Astrocytic C–X–C motif chemokine ligand-1 mediates β-amyloid-induced synaptotoxicity

Background Pathological interactions between β-amyloid (Aβ) and tau drive synapse loss and cognitive decline in Alzheimer’s disease (AD). Reactive astrocytes, displaying altered functions, are also a prominent feature of AD brain. This large and heterogeneous population of cells are increasingly recognised as contributing to early phases of disease. However, the contribution of astrocytes to Aβ-induced synaptotoxicity in AD is not well understood. Methods We stimulated mouse and human astrocytes with conditioned medium containing concentrations and species of human Aβ that mimic those in human AD brain. Medium from stimulated astrocytes was collected and immunodepleted of Aβ before being added to naïve rodent or human neuron cultures. A cytokine, identified in unbiased screens of stimulated astrocyte media and in postmortem human AD brain lysates was also applied to neurons, including those pre-treated with a chemokine receptor antagonist. Tau mislocalisation, synaptic markers and dendritic spine numbers were measured in cultured neurons and organotypic brain slice cultures. Results We found that conditioned medium from stimulated astrocytes induces exaggerated synaptotoxicity that is recapitulated following spiking of neuron culture medium with recombinant C–X–C motif chemokine ligand-1 (CXCL1), a chemokine upregulated in AD brain. Antagonism of neuronal C–X–C motif chemokine receptor 2 (CXCR2) prevented synaptotoxicity in response to CXCL1 and Aβ-stimulated astrocyte secretions. Conclusions Our data indicate that astrocytes exacerbate the synaptotoxic effects of Aβ via interactions of astrocytic CXCL1 and neuronal CXCR2 receptors, highlighting this chemokine–receptor pair as a novel target for therapeutic intervention in AD.

Focused Ultrasound Neuromodulation On A Multiwell MEA

Background: Microelectrode arrays (MEA) enable the measurement and stimulation of the electrical activity of cultured cells. The integration of other neuromodulation methods will greatly enhance the application range of MEAs to study their effects on neurons. A neuromodulation method that is recently gaining more attention is focused ultrasound neuromodulation (FUS), which has the potential to treat neurological disorders reversibly and precisely. Methods: In this work, we present the integration of a focused ultrasound delivery system with a multi-well MEA plate. Results: The ultrasound delivery system was characterised by ultrasound pressure measurements, and the integration with the MEA plate was modelled with finite-element simulations of acoustic field parameters. The results of the simulations were validated with experimental visualisation of the ultrasound field with Schlieren imaging. In addition, the system was tested on a murine primary hippocampal neuron culture, showing that ultrasound can influence the activity of the neurons. Conclusions: Our system was demonstrated to be suitable for studying the effect of focused ultrasound on neuronal cultures. The system allows reproducible experiments across the wells due to its robustness and simplicity of operation.

Astrocytic C-X-C motif chemokine ligand-1 mediates β-amyloid-induced synaptotoxicity

BackgroundPathological interactions between β-amyloid (Aβ) and tau drive the synapse loss that underlies neural circuit disruption and cognitive decline in Alzheimer’s disease (AD). Reactive astrocytes, displaying altered functions, are also a prominent feature of AD brain. This large and heterogeneous population of cells are increasingly recognised as contributing to early phases of disease. However, the contribution of astrocytes to detrimental Aβ-tau interactions in AD is not well understood.MethodsMouse and human astrocyte cultures were stimulated with concentrations and species of human Aβ that mimic those in human AD brain. Astrocyte conditioned medium was collected and immunodepleted of Aβ before being added to rodent or human neuron cultures. Cytokines, identified in unbiased screens were also applied to neurons, including following the pre-treatment of neurons with chemokine receptor antagonists. Tau mislocalisation, synaptic markers and dendritic spine numbers were measured in cultured neurons and organotypic brain slice cultures.ResultsConditioned medium from astrocytes stimulated with Aβ induces tau mislocalisation and exaggerated synaptotoxicity that is recapitulated following spiking of neuron culture medium with recombinant C-X-C motif chemokine ligand-1 (CXCL1), a chemokine we show to be upregulated in Alzheimer’s disease brain. Antagonism of neuronal C-X-C motif chemokine receptor 2 (CXCR2) prevented tau mislocalisation and synaptotoxicity in response to CXCL1 and Aβ-stimulated astrocyte secretions.ConclusionsOur data indicate that astrocytes exacerbate tau mislocalisation and the synaptotoxic effects of Aβ via interactions of astrocytic CXCL1 and neuronal CXCR2 receptors, highlighting this chemokine-receptor pair as a novel target for therapeutic intervention in AD.

Optimization of long-term human iPSC-derived spinal motor neuron culture using a dendritic polyglycerol amine-based substrate

Human induced pluripotent stem cells (h-iPSCs) derived from healthy and diseased individuals can give rise to many cell types, facilitating the study of mechanisms of development, human disease modeling, and early drug target validation. In this context, experimental model systems based on h-iPSC-derived motor neurons (MNs) have been used to study MN diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis. Modeling MN disease using h-iPSC-based approaches requires culture conditions that can recapitulate in a dish the events underlying differentiation, maturation, aging, and death of MNs. Current h-iPSC-derived MN-based applications are often hampered by limitations in our ability to monitor MN morphology, survival, and other functional properties over a prolonged timeframe, underscoring the need for improved long-term culture conditions. Here we describe a cytocompatible dendritic polyglycerol amine (dPGA) substrate-based method for prolonged culture of h-iPSC-derived MNs. We provide evidence that MNs cultured on dPGA-coated dishes are more amenable to long-term study of cell viability, molecular identity, and spontaneous network electrophysiological activity. The present study has the potential to improve iPSC-based studies of human MN biology and disease.

Spatiotemporally resolved subcellular phosphoproteomics

Proteome-wide profiling of protein phosphorylation has been widely used to reveal the underlying mechanism of diverse cellular signaling events. Yet, characterizing subcellular phosphoproteome with high spatial–temporal resolution has remained challenging. Herein, we developed a subcellular-specific uncaging-assisted biotinylation and mapping of phosphoproteome (SubMAPP) strategy to monitor the phosphorylation dynamics of subcellular proteome in living cells and animals. Our method capitalizes on the genetically encoded bioorthogonal decaging strategy, which enables the rapid activation of subcellular localized proximity labeling biotin ligase through either light illumination or small-molecule triggers. By further adopting an integrated orthogonal pull-down strategy with quantitative mass spectrometry, SubMAPP allowed for the investigation of subcellular phosphoproteome dynamics, revealing the altered phosphorylation patterns of endoplasmic reticulum (ER) luminal proteins under ER stress. Finally, we further expanded the scope of the SubMAPP strategy to primary neuron culture and living mice.

The anesthetic sevoflurane induces tau trafficking from neurons to microglia

Accumulation and spread of tau in Alzheimer’s disease and other tauopathies occur in a prion-like manner. However, the mechanisms and downstream consequences of tau trafficking remain largely unknown. We hypothesized that tau traffics from neurons to microglia via extracellular vesicles (EVs), leading to IL-6 generation and cognitive impairment. We assessed mice and neurons treated with anesthetics sevoflurane and desflurane, and applied nanobeam-sensor technology, an ultrasensitive method, to measure tau/p-tau amounts. Sevoflurane, but not desflurane, increased tau or p-tau amounts in blood, neuron culture medium, or EVs. Sevoflurane increased p-tau amounts in brain interstitial fluid. Microglia from tau knockout mice took up tau and p-tau when treated with sevoflurane-conditioned neuron culture medium, leading to IL-6 generation. Tau phosphorylation inhibitor lithium and EVs generation inhibitor GW4869 attenuated tau trafficking. GW4869 mitigated sevoflurane-induced cognitive impairment in mice. Thus, tau trafficking could occur from neurons to microglia to generate IL-6, leading to cognitive impairment.

Methods and Applications of Campenot Trichamber Neuronal Cultures for the Study of Neuroinvasive Viruses

The development of compartmentalized neuron culture systems has been invaluable in the study of neuroinvasive viruses, including the alpha herpesviruses Herpes Simplex Virus 1 (HSV-1) and Pseudorabies Virus (PRV). This chapter provides updated protocols for assembling and culturing rodent embryonic superior cervical ganglion (SCG) and dorsal root ganglion (DRG) neurons in Campenot trichamber cultures. In addition, we provide several illustrative examples of the types of experiments that are enabled by Campenot cultures: 1. Using fluorescence microscopy to investigate axonal outgrowth/extension through the chambers, and alpha herpesvirus infection, intracellular trafficking, and cell-cell spread via axons. 2. Using correlative fluorescence microscopy and cryo electron tomography to investigate the ultrastructure of virus particles trafficking in axons.