Clinical and epidemiological characteristics of COVID-19 during the early phase of the SARS-CoV-2 pandemic: a cross-sectional study among medical school physicians and residents employed in a regional reference teaching hospital in Northern Italy.

OBJECTIVES: The aim of the study was to evaluate the clinical presentation and burden of SARS-CoV-2 infections among medical school physicians and residents, mainly young medical doctors. The awareness of COVID­19 clinical manifestations can improve the early detection of mild cases, possibly reducing further transmission to colleagues and patients. MATERIAL AND METHODS: The study was carried out in March-May 2020, involving medical school physicians in a teaching hospital in northern Italy, with a working population of 881 medical doctors. Data collection was performed using a structured form investigating clinical and epidemiological information. RESULTS: One hundred sixty-two medical doctors contacted the Occupational Health Service reporting acute respiratory symptoms or close contact exposure to a confirmed COVID­19 case. Among the confirmed COVID­19 cases, most were male doctors during residency, and 85% presented a mild clinical picture. Fever (70.3%) and cough (51.4%) represented the most prevalent symptoms of COVID­19. As revealed by the univariate analysis, the prevalence of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) positivity increased with age (OR = 1.08, 95% CI: 1.02-1.14, p = 0.012), working in a COVID­19 ward (OR = 3.33, 95% CI: 1.09-10.21, p = 0.031), presenting alteration or loss of smell/taste (OR = 10.00, 95%CI: 2.80-35.69, p < 0.001) and myalgia (OR = 3.20, 95% CI: 1.00-10.26, p = 0.046), while being a resident (OR = 0.20, 95% CI: 0.05-0.80, p = 0.030) was associated with reduced odds of being infected, compared to staff physicians. Age and loss of smell/taste were the only factors independently associated with RT-PCR positivity. CONCLUSIONS: The majority of COVID­19 cases showed a mild clinical syndrome, ranging from absence or paucity of symptoms to common cold or influenza-like symptoms. The findings of the present study increase the accuracy of the clinical diagnosis for the prompt identification and management of suspected COVID­19 cases, being particularly useful during resurges of the SARS-CoV-2 pandemic. Int J Occup Med Environ Health. 2021;34(2):189-201.

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-beta peptides the cause of neurodegeneration. In this view, APP is merely a substrate, cleaved by the gamma-secretase complex to form toxic amyloid peptides, PSs are key players in gamma-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 and Presenilin 1 interaction studied by FRET in human H4 cells.

The mayor pathologic hallmarks of Alzheimer's disease (AD) are senile plaque and neurofibrillary tangles. Senile plaque are primarily made up of deposits of amyloid-beta protein, a proteolytic product derived from the amyloid precursor protein (APP). APP is a transmembrane protein detected into the endoplasmic reticulum, in the Golgi apparatus, at the cell surface, recycled by endocytosis to endosomes, whose physiological function is unclear. Presenilins (PS), are a component of gamma-secretase complex that cleave alpha-CTFs (carboxy-terminal fragment), or beta-CTFs, leaving 40 or 42 amino acids amyloid-beta peptides and 58 or 56 amino acids intracellular domains (AICD). Where the amyloid-beta peptides is generated is not clear. The study of APP-PS interaction in specific cell compartments provides a good opportunity to light upon the molecular mechanisms regulating the activity of the "gamma-secretase complex," and where beta-amyloid is generated. In our study we used a biophysical assay of protein proximity: fluorescence resonance energy transfer (FRET), that can provide information about molecular interactions when two proteins are in the close proximity (<10 nm), to examine the subcellular localization and interaction between APP and PS1 in human neuroglioma cells (H4). Confocal microscopic analysis reveals extensive colocalization in different cells' compartment, and centrosomal or microtubule organizing center (MTOC) localization of APP and PS1, but not necessarily a close molecular interaction. We used FRET to determine if APP and PS1 interact at the cell centrosome. FRET data suggest a close interaction between APP and PS1 in subcellular compartments and at the centrosome of H4 cells. Using this approach we show that APP and PS1 are closely associated in the centrosomes of the H4 cell.

Amyloid precursor protein and Presenilin1 interact with the adaptor GRB2 and modulate ERK 1,2 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-beta 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-beta 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.

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

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 Alzheimer's disease (AD) through the formation of membrane-bound C-terminal fragments (CTFs) and of beta-amyloid peptides (Abeta). 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.

The amyloid precursor protein and its network of interacting proteins: physiological and pathological implications.

The amyloid precursor protein (APP) is an ubiquitous receptor-like molecule involved in the pathogenesis of Alzheimer's disease that generates beta-amyloid peptides and causes plaque formation. APP and some of its C-terminal proteolytic fragments (CTFs) have also been shown to be in the center of a complex protein-protein network, where selective phosphorylation of APP C-terminus may regulate the interaction with cytosolic phosphotyrosine binding (PTB) domain or Src homology 2 (SH2) domain containing proteins involved in cell signaling. We have recently described an interaction between tyrosine-phosphorylated CTFs and ShcA adaptor protein which is highly enhanced in AD brain, and a new interaction between APP and the adaptor protein Grb2 both in human brain and in neuroblastoma cultured cells. These data suggest a possible role in cell signaling for APP and its CTFs, in a manner similar to that previously reported for other receptors, through a tightly regulated coupling with intracellular adaptors to control the signaling of the cell. In this review, we discuss the significance of these novel findings for AD development.

Apoptotic cell death and amyloid precursor protein signaling in neuroblastoma SH-SY5Y cells.

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 Alzheimer's 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 Alzheimer's disease and Down's 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 Alzheimer's disease.

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.

The processing of the amyloid precursor protein (APP) through the formation of C-terminal fragments (CTFs) and the production of beta-amyloid, are events likely to influence the development and the progression of Alzheimer's 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 Alzheimer's 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.

Pyroglutamate-modified amyloid beta-peptides--AbetaN3(pE)--strongly affect cultured neuron and astrocyte survival.

N-terminally truncated amyloid-beta (Abeta) peptides are present in early and diffuse plaques of individuals with Alzheimer's 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 Down's syndrome (DS). The pyroglutamate-containing isoforms at position 3 [AbetaN3(pE)-40/42] represent the prominent form among the N-truncated species, and may account for more than 50% of Abeta accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Abeta1-40, Abeta1-42, AbetaN3(pE)-40 and AbetaN3(pE)-42. Our data show that fibre morphology of Abeta peptides is greatly influenced by the C-terminus while toxicity, interaction with cell membranes and degradation are influenced by the N-terminus. AbetaN3(pE)-40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AbetaN3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AbetaN3(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 beta-amyloid aggregation and toxicity, likely worsening the onset and progression of the disease.