Establishment and Comprehensive Molecular Characterization of an Immortalized Glioblastoma Cell Line from a Brazilian Patient.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with few effective treatment strategies. The research on the development of new treatments is often constrained by the limitations of preclinical models, which fail to accurately replicate the disease's essential characteristics. Herein, we describe the obtention, molecular, and functional characterization of the GBM33 cell line. This cell line belongs to the GBM class according to the World Health Organization 2021 Classification of Central Nervous System Tumors, identified by methylation profiling. GBM33 expresses the astrocytic marker GFAP, as well as markers of neuronal origin commonly expressed in GBM cells, such as ßIII-tubulin and neurofilament. Functional assays demonstrated an increased growth rate when compared to the U87 commercial cell line and a similar sensitivity to temozolamide. GBM33 cells retained response to serum starvation, with reduced growth and diminished activation of the Akt signaling pathway. Unlike LN-18 and LN-229 commercial cell lines, GBM33 is able to produce primary cilia upon serum starvation. In summary, the successful establishment and comprehensive characterization of this GBM cell line provide researchers with invaluable tools for studying GBM biology, identifying novel therapeutic targets, and evaluating the efficacy of potential treatments.

Adiponectin reverses ß-Cell damage and impaired insulin secretion induced by obesity.

Obesity significantly decreases life expectancy and increases the incidence of age-related dysfunctions, including ß-cell dysregulation leading to inadequate insulin secretion. Here, we show that diluted plasma from obese human donors acutely impairs ß-cell integrity and insulin secretion relative to plasma from lean subjects. Similar results were observed with diluted sera from obese rats fed ad libitum, when compared to sera from lean, calorically restricted, animals. The damaging effects of obese circulating factors on ß-cells occurs in the absence of nutrient overload, and mechanistically involves mitochondrial dysfunction, limiting glucose-supported oxidative phosphorylation and ATP production. We demonstrate that increased levels of adiponectin, as found in lean plasma, are the protective characteristic preserving ß-cell function; indeed, sera from adiponectin knockout mice limits ß-cell metabolic fluxes relative to controls. Furthermore, oxidative phosphorylation and glucose-sensitive insulin secretion, which are completely abrogated in the absence of this hormone, are restored by the presence of adiponectin alone, surprisingly even in the absence of other serological components, for both the insulin-secreting INS1 cell line and primary islets. The addition of adiponectin to cells treated with plasma from obese donors completely restored ß-cell functional integrity, indicating the lack of this hormone was causative of the dysfunction. Overall, our results demonstrate that low circulating adiponectin is a key damaging element for ß-cells, and suggest strong therapeutic potential for the modulation of the adiponectin signaling pathway in the prevention of age-related ß-cell dysfunction.

Progress in Development of Magadiite to Produce Multifunctional Lamellar Materials.

The attractive properties of magadiite, a lamellar and crystalline material, could give rise to new industrial processes due to its unique and modulating intrinsic properties. In this context, the high degree of expansion of its lamellae, a key factor for its potential use in several areas of scientific research, has attracted the attention of several researchers. The aim of this review is to provide a historical overview of the hypothetical models developed to explain the magadiite crystalline structure. Furthermore, different synthesis strategies for the preparation of magadiites as sodic, protonic, and hybrid (inorganic-inorganic and inorganic-organic) materials are discussed along with several routes for obtaining modified magadiites. Also, the use of magadiite in catalytic reactions, notably in ethanol dehydration and fructose conversion reactions, is a growing area of research. Other potential applications include the adsorption and absorption of environmental pollutants (e.g., phenol and methylene blue in wastewater), use as a photocatalyst in the oxidation of toluene, and use in medicine (e.g., as a drug delivery or antibacterial/antifungal agent). This highlights the many opportunities for the development of new synthesis methods to obtain multifunctional materials in the search for new applications.

Sleep deprivation regulates availability of PrPC and Aß peptides which can impair interaction between PrPC and laminin and neuronal plasticity.

PrPC is a glycoprotein capable to interact with several molecules and mediates diverse signaling pathways. Among numerous ligands, laminin (LN) is known to promote neurite outgrowth and memory consolidation, while amyloid-beta oligomers (Aßo) trigger synaptic dysfunction. In both pathways, mGluR1 is recruited as co-receptor. The involvement of PrPC /mGluR1 in these opposite functions suggests that this complex is a key element in the regulation of synaptic activity. Considering that sleep-wake cycle is important for synaptic homeostasis, we aimed to investigate how sleep deprivation affects the expression of PrPC and its ligands, laminin, Aßo, and mGluR1, a multicomplex that can interfere with neuronal plasticity. To address this question, hippocampi of control (CT) and sleep deprived (SD) C57BL/6 mice were collected at two time points of circadian period (13 hr and 21 hr). We observed that sleep deprivation reduced PrPC and mGluR1 levels with higher effect in active state (21 hr). Sleep deprivation also caused accumulation of Aß peptides in rest period (13 hr), while laminin levels were not affected. In vitro binding assay showed that Aßo can compete with LN for PrPC binding. The influence of Aßo was also observed in neuritogenesis. LN alone promoted longer neurite outgrowth than non-treated cells in both Prnp+/+ and Prnp0/0 genotypes. Aßo alone did not show any effects, but when added together with LN, it attenuated the effects of LN only in Prnp+/+ cells. Altogether, our findings indicate that sleep deprivation regulates the availability of PrPC and Aß peptides, and based on our in vitro assays, these alterations induced by sleep deprivation can negatively affect LN-PrPC interaction, which is known to play roles in neuronal plasticity.

Evidence of Extracellular Vesicles Biogenesis and Release in Mouse Embryonic Stem Cells.

Extracellular vesicles (EVs) released by mouse embryonic stem cells (mESCs) are considered a source of bioactive molecules that modulate their microenvironment by acting on intercellular communication. Either intracellular endosomal machinery or their derived EVs have been considered a relevant system of signal circuits processing. Herein, we show that these features are found in mESCs. Ultrastructural analysis revealed structures and organelles of the endosomal system such as coated pits and endocytosis-related vesicles, prominent rough endoplasmic reticulum and Golgi apparatus, and multivesicular bodies (MVBs) containing either few or many intraluminal vesicles (ILVs) that could be released as exosomes to extracellular milieu. Besides, budding vesicles shed from the plasma membrane to the extracellular space is suggestive of microvesicle biogenesis in mESCs. mESCs and mouse blastocyst express specific markers of the Endosomal Sorting Complex Required for Transport (ESCRT) system. Ultrastructural analysis and Nanoparticle Tracking Analysis (NTA) of isolated EVs revealed a heterogeneous population of exosomes and microvesicles released by mESCs. These vesicles contain Wnt10b and the Notch ligand Delta-like 4 (DLL4) and also the co-chaperone stress inducible protein 1 (STI1) and its partner Hsp90. Wnt10b and Dll4 colocalize with EVs biogenesis markers in mESCs. Overall, the present study supports the function of the mESCs endocytic network and their EVs as players in stem cell biology.

Loss of prion protein is associated with the development of insulin resistance and obesity.

Prion protein (PrPC) was initially described due to its involvement in transmissible spongiform encephalopathies. It was subsequently demonstrated to be a cell surface molecule involved in many physiological processes, such as vesicle trafficking. Here, we investigated the roles of PrPC in the response to insulin and obesity development. Two independent PrPC knockout (KO) and one PrPC overexpressing (TG20) mouse models were fed high-fat diets, and the development of insulin resistance and obesity was monitored. PrPC KO mice fed high-fat diets presented all of the symptoms associated with the development of insulin resistance: hyperglycemia, hyperinsulinemia, and obesity. Conversely, TG20 animals fed high-fat diets showed reduced weight and insulin resistance. Accordingly, the expression of peroxisome proliferator-activated receptor gamma (PPARγ) was reduced in PrPC KO mice and increased in TG20 animals. PrPC KO cells also presented reduced glucose uptake upon insulin stimulation, due to reduced translocation of the glucose transporter Glut4. Thus, our results suggest that PrPC reflects susceptibility to the development of insulin resistance and metabolic syndrome.

A comprehensive characterization of cell cultures and xenografts derived from a human verrucous penile carcinoma.

This study aimed to establish and characterize primary cell cultures and xenografts derived from penile carcinoma (PeCa) in order to provide experimental models for cellular processes and efficacy of new treatments. A verrucous squamous cell carcinoma (VSCC) was macrodissected, dissociated, and cultivated in KSFM/DF12 medium. Cell cultures were evaluated at passage 5 (P5) using migration and invasion assays and were serially propagated, in vivo, in BALB/c nude mice until passage 3 (X1-X3). Immunophenotypic characterization of cultures and xenografts was performed. Genomic (CytoScan HD, Affymetrix) and transcriptomic profiles (HTA 2.0 platform, Affymetrix) for VSCC, cell cultures, and xenografts were assessed. P5 cells were able to migrate, invade the Matrigel, and produce tumors in immunodeficient mice, demonstrating their malignant potential. The xenografts unexpectedly presented a sarcomatoid-like carcinoma phenotype. Genomic analysis revealed a high similarity between the VSCC and tumor-derived xenograft, confirming its xenograft origin. Interestingly, a subpopulation of P5 cells presented stem cell-related markers (CD44(+)CD24(-) and ALDH1(high)) and sphere-forming capacity, suggesting their potential xenograft origin. Cell cultures and xenografts retained the genomic alterations present in the parental tumor. Compared to VSCC, differentially expressed transcripts detected in all experimental conditions were associated with cellular morphology, movement, and metabolism and organization pathways. Malignant cell cultures and xenografts derived from a verrucous penile carcinoma were established and fully characterized. Nevertheless, xenograft PeCa models must be used with caution, taking into consideration the selection of specific cell populations and anatomical sites for cell/tumor implantation.

Targeting prion protein interactions in cancer.

In recent years, prion protein (PrP(C)) has been considered as a promising target molecule for cancer therapies, due its direct or indirect participation in tumor growth, metastasis, and resistance to cell death induced by chemotherapy. PrP(C) functions as a scaffold protein, forming multiprotein complexes on the plasma membrane, which elicits distinct signaling pathways involved in diverse biological phenomena and could be modulated depending on the cell type, complex composition, and organization. In addition, PrP(C) and its partners participate in self-renewal of embryonic, tissue-specific stem cells and cancer stem cells, which are suggested to be responsible for the origin, maintenance, relapse, and dissemination of tumors. Interference with protein-protein interaction has been recognized as an important therapeutic strategy in cancer; indeed, the possible interference in PrP(C) engagement with specific partners is a novel strategy. Recently, our group successfully used that approach to interfere with the interaction between PrP(C) and HSP-90/70 organizing protein (HOP, also known as stress-inducible protein 1 - STI1) to control the growth of human glioblastoma in animal models. Thus, PrP(C)-organized multicomplexes have emerged as feasible candidates for anti-tumor therapy, warranting further exploration.

Dopamine induces the accumulation of insoluble prion protein and affects autophagic flux.

Accumulation of protein aggregates is a histopathological hallmark of several neurodegenerative diseases, but in most cases the aggregation occurs without defined mutations or clinical histories, suggesting that certain endogenous metabolites can promote aggregation of specific proteins. One example that supports this hypothesis is dopamine and its metabolites. Dopamine metabolism generates several oxidative metabolites that induce aggregation of α-synuclein, and represents the main etiology of Parkinson's diseases. Because dopamine and its metabolites are unstable and can be highly reactive, we investigated whether these molecules can also affect other proteins that are prone to aggregate, such as cellular prion protein (PrP(C)). In this study, we showed that dopamine treatment of neuronal cells reduced the number of viable cells and increased the production of reactive oxygen species (ROS) as demonstrated in previous studies. Overall PrP(C) expression level was not altered by dopamine treatment, but its unglycosylated form was consistently reduced at 100 µM of dopamine. At the same concentration, the level of phosphorylated mTOR and 4EBP1 was also reduced. Moreover, dopamine treatment decreased the solubility of PrP(C), and increased its accumulation in autophagosomal compartments with concomitant induction of LC3-II and p62/SQSTM1 levels. In vitro oxidation of dopamine promoted formation of high-order oligomers of recombinant prion protein. These results suggest that dopamine metabolites alter the conformation of PrP(C), which in turn is sorted to degradation pathway, causing autophagosome overload and attenuation of protein synthesis. Accumulation of PrP(C) aggregates is an important feature of prion diseases. Thus, this study brings new insight into the dopamine metabolism as a source of endogenous metabolites capable of altering PrP(C) solubility and its subcellular localization.

The growth of glioblastoma orthotopic xenografts in nude mice is directly correlated with impaired object recognition memory.

Cognitive dysfunction is found in patients with brain tumors and there is a need to determine whether it can be replicated in an experimental model. In the present study, the object recognition (OR) paradigm was used to investigate cognitive performance in nude mice, which represent one of the most important animal models available to study human tumors in vivo. Mice with orthotopic xenografts of the human U87MG glioblastoma cell line were trained at 9, 14, and 18days (D9, D14, and D18, respectively) after implantation of 5×10(5) cells. At D9, the mice showed normal behavior when tested 90min or 24h after training and compared to control nude mice. Animals at D14 were still able to discriminate between familiar and novel objects, but exhibited a lower performance than animals at D9. Total impairment in the OR memory was observed when animals were evaluated on D18. These alterations were detected earlier than any other clinical symptoms, which were observed only 22-24days after tumor implantation. There was a significant correlation between the discrimination index (d2) and time after tumor implantation as well as between d2 and tumor volume. These data indicate that the OR task is a robust test to identify early behavior alterations caused by glioblastoma in nude mice. In addition, these results suggest that OR task can be a reliable tool to test the efficacy of new therapies against these tumors.

The role of phytophysiognomies and seasonality on the structure of ground-dwelling anuran (Amphibia) in the Pampa biome, Southern Brazil.

Considering that habitat use by amphibians is related both with climate and environmental features, we tested the hypothesis that anuran assemblages found in different phytophysiognomies and in different seasons vary in structure. Additionally, we searched for species which can be indicators of habitat and seasons. The study was conducted in the Pampa biome, southern Brazil. Sampling was done through pitfall traps placed in three phytophysiognomies: grassland, ecotone grassland/forest; and forest. The seasonality factor was created by grouping months in warn and cold seasons. Sixteen species were found and the assemblages were influenced both by phytophysiognomies and climatic seasonality. In a paired comparison, the three phytophysiognomies differed in structure of assemblage from each other. Physalaemus henselii, P. riograndensis, Pseudopaludicola falcipes and Pseudis minuta were indicators of ecotone. Leptodactylus gracilis and Physalaemus biligonigerus were indicators of grassland. None species was indicator of forest. Most of the species were indicators of warm season: Elachistocleis bicolor, Leptodactylus fuscus, L. gracilis, L. latinasus, L. latrans, L. mystacinus, Physalaemus biligonigerus, P. cuvieri and Pseudis minuta. None species was indicator of cold season. We found that even for species of open areas, as Pampa, heterogeneous phytophysiognomies are important for maintaining abundance and constancy of populations of anuran.

Stress-inducible phosphoprotein 1 has unique cochaperone activity during development and regulates cellular response to ischemia via the prion protein.

Stress-inducible phosphoprotein 1 (STI1) is part of the chaperone machinery, but it also functions as an extracellular ligand for the prion protein. However, the physiological relevance of these STI1 activities in vivo is unknown. Here, we show that in the absence of embryonic STI1, several Hsp90 client proteins are decreased by 50%, although Hsp90 levels are unaffected. Mutant STI1 mice showed increased caspase-3 activation and 50% impairment in cellular proliferation. Moreover, placental disruption and lack of cellular viability were linked to embryonic death by E10.5 in STI1-mutant mice. Rescue of embryonic lethality in these mutants, by transgenic expression of the STI1 gene, supported a unique role for STI1 during embryonic development. The response of STI1 haploinsufficient mice to cellular stress seemed compromised, and mutant mice showed increased vulnerability to ischemic insult. At the cellular level, ischemia increased the secretion of STI1 from wild-type astrocytes by 3-fold, whereas STI1 haploinsufficient mice secreted half as much STI1. Interesting, extracellular STI1 prevented ischemia-mediated neuronal death in a prion protein-dependent way. Our study reveals essential roles for intracellular and extracellular STI1 in cellular resilience.

Laminin-γ1 chain and stress inducible protein 1 synergistically mediate PrPC-dependent axonal growth via Ca2+ mobilization in dorsal root ganglia neurons.

Prion protein (PrP(C)) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrP(C) interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimer's diseases. PrP(C) co-opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation. However, potential cross-talk between these protein complexes and their role in peripheral neurons has never been addressed. To explore this issue, we investigated PrP(C)-mediated axonogenesis in peripheral neurons in response to STI1 and laminin-γ1 chain-derived peptide (Ln-γ1). STI1 and Ln-γ1 promoted robust axonogenesis in wild-type neurons, whereas no effect was observed in neurons from PrP(C) -null mice. PrP(C) binding to Ln-γ1 or STI1 led to an increase in intracellular Ca(2+) levels via distinct mechanisms: STI1 promoted extracellular Ca(2+) influx, and Ln-γ1 released calcium from intracellular stores. Both effects depend on phospholipase C activation, which is modulated by mGluR1/5 for Ln-γ1, but depends on, C-type transient receptor potential (TRPC) channels rather than α7nAChR for STI1. Treatment of neurons with suboptimal concentrations of both ligands led to synergistic actions on PrP(C)-mediated calcium response and axonogenesis. This effect was likely mediated by simultaneous binding of the two ligands to PrP(C). These results suggest a role for PrP(C) as an organizer of diverse multiprotein complexes, triggering specific signaling pathways and promoting axonogenesis in the peripheral nervous system.

Disease-associated mutations in the prion protein impair laminin-induced process outgrowth and survival.

Prions, the agents of transmissible spongiform encephalopathies, require the expression of prion protein (PrP(C)) to propagate disease. PrP(C) is converted into an abnormal insoluble form, PrP(Sc), that gains neurotoxic activity. Conversely, clinical manifestations of prion disease may occur either before or in the absence of PrP(Sc) deposits, but the loss of normal PrP(C) function contribution for the etiology of these diseases is still debatable. Prion disease-associated mutations in PrP(C) represent one of the best models to understand the impact of PrP(C) loss-of-function. PrP(C) associates with various molecules and, in particular, the interaction of PrP(C) with laminin (Ln) modulates neuronal plasticity and memory formation. To assess the functional alterations associated with PrP(C) mutations, wild-type and mutated PrP(C) proteins were expressed in a neural cell line derived from a PrP(C)-null mouse. Treatment with the laminin γ1 chain peptide (Ln γ1), which mimics the Ln binding site for PrP(C), increased intracellular calcium in cells expressing wild-type PrP(C), whereas a significantly lower response was observed in cells expressing mutated PrP(C) molecules. The Ln γ1 did not promote process outgrowth or protect against staurosporine-induced cell death in cells expressing mutated PrP(C) molecules in contrast to cells expressing wild-type PrP(C). The co-expression of wild-type PrP(C) with mutated PrP(C) molecules was able to rescue the Ln protective effects, indicating the lack of negative dominance of PrP(C) mutated molecules. These results indicate that PrP(C) mutations impair process outgrowth and survival mediated by Ln γ1 peptide in neural cells, which may contribute to the pathogenesis of genetic prion diseases.

Prion potency in stem cells biology.

Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation, and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.

Biomineralization of superhydrophilic vertically aligned carbon nanotubes.

Vertically aligned carbon nanotubes (VACNT) promise a great role for the study of tissue regeneration. In this paper, we introduce a new biomimetic mineralization routine employing superhydrophilic VACNT films as highly stable template materials. The biomineralization was obtained after VACNT soaking in simulated body fluid solution. Detailed structural analysis reveals that the polycrystalline biological apatites formed due to the -COOH terminations attached to VACNT tips after oxygen plasma etching. Our approach not only provides a novel route for nanostructured materials, but also suggests that COOH termination sites can play a significant role in biomimetic mineralization. These new nanocomposites are very promising as nanobiomaterials due to the excellent human osteoblast adhesion.

Pampean lizard assemblage from subtropical Brazil: a temporal analysis.

The increasing human occupation of natural environments is one of the greatest threats to biodiversity. To mitigate the negative anthropogenic effects, it is necessary to understand the characteristics of natural populations and the natural history of species. A study was conducted with an assemblage of lizards from a disturbed area of the Pampa biome, from February 2001 to January 2004. The assemblage showed a unimodal seasonal pattern, with the recruitment period occurring during the warmer months. The captures were seasonal for two of the three monitored years, and concentrated within warmer months. The minimum temperature explained the number of catches for the assemblage as a whole. However, when the species were analyzed individually, the temperature only explained the seasonal occurrence of Teius oculatus. The abundance of species was significantly different in the third year of study for Cercosaura schreibersii and Ophiodes striatus. This latter species was no longer registered in the study area from May 2003 until the end of the study. Therefore, O. striatus may be more sensitive to environmental changes, considering the events of change in vegetation during the study. With frequent and increasing environmental disturbances, it is necessary to take conservation measures and encourage the increase of knowledge on Pampean lizards.

Pampean lizard assemblage from subtropical Brazil: a temporal analysis

The increasing human occupation of natural environments is one of the greatest threats to biodiversity. To mitigate the negative anthropogenic effects, it is necessary to understand the characteristics of natural populations and the natural history of species. A study was conducted with an assemblage of lizards from a disturbed area of the Pampa biome, from February 2001 to January 2004. The assemblage showed a unimodal seasonal pattern, with the recruitment period occurring during the warmer months. The captures were seasonal for two of the three monitored years, and concentrated within warmer months. The minimum temperature explained the number of catches for the assemblage as a whole. However, when the species were analyzed individually, the temperature only explained the seasonal occurrence of Teius oculatus. The abundance of species was significantly different in the third year of study for Cercosaura schreibersii and Ophiodes striatus. This latter species was no longer registered in the study area from May 2003 until the end of the study. Therefore, O. striatus may be more sensitive to environmental changes, considering the events of change in vegetation during the study. With frequent and increasing environmental disturbances, it is necessary to take conservation measures and encourage the increase of knowledge on Pampean lizards.

O crescimento da ocupação humana sobre ambientes naturais é uma das maiores ameaças à biodiversidade. Para amenizar os efeitos negativos antropogênicos, é necessário entender as características das populações naturais, e a história natural das espécies. Um estudo foi conduzido com uma assembeia de lagartos de uma área perturbada do Pampa, de fevereiro de 2002 a janeiro de 2004. A assembleia apresentou padrão sazonal unimodal, com recrutamento ocorrendo durante os meses mais quentes. As capturas foram sazonais durante dois dos três anos monitorados, e concentradas nos meses mais quentes. A temperatura mínima explica o número de capturas para a assembleia como um todo. Entretanto, quando as espécies foram analisadas individualmente, a variável explica apenas a ocorrência sazonal de Teius oculatus. A abundância das espécies foi significativamente distinta no terceiro ano de estudo para Cercosaura schreibersii e Ophiodes striatus. Essa última espécie não teve mais registros na área de maio de 2003 até o final do estudo. Assim, O. striatus pode ser mais sensível às udanças abientais, considerando os eventos de udança na vegetação ocorridas durante o estudo. Com as frequentes e crescentes perturbações ambientais que o bioma vem sofrendo, são necessárias ações de conservação, e dar suporte para o aumento do conhecimento dos lagartos pampeanos.

Enhanced neural progenitor/stem cells self-renewal via the interaction of stress-inducible protein 1 with the prion protein.

Prion protein (PrP(C) ), when associated with the secreted form of the stress-inducible protein 1 (STI1), plays an important role in neural survival, neuritogenesis, and memory formation. However, the role of the PrP(C) -STI1 complex in the physiology of neural progenitor/stem cells is unknown. In this article, we observed that neurospheres cultured from fetal forebrain of wild-type (Prnp(+/+) ) and PrP(C) -null (Prnp(0/0) ) mice were maintained for several passages without the loss of self-renewal or multipotentiality, as assessed by their continued capacity to generate neurons, astrocytes, and oligodendrocytes. The homogeneous expression and colocalization of STI1 and PrP(C) suggest that they may associate and function as a complex in neurosphere-derived stem cells. The formation of neurospheres from Prnp(0/0) mice was reduced significantly when compared with their wild-type counterparts. In addition, blockade of secreted STI1, and its cell surface ligand, PrP(C) , with specific antibodies, impaired Prnp(+/+) neurosphere formation without further impairing the formation of Prnp(0/0) neurospheres. Alternatively, neurosphere formation was enhanced by recombinant STI1 application in cells expressing PrP(C) but not in cells from Prnp(0/0) mice. The STI1-PrP(C) interaction was able to stimulate cell proliferation in the neurosphere-forming assay, while no effect on cell survival or the expression of neural markers was observed. These data suggest that the STI1-PrP(C) complex may play a critical role in neural progenitor/stem cells self-renewal via the modulation of cell proliferation, leading to the control of the stemness capacity of these cells during nervous system development.

Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin γ1 chain.

The prion protein (PrP(C)) is highly expressed in the nervous system, and its abnormal conformer is associated with prion diseases. PrP(C) is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP(C)-mediated intracellular signaling. Binding of laminin (Ln) to PrP(C) modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP(C)-Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP(C)-Ln signals. The Ln γ1-chain peptide, which contains the Ln binding site for PrP(C), induced neuritogenesis through activation of phospholipase C (PLC), Ca(2+) mobilization from intracellular stores, and protein kinase C and extracellular signal-regulated kinase (ERK1/2) activation in primary cultures of neurons from wild-type, but not PrP(C)-null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluR1/5) associate with PrP(C). Expression of either mGluR1 or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP(C)-Ln γ1 peptide interaction. Specific inhibitors of these receptors impaired PrP(C)-Ln γ1 peptide-induced signaling and neuritogenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP(C)-Ln, and they support the notion that PrP(C) participates in the assembly of multiprotein complexes with physiological functions on neurons.