Dysregulated copper transport in multiple sclerosis may cause demyelination via astrocytes

Demyelination is a key pathogenic feature of multiple sclerosis (MS). Here, we evaluated the astrocyte contribution to myelin loss and focused on the neurotrophin receptor TrkB, whose up-regulation on the astrocyte finely demarcated chronic demyelinated areas in MS and was paralleled by neurotrophin loss. Mice lacking astrocyte TrkB were resistant to demyelination induced by autoimmune or toxic insults, demonstrating that TrkB signaling in astrocytes fostered oligodendrocyte damage. In vitro and ex vivo approaches highlighted that astrocyte TrkB supported scar formation and glia proliferation even in the absence of neurotrophin binding, indicating TrkB transactivation in response to inflammatory or toxic mediators. Notably, our neuropathological studies demonstrated copper dysregulation in MS and model lesions and TrkB-dependent expression of copper transporter (CTR1) on glia cells during neuroinflammation. In vitro experiments evidenced that TrkB was critical for the generation of glial intracellular calcium flux and CTR1 up-regulation induced by stimuli distinct from neurotrophins. These events led to copper uptake and release by the astrocyte, and in turn resulted in oligodendrocyte loss. Collectively, these data demonstrate a pathogenic demyelination mechanism via the astrocyte release of copper and open up the possibility of restoring copper homeostasis in the white matter as a therapeutic target in MS.

A novel Ca2+ indicator for long-term tracking of intracellular calcium flux

The major drawback of using Fluo-4 AM is that it requires an organic anion transporter inhibitor, such as probenecid, to prevent leakage. This can hinder the studies that require extended monitoring time and longer cellular retention. To address the issue, a novel Ca2+ indicator, Calbryte 520 AM, was developed. We compared the performance of Fluo-4 AM and Calbryte 520 AM following prolonged incubation periods after cell loading. Cells loaded with Calbryte 520 AM retained the dye for up to 24 h while exhibiting significant fluorescence brightness and superior Fmax/F0 ratios (Fmax: fluorescence intensity upon stimulation; F0: intensity before stimulation). It demonstrated that the longer retention of Calbryte 520 AM can be exploited to accommodate for the extended time required when monitoring calcium dynamics.

Insulin-like Growth Factor-1 Regulates the Mechanosensitivity of Chondrocytes by Modulating TRPV4

ABSTRACTBoth mechanical and IGF-1 stimulation are required for normal articular cartilage development and maintenance of the extracellular matrix. While much effort has been made to define the signaling pathways associated with these anabolic stimuli, we focused on how these pathways interact to regulate chondrocyte function. The Transient Receptor Potential Vanilloid 4 (TRPV4) channel is central to chondrocyte mechanotransduction and regulation of cartilage homeostasis. However, the mechanism by which TRPV4 is mechanically gated or regulated is not clear. In this study we propose that insulin-like growth factor 1 (IGF-1), which is important in regulating matrix production during mechanical load, modulates TRPV4 channel activity. Our studies indicate that IGF-1 reduces hypotonic-induced TRPV4 currents, and intracellular calcium flux by increasing stress fiber formation and apparent cell stiffness. Disruption of F-actin following IFG-1 treatment results in the return of the intracellular calcium response to hypotonic swelling. Furthermore, we highlight that IGF-1 suppresses TRPV4 mediated calcium flux through the MAP7 binding domain (aa. 798-809), where actin binds to the TRPV4 channel. IGF-1 treatment differentially influences the intracellular calcium flux of HEK 293 cells stably expressing either wild-type or mutant (P799L or G800D) TRPV4 during hypotonic challenge. A key down-stream response to mechanical stimulation of chondrocytes is ATP release. Data here indicate that activation of TRPV4 through hypotonic swelling induces ATP release, but this release is greatly reduced with IGF-1 treatment. Taken together this study indicates that IGF-1 modulates TRPV4 channel response to mechanical stimulation by increasing cell stiffness. As chondrocyte response to mechanical stimulation is greatly altered during OA progression, IGF-1 presents as a promising candidate for prevention and treatment of articular cartilage damage.

Heat induces interleukin-6 in skeletal muscle cells via TRPV1/PKC/CREB pathways

Interleukin-6 (IL-6) is released from skeletal muscle cells and induced by exercise, heat, catecholamine, glucose, lipopolysaccharide, reactive oxygen species, and inflammation. However, the mechanism that induces release of IL-6 from skeletal muscle cells remains unknown. Thermosensitive transient receptor potential (TRP) proteins such as TRPV1–4 play vital roles in cellular functions. In this study we hypothesized that TRPV1 senses heat, transmits a signal into the nucleus, and produces IL-6. The purpose of the present study is to investigate the underlying mechanisms whereby skeletal muscle cells sense and respond to heat. When mouse myoblast cells were exposed to 37–42°C for 2 h, mRNA expression of IL-6 increased in a temperature-dependent manner. Heat also increased IL-6 secretion in myoblast cells. A fura 2 fluorescence dual-wavelength excitation method showed that heat increased intracellular calcium flux in a temperature-dependent manner. Intracellular calcium flux and IL-6 mRNA expression were increased by the TRPV1 agonists capsaicin and N-arachidonoyldopamine and decreased by the TRPV1 antagonists AMG9810 and SB366791 and siRNA-mediated knockdown of TRPV1. TRPV2, 3, and 4 agonists did not change intracellular calcium flux. Western blotting with inhibitors demonstrated that heat increased phosphorylation levels of TRPV1, followed by PKC and cAMP response element-binding protein (CREB). PKC inhibitors, Gö6983 and staurosporine, CREB inhibitors, curcumin and naphthol AS-E, and knockdown of CREB suppressed the heat-induced increases in IL-6. These results indicate that heat increases IL-6 in skeletal muscle cells through the TRPV1, PKC, and CREB signal transduction pathway.NEW & NOTEWORTHY Heat increases the release of interleukin-6 (IL-6) from skeletal muscle cells. IL-6 has been shown to serve immune responses and metabolic functions in muscle. It can be anti-inflammatory as well as proinflammatory. However, the mechanism that induces release of IL-6 from skeletal muscle cells remains unknown. Here we show that heat increases IL-6 in skeletal muscle cells through the transient receptor potential vannilloid 1, PKC, and cAMP response element-binding protein signal transduction pathway.

Force-Induced Cooperative Unfolding of Two Distinctive Domains in a Single Gpibalpha Molecule

GPIbα, a major member of the GPIb-IX-V complex, initiates a mechano-signaling pathway that leads to platelet intracellular calcium flux when binding to VWF at the A1 domain1. Exactly how this signal is transduced across the membrane is unknown. A recent work identifying the unfolding of a jextamembrane mechanosensitive domain (MSD) (Fig.1 A,C) suggested that this unfolding might play a role in the signal transduction of GPIbα2. Using molecular dynamics (MD) simulations to pull the GPIbα leucine-rich repeat domain (LRRD) from the VWF A1 domain, we observed unfolding of the LRRD (Fig.1 A) (manuscript under review). Here we used a biomembrane force probe (BFP) to study single GPIbα unfolding (Fig. 1E,F). Platelet GPIbα was pulled by A1 coated on the probe through a ramping phase (pink, Fig. 1G) to a clamping phase to wait for bond dissociation under a 25-pN force (red, Fig. 1G). Two unfolding signatures were identified: i) ramped unfolding, featured by a sudden force kink in the ramping phase (dashed circle, Fig. 1G); ii) clamped unfolding, featured by an abrupt force drop in the clamping phase (dashed ellipse, Fig. 1G). Unfolding lengths from both signatures were measured from the force vs. time data (Fig. 1G), based on which the unfolding events of MSD (7-25 nm), LRRD (25-56 nm) or both together (56-80 nm) were differentiated. Intriguingly, LRRD unfolding was only observed in the ramping phase, while MSD could unfold in both ramping and clamping phases. The frequency of observing both LRRD and MSD unfolding in the same cycle was much higher than the product of the frequencies for LRRD and MSD to unfold (the joint probability for both to unfold if they were independent), suggesting that the two GPIbα domains unfolded cooperatively (Fig 2A). Chi-square tests also rejected the hypothesis that MSD and LRRD unfolded independently (p =5.24×10-5). Separating the ramped and clamped unfolding of MSD and evaluating their respective cooperativity with LRRD unfolding demonstrated similar results (Fig 2A). Agreeing with these, both the ramped and clamped unfolding of MSD occurred much more frequently in the presence of LRRD unfolding (Fig 2B, left), and vice versa for the unfolding of LRRD (Fig 2B, right). Cooperative unfolding of LRRD and MSD may play a key role in transducing signals across platelet membrane. References 1 Nesbitt, W. S. et al. The Journal of biological chemistry277, 2965-2972, doi:10.1074/jbc.M110070200 (2002). 2 Zhang, W. et al. Blood125, 562-569, doi:10.1182/blood-2014-07-589507 (2015). Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Identification and Characterization of Integrin alphaIIbbeta3 Intermediate Affinity State Induced By Gpibalpha Mechanotransduction

During arterial haemostasis, platelets tether to and translocate on disrupted vascular surface via binding of GPIbα to the VWF A1 domain, which triggers activation signals that induce intracellular Ca2+ release and up-regulate integrin αIIbβ3 binding capacity1,2. Inhibition of this signaling pathway or blockade of αIIbβ3 binding has a devastating impact on platelet firm adhesion and aggregate formation1,3,4. We used a newly developed switch biomembrane force probe (BFP) assay to characterize the cooperativity between GPIbα and αIIbβ3 on single-molecular level. VWF-A1 and fibronectin fragment (FNIII7-10) were separately coated on two different probes, enabling controlled presentation of two ligands in separate space and time (Fig. A). Pulling GPIbα by A1 on the first probe with a durable force activated the discoid platelet (Fig. A, upper panel), as revealed by an intracellular calcium flux. Upon switching to the FNIII7-10-bearing bead, the A1-triggered platelet was interrogated for the activity of its surface αIIbβ3 (Fig. A, lower panel). A 25-pN force of >2s duration on a single GPIbα-A1 bond was found to activate αIIbβ3 into an intermediate affinity state, as reflected by a ~30% adhesion frequency with a FNIII7-10 bead, significantly higher than binding to low affinity αIIbβ3 on platelets without A1-triggering but much lower than binding to high affinity αIIbβ3 on platelets pre-incubated with ADP or thrombin (Fig. B), two strong platelet-activating agonists. This submaximal activation agreed with the previous immuno-staining findings2. αIIbβ3 activation and outside-in signaling require the sequential engagement of talin and Gα13 to β3 cytoplasmic tail, respectively5,6. Blocking Gα13-β3 engagement by a synthetic peptide mP6 (gift from Xiaoping Du, UIC)6 had no effect on FNIII7-10 binding of resting and A1-triggered platelets, but significantly suppressed the FNIII7-10 binding of platelets pre-incubated with ADP or thrombin by lowering their adhesion frequencies to a level comparable with A1-triggered platelets (Fig. B). The control peptide mP6Scr showed no effect on the ADP or thrombin stimulated platelets (Fig. B). In addition to binding affinity, bond lifetimes were measured by force-clamp experiment. Platelets with no treatment, A1 triggering and ADP stimulation formed αIIbβ3-FNIII7-10 (Fig. C) and αIIbβ3-fibrinogen (Fig. D) bonds with short, intermediate and long lifetimes, respectively. Interactions with fibrinogen exhibited catch-slip bonds regardless of the αIIbβ3 affinity state. By comparison, only high affinity αIIbβ3 exhibited a catch-slip bond with FNIII7-10 as slip-only bonds were observed for FNIII7-10 interactions with intermediate and low affinity αIIbβ3. These data suggest the existence of three distinct integrin states that correspond to the three affinities and force-dependent bond lifetime patterns, in which the intermediate state was induced by the GPIbα mechanotransduction (Fig. E). Gα13 engagement was not required for the intermediate state, but necessary for the high affinity state (Fig. E). References: 1. Nesbitt, W. S. et al. The Journal of biological chemistry277, 2965-2972, doi:10.1074/jbc.M110070200 (2002). 2. Kasirer-Friede, A. et al. Blood103, 3403-3411, doi:10.1182/blood-2003-10-3664 (2004). 3. Savage, B., Saldivar, E. & Ruggeri, Z. M. Cell84, 289-297 (1996). 4. Warwick, S. N. et al. Nature Medicine15, doi:10.1038/nm.1955 (2009). 5. Gong, H. et al. Science327, 340-343, doi:10.1126/science.1174779 (2010). 6. Shen, B. et al. Nature503, 131-135, doi:10.1038/nature12613 (2013). Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.