Epilepsy, one of the most common neurological diseases, affects approximately 1 percent of the U.S. population and is characterized by recurrent, unprovoked seizures (Stafstrom and Carmant, 2015). During seizures, neurons synchronize in one region of the brain and the aberrant neural activity can spread to other regions (Staley, 2015). Thus, epilepsy can arise when the balance between neural excitation and neural inhibition (E/I balance) is disrupted. Current therapeutic options aim to either lower excitatory activity or increase inhibitory activity in the brain, to restore the E/I balance. Nevertheless, one-third of epileptic individuals will not respond to currently available treatments (Stafstrom and Carmant, 2015), indicating the need to understand the underlying mechanisms that lead to neuronal hyperexcitability.
Excitatory activity in the brain primarily relies on the neurotransmitter glutamate, a chemical signal that crosses the synapse and binds AMPA receptors (AMPARs) on the post-synaptic cell (Hollmann and Heinemann, 1994). AMPAR quantity is proportional to the strength of the excitatory synapse. Regulating AMPAR quantity is a highly dynamic process, where AMPARs are modified at the protein level to either be inserted into the synapse (becoming functional) or to remain inside the cell (remaining inactive) (Shepherd and Huganir, 2007). At the protein level, these modifications include: addition of a phosphate group to the AMPAR protein (phosphorylation) to deliver it to the synapse (Rakhade et al., 2012); or, addition of palmitic acid lipids to the AMPAR (palmitoylation) to remove it from the synapse (Hayashi et al., 2005). Elucidating the role of AMPAR palmitoylation in vivo can inform how AMPARs regulate network excitability and how this process is affected in hyperexcitable states such as epilepsy.
Itoh et al. (2018) seek to understand how AMPAR palmitoylation regulates neuronal physiology and synaptic plasticity, whereby activity-dependent changes strengthen or weaken synapses. While prior work in vitro suggests that palmitoylation regulates whether AMPARs are removed from the synapse, little is known in vivo. Itoh et al. generated mice with mutated AMPAR subunits (GluA1) that lacked the main palmitoylation site, which prevents palmitoylation at GluA1 subunits throughout the brain (Fig., 1C).
Itoh et al. find new roles for AMPARs in regulating neuronal excitation. To begin with, their results suggest that palmitoylation and phosphorylation normally work in opposition to regulate AMPAR levels in vivo. The CS mutant mice have decreased palmitoylation in the hippocampus as expected, but display increased phosphorylation of GluA1 subunits and increased total quantity of GluA1 subunits at synapses in the hippocampus, a key brain region for learning and memory (Fig. 2B, 2C). Thus, palmitoylation is a protein modification that normally helps to decrease AMPARs at the synapse in vivo, thereby keeping neural excitation under control.
Furthermore, Itoh et al. propose that synaptic plasticity is regulated by palmitoylation. One form of synaptic plasticity is long-term potentiation (LTP) in neurons, which governs strengthening of synapses by AMPAR insertion following a strong stimulation analogous to a seizure. The CS mutant mice show a drastic increase in the size of dendritic spines, which are small protrusions along dendrites where synapses occur (Fig. 4, 5C). Taken together, palmitoylation may normally be required to prevent excessive dendritic spine growth during LTP.
Moreover, the authors discover that palmitoylation is a key protein modification to decrease the likelihood of aberrant neural activity, as seen in seizures. When the inhibitory (GABA-A) receptors were blocked to increase neural excitation, CS mutants showed greater susceptibility to develop generalized convulsive seizures, compared to controls (Fig. 6E). These tonic-clonic seizures recur in the CS mutants and show a sustained response (Fig. 6F-H). When anticonvulsants are administered to the CS mutant mice, the mutant mice are more resistant to common antiseizure medications (Figure 9). Therefore, palmitoylation is normally required to maintain the E/I balance to prevent hyper-excitable states and epilepsy.
Itoh et al. (2018) investigated the role of palmitoylation at AMPAR GluA1 subunits in vivo, to better understand how such protein-level modifications can have significant effects on regulating the balance of excitation and inhibition in the brain. Mutating one of the key palmitoylation sites of GluA1 subunits resulted in greater AMPAR levels at synapses, increased dendritic spine volumes, and a lower threshold (greater likelihood) for seizure-inducing activity (Figure 1). While this work makes a substantial advance in our understanding of molecular-level changes that contribute to epilepsy, several questions remain. For instance, GluA1s have other palmitoylation sites that may regulate AMPARs differently, or may amplify the effects at the palmitoylation site studied in this paper. Also, it remains unknown how palmitoylation of other AMPAR subunits (i.e. GluA2/3) contributes to regulation of the receptor, as this study focused only on GluA1 subunits. Itoh et al. show that palmitoylation of GluA1 subunits is important for maintaining the excitation/inhibition balance in the brain that, if disrupted, leads to hyperexcitable states and epilepsy, which advances our understanding of the molecular basis of this disease.
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