• Brain-derived cellular microparticles induce systemic coagulopathy in traumatic brain injury.• Platelets facilitate the transmigration of brain microparticles through the endothelial barrier into the circulation.Traumatic brain injury (TBI) is associated with coagulopathy, although it often lacks 2 key risk factors: severe bleeding and significant fluid resuscitation associated with hemorrhagic shock. The pathogenesis of TBI-associated coagulopathy remains poorly understood. We tested the hypothesis that brain-derived microparticles (BDMPs) released from an injured brain induce a hypercoagulable state that rapidly turns into consumptive coagulopathy. Here, we report that mice subjected to fluid percussion injury (1.9 6 0.1 atm) developed a BDMP-dependent hypercoagulable state, with peak levels of plasma glial cell and neuronal BDMPs reaching 17 496 6 4833/mL and 18 388 6 3657/mL 3 hours after TBI, respectively. Uninjured mice injected with BDMPs developed a dosedependent hyper-turned hypocoagulable state measured by a progressively prolonged clotting time, fibrinogen depletion, and microvascular fibrin deposition in multiple organs. The BDMPs were 50 to 300 nm with intact membranes, expressing neuronal or glial cell markers and procoagulant phosphatidylserine and tissue factor. Their procoagulant activity was greater than platelet microparticles and was dose-dependently blocked by lactadherin. Microparticles were produced from injured hippocampal cells, transmigrated through the disrupted endothelial barrier in a platelet-dependent manner, and activated platelets. These data define a novel mechanism of TBI-associated coagulopathy in mice, identify early predictive markers, and provide alternative therapeutic targets. (Blood. 2015;125(13):2151-2159

To examine the diversity of astrocytes in the human brain, we immunostained surgical specimens of temporal cortex and hippocampus and autopsy brains for CD44, a plasma membrane protein and extracellular matrix receptor. CD44 antibodies outline the details of astrocyte morphology to a degree not possible with glial fibrillary acidic protein (GFAP) antibodies. CD44ϩ astrocytes could be subdivided into two groups. First, CD44ϩ astrocytes with long processes were consistently found in the subpial area ("interlaminar" astrocytes), the deep isocortical layers, and the hippocampus. Many of these processes ended on blood vessels. Some were also found adjacent to large blood vessels, from which they extended long processes. We observed these CD44ϩ, long-process astrocytes in every brain we examined, from fetal to adult. These astrocytes generally displayed high immunostaining for GFAP, S100␤, and CD44, but low immunostaining for glutamine synthetase, excitatory amino-acid transporter 1 (EAAT1), and EAAT2. Aquaporin 4 (AQP4) appeared distributed all over the cell bodies and processes of the CD44ϩ astrocytes, while, in contrast, AQP4 localized to perivascular end feet in the CD44Ϫ protoplasmic astrocytes. Second, there were CD44ϩ astrocytes without long processes in the cortex. These were not present during gestation or at birth, and in adult brains varied substantially in number, shape, and immunohistochemical phenotype. Many of these displayed a "mixed" morphological and immunocytochemical phenotype between protoplasmic and fibrous astrocytes. We conclude that the diversity of astrocyte populations in the isocortex and archicortex in the human brain reflects both intrinsic and acquired phenotypes, the latter perhaps representing a shift from CD44Ϫ "protoplasmic" to CD44ϩ "fibrous"-like astrocytes.

Key Points Mitochondria were released from traumatically injured brain into systemic circulation and exposed CL on their surface. CL-exposed mitochondria are highly procoagulant and induced traumatic brain injury–associated coagulopathy.

Normal axonal mitochondrial transport and function is essential for the maintenance of synaptic function. Abnormal mitochondrial motility and mitochondrial dysfunction within axons are critical for amyloid β (Aβ)-induced synaptic stress and the loss of synapses relevant to the pathogenesis of Alzheimer’s disease (AD). However, the mechanisms controlling axonal mitochondrial function and transport alterations in AD remain elusive. Here, we report an unexplored role of cyclophilin D (CypD)-dependent mitochondrial permeability transition pore (mPTP) in Aβ-impaired axonal mitochondrial trafficking. Depletion of CypD significantly protects axonal mitochondrial motility and dynamics from Aβ toxicity as shown by increased axonal mitochondrial density and distribution and improved bidirectional transport of axonal mitochondria. Notably, blockade of mPTP by genetic deletion of CypD suppresses Aβ-mediated activation of the p38 mitogen-activated protein kinase signaling pathway, reverses axonal mitochondrial abnormalities, improves synaptic function, and attenuates loss of synapse, suggesting a role of CypD-dependent signaling in Aβ-induced alterations in axonal mitochondrial trafficking. The potential mechanisms of the protective effects of lacking CypD on Aβ-induced abnormal mitochondrial transport in axon are increased axonal calcium buffer capability, diminished reactive oxygen species (ROS), and suppressing downstream signal transduction P38 activation. These findings provide new insights into CypD-dependent mitochondrial mPTP and signaling on mitochondrial trafficking in axons and synaptic degeneration in an environment enriched for Aβ.

Multidrug resistance-associated protein 3 (MRP3, ABCC3) plays an important role in protecting hepatocytes and other tissues by excreting an array of toxic organic anion conjugates, including bile salts. MRP3/ABCC3 expression is increased in the liver of some cholestatic patients, but the molecular mechanism of this up-regulation remains elusive. In this report, we assessed liver MRP3/ABCC3 expression in patients (n=22) with obstructive cholestasis due to gallstones blockage of bile ducts and non-cholestatic patient controls (n=22). MRP3/ABCC3 mRNA and protein expression were significantly increased 3.4- and 4.6- fold, respectively in these cholestatic patients where elevated plasma TNFα (4.7-fold, P<0.01) and hepatic SP1 and LRH-1 expression (3.1- and 2.1-fold at mRNA level, 3.5- and 2.5-fold at protein level, respectively) were also observed. The induction of hepatic MRP3/ABCC3 mRNA expression is significantly positively correlated with the level of plasma TNFα in these patients. In HepG2 cells, TNFα treatment induced SP1 and MRP3/ABCC3 expression in a dose- and time-dependent manner, where increased phosphorylation of JNK/SAPK was also detected. These inductions were significantly reduced in the presence of the JNK inhibitor SP600125. TNFα treatment enhanced HepG2 cell nuclear extract binding activity to the MRP3/ABCC3 promoter, but was abolished by SP600125 as demonstrated by EMSA. An increase in nuclear protein binding activity to the MRP3/ABCC3 promoter consisting primarily of SP1 was also seen in liver samples from cholestatic patients as assessed by supershift EMSA assays. Conclusions Our findings indicate that up-regulation of hepatic MRP3/ABCC3 expression in human obstructive cholestasis is likely triggered by TNFα, mediated by activations of JNK/SAPK and SP1.

Like many other cell surface receptors, transforming growth factor ␤ (TGF-␤) receptors are internalized upon ligand stimulation. Given that the signaling-facilitating molecules Smad anchor for receptor activation (SARA) and Hrs are mainly localized in early endosomes, it was unclear whether receptor internalization is required for Smad2 activation. Using reversible biotin labeling, we directly monitored internalization of the TGF-␤ type I receptor. Our data indicate that TGF-␤ type I receptor is endocytosed via a clathrin-dependent mechanism and is effectively blocked by depletion of intracellular potassium or by expression of a mutant dynamin (K44A). However, blockage of receptor endocytosis by these two means has no effect on TGF-␤-mediated Smad2 activation. Furthermore, TGF-␤-induced Smad2 activation was unaffected by inhibition of hVPS34 activity with wortmannin or inhibitory anti-hVPS34 antibodies. Finally, we demonstrated that Smad2 interacted with cell surface receptors and that a SARA binding-deficient Smad2 mutant was phosphorylated by the receptors. Thus, our findings suggest that receptor endocytosis is dispersible for TGF-␤-mediated activation of Smad2 and that this activation can be mediated by both SARA-dependent and -independent mechanisms.Receptor endocytosis has long been regarded as an attenuation mechanism to switch off receptor signaling. However, accumulating evidence suggests that endocytosis may facilitate signaling by targeting signaling complexes to specific subcellular localization, either to increase access of activated receptor kinases to their substrates or to compartmentalize signaling complexes (1-3). Consistent with this idea, blocking of clathrinmediated endocytosis was found to attenuate the activation of the extracellular signal-regulated kinases by receptor tyrosine kinases and G-protein-coupled receptors (4 -7). Upon the activation of G-protein-coupled proteinase-activated receptor 2, a multiprotein signaling complex that contains ␤-arrestin 1, Raf-1, and extracellular signal-regulated kinases is formed on endocytic vesicles as well (8).TGF-␤ 1 binds to its cell surface receptors, resulting in the formation of type I and type II receptor complexes. In the complex, the TGF-␤ type II receptor (T␤RII) phosphorylates and activates the TGF-␤ type I receptor (T␤RI), which in turn phosphorylates the C-terminal serine residues of Smad proteins Smad2 and Smad3. As a result, Smad proteins accumulate in the nucleus, bind to DNA, and regulate transcription. The ligand-stimulated receptor complexes undergo endocytosis and are eventually degraded in an ubiquitin/lysosome-dependent pathway (9, 10).The FYVE domain-containing proteins Smad anchor for receptor activation (SARA) and Hrs (the hepatocyte growth factor-regulated tyrosine kinase substrate) have been suggested to facilitate Smad2 phosphorylation by bringing Smad2 to TGF-␤ or activin receptors (11,12). Interestingly, immunofluorescence studies revealed that both SARA and Hrs are predominantly localized in early endosomes (11,13). Thi...

von Willebrand factor (VWF) is an adhesive ligand, and its activity is proteolytically regulated by the metalloprotease ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat 13). An elevated level of plasma VWF has been widely considered a marker for endothelial cell activation in trauma and inflammation, but its causal role in these pathological conditions remains poorly defined. Using a fluid percussion injury mouse model, we demonstrated that VWF released during acute traumatic brain injury (TBI) was activated and became microvesicle-bound. The VWF-bound microvesicles promoted vascular leakage and systemic coagulation. Recombinant ADAMTS-13 given either before or after TBI reduced the VWF reactivity with minimal influence on VWF secretion. rADAMTS-13 protected the integrity of endothelial cell barriers and prevented TBI-induced coagulopathy by enhancing VWF cleavage without impairing basal hemostasis. Promoting microvesicle clearance by lactadherin had efficacy similar to that of rADAMTS-13. This study uncovers a novel synergistic action between VWF and cellular microvesicles in TBI-induced vascular leakage and coagulopathy and demonstrates protective effects of rADAMTS-13.

SUMMARYPurpose: Cortical tubers are epileptogenic lesions in patients with tuberous sclerosis complex (TSC). Giant cells and dysplastic neurons are pathological hallmarks of cortical tubers. Severe astrogliosis, which is invariably present in tubers, has attracted much less attention. We hypothesize that the development of astrogliosis in cortical tubers constitutes a primary pathology of astrocytes and is directly related to TSC 1/2 mutations. Methods: To begin to test this hypothesis, we performed immunohistochemical and electron microscopic analysis of brain tuber tissue resected from epileptic TSC patients. We compared alterations in tuber astrocytes to those found in other acute and chronic human epilepsy pathologies. Results: We found that astrogliosis in tubers is comprised of a mixture of "gliotic" and "reactive" astrocytes. The majority of tuber astrocytes are "gliotic" astrocytes that are morphologically and immunophenotypically similar to astrocytes in areas of gliosis in hippocampal sclerosis (HS). However, specific immunostaining features differentiate TSC gliosis from HS gliosis. "Reactive" tuber astrocytes are large-sized, vimentin positive cells in the vicinity of giant cells that show activation of the mammalian target of rapamycin (mTOR) pathway, consistent with mutated TSC gene function. These cells resemble acutely reactive human astrocytes seen in tissue resected from depth electrode implantation patients. Oligodendrocytes and NG2 expressing glial cells do not have any detectable alterations within tubers. Conclusion: We conclude that astrocytes are the type of glial cell selectively impacted in cortical tuber pathology. We propose that tubers may be dynamic lesions, with progression of astrocytes over time from "reactive" to "gliotic." Tuber astrogliosis in TSC may represent a genetic "model" of gliosis that is phenotypically similar to gliosis seen in acquired human pathologies.