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.

Gold nanostructures: synthesis, properties, and neurological applications.

Recent advances in technology are expected to increase our current understanding of neuroscience. Nanotechnology and nanomaterials can alter and control neural functionality in both in vitro and in vivo experimental setups. The intersection between neuroscience and nanoscience may generate long-term neural interfaces adapted at the molecular level. Owing to their intrinsic physicochemical characteristics, gold nanostructures (GNSs) have received much attention in neuroscience, especially for combined diagnostic and therapeutic (theragnostic) purposes. GNSs have been successfully employed to stimulate and monitor neurophysiological signals. Hence, GNSs could provide a promising solution for the regeneration and recovery of neural tissue, novel neuroprotective strategies, and integrated implantable materials. This review covers the broad range of neurological applications of GNS-based materials to improve clinical diagnosis and therapy. Sub-topics include neurotoxicity, targeted delivery of therapeutics to the central nervous system (CNS), neurochemical sensing, neuromodulation, neuroimaging, neurotherapy, tissue engineering, and neural regeneration. It focuses on core concepts of GNSs in neurology, to circumvent the limitations and significant obstacles of innovative approaches in neurobiology and neurochemistry, including theragnostics. We will discuss recent advances in the use of GNSs to overcome current bottlenecks and tackle technical and conceptual challenges.

Microglia integration into human midbrain organoids leads to increased neuronal maturation and functionality.

The human brain is a complex, three-dimensional structure. To better recapitulate brain complexity, recent efforts have focused on the development of human-specific midbrain organoids. Human iPSC-derived midbrain organoids consist of differentiated and functional neurons, which contain active synapses, as well as astrocytes and oligodendrocytes. However, the absence of microglia, with their ability to remodel neuronal networks and phagocytose apoptotic cells and debris, represents a major disadvantage for the current midbrain organoid systems. Additionally, neuroinflammation-related disease modeling is not possible in the absence of microglia. So far, no studies about the effects of human iPSC-derived microglia on midbrain organoid neural cells have been published. Here we describe an approach to derive microglia from human iPSCs and integrate them into iPSC-derived midbrain organoids. Using single nuclear RNA Sequencing, we provide a detailed characterization of microglia in midbrain organoids as well as the influence of their presence on the other cells of the organoids. Furthermore, we describe the effects that microglia have on cell death and oxidative stress-related gene expression. Finally, we show that microglia in midbrain organoids affect synaptic remodeling and increase neuronal excitability. Altogether, we show a more suitable system to further investigate brain development, as well as neurodegenerative diseases and neuroinflammation.

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.

Parkinson's Disease Phenotypes in Patient Neuronal Cultures and Brain Organoids Improved by 2-Hydroxypropyl-ß-Cyclodextrin Treatment.

BACKGROUND: The etiology of Parkinson's disease (PD) is only partially understood despite the fact that environmental causes, risk factors, and specific gene mutations are contributors to the disease. Biallelic mutations in the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) gene involved in mitochondrial homeostasis, vesicle trafficking, and autophagy are sufficient to cause PD. OBJECTIVES: We sought to evaluate the difference between controls' and PINK1 patients' derived neurons in their transition from neuroepithelial stem cells to neurons, allowing us to identify potential pathways to target with repurposed compounds. METHODS: Using two-dimensional and three-dimensional models of patients' derived neurons we recapitulated PD-related phenotypes. We introduced the usage of midbrain organoids for testing compounds. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), we corrected the point mutations of three patients' derived cells. We evaluated the effect of the selected compound in a mouse model. RESULTS: PD patient-derived cells presented differences in their energetic profile, imbalanced proliferation, apoptosis, mitophagy, and a reduced differentiation efficiency to tyrosine hydroxylase positive (TH+) neurons compared to controls' cells. Correction of a patient's point mutation ameliorated the metabolic properties and neuronal firing rates as well as reversing the differentiation phenotype, and reducing the increased astrocytic levels. Treatment with 2-hydroxypropyl-ß-cyclodextrin increased the autophagy and mitophagy capacity of neurons concomitant with an improved dopaminergic differentiation of patient-specific neurons in midbrain organoids and ameliorated neurotoxicity in a mouse model. CONCLUSION: We show that treatment with a repurposed compound is sufficient for restoring the impaired dopaminergic differentiation of PD patient-derived cells. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Histamine induces microglia activation and dopaminergic neuronal toxicity via H1 receptor activation.

BACKGROUND: Histamine is an amine widely known as a peripheral inflammatory mediator and as a neurotransmitter in the central nervous system. Recently, it has been suggested that histamine acts as an innate modulator of microglial activity. Herein, we aimed to disclose the role of histamine in microglial phagocytic activity and reactive oxygen species (ROS) production and to explore the consequences of histamine-induced neuroinflammation in dopaminergic (DA) neuronal survival. METHODS: The effect of histamine on phagocytosis was assessed both in vitro by using a murine N9 microglial cell line and primary microglial cell cultures and in vivo. Cells were exposed to IgG-opsonized latex beads or phosphatidylserine (PS) liposomes to evaluate Fcγ or PS receptor-mediated microglial phagocytosis, respectively. ROS production and protein levels of NADPH oxidases and Rac1 were assessed as a measure of oxidative stress. DA neuronal survival was evaluated in vivo by counting the number of tyrosine hydroxylase-positive neurons in the substantia nigra (SN) of mice. RESULTS: We found that histamine triggers microglial phagocytosis via histamine receptor 1 (H1R) activation and ROS production via H1R and H4R activation. By using apocynin, a broad NADPH oxidase (Nox) inhibitor, and Nox1 knockout mice, we found that the Nox1 signaling pathway is involved in both phagocytosis and ROS production induced by histamine in vitro. Interestingly, both apocynin and annexin V (used as inhibitor of PS-induced phagocytosis) fully abolished the DA neurotoxicity induced by the injection of histamine in the SN of adult mice in vivo. Blockade of H1R protected against histamine-induced Nox1 expression and death of DA neurons in vivo. CONCLUSIONS: Overall, our results highlight the relevance of histamine in the modulation of microglial activity that ultimately may interfere with neuronal survival in the context of Parkinson's disease (PD) and, eventually, other neurodegenerative diseases which are accompanied by microglia-induced neuroinflammation. Importantly, our results also open promising new perspectives for the therapeutic use of H1R antagonists to treat or ameliorate neurodegenerative processes.

Generation of two induced pluripotent stem cell lines and the corresponding isogenic controls from Parkinson's disease patients carrying the heterozygous mutations c.1290A > G (p.T351A) or c.2067A > G (p.T610A) in the RHOT1 gene encoding Miro1.

Primary skin fibroblasts from two Parkinson's disease (PD) patients carrying distinct heterozygous mutations in the RHOT1 gene encoding Miro1, namely c.1290A > G (Miro1 p.T351A) and c.2067A > G (Miro1 p.T610A), were converted into induced pluripotent stem cells (iPSCs) by episomal reprogramming. The corresponding isogenic gene-corrected lines have been generated using CRISPR/Cas9 technology. Here, we provide a comprehensive characterization and quality assurance of both isogenic pairs, which will be used to study Miro1-related molecular mechanisms underlying neurodegeneration in iPSC-derived neuronal models (e.g., midbrain dopaminergic neurons and astrocytes).

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.

Impaired neuron differentiation in GBA-associated Parkinson's disease is linked to cell cycle defects in organoids.

The mechanisms underlying Parkinson's disease (PD) etiology are only partially understood despite intensive research conducted in the field. Recent evidence suggests that early neurodevelopmental defects might play a role in cellular susceptibility to neurodegeneration. To study the early developmental contribution of GBA mutations in PD we used patient-derived iPSCs carrying a heterozygous N370S mutation in the GBA gene. Patient-specific midbrain organoids displayed GBA-PD relevant phenotypes such as reduction of GCase activity, autophagy impairment, and mitochondrial dysfunction. Genome-scale metabolic (GEM) modeling predicted changes in lipid metabolism which were validated with lipidomics analysis, showing significant differences in the lipidome of GBA-PD. In addition, patient-specific midbrain organoids exhibited a decrease in the number and complexity of dopaminergic neurons. This was accompanied by an increase in the neural progenitor population showing signs of oxidative stress-induced damage and premature cellular senescence. These results provide insights into how GBA mutations may lead to neurodevelopmental defects thereby predisposing to PD pathology.

Efficient spatially targeted gene editing using a near-infrared activatable protein-conjugated nanoparticle for brain applications.

Spatial control of gene expression is critical to modulate cellular functions and deconstruct the function of individual genes in biological processes. Light-responsive gene-editing formulations have been recently developed; however, they have shown limited applicability in vivo due to poor tissue penetration, limited cellular transfection and the difficulty in evaluating the activity of the edited cells. Here, we report a formulation composed of upconversion nanoparticles conjugated with Cre recombinase enzyme through a photocleavable linker, and a lysosomotropic agent that facilitates endolysosomal escape. This formulation allows in vitro spatial control in gene editing after activation with near-infrared light. We further demonstrate the potential of this formulation in vivo through three different paradigms: (i) gene editing in neurogenic niches, (ii) gene editing in the ventral tegmental area to facilitate monitoring of edited cells by precise optogenetic control of reward and reinforcement, and (iii) gene editing in a localized brain region via a noninvasive administration route (i.e., intranasal).

Generalising from conventional pipelines using deep learning in high-throughput screening workflows.

The study of complex diseases relies on large amounts of data to build models toward precision medicine. Such data acquisition is feasible in the context of high-throughput screening, in which the quality of the results relies on the accuracy of the image analysis. Although state-of-the-art solutions for image segmentation employ deep learning approaches, the high cost of manually generating ground truth labels for model training hampers the day-to-day application in experimental laboratories. Alternatively, traditional computer vision-based solutions do not need expensive labels for their implementation. Our work combines both approaches by training a deep learning network using weak training labels automatically generated with conventional computer vision methods. Our network surpasses the conventional segmentation quality by generalising beyond noisy labels, providing a 25% increase of mean intersection over union, and simultaneously reducing the development and inference times. Our solution was embedded into an easy-to-use graphical user interface that allows researchers to assess the predictions and correct potential inaccuracies with minimal human input. To demonstrate the feasibility of training a deep learning solution on a large dataset of noisy labels automatically generated by a conventional pipeline, we compared our solution against the common approach of training a model from a small manually curated dataset by several experts. Our work suggests that humans perform better in context interpretation, such as error assessment, while computers outperform in pixel-by-pixel fine segmentation. Such pipelines are illustrated with a case study on image segmentation for autophagy events. This work aims for better translation of new technologies to real-world settings in microscopy-image analysis.

New insights into the regulatory roles of microRNAs in adult neurogenesis.

Adult neurogenesis, the process of generation of new functional neurons from neural stem cells, occurs in the subventricular zone and the subgranular zone neurogenic niches. This neurogenic process is tightly controlled by several intrinsic factors, including microRNAs (miRNAs), a class of small non-coding RNAs, which control protein translation. MiRNAs have emerged as important regulators of both embryonic and adult neural stem cells self-renewal and proliferation, neuronal differentiation, migration, maturation and integration into the complex neuronal circuitry. Herein, we will provide a review of the most prominent and recent findings underlying the physiological regulatory role of several miRNAs during adult neurogenesis.

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.

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.

MicroRNA-124-loaded nanoparticles increase survival and neuronal differentiation of neural stem cells in vitro but do not contribute to stroke outcome in vivo.

There is a high quest for novel therapeutic strategies to enhance recovery after stroke. MicroRNA-124 (miR-124) has been described as neuroprotective and anti-inflammatory molecule. Moreover, miR-124 is a well described enhancer of adult neurogenesis that could offer potentially beneficial effects. Herein, we used miR-124-loaded nanoparticles (miR-124 NPs) to evaluate their therapeutic potential in an in vitro and in vivo model of stroke. For that, neuroprotective and neurogenic responses were assessed in an in vitro model of stroke. Here, we found that miR-124 NPs decreased cell death and improved neuronal differentiation of subventricular zone (SVZ) neural stem cell cultures after oxygen and glucose deprivation. In contrast, intravenous injection of miR-124 NPs immediately after permanent focal ischemia induced by photothrombosis (PT) did not provide a better neurological outcome. In addition, treatment did not affect the number of 5-bromo-2'-deoxyuridine (BrdU)- and doublecortin/BrdU- positive cells in the SVZ at the study endpoint of 14 days after PT. Likewise, the ischemic insult did not affect the numbers of neuronal progenitors in the SVZ. However, in PT mice miR-124 NPs were able to specifically augment interleukin-6 levels at day 2 post-stroke. Furthermore, we also showed that NPs reached the brain parenchyma and were internalized by brain resident cells. Although, promising in vitro data could not be verified in vivo as miR-124 NPs treatment did not improve functional outcome nor presented beneficial actions on neurogenesis or post-stroke inflammation, we showed that our NP formulation can be a safe alternative for drug delivery into the brain.

MicroRNA: Basic concepts and implications for regeneration and repair of neurodegenerative diseases.

MicroRNAs (miRNA) are small non-coding molecules that revolutionized our knowledge about the regulation of gene expression. Capable to target a large number of mRNA, miRNA are thought to regulate around 30% of the entire human genome. Therefore, these molecules are able to regulate several biological processes, including neuronal survival, differentiation and regeneration. Additionally, miRNA might act as valuable clinical agents in brain pathological conditions. Their specific expression patterns in the brain parenchyma and/or in circulating fluids have been highlighted as potential biomarkers, while the modulation of their activity may have therapeutic value for several neurodegenerative diseases. In this review, we describe miRNA biogenesis, signaling and regulation as well as the role of miR-9, miR-124, miR-132 and miR-137 in both adult neurogenesis and neurodegeneration, namely in Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. The relationship between miRNA, neurodegeneration and neurogenesis will be highlighted. Moreover, the benefits, outcomes and limitations of therapies using miRNA technology for neurodegenerative disorders will also be discussed.

Blue light potentiates neurogenesis induced by retinoic acid-loaded responsive nanoparticles.

Neurogenic niches constitute a powerful endogenous source of new neurons that can be used for brain repair strategies. Neuronal differentiation of these cells can be regulated by molecules such as retinoic acid (RA) or by mild levels of reactive oxygen species (ROS) that are also known to upregulate RA receptor alpha (RARα) levels. Data showed that neural stem cells from the subventricular zone (SVZ) exposed to blue light (405nm laser) transiently induced NADPH oxidase-dependent ROS, resulting in ß-catenin activation and neuronal differentiation, and increased RARα levels. Additionally, the same blue light stimulation was capable of triggering the release of RA from light-responsive nanoparticles (LR-NP). The synergy between blue light and LR-NP led to amplified neurogenesis both in vitro and in vivo, while offering a temporal and spatial control of RA release. In conclusion, this combinatory treatment offers great advantages to potentiate neuronal differentiation, and provides an innovative and efficient application for brain regenerative therapies. STATEMENT OF SIGNIFICANCE: Controlling the differentiation of stem cells would support the development of promising brain regenerative therapies. Blue light transiently increased reactive oxygen species, resulting in neuronal differentiation and increased retinoic acid receptor (RARα) levels. Additionally, the same blue light stimulation was capable of triggering the release of RA from light-responsive nanoparticles (LR-NP). The synergy between blue light and LR-NP led to amplified neurogenesis, while offering a temporal and spatial control of RA release. In this sense, our approach relying on the modulation of endogenous stem cells for the generation of new neurons may support the development of novel clinical therapies.

Traceable microRNA-124 loaded nanoparticles as a new promising therapeutic tool for Parkinson's disease.

Parkinson's disease (PD), a neurodegenerative disorder characterized by the selective degeneration of the nigrostriatal dopaminergic pathway, is a major socio-economic burden in modern society. While there is presently no cure for PD, enhancing the number of neural stem cells (NSCs) and/or stimulating their differentiation into new neurons are promising therapeutic strategies. Many proneurogenic factors have been implicated in controlling NSCs activity, including the microRNA (miR)-124. However, current strategies described for the intracellular delivery of miR involve mostly unspecific or inefficient platforms. In Saraiva et al. we developed miR-124 loaded nanoparticles (NPs) able to efficiently deliver miR-124 into neural stem/progenitor cells and boost neuronal differentiation and maturation in vitro. In vivo, the intracerebroventricular injection of miR-124 NPs increased the number of new neurons in the olfactory bulb of healthy and 6-hydroxidopamine (6-OHDA) lesioned mice, a model for PD. Importantly, miR-124 NPs enhanced the migration of new neurons into the 6-OHDA lesioned striatum, culminating in motor function improvement. Given the recent advent of clinical trials for miR-based therapies and the theranostic applications of our NPs, we expect to support the clinical translation of our delivery platform in the context of PD and other neurodegenerative diseases which may benefit from enhancing miR levels.

Nanomedicine Approaches to Modulate Neural Stem Cells in Brain Repair.

We explore the concept of modulating neural stem cells and their niches for brain repair using nanotechnology-based approaches. These approaches include stimulating cell proliferation, recruitment, and differentiation to functionally recover damaged areas. Nanoscale-engineered materials potentially overcome limited crossing of the blood-brain barrier, deficient drug delivery, and cell targeting.