Neurons increase astrocytic glutamate transporter expression and uptake capacity via Notch signalling*

Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh, EH8 9XD, UK .(ACT, PH, OD, PSB, DJAW & GEH); Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, National Centre for Biological Sciences, Bangalore 560065, India. (OD)

Astrocytes in mono-culture lack the complex highly branched structure of in vivo astrocytes (Rodnight and Gottfried (2013)). The addition of neurons to astrocyte cultures has been shown to induce astrocytes to possess a more in vivo-like morphology, however only a small number of astrocytic genes have found to be altered by the presence of neurons (Matsutani and Yamamoto, 1997; Swanson et al., 1997; Nakagawa and Schwartz, 2004). One example is the robust induction of the astrocytic glutamate transporters EAAT1/GLAST (Slc1a3) and EAAT2/Glt-1 (Slc1a2) (Gegelashvili et al. (1997); Ghosh et al. (2016)).  Although various mechanisms have been implicated in the neuron-induced upregulation of glutamate transporter expression in astrocytes, the exact pathway remains unknown. We first sought to determine the effects of neurons on astrocytic gene expression using a novel mixed-species protocol. RNAseq analysis of primary cultures of pure mouse astrocytes (mono-culture) or mouse astrocytes grown in the presence of rat neurons (co-culture) showed induction of many astrocytic (mouse) genes upon co-culture with neurons. These included genes involved in glutamate uptake and metabolism: Slc1a2 and Slc1a3, glutamine synthetase (Glul) and glutamate dehydrogenase (Glud1). Electrophysiological assessment confirmed that the neuronal induction of astrocytic glutamate transporter expression was associated with an increase in glutamate uptake capacity, with co-cultured astrocytes exhibiting a greater response to the EAAT transporter agonist L-aspartate than mono-cultured cells. Having found the downstream Notch signalling pathway genes Hes5 and Hey2 to be upregulated in co-cultured astrocytes, we next investigated the role of neuron-induced Notch signalling in the driving astrocytic gene expression. The neuronal induction of many astrocytic genes was found to be suppressed in co-cultured astrocytes by the addition of the Notch pathway inhibitor DAPT. These included Slc1a2 and Slc1a3, with glutamate transporter currents likewise significantly reduced. Conversely, transfection of mono-cultured astrocytes with a constitutively active form of the Notch effector CBF1 resulted in an increase in astrocytic glutamate transporter currents. Taken together these results show that neurons induce the expression of the astrocytic glutamate transporters via a Notch-dependent signalling cascade.

References

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Funded by: The Euan MacDonald Centre for Motor Neurone Disease Research, Edinburgh

* entered into the PhD student poster competition