Analysis of Synaptic Glutamate Clearance as a Possible Indicator of Synaptic Health in the Degenerating Rodent Brain

Abstract

Neurodegeneration may present multiple challenges when one tries to quantify the astrocytic contribution to synaptic reorganization in a diseased animal. It is therefore desirable to apply tests that would detect alterations occurring in vivo but at sufficiently elementary level of defined cellular interactions. With reliable tools to identify a given synapse with respect to the pathway of origin, one could examine the specific impact of experimentally induced modifications in adjacent astrocytes. Ideally, one might be able study the properties of individual synapses in contact with a predefined type of individual astrocyte.

In our lab, respective techniques for single synapse imaging and analysis of synapse-astrocyte relationships were established in the course of a preclinical research project on Huntington’s disease (HD). We aimed at exploring new possibilities of functional rescue by expression of a modified version of the glutamate transporter EAAT2, the type 2 excitatory amino acid transporter. Our focus was on improvement of glutamate clearance, since it has repeatedly been suggested that there might be an excitotoxic component in the pathogenesis of HD. From the very outset, we therefore sought to establish and to verify indicators suitable for independent evaluation of glutamate uptake as opposed to glutamate release.

HD is a fatal neurodegenerative disease of monogenic origin. Expression of mutant huntingtin (mHTT) damages the afflicted cells (both neurons and astrocytes) in multiple ways. The most vulnerable brain area would be the dorsal striatum (caudate nucleus in humans), and the most afflicted input is the corticostriatal pathway, The latter is known to control the initiation of movements. Its damage has been associated with symptoms of hypokinesia, i.e., less frequent and slowed spontaneous movement activity, loss of neuropil, increased neuronal excitability, and reduced capacity of glutamate uptake.

In the murine striatum, most of the synaptic glutamate uptake is carried out by the excitatory amino acid transporter type 2, EAAT2, but overexpression of native EAAT2 in HD mice provided little evidence for functional rescue. We therefore explored the effects of genetically modified forms of EAAT2 and were successful with a C-terminal-truncated version of EAAT2 (EAAT2-S506X) expressed in striatal astrocytes under the control of a GFAP-promoter. This intervention not only restored the glutamate uptake of transduced astrocytes but also ameliorated the symptoms of hypokinesia in treated HD mice. We therefore wanted to know to what extent the function of individual corticostriatal synapses is shaped by their astroglial environment.

The following description of respective experiments is divided in two parts. Part 1 addresses some basic requirements to be considered for the successful implementation of single-synapse glutamate imaging as such. Part 2 will show how single-synapse imaging can be combined with single-astrocyte imaging to characterize glutamatergic synaptic transmission in its dependence on astrocyte activity in HD and the disease-changing effects of astrocytic EAAT2 modifications. It will be demonstrated that the expression of a modified EAAT2 transgene can change the glutamate clearance characteristics of the contacted synapses.

Although the outlined applications of single-synapse imaging to preclinical research in HD will require a certain level of experimental skills and good organization of animal supply, they have the advantage that they can be performed with a relatively low-cost equipment. This should make our approach attractive for forthcoming projects on other types of neurodegeneration.