ARTICLE |
Correspondence to: Jorge Tolivia, Dept. Morfología y Biología Celular, Facultad de Biología y Medicina, Universidad de Oviedo, Julián Clavería s/n, Oviedo 33006, Spain. E-mail: jtolivia@correo.uniovi.es
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Astrocytosis is a hallmark of damage that frequently occurs during aging in human brain. Astrocytes proliferate in elderly subjects, becoming hypertrophic and highly immunoreactive for glial fibrillary acidic protein (GFAP). These cells are one type that actively responds in the repair and reorganization of damage to the neural parenchyma and are a source of several peptides and growth factors. One of these biomolecules is apolipoprotein D (apo D), a member of the lipocalin family implicated in the transport of small hydrophobic molecules. Although the role of apo D is unknown, increments in brain apo D expression have been observed in association with aging and with some types of neuropathology. We have found an overexpression of apo D mRNA in reactive astrocytes by in situ hybridization in combination with immunohistochemistry for apo D in normal aged human brains. The number of double-labeled cells varied according to the cerebral area and the gliosis grade. The possible significance of this increased synthesis of apo D in reactive astrocytes is discussed in relation to the role of apo D in aging and in glial function.
(J Histochem Cytochem 51:12851290, 2003)
Key Words: apolipoprotein D, aging, astrocytes, human, hybridocytochemistry, immunocytochemistry
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DURING AGING, a number of morphological and physiological changes occur in the mammalian brain (
Apolipoprotein D (apo D) is a lipocalin superfamily member, which binds and transports small hydrophobic ligands. Apo D is a component of high-density plasma lipoproteins (HDLs) and it is also present in breast cyst fluid, lachrimal secretions, apocrine axillary secretions, cerebrospinal fluid (CSF), and urine (
The increased apo D expression, compared with apo E in some neuropathologies (
The aim of this work was to detect the location and synthesis of apo D in reactive astrocytes of human cerebral cortex during normal aging by double immunocytochemistry (ICC) and in situ hybridization (ISH). Apo D synthesis by astrocytes has not been previously shown in human brain.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human tissues were provided by the Pathologic Anatomy Department at General Hospital of Asturias. Twelve elderly subjects (between 82 and 88 years) and eight young subjects (between 30 and 44 years) without neurological, psychological, or neuropathological disorders were used in this study. Pieces of human cerebral cortex from different areas were fixed by immersion for about 4 hr in 4% paraformaldehyde in PBS (pH 7.4). After fixation, pieces were dehydrated, cleared in butylacetate, and embedded in paraffin. Double immunohybridocytochemical staining for apo D protein and its mRNA was carried out according to the following protocol. Sections were dehydrated, dried at room temperature for 5 min, digested with 2 µg/µl proteinase K in 0.001 M PBS at 37C for 5 min, and rinsed in 0.001 M PBS. Twenty µl of ISH solution (Sigma Hybridization Solution, H-7782; St Louis, MO) was applied on each section for 10 min. Twenty µl of digoxigenin-labeled RNA probe for apo D (obtained from Dr. Carlos López-Otin, Dpto. Bioquímica y Biología Molecular, Universidad de Oviedo; see
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In addition to confirming the presence of astrogliosis by its morphological changes, consisting of swelling of the nucleus that appears in a eccentric position and/or hypertrophy of the cytoplasm with vigorous processes, we performed a double IHC technique for GFAP (red) and apo D (blue) (Fig 1A1C). As expected, an increment of GFAP was found in aging (Fig 1A and Fig 1B). The number of apo D-immunoreactive astrocytes was greater in old brains (Fig 1B) with respect to young brains (Fig 1A). The reactive astrocytes showed a strong positive signal for GFAP and strong staining for apo D (Fig 1C). When the double immunohybridocytochemical staining for apo D was achieved on sections, the cells identified as reactive astroglial cells exhibited the presence of apo D protein (brown) and its mRNA (dark blue) (Fig 1D1H). On the other hand, we observed that increases of apo D immunoreactivity during aging appeared to be due to astrocytes, mainly reactive ones. The deposits of apo D appeared principally in the cell somata in perinuclear position at first, and they could occupy the cytoplasm and even the initial tree of processes when apo D staining increased (Fig 1D1H). By ISH, apo D mRNA was clearly observed in many astroglial cells in all cases. This signal seems to be more intense in the astrogliotic areas present in the cerebral cortex of aged subjects (Fig 1D1H). In addition to the apo D location, the increment was linked to reactive astrocytes (Fig 1D1H). The mRNA signaling appeared to be principally located in a perinuclear position, but some positive granules were also observed in other cytoplasm regions (Fig 1G and Fig 1H). Apo D mRNA signal principally adopted a crescent form near the nucleus, while the apo D protein showed a more uniform localization in cytoplasm (Fig 1F and Fig 1H).
|
We have found that there are increments of apo D protein and apo D mRNA in aged cerebral cortex, probably due to the increased number of astrocytes and the higher levels of expression of apo D in reactive glia.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several observations have shown that the aging process causes dramatic changes in many neuronal markers (
Astroglial apo D synthesis and secretion in cultured astrocytes were reported by
![]() |
Acknowledgments |
---|
Supported by grants from the Fondo de Investigación Sanitaria Española (PI020324 and 03-RED-C03/06).
Received for publication January 29, 2003; accepted May 27, 2003.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams JH, Duchen LW (2002) Greenfield's Neuropathology. 7th ed New York, Oxford University Press
Amenta F, Bongrani S, Cadel S, Ricci A, Valsecchi B, Zeng YC (1994) Neuroanatomy of aging brain. Ann NY Acad Sci 717:33-44[Abstract]
Beffert U, Danik M, Krzywkowsky P, Ramassamy C, Berrada F, Poirier J (1998) The neurobiology of apolipoproteins and their receptors in the CNS and Alzheimer's disease. Brain Res Rev 27:119-142[Medline]
Belloir B, Kovari E, SuriniDemiri M, Savioz A (2001) Altered apolipoprotein D expression in the brain of patients with Alzheimer disease. J Neurosci Res 64:61-69[Medline]
Berciano MT, Andres MA, Calle E, Lafarga M (1995) Age-induced hypertrophy of astrocytes in rat supraoptic nucleus: a cytological, morphometric and immunocytochemical study. Anat Record 243:129-144[Medline]
Blais Y, Sugimoto K, Carrière M-C, Haagensen DE, Labrie F, Simard J (1995) Interleukin-6 inhibits the potent stimulatory action of androgens, glucocorticoids and interleukin-1 on apolipoprotein D and gross cystic disease fluid protein-15 expression in human breast cancer cells. Int J Cancer 62:732-737[Medline]
Boyles JK, Notterpeck LM, Wardell MR, Patel SC (1990) Rat identification, characterization and tissue distribution of apolipoprotein D in rat. J Lipid Res 31:2057-2065[Abstract]
DandoyDron F, Guillo F, Benboudjema L, Deslis J-P, Lasmezas C, Dormaont D, Tovey MG, Dron M (1998) Gene expression in scrapie: cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts. J Biol Chem 273:7691-7697
Dickson DW (1997) The pathogenesis of senile plaques. J Neuropathol Exp Neurol 56:321-339[Medline]
DíezItza I, Vizoso F, Merino A, Sánchez LM, Tolivia J, Fernández J, Ruibal A et al. (1994) Expression and prognostic significance of apolipoprotein D in breast cancer. Am J Pathol 144:310-320[Abstract]
Eng LF (1988) Regulation of glial intermediate filaments in astrogliosis. In Norenberg MD, Hertz L, Schousboe A, eds. The Biochemical Pathology of Astrocytes. New York, Alan R. Liss, 79-90
Franz G, Reindl M, Patel SC, Beer I, Unterrischter I, Berger T, Schmutzhard E et al. (1999) Increased expression of apolipoprotein D following experimental traumatic brain injury. J Neurochem 73:1615-1625[Medline]
Ghirnikar RS, Lee YL, Eng LF (1998) Inflammation in traumatic brain injury: role of cytokines and chemokines. Neurochem Res 23:329-340[Medline]
Ghiso J, Matsubara E, Koudinov A, ChoiLeuba G, Kraftsik R (1994) Visual cortex in Alzheimer's disease: occurrence of neuronal death and glial proliferation, and correlation with pathological hallmarks. Neurobiol Aging 15:29-43[Medline]
Giulian D, Woodward J, Young DG, Krebs JF, Lachman LB (1988) Interleukin-1 injected into mammalian brain stimulates astrogliosis and neovascularization. J Neurosci 8:2485-2490[Abstract]
Hopkins SJ, Rothwell NJ (1995) Cytokines and the nervous system I: expression and recognition. Trends Neurosci 18:83-88[Medline]
Kalman J, McConathy W, Araoz C, Kasa P, Lacko AG (2000) Apolipoprotein D in the aging brain and in Alzheimer's dementia. Neurol Res 22:330-336[Medline]
Lan HU, Mu W, NikolicPaterson DJ, Atkins RC (1995) A novel, simple, reliable, and sensitive method for multiple immunoenzyme staining: use of microwave oven heating to block antibody crossreactivity and retrieve antigens. J Histochem Cytochem 43:97-102
Landis DMD (1994) The early reactions of non-neuronal cells to brain injury. Annu Rev Neurosci 17:133-151[Medline]
Laping NJ, Teter B, Nichols NR, Rozovsky I, Finch CE (1994) Glial fibrillary acid protein: regulation by hormones, cytokines and growth factors. Brain Pathol 4:259-275[Medline]
LópezBoado YS, Tolivia J, LópezOtín C (1994) Apolipoprotein D gene induction by retinoic acid is concomitant with growth arrest and cell differentiation in human breast cancer cells. J Biol Chem 269:26871-26878
Meda L, Baron P, Scarlato G (2001) Glial activation in Alzheimer's disease: the role of abeta and its associated proteins. Neurobiol Aging 6:885-893
Montpied P, de Bock F, LernerNatoli M, Bockaert J, Rondouin G (1999) Hippocampal alterations of apolipoprotein E and D mRNA levels in vivo and in vitro following kainate excitotoxicity. Epilepsy Res 35:135-146[Medline]
Morgan DG, May PC (1990) Age-related changes in synaptic neurochemistry. In Schneider EL, Rowe JW, eds. Handbook of the Biology of Aging. 3rd ed New York, Academic Press, 219-254
Nathanial JH, Nathanial DR (1981) The reactive astrocyte. Adv Cell Neurobiol 2:249-301
Navarro A, Astudillo A, del Valle E, González del Rey C, Tolivia J (2001) Immunohistochemical presence of apolipoprotein D in senile plaques. J Histotech 24:45-48
Navarro A, Tolivia J, Astudillo A, del Valle E (1998) Pattern of apolipoprotein D immunoreactivity in human brain. Neurosci Lett 254:17-20[Medline]
Nichols NR (1999) Glial responses to steroids as markers of brain aging. J Neurobiol 40:585-601[Medline]
Norenberg MD (1994) Astrocyte responses to CNS injury. J Neuropathol Exper Neurol 53:213-220[Medline]
Ong WY, He Y, Suresh S, Patel SC (1997) Differential expression of apolipoprotein D and apolipoprotein E in the kainic acid-lesioned rat hippocampus. Neuroscience 79:359-367[Medline]
Patel SC, Asotra K, Patel YC, McConathy WJ, Patel RC, Suresh S (1995) Astrocytes synthesize and secrete the lipophilic ligand carried apolipoprotein D. Neuroreport 6:653-657[Medline]
Podor TJ, Joshua P, Butcher M, Seiffert D, Loskutoff D, Gauldie J (1992) Accumulation of type 1 plasminogen activator inhibitor and vitronectin at sites of cellular necrosis and inflammation. Ann NY Acad Sci 667:173-177[Medline]
Provost PR, Weech PK, Tremblay NM, Marcel YL, Rassart É (1990) Molecular characterization and differential mRNA distribution of rabbit apolipoprotein D. J Lipid Res 3:2057-2065
Rassart E, Bedirian A, Do Carmo S, Guinard O, Sirois J, Terrisse L, Milne R (2000) Apolipoprotein D. Biochim Biophys Acta 1482:185-198[Medline]
Séguin D, Desforges M, Rassart É (1994) Molecular characterization and differential mRNA tissue distribution of mouse apolipoprotein D. Mol Brain Res 30:242-250
Simard J, Veilleux R, de Launoit Y, Haagensen DE, Labrie F (1991) Stimulation of apolipoprotein D secretion by steroids coincides with inhibition of cell proliferation in human LNCaP prostate cancer cells. Cancer Res 51:4336-4341[Abstract]
Smith KH, Lawn RM, Wilcox JN (1990) Cellular localization of apolipoprotein D and lecithin: cholesterol acyltransferase mRNA in rhesus monkey tissues by in situ hybridization. J Lipid Res 31:995-1004[Abstract]
Spreyer P, Schaal H, Kuhn G, Rothe T, Unterbeck A, Oleck K, Muller HW (1990) Regeneration-associated high levels expression of apolipoprotein D mRNA in endoneural fibroblast of peripheral nerve. EMBO J 9:2479-2484[Abstract]
Suresh S, Yan Z, Patel PC, Patel YC, Patel SC (1998) Cellular cholesterol storage in the Niemann-Pick disease type C mouse is associated with increased expression and defective processing of apolipoprotein D. J Neurochem 70:242-251[Medline]
Terrisse L, Poirier J, Bertrand P, Merched A, Visvikis S, Siest G, Milne R et al. (1998) Increased levels of apolipoprotein D in cerebrospinal fluid and hippocampus of Alzheimer's patients. J Neurochem 71:1643-1650[Medline]
Terrisse L, Séguin D, Bertrand P, Poirier J, Milne R, Rassart É (1999) Modulation of apolipoprotein D and apolipoprotein E expression in rat hippocampus after entorhinal cortex lesion. Mol Brain Res 70:26-35[Medline]
Thomas EA, Danielson PE, Nelson PA, Pribyl TM, Hilbush BS, Hasel KW, Sutcliffe JG (2001a) Clozapine increases apolipoprotein D expression in rodent brain: towards a mechanism for neuroleptic pharmacotherapy. J Neurochem 76:789-796[Medline]
Thomas EA, Dean B, Pavey G, Sutcliffe JG (2001b) Increased CNS levels of apolipoprotein D in schizophrenic and bipolar subjects: implications for the pathophysiology of psychiatric disorders. Proc Natl Acad Sci USA 98:4066-4071
Thomas EA, Sautkulis LN, Criado JR, Games D, Sutcliffe JG (2001c) Apolipoprotein D mRNA expression is elevated in PDAPP transgenic mice. J Neurochem 79:1059-1064[Medline]
Topp KS, Faddis BT, Vijayan VK (1989) Trauma-induced proliferation of astrocytes in the brains of young and aged rats. Glia 2:201-211[Medline]
Yong VW, Krekoski CA, Forsyth PA, Bell R, Edwards DR (1998) Matrix metalloproteinases and disease of the CNS. Trends Neurosci 21:74-80