The Impact of Experimental Conditions on Cell Mechanics as Measured with Nanoindentation.

The evaluation of cell elasticity is becoming increasingly significant, since it is now known that it impacts physiological mechanisms, such as stem cell differentiation and embryogenesis, as well as pathological processes, such as cancer invasiveness and endothelial senescence. However, the results of single-cell mechanical measurements vary considerably, not only due to systematic instrumental errors but also due to the dynamic and non-homogenous nature of the sample. In this work, relying on Chiaro nanoindenter (Optics11Life), we characterized in depth the nanoindentation experimental procedure, in order to highlight whether and how experimental conditions could affect measurements of living cell stiffness. We demonstrated that the procedure can be quite insensitive to technical replicates and that several biological conditions, such as cell confluency, starvation and passage, significantly impact the results. Experiments should be designed to maximally avoid inhomogeneous scenarios to avoid divergences in the measured phenotype.

Patient's dermal fibroblasts as disease markers for visceral myopathy.

Visceral myopathy (VSCM) is a rare genetic disease, orphan of pharmacological therapy. VSCM diagnosis is not always straightforward due to symptomatology similarities with mitochondrial or neuronal forms of intestinal pseudo-obstruction. The most prevalent form of VSCM is associates with variants in the gene ACTG2, encoding the protein gamma-2 actin. Overall, VSCM is a mechano-biological disorder, in which different genetic variants lead to similar alterations to the contractile phenotype of enteric smooth muscles, resulting in the emergence of life-threatening symptoms. In this work we analyzed the morpho-mechanical phenotype of human dermal fibroblasts from patients affected with VSCM, demonstrating that they retain a clear signature of the disease when compared with different controls. We evaluated several biophysical traits of fibroblasts, and we show that a measure of cellular traction forces can be used as a non-specific biomarker of the disease. We propose that a simple assay based on traction forces could be designed to provide a valuable support for clinical decision or pre-clinical research.

Cellular Mechanosensitivity: Validation of an Adaptable 3D-Printed Device for Microindentation.

Mechanotransduction refers to the cellular ability to sense mechanical stimuli from the surrounding environment and convert them into biochemical signals that regulate cellular physiology and homeostasis. Mechanosensitive ion channels (MSCs), especially ones of Piezo family (Piezo1 and Piezo2), play a crucial role in mechanotransduction. These transmembrane proteins directly react to mechanical cues by triggering the onset of an ionic current. The relevance of this mechanism in driving physiology and pathology is emerging, and there is a growing need for the identification of an affordable and reliable assay to measure it. Setting up a mechanosensitivity assay requires exerting a mechanical stimulus on single cells while observing the downstream effects of channels opening. We propose an open-hardware approach to stimulate single adherent cells through controlled microindentation, using a 3D-printed actuation platform. We validated the device by measuring the mechanosensitivity of a neural mice cell line where the expression level and activity of Piezo1 were genetically and pharmacologically manipulated. Moreover, this extremely versatile device could be integrated with different read-out technologies, offering a new tool to improve the understanding of mechanotransduction in living cells.

Proteostasis unbalance in prion diseases: Mechanisms of neurodegeneration and therapeutic targets.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are progressive neurodegenerative disorders of the central nervous system that affect humans and animals as sporadic, inherited, and infectious forms. Similarly to Alzheimer's disease and other neurodegenerative disorders, any attempt to reduce TSEs' lethality or increase the life expectancy of affected individuals has been unsuccessful. Typically, the onset of symptoms anticipates the fatal outcome of less than 1 year, although it is believed to be the consequence of a decades-long process of neuronal death. The duration of the symptoms-free period represents by itself a major obstacle to carry out effective neuroprotective therapies. Prions, the infectious entities of TSEs, are composed of a protease-resistant protein named prion protein scrapie (PrPSc) from the prototypical TSE form that afflicts ovines. PrPSc misfolding from its physiological counterpart, cellular prion protein (PrPC), is the unifying pathogenic trait of all TSEs. PrPSc is resistant to intracellular turnover and undergoes amyloid-like fibrillation passing through the formation of soluble dimers and oligomers, which are likely the effective neurotoxic entities. The failure of PrPSc removal is a key pathogenic event that defines TSEs as proteopathies, likewise other neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease, characterized by alteration of proteostasis. Under physiological conditions, protein quality control, led by the ubiquitin-proteasome system, and macroautophagy clears cytoplasm from improperly folded, redundant, or aggregation-prone proteins. There is evidence that both of these crucial homeostatic pathways are impaired during the development of TSEs, although it is still unclear whether proteostasis alteration facilitates prion protein misfolding or, rather, PrPSc protease resistance hampers cytoplasmic protein quality control. This review is aimed to critically analyze the most recent advancements in the cause-effect correlation between PrPC misfolding and proteostasis alterations and to discuss the possibility that pharmacological restoring of ubiquitin-proteasomal competence and stimulation of autophagy could reduce the intracellular burden of PrPSc and ameliorate the severity of prion-associated neurodegeneration.

Emerging Role of Cellular Prion Protein in the Maintenance and Expansion of Glioma Stem Cells.

Cellular prion protein (PrPC) is a membrane-anchored glycoprotein representing the physiological counterpart of PrP scrapie (PrPSc), which plays a pathogenetic role in prion diseases. Relatively little information is however available about physiological role of PrPC. Although PrPC ablation in mice does not induce lethal phenotypes, impairment of neuronal and bone marrow plasticity was reported in embryos and adult animals. In neurons, PrPC stimulates neurite growth, prevents oxidative stress-dependent cell death, and favors antiapoptotic signaling. However, PrPC activity is not restricted to post-mitotic neurons, but promotes cell proliferation and migration during embryogenesis and tissue regeneration in adult. PrPC acts as scaffold to stabilize the binding between different membrane receptors, growth factors, and basement proteins, contributing to tumorigenesis. Indeed, ablation of PrPC expression reduces cancer cell proliferation and migration and restores cell sensitivity to chemotherapy. Conversely, PrPC overexpression in cancer stem cells (CSCs) from different tumors, including gliomas-the most malignant brain tumors-is predictive for poor prognosis, and correlates with relapses. The mechanisms of the PrPC role in tumorigenesis and its molecular partners in this activity are the topic of the present review, with a particular focus on PrPC contribution to glioma CSCs multipotency, invasiveness, and tumorigenicity.

Interleukin 4 modulates microglia homeostasis and attenuates the early slowly progressive phase of amyotrophic lateral sclerosis.

Microglia activation is a commonly pathological hallmark of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), a devastating disorder characterized by a selective motor neurons degeneration. Whether such activation might represent a causal event rather than a secondary epiphenomenon remains elusive. Here, we show that CNS-delivery of IL-4-via a lentiviral-mediated gene therapy strategy-skews microglia to proliferate, inducing these cells to adopt the phenotype of slowly proliferating cells. Transcriptome analysis revealed that IL-4-treated microglia express a broad number of genes normally encoded by embryonic microglia. Since embryonic microglia sustain CNS development, we then hypothesized that turning adult microglia to acquire such phenotype via IL-4 might be an efficient in vivo strategy to sustain motor neuron survival in ALS. IL-4 gene therapy in SOD1G93A mice resulted in a general amelioration of clinical outcomes during the early slowly progressive phase of the disease. However, such approach did not revert neurodegenerative processes occurring in the late and fast progressing phase of the disease.