Modeling of the mechano-chemical behavior of the nuclear pore complex: current research and perspectives

Recent evidence suggests that mechanical deformation of the cell nucleus regulates the nuclear import of the transcriptional activators of genes involved in primary physiological cell responses such as stem cell differentiation. In addition, this nuclear mechanosensing response is de-regulated in pathological states, such as cancer and neurodegeneration. One hypothesis that could greatly advance the field is that the deformation of the nuclear envelope activates nuclear pore complexes through a direct mechanical link. The understanding of this possible mechanism for nuclear pore complex stretch-activation entails studying the mechanical connection of this complex to the nuclear envelope at the nanoscale. The nanomechanics of the nuclear pore complex is thus emerging as a novel research field, bridging nanoscience with nanotechnology. This review examines the frontier of research methodologies that are potentially useful for building a computational model of this interaction. This includes, for example, electron tomography to assess the geometrical features of the nuclear pore complex and nanoindentation to estimate its mechanical properties and that of the nuclear envelope. In order to summarize the state-of-the-art and perspectives in the field of NPC nanomechanics, this review covers highly interdisciplinary experimental and theoretical research methodologies pertaining to the fields of physics, chemistry, biology, materials and mechanics

Lamin A/C Mechanotransduction in Laminopathies

Mechanotransduction translates forces into biological responses and regulates cell functionalities. It is implicated in several diseases, including laminopathies which are pathologies associated with mutations in lamins and lamin-associated proteins. These pathologies affect muscle, adipose, bone, nerve, and skin cells and range from muscular dystrophies to accelerated aging. Although the exact mechanisms governing laminopathies and gene expression are still not clear, a strong correlation has been found between cell functionality and nuclear behavior. New theories base on the direct effect of external force on the genome, which is indeed sensitive to the force transduced by the nuclear lamina. Nuclear lamina performs two essential functions in mechanotransduction pathway modulating the nuclear stiffness and governing the chromatin remodeling. Indeed, A-type lamin mutation and deregulation has been found to affect the nuclear response, altering several downstream cellular processes such as mitosis, chromatin organization, DNA replication-transcription, and nuclear structural integrity. In this review, we summarize the recent findings on the molecular composition and architecture of the nuclear lamina, its role in healthy cells and disease regulation. We focus on A-type lamins since this protein family is the most involved in mechanotransduction and laminopathies

Influence of the static magnetic field on cell response in a miniaturized optically accessible bioreactor for 3D cell culture

Hydraulic sealing is a crucial condition for the maintenance of sterility during long term operation of microfluidic bioreactors. We developed a miniaturized optically accessible bioreactor (MOAB) allowing perfused culture of 3D cellularised constructs. In the MOAB, the culture chambers are sealed by magnets that generate a weak static magnetic field (SMF). Here, we predicted computationally the exact level of SMF to which cells are subjected during culture in the MOAB and we assessed its influence on the viability, metabolic activity and gene expression of neuroblastoma-derived cells cultured up to seven days. The predicted SMF ranged from 0.32 to 0.57 T using an axial-symmetric model of a single chamber, whereas it ranged from 0.35 to 0.62 T using a 3D model of the complete device. Cell function was evaluated in SH-SY5Y neuroblastoma cells at 2 and 7 days of culture in the MOAB, compared to 2D monolayer, 3D non-perfused constructs, and 3D perfused constructs cultured in a modified MOAB with magnet-free sealing. We measured the cell metabolic activity normalized by the DNA content and the expression levels of heat-shock protein 70 (Hsp-70), Bcl-2 and Bax. We found that the level of SMF applied to cells in the MOAB did not influence their metabolic activity and exerted a stressful effect in 2D monolayer, not confirmed in 3D conditions, neither static not perfused. Instead, the magnets provided a significantly greater hydraulic sealing in long-term culture, thus the MOAB might be potentially exploitable for the development of reliable in vitro models of neurodegeneration

Pectin-based bioinks for 3D models of neural tissue produced by a pH-controlled kinetics

Introduction:In the view of 3D-bioprinting with cell models representative of neural cells, we produced inks to mimic the basic viscoelastic properties of brain tissue. Moving from the concept that rheology provides useful information to predict ink printability, this study improves and expands the potential of the previously published 3D-reactive printing approach by introducing pH as a key parameter to be controlled, together with printing time. Methods:The viscoelastic properties, printability, and microstructure of pectin gels crosslinked with CaCO3 were investigated and their composition was optimized (i.e., by including cell culture medium, HEPES buffer, and collagen). Different cell models representative of the major brain cell populations (i.e., neurons, astrocytes, microglial cells, and oligodendrocytes) were considered. Results and Discussion:The outcomes of this study propose a highly controllable method to optimize the printability of internally crosslinked polysaccharides, without the need for additives or post-printing treatments. By introducing pH as a further parameter to be controlled, it is possible to have multiple (pH-dependent) crosslinking kinetics, without varying hydrogel composition. In addition, the results indicate that not only cells survive and proliferate following 3D-bioprinting, but they can also interact and reorganize hydrogel microstructure. Taken together, the results suggest that pectin-based hydrogels could be successfully applied for neural cell culture

Oxygen Sensing in Neurodegenerative Diseases: Current Mechanisms, Implication of Transcriptional Response, and Pharmacological Modulation

: Significance: Oxygen (O2) sensing is the fundamental process through which organisms respond to changes in O2 levels. Complex networks exist allowing the maintenance of O2 levels through the perception, capture, binding, transport, and delivery of molecular O2. The brain extreme sensitivity to O2 balance makes the dysregulation of related processes crucial players in the pathogenesis of neurodegenerative diseases (NDs). In this study, we wish to review the most relevant advances in O2 sensing in relation to Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Recent Advances: Over the years, it has been clarified that most NDs share common pathways, a great number of which are in relation to O2 imbalance. These include hypoxia, hyperoxia, reactive oxygen species production, metabolism of metals, protein misfolding, and neuroinflammation. Critical Issues: There is still a gap in knowledge concerning how O2 sensing plays a role in the above indicated neurodegenerations. Specifically, O2 concentrations are perceived in body sites that are not limited to the brain, but primarily reside in other organs. Moreover, the mechanisms of O2 sensing, gene expression, and signal transduction seem to correlate with neurodegeneration, but many aspects are mechanistically still unexplained. Future Directions: Future studies should focus on the precise characterization of O2 level disruption and O2 sensing mechanisms in NDs. Moreover, advances need to be made also concerning the techniques used to assess O2 sensing dysfunctions in these diseases. There is also the need to develop innovative therapies targeting this precise mechanism rather than its secondary effects, as early intervention is necessary. Antioxid. Redox Signal. 38, 160-182

Optimization of femtosecond laser polymerized structural niches to control mesenchymal stromal cell fate in culture.

We applied two-photon polymerization to fabricate 3D synthetic niches arranged in complex patterns to study the effect of mechano-topological parameters on morphology, renewal and differentiation of rat mesenchymal stromal cells. Niches were formed in a photoresist with low auto-fluorescence, which enabled the clear visualization of the fluorescence emission of the markers used for biological diagnostics within the internal niche structure. The niches were structurally stable in culture up to three weeks. At three weeks of expansion in the niches, cell density increased by almost 10-fold and was 67% greater than in monolayer culture. Evidence of lineage commitment was observed in monolayer culture surrounding the structural niches, and within cell aggregates, but not inside the niches. Thus, structural niches were able not only to direct stem cell homing and colony formation, but also to guide aggregate formation, providing increased surface-to-volume ratios and space for stem cells to adhere and renew, respectively

Identification of a novel pathway in sporadic Amyotrophic Lateral Sclerosis mediated by the long non-coding RNA ZEB1-AS1

Background: Deregulation of transcription in the pathogenesis of sporadic Amyotrophic Lateral Sclerosis (sALS) is taking central stage with RNA-sequencing analyses from sALS patients tissues highlighting numerous deregulated long non-coding RNAs (lncRNAs). The oncogenic lncRNA ZEB1-AS1 is strongly downregulated in peripheral blood mononuclear cells of sALS patients. In addition, in cancer-derived cell lines, ZEB1-AS1 belongs to a negative feedback loop regulation with hsa-miR-200c, acting as a molecular sponge for this miRNA. The role of the lncRNA ZEB1-AS1 in sALS pathogenesis has not been characterized yet, and its study could help identifying a possible disease-modifying target. Methods: the implication of the ZEB1-AS1/ZEB1/hsa-miR-200c/BMI1 pathway was investigated in multiple patients-derived cellular models (patients-derived peripheral blood mononuclear cells and induced pluripotent stem cells-derived neural stem cells) and in the neuroblastoma cell line SH-SY5Y, where its function was inhibited via RNA interference. Molecular techniques such as Real Time PCR, Western Blot and Immunofluorescence were used to assess the pathway dysregulation. Results: Our results show a dysregulation of a signaling pathway involving ZEB1-AS1/hsa-miR-200c/β-Catenin in peripheral blood mononuclear cells and in induced pluripotent stem cells-derived neural stem cells from sALS patients. These results were validated in vitro on the cell line SH-SY5Y with silenced expression of ZEB1-AS1. Moreover, we found an increase for ZEB1-AS1 during neural differentiation with an aberrant expression of β-Catenin, highlighting also its aggregation and possible impact on neurite length. Conclusions: Our results support and describe the role of ZEB1-AS1 pathway in sALS and specifically in neuronal differentiation, suggesting that an impairment of β-Catenin signaling and an alteration of the neuronal phenotype are taking place