Pia Carlo

Amyloid precursor protein and presenilin1 interact with the adaptor GRB2 and modulate ERK1,2 signaling

The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-β peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-β peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.

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.

A circular channel crucible oscillating viscometer: Detection of DNA damage induced in vivo by exceedingly small doses of dimethylnitrosamine

A new oscillating crucible viscometer, having a U-shaped circular channel, is described. The damping coefficient δ is lowered by an increase of the viscosity η. The instrument described here allows the solution to come in contact with inert plastic only. At all steps of its preparation and during viscosity measurements, giant DNA from rat liver nuclei was maintained at shear stresses around 10−4 dynes cm−2. Viscosity was studied as a function of surface tension, DNA concentration and shear stress. It was found that under our experimental conditions it was possible to obtain meaningful values for reduced viscosity, ηred, practically identical to intrinsic viscosity [η]. Rat liver nuclei are incubated in an alkaline lysing solution (pH 12.5; 22 °C): they are lysed immediately and the released DNA starts to uncoil. The viscosity of solutions of this giant DNA increases very slowly with time, reaching a maximum only after about ten hours. The process was accelerated by single-stranded breaks arising from methylation of DNA in vivo with dimethylnitrosamine. It was found that the time of DNA disentanglement was sensitive to an exceedingly small number of breaks. We think that we were able to measure molecular weights around the length of the single strand of an average chromosome (Mn 5 × 1010). An empirical relation between molecular weight and reduced viscosity after complete disentanglement was also established, as a linear log-log plot, covering a molecular weight range between 108 and 2.5 × 1010. It is suggested that the viscosimetric evaluation of DNA disentanglement is probably the most sensitive method for studying DNA damage induced “in vivo” by chemical carcinogens.

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.

Viscoelastic properties of native DNA from intact nuclei of mammalian cells. Higher-order DNA packing and cell function.

Abstract Changes in reduced viscosity of nuclear lysates from rat liver cells have been studied, in conditions of very low shear stress by the use of an oscillating viscometer, as a function of incubation time in alkaline (pH 12.5) and neutral (pH 8.0) solutions. In non-denaturing conditions, nuclear DNA showed a stepwise, time-dependent increase of reduced viscosity, which suggests that it behaves as a single hydrodynamic unit that progressively changes its radius and viscoelastic properties because of a very slow unfolding, through discrete successive transitions, from a highly superpacked structure toward a linear relaxed B -form fiber. Experimental conditions shown to reduce chromatin-DNA superpacking without changing DNA length (e.g. G 1 cycling versus G 0 non-cycling liver cells, or young versus old rat liver cells) dramatically increased the initial value of reduced viscosity and its time-dependent increment. Conversely, in denaturing conditions, reduced viscosity increased in the initial phase (probably because DNA unfolding prevails on DNA unwinding), then exhibited a plateau level (when unfolding balances unwinding), and subsequently decreased progressively to the value of sheared DNA (when unwinding becomes more rapid due to the progressive breakage of phosphodiester bridges in alkali). Experimental conditions known to induce DNA single- or double-strand breaks (i.e. the use of liver cells from rats treated with dimethylnitrosamine or 2-acetylaminofluorene, or of liver cells exposed to X-rays) caused in both neutral and alkaline solution an increment in the initial reduced viscosity and in the slope of its time-dependent increase, which may be related to a reduction of chromatin-DNA superpacking. Moreover, it became evident in denaturing conditions that a decrease of the maximum viscosity and of the time taken to reach it both related to a reduced DNA length. These viscoelastic properties are constantly correlated with independent DNA structural measurements on the same nuclear lysates, to discriminate the effect due to mere aggregation and disaggregation.

Amyloid Precursor Protein and Presenilin Involvement in Cell Signaling

To date the most relevant role for the amyloid precursor protein (APP) and for the presenilins (PSs) on Alzheimer’s disease (AD) genesis is linked to the ‘amyloid hypothesis’, which considers an aberrant formation of amyloid-β peptides the cause of neurodegeneration. In this view, APP is merely a substrate, cleaved by the γ-secretase complex to form toxic amyloid peptides, PSs are key players in γ-secretase complex, and corollary or secondary events are Tau-linked pathology and gliosis. A second theory, complementary to the amyloid hypothesis, proposes that APP and PSs may modulate a yet unclear cell signal, the disruption of which may induce cell-cycle abnormalities, neuronal death, eventually amyloid formation and finally dementia. This hypothesis is supported by the presence of a complex network of proteins, with a clear relevance for signal transduction mechanisms, which interact with APP or PSs. In this scenario, the C-terminal domain of APP has a pivotal role due to the presence of the 682YENPTY687 motif that represents the docking site for multiple interacting proteins involved in cell signaling. In this review we discuss the significance of novel findings related to cell signaling events modulated by APP and PSs for AD development.

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.

Apoptotic Cell Death and Amyloid Precursor Protein Signaling in Neuroblastoma SH‐SY5Y Cells

Abstract: We have recently shown that the amyloid precursor protein (APP) and a subset of its C‐terminal fragments (CTFs) are tyrosine phosphorylated in human brain and in cultured cells. Tyrosine phosphorylation generates a substrate that is sequentially bound by the adaptor proteins ShcA and Grb2, and this interaction is significantly enhanced in Alzheimers disease brains. Here we have studied the APP/CTFs phosphorylation and ShcA activation in a human neuroblastoma cell line, SH‐SY5Y, under basal and apoptotic conditions. To commit these cells to apoptosis, we used staurosporin, a well‐known apoptotic inducer and protein kinase C blocker. Our data suggest the following: (1) in normally proliferating SH‐SY5Y cells, full‐length APP is complexed with Grb2[Q3], likely through its SH2 domain; (2) upon induction of apoptosis, APP is degraded and ShcA‐Grb2 coimmunoprecipitates with CTFs recognized by anti‐APP antibodies; and (3) caspase inhibitors partially block the degradation of APP and the coprecipitation of CTFs with ShcA‐Grb2 adaptors. In summary, our data suggest that in SH‐SY5Y cells, tyrosine‐phosphorylated APP is involved in a complex with ShcA‐Grb2 adaptors that is disrupted during apoptosis. The abnormal degradation of APP and consequent increased levels of CTFs (as has been observed in Alzheimers disease and Downs syndrome) generate a complex between tyrosine‐phosphorylated CTFs and intracellular adaptors. The signaling through APP and its CTFs may have significant relevance for apoptotic cell death in Alzheimers disease.

Absence of liver DNA fragmentation in rats treated with high oral doses of 32 benzodiazepine drugs

Literature data on mutagenic-carcinogenic activity of benzodiazepines are scarce, restricted to few of them, and contradictory. Consequently, in order to provide additional information for the assessment of the genotoxic risk connected with the use of this family of drugs, 32 benzodiazepines of various chemical structure have been tested for their capability to induce DNA damage in vivo, which is considered a sensitive index of potential mutagenic-carcinogenic activity. The frequency of DNA single-strand breaks and/or alkali-labile sites was checked in the liver of rats given orally a single dose (1 mmol/kg) or 15 successive daily doses (0.2 mmol/kg) by the use of a new viscometric technique capable of detecting one DNA lesion per 10(10) Da. Statistically significant changes of viscometric parameters indicative of liver DNA fragmentation were absent with all 32 benzodiazepines, after both acute and subacute treatments. Since the doses tested in rats were from 100 to more than 5000 times higher than doses usually administered to humans, these negative results are in favor of the absence of mutagenic-carcinogenic effects in patients taking benzodiazepines.