Synaptic Regulator α-Synuclein in Dopaminergic Fibers Is Essentially Required for the Maintenance of Subependymal Neural Stem Cells.

Synaptic protein α-synuclein (α-SYN) modulates neurotransmission in a complex and poorly understood manner and aggregates in the cytoplasm of degenerating neurons in Parkinson's disease. Here, we report that α-SYN present in dopaminergic nigral afferents is essential for the normal cycling and maintenance of neural stem cells (NSCs) in the brain subependymal zone of adult male and female mice. We also show that premature senescence of adult NSCs into non-neurogenic astrocytes in mice lacking α-SYN resembles the effects of dopaminergic fiber degeneration resulting from chronic exposure to 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine or intranigral inoculation of aggregated toxic α-SYN. Interestingly, NSC loss in α-SYN-deficient mice can be prevented by viral delivery of human α-SYN into their sustantia nigra or by treatment with l-DOPA, suggesting that α-SYN regulates dopamine availability to NSCs. Our data indicate that α-SYN, present in dopaminergic nerve terminals supplying the subependymal zone, acts as a niche component to sustain the neurogenic potential of adult NSCs and identify α-SYN and DA as potential targets to ameliorate neurogenic defects in the aging and diseased brain.SIGNIFICANCE STATEMENT We report an essential role for the protein α-synuclein present in dopaminergic nigral afferents in the regulation of adult neural stem cell maintenance, identifying the first synaptic regulator with an implication in stem cell niche biology. Although the exact role of α-synuclein in neural transmission is not completely clear, our results indicate that it is required for stemness and the preservation of neurogenic potential in concert with dopamine.

Disruption of a ciliary B9 protein complex causes Meckel syndrome.

Nearly every ciliated organism possesses three B9 domain-containing proteins: MKS1, B9D1, and B9D2. Mutations in human MKS1 cause Meckel syndrome (MKS), a severe ciliopathy characterized by occipital encephalocele, liver ductal plate malformations, polydactyly, and kidney cysts. Mouse mutations in either Mks1 or B9d2 compromise ciliogenesis and result in phenotypes similar to those of MKS. Given the importance of these two B9 proteins to ciliogenesis, we examined the role of the third B9 protein, B9d1. Mice lacking B9d1 displayed polydactyly, kidney cysts, ductal plate malformations, and abnormal patterning of the neural tube, concomitant with compromised ciliogenesis, ciliary protein localization, and Hedgehog (Hh) signal transduction. These data prompted us to screen MKS patients for mutations in B9D1 and B9D2. We identified a homozygous c.301A>C (p.Ser101Arg) B9D2 mutation that segregates with MKS, affects an evolutionarily conserved residue, and is absent from controls. Unlike wild-type B9D2 mRNA, the p.Ser101Arg mutation failed to rescue zebrafish phenotypes induced by the suppression of b9d2. With coimmunoprecipitation and mass spectrometric analyses, we found that Mks1, B9d1, and B9d2 interact physically, but that the p.Ser101Arg mutation abrogates the ability of B9d2 to interact with Mks1, further suggesting that the mutation compromises B9d2 function. Our data indicate that B9d1 is required for normal Hh signaling, ciliogenesis, and ciliary protein localization and that B9d1 and B9d2 are essential components of a B9 protein complex, disruption of which causes MKS.

Role of quercetin on sterigmatocystin-induced oxidative stress-mediated toxicity.

Oxidative stress appears to be a common trigger for many of the effects associated with the exposure to various mycotoxins, including sterigmatocystin (STE). However, studies to alleviate STE toxicity through the use of natural antioxidants are sparsely reported in literature. In the present study, the cytoprotective effect of quercetin (QUE) was tested in SH-SY5Y cells against STE-induced oxidative stress and cytotoxicity. The MTT assay revealed that STE decreased cell viability, whereas pre-treatment of cells with QUE restored it. The QUE was also found to counteract STE-induced ROS generation and decrease STE-induced up-regulation of the expression of the stress-inducible enzymes HO-1 and NOS-2. Pre-treatment with QUE also prevented STE-induced nuclear translocation of NF-κB, as measured by immunofluorescence. Finally, considering the key role played by NF-κB in the regulation of inflammation, the effect of STE on the pro-inflammatory cytokines TNF-α and IL-6 expression was evaluated. Our results showed the down-regulation of TNF-α and IL-6 following STE exposure, suggesting a negative immunomodulatory effect of STE. In QUE pre-treated samples, TNF-α and IL-6 were significantly further reduced, indicating the anti-inflammatory role of QUE. The results of the present study demonstrate for the first time that QUE exerts a cytoprotective role in STE-induced toxicity.

NT3/TrkC Pathway Modulates the Expression of UCP-1 and Adipocyte Size in Human and Rodent Adipose Tissue.

Neurotrophin-3 (NT3), through activation of its tropomyosin-related kinase receptor C (TrkC), modulates neuronal survival and neural stem cell differentiation. It is widely distributed in peripheral tissues (especially vessels and pancreas) and this ubiquitous pattern suggests a role for NT3, outside the nervous system and related to metabolic functions. The presence of the NT3/TrkC pathway in the adipose tissue (AT) has never been investigated. Present work studies in human and murine adipose tissue (AT) the presence of elements of the NT3/TrkC pathway and its role on lipolysis and adipocyte differentiation. qRT-PCR and immunoblot indicate that NT3 (encoded by NTF3) was present in human retroperitoneal AT and decreases with age. NT3 was also present in rat isolated adipocytes and retroperitoneal, interscapular, perivascular, and perirenal AT. Histological analysis evidences that NT3 was mainly present in vessels irrigating AT close associated to sympathetic fibers. Similar mRNA levels of TrkC (encoded by NTRK3) and ß-adrenoceptors were found in all ATs assayed and in isolated adipocytes. NT3, through TrkC activation, exert a mild effect in lipolysis. Addition of NT3 during the differentiation process of human pre-adipocytes resulted in smaller adipocytes and increased uncoupling protein-1 (UCP-1) without changes in ß-adrenoceptors. Similarly, transgenic mice with reduced expression of NT3 (Ntf3 knock-in lacZ reporter mice) or lacking endothelial NT3 expression (Ntf3flox1/flox2;Tie2-Cre+/0) displayed enlarged white and brown adipocytes and lower UCP-1 expression. Conclusions: NT3, mainly released by blood vessels, activates TrkC and regulates adipocyte differentiation and browning. Disruption of NT3/TrkC signaling conducts to hypertrophied white and brown adipocytes with reduced expression of the thermogenesis marker UCP-1.

Resistance of subventricular neural stem cells to chronic hypoxemia despite structural disorganization of the germinal center and impairment of neuronal and oligodendrocyte survival.

Chronic hypoxemia, as evidenced in de-acclimatized high-altitude residents or in patients with chronic obstructive respiratory disorders, is a common medical condition that can produce serious neurological alterations. However, the pathogenesis of this phenomenon is unknown. We have found that adult rodents exposed for several days/weeks to hypoxia, with an arterial oxygen tension similar to that of chronically hypoxemic patients, manifest a partially irreversible structural disarrangement of the subventricular neurogenic niche (subventricular zone) characterized by displacement of neurons and myelinated axons, flattening of the ependymal cell layer, and thinning of capillary walls. Despite these abnormalities, the number of neuronal and oligodendrocyte progenitors, neuroblasts, and neurosphere-forming cells as well as the proliferative activity in subventricular zone was unchanged. These results suggest that neural stem cells and their undifferentiated progeny are resistant to hypoxia. However, in vivo and in vitro experiments indicate that severe chronic hypoxia decreases the survival of newly generated neurons and oligodendrocytes, with damage of myelin sheaths. These findings help explain the effects of hypoxia on adult neurogenesis and provide new perspectives on brain responsiveness to persistent hypoxemia.

GFP immunogold staining, from light to electron microscopy, in mammalian cells.

GFP has emerged as an important reporter for monitoring gene expression, protein localization, cell transformation and cell lineage. The development of GFP as a marker in many different biological systems has emphasized the need to image GFP at high resolution. GFP immunogold labeling with colloidal gold particles becomes essential for electron microscopy (EM) ultrastructural detection. Because of the small size, colloidal gold particles require silver enhancement, a procedure to increase the size of the particle as well as gold toning to stabilize the silver layer. GFP preembedding immunogold staining enables high quality cellular-ultrastructural EM analysis mainly for two reasons, on one hand it allows adequate fixation for EM analysis maintaining GFP antigenicity, on the other hand it also enables the epoxy resins inclusion after immunogold staining. Both of them help to preserve better the ultrastructure. However GFP immunogold staining presents some drawbacks, such as the progressive decrease in immunogold labeling with tissue depth. Special attention must be taken when using GFP-tagged protein, since the fusion could interfere with their localization and function. In this review we provide a detailed protocol of the GFP immunogold staining, their main applications for EM and possible troubles.