Unc

orre

cted

Aut

hor P

roof

Journal of Alzheimer’s Disease xx (20xx) x–xxDOI 10.3233/JAD-150317IOS Press

1

Astrocytes Release HspB1 in Response toAmyloid-� 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 potentialextracellular functions of heat shock proteins have been the focus of an increasing number of studies. Our particular interest isHspB1 (Hsp25/27) and as astrocytes are an in vivo source of HspB1 it is a reasonable possibility they could release HspB1 inresponse to local stresses. Using primary cultures of rat cortical astrocytes, we investigated the extracellular release of HspB1with exposure to amyloid-� (A�). In order to assess potential mechanisms of release, we cotreated the cells with compoundsthat 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 otherinhibitors had any detectable influence. Protease protection assays indicated that some of the released HspB1 was associatedwith 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�. Insummary, A� can stimulate release of HspB1 from astrocytes, this release is insensitive to Golgi or lipid raft disruption, andHspB1 can be found either free in the medium or associated with exosomes. This release suggests that there is a potential forextracellular HspB1 to be able to bind and sequester extracellular A�.

7

8

9

10

11

12

13

14

15

16

17

18

19

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 PrincePhilip Drive, St. John’s, NL A1B 3V6, Canada. Tel.: +1 709 7776416; 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

Unc

orre

cted

Aut

hor P

roof

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

Unc

orre

cted

Aut

hor P

roof

F. Nafar et al. / HspB1 Release from Astrocytes 3

5 min at 4◦C to discard any cell debris. Cell lysates were148

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

lysis buffer (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 (Santa Cruz SC8354); 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

dase labeled secondary antibodies (1:5000 – 1:10000 in184

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

Unc

orre

cted

Aut

hor P

roof

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® chamber slides (Lab-Tek®). Cells were fixed258

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 andtreated with A� (10 �M), vehicle control (DMSO), or scrambledpeptide (10 �M) as described in the Methods. Conditioned medium(CM) was collected, concentrated, and equivalent protein amountssubjected to western blotting. In some experiments, the correspond-ing cell lysates were also collected for analyses. A) Western blotof 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 astrocytelysates following the various treatments (C-control; D-DMSO vehi-cle control; A�; Scr- scrambled peptide) showing little change inthe 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

sure toA�, and if sowhether that stimuluswasselective. 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

no treatment control condition), with quantitation of the 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

Unc

orre

cted

Aut

hor P

roof

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

probed with anti-A� (6E10) to show the amount of pep-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 theCM 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 fromcorresponding cultures were probed with anti-HspB1 and GAPDHas a loading control. C) Quantitation of the amount of HspB1 inthe medium expressed relative to the control expression at 2 h. Thelonger 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, butrelease is not blocked by BFA. In these experiments, cells weretreated with cycloheximide (CHX, 1�M) or Brefeldin A (BFA,10�M) for 1 h prior to the heat stress (HS). HS resulted in an increasein extracellular HspB1 in the CM (A, B) and also increased cellu-lar HspB1 (C). Treatment with CHX resulted in a large increase inreleased HspB1 (A, B), likely due to passive release following celldamage, but BFA had little effect on release. ∗∗∗p < 0.001 comparedto control; ∗p < 0.05 compared to HS alone; tp < 0.05 compared toBFA 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

Unc

orre

cted

Aut

hor P

roof

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 (inhibitor of protein synthesis) attenuated the HS-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 variousinhibitors, 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 aknown cell surface protein (integrin �6) and a known secretory protein (clusterin). Blots were cut into three pieces and exposed to the differentprimary antibodies concomitantly. Here the same samples are probed with the different antibodies, and the displayed blot is representative ofthree separate complete experiments; however, the n values for each treatment varied from 7–12 as the individual treatments were often doneseparately (though always in combination with the appropriate control). C) Densitometric quantitation of the effects of treatments on HspB1release. In the absence of A�, BFA results in significantly increased HspB1 release (p < 0.001); none of the other treatments were significantlydifferent from the vehicle control. A� treatment significantly increases extracellular HspB1 compared to the vehicle control (+p < 0.05), andnone 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�.

Unc

orre

cted

Aut

hor P

roof

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 wereexposed to different concentrations of Proteinase K at room tem-perature for 2 h; the reaction was quenched by the addition ofloading buffer followed by electrophoresis and western blotting withthe indicated antibodies. The probes are from the same blot cut inthree pieces and probed concomitantly. Note that the HspB1 is moreresistant 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

tron microscopy (Fig. 6A, B), as well as by the presence 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

Unc

orre

cted

Aut

hor P

roof

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 outto confirm the presence of exosomes (A – control CM; B - A�-treated CM). C) Conditioned medium (CM, 100 �g protein), the supernatatantfrom the exosomal preparation (Sup, 100 �g protein), recombinant human HspB1 (H, 2 �g), the Exosomal fraction (Exo, 35 �g protein), andthe microparticle fraction (MP, 5 �g protein) were run on the same blot and probed for HspB1 (C, D). Faint bands detectable in a lower exposureare clearly observed in a longer exposure (arrow). E) The same blot probed for TSG101, an exosomal marker. F. The ponceau red stained blotto 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

Unc

orre

cted

Aut

hor P

roof

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� orscrambled 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 doesnot 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

blots probed sequentially with anti-A� (6E10) and then466

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

Unc

orre

cted

Aut

hor P

roof

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

reported to bind HspB1 include the scavenger receptors 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

Unc

orre

cted

Aut

hor P

roof

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

sequester toxic oligomers of A� and attenuate neuronal625

death. Interestingly, the authors questioned how an626

intracellular chaperone could act on externally added627

A�, and comment that this problem could be solved if628

the glial HspB1 were externalized [77]. Our results pro-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

REFERENCES651

[1] Evans CG, Wisen S, Gestwicki JE (2006) Heat shock pro-652

teins 70 and 90 inhibit early stages of amyloid beta-(1-42)653

aggregation in vitro. J Biol Chem 281, 33182-33191.654

[2] Kannan R, Sreekumar PG, Hinton DR (2012) Novel roles for655

alpha-crystallins in retinal function and disease. Prog Retin656

Eye Res 31, 576-604.657

[3] Stege GJ, Renkawek K, Overkamp PS, Verschuure P, van658

Rijk AF, Reijnen-Aalbers A, Boelens WC, Bosman GJ, de659

Jong WW (1999) The molecular chaperone alphaB-crystallin660

enhances amyloid beta neurotoxicity. Biochem Biophys Res661

Commun 262, 152-156.662

[4] Xi D, Dong X, Deng W, Lai L (2011) Dynamic behavior of663

small heat shock protein inhibition on amyloid fibrillization of664

a small peptide (SSTSAA) from RNase A. Biochem Biophys665

Res Commun 416, 130-134.666

[5] Boncoraglio A, Minoia M, Carra S (2012) The family of 667

mammalian small heat shock proteins (HSPBs): Implications 668

in protein deposit diseases and motor neuropathies. Int J 669

Biochem Cell Biol 44, 1657-1669. 670

[6] Garrido C, Paul C, Seigneuric R, Kampinga HH (2012) The 671

small heat shock proteins family: The long forgotten chaper- 672

ones. Int J Biochem Cell Biol 44, 1588-1592. 673

[7] Franklin TB, Krueger-Naug AM, Clarke DB, Arrigo AP, 674

Currie RW (2005) The role of heat shock proteins Hsp70 and 675

Hsp27 in cellular protection of the central nervous system. Int 676

J Hyperthermia 21, 379-392. 677

[8] Latchman DS (2005) HSP27 and cell survival in neurones. 678

Int J Hyperthermia 21, 393-402. 679

[9] Smith RC, Rosen KM, Pola R, Magrane J (2005) Stress pro- 680

teins in Alzheimer’s disease. Int J Hyperthermia 21, 421-431. 681

[10] Brown IR (2007) Heat shock proteins and protection of the 682

nervous system. Ann N Y Acad Sci 1113, 147-158. 683

[11] Huot J, Houle F, Marceau F, Landry J (1997) Oxidative 684

stress-induced actin reorganization mediated by the p38 685

mitogen-activated protein kinase/heat shock protein 27 path- 686

way in vascular endothelial cells. Circ Res 80, 383-392. 687

[12] Theriault JR, Lambert H, Chavez-Zobel AT, Charest G, 688

Lavigne P, Landry J (2004) Essential role of the NH2-terminal 689

WD/EPF motif in the phosphorylation-activated protective 690

function of mammalian Hsp27. J Biol Chem 279, 23463- 691

23471. 692

[13] Perng MD, Cairns L, van den IP, Prescott A, Hutcheson AM, 693

Quinlan RA (1999) Intermediate filament interactions can be 694

altered by HSP27 and alphaB-crystallin. J Cell Sci 112(Pt 13), 695

2099-2112. 696

[14] Clarke JP, Mearow KM (2013) Cell stress promotes the asso- 697

ciation of phosphorylated HspB1 with F-actin. PLoS One 8, 698

e68978. 699

[15] Wilhelmus MM, Otte-Holler I, Wesseling P, de Waal RM, 700

Boelens WC, Verbeek MM (2006) Specific association of 701

small heat shock proteins with the pathological hallmarks of 702

Alzheimer’s disease brains. Neuropathol Appl Neurobiol 32, 703

119-130. 704

[16] King M, Nafar F, Clarke J, Mearow K (2009) The small 705

heat shock protein Hsp27 protects cortical neurons against 706

the toxic effects of beta-amyloid peptide. J Neurosci Res 87, 707

3161-3175. 708

[17] Conway M, Nafar F, Straka T, Mearow K (2014) Modula- 709

tion of amyloid-beta protein precursor expression by HspB1. 710

J Alzheimers Dis 42, 435-450. 711

[18] Wyttenbach A, Sauvageot O, Carmichael J, Diaz-Latoud C, 712

Arrigo AP, Rubinsztein DC (2002) Heat shock protein 27 713

prevents cellular polyglutamine toxicity and suppresses the 714

increase of reactive oxygen species caused by huntingtin. Hum 715

Mol Genet 11, 1137-1151. 716

[19] Akbar MT, Lundberg AM, Liu K, Vidyadaran S, Wells KE, 717

Dolatshad H, Wynn S, Wells DJ, Latchman DS, de Belleroche 718

J (2003) The neuroprotective effects of heat shock protein 27 719

overexpression in transgenic animals against kainate-induced 720

seizures and hippocampal cell death. J Biol Chem 278, 19956- 721

19965. 722

[20] Kalwy SA, Akbar MT, Coffin RS, de Belleroche J, Latch- 723

man DS (2003) Heat shock protein 27 delivered via a herpes 724

simplex virus vector can protect neurons of the hippocampus 725

against kainic-acid-induced cell loss. Brain Res Mol Brain 726

Res 111, 91-103. 727

[21] Sharp P, Krishnan M, Pullar O, Navarrete R, Wells D, de Belle- 728

roche J (2006) Heat shock protein 27 rescues motor neurons 729

following nerve injury and preserves muscle function. Exp 730

Neurol 198, 511-518. 731

Unc

orre

cted

Aut

hor P

roof

12 F. Nafar et al. / HspB1 Release from Astrocytes

[22] Sharp PS, Akbar MT, Bouri S, Senda A, Joshi K, Chen HJ,732

Latchman DS, Wells DJ, de Belleroche J (2008) Protective733

effects of heat shock protein 27 in a model of ALS occur734

in the early stages of disease progression. Neurobiol Dis 30,735

42-55.736

[23] Perrin V, Regulier E, Abbas-Terki T, Hassig R, Brouillet E,737

Aebischer P, Luthi-Carter R, Deglon N (2007) Neuroprotec-738

tion by Hsp104 and Hsp27 in lentiviral-based rat models of739

Huntington’s disease. Mol Ther 15, 903-911.740

[24] Zourlidou A, Payne Smith MD, Latchman DS (2004) HSP27741

but not HSP70 has a potent protective effect against alpha-742

synuclein-induced cell death in mammalian neuronal cells.743

J Neurochem 88, 1439-1448.744

[25] Bechtold DA, Brown IR (2000) Heat shock proteins Hsp27745

and Hsp32 localize to synaptic sites in the rat cerebellum746

following hyperthermia. Brain Res Mol Brain Res 75, 309-747

320.748

[26] Seibert TA, Hibbert B, Chen YX, Rayner K, Simard T, Hu T,749

Cuerrier CM, Zhao X, de Belleroche J, Chow BJ, Hawken S,750

Wilson KR, O’Brien ER (2013) Serum heat shock protein 27751

levels represent a potential therapeutic target for atheroscle-752

rosis: Observations from a human cohort and treatment of753

female mice. J Am Coll Cardiol 62, 1446-1454.754

[27] Rayner K, Chen YX, McNulty M, Simard T, Zhao X, Wells755

DJ, de Belleroche J, O’Brien ER (2008) Extracellular release756

of the atheroprotective heat shock protein 27 is mediated by757

estrogen and competitively inhibits acLDL binding to scav-758

enger receptor-A. Circ Res 103, 133-141.759

[28] Miller-Graziano CL, De A, Laudanski K, Herrmann T,760

Bandyopadhyay S (2008) HSP27: An anti-inflammatory and761

immunomodulatory stress protein acting to dampen immune762

function. Novartis Found Symp 291, 196-208; discussion 208-763

111, 221-194.764

[29] Yonekura K, Yokota S, Tanaka S, Kubota H, Fujii N,765

Matsumoto H, Chiba S (2004) Prevalence of anti-766

heat shock protein antibodies in cerebrospinal fluids of767

patients with guillain-barre syndrome. J Neuroimmunol 156,768

204-209.769

[30] Mearow KM, Mill JF, Freese E (1990) Neuron-glial interac-770

tions involved in the regulation of glutamine synthetase. Glia771

3, 385-392.772

[31] McCarthy KD, de Vellis J (1980) Preparation of separate773

astroglial and oligodendroglial cell cultures from rat cerebral774

tissue. J Cell Biol 85, 890-902.775

[32] Noble M, Murray K (1984) Purified astrocytes promote the776

in vitro division of a bipotential glial progenitor cell. EMBO777

J 3, 2243-2247.778

[33] Evdokimovskaya Y, Skarga Y, Vrublevskaya V, Morenkov779

O (2010) Secretion of the heat shock proteins HSP70 and780

HSC70 by baby hamster kidney (BHK-21) cells. Cell Biol Int781

34, 985-990.782

[34] Nickel W (2010) Pathways of unconventional protein secre-783

tion. Curr Opin Biotechnol 21, 621-626.784

[35] Lancaster GI, Febbraio MA (2005) Exosome-dependent traf-785

ficking of HSP70: A novel secretory pathway for cellular786

stress proteins. J Biol Chem 280, 23349-23355.787

[36] Mearow KM, Dodge ME, Rahimtula M, Yegappan C (2002)788

Stress-mediated signaling in PC12 cells - the role of the small789

heat shock protein, Hsp27, and Akt in protecting cells from790

heat stress and nerve growth factor withdrawal. J Neurochem791

83, 452-462.792

[37] Williams KL, Mearow KM (2011) Phosphorylation status of793

heat shock protein 27 influences neurite growth in adult dorsal794

root ganglion sensory neurons in vitro. J Neurosci Res 89,795

1160-1172.796

[38] Thery C, Amigorena S, Raposo G, Clayton A (2006) Isolation 797

and characterization of exosomes from cell culture super- 798

natants and biological fluids. Curr Protoc Cell Biol Chapter 799

3, Unit 3 22. 800

[39] Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, 801

Harding CV, Melief CJ, Geuze HJ (1996) B lymphocytes 802

secrete antigen-presenting vesicles. J Exp Med 183, 1161- 803

1172. 804

[40] Clayton A, Turkes A, Navabi H, Mason MD, Tabi Z (2005) 805

Induction of heat shock proteins in B-cell exosomes. J Cell 806

Sci 118, 3631-3638. 807

[41] Graner MW, Cumming RI, Bigner DD (2007) The heat shock 808

response and chaperones/heat shock proteins in brain tumors: 809

Surface expression, release, and possible immune conse- 810

quences. J Neurosci 27, 11214-11227. 811

[42] Guzhova I, Kislyakova K, Moskaliova O, Fridlanskaya I, 812

Tytell M, Cheetham M, Margulis B (2001) In vitro studies 813

show that Hsp70 can be released by glia and that exogenous 814

Hsp70 can enhance neuronal stress tolerance. Brain Res 914, 815

66-73. 816

[43] Mambula SS, Calderwood SK (2006) Heat shock protein 70 is 817

secreted from tumor cells by a nonclassical pathway involving 818

lysosomal endosomes. J Immunol 177, 7849-7857. 819

[44] Mambula SS, Stevenson MA, Ogawa K, Calderwood SK 820

(2007) Mechanisms for Hsp70 secretion: Crossing mem- 821

branes without a leader. Methods 43, 168-175. 822

[45] Sbai O, Ould-Yahoui A, Ferhat L, Gueye Y, Bernard A, 823

Charrat E, Mehanna A, Risso JJ, Chauvin JP, Fenouillet 824

E, Rivera S, Khrestchatisky M (2010) Differential vesicu- 825

lar distribution and trafficking of MMP-2, MMP-9, and their 826

inhibitors in astrocytes. Glia 58, 344-366. 827

[46] Taylor AR, Robinson MB, Gifondorwa DJ, Tytell M, Milligan 828

CE (2007) Regulation of heat shock protein 70 release in 829

astrocytes: Role of signaling kinases. Dev Neurobiol 67, 1815- 830

1829. 831

[47] Chirico WJ (2011) Protein release through nonlethal oncotic 832

pores as an alternative nonclassical secretory pathway. BMC 833

Cell Biol 12, 46. 834

[48] De Maio A (2011) Extracellular heat shock proteins, cellular 835

export vesicles, and the Stress Observation System: A form 836

of communication during injury, infection, and cell damage. 837

It is never known how far a controversial finding will go!; 838

Dedicated to Ferruccio Ritossa. Cell Stress Chaperones 16, 839

235-249. 840

[49] Giuliano JS Jr., Lahni PM, Wong HR, Wheeler DS (2011) 841

Pediatric sepsis - Part V: Extracellular heat shock proteins: 842

Alarmins for the host immune system. Open Inflamm J 4, 49- 843

60. 844

[50] Henderson B, Henderson S (2009) Unfolding the relation- 845

ship between secreted molecular chaperones and macrophage 846

activation states. Cell Stress Chaperones 14, 329-341. 847

[51] Arrigo AP (2012) Pathology-dependent effects linked to small 848

heat shock proteins expression: An update. Scientifica (Cairo) 849

2012, 185641. 850

[52] Schmitt E, Gehrmann M, Brunet M, Multhoff G, Garrido C 851

(2007) Intracellular and extracellular functions of heat shock 852

proteins: Repercussions in cancer therapy. J Leukoc Biol 81, 853

15-27. 854

[53] Rabouille C, Malhotra V, Nickel W (2012) Diversity in uncon- 855

ventional protein secretion. J Cell Sci 125, 5251-5255. 856

[54] Prudovsky I (2013) Nonclassically secreted regulators of 857

angiogenesis. Angiol Open Acces 1, 1000101. 858

[55] Nickel W, Rabouille C (2009) Mechanisms of regulated 859

unconventional protein secretion. Nat Rev Mol Cell Biol 10, 860

148-155. 861

Unc

orre

cted

Aut

hor P

roof

F. Nafar et al. / HspB1 Release from Astrocytes 13

[56] Nickel W, Seedorf M (2008) Unconventional mechanisms of862

protein transport to the cell surface of eukaryotic cells. Annu863

Rev Cell Dev Biol 24, 287-308.864

[57] Lee YJ, Lee HJ, Choi SH, Jin YB, An HJ, Kang JH, Yoon865

SS, Lee YS (2012) Soluble HSPB1 regulates VEGF-mediated866

angiogenesis through their direct interaction. Angiogenesis867

15, 229-242.868

[58] Sumizawa T, Igisu H (2008) Release of heat shock pro-869

teins from human neuroblastoma cells exposed to acrylamide.870

J Toxicol Sci 33, 117-122.871

[59] Dodge ME, Wang J, Guy C, Rankin S, Rahimtula M, Mearow872

KM (2006) Stress-induced heat shock protein 27 expression873

and its role in dorsal root ganglion neuronal survival. Brain874

Res 1068, 34-48.875

[60] Williams KL, Rahimtula M, Mearow KM (2005) Hsp27 and876

axonal growth in adult sensory neurons in vitro. BMC Neu-877

rosci 6, 24.878

[61] Williams KL, Rahimtula M, Mearow KM (2006) Heat shock879

protein 27 is involved in neurite extension and branching of880

dorsal root ganglion neurons in vitro. J Neurosci Res 84, 716-881

723.882

[62] Verdier Y, Zarandi M, Penke B (2004) Amyloid beta-peptide883

interactions with neuronal and glial cell plasma membrane:884

Binding sites and implications for Alzheimer’s disease. J Pept885

Sci 10, 229-248.886

[63] Patel AN, Jhamandas JH (2012) Neuronal receptors as targets887

for the action of amyloid-beta protein (Abeta) in the brain.888

Expert Rev Mol Med 14, e2.889

[64] Williams TL, Serpell LC (2011) Membrane and surface890

interactions of Alzheimer’s Abeta peptide–insights into the891

mechanism of cytotoxicity. FEBS J 278, 3905-3917.892

[65] De AK, Kodys KM, Yeh BS, Miller-Graziano C (2000)893

Exaggerated human monocyte IL-10 concomitant to mini-894

mal TNF-alpha induction by heat-shock protein 27 (Hsp27)895

suggests Hsp27 is primarily an antiinflammatory stimulus.896

J Immunol 165, 3951-3958.897

[66] Bechtold DA, Brown IR (2003) Induction of Hsp27 and898

Hsp32 stress proteins and vimentin in glial cells of the rat899

hippocampus following hyperthermia. Neurochem Res 28,900

1163-1173.901

[67] Currie RW, Ellison JA, White RF, Feuerstein GZ, Wang902

X, Barone FC (2000) Benign focal ischemic precondition-903

ing induces neuronal Hsp70 and prolonged astrogliosis with904

expression of Hsp27. Brain Res 863, 169-181.905

[68] Hecker JG, McGarvey M (2011) Heat shock proteins as906

biomarkers for the rapid detection of brain and spinal cord907

ischemia: A review and comparison to other methods of detec-908

tion in thoracic aneurysm repair. Cell Stress Chaperones 16,909

119-131.910

[69] Kirschstein T, Mikkat S, Mikkat U, Bender R, Kreutzer M,911

Schulz R, Kohling R, Glocker MO (2012) The 27-kDa heat

shock protein (HSP27) is a reliable hippocampal marker of 912

full development of pilocarpine-induced status epilepticus. 913

Epilepsy Res 98, 35-43. 914

[70] Schwarz L, Vollmer G, Richter-Landsberg C (2010) The small 915

heat shock protein HSP25/27 (HspB1) is abundant in cul- 916

tured astrocytes and associated with astrocytic pathology in 917

progressive supranuclear palsy and corticobasal degeneration. 918

Int J Cell Biol 2010, 717520. 919

[71] Villapol S, Acarin L, Faiz M, Castellano B, Gonzalez B (2008) 920

Survivin and heat shock protein 25/27 colocalize with cleaved 921

caspase-3 in surviving reactive astrocytes following excito- 922

toxicity to the immature brain. Neuroscience 153, 108-119. 923

[72] Wilhelmus MM, Boelens WC, Otte-Holler I, Kamps B, 924

Kusters B, Maat-Schieman ML, de Waal RM, Verbeek MM 925

(2006) Small heat shock protein HspB8: Its distribution in 926

alzheimer’s disease brains and its inhibition of amyloid- 927

beta protein aggregation and cerebrovascular amyloid-beta 928

toxicity. Acta Neuropathol 111, 139-149. 929

[73] Renkawek K, Stege GJ, Bosman GJ (1999) Dementia, glio- 930

sis and expression of the small heat shock proteins hsp27 931

and alpha B-crystallin in Parkinson’s disease. Neuroreport 932

10, 2273-2276. 933

[74] Stetler RA, Gan Y, Zhang W, Liou AK, Gao Y, Cao G, Chen J 934

(2010) Heat shock proteins: Cellular and molecular mech- 935

anisms in the central nervous system. Prog Neurobiol 92, 936

184-211. 937

[75] Abisambra JF, Jinwal UK, Jones JR, Blair LJ, Koren J, 3rd, 938

Dickey CA (2011) Exploiting the diversity of the heat-shock 939

protein family for primary and secondary tauopathy therapeu- 940

tics. Curr Neuropharmacol 9, 623-631. 941

[76] Kampinga HH, Garrido C (2012) HSPBs: Small proteins with 942

big implications in human disease. Int J Biochem Cell Biol 44, 943

1706-1710. 944

[77] Ojha J, Masilamoni G, Dunlap D, Udoff RA, Cashikar AG 945

(2011) Sequestration of toxic oligomers by HspB1 as a cyto- 946

protective mechanism. Mol Cell Biol 31, 3146-3157. 947

[78] Kudva YC, Hiddinga HJ, Butler PC, Mueske CS, Eberhardt 948

NL (1997) Small heat shock proteins inhibit in vitro A beta(1- 949

42) amyloidogenesis. FEBS Lett 416, 117-121. 950

[79] Lee S, Carson K, Rice-Ficht A, Good T (2006) Small heat 951

shock proteins differentially affect Abeta aggregation and 952

toxicity. Biochem Biophys Res Commun 347, 527-533. 953

[80] Wilhelmus MM, Boelens WC, Otte-Holler I, Kamps B, de 954

Waal RM, Verbeek MM (2006) Small heat shock proteins 955

inhibit amyloid-beta protein aggregation and cerebrovascular 956

amyloid-beta protein toxicity. Brain Res 1089, 67-78. 957

[81] Toth ME, Szegedi V, Varga E, Juhasz G, Horvath J, 958

Borbely E, Csibrany B, Alfoldi R, Lenart N, Penke B, Santha 959

M (2013) Overexpression of Hsp27 ameliorates symptoms of 960

Alzheimer’s disease in APP/PS1 mice. Cell Stress Chaper- 961

ones 18, 759-771. 962