The Prion Protein Controls Polysialylation of Neural Cell Adhesion Molecule 1 during Cellular Morphogenesis

The cellular prion protein (PrPC) is one of the most studied mammalian proteins. Despite the ongoing efforts and the ubiquitous expression of PrPC in vertebrate cells, the main function of this protein has remained enigmatic to date. Several lines of evidence pointed towards the involvement of PrPC in the morphogenetic reprogramming underlying the epithelial-tomesenchymal transition (EMT). To address this, we applied CRISPR-Cas9 genome engineering technique to establish multiple PrPC knockout cell models (Chapter 2). The follow-up studies uncovered a critical role of PrPC in EMT and showed that PrPC controls the polysialic acid (polySia) modification of neural cell adhesion molecule 1 (NCAM1). Surprisingly, this effect of PrPC on NCAM1 polysialylation relies on a novel signaling loop that modulates expression of ST8SIA2, one of the two polysialyltransferase enzymes responsible for adding polySia on NCAM1 (Chapter 3). Intriguingly, a comparative analysis revealed that PrPC levels correlate directly or inversely with ST8SIA2 levels in different cell models. Deep global proteome analyses of multiple PrP-deficient models not only uncovered surprising cell model-specific proteome shifts but also showed robust changes in the levels of MARCKS and BASP protein families at the plasma membrane. Further investigation of their involvement in a biology that controls NCAM1 polysialylation revealed that reduced levels of MARCKSL1 also led to a significant reduction of polySia-modified NCAM1 (Chapter 4). Data from this study emphasize a iii thus far underappreciated coordinated biology of PrP and NCAM1. A close comparison of knockout phenotypes in mice is consistent with a model, whereby several of the known phenotypes in PrP-deficient mice might be explained by the contribution of PrPC to NCAM1 polysialylation. Attempts to validate this model and to assert its significance for determining if perturbed PrPC signaling contributes to toxicity in prion diseases are ongoing and have not been included in this thesis at this time.Ph.D

NCAM1 Polysialylation

Much confusion surrounds the physiological function of the cellular prion protein (PrP C ). It is, however, anticipated that knowledge of its function will shed light on its contribution to neurodegenerative diseases and suggest ways to interfere with the cellular toxicity central to them. Consequently, efforts to elucidate its function have been all but exhaustive. Building on earlier work that uncovered the evolutionary descent of the prion founder gene from an ancestral ZIP zinc transporter, we recently investigated a possible role of PrP C in a morphogenetic program referred to as epithelial-to-mesenchymal transition (EMT). By capitalizing on PrP C knockout cell clones in a mammalian cell model of EMT and using a comparative proteomics discovery strategy, neural cell adhesion molecule-1 emerged as a protein whose upregulation during EMT was perturbed in PrP C knockout cells. Follow-up work led us to observe that PrP C regulates the polysialylation of the neural cell adhesion molecule NCAM1 in cells undergoing morphogenetic reprogramming. In addition to governing cellular migration, polysialylation modulates several other cellular plasticity programs PrP C has been phenotypically linked to. These include neurogenesis in the subventricular zone, controlled mossy fiber sprouting and trimming in the hippocampal formation, hematopoietic stem cell renewal, myelin repair and maintenance, integrity of the circadian rhythm, and glutamatergic signaling. This review revisits this body of literature and attempts to present it in light of this novel contextual framework. When approached in this manner, a coherent model of PrP C acting as a regulator of polysialylation during specific cell and tissue morphogenesis events comes into focus

SPG11 Presenting with Tremor

<div id="ojsAbstract"><p><strong>Background:</strong>&nbsp;Hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurological diseases, which typically present with progressive lower extremity weakness and spasticity causing progressive walking difficulties. Complicating neurological or extraneurological features may be present.</p><p><strong>Case Report:</strong>&nbsp;We describe a 19-year-old male who was referred because of an action tremor of the hands; he later developed walking difficulties. Callosal atrophy was present on his cerebral magnetic resonance imaging scan, prompting genetic testing for SPG11, which revealed homozygous mutations.</p><p><strong>Discussion:</strong>&nbsp;The clinical features, differential diagnosis and management of SPG11, the most common form of autosomal recessive complicated HSP with a thin corpus callosum are discussed.</p></div><p>&nbsp;</p

The Prion Protein Controls Polysialylation of Neural Cell Adhesion Molecule 1 during Cellular Morphogenesis.

Despite its multi-faceted role in neurodegenerative diseases, the physiological function of the prion protein (PrP) has remained elusive. On the basis of its evolutionary relationship to ZIP metal ion transporters, we considered that PrP may contribute to the morphogenetic reprogramming of cells underlying epithelial-to-mesenchymal transitions (EMT). Consistent with this hypothesis, PrP transcription increased more than tenfold during EMT, and stable PrP-deficient cells failed to complete EMT in a mammalian cell model. A global comparative proteomics analysis identified the neural cell adhesion molecule 1 (NCAM1) as a candidate mediator of this impairment, which led to the observation that PrP-deficient cells fail to undergo NCAM1 polysialylation during EMT. Surprisingly, this defect was caused by a perturbed transcription of the polysialyltransferase ST8SIA2 gene. Proteomics data pointed toward β-catenin as a transcriptional regulator affected in PrP-deficient cells. Indeed, pharmacological blockade or siRNA-based knockdown of β-catenin mimicked PrP-deficiency in regards to NCAM1 polysialylation. Our data established the existence of a PrP-ST8SIA2-NCAM signaling loop, merged two mature fields of investigation and offer a simple model for explaining phenotypes linked to PrP

PrP-deficiency affects expression of a subset of proteins undergoing pronounced expression levels changes during EMT.

<p>List of proteins exhibiting >20% level differences in comparison of global proteomes of TGFB1-treated stable PrP kd versus wt NMuMG cells (dataset II). Coverage: percentages of primary structure of covered by peptide-to-spectrum matches; # Peptides: number of peptides matched to a given protein entry (note that instances of the same peptide being identified with different modifications counted separately in this tally); Count: number of TMT signature ion distributions, which passed stringent filtering criteria and were used for relative quantitation. Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133741#pone.0133741.s003" target="_blank">S1 Table</a> for complete list of proteins identified, including control samples, confidence scores and statistical measures.</p

Quantitative mass spectrometry identifies perturbed ‘response to metal ions’ and EMT markers, including NCAM1, affected in PrP-deficient cells.

<p>(a) Design of quantitative global proteome comparisons giving rise to datasets I to III. (b) Workflow of global proteome analyses conducted by comparative mass spectrometry. Note that this workflow was executed 3 times to generated datasets I to III, with the ‘x’ being replaced by the respective condition specified at the top of this panel. To facilitate comparison of datasets, the three experiments differed in the biological samples which were labeled with even-numbered TMT reagents. All three datasets shared the use of wt NMuMG cell extracts following 48 h TGFB1 exposure as reference samples labeled with odd-numbered TMT reagents. (c) Example graph depicting post-acquisition filtering of datasets and benchmarks of mass spectrometry analysis (shown for dataset I). (d) Profound overlap amongst top 200 proteins whose levels are most changed during EMT or following stable PrP kd. (e) Exposure of NMuMG cells to TGFB1 causes changes to proteins whose KEGG annotations identify them as players in pathways that contribute to ‘focal adhesion’ formation and ‘actin cytoskeleton regulation’. (f) Direct comparison of global proteomes of wt and stable PrP kd NMuMG cells following TGFB1 exposure identifies highly significant perturbations in biological processes with ‘response to inorganic substance’ and ‘response to metal ions’ GO annotations.</p