Elisabetta Violani

Pyroglutamate‐modified amyloid β‐peptides – AβN3(pE) – strongly affect cultured neuron and astrocyte survival

N‐terminally truncated amyloid‐β (Aβ) peptides are present in early and diffuse plaques of individuals with Alzheimers disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Downs syndrome (DS). The pyroglutamate‐containing isoforms at position 3 [AβN3(pE)−40/42] represent the prominent form among the N‐truncated species, and may account for more than 50% of Aβ accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Aβ1–40, Aβ1–42, AβN3(pE)−40 and AβN3(pE)−42. Our data show that fibre morphology of Aβ peptides is greatly influenced by the C‐terminus while toxicity, interaction with cell membranes and degradation are influenced by the N‐terminus. AβN3(pE)−40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AβN3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AβN3(pE)−40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while full‐length peptides resulted partially degraded. These findings suggest that formation of N‐terminally modified peptides may enhance β‐amyloid aggregation and toxicity, likely worsening the onset and progression of the disease.

Quantitation of low abundance mRNAs in glial cells using different polymerase chain reaction (PCR)-based methods

The conventional methods for mRNA quantitation such as Northern blotting or ribonuclease protection assay sometimes lack enough sensitivity to study low abundance mRNAs or to work with limited amounts of biological samples. The sensitivity of the polymerase chain reaction (PCR) linked to reverse transcription (RT-PCR) has proven useful in amplifying specific mRNAs, especially those present in low copy number. Though, the quantitation of nucleic acids by means of PCR has proven problematic. The main constraint in obtaining quantitative data is inherent in the amplification reaction. Because amplification is an exponential process, small variations in the efficiency of amplification may significantly affect the final yield of the PCR product. The variables that influence the rate of the PCR include the abundance of the mRNA present in the starting material, the concentrations of the Taq DNA polymerase, dNTPs and magnesium ions, the annealing and elongation conditions, the ramping temperatures and the formation of primer secondary structures. Moreover, with the progression of the PCR cycles, reagents are consumed and inhibitors generated, leading to non-linear synthesis of DNA. Finally, tube-to-tube variations sometimes preclude accurate quantitation. Most of the above-mentioned problems can be overcome by the choice of adequate internal controls. The present report reviews two recently developed methods for RNA quantitation, the semi-quantitative PCR and the quantitative PCR illustrated for the measurement of monoamine oxidase (MAO) A and B mRNAs and the estrogen receptor (ER) mRNA respectively, with a particular emphasis on the design of appropriate internal controls to compensate for the intra- and inter-assay variability inherent to RT-PCR.

Monoamine oxidase B expression is selectively regulated by dexamethasone in cultured rat astrocytes

The influence of dexamethasone on monoamine oxidase (MAO) A and B expression and activity was investigated in primary cultures of rat type 1 astrocytes cultured under serum free, defined conditions. Dexamethasone treatment resulted in a dose- and time-dependent induction of MAO-B, but not of MAO-A, activity. The selective MAO-B increase was substantially reduced by the antagonist RU 486, thus suggesting a glucocorticoid receptor-mediated action of the hormone. Kinetic analysis showed an increase in Vmax of MAO-B with no change in apparent K(m). The dexamethasone-induced selective rise in MAO-B activity appeared to be due to enhanced enzyme synthesis, since MAO-B mRNA was markedly increased by dexamethasone treatment and the recovery of MAO-B activity after its irreversible inhibition by deprenyl was more pronounced in the presence than in the absence of the hormone. Furthermore, the dexamethasone effect was abolished by the protein synthesis inhibitors actinomycin D or cycloheximide. The present study demonstrates that dexamethasone is able to selectively induce MAO-B in type 1 astrocytes and leads to speculation of a possible role for glucocorticoids in the increase in brain MAO-B associated with neurodegenerative disorders, such as Parkinsons and Alzheimers diseases.

Estrogenic control of monoamine oxidase A activity in human neuroblastoma cells expressing physiological concentrations of estrogen receptor

Several lines of evidence support the hypothesis of a role played by estrogens in the manifestation of affective disorders in women. The analysis of the mechanism of action of a number of antidepressant drugs clearly demonstrated the involvement of the catecholaminergic system in the etiology of these complex behavioral pathologies. The present in vitro study was therefore undertaken to investigate the presence of a functional link between estrogen and catecholamine metabolism in cells of neural origin. The model system utilized was a human neuroblastoma cell line which was obtained by stable transfection of the estrogen receptor cDNA (SK-ER3). The present study shows that in SK-ER3 activation of the estrogen receptor correlates with a marked decrease in monoamine oxidase A activity. This effect is observed following treatment with a physiological concentration of 17 beta-estradiol and can be blocked by the specific antagonist of the steroid receptor, ICI 182,780. Dibutyryl cyclic AMP acting, like estrogens, on the state of differentiation of SK-ER3 cells did not affect monoamine oxidase A activity. The present study provides strong evidence of a strict relationship between estrogen receptor and monoamine oxidase A activity in human cells of neural origin, thus favoring the hypothesis of an antidepressive effect of estrogens exerted via inhibition of the monoamine oxidative pathway.

Signal transduction through tyrosine-phosphorylated carboxy-terminal fragments of APP via an enhanced interaction with Shc/Grb2 adaptor proteins in reactive astrocytes of Alzheimer's disease brain.

Abstract: The processing of the amyloid precursor protein (APP) through the formation of C‐terminal fragments (CTFs) and the production of β‐amyloid, are events likely to influence the development and the progression of Alzheimers disease (AD). APP is a transmembrane protein similar to a cell‐surface receptor with the intraluminal NPTY motif in the cytosolic C terminus. Although APP holoprotein can be bound to intracellular proteins like Fe65, X11, and mDab, the ultimate function and the mechanisms through which this putative receptor transfers its message are unclear. Here it is shown that in human brain, a subset of tyrosine‐phosphorylated CTFs represent docking sites for the adaptor protein ShcA. ShcA immunoreactivity is greatly enhanced in Alzheimers patients; it is mainly localized to glial cells and occurs at reactive astrocytes surrounding cerebral vessels and amyloid plaques. Grb2 also is involved in complexes with ShcA and tyrosine‐phosphorylated CTFs, and in AD brain the interaction between Grb2‐ShcA and CTFs is enhanced. Also, a higher amount of phospho‐ERK1,2 is present in AD brain in comparison with control cases, likely as a result of the ShcA activation. In vitro experiments show that the ShcA‐CTFs interaction is strictly confined to glial cells when treated with thrombin, which is a well‐known ShcA and ERK1,2 activator, mitogen, and regulator of APP cleavage. In untreated cells ShcA does not interact with either APP or CTFs, although they are normally produced. Altogether these data suggest that CTFs are implicated in cell signaling via Shc transduction machinery, likely influencing MAPK activity and glial reaction in AD patients.

Amyloid precursor protein modulates ERK-1 and -2 signaling.

Abstract:  The amyloid precursor protein (APP) is a transmembrane protein with a short cytoplasmic tail whose physiological function is unclear, although it is well documented that the proteolytic processing of APP could influence the development of Alzheimers disease (AD) through the formation of membrane‐bound C‐terminal fragments (CTFs) and of β‐amyloid peptides (Aβ). We have recently shown that tyrosine‐phosphorylated APP and CTFs may interact with Grb2 and ShcA adaptor proteins and that this coupling occurs at a higher extent in AD subjects only. To study the interaction between APP or CTFs and ShcA/Grb2 and to investigate their molecular target we have used as experimental model two different cell lines: H4 human neuroglioma cells and APP/APLP null mouse embryonic fibroblast cells (MEFs). Here we show that in H4 cells APP interacts with Grb2; conversely in APP/APLP‐null MEF cells this interaction is possible only after the reintroduction of human APP by transfection. We have also shown that in MEF cells the transfection of a plasmid encoding for human APP wild‐type enhances the phosphorylation of ERK‐1 and ‐2 as revealed by Western blotting and immunofluorescence experiments. Finally, also in H4 cells the overexpression of APP upregulates the levels of phospho‐ERK‐1 and ‐2. In summary our data suggest that APP may influence phospho‐ERK‐1 and ‐2 signaling through its binding with Grb2 and ShcA adaptors. The meaning of this event is not clear, but APP interaction with these adaptors could be relevant to regulate mitogenic pathway.