Panagiotis K. Politis

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.

Plasticity in human motor cortex is in part genetically determined

Neuronal plasticity refers to the ability of the brain to change in response to different experiences. Plasticity varies between people, but it is not known how much of this variability is due to differences in their genes. In humans, plasticity can be probed by a protocol termed paired associative stimulation and the changes in the motor system that are brought about by such stimulation are thought to be due to strengthening synapses which connect different neurons. We examined pairs of sisters which were either genetically identical (monozygotic) or different (dizygotic). We found that the variability within the monozygotic sister pairs was less than the variability within the dizygotic sister pairs. That plasticity in human motor cortex is in a substantial part genetically determined may be relevant for motor learning and neurorehabilitation, such as after stroke.

Altered expression of calreticulin during the development of fibrosis

Tissue damage following injury leads to inflammation and fibrosis. To understand the molecular mechanisms and the proteins involved in the fibrotic process, we used the well‐established unilateral ureteric obstruction rat model and we analyzed the alterations at early and late time intervals using a classical proteomic approach. Data analysis demonstrates a correlation between calreticulin up‐regulation and progression of fibrosis. Calreticulin is involved in Ca++ homeostasis but has not been previously implicated in animal models of fibrosis. Proteomic analysis consistently revealed up‐regulation of calreticulin in both early and late time intervals. These findings were further confirmed by biochemical and morphological approaches. Next, animal models of lung fibrosis (bleomycin‐induced) and heart fibrosis (desmin‐null) were examined. In the lung model, calreticulin expression was up‐regulated from early time intervals, whereas in the heart model no change in the expression of calreticulin was observed. In addition, TGF‐β, a well known major contributing factor in several fibrotic processes, was found to up‐regulate calreticulin in cultured human proximal tubule epithelial cells. The above observations suggest that calreticulin might be involved in fibrotic processes; however the mechanism(s) underlying its possible involvement are yet unresolved.

BM88 is a dual function molecule inducing cell cycle exit and neuronal differentiation of neuroblastoma cells via cyclin D1 down-regulation and retinoblastoma protein hypophosphorylation.

Control of cell cycle progression/exit and differentiation of neuronal precursors is of paramount importance during brain development. BM88 is a neuronal protein associated with terminal neuron-generating divisions in vivo and is implicated in mechanisms underlying neuronal differentiation. Here we have used mouse neuroblastoma Neuro 2a cells as an in vitro model of neuronal differentiation to dissect the functional properties of BM88 by implementing gain- and loss-of-function approaches. We demonstrate that stably transfected cells overexpressing BM88 acquire a neuronal phenotype in the absence of external stimuli, as judged by enhanced expression of neuronal markers and neurite outgrowth-inducing signaling molecules. In addition, cell cycle measurements involving cell growth assays, BrdUrd incorporation, and fluorescence-activated cell sorting analysis revealed that the BM88-transfected cells have a prolonged G1 phase, most probably corresponding to cell cycle exit at the G0 restriction point, as compared with controls. BM88 overexpression also results in increased levels of the cell cycle regulatory protein p53, and accumulation of the hypophosphorylated form of the retinoblastoma protein leading to cell cycle arrest, with concomitant decreased levels and, in many cells, cytoplasmic localization of cyclin D1. Conversely, BM88 gene silencing using RNA interference experiments resulted in acceleration of cell proliferation accompanied by impairment of retinoic acid-induced neuronal differentiation of Neuro 2a cells. Taken together, our results suggest that BM88 plays an essential role in regulating cell cycle exit and differentiation of Neuro 2a cells toward a neuronal phenotype and further support its involvement in the proliferation/differentiation transition of neural stem/progenitor cells during embryonic development.

Sox1 Maintains the Undifferentiated State of Cortical Neural Progenitor Cells via the Suppression of Prox1-Mediated Cell Cycle Exit and Neurogenesis†‡§

Neural stem/progenitor cells maintain their identity via continuous self‐renewal and suppression of differentiation. Gain‐of‐function experiments in the chick revealed an involvement for Sox1‐3 transcription factors in the maintenance of the undifferentiated neural progenitor (NP) identity. However, the mechanism(s) employed by each factor has not been resolved. Here, we derived cortical neural/stem progenitor cells from wild‐type and Sox1‐null mouse embryos and found that Sox1 plays a key role in the suppression of neurogenic cell divisions. Loss of Sox1 leads to progressive depletion of self‐renewing cells, elongation of the cell cycle of proliferating cells, and significant increase in the number of cells exiting the cell cycle. In proliferating NP cells, Sox1 acts via a prospero‐related homeobox 1 (Prox1)‐mediated pathway to block cell cycle exit that leads to neuronal differentiation in vivo and in vitro. Thus, our results demonstrate that Sox1 regulates the size of the cortical NP pool via suppression of Prox1‐mediated neurogenic cell divisions. STEM CELLS 2011;29:89–98

BM88/CEND1 coordinates cell cycle exit and differentiation of neuronal precursors

During development, coordinate regulation of cell cycle exit and differentiation of neuronal precursors is essential for generation of appropriate number of neurons and proper wiring of neuronal circuits. BM88 is a neuronal protein associated in vivo with terminal neuron-generating divisions, marking the exit of proliferative cells from the cell cycle. Here, we provide functional evidence that BM88 is sufficient to initiate the differentiation of spinal cord neural precursors toward acquisition of generic neuronal and subtype-specific traits. Gain-of-function approaches show that BM88 negatively regulates proliferation of neuronal precursors, driving them to prematurely exit the cell cycle, down-regulate Notch1, and commit to a neuronal differentiation pathway. The combined effect on proliferation and differentiation results in precocious induction of neurogenesis and generation of postmitotic neurons within the ventricular zone. The dual action of BM88 is not recapitulated by the cell cycle inhibitor p27Kip1, suggesting that cell cycle exit does not induce differentiation by default. Mechanistically, induction of endogenous BM88 by forced expression of the proneural gene Mash1 indicates that BM88 is part of the differentiation program activated by proneural genes. Furthermore, BM88 gene silencing conferred by small interfering RNA in spinal cord neural progenitor cells enhances cell cycle progression and impairs neuronal differentiation. Our results implicate BM88 in the synchronization of cell cycle exit and differentiation of neuronal precursors in the developing nervous system.

Coordination of cell cycle exit and differentiation of neuronal progenitors

During development, co-ordinate regulation of cell cycle exit and differentiation of neuronal precursors is essential for generation of appropriate number of neurons and proper wiring of neuronal circuits. Recent studies have identified some of the molecules implicated in the regulation of these cellular events, but the complex machinery that orchestrates these processes into a coherent developmental program remains unclear. BM88/Cend1 is a neuronal protein associated in vivo with terminal neuron-generating divisions, marking the exit of proliferative cells from the cell cycle. Genetic studies in neural cell lines, neural stem/progenitor cells using the neurosphere system and in the developing chicken neural tube in vivo have shown that BM88/Cend1 is a dual function molecule co-ordinating cell cycle exit and differentiation of neuronal progenitors. These studies have thus shed light on a molecular determinant that participates, along with other known and possibly still unknown regulators, in the complex processes by which a progenitor cell becomes a mature neuron.

Whole-transcriptome analysis of UUO mouse model of renal fibrosis reveals new molecular players in kidney diseases

Transcriptome analysis by RNA-seq technology allows novel insights into gene expression and regulatory networks in health and disease. To better understand the molecular basis of renal fibrosis, we performed RNA-seq analysis in the Unilateral Ureteric Obstruction (UUO) mouse model. We analysed sham operated, 2- and 8-day post-ligation renal tissues. Thousands of genes with statistical significant changes in their expression were identified and classified into cellular processes and molecular pathways. Many novel protein-coding genes were identified, including critical transcription factors with important regulatory roles in other tissues and diseases. Emphasis was placed on long non-coding RNAs (lncRNAs), a class of molecular regulators of multiple and diverse cellular functions. Selected lncRNA genes were further studied and their transcriptional activity was confirmed. For three of them, their transcripts were also examined in other mouse models of nephropathies and their up- or down-regulation was found similar to the UUO model. In vitro experiments confirmed that one selected lncRNA is independent of TGFβ or IL1b stimulation but can influence the expression of fibrosis-related proteins and the cellular phenotype. These data provide new information about the involvement of protein-coding and lncRNA genes in nephropathies, which can become novel diagnostic and therapeutic targets in the near future.

GAD65 epitope mapping and search for novel autoantibodies in GAD-associated neurological disorders.

Antibodies against Glutamic-acid-decarboxylase (GAD65) are seen in various CNS excitability disorders including stiff-person syndrome, cerebellar ataxia, encephalitis and epilepsy. To explore pathogenicity, we examined whether distinct epitope specificities or other co-existing antibodies may account for each disorder. The epitope recognized by all 27 tested patients, irrespective of clinical phenotype, corresponded to the catalytic core of GAD. No autoantibodies against known GABAergic antigens were found. In a screen for novel specificities using live hippocampal neurons, three epilepsy patients, but no other, were positive. We conclude that no GAD-specific epitope defines any neurological syndrome but other antibody specificities may account for certain phenotypes.

The role of nuclear receptors in controlling the fine balance between proliferation and differentiation of neural stem cells

In the central nervous system (CNS) of vertebrates a large variety of cell types are specified from a pool of highly plastic neural stem/progenitor cells (NSCs) via a combined action of extrinsic morphogenetic cues and intrinsic transcriptional regulatory networks. Nuclear receptors and their ligands are key regulators of fate decisions in NSCs during development and adulthood, through their ability to control transcription of downstream genes. In the last few years considerable progress has been made towards the understanding of the actions of nuclear receptors in NSCs as well as elucidating the mechanistic basis for these actions. Here we summarize recent progress in the role of nuclear receptors in the biology of NSCs. These studies highlight the importance of this family of transcriptional regulators in CNS development and function in health and disease. Furthermore, they raise the intriguing possibility of using nuclear receptors as therapeutic targets for nervous system related diseases and traumas.