Comparison of the heat shock response in cultured cortical neurons and astrocytes

transcript

Molecular Brain Research, 9 (1991) 39-45 39 Elsevier

BRESM 70247

R.N. Nishimura 1'2, B.E. Dwyer 1'2'4, K. Clegg 1, R. Cole 3 and J. de Vellis 3'4

1Veterans Administration Hospital, Sepulveda, CA 91343 (U.S.A.), 2Department of Neurology, UCLA, Los Angeles, CA 90024 (U.S.A.), 3UCLA Mental Retardation Center, Los Angeles, CA 90024 (U.S.A.), and ~UCLA Brain Research Institute, Los Angeles, CA 90024 (U.S.A.)

(Accepted 19 June 1990)

Key words: Heat shock; Heat shock protein; Tissue culture; Astroglia; Neuron

Cultured cortical neurons and astrocytes were compared for synthesis of the major inducible 68 kDa heat shock protein. By one- and two-dimensional electrophoresis the inducible 68 kDa protein appeared similar, but astrocytes produced greater amounts of the protein by 3 h than did neurons. Antibodies raised against HeLa cell inducible 72 and constitutive 73 kDa heat shock proteins were used to characterizes the inducible heat shock proteins in neurons and astrocytes. Unlike the gels, major differences were noted of the major inducible heat shock protein in astrocytes compared with neurons when analyzed by Western immunoblots. Heat shock protein 68 kDa mRNA induction in neurons was less than astrocytes suggesting an attenuated inducible 68 kDa heat shock protein response in neurons. The neuronal protein may be a different isoform of the 70 kDa family of heat shock proteins.

INTRODUCTION MATERIALS AND METHODS

Heat shock responses are found in bacteria to mam-

mals (see reviews in refs. 7, 17). In accordance with the

recommendat ion of Subjeck and Shyy, we will refer to

members of the 70 kDa family of heat shock proteins

(HSPs) as HSP 68, the major inducible heat shock

protein, and HSP 70, the constitutively expressed heat

shock protein. A major interest lies in the association of

heat tolerance with the induction and synthesis of these

proteins, especially the inducible 68 kDa protein. The

inability to synthesize these proteins, specifically the 68

kDa inducible protein, is associated with heat intolerance and cell death 6A7.

Primary cultured glial cells and cortical neurons syn-

thesized HSP 68 after heat stress 12'13. However, by in situ

hybridization very little inducible HSP 68 m R N A was

detected in the rabbit cerebral cortex although a marked

induction of HSP 70 m R N A was noted in cerebellar

granule cells 16. In that study oligodendroglia synthesized

the inducible HSP 68 m R N A adjacent to uninduced

neurons. This finding suggested that neurons and glial

cells have different thresholds for the major inducible

HSP induction or that forebrain cortical neurons did not

synthesize the HSP 68 under those conditions. The

present study was designed to investigate the differences

in the heat shock response of cortical neurons compared

to astrocytes. A preliminary report of these findings was

previously reported 13.

Cultures Purified cortical neurons were prepared by the method of Syapin

et ai. TM. Briefly cerebral hemispheres from 16 embryonic day fetal rats were collected and mechanically dissociated and cultured in poly-lysine-coated plastic dishes or flasks for 5 days in vitro (DIV). At that time cytosine arabinoside (1 x 105) was added to the feeding medium to rid the cultures of glial cells and other dividing cells. This was continued until the neurons were used in experiments at approximately 10 DIV. Purity was assessed at greater than 90% by immunostaining with neuron-specific enolase. Only 2-5% of cells immunostained with glial fibrillary acidic protein (GFAP), an astrocyte marker.

Purified primary astrocyte cultures were prepared as described by McCarthy and de Vellis 1° except that a mechanical rather than trypsin dissociation was utilized (Lu et al.9). Primary astrocyte cultures were trypsinized and dissociated as previously described ~2. These secondary cultures were grown for 14-28 DIV in serum- supplemented (10%) Dulbecco's modified Eagles' medium (DMEM)/Ham's F-12 (1:1) before use in experiments. Purity of cultures was assessed at greater than 98% by GFAP staining.

Heat stress and labeling Neurons and astrocytes were changed to DMEM/Ham's F-12

(1:1) warmed to 37 °C. Cultures were returned to a 37 °C incubator for 15 min then heat-stressed at 43 °C or 45 °C in a water bath and returned to a 37 °C incubator. Before radioactive labeling, cultures were changed to methionine-deficient medium (prewarmed to 37 °C), then labeled with trans-label 35S (ICN), 30/~Ci/ml, for 3 h at 37 °C. The incorporation of 35S was terminated by removing the medium. Cells were washed twice with ice-cold 0.1 M phosphate- buffered saline (PBS), pH 7.4, and protein was precipitated with 10% trichloroacetic acid (TCA) containing 0.1% methionine. Cultures were washed twice with PBS and the cells were scraped from the flasks and homogenized in 0.1% sodium dodecylsulfate (SDS). Aliquots were removed for measurement of acid-insoluble radioactivity and protein s .

Correspondence: R.N. Nishimura, VA Medical Center, 111N-1, 16111 Plummer St., Sepulveda, CA 91343, U.S.A.

Gel electrophoresis and immunostaining of cellular proteins One- and two-dimensional gels were prepared as previously

described ~ except that the sample preparation for the two- dimensional gels consisted of solubilization into Nonidet P-40 solution without precipitation in TCA. Western blots were prepared by the method of Towbin et al. 19 and immunostaining was performed as previously described ~2. Antibodies for the character- ization of the HSP 68 (C-92, N4-F3, 4, and polyclonal antibody) were kindly provided by W. Welch, University of California, San Francisco, CA. The monoclonal C-92 and polyclonal antibody were specific for the inducible HSP 72 in HeLa cells 21 . The C-92 antibody was previously shown to stain the inducible 68 kDa in cultured glial cells ~2. The N4-F3, 4 monoclonal antibody was specific for both the inducible HSP 72 and the constitutive HSP 73 (comparable to HSP 68 and HSP 70, respectively).

RNA preparation Sterile plasticware or acid-washed and baked glassware was used

for all operations. Buffers were treated with diethylpyrocarbonate (DEPC) (0.1%) and autoclaved or were prepared from DEPC- treated sterile water. RNA was prepared by the method of Chomczyski and Sacci 3. Rat astrocytes (1 x 107cells) were removed from flasks by trypsinization and suspended in 5 ml of ice-cold 0.1 M PBS and centrifuged at 800 rpm for 5 min to obtain a pellet. The pellet was extracted with GIT buffer (4 M guanidinium isothiocya- nate, 25 mM sodium citrate, pH 7.0, 0.5% sarkosyl, 0.1 M 2-mercaptoethanol). To 1 ml of GIT extract was added 0.1 ml of 2.5 M sodium acetate, pH 4.0, 1 ml of water saturated phenol, and 0.2 ml chloroform. This suspension was shaken vigorously and cooled on ice. Phase separation was achieved by centrifugation at 4 °C and the RNA-containing upper phase was removed and diluted with an equal volume of isopropyl alcohol. RNA was precipitated by storing the extract at -20 °C overnight and collected by centrifugation. The resultant RNA pellet was redissolved in 0.3 ml GIT buffer and RNA was reprecipitated at -20 °C with isopropyl alcohol. The pellet was washed 3 times with 80% ethanol and stored at -80 °C until used. RNA prepared this way was essentially free of contaminating protein (A260/280 > 1.70)

RNA hybridization RNA samples (4.4/~g) were diluted to 55/zl with sterile water.

Thirty-three/~1 of 20x SSC (3 M NaCI, 0.3 M sodium citrate, pH 7.0), and 22 #1 of 37% formaldehyde were added and the solution was heated to 65 °C for 15 min. Two/~g of RNA per well (50/d) was blotted under vacuum onto nitrocellulose membranes (pre-washed with sterile water and 20× SSC) in a BRL (Bethesda Research Lab.) blotting apparatus. Each well was washed twice with 50/~1 of 20× SSC and membranes were dried for 2 h under vacuum. The membranes were prehybridized at 55-60 °C for 2-3 h in a sealed plastic bag containing 50% formamide, 5× SSC, 50 mM sodium phosphate buffer, pH 7.0, 0.1 mg/mt salmon sperm DNA, 5x Denhardts' solution (50x Denhardts = 5 g polyvinylpyrrolidone, 5 g bovine serum albumin, 5 g Ficoll, diluted to 500 ml), and 0.2% SDS. 3zp-labeled HSP 68 riboprobe (see below) was injected (8 x 10 6 cpm in 0.5 ml prehybridization buffer per two blots) and the bag was resealed and incubated at 55-60 °C overnight. Membranes were sequentially washed with 2x SSC containing 0.1% SDS (3 times for 20 min at 37 °C), 2x SSC containing 1/~g/mi RNase A (once for 15 min at room temperature) and 2x SSC containing 0.1% SDS (twice). Membranes were air dried and exposed to Kodak X-OMAT AR-5 film for autoradiography.

HSP 68 probe preparation Cloned genomic DNA to the major inducible heat shock protein

(plasmid pH 2.3, Wu et al. 22) was kindly provided by Dr. Richard Morimoto, Northwestern University, Evanston, IL. The Hindlll- BamH1 fragment cut from the original vector was directionatly subcloned into plasmid pT7/T3-19, a dual promoter vector (BRL). 32P-labelled riboprobes were prepared using the Riboprobe Gemini System (Promega Biological Research Products, Madison, WI) and

recovered from the reaction mixture by column chromatography on NENSORB columns (New England Nuclear, Boston, MA). Unla- belled RNA complementary to the probe was also prepared and used as a standard ladder for RNA blot experiments.

Northern blot hybridization Ten /tg total RNA was evaporated to dryness by vacuum

centrifugation and dissolved in 8 /~1 of denaturing buffer (1 M glyoxal (deionized), 50% dimethyl sulfoxide (DMSO), 10 mM sodium phosphate, pH 7.0). The sample was heated at 50 °C for 1 h and cooled on ice. Two/~1 of a solution containing 10 mM sodium phosphate, pH 7.0, 50% glycerol and 0.4% Bromophenol blue was added to each sample. RNA was analyzed on 1.4% agarose gels run at 70 V in a 4 °C cold room on a Bio-Rad mini subcell apparatus. The running buffer was 10 mM sodium phosphate, pH 7.0, changed every 30 min. RNA standards (BRL) were run along with astrocyte samples. At the end of the run, standard lanes were cut from the gel, stained with acridine orange (30 /~l/ml in 10 mM sodium phosphate, pH 7.0), and destained in hot running water for 5 min. RNA was transferred to nitrocellulose membranes using 10x SSC as the blotting buffer. Membranes were hybridized to [3ep]HSP riboprobe as described above.

In situ hybridization In situ hybridization was performed according to published

procedures 1. Cells grown on glass coverslips were fixed in 4% buffered paraformaldehyde (0.1 M phosphate, pH 7.0) for 30 min at room temperature (R.T.). Coverslips were washed in 0.1 M phosphate buffered saline (PBS) 3 times for 5 min each with mild agitation. Cells were pretreated with 0.2 N HCI for 20 min at R.T. then washed twice in PBS. Digestion was performed with proteinase K (Sigma) (1/~g/ml in 100 ml Tris-HCl, 50 mM EDTA, pH 8.0, at 37 °C for 30 min). Cells were washed twice with PBS, rinsed with

distilled H20. Cells were acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0, for 10 min at R.T. Coverslips were washed 3 times in 2x SSC. Hybridization was performed in buffer (50% formamide, 1 mM EDTA, 2x SSC, yeast tRNA 500 #g/ml, 1 x Denhardts, 10% dextran sulfate, 0.1 mg/ml salmon sperm DNA, 100 mM dithiothreitol, 0.2% sodium dodecyl sulfate, 0.1 U/ml RNasin and 25% sterile water). The tritiated labeled riboprobe (prepared as per 32p-labeled probe) was added to the buffer and warmed to 65 °C for 5-10 min. Approximately 60 /~l of the prewarmed probe solution was added to each coverslip at 45 °C for 16-18 h in a humidified chamber. After hybridization, cells were washed 10-15 times with 4x SSC at R.T. RNasin digestion (RNasin A 100/~g/ml in 0.5 M NaCl, 1 mM EDTA, 10 mM Tris, pH 8.0) was performed at 37 °C for 30 min with agitation. Cells were washed in RNase buffer twice at 37 °C for 15 min, 10 times in 2x SSC at R.T. for 5 min each, 3 times in 0.1x SSC at 50 °C for 10 min, then two times in 0.1x SSC at R.T. for 10 min. Dehydration was performed in successive graded ethanol washes of 30, 50, 70 and 95%. Ammonium acetate (300 raM) was added to each wash. The slides were then air dried. Coverslips were dipped in NTB-2 Kodak emulsion and exposed for two months. Kodak D-19 developer was used to develop the exposed slides. Cells were stained in 0.01% Toluidine blue for 3 min, washed in water till dear, then air-dried and mounted on glass slides for viewing.

RESULTS

The heat shock response of rat cortical neurons was

compared with that of rat cortical astrocytes. After a

43 °C, 15 min heat shock, neurons did not synthesize the

major inducible 68 kDa heat shock protein (HPS 68) as

vigorously as astrocytes (Fig. 1). Because neurons might

have a higher threshold for HSP 68 induction, neurons

were subjected to a 45 °C, 10 min heat shock (Fig. 1).

However, under these conditions only minimal induction

of HSP 68 was observed in neurons as compared to

astrocytes.

HSP 68 synthesized by neurons and astrocytes mi-

grated similarly on two dimensional polyacrylamide gels

(Fig. 2). However, Western blot analysis revealed major

differences in antibody staining between neurons and

astrocytes. Since the gels were run with an equal number

of cpms per lane, the Western blots were standardized by

Fig. 2. Autoradiograph of two-dimensional polyacrylamide gels of astrocytes compared to neurons after 45 °C for 10 min heat shock and harvested after 3 h of recovery. Astrocytes are in a (control) and b (heat shock). Neurons are c (control) and d (heat shock). Each sample r e p r e s e n t s 10 6 counts.

Fig. 3. Western blot and immunostaining of total neuronal proteins after heat shock of 45 °C for 10 min and 3 h of recovery. Lanes 1 (control) and 2 (heat shock) were stained with the monoclonal antibody, C-92. Lanes la and 2a represent the same amounts of sample of lanes 1 and 2 except both were stained with a polyclonal antibody specific for the inducible 68 kDa HSP. Each lane represents equal numbers of cpms. Lanes 2 and 2a represent approximately three times more total cellular protein per lane than lanes 1 and la.

Fig. 5. Northern blot of mRNA derived from control and heat shocked neurons and astrocytes. Lanes a (control) and b (heat- shocked 45 °C for 10 min) represent total cellular RNA from astrocytes, and lanes c (control) and d (heat-shocked at 45 °C for 10 min) represent total cellular RNA from neurons. Lanes a and b represent 1 #g of RNA and c and d represent 5 pg of RNA. Lanes b and d demonstrate the hybridization of the 2.3 kb probe with the 2.5 and 2.8 kb messages. O represents the origin and E is the end of the run. RNA size (kb) is noted in the right margin.

Fig. 4. Western blot and immunostaining of total cellular neuronal and astrocyte protein. The top panel was immunostained using a monoclonal antibody (N4-F 3,4; specific for the inducible 68 kDa and constitutive 70 kDa HSPs). Lanes 1 and 2 are control and heat shocked (45 °C for 10 min and 3 h of recovery) astrocytes, respectively. Control neurons in lane 3 are compared with heat- shocked neurons lane 4 (heat-shocked 45 °C for 10 min and 3 h of recovery). Note that only the 68 kDa HSP stains in heat-shocked astrocytes while the 70 kDa HSP stains in all samples. The lower panel represents equal amounts of the same samples from the top panel excopt that they were stained with a polyclonal antibody specific for the inducible 68 kDa HSP. The astrocyte control stains heavily but less than the heat-shocked astrocytes. The heat-shocked neurons stains lightly for the 68 kDa protein. In addition the 70 kDa protein stains in the neurons. Each lane represents an equal number of cpm. Lanes 3, 3a, 4, and 4a represent more protein per lane than lanes 1, la, 2, and 2a.

us ing t he s a m e n u m b e r of c p m s p e r l ane . T h e m o n o c l o n a l

a n t i b o d y C-92 r ead i ly d e t e c t s H S P 68 in a s t rocy t e s ~2, bu t

e f fo r t s to i m m u n o s t a i n H S P 68 in n e u r o n s r e p e a t e d l y

w e r e n e g a t i v e (Fig. 3). U s e o f a n o t h e r m o n o c l o n a l

a n t i b o d y N4-F3 ,4 ( w h i c h r e c o g n i z e s b o t h t he 68 a n d 70

k D a H S P s ) a p p e a r e d to s t a in b o t h t h e cons t i t u t i ve ly

syn the s i zed 70 k D a H S P a n d t h e i n d u c i b l e H S P 68 of

a s t rocy te s bu t on ly t h e 70 k D a H S P of n e u r o n s (Fig. 4).

F ina l ly , i m m u n o s t a i n i n g w i th a p o l y c l o n a l a n t i b o d y spe-

1 2 3 4 5 6 7 8 9 10 11 Fig. 6. Slot blot using the 32p riboprobe for HSP 68 of neurons and astrocytes. Lanes 1 and 2 are control astrocytes and neurons respectively. Lanes 3 and 4 are heat shocked (43 °C for 15 rain and 3 h of recovery) astrocytes and neurons, respectively. Lanes 5 and 6 are heat-shocked (45 °C for 10 min and 3 h of recovery) astrocytes and neurons respectively. Each slot represents 2 ~g of total cellular RNA. HSP 68 standards are in slots 7-11 and represent 0.05, 0.1, 0.5, 1.0, and 2.5 #g of RNA, respectively.

Fig. 7. In situ hybridization of cortical neurons and astrocytes after heat shock. Astrocyte and neuron experiments were run separately and do not represent a direct comparison of astrocytes with neurons after heat shock. Fig. a and c are control astrocytes and neurons, respectively. Fig. b and d represent astrocytes and neurons 3 h after a heat shock of 45 °C for 10 min. Each photograph was 400x.

cific for the HSP 68 resulted in staining the constitutive and inducible 70 and 68 kDa HSPs, respectively, in neurons but only the inducible HSP 68 in astrocytes (Figs. 3 and 4). It is notable that HSP 68 was found constitutively in astrocytes using the polyclonal antibody. Recent studies show that astrocytes accumulate HSP 68 in serum-supplemented medium over 4 weeks in vitro by immunostaining Western blots with the C-92 antibody

(unpublished observation). Because of the decreased synthesis of an inducible 68

kDa HSP that was different by its reactivity with various antibodies, transcription of HSP 68 was studied in neurons and astrocytes. 32p-labelled riboprobe hybridized to two

faint RNA bands of about 2.5 and 2.8 kb in neurons (Fig. 5). Astrocytes showed pronounced hybridization for the same two bands (Fig. 5). This probe was shown to bind to two bands of astrocyte mRNA in previous work 5. RNA blot analysis revealed relatively poor induction of HSP 68 mRNA in neurons compared to astrocytes and suggested that weak induction of HSP 68 in neurons may reflect

transcriptional regulatory mechanisms (Fig. 6). The decreased synthesis of an inducible HSP 68 in

neurons was further studied by in situ hybridization. Fig. 7 showed hybridization of the inducible HSP 72 human probe to heat shocked astrocytes and neurons but not controls in separate experiments. Each photograph demonstrated representative areas of control and heat shocked neurons or astrocytes with comparable back- ground hybridization in control cells, respectively. This data demonstrated that the low mRNA levels in neurons were not from contaminating cells in the culture, in particular, astrocytes. The hybridization studies suggested that there was marked homology between HSP 68 mRNA from neurons and astrocytes. However, the antibody staining data raised the possibility that several proteins characterized as HSP 68 may exist in brain cells.

DISCUSSION

Cultured cortical neurons were much less capable of HSP 68 synthesis than cultured cortical astrocytes. One- and two-dimensional gel electrophoresis analysis suggested that the inducible HSP 68 in both neurons and astrocytes may be the same protein. However, Western blot analysis showed major differences between neurons

and astrocytes when the inducible HSP 68 was stained with mono- and polyclonal antibodies. Other laboratories have reported that the major inducible HSP exists as several isoforms 4,2°. The possibility that different HSP 68

isoforms exist in neurons and astrocytes, and the possi-

bility that they immunostain differently is currently under investigation. We observed several HSP 68 isoforms on

2-D gels when cell protein was precipitated with trichlo-

roacetic acid. When cell protein was solubilized directly into Nonidet P-40 only a single HSP 68 spot was

observed. Still it is possible that the HSP 68 in the neurons and astrocytes differ in a minor way. Both of the

monoclonal antibodies used in these studies are thought

to react with the carboxy-terminal end of the human inducible HSP 7211. It follows that the carboxy-terminal

end of the neuronal HSP 68 may be altered or modified

since it did not stain with either monoclonal antibody,

unlike the astrocytes. The nature of the difference is unknown and under investigation.

The R N A analysis suggested that sufficient homology

exists between human and rat HSP 68 m R N A for it to be

useful in detecting the induction of HSP 68 in neurons and astrocytes. Based on the low amount of [35S]

methionine incorporated into HSP 68 in heat-shocked neurons it is concluded that neurons exposed to the same

stress as astrocytes synthesized significantly less HSP 68.

The data showed that the attenuated synthesis of neu-

ronal heat shock protein correlated with the low level of specific m R N A induction. Despite the low level of

induction, in situ hybridization demonstrated that the

neurons synthesized HSP 68 m R N A after heat shock.

However, the R N A analysis did not allow conclusions on the presence of isoforms of HSP 68 or minor changes in the m R N A from each cell type.

The importance of these results remains to be seen.

Heat shock proteins presumably serve to protect cells after stress and promote cell recovery 17. Rat retina was

protected from light damage when the hyperthermia preceded the light exposure 2. Cells injected with anti-

bodies, which impaired the function of the 70 kDa HSPs and impaired the translocation of the HSPs into the

nucleus, failed to survive a heat shock that control cells survived 15. Johnston and Kucey showed impaired syn-

thesis of the inducible HSP 70 by transfecting cells with

HSP 70 promoter region DNA. The transfected cells

failed to synthesize the inducible HSP 70 and demonstrated increased thermosensitivity and subsequent cell

death. The present study of cerebral cortical neurons

demonstrated decreased synthesis of the inducible HSP 68 after heat shock, It follows that neurons would be

poorly tolerant of heat stress and perhaps other stresses since absence of the HSP 68 was associated with heat intolerance and cell death 6'15. Preliminary morphological

studies of cortical neurons support this hypothesis 14. The possible existence of cell specific HSP 68 isoforms which can be identified by immunostaining raises the possibility

that certain cells have a specific response to stress tailored to their phenotypes.

Acknowledgements. This research was supported by the Research Service of the Veterans Administration. The technical contribution of Karen Picard is gratefully acknowledged.

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Comparison of the heat shock response in cultured cortical neurons and astrocytes

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