A KCNQ2 variant causing Early Onset Epileptic Encephalopathy increases spontaneous network-driven activity and excitability of pyramidal cells in the layer II/III and V of the motor cortex during a limited period of development

SummaryDe novo variants in the KCNQ2 gene encoding the Kv7.2 subunit of the voltage-gated potassium Kv7/M channel are the main cause of Early Onset Epileptic Encephalopathy (EOEE) with suppression burst suggesting that this channel plays an important role for proper brain development. Functional analysis of these variants in heterologous cells has shown that most of them are loss of function leading to a reduction of M current. However the cellular mechanism of the neuronal network dysfunctionning is still not known. Here we characterized the electrophysiological properties of developing pyramidal cells of the layer II/III and V and analyzed spontaneous synaptic activity in these layers in motor cortical slices obtained from a recently generated heterozygous knock-in mouse harboring the loss-of-function pathogenic p.T274M variant. Experiments were performed on animals aged one week, three weeks and four-five weeks, and the results were compared with those of pyramidal cells recorded in slices from wild-type mice untreated or treated with the Kv7 channel blocker XE-991. We showed that the variant led to a hyperexcitability of pyramidal cells of layer II/III in cortical slices from animal aged 1 week and 3 weeks and to a level that was similar to the effect of XE-911. In layer V the impact of the variant was observed in slices from animal aged 3 weeks but not earlier and to a level that was lower to the effect of XE-991. However, in cortical slices from animal aged 4-5 weeks electrophysiological properties of pyramidal cells of layers II/III and V were no more affected by the variant but still sensitive to XE-991. The recovery of the electrophysiological responses in knock-in animals was associated with a slight but significant distal shift of the axonal initial segment (AIS) from the soma of pyramidal cells of layer II/III and V. Recordings of spontaneous synaptic activity in these layers revealed the presence of recurrent GABAergic network activities (RGNA) that were mainly observed during the three first postnatal weeks of life and which occurrence and frequency were increased in pyramidal cells of the layer II/III but not of the layer V of the knock-in mouse. There were no significant differences in synaptic activities mediated by GABA and glutamate receptors in cortical slices from animal aged 4-5 weeks. Together our data provided evidences that the heterozygous p.T274M variant impacts the activity of pyramidal cells and probably of Gabaergic interneurons during a limited period of development. Our data also indicated that neurons of the layer II/III are more sensitive to the variant than those located in the layer V in terms of age of onset, neuronal firing and spontaneous synaptic activities. Moreover our data suggest that a compensatory mechanism might take place in the knock-in mice aged 4-5 weeks allowing the recovery of control activity at cellular and network levels and which is associated with a slight displacement of the AIS. Thus, the effect of the variant on neuronal activity is developmentally regulated and is reminiscent to some characteristics of KCNQ2-related EOEE.

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The patterns and synaptic properties of horizontal intracortical connections in the rat motor cortex

Journal of Neurophysiology ◽

10.1152/jn.1993.70.4.1553 ◽

1993 ◽

Vol 70 (4) ◽

pp. 1553-1569 ◽

Cited By ~ 62

Author(s):

V. A. Aroniadou ◽

A. Keller

Keyword(s):

Motor Cortex ◽

Deep Layer ◽

Pyramidal Cells ◽

Middle Layer ◽

Synaptic Activity ◽

Adult Rats ◽

Retrograde Labeling ◽

Connectivity Patterns ◽

Layer V ◽

Dendritic Fields

1. The laminar distribution of synaptic activity in the primary motor cortex, elicited by stimulation of intracortical, horizontal afferents, was studied in young (12-17 days old) and adult rats using the in vitro brain slice preparation. Connectivity patterns were deduced from current-source density (CSD) analyses of field potential depth profiles and were confirmed by anatomic data of retrograde cell labeling after focal injections of a fluorescent tracer. 2. According to the CSD distributions, horizontal axons in layer II/III provide strong monosynaptic input to dendrites of layer II and III pyramidal cells in a distant column, and weaker monosynaptic input to layer V and VI cells by synapsing on dendritic fields at the border of layer III and V and in deep layer V. When these pathways are activated, layer II/III cells may relay excitatory activity to upper and deep layer V, as well as to other cells in layer II/III of the same column. Axons arising from layer V provide monosynaptic input to pyramidal cells in all layers of neighboring columns, by synapsing in two dendritic fields: one in the superficial layers and the other in middle layer V. Activation of these pathways may generate a disynaptic intracolumnar input from layer II/III cells to middle layer V, as well as to other cells in layer II/III. Similar patterns of synaptic activity were elicited by stimulation from 0.45 to 2 mm distal to the recorded column. There were no apparent differences between young and adult rats in the connectivity patterns revealed by the CSD analyses. 3. Tracer injections in layer III resulted in retrograde labeling of cells in layers II/III and V, at distances > 2 mm from the injection site, whereas injections in layer V resulted in retrograde labeling of cells at long distances in layer V and to a lesser extent in layer II/III. These findings indicate that neurons in layer V project, via horizontal axon collaterals, for long distances within layers III and V, whereas the horizontal axon collaterals of layer III cells are restricted, for the most part, to the superficial layers. 4. Suppression of inhibitory activity by bath application of the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline methiodide (BMI) did not alter the pattern of the CSD distributions. All synaptic currents present in the control medium were enhanced by application of BMI, although the effect was more pronounced on the polysynaptic components.(ABSTRACT TRUNCATED AT 400 WORDS)

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Loss-of-Function Alanyl-tRNA Synthetase Mutations Cause an Autosomal-Recessive Early-Onset Epileptic Encephalopathy with Persistent Myelination Defect

The American Journal of Human Genetics ◽

10.1016/j.ajhg.2015.02.012 ◽

2015 ◽

Vol 96 (4) ◽

pp. 675-681 ◽

Cited By ~ 54

Author(s):

Cas Simons ◽

Laurie B. Griffin ◽

Guy Helman ◽

Gretchen Golas ◽

Amy Pizzino ◽

...

Keyword(s):

Early Onset ◽

Autosomal Recessive ◽

Trna Synthetase ◽

Epileptic Encephalopathy ◽

Loss Of Function

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Synaptic Activity in Chronically Injured, Epileptogenic Sensory-Motor Neocortex

Journal of Neurophysiology ◽

10.1152/jn.00507.2001 ◽

2002 ◽

Vol 88 (1) ◽

pp. 2-12 ◽

Cited By ~ 76

Author(s):

Huifang Li ◽

David A. Prince

Keyword(s):

Patch Clamp ◽

Pyramidal Neurons ◽

Critical Role ◽

Pyramidal Cells ◽

Average Frequency ◽

Synaptic Activity ◽

Cortical Hyperexcitability ◽

Significant Difference ◽

Sensory Motor ◽

Layer V

We recorded spontaneous and evoked synaptic currents in pyramidal neurons of layer V in chronically injured, epileptogenic neocortex to assess changes in the efficacy of excitatory and inhibitory neurotransmission that might promote cortical hyperexcitability. Partial sensory-motor neocortical isolations with intact blood supply (“undercuts”) were made in 20 rats on postnatal day 21–25 and examined 2–6 wk later in standard brain slice preparations using whole cell patch-clamp techniques. Age-matched, uninjured naive rats ( n = 20) were used as controls. Spontaneous and miniature excitatory and inhibitory postsynaptic currents (s- and mEPSCs; s- and mIPSCs) were recorded using patch-clamp techniques. The average frequency of s- and mEPSCs was significantly higher, while that of s- and mIPSCs was significantly lower in neurons of undercuts versus controls. The increased frequency of excitatory events was due to an increase in both s- and mEPSC frequency, suggesting an increased number of excitatory contacts and/or increased release probability at excitatory terminals. No significant difference was observed in 10–90% rise time of these events. The input-output slopes of fast, short-latency, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate (AMPA/KA) receptor-mediated components of evoked EPSCs were steeper in undercuts than in controls. The peak amplitude of the AMPA/KA component of EPSCs evoked by supra-threshold stimuli was significantly greater in the partially isolated neocortex. In contrast, the N-methyl-d-aspartate receptor-mediated component of evoked EPSCs was not significantly different in neurons of injured versus control cortex, suggesting that the increased AMPA/KA component was due to postsynaptic alterations. Results support the conclusion that layer V pyramidal neurons receive increased AMPA/KA receptor-mediated excitatory synaptic drive and decreased GABAA receptor-mediated inhibition in this chronically injured, epileptogenic cortex. This shift in the balance of excitatory and inhibitory synaptic activation of layer V pyramidal cells toward excitation might be maladaptive and play a critical role in epileptogenesis.

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Background Synaptic Conductance and Precision of EPSP-Spike Coupling at Pyramidal Cells

Journal of Neurophysiology ◽

10.1152/jn.01027.2004 ◽

2005 ◽

Vol 93 (6) ◽

pp. 3248-3256 ◽

Cited By ~ 26

Author(s):

Veronika Zsiros ◽

Shaul Hestrin

Keyword(s):

Time Course ◽

Pyramidal Cells ◽

Spike Timing ◽

Synaptic Activity ◽

Synaptic Conductance ◽

Temporal Precision ◽

Voltage Dependent ◽

Cortical Slices

The temporal precision of converting excitatory postsynaptic potentials (EPSPs) into spikes at pyramidal cells is critical for the coding of information in the cortex. Several in vitro studies have shown that voltage-dependent conductances in pyramidal cells can prolong the EPSP time course resulting in an imprecise EPSP-spike coupling. We have used dynamic-clamp techniques to mimic the in vivo background synaptic conductance in cortical slices and investigated how the ongoing synaptic activity may affect the EPSP time course near threshold and the EPSP spike coupling. We report here that background synaptic conductance dramatically diminished the depolarization related prolongation of the EPSPs in pyramidal cells and improved the precision of spike timing. Furthermore, we found that background synaptic conductance can affect the interaction among action potentials in a spike train. Thus the level of ongoing synaptic activity in the cortex may regulate the capacity of pyramidal cells to process temporal information.

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Astroglial connexin 43 sustains glutamatergic synaptic efficacy

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0596 ◽

2014 ◽

Vol 369 (1654) ◽

pp. 20130596 ◽

Cited By ~ 40

Author(s):

Oana Chever ◽

Ulrike Pannasch ◽

Pascal Ezan ◽

Nathalie Rouach

Keyword(s):

Connexin 43 ◽

Neuronal Excitability ◽

Pyramidal Cells ◽

Active Role ◽

Synaptic Activity ◽

Membrane Properties ◽

Synaptic Efficacy ◽

Dynamic Interactions ◽

Electrophysiological Properties ◽

Ca1 Pyramidal Cells

Astrocytes dynamic interactions with neurons play an active role in neurotransmission. The gap junction (GJ) subunits connexins 43 and 30 are strongly expressed in astrocytes and have recently been shown to regulate synaptic activity and plasticity. However, the specific role of connexin 43 in the morphological and electrophysiological properties of astrocytes in situ as well as in synaptic transmission remains unknown. Here, we show that connexin 43, a major determinant of astroglial GJ coupling, regulates astrocyte cell volume, but has no impact on astroglial passive membrane properties. Furthermore, we demonstrate that connexin 43 modulates glutamatergic synaptic activity of hippocampal CA1 pyramidal cells. This regulation involves changes in synaptically released glutamate, with no alteration in neuronal excitability or postsynaptic function. These results reveal connexin 43 as a critical player in neuroglial interactions by supporting synaptic efficacy.

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Faculty Opinions recommendation of Early onset combined immunodeficiency and autoimmunity in patients with loss-of-function mutation in LAT.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726389874.793520787 ◽

2016 ◽

Author(s):

Amnon Altman

Keyword(s):

Early Onset ◽

Combined Immunodeficiency ◽

Loss Of Function

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Faculty Opinions recommendation of Loss-of-Function and Gain-of-Function Mutations in KCNQ5 Cause Intellectual Disability or Epileptic Encephalopathy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727772380.793535929 ◽

2017 ◽

Author(s):

Mitsuhiro Kato

Keyword(s):

Intellectual Disability ◽

Epileptic Encephalopathy ◽

Loss Of Function ◽

Gain Of Function

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ATP13A2 Regulates Cellular α-Synuclein Multimerization, Membrane Association, and Externalization

International Journal of Molecular Sciences ◽

10.3390/ijms22052689 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2689

Author(s):

Jianmin Si ◽

Chris Van den Haute ◽

Evy Lobbestael ◽

Shaun Martin ◽

Sarah van Veen ◽

...

Keyword(s):

Membrane Integrity ◽

Ubiquitin Proteasome System ◽

Regulatory Function ◽

Membrane Association ◽

Loss Of Function ◽

Atypical Form ◽

Polyamine Transport ◽

Independent Functions ◽

Ubiquitin Proteasome ◽

The Impact

ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson’s disease and Kufor–Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions.

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A Melanocortin-4 Receptor Agonist Induces Skin and Hair Pigmentation in Patients with Monogenic Mutations in the Leptin-Melanocortin Pathway

Skin Pharmacology and Physiology ◽

10.1159/000516282 ◽

2021 ◽

pp. 1-10

Author(s):

Varvara Kanti ◽

Lia Puder ◽

Irina Jahnke ◽

Philipp Maximilian Krabusch ◽

Jan Kottner ◽

...

Keyword(s):

Leptin Receptor ◽

Skin Pigmentation ◽

Gene Mutations ◽

Continuous Treatment ◽

Whole Body ◽

Hair Color ◽

Phase 2 ◽

Loss Of Function ◽

The Impact ◽

Over Time

Background and Objectives: Gene mutations within the leptin-melanocortin signaling pathway lead to severe early-onset obesity. Recently, a phase 2 trial evaluated new pharmacological treatment options with the MC4R agonist setmelanotide in patients with mutations in the genes encoding proopiomelanocortin (POMC) and leptin receptor (LEPR). During treatment with setmelanotide, changes in skin pigmentation were observed, probably due to off-target effects on the closely related melanocortin 1 receptor (MC1R). Here, we describe in detail the findings of dermatological examinations and measurements of skin pigmentation during this treatment over time and discuss the impact of these changes on patient safety. Methods: In an investigator-initiated, phase 2, open-label pilot study, 2 patients with loss-of-function POMC gene mutations and 3 patients with loss-of-function variants in LEPR were treated with the MC4R agonist setmelanotide. Dermatological examination, dermoscopy, whole body photographic documentation, and spectrophotometric measurements were performed at screening visit and approximately every 3 months during the course of the study. Results: We report the results of a maximum treatment duration of 46 months. Skin pigmentation increased in all treated patients, as confirmed by spectrophotometry. During continuous treatment, the current results indicate that elevated tanning intensity levels may stabilize over time. Lips and nevi also darkened. In red-haired study participants, hair color changed to brown after initiation of setmelanotide treatment. Discussion: Setmelanotide treatment leads to skin tanning and occasionally hair color darkening in both POMC- and LEPR-deficient patients. No malignant skin changes were observed in the patients of this study. However, the results highlight the importance of regular skin examinations before and during MC4R agonist treatment.

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