 |
||||
|
||||
|
||||
|
||||
|
||||
The Journal of Neuroscience, July 2, 2003, 23(13):5789-5798
Previous Article | Next Article
High Threshold for Induction of the Stress Response in Motor Neurons Is Associated with Failure to Activate HSF1
Zarah Batulan,1 Gayle A. Shinder,1 Sandra Minotti,1 Bei Ping He,2 Mohammad M. Doroudchi,1 Josephine Nalbantoglu,1 Michael J. Strong,2,3 andHeather D. Durham1 1Montreal Neurological Institute and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada H3A 2B4, 2Neurodegeneration Research Group, The John P. Robarts Research Institute, and 3Department of Clinical Neurological Sciences, The University of Western Ontario, London, Ontario, Canada N6A 5A5
Abstract
Top
Abstract
Introduction
Heat shock protein 70 (Hsp70) protects cultured motor neurons from the toxic effects of mutations in Cu/Zn-superoxide dismutase (SOD-1), which is responsible for a familial form of the disease, amyotrophic lateral sclerosis (ALS). Here, the endogenous heat shock response of motor neurons was investigated to determine whether a high threshold for activating this protective mechanism contributes to their vulnerability to stresses associated with ALS. When heat shocked, cultured motor neurons failed to express Hsp70 or transactivate a green fluorescent protein reporter gene driven by the Hsp70 promoter, although Hsp70 was induced in glial cells. No increase in Hsp70 occurred in motor neurons after exposure to excitotoxic glutamate or expression of mutant SOD-1 with a glycinealanine substitution at residue 93 (G93A), nor was Hsp70 increased in spinal cords of G93A SOD-1 transgenic mice or sporadic or familial ALS patients. In contrast, strong Hsp70 induction occurred in motor neurons with expression of a constitutively active form of heat shock transcription factor (HSF)-1 or when proteasome activity was sufficiently inhibited to induce accumulation of an alternative transcription factor HSF2. These results indicate that the high threshold for induction of the stress response in motor neurons stems from an impaired ability to activate the main heat shock–stress sensor, HSF1.
Key words: ALS; amyotrophic lateral sclerosis; heat shock; HSF1; HSF2; Hsp70; motor neuron; proteasome inhibition
Introduction
The heat shock response is an evolutionarily conserved, cytoprotective mechanism mediated by stress-induced transcription of genes encoding heat shock–stress proteins (HSP) (for review, see Morimoto et al., 1997). Families of stress-induced HSPs and constitutively expressed heat shock cognate proteins (HSC) facilitate nascent protein folding, refolding, or degradation of abnormally folded proteins, protein targeting, and cellular signaling (Gabai et al., 1997
HSPs can protect neural cells from various stresses including hyperthermia, ischemia, oxidative stress, and excitotoxicity (Lowenstein et al., 1991
Toxicity of mutant SOD-1 is attributable to a gain of function rather than a loss of the primary enzymatic function, dismutation of superoxide to hydrogen peroxide. One proposed mechanism of disease pathogenesis is that mutations in SOD-1 confer structural changes in the protein, resulting in altered solubility and aggregation (Deng et al., 1993
B-crystallin and an increase in chaperoning activity. Expression of mutant SOD-1 in motor neurons of dissociated murine spinal cord cultures resulted in aggregation of this protein into inclusions and loss of viability (Durham et al., 1997
Materials and Methods
Spinal cord cultures. Primary cultures of dissociated spinal cord and dorsal root ganglia (DRG) were prepared from embryonic day 13 CD1 mice and cultured on poly-D-lysine-coated glass coverslips as described previously (Roy et al., 1998Transgenic mice. The following lines were maintained in our animal facilities: B6SJL-TgN(SOD1–G93A)1Gur (expressing G93A mutant human SOD-1; hemizygotes become paralyzed in one or more limbs at
4 months of age), B6.Cg-TgN(SOD1–G93A) dl1Gur (hemizygotes develop clinical disease at
Human spinal cord sections. Archival paraffin-embedded cervical spinal cord sections were studied from control (two cases; neurologically normal), sporadic ALS (three cases), and autosomal dominant FALS (three cases, one SOD-1 A4V mutation and two without linkage to SOD-1).
Exposure of spinal cord DRG cultures to thermal and excitotoxic stress. For heat shock, spinal cord cultures were placed in minimum essential medium enriched with 5 gm of glucose without sodium bicarbonate, pH 7.2, heat shocked in a temperature controlled water bath, and recovered for variable periods at 37°C–5% CO2 before analysis of HSP expression. To induce excitotoxic stress, an appropriate volume of a 100 mM stock of glutamic acid (Sigma, Oakville, ON) was added.
Microinjection of plasmid vectors into cultured motor neurons. To study the effect of G93A mutant and wild-type human SOD-1 on Hsp70 induction in motor neurons, cDNAs were subcloned into pCEP4 and microinjected into motor neuron nuclei at 200 µg/ml in Tris-EDTA as described previously (Durham et al., 1997
Treatment with proteasomal inhibitors. Spinal cord cultures were transferred to CO2-equilibrated medium containing either MG132 (at final concentrations of 0.1, 1, 2.5, or 5 µM; Peptide Institute, Minoh, Japan) or DMSO carrier and were incubated overnight.
Antibodies. Primary antibodies were used against the following: SOD-1 (clone SD–G6, 1:300, Sigma; SOD-100, 1:100, Stressgen Biotechnologies, Victoria, BC), Hsp70 (SPA-810 for inducible Hsp70, 1:1000 for blots, 1:100 for immunolabeling of cultures and mouse tissue sections, 1:500 for human tissue sections, Stressgen Biotechnologies; W27 for both inducible and constitutive Hsp70, 1:100 Santa Cruz Biotechnology, Santa Cruz, CA), Hsc70 (K19, 1:1000 for blots; Santa Cruz Biotechnology), Hsp25 (SPA-801 for rodent Hsp25 with no cross reaction to Hsp27 primate isoform, 1:500/1000 for blots; Stressgen Biotechnologies), Hsp27 (M20, recognizes primate Hsp27 and rodent Hsp25, 1:100 for immunolabeling of cultures, 1:1000 for sections; Santa Cruz Biotechnology),
Sample preparation for Western blotting. Spinal cord cultures were harvested in lysis buffer containing the following: 20 mM HEPES, pH 7.5, 100 mM KCl, 5% glycerol, 0.1% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride (PMSF). After 30 min of incubation on ice, cell lysates were centrifuged at 12,700 x g for 15 min at 4°C. Lumbar spinal cords from mice were homogenized in 500 µl of the following lysis buffer: PBS, pH 8.0, containing 0.5% Nonidet-P40, 0.2% digitonin, and 0.23 mM PMSF. Cellular debris was pelleted by centrifugation at 15,400 x g for 15 min at 4°C. Supernatant fractions of culture or spinal cord homogenates were collected and protein concentrations were determined using the DC Protein Assay (Bio-Rad, Mississauga, Canada).
Western blotting. For spinal cord culture samples, 10 µg of protein were mixed with an equal volume of sample buffer (130 mM Tris, pH 6.8, 20% glycerol, 2% SDS, 10%
-mercaptoethanol, 0.08% bromophenol blue), boiled for 2 min, electrophoresed on 10% SDS–polyacrylamide gels, and transferred to Bio-Rad trans-blot transfer medium. Membranes were blocked for 1 hr with 5% milk in TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) and then incubated for 2 hr at room temperature (RT) or overnight at 4°C with primary antibody diluted in TBST containing 5% milk and 0.02% NaN3. After washing in TBST, blots were incubated in HRP-conjugated secondary antibody–5% milk–TBST for 2 hr at RT. Protein bands were visualized on x-ray film after incubation of the blots in the Renaissance Western blot chemiluminescence reagent (NEN, Boston, MA). Films were scanned using Microtek (Torrance, CA) Scan Wizard. Expression of HSPs was normalized to actin by quantitating band density on films using ImageQuant software (Amersham Biosciences, Baie D'Urfe, Quebec, Canada).
For lumbar spinal cord homogenates, 10 µg of protein were mixed with an equal volume of sample buffer, boiled for 5 min, electrophoresed on 12.5% SDS–polyacrylamide gels, and transferred to nitrocellulose. Membranes were blocked overnight at 4°C in TBST-milk. Incubations with primary and secondary antibodies were 1 hr at RT. Protein bands were detected as described for culture samples. Hsp25 and
Immunocytochemistry. Immunocytochemistry using fluorescent dye-conjugated secondary antibodies was performed as described previously (Roy et al., 1998
Immunohistochemistry. Lumbar spinal cords from transgenic mice were placed in Histo-Prep frozen tissue-embedding media (Fisher Scientific, Ottawa, Canada) and frozen in isopentane. Twenty micrometer cross sections were mounted onto glass slides and fixed in 3% paraformaldehyde for 30 min, permeabilized in 0.5% Nonidet-P40–PBS for 5 min, fixed in 3% paraformaldehyde for 2 min, and blocked for 3 hr at RT. To prevent nonspecific labeling with mouse monoclonal primary antibodies, FAB fragment anti-mouse IgG H+L (Jackson ImmunoResearch) was added for 30 min. Endogenous peroxidase activity was quenched by 5 min incubation with 2% H2O2. Primary antibodies were incubated overnight and detected using biotinylated secondary antibodies (1 hr at RT), Vectastain ABC peroxidase system, and 0.05% DAB. Sections were dehydrated in ethanol, dipped in xylene, and mounted in Permount (Fisher Scientific).
Human ALS spinal cord sections were deparaffinized using routine techniques. Antigen retrieval was performed at pH 10.0, and slides were incubated with 3% H2O2 for 5 min, 5% BSA for 20 min, and 0.1% trypsin–0.1 M PBS, pH 7.2, for 15 min at 37°C. Sections were labeled with either SPA-810 monoclonal antibody recognizing Hsp70 (1:500) or a goat antibody recognizing Hsp27 (M20; 1:1,000) overnight at 4°C and detected using HRP-conjugated secondary antibodies and the Elite Vectastain ABC peroxidase system (Vector Laboratories) with 0.05% DAB.
Results
Heat shock-induced Hsp70 expression in glial cells but not motor neuronsBecause induction of Hsp70 is a well characterized feature of the cellular response to hyperthermia, Hsp70 expression was used as the primary marker to investigate the heat shock–stress response in spinal cord cells. Primary spinal cord cultures were exposed for 1 hr to increasing temperatures (40–42°C) and then incubated for 2 hr at 37°C to allow for protein synthesis. Initially, HSP expression was examined by Western blotting to screen for thermally induced changes (Fig. 1). Inducible Hsp70 was minimal to nondetectable in cultures maintained at the 37°C control temperature, but increasing levels were observed in cultures exposed to temperatures above 40°C. When normalized to levels of actin, an approximate twofold increase in Hsp70 occurred at 42°C compared with 41°C. Expression of other HSPs, Hsp25, Hsc70, and
View larger version (62K):
[in this window]
[in a new window]
Figure 1. Heat shock induces Hsp70 in spinal cord cultures. Spinal cord cultures were subjected to increasing heat shock temperatures (40–42°C) for 1 hr, followed by 2 hr recovery at 37°C. Western blots were probed with antibodies to HSPs [Hsp70 (SPA-810), Hsp25 (SPA-801), Hsc70 (K19), and
To determine the cell types in spinal cord cultures that upregulated Hsp70 after heat shock, immunocytochemistry was performed (Fig. 2). After heat shock for 1 hr at 42°C and 2 hr of recovery at 37°C, numerous non-neuronal cells expressed Hsp70, whereas motor neurons did not (Fig. 2B). Double labeling of cultures with antibodies to Hsp70 and the high molecular weight neurofilament protein confirmed the lack of Hsp70 in large neurons with motor neuron morphology as well as DRG and other neurons in the culture at this heat shock condition (our unpublished data). Double labeling with antibody against the astrocytic marker GFAP confirmed that astrocytes expressed Hsp70 after heat shock (Fig. 2C,D).
View larger version (135K):
[in this window]
[in a new window]
Figure 2. Glial cells, but not motor neurons, upregulate Hsp70 after heat shock. A–D, Spinal cord cultures were subjected to 37°C control temperature (A) or 42°C heat shock (B–D) for 1 hr, followed by 2 hr recovery at 37°C. Hsp70 induction was assessed by immunocytochemistry using biotin-conjugated secondary antibodies and DAB as substrate (A, B) or by fluorescence-conjugated secondary antibodies (C). Heat-shocked spinal cord cultures were also double labeled with antibodies against GFAP (D) to verify that glial cells induced Hsp70. The arrow in A points to a motor neuron. Scale bars: A, B, 50 µm; C, D, 20 µm.
The absence of Hsp70 induction under these conditions of heat shock could be attributable to sufficient levels of constitutively expressed HSPs, such as Hsc70 or Hsp25, thereby obviating induction of the Hsp70 gene. Thus, different paradigms of heat shock were examined, including higher temperatures (43°C and 44°C), increasing durations of hyperthermia (up to 8 hr), and prolonged recovery times (up to 6 hr). Some of these extreme heat shock conditions caused motor neuron death; however, surviving motor neurons still failed to induce Hsp70, although expression was detected in other neurons, including DRG neurons, under these conditions (our unpublished data). Together, these results indicate that motor neurons have either a weak or nonexistent stress response after exposure to thermal stress.
Glutamate excitotoxicity did not induce Hsp70 in motor neurons
Glutamate excitotoxicity has been implicated in the pathogenesis of ALS (for review, see Cleveland and Rothstein, 2001
View larger version (130K):
[in this window]
[in a new window]
Figure 3. Motor neurons failed to express Hsp70 in response to glutamate. A–F, Spinal cord cultures were treated with 50µM glutamic acid (B, D,F) or vehicle (A, C,E) for 30 min and then assessed for Hsp70 induction by immunocytochemistry (SPA-810). Nitrotyrosine labeling demonstrated that glutamate treatment had an effect on motor neurons (E, F). Scale bar, 20 µm
Expression of G93A SOD-1 in motor neurons did not induce Hsp70
To determine whether expression of the disease-causing mutant protein G93A SOD-1 results in Hsp70 induction in cultured motor neurons, plasmid expression vectors encoding wildtype or G93A SOD-1 were microinjected into the nuclei of motor neurons in primary spinal cord cultures. In this model, motor neuron loss begins
View this table:
[in this window]
[in a new window]
Table 1. Expression of mutant SOD-1 fails to induce Hsp70 in cultured motor neurons
Hsp70 expression in motor neurons was also investigated in B6SJL-TgN(SOD1-G93A)1Gur transgenic mice. Similar to findings in the FALS culture model, Hsp70 was not detected in motor neurons in lumbar spinal cord sections by immunohistochemistry at 80, 92, or 115 d of age (Fig. 4A–C). Obvious loss of motor neurons in the ventral horn was apparent between 92 and 115 d as documented in previous studies of these mice (Dal Canto and Gurney, 1994
View larger version (103K):
[in this window]
[in a new window]
Figure 4. Expression of HSPs in lumbar spinal cord of G93A mutant SOD-1 transgenic mice. The 20 µm sections of lumbar spinal cord from 80, 92, and 115-d-old mice were labeled with antibodies to Hsp70 (SPA-810), Hsp25 (M20; recognizes primate Hsp27 and rodent Hsp25), GFAP, MAC-1, or
Reactive astrocytes in spinal cords of mutant SOD-1 transgenic mice upregulated Hsp25
By Western blotting, a significant increase in Hsp25 was observed in lumbar spinal cord homogenates from symptomatic G93A SOD-1 transgenic mice relative to age-matched nontransgenic and wild-type SOD-1 transgenic animals (Fig. 5A). The level of Hsp25 was also greater in symptomatic, relative to nonsymptomatic, G93A SOD-1 littermates killed at 115 d of age. Labeling of adjacent spinal cord sections with antibodies against Hsp25 and GFAP determined that Hsp25 was localized to reactive astrocytes (Fig. 4G–L). This pattern of Hsp25 labeling was distinct from the microglial marker MAC-1, which also increased with disease progression (Fig. 4M–O). The number of Hsp25-expressing astrocytes and intensity of labeling increased between 80 and 115 d of age, paralleling total levels of Hsp25 in spinal cord homogenates (Fig. 5A) and the time course of motor neuron loss, microglial infiltration, and clinical progression of disease (vide supra). At early stages, astrogliosis was restricted to the ventral gray matter (preclinical phase) and increased progressively until the end stage of disease (
View larger version (29K):
[in this window]
[in a new window]
Figure 5. Increase in Hsp25 with age and disease progression in G93A SOD-1 transgenic mice. A, Quantitation of Hsp25 on Western blots of spinal cord homogenates from wild-type and G93A SOD-1 mice. Ratios of Hsp25 to tubulin in samples from transgenic mice were normalized to ratios in matched samples of nontransgenic littermates. Note the threefold increase in Hsp25 in symptomatic G93A SOD-1 mice relative to those at 44 d of age. The mean Hsp25 level in the spinal cord of symptomatic animals was also higher than in samples from nonsymptomatic littermates. No inducible Hsp70 was detected on immunoblots (our unpublished data). Shown are means ± SD for three to nine animals per group. The asterisk indicates a significant difference from all other values; p 0.05 (t test; one-tailed; unequal variance). B, Quantitation of
The above findings in the B6SJL-TgN(SOD1-G93A)1Gur transgenic mouse line were confirmed in mice from the B6.Cg-TgN(SOD1-G93A)dl 1Gur [PDB] lineage which exhibits slower disease progression (our unpublished data).
Neither Hsp70 nor Hsp27 are upregulated in spinal cords from familial and sporadic ALS patients
To determine whether the findings in transgenic mice could be extended to ALS patients, HSP expression was assessed by immunohistochemistry in the cervical spinal cord obtained at autopsy from patients with sporadic ALS (three cases), FALS (three cases, one of which harbored the A4V SOD-1 mutation), and two control cases. Motor neurons in sections of the cervical spinal cord obtained from ALS patients (Fig. 6 D) were not labeled by anti-Hsp70. Glial immunoreactivity was occasionally observed in other parts of neuropil, indicating that this antibody can recognize Hsp70 when used for this procedure (our unpublished data).
View larger version (173K):
[in this window]
[in a new window]
Figure 6. HSP expression in cervical spinal cord of control, sporadic, and FALS (A4V SOD-1 mutation). Hsp27 was detected in spinal motor neurons and neuritic processes in the anterior horn of control (A), sporadic (B), and FALS (C) patients; the arrow in C points to a spinal motor neuron containing a Bunina body. Hsp70 immunolabeling was not observed in the cytoplasm of motor neurons of FALS (D) or sporadic ALS (our unpublished data). Hsp27 labeling in the neuropil (E) does not correspond to the pattern of GFAP immunoreactivity (F). Scale bars: A, 100 µm; B, 40 µm; C–F, 20 µm.
Consistent with observations in transgenic mice, constitutive expression of the human Hsp27 isoform, equivalent to mouse Hsp25, was observed in motor neurons of control (Fig. 6A), sporadic ALS (Fig. 6B), and FALS (Fig. 6C) cases. However, unlike the G93A SOD-1 transgenic mice, high expression of Hsp27 in reactive astrocytes was not detected in spinal cords from either sporadic or FALS patients (Fig. 6C). Hsp27 immunoreactivity appeared localized to motor neuronal cell bodies and neurites rather than coincident with areas of reactive gliosis. Analysis of the background neuropil indicated that Hsp27 labeling (Fig. 6E) did not arise from morphologically typical astrocytes immunolabeled with anti-GFAP (Fig. 6F).
Thus, motor neurons of ALS patients failed to express Hsp70, consistent with findings in primary culture and transgenic mouse experimental models. However, the increase in Hsp25 in reactive astrocytes in G93A SOD-1 transgenic mice was not replicated in the spinal cord of patients with familial or sporadic ALS.
Motor neurons expressed the major heat shock transcription factors HSF1 and HSF2
To determine whether the failure of motor neurons to induce Hsp70 after heat shock, glutamate, or mutant SOD-1 expression was attributable to lack of HSF, expression of the two major mammalian HSFs, HSF1 and HSF2, was assessed in spinal cord cultures. HSF1 primarily controls transcriptional activation of HSP genes in response to environmental stress by binding to HSEs on HSP promoters as trimers and undergoing hyperphosphorylation (Morimoto, 1998
In untreated spinal cord cultures, motor neurons and other cells were labeled by a monoclonal antibody to HSF1, which preferentially labeled nuclei (Fig. 7A). HSF2 was not detected in untreated cultures but accumulated in the nuclei of motor neurons and other spinal cord cells after overnight incubation with proteasome inhibitor MG132 (0.1–1 µM) (Fig. 7B2) as reported previously in cell lines (Mathew et al., 1998
View larger version (91K):
[in this window]
[in a new window]
Figure 7. Motor neurons express the heat shock transcription factors HSF1 and HSF2. A, Motor neurons and other cells in primary spinal cord cultures were labeled by a monoclonal antibody to HSF1 (SPA-950). B, Overnight treatment with the proteasomal inhibitor MG132 (0.1 µM) caused HSF2 to accumulate and translocate into the nuclei of spinal cord cells, including motor neurons (1,2).In motor neurons and other cells treated with MG132 (1µM), strong Hsp70 (SPA-810) expression was also detected by immunocytochemistry using DAB as substrate (3,4). Arrows indicate motor neurons. Scale bar, 30 µm.
Activation of HSF1 caused strong induction of markers of the heat shock–stress response in motor neurons
To address the possibility that motor neurons do not have sufficient amounts of HSF1 to mount a heat shock response, HSF1wt was overexpressed in motor neurons, and Hsp70 induction was assessed after heat stress. Motor neurons were microinjected with a plasmid-encoding HSF1wt, allowed to recover for 48 hr, and incubated at 42°C or 37°C for 1 hr and then 2 hr at 37°C. These conditions of heat shock were the same as those used in experiments shown in Figure 2, but again, no increase in Hsp70 expression was detected before or after heat shock, despite clear overexpression of HSF1wt in motor neurons (Fig. 8A1,5). In contrast, robust Hsp70 expression was observed at 37°C in motor neurons injected with plasmid encoding a constitutively activated form of HSF1 (HSF1(+)) (Fig. 8A3,7) but not an inactivatable form (HSF1(-)) (Fig. 8A4,8). HSF1(+) has a deletion of amino acids 202–316, which changes the conformation of the protein rendering HSF1 constitutively active and eliminating the requirement for phosphorylation of serine residues in this region. HSF1(-) has a deletion of amino acids 453–523 spanning the transcriptional activation domain. Hsp70 induction in motor neurons injected with HSF1(+) was consistently intense (Fig. 8A7, B, top,
). In the occasional Hsp70-immunopositive motor neuron expressing HSF1wt or HSF1(-), labeling was of relatively low intensity and frequently localized to the nucleus (Fig. 8B, middle,
). Not only did HSF1(+) cause stronger expression of Hsp70 (Fig. 8A7), it also caused a significant increase in the number of cells that induced Hsp70 (Fig. 8B).
View larger version (51K):
[in this window]
[in a new window]
Figure 8. Expression of activated HSF1, but not HSF1 wt, by gene transfer induces HSP70 expression in motor neurons. A, B, Motor neurons were microinjected with plasmid expression vectors encoding HSF1 wt, constitutively activated HSF1 (HSF1 (+)), or nonactivatable HSF1 (HSF1 (-)). After 48 hr, motor neurons injected with HSF1 wt were heat shocked (42°C; 1 hr) or subjected to control conditions (37°C; 1 hr) and recovered at 37°C for 2 hr. All cultures were double labeled with antibodies against HSF1 and Hsp70. A, Although motor neurons expressed HSF1 wt (1,2) at levels comparable with HSF1 (+) (3), only HSF1 (+) induced Hsp70 (5–7). B, Percentage of motor neurons coexpressing HSF1 and Hsp70 (n 4 cultures per group). Intense Hsp70 immunolabeling (
Similar results were obtained using a GFP reporter gene driven by the human Hsp70 promoter (pEGFP-HSE-GFP). Motor neurons were coinjected with this vector along with the red fluorescent marker, 70 kDa dextran TMR. The following day, motor neurons were imaged for TMR and GFP fluorescence (Fig. 8C) before heat shock (42°C, 1 hr) and (in the same cells) after 2, 10, and 24 hr recovery at 37°C. GFP expression was never observed in motor neurons at any of the recovery times (Fig. 8C4,5). However, when HSF1(+) vector was coinjected with pEGFP-HSE-GFP, strong induction of GFP was observed in motor neurons (Fig. 8C6). Thus, even with a high level of expression of wtHSF1, thermal stress failed to activate heat shock gene transcription, but bypassing the normal mechanisms of transcriptional activation resulted in expression of two different proteins under the control of the Hsp70 promoter.
Discussion
The results demonstrate that motor neurons have a high threshold for induction of the heat shock response. Although glia in primary spinal cord cultures upregulated the major stress-inducible protein Hsp70, after heat shock, motor neurons failed to express Hsp70 when subjected to hyperthermia, glutamate excitotoxicity, or expression of mutant SOD-1. Also, cultured motor neurons microinjected with a GFP reporter gene driven by the Hsp70 promoter failed to induce GFP after heat shock. No induction of Hsp70 was observed in spinal motor neurons in two lines of G93A mutant SOD-1 transgenic mice or in patients with ALS.Because different stressors can induce expression of different HSPs, Hsp25 and
Neurons, in general, show a higher threshold for induction of the stress response than astrocytes; yet, evidence points to motor neurons having a particularly high threshold for induction of Hsp70. DRG sensory neurons and other neurons in spinal cord–DRG culture expressed Hsp70 after heat shock at temperatures >42°C but motor neurons did not (our unpublished data). Other studies have shown that spinal motor neurons in situ failed to induce Hsp70 after heat shock (Manzerra and Brown, 1992
Exposure of motor neurons to hyperthermia would be expected to activate heat shock gene expression primarily through HSF1 (Morimoto, 1998
The failure of hippocampal neurons to induce Hsp70 after heat shock was attributed to a lack of HSF1 (Marcuccilli et al., 1996
Previous studies had implicated faster disassembly of hyperphosphorylated HSF1 trimers in the failure of Scrapie to induce Hsp70 in ScN2a neuroblastoma cells (Winklhofer et al., 2001
In addition to Hsp70, HSF1(+) induced expression of a GFP reporter under the control of the Hsp70 promoter. This result and the increase in Hsp70 after HSF1(+) gene transfer indicate that once the HSF–HSE complex was activated, transcription and translation proceeded.
The most likely cause of the failure of motor neurons to induce a stress response involves alterations in the signaling pathways that regulate HSF1 activation. HSF1(+) has a deletion spanning amino acids 202–316, a region harboring numerous serine residues, the phosphorylation states of which regulate the transactivational competence of HSF1 by altering its conformation (Zuo et al., 1995
In contrast to heat shock, glutamate excitotoxicity, and expression of mutant SOD-1, peptide inhibitors of proteasome activity induced robust expression of Hsp70 in motor neurons concomitant with accumulation and nuclear localization of the transcription factor HSF2. This result is consistent with the role for HSF2 in HSP gene transcription (Mathew et al., 1998
In conclusion, our results indicate that the impaired ability of motor neurons to mount a heat shock–stress response occurs at the level of activation of HSF1, the major cellular stress sensor. This feature, in addition to other factors, such as neurofilament organization, high level of glutamatergic input, expression of calcium-permeable AMPA receptors, and low levels of free radical scavengers and cytosolic calcium-binding proteins (for review, see Roy et al., 1998
Footnotes
Received Mar. 3, 2003; revised Apr. 14, 2003; accepted Apr. 21, 2003.This research was supported by the Canadian Institutes for Health Research, the ALS Society of Canada, and the ALS Association. We thank Dr. R. I. Morimoto for helpful discussion, the HSF2 antibody, and HSE–GFP reporter construct, Dr. R. Voellmy for the HSF1 constructs, Dr. S. David for the MAC-1 antibody, Emily Tam for assistance with MG132 experiments, and Dr. P. McPherson for assistance with GFP imaging.
Correspondence should be addressed to Dr. Heather D. Durham, Montreal Neurological Institute, 3801 University Street, Montreal, Quebec, Canada H3A 2B4. E-mail: heather.durham{at}mcgill.ca.
Copyright © 2003 Society for Neuroscience 0270-6474/03/235789-10$15.00/0
References
Bechtold DA, Rush SJ, Brown IR (2000) Localization of the heat-shock protein Hsp70 to the synapse following hyperthermic stress in the brain. J Neurochem 74: 641-646.[Medline]
Bence NF, Sampat RM, Kopito RR (2001) Impairment of the ubiquitin-proteasome system by protein aggregation. Science 292: 1552-1555.
[Abstract/Free Full Text] Benn SC, Perrelet D, Kato AC, Scholz J, Decosterd I, Mannion RJ, Bakowska JC, Woolf CJ (2002) Hsp27 upregulation and phosphorylation is required for injured sensory and motor neuron survival. Neuron 36: 45-56.[Web of Science][Medline]
Brown IR, Rush SJ (1999) Cellular localization of the heat shock transcription factors HSF1 and HSF2 in the rat brain during postnatal development and following hyperthermia. Brain Res 821: 333-340.[Medline]
Bruening W, Roy J, Giasson B, Figlewicz DA, Mushynski WE, Durham HD (1999) Up-regulation of protein chaperones preserves viability of cells expressing toxic Cu/Zn-superoxide dismutase mutants associated with amyotrophic lateral sclerosis. J Neurochem 72: 693-699.[Web of Science][Medline]
Bruijn LI, Becher MW, Lee MK, Anderson KL, Jenkins NA, Copeland NG, Sisodia SS, Rothstein JD, Borchelt DR, Price DL, Cleveland DW (1997) ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions. Neuron 18: 327-338.[Web of Science][Medline]
Chiu AY, Zhai P, Dal Canto MC, Peters TM, Kwon YW, Prattis SM, Gurney ME (1995) Age-dependent penetrance of disease in a transgenic mouse model of familial amyotrophic lateral sclerosis. Mol Cell Neurosci 6: 349-362.[Web of Science][Medline]
Chu B, Soncin F, Price BD, Stevenson MA, Calderwood SK (1996) Sequential phosphorylation by mitogen-activated protein kinase and glycogen synthase kinase 3 represses transcriptional activation by heat shock factor-1. J Biol Chem 271: 30847-30857.
[Abstract/Free Full Text] Cleveland DW, Rothstein JD (2001) From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS. Nat Rev Neurosci 2: 806-819.[Web of Science][Medline]
Costigan M, Mannion RJ, Kendall G, Lewis SE, Campagna JA, Coggeshall RE, Meridith-Middleton J, Tate S, Woolf CJ (1998) Heat shock protein 27: developmental regulation and expression after peripheral nerve injury. J Neurosci 18: 5891-5900.
[Abstract/Free Full Text] Cotto J, Fox S, Morimoto R (1997) HSF1 granules: a novel stress-induced nuclear compartment of human cells. J Cell Sci 110: 2925-2934.[Abstract]
Currie RW, Ellison JA, White RF, Feuerstein GZ, Wang X, Barone FC (2000) Benign focal ischemic preconditioning induces neuronal Hsp70 and prolonged astrogliosis with expression of Hsp27. Brain Res 863: 169-181.[Web of Science][Medline]
Dal Canto MC, Gurney ME (1994) Development of central nervous system pathology in a murine transgenic model of human amyotrophic lateral sclerosis. Am J Pathol 145: 1271-1279.[Abstract]
Dal Canto MC, Gurney ME (1995) Neuropathological changes in two lines of mice carrying a transgene for mutant human Cu, Zn SOD, and in mice overexpressing wild type human SOD: a model of familial amyotrophic lateral sclerosis (FALS). Brain Res 676: 25-40.[Web of Science][Medline]
Deng H-X, Hentati A, Tainer JA, Iqbal Z, Cayabyab A, Hung W-Y, Getzoff ED, Hu P, Herzfeldt B, Roos RP, Warner C, Deng G, Soriano E, Smyth C, Parge HE, Ahmed A, Roses AD, Hallewell RA, Pericak-Vance MA, Siddique T (1993) Amyotrophic lateral sclerosis and structural defects in Cu, Zn Superoxide dismutase. Science 261: 1047-1051.
[Abstract/Free Full Text] Doroudchi MM, Minotti S, Figlewicz DA, Durham HD (2001) Nitrotyrosination contributes minimally to toxicity of mutant SOD1 associated with ALS. NeuroReport 12: 1239-1243.[Medline]
Durham HD, Roy J, Dong L, Figlewicz DA (1997) Aggregation of mutant Cu/Zn superoxide dismutase proteins in a culture model of ALS. J Neuropathol Exp Neurol 56: 523-530.[Web of Science][Medline]
Fink SL, Chang LK, Ho DY, Sapolsky RM (1997) Defective herpes simplex virus vectors expressing the rat brain stress-inducible heat shock protein 72 protect cultured neurons from severe heat shock. J Neurochem 68: 961-969.[Web of Science][Medline]
Gabai VL, Meriin AB, Mosser DD, Caron AW, Rits S, Shifrin VI, Sherman MY (1997) Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance. J Biol Chem 272: 18033-18037.
[Abstract/Free Full Text] Goodson ML, Hong Y, Rogers R, Matunis MJ, Park-Sarge OK, Sarge KD (2001) Sumo-1 modification regulates the DNA binding activity of heat shock transcription factor 2, a promyelocytic leukemia nuclear body associated transcription factor. J Biol Chem 276: 18513-18518.
[Abstract/Free Full Text] Hall ED, Oostveen JA, Gurney ME (1998) Relationship of microglial and astrocytic activation to disease onset and progression in a transgenic model of familial ALS. Glia 23: 249-256.[Web of Science][Medline]
Hoffman EK, Wilcox HM, Scott RW, Siman R (1996) Proteasome inhibition enhances the stability of mouse Cu/Zn superoxide dismutase with mutations linked to familial amyotrophic lateral sclerosis. J Neurol Sci 139: 15-20.[Web of Science][Medline]
Holmberg CI, Hietakangas V, Mikhailov A, Rantanen JO, Kallio M, Meinander A, Hellman J, Morrice N, MacKintosh C, Morimoto RI, Eriksson JE, Sistonen L (2001) Phosphorylation of serine 230 promotes inducible transcriptional activity of heat shock factor 1. EMBO J 20: 3800-3810.[Web of Science][Medline]
Hyun DH, Lee MH, Halliwell B, Jenner P (2002) Proteasomal dysfunction induced by 4-hydroxy-2, 3-trans-nonenal, an end-product of lipid peroxidation: a mechanism contributing to neurodegeneration? J Neurochem 83: 360-370.[Web of Science][Medline]
Johnston JA, Dalton MJ, Gurney ME, Kopito RR (2000) Formation of high molecular weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis. Proc Natl Acad Sci USA 97: 12571-12576.
[Abstract/Free Full Text] Jolly C, Konecny L, Grady DL, Kutskova YA, Cotto JJ, Morimoto RI, Vourc'h C (2002) In vivo binding of active heat shock transcription factor 1 to human chromosome 9 heterochromatin during stress. J Cell Biol 156: 775-781.
[Abstract/Free Full Text] Kong J, Xu Z (1998) Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1. J Neurosci 18: 3241-3250.
[Abstract/Free Full Text] Krueger AM, Armstrong JN, Plumier J, Robertson HA, Currie RW (1999) Cell specific expression of Hsp70 in neurons and glia of the rat hippocampus after hyperthermia and kainic acid-induced seizure activity. Brain Res Mol Brain Res 71: 265-278.[Medline]
Lowenstein DH, Chan PH, Miles MF (1991) The stress protein response in cultured neurons: characterization and evidence for a protective role in excitotoxicity. Neuron 7: 1053-1060.[Web of Science][Medline]
Ludolph AC, Meyer T, Riepe MW (2000) The role of excitotoxicity in ALS–what is the evidence? J Neurol 247: 7-16.[Medline]
Manzerra P, Brown IR (1992) Expression of heat shock genes (hsp70) in the rabbit spinal cord: localization of constitutive and hyperthermia-inducible mRNA species. J Neurosci Res 31: 606-615.[Web of Science][Medline]
Marcuccilli CJ, Mathur SK, Morimoto RI, Miller RJ (1996) Regulatory differences in the stress response of hippocampal neurons and glial cells after heat shock. J Neurosci 16: 478-485.
[Abstract/Free Full Text] Mathew A, Mathur SK, Morimoto RI (1998) Heat shock response and protein degradation: regulation of HSF2 by the ubiquitin-proteasome pathway. Mol Cell Biol 18: 5091-5098.
[Abstract/Free Full Text] Mathur SK, Sistonen L, Brown IR, Murphy SP, Sarge KD, Morimoto RI (1994) Deficient induction of human hsp70 heat shock gene transcription in Y79 retinoblastoma cells despite activation of heat shock factor 1. Proc Natl Acad Sci USA 91: 8695-8699.
[Abstract/Free Full Text] Mautes AE, Noble LJ (2000) Co-induction of HSP70 and heme oxygenase-1 in macrophages and glia after spinal cord contusion in the rat. Brain Res 883: 233-237.[Medline]
Meriin AB, Yaglom JA, Gabai VL, Zon L, Ganiatsas S, Mosser DD, Zon L, Sherman MY (1999) Protein-damaging stresses activate c-Jun N-terminal kinase via inhibition of its dephosphorylation: a novel pathway controlled by HSP72. Mol Cell Biol 19: 2547-2555.
[Abstract/Free Full Text] Min JN, Han MY, Lee SS, Kim KJ, Park YM (2000) Regulation of rat heat shock factor 2 expression during the early organogenic phase of embryogenesis. Biochim Biophys Acta 1494: 256-262.[Medline]
Morimoto RI (1998) Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators [Rev]. Genes Dev 12: 3788-3796.
[Free Full Text] Morimoto RI, Santoro MG (1998) Stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection. Nat Biotechnol 16: 833-838.[Web of Science][Medline]
Morimoto RI, Kline MP, Bimston DN, Cotto JJ (1997) The heat-shock response: regulation and function of heat-shock proteins and molecular chaperones. Essays Biochem 32: 17-29.[Web of Science][Medline]
Pieri I, Cifuentes-Diaz C, Oudinet JP, Blondet B, Rieger F, Gonin S, Arrigo AP, Thomas Y (2001) Modulation of HSP25 expression during anterior horn motor neuron degeneration in the paralyse mouse mutant. J Neurosci Res 65: 247-253.[Medline]
Plumier JC, Hopkins DA, Robertson HA, Currie RW (1997) Constitutive expression of the 27-kDa heat shock protein (Hsp27) in sensory and motor neurons of the rat nervous system. J Comp Neurol 384: 409-428.[Web of Science][Medline]
Renkawek K, Voorter CE, Bosman GJ, van Workum FP, de Jong WW (1994) Expression of alpha B-crystallin in Alzheimer's disease. Acta Neuropathol (Berl) 87: 155-160.[Medline]
Rordorf G, Koroshetz WJ, Bonventre JV (1991) Heat shock protects cultured neurons from glutamate toxicity. Neuron 7: 1043-1051.[Web of Science][Medline]
Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O'Regan JP, Deng HX (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362: 59-62.[Medline]
Rothstein JD, Van Kammen M, Levey AI, Martin LJ, Kuncl RW (1995) Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Ann Neurol 38: 73-84.[Web of Science][Medline]
Roy J, Minotti S, Dong L, Figlewicz DA, Durham HD (1998) Glutamate potentiates the toxicity of mutant Cu/Zn-superoxide dismutase in motor neurons by postsynaptic calcium-dependent mechanisms. J Neurosci 18: 9673-9684.
[Abstract/Free Full Text] Sato K, Matsuki N (2002) A 72 kDa heat shock protein is protective against the selective vulnerability of CA1 neurons and is essential for the tolerance exhibited by CA3 neurons in the hippocampus. Neuroscience 109: 745-756.[Web of Science][Medline]
Satoh JI, Kim SU (1995) Differential expression of heat shock protein HSP27 in human neurons and glial cells in culture. J Neurosci Res 41: 805-818.[Medline]
Sherman MY, Goldberg AL (2001) Cellular defenses against unfolded proteins: a cell biologist thinks about neurodegenerative diseases. Neuron 29: 15-32.[Web of Science][Medline]
Shinder GA, Lacourse MC, Minotti S, Durham HD (2001) Mutant Cu/Zn-superoxide dismutase proteins have altered solubility and interact with heat shock/stress proteins in models of amyotrophic lateral sclerosis. J Biol Chem 276: 12791-12796.
[Abstract/Free Full Text] Shinohara H, Inaguma Y, Goto S, Inagaki T, Kato K (1993) Alpha B crystallin and HSP28 are enhanced in the cerebral cortex of patients with Alzheimer's disease. J Neurol Sci 119: 203-208.[Web of Science][Medline]
Snider BJ, Choi DW (1996) Heat stress reduces glutamate toxicity in cultured neurons without hsp70 expression. Brain Res 729: 273-276.[Medline]
Uney JB, Kew JN, Staley K, Tyers P, Sofroniew MV (1993) Transfection-mediated expression of human Hsp70i protects rat dorsal root ganglian neurones and glia from severe heat stress. FEBS Lett 334: 313-316.[Medline]
Urushitani M, Kurisu J, Tsukita K, Takahashi R (2002) Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis. J Neurochem 83: 1030-1042.[Web of Science][Medline]
Vleminckx V, Van Damme P, Goffin K, Delye H, Van Den BL, Robberecht W (2002) Upregulation of HSP27 in a transgenic model of ALS. J Neuropathol Exp Neurol 61: 968-974.[Web of Science][Medline]
Winklhofer KF, Reintjes A, Hoener MC, Voellmy R, Tatzelt J (2001) Geldanamycin restores a defective heat shock response in vivo. J Biol Chem 276: 45160-45167.
[Abstract/Free Full Text] Yenari MA, Fink SL, Sun GH, Chang LK, Patel MK, Kunis DM, Onley D, Ho DY, Sapolsky RM, Steinberg GK (1998) Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy. Ann Neurol 44: 584-591.[Web of Science][Medline]
Zuo J, Rungger D, Voellmy R (1995) Multiple layers of regulation of human heat shock transcription factor 1. Mol Cell Biol 15: 4319-4330.[Abstract]
This article has been cited by other articles:
C. L. Simpson, R. Lemmens, K. Miskiewicz, W. J. Broom, V. K. Hansen, P. W.J. van Vught, J. E. Landers, P. Sapp, L. Van Den Bosch, J. Knight, et al.
Variants of the elongator protein 3 (ELP3) gene are associated with motor neuron degeneration
Hum. Mol. Genet., February 1, 2009; 18(3): 472 - 481.
[Abstract] [Full Text] [PDF]
R. I. Morimoto
Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging
Genes & Dev., June 1, 2008; 22(11): 1427 - 1438.
[Abstract] [Full Text] [PDF]
E. V. Ilieva, V. Ayala, M. Jove, E. Dalfo, D. Cacabelos, M. Povedano, M. J. Bellmunt, I. Ferrer, R. Pamplona, and M. Portero-Otin
Oxidative and endoplasmic reticulum stress interplay in sporadic amyotrophic lateral sclerosis
Brain, December 1, 2007; 130(12): 3111 - 3123.
[Abstract] [Full Text] [PDF]
D. J. Gifondorwa, M. B. Robinson, C. D. Hayes, A. R. Taylor, D. M. Prevette, R. W. Oppenheim, J. Caress, and C. E. Milligan
Exogenous Delivery of Heat Shock Protein 70 Increases Lifespan in a Mouse Model of Amyotrophic Lateral Sclerosis
J. Neurosci., November 28, 2007; 27(48): 13173 - 13180.
[Abstract] [Full Text] [PDF]
D. M. Taylor, B. F. Gibbs, E. Kabashi, S. Minotti, H. D. Durham, and J. N. Agar
Tryptophan 32 Potentiates Aggregation and Cytotoxicity of a Copper/Zinc Superoxide Dismutase Mutant Associated with Familial Amyotrophic Lateral Sclerosis
J. Biol. Chem., June 1, 2007; 282(22): 16329 - 16335.
[Abstract] [Full Text] [PDF]
A. Falk, T. E. Karlsson, S. Kurdija, J. Frisen, and J. Zupicich
High-Throughput Identification of Genes Promoting Neuron Formation and Lineage Choice in Mouse Embryonic Stem Cells
Stem Cells, June 1, 2007; 25(6): 1539 - 1545.
[Abstract] [Full Text] [PDF]
G. MILLER and R. MITTLER
Could Heat Shock Transcription Factors Function as Hydrogen Peroxide Sensors in Plants?
Ann. Bot., August 1, 2006; 98(2): 279 - 288.
[Abstract] [Full Text] [PDF]
K. Phanvijhitsiri, M. W. Musch, M. J. Ropeleski, and E. B. Chang
Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells
Am J Physiol Cell Physiol, August 1, 2006; 291(2): C290 - C299.
[Abstract] [Full Text] [PDF]
B. J. Traynor, L. Bruijn, R. Conwit, F. Beal, G. O'Neill, S. C. Fagan, and M. E. Cudkowicz
Neuroprotective agents for clinical trials in ALS: A systematic assessment.
Neurology, July 11, 2006; 67(1): 20 - 27.
[Abstract] [Full Text] [PDF]
H.-Y. Shen, J.-C. He, Y. Wang, Q.-Y. Huang, and J.-F. Chen
Geldanamycin Induces Heat Shock Protein 70 and Protects against MPTP-induced Dopaminergic Neurotoxicity in Mice
J. Biol. Chem., December 2, 2005; 280(48): 39962 - 39969.
[Abstract] [Full Text] [PDF]
R. B. Choudry and M. E. Cudkowicz
Clinical Trials in Amyotrophic Lateral Sclerosis: The Tenuous Past and the Promising Future
J. Clin. Pharmacol., December 1, 2005; 45(12): 1334 - 1344.
[Abstract] [Full Text] [PDF]
J. Magrane, K. M. Rosen, R. C. Smith, K. Walsh, G. K. Gouras, and H. W. Querfurth
Intraneuronal {beta}-Amyloid Expression Downregulates the Akt Survival Pathway and Blunts the Stress Response
J. Neurosci., November 23, 2005; 25(47): 10960 - 10969.
[Abstract] [Full Text] [PDF]
M. Katsuno, C. Sang, H. Adachi, M. Minamiyama, M. Waza, F. Tanaka, M. Doyu, and G. Sobue
Pharmacological induction of heat-shock proteins alleviates polyglutamine-mediated motor neuron disease
PNAS, November 15, 2005; 102(46): 16801 - 16806.
[Abstract] [Full Text] [PDF]
A. Tiwari, Z. Xu, and L. J. Hayward
Aberrantly Increased Hydrophobicity Shared by Mutants of Cu,Zn-Superoxide Dismutase in Familial Amyotrophic Lateral Sclerosis
J. Biol. Chem., August 19, 2005; 280(33): 29771 - 29779.
[Abstract] [Full Text] [PDF]
H. Tummala, C. Jung, A. Tiwari, C. M. J. Higgins, L. J. Hayward, and Z. Xu
Inhibition of Chaperone Activity Is a Shared Property of Several Cu,Zn-Superoxide Dismutase Mutants That Cause Amyotrophic Lateral Sclerosis
J. Biol. Chem., May 6, 2005; 280(18): 17725 - 17731.
[Abstract] [Full Text] [PDF]
K. Fukada, F. Zhang, A. Vien, N. R. Cashman, and H. Zhu
Mitochondrial Proteomic Analysis of a Cell Line Model of Familial Amyotrophic Lateral Sclerosis
Mol. Cell. Proteomics, December 1, 2004; 3(12): 1211 - 1223.
[Abstract] [Full Text] [PDF]
This Article Abstract 
Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager 
Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (79) Citing Articles via Google Scholar Google Scholar Articles by Batulan, Z. Articles by Durham, H. D. Search for Related Content PubMed PubMed Citation Articles by Batulan, Z. Articles by Durham, H. D.
|
|
|
|
 |
|