Nouhad Kassem

Age-related changes in choroid plexus and blood–cerebrospinal fluid barrier function in the sheep

Dysfunction of the choroid plexuses (CPs) and the blood-cerebrospinal fluid barrier (BCSFB) might contribute to age-related cognitive decline and neurodegenerative disease. We used the CPs from young (1-2 years), middle-aged (3-6 years) and old (7-10 years) sheep to explore effects of aging on various aspects of CP and BCSFB functions. Total protein in the cerebrospinal fluid (CSF) was significantly higher in old compared to young sheep and CSF secretion by the CP perfused in situ was significantly lower in both old and middle-aged when compared to young sheep, which correlated with reduced (22)Na(+) uptake and efflux by the CP. Steady-state extractions of a low and medium size molecular weight extracellular space marker, (14)C-mannitol and (3)H-polyethylene glycol, respectively, were significantly higher in CPs from old compared to young animals; however, there was no significant difference in steady-state extraction of a high molecular weight marker, (125)I-bovine serum albumin. This indicates increased passive BCSFB permeability for small and medium sized molecules in old sheep. CP redox activity was significantly lower in the old animals as assessed by the MTT assay, however, there was no significant difference in ATP content and energy charge of the CP with age suggesting adequate baseline energy reserve capacity. These data indicate that normal aging processes alter protein content in the CSF, CSF secretion, integrity of the BCSFB and Na(+) flux in the epithelial layer, which could impact on CSF homeostasis and turnover.

Thyroxine (T4) transfer from CSF to choroid plexus and ventricular brain regions in rabbit: Contributory role of P-glycoprotein and organic anion transporting polypeptides

This study investigated the transfer of T4 from cerebrospinal fluid (CSF) into the choroid plexuses (CP) and ventricular brain regions, and the role of P-glycoprotein (P-gp), multidrug resistance protein 1 (mrp1) and organic anion transporting polypeptides (oatps). During in vivo ventriculo-cisternal (V-C) perfusion in the anesthetized rabbit (meditomidine hydrochloride 0.5 mg kg(-1), pentobarbitone 10 mg kg(-1) i.v.), 125I-T4 was perfused continuously into ventricular CSF with reference molecules 14C-mannitol and blue dextran. Over 2 h, 36.9+/-4.6% 125I-T4 was recovered in cisternal CSF. Addition of P-gp substrate verapamil increased CSF 125I-T4 recovery to 51.4+/-2.8%, although mrp1 and oatp substrates had no significant effect. In brain, 125I-T4 showed greatest accumulation in the CP (1.52+/-0.31 ml g(-1)), followed by ventricular regions (caudate putamen, ependyma, hippocampus, 0.05-0.14 ml g(-1)). At the CP, verapamil and probenecid (but not indomethacin) significantly increased 125I-T4 accumulation, implicating a role for P-gp and oatps. Of other brain regions, all three drugs increased accumulation in caudate putamen 3-5 times, and indomethacin and probenecid increased accumulation in ependyma 4-5 times. The role of P-gp was investigated further in isolated incubated CPs using 5 microg/ml C219 anti-P-gp antibody. Both 125I-T4 and 3H-cyclosporin accumulation increased by 80%, suggesting that P-gp is functional in the CP and has a role in removal of T4. Combined with the in vivo results, these studies suggest that P-gp provides a local homeostatic mechanism, maintaining CSF T4 levels. We conclude that P-gp and oatps contribute to the transfer of 125I-T4 between the CSF, CP and brain, hence regulating 125I-T4 availability in CSF.

Dose-dependent transthyretin inhibition of T4 uptake from cerebrospinal fluid in sheep

Transthyretin (TTR), synthesized by the choroid plexuses (CP) has an important role in transporting thyroxine from blood to cerebrospinal fluid (CSF). However, the role of TTR on thyroxine transport from CSF to either blood or brain is not clear. By using the incubated isolated ovine brain tissues technique, we found the CP accumulated most 125I-T4 compared to ventricular ependymal, frontal cortex or cerebellum. The accumulation was higher in the young CP than the old. There was dose-dependent inhibition by TTR on 125I-T4 accumulation in the brain tissues, and kinetics of T4 accumulation in presence of TTR was obtained by plotting a double reciprocal of B (bound) versus TTR concentration curve. The KD of 125I-T4 binding to TTR was higher in the CP compared to other tissues, suggesting that CP competes with TTR for T4 binding to a greater extent than the other tissues. Using the isolated perfused CP preparation, TTR significantly inhibited 125I-T4 efflux across CP from the CSF to blood side. Bovine serum albumin (BSA) was also able to inhibit 125I-T4 accumulation in the incubated tissues, but required higher concentrations to reach the level of inhibition seen with TTR. In conclusion, this study found a significant role for CSF TTR in preventing T4 loss to blood across the CP, and TTR inhibited both CP and selected brain tissue uptake in a dose-dependent manner. The physiological relevance of TTR may relate to preventing T4 loss from CSF and encouraging redistribution of hormone around the brain in CSF.

EGF-Induced VEGF Exerts a PI3K-Dependent Positive Feedback on ERK and AKT through VEGFR2 in Hematological In Vitro Models.

EGFR and VEGFR pathways play major roles in solid tumor growth and progression, however, little is known about these pathways in haematological tumors. This study investigated the crosstalk between EGFR and VEGFR2 signaling in two hematological in vitro models: THP1, a human monocytic leukemia, and Raji, a Burkitt’s lymphoma, cell lines. Results showed that both cell lines express EGFR and VEGFR2 and responded to EGF stimulation by activating EGFR, triggering VEGF production and phosphorylating ERK, AKT, and p38 very early, with a peak of expression at 10–20min. Blocking EGFR using Tyrphostin resulted in inhibiting EGFR induced activation of ERK, AKT, and p38. In addition, EGF stimulation caused a significant and immediate increase, within 1min, in pVEGFR2 in both cell lines, which peaked at ~5–10 min after treatment. Selective inhibition of VEGFR2 by DMH4, anti-VEGFR2 antibody or siRNA diminished EGF-induced pAKT and pERK, indicating a positive feedback exerted by EGFR-induced VEGF. Similarly, the specific PI3K inhibitor LY294002, suppressed AKT and ERK phosphorylation showing that VEGF feedback is PI3K-dependent. On the other hand, phosphorylation of p38, initiated by EGFR and independent of VEGF feedback, was diminished using PLC inhibitor U73122. Moreover, measurement of intracellular [Ca2+] and ROS following VEGFR2 inhibition and EGF treatment proved that VEGFR2 is not implicated in EGF-induced Ca2+ release whereas it boosts EGF-induced ROS production. Furthermore, a significant decrease in pAKT, pERK and p-p38 was shown following the addition of the ROS inhibitor NAC. These results contribute to the understanding of the crosstalk between EGFR and VEGFR in haematological malignancies and their possible combined blockade in therapy.

ROS mediates interferon gamma induced phosphorylation of Src, through the Raf/ERK pathway, in MCF-7 human breast cancer cell line.

Interferon gamma (IFN-ɣ) is a pleiotropic cytokine which plays dual contrasting roles in cancer. Although IFN-ɣ has been clinically used to treat various malignancies, it was recently shown to have protumorigenic activities. Reactive oxygen species (ROS) are overproduced in cancer cells, mainly due to NADPH oxidase activity, which results into several changes in signaling pathways. In this study, we examined IFN-ɣ effect on the phosphorylation levels of key signaling proteins, through ROS production, in the human breast cancer cell line MCF-7. After treatment by IFN-ɣ, results showed a significant increase in the phosphorylation of STAT1, Src, raf, AKT, ERK1/2 and p38 signaling molecules, in a time specific manner. Src and Raf were found to be involved in early stages of IFN-ɣ signaling since their phosphorylation increased very rapidly. Selective inhibition of Src-family kinases resulted in an immediate significant decrease in the phosphorylation status of Raf and ERK1/2, but not p38 and AKT. On the other hand, IFN-ɣ resulted in ROS generation, through H2O2 production, whereas pre-treatment with the ROS inhibitor NAC caused ROS inhibition and a significant decrease in the phosphorylation levels of AKT, ERK1/2, p38 and STAT1. Moreover, pretreatment with a selective NOX1 inhibitor resulted in a significant decrease of AKT phosphorylation. Finally, no direct relationship was found between ROS production and calcium mobilization. In summary, IFN-ɣ signaling in MCF-7 cell line is ROS-dependent and follows the Src/Raf/ERK pathway whereas its signaling through the AKT pathway is highly dependent on NOX1.

Stem cells and combination therapy for the treatment of traumatic brain injury

HighlightsThis review covers existing treatments in TBI therapy, with a focus on their potential applications to improve the functional outcomes of TBIComplex pathophysiology and molecular mechanisms of TBI are reviewed.Stem cell therapy as an optimal solution to treat TBIis reviewed, including endogenous or exogenous stem cells from embryonic, induced pluripotent, mesenchymal, and neural origin.Combination therapy is discussed as a novel method to treat TBI. Two approaches are highlighted, growth factors and ROCK inhibitors.Emphasis is given for improvements in motor and cognitive functions after stem cell therapy. ABSTRACT TBI is a nondegenerative, noncongenital insult to the brain from an external mechanical force; for instance a violent blow in a car accident. It is a complex injury with a broad spectrum of symptoms and has become a major cause of death and disability in addition to being a burden on public health and societies worldwide. As such, finding a therapy for TBI has become a major health concern for many countries, which has led to the emergence of many monotherapies that have shown promising effects in animal models of TBI, but have not yet proven any significant efficacy in clinical trials. In this paper, we will review existing and novel TBI treatment options. We will first shed light on the complex pathophysiology and molecular mechanisms of this disorder, understanding of which is a necessity for launching any treatment option. We will then review most of the currently available treatments for TBI including the recent approaches in the field of stem cell therapy as an optimal solution to treat TBI. Therapy using endogenous stem cells will be reviewed, followed by therapies utilizing exogenous stem cells from embryonic, induced pluripotent, mesenchymal, and neural origin. Combination therapy is also discussed as an emergent novel approach to treat TBI. Two approaches are highlighted, an approach concerning growth factors and another using ROCK inhibitors. These approaches are highlighted with regard to their benefits in minimizing the outcomes of TBI. Finally, we focus on the consequent improvements in motor and cognitive functions after stem cell therapy. Overall, this review will cover existing treatment options and recent advancements in TBI therapy, with a focus on the potential application of these strategies as a solution to improve the functional outcomes of TBI.

Thyroxine transfer from cerebrospinal fluid into choroid plexus and brain is affected by brefeldin A, low sodium, BCH, and phloretin, in ventriculo-cisternal perfused rabbits.

Background: Thyroxine (T4) hormone is synthesized by the thyroid gland and then released into the systemic circulation where it binds to a number of proteins. Dysfunction in T4 transport mechanisms has been demonstrated in multiple central nervous system (CNS) diseases including Alzheimers disease. In the presence of different compounds that inhibit potential T4 transport mechanisms, this study investigated the transfer of T4 from cerebrospinal fluid (CSF) into Choroid Plexus (CP) and other brain tissues. The compounds used were brefeldin A, low sodium artificial CSF (aCSF), BCH, phloretin, and taurocholate (TA). Methods: Radiolabeled T4 (125I-T4) was perfused continuously into the CSF and was assessed in several brain compartments with reference molecule 14C-mannitol and blue dextran, using the in vivo ventriculo-cisternal perfusion (V-C) technique in the rabbit. The aCSF containing the drug of interest was infused after 1 h of perfusion. Drugs were applied independently to the aCSF after 1 h of control perfusion. Results: Of interest, in presence of low sodium or BCH, the percentage recovery of 125I-T4, was increased compared to controls, with concomitant increase in T4 clearance. Conversely, brefeldin A, phloretin, and TA did not exert any significant effect on the recovery and clearance of 125I-T4 assessed in aCSF. On the other hand, the uptake of 125I-T4 into CP was raised by 18 fold compared to controls in the presence of brefeldin A. In addition, low sodium, BCH, or phloretin alone, enhanced the uptake of 125I-T4 by almost 3-fold, whereas TA did not show any significant effect. Finally, the uptake and distribution of 125I-T4 into other brain regions including ependymal region (ER) and caudate putamen (CAP) were significantly higher than in controls. Conclusion: Our study suggests the involvement of different mechanisms for the transfer of 125I-T4 from CSF into CP and other brain regions. This transfer may implicate sodium-dependent mechanisms, amino acid “L” system, or organic anion transporting polypeptide (OATP).

Thyroxine (T4) Transfer from Blood to Cerebrospinal Fluid in Sheep Isolated Perfused Choroid Plexus: Role of Multidrug Resistance-Associated Proteins and Organic Anion Transporting Polypeptides

Thyroxine (T4) enters the brain either directly across the blood–brain barrier (BBB) or indirectly via the choroid plexus (CP), which forms the blood–cerebrospinal fluid barrier (B-CSF-B). In this study, using isolated perfused CP of the sheep by single-circulation paired tracer and steady-state techniques, T4 transport mechanisms from blood into lateral ventricle CP has been characterized as the first step in the transfer across the B-CSF-B. After removal of sheep brain, the CPs were perfused with 125I-T4 and 14C-mannitol. Unlabeled T4 was applied during single tracer technique to assess the mode of maximum uptake (Umax) and the net uptake (Unet) on the blood side of the CP. On the other hand, in order to characterize T4 protein transporters, steady-state extraction of 125I-T4 was measured in presence of different inhibitors such as probenecid, verapamil, BCH, or indomethacin. Increasing the concentration of unlabeled-T4 resulted in a significant reduction in Umax%, which was reflected by a complete inhibition of T4 uptake into CP. In fact, the obtained Unet% decreased as the concentration of unlabeled-T4 increased. The addition of probenecid caused a significant inhibition of T4 transport, in comparison to control, reflecting the presence of a carrier mediated process at the basolateral side of the CP and the involvement of multidrug resistance-associated proteins (MRPs: MRP1 and MRP4) and organic anion transporting polypeptides (Oatp1, Oatp2, and Oatp14). Moreover, verapamil, the P-glycoprotein (P-gp) substrate, resulted in ~34% decrease in the net extraction of T4, indicating that MDR1 contributes to T4 entry into CSF. Finally, inhibition in the net extraction of T4 caused by BCH or indomethacin suggests, respectively, a role for amino acid “L” system and MRP1/Oatp1 in mediating T4 transfer. The presence of a carrier-mediated transport mechanism for cellular uptake on the basolateral membrane of the CP, mainly P-gp and Oatp2, would account for the efficient T4 transport from blood to CSF. The current study highlights a carrier-mediated transport mechanism for T4 movement from blood to brain at the basolateral side of B-CSF-B/CP, as an alternative route to BBB.

Signaling pathways activated by PACAP in MCF-7 breast cancer cells

PACAP has opposing roles ranging from activation to inhibition of tumor growth and PACAP agonists/antagonists could be used in tumor therapy. In this study, the effect of PACAP stimulation on signaling pathways was investigated in MCF-7 human adenocarcinoma breast cancer cells. Results showed that MCF-7 cells express VPAC1 and VPAC2, but not PAC1, receptors. In addition, PACAP increased the phosphorylation levels of STAT1, Src and Raf within seconds, confirming their involvement in early stages of PACAP signaling whereas maximal phosphorylation of AKT, ERK and p38 was reached 10 to 20 min later. Moreover, selective inhibition of Src or PI3K resulted in a significant decrease in the phosphorylation of ERK and AKT, but not p38, demonstrating that PACAP signaling follows Src/Raf/ERK and PI3K/AKT pathways. On the other hand, selective inhibition of PLC or PKA resulted in a significant decrease in the phosphorylation of p38, but not AKT or ERK, indicating that PACAP signaling also follows the PLC and PKA/cAMP pathways. Furthermore, PACAP induced ROS through H₂O₂ production whereas pretreatment with NAC inhibitor decreased AKT and ERK phosphorylation, but not p38. Selective NOX2 inhibition affected Src/Raf/Erk and PI3K/Akt pathways, without affecting the p38/PLC/PKA pathway whereas other inhibitors (ML171, VAS2870) had no effect on PACAP induced ROS generation. On the other hand, PACAP induced calcium release, which was decreased by pretreatment with PLC inhibitor. Finally, PACAP stimulation promoted apoptosis by increasing Bax and decreasing Bcl2 expression. In conclusion, we demonstrated that PACAP signaling in MCF-7 cells follows the Src/Raf/ERK and PI3K/AKT pathways and is VPAC1 dependent in a ROS dependent manner, whereas it follows PLC and PKA/cAMP pathways and is VPAC2 dependent through p38 MAP kinase activation involving calcium.