National Institute for Basic Biology

Neurobiology

DIVISION OF MOLECULAR NEUROBIOLOGYProfessor

Associate Professor

NODA, Masaharu

SHINTANI, Takafumi

Assistant Professor:

SAKUTA, Hiraki
HIYAMA, Takeshi
Technical Staff:
TAKEUCHI, Yasushi
NIBB Research Fellow:
MATSUDA, Takashi
NOMURA, Kengo*
Postdoctoral Fellow:
FUJIKAWA, Akihiro
SUZUKI, Ryoko
KUBOYAMA, Kazuya
LIN, Chia-Hao
NOMURA, Kengo
YU, Yang
SOKENDAI Graduate Student: MATSUDA, Takashi*
YU, Yang*
TANGA, Naomi
HIGASHI, Satoru
Technical Assistant:
MIURA, Seiko
NAKANISHI, Norie
WADA, Kotoe
KONISHI, MieISOSHIMA, Yoshiko
HASHIMOTO, Terumi
Secretary:KODAMA, Akiko

The scope of our interests encompasses underlying mechanisms for the development of the vertebrate central nervoussystem (CNS) and various functions of the mature brain,including body fluid homeostasis, blood pressure control,
food intake 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 by intellectual disability and a combination of typical facial featuresand large head circumference. Sotos syndrome has been
known to be caused by haploinsufficiency in the NSD1 gene.
Our knockdown experiments revealed that the expression of
APC2 in the nervous system was under the control of 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 by examining
Nsd1-KO mice.

II.Physiological roles of receptor-like protein
tyrosine phosphatases
Protein tyrosine phosphorylation plays crucial roles invarious 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

38

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, play pivotal roles in the development of several tissues including the vascular and nervous
systems. Eph receptor tyrosine kinases are known to playindispensable roles in the topographic central projectionof retinal ganglion cell (RGC) axons. Using the chick retinotectal projection system, we previously showed thatPTPRO controls the sensitivity of retinal axons to ephrins
and thereby has a crucial role in the establishment of topographic projections. We also demonstrated that R3 RPTPs
dephosphorylate Eph receptors. Among R3 RPTPs, PTPRJ
and PTPRO were expressed in developing mouse RGCs.
We are now investigating the relationship between roles ofPTPRJ and PTPRO in the central projection of the retinal
axons by using gene knockout mice.
Leptin, an adipocyte-derived hormone, is a critical factor
controlling food intake: it strongly inhibits food intake
through the regulation of neuronal activities in the hypothalamic arcuate nucleus (ARC). Most obese individuals
have an increased food intake despite high circulating leptin
levels, which is referred to as leptin resistance. However,
the exact mechanisms underlying leptin resistance in obese
patients have yet to be elucidated. We recently demonstrated
that PTPRJ is a physiological enzyme attenuating insulin
signaling in vivo. In Ptprj-KO mice, the activation of insulin
receptor (IR) kinase and Akt (or protein kinase B) wasenhanced, and glucose and insulin tolerance was improved.
Furthermore, Ptprj-KO mice exhibited lower weight gain
associated with lesser food intake compared with wild-type
mice.
We have now found that PTPRJ is involved in the regulation of leptin signaling in the ARC through the dephosphorylation of JAK2, the primary tyrosine kinase in leptin signaling. In line with these results, leptin signaling is enhancedin Ptprj-KO mice. Diet-induced obesity up-regulated PTPRJ
expression in the hypothalamus, and induction of leptin resistance was strongly attenuated in Ptprj-KO mice. Upon leptin
administration, Ptprj-KO mice fed a high-fat/high-sucrose

Figure 1. Ptprj-KO mice are protected from the development of dietinduced leptin resistance. (A) Daily change in food intake by and (B)Daily change in body weights of wild-type (WT) and Ptprj-KO mice fed
high-fat/high-sucrose diet (HF/HSD for 14 weeks) upon daily administration of leptin. Leptin (500 ng) or vehicle was i.c.v. injected during
the indicated period. ANOVA was used to detect significant differencesat the same time point between the two groups (n = 8 each; *P < 0.05,**P < 0.01, ***P < 0.001).

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2017. The former title isindicated by an asterisk (*).

diet showed significant reductions in food intake and bodyweight as compared with corresponding WT mice (Figure1). Moreover, the overexpression of PTPRJ in the ARC in
non-obese mice with a viral vector induced significant leptin
resistance. Overall, our results indicated that PTPRJ playscritical roles in the development of leptin resistance in vivo.

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 gene: twotransmembrane isoforms, PTPRZ-A and PTPRZ-B, and one
secretory isoform, PTPRZ-S (or phosphacan). All isoforms
are heavily modified with chondroitin sulfate (CS) chains,and identified as chondroitin sulfate proteoglycans (CSPGs)
in the CNS. The CS moiety on their extracellular domain of
PTPRZ is essential for achieving high-affinity binding sites
for the endogenous ligands such as pleiotrophin (PTN). We
have proposed that PTPRZ is a new molecular target for drug
development in glioblastoma and demyelinating diseases
such as multiple sclerosis (MS).
CSPGs enriched in demyelinating plaques are known to
impair remyelination by inhibiting the migration and differentiation of OPCs in MS patients. This year, we revealed
that protamine (PRM) effectively neutralizes the inhibitory
activities of CSPGs, thereby enhancing OPC differentiationand (re)myelination in mice. Cell-based assays revealed
that PRM exerted masking effects on extracellular CSPGs,
and also acted as a ligand mimetic for PTPRZ-A/B, thereby
improving oligodendrocyte differentiation even under unfavorable differentiation conditions with CSPGs (Figure 2).
Intranasal administration of PRM accelerated myelination in
the developing mouse brain, and its intracerebroventricularadministration stimulated remyelination after cuprizoneinduced demyelination.

transcription factors such as SOX2, OLIG2, and POU3F2,
along with decreased sphere-forming abilities of these cells.
We identified an allosteric PTPRZ inhibitor, NAZ2329(Figure 3A) for the first time. This compound efficiently
reduced the expression of SOX2 in C6 and U251 cells,
and abrogated the sphere-forming abilities of these cells.
Furthermore, tumor growth in the C6 xenograft mouse model
was significantly slower with the co-treatment of NAZ2329
with temozolomide, an alkylating agent, than with the individual treatments (Figure 3B). These results indicate thatPTPRZ is a promising molecular target for differentiationinducing therapy of malignant gliomas.

Figure 3. Identification of a new allosteric inhibitor, NAZ2329. A, X-ray
structure of PTPRZ1-D1 complexed with NAZ2329. Cys1933 at theactive site and Val-1911 at the bottom of the allosteric pocket are indicated in red and yellow, respectively. B, Antitumor effect of NAZ2329.
Nude mice subcutaneously implanted with C6 glioblastoma cells weretreated with DMSO, NAZ2329, temozolomide (TMZ), or the combination of NAZ2329 with TMZ until the humane endpoint (>3,000 mm3tumor size or 40 days after the treatment). Kaplan-Meier analysis of thefour treatment groups.

III. Brain systems for body-fluid homeostasis
We have shown that Na x, which structurally resembles
voltage-gated sodium channels (Nav1.1–1.9), is the brain[Na+] sensor to detect increases in [Na+] in body fluids. Nax
is preferentially expressed in specific glial cells of sensory
circumventricular organs (sCVOs) including the subfornical organ (SFO) and organum vasculosum lamina terminalis (OVLT). We have reported that Nax signals in these brain
regions deficient in a blood-brain barrier are involved in thecontrol of water and salt intake.

3-1 Thirst control by Nax and TRPV4

Figure 2. A possible regeneration model: Protamine may neutralize
CSPG-mediated inhibition of migration of OPCs into demyelinatingplaques and differentiation of OPCs to oligodendrocytes (see text).

A recent study on primary human glioblastomas suggested aclose association between PTPRZ1 (human PTPRZ) expression and cancer stemness. However, the functional roles ofPTPRZ in glioma stem cells have remained unclear. As wasexpected, sphere-forming cells from separated rat C6 andhuman U251 glioblastoma cell lines showed high expressionof PTPRZ-B, the short receptor isoform of PTPRZ. Stable
PTPRZ knockdown altered the expression levels of stem cell

We previously demonstrated that Nax signals in Nax-positive
glial cells leads to the activation of TRPV4-positive neurons
by using epoxyeicosatrienoic acids as gliotransmitters insCVOs to stimulate water intake. Also, we suggested that
another [Na+]-dependent signal is needed besides the NaxTRPV4 signal to explain the whole amount of water intake.
In order to identify the sensor molecule which generates theunknown [Na+]-dependent signal, we performed RNA-seq
analysis of sCVOs and identified several candidates. We arenow examining the functional roles of these candidates inwater intake.

3-2 Thirst and salt-appetite control by angiotensin II,[Na+], and cholecystokinin
Angiotensin II (Ang II) is known to drive both thirst andsalt appetite, and Ang II levels in blood are increased underboth water- and salt-depleted conditions. However, we selec-39National Institute for Basic Biology

Neurobiology

tively feel a thirst or salt appetite dependent on body fluid
conditions. The brain mechanisms by which we properly
take in water or salt have not been fully elucidated. Our
local deletion experiments of AT1a, an Ang II receptor gene,indicated that AT1a signals in the SFO are involved in bothwater and salt intakes, whereas those in the OVLT are onlyinvolved in water intake. AT1a-positive neurons in the SFO
project to several nuclei, including the OVLT and ventral
part of the bed nucleus of the stria terminalis (vBNST).
By using optogenetics, we demonstrated that thirst andsalt appetite are driven by distinct groups of AT1a-positive
neurons in the SFO: Neurons projecting to the OVLT control
water intake, while those projecting to the vBNST control
salt intake. We named these two different driver neurons‘water neurons’ and ‘salt neurons’, respectively (Figure 4).
‘Water neurons’ were suppressed under Na-depleted conditions through cholecystokinin (CCK)-mediated activation ofGABA(a-aminobutyric acid)ergic neurons. As was expected,CCK levels in the SFO were increased under Na-depleted(water-satiated) conditions. The control mechanism of CCK
secretion in the SFO remains to be elucidated. We hadreported that Nax-KO mice do not stop salt intake even underwater-depleted conditions. We herein revealed that ‘salt
neurons’ were suppressed under the water-depleted conditions through activation of another population of GABAergic
neurons by Nax signals. These studies thus provided new
insights into the central mechanisms by which body fluid
conditions control thirst and salt appetite. On the other hand,
it is also known that aldosterone enhances salt appetite
besides Ang II. Our next goal is integration mechanisms ofsalt-appetite signals from ‘salt neurons’ and ‘aldosteronesensitive neurons’ in the vBNST.
<Water-depleted condition><Na-depleted condition>

SFO

SFO

Ang II

Ang II
Na+

GABA
Water

CCK

GABA
Glia

Salt

GABA

Nax

Water

Na+

3rd ventricle

OVLT

Na+

GABA
Glia

Nax

Salt

3rd ventriclevBNST

OVLTvBNST

ThirstSalt appetite

Salt appetite

Water intake

Salt intake

Salt intake

Figure 4. A schematic overview of control mechanisms for thirst andsalt appetite in the SFO. Under the water-depleted condition (left), [AngII] and [Na+], but not [CCK], increase in the SFO, and “water neurons”
innervating the OVLT are selectively activated. Under the Na-depleted
condition (right), [Ang II] and [CCK], but not [Na+], increase, and “saltneurons” innervating the vBNST are selectively activated. These two
inhibitory systems by distinct GABAergic neurons are responsible for thespecific activation of “water neurons” or “salt neurons” dependent on thebody-fluid conditions. On the other hand, both water and Na intakes are
simultaneously stimulated under the water- and Na-depleted condition,
because the both neurons are active by Ang II.

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Publication List:〔Original papers〕
A., Chow, J.P.H., Matsumoto, M., Suzuki, R., Kuboyama, K.,• Fujikawa,Yamamoto, N., and Noda, M. (2017). Identification of novel splicing•
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•
•
•
•
•

variants of protein tyrosine phosphatase receptor type Z. J. Biochem.
162, 381-390.
Fujikawa, A., Sugawara, H., Tanaka, T., Matsumoto, M., Kuboyama, K.,Suzuki, R., Tanga, N., Ogata, A., Masumura, M., and Noda, M. (2017).
Targeting PTPRZ inhibits stem cell-like properties and tumorigenicity
in glioblastoma cells. Sci. Rep. 7, 5609.
Hiyama, T.Y., Utsunomiya, A.N., Matsumoto, M., Fujikawa, A., Lin,C.-H., Hara, K., Kagawa, R., Okada, S., Kobayashi, M., Ishikawa, M.,
Anzo, M., Cho, H., Takayasu, S., Nigawara, T., Daimon, M., Sato, T.,
Terui, K., Ito, E., and Noda, M. (2017). Adipsic hypernatremia without
hypothalamic lesions accompanied by autoantibodies to subfornicalorgan. Brain Pathol. 27, 323-331.
Kuboyama, K., Tanga, N., Suzuki, R., Fujikawa, A., and Noda, M.
(2017). Protamine neutralizes chondroitin sulfate proteoglycanmediated inhibition of oligodendrocyte differentiation. PLoS ONE 12,e0189164.
Matsuda, T., Hiyama, T.Y., Niimura, F., Matsusaka, T., Fukamizu, A.,
Kobayashi, K., Kobayashi, K., and Noda, M. (2017). Distinct neuralmechanisms for the control of thirst and salt appetite in the subfornicalorgan. Nat. Neurosci. 20, 230-241.
Nakayama, K., Ohashi, R., Shinoda, Y., Yamazaki, M., Abe, M.,Fujikawa, A., Shigenobu, S., Futatsugi, A., Noda, M., Mikoshiba, K.,Furuichi, T., Sakimura, K., and Shiina, N. (2017). RNG105/caprin1, an
RNA granule protein for dendritic mRNA localization, is essential for
long-term memory formation. eLife 6, e29677.
Shintani, T., Higashi, S., Suzuki, R., Takeuchi, Y., Ikaga, R., Yamazaki,T., Kobayashi, K., and Noda, M. (2017). PTPRJ inhibits leptin
signaling, and induction of PTPRJ in the hypothalamus is a cause of thedevelopment of leptin resistance. Sci. Rep. 7, 11627.
Utsunomiya, A.N., Okada, S., Hiyama, T.Y., Noda, M., and Kobayashi,
M. (2017). Clinical characteristic features of adipsic hypernatremia
patients wit subfornical organ-targeting antibody. Clin. Pediatr.
Endocrinol. 26, 197-205.