2020 EIIRISプロジェクト研究成果報告会

The epigenetic change of granule cell precursors causes excess
folding of cerebellar lobules to induce ASD-like malformation
⾃閉症モデル動物における神経細胞の変化とエピジェネティクス
応⽤化学・⽣命⼯学系 吉⽥祥⼦

The Schematic diagram of the cerebellar development
Immature

MatureJuvenile

Granular cell

EGL

EGL

ML

Purkinje cell

ML
PL

PL

PL

IGL

IGL

Golgi cell
Stellate cell

Materials & Methods

B

O
H 3C

A

1. 3B

O-NaML
PL

EGL
ML
PL

IGL

IGL

P16 control Cb

CD

VPA-ad Cb

E1

1. 110. 90. 8VH

E 16V PA 20 0E 16V PA 30 0E 16V PA 40 00. 8E 16
V PA 60 0GVHE 14E 16E 18

V PA 60 0

V PA 60 0

V PA 60 0

Excess folding was observed at the primaryfissure between the V and VI lobules in VPAad Cb (B and D) and appeared in the Cbadministrated the VPA 400 mg/kg above with
P14 VPA Cb
P14 control Cb
dose-dependent manner (E).
The E14 VPA 600 mg/kg administrated rat pups showed similar abnormal
development of the cerebellar cortex which was observed in the VPA 300
and 400 mg/kg administrated rat, however, the E18 VPA administrated pups
did not (F).

Result 2. Epigenetic changes in Cb early development
AIBB

AJEGL
PL
P3 control

P3 VPADCLKEEGLPL

P5 control

F

P3 control

P3 VPA

CDEGL

EGL

P5 VPA+P7 control

Valproate (VPA)
200-600mg/kg VPA was dosed P.O. to pregnant rat at E16, sometimes at E14 or E18.
Offspring was fixed at P3 to P21, and observed its cerebellum (Cb) stained with antiCalbindin D-28k, anti-glast, anti-GFAP, anti-reelin and anti-H3K27 antibodies.
In Hematoxylin-Eosin image, we calculated the ratio of the length of Purkinje layer (redline) to the depth of gyrus (green line).

PCs in P14 control Cb PCs in P14 VPA-ad Cb

1. 1

Fig 2. Excess folding in VPA-administrated Cb and dose- or timing-dependencies.

CH 3

DF

1. 2

0. 9

VPA(600mg/kg)

C

1. 3

E

1. 2Basket cell

After birth, the two layers, the External Granular Layer (EGL) and the Purkinje Layer (PL)are in the cerebellar cortex. With maturing, many granule cells migrate down to the InternalGranular Layer (IGL) and extend their axons towards the Molecular Layer (ML), andPurkinje cells become developed with elongation of their dendrites. Other interneurons
migrate up to the ML to develop the complex neuronal circuits with each other.

AResult
1. Excess foldingof cerebellar lobulesCB
N o rm a liz e d fo ld in g ra teA

Valproate (VPA), the popular anticonvulsant and mood stabilizer, isknown as an inducer of autism. It has many kinds of physiologicalproperties, including the inhibition of histone deacetylase (HDAC).
Recently we reported VPA administration to rat fetus caused
developmental changes and malformation in the cerebellar cortex,correlated with autism. In the VPA-administrated cerebellum, thedendrites of Purkinje cells were elongated earlier than in vehicles andimmature granule cells were left in the external germinal layer even in
P16. Additionally, VPA-administrated rat showed the excess folding
between the V to VI lobules of cerebellar vermis within two weeks afterF
birth with dose-dependent and administrated-period dependent
mannerand maintained in adult. This alteration would be deeply related withepigenetic change of neuronal development due to VPA.

N o rm a liz e d fo ld in g ra teIntroduction

In VPA-ad Cb, Purkinje cells (PCs)showed the abnormal form of dendrites
and broken lines of soma (C: control Cb,
D: VPA-ad Cb both in P14).

P7 VPA

PLIGL

PL
P7 control

IGL

P7 VPAFig 3. Excess folding in VPA-administrated earlydeveloping Cb. A, C, E: control, B, D, F: VPA-ad.

EFEven in P3 VPA-ad Cb,
acetylated H3K27 positive
cells were observed in theEGL(Fig.4B),whereas excess
EGL
folding was observed after EGL
PLP5 (Fig.3). As VPA is known PLIGL
P7 control
P7 VPAasnon-specific
HDAC IGL
inhibitor, it would make Fig 4 Distribution of acetylated H3K27 positive cells ( A, B), Calbindin D28k (green)
and reelin (red) (C, D) and GFAP (green) and GLAST (red) (E, F).
granular neuron precursors
deacetylate and develop. Reelin expressed higher in the EGL in P7 VPA-Cbanimals (Fig.4D) than control animals (Fig.4C), and GLAST, glutamate
transporter on astrocytes was developed earlier in P7 VPA-ad Cb (Fig.4F)
than control (Fig.4E).

Conclusion
VPA is a famous antiepileptic agent and a well-known HDAC inhibitor which causes wide-spread epigenetic change. Even in P3
cerebellum of VPA-administrated rat, H3K27-acetylated granule cells appeared in the EGL and the irregular folding appeared in P5. EGL
Epigenetic-changed granule cells expressed reelin earlier than control and migrated down to the Internal granular layer making thePLline for the excess folding. The expression of the astrocyte-specific glutamate transporter, GLAST was increased in P7 cerebellar
IGLcortex of VPA-admin.
In the developing cerebellum, the granule cells in the IGL secrete Reelin to regulate the position of Purkinje neurons. We suggest
H3K27 positive cells
the epigenetic alteration of granule cells with VPA would induce excess reelin expression and early development of granule cells.

P7 VPA