Die Rolle des Neuropeptids VGF in der Glioblastoma multiforme Mikroumgebung und seine Auswirkungen auf die zellulären Funktionen der Mikroglia

VGF is a neuropeptide precursor that regulates several aspects of brain function and many other endocrine processes involved in metabolism. Accordingly, the dysregulation of the different VGF-derived peptides has been associated with many metabolic and neurological disorders. In this regards VGF has been also proposed as a biomarker for secretory cancers, but the literature in this topic is still scarce. Glioblastomas are very malignant brain tumors that have different molecular subtypes, which present different clinical behavior. Factors involved in the communication of the tumor cells with their associated microenvironment can be target for tumor therapy as they are essential for dictating the course of the pathology. Microglia cells were already shown to express VGF-derived peptide TLQP-21 receptors, but little is known about how they respond to it and what are the effects of the activation of these receptors on their biological innate functions. This work showed for the first time VGF and its C-terminal peptides involvement in the glioma pathology, affecting the tumor cellular proliferation which could be associated with decreased survival of human patients, as it was demonstrated by TCGA dataset analysis that high VGF expression in GBM predicts poor survival. This data was confirmed in a mouse model of GBM lacking VGF, which resulted in a drastic increase in median survival compared to wild-types. In the wild-type mouse model high expression VGF was confirmed in both mRNA and protein levels. Furthermore, regions of the tumor with higher levels of VGF presented also higher infiltration or activation of microglia/macrophages, indicating the VGF could participate in the crosstalk between TAM and tumor cells. It was demonstrated that in culture tumor cells lose VGF expression, which is probably due to the loss of microglia cells, which do not survive in serum-free cultures. Treating the GBM primary cultures with neonatal microglia-conditioned medium was sufficient to partially recover VGF expression, indicating that these cells indeed regulate VGF expression by the tumor cells. It was therefore proposed a positive feedback loop as VGF being secreted from the tumor and attracting microglia cells (MG), which in turn trigger an increase in VGF expression by the GBM cells, possibly by secretion of VGF-producer factors such as IL-6, EGF or BNDF (according to the literature) and thus potentiate their invasiveness and proliferation. In vitro assays with VGF-derived peptide TLQP-21 confirmed that it is a potent chemoattractant for MG cells and increases MG phagocytosis. TLQP-21 nevertheless, did not efficiently alter the immunomodulatory capacities of microglia cells measured by the secretion of IL1-beta, IL6, TNF-alpha and CCL2 cytokines and chemokines. We sought to further characterize TLQP-21 response in microglia cells and therefore we performed whole-cell patch clamp during TLQP-21 treatment. We observed the presence of outward rectifying potassium currents, which reduced following ATP response, indicating a crosstalk with the ATP-associated signaling. The response was blocked by C3AR1 antagonist showing that this receptor mediates TLQP- 21 currents. Stimulation of microglia with TLQP-21 also increased intracellular calcium concentration and this was also blocked by the C3AR1 antagonist. Therefore in microglia cells the biological effects of TLQP-21 are mediated by C3AR1. Is possible that both the calcium signaling and potassium currents are related and involved in microglia innate functions. Further investigation on its multiple biological functions and many derived peptides and receptor will help clarifying the questions were here left opened. Overall the results make VGF a promising target for glioma therapy, more specifically for proneural GBM patients. We hope that new studies will further analyze the impact of VGF-based therapy on other animal models and in the future on human GBM patients.VGF ist ein Neuropeptid-Vorläufer, der am Stoffwechsel beteiligt ist und verschiedene Aspekte der Gehirnfunktion und viele andere endokrine Prozesse reguliert. Dementsprechend kann die Fehlregulation der verschiedenen VGF- abgeleiteten Peptide mit vielen metabolischen und neurologischen Störungen in Verbindung gebracht werden. In diesem Zusammenhang wurde VGF für sekretorischen Krebs auch als Biomarker vorgeschlagen, aber die Literatur zu diesem Thema ist nach wie vor spärlich. Glioblastome sind sehr bösartige Hirntumoren mit verschiedenen molekularen Subtypen und verschiedene klinischen Ausprägungen. Faktoren die an der Kommunikation zwischen Tumorzellen und den Zellen ihrer jeweiligen Mikroumgebung beteiligt sind, können für die Tumortherapie wichtige und neue Ziele darstellen. Es wurde bereits gezeigt, dass Mikroglia-Zellen Rezeptoren für das VGF- abgeleitete Peptid TLQP-21 exprimieren, aber es ist wenig darüber bekannt, wie sie darauf reagieren und welche die Wirkungen die Aktivierung dieser Rezeptoren auf die biologischen Funktionen dieser Zellen hat. Diese Arbeit zeigt zum ersten Mal die Beteiligung von VGF und seine C- terminalen Peptide in der Gliome Pathologie. Wir zeigen, dass VGF die Tumorzellproliferation beeinflusst, die mit einer verringerten Überleben von Gliome Patienten in Verbindung gebracht werden könnten. Mit Hilfe der TCGA Datensatzes Analyse haben wir gezeigt, dass Patienten mit hoher VGF-Expression schneller sterben als Patienten mit geringer VGF-Expression. Diese Daten wurden in einem Mausmodell eines VGF knock out Tumors bestätigt , da die Mäuse mit dem VGF knock out Tumor länger überlebten als die Tiere mit einem wildtyp Tumor. Die Mäuse mit wildtyp Tumor zeigten eine hohe Expression von VGF auf mRNA und Protein-Ebene. Regionen des Tumors mit einem höheren VGF zeigten auch eine höhere Anzahl von Mikroglia / Makrophagen. Es wurde gezeigt, dass frisch isolierte Tumorzellen mit fortschreitender Zeit in Kultur VGF-Expression verlieren, was wahrscheinlich auf den Verlust von Mikroglia-Zellen zurückzuführen ist, die in serumfreien Kulturen nicht überleben. Behandelten wir die Tumor Primärkulturen mit neontal Mikroglia-konditionierte Medium wurde die VGF- Expression teilweise wieder hergestellt. Wir schlagen daher vor, dass VGF über eine positive Rückkopplungsschleife aus dem Tumor sezerniert wird und die Mikroglia-Zellen (MG) vom Tumor über VGF angezogen werden, was wiederum eine Erhöhung der VGF-Expression durch den GBM Zellen auslöst, die ihrerseits durch Sekretion von VGF-produzierenden Faktoren wie IL-6 , EGF oder BNDF (entsprechend der Literatur) die Invasivität und Proliferation des Tumors potenzieren. In-vitro-Assays mit dem VGF-abgeleitetes Peptid TLQP-21 bestätigen, dass es ein potentes Chemoattraktant für MG-Zellen und erhöht MG Phagozytose ist. Andererseits verändert TLQP-21 die Sekretion von IL1-beta, IL-6, TNF-alpha und CCL2 durch Mikroglia nicht . Whole-cell Patch- Clamp Studien mit TLQP-21-führten zu nach außen gerichteten Kaliumströme, die die folgenden ATP Reaktion reduzierten und somit eine Beteiligung an ATP- assoziierten Signalwegen nahe legen. Die Reaktion wurde durch C3AR1 Antagonist blockiert, was zeigt, dass dieser Rezeptor TLQP-21 Ströme vermittelt. Stimulation der Mikroglia mit TLQP-21 erhöhte auch die intrazelluläre Calciumkonzentration , welche auch durch den C3AR1 Antagonisten blockiert werden konnte. Daher denken wir, dass in Mikroglia-Zellen die biologischen Wirkungen von TLQP-21 durch C3AR1 vermittelt werden. Es ist möglich, dass sowohl die Calcium- Signalgebung und Kaliumströme verbunden und an Funktionen der Mikroglia beteiligt sind. Viele biologische Funktionen von VGF gilt es noch aufzudecken, ebenso wie verschiedenen Zelltypen auf VGF-abgeleitete Peptide reagieren. Noch viel weniger ist über die Beteiligung von VGF an der Krebspathologie bekannt- Zusammenfassend sind unsere Befunde nicht nur wichtig für die Krebsforschung, sonder auch für andere Bereiche der VGF Forschung, da sie die funktionellen Veränderungen der Hirnmakrophagen nach VGF-Peptid- Stimulation zeigen, welche bei anderen Krankheitsbildern wie Alzheimer oder Schizophrenie eine große Rolle spielen könnten

Redução da proliferação celular e aumento da expressão de marcadores neurais de células-tronco de glioblastoma humano expostas a um inibidor de histona deacetilase

Os glioblastomas multiforme (GBM), são tumores cerebrais, que por sua malignidade, aliada ao seu rápido crescimento e frequente recorrência, exigem uma maior investigação da comunidade científica. Novas terapias devem afetar as células-tronco tumorais (CSC), as quais são responsáveis pela resistência e progressão tumoral. Neste trabalho fizemos o uso da Tricostatina A (TSA), um inibidor de histonas deacetilase, para se obter a modulação epigenética, e portanto, manipulação da expressão gênica, da linhagem celular U87-MG de GBM, utilizada aqui como um modelo para a pesquisa com CSC. Observamos a redução da proliferação e sobrevivência das tumoresferas de U87, as quais são formadas por CSC, seguida de alterações morfológicas nas células tratadas. A diferenciação das U87 foi confirmada pelo aumento dos níveis de marcadores neuronais e gliais, tais como NeuN e GFAP. Além disso, mostramos evidências de senescência celular após o tratamento com TSA. Nenhum efeito sobre a migração celular foi encontrado após a modulação epigenética. Portanto, os nossos resultados mostram a influência da TSA na diferenciação, proliferação e sobrevivência de CSC de glioma e também na indução da senescência celular, demonstrando o potencial da TSA na terapia dos tumores cerebrais.Glioblastoma multiforme (GBM), because of its fast growth and recurrence, require further investigation by the scientific community in order to find promising new therapies for these tumors, specially affecting their Cancer stem cells (CSC), which drive many tumorigenic processes. In this work we have made use of the HDAC inhibitor Trichostatin A to achieve the epigenetic modulation of the U87-MG GBM cell line, as a model for CSC research. We have observed reduction of the U87 tumorspheres, which are enriched for CSC, proliferation and survival followed by morphological changes both in the treated tumorspheres and in single cells. Enhanced on the U87 differentiation was confirmed by increased levels of neuronal and glial markers such as NeuN and GFAP. Furthermore we showed evidences of cellular senescence after the TSA treatment. No effect on cell migration was found after TSA treatment. Therefore, these results demonstrate a plethora effects on differentiation, proliferation, survival of glioma cells and induction of cellular senescence by TSA, making TSA a promising agent for glioma therapy

Glioma Revisited: From Neurogenesis and Cancer Stem Cells to the Epigenetic Regulation of the Niche

Gliomas are the most incident brain tumor in adults. This malignancy has very low survival rates, even when combining radio- and chemotherapy. Among the gliomas, glioblastoma multiforme (GBM) is the most common and aggressive type, and patients frequently relapse or become refractory to conventional therapies. The fact that such an aggressive tumor can arise in such a carefully orchestrated organ, where cellular proliferation is barely needed to maintain its function, is a question that has intrigued scientists until very recently, when the discovery of the existence of proliferative cells in the brain overcame such challenges. Even so, the precise origin of gliomas still remains elusive. Thanks to new advents in molecular biology, researchers have been able to depict the first steps of glioma formation and to accumulate knowledge about how neural stem cells and its progenitors become gliomas. Indeed, GBM are composed of a very heterogeneous population of cells, which exhibit a plethora of tumorigenic properties, supporting the presence of cancer stem cells (CSCs) in these tumors. This paper provides a comprehensive analysis of how gliomas initiate and progress, taking into account the role of epigenetic modulation in the crosstalk of cancer cells with their environment

The role of intraspinal sensory neurons in the control of quadrupedal locomotion

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