Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Brad:Mearow Lab Paper - September 28, 2015

  • Be the first to comment

  • Be the first to like this

Brad:Mearow Lab Paper - September 28, 2015

  1. 1. UncorrectedAuthorProof 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␤. 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 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
  2. 2. UncorrectedAuthorProof 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
  3. 3. UncorrectedAuthorProof 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
  4. 4. UncorrectedAuthorProof 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
  5. 5. UncorrectedAuthorProof 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
  6. 6. UncorrectedAuthorProof 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␤.
  7. 7. UncorrectedAuthorProof 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. 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. 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. 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. 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 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 increaseofreactiveoxygenspeciescausedbyhuntingtin.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] SharpP,KrishnanM,PullarO,NavarreteR,WellsD,deBelle- 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
  12. 12. UncorrectedAuthorProof 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:Roleofsignalingkinases.DevNeurobiol67,1815- 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
  13. 13. UncorrectedAuthorProof 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

    Be the first to comment

    Login to see the comments

Views

Total views

105

On Slideshare

0

From embeds

0

Number of embeds

1

Actions

Downloads

1

Shares

0

Comments

0

Likes

0

×