Verapamil Inhibits Ser202/Thr205 Phosphorylation of Tau by Blocking TXNIP/ROS/p38 MAPK Pathway

Purpose Oxidative stress is a hallmark of Alzheimer’s Disease (AD) and promotes tau phosphorylation. Since Thioredoxin Interacting protein (TXNIP), the inhibitor of the anti-oxidant system of Thioredoxin, is up regulated in the hippocampus of AD patients, we investigated whether TXNIP plays a role in promoting tau phosphorylation and whether Verapamil, an inhibitor of TXNIP expression, prevents TXNIP downstream effects. Methods We analyzed TXNIP expression and tau phosphorylation in the hippocampus of the 5xFAD mice in the absence and presence of a pharmacological treatment with Verapamil. Using SH-SY5Y cells, we verified the causative role of TXNIP in promoting tau phosphorylation atSer202/Thr205, by inducing TXNIP silencing. Results The amyloid beta peptide (Aβ1–42) leads to TXNIP over-expression in SH-SY5Y cells, which in turns induces oxidative stress and the activation of p38 MAPK, promoting tau phosphorylation at Ser202/Thr205. Silencing of TXNIP abolishes Aβ1–42-induced tau phosphorylation, p38 MAPK phosphorylation and subsequent tau phosphorylation. Verapamil prevents TXNIP expression as well as p38 MAPK and tau phosphorylation at Ser202/Thr205 in the hippocampus of the 5xFAD mice. Conclusions Our study unveil a novel pathway involved in AD progression that is inhibited by Verapamil, shedding new light on the understanding of the therapeutic potential of Verapamil in AD

Polymorphonuclear Cell Functional Impairment in Relapsing Remitting Multiple Sclerosis Patients: Preliminary Data

<div><p>Multiple Sclerosis patients run an increased risk of microbial infections, which leads to high rates of hospitalization and infection-related mortality. Although immunotherapy may increase infection risk in some cases, data as to the relationship among microbial factors, immunotherapy and alterations in the innate immunity of these patients are still scanty. On these grounds, this interdisciplinary study aims at investigating the role the functional activity of polymorphonuclear cells (PMNs) play in relapsing remitting multiple sclerosis at different stages. The <i>in vitro</i> ability of PMNs from patients, either untreated or treated with immunosuppressant or immunomodulatory drugs to kill <i>Klebsiella pneumonia</i> or <i>Candida albicans</i>, were investigated and compared to PMNs from healthy subjects. The release of various cytokines was also assessed, as was the production of reactive oxygen species and their ability to regulate apoptosis after microbial stimulation. Our results indicate that although patients have a normal number of PMNs, they have a statistically significant (p<0.05) reduction in intracellular killing activity. Although variations are strongly related to the therapeutic management of patients, they are independent from their disease stage. As no statistically significant differences were observed between patients and controls in cytokine release values, reactive oxygen species production or apoptosis, we came to the conclusion that other factors may be involved. Supportive validation of these results from further studies might well help in identifying a subset of patients at high risk of infection who could benefit from a closer follow-up and/or antibiotic prophylaxis.</p></div

The analysis of induced oxidative burst in MS patients.

<p>Whole blood from healthy subjects (HS; n = 12), from untreated-MS patients (n = 7), from patients treated with immunosuppressive drugs (IS-MS; n = 6) or with immunomodulatory drugs (IM-MS; n = 12), was stimulated with <i>K</i>. <i>pneumoniae</i> (<b>A</b>), <i>C</i>. <i>albicans</i> (<b>B</b>) or PMA (<b>C</b>) for 30 minutes. Percentage of positive cells for DHR123 (left panels) or average of fluorescence intensity (MFI) of DHR123 (right panels) were determined by flow cytometry. Data are represented as average ± SEM.</p

Proinflammatory cytokine release by PMNs.

<p>(<b>A</b>) PMNs from 16 healthy subjects (HS), from 4 untreated-MS patients, from 6 patients treated with immunosuppressive drugs (IS-MS) or from 13 patients treated with immunomodulatory drugs (IM-MS) were stimulated with <i>K</i>. <i>pneumoniae</i> for 30, 60, 90 and 180 minutes. (<b>B</b>) PMNs from 10 healthy subjects (HS), from 7 untreated-MS patients, from 7 patients treated with immunosuppressive drugs (IS-MS) or from 9 patients treated with immunomodulatory drugs (IM-MS) were stimulated with <i>C</i>. <i>albicans</i> for 30, 60, 90 and 180 minutes. Supernatants were evaluated for IL-8 (upper panels) and IL-1β release (lower panels). Data are represented as average ± SEM. *p<0.05.</p

The study of PMN apoptosis in MS patients.

<p>(<b>A</b>) The percentage of apoptotic purified PMNs (annexin V positive, PI negative) in healthy subjects (HS; n = 8), in untreated-MS patients (n = 6), in patients treated with immunosuppressive drugs (IS-MS; n = 5) or with immunomodulatory drugs (IM-MS; n = 10), after 3 hours of culture. (<b>B</b>) Early apoptotic cells were evaluated in HS (n = 5) and IM-MS (n = 5) PMNs after <i>K</i>. <i>pneumoniae</i> stimulation. Values were determined by flow cytometric analysis and data represented as average ± SEM. *p<0.05; **p<0.001</p

Demographic and clinical information on the healthy subjects (HSs) and MS patients.

<p>Average ± SEM are shown; EDSS: Kurtzke Expanded Disability Status Scale; RR: relapsing-remitting MS; UTIs: urinary tract infections; RTIs: respiratory tract infections</p><p>Demographic and clinical information on the healthy subjects (HSs) and MS patients.</p