Expression of nicotinic receptors on primary cultures of rat astrocytes and up-regulation of the α7, α4 and β2 subunits in response to nanomolar concentrations of the β-amyloid peptide1–42
Introduction
Alzheimer's disease, the most common cause of dementia, is characterized by loss of memory, confusion and impairment of cognitive function. There is evidence that the common pathological features of involving deposition of β-amyloid peptide and cholinergic degeneration may be related and play an important role in the pathogenesis of AD (Nordberg, 2001).
Nicotinic acetylcholine receptors belong to the super-family of ligand-gated ion channels and participate in a number of important functions of the brain, including cognitive functions, e.g., learning and memory (Galzi and Changeux, 1995, Nordberg, 2001, Paterson and Nordberg, 2000). The genes encoding the α2–α10 and β2–β4 subunits of neuronal nAChR have been cloned and various combinations of different α and β subunits form different nAChR subtypes that exhibit distinct functions and distributions in the brain (Paterson and Nordberg, 2000). The α4, β2 and α7 subtypes appear to be the most common forms of nAChRs in the brain (Chen and Patrick, 1997, Flores et al., 1992, Peng et al., 1999). Although the expression of nAChRs on neurons is well characterized, expression of these receptors on non-neuronal cells in the nervous system remains to be elucidated.
Losses of nAChRs, as reflected in reductions in the number of ligand-binding sites and levels of subunit proteins have been observed in several regions of the brains of AD patients (Guan et al., 2000, Martin-Ruiz et al., 1999, Marutle et al., 1999, Wevers et al., 2000, Yu et al., 2003). Furthermore, Aβs have been found to suppress the expression of nAChRs (in terms of ligand-binding, protein and mRNA levels) by PC12 cells (Guan et al., 2001, Guan et al., 2003), suggesting that these receptors are important targets for the pathological effects of Aβs in connection with AD.
Interestingly, the level of the α subunit protein of nAChR on astrocytes in brains with sporadic AD is high (Teaktong et al., 2003). Furthermore, the level of the α7 subunit protein on astrocytes is elevated, while that of α7 and α4 on neurons is decreased in the brains of patients carrying the Swedish amyloid precursor protein (APP) 670/671 mutation (Yu et al., 2005). There are indications that the level of expression of α7 on cells demonstrating astrocytic profiles in the AD brain might be associated with the formation of amyloid plaques (Wevers et al., 1999). Thus, Aβ1–42-positive astrocytes within the molecular layer of the cortex of AD patients exhibit intense and specific immunostaining for the α7 subunit of nAChR (Nagele et al., 2003). However, little is currently known concerning the expression of the genes coding for the different subunits of nAChRs by cultured astrocytes or about the direct influence of Aβs on the nAChRs in these cells.
Astrocytes perform a number of key structural and physiological functions in the normal brain and are affected by the pathological processes underlying the development of AD (Nagele et al., 2003). These cells can be activated both by aggregated Aβ and by the intact cores of amyloid plaques isolated from AD brain (DeWitt et al., 1998). In addition, astrocytes spreading throughout the entorhinal cortex of AD patients gradually accumulate Aβ1–42-positive material (Nagele et al., 2003). Moreover, the astrocytes that accumulate around deposits of Aβs can degrade these peptides (Wyss-Coray et al., 2003). A recent report revealed that astrocytes can protect neurons from the neurotoxic effect of Aβs (Paradisi et al., 2004).
In the present investigation, we have characterized the expression of the α2, α3, α4, α7, β2 and β3 subunits of nAChR by rat astrocytes in primary culture at both the protein and mRNA levels, examined stimulation of the calcium channel in these cells by nAChR agonists or antagonists; and studied the direct influence of Aβs on their expression of nAChRs.
Section snippets
Materials
Goat polyclonal antibodies directed towards the α2 (SC1770), α3 (SC1771), α4 (SC1772), α7 (SC1447), β2 (SC1449) and β3 (SC9346) subunits of nAChRs, anti-goat IgG conjugated with horseradish peroxidase and Cruz Marker (protein size markers) (Santa Cruz Biotechnology Inc.); rabbit polyclonal antibody against glial fibrillary acidic protein (GFAP) and anti-rabbit IgG labeled with CY-3 (Vector Company, Sweden); the TOTALLY RNA isolation kit (Invitrogen); the cDNA synthesis kit (Access-RT-PCR
The purity of primary cultures of astrocytes isolated from rat brain
Staining of primary cell cultures originating from newborn rat brain using a specific antibody directed towards GFAP revealed that more than 95% of the cells were positive and could therefore be identified as astrocytes (Fig. 1).
Expression of nAChR subunit proteins and corresponding mRNA by astrocytes
The levels of mRNA species coding for the α2, α3, α4, α7, β2 and β3 subunits of nAChR in astrocytes from the cortex and hippocampus of newborn rats were measured by RT-PCR. The α4, α7, β2 and β3 mRNAs were detected in both of these regions of the brain (Fig. 2A–D);
Discussion
The expression of subunits of nAChRs at both mRNA and protein levels by primary cultures of astrocytes has been characterized here. To date, nine such subunits have been identified and cloned from human brain, i.e., α2–α7 and β2–β4 (Paterson and Nordberg, 2000). Expression of the genes encoding the α2–α4, α7 and β2 subunits in rat brain has been indicated by the ribonuclease protection assay (Zhang et al., 1998). In addition, in situ hybridization has revealed the presence of the α3–α7 and
Acknowledgements
This work was supported financially by grants from the Swedish Medical Research Council (project no 05817), the Loo and Hans Oysterman's Foundation, the Alzheimer Foundation in Sweden, the Karolinska Institute Foundation, the Foundation for Geriatric Diseases at Karolinska Institute, the Old Servants Foundation, the Gun and Bertil Stohne's Foundation and the Tercentenary Foundation in Bank of Sweden.
References (41)
- et al.
Selective nicotinic receptor consequences in APP(SWE) transgenic mice
Mol. Cell. Neurosci.
(2002) - et al.
Overexpression of superoxide dismutase 1 protects against beta-amyloid peptide toxicity: effect of estrogen and copper chelators
Neurochem. Int.
(2004) - et al.
The alpha-bungarotoxin-binding nicotinic acetylcholine receptor from rat brain contains only the alpha7 subunit
J. Biol. Chem.
(1997) - et al.
Astrocytes regulate microglial phagocytosis of senile plaque cores of Alzheimer's disease
Exp. Neurol.
(1998) - et al.
Beta-amyloid 25–35 inhibits glutamate uptake in cultured neurons and astrocytes: modulation of uptake as a survival mechanism
Neurobiol. Dis.
(2004) - et al.
Neuronal nicotinic receptors: molecular organization and regulations
Neuropharmacology
(1995) - et al.
Differential nicotinic acetylcholine receptor subunit expression in the human hippocampus
J. Chem. Neuroanat.
(2003) - et al.
Regional distribution of nicotinic receptor subunit mRNAs in human brain: comparison between Alzheimer and normal brain
Brain Res. Mol. Brain Res.
(1999) - et al.
Astrocytes accumulate A beta 42 and give rise to astrocytic amyloid plaques in Alzheimer disease brains
Brain. Res.
(2003) Nicotinic receptor abnormalities of Alzheimer's disease: therapeutic implications
Biol. Psychiatry
(2001)
Neuronal nicotinic receptors in the human brain
Prog. Neurobiol.
Inducible, heterologous expression of human alpha7-nicotinic acetylcholine receptors in a native nicotinic receptor-null human clonal line
Brain Res.
Amyloid beta-protein and the genetics of Alzheimer's disease
J. Biol. Chem.
Amyloid-beta and tau serve antioxidant functions in the aging and Alzheimer brain
Free Radic. Biol. Med.
Expression of nicotinic acetylcholine receptors in Alzheimer's disease: postmortem investigations and experimental approaches
Behav. Brain Res.
High selective expression of alpha7 nicotinic receptors on astrocytes in the brains of patients with sporadic Alzheimer's disease and patients carrying Swedish APP 670/671 mutation: a possible association with neuritis plaques
Exp. Neurol.
Correlation of oxidative stress and the loss of the nicotinic receptor alpha 4 subunit in the temporal cortex of patients with Alzheimer's disease
Neurosci. Lett.
Postnatal changes of nicotinic acetylcholine receptor alpha 2, alpha 3, alpha 4, alpha 7 and beta 2 subunits genes expression in rat brain
Int. J. Dev. Neurosci.
Beta-amyloid peptides induce mitochondrial dysfunction and oxidative stress in astrocytes and death of neurons through activation of NADPH oxidase
J. Neurosci.
Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons
J. Comp. Neurol.
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