Spatiotemporal oscillations of Notch1, Dll1 and NICD are coordinated across the mouse PSM

During somitogenesis, epithelial somites form from the pre-somitic mesoderm (PSM) in a periodic manner. This periodicity is regulated by a molecular oscillator, known as the ‘segmentation clock’, that is characterised by an oscillatory pattern of gene expression that sweeps the PSM in a caudal-rostral direction. Key components of the segmentation clock are intracellular components of the Notch, Wnt and FGF pathways, and it is widely accepted that intracellular negative-feedback loops regulate oscillatory gene expression. However, an open question in the field is how intracellular oscillations are coordinated, in the form of spatiotemporal waves of expression, across the PSM. In this study, we provide a potential mechanism for this process. We show at the mRNA level that the Notch1 receptor and Delta-like 1 (Dll1) ligand vary dynamically across the PSM of both chick and mouse. Remarkably, we also demonstrate similar dynamics at the protein level; hence, the pathway components that mediate intercellular coupling themselves exhibit oscillatory dynamics. Moreover, we quantify the dynamic expression patterns of Dll1 and Notch1, and show they are highly correlated with the expression patterns of two known clock components [Lfng mRNA and the activated form of the Notch receptor (cleaved Notch intracellular domain, NICD)]. Lastly, we show that Notch1 is a target of Notch signalling, whereas Dll1 is Wnt regulated. Regulation of Dll1 and Notch1 expression thus links the activity of Wnt and Notch, the two main signalling pathways driving the clock

Ascl1 coordinately regulates gene expression and the chromatin landscape during neurogenesis

The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program

Long Non-coding RNAs Associated With Neurodegeneration-Linked Genes Are Reduced in Parkinson’s Disease Patients

Transcriptome analysis has identified a plethora of long non-coding RNAs (lncRNAs) expressed in the human brain and associated with neurological diseases. However, whether lncRNAs expression levels correlate with Parkinson’s disease (PD) pathogenesis remains unknown. Herein, we show that a number of lncRNA genes encompassing transcriptional units in close proximity to PD-linked protein-coding genes, including SNCA, LRRK2, PINK1, DJ-1, UCH-L1, MAPT and GBA1, are expressed in human dopaminergic cells and post-mortem material, such as cortex, Substantia Nigra and cerebellum. Interestingly, these lncRNAs are upregulated during neuronal differentiation of SH-SY5Y cells and of dopaminergic neurons generated from human fibroblast-derived induced pluripotent stem cells. Importantly, six lncRNAs are found under-expressed in the nigra and three in the cerebellum of PD patients compared to controls. Simultaneously, SNCA mRNA levels are increased in the nigra, while LRRK2 and PINK1 mRNA levels are decreased both in the nigra and the cerebellum of PD subjects compared to controls, indicating a possible correlation between the expression profile of the respective lncRNAs with their adjacent coding genes. Interestingly, all dysregulated lncRNAs are also detected in human peripheral blood mononuclear cells and four of them in exosomes derived from human cerebrospinal fluid, providing initial evidence for their potential use as diagnostic tools for PD. Our data raise the intriguing possibility that these lncRNAs may be involved in disease pathogenesis by regulating their neighboring PD-associated genes and may thus represent novel targets for the diagnosis and/or treatment of PD or related diseases

The Glial Regenerative Response to Central Nervous System Injury Is Enabled by Pros-Notch and Pros-NFκB Feedback

Organisms are structurally robust, as cells accommodate changes preserving structural integrity and function. The molecular mechanisms underlying structural robustness and plasticity are poorly understood, but can be investigated by probing how cells respond to injury. Injury to the CNS induces proliferation of enwrapping glia, leading to axonal re-enwrapment and partial functional recovery. This glial regenerative response is found across species, and may reflect a common underlying genetic mechanism. Here, we show that injury to the Drosophila larval CNS induces glial proliferation, and we uncover a gene network controlling this response. It consists of the mutual maintenance between the cell cycle inhibitor Prospero (Pros) and the cell cycle activators Notch and NFκB. Together they maintain glia in the brink of dividing, they enable glial proliferation following injury, and subsequently they exert negative feedback on cell division restoring cell cycle arrest. Pros also promotes glial differentiation, resolving vacuolization, enabling debris clearance and axonal enwrapment. Disruption of this gene network prevents repair and induces tumourigenesis. Using wound area measurements across genotypes and time-lapse recordings we show that when glial proliferation and glial differentiation are abolished, both the size of the glial wound and neuropile vacuolization increase. When glial proliferation and differentiation are enabled, glial wound size decreases and injury-induced apoptosis and vacuolization are prevented. The uncovered gene network promotes regeneration of the glial lesion and neuropile repair. In the unharmed animal, it is most likely a homeostatic mechanism for structural robustness. This gene network may be of relevance to mammalian glia to promote repair upon CNS injury or disease

Prox1 Regulates the Notch1-Mediated Inhibition of Neurogenesis

During development of the spinal cord, Prox1 controls the balance between proliferation and differentiation of neural progenitor cells via suppression of Notch1 gene expression

Retinoic Acid-Dependent Signaling Pathways and Lineage Events in the Developing Mouse Spinal Cord

Studies in avian models have demonstrated an involvement of retinoid signaling in early neural tube patterning. The roles of this signaling pathway at later stages of spinal cord development are only partly characterized. Here we use Raldh2-null mouse mutants rescued from early embryonic lethality to study the consequences of lack of endogenous retinoic acid (RA) in the differentiating spinal cord. Mid-gestation RA deficiency produces prominent structural and molecular deficiencies in dorsal regions of the spinal cord. While targets of Wnt signaling in the dorsal neuronal lineage are unaltered, reductions in Fibroblast Growth Factor (FGF) and Notch signaling are clearly observed. We further provide evidence that endogenous RA is capable of driving stem cell differentiation. Raldh2 deficiency results in a decreased number of spinal cord derived neurospheres, which exhibit a reduced differentiation potential. Raldh2-null neurospheres have a decreased number of cells expressing the neuronal marker β-III-tubulin, while the nestin-positive cell population is increased. Hence, in vivo retinoid deficiency impaired neural stem cell growth. We propose that RA has separable functions in the developing spinal cord to (i) maintain high levels of FGF and Notch signaling and (ii) drive stem cell differentiation, thus restricting both the numbers and the pluripotent character of neural stem cells

The role of the transcription factor Prox1 in the regulation of differentiation and maturation of neuronal cells during the development of central nervous system

During central nervous system (CNS) development, proper and timely induction of neurite and axon elongation is critical for generating functional, mature neurons and neuronal networks. Despite the wealth of information on the action of extracellular cues, little is known about the intrinsic gene regulatory factors that control this developmental decision. Here, we report the identification of Prox1, a homeobox transcription factor, as a key player in inhibiting neurite and axon elongation. Although Prox1 promotes acquisition of early neuronal identity and is expressed in nascent post-mitotic neurons, it is heavily down-regulated in the majority of terminally differentiated neurons, indicating a regulatory role in delaying axon outgrowth in newly formed neurons. Consistently, we show that Prox1 is sufficient to inhibit neurite extension in neuroblastoma cell lines. Furthermore, shRNA-mediated knock-down of Prox1 in Neuro2A cells induces the extension of neurites. More importantly, Prox1 overexpression suppresses axon elongation in primary neuronal cultures as well as in the developing mouse brain, while Prox1 knock-down promotes axon outgrowth. Mechanistically, RNA-Seq analysis reveals that Prox1 affects critical pathways for neuronal maturation and neurite extension. Interestingly, Prox1 strongly inhibits many components of Ca2+ signaling pathway, an important mediator of neurite and axon extension and neuronal maturation. In accordance, Prox1 represses Ca2+ entry upon KClmediated depolarization and reduce CREB phosphorylation. These observations suggest that Prox1 acts as a potent suppressor of neurite and axon elongation by inhibiting Ca2+ signaling pathway. This action may provide the appropriate time window for nascent neurons to find the correct position in the CNS prior to initiation of axon and/or neurite elongation.Κατά τη διάρκεια της ανάπτυξης του κεντρικού νευρικού συστήματος, η κατάλληλη και στο σωστό χρόνο επαγωγή της επιμήκυνσης των νευριτών και ειδικότερα του άξονα είναι πολύ σημαντική για την ανάπτυξη λειτουργικών, ώριμων νευρώνων και νευρωνικών δικτύων. Παρόλο την πληθώρα των πληροφοριών για τη δράση εξωκυττάριων παραγόντων, λίγα είναι γνωστά για τους ενδοκυτταρικούς μηχανισμούς μεταγραφικής ρύθμισης που επιδρούν σε αυτή την αναπτυξιακή διαδικασία. Στην παρούσα εργασία υποδεικνύεται ότι ο Prox1 ομοιοτικός(homeobox) μεταγραφικός παράγοντας δρα ως ένας βασικός παράγοντας για την καταστολή της επέκτασης του νευρικού άξονα και ίσως και των υπόλοιπων νευριτών. Αν και ο Prox1 είναι υπεύθυνος για την επαγωγή της ταυτότητας των νεοσχηματιζόμενων μετα-μιτωτικών νευρικών κυττάρων στα οποία και εκφράζεται, η έκφραση του καταστέλλεται έντονα στην πλειοψηφία των τελικά διαφοροποιημένων νευρικών κυττάρων. Το γεγονός αυτό υποδεικνύει έναν αρνητικό ρυθμιστικό ρόλο του Prox1 στην επιμήκυνση του άξονα και των νευριτών στα νεαρά νευρικά κύτταρα. Σε συμφωνία με αυτόν το ρόλο, η παρούσα εργασία υπέδειξε ότι ο Prox1 είναι ικανός να μειώσει την επέκταση των νευριτών σε κύτταρα νευροβλαστώματος. Αντίθετα, παρατηρήθηκε αύξηση της επέκτασης των νευριτών σε κύτταρα νευροβλαστώματος όπου είχε κατασταλεί η έκφραση του Prox1 με τη χρήση shRNA πλασμιδιακού φορέα. Επιπλέον, η υπερέκφραση του Prox1 κατέστειλε την επιμήκυνση του άξονα σε πρωτογενή νευρικά κύτταρα και στον αναπτυσσόμενο εγκέφαλο μυός, ενώ η μείωση της έκφρασης οδήγησε σε επαγωγή. Για τη διερεύνηση του μηχανισμού δράσης του Prox1 πραγματοποιήθηκε RNA-Seq ανάλυση με την οποία διαπιστώθηκε ότι ο Prox1 επιδρά σε πολλά μονοπάτια που σχετίζονται με την παραπάνω διεργασία και γενικότερα με τη νευρική ωρίμανση. Επίσης, σημαντικό ήταν ότι τα αποτελέσματα του RNA-Seq ανέδειξαν ότι ο Prox1 επηρεάζει πολλούς παράγοντες που λαμβάνουν μέρος στο μονοπάτι του ασβεστίου, το οποίο αποτελεί σημαντικό διαμεσολαβητή της επιμήκυνσης του άξονα και της νευρικής ωρίμανσης. Τα παραπάνω είναι σε συμφωνία με μία σειρά πειραμάτων όπου υποδείχθηκε ότι ο Prox1 οδηγεί σε μείωση της εισροής ιόντων ασβεστίου μετά από εκπόλωση της μεμβράνης των νευρικών κυττάρων μέσω της διέγερσης με KCl. Παράλληλα μειώνεται και η φωσφορυλίωση του CREB παράγοντα, βασικού καταρροϊκού στόχου του μονοπατιού του Ca2+. Συνολικά, όλες οι παραπάνω παρατηρήσεις υποδηλώνουν μια ισχυρή κατασταλτική δράση του Prox1 στην επέκταση του άξονα και ίσως και των υπόλοιπων νευριτών μέσω της καταστολής του μονοπατιού του ασβεστίου. Αυτή η δράση θα μπορούσε να παρέχει το κατάλληλο χρονικό διάστημα ώστε τα νεαρά νευρικά κύτταρα να βρουν την τελική τους θέση στο κεντρικό νευρικό σύστημα, πριν την έναρξη της επιμήκυνσης του άξονα και/ή των υπόλοιπων νευριτών

Compound heterozygosity of a frameshift mutation in the coding region and a single base substitution in the promoter of the ACTH receptor gene in a family with isolated glucocorticoid deficiency

Isolated glucocorticoid deficiency (IGD) is an autosomal recessive syndrome characterized by glucocorticoid insufficiency without mineralocorticoid deficiency. Mutations in the coding region of the ACTH receptor (MC2R) have been reported in several families with IGD. We amplified and sequenced the entire MC2R coding region in a new family with IGD. The proband was found to be heterozygous (paternal allele) for the mutation Gly217fs, which changes the open reading frame of the MC2R protein resulting in a truncated receptor. No other abnormality was found in the MC2R coding region. However, sequencing of the promoter region of the MC2R gene (-1017/44 bp) of the proband revealed a heterozygous T→C substitution in the maternal allele at -2 bp position from initiation of the transcription start site. This substitution was found in only 6.5% in a healthy unrelated population. Constructs containing this polymorphism consistently showed a significant 15% decrease in promoter activity compared to wild type. In conclusion, we provide evidence that the IGD in this previously unreported family with ACTH resistance appears to be secondary to compound heterozygosity of a coding region and a promoter mutation in the MC2R gene. © Freund Publishing House Ltd., London

Basement membrane peptides: Functional considerations and biomedical applications in autoimmunity

Basement membranes are specialized extracellular matrices that surround certain cell types (muscle cells, adipose cells, etc) and are present under the basal surface of cells exhibiting polarity (epithelial, endothelial and mesothelial cells). They have a unique macromolecular composition, consisting mainly of type IV collagen isoforms, laminin isoforms, entactin/nidogen, and perlecan. These components self associate and interact with each other to form networks. Other macromolecules may be found in specialized basement membranes. In this short review, the role of selected basement membrane proteins in autoimmune diseases will be highlighted. As an example, Goodpasture’s syndrome will be presented and the relatively long quest for identification of the antigenic epitope on specific domains of the alpha 3(IV)NC1 will be summarized. Chagas disease will be discussed as an example of laminin-mediated autoimmunity, with emphasis on the role of sugar-based antigenic epitope(s) will be presented. Immune-mediated tubulointerstitial nephritis will be introduced and the role of a synthetic peptide in detecting proximal tubule damage in acute renal failure will be discussed. Auto-immune diseases where other basement membrane macromolecules are involved will be mentioned. Finally, the importance of understanding the functions served by domains at close proximity to the antigenic epitope(s) will be highlighted