Prion diseases are fatal, transmissible, neurodegenerative diseases caused by the misfolding of the prion protein (PrP). At present, the molecular pathways underlying prion-mediated neurotoxicity are largely unknown. We hypothesized that the transcriptional regulator of the stress response, heat shock factor 1 (HSF1), would play an important role in prion disease. Uninoculated HSF1 knockout (KO) mice used in our study do not show signs of neurodegeneration as assessed by survival, motor performance, or histopathology. When inoculated with Rocky Mountain Laboratory (RML) prions HSF1 KO mice had a dramatically shortened lifespan, succumbing to disease ≈20% faster than controls. Surprisingly, both the onset of home-cage behavioral symptoms and pathological alterations occurred at a similar time in HSF1 KO and control mice. The accumulation of proteinase K (PK)-resistant PrP also occurred with similar kinetics and prion infectivity accrued at an equal or slower rate. Thus, HSF1 provides an important protective function that is specifically manifest after the onset of behavioral symptoms of prion disease

Aguzzi, Adriano

Borkowski, Andrew W.

Heppner, Frank L.

Hutter, Gregor

Jackson, Walter S.

King, Oliver D.

Lindquist, Susan

Raymond, Gregory J.

Steele, Andrew D.

English

PubMed

Caltech Authors - Main

Supporting Information
Steele et al. 10.1073/pnas.0806319105
SI Methods
Prion Inoculations. For prion injections, mice were injected with
varying titers (indicated in Table S1) of RML ‘‘Chandler’’ strain
of murine adapted scrapie (30 l for IC or 100 l for IP). Normal
brain homogenate (1%) controls were included in several ex-
periments, such as histological samples and analysis of the
survival of uninfected mice. The titering of the infectivity of
samples (pooled from n  2 HSF1 WT and n  2 HSF1 KO)
taken at 2, 3, 4, and 5 MPI used Tga20 recipient mice that had
been backcrossed to C57BL/6J for 5–6 generations and bred
onto a WT PrP genome (1). CD1 recipients were used for titering
the infectivity of terminal brain samples taken pooled from
HSF1 WT (n  4) and HSF1 KO (n  4). Tga20 mice surviving
200 DPI and CD1 mice surviving 300 DPI were considered
uninfected. For survival analyses, the time of death represents
the actual death or the euthanasia when the mouse could no
longer right itself when placed on its back.
Statistical Analysis. We used bootstrap resampling (2) for the
statistical analysis of prion titers. First, we examined only a single
population of mice at a time, drawing 10,000 bootstrapped
samples from the population. For example, if there were N mice
in the population, we randomly selected n mice from the
population, but with replacement, and this was done 10,000
times. Then the titer was computed for each of these 10,000
randomly resampled populations, and these values were used to
estimate 95% confidence intervals for the titer, by eliminating
the 2.5% of extreme values from each end of the distribution of
titers. The two populations, HSF1 WT and HSF1 KO, were
separately simulated and bootstrap confidence intervals were
computed for each of them. To compute P values for differences
between populations, one with n mice and the other with m mice,
we first computed the titers for each population separately. We
then pooled all of the mice together into a population of size n
 M. Then, 10,000 times, we resampled (again with replace-
ment) one group of n mice and one group of m mice from this
pooled population, and computed the difference in titers for
these two populations. The fraction of times for which this
difference was bigger than the difference between the two
original nonresampled titers gives the empirical P value.
Other statistical calculations were performed using Graphpad
Instat (Wilcoxon rank sum) for behavioral data or Graphpad
Prism (log rank test) for survival data.
Western Blot Analysis. Brain homogenates were prepared in glass
dounce homogenizers as 10% homogenates (wt/vol) of whole
brain in PBS from samples frozen at indicated time points. After
sonication, large debris was pelleted by low-speed centrifugation.
Further dilutions were made into lysis buffer containing (PBS 
1% Tween 20  1% Triton X 100 and 150 mM NaCl). The
equivalent of 30–50 g of total protein was loaded onto 10%
Bis-Tris gels (Invitrogen), transferred to nitrocellulose mem-
branes, and analyzed by Western blotting using the monoclonal
antibody SAF83 for PrP (Cayman Chemical). A monoclonal
antibody against -tubulin was used as a loading control (Sigma).
Using infrared-conjugated secondary antibodies, protein was
visualized using the Licor Odyssey scanner.
Behavioral Analysis. Video-based software analysis of behavior
was similar to our prior study (3). Briefly, mice that were
normally group housed (n  2–5) were singly caged and video
recorded for 24 h, a complete light–dark cycle (dim red lights
were used for recording in the dark phase). The videos were
analyzed using the definitions and settings described in (3) using
HomeCageScan 2.0 software (Clever Systems) with the excep-
tion that ‘‘hang vertically’’ and ‘‘hang upside down’’ are merged
into the category ‘‘hanging.’’
Neuropathological Analysis. Brains were immersion fixed in for-
malin and treated with 98% formic acid for 1 h, then postfixed
in formalin for 24 h before paraffin embedding. Two-
micrometer-thick sections were cut onto positively charged glass
slides and stained with hematoxylin and eosin, or immuno-
stained. For prion aggregate staining, sections were deparaf-
finized and incubated for 6 min in 98% formic acid, then washed
in distilled water for 5 min. For antigen retrieval, sections were
heated to 100°C in a pressure cooker in citrate buffer (pH 6.0),
cooled for 3 min, then washed in distilled water for 5 min.
Immunohistochemical stains were performed on an automated
Nexus staining apparatus (VentanaMedical Systems) using an
IVIEW DAB Detection Kit (Ventana ). After incubation with
protease 1 (Ventana ) for 16 min, sections were incubated with
anti-PrP SAF-84 (SPI bio; 1:200) for 32 min. Sections were
counterstained with hematoxylin. GFAP (1:1000 for 24 min;
DAKO) immunohistochemistry for astrocytes and Iba1 (1:2500
for 32 min; Wako Chemicals) for microglia was performed
similarly, however with antigen retrieval by heating to 100°C in
EDTA buffer (pH 8.0) within a steamer. Analysis of vacuolation
and gliosis in the hippocampus, thalamus, striatum, cortex, and
cerebellum of prion-inoculated HSF1 WT and HSF1 KO brain
sections was conducted blindly.
1. Steele AD, Emsley JG, Ozdinler PH, Lindquist S, Macklis JD (2006) Prion protein (PrPc)
positively regulates neural precursor proliferation during developmental and adult
mammalian neurogenesis. Proc Natl Acad Sci USA 103:3416–3421.
2. Efron B, Tibshirani RJ (1994) An Introduction to Boostrapping. Chapman and Hall/CRC.
3. Steele AD, Jackson WS, King OD, Lindquist S (2007) The power of automated high-
resolution behavior analysis revealed by its application to mouse models of Hunting-
ton’s and prion diseases. Proc Natl Acad Sci USA 104:1983–1988.
Steele et al. www.pnas.org/cgi/content/short/0806319105 1 of 5
Fig. S1. Histological characterization of uninoculated HSF1 KO and control brain sections. Hematoxylin and eosin (H&E), Luxon nissl, and GFAP staining of HSF1
WT brain samples (A) and HSF1 KO brain samples (B). H&E staining of HSF1 knockout brains showed a variable amount of vacuolation and one representative
sample (out of 6 analyzed) is shown. This phenotype was present in HSF1 WT mice but at a much lower frequency. Luxon-Nissl myelin staining is slightly weaker
in the cerebellum of HSF1 KO mice (B). Gliosis measured with anti-GFAP staining showed a subtle increase in HSF1 KO cerebellum compared to HSF1 WT
cerebellum (A and B). (C and D) CNPase staining shown at 1 magnification (scale bar corresponds to 1 mm) of two HSF1 WT and two HSF1 KO brains to show
a largely intact myelination. (E and F) Higher magnification of the internal capsule of HSF1 WT (E) and HSF1 KO (F) sections is also shown. (G and H)
Immunohistochemistry for the oligodendrocyte antigen cyclic nucleotide phosphodiesterase (CNPase) of cerebella from HSF1 WT (G) and HSF1 KO (H) shown
at low magnification (scale bar corresponds to 100 m) and at higher magnification suggest ample expression of this antigen in both HSF1 WT and KO brains.
Steele et al. www.pnas.org/cgi/content/short/0806319105 2 of 5
Table S1. Summary of RML prion inoculations in HSF1 WT and KO mice
RML inoculum
dose/route
Median survival (days)
and no. of animals
Difference in median
survival (WT–KO) (days)
P value
(log rank test)
6.5 logLD50 IC WT (n  18) 177 KO (n  12) 144 33 0.0001
5.5 logLD50 IC WT (n  16) 184 KO (n  16) 146 38 0.0001
4.5 logLD50 IC WT (n  18) 201 KO (n  15) 165 36 0.0001
3.5 logLD50 IC WT (n  38) 202 KO (n  12) 181 22 0.0001
2.5 logLD50 IC WT (n  21) 225 KO (n  10) 186 39 0.0003
5.5 logLD50 IP WT (n  19) 254 KO (n  8) 201 53 0.0001
3.5 logLD50 IP WT (n  13) 272 KO (n  7) 228 44 0.0001
RML, Rocky Mountain Laboratory; IC, intracranial; IP, intraperitoneal
Steele et al. www.pnas.org/cgi/content/short/0806319105 3 of 5
Table S2. Summary of titration experiments from prion-inoculated HSF1 WT and KO brains over a time course of prion disease
Time point Dilution
Survival of recipients
(days postinoculation)
Titer
(logLD50/30 l)
Bootstrapped
P values
HSF1 WT 2 MPI (n  2) 102 72, 73, 74, 75, 76 4.1 0.019
103 94, 94, 96, 98, 99
104 112, 202, 202, 202, 202
105 123, 129, 202, 202, 202
106 202, 202, 202, 202, 202
107 202, 202, 202, 202, 202, 202
HSF1 KO 2 MPI (n  2) 102 79, 93, 94, 96 3.1
103 104, 109, 129, 202, 202
104 202, 202, 202, 202, 202, 202
105 202, 202, 202, 202, 202, 202
106 202, 202, 202, 202, 202, 202
107 202, 202, 202, 202, 202, 202
HSF1 WT 3 MPI (n  2) 102 62, 66, 74, 75
103 75, 75, 76, 77 6.25 0.320
104 73, 81, 83, 84
105 89, 99, 100, 101
106 90, 98, 201, 201
107 105, 201, 201, 201
108 201, 201, 201, 201
HSF1 KO 3 MPI (n  2) 102 66, 68, 69, 72 6.5
103 63, 76, 78, 79
104 85, 87, 90, 97
105 94, 94, 104, 107
106 120, 124, 139, 144
107 201, 201, 201, 201
108 201, 201, 201, 201
HSF1 WT 4 MPI (n  2) 103 74, 74, 75, 75, 76 8.25 0.055
104 74, 74, 83, 83
105 84, 89, 90, 91
106 90, 105, 124, 133
107 91, 124, 131, 200
108 125, 133, 200, 200
109 105, 179, 200, 200
HSF1 KO 4 MPI (n  2) 103 70, 70, 71, 74 6.65
104 83, 84, 87, 90
105 84, 90, 93, 96
106 93, 131, 200, 200
107 131, 200, 200, 200, 200
108 124, 200, 200, 200, 200
109 106, 200, 200, 200
HSF1 WT 5 MPI (n  2) 104 70, 82, 83, 85 6.75 0.547
105 82, 83, 83, 84, 85
106 96, 97, 103, 111
107 113, 200, 200, 200
108 200, 200, 200, 200
109 200, 200, 200, 200
1010 200, 200, 200, 200
HSF1 KO 5 MPI (n  2) 104 75, 76, 81, 81 6.9
105 87, 92, 98, 109
106 109, 130, 188
107 119, 122, 200, 200, 200
108 200, 200, 200, 200, 200
109 200, 200, 200, 200
1010 200, 200, 200, 200
HSF1 WT* terminal (n  4) 103 160, 163, 166, 166 8.1 0.016
104 153, 164, 170, 170, 176
105 166, 174, 174, 174, 174
106 174, 182, 182, 188, 188
107 192, 195, 210, 226, 226
108 239, 266, 300, 300, 300
109 239, 300, 300, 300, 300
HSF1 KO* terminal (n  4) 103 153, 153, 160, 161, 161 6.9
104 170, 170, 172, 175, 177
Steele et al. www.pnas.org/cgi/content/short/0806319105 4 of 5
Time point Dilution
Survival of recipients
(days postinoculation)
Titer
(logLD50/30 l)
Bootstrapped
P values
105 160, 175, 179, 182, 184
106 180, 182, 196, 198, 300
107 182, 205, 223, 300, 300
108 300, 300, 300, 300
109 300, 300, 300, 300
mpi, months postinoculation.
*CD1 recipient mice were used for determining these titers whereas Tga20 mice were used for all other titrations. The threshold for determining disease-free
status was a symptom-free mouse at 200 DPI for Tga20 and 300 DPI for CD1 mice.
Steele et al. www.pnas.org/cgi/content/short/0806319105 5 of 5


Heat shock factor 1 regulates lifespan as distinct from disease onset in prion disease

Prion diseases are fatal, transmissible, neurodegenerative diseases caused by the misfolding of the prion protein (PrP). At present, the molecular pathways underlying prion-mediated neurotoxicity are largely unknown. We hypothesized that the transcriptional regulator of the stress response, heat shock factor 1 (HSF1), would play an important role in prion disease. Uninoculated HSF1 knockout (KO) mice used in our study do not show signs of neurodegeneration as assessed by survival, motor performance, or histopathology. When inoculated with Rocky Mountain Laboratory (RML) prions HSF1 KO mice had a dramatically shortened lifespan, succumbing to disease approximately 20% faster than controls. Surprisingly, both the onset of home-cage behavioral symptoms and pathological alterations occurred at a similar time in HSF1 KO and control mice. The accumulation of proteinase K (PK)-resistant PrP also occurred with similar kinetics and prion infectivity accrued at an equal or slower rate. Thus, HSF1 provides an important protective function that is specifically manifest after the onset of behavioral symptoms of prion disease

Heat shock factor 1 regulates lifespan as distinct from disease onset in prion disease

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