ORIGINAL PAPER

Phosphorylation-dependent subcellular localization of the smallheat shock proteins HspB1/Hsp25 and HspB5/aB-crystallinin cultured hippocampal neurons

Thomas Schmidt • Britta Bartelt-Kirbach •

Nikola Golenhofen

Accepted: 28 April 2012 / Published online: 23 May 2012

� Springer-Verlag 2012

Abstract The so-called stress response involving upreg-

ulation of heat shock proteins (Hsps) is a powerful mech-

anism of cells to deal with harmful conditions to which

they are exposed throughout life, such as hyperthermia,

hypoxia or oxidative stress. To gain more information

about the molecular targets by which HspB1 (Hsp25) and

HspB5 (aB-crystallin) might exert their neuroprotective

effect we investigated the subcellular localization of

unphosphorylated and phosphorylated HspB1 and B5 in

neurons by immunocytochemistry and subcellular frac-

tionation. In cultured hippocampal neurons, the unphos-

phorylated forms of both Hsps were localized in the

perikaryon and nucleus, whereas the phosphorylated forms

were recruited into neuronal processes. pHspB1-Ser15 and

-Ser 86 were found within dendrites with a punctate dis-

tribution pattern partially colocalizing with the synaptic

marker vGlut-1. pHspB5-Ser19 and -Ser45 localized to

axons and dendrites with a filamentous-like staining pat-

tern, whereas pHspB5-Ser59 was found in dendrites,

especially along the plasma membrane and in spines.

Biochemical analysis, i.e. subcellular fractionation of rat

brain with subsequent Western blotting supported these

localizations. These data show that in neurons HspB1 and

B5 may have various molecular interaction partners at

synapses, within dendrites and axons and that this inter-

action is likely to be regulated by phosphorylation. Stress-

induced phosphorylation of HspB1 and B5 may lead to

binding of these Hsps to their targets at synapses and

neuronal processes which might provide one important

mechanism of how they exert their neuroprotective effect.

Keywords Stress tolerance � Neuroprotection �Hippocampus � Small heat shock proteins �a-Crystallin � Synapse

Introduction

In the brain as well as in other organs sublethal insults lead

to the development of stress tolerance meaning that the

resistance to a second potentially lethal stimulus is

increased. This adaptive cytoprotection is a powerful

endogenous mechanism for cells to survive exposure to

recurrent stress events (Kitagawa et al. 1990; Obrenovitch

2008). It has become clear that multiple effectors contrib-

ute to this phenomenon among them the upregulation of a

selected group of proteins, called heat shock proteins

(Hsps), functioning as molecular chaperones and promot-

ing cellular survival (Ritossa 1962; Vos et al. 2008; Welch

1992).

The small heat shock proteins (sHsps, 12–43 kDa)

consist of 11 family members, named HspB1 to HspB11 in

accordance with the guidelines of the HUGO Gene

Nomenclature Committee (Bellyei et al. 2007; Kappe et al.

2003). The best known representatives of the sHsp family

are HspB1 (also named Hsp25 or Hsp27) and HspB5 (also

named aB-crystallin). sHsps exert their cytoprotective

function mainly via their chaperone-like activity, namely

they are able to prevent denaturation and aggregation of

other proteins (Haslbeck et al. 2005; Sun and MacRae

2005). However, it has been shown that sHsps have addi-

tionally other specific cellular functions, such as inhibition

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00418-012-0964-x) contains supplementarymaterial, which is available to authorized users.

T. Schmidt � B. Bartelt-Kirbach � N. Golenhofen (&)

Institute of Anatomy and Cell Biology, University of Ulm,

Albert-Einstein-Allee 11, 89081 Ulm, Germany

e-mail: nikola.golenhofen@uni-ulm.de

123

Histochem Cell Biol (2012) 138:407–418

DOI 10.1007/s00418-012-0964-x

of apoptosis and binding to cytoskeletal proteins which

contribute to their cytoprotective effect (Garrido et al.

2001; Golenhofen et al. 1998, 2002; Gusev et al. 2002;

Verschuure et al. 2002). HspB1 and HspB5 not only

become upregulated in response to stress conditions, but

also phosphorylated and it is generally believed that

phosphorylation increases the protective effect. Rat HspB1

has two potential phosphorylation sites at Ser 15 and Ser

86, whereas HspB5 has three phosphorylation sites at Ser

19, Ser 45 and Ser 59 (Chiesa et al. 1987; Gaestel et al.

1991; Ito et al. 1997; Landry et al. 1992). Phosphorylation

is mediated mainly by the p38 MAP-kinase pathway but

other kinases, such as PKA, PKC or Akt may be also

involved (Kato et al. 1998; Kostenko and Moens 2009;

Stokoe et al. 1992). Interestingly, phosphorylation of

HspB1 and B5 leads to a decreased chaperone function

which is probably mediated by a dissociation of the large

multimers formed by sHsps (Ito et al. 2001; Kamei et al.

2001). This is in contrast to the observed increased cyto-

protective effect of phosphorylated sHsps which leads to

the hypothesis that the specific (non-chaperone) functions

of sHsps are regulated by phosphorylation resulting in

increased cell viability. For example, phosphorylation

increases the binding of HspB1 to F-actin and promotes the

anti-apoptotic effect of HspB1 (Charette et al. 2000;

Wieske et al. 2001).

Many sHsps show high expression in muscle tissue

where they have been studied extensively and their car-

dioprotective role has clearly been identified (Danan et al.

2007; Fan et al. 2005; Golenhofen et al. 2006; Hollander

et al. 2004; Ray et al. 2001). In the nervous system sHsps

are expressed to a lower extent and are found in glial cells

as well as in neurons (Bartelt-Kirbach and Golenhofen

2011; Quraishe et al. 2008; Verschuure et al. 2003). Yet

little is known about their functions in neurons. Several

sHsps are upregulated in mouse models of neurodegener-

ative diseases (Wang et al. 2008) and have been shown to

be components of amyloid plaques in Alzheimer‘s disease

(Renkawek et al. 1994; Wilhelmus et al. 2006). However,

the function of sHsps in these plaques (inhibition or

enhancement of amyloid-b-mediated toxicity) is not clear

(Narayanan et al. 2006; Stege et al. 1999; Wilhelmus et al.

2006). Further evidence for an important role of sHsps in

the nervous system comes from the fact that mutations of

HspB1, HspB3 and HspB8 genes are associated with distal

hereditary motor neuropathy or Charcot–Marie–Tooth

disease (Evgrafov et al. 2004; Irobi et al. 2004; Kolb et al.

2010). In addition, a neuroprotective function for HspB1

was shown in vivo using HspB1 transgenic animals in

which neuronal cell death was significantly reduced after

kainate-induced seizures (Akbar et al. 2003). All these data

support the importance of sHsps in brain function at

physiological as well as pathophysiological conditions. It

remains to be elucidated which specific molecular mech-

anisms are involved in sHsp functions in neurons and if

these functions are regulated by phosphorylation.

This study was undertaken to investigate if phosphory-

lation of the small heat shock proteins HspB1 and HspB5

might be an important mechanism by which the specific

(non-chaperone) functions of these two proteins in neurons

are regulated. If phosphorylation of HspB1 and B5 leads to

binding of these sHsps to specific target proteins one would

expect different subcellular distributions of the phosphor-

ylated and unphosphorylated forms. By immunocyto-

chemistry, we found indeed that phosphorylation of HspB1

and B5 at different phosphorylation sites led to recruitment

of these proteins to various subcellular compartments

within neuronal processes. This hints at different molecular

targets of the various phosphorylated forms of HspB1 and

B5 in neurons.

Materials and methods

Animal experiments

All animal experiments were performed in compliance

with the guidelines for the welfare of experimental animals

issued by the Federal Government of Germany, the

National Institute of Health and the Max Planck Society.

The described experiments with rat tissue and cultured

cells/neurons are approved by the local ethics committee

(Ulm University). ID Number: O.103.

Cell culture

Sprague–Dawley rats (Crl:CD, Charles-River Lab. Int.

Inc., Wilmington, MA, USA) at 18 or 19 days of gestation

were killed by CO2. After dissection, the embryonal hip-

pocampi were dissociated with 0.25 % trypsin in HBSS

(PAA, Pasching, Austria) followed by treatment with

0.01 % DNase I (Invitrogen, Darmstadt, Germany). The

cell suspension was then passed through a 100-lm sieve,

counted and plated at a density of 7,500/cm2 on poly(L)

lysine-coated petri dishes in DMEM medium with 10 %

FCS. After 3 h medium was changed to neurobasal med-

ium (Invitrogen, Darmstadt, Germany) supplemented with

B-27. The cells were allowed to grow for 21 days at 37 �C,

5 % CO2, 95 % air in a humidified atmosphere. At least

three biological replicates were carried out for each

experimental series.

Immunocytochemistry

Cultured hippocampal neurons (21 days in vitro) grown on

cover slips at a density of 7,500 cells/cm2 were fixed with

408 Histochem Cell Biol (2012) 138:407–418

123

cooled methanol on ice for 3 min. After washing once with

PBS, cells were blocked for unspecific binding with 3 %

bovine serum albumin and 1 % normal goat serum in PBS

for 30–60 min at RT. The primary antibodies were applied

at 4 �C overnight. Rabbit anti-HspB1 (Stressgen SPA-801,

VIC, Canada), anti-HspB5 (Stressgen SPA-223), anti-

phospho(Ser16)HspB1 (Acris, San Diego, CA, USA), anti-

phospho(Ser86)HspB1 (Acris), anti-phospho(Ser19)HspB5

(Stressgen), anti-phospho(Ser45)HspB5 (Stressgen) were

used at a dilution of 1:100, rabbit anti-phospho

(Ser59)HspB5 (Stressgen) at a final dilution of 1:200,

guinea pig anti-vesicular glutamatetransporter 1 (Chem-

icon) and mouse pan-axonal neurofilament marker anti-

body (SMI 312, Covance, Emeryville, CA, USA) at a final

dilution of 1:2,000. After further washing, cells were

incubated with a mixture of goat anti-rabbit Alexa Fluor

568 and goat anti-mouse or goat anti-guinea pig Alexa

Fluor 488 (dilutions 1:900, Molecular Probes, USA) for

45 min at RT. After final rinsing for 3 9 5 min with PBS

cells were mounted in Mowiol 4.88 (Hoechst, Germany)

containing 1 lg/ml DAPI (Applichem, Darmstadt,

Germany). Images were acquired on a Zeiss Axioscope2

microscope equipped with an AxioCam MRm camera

(Zeiss, Oberkochen, Germany) and analyzed using Axio-

Vision software (Zeiss) and Adobe Photoshop (Adobe

Systems, USA). Representative images from at least three

independent experiments are shown. Controls were per-

formed by omission of the primary antibodies. For further

control of the phospho-specific antibodies, preabsorption

experiments were performed. Antibodies were incubated

with an excess of the respective synthetic phosphopeptide

used for immunization (5 lg peptide/1 lg antibody) for

2 h at RT with overhead rotation prior to application to

the cells. The following peptides were used: LLRGP(pS)

WDPFRC (Acris) for anti-pHspB1-Ser16, RQL(pS)GVSEIR

for anti-pHspB1-Ser86 (Abgent, San Diego, USA), FFPFH

(pS)PSRLFD for anti-pHspB5-Ser19 (Abgent), L(pS)PFYLR

PPSF for anti-pHspB5-Ser45 (Abgent) and FLRAP(pS)WID

TG for anti-pHspB5-Ser59 (Abgent).

Subcellular fractionation

Subcellular fractionation and isolation of synaptic fractions

from rat brain were performed as described recently (Lie-

bau et al. 2009) based on the method published by Carlin

et al. (1980). Briefly, brains of 4-month old rats (Sprague–

Dawley, see above) were dissected, the cerebellum and

brain stem removed and the remaining brain homogenized

in a buffer containing 320 mM sucrose, 5 mM HEPES pH

7.4 and a protease inhibitor cocktail (Roche Applied Sci-

ences, Mannheim, Germany). Nuclei and cell debris were

pelleted (1,000g, 10 min). The supernatant was subjected

to further centrifugation at 12,000g for 20 min. The

resulting supernatant containing the cytoplasm was saved

and the pellet resembling a crude synaptosome fraction was

resuspended (buffer: 320 mM sucrose, 5 mM Tris pH 8.1)

and loaded on a step sucrose gradient (0.85/1.0/1.2 M).

After centrifugation (85,000g for 2 h) myelin, membrane

and synaptosome fractions were harvested at the inter-

phases and the mitochondrial fraction was obtained as

pellet. The synaptosomes were further fractionated by lysis

in a hypotonic buffer (1 mM Tris, pH 8.1) for 30 min and

centrifugation at 33,000g for 30 min. The resulting pellet

was subjected to a second step sucrose gradient (0.85/1.0/

1.2 M) and the synaptic membrane fraction harvested at the

interphase between 1.0 and 1.2 M sucrose. To release the

postsynaptic density (PSD) from the membranes, this

fraction was resuspended in a detergent containing buffer

(320 mM sucrose, 5 mM Tris (pH 8.1), 0.5 % (v/v) Tri-

tonX-100). The PSD was pelleted by final centrifugation at

33,000g for 30 min. All steps were performed at 4 �C. The

purity of the synaptic fractions and the PSD was deter-

mined by Western blot and electron microscopy.

Western blot

Laemmli protein sample buffer was added to the different

subcellular fractions (Laemmli 1970). Samples were then

sonicated and heated to 95 �C for 5 min. Protein concen-

tration was measured with amidoblack staining (Heinzel

et al. 1965). Equal amounts of protein were separated on

15 % SDS–polyacrylamide gels followed by semidry

blotting onto Hybond PVDF membranes (Amersham, GE

Healthcare, Freiburg, Germany) with 0.8 mA/cm2 for

90 min in a trans-blot SD chamber (Bio-Rad, Munich,

Germany) with Kyhse–Andersen transfer buffer (1984).

For analysis of phosphorylated proteins wet transfer with

90 V for 150 min at 4 �C was performed using the same

buffer and membrane. Membranes were blocked with 5 %

(w/v) low fat milk in Tris buffered saline (50 mM Tris,

150 mM NaCl, pH 7.5) containing 0.05 % (v/v) Tween 20

for 1 h, followed by incubation with the primary antibody

(diluted in the blocking solution) at 4 �C overnight. Poly-

clonal antibodies were used for the detection of HspB1,

HspB5 and their phosphorylated forms (described in

‘‘Immunocytochemistry’’) and monoclonal antibodies for

detection of PSD-95 (Chemicon, MAB 1598) and synap-

tophysin (Sigma, SVP-38). All antibodies were applied at a

1:1,000 dilution. Secondary antibodies were horseradish

peroxidase-labelled goat anti-rabbit or anti-mouse IgG

(Jackson Immunoresearch, West Grove, PA), used at a

dilution of 1:3,000 in the blocking solution. Washing was

carried out 3 times for 5 min in TBS with 0.05 % Tween

20. Bound secondary antibodies were detected with the

enhanced chemiluminescence system. Quantification of

band intensity was carried out on scanned Western blot

Histochem Cell Biol (2012) 138:407–418 409

123

images with ImageJ software (National Institutes of Health)

from blots of three to five independent experiments.

Statistics

Data are presented as mean ± standard error of the mean.

The non-parametric Mann–Whitney U test was used for

statistical analysis. Statistical significance was assumed for

p \ 0.05.

Results

Subcellular localization of HspB1 and its

phosphorylated forms in cultured hippocampal neurons

To find out if unphosphorylated and phosphorylated forms

of HspB1 might have different molecular targets, we

investigated the subcellular localization of HspB1 and its

phosphorylated forms in cultured hippocampal neurons

grown for 21 days in vitro by immunocytochemistry.

Figure 1 shows double stainings for HspB1 or phospho-

HspB1 and the vesicular glutamatetransporter 1 (vGlut-1), a

marker for synapses. For labelling of HspB1 antibodies

recognizing specifically the respective phosphorylation site

or an antibody which recognizes HspB1 independent of its

phosphorylation status were used. In the latter case the im-

munosignal will be generated mostly by the unphosphory-

lated form because it is known that the major part of total

cellular HspB1 is not phosphorylated. Figure 1 shows that

HspB1 is mainly localized in the perikaryon and nucleus of

cultured hippocampal neurons. In contrast, both phosphor-

ylated forms of HspB1 were additionally found in neuronal

processes. Within the dendrites, a punctate staining pattern

could be observed for pHspB1-Ser15 and even more clearly

for pHspB1-Ser86. Colocalization of some of these pHspB1

positive puncta with vGlut-1 hint at synaptic localization of

pHspB1. Specificity of the phospho-specific antibodies was

Fig. 1 Cultured hippocampal neurons immunolabelled for HspB1 or

phospho-HspB1 (red) and vesicular glutamatetransporter-1 (vGlut-1

green). Whereas total HspB1 (unphosphorylated and phosphorylated)

was mainly localized in the perikaryon and nucleus both phosphor-

ylated forms of HspB1 were additionally found in neuronal processes.

Note the punctate staining pattern within the dendrites for pHspB1-

Ser15 and even more clearly for pHspB1-Ser86 which partially

colocalized with vGlut-1 (arrows) hinting at a synaptic localization of

pHspB1. Bar 20 lm

410 Histochem Cell Biol (2012) 138:407–418

123

controlled by preabsorption of the antibodies with their

immunogens which blocked the immunosignals (Fig. 1,

supplementary electronic material). To investigate if

pHspB1 can also be found in axons double labelling for

pHspB1 and for axonal-specific neurofilaments (NF) was

performed. Figure 2 shows that pHspB1-Ser15 and pHspB1-

Ser86 were hardly detectable in axons whereas dendrites

were prominently labelled. Only thick axonal bundles

showed a faint staining for pHspB1 demonstrating that

pHspB1 is localized in axons, but to a much lower extent

than in dendrites. These data indicate that phosphorylation

of HspB1 leads to a recruitment of this protein to dendrites

and especially synaptic sites within them.

Subcellular localization of HspB5 and its phosphorylated

forms in cultured hippocampal neurons

To investigate if the subcellular localization of HspB5 is

regulated by phosphorylation as observed for HspB1, we

performed immunostaining for HspB5 and its phosphory-

lated forms in cultured hippocampal neurons. HspB5 has

three phosphorylation sites, i.e. Ser19, Ser45 and Ser59.

Using an antibody (Pan-HspB5) recognizing the unphos-

phorylated and phosphorylated forms HspB5 was found to

be localized in the nucleus and in the perikaryon (Fig. 3).

However, using antibodies specific for each phosphoryla-

tion site of HspB5 strong immunosignals for all three

phosphoforms were additionally observed in neuronal

processes. Thus, the immunosignal of the Pan-HspB5

antibody will most likely resemble the localization of

the unphosphorylated form which is known to be the pre-

dominant form at control (non-stress) conditions. pHspB5-

Ser45 is localized within the dendrites (which can be

identified by their decoration with synapses labeled with

vGlut-1) in a filamentous-like pattern (Fig. 3). This stain-

ing pattern could also be observed for pHspB5-Ser19,

however, pHspB5-Ser19 was additionally found at small

discrete dots in the periphery of dendrites which colocalize

with vGlut-1. Compared with these two phosphorylated

forms pHspB5-Ser59 was localized in a complete different

compartment within the dendrites. Staining within the

center of dendrites was very weak, but strong along the

plasma membrane and in puncta at the periphery of den-

drites. These puncta most likely resemble spines since

often at the tip of these spines a vGlut-1 positive dot could

be observed. Thus, pHspB5-Ser59 seems to be localized at

the plasma membrane or the underlying cytoskeleton and

in dendritic spines. The subcellular distribution of pHspB5-

Ser59 also differed from the other two phospho-forms of

HspB5 regarding the axons. pHspB5-Ser19 and -Ser45

were found to occur in axons (identified by labeling with

NF, Fig. 4), whereas pHspB5-Ser59 was nearly not

detectable in axons. Specificity of the phospho-specific

antibodies was controlled by preabsorption of the anti-

bodies with their immunogens which blocked the immu-

nosignals (Fig. 1, supplementary electronic material).

Thus, subcellular localization of HspB5 in neurons depends

on its phosphorylation status. Whereas the unphosphorylated

form was predominantly localized in the perikaryon and

nucleus the phosphorylated forms were found in different

compartments of neuronal processes which again varied

depending on which individual site was phosphorylated.

Subcellular fractionation

To further characterize the localization of HspB1 and B5 in

neurons rat brains were subjected to subcellular fraction-

ation with subsequent Western blotting. Rat brains instead

Fig. 2 Cultured hippocampal neurons immunolabelled for phospho-HspB1 (red) and axonal-specific neurofilament (NF green). Both pHspB1-

Ser15 and pHspB1-Ser86 were hardly detectable in axons (arrows), whereas dendrites were prominently labelled

Histochem Cell Biol (2012) 138:407–418 411

123

of cultured cells were used to obtain a high enough yield of

total protein in each fraction. Figure 5 shows representative

blots of three experiments and quantification of band

intensities are shown in Fig. 2 of the supplementary elec-

tronic material. First the fractions were characterized using

the postsynaptic marker PSD-95 and the presynaptic mar-

ker synaptophysin. Both proteins were enriched in synap-

tosomes and synaptic membranes, whereas as expected

only PSD-95 was enriched in the postsynaptic density

fraction (PSD). HspB1 was significantly enriched in the

nuclear fraction but was also present in many other frac-

tions such as the cytoplasm, the membrane fraction, syn-

aptosomes and synaptic membranes (Fig. 5). This confirms

the localization of HspB1 in the cytoplasm and nucleus as

supposed from the immunocytochemical experiments.

pHspB1-Ser86 showed a similar subcellular distribution,

Fig. 3 Cultured hippocampal neurons immunolabelled for HspB5 or

phospho-HspB5 (red) and vesicular glutamatetransporter-1 (vGlut-1

green). Whereas total HspB5 (unphosphorylated and phosphorylated)

was mainly localized in the perikaryon and nucleus, all three

phosphorylated forms of HspB5 were additionally found in neuronal

processes. pHspB5-Ser19 and -Ser45 showed a filamentous-like

distribution in the center of the dendrites, whereas pHspB5-Ser59 was

localized along the plasma membrane of dendrites and in puncta at

the periphery of dendrites. These puncta seem to resemble spines

since often at the tip of these spines a vGlut-1 positive dot could be

observed (arrows). In addition, pHspB5-Ser19 was found at small

discrete dots in the periphery of dendrites which colocalize with

vGlut-1 (arrows). Bar 20 lm

412 Histochem Cell Biol (2012) 138:407–418

123

however, it could be detected additionally in the postsyn-

aptic density fraction. pHspB1-S15 was enriched in the

membrane fraction, synaptosomes and synaptic junctions

and was detectable in little amount in the postsynaptic

densities. Interestingly, in these fractions, an immunore-

active double band could be seen which might reflect a

Fig. 4 Cultured hippocampal neurons immunolabelled for phospho-

HspB5 (red) and axonal-specific neurofilament (NF green). pHspB5-

Ser19 and-Ser45 were found to be localized in axons (NF positive

structures arrows) whereas pHspB5-Ser59 was nearly not detectable

in axons

Fig. 5 Subcellular fractionation of rat brains with subsequent

Western blotting for PSD-95, synaptophysin, HspB1, HspB5 and

their phospho-forms. As expected enrichment of PSD-95 in the

postsynaptic density fraction and of synaptophysin in the synapto-

somes and synaptic membrane fraction could be observed. HspB1

was significantly enriched in nuclei, but was also present in many

other fractions such as the cytoplasm, the membrane fraction,

synaptosomes and synaptic membranes. pHspB1-Ser86 showed a

similar subcellular distribution, however, it could be detected

additionally in the postsynaptic density fraction. pHspB1-S15 was

enriched in the membrane fraction, synatosomes and synaptic

junctions and was detectable in little amount in the postsynaptic

densities. HspB5 was significantly enriched in the myelin, membrane

fraction and the PSD and to a lower extent detectable in the

cytoplasm, nuclear fraction, synaptosomes and synaptic membranes.

Note that pHspB5-Ser 19 was not present in any of the synaptic

fractions analyzed whereas pHspB5-Ser45 and -Ser59 were detectable

in low amounts in synaptosomes and synaptic membranes and to a

much higher amount in postsynaptic densities

Histochem Cell Biol (2012) 138:407–418 413

123

different phosphorylation status (mono- and biphosphory-

lated forms) or alternative splice variants. Thus, the bio-

chemical analysis showed that pHspB1 is present in

synaptosomes and supports the immunocytochemical data

of localization of pHspB1 at synapses. Since no enrichment

of pHspB1 in the postsynaptic density fraction could be

observed pHspB1 might interact most likely with presyn-

aptic structures.

HspB5 was enriched in the myelin, membrane fraction

and postsynaptic densities but was also present in the

cytoplasm, nuclear fraction, synaptosomes and synaptic

membranes. The enrichment in myelin fits to data in the

literature reporting expression of HspB5 in oligodendro-

cytes (see ‘‘Discussion’’). This cannot be compared with

our immunocytochemical data because myelinization

does not occur in neuronal cell cultures. The immunoblots

using the phospho-specific antibodies against HspB5

showed that HspB5 in the myelin fraction was also

strongly phosphorylated. Regarding the synaptic fractions

pHspB5-Ser 19 was not present in any of these fractions

analyzed. pHspB5-Ser45 and -Ser59 were detectable in

low amounts in synaptosomes and synaptic membranes

and to a much higher amount in postsynaptic densities.

Thus, these two phospho forms are clearly present at

synapses where they seem to be localized at the post-

synaptic site. These data support our immunocytochemi-

cal experiments showing pHspB5-Ser59 being localized

along the plasma membrane and at synapses but do not fit

exactly to the localization observed for pHspB5-Ser19

and -Ser45. These differences might be explained by the

fact that whole brains were used for subcellular frac-

tionation whereas cultured hippocampal neurons were

subjected to immunocytochemical analysis.

Thus, the biochemical analysis confirms the key finding

of synaptic localization of phospho-HspB1 and -B5

obtained by immunocytochemistry.

Discussion

Small heat shock proteins (sHsps) help the cell to survive

many harmful conditions to which the human body is

exposed throughout life such as hyperthermia, hypoxia or

oxidative stress. Despite the multitude of studies on sHsp

function in the heart, much less is known about their role in

the brain. In this study, we show in neurons for the first

time that phosphorylated HspB1 and HspB5 display dif-

ferent subcellular distributions compared to the unphos-

phorylated forms suggesting that phosphorylation of these

Hsps leads to interaction with specific molecular targets.

The distribution pattern of the respective pHsps hints at

filamentous structures within dendrites or axons and com-

ponents of synapses as possible targets.

Role of phosphorylation of HspB1 and B5

It is well known that HspB1 and B5 are phosphorylated in

response to various types of stresses in many different cell

types and that this process increases the development of

stress tolerance (Arrigo and Welch 1987; Ito et al. 1997;

Landry et al. 1991, 1992). For example mimicking phos-

phorylation of HspB5 on Ser59 protects cardiac myocytes

from apoptosis (Morrison et al. 2003). Such phosphoryla-

tion-dependent protective effects could also be observed in

neurons where phosphorylation of HspB1 stimulates neu-

rite outgrowth and cell survival after injury (Benn et al.

2002; Williams and Mearow 2011). However, these cyto-

protective effects of both sHsps are not mediated by their

chaperone function since phosphorylation leads to disso-

ciation of large sHsp oligomers to smaller sizes which in

turn decreases their chaperone activity in vitro (Ito et al.

2001; Kamei et al. 2001; Kato et al. 1994). It is believed

that phosphorylation of sHsps regulates their intracellular

distribution and binding to specific targets. This has been

investigated most intensively in heart where ischemia-

induced phosphorylation of sHsps leads to a translocation

from the cytosol to myofibrillar proteins (Barbato et al.

1996; Chiesi et al. 1990; Golenhofen et al. 1998, 2002,

2004). Yet such a translocation has not been reported in

neurons. Our data of localization of phospho-Hsps in dif-

ferent cellular compartments as compared to the unphos-

phorylated forms support the hypothesis that in neurons

intracellular localization of sHsps is also regulated by

phosphorylation. Most likely this is due to different

molecular binding partners of phosphorylated versus non-

phosphorylated sHsps. To our knowledge, this study is also

the first one showing different subcellular localization of

each individual form of the three phosphorylated forms of

HspB5. This hints at different molecular targets of the

individual phospho-forms of HspB5 and is in contrast to

phopho-HspB1 where both phospho-forms localized to

similar compartments.

Possible molecular targets of HspB1 and B5

Several reports in non-neuronal cells indicate that sHsps

interact with targets of many different cellular components,

such as nuclear proteins, the plasma membrane, the cyto-

skeleton and myofibrillar structures in muscle tissue. In this

study, we could confirm localization of HspB1 and B5 in

some of these compartments in hippocampal neurons but

found additionally hints at synaptic localization of these

two proteins.

Nuclear accumulation of both HspB1 and B5 is seen at

physiological conditions and especially in response to

various types of stress, such as heat shock, ischemia or

exposure to toxins where they are often found to be

414 Histochem Cell Biol (2012) 138:407–418

123

associated with nuclear speckles (Arrigo et al. 1988;

Bryantsev et al. 2007; Loktionova et al. 1996; McClaren

and Isseroff 1994; van den IJssel et al. 2003; Voorter et al.

1992). Our data show that nuclear localization of these two

sHsps can also be observed in cultured hippocampal neu-

rons even at control conditions. The biochemical analysis

suggests that HspB1 is highly enriched in the nuclear

fraction, whereas HspB5 content in cell nuclei is much

lower. Bryantsev et al. (2007) showed that pseudophosph-

orylated HspB1 is recruited to cell nuclei suggesting that

nuclear translocation is regulated by phosphorylation. The

function of HspB1 and B5 in the nucleus is not fully

understood. Colocalization of the two sHsps with splicing

factors hint at a role in mRNA processing, but the molecular

targets remain to be identified (Bryantsev et al. 2007; van

den IJssel et al. 2003; van Rijk et al. 2003).

Our finding of enrichment of HspB5 in the membrane

and myelin fraction supports previous data showing asso-

ciation of HspB5 with the plasma membrane and in vitro

studies demonstrating integration of HspB5 into lenticular

plasma membranes by hydrophobic interactions (Cobb and

Petrash 2000; Ifeanyi and Takemoto 1990). Two recent

reports have shown that HspB5 is found in lipid rafts of

retinal pigment epithelial cells and that it is secreted via

exosomes into the extracellular space (Gangalum et al.

2011; Sreekumar et al. 2010). If such a secretion takes also

place in neurons this might provide an explanation of the

observed extracellular deposits of HspB5 in various neu-

rodegenerative diseases such as association with amyloid

plaques in Alzheimer‘s disease. The role of phosphoryla-

tion in this process is yet unclear. Gangalum et al. (2011)

showed by 2D-gel-analysis that two forms of HspB5

occurred in the exosomes and that these are identical to the

intracellular ones. Our immunocytochemical data hint at a

membranal localization of especially pHspB5-Ser59 which

might form oligomers also with unphosphorylated HspB5

resulting in a mixture of different forms of HspB5 in the

membrane. In contrast to HspB5, HspB1 seems to be not

abundant in plasma membranes. We did not find HspB1 to

be enriched in membranal fractions of rat brain. Only few

reports describe HspB1 to be associated with membranes,

for example in tumor cell lines (Graner et al. 2007). One

special membrane fraction is the myelin fraction where

HspB5 is present in high concentrations as shown in this

and previous studies. It serves as a guardian protein pro-

tecting the myelin sheath and is a key autoantigen in

multiple sclerosis (Ousman et al. 2007; van Noort et al.

1995). This is another unique function of HspB5 which is

not exerted by HspB1.

In many cell types HspB1 and B5 are known to be able

to bind to various components of the cytoskeleton, such as

F-actin, intermediate filaments, tubulin and titin and are

involved in regulation of filament organization (Bullard

et al. 2004; Djabali et al. 1997; Nicholl and Quinlan 1994;

Panasenko et al. 2003; Perng et al. 1999; Wang and Spector

1996; Xi et al. 2006). In hippocampal neurons, we found

the phosphorylated forms of HspB1 and B5 being localized

in neuronal processes which are rich of filaments; espe-

cially, pHspB5-Ser 19 and -45 showed a filamentous-like

distribution within dendrites and axons. It remains to be

determined to which types of filaments they bind. pHspB1

likely binds to neurofilaments or microtubules since over-

expression of various mutants of HspB1 have been shown

to disrupt neurofilament assembly or to disturb microtubule

dynamics (Ackerley et al. 2006; Almeida-Souza et al.

2011; Evgrafov et al. 2004).

Most interesting is our new finding of synaptic locali-

zation of phosphorylated HspB1 and B5 in hippocampal

neurons. Until now only two studies exist which hint at a

synaptic localization of HspB1 and B5, however, they did

not consider the phosphorylation status (Bechtold and

Brown 2000; Quraishe et al. 2008). Bechtold and Brown

(2000) identified HspB1 in synapses of the cerebellum after

hyperthermia, but suggested that HspB1 was secreted by

glial cells as those surround synapses and contain high

amounts of HspB1 and that subsequently HspB1 is absor-

bed by the neurons directly at the synaptic sites. The data

of this study show clearly that in hippocampal cultures glial

cells contain negligible amounts of phospho-HspB1 and B5

and that unphosphorylated HspB1 and B5 are localized in

neuronal perikarya allowing us to conclude that synaptic

HspB1 and B5 were synthesized within the neurons

themselves. The second study (Quraishe et al. 2008)

showed HspB1 and B5 in synaptosomal fractions of mouse

brain with similar results as obtained in this study using rat

brain. In addition, we could show that synaptosomes con-

tain phosphorylated HspB1 and HspB5. Taken these data

together with our immunocytochemical experiments, we

can conclude that pHspB1-Ser15, -Ser86 and pHspB5-

Ser59 are localized at synapses. pHspB5-Ser19 and -Ser45

were only found to be synaptic by one of these two

methods used. One has to take into consideration that

whole rat brains were used for subcellular fractionation,

whereas cultured hippocampal neurons for immunocyto-

chemistry. Thus, the discrepancies in localization of some

pHsps observed with these two methods may be due to

different brain regions or due to the culture conditions

themselves. Interestingly, phospho-HspB1 and B5 differed

in their subsynaptic distribution. pHspB1 was present in

synaptosomes, synaptic membranes but only to little

amounts in the postsynaptic density fraction, whereas

pHspB5-Ser59 was also detected in the postsynaptic den-

sity hinting at a presynaptic localization of phospho-HspB1

and postsynaptic localization of phospho-HspB5-Ser59.

Histochem Cell Biol (2012) 138:407–418 415

123

Functional implications of HspB1 and B5 in neurons

Taken together, HspB1 and B5 occur in many different

subcellular compartments in neurons and thus seem to have

various molecular interaction partners. This interaction is

likely to be regulated by phosphorylation. Most interest-

ingly we could show here for the first time that phos-

phorylation of HspB5 at different sites leads to recruitment

to different subcellular structures. This suggests that dif-

ferential phosphorylation is one important mechanism of

how a specific interaction partner is selected.

By comparison with other cell types one might speculate

that phosphorylation of HspB1 and B5 is induced by cel-

lular stress conditions leading to binding of these Hsps to

their targets resulting in increased stress tolerance. How-

ever, after heat shock in cultured hippocampal neurons no

translocation of HspB1 or B5 could be observed by

immunocytochemistry using the Pan-HspB1 and -B5 anti-

bodies (Bartelt-Kirbach and Golenhofen 2011). Thus,

either heat shock in neurons does not display an adequate

cellular stress leading to significant increase of phosphor-

ylation and translocation of HspB1 and B5 or the sensi-

tivity of the used antibodies was not high enough to detect

the portion of translocated Hsps. Future experiments are

needed to measure the degree of phosphorylation in neu-

rons at control and after stress conditions to clarify this

issue. Most likely the process of culturing neurons itself

resembles some kind of cellular stress which allowed us to

detect the phospho-forms of HspB1 and B5 in untreated

cultured hippocampal neurons and making the investiga-

tion of their target structures possible. Especially interest-

ing is our finding of phopsho-HspB1 and B5 along

filamentous structures of axons and dendrites as well as

synaptic sites suggesting protection of these very stress-

sensitive structures during pathological situations. The

specific synaptic target molecules remain to be identified in

future studies.

Acknowledgments We thank Bianca Mekle and Silke Zschemisch

for their excellent technical assistance and the International Graduate

School in Molecular Medicine (University of Ulm, Germany) of

which Thomas Schmidt is a member.

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