Cav1.2 channelopathies causing autism: new hallmarks on Timothy syndrome.

Cav1.2 L-type calcium channels play key roles in long-term synaptic plasticity, sensory transduction, muscle contraction, and hormone release. De novo mutations in the gene encoding Cav1.2 (CACNA1C) causes two forms of Timothy syndrome (TS1, TS2), characterized by a multisystem disorder inclusive of cardiac arrhythmias, long QT, autism, and adrenal gland dysfunction. In both TS1 and TS2, the missense mutation G406R is on the alternatively spliced exon 8 and 8A coding for the IS6-helix of Cav1.2 and is responsible for the penetrant form of autism in most TS individuals. The mutation causes specific gain-of-function changes to Cav1.2 channel gating: a "leftward shift" of voltage-dependent activation, reduced voltage-dependent inactivation, and a "leftward shift" of steady-state inactivation. How this occurs and how Cav1.2 gating changes alter neuronal firing and synaptic plasticity is still largely unexplained. Trying to better understanding the molecular basis of Cav1.2 gating dysfunctions leading to autism, here, we will present and discuss the properties of recently reported typical and atypical TS phenotypes and the effective gating changes exhibited by missense mutations associated with long QTs without extracardiac symptoms, unrelated to TS. We will also discuss new emerging views achieved from using iPSCs-derived neurons and the newly available autistic TS2-neo mouse model, both appearing promising for understanding neuronal mistuning in autistic TS patients. We will also analyze and describe recent proposals of molecular pathways that might explain mistuned Ca2+-mediated and Ca2+-independent excitation-transcription signals to the nucleus. Briefly, we will also discuss possible pharmacological approaches to treat autism associated with L-type channelopathies.

Impaired chromaffin cell excitability and exocytosis in autistic Timothy syndrome TS2-neo mouse rescued by L-type calcium channel blockers.

KEY POINTS: Tymothy syndrome (TS) is a multisystem disorder featuring cardiac arrhythmias, autism and adrenal gland dysfunction that originates from a de novo point mutation in the gene encoding the Cav1.2 (CACNA1C) L-type channel. To study the role of Cav1.2 channel signals in autism, the autistic TS2-neo mouse has been generated bearing the G406R point-mutation associated with TS type-2. Using heterozygous TS2-neo mice, we report that the G406R mutation reduces the rate of inactivation and shifts leftward the activation and inactivation of L-type channels, causing marked increase of resting Ca2+ influx ('window' Ca2+ current). The increased 'window current' causes marked reduction of NaV channel density, switches normal tonic firing to abnormal burst firing, reduces mitochondrial metabolism, induces cell swelling and decreases catecholamine release. Overnight incubations with nifedipine rescue NaV channel density, normal firing and the quantity of catecholamine released. We provide evidence that chromaffin cell malfunction derives from altered Cav1.2 channel gating. ABSTRACT: L-type voltage-gated calcium (Cav1) channels have a key role in long-term synaptic plasticity, sensory transduction, muscle contraction and hormone release. A point mutation in the gene encoding Cav1.2 (CACNA1C) causes Tymothy syndrome (TS), a multisystem disorder featuring cardiac arrhythmias, autism spectrum disorder (ASD) and adrenal gland dysfunction. In the more severe type-2 form (TS2), the missense mutation G406R is on exon 8 coding for the IS6-helix of the Cav1.2 channel. The mutation causes reduced inactivation and induces autism. How this occurs and how Cav1.2 gating-changes alter cell excitability, neuronal firing and hormone release on a molecular basis is still largely unknown. Here, using the TS2-neo mouse model of TS we show that the G406R mutation altered excitability and reduced secretory activity in adrenal chromaffin cells (CCs). Specifically, the TS2 mutation reduced the rate of voltage-dependent inactivation and shifted leftward the activation and steady-state inactivation of L-type channels. This markedly increased the resting 'window' Ca2+ current that caused an increased percentage of CCs undergoing abnormal action potential (AP) burst firing, cell swelling, reduced mitochondrial metabolism and decreased catecholamine release. The increased 'window' Ca2+ current caused also decreased NaV channel density and increased steady-state inactivation, which contributed to the increased abnormal burst firing. Overnight incubation with the L-type channel blocker nifedipine rescued the normal AP firing of CCs, the density of functioning NaV channels and their steady-state inactivation. We provide evidence that CC malfunction derives from the altered Cav1.2 channel gating and that dihydropyridines are potential therapeutics for ASD.

Early Alterations of Hippocampal Neuronal Firing Induced by Abeta42.

We studied the effect of Amyloid ß 1-42 oligomers (Abeta42) on Ca2+ dependent excitability profile of hippocampal neurons. Abeta42 is one of the Amyloid beta peptides produced by the proteolytic processing of the amyloid precursor protein and participates in the initiating event triggering the progressive dismantling of synapses and neuronal circuits. Our experiments on cultured hippocampal network reveal that Abeta42 increases intracellular Ca2+ concentration by 46% and inhibits firing discharge by 19%. More precisely, Abeta42 differently regulates ryanodine (RyRs), NMDA receptors (NMDARs), and voltage gated calcium channels (VGCCs) by increasing Ca2+ release through RyRs and inhibiting Ca2+ influx through NMDARs and VGCCs. The overall increased intracellular Ca2+ concentration causes stimulation of K+ current carried by big conductance Ca2+ activated potassium (BK) channels and hippocampal network firing inhibition. We conclude that Abeta42 alters neuronal function by means of at least 4 main targets: RyRs, NMDARs, VGCCs, and BK channels. The development of selective modulators of these channels may in turn be useful for developing effective therapies that could enhance the quality of life of AD patients during the early onset of the pathology.

Bud extracts from Salix caprea L. inhibit voltage gated calcium channels and catecholamines secretion in mouse chromaffin cells.

BACKGROUND: Salix caprea L. is an ornamental plant with prominent antioxidant activity. In the last decades Salix caprea bud extracts (SCBEs) have been used for the treatment of oxidative stress related disorders. PURPOSE: A large part of cellular functions depends on the amount of intracellular Ca2+ concentration which in turn is mainly determined by Ca2+ ions movements across plasma membrane as well as by Ca2+ released from the stores. For better evaluating the mechanism of action of SCBEs, we focused on the effect of SCBEs on voltage gated Ca2+ channels (VGCCs) functioning and related catecholamines secretion in mouse chromaffin cells (MCCs). These latter are neuroendocrine cells that share a wide variety of functions with neurons. They are particularly interesting for studying the relationship between VGCCs activation and catecholamines secretion both in control and under stressful conditions. STUDY DESIGN AND METHODS: We focused on the effect of SCBEs on VGCCs being these latter considered one of the main pathway of Ca2+ influx across plasma membrane. Ca2+ currents and capacitance changes were measured in patch clamp experiments performed in voltage clamp configuration. RESULTS: We show that SCBEs inhibited VGCCs in a dose dependent manner. On average, the saturating concentration of SCBEs (SCBEsmax) is able to block 36% of the maximum Ca2+ current amplitude (ICa) without selectivity for L (ICa, L) or non-L type (ICa, non-L) Ca2+ channels. Furthermore, ICa inhibition is not followed by alteration of VGCCs gating kinetics, but is responsible for a marked decrease of Ca2+ dependent catecholamines secretion. CONCLUSION: We conclude that the ability of SCBEs to inhibit VGCCs function, known to be potentiated during oxidative stress, could contribute to the already known antioxidant properties of Salix caprea L. We finally suggest that the inhibitory effect of SCBEs on catecholamines secretion may contribute to treat stress dependent cellular dysfunctions.

Bud extracts from Tilia tomentosa Moench inhibit hippocampal neuronal firing through GABAA and benzodiazepine receptors activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Tilia tomentosa Moench bud extracts (TTBEs) is used in traditional medicine for centuries as sedative compound. Different plants belonging to the Tilia genus have shown their efficacy in the treatment of anxiety but still little is known about the mechanism of action of their bud extracts. AIM OF THE STUDY: To evaluate the action of TTBEs as anxiolytic and sedative compound on in vitro hippocampal neurons. MATERIAL AND METHODS: The anxiolytic effect of TTBEs was assayed by testing the effects of these compounds on GABAA receptor-activated chloride current of hippocampal neurons by means of the patch-clamp technique and microelectrode-arrays (MEAs). RESULTS: TTBEs acutely administered on mouse hippocampal neurons, activated a chloride current comparable to that measured in the presence of GABA (100 µM). Bicuculline (100 µM) and picrotoxin (100 µM) blocked about 90% of this current, while the remaining 10% was blocked by adding the benzodiazepine (BDZ) antagonist flumazenil (30 µM). Flumazenil alone blocked nearly 60% of the TTBEs activated current, suggesting that TTBEs binds to both GABAA and BDZ receptor sites. Application of high-doses of TTBEs on spontaneous active hippocampal neurons grown for 3 weeks on MEAs blocked the synchronous activity of these neurons. The effects were mimicked by GABA and prevented by picrotoxin (100µM) and flumazenil (30 µM). At minimal doses, TTBEs reduced the frequency of synchronized bursts and increased the cross-correlation index of synchronized neuronal firing. CONCLUSIONS: Our data suggest that TTBEs mimics GABA and BDZ agonists by targeting hippocampal GABAergic synapses and inhibiting network excitability by increasing the strength of inhibitory synaptic outputs. Our results contribute toward the validation of TTBEs as effective sedative and anxiolytic compound.

T-type channel-mediated neurotransmitter release.

Besides controlling a wide variety of cell functions, T-type channels have been shown to regulate neurotransmitter release in peripheral and central synapses and neuroendocrine cells. Growing evidence over the last 10 years suggests a key role of Cav3.2 and Cav3.1 channels in controlling basal neurosecretion near resting conditions and sustained release during mild stimulations. In some cases, the contribution of low-voltage-activated (LVA) channels is not directly evident but requires either the activation of coupled presynaptic receptors, block of ion channels, or chelation of metal ions. Concerning the coupling to the secretory machinery, T-type channels appear loosely coupled to neurotransmitter and hormone release. In neurons, Cav3.2 and Cav3.1 channels mainly control the asynchronous appearance of "minis" [miniature inhibitory postsynaptic currents (mIPSCs) and miniature excitatory postsynaptic currents (mEPSCs)]. The same loose coupling is evident from membrane capacity and amperometric recordings in chromaffin cells and melanotropes where the low-threshold-driven exocytosis possesses the same linear Ca(2+) dependence of the other voltage-gated Ca(2+) channels (Cav1 and Cav2) that is strongly attenuated by slow calcium buffers. The intriguing issue is that, despite not expressing a consensus "synprint" site, Cav3.2 channels do interact with syntaxin 1A and SNAP-25 and, thus, may form nanodomains with secretory vesicles that can be regulated at low voltages. In this review, we discuss all the past and recent issues related to T-type channel-secretion coupling in neurons and neuroendocrine cells.