, causing
the dimerization and inactivation of PTPase (see text).

Emerging data have indicated that PTPRZ is aberrantly
over-expressed in glioblastoma and it is a causative factorfor its malignancy. However, small molecules that selectivelyinhibit the catalytic activity of PTPRZ have not been developed so far. We identified SCB4380 as the first potent inhibitor for PTPRZ by in vitro screening of a chemical library.
We determined the crystal structure of the catalytic domainof PTPRZ. Furthermore, the structural basis of the bindingof SCB4380 elucidated by a molecular docking method
was validated by site-directed mutagenesis studies (Figure2A). The intracellular delivery of SCB4380 by liposome
carriers inhibited PTPRZ activity in C6 glioblastoma cells,and thereby suppressed their migration and proliferation invitro and tumor growth in a rat allograft model (Figure 2B).
Therefore, selective inhibition of PTPRZ represents a promising approach for glioma therapy.
ABvehicleOVLT (SFO)Nax

SCB4380

2.0

[Na+]

Na+

*

Na+-dependent signal

Water-intake
control neuron

Water intake

Ca2+
TRPV4vehicle

SCB4380

0AA

Figure 2. Development of SCB4380 and its application to a rat glioblastoma model. A, whole view of the best docking pose of SCB4380 shownby stick representation. B, rat C6 glioblastoma cells transplanted into rat
brains were allowed to grow for 7 days. One day after surgery, SCB4380/
liposome, or vehicle was icv injected daily for 5 days. Representative
sections are shown together with an enlargement of the boxed area. Thegraph shows tumor volume. The horizontal bars indicate the average ofeach group. *P < 0.05, by the Student’s t-test.

III. Brain systems for body-fluid homeostasis

We have previously shown that Na x, which structurally
resembles voltage-gated sodium channels (Nav1.1–1.9), isa Na+-concentration ([Na+])-sensitive Na channel with agating threshold of ~150 mM for extracellular [Na+] ([Na+]o)in vitro. Nax is preferentially expressed in the glial cells ofsensory circumventricular organs (sCVOs) including the36

Na+EETs

1.0

active center

Astrocytes
EETs

BloodVessel

volume (mm3)SCB4380

We found that Nax is also involved in thirst control indehydrated animals (Figure 3). We investigated voluntarywater intake immediately induced after an intracerebroventricular (icv) administration of a hypertonic NaCl solution
in mice. Because the transient receptor potential vanilloid(TRPV) channels, TRPV1 and TRPV4, have been proposed
to function as osmosensors, we examined TRPV1-, TRPV4-,Na x-, and their double-gene KO mice. The induction ofwater intake by TRPV1-KO mice was normal, whereas thatby TRPV4-KO and Nax-KO mice was significantly less thanthat by WT mice. Water intake by Nax/TRPV4-double KO
mice was similar to that by the respective single KO mice.
When TRPV4 activity was blocked with a specific antagonist(HC-067047), water intake by WT mice was significantly
reduced, whereas that by TRPV4-KO and Nax-KO mice wasnot. The same results were obtained by the administration ofmiconazole, which inhibits the biosynthesis of epoxyeicosatrienoic acids (EETs), endogenous agonists for TRPV4, fromarachidonic acid (AA). Moreover, icv injection of hypertonic
NaCl with AA or 5,6-EET restored water intake by Nax-KO
mice to the WT level, but not that by TRPV4-KO mice.
These results suggest that the Na+ signal generated in Naxpositive glial cells leads to the activation of TRPV4-positiveneurons in sCVOs in order to stimulate water intake by using
EETs as gliotransmitters.

Nax[Na+]Ependymal cells

3rd Ventricle

Figure 3. A possible signaling pathway in sCVOs underlying the induction of water intake. When [Na+]o in plasma and cerebrospinal fluid
increases, Nax channels (red) in glial cells (astrocytes and ependymalcells) in the SFO or OVLT are activated, leading to the synthesis of EETs.
EETs released from glial cells activate TRPV4 channels (blue) in theneighboring neurons, which conceivably control water-intake behavior.
The additional activation signal of an unknown Na+-dependent mechanism is required for the Nax/TRPV4-dependent induction of water intake.

3-2 Thirst and salt-appetite control in the brain byangiotensin II, [Na+], and cholecystokinin

Angiotensin II (Ang II) is known to drive both thirst andsalt appetite; however, the neural mechanisms underlying selective water- and/or salt-intake behaviors remain
unknown. Using optogenetics, we showed that thirst andsalt appetite are driven by distinct groups of angiotensinII receptor type 1a-positive excitatory neurons in the SFO(Figure 4). Neurons projecting to the OVLT control water
intake, while those projecting to the ventral part of the bed
nucleus of the stria terminalis (vBNST) control salt intake.
Thirst-driving neurons are suppressed under sodium-depleted
conditions through cholecystokinin (CCK)-mediated activation of GABAergic neurons. In contrast, the salt-appetitedriving neurons were suppressed under dehydrated conditions through activation of another population of GABAergic
neurons by Nax signals. These distinct mechanisms in theSFO may underlie the selective intakes of water and/or salt
and may contribute to body fluid homeostasis.
<Water-depleted condition><Na-depleted condition>

SFO

SFO

Ang IIGABA
Water

CCKGABA
Nax
Glia

GABA

Salt

Na+Water

3rd ventricle

OVLTvBNSTOVLT•〔Original papers (E-publication ahead of print)〕

GABA
Nax
Glia

Salt

3rd ventricle

Na+•Uchiyama, S., Tomoo, T., Ogata, A., Masumura, M., and Noda, M.
(2016). Small-molecule inhibition of PTPRZ reduces tumor growth in arat model of Glioblastoma. Sci. Rep. 6, 20473.
Kuboyama, K., Fujikawa, A., Suzuki, R., Tanga, N., and Noda, M.
(2016). Role of chondroitin sulfate (CS) modification in the regulationof protein tyrosine phosphatase receptor type Z (PTPRZ) activity:
Pleiotrophin-PTPRZ-A signaling is involved in oligodendrocyte
differentiation. J. Biol. Chem. 291, 18117-18128.
Sakuta, H., Nishihara, E., Hiyama, T.Y., Lin, C.-H., and Noda, M.
(2016). Nax signaling evoked by an increase in [Na+] in CSF induces
water intake via EET-mediated TRPV4 activation. Am. J. Physiol.
Regul. Integr. Comp. Physiol. 311, R299-R306.

T.Y., Utsunomiya, A.N., Matsumoto, M., Fujikawa, A., Lin,• Hiyama,C.-H., Hara, K., Kagawa, R., Okada, S., Kobayashi, M., Ishikawa, M.,

Ang II

Na+A., Nagahira, A., Sugawara, H., Ishii, K., Imajo, S.,• Fujikawa,Matsumoto, M., Kuboyama, K., Suzuki, R., Tanga, N., Noda, M.,vBNST

ThirstSalt appetite

Salt appetite

Water intake

Salt intake

Salt intake

Figure 4. A schematic overview of controls for thirst and salt appetitefrom SFO. Under the water-depleted condition (left), both Ang II and[Na+] increase in SFO. “Water neurons” innervating OVLT are selectivelyactivated under the water-depleted condition. Under the Na-depleted condition (right), both Ang II and CCK increase in SFO, and “salt neurons”
which innervate vBNST are selectively activated. Under the water- andNa-depleted condition, both water and Na intakes are stimulated, because
neither the Nax signal nor CCK signal is induced.

•Anzo, M., Cho, H., Takayasu, S., Nigawara, T., Daimon, M., Sato,T., Terui, K., Ito, E., and Noda, M. Adipsic hypernatremia without
hypothalamic lesions accompanied by autoantibodies to subfornicalorgan. Brain Pathol. 2016 Aug 2.
Matsuda, T., Hiyama, T.Y., Niimura, F., Matsusaka, T., Fukamizu,A., Kobayashi, K., Kobayashi, K., and Noda, M. Distinct neuralmechanisms for the control of thirst and salt appetite in the subfornicalorgan. Nat. Neurosci. 2016 Dec 19.

〔Review articles〕

A., and Noda, M. (2016). Role of pleiotrophin-protein• Fujikawa,tyrosine phosphatase receptor type Z signaling in myelination. NeuralRegen. Res. 11, 549-551.

T.Y., and Noda, M. (2016). Sodium sensing in the subfornical• Hiyama,organ and body-fluid homeostasis. Neurosci. Res. 113, 1-11.

3-3 Adipsic hypernatremia accompanied by autoantibodies to the SFO

Adipsic (or essential) hypernatremia is a rare hypernatremia
caused by a deficiency in thirst regulation and vasopressin
release. In 2010, we reported a case in which autoantibodies
targeting Nax caused adipsic hypernatremia without hypothalamic structural lesions demonstrable by magnetic resonanceimaging (MRI). This year, we reported three newly identified patients with similar symptoms. Immunohistochemical
analyses using the sera of these patients revealed that antibodies specifically reactive to the mouse SFO were present
in the sera of all cases. Passive transfer to mice of the patient
immunoglobulin revealed that autoimmune destruction of
the SFO may be the cause of the adipsic hypernatremia. This
study provides a possible explanation for the pathogenesis ofadipsic hypernatremia without demonstrable hypothalamuspituitary lesions.

Publication List:〔Original papers〕

T., Kamiya, M., Obata, F., Yamaguchi, Y., Hiyama, T.Y.,• Doura,Matsuda, T., Fukamizu, A., Noda, M., Miura, M., and Urano, Y.

(2016). Detection of lacZ-positive cells in living tissue with single-cellresolution. Angew. Chem. Int. Ed. Engl. 55, 9620–9624.

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