Biomolecules to Biomarkers? U87MG Marker Evaluation on the Path towards Glioblastoma Multiforme Pathogenesis.

The heterogeneity of the glioma subtype glioblastoma multiforme (GBM) challenges effective neuropathological treatment. The reliance on in vitro studies and xenografted animal models to simulate human GBM has proven ineffective. Currently, a dearth of knowledge exists regarding the applicability of cell line biomolecules to the realm of GBM pathogenesis. Our study's objectives were to address this preclinical issue and assess prominin-1, ICAM-1, PARTICLE and GAS5 as potential GBM diagnostic targets. The methodologies included haemoxylin and eosin staining, immunofluorescence, in situ hybridization and quantitative PCR. The findings identified that morphology correlates with malignancy in GBM patient pathology. Immunofluorescence confocal microscopy revealed prominin-1 in pseudo-palisades adjacent to necrotic foci in both animal and human GBM. Evidence is presented for an ICAM-1 association with degenerating vasculature. Significantly elevated nuclear PARTICLE expression from in situ hybridization and quantitative PCR reflected its role as a tumor activator. GAS5 identified within necrotic GBM validated this potential prognostic biomolecule with extended survival. Here we present evidence for the stem cell marker prominin-1 and the chemotherapeutic target ICAM-1 in a glioma animal model and GBM pathology sections from patients that elicited alternative responses to adjuvant chemotherapy. This foremost study introduces the long non-coding RNA PARTICLE into the context of human GBM pathogenesis while substantiating the role of GAS5 as a tumor suppressor. The validation of GBM biomarkers from cellular models contributes to the advancement towards superior detection, therapeutic responders and the ultimate attainment of promising prognoses for this currently incurable brain cancer.

Cerebellar demyelination and neurodegeneration associated with mTORC1 hyperactivity may contribute to the developmental onset of autism-like neurobehavioral phenotype in a rat model.

The cerebellum hosts more than half of all neurons of the human brain, with their organized activity playing a key role in coordinating motor functions. Cerebellar activity has also been implicated in the control of speech, communication, and social behavior, which are compromised in autism spectrum disorders (ASD). Despite major research advances, there is a shortage of mechanistic data relating cellular and molecular changes in the cerebellum to autistic behavior. We studied the impact of tuberous sclerosis complex 2 haploinsufficiency (Tsc2+/-) with downstream mTORC1 hyperactivity on cerebellar morphology and cellular organization in 1, 9, and 18 m.o. Eker rats, to determine possible structural correlates of an autism-like behavioural phenotype in this model. We report a greater developmental expansion of the cerebellar vermis, owing to enlarged white matter and thickened molecular layer. Histochemical and immunofluorescence data suggest age-related demyelination of central tract of the vermis, as evident from reduced level of myelin-basic protein in the arbora vitae. We also observed a higher number of astrocytes in Tsc2+/- rats of older age while the number of Purkinje cells (PCs) in these animals was lower than in wild-type controls. Unlike astrocytes and PCs, Bergmann glia remained unaltered at all ages in both genotypes, while the number of microglia was higher in Tsc2+/- rats of older age. The convergent evidence for a variety of age-dependent cellular changes in the cerebellum of rats associated with mTORC1 hyperactivity, thus, predicts an array of functional impairments, which may contribute to the developmental onset of an autism-like behavioral phenotype in this model. LAY SUMMARY: This study elucidates the impact of constitutive mTORC1 hyperactivity on cerebellar morphology and cellular organization in a rat model of autism and epilepsy. It describes age-dependent degeneration of Purkinje neurons, with demyelination of central tract as well as activation of microglia, and discusses the implications of these changes for neuro-behavioral phenotypes. The described changes provide new indications for the putative mechanisms underlying cerebellar impairments with their age-related onset, which may contribute to the pathobiology of autism, epilepsy, and related disorders.

Trophic factors as potential therapies for treatment of major mental disorders.

Notwithstanding major advances in psychotherapeutics, their efficacy and specificity remain limited. The slow onset of beneficial outcomes and numerous adverse effects of widely used medications remain of chief concern, warranting in-depth studies. The majority of frontline therapies are thought to enhance the endogenous monoaminergic drive, to initiate a cascade of molecular events leading to lasting functional and structural plasticity. They also involve alterations in trophic factor signalling, including brain-derived neurotrophic factor (BDNF), VGF (non-acronymic), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), glial cell-derived neurotrophic factor (GDNF), and others. In several major mental disorders, emerging data suggest protective and restorative effects of trophic factors in preclinical models, when applied on their own. Antidepressant outcomes of VGF and FGF2, for instance, were shown in experimental animals, while BDNF and GDNF prove useful in the treatment of addiction, schizophrenia, and autism spectrum disorders. The main challenge with the effective translation of these and other findings in the clinic is the knowledge gap in action mechanisms with potential risks, as well as the lack of effective platforms for validation under clinical settings. Herein, we review the state-of-the-art and advances in the therapeutic use of trophic factors in several major neuropsychiatric disorders.

All-Trans Retinoic Acid Fosters the Multifarious U87MG Cell Line as a Model of Glioblastoma.

Glioblastoma multiforme (GBM) is a primary brain cancer of poor prognosis, with existing treatments remaining essentially palliative. Current GBM therapy fails due to rapid reappearance of the heterogeneous neoplasm, with models suggesting that the recurrent growth is from treatment-resistant glioblastoma stem-like cells (GSCs). Whether GSCs depend on survival/proliferative cues from their surrounding microenvironmental niche, particularly surrounding the leading edge after treatment remains unknown. Simulating human GBM in the laboratory relies on representative cell lines and xenograft models for translational medicine. Due to U87MG source discrepancy and differential proliferation responses to retinoic acid treatment, this study highlights the challenges faced by laboratory scientists working with this representative GBM cell line. Investigating the response to all trans-retinoic acid (ATRA) revealed its sequestering of the prominin-1 stem cell marker. ICAM-1 universally present throughout U87MG was enhanced by ATRA, of interest for chemotherapy targeting studies. ATRA triggered diverse expression patterns of long non-coding RNAs PARTICLE and GAS5 in the leading edge and established monolayer growth zone microenvironment. Karyotyping confirmed the female origin of U87MG sourced from Europe. Passaging U87MG revealed the presence of chromosomal anomalies reflective of structural genomic alterations in this glioblastoma cell line. All evidence considered, this study exposes further phenotypic nuances of U87MG which may belie researchers seeking data contributing towards the elusive cure for GBM.

Revisiting Brain Tuberous Sclerosis Complex in Rat and Human: Shared Molecular and Cellular Pathology Leads to Distinct Neurophysiological and Behavioral Phenotypes.

Tuberous sclerosis complex (TSC) is a dominant autosomal genetic disorder caused by loss-of-function mutations in TSC1 and TSC2, which lead to constitutive activation of the mammalian target of rapamycin C1 (mTORC1) with its decoupling from regulatory inputs. Because mTORC1 integrates an array of molecular signals controlling protein synthesis and energy metabolism, its unrestrained activation inflates cell growth and division, resulting in the development of benign tumors in the brain and other organs. In humans, brain malformations typically manifest through a range of neuropsychiatric symptoms, among which mental retardation, intellectual disabilities with signs of autism, and refractory seizures, which are the most prominent. TSC in the rat brain presents the first-rate approximation of cellular and molecular pathology of the human brain, showing many instructive characteristics. Nevertheless, the developmental profile and distribution of lesions in the rat brain, with neurophysiological and behavioral manifestation, deviate considerably from humans, raising numerous research and translational questions. In this study, we revisit brain TSC in human and Eker rats to relate their histopathological, electrophysiological, and neurobehavioral characteristics. We discuss shared and distinct aspects of the pathology and consider factors contributing to phenotypic discrepancies. Given the shared genetic cause and molecular pathology, phenotypic deviations suggest an incomplete understanding of the disease. Narrowing the knowledge gap in the future should not only improve the characterization of the TSC rat model but also explain considerable variability in the clinical manifestation of the disease in humans.

Tuberous Sclerosis (tsc2+/-) Model Eker Rats Reveals Extensive Neuronal Loss with Microglial Invasion and Vascular Remodeling Related to Brain Neoplasia.

Tuberous sclerosis complex (TSC) is a genetic disorder characterized by frequent noncancerous neoplasia in the brain, which can induce a range of severe neuropsychiatric symptoms in humans, resulting from out of control tissue growth. The causative spontaneous loss-of-function mutations have been also identified in rats. Herein, we studied histopathological and molecular changes in brain lesions of the Eker rat model carrying germline mutation of the tsc2 gene, predisposed to multiple neoplasias. Predominant subcortical tumors were analyzed, along with a rare form occurring within the pyriform lobe. The uniform composition of lesions supports the histochemical parity of malformations, with immunofluorescence data supporting their neuro-glial origin. Massive depletion of mature neurons and axonal loss were evident within lesions, with occasional necrotic foci implying advanced stage of pathology. Enrichment of mesenchymal-derived cell markers with hallmarks of neurogenesis and active microglia imply enhanced cell proliferation, with local immune response. The depletion of capillaries within the core was complemented by the formation of dense mesh of nascent vessels at the interface of neoplasia with healthy tissue, implying large-scale vascular remodeling. Taken as a whole, these findings present several novel features of brain tumors in Eker rat model, rendering it suitable for studies of the pathobiology and progression of primary brain tumors, with therapeutic interventions.

Peroral administration of 5-bromo-2-deoxyuridine in drinking water is not a reliable method for labeling proliferating S-phase cells in rats.

In rodents, peroral (p.o.) administration of 5-bromo-2'-deoxyuridine (BrdU) dissolved in drinking water is a widely used method for labeling newly formed cells over a prolonged time-period. Despite the broad applicability of this method, the pharmacokinetics of BrdU in rats or mice after p.o. administration remains unknown. Moreover, the p.o. route of administration may be limited by the relatively low amount of BrdU consumed over 24h and the characteristic drinking pattern of rats, with water intake being observed predominantly during the dark phase. Therefore, we investigated the reliability of staining proliferating S-phase cells with BrdU after p.o. administration (1mg/ml) to rats using both in vitro and in vivo conditions. Flow cytometric analysis of tumor cells co-cultivated with sera from experimental animals exposed to BrdU dissolved in drinking water or 25% orange juice revealed that the concentration of BrdU in the blood sera of rats throughout the day was below the detection limits of our assay. Ingested BrdU was only sufficient to label approximately 4.2±0.3% (water) or 4.2±0.3% (25% juice) of all S-phase cells. Analysis of data from in vivo conditions indicates that only 7.6±3.3% or 15.5±2.3% of all S-phase cells in the dentate gyrus of the hippocampus was labeled in animals administered drinking water containing BrdU during the light and dark phases of the day. In addition, the intensity of BrdU-positive nuclei in animals receiving p.o. administration of BrdU was significantly lower than in control animals intraperitoneally injected with BrdU. Our data indicate that the conventional approach of p.o. administration of BrdU in the drinking water to rats provides strongly inaccurate information about the number of proliferating cells in target tissues. Therefore other administration routes, such as osmotic mini pumps, should be considered for labeling of proliferating cells over a prolonged time-period.