Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS.

Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.

Bioengineered 3D Glial Cell Culture Systems and Applications for Neurodegeneration and Neuroinflammation.

Neurodegeneration and neuroinflammation are key features in a range of chronic central nervous system (CNS) diseases such as Alzheimer's and Parkinson's disease, as well as acute conditions like stroke and traumatic brain injury, for which there remains significant unmet clinical need. It is now well recognized that current cell culture methodologies are limited in their ability to recapitulate the cellular environment that is present in vivo, and there is a growing body of evidence to show that three-dimensional (3D) culture systems represent a more physiologically accurate model than traditional two-dimensional (2D) cultures. Given the complexity of the environment from which cells originate, and their various cell-cell and cell-matrix interactions, it is important to develop models that can be controlled and reproducible for drug discovery. 3D cell models have now been developed for almost all CNS cell types, including neurons, astrocytes, microglia, and oligodendrocyte cells. This review will highlight a number of current and emerging techniques for the culture of astrocytes and microglia, glial cell types with a critical role in neurodegenerative and neuroinflammatory conditions. We describe recent advances in glial cell culture using electrospun polymers and hydrogel macromolecules, and highlight how these novel culture environments influence astrocyte and microglial phenotypes in vitro, as compared to traditional 2D systems. These models will be explored to illuminate current trends in the techniques used to create 3D environments for application in research and drug discovery focused on astrocytes and microglial cells.

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier.

BACKGROUND: Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. METHODS: Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. RESULTS: Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. CONCLUSIONS: Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery.

Nitric oxide and histone deacetylases: A new relationship between old molecules.

Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from a range of nuclear and cytoplasmic proteins. Recently, we described a novel route to neurotrophin-dependent gene activation in neurons, which requires the S-nitrosylation of nuclear HDAC2 by the gaseous molecule nitric oxide (NO).1 We have further investigated the NO-dependent regulation of HDACs in neurons. Using a fluorogenic deacetylation assay, we show that NO decreases the enzymatic activity of a subgroup of neuronal HDACs in vitro and that this inhibition is not due to damaging modifications such as oxidation or tyrosine nitration. The neuronal HDACs whose catalytic activity is inhibited by NO are entirely those that are localized in the cytoplasm. These observations support and extend the concept that nitric oxide is a key regulator of HDAC function in mammalian neurons.

Integrin-binding RGD peptides induce rapid intracellular calcium increases and MAPK signaling in cortical neurons.

Integrins mediate cell adhesion to the extracellular matrix and initiate intracellular signaling. They play key roles in the central nervous system (CNS), participating in synaptogenesis, synaptic transmission and memory formation, but their precise mechanism of action remains unknown. Here we show that the integrin ligand-mimetic peptide GRGDSP induced NMDA receptor-dependent increases in intracellular calcium levels within seconds of presentation to primary cortical neurons. These were followed by transient activation and nuclear translocation of the ERK1/2 mitogen-activated protein kinase. RGD-induced effects were reduced by the NMDA receptor antagonist MK801, and ERK1/2 signaling was specifically inhibited by ifenprodil and PP2, indicating a functional connection between integrins, Src and NR2B-containing NMDA receptors. GRGDSP peptides were not significantly neuroprotective against excitotoxic insults. These results demonstrate a previously undescribed, extremely rapid effect of RGD peptide binding to integrins on cortical neurons that implies a close, functionally relevant connection between adhesion receptors and synaptic transmission.