Transcript Prion disease

The Role of Cellular Chaperones and
the Unfolded Protein Response in
GSS Syndrome
Julie Moreno PhD
Telling Lab
Prion protein (PrP):
a known cellular factor
• PrP null mice are unable to replicate and produce
disease when exposed to infectious prion
materials
(Brandner et al., 1996; Bueler et al., 1993; Fischer et al., 1996; Manson et al., 1994; Prusiner et al., 1993)
– Removal of cellular PrP (PrPC) can reverse
pathogenesis and is neuroprotective
(Mallucci et al., 2003; 2007; White et al., 2008)
• However, not all cells are susceptible to prion
infection even with PrP expression
– Some prion susceptible cells have sub populations
that are resistant as well
Hypothesis
• In addition to the prion protein, additional
unidentified cellular factors regulate disease
susceptibility and pathogenesis
Generation and Identification of
susceptible (S) and resistant (R) cells
Prions
Uninfected
Experimental Set-up: Further Analysis
of uninfected S and R cells
1.) Uninfected
Passage
Known S and R
Archive
Passage
2.) Prions
Protein
lysates
RNA
lysates
Further Analysis
Questions
– What genes/cellular pathways are responsible for
prion susceptibility?
– Is there a unique genetic signature dependent on
the specific PrP strain? Or generic?
Identification of differences between S
and R cells
RK13-ElkPrP differential
expressed genes
RK13-Deer PrP differential
expressed genes
108 known genes (80%)
28 ‘new’genes (20%)
Combined RK13- Deer, Elk and OvinePrP differential
expressed genes
Small heat shock protein or
chaperone: HSPB8
• With the co-chaperone BAG3 is known to activate and
recruit autophagic machinery in protein folding
disorders (Carra et al, 2008 JBC ; Crippa et al., Hum Mol Gen 2010)
• Found upregulated in Alzheimer’s (AD), Parkinson’s
(PD), Huntington’s (HD) and spinocerebellar ataxia type
3 (SCA3) diseased patient brains specifically in
astrocytes (Seidel et al., 2012 Neuropath and Appl Neurobio.)
• Inhibits protein synthesis though the P-eIF2a
stimulating autophagy (Carra et al, 2009 JBC)
Participation of HSPB8 in regulated
proteolysis of misfolded proteins
Prion disease
©2011 by American Physiological Society
Mymrikov E V et al. Physiol Rev 2011;91:1123-1159
Objectives
• Assess cellular changes of HSPB8 and the UPR
during disease
• Determine the role of HSPB8 on prion protein
misfolding and pathogenesis
• Investigate the modulation of HSPB8 levels in
cells and TgGSS mice
Spontaneous neurodegenerative
disease in Tg(GSS)mice
Tg(MoPrP-P101L)-Tg(GSS)22
134±1 (8/8)
Tg(MoPrP)
100
>700 (0/20)
75
50
25
0
1
101
201
301
401
501
601
701
Tg(MoPrP-P101L)
220±10 (18/18)
Spongiform degeneration
Astrocytic Gliosis
Amyloid deposition
Nazor et al., 2005
Spontaneous neurodegenerative
disease in Tg(GSS)mice
Cerebellum
Cortex-GFAP
Nazor et al., 2007
Cortex
HSPB8 protein levels rise in susceptible
cells and with clinical disease
1
HSPB8
GAPDH
60
40
0
Preclinical
103 dpi
117 dpi
**
20
DeerPrP-S
Clinical
50
139 dpi
40
HSPB8
GAPDH
HSPB8 protein levels
(relative to GAPDH)
2 3
Tm
2 3 RKV D+ 24hr
HSPB8 protein levels
(relative to GAPDH)
1
80
R cells
S cells
DeerPrP-R
**
30
20
10
0
UI
103
117
139
HSPB8 levels increase overtime in
Tg(MoPrP-P101L) GSS mice
31
HSPB8
40
GAPDH
Role of HSPB8 in susceptibility and
disease
• HSPB8 is increased in:
– cells susceptible to prions
– Mice infected with prions
– Tg(GSS) genetic mouse model of prion disease
• What is the mechanism behind HSPB8
changes?
HSPB8 and Unfolded
Protein Response (UPR)
HSPB8
P
eIF2a
Gene-Specific
Translation
BACE1
ATF4
Ab
CREB-P
General
Translation
Rplp0
Rpl27
SNAP29
PPARG
C1A
Memory deficits/ Neurodegeneration
Conclusions
• Established resistant cell lines that express PrP
however lack ability to be infected
– Identified candidate genes and pathways
• HSPB8 levels rise in susceptible cells, prion
infection and during Tg(GSS) disease progression
• eIF2a-P or activation occurs during disease
progression in Tg(GSS) mice
Future directions
• Continue analysis of HSBP8 and the UPR in
Tg(GSS) mice
• Modulate genes/pathways using genome
editing and expression constructs
• Continue to identify genes important
to prion susceptibility
Acknowledgements
Glenn Telling
Richard Bessen
Telling Lab:
Current members
Jifeng Bian
Hae Eun Kong
Sehun Kim
Vannessa Selwyn
Delaney Swindle
Matt Sabel
Michael Young
James Dilisio
Colin Hastings
Tim Koh
Colin Wakeman
Maddie Harmon
Former members
Jeff Christiansen
Crystal Reid
Vadim Khaychuk
Carla Calvi
Funding
CJD Foundation Grant
R21 NS091027