RESUMO
The retina is the sensory tissue responsible for the first stages of visual processing, with a conserved anatomy and functional architecture among vertebrates. To date, retinal eye diseases, such as diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, glaucoma, and others, affect nearly 170 million people worldwide, resulting in vision loss and blindness. To tackle retinal disorders, the developing retina has been explored as a versatile model to study intercellular signaling, as it presents a broad neurochemical repertoire that has been approached in the last decades in terms of signaling and diseases. Retina, dissociated and arranged as typical cultures, as mixed or neuron- and glia-enriched, and/or organized as neurospheres and/or as organoids, are valuable to understand both neuronal and glial compartments, which have contributed to revealing roles and mechanisms between transmitter systems as well as antioxidants, trophic factors, and extracellular matrix proteins. Overall, contributions in understanding neurogenesis, tissue development, differentiation, connectivity, plasticity, and cell death are widely described. A complete access to the genome of several vertebrates, as well as the recent transcriptome at the single cell level at different stages of development, also anticipates future advances in providing cues to target blinding diseases or retinal dysfunctions.
Assuntos
Doenças Retinianas , Animais , Humanos , Cegueira , Nível de Saúde , Neuroglia , Neurônios , RetinaRESUMO
The vascular and the nervous systems share similarities in addition to their complex role in providing oxygen and nutrients to all cells. Both are highly branched networks that frequently grow close to one another during development. Vascular patterning and neural wiring share families of guidance cues and receptors. Most recently, this relationship has been investigated in terms of peripheral nervous system (PNS) regeneration, where nerves and blood vessels often run in parallel so endothelial cells guide the formation of the Büngner bands which support axonal regeneration. Here, we characterized the vascular response in regenerative models of the central and peripheral nervous system. After sciatic nerve crush, followed by axon regeneration, there was a significant increase in the blood vessel density 7 days after injury. In addition, the optic nerve crush model was used to evaluate intrinsic regenerative potential activated with a combined treatment that stimulated retinal ganglion cells (RGCs) regrowth. We observed that a 2-fold change in the total number of blood vessels occurred 7 days after optic nerve crush compared to the uncrushed nerve. The difference increased up to a 2.7-fold change 2 weeks after the crush. Interestingly, we did not observe differences in the total number of blood vessels 2 weeks after crush, compared to animals that had received combined treatment for regeneration and controls. Therefore, the vascular characterization showed that the increase in vascular density was not related to the efficiency of both peripheral and central axonal regeneration.
Assuntos
Axônios , Regeneração Nervosa , Camundongos , Animais , Axônios/fisiologia , Regeneração Nervosa/fisiologia , Células Endoteliais , Nervo Óptico/fisiologia , Células Ganglionares da Retina/fisiologia , Compressão NervosaRESUMO
The endocannabinoid system (eCS) is widely distributed in mammalian tissues and it is classically formed by cannabinoid receptors, endogenous bioactive lipids and its synthesis and degradation enzymes. Due to the modulatory role of eCS in synaptic activity in the Central Nervous System (CNS), phytocannabinoids have been increasingly used for the treatment of neurological disorders, even though little is known in terms of the long-term effect of these treatments on CNS development, mainly in the timeframe that comprises childhood and adolescence. Furthermore, an increased number of clinical trials using full-spectrum Cannabis extracts has been seen, rather than the isolated form of phytocannabinoids, when exploring the therapeutical benefits of the Cannabis plant. Thus, this study aims to evaluate the effect of cannabidiol (CBD)-enriched Cannabis extract on synaptic components in the hippocampus of rats from adolescence to early adulthood (postnatal day 45 to 60). Oral treatment of healthy male Wistar rats with a CBD-enriched Cannabis extract (3 mg/kg/day CBD) during 15 days did not affect food intake and water balance. There was also no negative impact on locomotor behaviour and cognitive performance. However, the hippocampal protein levels of GluA1 and GFAP were reduced in animals treated with the extract, whilst PSD95 levels were increased, which suggests rearrangement of glutamatergic synapses and modulation of astrocytic features. Microglial complexity was reduced in CA1 and CA3 regions, but no alterations in their phagocytic activity have been identified by Iba-1 and LAMP2 co-localization. Collectively, our data suggest that CBD-enriched Cannabis treatment may be safe and well-tolerated in healthy subjects, besides acting as a neuroprotective agent against hippocampal alterations related to the pathogenesis of excitatory and astrogliosis-mediated disorders in CNS.
Assuntos
Canabidiol , Cannabis , Alucinógenos , Ratos , Animais , Canabidiol/uso terapêutico , Cannabis/metabolismo , Ratos Wistar , Endocanabinoides , Agonistas de Receptores de Canabinoides , Extratos Vegetais/uso terapêutico , Hipocampo/metabolismo , Mamíferos/metabolismoRESUMO
The endocannabinoid system (ECS) refers to a complex cell-signaling system highly conserved among species formed by numerous receptors, lipid mediators (endocannabinoids) and synthetic and degradative enzymes. It is widely distributed throughout the body including the CNS, where it participates in synaptic signaling, plasticity and neurodevelopment. Besides, the olfactory ensheathing glia (OEG) present in the olfactory system is also known to play an important role in the promotion of axonal growth and/or myelination. Therefore, both OEG and the ECS promote neurogenesis and oligodendrogenesis in the CNS. Here, we investigated if the ECS is expressed in cultured OEG, by assessing the main markers of the ECS through immunofluorescence, western blotting and qRT-PCR and quantifying the content of endocannabinoids in the conditioned medium of these cells. After that, we investigated whether the production and release of endocannabinoids regulate the differentiation of oligodendrocytes co-cultured with hippocampal neurons, through Sholl analysis in oligodendrocytes expressing O4 and MBP markers. Additionally, we evaluated through western blotting the modulation of downstream pathways such as PI3K/Akt/mTOR and ERK/MAPK, being known to be involved in the proliferation and differentiation of oligodendrocytes and activated by CB1, which is the major endocannabinoid responsive receptor in the brain. Our data show that OEG expresses key genes of the ECS, including the CB1 receptor, FAAH and MAGL. Besides, we were able to identify AEA, 2-AG and AEA related mediators palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in the conditioned medium of OEG cultures. These cultures were also treated with URB597 10-9 M, a FAAH selective inhibitor, or JZL184 10-9 M, a MAGL selective inhibitor, which led to the increase in the concentrations of OEA and 2-AG in the conditioned medium. Moreover, we found that the addition of OEG conditioned medium (OEGCM) enhanced the complexity of oligodendrocyte process branching in hippocampal mixed cell cultures and that this effect was inhibited by AM251 10-6 M, a CB1 receptor antagonist. However, treatment with the conditioned medium enriched with OEA or 2-AG did not alter the process branching complexity of premyelinating oligodendrocytes, while decreased the branching complexity in mature oligodendrocytes. We also observed no change in the phosphorylation of Akt and ERK 44/42 in any of the conditions used. In conclusion, our data show that the ECS modulates the number and maturation of oligodendrocytes in hippocampal mixed cell cultures.
RESUMO
Diabetic retinopathy is a neurovascular complication of diabetes and the main cause of vision loss in adults. Glial cells have a key role in maintenance of central nervous system homeostasis. In the retina, the predominant element is the Müller cell, a specialized cell with radial morphology that spans all retinal layers and influences the function of the entire retinal circuitry. Müller cells provide metabolic support, regulation of extracellular composition, synaptic activity control, structural organization of the blood-retina barrier, antioxidant activity, and trophic support, among other roles. Therefore, impairments of Müller actions lead to retinal malfunctions. Accordingly, increasing evidence indicates that Müller cells are affected in diabetic retinopathy and may contribute to the severity of the disease. Here, we will survey recently described alterations in Müller cell functions and cellular events that contribute to diabetic retinopathy, especially related to oxidative stress and inflammation. This review sheds light on Müller cells as potential therapeutic targets of this disease.
RESUMO
Despite the importance of pain as a warning physiological system, chronic neuropathic pain is frequently caused by damage in the nervous system, followed by persistence over a long period, even in the absence of dangerous stimuli or after healing of injuries. Chronic neuropathic pain affects hundreds of millions of adults worldwide, creating a direct impact on quality of life. This pathology has been extensively characterized concerning its cellular and molecular mechanisms, and the endocannabinoid system (eCS) is widely recognized as pivotal in the development of chronic neuropathic pain. Scientific evidence has supported that phyto-, synthetic and endocannabinoids are efficient for pain management, while strong data arise from the therapeutic use of Cannabis-derived products. The use of medicinal Cannabis products is directed toward not only relieving symptoms of chronic pain, but also improving several aspects of patients' welfare. Here, we review the involvement of eCS, along with other cellular and molecular elements, in chronic neuropathic pain pathology and how this system can be targeted for pain management.
RESUMO
The limited access to functional human brain tissue has led to the development of stem cell-based alternative models. The differentiation of human pluripotent stem cells into cerebral organoids with self-organized architecture has created novel opportunities to study the early stages of the human cerebral formation. Here we applied state-of-the-art label-free shotgun proteomics to compare the proteome of stem cell-derived cerebral organoids to the human fetal brain. We identified 3,073 proteins associated with different developmental stages, from neural progenitors to neurons, astrocytes, or oligodendrocytes. The major protein groups are associated with neurogenesis, axon guidance, synaptogenesis, and cortical brain development. Glial cell proteins related to cell growth and maintenance, energy metabolism, cell communication, and signaling were also described. Our data support the variety of cells and neural network functional pathways observed within cell-derived cerebral organoids, confirming their usefulness as an alternative model. The characterization of brain organoid proteome is key to explore, in a dish, atypical and disrupted processes during brain development or neurodevelopmental, neurodegenerative, and neuropsychiatric diseases.
RESUMO
Neurospheres prepared from multipotent progenitors in the retina obtained from postnatal mice differentiate into neurons and Müller glia (De Melo Reis et al., in Cell Mol Neurobiol 31:835-846, 2011). Here, we investigated whether neurospheres prepared from adult chickens (ciliary marginal zone, CMZ) or (ciliary body) retina could also lead to differentiated neurons and glia. Neurospheres were prepared from post-hatched chickens or from adult mice after 7 days in the presence of mitogenic factors (FGFb, insulin, and EGF), generating neurons and glial cells. In addition, Müller (2M6 or glutamine synthetase positive cells) derived from post-hatch chicken CMZ neurospheres displayed the dopaminergic phenotype. Furthermore, we observed that Müller cells derived from adult chickens and mice retina neurospheres released significant amounts of dopamine as well as of its metabolites. Taken together, our data lead us to conclude that as for embryonic (chick) or newborn (mouse), the dopaminergic phenotype is a default condition of Müller glial cells obtained from neurospheres prepared from mature retina. Our data raise the possibility that Müller cells from differentiated tissue could be used to ameliorate neurodegenerative diseases involving dopaminergic dysfunction as in Parkinson's disease as shown previously (Stutz et al., in J Neurochem 128:829-840, 2014).
Assuntos
Envelhecimento/metabolismo , Dopamina/metabolismo , Células Ependimogliais/citologia , Retina/citologia , Esferoides Celulares/citologia , Animais , Animais Recém-Nascidos , Separação Celular , Células Cultivadas , Galinhas , Células Ependimogliais/metabolismo , Metaboloma , Camundongos Endogâmicos C57BL , Fenótipo , Esferoides Celulares/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Degenerative retinopathies are the leading causes of irreversible visual impairment in the elderly, affecting hundreds of millions of patients. Müller glia cells (MGC), the main type of glia found in the vertebrate retina, can resume proliferation in the rodent adult injured retina but contribute weakly to tissue repair when compared to zebrafish retina. However, postnatal and adult mouse MGC can be genetically reprogrammed through the expression of the transcription factor (TF) Achaete-scute homolog 1 (ASCL1) into induced neurons (iNs), displaying key hallmarks of photoreceptors, bipolar and amacrine cells, which may contribute to regenerate the damaged retina. Here, we show that the TF neurogenin 2 (NEUROG2) is also sufficient to lineage-reprogram postnatal mouse MGC into iNs. The efficiency of MGC lineage conversion by NEUROG2 is similar to that observed after expression of ASCL1 and both TFs induce the generation of functionally active iNs. Treatment of MGC cultures with EGF and FGF2 prior to Neurog2 or Ascl1 expression enhances reprogramming efficiencies, what can be at least partially explained by an increase in the frequency of MGCs expressing sex determining region Y (SRY)-box 2 (SOX2). Transduction of either Neurog2 or Ascl1 led to the upregulation of key retina neuronal genes in MGC-derived iNs, but only NEUROG2 induced a consistent increase in the expression of putative retinal ganglion cell (RGC) genes. Moreover, in vivo electroporation of Neurog2 in late progenitors from the neonatal rat retina, which are transcriptionally similar to MGCs, also induced a shift in the generation of retinal cell subtypes, favoring neuronal differentiation at the expense of MGCs and resuming the generation of RGCs. Altogether, our data indicate that NEUROG2 induces lineage conversion of postnatal rodent MGCs into RGC-like iNs in vitro and resumes the generation of this neuronal type from late progenitors of the retina in vivo.
RESUMO
The avian retina has been used as a model to study signaling by different neuro- and gliotransmitters. It is unclear how dopaminergic and cannabinoid systems are related in the retina. Here we studied the expression of type 1 and 2 cannabinoid receptors (CB1 and CB2), as well as monoacylglycerol lipase (MAGL), the enzyme that degrades 2-arachidonoylglycerol (2-AG), during retina development. Our data show that CB1 receptor is highly expressed from embryonic day 5 (E5) until post hatched day 7 (PE7), decreasing its levels throughout development. CB1 is densely found in the ganglion cell layer (GCL) and inner plexiform layer (IPL). CB2 receptor was also found from E5 until PE7 with a decrease in its contents from E9 afterwards. CB2 was mainly present in the lamination of the IPL at PE7. MAGL is expressed in all retinal layers, mainly in the IPL and OPL from E9 to PE7 retina. CB1 and CB2 were found both in neurons and glia cells, but MAGL was only expressed in Müller glia. Older retinas (PE7) show CB1 positive cells mainly in the INL and co-expression of CB1 and tyrosine hydroxylase (TH) are shown in a few cells when both systems are mature. CB1 co-localized with TH and was heavily associated to D1 receptor labeling in primary cell cultures. Finally, cyclic AMP (cAMP) was activated by the selective D1 agonist SKF38393, and inhibited when cultures were treated with WIN55, 212-2 (WIN) in a CB1 dependent manner. The results suggest a correlation between the endocannabinoid and dopaminergic systems (DSs) during the avian retina development. Activation of CB1 limits cAMP accumulation via D1 receptor activation and may influence embryological parameters during avian retina differentiation.
RESUMO
Müller cells constitute the main glial cell type in the retina where it interacts with virtually all cells displaying relevant functions to retinal physiology. Under appropriate stimuli, Müller cells may undergo dedifferentiation, being able to generate other neural cell types. Here, we show that purified mouse Müller cells in culture express a group of proteins related to the dopaminergic phenotype, including the nuclear receptor-related 1 protein, required for dopaminergic differentiation, as well the enzyme tyrosine hydroxylase. These dopaminergic components are active, since Müller cells are able to synthesize and release dopamine to the extracellular medium. Moreover, Müller-derived tyrosine hydroxylase can be regulated, increasing its activity because of phosphorylation of serine residues in response to agents that increase intracellular cAMP levels. These observations were extended to glial cells obtained from adult monkey retinas with essentially the same results. To address the potential use of dopaminergic Müller cells as a source of dopamine in cell therapy procedures, we used a mouse model of Parkinson's disease, in which mouse Müller cells with the dopaminergic phenotype were transplanted into the striatum of hemi-parkinsonian mice generated by unilateral injection of 6-hydroxydopamine. These cells fully decreased the apomorphine-induced rotational behavior and restored motor functions in these animals, as measured by the rotarod and the forelimb-use asymmetry (cylinder) tests. The data indicate local restoration of dopaminergic signaling in hemi-parkinsonian mice confirmed by measurement of striatal dopamine after Müller cell grafting.