Techniques for evaluating the ATP-gated ion channel P2X7 receptor function in macrophages and microglial cells.

Resident macrophages are tissue-specific innate immune cells acting as sentinels, constantly patrolling their assigned tissue to maintain homeostasis, and quickly responding to pathogenic invaders or molecular danger signals molecules when necessary. Adenosine triphosphate (ATP), when released to the extracellular medium, acts as a danger signal through specific purinergic receptors. Interaction of ATP with the purinergic receptor P2X7 activates macrophages and microglial cells in different pathological conditions, triggering inflammation. The highly expressed P2X7 receptor in these cells induces cell membrane permeabilization, inflammasome activation, cell death, and the production of inflammatory mediators, including cytokines and nitrogen and oxygen-reactive species. This review explores the techniques to evaluate the functional and molecular aspects of the P2X7 receptor, particularly in macrophages and microglial cells. Polymerase chain reaction (PCR), Western blotting, and immunocytochemistry or immunohistochemistry are essential for assessing gene and protein expression in these cell types. Evaluation of P2X7 receptor function involves the use of ATP and selective agonists and antagonists and diverse techniques, including electrophysiology, intracellular calcium measurements, ethidium bromide uptake, and propidium iodide cell viability assays. These techniques are crucial for studying the role of P2X7 receptors in immune responses, neuroinflammation, and various pathological conditions. Therefore, a comprehensive understanding of the functional and molecular aspects of the P2X7 receptor in macrophages and microglia is vital for unraveling its involvement in immune modulation and its potential as a therapeutic target. The methodologies presented and discussed herein offer valuable tools for researchers investigating the complexities of P2X7 receptor signaling in innate immune cells in health and disease.

Age-dependent memory impairment induced by co-exposure to nicotine and a synthetic cannabinoid in mice.

Co-use of marijuana and tobacco products is the second most common drug combination among adolescents. Nicotine (NIC) and cannabinoid use during adolescence induce similar detrimental changes, raising the hypothesis that simultaneous exposure could result in even more severe outcomes. Thus, we investigated whether the co-exposure to NIC and the synthetic cannabinoid WIN 55,212-2 (WIN) in adolescent mice causes behavioral outcomes different from those observed after exposure to a single drug. Male Swiss mice were exposed twice daily to NIC, WIN, or NIC + WIN during adolescence (PND28-47) or adulthood (PND70-89). Drug combination led to a greater reduction in weight gain in adolescent mice, while NIC-induced weight loss was observed in adults. During administration, NIC provoked hypothermia, and WIN produced hyperlocomotion in adolescent and adult mice. Animals exposed to NIC + WIN presented a profile of changes similar to those exposed to NIC. After drug exposure, changes in locomotion, thigmotaxis, social preference, prepulse inhibition, and working and recognition memory were evaluated. Adolescent but not adult mice exposed to NIC showed withdrawal-related hyperlocomotion unaffected by WIN co-administration. An age-specific impairment in object recognition memory was induced only by drug co-exposure during adolescence, which resolved spontaneously before reaching early adulthood. A transient decrease in hippocampal α7 nAChR subunit and CB1 receptor mRNA levels was induced by NIC exposure, which may be involved but is not enough to explain the memory impairment. Our work confirms the potential of NIC and cannabinoids association to aggravate some of the individual drug effects during critical neurodevelopmental periods.

NMDA Receptor Activation and Ca2+/PKC Signaling in Nicotine-Induced GABA Transport Shift in Embryonic Chick Retina.

Nicotinic receptors are present in the retina of different vertebrates, and in the chick retina, it is present during early development throughout to post-hatching. These receptors are activated by nicotine, an alkaloid with addictive and neurotransmitter release modulation properties, such as GABA signaling. Here we evaluated the mechanisms of nicotine signaling in the avian retina during the development of neuron-glia cells at a stage where synapses are peaking. Nicotine almost halved [3H]-GABA uptake, reducing it by 45% whilst increasing more than two-fold [3H]-GABA release in E12 embryonic chick retinas. Additionally, nicotine mediated a 33% increase in [3H]-D-aspartate release. MK-801 50 µM blocked 66% of nicotine-induced [3H]-GABA release and Gö 6983 100 nM prevented the nicotine-induced reduction in [3H]-GABA uptake by rescuing 40% of this neurotransmitter uptake, implicating NMDAR and PKC (respectively) in the nicotinic responses. In addition, NO-711 prevented [3H]-GABA uptake and release induced by nicotine. Furthermore, the relevance of calcium influx for PKC activation was evidenced through fura-2 imaging. We conclude that the shift of GABA transport mediated by nicotine promotes GABA release by inducing transporter reversal via nicotine-induced EAA release through EAATs, or by a direct effect of nicotine in activating nicotinic receptors permeable to calcium and promoting PKC pathway activation and shifting GAT-1 activity, both prompting calcium influx, and activation of the PKC pathway and shifting GAT-1 activity.

Synthesis and biological evaluation of 4-hydroxy-methylpiperidinyl-N-benzyl-acylarylhydrazone hybrids designed as novel multifunctional drug candidates for Alzheimer's disease.

The search for new drug candidates against Alzheimer's disease (AD) remains a complex challenge for medicinal chemists due to its multifactorial pathogenesis and incompletely understood physiopathology. In this context, we have explored the molecular hybridization of pharmacophore structural fragments from known bioactive molecules, aiming to obtain a novel molecular architecture in new chemical entities capable of concomitantly interacting with multiple targets in a so-called multi-target directed ligands (MTDLs) approach. This work describes the synthesis of 4-hydroxymethyl)piperidine-N-benzyl-acyl-hydrazone derivatives 5a-l, designed as novel MTDLs, showing improved multifunctional properties compared to the previously reported parent series of N-benzyl-(3-hydroxy)piperidine-acyl-hydrazone derivatives 4. The new improved derivatives were studied in silico, regarding their mode of interaction with AChE enzyme, and in vitro, for evaluation of their effects on the selective inhibition of cholinesterases, cellular antioxidant, and neuroprotective activities as their cytotoxicity in human neuroblastoma (SH-SY5Y) cells. Overall, compound PQM-181 (5 k) showed the best balanced selective and non-competitive inhibition of AChE (IC50 = 5.9 µM, SI > 5.1), with an additional antioxidant activity (IC50 = 7.45 µM) against neuronal t-BOOH-induced oxidative stress and neuroprotective ability against neurotoxicity elicited by both t-BOOH and OAß1-42, and a moderate ability to interfere in Aß1-42 aggregates, with low cytotoxicity and good predictive druggability properties, suggesting a multifunctional pharmacological profile suitable for further drug development against AD.

Design, synthesis, and biological evaluation of new thalidomide-donepezil hybrids as neuroprotective agents targeting cholinesterases and neuroinflammation.

A new series of eight multifunctional thalidomide-donepezil hybrids were synthesized based on the multi-target-directed ligand strategy and evaluated as potential neuroprotective, cholinesterase inhibitors and anti-neuroinflammatory agents against neurodegenerative diseases. A molecular hybridization approach was used for structural design by combining the N-benzylpiperidine pharmacophore of donepezil and the isoindoline-1,3-dione fragment from the thalidomide structure. The most promising compound, PQM-189 (3g), showed good AChE inhibitory activity with an IC50 value of 3.15 µM, which was predicted by docking studies as interacting with the enzyme in the same orientation observed in the AChE-donepezil complex and a similar profile of interaction. Additionally, compound 3g significantly decreased iNOS and IL-1ß levels by 43% and 39%, respectively, after 24 h of incubation with lipopolysaccharide. In vivo data confirmed the ability of 3g to prevent locomotor impairment and changes in feeding behavior elicited by lipopolysaccharide. Moreover, the PAMPA assay evidenced adequate blood-brain barrier and gastrointestinal tract permeabilities with an Fa value of 69.8%. Altogether, these biological data suggest that compound 3g can treat the inflammatory process and oxidative stress resulting from the overexpression of iNOS and therefore the increase in reactive nitrogen species, and regulate the release of pro-inflammatory cytokines such as IL-1ß. In this regard, compound PQM-189 (3g) was revealed to be a promising neuroprotective and anti-neuroinflammatory agent with an innovative thalidomide-donepezil-based hybrid molecular architecture.

P2X7 Receptor Triggers Lysosomal Leakage Through Calcium Mobilization in a Mechanism Dependent on Pannexin-1 Hemichannels.

The P2X7 receptor is a critical purinergic receptor in immune cells. Its activation was associated with cathepsin release into macrophage cytosol, suggesting its involvement in lysosomal membrane permeabilization (LMP) and leakage. Nevertheless, the mechanisms by which P2X7 receptor activation induces LMP and leakage are unclear. This study investigated cellular mechanisms associated with endosomal and lysosomal leakage triggered by P2X7 receptor activation. We found that ATP at 500 µM and 5 mM (but not 50 µM) induced LMP in non-stimulated peritoneal macrophages. This effect was not observed in P2X7-deficient or A740003-pretreated macrophages. We found that the P2X7 receptor and pannexin-1 channels mediate calcium influx that might be important for activating specific ion channels (TRPM2 and two-pore channels) on the membranes of late endosomes and lysosomes leading to LMP leakage and consequent cathepsin release. These findings suggest the critical role of the P2X7 receptor in inflammatory and infectious diseases via lysosomal dysfunction.

Design, Synthesis and Biological Evaluation of Novel Triazole N-acylhydrazone Hybrids for Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder that involves different pathogenic mechanisms. In this regard, the goal of this study was the design and synthesis of new compounds with multifunctional pharmacological activity by molecular hybridization of structural fragments of curcumin and resveratrol connected by an N-acyl-hydrazone function linked to a 1,4-disubstituted triazole system. Among these hybrid compounds, derivative 3e showed the ability to inhibit acetylcholinesterase activity, the intracellular formation of reactive oxygen species as well as the neurotoxicity elicited by Aß42 oligomers in neuronal SH-SY5Y cells. In parallel, compound 3e showed a good profile of safety and ADME parameters. Taken together, these results suggest that 3e could be considered a lead compound for the further development of AD therapeutics.

Effects of combined 5-HT2A and cannabinoid receptor modulation on a schizophrenia-related prepulse inhibition deficit in mice.

RATIONALE: Prepulse inhibition of the startle reflex (PPI) is disrupted in several psychiatric disorders including schizophrenia. Understanding PPI pharmacology may help elucidate the pathophysiology of these disorders and lead to better treatments. Given the advantages of multi-target approaches for complex mental illnesses treatment, we have investigated the interaction between receptors known to modulate PPI (5-HT1A and 5-HT2A) and the neuromodulatory endocannabinoid system. OBJECTIVES: To investigate serotonin and cannabinoid receptor (CBR) co-modulation in a model of PPI disruption relevant to schizophrenia METHODS: Male Swiss mice were pretreated with WIN 55,212-2 (CBR agonist), rimonabant (CB1R inverse agonist), 8-OH-DPAT (5-HT1A/7 agonist), and volinanserin (5-HT2A antagonist) or with a combination of a cannabinoid and a serotonergic drug. PPI disruption was induced by acute administration of MK-801. RESULTS: WIN 55,212-2 and rimonabant did not change PPI nor block MK-801-induced deficits. 8-OH-DPAT increased PPI in control mice and, in a higher dose, inhibited MK-801-induced impairments. Volinanserin also increased PPI in control and MK-801-treated mice, presenting an inverted U-shaped dose-response curve. Co-administration of either cannabinoid ligand with 8-OH-DPAT did not change PPI; however, the combination of volinanserin with rimonabant increased PPI in both control and MK-801-exposed mice. CONCLUSIONS: WIN 55,212-2 and rimonabant had similar effects in PPI. Moreover, serotonin and cannabinoid receptors interact to modulate PPI. While co-modulation of CBR and 5-HT1A receptors did not change PPI, a beneficial effect of 5-HT2A and CB1R antagonist combination was detected, possibly mediated through potentiation of 5-HT2A blockade effects by concomitant CB1R blockade.

Live Cell Imaging Supports a Key Role for Histone Deacetylase as a Molecular Target during Glioblastoma Malignancy Downgrade through Tumor Competence Modulation.

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system, and the identification of the mechanisms underlying the biological basis of GBM aggressiveness is essential to develop new therapies. Due to the low prognosis of GBM treatment, different clinical studies are in course to test the use of histone deacetylase inhibitors (iHDACs) in anticancer cocktails. Here, we seek to investigate the impact of HDAC activity on GBM cell behavior and plasticity by live cell imaging. We pharmacologically knock down HDAC activity using two different inhibitors (TSA and SAHA) in two different tumor cell types: a commercial GBM cell line (U87-MG) and primary tumor (GBM011). Upon 72 hours of in vitro iHDAC treatment, GBM cells presented a very unusual elongated cell shape due to tunneling tube formation and independent on TGF-ß signaling epithelial to mesenchymal transition. Live cell imaging revealed that voltage-sensitive Ca++ signaling was disrupted upon HDAC activity blockade. This behavior was coupled to vimentin and connexin 43 gene expression downregulation, suggesting that HDAC activity blockade downgrades GBM aggressiveness mostly due to tumor cell competence and plasticity modulation in vitro. To test this hypothesis and access whether iHDACs would modulate tumor cell behavior and plasticity to properly respond to environmental cues in vivo, we xenografted GBM oncospheres in the chick developing the neural tube. Remarkably, upon 5 days in the developing neural tube, iHDAC-treated GBM cells ectopically expressed HNK-1, a tumor-suppressor marker tightly correlated to increased survivor of patients. These results describe, for the first time in the literature, the relevance of iHDACs for in vivo tumor cell morphology and competence to properly respond to environmental cues. Ultimately, our results highlight the relevance of chromatin remodeling for tumor cell plasticity and shed light on clinical perspectives aiming the epigenome as a relevant therapeutic target for GBM therapy.

Zika virus replicates in adult human brain tissue and impairs synapses and memory in mice.

Neurological complications affecting the central nervous system have been reported in adult patients infected by Zika virus (ZIKV) but the underlying mechanisms remain unknown. Here, we report that ZIKV replicates in human and mouse adult brain tissue, targeting mature neurons. ZIKV preferentially targets memory-related brain regions, inhibits hippocampal long-term potentiation and induces memory impairment in adult mice. TNF-α upregulation, microgliosis and upregulation of complement system proteins, C1q and C3, are induced by ZIKV infection. Microglia are found to engulf hippocampal presynaptic terminals during acute infection. Neutralization of TNF-α signaling, blockage of microglial activation or of C1q/C3 prevent synapse and memory impairment in ZIKV-infected mice. Results suggest that ZIKV induces synapse and memory dysfunction via aberrant activation of TNF-α, microglia and complement. Our findings establish a mechanism by which ZIKV affects the adult brain, and point to the need of evaluating cognitive deficits as a potential comorbidity in ZIKV-infected adults.

Generation of iPSC-Derived Human Peripheral Sensory Neurons Releasing Substance P Elicited by TRPV1 Agonists.

Neural crest stem cells (NCPCs) have been shown to differentiate into various cell types and tissues during embryonic development, including sensory neurons. The few studies addressing the generation of NCPCs and peripheral sensory neurons (PSNs) from human induced pluripotent stem cells (hiPSCs), generated sensory cells without displaying robust activity. Here, we describe an efficient strategy for hiPSCs differentiation into NCPCs and functional PSNs using chemically defined media and factors to achieve efficient differentiation, confirmed by the expression of specific markers. After 10 days hiPSCs differentiated into NCPCs, cells were then maintained in neural induction medium containing defined growth factors for PSNs differentiation, followed by 10 days in neonatal human epidermal keratinocytes- (HEKn-) conditioned medium (CM). We observed a further increase in PSN markers expression and neurites length after CM treatment. The resulting neurons elicited action potentials after current injection and released substance P (SP) in response to nociceptive agents such as anandamide and resiniferatoxin. Anandamide induced substance P release via activation of TRPV1 and not CB1. Transcriptomic analysis of the PSNs revealed the main dorsal root ganglia neuronal markers and a transcriptional profile compatible with C fiber-low threshold mechanoreceptors. TRPV1 was detected by immunofluorescence and RNA-Seq in multiple experiments. In conclusion, the developed strategy generated PSNs useful for drug screening that could be applied to patient-derived hiPSCs, consisting in a powerful tool to model human diseases in vitro.

Design, synthesis and pharmacological evaluation of N-benzyl-piperidinyl-aryl-acylhydrazone derivatives as donepezil hybrids: Discovery of novel multi-target anti-alzheimer prototype drug candidates.

A new series of sixteen multifunctional N-benzyl-piperidine-aryl-acylhydrazones hybrid derivatives was synthesized and evaluated for multi-target activities related to Alzheimer's disease (AD). The molecular hybridization approach was based on the combination, in a single molecule, of the pharmacophoric N-benzyl-piperidine subunit of donepezil, the substituted hydroxy-piperidine fragment of the AChE inhibitor LASSBio-767, and an acylhydrazone linker, a privileged structure present in a number of synthetic aryl- and aryl-acylhydrazone derivatives with significant AChE and anti-inflammatory activities. Among them, compounds 4c, 4d, 4g and 4j presented the best AChE inhibitory activities, but only compounds 4c and 4g exhibited concurrent anti-inflammatory activity in vitro and in vivo, against amyloid beta oligomer (AßO) induced neuroinflammation. Compound 4c also showed the best in vitro and in vivo neuroprotective effects against AßO-induced neurodegeneration. In addition, compound 4c showed a similar binding mode to donepezil in both acetylated and free forms of AChE enzyme in molecular docking studies and did not show relevant toxic effects on in vitro and in vivo assays, with good predicted ADME parameters in silico. Overall, all these results highlighted compound 4c as a promising and innovative multi-target drug prototype candidate for AD treatment.

Design, synthesis and evaluation of novel feruloyl-donepezil hybrids as potential multitarget drugs for the treatment of Alzheimer's disease.

A novel series of feruloyl-donepezil hybrid compounds were designed, synthesized and evaluated as multitarget drug candidates for the treatment of Alzheimer's Disease (AD). In vitro results revealed potent acetylcholinesterase (AChE) inhibitory activity for some of these compounds and all of them showed moderate antioxidant properties. Compounds 12a, 12b and 12c were the most potent AChE inhibitors, highlighting 12a with IC50 = 0.46 µM. In addition, these three most promising compounds exhibited significant in vivo anti-inflammatory activity in the mice paw edema, pleurisy and formalin-induced hyperalgesy models, in vitro metal chelator activity for Cu2+ and Fe2+, and neuroprotection of human neuronal cells against oxidative damage. Molecular docking studies corroborated the in vitro inhibitory mode of interaction of these active compounds on AChE. Based on these data, compound 12a was identified as a novel promising drug prototype candidate for the treatment of AD with innovative structural feature and multitarget effects.

Dopamine promotes NMDA receptor hypofunction in the retina through D1 receptor-mediated Csk activation, Src inhibition and decrease of GluN2B phosphorylation.

Dopamine and glutamate are critical neurotransmitters involved in light-induced synaptic activity in the retina. In brain neurons, dopamine D1 receptors (D1Rs) and the cytosolic protein tyrosine kinase Src can, independently, modulate the behavior of NMDA-type glutamate receptors (NMDARs). Here we studied the interplay between D1Rs, Src and NMDARs in retinal neurons. We reveal that dopamine-mediated D1R stimulation provoked NMDAR hypofunction in retinal neurons by attenuating NMDA-gated currents, by preventing NMDA-elicited calcium mobilization and by decreasing the phosphorylation of NMDAR subunit GluN2B. This dopamine effect was dependent on upregulation of the canonical D1R/adenylyl cyclase/cAMP/PKA pathway, of PKA-induced activation of C-terminal Src kinase (Csk) and of Src inhibition. Accordingly, knocking down Csk or overexpressing a Csk phosphoresistant Src mutant abrogated the dopamine-induced NMDAR hypofunction. Overall, the interplay between dopamine and NMDAR hypofunction, through the D1R/Csk/Src/GluN2B pathway, might impact on light-regulated synaptic activity in retinal neurons.

Cardanol-derived AChE inhibitors: Towards the development of dual binding derivatives for Alzheimer's disease.

Cardanol is a phenolic lipid component of cashew nut shell liquid (CNSL), obtained as the byproduct of cashew nut food processing. Being a waste product, it has attracted much attention as a precursor for the production of high-value chemicals, including drugs. On the basis of these findings and in connection with our previous studies on cardanol derivatives as acetylcholinesterase (AChE) inhibitors, we designed a novel series of analogues by including a protonable amino moiety belonging to different systems. Properly addressed docking studies suggested that the proposed structural modifications would allow the new molecules to interact with both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE, thus being able to act as dual binding inhibitors. To disclose whether the new molecules showed the desired profile, they were first tested for their cholinesterase inhibitory activity towards EeAChE and eqBuChE. Compound 26, bearing an N-ethyl-N-(2-methoxybenzyl)amine moiety, showed the highest inhibitory activity against EeAChE, with a promising IC50 of 6.6 µM, and a similar inhibition profile of the human isoform (IC50 = 5.7 µM). As another positive feature, most of the derivatives did not show appreciable toxicity against HT-29 cells, up to a concentration of 100 µM, which indicates drug-conform behavior. Also, compound 26 is capable of crossing the blood-brain barrier (BBB), as predicted by a PAMPA-BBB assay. Collectively, the data suggest that the approach to obtain potential anti-Alzheimer drugs from CNSL is worth of further pursuit and development.

Design, synthesis, and evaluation of guanylhydrazones as potential inhibitors or reactivators of acetylcholinesterase.

Analogs of pralidoxime, which is a commercial antidote for intoxication from neurotoxic organophosphorus compounds, were designed, synthesized, characterized, and tested as potential inhibitors or reactivators of acetylcholinesterase (AChE) using the Ellman's test, nuclear magnetic resonance, and molecular modeling. These analogs include 1-methylpyridine-2-carboxaldehyde hydrazone, 1-methylpyridine-2-carboxaldehyde guanylhydrazone, and six other guanylhydrazones obtained from different benzaldehydes. The results indicate that all compounds are weak AChE reactivators but relatively good AChE inhibitors. The most effective AChE inhibitor discovered was the guanylhydrazone derived from 2,4-dinitrobenzaldehyde and was compared with tacrine, displaying similar activity to this reference material. These results indicate that guanylhydrazones as well as future similar derivatives may function as drugs for the treatment of Alzheimer's disease.

Waveform Similarity Analysis: A Simple Template Comparing Approach for Detecting and Quantifying Noisy Evoked Compound Action Potentials.

Experimental electrophysiological assessment of evoked responses from regenerating nerves is challenging due to the typical complex response of events dispersed over various latencies and poor signal-to-noise ratio. Our objective was to automate the detection of compound action potential events and derive their latencies and magnitudes using a simple cross-correlation template comparison approach. For this, we developed an algorithm called Waveform Similarity Analysis. To test the algorithm, challenging signals were generated in vivo by stimulating sural and sciatic nerves, whilst recording evoked potentials at the sciatic nerve and tibialis anterior muscle, respectively, in animals recovering from sciatic nerve transection. Our template for the algorithm was generated based on responses evoked from the intact side. We also simulated noisy signals and examined the output of the Waveform Similarity Analysis algorithm with imperfect templates. Signals were detected and quantified using Waveform Similarity Analysis, which was compared to event detection, latency and magnitude measurements of the same signals performed by a trained observer, a process we called Trained Eye Analysis. The Waveform Similarity Analysis algorithm could successfully detect and quantify simple or complex responses from nerve and muscle compound action potentials of intact or regenerated nerves. Incorrectly specifying the template outperformed Trained Eye Analysis for predicting signal amplitude, but produced consistent latency errors for the simulated signals examined. Compared to the trained eye, Waveform Similarity Analysis is automatic, objective, does not rely on the observer to identify and/or measure peaks, and can detect small clustered events even when signal-to-noise ratio is poor. Waveform Similarity Analysis provides a simple, reliable and convenient approach to quantify latencies and magnitudes of complex waveforms and therefore serves as a useful tool for studying evoked compound action potentials in neural regeneration studies.

Mitomycin-treated undifferentiated embryonic stem cells as a safe and effective therapeutic strategy in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is an incurable progressive neurodegenerative disorder. Clinical presentation of PD stems largely from the loss of dopaminergic neurons in the nigrostriatal dopaminergic pathway, motivating experimental strategies of replacement based on cell therapy. Transplantation of dopaminergic neurons derived from embryonic stem cells significantly improves motor functions in rodent and non-human primate models of PD. However, protocols to generate dopaminergic neurons from embryonic stem cells generally meet with low efficacy and high risk of teratoma formation upon transplantation. To address these issues, we have pre-treated undifferentiated mouse embryonic stem cells (mESCs) with the DNA alkylating agent mitomycin C (MMC) before transplantation. MMC treatment of cultures prevented tumorigenesis in a 12 week follow-up after mESCs were injected in nude mice. In 6-OH-dopamine-lesioned mice, intrastriatal injection of MMC-treated mESCs markedly improved motor function without tumor formation for as long as 15 months. Furthermore, we show that halting mitotic activity of undifferentiated mESCs induces a four-fold increase in dopamine release following in vitro differentiation. Our findings indicate that treating mESCs with MMC prior to intrastriatal transplant is an effective to strategy that could be further investigated as a novel alternative for treatment of PD.

Astrocyte transforming growth factor beta 1 promotes inhibitory synapse formation via CaM kinase II signaling.

The balance between excitatory and inhibitory synaptic inputs is critical for the control of brain function. Astrocytes play important role in the development and maintenance of neuronal circuitry. Whereas astrocytes-derived molecules involved in excitatory synapses are recognized, molecules and molecular mechanisms underlying astrocyte-induced inhibitory synapses remain unknown. Here, we identified transforming growth factor beta 1 (TGF-ß1), derived from human and murine astrocytes, as regulator of inhibitory synapse in vitro and in vivo. Conditioned media derived from human and murine astrocytes induce inhibitory synapse formation in cerebral cortex neurons, an event inhibited by pharmacologic and genetic manipulation of the TGF-ß pathway. TGF-ß1-induction of inhibitory synapse depends on glutamatergic activity and activation of CaM kinase II, which thus induces localization and cluster formation of the synaptic adhesion protein, Neuroligin 2, in inhibitory postsynaptic terminals. Additionally, intraventricular injection of TGF-ß1 enhanced inhibitory synapse number in the cerebral cortex. Our results identify TGF-ß1/CaMKII pathway as a novel molecular mechanism underlying astrocyte control of inhibitory synapse formation. We propose here that the balance between excitatory and inhibitory inputs might be provided by astrocyte signals, at least partly achieved via TGF-ß1 downstream pathways. Our work contributes to the understanding of the GABAergic synapse formation and may be of relevance to further the current knowledge on the mechanisms underlying the development of various neurological disorders, which commonly involve impairment of inhibitory synapse transmission.

Erythrina mulungu alkaloids are potent inhibitors of neuronal nicotinic receptor currents in mammalian cells.

Crude extracts and three isolated alkaloids from Erythrina mulungu plants have shown anxiolytic effects in different animal models. We investigated whether these alkaloids could affect nicotinic acetylcholine receptors and if they are selective for different central nervous system (CNS) subtypes. Screening experiments were performed using a single concentration of the alkaloid co-applied with acetylcholine in whole cell patch-clamp recordings in three different cell models: (i) PC12 cells natively expressing α3* nicotinic acetylcholine receptors; (ii) cultured hippocampal neurons natively expressing α7* nicotinic acetylcholine receptors; and (iii) HEK 293 cells heterologoulsy expressing α4ß2 nicotinic acetylcholine receptors. For all three receptors, the percent inhibition of acetylcholine-activated currents by (+)-11á-hydroxyerysotrine was the lowest, whereas (+)-erythravine and (+)-11á-hydroxyerythravine inhibited the currents to a greater extent. For the latter two substances, we obtained concentration-response curves with a pre-application protocol for the α7* and α4ß2 nicotinic acetylcholine receptors. The IC50 obtained with (+)-erythravine and (+)-11á-hydroxyerythravine were 6 µM and 5 µM for the α7* receptors, and 13 nM and 4 nM for the α4ß2 receptors, respectively. Our data suggest that these Erythrina alkaloids may exert their behavioral effects through inhibition of CNS nicotinic acetylcholine receptors, particularly the α4ß2 subtype.