studies

308A AASLD ABSTRACTS HEPATOLOGY, October, 2015
196
A functional classification of ABCB4 missense variations
identified in progressive familial intrahepatic cholestasis
type 3
Jean-Louis Delaunay 1 , Anne-Marie Durand-Schneider 1 , Claire Dossier
1 , Thomas Falguières 1 , Julien Gautherot 1 , Tounsia Aït-Slimane 1 ,
Chantal Housset 1 , Emmanuel Jacquemin 2 , Michèle Maurice 1 ;
1 UMR_S 938, UPMC & Inserm, Paris Cedex 12, France; 2 Hépatologie
Pédiatrique, APHP, Hôpital Bicêtre, Kremlin-Bicêtre, France
Progressive familial intrahepatic cholestasis type 3 (PFIC3) is
caused by bi-allelic ABCB4 variations. More than 70 % of
disease-causing ABCB4 variations are missense variations.
The aims of the study were i) to propose a functional classification
of ABCB4 missense variations; ii) to test the rescue of
trafficking-defective mutants by cyclosporins. Methods: Twelve
single-nucleotide missense variations that were identified in 9
PFIC3 patients (6 homozygous, 3 composite heterozygous)
and located in or close to transmembrane domains (F357L,
S346I, P726L, T775M, Q855L, G954S), first intracellular loop
(T175A), first nucleotide-binding domain (T424A, N510S,
I541F, L556R), and linker region (R652G), were reproduced in
the ABCB4 cDNA. The mutants thus obtained were expressed
in HepG2 (transiently) and HEK 293 cells (stably), and compared
with wild type ABCB4, to determine their impact on the
ABCB4 expression or phosphatidylcholine transport activity,
measured by a fluorimetric assay. Results: Four mutants were
either fully (I541F and L556R) or partially (S346I and Q855L)
retained in the endoplasmic reticulum, in an immature endoglycosidase
H-sensitive form, of lower molecular weight than
the mature N-glycosidase F-sensitive form. Phosphatidylcholine
secretion by these mutants was less than 10 % that of wild
type, except for Q855L (30 %). Rescue of these trafficking
defects, i.e. exit from the endoplasmic reticulum and increase
of the mature form at the bile canaliculi, was obtained by cell
treatments with cyclosporins A or C, and to a lesser extent
B, D or H. Four mutations with little or no effect on ABCB4
expression at the bile canaliculi, caused a significant reduction
(F357L, T775M and G954S) or absence (P726L) of phosphatidylcholine
secretion. Two mutants (T424A and N510S) were
normally processed and expressed at the bile canaliculi but
their stability, assessed by protein decay, was reduced. We
found no defect of the T175A mutant, nor of R652G, previously
described as a polymorphism, which herein co-existed
with another variant (G954S) on the same allele, in two siblings.
In patients, the most severe phenotypes appreciated by
the duration of transplant-free survival, were caused by ABCB4
variants that were markedly retained in the endoplasmic reticulum,
and expressed in a homozygous status. In conclusion,
ABCB4 variations can be classified as follows: nonsense variations
(I), and on the basis of current findings, missense variations
that primarily affect the traffic (II), activity (III) or stability
(IV) of the protein. Among missense variants, those in class II
cause the most severe clinical phenotypes and can be rescued
by pharmacological therapies.
Disclosures:
The following authors have nothing to disclose: Jean-Louis Delaunay, Anne-Marie
Durand-Schneider, Claire Dossier, Thomas Falguières, Julien Gautherot, Tounsia
Aït-Slimane, Chantal Housset, Emmanuel Jacquemin, Michèle Maurice
197
Liver-specific deletion of the insulin receptor profoundly
reduces accumulation and proteotoxicity of misfolded
α 1
-antitrypsin Z (ATZ) in a mouse model
Andrew Chu 1 , Tunda Hidvegi 1 , Souvik Chakraborty 1 , Micheal
Ewing 1 , Amitava Mukherjee 1 , Patrick Araya 2 , Pamela Hale 1 ,
Christine Dippold 1 , Yan Wang 1 , Jie LI 1 , Evelyn Akpadock 1 , Hou
Ming Cai 1 , Stephen C. Pak 1 , Donna B. Stolz 2 , Gary A. Silverman
1 , David H. Perlmutter 1 ; 1 Pediatrics - Gastroenterology, Children’s
Hospital of Pittsburgh of UPMC; University of Pittsburgh,
Pittsburgh, PA; 2 University of Pittsburgh, Pittsburgh, PA
Alpha-1-antitrypsin deficiency (ATD)-associated liver disease
is an important cause of cirrhosis and hepatocellular carcinoma,
with liver transplantation currently the only treatment
for advanced disease. Insulin signaling is a key regulator of
proteostasis mechanisms, with inhibition of insulin signaling
linked to increased longevity in animals. The purpose of our
study was to determine whether abrogation of insulin signaling
reduces ATZ accumulation and proteotoxicity in a mouse
model of ATD-associated liver disease. To investigate the effect
of loss of insulin signaling, we generated double transgenic
mice (IRZZ) by crossing liver-specific insulin receptor knockout
mice (LIRKO) to the PiZ mouse model of ATD-associated
liver disease. Histological analysis of livers from male mice
at 6 months and 12-14 months demonstrated a 60 percent
reduction of intracellular ATZ globules by PAS-D staining and
a 50 percent reduction in hepatic fibrosis by Sirius Red staining
in IRZZ mice compared with PiZ mice. There was also a
complete elimination of nodular regeneration in the IRZZ liver.
This is consistent with results from a C. elegans model of ATD
in which RNAi corresponding to several intermediates in the
insulin signaling pathway reduce intracellular ATZ accumulation.
To determine the mechanism of reduced hepatic ATZ load
in the IRZZ mice, we carried out pulse-chase radiolabelling on
isolated hepatocytes and found a significant increase in intracellular
degradation of ATZ in the IRZZ compared to the PiZ
hepatocytes. To determine whether specific gene expression
signatures or signaling pathways were involved in the effect
of insulin receptor deletion, we used transcriptomic analysis to
compare hepatic RNA levels in IRZZ to PiZ and control (LIRKO
and wild-type) mice. Compared to PiZ, the gene expression
pattern in IRZZ mice showed downregulation of collagen V
consistent with the marked decrease in Sirius Red staining,
upregulation of genes associated with hepatocellular proliferation/regeneration
(prominin 1 and 2), and upregulation of
genes involved in lysosomal function (lysosomal ATPase, H +
transporting, V0 subunit D isoform, Atp6v0d2). Furthermore,
using immunofluorescence for the lysosomal membrane protein
LAMP2 and transmission electron microscopy, we found a
marked increase in number of lysosomes in the IRZZ as compared
to the PiZ liver, suggesting activation of the autophagolysosomal
system in the IRZZ model. These results indicate that
the insulin signaling pathway suppresses a key mechanism by
which the liver attempts to protect itself from misfolded ATZ and
suggest that activation of the autophagolysosomal system is the
target of this mechanism.
Disclosures:
The following authors have nothing to disclose: Andrew Chu, Tunda Hidvegi,
Souvik Chakraborty, Micheal Ewing, Amitava Mukherjee, Patrick Araya, Pamela
Hale, Christine Dippold, Yan Wang, Jie LI, Evelyn Akpadock, Hou Ming Cai,
Stephen C. Pak, Donna B. Stolz, Gary A. Silverman, David H. Perlmutter