Carbon nanotubes have been applied in several areas of nerve tissue engineering to probe and augment cell behaviour, to label and track subcellular components, and to study the growth and ...organization of neural networks. Recent reports show that nanotubes can sustain and promote neuronal electrical activity in networks of cultured cells, but the ways in which they affect cellular function are still poorly understood. Here, we show, using single-cell electrophysiology techniques, electron microscopy analysis and theoretical modelling, that nanotubes improve the responsiveness of neurons by forming tight contacts with the cell membranes that might favour electrical shortcuts between the proximal and distal compartments of the neuron. We propose the 'electrotonic hypothesis' to explain the physical interactions between the cell and nanotube, and the mechanisms of how carbon nanotubes might affect the collective electrical activity of cultured neuronal networks. These considerations offer a perspective that would allow us to predict or engineer interactions between neurons and carbon nanotubes.
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IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
We demonstrate the possibility of using carbon nanotubes (CNTs) as potential devices able to improve neural signal transfer while supporting dendrite elongation and cell adhesion. The results ...strongly suggest that the growth of neuronal circuits on a CNT grid is accompanied by a significant increase in network activity. The increase in the efficacy of neural signal transmission may be related to the specific properties of CNT materials, such as the high electrical conductivity.
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Complement component C1q can act as a pro-tumorigenic factor in the tumor microenvironment (TME). The TME in malignant pleural mesothelioma (MPM) is rich in C1q and hyaluronic acid (HA), whose ...interaction enhances adhesion, migration and proliferation of malignant cells. HA-bound C1q is also capable of modulating HA synthesis. Thus, we investigated whether HA-C1q interaction would affect HA degradation, analyzing the main degradation enzymes, hyaluronidase (HYAL)1 and HYAL2, and a C1q receptor candidate. We first proceeded with the characterization of HYALs in MPM cells, especially HYAL2, since bioinformatics survival analysis revealed that higher
mRNA levels have an unfavorable prognostic index in MPM patients. Interestingly, Real-Time quantitative PCR, flow cytometry and Western blot highlighted an upregulation of HYAL2 after seeding of primary MPM cells onto HA-bound C1q. In an attempt to unveil the receptors potentially involved in HA-C1q signaling, a striking co-localization between HYAL2 and globular C1q receptor/HABP1/p32 (gC1qR) was found by immunofluorescence, surface biotinylation and proximity ligation assays. RNA interference experiments revealed a potentially regulatory function exerted by gC1qR on HYAL2 expression, since
(gene for gC1qR) silencing unexpectedly caused HYAL2 downregulation. In addition, the functional blockage of gC1qR by a specific antibody hindered HA-C1q signaling and prevented HYAL2 upregulation. Thus, C1q-HA interplay is responsible for enhanced HYAL2 expression, suggesting an increased rate of HA catabolism and the release of pro-inflammatory and pro-tumorigenic HA fragments in the MPM TME. Our data support the notion of an overall tumor-promoting property of C1q. Moreover, the overlapping localization and physical interaction between HYAL2 and gC1qR suggests a potential regulatory effect of gC1qR within a putative HA-C1q macromolecular complex.
Increased hyaluronic acid (HA) production is often associated with cancer progression. In malignant pleural mesothelioma (MPM), HA is found at elevated levels in pleural effusions and sera of ...patients, and it has been widely debated whether MPM cells are able to produce HA by themselves or through the release of growth factors stimulating other cells. Another key component of the MPM microenvironment is C1q, which can act as a pro-tumorigenic factor favoring cell adhesion, migration and proliferation. The aim of the current study was to prove that MPM primary cells are able to synthesize HA and to inquire the stimulus given by C1q-HA matrix to HA synthesis. We confirmed the presence of a HA coat and cable-like structures around MPM primary cells, as well as an intracellular pool, mainly localized in the cytoplasmic and perinuclear region. After evaluating HA synthase (HAS) enzymes' basal expression in MPM primary cells, we found that C1q bound to HA was able to impinge upon HA homeostasis by upregulating HAS3 both at the mRNA and the protein levels. High expression of
has been correlated with a shorter life expectancy in MPM by bioinformatical analysis. These data confirmed that C1q bound to HA may exert pro-tumorigenic activity and identified HAS3 as a potential target in MPM.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Tauopathies are prevalent, invariably fatal brain diseases for which no cure is available. Tauopathies progressively affect the brain through cell‐to‐cell transfer of tau protein amyloids, yet the ...spreading mechanisms remain unknown. Here we show that the cellular prion protein (PrPC) facilitates the uptake of tau aggregates by cultured cells, possibly by acting as an endocytic receptor. In mouse neuroblastoma cells, pull‐down experiments revealed that tau amyloids bind to PrPC. Confocal images of both wild‐type and PrPC ‐knockout N2a cells treated with fluorescently labeled synthetic tau fibrils showed that the internalization was reduced in isogenic cells devoid of the gene encoding PrPC. Pre‐treatment of the same cells with antibodies against N‐proximal epitopes of PrPC impaired the binding of tau amyloids and decreased their uptake. Surprisingly, exposure of chronically prion‐infected cells to tau amyloids reduced the accumulation of aggregated prion protein and this effect lasted for more than 72 hr after amyloid removal. These results point to bidirectional interactions between the two proteins: while PrPC mediates the entrance of tau fibrils in cells, PrPSc buildup is greatly reduced in their presence, possibly because of an impairment in the prion conversion process.
We propose the following interplay between tau and the prion protein in the brain: the direct binding of tau amyloids to the physiological form of the prion protein (PrPC) facilitates the cellular uptake of the amyloids, which in turn increase the levels of PrPC probably by interfering with its degradation. Tau amyloids hamper also the conversion of PrPC into its pathogenic conformer PrPSc. This effect is independent of the prion strain and proceeds through the promotion of the α‐cleavage of PrPC, which disrupts the region of the prion protein essential for the conversion.
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Although ATP is important for intercellular communication, little is known about the mechanism of endogenous ATP release due
to a dearth of suitable models. Using PC12 cells known to express the P2X ...2 subtype of ATP receptors and to store ATP with catecholamines inside dense-core vesicles, we found that clusters of PC12
cells cultured for 3â7 days generated small transient inward currents (STICs) after an inward current elicited by exogenous
ATP. The amplitude of STICs in individual cells correlated with the peak amplitude of ATP-induced currents. STICs appeared
as asynchronous responses (approximately 20 pA average amplitude) for 1â20 s and were investigated with a combination of patch
clamping, Ca 2+ imaging, biochemistry and electron microscopy. Comparable STICs were produced by focal KCl pulses and were dependent on extracellular
Ca 2+ . STICs were abolished by the P2X antagonist PPADS and potentiated by Zn 2+ , suggesting they were mediated by P2X 2 receptor activation. The highest probability of observing STICs was after the peak of intracellular Ca 2+ increase caused by KCl. Biochemical measurements indicated that KCl application induced a significant release of ATP from
PC12 cells. Electron microscopy studies showed narrow clefts without âsynaptic-likeâ densities between clustered cells. Our
data suggest that STICs were caused by quantal release of endogenous ATP by depolarized PC12 cells in close juxtaposition
to the recorded cell. Thus, STICs may be a new experimental model to characterize the physiology of vesicular release of ATP
and to study the kinetics and pharmacology of P2X 2 receptor-mediated quantal currents.
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It is widely accepted that nicotinic acetylcholine receptor (nAChR) channel activity controls myoblast fusion into myotubes
during myogenesis. In this study we explored the possible role of nAChR ...channels after cell fusion in a murine cell model.
Using videoimaging techniques we showed that embryonic muscle nAChR channel openings contribute to the spontaneous transients
of intracellular concentration of Ca 2 + (Ca 2 + i ) and to twitches characteristic of developing myotubes before innervation. Moreover, we observed a choline acetyltransferase
immunoreactivity in the myotubes and we detected an acetylcholine-like compound in the extracellular solution. Therefore,
we suggest that the autocrine activation of nAChR channels gives rise to Ca 2 + i spikes and contractions. Spontaneous openings of the nAChR channels may be an alternative, although less efficient, mechanism.
We report also that blocking the nAChRs causes a significant reduction in cell survival, detectable as a decreased number
of myotubes in culture. This led us to hypothesize a possible functional role for the autocrine activation of the nAChRs.
By triggering mechanical activity, such activation could represent a strategy to ensure the trophism of myotubes in the absence
of nerves.
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Alginate/hydroxyapatite composite scaffolds were developed using a novel production design. Hydroxyapatite (HAp) was incorporated into an alginate solution and internal gelling was induced by ...addition of slowly acid hydrolyzing d-gluconic acid δ-lactone (GDL) for the direct release of calcium ions from HAp. Hydrogels were then freeze-casted to produce a three-dimensional isotropic porous network. Scanning electron microscopy (SEM) observations, confocal laser scanning microscopy (CLSM) and microcomputed tomography (μ-CT) analysis of the scaffolds showed an optimal interconnected porous structure with pore sizes ranging between 100 and 300 μm and over 88% porosity. Proliferation assay and SEM observations demonstrated that human osteosarcoma cell lines were able to proliferate, maintain osteoblast-like phenotype and massively colonize the scaffold structure. Overall, these combined results indicate that the novel alginate based composites efficiently support the adhesion and proliferation of cells showing at the same time adequate structural and physical-chemical properties for being used as scaffolds in bone tissue engineering strategies.
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In the presence of certain excitatory substances the rat isolated spinal cord generates rhythmic oscillations believed to
be an in-built locomotor programme (fictive locomotion). However, it is ...unknown whether a long-term culture of the same tissue
can express rhythmic activity. Such a simplified model system would provide useful data on the minimal circuitry involved
and the cellular mechanisms mediating this phenomenon. For this purpose we performed patch clamp recording (under whole-cell
voltage or current clamp conditions) from visually identified ventral horn interneurones of an organotypic slice culture of
the rat spinal cord.
Ventral horn interneurones expressed rhythmic bursting when the extracellular K + was raised from 4 to 6-7 mM. Under voltage clamp this activity consisted of composite synaptic currents grouped into bursts
lasting 0.9 ± 0.5 s (2.8 ± 1.5 s period) and was generated at network level as it was blocked by tetrodotoxin or low-Ca 2+ -high-Mg 2+ solution and its periodicity was unchanged at different potential levels.
In current clamp mode bursting was usually observed as episodes comprising early depolarizing potentials followed by hyperpolarizing
events with tight temporal patterning. Bursting was fully suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and reduced
in amplitude and duration by N -methyl-D-aspartate (NMDA) receptor antagonism without change in periodicity. Extracellular field recording showed bursting
activity over a wide area of the ventral horn.
Regular, rhythmic activity similar to that induced by K + also appeared spontaneously in Mg 2+ -free solution. The much slower rhythmic pattern induced by strychnine and bicuculline was also accelerated by high-K + solution.
The fast and regular rhythmic activity of interneurones in the spinal organotypic culture is a novel observation which suggests
that the oversimplified circuit present in this culture is a useful model for investigating spinal rhythmic activity.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Tau protein is the major component of intracellular aggregates that characterize a number of neurodegenerative disorders collectively called tauopathies. Tau amyloids propagate from cell to cell ...through different routes, such as dynamin‐dependent endocytosis, actin‐dependent macropinocytosis, tunneling nanotubes. Here, we evaluate the role of the prion protein PrPC in promoting the internalization of synthetic tau aggregates in cultured cells. As previously found by our group, the prion protein facilitates the uptake of synuclein fibrils in neuroblastoma cell lines, in primary neurons and in mice. Using confocal microscopy, we show that also tau fibrils take advantage of the presence of the prion protein on the plasma membrane to gain entrance to the cell, and that antibodies directed against the binding site of the fibrils on PrPC can decrease the internalization of the aggregates. Moreover, tau fibrils interact with both the cellular form PrPC and the pathological conformer PrPSc, inducing clearance of the latter through a combination of lysosomal activation and inhibition of prion conversion. Our results indicate that the prion protein can act as a receptor for aggregated proteins (synuclein, tau), and that targeting PrPC could be a therapeutic strategy to slow down the spreading of tau pathology.
Support or Funding Information
This work was supported by the Program FIRB (Prot. RBAP11FRE9_001).
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
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