Astrocytes were exposed to amyloid-beta (Aβ) to investigate the extracellular release of heat shock protein B1 (HspB1). Exposure to Aβ resulted in the selective release of HspB1 from astrocytes. Some of the released HspB1 was associated with exosomes or membrane fractions. Release was insensitive to inhibitors of classical protein secretion pathways. Immunoprecipitation experiments showed the extracellular HspB1 could interact with extracellular Aβ. The study demonstrates that Aβ can stimulate the non-classical release of HspB1 from astrocytes, which may function to bind and sequester extracellular Aβ.

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Journal of Alzheimer’s Disease xx (20xx) x–xx
DOI 10.3233/JAD-150317
IOS Press
1
Astrocytes Release HspB1 in Response to
Amyloid-␤ Exposure in vitro
1
2
Firoozeh Nafar, J. Bradley Williams and Karen M. Mearow∗
3
Division of BioMedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John’s,
NL, Canada
4
5
Handling Associate Editor: Jose Abisambra6
Accepted 12 August 2015
Abstract. Although heat shock proteins are thought to function primarily as intracellular chaperones, the release and potential
extracellular functions of heat shock proteins have been the focus of an increasing number of studies. Our particular interest is
HspB1 (Hsp25/27) and as astrocytes are an in vivo source of HspB1 it is a reasonable possibility they could release HspB1 in
response to local stresses. Using primary cultures of rat cortical astrocytes, we investigated the extracellular release of HspB1
with exposure to amyloid-␤ (A␤). In order to assess potential mechanisms of release, we cotreated the cells with compounds
that can modulate protein secretion including Brefeldin A, Methyl ␤-cyclodextrin, and MAP kinase inhibitors. Exposure to A␤
(0.1, 1.0, 2.0 ␮M) for 24–48 h resulted in a selective release of HspB1 that was insensitive to BFA treatment; none of the other
inhibitors had any detectable influence. Protease protection assays indicated that some of the released HspB1 was associated
with a membrane bound fraction, and analysis of exosomal preparations indicated the presence of HspB1 in exosomes. Finally,
immunoprecipitation experiments demonstrated that the extracellular HspB1 was able to interact with extracellular A␤. In
summary, A␤ can stimulate release of HspB1 from astrocytes, this release is insensitive to Golgi or lipid raft disruption, and
HspB1 can be found either free in the medium or associated with exosomes. This release suggests that there is a potential for
extracellular HspB1 to be able to bind and sequester extracellular A␤.
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Keywords: Amyloid, astrocytes, extracellular heat shock protein B1 (Hsp27)20
INTRODUCTION21
Heat shock proteins (HSPs) are a family of chap-22
erone proteins that can be upregulated in response23
to various cellular or environmental stressors. The24
particular HSP family member induced may differ25
among different cells and in response to differing26
stresses. These proteins can act as cellular chaperones27
promoting appropriate protein folding and removing28
misfolded or aggregated proteins [1–4]. We have been29
investigating the small heat shock protein, HspB130
(Hsp25/27), which unlike some of the other HSPs,31
functions in an ATP-independent manner and does not
∗Correspondence to: Dr. Karen M. Mearow, Division of BioMed-
ical Sciences, Memorial University of Newfoundland, 300 Prince
Philip Drive, St. John’s, NL A1B 3V6, Canada. Tel.: +1 709 777
6416; Fax: +1 709 777 8281; E-mail: kmearow@mun.ca.
participate in protein folding per se [5, 6]. HspB1 can 32
play a protective role in neurons but its effects may 33
differ from those of Hsp70 and other HSPs (reviewed 34
in [7–10]). HspB1 can act as a chaperone enabling 35
the sequestration of misfolded proteins, but is also 36
important in regulating cytoskeletal elements [11–14]. 37
HspB1 has been noted to be increased in AD brains 38
along with accumulation of HSPs in plaques, neurofib- 39
rillary tangles, and Lewy bodies [3, 9, 15]. We have 40
previously reported that HspB1 can protect cortical 41
neurons from the deleterious effects of A␤ exposure in 42
vitro [16] pointing to a possible protective mechanism. 43
In a recent study, we explored the potential effects of 44
the HspB1 on amyloid-␤ precursor protein (A␤PP) 45
processing and distribution within HEK293 stable cell 46
lines expressing either A␤PPwt or A␤PPsw. Expres- 47
sion of HspB1 was observed to alter A␤PP expression 48
ISSN 1387-2877/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved

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2 F. Nafar et al. / HspB1 Release from Astrocytes
and processing in these cell lines, and furthermore, the49
presence of HspB1 decreased the amount of amyloid-␤50
(A␤)42 released by the cell lines [17].51
Despite the lack of endogenous neuronal expres-52
sion, a number of studies have shown that exogenous53
expression of HspB1 can provide a protective influ-54
ence in a variety of disease-related models including55
ischemia, stroke, amyotrophic lateral sclerosis, and56
Huntington’s disease [18–24]. Although it was nec-57
essary in our experiments, and those cited above, to58
express exogenous HspB1, there are potentially other59
ways in which endogenous HspB1 might protect neu-60
rons in vivo. One possibility is that glial cells could61
release HspB1 that can be then taken up by adjacent62
neurons [25], or alternatively act to sequester A␤. We63
have previously observed that HspB1 can be released64
into the medium of cultured cells [17] and HspB1 is65
also found in the cerebrospinal fluid and serum in vivo66
[26–29].67
Our hypothesis is that astrocytes are able to release68
HspB1 in response to a local stimulus (for example,69
local accumulation or release of A␤) that would then70
be available to provide a protective effect by either71
sequestering amyloid or being available to be taken up72
by other cells, such as neurons. Here we report that73
treatment of primary astrocytes with A␤ results in the74
release of HspB1, and that this release appears to occur75
via a non-classical method of secretion.76
METHODS77
Cell culture78
Dissection and dissociation79
Monolayer cultures of astrocytes were prepared80
from P1-P2 rat brain cortex according to estab-81
lished protocols [30, 31]. All animal usage was82
approved by the Institutional Animal Care Committee83
(IACC protocol KM-14-10). Briefly, the brain was84
removed and placed in ice cold Hanks Balanced Salt85
Solution (HBSS, Invitrogen/Gibco) containing 1%86
Penicillin/Streptomycin, and 0.2% HEPES (Invitro-87
gen/Gibco), and the cortex was dissected from the88
brain. The hippocampus, meninges, and blood vessels89
were then peeled away from the cortex, and corti-90
cal tissue was enzymatically dissociated. The tissue91
was centrifuged, resuspended in Dulbecco’s modi-92
fied Eagle Medium (DMEM, Gibco) with 10% FCS,93
and 1% Pen/Strep/glutamine and subjected to tritu-94
ration. The cell suspension was centrifuged at 130095
RPM for 5 min and the resulting cell pellet was sus-96
pended in 10 ml of medium for plating in T-75 flasks.97
Cells were cultured for 5–7 days and confluent cul- 98
tures were subsequently shaken overnight at 37◦C on 99
a platform rotary shaker (150–170 rpm) to remove 100
microglia, oligodendrocytes, and neurons [32]; the 101
medium was then replaced with fresh medium and the 102
flasks returned to the incubator for 24 h to allow cells 103
to recover. The remaining cells were then removed 104
from the flasks with 0.025% trypsin and replated in 105
DMEM-FCS in T-25 flasks, 6-well plates, or onto 106
collagen-coated 16-well glass slides. The medium was 107
changed every 3 days and also 24 h prior to any experi- 108
mental manipulations. In some cases, the medium was 109
changed to a low serum formulation (DMEM with 110
1% exosome-free FCS). These cultures were >95% 111
astrocytes as assessed by immunocytochemistry (ICC) 112
with anti-GFAP. Secondary passage astrocytes were 113
employed for all experimental procedures. 114
Culture treatments 115
Prior to experimental treatments, medium was 116
changed to a low-serum (1% exosome-free FCS) 117
medium. Cultures were treated with A␤ at varying 118
concentrations (0.1, 1, or 10 ␮M) or times of expo- 119
sure (24 or 48 h); scrambled peptide was employed 120
as a control. To assess for release of proteins into the 121
medium, culture medium was collected at 24 and 48 h 122
after A␤ addition. Medium was centrifuged to remove 123
any cellular debris and then concentrated using Ami- 124
con concentrators (10 KD cutoff). 125
For inhibitor studies, the inhibitors were added 1 h 126
prior to A␤ treatment and cells were cultured for a 127
further 24–48 h prior to cell or conditioned medium 128
sampling. Brefeldin A (BFA, Calbiochem; 10 ␮M) 129
blocks protein export from the endoplasmic reticulum 130
(ER) and disrupts the Golgi apparatus, and blocks the 131
classical protein secretion mechanism [33–35]. Methyl 132
␤-cyclodextrin (MBC, Calbiochem; 10 ␮M) depletes 133
membrane cholesterol and disrupts lipid rafts [33, 35]. 134
Cycloheximide (CHX, 1 ␮M) blocks de novo protein 135
synthesis and was employed to assess whether release 136
requires de novo protein synthesis [33]. To test involve- 137
ment of protein kinases, we employed inhibitors of p38 138
MAPK (SB20315, Calbiochem; 10 ␮M) and of p42/44 139
MAPK (U0126, Calbiochem; 10 ␮M) [36, 37]. Vehicle 140
(DMSO) controls were included in all experiments. 141
Protein and conditioned media collection 142
Cell lysates and conditioned medium were collected 143
24 and 48 h post-treatment. Conditioned medium was 144
collected on ice with protease inhibitor cocktail tablet 145
(Roche Diagnostics, Laval, QC) added immediately 146
upon collection. Media was centrifuged at 14,000 g for 147

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F. Nafar et al. / HspB1 Release from Astrocytes 3
5 minat4◦Ctodiscardanycelldebris.Celllysateswere148
collected by adding 1 ml ice cold TBS with 200 mM149
sodium vanadate and scraping cells off the plate with150
a rubber policeman. Cells were pelleted for 5 min at151
4,000 g at 4◦C and resuspended in ice-cold protein152
lysisbuffer(1%NP40,10%glycerol,O-␤thioglucopy-153
ranoside, protease inhibitor tablet, 200 mM sodium154
vanadate, sodium fluoride and magnesium chloride in155
TBS) and stored at –80◦C until analysis.156
Western blot analysis157
Western blot analysis was performed with sam-158
ples of total cellular lysate and conditioned medium.159
Protein concentrations were determined using a BSA160
protein assay kit (Pierce Chemicals, Rockford, IL).161
Laemmli sample buffer (10% SDS, glycerol, 1M Tris162
pH 6.8, dH20, 0.01% Bromophenol blue) containing163
fresh ␤-mercaptoethanol (BME) was added to 50 ␮g of164
cellular lysate or 200 ␮g of conditioned media protein,165
boiled and separated on an pre-cast 4–20% gradient166
Tris-glycine gel using the X-Cell Surelock System167
(Invitrogen). Separated protein was then transferred168
to a nitrocellulose membrane and after transfer, blots169
were stained with Ponceau Red to assess equivalency170
of protein loading. Blots were washed with TBS-T171
(1M Tris base, 2.5M NaCl, 50% Tween) to remove172
Ponceau Red and blocked with either 3% milk or BSA173
depending on the primary antibody dilution conditions174
for 1 h to prevent non-specific binding. Once blocked,175
blots were incubated with antibodies to one or more of176
the following proteins overnight at 4◦C on a shaking177
platform: HspB1 (SPA-801, Enzo Life Sciences); clus-178
terin/ApoJ(SantaCruzSC8354);actin(Sigma-Aldrich179
A2066); GAPDH (Abcam ab9485); Integrin a6 (DHB180
P2C62C4); Hsc70/Hsp70 (SMC104A, Enzo Life Sci-181
ences); TSG101 (Santa Cruz SC7964). Following182
washing, signal was detected with horseradish peroxi-183
daselabeledsecondaryantibodies(1:5000–1:10000in184
3% milk) and Super Signal West Pico chemilumines-185
cence substrate (ECL; Thermo Scientific, Rockford,186
IL) for 5 min and developed using films. Densitom-187
etry analysis was performed using ImageJ software188
and images prepared with Adobe Photoshop graphics189
software.190
Immunoprecipitation (IP)191
Conditioned medium samples were used for IP with192
either anti-HspB1 or anti-A␤ (clone 6E10, Covance).193
10–15 ml of medium was concentrated 2–3 fold using194
3 KD Amicon centrifuge filters, protein concentration195
was determined and 200 ␮g of protein was used for IP196
experimentation. Either anti-HspB1or 6E10 was added197
to the medium samples and incubated for 1 h with rota- 198
tion followed by addition of 20 ␮l of magnetic protein 199
A/G beads overnight to immunoprecipitate any A␤ and 200
HspB1 complexes that formed. Samples were exposed 201
to a magnet to separate the immunoprecipitates (mag- 202
netic A/G beads, antibody and any protein complexes 203
attached) from supernatant. Protein concentration of 204
supernatant was determined and 30 ␮g was added to 205
5X Laemmli sample buffer with fresh dithiothreitol 206
(DTT). The IP sample was resuspended with 40 ␮l of 207
2X Laemmli sample buffer with fresh DTT and both 208
supernatant and IP samples were electrophoresed as 209
per our western blot protocol [17]. Blots were probed 210
with 6E10 and anti HspB1 (either rabbit (SPA-801) or 211
goat (SC polyclonal) antibodies). 212
Proteinase protection assay 213
Conditioned medium (CM) was collected and 214
aliquots (50–100 ␮g protein) were treated with Pro- 215
teinase K. Briefly, 120 ␮l of CM was treated with 4 ␮l 216
of PK (100, 10 or 1 mg/ml in 50 mM Tris-HCl pH8, 217
10 mM CaCl2) and incubated on ice or at 25°C for 218
2 h. The reaction was quenched by addition of loading 219
buffer, samples boiled and electrophoresed. Subse- 220
quent blots were probed with anti-HspB1, Integrin ␣6, 221
or clusterin/ApoJ. 222
Exosome isolation 223
Exosomes were isolated from conditioned medium 224
via a standard ultracentrifugation protocol [38]. As 225
noted above, astrocytes were cultured in a low-serum 226
medium containing 1% exosome-free FBS, and treated 227
for 24 h with either A␤ or vehicle control (DMSO). The 228
CM was then collected and cleared of cellular debris 229
by two rounds of low speed centrifugation (2000 g, 230
10 min, and 10,000 g for 30 min). The supernatant was 231
then centrifuged at 100,000 g for 3 h; the resulting pel- 232
lets were washed (x 2) with PBS and re-centrifuged at 233
100,000 g for 1 h [38, 39]. Pellets were resuspended 234
in 50–150 ␮l of PBS and either analysed immedi- 235
ately or stored at –80°C until use. For western blot 236
analyses, 50–200 ␮g protein were electrophoresed and 237
blots probed with anti-HspB1, TSG101, Hsc/Hsp70, 238
clusterin/ApoJ. 239
Electron microscopy 240
5–10 ␮l of exosomes was mixed with an equivalent 241
volume of 1% LMP agarose, fixed with Karnovsky fix- 242
ative for 24 h and stored in 0.1M Na cacodylate buffer 243
until processed. The specimens were osmicated, dehy- 244
drated using graded alcohol and acetone followed by 245
infiltration with EPON resin, embedded in molds, and 246

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4 F. Nafar et al. / HspB1 Release from Astrocytes
polymerized overnight at 70°C. Thin sections were cut247
using Reichert ultra-cut S at 85 nm using a Diatome248
diamond knife, mounted on 300 mesh copper grids and249
dried for 30 min. Grids were then stained with uranyl250
acetate followed by lead citrate stain. Grids were then251
examined in a JOEL 1200EX electron microscope and252
images captured using a SIA –L3 C digital camera;253
the camera images were calibrated using a carbon line254
grating.255
Immunocytochemistry256
Astrocytes were plated on collagen-coated 16-well257
Lab-Tek® chamberslides(Lab-Tek®).Cellswerefixed258
in 4% paraformaldehyde in phosphate buffered saline259
(PBS) for 15 min, washed with PBS and permeabilized260
with 0.1% Triton X and blocked with 5% donkey serum261
for 1 h. Primary antibodies included GFAP (Chemicon262
MAB360), HspB1 (SPA-801, (Assay Designs), Golgi263
58K (Abcam ab9845). Cells were incubated in pri-264
mary antibodies overnight (20 h) at 4◦C, washed in265
PBS and incubated with secondary antibodies for 1 h266
in the dark. Secondary antibodies were: DylightTM267
488-conjugated AffiniPure donkey anti-mouse IgG;268
Dylight 649-conjugate AffiniPure donkey anti-rabbit269
(1:250, Jackson Laboratories, West Grove, PA). Cells270
were rinsed with TBS-½T (Tris-buffered saline with271
0.25% Tween) and in some cases, were stained with272
DAPI in TBS for 5 min. Cells were again washed with273
TBS-½T and cover-slipped using polyvinyl alcohol274
mounting medium with DABCO® (Sigma–Aldrich).275
Images were routinely acquired in three channels (488,276
549, 647) using confocal scanning microscopy with277
sequential Z-stage scanning (Olympus Fluoview 1000278
microscope).279
Statistical analysis280
Statistical analysis was performed in GraphPad281
Prism 6.0 (GraphPad Software Inc., La Jolla, CA).282
Figures are shown with mean values ± SEM with283
significance determined by either one-way ANOVA284
testing followed by Tukey post-hoc tests or t-tests to285
compare two groups, for example ± A␤. Significance286
was determined at p < 0.05 unless otherwise stated.287
RESULTS288
Astrocytes express and release HspB1289
Astrocytes isolated from neonatal rat cortex were290
cultured and expression of HspB1 was assessed by291
western blotting and ICC. Astrocytes robustly express292
HspB1 (see Fig. 1B; see also Supplementary Figure 1)293
Fig. 1. Exposure of astrocytes to A␤ results in an increase of extra-
cellular HspB1 release. Rat primary astrocytes were cultured and
treated with A␤ (10 ␮M), vehicle control (DMSO), or scrambled
peptide (10 ␮M) as described in the Methods. Conditioned medium
(CM) was collected, concentrated, and equivalent protein amounts
subjected to western blotting. In some experiments, the correspond-
ing cell lysates were also collected for analyses. A) Western blot
of CM comparing release of HspB1 in the vehicle control, scram-
bled peptide, and A␤ treated cells. B) Quantitation of western blots
(n = 4–6 experiments) showing significant increase in extracellu-
lar HspB1 following A␤ treatment. C) Western blot of astrocyte
lysates following the various treatments (C-control; D-DMSO vehi-
cle control; A␤; Scr- scrambled peptide) showing little change in
the expression of cellular HspB1 over the course of the experiment.
∗∗∗p < 0.001.
and we were interested in determining whether HspB1 294
would be released into the culture medium with expo- 295
suretoA␤,andifsowhetherthatstimuluswasselective. 296
We exposed cells to A␤ (10 ␮M), scrambled control 297
peptide, and the vehicle (DMSO), and collected the 298
medium and the cells 24 h after exposure. As shown 299
in Fig. 1A, the CM contained HspB1 with increasing 300
amounts observed with A␤ exposure (compared to the 301
notreatmentcontrolcondition),withquantitationofthe 302
extracellular HspB1 under control, vehicle, scrambled 303
peptide, and A␤ (10 ␮M) treatments displayed graph- 304
ically. Expression of HspB1 in total cell lysates does 305
not appear to be influenced by any of the treatments 306
(Fig. 1B). 307

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F. Nafar et al. / HspB1 Release from Astrocytes 5
To further investigate the effects of A␤, we carried308
out experiments testing release of HspB1 with 0.1, 1.0,309
and 2.0 ␮M A␤ for 24 and 48 h. The medium and310
cells were collected at either 24 or 48 h of treatment311
and resulting western blots probed for expression of312
HspB1. As shown in Fig. 2A, HspB1 in the medium313
accumulates with longer exposure; the blots were also314
probedwithanti-A␤(6E10)toshowtheamountofpep-315
tide detectable in the medium. Based upon the results316
of these experiments, we chose 1.0 ␮M A␤ as the con-317
centration to use in subsequent experiments.318
Mechanism of HspB1 release319
To gain insight into whether this release was passive320
(perhaps due to cell damage) or via a secretory path-321
Fig. 2. HspB1 release is increased with time in culture. A) Rat pri-
mary astrocytes were treated with 0.1, 1.0, or 2.0 ␮M A␤ and the
CM collected at 24 or 48 h. Blots were also probed with anti-A␤
to confirm the presence of A␤ in the medium. B) Cell lysates from
corresponding cultures were probed with anti-HspB1 and GAPDH
as a loading control. C) Quantitation of the amount of HspB1 in
the medium expressed relative to the control expression at 2 h. The
longer exposure to A␤ results in further increases in released HspB1.
∗∗p < 0.05; ∗∗∗p < 0.001. (n = 3 separate experiments).
Fig. 3. Extracellular HspB1 is increased following a heat stress, but
release is not blocked by BFA. In these experiments, cells were
treated with cycloheximide (CHX, 1␮M) or Brefeldin A (BFA,
10␮M) for 1 h prior to the heat stress (HS). HS resulted in an increase
in extracellular HspB1 in the CM (A, B) and also increased cellu-
lar HspB1 (C). Treatment with CHX resulted in a large increase in
released HspB1 (A, B), likely due to passive release following cell
damage, but BFA had little effect on release. ∗∗∗p < 0.001 compared
to control; ∗p < 0.05 compared to HS alone; tp < 0.05 compared to
BFA alone.
way, we carried out a series of experiments initially 322
using heat shock as the stress stimulus and treatments 323
with several different inhibitors of protein synthe- 324
sis or transport within the cell. Heat stress has been 325
routinely used to stimulate extracellular release of sev- 326
eral Hsps in tumor cell lines as well as in primary 327
cells [35, 40–43]. Figure 3 shows that HS resulted 328
in an increase in the amount of HspB1 released into 329
the medium, as well as a modest increase in cellular 330
expression (averaged over three separate experiments). 331
In order to determine whether this was regulated in 332
any way, we exposed the cells to several treatments 333
that have been shown to alter protein secretion. Astro- 334
cytes were treated with the chemicals for 1 h prior 335
to the stress stimulus (control cells were similarly 336

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6 F. Nafar et al. / HspB1 Release from Astrocytes
treated but not exposed to the heat stress) and then the337
medium and cells were collected 24 h later. Although338
CHX(inhibitorofproteinsynthesis)attenuatedtheHS-339
induced increase in cellular expression as expected,340
there was a significant increase in extracellular HspB1,341
which was likely due to cellular damage since the cul-342
tures did not appear to be particularly healthy with the343
CHX treatment. Treatment with BFA (which blocks344
protein export from the ER and disrupts Golgi func-345
tion and is considered an inhibitor of classical protein346
secretion [34]), had no significant effect in the non-347
heat stressed cells, in particular there was no decrease348
in release. While there was an increase compared to349
control, this was not significantly different from the350
HS condition. This result is similar to that reported351
for Hsp70 and Hsp90, as well as other non-classically352
secreted proteins such as IL-6 or FGF [33, 44].353
Release of HspB1 with Aβ and inhibitors 354
We next examined release of HspB1 in astrocytes 355
treated with 1 ␮M A␤ and the various agents. We 356
employed BFA to block the classical secretion path- 357
way and MBC to disrupt lipid rafts [33, 45] (Fig. 4). 358
Prior reports had suggested that activation of protein 359
kinases by heat stress were involved in regulating the 360
release of Hsp70 from astrocytes [46], so we employed 361
an inhibitor of MAPK (U0126) as well as a p38MAPK 362
inhibitor (SB20358); the latter is of particular inter- 363
est since p38 phosphorylates HspB1 and is involved 364
in its interaction with actins, which could potentially 365
be involved in regulating release [14]. Both EDTA 366
and MgCl2 have been shown to influence release of 367
other leaderless proteins by chelation of calcium and 368
ATP [33, 43, 47]. Figure 4A presents a representative 369
Fig. 4. HspB1 release does not appear to involve conventional protein secretion pathway. Astrocytes were pretreated for 1 h with the various
inhibitors, followed by no further treatment (A) or A␤ (1␮M) for 24 h (B). We compared the effects of the treatments on HspB1 release with a
known cell surface protein (integrin ␣6) and a known secretory protein (clusterin). Blots were cut into three pieces and exposed to the different
primary antibodies concomitantly. Here the same samples are probed with the different antibodies, and the displayed blot is representative of
three separate complete experiments; however, the n values for each treatment varied from 7–12 as the individual treatments were often done
separately (though always in combination with the appropriate control). C) Densitometric quantitation of the effects of treatments on HspB1
release. In the absence of A␤, BFA results in significantly increased HspB1 release (p < 0.001); none of the other treatments were significantly
different from the vehicle control. A␤ treatment significantly increases extracellular HspB1 compared to the vehicle control (+p < 0.05), and
none of the treatments has any further significant effect on this release. D) BFA clearly blocks the secretion of clusterin (B, D) ∗∗∗p < 0.001,
compared to the paired control. As expected, none of the treatments had any detectable influence on ␣6 integrin expression. Lower dotted line
– denotes the vehicle control expression; upper dotted line - expression in the presence of A␤.

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F. Nafar et al. / HspB1 Release from Astrocytes 7
western blot for vehicle control samples treated with370
the various compounds, while Fig. 4B shows a rep-371
resentative blot for the A␤ samples with the same372
compounds. In this experimental series, we also373
assessed release of clusterin (a known secretory pro-374
tein, secretion of which should be blocked by BFA) to375
compare with the HspB1, and integrin ␣6 (which is an376
integral membrane protein often found on the surface377
of released microvesicles).378
The blots show quite clearly that although clusterin379
release is blocked by BFA, that of HspB1 is enhanced380
both in the vehicle control and the A␤-treated samples;381
note that these are the same samples probed sequen-382
tially. Although the reason for the increase with BFA383
is unclear, ICC assessment of cells under each of these384
different conditions did not show any obvious cell385
death that could result in enhanced passive release (see386
Supplementary Figure 1).387
Figure 4C presents the graphical analysis of HspB1388
release with the various treatments. A␤ results in389
increased release in all samples compared to the vehi-390
cle control samples. Cotreatment with BFA results391
in increased release in both the vehicle and A␤-392
treated samples, although none of the other inhibitor393
cotreatments results in increased release over the A␤394
treatment. Treatment with EDTA resulted in cellu-395
lar detachment from the substrate and thus was not396
continued. We also tested the influence of KCl (to397
induce depolarization) and glibenclamide (an ABC398
transporter inhibitor), although neither of these had399
a significant effect on the detection of extracellular400
HspB1. None of the treatments had any apparent effect401
on cellular levels of HspB1 or clusterin (data not402
shown).403
Extracellularly released HspB1 is resistant to404
proteinase K treatment405
The results so far indicated that HspB1 did not406
appear to be released via a classical secretion pathway.407
Some non-classically secreted proteins are packaged408
into vesicles, although other possibilities include pas-409
sive release (perhaps related to membrane disruption),410
lysosomal secretion, blebbing, or exosomal release.411
One way to assess whether the protein is free in solu-412
tion or associated with a membrane-bound structure413
is to carry out a protease protection assay (to test for414
vesicle-independent release [47]). We tested this pos-415
sibility by treating aliquots of the CM with proteinase416
K (PK) and assessing protein degradation by western417
blotting. As shown in Fig. 5, treatment of the CM with418
1 ␮g/ml of PK completely abolishes the signal for inte-419
Fig. 5. Extracellular HspB1 is resistant to protease degradation.
A protease protection assay was carried out on CM samples
(50–100 ␮g protein) as outlined in the Methods. Samples were
exposed to different concentrations of Proteinase K at room tem-
perature for 2 h; the reaction was quenched by the addition of
loading buffer followed by electrophoresis and western blotting with
the indicated antibodies. The probes are from the same blot cut in
three pieces and probed concomitantly. Note that the HspB1 is more
resistant to the proteinase K than either of the other proteins.
grin ␣6 (used as a marker for an integral membrane 420
protein associated with the outer surface of membrane 421
vesicles). The signal for clusterin is somewhat dimin- 422
ished at 1 ␮g/ml, but abolished with 10 ␮g/ml of PK. In 423
contrast, degradation of the HspB1 requires the highest 424
PK concentration. This is suggestive of at least some 425
of the released HspB1 being protected by inclusion in 426
a membrane bound structure. 427
Presence of HspB1 in exosomes 428
To determine whether HspB1 might be being 429
released in vesicles, we then isolated exosomes from 430
CM from vehicle- and A␤-treated astrocytes. Presence 431
of exosomes in the preparations was confirmed by elec- 432
tronmicroscopy(Fig.6A,B),aswellasbythepresence 433
of TSG101 (an exosomal marker, Fig. 6E) in western 434
blots of exosomal preparations. In Fig. 6C-E, a west- 435
ern blot probed sequentially for HspB1 and TSG101 436
is presented. Here the exosomal fraction (exo), the 437
concentrated conditioned medium before exosomal 438
fractionation (CM) and the supernatant from the exo- 439
somal pellet (Sup) have been run on the same blot. The 440
top panel (6C) is a short exposure showing HspB1 in 441
the CM and in the exosomal supernatant; faint bands 442
can be observed in the exosomal fraction lanes (arrow). 443
With a longer exposure, the presence of HspB1 in 444

8. UncorrectedAuthorProof
8 F. Nafar et al. / HspB1 Release from Astrocytes
Fig. 6. Released HspB1 is associated with exosomes. Exosomes were isolated from CM as outlined in the Methods. A, B) EM was carried out
to confirm the presence of exosomes (A – control CM; B - A␤-treated CM). C) Conditioned medium (CM, 100 ␮g protein), the supernatatant
from the exosomal preparation (Sup, 100 ␮g protein), recombinant human HspB1 (H, 2 ␮g), the Exosomal fraction (Exo, 35 ␮g protein), and
the microparticle fraction (MP, 5 ␮g protein) were run on the same blot and probed for HspB1 (C, D). Faint bands detectable in a lower exposure
are clearly observed in a longer exposure (arrow). E) The same blot probed for TSG101, an exosomal marker. F. The ponceau red stained blot
to show protein loading.
the exosomal fraction is more obvious (note that this445
HspB1 antibody tends to show a doublet for HspB1446
particularly in CM samples). TSG101 is also clearly447
detectable in the exosomal fraction although because448
of the amount of BSA and other medium components449
in the concentrated medium, detection of TSG101 in450
the medium fractions is precluded. The ponceau red451
stained blot image is shown in Fig. 6F. These results452
suggest that HspB1 can be detected in exosomes, but453
HspB1 free in the medium is likely more abundant,454
since the amount in the supernatant after exosome iso- 455
lation is not depleted (Fig. 6C). 456
Extracellular HspB1 interacts with Aβ 457
We then tested whether extracellular HspB1 could 458
interact with A␤ present in the medium. We have pre- 459
viously reported that HspB1 can interact directly with 460
A␤, based upon IP experiments [17]. Here CM sam- 461
ples were subjected to IP with either anti-HspB1or 462

9. UncorrectedAuthorProof
F. Nafar et al. / HspB1 Release from Astrocytes 9
Fig. 7. Extracellular HspB1 can interact with extracellular A␤. Representative blots of CM samples from 24 or 48 h cultures treated with A␤ or
scrambled peptide were subjected to immunoprecipitation (IP) with anti-HspB1 (A) or anti-A␤ (B), and subsequent blots probed with anti-A␤
first, followed by anti-HspB1 (A) or anti-HspB1 first, followed by anti-A␤ (B). IP with HspB1 co-precipitates A␤, although IP with 6E10 does
not appear to bring down any HspB1. This could potentially due to the large excess of A␤ in the medium compared to the amount of HspB1.
anti-A␤ (6E10). Representative western blots of four463
separate IP experiments are presented in Fig. 7. In464
Figure 7A, HspB1 has been subjected to IP and the465
blotsprobedsequentiallywithanti-A␤(6E10)andthen466
anti-HspB1 (after stripping). The top panel shows that467
A␤ is co-precipitated with HspB1, while the bottom468
panel shows the blot probed with anti-HspB1. The bot-469
tom band appears to be the specific HspB1 band that470
can be detected just below the light chain IgG, based on471
the positive control of r-HspB1 (last lane). 7B shows a472
corresponding IP of A␤ (with 6E10) probed with anti-473
HspB1 first, followed by anti-A␤; in this case, there is474
little detectable HspB1 in the IP samples.475
Our data thus show that astrocytes can release476
HspB1 extracellularly, that this release is increased by477
stress and by exposure to A␤ in particular, occurs via a478
non-classical mechanism that involves some exosomal479
release. Furthermore, the released HspB1 appears to be480
able to interact with the A␤ present in the medium.481
DISCUSSION482
Astrocytes robustly express HspB1, which can be483
upregulated by heat stress and released into the extra-484
cellular milieu. Our results show that A␤ can elicit485
increased HspB1 release compared to the vehicle486
control treatment. The release in both the vehicle487
and A␤-treated conditions was not inhibited by BFA 488
treatment. 489
Although HSPs are thought to function primarily 490
as intracellular chaperones, the release and potential 491
extracellular functions of HSPs have been the focus 492
of an increasing number of studies (reviewed in [44, 493
48–52]. 494
Most secreted proteins possess an N-terminal sig- 495
nal peptide that directs their sorting to the ER and 496
subsequently through the ER-Golgi compartment for 497
release via conventional ER-Golgi secretory pathway 498
[34, 53, 54]. There are, however, a large number of pro- 499
teins that have been shown to exit cells via pathways 500
independent of the conventional secretory pathway. 501
Generally these proteins lack the signal peptide and 502
their release is not blocked by BFA [53, 54]. This 503
unconventional release is regulated to some degree and 504
often induced by stress, and many of the proteins (both 505
cytosolic and nuclear) secreted in by non-conventional 506
pathways have roles in inflammation, tissue repair and 507
angiogenesis. Several different categories of release 508
have been described including direct translocation of 509
proteins through the plasma membrane to the extracel- 510
lular compartment and release via lysosome, exosome 511
orbymembraneblebbingandvesicleshedding[53–56]. 512
Heat shock proteins lack the classic N-terminal 513
leader sequence normally associated with the classical 514

10. UncorrectedAuthorProof
10 F. Nafar et al. / HspB1 Release from Astrocytes
secretion pathway, and reported mechanisms under-515
lying their release into the extracellular environment516
tend to be dependent on cell type and context [44,517
49–51]. HspB1 has been reported to be released by518
a variety of cell types including glial tumor cells (exo-519
somes) [41], vascular endothelial cells (soluble) [57],520
B cells (exosomes) [40], macrophages (lysosome-521
like vesicles) [27], neuroblastoma cells [58], HEK293522
cells [17]. HspB1 release from endothelial cells was523
noted as being soluble, since the secreted HspB1524
interacted with soluble VEGF to regulate angiogen-525
esis; interestingly phosphorylation of HspB1 inhibited526
its release in these experiments [57]. Secretion of527
HspB1 from macrophages was regulated by estrogen528
and intracellular colocalization of HspB1 and LAMP529
in lysosomal vesicles was observed [27]. We have530
previously reported that overexpression of HspB1 in531
HEK293 results in HspB1 release into the culture532
medium although in that study we did not investigate533
release mechanisms [17]. The acrylamide-induced534
increase in extracellular Hsps in neuroblastoma cells535
may have been a result of passive release following536
increased cellular toxicity [58].537
In our prior studies, we have shown that HspB1 pro-538
motes survival in PC12 cells [36], primary peripheral539
[59] and central neurons [16]. HspB1 also plays a role540
in axonal initiation and extension and branching of pri-541
mary neuron axons [16, 37, 60, 61]. HspB1 is generally542
found localized in the cytosol in a diffuse or granular543
appearance, while in migrating cells and growth cones544
it is found along the leading edge of lamellopodia asso-545
ciated with actin [14, 60]. Heat stress in PC12 cells546
results in the redistribution of HspB1 from the cytosol547
to the cytoskeletal fraction, particularly increasing its548
association with actin, but also causes membrane bleb-549
bing, with blebs displaying localization of HspB1 and550
actin [14]. In the current study, we detected sporadic551
cells with blebs following treatment with A␤ and BFA,552
although given the sporadic nature of this occurrence553
we do not think it likely that it could fully account554
for the increased HspB1 release we observed. It is,555
however, possible that the BFA treatment resulted in556
membrane leakiness, although this would not explain557
the reduction in the release of clusterin.558
How does A␤ stimulate HspB1 release? Our results559
suggest that A␤ selectively increases HspB1 release,560
but how this comes about is not clear. A␤ can bind to561
phospholipids in the cell membrane and to a variety562
of cellular receptors including p75, nicotinic AChRs,563
glutamate receptors [62, 63]. A␤ can also form stable564
membrane pores and channels with resulting dysreg-565
ulation of Ca flux and which could promote protein566
release across the membrane or via vesicular release. 567
[64]. In our experiments, treatment of the cells with 568
KCl (to induce membrane depolarization) had little 569
effect on HspB1 release. 570
The extracellular function of HspB1 is not entirely 571
clear. Like other Hsps, HspB1 has been suggested to 572
play a role in immunomodulation [49], with a num- 573
ber of studies reporting an anti-inflammatory action by 574
increasing production of anti-inflammatory cytokines 575
by monocytes and macrophages [27, 65]. Lee and 576
colleagues have recently reported that soluble HspB1 577
inhibits the function of VEGF by a direct interaction 578
with VEGF which can result in decreased angiogene- 579
sis and tumor metastasis; they also suggest that VEGF 580
itself can inhibit HspB1 release and thus regulate 581
angiogenesis [57]. Cellular receptors that have been 582
reportedtobindHspB1includethescavengerreceptors 583
and toll-like receptors. In preliminary studies we have 584
exposed cortical neurons and astrocytes to recombi- 585
nant HspB1. We did not see any detectable influence on 586
neuron survival nor internalization of HspB1 over the 587
course of these short-term experiments (10 min-6 h). In 588
the astrocyte cultures exposed to rHspB1 (endotoxin- 589
free), we observed activation of signaling pathways, in 590
particular MAPK and Akt; however, we also noted that 591
the act of changing the medium results in pathway acti- 592
vation although this was enhanced when rHspB1 was 593
also provided. Further study is required to determine 594
what cellular receptors HspB1 might bind to, what sig- 595
naling pathways are activated, and whether there is any 596
influence on cellular survival or local inflammatory 597
responses. 598
There have been numerous studies reporting upreg- 599
ulation of glial HspB1 in response to various stimuli, 600
including heat, excitotoxicity, and ischemia both 601
in vitro and in vivo [16, 36, 66–71]. Increased expres- 602
sion of HspB1 is reported in several neurodegenerative 603
disorders (e.g., [72–76]), however, there have been 604
no reports of extracellular release of HspB1 from 605
glial cells under these conditions. HspB1 promotes 606
neuronal survival in response to various stresses, 607
and it could be acting intracellularly (to chaper- 608
one the cytoskeleton or protein aggregates [14, 16, 609
37, 61, 77], or alternatively it could be released into 610
the extracellular space where it could potentially be 611
sequestering amyloid [17, 78–80]. Overexpression of 612
HspB1 in A␤PPswe/PS1dE9 transgenic mice resulted 613
in decreased appearance of amyloid plaques, as well 614
as attenuating the behavioral deficits associated with 615
this mouse model [81]. 616
A number of studies have reported that small Hsps 617
including HspB1 can influence A␤ aggregation and 618

11. UncorrectedAuthorProof
F. Nafar et al. / HspB1 Release from Astrocytes 11
toxicity as well as sequester toxic oligomers [77, 78,619
80]. HspB1 has been localized to plaques in AD brain620
samples [15] as well as in transgenic mouse mod-621
els of AD [77]. In the latter study, HspB1 was not622
only localized in plaques in AD mouse model brains,623
but HspB1 added to culture medium was shown to624
sequestertoxicoligomersofA␤andattenuateneuronal625
death. Interestingly, the authors questioned how an626
intracellular chaperone could act on externally added627
A␤, and comment that this problem could be solved if628
theglialHspB1wereexternalized[77].Ourresultspro-629
vide evidence that HspB1 can indeed be released from630
glial cells, and relevantly, in response to extracellularly631
added A␤.632
In summary, our results show that relatively low633
concentrations of A␤ can stimulate release of HspB1634
from astrocytes, via a non-classical secretion mecha-635
nism. HspB1 can be found either free in the medium or636
associated with exosomes. Further, HspB1 and A␤ in637
the medium can interact, in the sense that IP of either638
HspB1 or A␤ coprecipitates the other component.639
ACKNOWLEDGMENTS640
Funding for this work was provided by a partner-641
ship grant from the Canadian Institutes of Health and642
the Research and Development Corporation of New-643
foundland and Labrador.644
Authors’ disclosures available online (http://j-alz.645
com/manuscript-disclosures/15-0317r2).646
SUPPLEMENTARY MATERIAL647
The supplementary material is available in the648
electronic version of this article: http://dx.doi.org/649
10.3233/JAD-150317.650
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