Accumulation of amyloid-ß in the cerebellar cortex of essential tremor patients.

The accumulation of insoluble amyloid-beta (Aß) peptides is associated with neurodegenerative disorders, such as Alzheimer's disease (AD). As essential tremor (ET) could involve neurodegenerative processes in the cerebellum, we quantified soluble and insoluble Aß in cerebellar cortices from patients diagnosed with ET (n=9), compared to Controls (n=16) or individuals with Parkinson's disease (n=10). Although ante-mortem cognitive performance was not documented, all individuals included had the diagnosis of AD ruled out by a neuropathologist. ELISA-determined concentrations of insoluble Aß42 in ET patients displayed a bimodal distribution, with a median 246-fold higher than in Controls (P<0.01, Kruskal-Wallis). Higher Aß42 concentrations were measured in the parietal cortex of the same ET patients, compared to Controls (107-fold median increase, P<0.01, Kruskal-Wallis), but similar phosphorylated tau levels were detected. The rise in cerebellar insoluble Aß42 concentrations is not associated to APP expression and processing or the ApoE4 status. However, Aß42 levels in ET individuals were correlated with cerebellar insoluble phosphorylated tau (r(2)=0.71, P=0.005), unphosphorylated neurofilament heavy chain (NF-H; r(2)=0.50, P=0.030) and Lingo-1 (r(2)=0.73, P=0.007), indicative of a generalized neurodegenerative process involving the cerebellum. Our results suggest prevalent accumulations of insoluble Aß42 in the cerebellum of ET, but not in age-matched PD. Whether this anomaly plays a role in ET symptoms warrants further investigations.

STIM1 participates in the contractile rhythmicity of HL-1 cells by moderating T-type Ca(2+) channel activity.

STIM1 plays a crucial role in Ca(2+) homeostasis, particularly in replenishing the intracellular Ca(2+) store following its depletion. In cardiomyocytes, the Ca(2+) content of the sarcoplasmic reticulum must be tightly controlled to sustain contractile activity. The presence of STIM1 in cardiomyocytes suggests that it may play a role in regulating the contraction of cardiomyocytes. The aim of the present study was to determine how STIM1 participates in the regulation of cardiac contractility. Atomic force microscopy revealed that knocking down STIM1 disrupts the contractility of cardiomyocyte-derived HL-1 cells. Ca(2+) imaging also revealed that knocking down STIM1 causes irregular spontaneous Ca(2+) oscillations in HL-1 cells. Action potential recordings further showed that knocking down STIM1 induces early and delayed afterdepolarizations. Knocking down STIM1 increased the peak amplitude and current density of T-type voltage-dependent Ca(2+) channels (T-VDCC) and shifted the activation curve toward more negative membrane potentials in HL-1 cells. Biotinylation assays revealed that knocking down STIM1 increased T-VDCC surface expression and co-immunoprecipitation assays suggested that STIM1 directly regulates T-VDCC activity. Thus, STIM1 is a negative regulator of T-VDCC activity and maintains a constant cardiac rhythm by preventing a Ca(2+) overload that elicits arrhythmogenic events.

Type 1 inositol-1,4,5-trisphosphate receptor is a late substrate of caspases during apoptosis.

Apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. One of them, the type 1 inositol-1,4,5-trisphosphate receptor (IP(3) R-1), a multimeric receptor located on the endoplasmic reticulum (ER) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (STS) induced-apoptosis in Jurkat cells. Because the reported cleavage site separates the IP(3) binding site from the channel moiety, its cleavage would shut down a critical signaling pathway that is common to several cellular processes. Here we show that IP(3) R-1 is not cleaved in 293 cells treated with STS, TNFα, Trail, or ultra-violet (UV) irradiation. Further, it is not cleaved in Hela or Jurkat cells induced to undergo apoptosis with Trail, TNFα, or UV. In accordance with previous reports, we demonstrate that it is cleaved in a Jurkat cell line treated with STS. However its cleavage occurs only after poly(ADP-ribose) polymerase (PARP), which cleavage is a hallmark of apoptosis, and p23, a poor caspase-7 substrate, are completely cleaved, suggesting that IP(3) R-1 is a relatively late substrate of caspases. Nevertheless, the receptor is fully accessible to proteolysis in cellulo by ectopically overexpressed caspase-7 or by the tobacco etch virus (TEV) protease. Finally, using recombinant caspase-3 and microsomal fractions enriched in IP(3) R-1, we show that the receptor is a poor caspase-3 substrate. Consequently, we conclude that IP(3) R-1 is not a key death substrate.

The activation state of the inositol 1,4,5-trisphosphate receptor regulates the velocity of intracellular Ca2+ waves in bovine aortic endothelial cells.

Ca(2+) is a highly versatile second messenger that plays a key role in the regulation of many cell processes. This versatility resides in the fact that different signals can be encoded spatio-temporally by varying the frequency and amplitude of the Ca(2+) response. A typical example of an organized Ca(2+) signal is a Ca(2+) wave initiated in a given area of a cell that propagates throughout the entire cell or within a specific subcellular region. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP(3) R) is responsible for the release of Ca(2+) from the endoplasmic reticulum. IP(3) R activity can be directly modulated in many ways, including by interacting molecules, proteins, and kinases such as PKA, PKC, and mTOR. In the present study, we used a videomicroscopic approach to measure the velocity of Ca(2+) waves in bovine aortic endothelial cells under various conditions that affect IP(3) R function. The velocity of the Ca(2+) waves increased with the intensity of the stimulus while extracellular Ca(2+) had no significant impact on wave velocity. Forskolin increased the velocity of IP(3) R-dependent Ca(2+) waves whereas PMA and rapamycin decreased the velocity. We used scatter plots and Pearson's correlation test to visualize and quantify the relationship between the Ca(2+) peak amplitude and the velocity of Ca(2+) waves. The velocity of IP(3) R-dependent Ca(2+) waves poorly correlated with the amplitude of the Ca(2+) response elicited by agonists in all the conditions evaluated, indicating that the velocity depended on the activation state of IP(3) R, which can be modulated in many ways.

Microfilament and microtubule assembly is required for the propagation of inositol trisphosphate receptor-induced Ca2+ waves in bovine aortic endothelial cells.

Ca2+ is a highly versatile second messenger that plays a key role in the regulation of numerous cell processes. One-way cells ensure the specificity and reliability of Ca2+ signals is by organizing them spatially in the form of waves that propagate throughout the cell or within a specific subcellular region. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is responsible for the release of Ca2+ from the endoplasmic reticulum. The spatial aspect of the Ca2+ signal depends on the organization of various elements of the Ca2+ signaling toolkit and varies from tissue to tissue. Ca2+ is implicated in many of endothelium functions that thus depend on the versatility of Ca2+ signaling. In the present study, we showed that the disruption of caveolae microdomains in bovine aortic endothelial cells (BAEC) with methyl-beta-cyclodextrin was not sufficient to disorganize the propagation of Ca2+ waves when the cells were stimulated with ATP or bradykinin. However, disorganizing microfilaments with latrunculin B and microtubules with colchicine both prevented the formation of Ca2+ waves. These results suggest that the organization of the Ca2+ waves mediated by IP3R channels does not depend on the integrity of caveolae in BAEC, but that microtubule and microfilament cytoskeleton assembly is crucial.

Sphingosine-1-phosphate induces the association of membrane-type 1 matrix metalloproteinase with p130Cas in endothelial cells.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in sphingosine-1-phosphate(S1P)-dependent migration of endothelial cells but the underlying mechanisms remain largely unknown. Herein, we show that S1P promotes the relocalization of MT1-MMP to peripheral actin-rich membrane ruffles that is coincident with its association with the adaptor protein p130Cas at the leading edge of migrating cells. Immunoprecipitation and confocal microscopy analyses suggest that this interaction required the tyrosine phosphorylation of p130Cas and also involves S1P-dependent phosphorylation of MT1-MMP within its cytoplasmic sequence. The interaction of MT1-MMP with p130Cas at the cell periphery suggests the existence of a close interplay between pericellular proteolysis and signaling pathways involved in EC migration.

Antibody-conjugated mesoporous silica nanoparticles for brain microvessel endothelial cell targeting.

Brain microvessel endothelial cells (BMECs) are the main structural and dynamic components of the blood-brain barrier (BBB), preventing the majority of drugs from reaching the brain. Since BMECs are involved in a wide range of central nervous system diseases, the development of nanocarriers that trigger receptor-mediated uptake in these cells has been suggested as a promising approach to an increased drug delivery to the brain. Here, we report the size and the bioconjugation effects of antibody-conjugated mesoporous silica nanoparticles (MSNs) on in vitro and in vivo targeting ability to BMECs. For this, Ri7 antibody was conjugated to MSNs of two different sizes (50 nm and 160 nm in diameter) through a polyethylene glycol (PEG) linker. The particles were also functionalized with a MRI contrast agent (gadolinium chelate) and with a fluorescent label. The functionalized MSN suspensions showed good colloidal stability. The Ri7 antibody immobilized on the MSN surface maintained its high specific activity and high binding affinity, as demonstrated in vitro. Cells incubated with gadolinium-chelated Ri7-MSNs showed a significant MRI positive contrast enhancement, highlighting the potential of such nanoparticles for theranostic applications. To measure the uptake and affinity of Ri7-MSNs to brain endothelial and neuronal cells, cell uptake studies were performed and a quantitative cellular assay was developed. The results revealed that endocytosis of nanoparticles is mediated by transferrin receptors and that Ri7-MSN cellular uptake is size- and time-dependent. A highest specific uptake was found with 50 nm Ri7-MSNs. Upon intravenous injection, 50 nm Ri7-MSNs were specifically accumulated in BMECs, suggesting the strong potential of antibody-coated nanoparticles for targeting BMECs in vivo. These findings open the door to therapeutic targeting of BMECs, enabling potential therapeutic drug delivery to the brain.

STIM1 positively regulates the Ca2+ release activity of the inositol 1,4,5-trisphosphate receptor in bovine aortic endothelial cells.

The endothelium is actively involved in many functions of the cardiovascular system, such as the modulation of arterial pressure and the maintenance of blood flow. These functions require a great versatility of the intracellular Ca2+ signaling that resides in the fact that different signals can be encoded by varying the frequency and the amplitude of the Ca2+ response. Cells use both extracellular and intracellular Ca2+ pools to modulate the intracellular Ca2+ concentration. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R), located on the endoplasmic reticulum (ER), is responsible for the release of Ca2+ from the intracellular store. The proteins STIM1 and STIM2 are also located on the ER and they are involved in the activation of a store-operated Ca2+ entry (SOCE). Due to their Ca2+ sensor property and their close proximity with IP3Rs on the ER, STIMs could modulate the activity of IP3R. In this study, we showed that STIM1 and STIM2 are expressed in bovine aortic endothelial cells and they both interact with IP3R. While STIM2 appears to play a minor role, STIM1 plays an important role in the regulation of agonist-induced Ca2+ mobilization in BAECs by a positive effect on both the SOCE and the IP3R-dependent Ca2+ release.