Oligodendrocytes express different isoforms of ?-amyloid precursor protein in chemically defined cell culture conditions: In situ hybridization and immunocytochemical detection

Abstract

The expression of beta-amyloid precursor protein (betaAPP) by astrocytes is well documented; however, data concerning oligodendrocytes remain controversial. The main goal of the present study was to determine whether or not oligodendrocytes in culture constitutively express the different betaAPP isoforms. Oligodendrocytes were cultured in a chemically defined medium that avoids putative effects of unknown serum factors on oligodendrocyte development. We have employed immunocytochemistry and in situ hybridization with antibodies and synthetic oligonucleotides recognizing, respectively, specific protein epitopes and mRNA transcripts of rat betaAPP isoforms. Oligodendrocytes, in both mixed primary cultures in the presence of serum or in secondary cultures in defined medium, were clearly labeled by antibodies directed to different betaAPP sequences. Antibodies against the serine protease inhibitor domain of betaAPP, also strongly labelled oligodendrocytes. Immunohistochemistry and in situ hybridization were combined to determine precisely the expression of different isoforms of betaAPP. In situ hybridization revealed the presence in oligodendrocytes of mRNA transcripts coding not only for betaAPP695 but also for betaAPP770 and betaAPP751. This indicates that betaAPP immunoreactivity found in oligodendrocytes corresponds to constitutive expression of betaAPP. Oligodendrocyte cultured in chemically defined medium are able to express not only betaAPP695 but also betaAPP770, betaAPP751 isoforms containing the Kunitz protease inhibitor domain. Although the role of betaAPP in the pathological processes of Alzheimer's disease (AD) remains unknown, possible disturbances of betaAPP processing and/or synthesis in oligodendrocytes may account for some myelin disorders observed in AD and other senile dementias.

Oligodendrocytes are a Novel Source of Amyloid Peptide Generation

“…Apart from the fact that the commonly used Swedish mutant APP alters APP cleavage, it is difficult to draw direct comparisons between APP over-expressing cell lines and cultured primary CNS cells expressing physiological levels of APP. It is important to keep in mind, as well, that primary glial cells [19], including oligodendrocytes [21,23] express multiple APP isoforms in contradistinction to ectopic expression of a single APP isoform in a cell line.…”

“…These APP isoforms are expressed in CNS and in other tissues, although in different proportions, with APP 695 predominating in brain neurons and altered in AD [44]. Garcia-Ladona et al [21] demonstrated that antibodies directed against different sequences of APP labeled oligodendrocytes in mixed primary cultures and in secondary oligodendrocyte-enriched cultures (mature and immature) maintained in a chemically defined medium. In situ hybridisation revealed the presence in oligodendrocytes of mRNA transcripts coding not only for APP 695 but also for APP 751 andAPP 770 , isoforms containing the Kunitz protease inhibitor domain [45,46].…”

“…In situ hybridisation revealed the presence in oligodendrocytes of mRNA transcripts coding not only for APP 695 but also for APP 751 andAPP 770 , isoforms containing the Kunitz protease inhibitor domain [45,46]. Our findings agree with data demonstrating the presence of APP-positive oligodendrocytes in rodent brain [13,14,24] and in cell culture [21][22][23].…”

“…It remains a matter of debate whether or not glial cells or their own APP may play a role in AD [15][16][17]. The expression of APP in astrocytes is well-documented [18][19][20], although data for oligodendrocytes remain equivocal [18,[21][22][23]. Immunohistochemical studies in situ have reported the presence of APP-immunoreactive oligodendrocytes in brain white matter [14,24], while in situ hybridisation suggests the presence of APP mRNA transcripts in oligodendrocytes [11,14,[24][25][26].…”

Causes and Diagnosis of Alzheimers Disease: A Proteomics Approach

“…Cells in culture represent a convenient vehicle for both genetic and environmental manipulations, and a wide array of central nervous system cell types are available, including astrocytes (Fedoroff et al, 1984;Greenwood, 1991;Johnstone et al, 1986;Kennedy and Lisak, 1980), oligodendrocytes (Gebicke-(Harter et al, 1981;Kennedy and Lisak, 1980), ependymal cells (Laabich et al, 1992), microglia (Dobrenis, 1998), primary neuronal cultures (Wainer, et al, 1991) and cerebral vascular endothelium (Dorovini-Zis et al, 1991;Greenwood, 1991). Some of these have been used as convenient vehicles for studying AD pathology, including the effects of genetic manipulation (Czech et al, 1998;Ko et al, 2002), hypothesis testing (Garcia-Ladona et al, 1997;Harada and Sugimoto, 1999;Jordan et al, 1998;Michikawa and Yanagisawa, 1999) and evaluating drug effects (Akasofu et al, 2003;Moriguchi et al, 2003;Moriguchi et al, 2005;Seyb et al, 2006;Shaughnessy et al, 2004). Cell culture also allows flexibility in experimental design, such as co-culturing mixed cell populations to study their interactions and simulate organ systems.…”