Pellegrino Lippiello

Molecular Pharmacology of the Amiloride Analog 3-Amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)-amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) as a Pan Inhibitor of the Na+-Ca2+ Exchanger Isoforms NCX1, NCX2, and NCX3 in Stably Transfected Cells

With the help of single-cell microflorimetry, 45Ca2+ radiotracer fluxes, and patch-clamp in whole-cell configuration, we examined the effect of the amiloride derivative 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) on the activity of the three isoforms of the Na+/Ca2+ exchanger (NCX) and on several other membrane currents including voltage- and pH-sensitive ones. This amiloride analog suppressed the bidirectional activity of all NCX isoforms in a concentration-dependent manner. The IC50 values of CB-DMB were in the nanomolar range for the outward and the inward components of the bidirectional NCX1, NCX2, and NCX3 activity. Deletion mutagenesis showed that CB-DMB inhibited NCX activity mainly at level of the f-loop but not through the interaction with Gly833 located at the level of the α2 repeat. On the other hand, CB-DMB suppressed in the micromolar range the other plasma membrane currents encoded by voltage-dependent Ca2+ channels, tetrodotoxin-sensitive Na+ channels, and pH-sensitive ASIC1a. Collectively, the data of the present study showed that CB-DMB, when used in the nanomolar range, is one of the most potent compounds that can block the activity of the three NCX isoforms when they work both in the forward and in the reverse modes of operation without interfering with other ionic channels.

The 5-HT7 receptor triggers cerebellar long-term synaptic depression via PKC-MAPK.

The 5-HT7 receptor (5-HT7R) mediates important physiological effects of serotonin, such as memory and emotion, and is emerging as a therapeutic target for the treatment of cognitive disorders and depression. Although previous studies have revealed an expression of 5-HT7R in cerebellum, particularly at Purkinje cells, its functional role and signaling mechanisms have never been described. Using patch-clamp recordings in cerebellar slices of adult mice, we investigated the effects of a selective 5-HT7R agonist, LP-211, on the main plastic site of the cerebellar cortex, the parallel fiber-Purkinje cell synapse. Here we show that 5-HT7R activation induces long-term depression of parallel fiber-Purkinje cell synapse via a postsynaptic mechanism that involves the PKC-MAPK signaling pathway. Moreover, a 5-HT7R antagonist abolished the expression of PF-LTD, produced by pairing parallel fiber stimulation with Purkinje cell depolarization; whereas, application of a 5-HT7R agonist impaired LTP induced by 1 Hz parallel fiber stimulation. Our results indicate for the first time that 5-HT7R exerts a fine regulation of cerebellar bidirectional synaptic plasticity that might be involved in cognitive processes and neuropsychiatric disorders involving the cerebellum.

Modulation, Plasticity and Pathophysiology of the Parallel Fiber-Purkinje Cell Synapse

The parallel fiber-Purkinje cell (PF-PC) synapse represents the point of maximal signal divergence in the cerebellar cortex with an estimated number of about 60 billion synaptic contacts in the rat and 100,000 billions in humans. At the same time, the Purkinje cell dendritic tree is a site of remarkable convergence of more than 100,000 parallel fiber synapses. Parallel fiber activity generates fast postsynaptic currents via α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and slower signals, mediated by mGlu1 receptors, resulting in Purkinje cell depolarization accompanied by sharp calcium elevation within dendritic regions. Long-term depression (LTD) and long-term potentiation (LTP) have been widely described for the PF-PC synapse and have been proposed as mechanisms for motor learning. The mechanisms of induction for LTP and LTD involve different signaling mechanisms within the presynaptic terminal and/or at the postsynaptic site, promoting enduring modification in the neurotransmitter release and change in responsiveness to the neurotransmitter. The PF-PC synapse is finely modulated by several neurotransmitters, including serotonin, noradrenaline and acetylcholine. The ability of these neuromodulators to gate LTP and LTD at the PF-PC synapse could, at least in part, explain their effect on cerebellar-dependent learning and memory paradigms. Overall, these findings have important implications for understanding the cerebellar involvement in a series of pathological conditions, ranging from ataxia to autism. For example, PF-PC synapse dysfunctions have been identified in several murine models of spino-cerebellar ataxia (SCA) types 1, 3, 5 and 27. In some cases, the defect is specific for the AMPA receptor signaling (SCA27), while in others the mGlu1 pathway is affected (SCA1, 3, 5). Interestingly, the PF-PC synapse has been shown to be hyper-functional in a mutant mouse model of autism spectrum disorder, with a selective deletion of Pten in Purkinje cells. However, the full range of methodological approaches, that allowed the discovery of the physiological principles of PF-PC synapse function, has not yet been completely exploited to investigate the pathophysiological mechanisms of diseases involving the cerebellum. We, therefore, propose to extend the spectrum of experimental investigations to tackle this problem.

Nitric Oxide Stimulates NCX1 and NCX2 but Inhibits NCX3 Isoform by Three Distinct Molecular Determinants

In this study, the role of nitric oxide (NO) in the modulation of the activity of NCX1, NCX2, and NCX3 exchangers was investigated in baby hamster kidney cells singly transfected with each of these isoforms by single-cell Fura-2-microfluorometry and patch clamp. Furthermore, the molecular determinants of NO on each isoform were identified by deletion, site-directed mutagenesis, and chimera strategies. Our data revealed four main findings. First, the NO-donor S-nitroso-N-acetylpenicillamine (SNAP; 10 nM) and the NO-precursor l-arginine (10 mM) were both able to increase NCX1 activity in a cGMP-independent way. Moreover, within the amino acid sequence 723 to 734 of the f-loop, Cys730 resulted as the target of NO on NCX1. Second, SNAP and l-arginine were able to increase NCX2 activity, but this effect was prevented by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In addition, the membrane-permeable 8-bromoguanosine-cGMP alone was able to mimic the stimulatory effect of the gaseous mediator, suggesting the involvement of a cGMP-dependent mechanism. Within the amino acid sequence 699 to 744 of the f-loop, Ser713 was the NO molecular determinant on the NCX2 protein; Third, NCX3 activity was instead down-regulated by NO in a cGMP-independent manner. This NO-inhibitory action was exerted at the level of Cys156 in the α1-region outside the f-loop. Finally, the activity of the two NCX3 chimeras—obtained by the replacement of the NO-insensitive NCX3 region with the homologous NO-sensitive segments of NCX1 or NCX2—was potentiated by SNAP. Together, the present data demonstrate that NO differently regulates the activity of the three gene products NCX1, NCX2, and NCX3 by modulating specific molecular determinants.

Noradrenergic modulation of the parallel fiber-Purkinje cell synapse in mouse cerebellum.

The signals arriving to Purkinje cells via parallel fibers are essential for all tasks in which the cerebellum is involved, including motor control, learning new motor skills and calibration of reflexes. Since learning also requires the activation of adrenergic receptors, we investigated the effects of adrenergic receptor agonists on the main plastic site of the cerebellar cortex, the parallel fiber-Purkinje cell synapse. Here we show that noradrenaline serves as an endogenous ligand for both α1-and α2-adrenergic receptors to produce synaptic depression between parallel fibers and Purkinje cells. On the contrary, PF-EPSCs were potentiated by the β-adrenergic receptor agonist isoproterenol. This short-term potentiation was postsynaptically expressed, required protein kinase A, and was mimicked by the β2-adrenoceptor agonist clenbuterol, suggesting that the β2-adrenoceptors mediate the noradrenergic facilitation of synaptic transmission between parallel fibers and Purkinje cells. Moreover, β-adrenoceptor activation lowered the threshold for cerebellar long-term potentiation induced by 1 Hz parallel fiber stimulation. The presence of both α and β adrenergic receptors on Purkinje cells suggests the existence of bidirectional mechanisms of regulation allowing the noradrenergic afferents to refine the signals arriving to Purkinje cells at particular arousal states or during learning.

Cysteine Prevents the Reduction in Keratin Synthesis Induced by Iron Deficiency in Human Keratinocytes

l‐cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up‐regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up‐regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia. J. Cell. Biochem. 117: 402–412, 2016.

Down regulation of pro-inflammatory pathways by tanshinone IIA and cryptotanshinone in a non-genetic mouse model of Alzheimer's disease

Graphical abstract Figure. No caption available. &NA; Alzheimers disease (AD) is a common form of dementia mainly characterized by the deposition of neurofibrillary tangles and &bgr;‐amyloid (A&bgr;) peptides in the brain. Additionally, increasing evidence demonstrates that a neuro‐inflammatory state plays a key role in the development of this disease. Beside synthetic drugs, the use of natural compounds represents an alternative for the development of new potential drugs for the treatment of AD. Among these, the root of Salvia miltiorhiza Bunge (also known as Danshen) used for the treatment of cardiovascular, cerebrovascular disease and CNS functional decline in Chinese traditional medicine is one of the most representative examples. We therefore evaluated the effects of tanshinone IIA (TIIA) and cryptotanshinone (CRY) (the two major lipophilic compounds of Danshen) in a non‐genetic mouse model of &bgr;‐amyloid (A&bgr;)‐induced AD, which is mainly characterized by reactive gliosis and neuro‐inflammation in the brain. To this aim, mice were injected intracerebroventricularly (i.c.v.) with A&bgr;1–42 peptide (3 &mgr;g/3 &mgr;l) and after with TIIA and CRY (1, 3, or 10 mg/kg) intraperitoneally (i.p.) 3 times weekly for 21 days following the induction of experimental AD. Spatial working memory was assessed as a measure of short‐term memory in mice, whereas the level of GFAP, S100&bgr;, COX‐2, iNOS and NF‐kBp65 monitored by western blot and ELISA assay, were selected as markers of reactive gliosis and neuro‐inflammation. Finally, by docking studies, the modulation of key pro‐inflammatory enzymes and pathways involved in the AD‐related neuro‐inflammation were also investigated. Results indicate that TIIA and CRY alleviate memory decline in A&bgr;1‐42‐injected mice, in a dose dependent manner. Moreover, the analysis of gliosis‐related and neuro‐inflammatory markers in the hippocampal tissues reveal a remarkable reduction in the expression of GFAP, S100&bgr;, COX‐2, iNOS and NF‐kBp65 after CRY (10 mg/kg) treatment. These effects were less evident, but still significant, after TIIA (10 mg/kg). Finally, in silico analysis also revealed that both compounds were able to interact with the binding sites of NF‐kBp65 endorsing the data from biochemical analysis. We conclude that TIIA and CRY display anti‐inflammatory and neuroprotective effect in a non‐genetic mouse model of AD, thus playing a role in slowing down the course and onset of AD.

Everolimus improves memory and learning while worsening depressive- and anxiety-like behavior in an animal model of depression

Everolimus (EVR) is an orally-administered rapamycin analog that selectively inhibits the mammalian target of rapamycin (mTOR) kinase (mainly mTORC1 and likely mTORC2) and the related signaling pathway. mTOR is a serine/threonine protein kinase regulating multiple important cellular functions; dysfunction of mTOR signaling has also been implicated in the pathophysiology of several neurological, neurodegenerative, developmental and cognitive disorders. EVR is widely used as an anti-neoplastic therapy and more recently in children with tuberous sclerosis complex (TSC). However, no clear correlation exists between EVR use and development of central side effects e.g. depression, anxiety or cognitive impairment. We studied the effects of a 3 weeks administration of EVR in mice chronically treated with betamethasone 21-phosphate disodium (BTM) as a model of depression and cognitive decline. EVR treatment had detrimental effects on depressive- and anxiety-like behavior while improving cognitive performance in both control (untreated) and BTM-treated mice. Such effects were accompanied by an increased hippocampal neurogenesis and synaptogenesis. Our results therefore might support the proposed pathological role of mTOR dysregulation in depressive disorders and confirm some previous data on the positive effects of mTOR inhibition in cognitive decline. We also show that EVR, possibly through mTOR inhibition, may be linked to the development of anxiety. The increased hippocampal neurogenesis by EVR might explain its ability to improve cognitive function or protect from cognitive decline. Our findings suggest some caution in the use of EVR, particularly in the developing brain; patients should be carefully monitored for their psychiatric/neurological profiles in any clinical situation where an mTOR inhibitor and in particular EVR is used e.g. cancer treatment, TSC or immunosuppression.

Maturation, Refinement, and Serotonergic Modulation of Cerebellar Cortical Circuits in Normal Development and in Murine Models of Autism

The formation of the complex cerebellar cortical circuits follows different phases, with initial synaptogenesis and subsequent processes of refinement guided by a variety of mechanisms. The regularity of the cellular and synaptic organization of the cerebellar cortex allowed detailed studies of the structural plasticity mechanisms underlying the formation of new synapses and retraction of redundant ones. For the attainment of the monoinnervation of the Purkinje cell by a single climbing fiber, several signals are involved, including electrical activity, contact signals, homosynaptic and heterosynaptic interaction, calcium transients, postsynaptic receptors, and transduction pathways. An important role in this developmental program is played by serotonergic projections that, acting on temporally and spatially regulated postsynaptic receptors, induce and modulate the phases of synaptic formation and maturation. In the adult cerebellar cortex, many developmental mechanisms persist but play different roles, such as supporting synaptic plasticity during learning and formation of cerebellar memory traces. A dysfunction at any stage of this process can lead to disorders of cerebellar origin, which include autism spectrum disorders but are not limited to motor deficits. Recent evidence in animal models links impairment of Purkinje cell function with autism-like symptoms including sociability deficits, stereotyped movements, and interspecific communication by vocalization.

From Cannabis to Cannabidiol to Treat Epilepsy, Where Are We?

BACKGROUND Several antiepileptic drugs (AEDs), about 25, are currently clinically available for the treatment of patients with epilepsy. Despite this armamentarium and the many recently introduced AEDs, no major advances have been achieved considering the number of drug resistant patients, while many benefits have been indeed obtained for other clinical outcomes (e.g. better tolerability, less interactions). Cannabinoids have long been studied for their potential therapeutical use and more recently phytocannabinoids have been considered a valuable tool for the treatment of several neurological disorders including epilepsy. Among this wide class, the most studied is cannabidiol (CBD) considering its lack of psychotropic effects and its anticonvulsant properties. OBJECTIVE Analyse the currently available literature on CBD also in light of other data on phytocannabinoids, reviewing data spanning from the mechanism of action, pharmacokinetic to clinical evidences. RESULTS Several preclinical studies have tried to understand the mechanism of action of CBD, which still remains largely not understood. CBD has shown significant anticonvulsant effects mainly in acute animal models of seizures; beneficial effects were reported also in animal models of epileptogenesis and chronic models of epilepsy, although not substantial. In contrast, data coming from some studies raise questions on the effects of other cannabinoids and above all marijuana. CONCLUSION There is indeed sufficient supporting data for clinical development and important antiepileptic effects and the currently ongoing clinical studies will permit the real usefulness of CBD and possibly other cannabinoids. Undoubtedly, several issues also need to be addressed in the next future (e.g. better pharmacokinetic profiling). Finally, shading light on the mechanism of action and the study of other cannabinoids might represent an advantage for future developments.