COB231 targets amyloid plaques in post-mortem human brain tissue and in an Alzheimer mouse model.

Previous works have shown the interest of naturally fluorescent proflavine derivatives to label Abeta deposits in vitro. This study aimed to further characterize the properties of the proflavine 3-acetylamino-6-[3-(propargylamino)propanoyl]aminoacridine (COB231) derivative as a probe. This compound was therefore evaluated on human post-mortem and mice brain slices and in vivo in 18-month-old triple transgenic mice APPswe, PS1M146V and tauP301L (3xTgAD) mice presenting the main characteristics of Alzheimer's disease (AD). COB231 labelled amyloid plaques on brain slices of AD patients, and 3xTgAD mice at 10 and 0.1 µM respectively. However, no labelling of the neurofibrillary tangle-rich areas was observed either at high concentration or in the brain of fronto-temporal dementia patients. The specificity of this mapping was attested in mice using Thioflavin S and IMPY as positive controls of amyloid deposits. After intravenous injection of COB231 in old 3xTgAD mice, fluorescent amyloid plaques were detected in the cortex and hippocampus, demonstrating COB231 blood­brain barrier permeability. We also controlled the cellular localization of COB231 on primary neuronal cultures and showed that COB231 accumulates into the cytoplasm and not into the nucleus. Finally, using a viability assay, we only detected a slight cytotoxic effect of COB231 (< 10%) for the highest concentration (100 µM).

Cellular diversity within embryonic stem cells: pluripotent clonal sublines show distinct differentiation potential.

Embryonic stem cells (ESC), derived from the early inner cell mass (ICM), are constituted of theoretically homogeneous pluripotent cells. Our study was designed to test this concept using experimental approaches that allowed characterization of progenies derived from single parental mouse ESC. Flow cytometry analysis showed that a fraction of ESC submitted to neural differentiation generates progenies that escape the desired phenotype. Live imaging of individual cells demonstrated significant variations in the capacity of parental ESC to generate neurons, raising the possibility of clonal diversity among ESC. To further substantiate this hypothesis, clonal sublines from ESC were generated by limit dilution. Transcriptome analysis of undifferentiated sublines showed marked differences in gene expression despite the fact that all clones expressed pluripotency markers. Sublines showed distinct differentiation potential, both in phenotypic differentiation assays and with respect to gene expression in embryoid bodies. Clones generated from another ESC line also showed individualities in their differentiation potential, demonstrating the wider applicability of these findings. Taken together, our observations demonstrate that pluripotent ESC consist of individual cell types with distinct differentiation potentials. These findings identify novel elements for the biological understanding of ESC and provide new tools with a major potential for their future in vitro and in vivo use.

The NADPH oxidase NOX2 controls glutamate release: a novel mechanism involved in psychosis-like ketamine responses.

Subanesthetic doses of NMDA receptor antagonist ketamine induce schizophrenia-like symptoms in humans and behavioral changes in rodents. Subchronic administration of ketamine leads to loss of parvalbumin-positive interneurons through reactive oxygen species (ROS), generated by the NADPH oxidase NOX2. However, ketamine induces very rapid alterations, in both mice and humans. Thus, we have investigated the role of NOX2 in acute responses to subanesthetic doses of ketamine. In wild-type mice, ketamine caused rapid (30 min) behavioral alterations, release of neurotransmitters, and brain oxidative stress, whereas NOX2-deficient mice did not display such alterations. Decreased expression of the subunit 2A of the NMDA receptor after repetitive ketamine exposure was also precluded by NOX2 deficiency. However, neurotransmitter release and behavioral changes in response to amphetamine were not altered in NOX2-deficient mice. Our results suggest that NOX2 is a major source of ROS production in the prefrontal cortex controlling glutamate release and associated behavioral alterations after acute ketamine exposure. Prolonged NOX2-dependent glutamate release may lead to neuroadaptative downregulation of NMDA receptor subunits.

Calnexin deficiency leads to dysmyelination.

Calnexin is a molecular chaperone and a component of the quality control of the secretory pathway. We have generated calnexin gene-deficient mice (cnx(-/-)) and showed that calnexin deficiency leads to myelinopathy. Calnexin-deficient mice were viable with no discernible effects on other systems, including immune function, and instead they demonstrated dysmyelination as documented by reduced conductive velocity of nerve fibers and electron microscopy analysis of sciatic nerve and spinal cord. Myelin of the peripheral and central nervous systems of cnx(-/-) mice was disorganized and decompacted. There were no abnormalities in neuronal growth, no loss of neuronal fibers, and no change in fictive locomotor pattern in the absence of calnexin. This work reveals a previously unrecognized and important function of calnexin in myelination and provides new insights into the mechanisms responsible for myelin diseases.

Proflavine derivatives as fluorescent imaging agents of amyloid deposits.

A series of proflavine derivatives for use to further image Aß amyloid deposits were synthesized and characterized. Aged 3xTg-AD (23 months old) mice hippocampus sections incubated with these derivatives revealed preferential labeling of amyloid plaques. Furthermore an in vitro binding study showed an inhibitory effect, although moderate, of these compounds on Aß(40) fibril formation. This study highlights the potential of proflavine as a molecular scaffold for designing new Aß imaging agents, its native fluorescence allowing in vitro neuropathological staining in AD damaged brain sections.

Stem cell-derived neurons grafted in the striatum are expelled out of the brain after chronic cortical stroke.

BACKGROUND AND PURPOSE: In humans and rodents, cortical stroke can lead to cortex atrophy in long-term survivors. In the rodent, fetal brain neural precursors or stem cell-derived neurons grafted in the stroke-lesioned brain integrate successfully and reduce infarct in the short term. We have examined the fate, in the long term, of mouse embryonic stem cell-derived neural precursors grafted after permanent middle cerebral artery occlusion in mice. METHODS: Green fluorescent protein-labeled neural precursors were grafted in the striatum of control and lesioned mice and their fate examined 9 months later. RESULTS: In control mice, the neuronal progeny of mouse embryonic stem cells innervated distant brain structures, in a way remarkably similar between animals, displayed a laterality preference and remained polysialated neural cell adhesion molecule-immunoreactive. In lesioned mice, grafted cells were expelled out of the brain. CONCLUSIONS: Stroke-related brain atrophy and reshaping were not prevented by cell grafting and, eventually, led to the expulsion of the graft.

Development of human nervous tissue upon differentiation of embryonic stem cells in three-dimensional culture.

Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interface-based culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues.

Fate of undifferentiated mouse embryonic stem cells within the rat heart: role of myocardial infarction and immune suppression.

Abstract It has recently been suggested that the infarcted rat heart microenvironment could direct pluripotent mouse embryonic stem cells to differentiate into cardiomyocytes through an in situ paracrine action. To investigate whether the heart can function as a cardiogenic niche and confer an immune privilege to embryonic stem cells, we assessed the cardiac differentiation potential of undifferentiated mouse embryonic stem cells (mESC) injected into normal, acutely or chronically infarcted rat hearts. We found that mESC survival depended on immunosuppression both in normal and infarcted hearts. However, upon Cyclosporin A treatment, both normal and infarcted rat hearts failed to induce selective cardiac differentiation of implanted mESC. Instead, teratomas developed in normal and infarcted rat hearts 1 week and 4 weeks (50% and 100%, respectively) after cell injection. Tight control of ESC commitment into a specific cardiac lineage is mandatory to avoid the risk of uncontrolled growth and tumourigenesis following transplantation of highly plastic cells into a diseased myocardium.

Genetic engineering of embryonic stem cells.

We describe a novel generation oflentiviral vectors that are particularly well suited for work with embryonic stem cells. The possibility of selecting cell lines with antibiotics and the rapid insertion of any combination of promoters and genes of interest makes them a powerful tool in the generation of transgenic ES cell lines. This vector can also greatly facilitate studies aimed at the improvement of neuronal engineering from ES cells, by making it possible to monitor the emergence and differentiation of neurons.

Genetic engineering of embryonic stem cells.

We describe a novel generation of lentiviral vectors that are particularly well suited for work with embryonic stem cells. The possibility of selecting cell lines with antibiotics and the rapid insertion of any combination of promoters and genes of interest makes them a powerful tool in the generation of transgenic ES cell lines. This vector can also greatly facilitate studies aimed at the improvement of neuronal engineering from ES cells, by making it possible to monitor the emergence and differentiation of neurons.

Deep brain stimulation-associated brain tissue imprints: a new in vivo approach to biological research in human Parkinson's disease.

BACKGROUND: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) or the internal segment of the globus pallidus (GPi) has been established as a highly effective symptomatic therapy for Parkinson's disease (PD). An intriguing biological aspect related to the DBS procedure is that a temporary contact establishes between surgical instruments and the surrounding brain tissue. In this exploratory study, we took advantage of this unique context to harvest brain material adhering to the stylet routinely used during surgery, and to examine the biological value of these samples, here referred to as "brain tissue imprints" (BTIs). RESULTS: Nineteen BTIs from 12 STN- or GPi-electrode implanted patients were obtained in vivo during DBS surgery, without any modification of the surgical procedure. Immunofluorescence analyses confirmed that our approach allowed the harvesting of many neural cells including neurons harboring distinct neurotransmitter markers. Shotgun proteomic and transcriptomic analyses provided for the first time molecular information from DBS-associated brain samples, and confirmed the compatibility of this new type of sample with poly-omic approaches. The method appears to be safe and results consistent. CONCLUSIONS: We here propose BTIs as original and highly valuable brain samples, and DBS-related brain imprinting as a new conceptual approach to biological research in living patients with PD.

Elimination of proliferating cells from CNS grafts using a Ki67 promoter-driven thymidine kinase.

Pluripotent stem cell (PSC)-based cell therapy is an attractive concept for neurodegenerative diseases, but can lead to tumor formation. This is particularly relevant as proliferating neural precursors rather than postmitotic mature neurons need to be transplanted. Thus, safety mechanisms to eliminate proliferating cells are needed. Here, we propose a suicide gene approach, based on cell cycle-dependent promoter Ki67-driven expression of herpes simplex virus thymidine kinase (HSV-TK). We generated a PSC line expressing this construct and induced neural differentiation. In vitro, proliferating PSC and early neural precursor cells (NPC) were killed by exposure to ganciclovir. In vivo, transplantation of PSC led to tumor formation, which was prevented by early ganciclovir treatment. Transplanted NPC did not lead to tumor formation and their survival and neural maturation were not affected by ganciclovir. In conclusion, the cell cycle promoter-driven suicide gene approach described in this study allows killing of proliferating undifferentiated precursor cells without expression of the suicide gene in mature neurons. This approach could also be of use for other stem cell-based therapies where the final target consists of postmitotic cells.

Human three-dimensional engineered neural tissue reveals cellular and molecular events following cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the most common cause of congenital infection of the central nervous system (CNS). To overcome the limited access to human neural tissue and stringent species specificity of HCMV, we used engineered neural tissues to: (i) provide a technical advance to mimick features of HCMV infection in a human neural fetal tissue in vitro and (ii) characterize the molecular and cellular phenomenon following HCMV infection in this tissue. Herein, we infected hESC-derived engineered neural tissues (ENTs) whose organization resembles fetal brain. Transcriptome analysis of ENTs demonstrated that HCMV infection displayed features of the infection with the expression of genes involved in lipid metabolism, growth and development, as well as stress and host-response in a time-dependent manner. Immunohistochemical analysis demonstrated that HCMV did not firstly infect neural tubes (i.e. radially organized, proliferating stem cell niches), but rather an adjacent side population of post-mitotic cells expressing nestin, doublecortin, Sox1, musashi and vimentin markers. Importantly, we observe the same tropism in naturally HCMV-infected fetal brain specimens. To the best of our knowledge this system represents the first human brain-like tissue able to provide a more physiologically model for studying HCMV infection.

Induction of enhanced green fluorescent protein expression in response to lesions in the nervous system.

We have generated a mouse strain carrying a transgene driven by a strong and ubiquitous promoter (human cytomegalovirus hCMV/beta-actin) and containing an enhanced green fluorescent protein (eGFP) coding sequence upstream of the 3' untranslated region (3'UTR) of tissue-type plasminogen activator (t-PA) mRNA. The 3'UTR of t-PA mRNA is known to be involved in the reversible deadenylation and translational repression of transcripts in mouse oocytes. hCMV/beta-actin-eGFP-3'UTR t-PA transgenic mice express eGFP mRNA in all brain structures analyzed but lack eGFP fluorescence, with the exception of blood vessels, choroid plexus, and Purkinje cells. Taking advantage of these features, we tested whether certain pathological conditions, in particular injuries of the nervous system, might trigger eGFP fluorescence in traumatized cells or neurons. From this perspective, we analyzed eGFP mRNA expression and eGFP fluorescence in experimental models of nervous system lesions, such as motoneuron axotomy and cerebral stroke induced by middle cerebral artery occlusion. We found an increase in eGFP fluorescence in specific brain areas in cells suffering or reacting to these injuries. This increased fluorescence is correlated with an increased transcription of eGFP in lesioned cells, presumably enhanced by a release of the translational silencing mediated by the 3'UTR region of the t-PA mRNA. This transgenic mouse model may prove useful to study the development of neurodegenerative lesions.

Chemokine-induced cell death in CCR5-expressing neuroblastoma cells.

CCR5 is expressed in neurons but its function in this cellular context is hitherto poorly understood. We have generated CCR5-expressing SH-SY5Y neuroblastoma cells. CCR5 ligands induced cell death in these cells, but not in control neuroblastoma cells or in CCR5-expressing fibroblasts. CCR5-dependent killing of neuroblastoma cells occurred through apoptosis, since it was accompanied by caspase-3 activation and could be prevented by a caspase-3 inhibitor. Finally, cell killing by activated microglia was more rapid and extensive in CCR5-expressing neuroblastoma cells than in control cells. In summary, CCR5 may act as a death receptor in cells of neuronal lineage and therefore be involved in inflammatory neurodegeneration.

Engineering of midbrain organoids containing long-lived dopaminergic neurons.

The possibility to generate dopaminergic (DA) neurons from pluripotent stem cells represents an unlimited source of material for tissue engineering and cell therapy for neurodegenerative disease. We set up a protocol based on the generation of size-calibrated neurospheres for a rapid production (3 weeks) of a high amount of DA neurons (>60%) oriented toward a midbrain-like phenotype, characterized by the expression of FOXA2, LMX1A, tyrosine hydroxylase (TH), NURR1, and EN1. By using γ-secretase inhibitors and varying culture time of neurospheres, we controlled maturation and cellular composition of a three-dimensional (3D) engineered nervous tissue (ENT). ENT contained neurons and glial cells expressing various markers of maturity, such as synaptophysin, neuronal nuclei-specific protein (NeuN), and glial fibrillary acidic protein (GFAP), and were electrophysiologically active. We found that 3-week-old neurospheres were optimal to generate 3D tissue containing DA neurons with typical A9 morphology. ENT generated from 4-week-old neurospheres launched glial cell type since astrocytes and myelin could be detected massively at the expense of TH-immunoreactive neurons. All γ-secretase inhibitors were not equivalent; compound E was more efficient than DAPT in generating DA neurons. This DA tissue provides a tool for drug screening, and toxicology. It should also become a useful biomaterial for studies on Parkinson's disease.

The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESCs) offer attractive prospective as potential source of neurons for cell replacement therapy in human neurodegenerative diseases. Besides, ESCs neural differentiation enables in vitro tissue engineering for fundamental research and drug discovery aimed at the nervous system. We have established stable and long-term three-dimensional (3D) culture conditions which can be used to model long latency and complex neurodegenerative diseases. Mouse ESCs-derived neural progenitor cells generated by MS5 stromal cells induction, result in strictly neural 3D cultures of about 120-mum thick, whose cells expressed mature neuronal, astrocytes and myelin markers. Neurons were from the glutamatergic and gabaergic lineages. This nervous tissue was spatially organized in specific layers resembling brain sub-ependymal (SE) nervous tissue, and was maintained in vitro for at least 3.5 months with great stability. Electron microscopy showed the presence of mature synapses and myelinated axons, suggesting functional maturation. Electrophysiological activity revealed biological signals involving action potential propagation along neuronal fibres and synaptic-like release of neurotransmitters. The rapid development and stabilization of this 3D cultures model result in an abundant and long-lasting production that is compatible with multiple and productive investigations for neurodegenerative diseases modeling, drug and toxicology screening, stress and aging research.

NOX4 expression in human microglia leads to constitutive generation of reactive oxygen species and to constitutive IL-6 expression.

Reactive oxygen species (ROS) generation by microglia is implicated in neuroinflammation and neurotoxicity, as well as in host defense, cell proliferation and excitatory amino acid release. Recent studies demonstrate that primary microglia preparations not only express the phagocyte NADPH oxidase NOX2, but also the NOX1 and NOX4 isoforms. Here we investigated the relationship between neuroinflammation and NOX isoform expression in the human microglia cell line clone 3 (HMC3). HMC3 cells are typical microglia, as suggested by the constitutive expression of Iba-1 and CD14, and IFN-gamma-induced expression of CD11b, CD68 and MHCII. However, the characteristics of NOX isoform expression and ROS generation by HMC3 cells were unexpected. RT-PCR demonstrated abundant expression of NOX4, but almost no NOX2 mRNA. ROS generation was constitutive and appeared predominantly intracellular, as superoxide was detected within intracellular vesicles, while the cell-permeable H(2)O(2) was found in the extracellular space. ROS generation by HMC3 was efficiently suppressed by siRNA directed against NOX4, but not by control siRNA. NOX4 suppression did not alter expression of the microglia-typical genes MHCII, CD68 and CD11b, nor did it affect the expression of iNOS, VEGF or TGF-beta. However, there was a marked decrease in IL-6 mRNA. Taken together, we demonstrate a constitutive NOX4-dependent ROS generation in a microglial cell line which leads to expression of IL-6 mRNA. The possibility that microglia could switch from tightly regulated NOX2-dependent ROS generation to constitutive NOX4-dependent ROS generation is of interest for the understanding of the role of microglia in maintaining the balance between neuroprotection and neuroinflammatory damage.

Involvement of NOX2 in the development of behavioral and pathologic alterations in isolated rats.

BACKGROUND: Social stress leads to oxidative stress in the central nervous system, contributing to the development of mental disorders. Loss of parvalbumin in interneurons is an important feature of these diseases. We studied the role of the superoxide-producing nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) in rats exposed to social isolation. METHODS: Male rats were kept for 7 weeks in group or in social isolation (n = 6-10 per group). Behavioral tests, immunohistochemistry, and analysis of NOX2 expression were performed at the end of social isolation. Apocynin was given in the drinking water (5 mg/kg/day). RESULTS: NOX2 was below detection level in the brains of control animals, whereas it was highly expressed in isolated rats, particularly in nucleus accumbens and prefrontal cortex. Indirect markers of oxidative stress (oxidized nucleic acid 8-hydroxy-2'-deoxyguanosine, redox-sensitive transcription factor c-fos, and hypoxia-inducible factor-1alpha) were increased after social isolation in brain areas with high NOX2 expression. An increase in immunoreactive microglia suggested that oxidative stress could be in part due to NOX2 activation in microglia. In response to social isolation, rats showed increased locomotor activity, decreased discrimination, signs of oxidative stress in neurons, and loss of parvalbumin-immunoreactivity. Treatment of isolated rats with the antioxidant/NOX inhibitor apocynin prevented the behavioral and histopathological alterations induced by social isolation. CONCLUSIONS: Our data suggest that NOX2-derived oxidative stress is involved in loss of parvalbumin immunoreactivity and development of behavioral alterations after social isolation. These results provide a molecular mechanism for the coupling between social stress and brain oxidative stress, as well as potential new therapeutic avenues.

Pluripotent stem cells as new drugs? The example of Parkinson's disease.

Cell replacement therapy is a widely discussed novel concept of medical treatment. The increased knowledge in the stem cell field, particularly pluripotent stem cells, potentially provides powerful tools for this therapeutic concept. A large number of disease characterized by the loss of functional cells are potential candidates for cell replacement therapy and, in this regards, Parkinson's disease is of particular interest. It is one of the most prevalent neurodegenerative diseases caused by the loss of dopaminergic neurons in the Substantia nigra pars compacta. Pharmacological therapies are valuable but suffer from the progressive decline of efficacy as the disease progresses. Cell therapy application has emerged about two decades ago as a valid therapeutic alternative and recent advances in stem cell research suggest that pluripotent stem cell transplantation may be a promising approach to replace degenerated neurons in Parkinson's disease. Various sources of pluripotent stem cells (PSC) currently tested in animal models of Parkinson's disease have proven their efficacy in relieving symptoms and restoring damaged brain function. This review summarizes and discusses the important challenges that actually must be solved before the first studies of PSC transplantation can be undertaken into humans.