S170 Abstracts / IBRO Reports 6 (2019) S54–S345
P09.07
A step towards demystifying sleep physiology:
Forward genetics approach in mice
Staci J. Kim 1 , Chika Miyoshi 1 , Takahiro Ezaki 2 ,
Aya Ikkyu 1 , Noriko Hotta-Hirashima 1 , Satomi
Kanno 1 , Miyo Kakizaki 1 , Mana Yamada 1 ,
Shigeharu Wakana 3 , Hiromasa Funato 1 , Masashi
Yanagisawa 1,∗
1 International Institute for Integrative Sleep
Medicine (WPI-IIIS), University of Tsukuba, Tsukuba,
Japan
2 RCH Center for Advanced Science and Technology,
The University of Tokyo, Tokyo, Japan
3 Institute of Biomedical Research and Innovation,
Kobe, Japan
Although the maintenance of good sleep is essential for the
physical and mental well-being, the fundamental mechanism of
sleep and wake behavior is largely unknown. In an attempt to
demystify the regulatory cascade of sleep and wake behavior, we
established a large-scale dominant screening system of randomly
mutagenized mice based on EEG and EMG polysomnography. This
study describes the details of the methodology and discusses the
theoretical basis of the forward genetics study in sleep. Two closely
related C57BL/6J and C57BL/6N mouse substrains were selected as
mutagenized and counter strains, respectively, based on their near
identical sleep/wake behavior. The reproducibility test of various
sleep/wake parameters indicated the total wake time, NREMS time
and the REMS episode duration to be the most suitable parameters
in phenotypic screening showing low variability, as indicated by
coefficient of variance. We performed simulations of LOD score as
a function of the screening size and show the relationship among
the achievable LOD score, statistical significance and the extent of
the sleep phenotype. In addition, a mutant pedigree carrying a het-
erozygous mutation in Cacna1a gene is described showing a mild
decrease in the total wake time. Our study provides basis for appli-
cation of the phenotypic screening in other types of behavior than
sleep and wakefulness.
https://doi.org/10.1016/j.ibror.2019.07.534
P09.08
Immune-tolerance to cytosolic neural antigens
biases the retinal environment towards a
neuroprotective profile
Esperanza Melendez Herrera 1,∗ , Lorena
Martínez-Alcantar 1 , Diana Karina
Talavera-Carrillo 1 , Jonhatan Uriel
Pineda-Salazar 1 , Miguel Avalos-Viveros 1 , Gabriel
Gutiérrez-Ospina 2 , Bryan Victor Phillips-Farfán 3 ,
Alma Lilia Fuentes-Farías 1
1 Universidad Michoacana de San Nicolás de
Hidalgo, Morelia, Mexico
2 Universidad Nacional Autónoma de México, Ciudad
de México, Mexico
3 Instituto Nacional de Pediatría, Ciudad de México,
Mexico
Retinal ganglion cells (RGC) die massively early after optic nerve
injury, not only as a result of forthright trauma to their axons, but
also due to secondary degeneration triggered by the pernicious
interplay between neural and immune cells. A chief factor driving
this secondary death is the establishment of chronic inflammatory
processes triggered by the continuous presentation of neural
tissue-derived debris to T cells, which acquire a pro-inflammatory
(type 1 T helper, Th1) phenotype and damage healthy neighboring
cells. Thus, reducing the Th1 response could protect the neural
tissue and avoid secondary death associated to injury. To test this
idea, systemic immune-tolerance to neural-tissue derived antigens
was induced via anterior chamber-associated immune deviation
(ACAID) prior to optic nerve crush injury. Different neural-tissue
derived extracts (WH: whole homogenate, CF: cytosolic fraction
and MF: membrane fraction) were used to induce ACAID. Their
potential to avoid or delay secondary death of RGC was tested 7
and 14 days after injury (dai). The results show that all extracts
induced an antigenic response in the delayed hypersensitivity test
as well as ACAID after anterior chamber inoculation, but only the
CF improved survival of RGC after injury. Additionally, to charac-
terize the environment that supports this neuroprotective effect,
mRNA expression of several immune mediators, neurotrophic
factors and phenotype markers was analyzed 2 and 7 dai in retinas
from CF immune-tolerant animals. The results showed increased
expression of anti-inflammatory mediators in CF immune-tolerant
rats at both evaluated times, as well as differential expression
of BDNF and NT4. Moreover, increased expression of markers
for regulatory T cells and alternatively activated macrophages
was observed at 7 dai. The data suggest that immune-tolerance
to neural-tissue derived cytosolic proteins induced by ACAID is
an efficient strategy to avoid or delay secondary degeneration
following traumatic injury to the central nervous system.
https://doi.org/10.1016/j.ibror.2019.07.535
P09.09
Nd1-L as a novel stress granules (SGs)
component that regulates stress granule
dynamics and associates with autophagy
components
Pureum Jeon 1 , Sang-Won Park 2 , Yong-Woo
Jun 2 , Mi-Hee Jun 1 , You-Kyung Lee 1 , Ha-Eun
Choi 1 , Deok-Jin Jang 2 , Jina Lee 1,∗
1 Hannam University, Daejeon, Republic of Korea
2 Kyungpook University, Sangju, Republic of Korea
Autophagy selectively or non-selectively degrades cytosolic
components in lysosome by LC3/GABARAP-mediated autophago-
some biogenesis or cargo recognition/recruitment. This process
is tightly regulated by many LC3/GABARAP-binding proteins with
a short hydrophobic LC3-interacting region (LIR) motif. To date,
although several putative LIR motifs from many LIR-containing
proteins have been identified, their specific binding properties for
LC3, or GABARAP proteins (LC3s, GABARAPs) and their functional
significances on autophagy remain elusive. Here, we developed
a novel method to detect the binding property of potential LIR-
motifs for LC3s or GABARAPs in live cells. Using this novel method,
among 88 putative LIR motifs from LIR-containing proteins. We
found that 22 LIR motifs had significant binding to LC3s, or (and)
GABARAPs. Among them, a canonical LIR motif from Nd1-L had
selective GABARAP/L1/L2 binding in a LIR-dependent manner. Very
interestingly, upon oxidative stress, Nd1-L is localized to TIA-1 pos-
itive stress granules together with GABARAPL1 and it associates
with p62, or ataxin2. Loss of Nd1-L reduces dynamics of SGs and
number of SGs while the size of SGs was increased indicating its
role on regulation of stress granule. Therefore, we propose Nd1-L
as a novel component of SGs and autophagy.
https://doi.org/10.1016/j.ibror.2019.07.536