Glial-fibrillary-acidic-protein (GFAP) has recently drawn significant attention from the clinical environment as a promising biomarker. The pathologies which can be linked to the presence of GFAP in ...blood severely affect the human central nervous system. These pathologies are glioblastoma multiforme (GBM), traumatic brain injuries (TBIs), multiple sclerosis (MS), intracerebral hemorrhage (ICH), and neuromyelitis optica (NMO). Here, we develop three different detection strategies for GFAP, among the most popular in the biosensing field and never examined side by side within the experimental frame. We compare their capability of detecting GFAP in a clean-buffer and serum-matrix by using gold-coated quartz-crystal-microbalance (QCM) sensors. All the three detection strategies are based on antibodies, and each of them focuses on a key aspect of the biosensing process. The first is based on a polyethylene glycol (PEG) chain for antifouling, the second on a protein-G linker for controlling antibody-orientation, and the third on antibody-splitting and direct surface immobilization for high-surface coverage. Then, we select the best-performing protocol and validate its detection performance with an ultra-high-frequency (UHF) surface-acoustic-wave (SAW) based lab-on-chip (LoC). GFAP successful detection is demonstrated in a clean-buffer and serum-matrix at a concentration of 35 pM. This GFAP level is compatible with clinical diagnostics. This result suggests the use of our technology for the realization of a point-of-care biosensing platform for the detection of multiple brain-pathology biomarkers.
•GFAP is linked to severe pathologies of the human central nervous system.•GFAP is a promising biomarker that can be found in circulating blood.•Three functionalization approaches for detecting GFAP with biosensors are developed.•The functionalization capability of detecting GFAP is firstly evaluated by QCM.•A new SAW biosensor is used for detecting diagnostic GFAP levels in serum-matrix.
Globoid cell leukodystrophy (GLD) is a metabolic disease caused by mutations in the galactocerebrosidase (GALC) gene. GALC is a lysosomal enzyme whose function is to degrade galacto-lipids, including ...galactosyl-ceramide and galactosyl-sphingosine (psychosine, PSY). GALC loss of function causes progressive intracellular accumulation of PSY. It is widely held that PSY is the main trigger for the degeneration of myelinating cells and progressive white-matter loss. However, still little is known about the molecular mechanisms by which PSY imparts toxicity. Here, we address the role of calcium dynamics during PSY-induced cell death. Using the human oligodendrocyte cell line MO3.13, we report that cell death by PSY is accompanied by robust cytosolic and mitochondrial calcium (Ca(2+)) elevations, and by mitochondrial reactive oxygen species (ROS) production. Importantly, we demonstrate that the reduction of extracellular calcium content by the chelating agent ethylenediaminetetraacetic acid can decrease intra-mitochondrial ROS production and enhance cell viability. Antioxidant administration also reduces mitochondrial ROS production and cell loss, but this treatment does not synergize with Ca(2+) chelation. Our results disclose novel intracellular pathways involved in PSY-induced death that may be exploited for therapeutic purposes to delay GLD onset and/or slow down its progression.
Correlative approaches are a powerful tool in the investigation of biological samples, but require specific preparation procedures to maintain the strength of the employed methods. Here we report the ...optimization of the embedding protocol of nervous system samples for a correlative synchrotron X-ray computed microtomography (micro-CT) and transmission electron microscopy (TEM) approach. We demonstrate that it is possible to locate, with the micrometric resolution of micro-CT, specific volumes of interest for a further ultrastructural characterization to be performed with TEM. This approach can be applied to samples of different size and morphology up to several cm. Our optimized method represents an invaluable tool for investigating those pathologies in which microscopic alterations are localized in few confined regions, rather than diffused in entire tissues, organs or systems. We present a proof of concept of our method in a mouse model of Globoid Cells Leukodistrophy.
During neuronal development, neuronal cells read extracellular stimuli from the micro/nano-environment within which they exist, retrieving essential directionality and wiring information. Here, focal ...adhesions (FAs-protein clusters anchoring integrins to cytoskeleton) act as sensors, by integrating signals from both the extracellular matrix environment and chemotactic factors, contributing to the final neuronal pathfinding and migration. In the processes that orchestrate neuronal development, the important function of ubiquitin E3A ligase (UBE3A) is emerging. UBE3A has crucial functions in the brain and changes in its expression levels lead to neurodevelopmental disorders: the lack of UBE3A leads to Angelman syndrome (AS, OMIN 105830), while its increase causes autisms (Dup15q-autism). By using nano/micro-structured anisotropic substrates we previously showed that UBE3A-deficient neurons have deficits in contact guidance (Tonazzini et al, Mol Autism 2019). Here, we investigate the adhesion and migration dynamics of UBE3A-silenced SH-SY5Y neuroblastoma cells in vitro by exploiting nano/micro-grooved substrates. We analyze the molecular processes regulating the development of FAs by transfection with EGFP-vector encoding for paxillin, a protein of FA clusters, and by live-cell total-internal-reflection-fluorescence microscopy. We show that UBE3A-silenced SH-SY5Y cells have impaired FA morphological development and pathway activation, which lead to a delayed adhesion and also explain the defective contact guidance in response to directional topographical stimuli. However, UBE3A-silenced SH-SY5Y cells show an overall normal migration behavior, in terms of speed and ability to follow the GRs directional stimulus. Only the collective cell migration upon cell gaps was slightly delayed for UBE3Ash SHs. Overall, the deficits of UBE3Ash SHS-SY5Y cells in FA maturation/sensing and in collective migration may have patho-physiological implications, in AS condition, considering the much more complex stimuli that neurons find in vivo during the neurodevelopment.
We investigate how multiscale morphology of functional thin films affects the in vitro behavior of human neural astrocytoma 1321N1 cells. Pentacene thin film morphology is precisely controlled by ...means of the film thickness, Θ (here expressed in monolayers (ML)). Fluorescence and atomic force microscopy allow us to correlate the shape, adhesion, and proliferation of cells to the morphological properties of pentacene films controlled by saturated roughness, σ, correlation length, ξ, and fractal dimension, df. At early incubation times, cell adhesion exhibits a transition from higher to lower values at Θ ≈ 10 ML. This is explained using a model of conformal adhesion of the cell membrane onto the growing pentacene islands. From the model fitting of the data, we show that the cell explores the surface with a deformation of the membrane whose minimum curvature radius is 90 (± 45) nm. The transition in the adhesion at ∼10 ML arises from the saturation of ξ accompanied by the monotonic increase of σ, which leads to a progressive decrease of the pentacene local radius of curvature and hence to the surface area accessible to the cell. Cell proliferation is also enhanced for Θ < 10 ML, and the optimum morphology parameter ranges for cell deployment and growth are σ ≤ 6 nm, ξ > 500 nm, and df > 2.45. The characteristic time of cell proliferation is τ ≈ 10 ± 2 h.
Recent discoveries indicate that during neuronal development the signaling processes that regulate extracellular sensing (e.g., adhesion, cytoskeletal dynamics) are important targets for ...ubiquitination-dependent regulation, in particular through E3 ubiquitin ligases. Among these, Ubiquitin E3a ligase (UBE3A) has a key role in brain functioning, but its function and how its deficiency results in the neurodevelopmental disorder Angelman syndrome is still unclear. Here, the role of UBE3A is investigated in neurite contact guidance during neuronal development, in vitro. The microtopography sensing of wild-type and Ube3a-deficient hippocampal neurons is studied by exploiting gratings with different topographical characteristics, with the aim to compare their capabilities to read and follow physical directional stimuli. It is shown that neuronal contact guidance is defective in Ube3a-deficient neurons, and this behavior is linked to an impaired activation of the focal adhesion signaling pathway. Taken together, the results suggest that the neuronal contact sensing machinery might be affected in Angelman syndrome.
Adenosine and ATP, via their specific P1 and P2 receptors, modulate a wide variety of cellular and tissue functions, playing a neuroprotective or neurodegenerative role in brain damage conditions. ...Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, recent data suggest the existence of a heterodimerization and a functional interaction between P1 and P2 receptors in the brain. In particular, interactions of adenosine A1 and P2Y1 receptors may play important roles in the purinergic signalling cascade. In the present work, we investigated the subcellular localization/co‐localization of the receptors and their functional cross‐talk at the membrane level in Wistar rat hippocampus. This is a particularly vulnerable brain area, which is sensitive to adenosine‐ and ATP‐mediated control of glutamatergic transmission. The postembedding immunogold electron microscopy technique showed that the two receptors are co‐localized at the synaptic membranes and surrounding astroglial membranes of glutamatergic synapses. To investigate the functional cross‐talk between the two types of purinergic receptors, we evaluated the reciprocal effects of their activation on their G protein coupling. P2Y1 receptor stimulation impaired the potency of A1 receptor coupling to G protein, whereas the stimulation of A1 receptors increased the functional responsiveness of P2Y1 receptors. The results demonstrated an A1–P2Y1 receptor co‐localization at glutamatergic synapses and surrounding astrocytes and a functional interaction between these receptors in hippocampus, suggesting ATP and adenosine can interact in purine‐mediated signalling. This may be particularly important during pathological conditions, when large amounts of these mediators are released.
The F11 hybridoma, a dorsal root ganglion-derived cell line, was used to investigate the response of nociceptive sensory neurons to nanotopographical guidance cues. This established this cell line as ...a model of peripheral sensory neuron growth for tissue scaffold design. Cells were seeded on substrates of cyclic olefin copolymer (COC) films imprinted via nanoimprint lithography (NIL) with a grating pattern of nano-scale grooves and ridges. Different ridge widths were employed to alter the focal adhesion formation, thereby changing the cell/substrate interaction. Differentiation was stimulated with forskolin in culture medium consisting of either 1 or 10% fetal bovine serum (FBS). Per medium condition, similar neurite alignment was achieved over the four day period, with the 1% serum condition exhibiting longer, more aligned neurites. Immunostaining for focal adhesions found the 1% FBS condition to also have fewer, less developed focal adhesions. The robust response of the F11 to guidance cues further builds on the utility of this cell line as a sensory neuron model, representing a useful tool to explore the design of regenerative guidance tissue scaffolds.
Brain levels of adenosine are elevated during hypoxia. Through effects on adenosine receptors (A(1), A(2A), A(2B) and A(3)) on astrocytes, adenosine can influence functions such as glutamate uptake, ...reactive gliosis, swelling, as well as release of neurotrophic and neurotoxic factors having an impact on the outcome of metabolic stress. We have studied the roles of these receptors in astrocytes by evaluating their susceptibility to damage induced by oxygen deprivation or exposure to the hypoxia mimic cobalt chloride (CoCl(2)). Hypoxia caused ATP breakdown and purine release, whereas CoCl(2) (0.8 mM) mainly reduced ATP by causing cell death in human D384 astrocytoma cells. Further experiments were conducted in primary astrocytes prepared from specific adenosine receptor knock-out (KO) and wild type (WT) mice. In WT cells purine release following CoCl(2) exposure was mainly due to nucleotide release, whereas hypoxia-induced intracellular ATP breakdown followed by nucleoside efflux. N-ethylcarboxamidoadenosine (NECA), an unselective adenosine receptor agonist, protected from cell death following hypoxia. Cytotoxicity was more pronounced in A(1)R KO astrocytes and tended to be higher in WT cells in the presence of the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Genetic deletion of A(2A) receptor resulted in less prominent effects. A(3)R KO glial cells were more affected by hypoxia than WT cells. Accordingly, the A(3) receptor agonist 2-chloro-N(6)-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (CL-IB-MECA) reduced ATP depletion caused by hypoxic conditions. It also reduced apoptosis in human astroglioma D384 cells after oxygen deprivation. In conclusion, the data point to a cytoprotective role of adenosine mediated by both A(1) and A(3) receptors in primary mouse astrocytes.
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