Extrasynaptic transmission and the diffusion parameters of the extracellular space

Extrasynaptic volume transmission, mediated by the diffusion of neuroactive substances in the extracellular space (ECS), plays an important role in short- and long-distance communication between nerve cells. The ability of a substance to reach extrasynaptic high-affinity receptors via diffusion depends on the ECS diffusion parameters, ECS volume fraction alpha (alpha=ECS volume/total tissue volume) and tortuosity lambda (lambda2=free/apparent diffusion coefficient), which reflects the presence of diffusion barriers represented by, e.g., fine astrocytic processes or extracellular matrix molecules. These barriers channel the migration of molecules in the ECS, so that diffusion may be facilitated in a certain direction, i.e. anisotropic. The diffusion parameters alpha and lambda differ in various brain regions, and diffusion in the CNS is therefore inhomogeneous. Changes in diffusion parameters have been found in many physiological and pathological states, such as development and aging, neuronal activity, lactation, ischemia, brain injury, degenerative diseases, tumor growth and others, in which cell swelling, glial remodeling and extracellular matrix changes are key factors influencing diffusion. Changes in ECS volume, tortuosity and anisotropy significantly affect the accumulation and diffusion of neuroactive substances and thus extrasynaptic transmission, neuron-glia communication, mediator "spillover" and synaptic crosstalk as well as, cell migration. The various changes occurring during pathological states can be important for diagnosis, drug delivery and treatment.     

Glial cells and volume transmission in the CNS

Although synaptic transmission is an important means of communication between neurons, neurons themselves and neurons and glia also communicate by extrasynaptic "volume" transmission, which is mediated by diffusion in the extracellular space (ECS). The ECS of the central nervous system (CNS) is the microenvironment of neurons and glial cells. The composition and size of ECS change dynamically during neuronal activity as well as during pathological states. Following their release, a number of neuroactive substances, including ions, mediators, metabolites and neurotransmitters, diffuse via the ECS to targets distant from their release sites. Glial cells affect the composition and volume of the ECS and therefore also extracellular diffusion, particularly during development, aging and pathological states such as ischemia, injury, X-irradiation, gliosis, demyelination and often in grafted tissue. Recent studies also indicate that diffusion in the ECS is affected by ECS volume inhomogeneities, which are the result of a more compacted space in certain regions, e.g. in the vicinity of oligodendrocytes. Besides glial cells, the extracellular matrix also changes ECS geometry and forms diffusion barriers, which may also result in diffusion anisotropy. Glial cells therefore play an important role in extrasynaptic transmission, for example in functions such as vigilance, sleep, depression, chronic pain, LTP, LTD, memory formation and other plastic changes in the CNS. In turn, ECS diffusion parameters affect neuron-glia communication, ionic homeostasis and movement and/or accumulation of neuroactive substances in the brain.     

Diffusion properties of the brain in health and disease

Extrasynaptic transmission between neurons and communication between neurons and glia are mediated by the diffusion of neuroactive substances in the extracellular space (ECS)--volume transmission. Diffusion in the CNS is inhomogeneous and often not uniform in all directions (anisotropic). Ionic changes and amino acid release result in cellular (particularly glial) swelling, compensated for by ECS shrinkage and a decrease in the apparent diffusion coefficients of neuroactive substances or water (ADCW). The diffusion parameters of the CNS in adult mammals (including humans), ECS volume fraction alpha (alpha = ECS volume/total tissue volume; normally 0.20-0.25) and tortuosity lambda (lambda2 = D/ADC; normally 1.5-1.6), hinder the diffusion of neuroactive substances and water. A significant decrease in ECS volume and an increase in diffusion barriers (tortuosity) and anisoptropy have been observed during stimulation, lactation or learning deficits during aging, due to structural changes such as astrogliosis, the re-arrangement of astrocytic processes and a loss of extracellular matrix. Decreases in the apparent diffusion coefficient of tetramethylammonium (ADCTMA) and ADCW due to astrogliosis and increased proteoglycan expression were found in the brain after injury and in grafts of fetal tissue. Tenascin-R and tenascin C-deficient mice also showed significant changes in ADCTMA and ADCW, suggesting an important role for extracellular matrix molecules in ECS diffusion. Changes in ECS volume, tortuosity and anisotropy significantly affect neuron-glia communication, the spatial relation of glial processes towards synapses, the efficacy of glutamate or GABA 'spillover' and synaptic crosstalk, the migration of cells, the action of hormones and the toxic effects of neuroactive substances and can be important for diagnosis, drug delivery and new treatment strategies.     

The Impact of Alpha-Syntrophin Deletion on the Changes in Tissue Structure and Extracellular Diffusion Associated with Cell Swelling under Physiological and Pathological Conditions

Aquaporin-4 (AQP4) is the primary cellular water channel in the brain and is abundantly expressed by astrocytes along the blood-brain barrier and brain-cerebrospinal fluid interfaces. Water transport via AQP4 contributes to the activity-dependent volume changes of the extracellular space (ECS), which affect extracellular solute concentrations and neuronal excitability. AQP4 is anchored by α-syntrophin (α-syn), the deletion of which leads to reduced AQP4 levels in perivascular and subpial membranes. We used the real-time iontophoretic method and/or diffusion-weighted magnetic resonance imaging to clarify the impact of α-syn deletion on astrocyte morphology and changes in extracellular diffusion associated with cell swelling in vitro and in vivo. In mice lacking α-syn, we found higher resting values of the apparent diffusion coefficient of water (ADC_W) and the extracellular volume fraction (α). No significant differences in tortuosity (λ) or non-specific uptake (k′), were found between α-syn-negative (α-syn −/−) and α-syn-positive (α-syn +/+) mice. The deletion of α-syn resulted in a significantly smaller relative decrease in α observed during elevated K⁺ (10 mM) and severe hypotonic stress (−100 mOsmol/l), but not during mild hypotonic stress (−50 mOsmol/l). After the induction of terminal ischemia/anoxia, the final values of ADC_W as well as of the ECS volume fraction α indicate milder cell swelling in α-syn −/− in comparison with α-syn +/+ mice. Shortly after terminal ischemia/anoxia induction, the onset of a steep rise in the extracellular potassium concentration and an increase in λ was faster in α-syn −/− mice, but the final values did not differ between α-syn −/− and α-syn +/+ mice. This study reveals that water transport through AQP4 channels enhances and accelerates astrocyte swelling. The substantially altered ECS diffusion parameters will likely affect the movement of neuroactive substances and/or trophic factors, which in turn may modulate the extent of tissue damage and/or drug distribution.     

Extracellular space diffusion and extrasynaptic transmission

The diffusion of neuroactive substances in the extracellular space (ECS) plays an important role in short- and long-distance communication between nerve cells and is the underlying mechanism of extrasynaptic (volume) transmission. The diffusion properties of the ECS are described by three parameters: 1. ECS volume fraction alpha (alpha=ECS volume/total tissue volume), 2. tortuosity lambda (lambda2=free/apparent diffusion coefficient), reflecting the presence of diffusion barriers represented by, e.g., fine neuronal and glial processes or extracellular matrix molecules and 3. nonspecific uptake k'. These diffusion parameters differ in various brain regions, and diffusion in the CNS is therefore inhomogeneous. Moreover, diffusion barriers may channel the migration of molecules in the ECS, so that diffusion is facilitated in a certain direction, i.e. diffusion in certain brain regions is anisotropic. Changes in the diffusion parameters have been found in many physiological and pathological states in which cell swelling, glial remodeling and extracellular matrix changes are key factors influencing diffusion. Changes in ECS volume, tortuosity and anisotropy significantly affect the accumulation and diffusion of neuroactive substances in the CNS and thus extrasynaptic transmission, neuron-glia communication, transmitter "spillover" and synaptic cross-talk as well as cell migration, drug delivery and treatment.     

Extracellular matrix as a contextual determinant of transforming growth factor-β signaling in epithelial-mesenchymal transition and in cancer

Extracellular matrix (ECM) provides both structural support and contextual information to cells within tissues and organs. The combination of biochemical and biomechanical signals from the ECM modulates responses to extracellular signals toward differentiation, proliferation, or apoptosis; alterations in the ECM are necessary for development and remodeling processes, but aberrations in the composition and organization of ECM are associated with disease pathology and can predispose to development of cancer. The primary cell surface sensors of the ECM are the integrins, which provide the physical connection between the ECM and the cytoskeleton and also convey biochemical information about the composition of the ECM. Transforming growth factor-β (TGF-β) is an extracellular signaling molecule that is a powerful controller of a variety of cellular functions, and that has been found to induce very different outcomes according to cell type and cellular context. It is becoming clear that ECM-mediated signaling through integrins is reciprocally influenced by TGF-β: integrin expression, activation, and responses are affected by cellular exposure to TGF-β, and TGF-β activation and cellular responses are in turn controlled by signaling from the ECM through integrins. Epithelial-mesenchymal transition (EMT), a physiological process that is activated by TGF-β in normal development and in cancer, is also affected by the composition and structure of the ECM. Here, we will outline how signaling from the ECM controls the contextual response to TGF-β, and how this response is selectively modulated during disease, with an emphasis on recent findings, current challenges, and future opportunities.     

Altered Astrocytic Swelling in the Cortex of α-Syntrophin-Negative GFAP/EGFP Mice

Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K⁺ and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4). As the removal of perivascular AQP4 via the deletion of α-syntrophin was shown to delay edema formation and K⁺ clearance, we aimed to elucidate the impact of α-syntrophin knockout on volume changes in individual astrocytes in situ evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α) in situ and in vivo in the mouse cortex employing the real-time iontophoretic method. RT-qPCR profiling was used to reveal possible differences in the expression of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. To visualize individual astrocytes in mice lacking α-syntrophin we crossbred GFAP/EGFP mice, in which the astrocytes are labeled by the enhanced green fluorescent protein under the human glial fibrillary acidic protein promoter, with α-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that α-syntrophin deletion results in significantly smaller astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD) or 50 mM K⁺. As for the mild stimuli, such as mild hypoosmotic or hyperosmotic stress or 10 mM K⁺, α-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative α changes showed a significantly smaller decrease in α-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of α-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K⁺.     

Integrin-mediated regulation of neurovascular development,physiology and disease

The mammalian central nervous system (CNS) is comprised of billions of neurons and glia that are intertwined with an elaborate network of blood vessels. These various neural and vascular cell types actively converse with one another to form integrated, multifunctional complexes, termed neurovascular units. Cell-cell communication within neurovascular units promotes normal CNS development and homeostasis, and abnormal regulation of these events leads to a variety of debilitating CNS diseases. This review will summarize (1) cellular and molecular mechanisms that regulate physiological assembly and maintenance of neurovascular units; and (2) signaling events that induce pathological alterations in neurovascular unit formation and function. An emphasis will be placed on neural-vascular cell adhesion events mediated by integrins and their extracellular matrix (ECM) ligands. I will highlight the role of a specific adhesion and signaling axis involving αvβ8 integrin, latent transforming growth factor β''s (TGFβ''s), and canonical TGFβ receptors. Possible functional links between components of this axis and other signal transduction cascades implicated in neurovascular development and disease will be discussed. Comprehensively understanding the pathways that regulate bidirectional neural-vascular cell contact and communication will provide new insights into the mechanisms of neurovascular unit development, physiology and disease.Key words: αvβ8 integrin, latent TGFβ, neurovascular unit, brain angiogenesis, cerebral hemorrhage     

Aquaporin-4–dependent K+ and water transport modeled in brain extracellular space following neuroexcitation

Potassium (K⁺) ions released into brain extracellular space (ECS) during neuroexcitation are efficiently taken up by astrocytes. Deletion of astrocyte water channel aquaporin-4 (AQP4) in mice alters neuroexcitation by reducing ECS [K⁺] accumulation and slowing K⁺ reuptake. These effects could involve AQP4-dependent: (a) K⁺ permeability, (b) resting ECS volume, (c) ECS contraction during K⁺ reuptake, and (d) diffusion-limited water/K⁺ transport coupling. To investigate the role of these mechanisms, we compared experimental data to predictions of a model of K⁺ and water uptake into astrocytes after neuronal release of K⁺ into the ECS. The model computed the kinetics of ECS [K⁺] and volume, with input parameters including initial ECS volume, astrocyte K⁺ conductance and water permeability, and diffusion in astrocyte cytoplasm. Numerical methods were developed to compute transport and diffusion for a nonstationary astrocyte–ECS interface. The modeling showed that mechanisms b–d, together, can predict experimentally observed impairment in K⁺ reuptake from the ECS in AQP4 deficiency, as well as altered K⁺ accumulation in the ECS after neuroexcitation, provided that astrocyte water permeability is sufficiently reduced in AQP4 deficiency and that solute diffusion in astrocyte cytoplasm is sufficiently low. The modeling thus provides a potential explanation for AQP4-dependent K⁺/water coupling in the ECS without requiring AQP4-dependent astrocyte K⁺ permeability. Our model links the physical and ion/water transport properties of brain cells with the dynamics of neuroexcitation, and supports the conclusion that reduced AQP4-dependent water transport is responsible for defective neuroexcitation in AQP4 deficiency.     

Syndecan-1-Induced ECM Fiber Alignment Requires Integrin αvβ3 and Syndecan-1 Ectodomain and Heparan Sulfate Chains

Expression of the cell surface proteoglycan syndecan-1 (Sdc1) is frequently induced in stromal fibroblasts of invasive breast carcinomas. We have recently identified a correlation between stromal Sdc1 expression and extracellular matrix (ECM) fiber alignment, both in vitro and in vivo. ECMs derived from Sdc1-positive human mammary fibroblasts (HMF) showed an aligned fiber architecture, which contrasted markedly with the more random fiber arrangement in the ECM produced by Sdc1-negative HMFs. We further demonstrated that aligned fiber architecture promotes the directional migration and invasion of breast carcinoma cells. To decipher the molecular mechanisms governing the formation of an aligned, invasion-permissive ECM, a series of Sdc1 mutants was introduced into HMF. We found that both the ectodomain and heparan sulfate chains of Sdc1 were required for full activity of Sdc1 in regulating ECM alignment, while transmembrane and cytoplasmic domains were dispensable. Sdc1 regulates the activities of several integrins via its ectodomain. Integrins are key players in the assembly of fibronectin-rich ECM. In addition, integrins are capable of regulating cell morphology and cell shape and orientation may affect ECM architecture. Therefore, we investigated the role of integrins in Sdc1-mediated ECM fiber alignment. Sdc1-overexpressing HMF gained an enhanced spindle-shaped morphology when cultured in an overconfluent state under conditions permissive for ECM production, which was partially reversed by siRNA-mediated silencing of β3 integrin expression. Moreover, suppression of αvβ3 integrin activity by a function-blocking antibody or β3 knockdown largely abolished the aligned ECM fiber architecture and consequently the invasion-permissive properties of the ECM induced by Sdc1. The results suggest that Sdc1 may modulate fibronectin fibrillogenesis and/or alter cell morphology during ECM production through αvβ3 integrin, thereby mediating ECM fiber alignment. Understanding the mechanisms governing ECM organization may lead to the development of novel stroma-targeted therapy for breast cancer, aiming at converting an invasion-permissive to an invasion-restrictive microenvironment.     

Hypoxia Inducible Factor-2α Regulates the Development of Retinal Astrocytic Network by Maintaining Adequate Supply of Astrocyte Progenitors

Here we investigate the role of hypoxia inducible factor (HIF)-2α in coordinating the development of retinal astrocytic and vascular networks. Three Cre mouse lines were used to disrupt floxed Hif-2α, including Rosa26^CreERT2, Tie2^Cre, and GFAP^Cre. Global Hif-2α disruption by Rosa26^CreERT2 led to reduced astrocytic and vascular development in neonatal retinas, whereas endothelial disruption by Tie2^Cre had no apparent effects. Hif-2α deletion in astrocyte progenitors by GFAP^Cre significantly interfered with the development of astrocytic networks, which failed to reach the retinal periphery and were incapable of supporting vascular development. Perplexingly, the abundance of strongly GFAP⁺ mature astrocytes transiently increased at P0 before they began to lag behind the normal controls by P3. Pax2⁺ and PDGFRα⁺ astrocytic progenitors and immature astrocytes were dramatically diminished at all stages examined. Despite decreased number of astrocyte progenitors, their proliferation index or apoptosis was not altered. The above data can be reconciled by proposing that HIF-2α is required for maintaining the supply of astrocyte progenitors by slowing down their differentiation into non-proliferative mature astrocytes. HIF-2α deficiency in astrocyte progenitors may accelerate their differentiation into astrocytes, a change which greatly interferes with the replenishment of astrocyte progenitors due to insufficient time for proliferation. Rapidly declining progenitor supply may lead to premature cessation of astrocyte development. Given that HIF-2α protein undergoes oxygen dependent degradation, an interesting possibility is that retinal blood vessels may regulate astrocyte differentiation through their oxygen delivery function. While our findings support the consensus that retinal astrocytic template guides vascular development, they also raise the possibility that astrocytic and vascular networks may mutually regulate each other''s development, mediated at least in part by HIF-2α.     

The brain's interstitial clefts and their glial walls

The heterogeneous contents of the CNS interstitial clefts and the configuration of their astrocytic walls may be regionally variable. Astrocytic processes of the glia limitans, in normal midbrain and in astroglial scars, form thin, parallel, concentric sheets comprising the walls of narrow interstitial clefts. There is a critical thickness of about 20 to 30 nm, below which astrocytic cell process or those of the fibroblast-like cells in the meninges, do not invaginate to form transcytotic vesicles. Large hydrophilic solutes cannot, therefore, pass across the thin portion of a cell process. Consequently, (a) the diffusion and convection paths of interstitial fluid and solutes are lengthened, (b) a solute will remain within the interstitial cleft between thin lamellae for a relatively long time and (c) if a ligand does bind to its receptor on the thin process's cell membrane, there can be no receptor-mediated transcytosis at that site. Interstitial clefts, themselves, vary in size, shape and content, including extracellular matrix and basal lamina. A common constituent of basal lamina and extracellular matrix, presumably including that at ependymal, astroglial and endothelial interfaces of the CNS, is heparan sulfate proteoglycans. As in other organs, these proteoglycans may store growth factors, growth inhibitors, cytokines and other modulators which can then be released enzymatically during, e.g., regeneration. Exogenous heparan sulfate proteoglycan might serve as a natural, intermittent-release matrix for delivery of trophic factors.     

Multilevel regulation of matrix metalloproteinases in tissue homeostasis indicates their molecular specificity in vivo

The matrix metalloproteinases (MMPs) play a crucial role in irreversible remodeling of the extracellular matrix (ECM) in normal homeostasis and pathological states. Accumulating data from various studies strongly suggest that MMPs are tightly regulated, starting from the level of gene expression all the way to zymogen activation and endogenous inhibition, with each level controlled by multiple factors. Recent in vivo findings indicate that cell–ECM and cell–cell interactions, as well as ECM bio-active products, contribute an additional layer of regulation at all levels, indicating that individual MMP expression and activity in vivo are highly coordinated and tissue specific processes.     

Placenta-derived mesenchymal stem cells improve memory dysfunction in an Aβ1–42-infused mouse model of Alzheimer's disease

Mesenchymal stem cells (MSCs) promote functional recoveries in pathological experimental models of central nervous system (CNS) and are currently being tested in clinical trials for neurological disorders, but preventive mechanisms of placenta-derived MSCs (PD-MSCs) for Alzheimer''s disease are poorly understood. Herein, we investigated the inhibitory effect of PD-MSCs on neuronal cell death and memory impairment in Aβ_1–42-infused mice. After intracerebroventrical (ICV) infusion of Aβ_1–42 for 14 days, the cognitive function was assessed by the Morris water maze test and passive avoidance test. Our results showed that the transplantation of PD-MSCs into Aβ_1–42-infused mice significantly improved cognitive impairment, and behavioral changes attenuated the expression of APP, BACE1, and Aβ, as well as the activity of β-secretase and γ-secretase. In addition, the activation of glia cells and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were inhibited by the transplantation of PD-MSCs. Furthermore, we also found that PD-MSCs downregulated the release of inflammatory cytokines as well as prevented neuronal cell death and promoted neuronal cell differentiation from neuronal progenitor cells in Aβ_1–42-infused mice. These data indicate that PD-MSC mediates neuroprotection by regulating neuronal death, neurogenesis, glia cell activation in hippocampus, and altering cytokine expression, suggesting a close link between the therapeutic effects of MSCs and the damaged CNS in Alzheimer''s disease.     

Microglia convert aggregated amyloid-β into neurotoxic forms through the shedding of microvesicles

Alzheimer''s disease (AD) is characterized by extracellular amyloid-β (Aβ) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that soluble pre-fibrillar Aβ species, rather than insoluble fibrils, are the most toxic forms of Aβ. Preventing soluble Aβ formation represents, therefore, a major goal in AD. We investigated whether microvesicles (MVs) released extracellularly by reactive microglia may contribute to AD degeneration. We found that production of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and in subjects with mild cognitive impairment and that AD MVs are toxic for cultured neurons. The mechanism responsible for MV neurotoxicity was defined in vitro using MVs produced by primary microglia. We demonstrated that neurotoxicity of MVs results from (i) the capability of MV lipids to promote formation of soluble Aβ species from extracellular insoluble aggregates and (ii) from the presence of neurotoxic Aβ forms trafficked to MVs after Aβ internalization into microglia. MV neurotoxicity was neutralized by the Aβ-interacting protein PrP and anti-Aβ antibodies, which prevented binding to neurons of neurotoxic soluble Aβ species. This study identifies microglia-derived MVs as a novel mechanism by which microglia participate in AD degeneration, and suggest new therapeutic strategies for the treatment of the disease.     

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis¹. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia^2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components³. Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential^4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.     

N-Ethylmaleimide-sensitive Factor Attachment Protein α (αSNAP) Regulates Matrix Adhesion and Integrin Processing in Human Epithelial Cells

Integrin-based adhesion to the extracellular matrix (ECM) plays critical roles in controlling differentiation, survival, and motility of epithelial cells. Cells attach to the ECM via dynamic structures called focal adhesions (FA). FA undergo constant remodeling mediated by vesicle trafficking and fusion. A soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein α (αSNAP) is an essential mediator of membrane fusion; however, its roles in regulating ECM adhesion and cell motility remain unexplored. In this study, we found that siRNA-mediated knockdown of αSNAP induced detachment of intestinal epithelial cells, whereas overexpression of αSNAP increased ECM adhesion and inhibited cell invasion. Loss of αSNAP impaired Golgi-dependent glycosylation and trafficking of β1 integrin and decreased phosphorylation of focal adhesion kinase (FAK) and paxillin resulting in FA disassembly. These effects of αSNAP depletion on ECM adhesion were independent of apoptosis and NSF. In agreement with our previous reports that Golgi fragmentation mediates cellular effects of αSNAP knockdown, we found that either pharmacologic or genetic disruption of the Golgi recapitulated all the effects of αSNAP depletion on ECM adhesion. Furthermore, our data implicates β1 integrin, FAK, and paxillin in mediating the observed pro-adhesive effects of αSNAP. These results reveal novel roles for αSNAP in regulating ECM adhesion and motility of epithelial cells.     

Random-walk model of diffusion in three dimensions in brain extracellular space: comparison with microfiberoptic photobleaching measurements

Diffusion through the extracellular space (ECS) in brain is important in drug delivery, intercellular communication, and extracellular ionic buffering. The ECS comprises ∼20% of brain parenchymal volume and contains cell-cell gaps ∼50 nm. We developed a random-walk model to simulate macromolecule diffusion in brain ECS in three dimensions using realistic ECS dimensions. Model inputs included ECS volume fraction (α), cell size, cell-cell gap geometry, intercellular lake (expanded regions of brain ECS) dimensions, and molecular size of the diffusing solute. Model output was relative solute diffusion in water versus brain ECS (D_o/D). Experimental D_o/D for comparison with model predictions was measured using a microfiberoptic fluorescence photobleaching method involving stereotaxic insertion of a micron-size optical fiber into mouse brain. D_o/D for the small solute calcein in different regions of brain was in the range 3.0-4.1, and increased with brain cell swelling after water intoxication. D_o/D also increased with increasing size of the diffusing solute, particularly in deep brain nuclei. Simulations of measured D_o/D using realistic α, cell size and cell-cell gap required the presence of intercellular lakes at multicell contact points, and the contact length of cell-cell gaps to be least 50-fold smaller than cell size. The model accurately predicted D_o/D for different solute sizes. Also, the modeling showed unanticipated effects on D_o/D of changing ECS and cell dimensions that implicated solute trapping by lakes. Our model establishes the geometric constraints to account quantitatively for the relatively modest slowing of solute and macromolecule diffusion in brain ECS.     

Galectin-1 stimulates motility of human umbilical cord blood-derived mesenchymal stem cells by downregulation of smad2/3-dependent collagen 3/5 and upregulation of NF-κB-dependent fibronectin/laminin 5 expression

Galectin-1 (Gal-1) belongs to a family of endogenous lectins with conserved carbohydrate recognition domains binding β-galactosidase sugars and plays a vital role in regulating stem cell functions including determination of cell fate. However, our understanding of the functional roles of Gal-1 in human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) is still fragmentary and incomplete. Gal-1 significantly increased motility after a 24-h incubation, and this effect was inhibited by β-lactose. We analyzed 17 extracellular matrix (ECM) genes in UCB-MSCs. Gal-1 decreased the expression of collagen genes COL3A1 (COL-3) and COL5A1 (COL-5) but increased the expression of fibronectin (FN) and laminin 5 (LM-5), that were reversed by β-lactose. Gal-1 increased protein kinase C (PKC), c-Src, and caveolin-1 (Cav-1) phosphorylation that was attenuated by β-lactose and the Src inhibitor PP2. In addition, pretreatment with the lipid raft disruptor Mβ-CD and the PKC inhibitors inhibited Gal-1-induced UCB-MSC motility. In addition, Gal-1 reduced smad2/3 phosphorylation and induced nuclear factor (NF)-κB phosphorylation. Pretreatment with Mβ-CD attenuated Gal-1-reduced smad2/3 phosphorylation, COL-3, and COL-5 expression but did not affect NF-κB phosphorylation, FN, or LM-5 expression. In contrast, PKC inhibitors only attenuated NF-κB phosphorylation, FN, and LM-5 expression. Reconstructing Gal-1-induced genetic changes by replacing it with siRNA specific for COL-3 or COL-5, or treatment of the cells with FN and LM-5 proteins, increased motility and its related proteins such as focal adhesion kinase, Akt, Erk, integrins, and matrix metalloproteinase-2. A combined treatment with COL-3/COL-5 siRNA or FN/LM-5 compared with that of single treatments was synergistic. However, a single Gal-1 treatment maximally stimulated motility and related protein phosphorylation/expression. These results demonstrate that Gal-1 stimulated human UCB-MSC motility by decreasing COL-3/COL-5 expression and increasing FN/LM-5 expression through a PKC-dependent NF-κB and c-Src/Cav-1-dependent smad2/3 pathway that was critical for governing the activation of FAK, Akt, Erk, integrins, and MMP2.