The sphingosine 1-phosphate analogue, FTY720, modulates the lipidomic signature of the mouse hippocampus.

The small-molecule drug, FTY720 (fingolimod), is a synthetic sphingosine 1-phosphate (S1P) analogue currently used to treat relapsing-remitting multiple sclerosis in both adults and children. FTY720 can cross the blood-brain barrier (BBB) and, over time, accumulate in lipid-rich areas of the central nervous system (CNS) by incorporating into phospholipid membranes. FTY720 has been shown to enhance cell membrane fluidity, which can modulate the functions of glial cells and neuronal populations involved in regulating behaviour. Moreover, direct modulation of S1P receptor-mediated lipid signalling by FTY720 can impact homeostatic CNS physiology, including neurotransmitter release probability, the biophysical properties of synaptic membranes, ion channel and transmembrane receptor kinetics, and synaptic plasticity mechanisms. The aim of this study was to investigate how chronic FTY720 treatment alters the lipid composition of CNS tissue in adolescent mice at a key stage of brain maturation. We focused on the hippocampus, a brain region known to be important for learning, memory, and the processing of sensory and emotional stimuli. Using mass spectrometry-based lipidomics, we discovered that FTY720 increases the fatty acid chain length of hydroxy-phosphatidylcholine (PCOH) lipids in the mouse hippocampus. It also decreases PCOH monounsaturated fatty acids (MUFAs) and increases PCOH polyunsaturated fatty acids (PUFAs). A total of 99 lipid species were up-regulated in the mouse hippocampus following 3 weeks of oral FTY720 exposure, whereas only 3 lipid species were down-regulated. FTY720 also modulated anxiety-like behaviours in young mice but did not affect spatial learning or memory formation. Our study presents a comprehensive overview of the lipid classes and lipid species that are altered in the hippocampus following chronic FTY720 exposure and provides novel insight into cellular and molecular mechanisms that may underlie the therapeutic or adverse effects of FTY720 in the central nervous system.

Mechanobiology of the brain in ageing and Alzheimer's disease.

Just as the epigenome, the proteome and the electrophysiological properties of a cell influence its function, so too do its intrinsic mechanical properties and its extrinsic mechanical environment. This is especially true for neurons of the central nervous system (CNS) as long-term maintenance of synaptic connections relies on efficient axonal transport machinery and structural stability of the cytoskeleton. Recent reports suggest that profound physical changes occur in the CNS microenvironment with advancing age which, in turn, will impact highly mechanoresponsive neurons and glial cells. Here, we discuss the complex and inhomogeneous mechanical structure of CNS tissue, as revealed by recent mechanical measurements on the brain and spinal cord, using techniques such as magnetic resonance elastography and atomic force microscopy. Moreover, ageing, traumatic brain injury, demyelination and neurodegeneration can perturb the mechanical properties of brain tissue and trigger mechanobiological signalling pathways in neurons, glia and cerebral vasculature. It is, therefore, very likely that significant changes in cell and tissue mechanics contribute to age-related cognitive decline and deficits in memory formation which are accelerated and magnified in neurodegenerative states, such as Alzheimer's disease. Importantly, we are now beginning to understand how neuronal and glial cell mechanics and brain tissue mechanobiology are intimately linked with neurophysiology and cognition.

Spatiotemporally Super-Resolved Volumetric Traction Force Microscopy.

Quantification of mechanical forces is a major challenge across biomedical sciences. Yet such measurements are essential to understanding the role of biomechanics in cell regulation and function. Traction force microscopy remains the most broadly applied force probing technology but typically restricts itself to single-plane two-dimensional quantifications with limited spatiotemporal resolution. Here, we introduce an enhanced force measurement technique combining 3D super-resolution fluorescence structural illumination microscopy and traction force microscopy (3D-SIM-TFM) offering increased spatiotemporal resolution, opening-up unprecedented insights into physiological three-dimensional force production in living cells.

Cold snapshots of DNA repair: Cryo-EM structures of DNA-PKcs and NHEJ machinery.

The proteins and protein assemblies involved in DNA repair have been the focus of a multitude of structural studies for the past few decades. Historically, the structures of these protein complexes have been resolved by X-ray crystallography. However, more recently with the advancements in cryo-electron microscopy (cryo-EM) ranging from optimising the methodology for sample preparation to the development of improved electron detectors, the focus has shifted from X-ray crystallography to cryo-EM. This methodological transition has allowed for the structural determination of larger, more complex protein assemblies involved in DNA repair pathways and has subsequently led to a deeper understanding of the mechanisms utilised by these fascinating molecular machines. Here, we review some of the key structural advancements that have been gained in the study of non-homologous end joining (NHEJ) by the use of cryo-EM, with a focus on assemblies composed of DNA-PKcs and Ku70/80 (Ku) and the various methodologies utilised to obtain these structures.

Hippocampus of the APPNL-G-F mouse model of Alzheimer's disease exhibits region-specific tissue softening concomitant with elevated astrogliosis.

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 APPNL-G-F mouse model. Several hippocampal regions in APPNL-G-F 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.

Evaluating the Impact of a Biomimetic Mechanical Environment on Cancer Invasion and Matrix Remodeling.

The stiffness of tumors and their host tissues is much higher than most hydrogels, which are conventionally used to study in vitro cancer progression. The tumoroid assay is an engineered 3D in vitro tumor model that allows investigation of cancer cell invasion in an environment that is biomimetic in terms of extracellular matrix (ECM) composition and stiffness. Using this model, the change in matrix stiffness by epithelial colorectal cancer cells is systematically characterized by atomic force microscopy indentation tests. Less invasive epithelial cancer cells stiffen the tumor microenvironment while highly aggressive epithelial cancer cells show significant softening of the tumor microenvironment. Changes in stiffness are attributed to both cell-generated active forces as well as ECM degradation and remodeling. The degradation is in part attributed to the enzymatic activity of matrix metalloproteinases (MMPs) as demonstrated by the significant expression of MMP-2 and MMP-9 at both gene and protein levels. Targeting MMP activity through broad-spectrum drug inhibition (BB-94) reverses the changes in stiffness and also decreases cancer cell invasion. These results promote the idea of using mechano-based cancer therapies such as MMP inhibition.

Corrigendum: Hippocampus of the APPNL-G-F mouse model of Alzheimer's disease exhibits region-specific tissue softening concomitant with elevated astrogliosis.

[This corrects the article DOI: 10.3389/fnagi.2023.1212212.].

Knock-in models related to Alzheimer's disease: synaptic transmission, plaques and the role of microglia.

BACKGROUND: Microglia are active modulators of Alzheimer's disease but their role in relation to amyloid plaques and synaptic changes due to rising amyloid beta is unclear. We add novel findings concerning these relationships and investigate which of our previously reported results from transgenic mice can be validated in knock-in mice, in which overexpression and other artefacts of transgenic technology are avoided. METHODS: AppNL-F and AppNL-G-F knock-in mice expressing humanised amyloid beta with mutations in App that cause familial Alzheimer's disease were compared to wild type mice throughout life. In vitro approaches were used to understand microglial alterations at the genetic and protein levels and synaptic function and plasticity in CA1 hippocampal neurones, each in relationship to both age and stage of amyloid beta pathology. The contribution of microglia to neuronal function was further investigated by ablating microglia with CSF1R inhibitor PLX5622. RESULTS: Both App knock-in lines showed increased glutamate release probability prior to detection of plaques. Consistent with results in transgenic mice, this persisted throughout life in AppNL-F mice but was not evident in AppNL-G-F with sparse plaques. Unlike transgenic mice, loss of spontaneous excitatory activity only occurred at the latest stages, while no change could be detected in spontaneous inhibitory synaptic transmission or magnitude of long-term potentiation. Also, in contrast to transgenic mice, the microglial response in both App knock-in lines was delayed until a moderate plaque load developed. Surviving PLX5266-depleted microglia tended to be CD68-positive. Partial microglial ablation led to aged but not young wild type animals mimicking the increased glutamate release probability in App knock-ins and exacerbated the App knock-in phenotype. Complete ablation was less effective in altering synaptic function, while neither treatment altered plaque load. CONCLUSIONS: Increased glutamate release probability is similar across knock-in and transgenic mouse models of Alzheimer's disease, likely reflecting acute physiological effects of soluble amyloid beta. Microglia respond later to increased amyloid beta levels by proliferating and upregulating Cd68 and Trem2. Partial depletion of microglia suggests that, in wild type mice, alteration of surviving phagocytic microglia, rather than microglial loss, drives age-dependent effects on glutamate release that become exacerbated in Alzheimer's disease.

A new framework for characterization of poroelastic materials using indentation.

To characterize a poroelastic material, typically an indenter is pressed onto the surface of the material with a ramp of a finite approach velocity followed by a hold where the indenter displacement is kept constant. This leads to deformation of the porous matrix, pressurization of the interstitial fluid and relaxation due to redistribution of fluid through the pores. In most studies the poroelastic properties, including elastic modulus, Poisson ratio and poroelastic diffusion coefficient, are extracted by assuming an instantaneous step indentation. However, exerting step like indentation is not experimentally possible and usually a ramp indentation with a finite approach velocity is applied. Moreover, the poroelastic relaxation time highly depends on the approach velocity in addition to the poroelastic diffusion coefficient and the contact area. Here, we extensively studied the effect of indentation velocity using finite element simulations which has enabled the formulation of a new framework based on a master curve that incorporates the finite rise time. To verify our novel framework, the poroelastic properties of two types of hydrogels were extracted experimentally using indentation tests at both macro and micro scales. Our new framework that is based on consideration of finite approach velocity is experimentally easy to implement and provides a more accurate estimation of poroelastic properties. STATEMENT OF SIGNIFICANCE: Hydrogels, tissues and living cells are constituted of a sponge-like porous elastic matrix bathed in an interstitial fluid. It has been shown that these materials behave according to the theory of 'poroelasticity' when mechanically stimulated in a way similar to that experienced in organs within the body. In this theory, the rate at which the fluid-filled sponge can be deformed is limited by how fast interstitial fluid can redistribute within the sponge in response to deformation. Here, we simulated indentation experiments at different rates and formulated a new framework that inherently captures the effects of stimulation speed on the mechanical response of poroelastic materials. We validated our framework by conducting experiments at different length-scales on agarose and polyacrylamide hydrogels.