Astrocyte neuropathology in autism: role of neuroinflammation & glutamatergic signalling

Abstract

Alterations in excitatory glutamatergic signalling together with increased astrocytic activation and neuroinflammation have been observed in the brain of individuals with autism. Astrocytes play important roles in developmental corticogenesis and neurogenesis, as well as regulating glutamate receptor signalling and intercellular glutamate levels at the glutamatergic tripartite synapse. The overall aim of my PhD was to investigate astrocytic neuropathology in autism and its potential interaction with disruptions in mGluR5 signalling. I conducted a post-mortem stereological investigation within the white matter of the dorsolateral prefrontal cortex (DLPFC) to assess density of astrocyte and other glia utilising the optical fractionator. In addition, astrocytic somal size was assessed via the nucleator and total astrocyte process length estimated utilising the spaceballs probe. Using an in-vitro approach I then explored the effect of Poly I:C mediated astrocyte activation on mGluR5 glutamatergic signalling. This included assessing levels of the pro-inflammatory markers IL-6 and Rantes using ELISA, gene expression of astrocytic and glutamatergic genes via qPCR, as well as mGluR5 activity using a radioligand binding assay. Finally, I characterised the gene and protein expression of astrocyte markers of human pluriporent stem cells (hPSC) derived astrocytes using qPCR and immunohistochemistry, as well as cytokines levels using ELISA. The current study revealed no change in astrocyte density or activation morphology within the white matter of the DLPFC in autism versus age matched controls. There was also no alteration in astrocyte cell somal size and total process length. In-vitro Poly I:C induced astrocyte activation demonstrated reduced mGluR5 binding and mRNA expression, with disruption to other astrocytic glutamatergic elements. A novel protocol for differentiating.

human pluripotent stem cells into astrocytes was developed, with hPSC-derived astrocytes displaying morphology similar to that of primary human foetal astrocytes and expressing mature astrocyte markers at the gene and protein level, as well as having the ability to be activated upon exposure to Poly I:C.

My findings suggest that astrocyte activation within the brain in autism may be less severe than previously appreciated, with the absence of severe astrocytic hypertrophy and increased proliferation not observed. Results from the mechanistic in-vitro studies suggest that mGluR5 and glutamatergic signalling dysregulation can occur as a result of astrocyte activation, and that modulation of mGluR5 through positive allosteric modulation may have potential benefits in reversing some of these astrocyte activation mediated glutamatergic disruptions. Finally, our improved protocol for hPSC astrocyte differentiation provides a simple and efficient method to derive mature and functional astrocytes as an in-vitro model for autism and other neurological disorders.