Impaired Ca-signaling in astrocytes from the Ts16 mouse model of Down syndrome

doi:10.1016/S0304-3940(97)13406-1

Neuroscience Letters

Volume 223, Issue 2, 21 February 1997, Pages 81-84

https://doi.org/10.1016/S0304-3940(97)13406-1 Get rights and content

Abstract

The trisomy 16 mouse model of Down syndrome has been used to compare calcium (Ca)-homeostasis and Ca-signaling in astrocytes from trisomic mice and from diploid littermates. Ratio calcium-imaging of Fura-2/AM loaded primary astroglial cultures prepared from the hippocampus shows that resting Ca levels are on average significantly higher in trisomic than in the control astrocytes (280 vs. 120 nM). Serotonin (3 μM) and glutamate (30–300 μM) evoked transient Ca-increases from 400 to 600 nM in euploid but from only 20 to 150 nM in trisomic astrocytes. Imaging of ATP-driven Ca-accumulation in cellular organelles revealed a significantly stronger uptake of Ca in trisomic astrocytes that might buffer cytosolic Ca-increases. Our results demonstrate major disturbances in Ca-signaling in trisomic astrocytes that are likely to be of pathophysiological relevance.

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Acknowledgements

We thank Dr. H. Winking, Medizinische Hochschule Lübeck, Germany for the kind gift of Ts16 Robertsonian mice and R. Schneider, K. Berlin and A. Düerkop for excellent technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany through a Heisenberg-Stipendium to W.M. and a research grant (SFB 507/C4) and by the Charité, Berlin, Germany.

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For instance, in FX-associated tremor/ataxia syndrome, astrocytes exhibit increased spontaneous asynchronous calcium oscillations [63]. In a DS mouse model, astrocytes basal calcium levels are also enhanced and evoked calcium responses are reduced, likely due to an accumulation in organelles that may buffer cytosolic calcium [64]. Finally, aberrant calcium signaling also occurs in DS patient-derived astroglia, with more frequent and increased spontaneous calcium fluctuations, independently of functional astroglial maturation [65].
Neurodevelopmental disorders, including those involving intellectual disability, are characterized by abnormalities in formation and functions of synaptic circuits. Traditionally, research on synaptogenesis and synaptic transmission in health and disease focused on neurons, however, a growing number of studies have highlighted the role of astrocytes in this context. Tight structural and functional interactions of astrocytes and synapses indeed play important roles in brain functions, and the repertoire of astroglial regulations of synaptic circuits is large and complex. Recently, genetic studies of intellectual disabilities have underscored potential contributions of astrocytes in the pathophysiology of these disorders. Here we review how alterations of astrocyte functions in disease may interfere with neuronal excitability and the balance of excitatory and inhibitory transmission during development, and contribute to intellectual disabilities.
In Vivo Restoration of Physiological Levels of Truncated TrkB.T1 Receptor Rescues Neuronal Cell Death in a Trisomic Mouse Model
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Thus, changes in Akt phosphorylation levels may be in part responsible for the premature neuronal cell death observed in Ts16 neurons. Ts16 neurons and astrocytes have increased levels of resting intracellular Ca2+ (Bambrick et al., 1997; Muller et al., 1997; Schuchmann et al., 1998). Recently, it was shown that BDNF induces an increase in cytoplasmic Ca2+ in astroglia.
Imbalances in neurotrophins or their high-affinity Trk receptors have long been reported in neurodegenerative diseases. However, a molecular link between these gene products and neuronal cell death has not been established. In the trisomy 16 (Ts16) mouse there is increased apoptosis in the cortex, and hippocampal neurons undergo accelerated cell death that cannot be rescued by administration of brain-derived neurotrophic factor (BDNF). Ts16 neurons have normal levels of the TrkB tyrosine kinase receptor but an upregulation of the TrkB.T1 truncated receptor isoform. Here we show that restoration of the physiological level of the TrkB.T1 receptor by gene targeting rescues Ts16 cortical cell and hippocampal neuronal death. Moreover, it corrects resting Ca²⁺ levels and restores BDNF-induced intracellular signaling mediated by full-length TrkB in Ts16 hippocampal neurons. These data provide a direct link between neuronal cell death and abnormalities in Trk neurotrophin receptor levels.
On the cause of mental retardation in Down syndrome: Extrapolation from full and segmental trisomy 16 mouse models
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Down syndrome (DS, trisomy 21, Ts21) is the most common known cause of mental retardation. In vivo structural brain imaging in young DS adults, and post-mortem studies, indicate a normal brain size after correction for height, and the absence of neuropathology. Functional imaging with positron emission tomography (PET) shows normal brain glucose metabolism, but fewer significant correlations between metabolic rates in different brain regions than in controls, suggesting reduced functional connections between brain circuit elements. Cultured neurons from Ts21 fetuses and from fetuses of an animal model for DS, the trisomy 16 (Ts16) mouse, do not differ from controls with regard to passive electrical membrane properties, including resting potential and membrane resistance. On the other hand, the trisomic neurons demonstrate abnormal active electrical and biochemical properties (duration of action potential and its rates of depolarization and repolarization, altered kinetics of active Na⁺, Ca²⁺ and K⁺ currents, altered membrane densities of Na⁺ and Ca²⁺ channels). Another animal model, the adult segmental trisomy 16 mouse (Ts65Dn), demonstrates reduced long-term potentiation and increased long-term depression (models for learning and memory related to synaptic plasticity) in the CA1 region of the hippocampus. Evidence suggests that the abnormalities in the trisomy mouse models are related to defective signal transduction pathways involving the phosphoinositide cycle, protein kinase A and protein kinase C. The phenotypes of DS and its mouse models do not involve abnormal gene products due to mutations or deletions, but result from altered expression of genes on human chromosome 21 or mouse chromosome 16, respectively. To the extent that the defects in signal transduction and in active electrical properties, including synaptic plasticity, that are found in the Ts16 and Ts65Dn mouse models, are found in the brain of DS subjects, we postulate that mental retardation in DS results from such abnormalities. Changes in timing and synaptic interaction between neurons during development can lead to less than optimal functioning of neural circuitry and signaling then and in later life.
Evidence for an interferon-related inflammatory reaction in the trisomy 16 mouse brain leading to caspase-1-mediated neuronal apoptosis
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The trisomy of human chromosome 21 (Down syndrome) is the leading genetic cause of learning difficulties in children, and predisposes this population to the early onset of the neurodegeneration of Alzheimer’s disease. Down syndrome is associated with increased interferon (IFN) sensitivity resulting in unexpectedly high levels of IFN inducible gene products including Fas, complement factor C3, and neuronal HLA I which could result in a damaging inflammatory reaction in the brain. Consistent with this possibility, we report here that the trisomy 16 mouse fetus has significantly increased whole brain IFN-γ and Fas receptor immunoreactivity and that cultured whole brain trisomy 16 mouse neurons have increased basal levels of caspase 1 activity and altered homeostasis of intracellular calcium and pH. The trisomic neurons also showed a heightened sensitivity to the increase in both Fas receptor levels and caspase 1 activity we observed when IFN-γ was added to the neuron culture media. Because of the autoregulatory nature of IFN activity, and the IFN inducing capability of caspase-1-activated cytokine activity, our data argue in favor of the possibility of an interferon-mediated, self-perpetuating, inflammatory response in the trisomy brain that could subserve the loss of neuron viability seen in this trisomy 16 mouse model for Down syndrome.
GFAP phosphorylation studied in digitonin-permeabilized astrocytes: Standardization of conditions
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Citation Excerpt :
Moreover, ATP has a permeabilizing effect itself in some cells such as mast cells and macrophages [7], pointing to major differences between different cell types. Nevertheless, the protective effect of ATP should be considered when interpreting data from studies of intracellular stores of Ca2+ in permeabilized astrocytes [22]. Also, ATP could be implicated in the low rates of permeabilization by digitonin in astrocytes using 20 μM digitonin for 15 min, as reported in Ref. [17].
Cycles of assembly/disassembly of the intermediate filaments of astrocytes are modulated by the phosphorylation of glial fibrillary acidic protein (GFAP). The sites on GFAP are localized at the N-terminal where they are phosphorylated by cAMP-dependent and Ca²⁺-dependent protein kinases. Phosphorylation of GFAP has been investigated in brain slices, astrocyte cultures, cytoskeletal fractions and purified systems. Here we describe a different approach to study GFAP phosphorylation. We show that permeabilization of astrocytes in culture with digitonin allows direct access to the systems phosphorylating GFAP. Conditions for the permeabilization were established with an assay based on the exclusion of Trypan blue. Incubation of permeabilized cells with cAMP and Ca²⁺ increased the phosphorylation state of GFAP. Immunocytochemistry with anti-GFAP showed that permeabilized astrocytes retained their typical flat, fibroblast morphology and exhibited well preserved glial filaments. On incubation with cAMP the filaments apparently condensed to form long processes. The results suggest the approach of studying structural changes in glial filaments in parallel to protein phosphorylation, in the presence of specific modulators of protein kinases and phosphatases has considerable potential.
Digitonin-permeabilization of astrocytes in culture monitored by trypan blue exclusion and loss of S100B by ELISA
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The present protocol details a procedure to permeabilize astrocytes in cultures with digitonin as well as to discuss some data about factors that interfere in permeabilization, particularly divalent cations and nucleotides. Two methods to assess astrocyte permeabilization are described: trypan blue exclusion and ELISA for S100B, a specific protein expressed by these cells. Digitonin-permeabilization of astrocytes has been used to investigate intracellular pools of Ca²⁺, internal stores of metabolites, phosphoinositide hydrolysis, and recently we standardized a procedure to study protein phosphorylation (Brain Res. 853 (2000) 32–40). A short incubation time (10 min) with 30 μM digitonin permeabilized at least 75% of cells. A range of media with different ionic nature can be used in cell permeabilization without affecting significantly the extent of permeabilization, but calcium and ATP of the order of 10⁻⁵ M induced a partial resealing which deserves to be considered in assays of permeabilized preparations of astrocytes.

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Impaired Ca-signaling in astrocytes from the Ts16 mouse model of Down syndrome

Abstract

Section snippets

Acknowledgements

Brain Res.

Brain Res. Bull.

Neuron

J. Biol. Chem.

Neuron

Neuron

Neuron

Trends Neurosci.

Trophic interactions between astroglial cells and hippocampal neurons in culture

Science

Rat hippocampal neurons in dispersed cell culture

Brain Res.

Sustained increase in intracellular calcium promotes neuronal survival

J. Neurosci.