PACAP and VIP increase the expression of myelin- markers in rat schwannoma cells

Defining the mechanisms regulating peripheral myelinogenesis in vitro remains a daunting task. One of the major complications is related to the inability of Schwann cells to produce myelin in vitro. However, a limited number of schwannoma cell lines are now being accepted as adequate model systems to study myelin expression since they present most of the features that characterize normal myelinating Schwann cells. PACAP and VIP peptides have been shown to play part in myelin maturation and synthesis in the central nervous system, but no data regarding their potential actions have been reported in the peripheral nervous system, particularly in vitro. In this study, we investigated the effects of both peptides on the expression of myelin-specific markers using the well-established rat RT4 schwannoma cell line. In addition, we endeavored to partly unravel the underlying molecular mechanisms involved. Expression of myelin-specific markers (MAG, MBP and MPZ, respectively) was assessed in cells grown either in the presence of normal serum (10% FBS) or serum starved and treated with or without 100nM PACAP or VIP. Effects of pharmacological blockade of PACAP and VIP receptors as well as of the main signaling pathways on the expression of myelin-markers were also determined. Our data shows that serum starvation (24h) was sufficient to induce a significant increase in the expression of myelin markers. This result was paralleled by a concurrent increase in the endogenous expression of both peptides, as well as of the non-specific PACAP/VIP receptor 2 (VPAC2), but not of VPAC1 or PAC1. Exogenous PACAP or VIP treatment further exacerbated starvation-driven expression of myelin markers, suggesting an active role of these peptides in myelin generation. Interestingly, stimulation with either peptides increased phosphorylation levels of Akt at Ser473 residue whereas they did not affect Erk1/2 activation. Treatment with the PAC1 receptor antagonist (PACAP6-38) had no effects on myelin markers expression while a non-specific VPAC receptor antagonist completely abrogated both starvation and/or starvation + peptide-induced expression of myelin markers. Similar effects were obtained in cell pretreated with the PKA inhibitor (H-89, 10μM), suggesting that peptide-driven induction of myelin markers occurs preferentially via the canonical PKA/Akt signaling pathway. In conclusion, the results here presented provide the basis for future studies on the role of these peptides in physiological myelination and in demyelinating pathologies such as Charcot-Mary-Tooth disease

Cell-to-Cell Adhesion and Neurogenesis in Human Cortical Development: A Study Comparing 2D Monolayers with 3D Organoid Cultures

SUMMARY Organoids (ORGs) are increasingly used as models of cerebral cortical development. Here, we compared transcriptome and cellular phenotypes between telencephalic ORGs and monolayers (MONs) generated in parallel from three biologically distinct induced pluripotent stem cell (iPSC) lines. Multiple readouts revealed increased proliferation in MONs, which was caused by increased integrin signaling. MONs also exhibited altered radial glia (RG) polarity and suppression of Notch signaling, as well as impaired generation of intermediate progenitors, outer RG, and cortical neurons, which were all partially reversed by reaggregation of dissociated cells. Network analyses revealed co-clustering of cell adhesion, Notch-related transcripts and their transcriptional regulators in a module strongly downregulated in MONs. The data suggest that ORGs, with respect to MONs, initiate more efficient Notch signaling in ventricular RG owing to preserved cell adhesion, resulting in subsequent generation of intermediate progenitors and outer RG, in a sequence that recapitulates the cortical ontogenetic process

Expression of Parkin isoforms in human lymphomonocyte

Parkin (PARK2) is one of the largest gene in the human genome. Its mutations cause a form of autosomal recessive juvenile-onset of Parkinson disease (ARJPD) (1). To date, a repertoire of 21 parkin alternative splice variants has been identified. In the past, the role of the full-length parkin protein was extensively investigated and assessed also in human blood samples (2, 3). In contrast, less attention has been put on the other isoforms. In the present study, we investigated for the first time, the expression profile of parkin isoforms in three lymphomonocyte (LMN) subpopulations: T lymphocyte (CD2+), monocyte (CD14+) and B lymphocyte (CD19+). The expression of H1/H5 and H6 isoforms has been observed in total LMN homogenate, whereas H20 and H1/H5 variants were detected in all three LMN subpopulations by western blot analysis. The cellular distribution of parkin isoforms has been evaluated by immunofluorescence analysis. Although parkin is predominantly expressed in the cytoplasm, immunoreactivity has also been detected at nuclear and perinuclear level. Our data suggest that, the discovery of a specific expression profile of these isoforms into LMN of ARJP patients might allow developing new diagnostic tools for this neurodegenerative disease

Interleukin-1 family members in the retina of streptozotocin-injected rats

Diabetic retinopathy (DR) is one of the most common complications of diabetes. It has been demonstrated that pro-inflammatory cytokines are increased in diabetic retina, including interleukin-1β (IL-1β) (Joussen et al., 2001), suggesting that this cytokine might play an important role in the pathogenesis of DR (Kowluru and Odenbach, 2004). The principal components of the IL-1 family are two secreted factors, IL-1α and IL-1β, two transmembrane receptors (IL-1RI and IL-1RII), and a natural antagonist receptor of IL-1 function (IL-1Ra). To date the molecular mechanisms mediated by IL-1 family members in DR have not been fully characterized. In the present study, to explore the role of IL-1, we analyzed the expression and distribution of IL-1α and IL-1β and relative receptors in a model in vivo of DR. Diabetes was induced in adult rats by intraperitoneal injection of streptozotocin (60 mg/kg). Protein expression and distribution of IL-1 members and relative receptors were analyzed in retinas of nondiabetic and diabetic rats three weeks after induction of diabetes by Western blot and confocal microscopy analyses. Hyperglycemia induced a significant increased in IL-1β protein expression levels and distribution as compared to nondiabetic animals. Specifically, IL-1β retinal immunoreactivity was found not only in the rod and cone layer (RCL), but also in the outer plexiform layer (OPL), inner plexiform (IPL) and in the ganglion cell layer (GCL) of diabetic rats. IL-1α transcription levels were unchanged in the retinas of both animal groups. Consistent with expression studies, IL-1α localization did not differ between diabetic and nondiabetic rats. IL-1RI, IL-1RII and IL-1Ra expression was significantly increased in the retina of diabetic rats when compared to controls. Accordingly, IL-1RI positiveness was thoroughly increased in all retinal layers of diabetic rats, while no evident changes were apparent for IL-1RII, which was localized in the RCL layer and in outer nuclear layer (ONL) of both diabetic and nondiabetic rats. These finding point to IL-1 family members as key elements in the pro-inflammatory cascade after hyperglycemia-induced retinal damage, and therefore support the implementation of novel therapeutic strategies aimed at reducing IL-1 production for the treatment of DR

Differentiation of Human iPSCs Into Telencephalic Neurons Using 3D Organoids and Monolayer Culture

Human induced pluripotent stem cells (hiPSCs) are emerging as a useful tool for modelling in vitro early brain development and neurological disorders. Molecular mechanisms and cell interactions that regulate the neurodevelopment at early stages remain unclear because of human brain’s complexity and limitations of functional studies. Two major culture methodologies are used to differentiate in vitro hiPSCs into neurons: monolayer (2D) and organoid (3D) cultures. Here we investigate the effect of cell dissociation and the loss of 3D organization during the early differentiation process of neuronal progenitors. Using the same culture media, we first differentiated hiPSCs into neural progenitor cells (NPCs) and then induced their differentiation into neurons in 3 different modalities: 3D undissociated organoids, dissociated NPCs followed by immediate re-aggregation into an organoid, and dissociated NPCs cultured as monolayer. We assessed neuronal differentiation efficiency of each method by immunocytochemistry, qPCR, western blot, and RNA-Seq analysis over a time course. Our data revealed substantial differences in gene and protein expression among the three systems, including genes of the Notch pathway (e.g. NEUROD1, NEUROG2), earliest determinants of cortical region differentiation (e.g. SOX1, FEZF1) as well as later transcriptional regulators that specify cortical neuron subtypes (e.g. TBR1, CTIP2), which were all downregulated in monolayer. Moreover, we found that genes and pathways mediating cell-to-cell interactions (e.g. CNTNs, CAMs) were mostly upregulated in the 3D culture systems, whereas cell-extracellular matrix interaction molecules (e.g. ITG, LAM) were mostly upregulated in 2D, indicating that cell surface molecules may be involved in specification of neuronal cell types. Our results address the methodological question of the appropriateness of a differentiation method for a particular experimental goal, and, beyond that, reveal important early determinants that exert a decisive influence on neuronal differentiation and regional specification of human neural stem cells.

Dopamine D3 receptor modulates tissue type plasminogen activator (tPA) activity in mouse brain

Growing amount of evidence points to the dopamine D3 receptor (D3R) as an important mediator in the broad array of events that regulate memory function, perhaps through the modulation of molecular pathways involved in neurotrophic factor activation. Tissue type plasminogen activator (tPA) is a proteolytic enzyme that cleaves the precursor of brain derived neurotrophic factor (proBDNF) into the biologically active form of mature BDNF. However, whether D3Rs modulate tPA activity on BDNF in brain has not been ascertained yet. Here in the present study, using D3R knock-out (D3-/-) mice, we demonstrate that receptor inactivation is associated with increased tPA expression both in prefrontal cortex and, to a greater extent, in the hippocampus, two regions associated with memory processes. The heightened tPA levels observed in D3-/- mice inversely correlated with proBDNF protein expression, whereas they positively correlated with both BDNF mRNA and mature BDNF protein levels. In conclusion, our finding strongly suggest that D3Rs might modulate tPAmediated post-transcriptional processing of BDNF in brain regions critical to memory function

Comparative transcriptome and gene regulation in human iPSC-derived organoids and donor-identical brain tissue

Modeling human brain development in vitro is critically important to understand the pathophysiology of neuropsychiatric disorders. As part of the PsychENCODE project, we generated human induced pluripotent stem cells (hiPSCs) from skin fibroblasts of three human specimens at 15, 16 and 17 postconceptional weeks. These hiPSC were differentiated into telencephalic organoids to study early genetic programs in forebrain development. By using RNA-seq and histone chromatin immunoprecipitation (ChIP-seq), we compared transcriptomes and epigenomes of hiPSCs-derived organoids to donor-identical cortical brain tissue. Immunocytochemical characterization of the organoids over a time course (TD0, TD11 and TD30) showed expression of radial glial markers and mature cortical neurons confirming telencephalic fate. Hierarchical clustering of the organoids’ transcriptomes demonstrated stage-specific patterns of gene expression during in vitro development. Mapping organoids’ transcriptomes against the BrainSpan dataset suggested highest correlations with neocortex and showed their correspondence to post-conceptional weeks 8-16 of human fetal development. We then inferred transcriptional alterations, by differential gene expression, between organoids and the two brain regions analyzed. We found ~5000 of differentially expressed genes (DEG) between TD0 and fetal cortex and a decreasing number of DEG at TD11 and TD30 suggesting a stronger, albeit incomplete similarity of the organoids to the cortex at later time points. ChIP-seq experiments identified H3K27ac and H3K4me3 peaks (putative promoters and enhancers) differentially active at different organoids developmental stages and between organoids and fetal brain. Overall, however, hierarchical clustering of H3K27ac and H3K4me3 peaks demonstrated clustering of organoids with human fetal brain samples from various databases, whereas neonatal and adult brain samples formed separate clusters. These data suggest that organoids recapitulate in part transcriptome and epigenome features of fetal human brain

Schizophrenia-associated somatic copy-number variants from 12,834 cases reveal recurrent NRXN1 and ABCB11 disruptions

While germline copy-number variants (CNVs) contribute to schizophrenia (SCZ) risk, the contribution of somatic CNVs (sCNVs)—present in some but not all cells—remains unknown. We identified sCNVs using blood-derived genotype arrays from 12,834 SCZ cases and 11,648 controls, filtering sCNVs at loci recurrently mutated in clonal blood disorders. Likely early-developmental sCNVs were more common in cases (0.91%) than controls (0.51%, p = 2.68e−4), with recurrent somatic deletions of exons 1–5 of the NRXN1 gene in five SCZ cases. Hi-C maps revealed ectopic, allele-specific loops forming between a potential cryptic promoter and non-coding cis-regulatory elements upon 5′ deletions in NRXN1. We also observed recurrent intragenic deletions of ABCB11, encoding a transporter implicated in anti-psychotic response, in five treatment-resistant SCZ cases and showed that ABCB11 is specifically enriched in neurons forming mesocortical and mesolimbic dopaminergic projections. Our results indicate potential roles of sCNVs in SCZ risk

Neuroprotective effects of PACAP, VIP and NAP against hyperglycaemic retinal damage

Diabetic retinopathy, one the most significant and disabling chronic complications of diabetes mellitus, has mainly been regarded as a microcirculatory disease of the retina. However, there is emerging evidence to suggest that a primary and early neurodegenerative process characterizes DR. The reason for the retinal degeneration resides in the metabolic changes caused by hyperglycemia and in the decreased capability of the retina to adapt to this environment. This situation leads to altered expression patterns of neuropeptides, growth and transcription factors, apoptosis, increased oxidative stress, inflammatory response, angiogenesis and disruption of the blood-retinal barrier (BRB). All major cell types of the retina are affected: neuronal as well as the glial cells and pigment epithelial cells. Pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and davunetide (NAP) are three pleiotropic related neuropeptides also expressed in the retina. Their protective role has been shown in different retinal injuries, but little is known about the relationship between these peptides and DR. The aim of the present thesis was to investigate, using both in vitro and in vivo model of DR, the role of these peptdides on retinal layers by monitoring apoptotic events by western blot and confocal microscopy analysis and measuring the integrity of the outer blood retinal barrier with permeability and transepithelial electrical resistance assay. The underlying signal transduction pathways activated by each peptide and the impact of hyperglicaemia on the expression and distribution at retinal levels of the inflammatory IL-1 mediators were also evaluated. Diabetes was mimicked in adult rats by intraperitoneal injection of streptozotocin (STZ) and human retinal pigment epithelial cells (ARPE19) were cultured for 26 days in high glucose (25mM of D-glucose) and IL-1beta. The results confirmed that hyperglycemia induced early apoptotic death in the cellular components of the neuroretina, breakdown of the outer blood retinal barrier (BRB) and dysregulation of several components of the metabolic and signaling pathways. The neuropeptides tested activate promising pathways useful for the treatment of this retinal degenerative disease. In particular, PACAP and VIP promote the integrity of the outer BRB, possibly through the modulation of proteins related to tight junctions. Davunetive is able to reduce apoptosis in the diabetic retina by activating the anti-apoptotic p-Akt, p-ERK1, p-ERK2, Bcl-2 and decreasing levels of the pro-apoptotic elements, such as cleaved caspase-3. These results raise the opportunity for the use of these peptides as a possible therapeutic or preventive methods in treating diabetes