Drosophila SPG12 ortholog, reticulon-like 1, governs presynaptic ER organization and Ca2+ dynamics.

Neuronal endoplasmic reticulum (ER) appears continuous throughout the cell. Its shape and continuity are influenced by ER-shaping proteins, mutations in which can cause distal axon degeneration in Hereditary Spastic Paraplegia (HSP). We therefore asked how loss of Rtnl1, a Drosophila ortholog of the human HSP gene RTN2 (SPG12), which encodes an ER-shaping protein, affects ER organization and the function of presynaptic terminals. Loss of Rtnl1 depleted ER membrane markers at Drosophila presynaptic motor terminals and appeared to deplete narrow tubular ER while leaving cisternae largely unaffected, thus suggesting little change in resting Ca2+ storage capacity. Nevertheless, these changes were accompanied by major reductions in activity-evoked Ca2+ fluxes in the cytosol, ER lumen, and mitochondria, as well as reduced evoked and spontaneous neurotransmission. We found that reduced STIM-mediated ER-plasma membrane contacts underlie presynaptic Ca2+ defects in Rtnl1 mutants. Our results show the importance of ER architecture in presynaptic physiology and function, which are therefore potential factors in the pathology of HSP.

Endoplasmic Reticulum Lumenal Indicators in Drosophila Reveal Effects of HSP-Related Mutations on Endoplasmic Reticulum Calcium Dynamics.

Genes for endoplasmic reticulum (ER)-shaping proteins are among the most commonly mutated in hereditary spastic paraplegia (HSP). Mutation of these genes in model organisms can lead to disruption of the ER network. To investigate how the physiological roles of the ER might be affected by such disruption, we developed tools to interrogate its Ca2+ signaling function. We generated GAL4-driven Ca2+ sensors targeted to the ER lumen, to record ER Ca2+ fluxes in identified Drosophila neurons. Using GAL4 lines specific for Type Ib or Type Is larval motor neurons, we compared the responses of different lumenal indicators to electrical stimulation, in axons and presynaptic terminals. The most effective sensor, ER-GCaMP6-210, had a Ca2+ affinity close to the expected ER lumenal concentration. Repetitive nerve stimulation generally showed a transient increase of lumenal Ca2+ in both the axon and presynaptic terminals. Mutants lacking neuronal reticulon and REEP proteins, homologs of human HSP proteins, showed a larger ER lumenal evoked response compared to wild type; we propose mechanisms by which this phenotype could lead to neuronal dysfunction or degeneration. Our lines are useful additions to a Drosophila Ca2+ imaging toolkit, to explore the physiological roles of ER, and its pathophysiological roles in HSP and in axon degeneration more broadly.

Rapid Release of Ca2+ from Endoplasmic Reticulum Mediated by Na+/Ca2+ Exchange.

Phototransduction in Drosophila is mediated by phospholipase C (PLC) and Ca2+-permeable TRP channels, but the function of endoplasmic reticulum (ER) Ca2+ stores in this important model for Ca2+ signaling remains obscure. We therefore expressed a low affinity Ca2+ indicator (ER-GCaMP6-150) in the ER, and measured its fluorescence both in dissociated ommatidia and in vivo from intact flies of both sexes. Blue excitation light induced a rapid (tau ∼0.8 s), PLC-dependent decrease in fluorescence, representing depletion of ER Ca2+ stores, followed by a slower decay, typically reaching ∼50% of initial dark-adapted levels, with significant depletion occurring under natural levels of illumination. The ER stores refilled in the dark within 100-200 s. Both rapid and slow store depletion were largely unaffected in InsP3 receptor mutants, but were much reduced in trp mutants. Strikingly, rapid (but not slow) depletion of ER stores was blocked by removing external Na+ and in mutants of the Na+/Ca2+ exchanger, CalX, which we immuno-localized to ER membranes in addition to its established localization in the plasma membrane. Conversely, overexpression of calx greatly enhanced rapid depletion. These results indicate that rapid store depletion is mediated by Na+/Ca2+ exchange across the ER membrane induced by Na+ influx via the light-sensitive channels. Although too slow to be involved in channel activation, this Na+/Ca2+ exchange-dependent release explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors exposed to Ca2+-free solutions.SIGNIFICANCE STATEMENT Phototransduction in Drosophila is mediated by phospholipase C, which activates TRP cation channels by an unknown mechanism. Despite much speculation, it is unknown whether endoplasmic reticulum (ER) Ca2+ stores play any role. We therefore engineered flies expressing a genetically encoded Ca2+ indicator in the photoreceptor ER. Although NCX Na+/Ca2+ exchangers are classically believed to operate only at the plasma membrane, we demonstrate a rapid light-induced depletion of ER Ca2+ stores mediated by Na+/Ca2+ exchange across the ER membrane. This NCX-dependent release was too slow to be involved in channel activation, but explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors bathed in Ca2+-free solutions.

Dynamic action potential clamp predicts functional separation in mild familial and severe de novo forms of SCN2A epilepsy.

De novo variants in SCN2A developmental and epileptic encephalopathy (DEE) show distinctive genotype-phenotype correlations. The two most recurrent SCN2A variants in DEE, R1882Q and R853Q, are associated with different ages and seizure types at onset. R1882Q presents on day 1 of life with focal seizures, while infantile spasms is the dominant seizure type seen in R853Q cases, presenting at a median age of 8 months. Voltage clamp, which characterizes the functional properties of ion channels, predicted gain-of-function for R1882Q and loss-of-function for R853Q. Dynamic action potential clamp, that we implement here as a method for modeling neurophysiological consequences of a given epilepsy variant, predicted that the R1882Q variant would cause a dramatic increase in firing, whereas the R853Q variant would cause a marked reduction in action potential firing. Dynamic clamp was also able to functionally separate the L1563V variant, seen in benign familial neonatal-infantile seizures from R1882Q, seen in DEE, suggesting a diagnostic potential for this type of analysis. Overall, the study shows a strong correlation between clinical phenotype, SCN2A genotype, and functional modeling. Dynamic clamp is well positioned to impact our understanding of pathomechanisms and for development of disease mechanism-targeted therapies in genetic epilepsy.

Sodium channels and the neurobiology of epilepsy.

Voltage-gated sodium channels (VGSCs) are integral membrane proteins. They are essential for normal neurologic function and are, currently, the most common recognized cause of genetic epilepsy. This review summarizes the neurobiology of VGSCs, their association with different epilepsy syndromes, and the ways in which we can experimentally interrogate their function. The most important sodium channel subunit of relevance to epilepsy is SCN1A, in which over 650 genetic variants have been discovered. SCN1A mutations are associated with a variety of epilepsy syndromes; the more severe syndromes are associated with truncation or complete loss of function of the protein. SCN2A is another important subtype associated with epilepsy syndromes, across a range of severe and less severe epilepsies. This subtype is localized primarily to excitatory neurons, and mutations have a range of functional effects on the channel. SCN8A is the other main adult subtype found in the brain and has recently emerged as an epilepsy gene, with the first human mutation discovered in a severe epilepsy syndrome. Mutations in the accessory ß subunits, thought to modulate trafficking and function of the α subunits, have also been associated with epilepsy. Genome sequencing is continuing to become more affordable, and as such, the amount of incoming genetic data is continuing to increase. Current experimental approaches have struggled to keep pace with functional analysis of these mutations, and it has proved difficult to build associations between disease severity and the precise effect on channel function. These mutations have been interrogated with a range of experimental approaches, from in vitro, in vivo, to in silico. In vitro techniques will prove useful to scan mutations on a larger scale, particularly with the advance of high-throughput automated patch-clamp techniques. In vivo models enable investigation of mutation in the context of whole brains with connected networks and more closely model the human condition. In silico models can help us incorporate the impact of multiple genetic factors and investigate epistatic interactions and beyond.

EEG dipole source localization of interictal spikes in non-lesional TLE with and without hippocampal sclerosis.

PURPOSE: A subgroup of patients with non-lesional temporal lobe epilepsy (NLTLE) have no evidence of hippocampal sclerosis (HS) on MRI or on histopathology. It is controversial whether this represents a different clinicopathological syndrome from NLTLE with HS, or whether both subgroups represent different ends of the spectrum of mesial TLE. Here the EEG source localization dipoles were compared between NLTLE patients with HS (HS+) and without HS (HS-), and the relationship with post-surgical outcome was investigated. METHODS: EEG dipole source localization of interictal epileptiform spikes recorded during prolonged video-EEG monitoring was performed from 22 consecutive HS+ and 12 HS- NLTLE patients. EEG was acquired using 29 scalp electrodes, including an inferior temporal row. Up to 13 spikes per patient were averaged and sources localized using a boundary element model based on the patients volumetric MRI. RESULTS: 21/34 patients (62%) had dipoles for the interictal spikes localized to the epileptogenic temporal lobe. The site of the intratemporal localization did not differ significantly between the HS+ and HS- patients, with the dipoles localizing to the mesial temporal region in 27% of HS+ and 25% of HS- patients. There was no significant relationship between the localization and orientation of the dipoles and the surgical outcome. CONCLUSION: The dipoles for interictal spikes do not differ between HS+ and HS- patients, suggesting that these subgroups of NLTLE patients do not have distinct cerebral generators.

Comparison of radiofrequency exposure of a mouse dam and foetuses at 900 MHz.

In vivo studies involving radiofrequency (RF) exposure of rodents require detailed dosimetric analysis to enable correct interpretation of biological outcomes. Detailed anatomical models of mice--a female, a pregnant female, a male and a foetus--have been developed for analyses using finite difference numerical techniques. The mouse models, consisting of 49 tissues, will be made freely available to the research community. In this note, the pregnant mouse model, which included eight mature foetuses, was utilized specifically to consider (a) the RF dosimetry in a radial cavity exposure system operated at a frequency of 900 MHz and (b) a 900 MHz plane wave exposure. A comparison was made between the exposure of the mouse dam and the foetuses as specified by the specific energy absorption rate (SAR) and the resultant temperature change. In general, the SAR levels in the foetuses were determined to be slightly lower (around 14% lower than the average values of the dam) and the peak temperature increase was significantly lower (45%) than the values in the dam.

The diagnostic value of oral lacerations and incontinence during convulsive "seizures".

PURPOSE: Oral lacerations and urinary incontinence have long been considered useful clinical features for the diagnosis of epileptic seizures; however, both are also reported in patients with psychogenic nonepileptic seizures (PNES). The aims of the study were (1) to investigate whether the presence and nature of oral lacerations or incontinence during convulsive seizures of patients with epilepsy differed from those with PNES, and (2) whether the side of the oral laceration has any correlation with the epilepsy syndrome or lateralization. METHODS: Eighty-four consecutive patients who experienced at least one convulsive event during video-EEG monitoring (VEM) were questioned and examined for oral lacerations and incontinence. Seizure classification was determined by a team of epileptologists based on the VEM findings and other clinical and investigational data, blinded to the oral laceration and incontinence information. RESULTS: The presence of oral lacerations among patients with epileptic seizures was 26% (17/66), in contrast it was 0% (0/18) with PNES (p = 0.01). Of the oral lacerations sustained by patients during an epileptic seizure, 14 were to the side of the tongue, one to the tip of the tongue, two to the cheek, and three to the lip. No significant relationships were observed between seizure lateralization and oral lacerations. Incontinence occurred in 23% (15/66) of epilepsy patients and 6% (1/18) of PNES patients (p = 0.09). There was no relationship between epilepsy type or lateralization and the prevalence of incontinence. CONCLUSIONS: Despite frequent reports of oral lacerations and incontinence by patients with PNES, objective evidence for this is highly specific to convulsive epileptic seizures.