NADPH Oxidases in Neurodegenerative Disorders: Mechanisms and Therapeutic Opportunities.

SIGNIFICANCE: The NADPH oxidase (NOX) enzyme family, located in the central nervous system (CNS), is recognized as a source of reactive oxygen species (ROS) in the brain. Despite its importance in cellular processes, excessive ROS generation leads to cell death and is involved in the pathogenesis of neurodegenerative disorders. RECENT ADVANCES: NOX enzymes contribute to the development of neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and stroke, highlighting their potential as targets for future therapeutic development. This review will discuss NOX's contribution and therapeutic targeting potential in neurodegenerative diseases, focusing on PD, AD, ALS, and Stroke. CRITICAL ISSUES: Homeostatic and physiological levels of ROS are crucial for regulating several processes, such as development, memory, neuronal signaling, and vascular homeostasis. However, NOX-mediated excessive ROS generation is deeply involved in the damage of DNA, proteins, and lipids, leading to cell death in the pathogenesis of a wide range of diseases, namely neurodegenerative diseases. FUTURE DIRECTIONS: It is essential to understand the role of NOX homologs in neurodegenerative disorders and the pathological mechanisms undergoing neurodegeneration mediated by increased levels of ROS. This further knowledge will allow the development of new specific NOX inhibitors and their application for neurodegenerative disease therapeutics.

MicroRNA-124-3p Modulates Alpha-Synuclein Expression Levels in a Paraquat-Induced in vivo Model for Parkinson's Disease.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease and the most common movement disorder. Although PD etiology is not fully understood, alpha (α)-synuclein is a key protein involved in PD pathology. MicroRNAs (miRNA), small gene regulatory RNAs that control gene expression, have been identified as biomarkers and potential therapeutic targets for brain diseases, including PD. In particular, miR-124 is downregulated in the plasma and brain samples of PD patients. Recently we showed that the brain delivery of miR-124 counteracts 6-hydroxydopamine-induced motor deficits. However, its role in α-synuclein pathology has never been addressed. Here we used paraquat (PQ)-induced rat PD model to evaluate the role of miR-124-3p in α-synuclein accumulation and dopaminergic neuroprotection. Our results showed that an intranigral administration of miR-124-3p reduced the expression and aggregation of α-synuclein in the substantia nigra (SN) of rats exposed to PQ. NADPH oxidases (NOX), responsible for reactive oxygen species generation, have been considered major players in the development of α-synuclein pathology. Accordingly, miR-124-3p decreased protein expression levels of NOX1 and its activator, small GTPase Rac1, in the SN of PQ-lesioned rats. Moreover, miR-124-3p was able to counteract the reduced levels of pituitary homeobox 3 (PITX3), a protein required for the dopaminergic phenotype, induced by PQ in the SN. This is the first study showing that miR-124-3p decreases PQ-induced α-synuclein levels and the associated NOX1/Rac1 signaling pathway, and impacts PITX3 protein levels, supporting the potential of miR-124-3p as a disease-modifying agent for PD and related α-synucleinopathies.

"Lipopolysaccharide-induced animal models for neuroinflammation - An overview."

Neuroinflammation is a pathological mechanism contributing to neurodegenerative diseases. For in-depth studies of neuroinflammation, several animal models reported reproducing behavioral dysfunctions and cellular pathological mechanisms induced by brain inflammation. One of the most popular models of neuroinflammation is the one generated by lipopolysaccharide exposure. Despite its importance, the reported results using this model show high heterogeneity, making it difficult to analyze and compare the outcomes between studies. Therefore, the current review aims to summarize the different experimental paradigms used to reproduce neuroinflammation by lipopolysaccharide exposure and its respective outcomes, helping to choose the model that better suits each specific research aim.

CtBP Neuroprotective Role in Toxin-Based Parkinson's Disease Models: From Expression Pattern to Dopaminergic Survival.

C-terminal binding proteins (CtBP) are transcriptional co-repressors regulating gene expression. CtBP promote neuronal survival through repression of pro-apoptotic genes, and may represent relevant targets for neurodegenerative disorders, such as Parkinson's disease (PD). Nevertheless, evidence of the role of CtBP1 and CtBP2 in neurodegeneration are scarce. Herein, we showed that CtBP1 and CtBP2 are expressed in neurons, dopaminergic neurons, astrocytes, and microglia in the substantia nigra (SN) and striatum of adult mice. Old mice showed a lower expression of CtBP1 in the SN and higher expression of CtPB2 in the SN and striatum compared with adult mice. In vivo models for PD (paraquat, MPTP, 6-OHDA) showed increased expression of CtBP1 in the SN and striatum while CtBP2 expression was increased in the striatum of paraquat-treated rats only. Moreover, an increased expression of both CtBP was found in a dopaminergic cell line (N27) exposed to 6-OHDA. In the 6-OHDA PD model, we found a dual effect using an unspecific ligand of CtBP, the 4-methylthio 2-oxobutyric acid (MTOB): higher concentrations (e.g. 2500 µM, 1000 µM) inhibited dopaminergic survival, while at 250 µM it counteracted cell death. In vitro, this latter protective role was absent after the siRNA silencing of CtBP1 or CtBP2. Altogether, this is the first report exploring the cellular and regional expression pattern of CtBP in the nigrostriatal pathway and the neuroprotective role in PD toxin-based models. CtBP could counteract dopaminergic cell death in the 6-OHDA PD model and, therefore, CtBP function and therapeutic potential in PD should be further explored.

Regulation of Αlpha-Synuclein Gene (SNCA) by Epigenetic Modifier TET1 in Parkinson Disease.

PURPOSE: Deregulation of SNCA encoding α-synuclein (α-SYN) has been associated with both the familial and sporadic forms of Parkinson disease (PD). Epigenetic regulation plays a crucial role in PD. The intron1 of SNCA harbors a large unmethylated CpG island. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1), a CpG island binding protein, can repress gene expression by occupying hypomethylated CpG-rich promoters, and therefore SNCA could be a target for TET1. We investigated whether TET1 binds to SNCA-intron1 and regulates gene expression. METHODS: The dopaminergic neuronal cell line, ReNcell VM, was used. Reverse transcription-polymerase chain reaction (RT-PCR), real time-quantitative PCR, Western blot, dot-blot, and Chromatin immunoprecipitation were conducted. The substantia nigra tissues of postmortem PD samples were used to confirm the level of TET1 expression. RESULTS: In the human dopaminergic cell line, ReNcell VM, overexpression of the DNA-binding domain of TET1 (TET1-CXXC) led to significant repression of α-SYN. On the contrary, knocking down of TET1 led to significantly higher expression of α-SYN. However, overexpression of the DNA-hydroxymethylating catalytic domain of TET1 failed to change the expression of α-SYN. Altogether, we showed that TET1 is a repressor for SNCA, and a CXXC domain of TET1 is the primary mediator for this repressive action independent of its hydroxymethylation activity. TET1 levels in PD patients are significantly lower than that in the controls. CONCLUSION: We identified that TET1 acts as a repressor for SNCA by binding the intron1 regions of the gene. As a high level of α-SYN is strongly implicated in the pathogenesis of PD, discovering a repressor for the gene encoding α-SYN is highly important for developing novel therapeutic strategies for the disease.

MicroRNA-124-3p-enriched small extracellular vesicles as a therapeutic approach for Parkinson's disease.

Parkinson's disease is a neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra with no effective cure available. MicroRNA-124 has been regarded as a promising therapeutic entity for Parkinson's disease due to its pro-neurogenic and neuroprotective roles. However, its efficient delivery to the brain remains challenging. Here, we used umbilical cord blood mononuclear cell-derived extracellular vesicles as a biological vehicle to deliver microRNA (miR)-124-3p and evaluate its therapeutic effects in a mouse model of Parkinson's disease. In vitro, miR-124-3p-loaded small extracellular vesicles induced neuronal differentiation in subventricular zone neural stem cell cultures and protected N27 dopaminergic cells against 6-hydroxydopamine-induced toxicity. In vivo, intracerebroventricularly administered small extracellular vesicles were detected in the subventricular zone lining the lateral ventricles and in the striatum and substantia nigra, the brain regions most affected by the disease. Most importantly, although miR-124-3p-loaded small extracellular vesicles did not increase the number of new neurons in the 6-hydroxydopamine-lesioned striatum, the formulation protected dopaminergic neurons in the substantia nigra and striatal fibers, which fully counteracted motor behavior symptoms. Our findings reveal a novel promising therapeutic application of small extracellular vesicles as delivery agents for miR-124-3p in the context of Parkinson's disease.

Argonaute-2 protects the neurovascular unit from damage caused by systemic inflammation.

BACKGROUND: The brain vasculature plays a pivotal role in the inflammatory process by modulating the interaction between blood cells and the neurovascular unit. Argonaute-2 (Ago2) has been suggested as essential for endothelial survival but its role in the brain vasculature or in the endothelial-glial crosstalk has not been addressed. Thus, our aim was to clarify the significance of Ago2 in the inflammatory responses elicited by these cell types. METHODS: Mouse primary cultures of brain endothelial cells, astrocytes and microglia were used to evaluate cellular responses to the modulation of Ago2. Exposure of microglia to endothelial cell-conditioned media was used to assess the potential for in vivo studies. Adult mice were injected intraperitoneally with lipopolysaccharide (LPS) (2 mg/kg) followed by three daily intraperitoneal injections of Ago2 (0.4 nM) to assess markers of endothelial disruption, glial reactivity and neuronal function. RESULTS: Herein, we demonstrated that LPS activation disturbed the integrity of adherens junctions and downregulated Ago2 in primary brain endothelial cells. Exogenous treatment recovered intracellular Ago2 above control levels and recuperated vascular endothelial-cadherin expression, while downregulating LPS-induced nitric oxide release. Primary astrocytes did not show a significant change in Ago2 levels or response to the modulation of the Ago2 system, although endogenous Ago2 was shown to be critical in the maintenance of tumor necrosis factor-α basal levels. LPS-activated primary microglia overexpressed Ago2, and Ago2 silencing contained the inflammatory response to some extent, preventing interleukin-6 and nitric oxide release. Moreover, the secretome of Ago2-modulated brain endothelial cells had a protective effect over microglia. The intraperitoneal injection of LPS impaired blood-brain barrier and neuronal function, while triggering inflammation, and the subsequent systemic administration of Ago2 reduced or normalized endothelial, glial and neuronal markers of LPS damage. This outcome likely resulted from the direct action of Ago2 over the brain endothelium, which reestablished glial and neuronal function. CONCLUSIONS: Ago2 could be regarded as a putative therapeutic agent, or target, in the recuperation of the neurovascular unit in inflammatory conditions.

Targeting senescent cells improves functional recovery after spinal cord injury.

Persistent senescent cells (SCs) are known to underlie aging-related chronic disorders, but it is now recognized that SCs may be at the center of tissue remodeling events, namely during development or organ repair. In this study, we show that two distinct senescence profiles are induced in the context of a spinal cord injury between the regenerative zebrafish and the scarring mouse. Whereas induced SCs in zebrafish are progressively cleared out, they accumulate over time in mice. Depletion of SCs in spinal-cord-injured mice, with different senolytic drugs, improves locomotor, sensory, and bladder functions. This functional recovery is associated with improved myelin sparing, reduced fibrotic scar, and attenuated inflammation, which correlate with a decreased secretion of pro-fibrotic and pro-inflammatory factors. Targeting SCs is a promising therapeutic strategy not only for spinal cord injuries but potentially for other organs that lack regenerative competence.

Correction: Simão, A.Y., et al. Evaluation of the Cytotoxicity of Ayahuasca Beverages. Molecules 2020, 25, 5594.

This paper [...].

Evaluation of the Cytotoxicity of Ayahuasca Beverages.

Ayahuasca is a beverage consumed at shamanic ceremonies and currently has gained popularity on recreational scenarios. It contains beta-carboline alkaloids and N,N-dimethyltryptamine, which possesses hallucinogenic effects. Only a few studies have elicited the psychoactive effects and the dose of such compounds on neurological dopaminergic cells or animals. In this work, we aimed to study the cytotoxic effects of these compounds present in ayahuasca beverages and on five different teas (Banisteriopsis caapi, Psychotria viridis, Peganum harmala, Mimosa tenuiflora and Dc Ab (commercial name)) preparations on dopaminergic immortalized cell lines. Moreover, a characterization of the derivative alkaloids was also performed. All the extracts were characterized by chromatographic systems and the effect of those compounds in cell viability and total protein levels were analyzed in N27 dopaminergic neurons cell line. This is the first article where cytotoxicity of ayahuasca tea is studied on neurological dopaminergic cells. Overall, results showed that both cell viability and protein contents decreased when cells were exposed to the individual compounds, as well as to the teas and to the two mixtures based on the traditional ayahuasca beverages.

Author Correction: A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of a Parkinson's disease rat model using an upgraded paraquat exposure paradigm.

Animal models of human diseases are crucial experimental tools to investigate the mechanisms involved in disease pathogenesis and to develop new therapies. In spite of the numerous animal models currently available that reproduce several neuropathological features of Parkinson disease (PD), it is challenging to have one that consistently recapitulates human PD conditions in both motor behaviors and biochemical pathological outcomes. Given that, we have implemented a new paradigm to expose rats to a chronic low dose of paraquat (PQ), using osmotic minipumps and characterized the developed pathologic features over time. The PQ exposure paradigm used lead to a rodent model of PD depicting progressive nigrostriatal dopaminergic neurodegeneration, characterized by a 41% significant loss of dopaminergic neuron in the substantia nigra pars compacta (SNpc), a significant decrease of 18% and 40% of dopamine levels in striatum at week 5 and 8, respectively, and a significant 1.5-fold decrease in motor performance. We observed a significant increase of microglia activation state, sustained levels of α-synucleinopathy and increased oxidative stress markers in the SNpc. In summary, this is an explorative study that allowed to characterize an improved PQ-based rat model that recapitulates cardinal features of PD and may represent an attractive tool to investigate several mechanisms underlying the various aspects of PD pathogenesis as well as for the validation of the efficacy of new therapeutic approaches that targets different mechanisms involved in PD neurodegeneration.

C-Terminal Binding Proteins Promote Neurogenesis and Oligodendrogenesis in the Subventricular Zone.

C-terminal binding proteins (CtBPs) are transcriptional modulators that can regulate gene expression through the recruitment of a corepressor complex composed of chromatin-modifying enzymes and transcriptional factors. In the brain, CtBPs have been described as regulators of cell proliferation, differentiation, and survival. Nevertheless, the role of CtBPs on postnatal neural stem cells (NSCs) fate is not known yet. Herein, we evaluate the expression and functions of CtBPs in postnatal NSCs from the subventricular zone (SVZ). We found that CtBPs were expressed in immature/progenitor cells, neurons and glial cells in the SVZ niche. Using the CtBPs modulator 4-methylthio 2-oxobutyric acid (MTOB), our results showed that 1 mM of MTOB induced cell death, while 5, 25, and 50 µM increased the number of proliferating neuroblasts, mature neurons, and oligodendrocytes. Interestingly, it also increased the dendritic complexity of immature neurons. Altogether, our results highlight CtBPs putative application for brain regenerative applications.

Toxicological Aspects and Determination of the Main Components of Ayahuasca: A Critical Review.

Ayahuasca is a psychoactive beverage prepared traditionally from a mixture of the leaves and stems of Psychotria viridis and Banisteriopsis caapi, respectively, being originally consumed by indigenous Amazonian tribes for ritual and medicinal purposes. Over the years, its use has spread to other populations as a means to personal growth and spiritual connection. Also, the recreational use of its isolated compounds has become prominent. The main compounds of this tea-like preparation are N,N-dimethyltryptamine (DMT), ß-Carbolines, and harmala alkaloids, such as harmine, tetrahydroharmine, and harmaline. The latter are monoamine-oxidase inhibitors and are responsible for DMT psychoactive and hallucinogenic effects on the central nervous system. Although consumers defend its use, its metabolic effects and those on the central nervous system are not fully understood yet. The majority of studies regarding the effects of this beverage and of its individual compounds are based on in vivo experiments, clinical trials, and even surveys. This paper will not only address the toxicological aspects of the ayahuasca compounds but also perform a comprehensive and critical review on the analytical methods available for their determination in biological and non-biological specimens, with special focus on instrumental developments and sample preparation approaches.

Customized tracheal design using 3D printing of a polymer hydrogel: influence of UV laser cross-linking on mechanical properties.

BACKGROUND: The use of 3D printing of hydrogels as a cell support in bio-printing of cartilage, organs and tissue has attracted much research interest. For cartilage applications, hydrogels as soft materials must show some degree of rigidity, which can be achieved by photo- or chemical polymerization. In this work, we combined chemical and UV laser polymeric cross-linkage to control the mechanical properties of 3D printed hydrogel blends. Since there are few studies on UV laser cross-linking combined with 3D printing of hydrogels, the work here reported offered many challenges. METHODS: Polyethylene glycol diacrylate (PEGDA), sodium alginate (SA) and calcium sulphate (CaSO4) polymer paste containing riboflavin (vitamin B2) and triethanolamine (TEOHA) as a biocompatible photoinitiator was printed in an extrusion 3D plotter using a coupled UV laser. The influence of the laser power on the mechanical properties of the printed samples was then examined in unconfined compression stress-strain tests of 1 × 1 × 1 cm3 sized samples. To evaluate the adhesion of the material between printed layers, compression measurements were performed along the parallel and perpendicular directions to the printing lines. RESULTS: At a laser density of 70 mW/cm2, Young's modulus was approximately 6 MPa up to a maximum compression of 20% in the elastic regime for both the parallel and perpendicular measurements. These values were within the range of biological cartilage values. Cytotoxicity tests performed with Vero cells confirmed the cytocompatibility. CONCLUSIONS: We printed a partial tracheal model using optimized printing conditions and proved that the materials and methods developed may be useful for printing of organ models to support surgery or even to produce customized tracheal implants, after further optimization.

Simultaneous Separation of Antioxidants and Carbohydrates From Food Wastes Using Aqueous Biphasic Systems Formed by Cholinium-Derived Ionic Liquids.

The food industry produces significant amounts of waste, many of them rich in valuable compounds that could be recovered and reused in the framework of circular economy. The development of sustainable and cost-effective technologies to recover these value added compounds will contribute to a significant decrease of the environmental footprint and economic burden of this industry sector. Accordingly, in this work, aqueous biphasic systems (ABS) composed of cholinium-derived bistriflimide ionic liquids (ILs) and carbohydrates were investigated as an alternative process to simultaneously separate and recover antioxidants and carbohydrates from food waste. Aiming at improving the biocompatible character of the studied ILs and proposed process, cholinium-derived bistriflimide ILs were chosen, which were properly designed by playing with the cation alkyl side chain and the number of functional groups attached to the cation to be able to create ABS with carbohydrates. These ILs were characterized by cytotoxicity assays toward human intestinal epithelial cells (Caco-2 cell line), demonstrating to have a significantly lower toxicity than other well-known and commonly used fluorinated ILs. The capability of these ILs to form ABS with a series of carbohydrates, namely monosaccharides, disaccharides and polyols, was then appraised by the determination of the respective ternary liquid-liquid phase diagrams at 25°C. The studied ABS were finally used to separate carbohydrates and antioxidants from real food waste samples, using an expired vanilla pudding as an example. With the studied systems, the separation of the two products occurs in one-step, where carbohydrates are enriched in the carbohydrate-rich phase and antioxidants are mainly present in the IL-rich phase. Extraction efficiencies of carbohydrates ranging between 89 and 92% to the carbohydrate-rich phase, and antioxidant relative activities ranging between 65 and 75% in the IL-rich phase were obtained. Furthermore, antioxidants from the IL-rich phase were recovered by solid-phase extraction, and the IL was recycled for two more times with no losses on the ABS separation performance. Overall, the obtained results show that the investigated ABS are promising platforms to simultaneously separate carbohydrates and antioxidants from real food waste samples, and could be used in further related applications foreseeing industrial food waste valorization.

A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals.

Spinal cord injury (SCI) is a complex condition, with limited therapeutic options, that results in sensory and motor disabilities. To boost discovery of novel therapeutics, we designed a simple and efficient drug screening platform. This innovative approach allows to determine locomotor rescue properties of small molecules in a zebrafish (Danio rerio) larval spinal cord transection model. We validated our screening platform by showing that Riluzole and Minocycline, two molecules that are in clinical trials for SCI, promote rescue of the locomotor function of the transected larvae. Further validation of the platform was obtained through the blind identification of D-Cycloserine, a molecule scheduled to enter phase IV clinical trials for SCI. Importantly, we identified Tranexamic acid and further showed that this molecule maintains its locomotor recovery properties in a rodent female contusion model. Our screening platform, combined with drug repurposing, promises to propel the rapid translation of novel therapeutics to improve SCI recovery in humans.

Histamine modulates hippocampal inflammation and neurogenesis in adult mice.

Evidence points to a dual role of histamine in microglia-mediated neuroinflammation, a key pathological feature of several neurodegenerative pathologies. Moreover, histamine has been suggested as a modulator of adult neurogenesis. Herein, we evaluated the effect of histamine in hippocampal neuroinflammation and neurogenesis under physiological and inflammatory contexts. For that purpose, mice were intraperitoneally challenged with lipopolysaccharide (LPS) followed by an intrahippocampal injection of histamine. We showed that histamine per se triggered glial reactivity and induced mild long-term impairments in neurogenesis, reducing immature neurons dendritic volume and complexity. Nevertheless, in mice exposed to LPS (2 mg/Kg), histamine was able to counteract LPS-induced glial activation and release of pro-inflammatory molecules as well as neurogenesis impairment. Moreover, histamine prevented LPS-induced loss of immature neurons complexity as well as LPS-induced loss of both CREB and PSD-95 proteins (essential for proper neuronal activity). Altogether, our results highlight histamine as a potential therapeutic agent to treat neurological conditions associated with hippocampal neuroinflammation and neurodegeneration.