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Heat-shock cognate 70 messenger RNA expression in postmortem human hippocampus: regional differences and age-related changes
ELSEVIER
Neuroscience Letters 196 (1995) 89-92
NHROSClENC[ LETTERS
Heat-shock cognate 70 messenger RNA expression in postmortem human hippocampus: regional differences and age-related changes H i d e o T o h g i a,*, K i m i a k i U t s u g i s a w a a, M a s a h i r o Y o s h i m u r a b, M u n e h i s a Y a m a g a t a a , Y u r i k o Nagane~ aDepartment of Neurology, lwate Medical University, lwate Medical University, 19-1, Uchimaru, Morioka, 020, Japan bDepartment of Neuropathology, Tokyo Metropolitan Medical Examiner's Office, Tokyo, Japan Received 29 May 1995; revised version received 14 July 1995; accepted 17 July 1995
Abstract
In situ hybridization of postmortem human brain tissue showed that constitutive heat-shock cognate 70 (hsc 70) mRNA was expressed in more than 50% of the pyramidal neurons in the hippocampal subfields. The ratio (%) of the hsc 70 mRNA-expressing neurons to the total neurons was significantly greater in CA3 and the hilus than in CA1 and CA2. The lower ratio in CA1 may be related to its vulnerability to various stresses. The ratio of hsc 70 mRNA-expressing neurons in CA1 was significantly greater in the older subjects than in the younger ones. This may reflect the up-regulated hsc 70 mRNA induction in response to a reduction in free hsc 70 because the binding of hsc 70 to aberrant proteins may be increased in aged persons.
Keywords:Heat-shock cognate 70 mRNA; In situ hybridization; Human hippocampus; Aging The hippocampus is mainly involved in memorization, and the damage to it iis a prominent feature (compared with other regions) in the brain of the aged and persons with Alzheimer type dementia (ATD). Furthermore, particular hippocampal sllbfields are selectively damaged after ischemia (CAD, in kainic acid-induced lesions (CA3) [20], and during aging (CA4 or hilus) [17,24]. This selective vulnerability of the hippocampus in stress- and age-related cell death has been ascribed to the metabolic characteristics of its neurons, synapses and the receptors on its pyramidal cells. Other potentially important mechanisms may involve differences in heat-shock protein induction. The strictly stress-inducible heat-shock protein 70 (hsp 70) and the constitutively expressed heat-shock cognate protein (hsc 70) are the members of the mammalian 70-kDa family of heat-shock proteins (hsp 70s). Both act as molecular chaperones [9] and contribute to cellular repair and to survival mechanisms. High levels of hsc 70 mRNA have been found in control animal brains, particularly in pyramidal neurons in the hippocampus and in cerebellar Purkinje neurons [6,18,21,23]. Previous reports on aging changes in hsp 70s in general have shown that hsp 70 synthesis and mRNA induc* Corresponding author, Fax: +81 196 54 9860.
tion after heat-shock decrease in old rats [4,21]; but, one report found that a greater amount of heat-shock proteins were synthesized in old than in young Drosophila flies [11]. Only a few studies done on humans have shown an increase in hsp 70s in Alzheimer's disease brains [13,14, 22]. To our knowledge, no study has been done on the age-related changes of hsp 70s in the human brain. We report the regional differences in and the effects of aging on hsc 70 mRNA expression in pyramidal neurons in postmortem human hippocampus. Brains were obtained at autopsy from 15 patients (3590 years; 12 men and 3 women) who had no history of brain disease. The subjects had been in good health except for some chronic diseases such as diabetes and hypertension which are common in old age. All died suddenly outside the medical institutions and underwent autopsy within 12 h of death. Their premortem physical and mental conditions, obtained through family and collateral interviews, were assessed by pathological examination. The brains were cut coronally at the level of the mammillary bodies. Brain tissues taken from the head of the hippocampus were fixed in 4% paraformaldehyde (in PBS treated with 0.1% diethylpyrocarbonate (DEPC))for 6 h, then rapidly frozen at -45°C in liquid nitrogen and stored at -70°C. Serial cryostat sections (15#m) were
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0 3 0 4 - 3 9 4 0 ( 9 5 ) 1 1 8 5 4 - M
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H. Tohgi et al. / Neuroscience Letters 196 (1995) 89-92
mounted on heat-dried silane slides. Sections were dehydrated in increasing concentrations of ethanol at 4°C, then dried, treated with prewarmed 40/~g/ml proteinase K in PBS for 5 min at 37°C and washed with PBS. They were then immersed in 4% paraformaldehyde in PBS, washed in PBS, again dehydrated in increasing concentrations of ethanol then dried. A specific oligonucleotide probe was synthesized in an automated DNA synthesizer. The probe was a 32 base single-stranded nucleotide with sequences of the antisense orientation. The nucleotide 1720-1751 (5'-TI'CTATGGTI'CTGACAAAGATGAAGGAAATTG-3')[8,19] was chosen for the human hsc 70-specific probe, which was biotin-labeled at the 3' end. Hybridization was done with buffer containing 50% formamide, 10% dextran sulfate, 4× SSC, 2× Denhardt's solution, and salmon sperm DNA 400/~g/ml. The antisense probes at final concentrations of 0.5/tg/ml were heated at 98°C for 10 min then quickly cooled on ice. The hybridization mixture (50/tl/slide) and 200mM of the vanadyl ribonucleoside complex (5/~l/slide) were pipetted onto the sections, which were then hybridized at 50°C for 12h. These slides were then washed with 0.2× SSC (3 min each 3 times at room temperature, and 30 min each twice at 50°C), and then treated with streptoavidinalkaline phosphatase conjugate. In situ signals were developed in NBT/BCIP solution. No specific hybridization was observed following ribonuclease or non-labelled oligonucleotide pretreatments. Nuclei were stained with methyl green. Immediately adjacent sections were stained with hematoxylin and eosin (HE). CA1, CA2, CA3 and the hilus were photographed at × 100 magnification. Neurons that were more intensely colored than the backgrounds were considered as positive for hsc 70 mRNA. Fig. la shows an example of hsc 70 mRNA-containing and -noncontaining neurons. The densities (number/mm 2) of the neurons considered to contain hsc 70 mRNA, and of the total number of neurons cut through the nucleus in the adjacent HE sections were counted, and the ratios (%) were calculated. Intergroup differences of the means were evaluated with Student's ttest. Correlations between age and histological parameters were tested with Spearman's rank correlation coefficients. P < 0.05 was considered significant. The hsc mRNA was expressed in more than 50% of the neurons in the hippocampal subfields. In the younger adults (< 65 years), the ratio of hsc mRNA-expressing neurons was significantly greater in CA3 (77 +_6%) and the hilus (68 + 8%) than in CA1 (59_+ 3%) and CA2 (52 +_4%) (P < 0.05) (Fig. 2). Similar significant regional differences were found in the aged (>65 years) (P < 0.05) (Fig. 2). In all the subfields studied, the ratio of the hsc 70 mRNA-expressing neurons was greater in the aged than in the younger adults, the difference being significant only for CA1 (P < 0.05) but approaching the significance level for CA3 (P = 0.06). The ratio of the hsc 70 mRNA-
expressing neurons was significantly correlated with age (r = 0.52, P = 0.05; Fig. 3). Figs. lb,c show the respective representative in situ hybridization histologies in younger and aged subjects. The ratio of hsc 70 mRNA-expressing neurons was not correlated with the interval between death and autopsy nor with the systemic pathological findings (data not shown).
Fig. 1. (a) Representative in situ hybridization histology showing neurons expressing (arrows) and not expressing (arrowheads) hse 70 mRNA x200 (bar 50/~m); (b,c) Representative in situ hybridization histology for hsc 70 mRNAin CA3 of a youngadult (b) in comparison with that in an aged person (c), x 100 (bar 100~m).
H. Tohgi et al. / Neuroscience Letters 196 (1995) 89-92
The possible influence of the premortem course on hsc 70 mRNA expression and its variation cannot be ruled out. It is unlikely, however, because the interval between the onset of the fatal episode and death was less than 24 h, and there was no evidence to suggest a premortem temperature elevation L>39°C which is required for hsc mRNA induction [19]. No previous studies [13,14,22] except one [12], found any correlation between the inducible hsp 70, hsx 70, or constitutive hsc 70 levels and premortem fever or coma in the brains of patients who died in hospitals, probably after a longer agonal state than our subjects experienced. The greater ratios of hsc 70 mRNA-expressing neurons found in the CA3 and the hilus than in CA1 and CA2 are in part similar to the results for rats in which the hsc 70 mRNA levels in CA3 were about twofold those in CA1 or CA4 [21]. The low hsc 70 mRNA levels in CA1 may partially be related to the area's known vulnerability to various stresses. Hsc 70, unlike hsp 70, is induced in response to mild stresses such as very brief periods of ischemia [16] and a low temperature elevation [19]. The low hsc 70 mRNA levels found in CA2, which is known to be stress-resistant, indicate that other protective mechanisms operate in individual hippocampal sectors. The mechanisms for the age-related increase in the ratio of hsc 70 mRNA-expressing neurons, particularly in CA1, are unknown. Increased hsx 70 mRNA expression in frontal cortex white matter in AD and other neurodegenerative diseases [14] and the increased synthesis of constitutive hsp 72/73 [22] and hsp 72 [13] in AD have been reported. In one s:mdy [13], hsp 72 was increased in neurons containing neurofibrillary tangles and in the neurites in senile plaque, whereas the constitutive glucose-regulated protein (grp) was increased in normal-
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CA1 CAg. CA3 Hilus *p<0.05, **p<0.001 vs. CA1 tp<0.05, t tp<0.001 vs. CA2 § p<0.05
Fig. 2. The ratio (%) of hsc 70 mRNA-expressing neurons to the total neurons in the hippocampal subfields of young adults (<65 years, n -- 6, open columns) and aged persons (>65 years, n = 9, hatched columns). * P < 0 . 0 5 , * * P < 0 . 0 0 1 corapared to CAI; t P < 0 . 0 5 , t t p < 0 . 0 0 1 compared to CA2.
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Age (years) Fig. 3. The ratio of the hsc 70 mRNA-expressing neurons in CAI as a function of age. Spearman's rank correlation coefficient (rs)= 0.52, P = 0.05.
appearing neurons in the AD brains. This suggests that hsp 72 is induced in order to facilitate the degradation of abnormal proteins, whereas grp protects neurons from AD-specific damage [13]. The increase in hsc 70 mRNA in the aged therefore may reflect the reduction in free hsc 70 which is a result of the increase in the amount of hsc 70 bound to the aberrant proteins that tend to accumulate with aging because the induction of hsp 70s is activated in a feedback manner by the reduction of their free forms [ 1,2]. If the brains of the aged are deficient in free hsc 70, unstable nascent proteins may unfold and accumulate [2], leading to general proteotoxicity [15]. Alternatively, the pronounced upregulation of hsc 70 transcription in the aged that is caused by unknown factors may simply prime the hippocampal neurons to translate additional protein when needed to protect neurons from stresses, a possibility that should be tested by determining hsc 70 protein levels. The ratio of hippocampal neuron loss with age is reported to be similar in all the subfields [7] or to be significant only for CA4 or the hilus [17,24]. Our findings therefore do not explain the mechanisms for age-related cell loss, but they may be indicative of different susceptibilities to stresses of the remaining neurons in the hippocampal subregions. In conclusion, the reduced hsc 70 mRNA expression seen in CA1 may, in part, explain its selective vulnerability to stresses. The increase with age of hsc 70 mRNA expression in CA1 may reflect upregulated transcription in response to a reduction in the amount of free hsc 70 available for the chaperoning of proteins. We thank Sakuyo Nagai for her technical assistance and Miharu Sawame for her secretarial help. This study was supported in part by grants-in-aid from the Ministry of Health and Welfare, Japan. [1] Baler, R., Welch, W.J. and Voellmy, R., Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp 70 as a potential autoregulatory factor, J. Cell. Biol., 117 (1992) 11511159.
92
H. Tohgi et al. /Neuroscience Letters 196 (1995) 89-92
[2] Beckmann, R.P., Lovett, M. and Welch, W.J., Examining the function and regulation of hsp 70 in cells subjected to metabolic stress, J. Cell. Biol., 117 (1992) 1137-1150. [3] Beckmann, R.P., Mizzen, L.A. and Welch, W.J., Interaction of hsp 70 with newly synthesized proteins: implications for protein folding and assembly, Science, 248 (1990) 850-854. [4] Blake, M.J., Fargnole, J., Gershon, D. and Holbroke, N.J., Concomitant decline in heat-induced hyperthermia and hsp 70 mRNA expression in aged rats, Am. J. Physiol., 260 (1991) R663-R667. [5] Brown, C.J., Martin, R.L., Hansen, W.J., Backman, R.P. and Welch, W.J., The constitutive and stress inducible forms of hsp 70 exhibit functional similarities and interact with one another in an ATP-dependent fashion, J. Cell. Biol., 120 (1993) 1101-1112. [6] Brown, I.R., Ruch, S.J., Expression of heat shock genes (hsp 70) in the mammalian brain: distinguishing constitutively expressed and hyperthermia-indueible mRNA species, J. Neurosci. Res., 25 (1990) 14-19. [7] Coleman, P.D., Flood, D.G., Neuron numbers and dendritic extent in normal aging and Alzheimer's disease, Neurobiol. Aging, 8 (1987) 521-545. [8] Dworniczak, B., Mirault M.-E., Structure and expression of a human gene coding for a 71 kd heat shock 'cognate' protein, Nucleic Acids Res., 15 (1987) 5181-5197. [9] Ellis, R.J., The molecular chaperone concept, Cell Biol. 1 (1990) 1-9. [10] Fargnoli, J., Kunisada, T., Fornace, A.J., Schneider, E.L. and Holbrook, N.J., Decreased expression of heat shock protein 70 mRNA and protein after heat treatment in cells of aged rats, Proc. Natl. Acad. Sci. USA, 87 (1990) 846-850. [11] Fleming, J.E., Walton, J.K., Dubitsky, R. and Bensch, K.G., Aging results in an unusual expression of Drosophila heat shock proteins, Proc. Natl. Acad. Sci. USA, 85 (1988) 4099-4103. [12] Guillemette, J.G., Wong, L., Crapper, McLachlan, D.R. and Lewis, P.N., Characterization of messenger RNA from the cerebral cortex of control and Alzheimer-afflicted brain, J. Neurochem., 47 (1986) 987-997. [13] Hamos, J.E., Oblas, B., Pulaski-Salo, D., Welch, W.J., Bole, D.G. and Drachman DA., Expression of heat shock proteins in Alzheimer's disease, Neurology, 41 (1991) 345-350.
[14] Harrison, P.J., Procter, A.W., Exworthy, T., Roberts, G.W., Najlerahim, A., Barton, A.J.L. and Pearson, R.C.A., Heat shock protein (hsx 70) mRNA expression in human brain: effects of neurodegenerative disease and agonal state, Neuropathol. Appl. Neurobiol., 19 (1993) 10-21. [15] Hightower, L.E., Heat shock stress proteins, chaperones, and proteotoxicity, Cell, 66 (1991) 191-197. [16] Kawagoe, J., Abe, K. and Kogure, K., Different thresholds of hsp 70 and hsc 70 heat shock mRNA induction in post-ischemic gerbil brain, Brain Res., 599 (1992) 197-203. [17] Mani, R.B., Lohr, J.B. and Jeste, D.V., Hippocampal pyramidal cells and aging in the human: a quantitative study of neuronal loss in sectors CAI to CA4, Exp. Neurol., 94 (1986) 29-40. [18] Manzerra, P., Ruch, S.J. and Brown, I.R., Temporal and spatial distribution of heat shock mRNA and protein (hsp 70) in the rabbit cerebellum in response to hyperthermia, J. Neurosci. Res., 36 (1993) 480-490. [19] Miller, E.K., Raese, J.D. and Morrison-Bogorad. M., Expression of heat shock protein 70 and heat shock cognate 70 messenger RNAs in rat cortex and cerebellum after heat shock or amphetamine treatment, J. Neurochem., 56 (1991 ) 2060-2071. [20] Onodera, H., Sato, G. and Kogure, K., Lesions to Schaffer collaterals prevent ischemic death of CA1 pyramidal cells, Neurosci. Lett., 68 (1986) 169-174. [21] Pardue, S., Groshan, K., Raese, J.D. and Morrison-Bogorad, M., Hsp 70 mRNA induction is reduced in neurons of aged rat hippocampus after thermal stress, Neurobiol. Aging, 13 (1992) 661672. [22] Perez, N., Sugar, J., Charya, S., Johnson, G., Merril, C., Bierer, L., Pet, D., Haroutunian, V. and Wallace, W., Increased synthesis and accumulation of heat shock 70 proteins in Alzheimer's disease, Mol. Brain Res., 11 (1991) 249-254. [23] Sprang, G.K., Brown, 1.R., Selective induction of a heat-shock gene in fibre tracts and cerebeltar neurons of the rabbit brain detected by in situ hybridization, Mol. Brain Res., 3 (1987) 89-93. [24] West, M.J. and Gurdersen, H.J.G., Unbiased stereological estimation of the number of neurons in the human hippocampus, J. Comp. Neurol., 296 (1990) 1-22.
Neuroscience Letters 196 (1995) 89-92
NHROSClENC[ LETTERS
Heat-shock cognate 70 messenger RNA expression in postmortem human hippocampus: regional differences and age-related changes H i d e o T o h g i a,*, K i m i a k i U t s u g i s a w a a, M a s a h i r o Y o s h i m u r a b, M u n e h i s a Y a m a g a t a a , Y u r i k o Nagane~ aDepartment of Neurology, lwate Medical University, lwate Medical University, 19-1, Uchimaru, Morioka, 020, Japan bDepartment of Neuropathology, Tokyo Metropolitan Medical Examiner's Office, Tokyo, Japan Received 29 May 1995; revised version received 14 July 1995; accepted 17 July 1995
Abstract
In situ hybridization of postmortem human brain tissue showed that constitutive heat-shock cognate 70 (hsc 70) mRNA was expressed in more than 50% of the pyramidal neurons in the hippocampal subfields. The ratio (%) of the hsc 70 mRNA-expressing neurons to the total neurons was significantly greater in CA3 and the hilus than in CA1 and CA2. The lower ratio in CA1 may be related to its vulnerability to various stresses. The ratio of hsc 70 mRNA-expressing neurons in CA1 was significantly greater in the older subjects than in the younger ones. This may reflect the up-regulated hsc 70 mRNA induction in response to a reduction in free hsc 70 because the binding of hsc 70 to aberrant proteins may be increased in aged persons.
Keywords:Heat-shock cognate 70 mRNA; In situ hybridization; Human hippocampus; Aging The hippocampus is mainly involved in memorization, and the damage to it iis a prominent feature (compared with other regions) in the brain of the aged and persons with Alzheimer type dementia (ATD). Furthermore, particular hippocampal sllbfields are selectively damaged after ischemia (CAD, in kainic acid-induced lesions (CA3) [20], and during aging (CA4 or hilus) [17,24]. This selective vulnerability of the hippocampus in stress- and age-related cell death has been ascribed to the metabolic characteristics of its neurons, synapses and the receptors on its pyramidal cells. Other potentially important mechanisms may involve differences in heat-shock protein induction. The strictly stress-inducible heat-shock protein 70 (hsp 70) and the constitutively expressed heat-shock cognate protein (hsc 70) are the members of the mammalian 70-kDa family of heat-shock proteins (hsp 70s). Both act as molecular chaperones [9] and contribute to cellular repair and to survival mechanisms. High levels of hsc 70 mRNA have been found in control animal brains, particularly in pyramidal neurons in the hippocampus and in cerebellar Purkinje neurons [6,18,21,23]. Previous reports on aging changes in hsp 70s in general have shown that hsp 70 synthesis and mRNA induc* Corresponding author, Fax: +81 196 54 9860.
tion after heat-shock decrease in old rats [4,21]; but, one report found that a greater amount of heat-shock proteins were synthesized in old than in young Drosophila flies [11]. Only a few studies done on humans have shown an increase in hsp 70s in Alzheimer's disease brains [13,14, 22]. To our knowledge, no study has been done on the age-related changes of hsp 70s in the human brain. We report the regional differences in and the effects of aging on hsc 70 mRNA expression in pyramidal neurons in postmortem human hippocampus. Brains were obtained at autopsy from 15 patients (3590 years; 12 men and 3 women) who had no history of brain disease. The subjects had been in good health except for some chronic diseases such as diabetes and hypertension which are common in old age. All died suddenly outside the medical institutions and underwent autopsy within 12 h of death. Their premortem physical and mental conditions, obtained through family and collateral interviews, were assessed by pathological examination. The brains were cut coronally at the level of the mammillary bodies. Brain tissues taken from the head of the hippocampus were fixed in 4% paraformaldehyde (in PBS treated with 0.1% diethylpyrocarbonate (DEPC))for 6 h, then rapidly frozen at -45°C in liquid nitrogen and stored at -70°C. Serial cryostat sections (15#m) were
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0 3 0 4 - 3 9 4 0 ( 9 5 ) 1 1 8 5 4 - M
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H. Tohgi et al. / Neuroscience Letters 196 (1995) 89-92
mounted on heat-dried silane slides. Sections were dehydrated in increasing concentrations of ethanol at 4°C, then dried, treated with prewarmed 40/~g/ml proteinase K in PBS for 5 min at 37°C and washed with PBS. They were then immersed in 4% paraformaldehyde in PBS, washed in PBS, again dehydrated in increasing concentrations of ethanol then dried. A specific oligonucleotide probe was synthesized in an automated DNA synthesizer. The probe was a 32 base single-stranded nucleotide with sequences of the antisense orientation. The nucleotide 1720-1751 (5'-TI'CTATGGTI'CTGACAAAGATGAAGGAAATTG-3')[8,19] was chosen for the human hsc 70-specific probe, which was biotin-labeled at the 3' end. Hybridization was done with buffer containing 50% formamide, 10% dextran sulfate, 4× SSC, 2× Denhardt's solution, and salmon sperm DNA 400/~g/ml. The antisense probes at final concentrations of 0.5/tg/ml were heated at 98°C for 10 min then quickly cooled on ice. The hybridization mixture (50/tl/slide) and 200mM of the vanadyl ribonucleoside complex (5/~l/slide) were pipetted onto the sections, which were then hybridized at 50°C for 12h. These slides were then washed with 0.2× SSC (3 min each 3 times at room temperature, and 30 min each twice at 50°C), and then treated with streptoavidinalkaline phosphatase conjugate. In situ signals were developed in NBT/BCIP solution. No specific hybridization was observed following ribonuclease or non-labelled oligonucleotide pretreatments. Nuclei were stained with methyl green. Immediately adjacent sections were stained with hematoxylin and eosin (HE). CA1, CA2, CA3 and the hilus were photographed at × 100 magnification. Neurons that were more intensely colored than the backgrounds were considered as positive for hsc 70 mRNA. Fig. la shows an example of hsc 70 mRNA-containing and -noncontaining neurons. The densities (number/mm 2) of the neurons considered to contain hsc 70 mRNA, and of the total number of neurons cut through the nucleus in the adjacent HE sections were counted, and the ratios (%) were calculated. Intergroup differences of the means were evaluated with Student's ttest. Correlations between age and histological parameters were tested with Spearman's rank correlation coefficients. P < 0.05 was considered significant. The hsc mRNA was expressed in more than 50% of the neurons in the hippocampal subfields. In the younger adults (< 65 years), the ratio of hsc mRNA-expressing neurons was significantly greater in CA3 (77 +_6%) and the hilus (68 + 8%) than in CA1 (59_+ 3%) and CA2 (52 +_4%) (P < 0.05) (Fig. 2). Similar significant regional differences were found in the aged (>65 years) (P < 0.05) (Fig. 2). In all the subfields studied, the ratio of the hsc 70 mRNA-expressing neurons was greater in the aged than in the younger adults, the difference being significant only for CA1 (P < 0.05) but approaching the significance level for CA3 (P = 0.06). The ratio of the hsc 70 mRNA-
expressing neurons was significantly correlated with age (r = 0.52, P = 0.05; Fig. 3). Figs. lb,c show the respective representative in situ hybridization histologies in younger and aged subjects. The ratio of hsc 70 mRNA-expressing neurons was not correlated with the interval between death and autopsy nor with the systemic pathological findings (data not shown).
Fig. 1. (a) Representative in situ hybridization histology showing neurons expressing (arrows) and not expressing (arrowheads) hse 70 mRNA x200 (bar 50/~m); (b,c) Representative in situ hybridization histology for hsc 70 mRNAin CA3 of a youngadult (b) in comparison with that in an aged person (c), x 100 (bar 100~m).
H. Tohgi et al. / Neuroscience Letters 196 (1995) 89-92
The possible influence of the premortem course on hsc 70 mRNA expression and its variation cannot be ruled out. It is unlikely, however, because the interval between the onset of the fatal episode and death was less than 24 h, and there was no evidence to suggest a premortem temperature elevation L>39°C which is required for hsc mRNA induction [19]. No previous studies [13,14,22] except one [12], found any correlation between the inducible hsp 70, hsx 70, or constitutive hsc 70 levels and premortem fever or coma in the brains of patients who died in hospitals, probably after a longer agonal state than our subjects experienced. The greater ratios of hsc 70 mRNA-expressing neurons found in the CA3 and the hilus than in CA1 and CA2 are in part similar to the results for rats in which the hsc 70 mRNA levels in CA3 were about twofold those in CA1 or CA4 [21]. The low hsc 70 mRNA levels in CA1 may partially be related to the area's known vulnerability to various stresses. Hsc 70, unlike hsp 70, is induced in response to mild stresses such as very brief periods of ischemia [16] and a low temperature elevation [19]. The low hsc 70 mRNA levels found in CA2, which is known to be stress-resistant, indicate that other protective mechanisms operate in individual hippocampal sectors. The mechanisms for the age-related increase in the ratio of hsc 70 mRNA-expressing neurons, particularly in CA1, are unknown. Increased hsx 70 mRNA expression in frontal cortex white matter in AD and other neurodegenerative diseases [14] and the increased synthesis of constitutive hsp 72/73 [22] and hsp 72 [13] in AD have been reported. In one s:mdy [13], hsp 72 was increased in neurons containing neurofibrillary tangles and in the neurites in senile plaque, whereas the constitutive glucose-regulated protein (grp) was increased in normal-
100~ zo~
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80-
[ ] < 65 years [ ] :> 65 years
t
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40
CA1 CAg. CA3 Hilus *p<0.05, **p<0.001 vs. CA1 tp<0.05, t tp<0.001 vs. CA2 § p<0.05
Fig. 2. The ratio (%) of hsc 70 mRNA-expressing neurons to the total neurons in the hippocampal subfields of young adults (<65 years, n -- 6, open columns) and aged persons (>65 years, n = 9, hatched columns). * P < 0 . 0 5 , * * P < 0 . 0 0 1 corapared to CAI; t P < 0 . 0 5 , t t p < 0 . 0 0 1 compared to CA2.
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Age (years) Fig. 3. The ratio of the hsc 70 mRNA-expressing neurons in CAI as a function of age. Spearman's rank correlation coefficient (rs)= 0.52, P = 0.05.
appearing neurons in the AD brains. This suggests that hsp 72 is induced in order to facilitate the degradation of abnormal proteins, whereas grp protects neurons from AD-specific damage [13]. The increase in hsc 70 mRNA in the aged therefore may reflect the reduction in free hsc 70 which is a result of the increase in the amount of hsc 70 bound to the aberrant proteins that tend to accumulate with aging because the induction of hsp 70s is activated in a feedback manner by the reduction of their free forms [ 1,2]. If the brains of the aged are deficient in free hsc 70, unstable nascent proteins may unfold and accumulate [2], leading to general proteotoxicity [15]. Alternatively, the pronounced upregulation of hsc 70 transcription in the aged that is caused by unknown factors may simply prime the hippocampal neurons to translate additional protein when needed to protect neurons from stresses, a possibility that should be tested by determining hsc 70 protein levels. The ratio of hippocampal neuron loss with age is reported to be similar in all the subfields [7] or to be significant only for CA4 or the hilus [17,24]. Our findings therefore do not explain the mechanisms for age-related cell loss, but they may be indicative of different susceptibilities to stresses of the remaining neurons in the hippocampal subregions. In conclusion, the reduced hsc 70 mRNA expression seen in CA1 may, in part, explain its selective vulnerability to stresses. The increase with age of hsc 70 mRNA expression in CA1 may reflect upregulated transcription in response to a reduction in the amount of free hsc 70 available for the chaperoning of proteins. We thank Sakuyo Nagai for her technical assistance and Miharu Sawame for her secretarial help. This study was supported in part by grants-in-aid from the Ministry of Health and Welfare, Japan. [1] Baler, R., Welch, W.J. and Voellmy, R., Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp 70 as a potential autoregulatory factor, J. Cell. Biol., 117 (1992) 11511159.
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