Stephanie Schorge

Studies of NMDA Receptor Function and Stoichiometry with Truncated and Tandem Subunits

The subunits that compose eukaryotic glutamate ion channel receptors have three transmembrane domains (TMs) and terminate with intracellular tails that are important for controlling channel expression and localization. Truncation of NMDA receptor subunits before the final TM showed that this TM and intracellular tail region are necessary to form functional channels. However, it is shown here that these truncated subunits may be partially rescued by coexpressing the final TM and tail as a separate protein. The whole-cell currents so produced are somewhat lower than with full-length subunits, and they do not show the sag characteristic of currents from channels containing NR1 and NR2A subunits in the continued presence of an agonist. In addition, these truncated subunits were joined to full-length subunits to generate tandems. The functional expression of these tandems confirmed the tetrameric structure of NMDA receptors and also suggested that the subunits making up NMDA receptors are arranged as a dimer of dimers in the receptors with a 1-1-2-2 orientation of the subunits in the channel, and not in an alternating pattern of subunits around the pore. These results may redirect future studies into the mechanism of binding and gating in these receptors toward schemes including dimers, and may also be relevant to studies of glutamate receptor ion channels in general.

Optogenetic and Potassium Channel Gene Therapy in a Rodent Model of Focal Neocortical Epilepsy

Light-activated gene therapy acutely suppresses seizures, and gene therapy with a potassium channel prevents epileptogenesis and treats established epilepsy in a rodent model. Casting Light on the Shadow of Epilepsy Epilepsy affects 1% of the population and is often resistant to medication. Surgery to remove the region of the brain that generates seizures is only feasible in a minority of cases because of risks to movement, language, vision, and other essential functions. There is an urgent need for alternative treatments. In a new study, Wykes et al. used a virus to express a therapeutic gene in a small number of brain neurons in the seizure-generating zone in a rat model of drug-resistant epilepsy. They also developed new wireless technology to monitor and detect seizures using a miniaturized implanted transmitter and advanced algorithms. They took two approaches to reduce brain circuit excitability and hence epileptic seizures in the rat model. First, to suppress neuronal firing acutely, they expressed the light-sensitive chloride transporter halorhodopsin in the seizure-generating zone. When laser light was delivered via an optic fiber to this region and the halorhodopsin was activated, they observed a decrease in electrical seizure activity. The success of this “optogenetic” approach implies that a device could be developed to detect and stop seizures “on demand” akin to an implantable defibrillator for heart rhythm disturbances. For longer-term suppression of epilepsy, the investigators overexpressed a brain potassium ion channel that normally regulates both neuronal excitability and neurotransmitter release. This gene therapy treatment fully prevented epilepsy from developing in the rat model. When it was applied during established epilepsy, potassium channel gene therapy progressively diminished the frequency of seizures until they stopped after a few weeks. Neither of the gene therapy approaches tested interfered with normal behavior, most likely because only a small number of neurons were targeted. Although still in the earliest stages of study, this gene therapy approach may hold promise for treating drug-resistant epilepsy. Neocortical epilepsy is frequently drug-resistant. Surgery to remove the epileptogenic zone is only feasible in a minority of cases, leaving many patients without an effective treatment. We report the potential efficacy of gene therapy in focal neocortical epilepsy using a rodent model in which epilepsy is induced by tetanus toxin injection in the motor cortex. By applying several complementary methods that use continuous wireless electroencephalographic monitoring to quantify epileptic activity, we observed increases in high frequency activity and in the occurrence of epileptiform events. Pyramidal neurons in the epileptic focus showed enhanced intrinsic excitability consistent with seizure generation. Optogenetic inhibition of a subset of principal neurons transduced with halorhodopsin targeted to the epileptic focus by lentiviral delivery was sufficient to attenuate electroencephalographic seizures. Local lentiviral overexpression of the potassium channel Kv1.1 reduced the intrinsic excitability of transduced pyramidal neurons. Coinjection of this Kv1.1 lentivirus with tetanus toxin fully prevented the occurrence of electroencephalographic seizures. Finally, administration of the Kv1.1 lentivirus to an established epileptic focus progressively suppressed epileptic activity over several weeks without detectable behavioral side effects. Thus, gene therapy in a rodent model can be used to suppress seizures acutely, prevent their occurrence after an epileptogenic stimulus, and successfully treat established focal epilepsy.

Voltage sensor charge loss accounts for most cases of hypokalemic periodic paralysis

Background: Several missense mutations of CACNA1S and SCN4A genes occur in hypokalemic periodic paralysis. These mutations affect arginine residues in the S4 voltage sensors of the channel. Approximately 20% of cases remain genetically undefined. Methods: We undertook direct automated DNA sequencing of the S4 regions of CACNA1S and SCN4A in 83 cases of hypokalemic periodic paralysis. Results: We identified reported CACNA1S mutations in 64 cases. In the remaining 19 cases, mutations in SCN4A or other CACNA1S S4 segments were found in 10, including three novel changes and the first mutations in channel domains I (SCN4A) and III (CACNA1S). Conclusions: All mutations affected arginine residues, consistent with the gating pore cation leak hypothesis of hypokalemic periodic paralysis. Arginine mutations in S4 segments underlie 90% of hypokalemic periodic paralysis cases.

Maximum likelihood fitting of single channel NMDA activity with a mechanism composed of independent dimers of subunits

Steady‐state single channel activity from NMDA receptors was recorded at a range of concentrations of both glutamate and glycine. The results were fitted with several plausible mechanisms that describe both binding and gating. The mechanisms we have tested were based on our present understanding of receptor structure, or based on previously proposed mechanisms for these receptors. The steady‐state channel properties appear to have virtually no dependence on the concentration of either ligand, other than the frequency of channel activations. This limited the ability to discriminate detail in the mechanism, and, along with the persistence of open–shut correlations in high agonist concentrations, suggests that NMDA channels, unlike other neurotransmitter receptors, cannot open unless all binding sites are occupied. As usual for analyses of NMDA channels, the applicability of our results to physiological observations is limited by uncertainties in synaptic zinc and hydrogen ion concentrations, both of these being known to affect the receptor. The mechanism that we propose, on the basis of steady‐state single channel recordings, predicts with fair accuracy the apparent open and shut‐time distributions in different concentrations of agonists, correlations between open and shut times, and both the rising and falling phases of the macroscopic response to concentration jumps, and can therefore account for the main features of synaptic currents.

Human ataxias: a genetic dissection of inositol triphosphate receptor (ITPR1)-dependent signaling

A persistent mystery about the ataxias has been why mutations in genes--many of which are expressed widely in the brain--primarily cause ataxia, and not, for example, epilepsy or dementia. Why should a polyglutamine stretch in the TATA-binding protein (that is important in all cells) particularly disrupt cerebellar coordination? We propose that advances in the genetics of cerebellar ataxias suggest a rational hypothesis for how so many different genes lead to predominantly cerebellar defects. We argue that the unifying feature of many genes involved in cerebellar ataxias is their impact on the signaling protein ITPR1 (inositiol 1,4,5-triphosphate receptor type 1), that underlies coincidence detection in Purkinje cells and could play an important role in cerebellar coordination.

Episodic Ataxia Type 1: A Neuronal Potassium Channelopathy

SummaryEpisodic ataxia type 1 is a paroxysmal neurological disorder characterized by short-lived attacks of recurrent midline cerebellar dysfunction and continuous motor activity. Mutations in KCN1A, the gene encoding Kv1.1, a voltage-gated neuronal potassium channel, are associated with the disorder. Although rare, the syndrome highlights the fundamental features of genetic ion-channel diseases and serves as a useful model for understanding more common paroxysmal disorders, such as epilepsy and migraine. This review examines our current understanding of episodic ataxia type 1, focusing on its clinical and genetic features, pathophysiology, and treatment.

Chemical–genetic attenuation of focal neocortical seizures

Focal epilepsy is commonly pharmacoresistant, and resective surgery is often contraindicated by proximity to eloquent cortex. Many patients have no effective treatment options. Gene therapy allows cell-type specific inhibition of neuronal excitability, but on-demand seizure suppression has only been achieved with optogenetics, which requires invasive light delivery. Here we test a combined chemical–genetic approach to achieve localized suppression of neuronal excitability in a seizure focus, using viral expression of the modified muscarinic receptor hM4Di. hM4Di has no effect in the absence of its selective, normally inactive and orally bioavailable agonist clozapine-N-oxide (CNO). Systemic administration of CNO suppresses focal seizures evoked by two different chemoconvulsants, pilocarpine and picrotoxin. CNO also has a robust anti-seizure effect in a chronic model of focal neocortical epilepsy. Chemical–genetic seizure attenuation holds promise as a novel approach to treat intractable focal epilepsy while minimizing disruption of normal circuit function in untransduced brain regions or in the absence of the specific ligand.

Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures.

The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy.

Genetic and functional characterisation of the P/Q calcium channel in episodic ataxia with epilepsy

Mutations in CACNA1A, which encodes the principal subunit of the P/Q calcium channel, underlie episodic ataxia type 2 (EA2). In addition, some patients with episodic ataxia complicated by epilepsy have been shown to harbour CACNA1A mutations, raising the possibility that P/Q channel dysfunction may be linked to human epilepsy. We undertook a review of all published CACNA1A EA2 cases and this showed that 7% have epilepsy – representing a sevenfold increased epilepsy risk compared to the background population risk (P < 0.001). We also studied a series of 17 individuals with episodic ataxia accompanied by epilepsy and/or clearly epileptiform electroencephalograms (EEGs). We screened the entire coding region of CACNA1A for point mutations and rearrangements to determine if genetic variation in the gene is associated with the epilepsy phenotype, and measured the functional impact of all missense variations on heterologously expressed P/Q channels. We identified two large scale deletions and two new missense mutations in CACNA1A. When expressed, L621R had little detectable effect on P/Q channel function, while the other missense change, G540R, caused an approximately 30% reduction in current density. In nine patients we also identified the previously reported non‐synonymous coding variants (E921D and E993V) which also resulted in impairment of P/Q channel function. Taken together, 12 of the 17 patients have genetic changes which decrease P/Q channel function. We conclude that variants in the coding region of CACNA1A that confer a loss of P/Q‐type channel function are associated with episodic ataxia and epilepsy. Our data suggest that functional stratification of all variants, including common polymorphisms, rare variants and novel mutations, may provide new insights into the mechanisms of channelopathies.

Large scale calcium channel gene rearrangements in episodic ataxia and hemiplegic migraine: implications for diagnostic testing

Background: Episodic ataxia type 2 (EA2) and familial hemiplegic migraine type 1 (FHM1) are autosomal dominant disorders characterised by paroxysmal ataxia and migraine, respectively. Point mutations in CACNA1A, which encodes the neuronal P/Q-type calcium channel, have been detected in many cases of EA2 and FHM1. The genetic basis of typical cases without CACNA1A point mutations is not fully known. Standard DNA sequencing methods may miss large scale genetic rearrangements such as deletions and duplications. The authors investigated whether large scale genetic rearrangements in CACNA1A can cause EA2 and FHM1. Methods: The authors used multiplex ligation dependent probe amplification (MLPA) to screen for intragenic CACNA1A rearrangements. Results: The authors identified five previously unreported large scale deletions in CACNA1A in seven families with episodic ataxia and in one case with hemiplegic migraine. One of the deletions (exon 6 of CACNA1A) segregated with episodic ataxia in a four generation family with eight affected individuals previously mapped to 19p13. In addition, the authors identified the first pathogenic duplication in CACNA1A in an index case with isolated episodic diplopia without ataxia and in a first degree relative with episodic ataxia. Conclusions: Large scale deletions and duplications can cause CACNA1A associated channelopathies. Direct DNA sequencing alone is not sufficient as a diagnostic screening test.