Vânia F. Prado

Analgesic effect in rodents of native and recombinant Phα1β toxin, a high-voltage-activated calcium channel blocker isolated from armed spider venom

Abstract Calcium influx through neuronal voltage‐sensitive calcium channels (VSCCS) mediates nociceptive information in the spinal dorsal horn. In fact, spinally administered VSCCS blockers, such as ω‐conotoxin MVIIA, have analgesic effect apart of their low therapeutic index and many side effects. Here we study the analgesic potential of Phα1β, a calcium channel blocker, in rodent models of acute and persistent pain. Spinally administered Phα1β showed higher efficacy and long‐lasting analgesia in a thermal model of pain, when compared with ω‐conotoxin MVIIA. Moreover, Phα1β was more effective and potent than ω‐conotoxin MVIIA not only to prevent, but especially to reverse, previously installed persistent chemical and neuropathic pain. Furthermore, the analgesic action of both toxins are related with the inhibition of Ca2+‐evoked release of pro‐nociceptive neurotransmitter, glutamate, from rat spinal cord synaptosomes and decrease of glutamate overflow in cerebrospinal fluid. When side effects were assessed, we found that Phα1β had a therapeutic index wider than ω‐ conotoxin MVIIA. Finally, recombinant Phα1β expressed in Escherichia coli showed marked analgesic activity similar to the native toxin. Taken together, the present study demonstrates that native and recombinant Phα1β have analgesic effects in rodent models of pain, suggesting that this toxin may have potential to be used as a drug in the control of persistent pathological pain.

Trafficking of green fluorescent protein tagged-vesicular acetylcholine transporter to varicosities in a cholinergic cell line: VAChT traffic in living cells

Synaptic vesicle proteins are suggested to travel from the trans‐Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)‐tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP–VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP–VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4‐64 and GFP–Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP–VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis‐defective GFP–VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin‐mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.

The absence of VGLUT3 predisposes to cocaine abuse by increasing dopamine and glutamate signaling in the nucleus accumbens.

Tonically active cholinergic interneurons (TANs) from the nucleus accumbens (NAc) are centrally involved in reward behavior. TANs express a vesicular glutamate transporter referred to as VGLUT3 and thus use both acetylcholine and glutamate as neurotransmitters. The respective roles of each transmitter in the regulation of reward and addiction are still unknown. In this study, we showed that disruption of the gene that encodes VGLUT3 (Slc17a8) markedly increased cocaine self-administration in mice. Concomitantly, the amount of dopamine (DA) release was strongly augmented in the NAc of VGLUT3−/− mice because of a lack of signaling by metabotropic glutamate receptors. Furthermore, dendritic spines and glutamatergic synaptic transmission on medium spiny neurons were increased in the NAc of VGLUT3−/− mice. Increased DA and glutamate signaling in the NAc are hallmarks of addiction. Our study shows that TANs use glutamate to reduce DA release and decrease reinforcing properties of cocaine in mice. Interestingly, we also observed an increased frequency of rare variations in SLC17A8 in a cohort of severe drug abusers compared with controls. Our findings identify VGLUT3 as an unexpected regulator of drug abuse.

Pulmonary Inflammation Is Regulated by the Levels of the Vesicular Acetylcholine Transporter

Acetylcholine (ACh) plays a crucial role in physiological responses of both the central and the peripheral nervous system. Moreover, ACh was described as an anti-inflammatory mediator involved in the suppression of exacerbated innate response and cytokine release in various organs. However, the specific contributions of endogenous release ACh for inflammatory responses in the lung are not well understood. To address this question we have used mice with reduced levels of the vesicular acetylcholine transporter (VAChT), a protein required for ACh storage in secretory vesicles. VAChT deficiency induced airway inflammation with enhanced TNF-α and IL-4 content, but not IL-6, IL-13 and IL-10 quantified by ELISA. Mice with decreased levels of VAChT presented increased collagen and elastic fibers deposition in airway walls which was consistent with an increase in inflammatory cells positive to MMP-9 and TIMP-1 in the lung. In vivo lung function evaluation showed airway hyperresponsiveness to methacholine in mutant mice. The expression of nuclear factor-kappa B (p65-NF-kB) in lung of VAChT-deficient mice were higher than in wild-type mice, whereas a decreased expression of janus-kinase 2 (JAK2) was observed in the lung of mutant animals. Our findings show the first evidence that cholinergic deficiency impaired lung function and produce local inflammation. Our data supports the notion that cholinergic system modulates airway inflammation by modulation of JAK2 and NF-kB pathway. We proposed that intact cholinergic pathway is necessary to maintain the lung homeostasis.

Vesicular acetylcholine transporter knock-down mice show sexual dimorphism on memory.

The key neural substrates involved in memory and cognitive tasks have been reported to receive important modulation from ovarian hormones. In fact, neurochemical systems associated with cognitive functions, such as the cholinergic system, are, at least in part, under modulation of estrogens. Here we show that vesicular acetylcholine transporter (VAChT) mutant mice, which express lower levels of the VAChT (VAChT KD) and reduced acetylcholine release, present sexual dimorphism on memory. We evaluate short- and long-term object recognition memories (STM and LTM) in both sexes. We have showed previously, and confirm here, that VAChT KDHET male mice present deficits in both STM and LTM object recognition memories in comparison with WT. In contrast, VAChT KDHET female mice present deficit in LTM, but not in STM. To test if the female hormones levels could be a determinant factor on sexual dimorphism observed, we submitted female mice to ovariectomy (OVX) or sham-surgery. After 1 week (1 w), we evaluate STM. Female hormone deprivation promotes STM impairment in VAChT KDHET, but not in WT female mice. Our results strongly suggest that the sexual dimorphism observed in VAChT KDHET mice on STM is due to modulation of cholinergic system by ovarian hormones.

Role of protein kinase C in the release of [3H]acetylcholine from myenteric plexus treated with vesamicol

The present experiments investigated the release of [3H]acetylcholine ([3H]ACh) from the guinea pig myenteric plexus treated with 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a drug that impairs ACh accumulation by synaptic vesicles. Ouabain, an Na+-K+ ATPase inhibitor, released [3H]ACh synthesised in the presence of (-)-vesamicol, while electrical field stimulation or KCl depolarisation were not effective to release the transmitter in this condition. The effect of ouabain was Ca2+-dependent and in the presence of (-)-vesamicol it was blocked by calphostin C, an inhibitor of protein kinase C (PKC). In addition, stimulation of kinase C activity by a phorbol ester, but not by its inactive isomer, prevented (-)-vesamicol from interfering with the release of [3H]ACh in electrically-stimulated myenteric plexus, similar to the effect of ouabain. We conclude that release of [3H]ACh induced by ouabain in the presence of (-)-vesamicol depends on PKC activation.

Sleep pattern and learning in knockdown mice with reduced cholinergic neurotransmission

Impaired cholinergic neurotransmission can affect memory formation and influence sleep-wake cycles (SWC). In the present study, we describe the SWC in mice with a deficient vesicular acetylcholine transporter (VAChT) system, previously characterized as presenting reduced acetylcholine release and cognitive and behavioral dysfunctions. Continuous, chronic ECoG and EMG recordings were used to evaluate the SWC pattern during light and dark phases in VAChT knockdown heterozygous (VAChT-KDHET, n=7) and wild-type (WT, n=7) mice. SWC were evaluated for sleep efficiency, total amount and mean duration of slow-wave, intermediate and paradoxical sleep, as well as the number of awakenings from sleep. After recording SWC, contextual fear-conditioning tests were used as an acetylcholine-dependent learning paradigm. The results showed that sleep efficiency in VAChT-KDHET animals was similar to that of WT mice, but that the SWC was more fragmented. Fragmentation was characterized by an increase in the number of awakenings, mainly during intermediate sleep. VAChT-KDHET animals performed poorly in the contextual fear-conditioning paradigm (mean freezing time: 34.4±3.1 and 44.5±3.3 s for WT and VAChT-KDHET animals, respectively), which was followed by a 45% reduction in the number of paradoxical sleep episodes after the training session. Taken together, the results show that reduced cholinergic transmission led to sleep fragmentation and learning impairment. We discuss the results on the basis of cholinergic plasticity and its relevance to sleep homeostasis. We suggest that VAChT-KDHET mice could be a useful model to test cholinergic drugs used to treat sleep dysfunction in neurodegenerative disorders.

Reduced vesicular acetylcholine transporter favors antidepressant behaviors and modulates serotonin and dopamine in female mouse brain

&NA; Depression is extremely harmful to modern society. Despite its complex spectrum of symptoms, previous studies have mostly focused on the monaminergic system in search of pharmacological targets. However, other neurotransmitter systems have also been linked to the pathophysiology of depression. In this study, we provide evidence for a role of the cholinergic system in depressive‐like behavior of female mice. We evaluated mice knockdown for the vesicular acetylcholine transporter (VAChT KD mice), which have been previously shown to exhibit reduced cholinergic transmission. Animals were subjected to the tail suspension and marble burying tests, classical paradigms to assess depressive‐like behaviors and to screen for novel antidepressant drugs. In addition, brain levels of serotonin and dopamine were measured by high performance liquid chromatography. We found that female homozygous VAChT KD mice spent less time immobile during tail suspension and buried less marbles, indicating a less depressive phenotype. These differences in behavior were reverted by central, but not peripheral, acetylcholinesterase inhibition. Moreover, female homozygous VAChT KD mice exhibited higher levels of dopamine and serotonin in the striatum, and increased dopamine in the hippocampus. Our study thus shows a connection between depressive‐like behaviors and the cholinergic system, and that the latter interacts with the monoaminergic system. HighlightsThe cholinergic hypothesis of depression is revisited through a transgenic animal with hipocholinergic function.VAChT knockdown mouse exhibits less depressive‐like behaviors.VAChT knockdown mouse has no alteration in locomotor activity and a trend towards reduced anxiety.VAChT knockdown mouse has increased levels of dopamine and serotonin.VAChT may be a promising target to develop antidepressant drugs.

Reduced expression of VAChT increases renal fibrosis

Chronic kidney disease (CKD) is associated with several other long-lasting conditions such as diabetes and cardiovascular diseases and it is a significant contributor to mortality worldwide. Obstructive kidney disease is one of the leading causes of CKD in children and may result from a wide variety of pathologic processes. Recent studies have shown that α7 nicotinic acetylcholine receptor (α7 nAChR) activation in the cholinergic anti-inflammatory pathway reduces production of inflammatory mediators and consequently prevents tissue injury and death. Here, we examined the role of endogenous release of acetylcholine on the development of fibrosis in renal tissue using a model of unilateral ureter obstruction (UUO)-induced CKD, in which obstruction promotes inflammation-mediated kidney damages. To interfere with acetylcholine secretion, we used mice in which the vesicular acetylcholine transporter is genetically reduced (VAChT KD(hom) mice). We observed a higher renal damage in VAChT mutant mice when compared to wild type controls, exemplified by higher proteinuria and increased amount of type 1 collagen in the kidney tissue, indicating accentuated fibrogenesis. These results were accompanied by enhanced localized kidney inflammation, with increased TH1/TH17 profile response. Administration of PNU-282987, a selective agonist of α7 nAChR, significantly attenuated kidney injury after UUO in VAChT KD(hom) mice, indicating that the lack of acetylcholine release decrease the action of the cholinergic anti-inflammatory pathway, promoting an up-regulation of pro-inflammatory and pro-fibrotic pathways. These results suggest that physiological activation of the cholinergic anti-inflammatory pathway regulates inflammatory responses in the kidney suggesting a new therapeutic approach for kidney disease.

Expression of a recombinant Phoneutria toxin active in calcium channels

PnTx3-4 is a toxin isolated from the venom of the spider Phoneutria nigriventer that blocks N-, P/Q-, and R-type voltage-gated calcium channels and has great potential for clinical applications. In this report we used the SUMO system to express large amounts of recombinant PnTx3-4 peptide, which was found in both soluble and insoluble fractions of bacterial extracts. We purified the recombinant toxin from both fractions and showed that the recombinant peptide showed biological activity similar to the native PnTx3-4. In silico analysis of the primary sequence of PnTx3-4 indicated that the peptide conforms to all the criteria of a knottin scaffold. Additionally, circular dichroism spectrum analysis of the recombinant PnTx3-4 predicted that the toxin structure is composed of approximately 53% turns/unordered, 31% α-helix and 16% β-strand, which is consistent with predicted model of the PnTx3-4 knottin scaffold available at the knottin database (http://knottin.cbs.cnrs.fr). These studies provide the basis for future large scale production and structure-function investigation of PnTx3-4.