Chemogenetic manipulation of astrocyte activity at the synapse- a gateway to manage brain disease

Astrocytes are the major glial cell type in the central nervous system (CNS). Initially regarded as supportive cells, it is now recognized that this highly heterogeneous cell population is an indispensable modulator of brain development and function. Astrocytes secrete neuroactive molecules that regulate synapse formation and maturation. They also express hundreds of G protein-coupled receptors (GPCRs) that, once activated by neurotransmitters, trigger intracellular signalling pathways that can trigger the release of gliotransmitters which, in turn, modulate synaptic transmission and neuroplasticity. Considering this, it is not surprising that astrocytic dysfunction, leading to synaptic impairment, is consistently described as a factor in brain diseases, whether they emerge early or late in life due to genetic or environmental factors. Here, we provide an overview of the literature showing that activation of genetically engineered GPCRs, known as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), to specifically modulate astrocyte activity partially mimics endogenous signalling pathways in astrocytes and improves neuronal function and behavior in normal animals and disease models. Therefore, we propose that expressing these genetically engineered GPCRs in astrocytes could be a promising strategy to explore (new) signalling pathways which can be used to manage brain disorders. The precise molecular, functional and behavioral effects of this type of manipulation, however, differ depending on the DREADD receptor used, targeted brain region and timing of the intervention, between healthy and disease conditions. This is likely a reflection of regional and disease/disease progression-associated astrocyte heterogeneity. Therefore, a thorough investigation of the effects of such astrocyte manipulation(s) must be conducted considering the specific cellular and molecular environment characteristic of each disease and disease stage before this has therapeutic applicability.This work was supported by the KU Leuven Research Council (C14/20/071) and the Research Foundation Flanders Belgium (FWO, G080821N) via research project funding. MGH is currently the ERA Chair (NCBio) at i3S Porto funded by the European Commission (H2020-WIDESPREAD-2018-2020-6; NCBio; 951923)

Simultaneous determination of total and extracellular concentrations of the amino acid neurotransmitters in cat visual cortex by microbore liquid chromatography and electrochemical detection

To investigate the influence of a partial sensory deprivation on the total and extracellular concentration of the amino acid neurotransmitters in cat visual cortex, two microbore HPLC methods were developed for the simultaneous determination of aspartate, glutamate, glycine, taurine and gamma-aminobutyric acid in cat brain extracts or microdialysis samples. For the determination of the total neurotransmitter concentrations in the visual cortex, the brains were quickly frozen and 200-microns cryostat sections were made. From these sections tissue samples of 2 x 2 mm2 containing the six cortical layers were dissected out of the central and peripheral parts of area 17. After homogenisation and centrifugation, the supernatants were used for quantitative amino acid analysis using an o-phthalaldehyde-tert.-butylthiol pre-column derivatisation HPLC gradient elution method on a microbore column (100 x 1 mm I.D.; C8) and single electrochemical detection. Microdialysis samples from area 17 were obtained every 15 min using 2-mm probes perfused with synthetic cerebrospinal fluid at a flow-rate of 1 microliter/min. After o-phthalaldehyde-tert.-butylthiol derivatisation they were analysed on a microbore column by isocratic elution and dual electrochemical detection. The instrumentation and the different separation parameters were optimised and standard curve, recovery, analytical precision and detection limits for each neurotransmitter were determined.status: publishe

Sweet Substitute: A software tool for in silico fragmentation of peptide-linked N-glycans

A software tool, Sweet Substitute, is described, which assists tandem mass spectrometry (MS/MS)-based glycosylation characterization from within a tryptic digest. The algorithm creates a virtual nanoelectrospray-quadrupole time-of-flight style-MS/MS spectrum of any user-defined N-linked glycan structure. An empirical peak height modeling routine is implemented in the program. By comparing the theoretical MS/MS data with the deconvoluted and deisotoped experimental MS/MS data, the user is able to quickly assess whether a proposed candidate oligosaccharide structure is a plausible one.status: publishe

Development and plasticity-related changes in protein expression patterns in cat visual cortex: A fluorescent two-dimensional difference gel electrophoresis approach

During early postnatal brain development, changes in visual input can lead to specific alteration of function and connectivity in mammalian visual cortex. in cat, this so-called critical period exhibits maximal sensory-driven adaptations around postnatal day 30 (P30), and ceases toward adulthood. We examined the molecular framework that directs age- and experience-dependent plasticity in cat visual cortex, by comparing protein expression profiles at eye opening (postnatal day 10 (P10), when experience-dependent plasticity starts), the peak of the critical period (P30), and in adulthood. Using 2-D DIGE, we performed comparisons of P10-P30 and P30-adult brain protein samples. Sixty protein spots showed statistically significant intensity changes in at least one comparison. Fifty-one spots were identified using quadrupole-TOF MS/MS or LC-MS/MS, containing 37 different proteins. The progressive increase or decrease in protein expression levels could be correlated to age-dependent postnatal brain development. Four spots containing transferrin, 14-3-3 alpha/beta and cypin, showed maximal protein expression levels at P30, thereby showing a positive correlation to critical period plasticity. Western analysis indeed revealed a clear effect of visual deprivation on cypin expression in cat visual cortex. Our results therefore demonstrate the power of 2-D DIGE as a tool toward understanding the molecular basis of nervous system development and plasticity.status: publishe

A Fair Assessment of Evaluation Tools for the Murine Microbead Occlusion Model of Glaucoma

Despite being one of the most studied eye diseases, clinical translation of glaucoma research is hampered, at least in part, by the lack of validated preclinical models and readouts. The most popular experimental glaucoma model is the murine microbead occlusion model, yet the observed mild phenotype, mixed success rate, and weak reproducibility urge for an expansion of available readout tools. For this purpose, we evaluated various measures that reflect early onset glaucomatous changes in the murine microbead occlusion model. Anterior chamber depth measurements and scotopic threshold response recordings were identified as an outstanding set of tools to assess the model’s success rate and to chart glaucomatous damage (or neuroprotection in future studies), respectively. Both are easy-to-measure, in vivo tools with a fast acquisition time and high translatability to the clinic and can be used, whenever judged beneficial, in combination with the more conventional measures in present-day glaucoma research (i.e., intraocular pressure measurements and post-mortem histological analyses). Furthermore, we highlighted the use of dendritic arbor analysis as an alternative histological readout for retinal ganglion cell density counts.status: publishe

Effect of a single in ovo injection of 2,3,7,8-tetra-chlorodibenzo-p-dioxin on protein expression in liver and ovary of the one-day-old chick analyzed by fluorescent two-dimensional difference gel electrophoresis and mass spectrometry

The polyhalogenated aromatic hydrocarbon 2,3,7,8-tetracliloroclibenzo-p-dioxin (TCDD) is an ubiquitously distributed environmental pollutant which can induce a broad spectrum of toxic responses in animals, including birds. in this study, we investigated the impact of 0 or 20 ng TCDD injections into the yolk of chicken eggs before start of development, on liver and ovarian protein expression in hatchlings using fluorescent two-dimensional difference gel electrophoresis (2-D-DIGE) under a pH range of 4-7, combined with MS. Despite considerable inter-individual variability, exposure to TCDD prior to the start of embryonic development resulted in significant changes in expression of a small set of proteins. Expression of fibrinogen gamma chain precursor in the liver and 60 kDa heat shock protein in the ovary were significantly higher as a result of the very early exposure to TCDD. NADH ubiquinone oxidoreductase (42 kDa subunit) and regucalcin expression was decreased by early TCDD treatment in the liver and ovary, respectively. These proteins could not be directly linked with drug metabolism per se but are involved in blood dotting, oxidative stress, electron transport, and calcium regulation. It remains to be elucidated how these changes in the hatchling might be linked to the observed long-term consequences during posthatch life of the chicken.status: publishe