HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes.

SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.

A Novel Modulator of Kv3 Potassium Channels Regulates the Firing of Parvalbumin-Positive Cortical Interneurons.

Kv3.1 and Kv3.2 high voltage-activated potassium channels, which display fast activation and deactivation kinetics, are known to make a crucial contribution to the fast-spiking phenotype of certain neurons. Pharmacological experiments show that the blockade of native Kv3 currents with low concentrations of tetraethylammonium or 4-aminopyridine impairs the expression of this firing phenotype. In particular, Kv3 channels are highly expressed by fast-spiking, parvalbumin-positive interneurons in corticolimbic brain circuits, which modulate the synchronization of cortical circuits and the generation of brain rhythms. Here, we describe a novel small molecule, (5R)-5-ethyl-3-(6-{[4-methyl-3-(methyloxy)phenyl]oxy}-3-pyridinyl)-2,4-imidazolidinedione (AUT1), which modulates Kv3.1 and Kv3.2 channels in human recombinant and rodent native neurons. AUT1 increased whole currents mediated by human Kv3.1b and Kv3.2a channels, with a concomitant leftward shift in the voltage dependence of activation. A less potent effect was observed on hKv3.3 currents. In mouse somatosensory cortex slices in vitro, AUT1 rescued the fast-spiking phenotype of parvalbumin-positive-fast-spiking interneurons following an impairment of their firing capacity by blocking a proportion of Kv3 channels with a low concentration of tetraethylammonium. Notably, AUT1 had no effect on interneuron firing when applied alone. Together, these data confirm the role played by Kv3 channels in the regulation of the firing phenotype of somatosensory interneurons and suggest that AUT1 and other Kv3 modulators could represent a new and promising therapeutic approach to the treatment of disorders associated with dysfunction of inhibitory feedback in corticolimbic circuits, such as schizophrenia.

Generation of hiPSC-Derived Functional Dopaminergic Neurons in Alginate-Based 3D Culture.

Human induced pluripotent stem cells (hiPSCs) represent an unlimited cell source for the generation of patient-specific dopaminergic (DA) neurons, overcoming the hurdle of restricted accessibility to disease-affected tissue for mechanistic studies on Parkinson's disease (PD). However, the complexity of the human brain is not fully recapitulated by existing monolayer culture methods. Neurons differentiated in a three dimensional (3D) in vitro culture system might better mimic the in vivo cellular environment for basic mechanistic studies and represent better predictors of drug responses in vivo. In this work we established a new in vitro cell culture system based on the microencapsulation of hiPSCs in small alginate/fibronectin beads and their differentiation to DA neurons. Optimization of hydrogel matrix concentrations and composition allowed a high viability of embedded hiPSCs. Neural differentiation competence and efficiency of DA neuronal generation were increased in the 3D cultures compared to a conventional 2D culture methodology. Additionally, electrophysiological parameters and metabolic switching profile confirmed increased functionality and an anticipated metabolic resetting of neurons grown in alginate scaffolds with respect to their 2D counterpart neurons. We also report long-term maintenance of neuronal cultures and preservation of the mature functional properties. Furthermore, our findings indicate that our 3D model system can recapitulate mitochondrial superoxide production as an important mitochondrial phenotype observed in neurons derived from PD patients, and that this phenotype might be detectable earlier during neuronal differentiation. Taken together, these results indicate that our alginate-based 3D culture system offers an advantageous strategy for the reliable and rapid derivation of mature and functional DA neurons from hiPSCs.

Modeling Parkinson's disease in midbrain-like organoids.

Modeling Parkinson's disease (PD) using advanced experimental in vitro models is a powerful tool to study disease mechanisms and to elucidate unexplored aspects of this neurodegenerative disorder. Here, we demonstrate that three-dimensional (3D) differentiation of expandable midbrain floor plate neural progenitor cells (mfNPCs) leads to organoids that resemble key features of the human midbrain. These organoids are composed of midbrain dopaminergic neurons (mDANs), which produce and secrete dopamine. Midbrain-specific organoids derived from PD patients carrying the LRRK2-G2019S mutation recapitulate disease-relevant phenotypes. Automated high-content image analysis shows a decrease in the number and complexity of mDANs in LRRK2-G2019S compared to control organoids. The floor plate marker FOXA2, required for mDAN generation, increases in PD patient-derived midbrain organoids, suggesting a neurodevelopmental defect in mDANs expressing LRRK2-G2019S. Thus, we provide a robust method to reproducibly generate 3D human midbrain organoids containing mDANs to investigate PD-relevant patho-mechanisms.

Ca2+ channels and synaptic transmission at the adult, neonatal, and P/Q-type deficient neuromuscular junction.

Different types of voltage-activated Ca(2+) channels have been established based on their molecular structure and pharmacological and biophysical properties. One of them, the P/Q-type, is the main channel involved in nerve-evoked neurotransmitter release at neuromuscular junctions and the immunological target in Eaton-Lambert Syndrome. At adult neuromuscular junctions, L- and N-type Ca(2+) channels become involved in transmitter release only under certain experimental or pathological conditions. In contrast, at neonatal rat neuromuscular junctions, nerve-evoked synaptic transmission depends jointly on both N- and P/Q-type channels. Synaptic transmission at neuromuscular junctions of the ataxic P/Q-type Ca(2+) channel knockout mice is also dependent on two different types of channels, N- and R-type. At both neonatal and P/Q knockout junctions, the K(+)-evoked increase in miniature endplate potential frequency was not affected by N-type channel blockers, but strongly reduced by both P/Q- and R-type channel blockers. These differences could be accounted for by a differential location of the channels at the release site, being either P/Q- or R-type Ca(2+) channels located closer to the release site than N-type Ca(2+) channels. Thus, Ca(2+) channels may be recruited to mediate neurotransmitter release where P/Q-type channels seem to be the most suited type of Ca(2+) channel to mediate exocytosis at neuromuscular junctions.

Interneurone bursts are spontaneously associated with muscle contractions only during early phases of mouse spinal network development: a study in organotypic cultures.

For a short time during development immature circuits in the spinal cord and other parts of the central nervous system spontaneously generate synchronous patterns of rhythmic activity. In the case of the spinal cord, it is still unclear how strongly synchronized bursts generated by interneurones are associated with motoneurone firing and whether the progressive decline in spontaneous bursting during circuit maturation proceeds in parallel for motoneurone and interneurone networks. We used organotypic cocultures of spinal cord and skeletal muscle in order to investigate the ontogenic evolution of endogenous spinal network activity associated with the generation of coordinate muscle fibre contractions. A combination of multiunit electrophysiological recordings, videomicroscopy and optical flow computation allowed us to measure the correlation between interneurone firing and motoneurone outputs after 1, 2 and 3 weeks of in vitro development. We found that, in spinal organotypic slices, there is a developmental switch of spontaneous activity from stable bursting to random patterns after the first week in culture. Conversely, bursting recorded in the presence of strychnine and bicuculline became increasingly regular with time in vitro. The time course of spontaneous activity maturation in organotypic slices is similar to that previously reported for the spinal cord developing in utero. We also demonstrated that spontaneous bursts of interneurone action potentials strongly correlate with muscular contractions only during the first week in vitro and that this is due to the activation of motoneurones via AMPA-type glutamate receptors. These results indicate the occurrence in vitro of motor network development regulating bursting inputs from interneurones to motoneurones.

Differential Ca2+-dependence of transmitter release mediated by P/Q- and N-type calcium channels at neonatal rat neuromuscular junctions.

N- and P/Q-type voltage dependent calcium channels (VDCCs) mediate transmitter release at neonatal rat neuromuscular junction (NMJ). Thus the neonatal NMJ allows an examination of the coupling of different subtypes of VDCCs to the release process at a single synapse. We studied calcium dependence of transmitter release mediated by each channel by blocking with omega-conotoxin GVIA the N-type channel or with omega-agatoxin IVA the P/Q-type channel while changing the extracellular calcium concentration ([Ca2+]o). Transmitter release mediated by P/Q-type VDCCs showed steeper calcium dependence than N-type mediated release (average slope 3.6 +/- 0.09 vs. 2.6 +/- 0.03, respectively). Loading the nerve terminals with 10 microm BAPTA-AM in the extracellular solution reduced transmitter release and occluded the blocking effect of omega-conotoxin GVIA (blockade -2 +/- 9%) without affecting the action of omega-agatoxin IVA (blockade 85 +/- 4%). Both VDCC blockers were able to reduce the amount of facilitation produced by double-pulse stimulation. In these conditions facilitation was restored by increasing [Ca2+]o. The facilitation index (fi) was also reduced by loading nerve terminals with 10 microm BAPTA-AM (fi = 1.2 +/- 0.1). The control fi was 2.5 +/- 0.1. These results show that P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than were N-type VDCCs at the neonatal neuromuscular junction. This difference could be accounted for by a differential location of these channels at the release site. In addition, our results indicate that space-time overlapping of calcium domains was required for facilitation.

Calcium channels involved in neurotransmitter release at adult, neonatal and P/Q-type deficient neuromuscular junctions (Review).

Different types of voltage-dependent calcium channels (VDCCs) have been recognized based on their molecular structure as well as their pharmacological and biophysical properties. One of these, the P/Q type, is the main channel involved in nerve evoked neurotransmitter release at neuromuscular junctions (NMJs) and many central nervous system synapses. However, under particular experimental or biological conditions, other channels can be involved. L-type VDCC presence at the NMJ has been demonstrated by the contribution to the perineural calcium currents (Ica) at adult mice Bapta-loaded NMJs. This is probably a result of a reduction in Ca(2+) inactivation. The L-type current was not coupled to neurotransmitter release, but became coupled, as demonstrated by the release of acetylcholine, after the inhibition of serine/threonine protein phosphatases with okadaic acid (OA). Thus, under these conditions, L-type channels were unmasked at Bapta- but not at Egta-loaded NMJs. This suggests that the speed, not the capacity, of the calcium chelator was decisive in preventing Ca(2+)-inactivation and facilitating the contribution to neurotransmitter release. At neonatal rat NMJs, N-type VDCCs were involved early during development whereas P/Q-type VDCCs play a main role at all stages of development. Furthermore, P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than N-type VDCCs. This difference could be accounted for by a differential location of these channels at the release site. Neuromuscular transmission in P/Q-type calcium channel knock out ataxic mice jointly depends on both N-type and R-type channels and shows several altered properties including low quantal content. Thus, calcium channels may be recruited to mediate neurotransmitter release with a functional hierarchy where the P/Q channel seems to be the channel most suited to mediate exocytosis at NMJs.