Neuronal subpopulations display differential vulnerabilities to disease, but the factors that determine their susceptibility are poorly understood. Toxic increases in intracellular calcium are a key ...factor in several neurodegenerative processes, with calcium-binding proteins providing an important first line of defense through their ability to buffer incoming calcium, allowing the neuron to quickly achieve homeostasis. Since neurons expressing different calcium-binding proteins have been reported to be differentially susceptible to degeneration, it can be hypothesized that rather than just serving as markers of different neuronal subpopulations, they might actually be a key determinant of survival. In this review, we will summarize some of the evidence that expression of the EF-hand calcium-binding proteins, calbindin, calretinin and parvalbumin, may influence the susceptibility of distinct neuronal subpopulations to disease processes.
Glutamate neurotransmission and metabolism are finely modulated by the retinal network, where the efficient processing of visual information is shaped by the differential distribution and composition ...of glutamate receptors and transporters. However, disturbances in glutamate homeostasis can result in glutamate excitotoxicity, a major initiating factor of common neurodegenerative diseases. Within the retina, glutamate excitotoxicity can impair visual transmission by initiating degeneration of neuronal populations, including retinal ganglion cells (RGCs). The vulnerability of RGCs is observed not just as a result of retinal diseases but has also been ascribed to other common neurodegenerative and peripheral diseases. In this review, we describe the vulnerability of RGCs to glutamate excitotoxicity and the contribution of different glutamate receptors and transporters to this. In particular, we focus on the
-methyl-d-aspartate (NMDA) receptor as the major effector of glutamate-induced mechanisms of neurodegeneration, including impairment of calcium homeostasis, changes in gene expression and signalling, and mitochondrial dysfunction, as well as the role of endoplasmic reticular stress. Due to recent developments in the search for modulators of NMDA receptor signalling, novel neuroprotective strategies may be on the horizon.
Glial glutamate transporters actively participate in neurotransmission and have a fundamental role in determining the ambient glutamate concentration in the extracellular space. Their expression is ...dynamically regulated in many diseases, including experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In EAE, a downregulation has been reported which may render neurons more susceptible to glutamate excitotoxicity. In this study, we have investigated the expression of GLAST (EAAT1) and GLT-1 (EAAT2) in the retina of Brown Norway rats following induction of myelin oligodendrocyte glycoprotein (MOG)-EAE, which results in retinal ganglion cell (RGC) degeneration and dysfunction. In addition, we tested whether AAV-mediated overexpression of GLAST in the retina can protect RGCs from degeneration. To address the impact of glutamate transporter modulation on RGCs, we performed whole-cell recordings and measured tonic NMDA receptor-mediated currents in the absence and presence of a glutamate-uptake blocker. We report that αOFF-RGCs show larger tonic glutamate-induced currents than αON-RGCs, in line with their greater vulnerability under neuroinflammatory conditions. We further show that increased AAV-mediated expression of GLAST in the retina does indeed protect RGCs from degeneration during the inflammatory disease. Collectively, our study highlights the neuroprotective role of glutamate transporters in the EAE retina and provides a characterization of tonic glutamate-currents of αRGCs. The larger effects of increased extracellular glutamate concentration on the αOFF-subtype may underlie its enhanced vulnerability to degeneration.
•Retinal glutamate transporter expression is reduced during pre-symptomatic EAE.•Overexpression of a glutamate transporter (GLAST) reduces ganglion cell loss in EAE.•Overexpression of GLAST reduces visual disturbances in EAE.•Effects of glutamate transport block are specific for different ganglion cell types.•Glutamate-induced tonic currents/oscillations involve NMDARs and Ca2+ channels.
Glutamate signalling is an essential aspect of neuronal communication involving many different glutamate receptors, and underlies the processes of memory, learning and synaptic plasticity. Despite ...neuroinflammatory diseases covering a range of maladies with very different biological causes and pathophysiologies, a central role for dysfunctional glutamate signalling is becoming apparent. This is not just restricted to the well-described role of glutamate in mediating neurodegeneration, but also includes a myriad of other influences that glutamate can exert on the vasculature, as well as immune cell and glial regulation, reflecting the ability of neurons to communicate with these compartments in order to couple their activity with neuronal requirements. Here, we discuss the role of pathophysiological glutamate signalling in neuroinflammatory disease, using both multiple sclerosis and Alzheimer’s disease as examples, and how current steps are being made to harness our growing understanding of these processes in the development of neuroprotective strategies. This review focuses in particular on
N
-methyl-
D
-aspartate (NMDA) and 2-amino-3-(3-hydroxy-5-methylisooxazol-4-yl) propionate (AMPA) type ionotropic glutamate receptors, although metabotropic, G-protein-coupled glutamate receptors may also contribute to neuroinflammatory processes. Given the indispensable roles of glutamate-gated ion channels in synaptic communication, means of pharmacologically distinguishing between physiological and pathophysiological actions of glutamate will be discussed that allow deleterious signalling to be inhibited whilst minimising the disturbance of essential neuronal function.
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterised by acute inflammation and subsequent neuro-axonal degeneration resulting in progressive ...neurological impairment. Aberrant immune system activation in the periphery and subsequent lymphocyte migration to the CNS contribute to the pathophysiology. Recent research has identified metabolic dysfunction as an additional feature of MS. It is already well known that energy deficiency in neurons caused by impaired mitochondrial oxidative phosphorylation results in ionic imbalances that trigger degenerative pathways contributing to white and grey matter atrophy. However, metabolic dysfunction in MS appears to be more widespread than the CNS. This review focuses on recent research assessing the metabolism and mitochondrial function in peripheral immune cells of MS patients and lymphocytes isolated from murine models of MS. Emerging evidence suggests that pharmacological modulation of lymphocytic metabolism may regulate their subtype differentiation and rebalance pro- and anti-inflammatory functions. As such, further understanding of MS immunometabolism may aid the identification of novel treatments to specifically target proinflammatory immune responses.
Objective
To explore the presence and consequences of tissue hypoxia in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).
Methods
EAE was induced in Dark ...Agouti rats by immunization with recombinant myelin oligodendrocyte glycoprotein and adjuvant. Tissue hypoxia was assessed in vivo using 2 independent methods: an immunohistochemical probe administered intravenously, and insertion of a physical, oxygen‐sensitive probe into the spinal cord. Indirect markers of tissue hypoxia (eg, expression of hypoxia‐inducible factor‐1α HIF‐1α, vessel diameter, and number of vessels) were also assessed. The effects of brief (1 hour) and continued (7 days) normobaric oxygen treatment on function were evaluated in conjunction with other treatments, namely administration of a mitochondrially targeted antioxidant (MitoQ) and inhibition of inducible nitric oxide synthase (1400W).
Results
Observed neurological deficits were quantitatively, temporally, and spatially correlated with spinal white and gray matter hypoxia. The tissue expression of HIF‐1α also correlated with loss of function. Spinal microvessels became enlarged during the hypoxic period, and their number increased at relapse. Notably, oxygen administration significantly restored function within 1 hour, with improvement persisting at least 1 week with continuous oxygen treatment. MitoQ and 1400W also caused a small but significant improvement.
Interpretation
We present chemical, physical, immunohistochemical, and therapeutic evidence that functional deficits caused by neuroinflammation can arise from tissue hypoxia, consistent with an energy crisis in inflamed central nervous system tissue. The neurological deficit was closely correlated with spinal white and gray matter hypoxia. This realization may indicate new avenues for therapy of neuroinflammatory diseases such as MS. Ann Neurol 2013;74:815–825
Neurexins are cell-surface molecules that bind neuroligins to form a heterophilic, Ca²⁺-dependent complex at central synapses. This transsynaptic complex is required for efficient neurotransmission ...and is involved in the formation of synaptic contacts. In addition, both molecules have been identified as candidate genes for autism. Here we performed mutagenesis experiments to probe for essential components of the neurexin/neuroligin binding interface at the single-amino acid level. We found that in neurexins the contact area is sharply delineated and consists of hydrophobic residues of the LNS domain that surround a Ca²⁺ binding pocket. Point mutations that changed electrostatic and shape properties leave Ca²⁺ coordination intact but completely inhibit neuroligin binding, whereas alternative splicing in α- and β-neurexins and in neuroligins has a weaker effect on complex formation. In neuroligins, the contact area appears less distinct because exchange of a more distant aspartate completely abolished binding to neurexin but many mutations of predicted interface residues had no strong effect on binding. Together with calculations of energy terms for presumed interface hot spots that complement and extend our mutagenesis and recent crystal structure data, this study presents a comprehensive structural basis for the complex formation of neurexins and neuroligins and their transsynaptic signaling between neurons.
Organ-on-chip technology is a powerful tool for in vitro modeling. Combining it with organoids overcomes lumen inaccessibility while preserving cellular diversity and function of the intestinal ...epithelium. Here, we present a protocol for generating and analyzing organ-on-chips using human and mouse intestinal organoids. This protocol covers organoid line establishment, single-cell dissociation, chip preparation, and seeding. It outlines procedures for permeability assays, RNA isolation, staining, and imaging. Additionally, we describe independent stimulation and sampling of the apical and basal side.
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•Generation of organ-on-chip using human and murine intestinal organoids•Analysis of barrier integrity by functional assay and microscopy•Independent and concurrent sampling and stimulation of apical and basal side•Tool to study transcriptome and secretome and implement co-culture
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Organ-on-chip technology is a powerful tool for in vitro modeling. Combining it with organoids overcomes lumen inaccessibility while preserving cellular diversity and function of the intestinal epithelium. Here, we present a protocol for generating and analyzing organ-on-chips using human and mouse intestinal organoids. This protocol covers organoid line establishment, single-cell dissociation, chip preparation, and seeding. It outlines procedures for permeability assays, RNA isolation, staining, and imaging. Additionally, we describe independent stimulation and sampling of the apical and basal side.
Tumour necrosis factor (TNF) is a pleiotropic cytokine and master regulator of the immune system. It acts through two receptors resulting in often opposing biological effects, which may explain the ...lack of therapeutic potential obtained so far in multiple sclerosis (MS) with non-receptor-specific anti-TNF therapeutics. Under neuroinflammatory conditions, such as MS, TNF receptor-1 (TNFR1) is believed to mediate the pro-inflammatory activities associated with TNF, whereas TNF receptor-2 (TNFR2) may instead induce anti-inflammatory effects as well as promote remyelination and neuroprotection. In this study, we have investigated the therapeutic potential of blocking TNFR1 whilst simultaneously stimulating TNFR2 in a mouse model of MS.
Experimental autoimmune encephalomyelitis (EAE) was induced with myelin oligodendrocyte glycoprotein (MOG
) in humanized TNFR1 knock-in mice. These were treated with a human-specific TNFR1-selective antagonistic antibody (H398) and a mouse-specific TNFR2 agonist (EHD2-sc-mTNF
), both in combination and individually. Histopathological analysis of spinal cords was performed to investigate demyelination and inflammatory infiltration, as well as axonal and neuronal degeneration. Retinas were examined for any protective effects on retinal ganglion cell (RGC) degeneration and neuroprotective signalling pathways analysed by Western blotting.
TNFR modulation successfully ameliorated symptoms of EAE and reduced demyelination, inflammatory infiltration and axonal degeneration. Furthermore, the combinatorial approach of blocking TNFR1 and stimulating TNFR2 signalling increased RGC survival and promoted the phosphorylation of Akt and NF-κB, both known to mediate neuroprotection.
These results further support the potential of regulating the balance of TNFR signalling, through the co-modulation of TNFR1 and TNFR2 activity, as a novel therapeutic approach in treating inflammatory demyelinating disease.
Tumour necrosis factor (TNF) signalling is mediated via two receptors, TNF-receptor 1 (TNFR1) and TNF-receptor 2 (TNFR2), which work antithetically to balance CNS immune responses involved in ...autoimmune diseases such as multiple sclerosis. To determine the therapeutic potential of selectively inhibiting TNFR1 in mice with experimental autoimmune encephalomyelitis, we used chimeric human/mouse TNFR1 knock-in mice allowing the evaluation of antagonistic anti-human TNFR1 antibody efficacy. Treatment of mice after onset of disease with ATROSAB resulted in a robust amelioration of disease severity, correlating with reduced central nervous system immune cell infiltration. Long-term efficacy of treatment was achieved by treatment with the parental mouse anti-human TNFR1 antibody, H398, and extended by subsequent re-treatment of mice following relapse. Our data support the hypothesis that anti-TNFR1 therapy restricts immune cell infiltration across the blood-brain barrier through the down-regulation of TNF-induced adhesion molecules, rather than altering immune cell composition or activity. Collectively, we demonstrate the potential for anti-human TNFR1 therapies to effectively modulate immune responses in autoimmune disease.
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