Abstract
Glutamate-activated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) mediate the majority of excitatory neurotransmission in brain and thus are major drug targets for diseases associated with hyperexcitability or neurotoxicity. Due to the critical nature of AMPA-Rs in normal brain function, typical AMPA-R antagonists have deleterious effects on cognition and motor function, highlighting the need for more precise modulators. A dramatic increase in the flip isoform of alternatively spliced AMPA-R GluA1 subunits occurs post-seizure in humans and animal models. GluA1-flip produces higher gain AMPA channels than GluA1-flop, increasing network excitability and seizure susceptibility. Splice modulating oligonucleotides (SMOs) bind to pre-mRNA to influence alternative splicing, a strategy that can be exploited to develop more selective drugs across therapeutic areas. We developed a novel SMO, GR1, which potently and specifically decreased GluA1-flip expression throughout the brain of neonatal mice lasting at least 60 days after single intracerebroventricular injection. GR1 treatment reduced AMPA-R mediated excitatory postsynaptic currents at hippocampal CA1 synapses, without affecting long-term potentiation or long-term depression, cellular models of memory, or impairing GluA1-dependent cognition or motor function in mice. Importantly, GR1 demonstrated anti-seizure properties and reduced post-seizure hyperexcitability in neonatal mice, highlighting its drug candidate potential for treating epilepsies and other neurological diseases involving network hyperexcitability.
Highlights
Glutamate-activated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) mediate the majority of brain fast excitatory neurotransmission
Peak reduction of GluA1-flip transcript was achieved with a GR1 dose of 2 μg per injection, such that GluA1-flip in the cortex and hippocampus was reduced by 97 ± 0.002 and 83 ± 0.02%, respectively, compared to saline-treated controls (Fig 2b)
The Splice modulating oligonucleotides (SMOs) developed in this study provided the opportunity to evaluate the impact of direct manipulation of GluA flip/flop isoform expression on AMPA-R mediated synaptic transmission in vivo, as a first step to understanding how GluA alternative splicing can be harnessed as an anti-seizure or neuroprotective therapeutic
Summary
Glutamate-activated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) mediate the majority of brain fast excitatory neurotransmission. Ionotropic AMPA-R GluA subunits are alternatively spliced at the flip/flop cassette, and GluA “flip” or “flop” splice variant composition confers substantially different channel properties in the brain [4,5]. GluA1-flip and GluA1-flop containing channels have similar desensitization kinetics, but GluA1-flip confers greater glutamate sensitivity, giving rise to larger amplitude responses to given concentrations of glutamate than GluA1-flop [4,5]. These characteristics make AMPA-Rs containing GluA-flip isoforms higher gain channels compared to GluA-flop. The contributions of GluA flip and flop isoforms to synaptic transmission and neuronal network properties are unknown, due to a lack of experimental compounds that modulate splice variants of individual GluAs
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