Regulation of calcium-permeable AMPA receptors by auxiliary subunits in cerebellar neurons

AMPA receptors (AMPARs) mediate the majority of fast excitatory synaptic transmission in the central nervous system. They exist as homo- or hetero-tetrameric assemblies of GluA1-4 subunits. Subunit composition is a crucial determinant of AMPAR biophysical and pharmacological properties. Notably, inclusion of the GluA2 subunit renders AMPARs impermeable to calcium ions as result of editing at the ‘Q/R site’. Despite the predominant expression of calcium-impermeable AMPARs (CI-AMPARs), calcium-permeable AMPARs (CP-AMPARs) play key roles in multiple physiological aspects of transmission and the influx of calcium ions through CP-AMPARs contributes to neuronal death in several neurological disorders. Given the critical roles played by CP-AMPARs in normal synaptic function and neurodegenerative processes, it is essential to understand their regulation. Neuronal AMPARs exist as complexes with accessory proteins such as transmembrane AMPAR regulatory proteins (TARPs), cornichons (CNIHs), cysteine-knot AMPA receptor-modulating proteins (CKAMPs/shisas) and GSG1L. These ancillary proteins not only govern receptor delivery to the cell membrane but also actively shape their biophysical and pharmacological properties. This thesis describes an investigation into subunit-specific AMPAR trafficking by TARPs in cerebellar granule cells (GCs). In the epileptic and ataxic stargazer mouse, a mutation in the stargazin (γ 2) gene and consequent loss of functional γ 2 protein results in a complete absence of AMPAR-mediated synaptic transmission at cerebellar mossy fibre (MF) to GC synapses. This led to the identification of γ 2 as the prototypical TARP. I used, siRNA constructs to manipulate the AMPAR subunit and TARP content in stargazer neurons to study the interplay between the type I TARP γ 2 and the less well understood type II TARP γ 7 in the synaptic targeting of CI- and CP-AMPARs. The last chapter of my thesis presents an investigation into the expression and function of AMPARs in a mouse model of juvenile Batten disease, a condition in which changes in cerebellar CP-AMPAR had been proposed by others. Overall, my results establish an important role of γ 7 in CP-AMPAR regulation in GCs and demonstrate a presynaptic rather than postsynaptic change, in the Batten disease model

Enhanced functional detection of synaptic calcium-permeable AMPA receptors using intracellular NASPM

Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic plasticity and pathology. They can be distinguished from GluA2-containing calcium-impermeable AMPARs by the inward rectification of their currents, which reflects voltage-dependent channel block by intracellular spermine. However, the efficacy of this weakly permeant blocker is differentially altered by the presence of AMPAR auxiliary subunits - including transmembrane AMPAR regulatory proteins, cornichons and GSG1L - which are widely expressed in neurons and glia. This complicates the interpretation of rectification as a measure of CP-AMPAR expression. Here we show that inclusion of the spider toxin analogue 1‑naphthylacetyl spermine (NASPM) in the intracellular solution results in complete block of GluA1-mediated outward currents irrespective of the type of associated auxiliary subunit. In neurons from GluA2-knockout mice expressing only CP-AMPARs, intracellular NASPM, unlike spermine, completely blocks outward synaptic currents. Thus, our results identify a functional measure of CP-AMPARs, that is unaffected by their auxiliary subunit content

TARP γ-7 selectively enhances synaptic expression of calcium-permeable AMPARs

Regulation of calcium-permeable AMPA receptors (CP-AMPARs) is crucial in normal synaptic function and neurological disease states. Although transmembrane AMPAR regulatory proteins (TARPs) such as stargazin (γ-2) modulate the properties of calcium-impermeable AMPARs (CI-AMPARs) and promote their synaptic targeting, the TARP-specific rules governing CP-AMPAR synaptic trafficking remain unclear. We used RNA interference to manipulate AMPAR-subunit and TARP expression in γ-2–lacking stargazer cerebellar granule cells—the classic model of TARP deficiency. We found that TARP γ-7 selectively enhanced the synaptic expression of CP-AMPARs and suppressed CI-AMPARs, identifying a pivotal role of γ-7 in regulating the prevalence of CP-AMPARs. In the absence of associated TARPs, both CP-AMPARs and CI-AMPARs were able to localize to synapses and mediate transmission, although their properties were altered. Our results also establish that TARPed synaptic receptors in granule cells require both γ-2 and γ-7 and reveal an unexpected basis for the loss of AMPAR-mediated transmission in stargazer mice