The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca(2+)]i) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.

Stroke induces the recruitment of neuronal precursors from the subventricular zone (SVZ) into the ischemic striatum. In injured areas, de-routed neuroblasts use blood vessels as a physical scaffold to their migration, in a process that resembles the constitutive migration seen in the rostral migratory stream (RMS). The molecular mechanism underlying injury-induced vasculature-mediated migration of neuroblasts in the post-stroke striatum remains, however, elusive. Using adult mice we now demonstrate that endothelial cells in the ischemic striatum produce brain-derived neurotrophic factor (BDNF), a neurotrophin that promotes the vasculature-mediated migration of neuronal precursors in the RMS, and that recruited neuroblasts maintain expression of p75NTR, a low-affinity receptor for BDNF. Reactive astrocytes, which are widespread throughout the damaged area, ensheath blood vessels and express TrkB, a high-affinity receptor for BDNF. Despite the absence of BDNF mRNA, we observed strong BDNF immunolabeling in astrocytes, suggesting that these glial cells trap extracellular BDNF. Importantly, this pattern of expression is reminiscent of the adult RMS, where TrkB-expressing astrocytes bind and sequester vasculature-derived BDNF, leading to the entry of migrating cells into the stationary phase. Real-time imaging of cell migration in acute brain slices revealed a direct role for BDNF in promoting the migration of neuroblasts to ischemic areas. We also demonstrated that cells migrating in the ischemic striatum display higher exploratory behavior and longer stationary periods than cells migrating in the RMS. Our findings suggest that the mechanisms involved in the injury-induced vasculature-mediated migration of neuroblasts recapitulate, at least partially, those observed during constitutive migration in the RMS.

Cell transplantation is a promising approach for the reconstruction of neuronal circuits after brain damage. Transplanted neurons integrate with remarkable specificity into circuitries of the mouse cerebral cortex affected by neuronal ablation. However, it remains unclear how neurons perform in a local environment undergoing reactive gliosis, inflammation, macrophage infiltration, and scar formation, as in traumatic brain injury (TBI). To elucidate this, we transplanted cells from the embryonic mouse cerebral cortex into TBI-injured, inflamed-only, or intact cortex of adult mice. Brain-wide quantitative monosynaptic rabies virus (RABV) tracing unraveled graft inputs from correct regions across the brain in all conditions, with pronounced quantitative differences: scarce in intact and inflamed brain versus exuberant after TBI. In the latter, the initial overshoot is followed by pruning, with only a few input neurons persisting at 3 months. Proteomic profiling identifies candidate molecules for regulation of the synaptic yield, a pivotal parameter to tailor for functional restoration of neuronal circuits.

Neural stem cells (NSCs) from the subventricular zone (SVZ) have been indicated as a source of new oligodendrocytes to use in regenerative medicine for myelin pathologies. Indeed, NSCs are multipotent cells that can self-renew and differentiate into all neural cell types of the central nervous system. In normal conditions, SVZ cells are poorly oligodendrogenic, nevertheless their oligodendrogenic potential is boosted following demyelination. Importantly, progressive restriction into the oligodendrocyte fate is specified by extrinsic and intrinsic factors, endocannabinoids being one of these factors. Although a role for endocannabinoids in oligodendrogenesis has already been foreseen, selective agonists and antagonists of cannabinoids receptors produce severe adverse side effects. Herein, we show that hemopressin (Hp), a modulator of CB1 receptors, increased oligodendroglial differentiation in SVZ neural stem/progenitor cell cultures derived from neonatal mice. The original results presented in this work suggest that Hp and derivates may be of potential interest for the development of future strategies to treat demyelinating diseases.

Histamine is commonly acknowledged as an inflammatory mediator in peripheral tissues, leaving its role in brain immune responses scarcely studied. Therefore, our aim was to uncover the cellular and molecular mechanisms elicited by this molecule and its receptors in microglia-induced inflammation by evaluating cell migration and inflammatory mediator release.

Retinoids regulate a wide spectrum of cellular functions from the embryo throughout adulthood, including cell differentiation, metabolic regulation, and inflammation. These traits make retinoids very attractive molecules for medical purposes. In light of some of the physicochemical limitations of retinoids, the development of drug delivery systems offers several advantages for clinical translation of retinoid-based therapies, including improved solubilization, prolonged circulation, reduced toxicity, sustained release, and improved efficacy. In this Review, we discuss advances in preclinical and clinical tests regarding retinoid formulations, specifically the ones based in natural retinoids, evaluated in the context of regenerative medicine, brain, cancer, skin, and immune diseases. Advantages and limitations of retinoid formulations, as well as prospects to push the field forward, will be presented.

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.

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.

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).

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.

Methamphetamine (METH) is a psychostimulant drug of abuse that causes severe brain damage. However, the mechanisms responsible for these effects are poorly understood, particularly regarding the impact of METH on hippocampal neurogenesis. Moreover, neuropeptide Y (NPY) is known to be neuroprotective under several pathological conditions. Here, we investigated the effect of METH on dentate gyrus (DG) neurogenesis, regarding cell death, proliferation and differentiation, as well as the role of NPY by itself and against METH-induced toxicity. DG-derived neurosphere cultures were used to evaluate the effect of METH or NPY on cell death, proliferation or neuronal differentiation. Moreover, the role of NPY and its receptors (Y(1), Y(2) and Y(5)) was investigated under conditions of METH-induced DG cell death. METH-induced cell death by both apoptosis and necrosis at concentrations above 10 nM, without affecting cell proliferation. Furthermore, at a non-toxic concentration (1 nM), METH decreased neuronal differentiation. NPY's protective effect was mainly due to the reduction of glutamate release, and it also increased DG cell proliferation and neuronal differentiation via Y(1) receptors. In addition, while the activation of Y(1) or Y(2) receptors was able to prevent METH-induced cell death, the Y(1) subtype alone was responsible for blocking the decrease in neuronal differentiation induced by the drug. Taken together, METH negatively affects DG cell viability and neurogenesis, and NPY is revealed to be a promising protective tool against the deleterious effects of METH on hippocampal neurogenesis.

Transplantation is a clinically relevant approach for brain repair, but much remains to be understood about influences of the disease environment on transplant connectivity. To explore the effect of amyloid pathology in Alzheimer's disease (AD) and aging, we examined graft connectivity using monosynaptic rabies virus tracing in APP/PS1 mice and in 16- to 18-month-old wild-type (WT) mice. Transplanted neurons differentiated within 4 weeks and integrated well into the host visual cortex, receiving input from the appropriate brain regions for this area. Unexpectedly, we found a prominent several-fold increase in local inputs, in both amyloid-loaded and aged environments. State-of-the-art deep proteome analysis using mass spectrometry highlights complement system activation as a common denominator of environments promoting excessive local input connectivity. These data therefore reveal the key role of the host pathology in shaping the input connectome, calling for caution in extrapolating results from one pathological condition to another.

COVID-19 is the most impacting global pandemic of all time, with over 600 million infected and 6.5 million deaths worldwide, in addition to an unprecedented economic impact. Despite the many advances in scientific knowledge about the disease, much remains to be clarified about the molecular alterations induced by SARS-CoV-2 infection. In this work, we present a hybrid proteomics and in silico interactomics strategy to establish a COVID-19 salivary protein profile. Data are available via ProteomeXchange with identifier PXD036571. The differential proteome was narrowed down by the Partial Least-Squares Discriminant Analysis and enrichment analysis was performed with FunRich. In parallel, OralInt was used to determine interspecies Protein-Protein Interactions between humans and SARS-CoV-2. Five dysregulated biological processes were identified in the COVID-19 proteome profile: Apoptosis, Energy Pathways, Immune Response, Protein Metabolism and Transport. We identified 10 proteins (KLK 11, IMPA2, ANXA7, PLP2, IGLV2-11, IGHV3-43D, IGKV2-24, TMEM165, VSIG10 and PHB2) that had never been associated with SARS-CoV-2 infection, representing new evidence of the impact of COVID-19. Interactomics analysis showed viral influence on the host immune response, mainly through interaction with the degranulation of neutrophils. The virus alters the host's energy metabolism and interferes with apoptosis mechanisms.

Several cell-based therapies are under pre-clinical and clinical evaluation for the treatment of ischemic diseases. Poor survival and vascular engraftment rates of transplanted cells force them to work mainly via time-limited paracrine actions. Although several approaches, including the use of soluble vascular endothelial growth factor (sVEGF)-VEGF165, have been developed in the last 10 years to enhance cell survival, they showed limited efficacy. Here, we report a pro-survival approach based on VEGF-immobilized microparticles (VEGF-MPs). VEGF-MPs prolong VEGFR-2 and Akt phosphorylation in cord blood-derived late outgrowth endothelial progenitor cells (OEPCs). In vivo, OEPC aggregates containing VEGF-MPs show higher survival than those treated with sVEGF. Additionally, VEGF-MPs decrease miR-17 expression in OEPCs, thus increasing the expression of its target genes CDKN1A and ZNF652. The therapeutic effect of OEPCs is improved in vivo by inhibiting miR-17. Overall, our data show an experimental approach to improve therapeutic efficacy of proangiogenic cells for the treatment of ischemic diseases.Soluble vascular endothelial growth factor (VEGF) enhances vascular engraftment of transplanted cells but the efficacy is low. Here, the authors show that VEGF-immobilized microparticles prolong survival of endothelial progenitors in vitro and in vivo by downregulating miR17 and upregulating CDKN1A and ZNF652.

Inflammatory mechanisms triggered by microglial cells are involved in the pathophysiology of several brain disorders, hindering repair. Herein, we propose the use of retinoic acid-loaded polymeric nanoparticles (RA-NP) as a means to modulate microglia response towards an anti-inflammatory and neuroprotective phenotype (M2). RA-NP were first confirmed to be internalized by N9 microglial cells; nanoparticles did not affect cell survival at concentrations below 100 μg/mL. Then, immunocytochemical studies were performed to assess the expression of pro- and anti-inflammatory mediators. Our results show that RA-NP inhibited LPS-induced release of nitric oxide and the expression of inducible nitric oxide synthase and promoted arginase-1 and interleukin-4 production. Additionally, RA-NP induced a ramified microglia morphology (indicative of M2 state), promoting tissue viability, particularly neuronal survival, and restored the expression of postsynaptic protein-95 in organotypic hippocampal slice cultures exposed to an inflammatory challenge. RA-NP also proved to be more efficient than the free equivalent RA concentration. Altogether, our data indicate that RA-NP may be envisioned as a promising therapeutic agent for brain inflammatory diseases.

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.

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.

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.

Retinoic acid (RA) plays an important role in the commitment, maturation and survival of neural cells. Recently, RA was pointed as a therapeutic option for some neurodegenerative diseases, including Parkinson's disease (PD). The administration of RA has been defying, and in this sense we have previously developed novel RA-loaded polymeric nanoparticles (RA-NPs) that ensure the efficient intracellular transport and controlled release of RA. Herein, we show that nanoformulation as an efficient neuroprotective effect on dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced mouse model for PD. The results showed that the RA-NPs administration induced a significant reduction of DA neuron loss in the substantia nigra (SN) as well as their neuronal fiber/axonal innervations in the striatum. Furthermore, we observed an increase in the expression levels of the transcription factors Pitx3 and Nurr1 induced by RA-NPs, showing its supportive effect on the development and functional maintenance of DA neurons in PD. This is the first study showing that RA-NPs can be an innovative strategy to halt the progression of PD pathogenesis, suggesting that this nanoformulation could be of particular interest for the development of new approaches for PD therapeutics.

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.