an-enzyme-serum-free-neural-stem-cell-culture-model-for-emt

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Prion diseases are characterized by the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(C), which is encoded by the Prnp gene. PrP(C) is highly expressed on neurons and its function is unknown. Recombinant PrP(C) was claimed to possess superoxide dismutase (SOD) activity, and it was hypothesized that abrogation of this function may contribute to neurodegeneration in prion diseases. We tested this hypothesis in vivo by studying copper/zinc and manganese SOD activity in genetically defined crosses of mice lacking the Sod1 gene with mice lacking PrP(C), and with hemizygous or homozygous tga20 transgenic mice overexpressing various levels of PrP(C). We failed to detect any influence of the Prnp genotype and gene dosage on SOD1 or SOD2 activity in heart, spleen, brain, and synaptosome-enriched brain fractions. Control experiments included crosses of mice lacking or overexpressing PrPc with mice overexpressing human Cu2+/Zn2+-superoxide dismutase, and confirmed that SOD enzymatic activity correlated exclusively with the gene dosage of bona fide human or murine SOD. We conclude that PrP(C) in vivo does not discernibly contribute to total SOD activity and does not possess an intrinsic dismutase activity.

No superoxide dismutase activity of cellular prion protein in vivo.

Abdominal seeding of an atypical teratoid/rhabdoid tumor of the pineal gland along a ventriculoperitoneal shunt catheter.

Prion infections induce severe disruption of the central nervous system with neuronal vacuolation and extensive glial reactions, and invariably lead to death of affected individuals. The molecular underpinnings of these events are not well understood. To better define the molecular consequences of prion infections, we analyzed the transcriptional response to persistent prion infection in a panel of three murine neural cell lines in vitro. Colony spot immunochemistry assays indicated that 65-100% of cells were infected in each line. Only the Nav1 gene was marginally modulated in one cell line, whereas transcripts previously reported to be derailed in prion-infected cells were not confirmed in the present study. We attribute these discrepancies to the experimental stringency of the current study, which was performed under conditions designed to minimize potential genetic drifts. These findings are at striking variance with gene expression studies performed on whole brains upon prion infections in vivo, suggesting that many of the latter changes represent secondary reactions to infection. We conclude that, surprisingly, there are no universal transcriptional changes induced by prion infection of neural cells in vitro.

Transcriptional stability of cultured cells upon prion infection.

Prion diseases are fatal, transmissible, neurodegenerative diseases caused by the misfolding of the prion protein (PrP). At present, the molecular pathways underlying prion-mediated neurotoxicity are largely unknown. We hypothesized that the transcriptional regulator of the stress response, heat shock factor 1 (HSF1), would play an important role in prion disease. Uninoculated HSF1 knockout (KO) mice used in our study do not show signs of neurodegeneration as assessed by survival, motor performance, or histopathology. When inoculated with Rocky Mountain Laboratory (RML) prions HSF1 KO mice had a dramatically shortened lifespan, succumbing to disease approximately 20% faster than controls. Surprisingly, both the onset of home-cage behavioral symptoms and pathological alterations occurred at a similar time in HSF1 KO and control mice. The accumulation of proteinase K (PK)-resistant PrP also occurred with similar kinetics and prion infectivity accrued at an equal or slower rate. Thus, HSF1 provides an important protective function that is specifically manifest after the onset of behavioral symptoms of prion disease.

Heat shock factor 1 regulates lifespan as distinct from disease onset in prion disease.

Prion-Krankheiten sind t&ouml;dlich, &uuml;bertragbare, Neurodegenerative Erkrankungen durch die misfolding das Prion-Protein (PrP). Derzeit sind die molekularen Bahnen zugrunde liegenden Prion-vermittelte Neurotoxizit&auml;t weitgehend unbekannt. Wir die Hypothese, dass die transkriptionelle Regulierungsbeh&ouml;rde f&uuml;r die Stress-Reaktion, Hitze-Schock-Faktor 1 (HSF1), Prion-Krankheit eine wichtige Rolle spielen w&uuml;rde. Uninoculated HSF1 Knockout (KO)-M&auml;use verwendet, die in unserer Studie zeigen nicht Zeichen der Neurodegeneration, die &uuml;berleben, Motorleistungen oder eine histopathologische Untersuchung zu bewerten. Wenn geimpft mit Rocky Mountain Laboratory (RML) Prionen HSF1 KO M&auml;usen hatte eine stark verk&uuml;rzte Lebensdauer, Krankheit erliegen ca. 20 % schneller als Steuerelemente. &Uuml;berraschenderweise ist sowohl der Beginn der Startseite-K&auml;fig behavioral Symptome und pathologische Ver&auml;nderungen zur gleichen Zeit in HSF1 KO und Kontrolle M&auml;use aufgetreten. Die Anh&auml;ufung von Proteinase K (PK)-best&auml;ndig PrP erfolgte auch mit &auml;hnlichen Kinetik und Prion-Infektiosit&auml;t abgegrenzt gleich oder langsamer Geschwindigkeit. HSF1 stellt daher eine wichtige Schutzfunktion, die nach Beginn der behavioral Symptome einer Prionenkrankheit speziell Manifest ist.

Hitze-Schock-Faktor 1 regelt Lebensdauer im Unterschied zu Beginn der Krankheit in Prion-Krankheit.

A central focus of aging research is to determine how calorie restriction (CR) extends lifespan and delays diseases of aging. SIRT1, the mammalian ortholog of Sir2 in yeast, is a longevity factor which mediates dietary restriction in diverse species. In addition, SIRT1 plays a protective role in several models of neurodegenerative disease. We tested the role of SIRT1 in mediating the effects of CR in a mouse model of prion disease. Prion diseases are protein misfolding disorders of the central nervous system with many similarities to other neurodegenerative diseases, including deposition of aggregated protein, gliosis, and loss of synapses and neurons. We report that the onset of prion disease is delayed by CR and in the SIRT1 KO mice fed ad libitum. CR exerts no further effect on the SIRT1 KO strain, suggesting the effects of CR and SIRT1 deletion are mechanistically coupled. In conjunction, SIRT1 is downregulated in certain brain regions of CR mice. The expression of PrP mRNA and protein is reduced in the brains of CR mice and in SIRT1 knockout mice, suggesting a possible mechanism for the delayed onset of disease, as PrP levels are a critical determinant of how quickly mice succumb to prion disease. Surprisingly, CR greatly shortens the duration of clinical symptoms of prion disease and ultimately shortens lifespan of prion-inoculated mice in a manner that is independent of SIRT1. Taken together, our results suggest a more complex interplay between CR, SIRT1, and neurodegenerative diseases than previously appreciated.

The role of calorie restriction and SIRT1 in prion-mediated neurodegeneration.

Ein zentraler Schwerpunkt der alternden Forschung ist zu bestimmen, wie die Kalorienrestriktion (CR) Lebensdauer und Verz&ouml;gerungen Erkrankungen des Alterns erweitert. Sirt1, der S&auml;ugetier-Ortholog des Sir2 in der Hefe, ist eine Langlebigkeit-Faktor, der di&auml;tetische Einschr&auml;nkung in vielf&auml;ltigen Arten vermittelt. SIRT1 spielt dar&uuml;ber hinaus eine sch&uuml;tzende Rolle in mehreren Modellen der Neurodegenerative Krankheit. Wir testeten die Rolle des SIRT1 bei der Vermittlung von der Effekten CR in einem Mausmodell der Prion-Krankheit. Prionenkrankheiten sind Protein misfolding Erkrankungen des zentralen Nervensystems mit vielen &Auml;hnlichkeiten zu anderen neurodegenerativen Krankheiten, einschlie&szlig;lich Absetzung von aggregierten Protein, Gliose und Verlust von Synapsen und Neuronen. Wir berichten, dass der Beginn der Prion-Krankheit durch ZE verz&ouml;gert ist und in das SIRT1-KO M&auml;usen Ad Libitum gef&uuml;ttert. CR &uuml;bt keine weiteren Auswirkungen auf das SIRT1 KO-Stamm, was darauf hindeutet, dass die Auswirkungen der CR und SIRT1 L&ouml;schung mechanistisch gekoppelt sind. In Verbindung ist SIRT1 Downregulated in bestimmten Hirnregionen CR-M&auml;use. Der Ausdruck der PrP-mRNA und Protein wird reduziert, in den Gehirnen der M&auml;use CR und SIRT1-Knockout-M&auml;use, einen m&ouml;glichen Mechanismus f&uuml;r den verz&ouml;gerten Beginn der Krankheit, was darauf hindeutet, wie PrP-Ebenen eine wichtige Determinante sind der wie schnell M&auml;use Prion-Krankheit erliegen. &Uuml;berraschenderweise CR stark verk&uuml;rzt die Dauer der klinischen Symptome der Prion-Krankheit und letztlich verk&uuml;rzt die Lebensdauer von M&auml;usen in einer Weise, die unabh&auml;ngig von SIRT1 Prion-geimpft. Zusammengenommen deuten unsere Ergebnisse eine komplexe Wechselspiel zwischen CR SIRT1 und Neurodegenerative Krankheiten als bisher angenommen.

Die Rolle der Kalorienrestriktion und SIRT1 im Prion-vermittelte Neurodegeneration.

Progressive accumulation of PrP(Sc), a hallmark of prion diseases, occurs when conversion of PrP(C) into PrP(Sc) is faster than PrP(Sc) clearance. Engulfment of apoptotic bodies by phagocytes is mediated by Mfge8 (milk fat globule epidermal growth factor 8). In this study, we show that brain Mfge8 is primarily produced by astrocytes. Mfge8 ablation induced accelerated prion disease and reduced clearance of cerebellar apoptotic bodies in vivo, as well as excessive PrP(Sc) accumulation and increased prion titers in prion-infected C57BL/6 × 129Sv mice and organotypic cerebellar slices derived therefrom. These phenotypes correlated with the presence of 129Sv genomic markers in hybrid mice and were not observed in inbred C57BL/6 Mfge8(-/-) mice, suggesting the existence of additional strain-specific genetic modifiers. Because Mfge8 receptors are expressed by microglia and depletion of microglia increases PrP(Sc) accumulation in organotypic cerebellar slices, we conclude that engulfment of apoptotic bodies by microglia may be an important pathway of prion clearance controlled by astrocyte-borne Mfge8.

Engulfment of cerebral apoptotic bodies controls the course of prion disease in a mouse strain-dependent manner.

Low-grade gliomas (LGGs) are rare brain neoplasms, with survival spanning up to a few decades. Thus, accurate evaluations on how biomarkers impact survival among patients with LGG require long-term studies on samples prospectively collected over a long period.

IDH/MGMT-driven molecular classification of low-grade glioma is a strong predictor for long-term survival.

Neural stem cells (NSCs) typically show efficient self-renewal and selective differentiation. Their invasion potential, however, is not well studied. In this study, Sox2-positive NSCs from the E14.5 rat cortex were found to be non-invasive and showed only limited migration in vitro. By contrast, FGF2-expanded NSCs showed a strong migratory and invasive phenotype in response to the combination of FGF2 and BMP4. Invasive NSCs expressed Podoplanin (PDPN) and p75NGFR (Ngfr) at the plasma membrane after exposure to FGF2 and BMP4. FGF2 and BMP4 together upregulated the expression of Msx1, Snail1, Snail2, Ngfr, which are all found in neural crest (NC) cells during or after epithelial-mesenchymal transition (EMT), but not in forebrain stem cells. Invasive cells downregulated the expression of Olig2, Sox10, Egfr, Pdgfra, Gsh1/Gsx1 and Gsh2/Gsx2. Migrating and invasive NSCs had elevated expression of mRNA encoding Pax6, Tenascin C (TNC), PDPN, Hey1, SPARC, p75NGFR and Gli3. On the basis of the strongest upregulation in invasion-induced NSCs, we defined a group of five key invasion-related genes: Ngfr, Sparc, Snail1, Pdpn and Tnc. These genes were co-expressed and upregulated in seven samples of glioblastoma multiforme (GBM) compared with normal human brain controls. Induction of invasion and migration led to low expression of differentiation markers and repressed proliferation in NSCs. Our results indicate that normal forebrain stem cells have the inherent ability to adopt a glioma-like invasiveness. The results provide a novel in vitro system to study stem cell invasion and a novel glioma invasion model: tumoral abuse of the developmental dorsoventral identity regulation.

Non-invasive neural stem cells become invasive in vitro by combined FGF2 and BMP4 signaling.

Prnp(-/-) mice lack the prion protein PrP(C) and are resistant to prion infections, but variable phenotypes have been reported in Prnp(-/-) mice and the physiological function of PrP(C) remains poorly understood. Here we examined a cell-autonomous phenotype, inhibition of macrophage phagocytosis of apoptotic cells, previously reported in Prnp(-/-) mice. Using formal genetic, genomic, and immunological analyses, we found that the regulation of phagocytosis previously ascribed to PrP(C) is instead controlled by a linked locus encoding the signal regulatory protein α (Sirpa). These findings indicate that control of phagocytosis was previously misattributed to the prion protein and illustrate the requirement for stringent approaches to eliminate confounding effects of flanking genes in studies modeling human disease in gene-targeted mice. The plethora of seemingly unrelated functions attributed to PrP(C) suggests that additional phenotypes reported in Prnp(-/-) mice may actually relate to Sirpa or other genetic confounders.

SIRPα polymorphisms, but not the prion protein, control phagocytosis of apoptotic cells.

In clinical trials, overexpression of neurokinin-1 receptors (NK1R) in gliomas has been exploited by intratumoral injection of its radiolabeled ligand, substance P (SP). However, despite proven NK1R expression, patients' response to the therapy was inhomogeneous. This study aims to identify the factors predicting response to NK1R-targeted glioma therapy, thereby allowing the discrimination between potential "responders" and "nonresponders" and thus a personalized therapeutic approach. Four widely used glioblastoma cell lines were examined concerning their RNA levels of full-length and truncated NK1R subtypes. Binding of SP to NK1R and internalization into glioma cells was studied by three different approaches using radiolabeled SP ((177)Lu-[DOTA, Thi(8), Met(O2)(11)]-SP), a fluorescence-labeled SP derivative (SP-FAM), and a toxin-SP conjugate (saporin-SP). While NK1R RNA was detected in all cases, receptor subtype analysis revealed impressive differences between the cell lines; LN319 exhibited the highest level of full-length NK1R RNA. Significant binding of SP conjugates to NK1R, cell internalization, and specific cell killing were only observed with the cell line LN319. Thus, different NK1R subtype profiles of glomerular basement membrane (GBM) cell lines appear to influence the binding of SP conjugates and their cell internalization properties. Both processes are crucial steps for NK1R-based targeted therapy. Pretherapeutic testing for NK1R subtype expression may therefore be advisable before initiation of this generally promising therapeutic modality.

Expression of different neurokinin-1 receptor (NK1R) isoforms in glioblastoma multiforme: potential implications for targeted therapy.

Cerebrospinal fluid leakage is an immanent risk of cranial surgery with dural opening. Recognizing the risk factors for this complication and improving the technique of dural closure may reduce the associated morbidity and its surgical burden. The aim of this paper was to investigate whether the addition of TachoSil on top of the dural suture reduces postoperative CSF leakage compared with dural suturing alone and to assess the frequency and risk factors for dural leakage and potentially related complications after elective craniotomy.

Risk factors for postoperative CSF leakage after elective craniotomy and the efficacy of fleece-bound tissue sealing against dural suturing alone: a randomized controlled trial.

The presence, characteristics, and potential clinical relevance of neural progenitor populations within the neural placodes of myelomeningocele patients remain to be studied. Neural stem cells are known to reside adjacent to ependyma-lined surfaces along the central nervous system axis.

Neural Placode Tissue Derived From Myelomeningocele Repair Serves as a Viable Source of Oligodendrocyte Progenitor Cells.

This is the first report of a primarily intracranial interdigitating dendritic cell sarcoma (IDCS).

Intracranial Interdigitating Dendritic Cell Sarcoma: First Case Report.

Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo.

Article	Year
No superoxide dismutase activity of cellular prion protein in vivo. Biological chemistry\| PubMed ID: 14515989	2003
Abdominal seeding of an atypical teratoid/rhabdoid tumor of the pineal gland along a ventriculoperitoneal shunt catheter. Acta neuropathologica\| PubMed ID: 16328512	2006
Transcriptional stability of cultured cells upon prion infection. Journal of molecular biology\| PubMed ID: 18082765	2008
Heat shock factor 1 regulates lifespan as distinct from disease onset in prion disease. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 18757733	2008
The role of calorie restriction and SIRT1 in prion-mediated neurodegeneration. Experimental gerontology\| PubMed ID: 18799131	2008
Engulfment of cerebral apoptotic bodies controls the course of prion disease in a mouse strain-dependent manner. The Journal of experimental medicine\| PubMed ID: 20837697	2010
IDH/MGMT-driven molecular classification of low-grade glioma is a strong predictor for long-term survival. Neuro-oncology\| PubMed ID: 23408861	2013
Non-invasive neural stem cells become invasive in vitro by combined FGF2 and BMP4 signaling. Journal of cell science\| PubMed ID: 23788430	2013
SIRPα polymorphisms, but not the prion protein, control phagocytosis of apoptotic cells. The Journal of experimental medicine\| PubMed ID: 24145514	2013
Expression of different neurokinin-1 receptor (NK1R) isoforms in glioblastoma multiforme: potential implications for targeted therapy. Cancer biotherapy & radiopharmaceuticals\| PubMed ID: 24552486	2014
Risk factors for postoperative CSF leakage after elective craniotomy and the efficacy of fleece-bound tissue sealing against dural suturing alone: a randomized controlled trial. Journal of neurosurgery\| PubMed ID: 25036199	2014
Neural Placode Tissue Derived From Myelomeningocele Repair Serves as a Viable Source of Oligodendrocyte Progenitor Cells. Neurosurgery\| PubMed ID: 26225855	2015
Intracranial Interdigitating Dendritic Cell Sarcoma: First Case Report. Neurosurgery\| PubMed ID: 26237342	2015
Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo. PloS one\| PubMed ID: 27092773	2016

Gregor Hutter

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