Background and Purpose
The sphingosine 1‐phosphate receptor subtype 1 (S1P1R) is modulated by phosphorylated FTY720 (pFTY720), which causes S1P1R internalization preventing lymphocyte migration thus ...limiting autoimmune response. Studies indicate that internalized S1P1Rs continue to signal, maintaining an inhibition of cAMP, thus raising question whether the effects of pFTY720 are due to transient initial agonism, functional antagonism and/or continued signalling. To further investigate this, the current study first determined if continued S1P1R activation is pathway specific.
Experimental Approach
Using human and rat astrocyte cultures, the effects of S1P1R activation on cAMP, pERK and Ca2+ signalling was investigated. In addition, to examine the role of S1P1R redistribution on these events, a novel biologic (MNP301) that prevented pFTY720‐mediated S1P1R redistribution was engineered.
Key Results
The data showed that pFTY720 induced long‐lasting S1P1R redistribution and continued cAMP signalling in rat astrocytes. In contrast, pFTY720 induced a transient increase of Ca2+ in astrocytes and subsequent antagonism of Ca2+ signalling. Notably, while leaving pFTY720‐induced cAMP signalling intact, the novel MNP301 peptide attenuated S1P1R‐mediated Ca2+ and pERK signalling in cultured rat astrocytes.
Conclusions and Implications
These findings suggested that pFTY720 causes continued cAMP signalling that is not dependent on S1P1R redistribution and induces functional antagonism of Ca2+ signalling after transient stimulation. To our knowledge, this is the first report demonstrating that pFTY720 causes continued signalling in one pathway (cAMP) versus functional antagonism of another pathway (Ca2+) and which also suggests that redistributed S1P1Rs may have differing signalling properties from those expressed at the surface.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Cancer cell extravasation, a key step in the metastatic cascade, involves cancer cell arrest on the endothelium, transendothelial migration (TEM), followed by the invasion into the subendothelial ...extracellular matrix (ECM) of distant tissues. While cancer research has mostly focused on the biomechanical interactions between tumor cells (TCs) and ECM, particularly at the primary tumor site, very little is known about the mechanical properties of endothelial cells and the subendothelial ECM and how they contribute to the extravasation process. Here, an integrated experimental and theoretical framework is developed to investigate the mechanical crosstalk between TCs, endothelium and subendothelial ECM during in vitro cancer cell extravasation. It is found that cancer cell actin‐rich protrusions generate complex push–pull forces to initiate and drive TEM, while transmigration success also relies on the forces generated by the endothelium. Consequently, mechanical properties of the subendothelial ECM and endothelial actomyosin contractility that mediate the endothelial forces also impact the endothelium's resistance to cancer cell transmigration. These results indicate that mechanical features of distant tissues, including force interactions between the endothelium and the subendothelial ECM, are key determinants of metastatic organotropism.
A combination of advanced engineering/material sciences, biological tools, and computational/ mathematical simulations are employed to study how the mechanical features of both endothelial cells and the subendothelial matrix at secondary organ sites may influence cancer metastasis and, in particular, the transendothelial migration of cancer cells, which is a major step during the extravasation process.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Over four decades ago, it was discovered that high-frequency stimulation of the dentate gyrus induces long-term potentiation (LTP) of synaptic transmission. LTP is believed to underlie how we process ...and code external stimuli before converting it to salient information that we store as 'memories'. It has been shown that rats performing spatial learning tasks display theta-frequency (3-12 Hz) hippocampal neural activity. Moreover, administering theta-burst stimulation (TBS) to hippocampal slices can induce LTP. TBS triggers a sustained rise in intracellular calcium Ca2+i in neurons leading to new protein synthesis important for LTP maintenance. In this study, we measured TBS-induced Ca2+i oscillations in thousands of cells at increasing distances from the source of stimulation. Following TBS, a calcium wave propagates radially with an average speed of 5.2 µm/s and triggers multiple and regular Ca2+i oscillations in the hippocampus. Interestingly, the number and frequency of Ca2+i fluctuations post-TBS increased with respect to distance from the electrode. During the post-tetanic phase, 18% of cells exhibited 3 peaks in Ca2+i with a frequency of 17 mHz, whereas 2.3% of cells distributed further from the electrode displayed 8 Ca2+i oscillations at 33 mHz. We suggest that these observed Ca2+i oscillations could lead to activation of transcription factors involved in synaptic plasticity. In particular, the transcription factor, NF-κB, has been implicated in memory formation and is up-regulated after LTP induction. We measured increased activation of NF-κB 30 min post-TBS in CA1 pyramidal cells and also observed similar temporal up-regulation of NF-κB levels in CA1 neurons following water maze training in rats. Therefore, TBS of hippocampal slice cultures in vitro can mimic the cell type-specific up-regulations in activated NF-κB following spatial learning in vivo. This indicates that TBS may induce similar transcriptional changes to spatial learning and that TBS-triggered Ca2+i oscillations could activate memory-associated gene expression.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Fingolimod (FTY720) is an oral therapy for relapsing remitting multiple sclerosis (MS) and targets sphingosine 1-phosphate receptors (S1PRs). FTY720 also rescues animals from experimental autoimmune ...encephalomyelitis (EAE), an animal model of MS. The protective effects of FTY720 in EAE are primarily scored manually by examining weight loss and limb paralysis that begins around 10-12 days after immunisation. To our knowledge, pre-clinical effects of FTY720 on animal behaviour early in EAE have not been explored. Here, we developed an automated behaviour monitoring system to examine the early effects of FTY720 on subtle pre-symptomatic behaviour of mice induced with EAE. Our automated home-cage monitoring system (AHC-MS) enabled non-contact detection of movement and ultrasonic vocalisations (USVs) of mice induced with EAE, thus allowing detection of subtle changes in mouse behaviour before paralysis occurs. Mice receiving FTY720 emit longer USVs and display higher levels of motor activity than vehicle-treated EAE mice before clinical symptoms become apparent. Importantly, this study promotes the 3Rs ethics (replacement, reduction and refinement) in the EAE animal model and may also improve pre-screening of potentially novel MS therapies. In addition, this is the first report showing the early effects of FTY720 in EAE which underscores its protective effects.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Cells maintain their volume through fine intracellular osmolarity regulation. Osmotic challenges drive fluid into or out of cells causing swelling or shrinkage, respectively. The dynamics of cell ...volume changes depending on the rheology of the cellular constituents and on how fast the fluid permeates through the membrane and cytoplasm. We investigated whether and how poroelasticity can describe volume dynamics in response to osmotic shocks. We exposed cells to osmotic perturbations and used defocusing epifluorescence microscopy on membrane-attached fluorescent nanospheres to track volume dynamics with high spatiotemporal resolution. We found that a poroelastic model that considers both geometrical and pressurization rates captures fluid-cytoskeleton interactions, which are rate-limiting factors in controlling volume changes at short timescales. Linking cellular responses to osmotic shocks and cell mechanics through poroelasticity can predict the cell state in health, disease, or in response to novel therapeutics.
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•Cell height changes can be finely captured by defocusing microscopy•Water permeation and cellular deformability regulate dynamics of cell volume changes•Poroelasticity describes the dynamics of cell volume changes•The response of cell to hypo or hyperosmotic shocks are modeled by poroelasticity
Cellular physiology; Cell biology; Biomechanics
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. ...However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine.
Upregulation of the transcription factor, REST, is neuroprotective in the ageing brain. We quantified changes in nuclear REST expression in the brains of ageing wild‐type (WT) and Alzheimer’s disease ...(AD) rats. REST increased in structures involved in learning and memory in WT but not AD rats, potentially explaining why these regions are vulnerable to synapse loss, neuroinflammation and astrogliosis.
In the brain, REST (Repressor Element‐1 Silencing Transcription factor) is a key regulator of neuron cell‐specific gene expression. Nuclear translocation of neuronal REST has been shown to be neuroprotective in a healthy ageing context. In contrast, inability to upregulate nuclear REST is thought to leave ageing neurons vulnerable to neurodegenerative stimuli, such as Alzheimer’s disease (AD) pathology. Hippocampal and cortical neurons are known to be particularly susceptible to AD‐associated neurodegeneration. However, REST expression has not been extensively characterised in the healthy ageing brain. Here, we examined the spatiotemporal immunolocalisation of REST in the brains of healthy ageing wild‐type Fischer‐344 and transgenic Alzheimer’s disease rats (TgF344‐AD). Nuclear expression of REST increased from 6 months to 18 months of age in the hippocampus, frontal cortex and subiculum of wild‐type rats, but not in TgF344‐AD rats. No changes in REST were measured in more posterior cortical regions or in the thalamus. Interestingly, levels of the presynaptic marker synaptophysin, a known gene target of REST, were lower in CA1 hippocampal neurons of 18‐month TgF344‐AD rats compared to 18‐month wild‐types, suggesting that elevated nuclear REST may protect against synapse loss in the CA1 of 18‐month wild‐type rats. High REST expression in ageing wild‐type rats did not, however, protect against axonal loss nor against astroglial reactivity in the hippocampus. Taken together, our data confirm that changes in nuclear REST expression are context‐, age‐ and brain region‐specific. Moreover, key brain structures involved in learning and memory display elevated REST expression in healthy ageing wild‐type rats but not TgF344‐AD rats.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
J. Neurochem. (2009) 112, 991-1004. Long-term memory is formed by alterations in glutamate-dependent excitatory synaptic transmission, which is in turn regulated by synaptosomal protein of 25 kDa ...(SNAP-25), a key component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex essential for exocytosis of neurotransmitter-filled synaptic vesicles. Both reduced and excessive SNAP-25 activity has been implicated in various disease states that involve cognitive dysfunctions such as attention deficit hyperactivity disorder, schizophrenia and Alzheimer's disease. Here, we over-express SNAP-25 in the adult rat dorsal hippocampus by infusion of a recombinant adenoassociated virus vector, to evaluate the consequence of late adolescent-adult dysfunction of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein in the absence of developmental disruption. We report a specific and significant increase in the levels of extracellular glutamate detectable by microdialysis and a reduction in paired-pulse facilitation in the hippocampus. In addition, SNAP-25 over-expression produced cognitive deficits, delaying acquisition of a spatial map in the water maze and impairing contextual fear conditioning, both tasks known to be dorsal hippocampal dependent. The high background transmission state and pre-synaptic dysfunction likely result in interference with requisite synapse selection during spatial and fear memory consolidation. Together these studies provide the first evidence that excess SNAP-25 activity, restricted to the adult period, is sufficient to mediate significant deficits in the memory formation process.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
A learning event initiates a cascade of altered gene expression leading to synaptic remodelling within the hippocampal dentate gyrus, a structure vital to memory formation. To illuminate this ...transcriptional program of synaptic plasticity we used microarrays to quantify mRNA from the rat dentate gyrus at increasing times following passive avoidance learning. Approximately, 500 known genes were transcriptionally regulated across the 24 h post‐training period. The 0–2 h period saw up‐regulation of genes involved in transcription while genes with a role in synaptic/cytoskeletal structure increased 0–6 h, consistent with structural rearrangements known to occur at these times. The most striking feature was the profound down‐regulation, across all functional groups, 12 h post‐training. Bioinformatics analysis identified the likely transcription factors controlling gene expression in each post‐training period. The role of NFκB, implicated in the early post‐training period was subsequently confirmed with activation and nuclear translocation seen in dentate granule neurons following training. mRNA changes for four genes, LRP3 (0 h), alpha actin (3 h), SNAP25 and NSF (6–12 h), were validated at message and/or protein level and shown to be learning specific. Thus, the memory‐associated transcriptional cascade supports the cardinal periods of synaptic loosening, reorganisation and selection thought to underpin the process of long‐term memory consolidation in the hippocampus.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Widespread neurodegeneration, enlargement of cerebral ventricles, and atrophy of cortical and hippocampal brain structures are classic hallmarks of Alzheimer's disease (AD). Prominent macroscopic ...disturbances to the cytoarchitecture of the AD brain occur alongside changes in the mechanical properties of brain tissue, as reported in recent magnetic resonance elastography (MRE) measurements of human brain mechanics. Whilst MRE has many advantages, a significant shortcoming is its spatial resolution. Higher resolution "cellular scale" assessment of the mechanical alterations to brain regions involved in memory formation, such as the hippocampus, could provide fresh new insight into the etiology of AD. Characterization of brain tissue mechanics at the cellular length scale is the first stepping-stone to understanding how mechanosensitive neurons and glia are impacted by neurodegenerative disease-associated changes in their microenvironment. To provide insight into the microscale mechanics of aging brain tissue, we measured spatiotemporal changes in the mechanical properties of the hippocampus using high resolution atomic force microscopy (AFM) indentation tests on acute brain slices from young and aged wild-type mice and the APP
mouse model. Several hippocampal regions in APP
mice are significantly softer than age-matched wild-types, notably the dentate granule cell layer and the CA1 pyramidal cell layer. Interestingly, regional softening coincides with an increase in astrocyte reactivity, suggesting that amyloid pathology-mediated alterations to the mechanical properties of brain tissue may impact the function of mechanosensitive astrocytes. Our data also raise questions as to whether aberrant mechanotransduction signaling could impact the susceptibility of neurons to cellular stressors in their microenvironment.
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