Anti-beta III Tubulin Antikörper [TU-20] (A86691)

Glioblastoma multiforme is an invasive malignant glial brain tumor with a poor prognosis for patients. The primary reasons that lead to the development of treatment resistance are associated with tumor cells infiltrating the brain parenchyma and the specific properties of tumor stem cells. A crucial research area in medical science is the search for effective agents that are able to act on these targets. Fascaplysin alkaloids possess potent antitumor activity. Modern methods for the targeted delivery of drugs reveal extensive possibilities in terms of the clinical use of these compounds. The aim of the present study was to establish effective concentrations of fascaplysin that inhibit the growth and kill the cells of glial tumors, as well as to perform a comparative analysis of fascaplysin's effectiveness in relation to other chemotherapy drugs. C6 glioma cells were utilized as an optimal model of glioblastoma. It was established that fascaplysin at 0.5 µM has a strong cytotoxic effect, which is subsequently replaced by tumor cell death via apoptosis as the length of drug exposure time is increased. Fascaplysin kills glioma cells at a dose higher than 0.5 µM. The efficiency of fascaplysin was observed to significantly exceed that of temozolomide. Therefore, a significant feature of fascaplysin is its ability to inhibit the growth of and kill multipotent tumor cells.

Spinal cord injury (SCI) can lead to locomotor deficits, and the repair of chronic SCI is considered one of the most challenging clinical problems. Although extensive studies have evaluated treatments for acute SCI in small animals, comparatively fewer studies have been conducted on large-animal SCI in the chronic phase, which is more clinically relevant. Here, we used a collagen-based biomaterial, named the NeuroRegen scaffold, loaded with human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in a canine chronic SCI model. To generate chronic SCI, the T8 spinal cord segment was removed by complete transection of the spinal cord. Two months later, glial scar tissue was removed and a NeuroRegen scaffold was transplanted into the lesion area. Functionalized NeuroRegen scaffold implantation promoted both locomotor recovery and endogenous neurogenesis in the lesion area. Moreover, some newly generated neurons successfully matured into 5-HT-positive neurons at 1 year post-injury. In addition, many regenerated axon fibers in the lesion area exhibited remyelination and synapse formation at 1 year post-injury in the functionalized NeuroRegen scaffold group. In conclusion, the NeuroRegen scaffold functionalized with hUC-MSCs is a promising potential therapeutic approach to chronic SCI that promotes neuronal regeneration, reduces glial scar formation, and ultimately improves locomotor recovery.

The dental follicle is an ectomesenchymal tissue surrounding the developing tooth germ. Human dental follicle cells (hDFCs) have the capacity to commit to differentiation into multiple cell types. Here we investigated the capacity of hDFCs to differentiate into neural cells and the efficiency of a two-step strategy involving floating neurosphere-like bodies for neural differentiation. Undifferentiated hDFCs showed a spindle-like morphology and were positive for neural markers such as nestin, ß-III-tubulin, and S100ß. The cellular morphology of several cells was neuronal-like including branched dendrite-like processes and neurites. Next, hDFCs were used for neurosphere formation in serum-free medium containing basic fibroblast growth factor, epidermal growth factor, and B27 supplement. The number of cells with neuronal-like morphology and that were strongly positive for neural markers increased with sphere formation. Gene expression of neural markers also increased in hDFCs with sphere formation. Next, gene expression of neural markers was examined in hDFCs during neuronal differentiation after sphere formation. Expression of Musashi-1 and Musashi-2, MAP2, GFAP, MBP, and SOX10 was upregulated in hDFCs undergoing neuronal differentiation via neurospheres, whereas expression of nestin and?ß-III-tubulin was downregulated. In conclusion, hDFCs may be another optimal source of neural/glial cells for cell-based therapies to treat neurological diseases.

Doxorubicin, a commonly used chemotherapy agent, induces severe cardio- and neurotoxicity. Molecular mechanisms of cardiotoxicity have been extensively studied, but mechanisms by which doxorubicin exhibits its neurotoxic properties remain unclear. Here, we show that doxorubicin impairs neuronal autophagy, leading to the accumulation of an autophagy substrate p62. Neurons treated with doxorubicin contained autophagosomes, damaged mitochondria, and lipid droplets. The brains from mice treated with pegylated liposomal doxorubicin exhibited autophagosomes, often with mitochondria, lipofuscin, and lipid droplets. Interestingly, lysosomes were less acidic in doxorubicin-treated neurons. Overexpression of the transcription factor EB (TFEB), which controls the autophagy-lysosome axis, increased survival of doxorubicin-treated neurons. 2-Hydroxypropyl-ß-cyclodextrin (HPßCD), an activator of TFEB, also promoted neuronal survival, decreased the levels of p62, and lowered the pH in lysosomes. Taken together, substantial changes induced by doxorubicin contribute to neurotoxicity, cognitive disturbances in cancer patients and survivors, and accelerated brain aging. The TFEB pathway might be a new approach for mitigating damage of neuronal autophagy caused by doxorubicin.

Glioblastoma multiforme is an aggressive malignant brain tumor with terminal consequences. A primary reason for its resistance to treatment is associated with cancer stem cells (CSCs), of which there are currently no effective ways to destroy. It remains unclear what cancer cells become a target of stem cell migration, what the role of this process is in oncogenesis and what stem cell lines should be used in developing antitumor technologies. Using modern post‑genome technologies, the present study investigated the migration of human stem cells to cancer cells in vitro, the comparative study of cell proteomes of certain stem cells (including CSCs) was conducted and stem cell migration in vivo was examined. Of all glioblastoma cells, CSCs have the stability to attract normal stem cells. Critical differences in cell proteomes allow the consideration of hematopoietic stem cells (HSCs) as an instrument for interaction with glioblastoma CSCs. Following injection into the bloodstream of animals with glioblastoma, the majority of HSCs migrated to the tumor‑containing brain hemisphere and penetrated the tumor tissue. HSCs therefore are of potential use in the development of methods to target CSCs.

Notch signaling plays a crucial role in controling the proliferation and differentiation of stem and progenitor cells during embryogenesis or organogenesis, but its regulation is incompletely understood. BLOS2, encoded by the Bloc1s2 gene, is a shared subunit of two lysosomal trafficking complexes, biogenesis of lysosome-related organelles complex-1 (BLOC-1) and BLOC-1-related complex (BORC). Bloc1s2^-/- mice were embryonic lethal and exhibited defects in cortical development and hematopoiesis. Loss of BLOS2 resulted in elevated Notch signaling, which consequently increased the proliferation of neural progenitor cells and inhibited neuronal differentiation in cortices. Likewise, ablation of bloc1s2 in zebrafish or mice led to increased hematopoietic stem and progenitor cell production in the aorta-gonad-mesonephros region. BLOS2 physically interacted with Notch1 in endo-lysosomal trafficking of Notch1. Our findings suggest that BLOS2 is a novel negative player in regulating Notch signaling through lysosomal trafficking to control multiple stem and progenitor cell homeostasis in vertebrates.

Chemotherapy-induced neurotoxicity can reduce the quality of life of patients by affecting their intelligence, senses and mobility. Ten percent of safety-related late-stage clinical failures are due to neurological side effects. Animal models are poor in predicting human neurotoxicity due to interspecies differences and most in vitro assays cannot distinguish neurotoxicity from general cytotoxicity for chemotherapeutics. We developed in vitro assays capable of quantifying the paediatric neurotoxic potential for cytotoxic drugs. Mixed cultures of human fetal brain cells were differentiated in monolayers and as 3D-neurospheres in the presence of non-neurotoxic chemotherapeutics (etoposide, teniposide) or neurotoxicants (methylmercury). The cytotoxic potency towards dividing progenitors versus differentiated neurons and astrocytes was compared using: (1) immunohistochemistry staining and cell counts in monolayers; (2) through quantitative Western blots in neurospheres; and (3) neurosphere migration assays. Etoposide and teniposide, were 5-10 times less toxic to differentiated neurons compared to the mix of all cells in monolayer cultures. In contrast, the neurotoxicant methylmercury did not exhibit selectivity and killed all cells with the same potency. In 3D neurospheres, etoposide and teniposide were 24 to 10 times less active against neurons compared to all cells. These assays can be used prioritise drugs for local drug delivery to brain tumours.

Collapsin response mediator protein-2 (CRMP2) regulates axonal growth cone extension, and increased CRMP2 phosphorylation may lead to axonal degeneration. Axonal and synaptic pathology is an important feature of Lewy body dementias (LBD), but the state of CRMP2 phosphorylation (pCRMP2) as well as its correlations with markers of neurodegeneration have not been studied in these dementias. Hence, we measured CRMP2 phosphorylation at Thr509, Thr514 and Ser522, as well as markers of ß-amyloid (Aß), tau-phosphorylation, a-synuclein and synaptic function in the postmortem neocortex of a longitudinally assessed cohort of LBD patients characterized by low (Parkinson's disease dementia, PDD) and high (dementia with Lewy bodies, DLB) burden of Alzheimer type pathology. We found specific increases of pCRMP2 at Thr514 in DLB, but not PDD. The increased CRMP2 phosphorylation correlated with fibrillogenic Aß as well as with losses of markers for axon regeneration (ß-III-tubulin) and synaptic integrity (synaptophysin) in LBD. In contrast, pCRMP2 alterations did not correlate with tau-phosphorylation or a-synuclein, and also appear unrelated to immunoreactivities of putative upstream kinases glycogen synthase kinase 3ß and cyclin-dependent kinase 5, as well as to protein phosphatase 2A. In conclusion, increased pCRMP2 may underlie the axonal pathology of DLB, and may be a novel therapeutic target. However, antecedent signaling events as well as the nature of pCRMP2 association with Aß and other neuropathologic markers require further study.

The development of antitumor medication based on autologous stem cells is one of the most advanced methods in glioblastoma multiforme (GBM) treatment. However, there are no objective criteria for evaluating the effectiveness of this medication on cancer stem cells (CSCs). One possible criterion could be a change in the number of microglial cells and their specific location in the tumor. The present study aimed to understand the interaction between microglial cells and CSCs in an experimental glioblastoma model. C6 glioma cells were used to create a glioblastoma model, as they have the immunophenotypic characteristics of CSCs. The glioma cells (0.2×10⁶) were stereotactically implanted into the brains of 60 rats. On the 10th, 20th and 30th days after implantation, the animals were 15 of the animals were sacrificed, and the obtained materials were analyzed by morphological and immunohistochemical analysis. Implantation of glioma cells into the rat brains caused rapid development of tumors characterized by invasive growth, angiogenesis and a high rate of proliferation. The maximum concentration of microglia was observed in the tumor nodule between days 10 and 20; a high proliferation rate of cancer cells was also observed in this area. By day 30, necrosis advancement was observed and the maximum number of microglial cells was concentrated in the invasive area; the invasive area also exhibited positive staining for CSC marker antibodies. Microglial cells have a key role in the invasive growth processes of glioblastoma, as demonstrated by the location of CSCs in the areas of microglia maximum concentration. Therefore, the present study indicates that changes in microglia position and corresponding suppression of tumor growth may be objective criteria for evaluating the effectiveness of biomedical treatment against CSCs.

The loss of gut epithelium integrity leads to translocation of microbes and microbial products resulting in immune activation and drives systemic inflammation in acquired immunodeficiency syndrome (AIDS) patients. Although viral loads in HIV patients are significantly reduced in the post-cART era, inflammation and immune activation persist and can lead to morbidity. Here, we determined the interactive effects of the viral protein HIV-1 Tat and lipopolysaccharide (LPS) on enteric neurons and glia. Bacterial translocation was significantly enhanced in Tat-expressing (Tat+) mice. Exposure to HIV-1 Tat in combination with LPS enhanced the expression and release of the pro-inflammatory cytokines IL-6, IL-1ß and TNF-a in the ilea of Tat+ mice and by enteric glia. This coincided with enhanced NF-?B activation in enteric glia that was abrogated in glia from TLR4 knockout mice and by knockdown (siRNA) of MyD88 siRNA in wild type glia. The synergistic effects of Tat and LPS resulted in a reduced rate of colonic propulsion in Tat+ mice treated with LPS. These results show that HIV-1 Tat interacts with the TLR4 receptor to enhance the pro-inflammatory effects of LPS leading to gastrointestinal dysmotility and enhanced immune activation.

The cat shark is increasingly used as a model for Chondrichthyes, an evolutionarily important sister group of the bony vertebrates that include teleosts and tetrapods. In the bony vertebrates, the first axon tracts form a highly conserved early axon scaffold. The corresponding structure has not been well characterised in cat shark and will prove a useful model for comparative studies. Using pan-neural markers, the early axon scaffold of the cat shark, Scyliorhinus canicula, was analysed. Like in other vertebrates, the medial longitudinal fascicle was the first axon tract to form from a small cluster of neurones in the ventral brain. Subsequently, additional neuronal clusters and axon tracts emerged which formed an array of longitudinal, transversal, and commissural axons tracts in the Scyliorhinus canicula embryonic brain. The first structures to appear after the medial longitudinal fascicle were the tract of the postoptic commissure, the dorsoventral diencephalic tract, and the descending tract of the mesencephalic nucleus of the trigeminal nerve. These results confirm that the early axon scaffold in the embryonic brain is highly conserved through vertebrate evolution.

Sensory neurons are particularly susceptible to neuronal damage in diabetes, and silent mating type information regulation 2 homolog 1 (Sirt1) has been recently identified as a key gene in neuroprotection and wound healing. We found that the expression of Sirt1 was downregulated in trigeminal sensory neurons of diabetic mice. A microRNA microarray analysis identified microRNA-182 (miR-182) as a Sirt1 downstream effector, and the expression level of miR-182 was increased by Sirt1 overexpression in trigeminal neurons; Sirt1 bound to the promoter of miR-182 and regulated its transcription. We also revealed that miR-182 enhanced neurite outgrowth in isolated trigeminal sensory neurons and overcame the detrimental effects of hyperglycemia by stimulating corneal nerve regeneration by decreasing the expression of one of its target genes, NOX4. Furthermore, the effects of miR-182 on corneal nerve regeneration are associated with a functional recovery of corneal sensation in hyperglycemic conditions. These data demonstrate that miR-182 is a key regulator in diabetic corneal nerve regeneration through targeting NOX4, suggesting that miR-182 might be a potential target for the treatment of diabetic sensory nerve regeneration and diabetic keratopathy.

© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Congenital infection by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae of the central nervous system, including sensorineural deafness, cerebral palsies or devastating neurodevelopmental abnormalities (0.1% of all births). To gain insight on the impact of HCMV on neuronal development, we used both neural stem cells from human embryonic stem cells (NSC) and brain sections from infected fetuses and investigated the outcomes of infection on Peroxisome Proliferator-Activated Receptor gamma (PPAR?), a transcription factor critical in the developing brain. We observed that HCMV infection dramatically impaired the rate of neuronogenesis and strongly increased PPAR? levels and activity. Consistent with these findings, levels of 9-hydroxyoctadecadienoic acid (9-HODE), a known PPAR? agonist, were significantly increased in infected NSCs. Likewise, exposure of uninfected NSCs to 9-HODE recapitulated the effect of infection on PPAR? activity. It also increased the rate of cells expressing the IE antigen in HCMV-infected NSCs. Further, we demonstrated that (1) pharmacological activation of ectopically expressed PPAR? was sufficient to induce impaired neuronogenesis of uninfected NSCs, (2) treatment of uninfected NSCs with 9-HODE impaired NSC differentiation and (3) treatment of HCMV-infected NSCs with the PPAR? inhibitor T0070907 restored a normal rate of differentiation. The role of PPAR? in the disease phenotype was strongly supported by the immunodetection of nuclear PPAR? in brain germinative zones of congenitally infected fetuses (N = 20), but not in control samples. Altogether, our findings reveal a key role for PPAR? in neurogenesis and in the pathophysiology of HCMV congenital infection. They also pave the way to the identification of PPAR? gene targets in the infected brain.

Autophagy is a conserved, intracellular, lysosomal degradation pathway. While mechanistic aspects of this pathway are increasingly well defined, it remains unclear how autophagy modulation impacts normal physiology. It is, however, becoming clear that autophagy may play a key role in regulating developmental pathways. Here we describe for the first time how autophagy impacts stem cell differentiation by degrading Notch1. We define a novel route whereby this plasma membrane-resident receptor is degraded by autophagy, via uptake into ATG16L1-positive autophagosome-precursor vesicles. We extend our findings using a physiologically relevant mouse model with a hypomorphic mutation in Atg16L1, a crucial autophagy gene, which shows developmental retention of early-stage cells in various tissues where the differentiation of stem cells is retarded and thus reveal how modest changes in autophagy can impact stem cell fate. This may have relevance for diverse disease conditions, like Alzheimer's Disease or Crohn's Disease, associated with altered autophagy.

Loss-of-function mutations in the gene encoding GBA (glucocerebrosidase, ß, acid), the enzyme deficient in the lysosomal storage disorder Gaucher disease, elevate the risk of Parkinson disease (PD), which is characterized by the misprocessing of SNCA/a-synuclein. However, the mechanistic link between GBA deficiency and SNCA accumulation remains poorly understood. In this study, we found that loss of GBA function resulted in increased levels of SNCA via inhibition of the autophagic pathway in SK-N-SH neuroblastoma cells, primary rat cortical neurons, or the rat striatum. Furthermore, expression of the autophagy pathway component BECN1 was downregulated as a result of the GBA knockdown-induced decrease in glucocerebrosidase activity. Most importantly, inhibition of autophagy by loss of GBA function was associated with PPP2A (protein phosphatase 2A) inactivation via Tyr307 phosphorylation. C2-ceramide (C2), a PPP2A agonist, activated autophagy in GBA-silenced cells, while GBA knockdown-induced SNCA accumulation was reversed by C2 or rapamycin (an autophagy inducer), suggesting that PPP2A plays an important role in the GBA knockdown-mediated inhibition of autophagy. These findings demonstrate that loss of GBA function may contribute to SNCA accumulation through inhibition of autophagy via PPP2A inactivation, thereby providing a mechanistic basis for the increased PD risk associated with GBA deficiency.

Adolescent rats are prone to impaired fear extinction, suggesting that mechanistic differences in extinction could exist in adolescent and adult rats. Since the infralimbic cortex (IL) is critical for fear extinction, we used PCR array technology to identify gene expression changes in IL induced by fear extinction in adolescent rats. Interestingly, the ephrin type B receptor 2 (EphB2), a tyrosine kinase receptor associated with synaptic development, was downregulated in IL after fear extinction. Consistent with the PCR array results, EphB2 levels of mRNA and protein were reduced in IL after fear extinction compared with fear conditioning, suggesting that EphB2 signaling in IL regulates fear extinction memory in adolescents. Finally, reducing EphB2 synthesis in IL with shRNA accelerated fear extinction learning in adolescent rats, but not in adult rats. These findings identify EphB2 in IL as a key regulator of fear extinction during adolescence, perhaps due to the increase in synaptic remodeling occurring during this developmental phase.

Copyright © 2015 the authors 0270-6474/15/3512394-10$15.00/0.

Expanded GGGGCC (G4C2) nucleotide repeats within the C9ORF72 gene are the most common genetic mutation associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Sense and antisense transcripts of these expansions are translated to form five dipeptide repeat proteins (DRPs). We employed primary cortical and motor neuron cultures, live-cell imaging, and transgenic fly models and found that the arginine-rich dipeptides, in particular Proline-Arginine (PR), are potently neurotoxic. Factors that anticipated their neurotoxicity included aggregation in nucleoli, decreased number of processing bodies, and stress granule formation, implying global translational dysregulation as path accountable for toxicity. Nuclear PR aggregates were also found in human induced motor neurons and postmortem spinal cord tissues from C9ORF72 ALS and ALS/FTD patients. Intronic G4C2 transcripts, but not loss of C9ORF72 protein, are also toxic to motor and cortical neurons. Interestingly, G4C2 transcript-mediated neurotoxicity synergizes with that of PR aggregates, suggesting convergence of mechanisms.

Copyright © 2014 Elsevier Inc. All rights reserved.

Lysine-specific demethylase 1 (LSD1) is involved in gene regulation and development; however, its precise function, molecular targets and underlying mechanisms during development are poorly understood. Here we show that LSD1 is required for neuronal progenitor cell (NPC) maintenance during cortical development. A ChIP-seq analysis identified a LSD1-binding site (LBAL) downstream of Atrophin1 (ATN1). Surprisingly, tranylcypromine (LSD1 inhibitor) treatment increased H3K4 methylation at LBAL, leading to ATN1 repression and NPC differentiation. Knockdown of LSD1 and ATN1 phenocopied each other in inducing NPC premature differentiation and depletion, which could be rescued by ATN1 overexpression, suggesting that LSD1 controls NPC differentiation via regulation of ATN1 methylation status and expression. The involvement of LSD1 in ATN1 expression and NPC maintenance were confirmed in knockout mice. These findings hint at the potential application for the clinical drug, tranylcypromine, in the prevention and/or treatment of ATN1-associated degenerative disease, dentatorubral-pallidoluysian atrophy.

Somatic cells can be reprogrammed to reach an embryonic stem cell-like state by overexpression of defined factors. Recent studies have greatly improved the efficiency of the reprogramming process but the underlying mechanisms regulating the transition from a somatic to a pluripotent state are still relatively unknown. MicroRNAs (miRs) are small noncoding RNAs that primarily regulate target gene expression post-transcriptionally. Here we present a systematic and comprehensive study of microRNAs in mouse embryonic fibroblasts (MEFs) during the early stage of cell fate decisions and reprogramming to a pluripotent state, in which significant transcriptional and epigenetic changes occur. One microRNA found to be highly induced during this stage of reprogramming, miR-135b, targeted the expression of extracellular matrix (ECM) genes including Wisp1 and Igfbp5. Wisp1 was shown to be a key regulator of additional ECM genes that serve as barriers to reprogramming. Regulation of Wisp 1 is likely mediated through biglycan, a glycoprotein highly expressed in MEFs that is silenced in reprogrammed cells. Collectively, this report reveals a novel link between microRNA-mediated regulation of ECM formation and somatic cell reprogramming, and demonstrates that microRNAs are powerful tools to dissect the intracellular and extracellular molecular mechanisms of reprogramming.

© 2014 Li et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Glutamine synthetase catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine, thus playing a pivotal role in glutamate and glutamine homoeostasis. Despite a plethora of studies on this enzyme, knowledge about the regional and cellular distribution of this enzyme in human brain is still fragmentary. Therefore, we mapped fourteen post-mortem brains of psychically healthy individuals for the distribution of the glutamine synthetase immunoreactive protein. It was found that glutamine synthetase immunoreactivity is expressed in multiple gray and white matter astrocytes, but also in oligodendrocytes, ependymal cells and certain neurons. Since a possible extra-astrocytic expression of glutamine synthetase is highly controversial, we paid special attention to its appearance in oligodendrocytes and neurons. By double immunolabeling of mouse brain slices and cultured mouse brain cells for glutamine synthetase and cell-type-specific markers we provide evidence that besides astrocytes subpopulations of oligodendrocytes, microglial cells and neurons express glutamine synthetase. Moreover, we show that glutamine synthetase-immunopositive neurons are not randomly distributed throughout human and mouse brain, but represent a subpopulation of nitrergic (i.e. neuronal nitric oxide synthase expressing) neurons. Possible functional implications of an extra-astrocytic localization of glutamine synthetase are discussed.

Copyright © 2014 Elsevier B.V. All rights reserved.

Achieving safe and readily accessible sources for cell replacement therapy in Parkinson's disease (PD) is still a challenging unresolved issue. Recently, a primitive neural crest stem cell population (hOMSC) was isolated from the adult human oral mucosa and characterized in vitro and in vivo. In this study we assessed hOMSC ability to differentiate into dopamine-secreting cells with a neuronal-dopaminergic phenotype in vitro in response to dopaminergic developmental cues and tested their therapeutic potential in the hemi-Parkinsonian rat model. We found that hOMSC express constitutively a repertoire of neuronal and dopaminergic markers and pivotal transcription factors. Soluble developmental factors induced a reproducible neuronal-like morphology in the majority of hOMSC, downregulated stem cells markers, upregulated the expression of the neuronal and dopaminergic markers that resulted in dopamine release capabilities. Transplantation of these dopaminergic-induced hOMSC into the striatum of hemi-Parkinsonian rats improved their behavioral deficits as determined by amphetamine-induced rotational behavior, motor asymmetry and motor coordination tests. Human TH expressing cells and increased levels of dopamine in the transplanted hemispheres were observed 10 weeks after transplantation. These results demonstrate for the first time that soluble factors involved in the development of DA neurons, induced a DA phenotype in hOMSC in vitro that significantly improved the motor function of hemiparkinsonian rats. Based on their neural-related origin, their niche accessibility by minimal-invasive procedures and their propensity for DA differentiation, hOMSC emerge as an attractive tool for autologous cell replacement therapy in PD.

Imprinted genes play a critical role in brain development and mental health, although the underlying molecular and cellular mechanisms remain incompletely understood. The family of basic helix-loop-helix (bHLH) proteins directs the proliferation, differentiation, and specification of distinct neuronal progenitor populations. Here, we identified the bHLH factor gene Tcf4 as a direct target gene of Zac1/Plagl1, a maternally imprinted transcriptional regulator, during early neurogenesis. Zac1 and Tcf4 expression levels concomitantly increased during neuronal progenitor differentiation; moreover, Zac1 interacts with two cis-regulatory elements in the Tcf4 gene locus, and these elements together confer synergistic activation of the Tcf4 gene. Tcf4 upregulation enhances the expression of the cyclin-dependent kinase inhibitor gene p57(Kip2), a paternally imprinted Tcf4 target gene, and increases the number of cells in G1 phase. Overall, we show that Zac1 controls cell cycle arrest function in neuronal progenitors through induction of p57(Kip2) via Tcf4 and provide evidence for cooperation between imprinted genes and a bHLH factor in early neurodevelopment.

Transcriptional dysregulation is a major pathological feature of Huntington's disease (HD). The goal of this study was to understand how p65/RelA co-regulated genes, specifically those of the cytokine and endocannabinoid systems, were affected in HD. p65/RelA levels were lower in human HD tissue and R6/2 HD mice, as were the levels of the type 1 cannabinoid receptor (CB1), IL-1ß, IL-8, CCL5, GM-CSF, MIP-1ß, and TNFa, all of which may be regulated by p65/RelA. Activation of p65/RelA restored CB1 and CCL5 expression in STHdh cell models of HD. Therefore, p65/RelA activation may normalize the expression of some genes in HD.

Copyright © 2013 Elsevier B.V. All rights reserved.

Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required.

Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation-sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type-specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types.

Drug addiction is a chronic brain disease that is a serious social problem and causes enormous financial burden. Because mitochondrial abnormalities have been associated with opiate addiction, we examined the effect of morphine on mtDNA levels in rat and mouse models of addiction and in cultured cells. We found that mtDNA copy number was significantly reduced in the hippocampus and peripheral blood of morphine-addicted rats and mice compared with control animals. Concordantly, decreased mtDNA copy number and elevated mtDNA damage were observed in the peripheral blood from opiate-addicted patients, indicating detrimental effects of drug abuse and stress. In cultured rat pheochromocytoma (PC12) cells and mouse neurons, morphine treatment caused many mitochondrial defects, including a reduction in mtDNA copy number that was mediated by autophagy. Knockdown of the Atg7 gene was able to counteract the loss of mtDNA copy number induced by morphine. The mitochondria-targeted antioxidant melatonin restored mtDNA content and neuronal outgrowth and prevented the increase in autophagy upon morphine treatment. In mice, coadministration of melatonin with morphine ameliorated morphine-induced behavioral sensitization, analgesic tolerance and mtDNA content reduction. During drug withdrawal in opiate-addicted patients and improvement of protracted abstinence syndrome, we observed an increase of serum melatonin level. Taken together, our study indicates that opioid addiction is associated with mtDNA copy number reduction and neurostructural remodeling. These effects appear to be mediated by autophagy and can be salvaged by melatonin.

The molecular mechanism underlying the selective vulnerability of certain neuronal populations associated with neurodegenerative diseases remains poorly understood. Basal autophagy is important for maintaining axonal homeostasis and preventing neurodegeneration. In this paper, we demonstrate that mice deficient in the metazoan-specific autophagy gene Epg5/epg-5 exhibit selective damage of cortical layer 5 pyramidal neurons and spinal cord motor neurons. Pathologically, Epg5 knockout mice suffered muscle denervation, myofiber atrophy, late-onset progressive hindquarter paralysis, and dramatically reduced survival, recapitulating key features of amyotrophic lateral sclerosis (ALS). Epg5 deficiency impaired autophagic flux by blocking the maturation of autophagosomes into degradative autolysosomes, leading to accumulation of p62 aggregates and ubiquitin-positive inclusions in neurons and glial cells. Epg5 knockdown also impaired endocytic trafficking. Our study establishes Epg5-deficient mice as a model for investigating the pathogenesis of ALS and indicates that dysfunction of the autophagic-endolysosomal system causes selective damage of neurons associated with neurodegenerative diseases.

The transcription factors Sox2, Oct4, and Nanog regulate within a narrow dose-range embryonic stem (ES) cell pluripotency and cell lineage commitment. Excess of Oct4 relative to Sox2 guides cells to mesoendoderm (ME), while abundance of Sox2 promotes neuroectoderm (NE) formation. Literature does not address whether ethanol interferes with these regulatory interactions during neural development. We hypothesized that ethanol exposure of ES cells in early differentiation causes an imbalance of Oct4 and Sox2 that diverts cells away from NE to ME lineage, consistent with the teratogenesis effects caused by prenatal alcohol exposure. Mouse ES cells were exposed to ethanol (0, 25, 50, and 100?mM) during retinoic acid (10?nM)-directed differentiation to NE for 0-6 days, and the expression of Sox2, Oct4, and Nanog was measured in single live cells by multiparametric flow cytometry, and the cellular phenotype was characterized by immunocytochemistry. Our data showed an ethanol dose- and time-dependent asymmetric modulation of Oct4 and Sox2 expression, as early as after 2 days of exposure. Single-cell analysis of the correlated expression of Sox2, Oct4, and Nanog revealed that ethanol promoted distinct subpopulations with a high Oct4/Sox2 ratio. Ethanol-exposed cells differentiated to fewer ß-III tubulin-immunoreactive cells with an immature neuronal phenotype by 4 days. We interpret these data as suggesting that ethanol diverted cells in early differentiation from the NE fate toward the ME lineage. Our results provide a novel insight into the mode of ethanol action and opportunities for discovery of prenatal biomarkers at early stages.

Adult stem cells from the dermis would be an attractive cell source for therapeutic purposes as well as studying the process of skin aging. Several studies have reported that human dermal stem/progenitor cells (hDSPCs) with multipotent properties exist within the dermis of adult human skin. However, these cells have not been well characterized, because methods for their isolation or enrichment have not yet been optimized. In the present study, we enriched high side scatter (SSC(high))-hDSPCs from normal human dermal fibroblasts using a structural characteristic, intracellular granularity, as a sorting parameter. The SSC(high)-hDSPCs had high in vitro proliferation properties and expressed high levels of SOX2 and S100B, similar to previously identified mouse SOX2+ hair follicle dermal stem cells. The SSC(high)-hDSPCs could differentiate into not only mesodermal cell types, for example, adipocytes, chondrocytes, and osteoblasts, but also neuroectodermal cell types, such as neural cells. In addition, the SSC(high)-hDSPCs exhibited no significant differences in the expression of nestin, vimentin, SNAI2, TWIST1, versican, and CORIN compared with non-hDSPCs. These cells are therefore different from the previously identified multipotent fibroblasts and skin-derived progenitors. In this study, we suggest that hDSPCs can be enriched by using characteristic of their high intracellular granularity, and these SSC(high)-hDSPCs exhibit high in vitro proliferation and differentiation potentials.

Doublecortin (Dcx) is the causative gene for X-linked lissencephaly, which encodes a microtubule-binding protein. Axon tracts are abnormal in both affected individuals and in animal models. To determine the reason for the axon tract defect, we performed a semiquantitative proteomic analysis of the corpus callosum in mice mutant for Dcx. In axons from mice mutant for Dcx, widespread differences are found in actin-associated proteins as compared with wild-type axons. Decreases in actin-binding proteins a-actinin-1 and a-actinin-4 and actin-related protein 2/3 complex subunit 3 (Arp3), are correlated with dysregulation in the distribution of filamentous actin (F-actin) in the mutant neurons with increased F-actin around the cell body and decreased F-actin in the neurites and growth cones. The actin distribution defect can be rescued by full-length Dcx and further enhanced by Dcx S297A, the unphosphorylatable mutant, but not with the truncation mutant of Dcx missing the C-terminal S/P-rich domain. Thus, the C-terminal region of Dcx dynamically regulates formation of F-actin features in developing neurons, likely through interaction with spinophilin, but not through a-actinin-4 or Arp3. We show with that the phenotype of Dcx/Doublecortin-like kinase 1 deficiency is consistent with actin defect, as these axons are selectively deficient in axon guidance, but not elongation.

Ei24 is a DNA damage response gene involved in growth suppression and apoptosis. The physiological function of Ei24, however, is poorly understood. Here we generated conditional knock-out mice of Ei24 and demonstrated that EI24 is an essential component of the basal autophagy pathway. Mice with neural-specific Ei24 deficiency develop age-dependent neurological abnormalities caused by massive axon degeneration and extensive neuron loss in brain and spinal cord. Notably, ablation of Ei24 leads to vacuolated oligodendroglial cells and demyelination of axons. Liver-specific depletion of Ei24 causes severe hepatomegaly with hepatocyte hypertrophy. Ei24 deficiency impairs autophagic flux, leading to accumulation of LC3, p62 aggregates, and ubiquitin-positive inclusions. Our study indicates that Ei24 is an essential autophagy gene and plays an important role in clearance of aggregate-prone proteins in neurons and hepatocytes.

Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increasingly lineage restricted. We examined retinoic acid-induced changes in the cellular proteoglycan composition of the well-characterized teratocarcinoma line NCCIT. Our analysis revealed changes in the abundance of transcripts for genes encoding core proteins, enzymes that are responsible for early and late linkage region biosynthesis, as well as enzymes for GAG chain extension and modification. Transcript levels for genes encoding core proteins used as backbones for polysaccharide synthesis revealed highly significant increases in expression of lumican and decorin, 1,500-fold and 2,800-fold, respectively. Similarly, glypican 3, glypican 5, versican and glypican 6 showed increases between 5 and 70-fold. Significant decreases in biglycan, serglycin, glypican 4, aggrecan, neurocan, CD74 and glypican 1 were observed. Disaccharide analysis of the glycans in heparin/heparan sulfate and chondroitin/dermatan sulfate revealed retinoic acid-induced changes restricted to chondroitin/dermatan sulfate glycans. Our study provides the first detailed analysis of changes in the glycosaminoglycan profile of human pluripotent cells upon treatment with the retinoic acid morphogen.

Somatic cells can be reprogrammed to form embryonic stem cell-like induced pluripotent stem cells (iPSCs), but the process suffers from low efficiency and the underlying molecular mechanisms that control reprogramming remain poorly understood. Here we perform an inhibitor screen to identify kinases that enhance, or present a barrier to, reprogramming. In particular, inhibitors of p38, inositol trisphosphate 3-kinase, and Aurora A kinase potently enhance iPSC generation, and iPSCs derived from inhibitor-treated somatic cells are capable of reaching a fully reprogrammed state. Knockdown of target kinases by short interfering RNAs confirms that they function as barrier genes. We show that Aurora A kinase, which functions in centrosome activity and spindle assembly, is highly induced during reprogramming and inhibits Akt-mediated inactivation of GSK3ß, resulting in compromised reprogramming efficiency. Together, our results not only identify new compounds that enhance iPSC generation but also shed new light on the function of Aurora A kinase in the reprogramming process.

The formation of advanced glycation end products is one of the major factors involved in diabetic neuropathy, aging, and neurodegenerative diseases. Reactive carbonyl compounds, such as methylglyoxal (MG), play a key role in cross-linking to various proteins in the extracellular matrix, especially in neurons, which have a high rate of oxidative metabolism. The MG effect was tested on dorsal root ganglia primary neurons in cultures from adult male Balb/c mice. Lower MG doses contribute to an increased adherence of neurons on their support and an increased glia proliferation, as proved by MTS assay and bright-field microscopy. Time-lapse fluorescence microscopy by Fura-2 was performed for monitoring the relative fluorescence ratio changes (?R/R(0)) upon depolarization and immunofluorescence staining for quantifying the degree of neurites extension. The relative change in fluorescence ratio modifies the amplitude and dispersion depending on the subtype of sensory neurons, the medium-sized neurons are more sensitive to MG treatment when compared to small ones. Low MG concentrations (0-150 µM) increase neuronal viability, excitability, and the capacity of neurite extension, while higher concentrations (250-750 µM) are cytotoxic in a dose-dependent manner. In our opinion, MG could be metabolized by the glyoxalase system inside sensory neurons up to a threshold concentration, afterwards disturbing the cell equilibrium. Our study points out that MG has a dual effect concentration dependent on the neuronal viability, excitability, and neurite outgrowth, but only the excitability changes are soma-sized dependent. In conclusion, our data may partially explain the distinct neuronal modifications in various neurodegenerative pathologies.

Lamins A and C, alternatively spliced products of the LMNA gene, are key components of the nuclear lamina. The two isoforms are found in similar amounts in most tissues, but we observed an unexpected pattern of expression in the brain. Western blot and immunohistochemistry studies showed that lamin C is abundant in the mouse brain, whereas lamin A and its precursor prelamin A are restricted to endothelial cells and meningeal cells and are absent in neurons and glia. Prelamin A transcript levels were low in the brain, but this finding could not be explained by alternative splicing. In lamin A-only knockin mice, where alternative splicing is absent and all the output of the gene is channeled into prelamin A transcripts, large amounts of lamin A were found in peripheral tissues, but there was very little lamin A in the brain. Also, in knockin mice expressing exclusively progerin (a toxic form of prelamin A found in Hutchinson-Gilford progeria syndrome), the levels of progerin in the brain were extremely low. Further studies showed that prelamin A expression, but not lamin C expression, is down-regulated by a brain-specific microRNA, miR-9. Expression of miR-9 in cultured cells reduced lamin A expression, and this effect was abolished when the miR-9-binding site in the prelamin A 3' UTR was mutated. The down-regulation of prelamin A expression in the brain could explain why mouse models of Hutchinson-Gilford progeria syndrome are free of central nervous system pathology.

Transplantation of human neural stem cells (hNSCs) is emerging as a viable treatment for stroke related brain injury. However, intraparenchymal grafts do not regenerate lost tissue, but rather integrate into the host parenchyma without significantly affecting the lesion cavity. Providing a structural support for the delivered cells appears important for cell based therapeutic approaches. The non-invasive monitoring of therapeutic methods would provide valuable information regarding therapeutic strategies but remains a challenge. Labeling transplanted cells with metal-based (1)H-magnetic resonance imaging (MRI) contrast agents affects the visualization of the lesion cavity. Herein, we demonstrate that a (19)F-MRI contrast agent can adequately monitor the distribution of transplanted cells, whilst allowing an evaluation of the lesion cavity and the formation of new tissue on (1)H-MRI scans. Twenty percent of cells labeled with the (19)F agent were of host origin, potentially reflecting the re-uptake of label from dead transplanted cells. Both T(2)- and diffusion-weighted MRI scans indicated that transplantation of hNSCs suspended in a gel form of a xenogeneic extracellular matrix (ECM) bioscaffold resulted in uniformly distributed cells throughout the lesion cavity. However, diffusion MRI indicated that the injected materials did not yet establish diffusion barriers (i.e. cellular network, fiber tracts) normally found within striatal tissue. The ECM bioscaffold therefore provides an important support to hNSCs for the creation of de novo tissue and multi-nuclei MRI represents an adept method for the visualization of some aspects of this process. However, significant developments of both the transplantation paradigm, as well as regenerative imaging, are required to successfully create new tissue in the lesion cavity and to monitor this process non-invasively.

Copyright © 2011 Elsevier Ltd. All rights reserved.

Smith-Lemli-Opitz syndrome (SLOS) is an inborn error of metabolism caused by defective cholesterol biosynthesis. Mutations within the gene encoding 7-dehydrocholesterol reductase (DHCR7), the last enzyme in the pathway, lead to the accumulation of 7-dehydrocholesterol (7-DHC) in the brain tissue and blood of the SLOS patients. The objective of this study was to determine the consequences of the accumulation of an immediate cholesterol precursor, 7-DHC and its oxysterol metabolite, 3ß,5a-dihydroxycholest-7-en-6-one (DHCEO), in the brain tissue of Dhcr7-KO mouse, a model for SLOS. We found that cholesterol, 7-DHC and DHCEO show region-specific distribution, suggesting that the midbrain and the cortex are the primary sites of vulnerability. We also report that neurons are ten fold more susceptible to a 7-DHC-derived oxysterol mixture than glial cells, and that DHCEO accelerates differentiation and arborization of cortical neurons. The overall results suggest that 7-DHC oxidative metabolites are critical contributors to altered neural development in SLOS. The future studies will test if antioxidant supplementation will ameliorate some of the clinical symptoms associated with this devastating disease.

Copyright © 2011 Elsevier Inc. All rights reserved.

Recent breakthroughs in creating induced pluripotent stem cells (iPSCs) provide alternative means to obtain embryonic stem-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. iPSCs are versatile tools for investigating early developmental processes and could become sources of tissues or cells for regenerative therapies. Here, for the first time, we describe a strategy to analyze genomics datasets of mouse embryonic fibroblasts (MEFs) and embryonic stem cells to identify genes constituting barriers to iPSC reprogramming. We further show that computational chemical biology combined with genomics analysis can be used to identify small molecules regulating reprogramming. Specific downregulation by small interfering RNAs (siRNAs) of several key MEF-specific genes encoding proteins with catalytic or regulatory functions, including WISP1, PRRX1, HMGA2, NFIX, PRKG2, COX2, and TGFß3, greatly increased reprogramming efficiency. Based on this rationale, we screened only 17 small molecules in reprogramming assays and discovered that the nonsteroidal anti-inflammatory drug Nabumetone and the anticancer drug 4-hydroxytamoxifen can generate iPSCs without Sox2. Nabumetone could also produce iPSCs in the absence of c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPSCs. In summary, we report a new concept of combining genomics and computational chemical biology to identify new drugs useful for iPSC generation. This hypothesis-driven approach provides an alternative to shot-gun screening and accelerates understanding of molecular mechanisms underlying iPSC induction.

Copyright © 2011 AlphaMed Press.

Although induced pluripotent stem cells (iPSCs) hold great promise for customized regenerative medicine, the molecular basis of reprogramming is largely unknown. Overcoming barriers that maintain cell identities is a critical step in the reprogramming of differentiated cells. Since microRNAs (miRNAs) modulate target genes tissue-specifically, we reasoned that distinct mouse embryonic fibroblast (MEF)-enriched miRNAs post-transcriptionally modulate proteins that function as reprogramming barriers. Inhibiting these miRNAs should influence cell signaling to lower those barriers. Here we show that depleting miR-21 and miR-29a enhances reprogramming efficiency in MEFs. We also show that the p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. In addition, we provide the first evidence that c-Myc enhances reprogramming partly by repressing MEF-enriched miRNAs, such as miR-21 and miR-29a. Our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in iPSC generation. These studies should facilitate development of clinically applicable reprogramming strategies.

Three-dimensional topology of DNA in the cell nucleus provides a level of transcription regulation beyond the sequence of the linear DNA. To study the relationship between the transcriptional activity and the spatial environment of a gene, we used allele-specific chromosome conformation capture-on-chip (4C) technology to produce high-resolution topology maps of the active and inactive X chromosomes in female cells. We found that loci on the active X form multiple long-range interactions, with spatial segregation of active and inactive chromatin. On the inactive X, silenced loci lack preferred interactions, suggesting a unique random organization inside the inactive territory. However, escapees, among which is Xist, are engaged in long-range contacts with each other, enabling identification of novel escapees. Deletion of Xist results in partial refolding of the inactive X into a conformation resembling the active X without affecting gene silencing or DNA methylation. Our data point to a role for Xist RNA in shaping the conformation of the inactive X chromosome at least partially independent of transcription.

Despite extensive research to develop an effective neuroprotective strategy for the treatment of ischemic stroke, therapeutic options remain limited. Although caspase-dependent death is thought to play a prominent role in neuronal injury, direct evidence of active initiator caspases in stroke and the functional relevance of this activity have not previously been shown. Using an unbiased caspase-trapping technique in vivo, we isolated active caspase-9 from ischemic rat brain within 1 h of reperfusion. Pathogenic relevance of active caspase-9 was shown by intranasal delivery of a novel cell membrane-penetrating highly specific inhibitor for active caspase-9 at 4 h postreperfusion (hpr). Caspase-9 inhibition provided neurofunctional protection and established caspase-6 as its downstream target. The temporal and spatial pattern of expression demonstrates that neuronal caspase-9 activity induces caspase-6 activation, mediating axonal loss by 12 hpr followed by neuronal death within 24 hpr. Collectively, these results support selective inhibition of these specific caspases as an effective therapeutic strategy for stroke.

Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative, clonogenic and multipotent stem cells with a neural crest cell origin. Additionally, they can be collected with minimal invasiveness in comparison with other sources of mesenchymal stem cells (MSCs). Therefore, SHED could be a desirable option for potential therapeutic applications. In this study, SHEDs were established from enzyme-disaggregated deciduous dental pulp obtained from 6 to 9 year-old children. The cells had typical fibroblastoid morphology and expressed antigens characteristic of MSCs, STRO1, CD146, CD45, CD90, CD106 and CD166, but not the hematopoietic and endothelial markers, CD34 and CD31, as assessed by FACS analysis. Differentiation assessment revealed a strong osteogenic and adipogenic potential of SHEDs. In order to further evaluate the in vitro differentiation potential of SHED into neural cells, a simple short time growth factor-mediated induction was used. Immunofluorescence staining and flow cytometric analysis revealed that SHED rapidly expressed nestin and b-III tubulin, and later expressed intermediate neural markers. In addition, the intensity and percentages of nestin and b-III tubulin and mature neural markers (PSA-NCAM, NeuN, Tau, TH, or GFAP) increased significantly following treatment. Moreover, RT-PCR and Western blot analyses showed that the neural markers were strongly up-regulated after induction. In conclusion, these results provide evidence that SHED can differentiate into neural cells by the expression of a comprehensive set of genes and proteins that define neural-like cells in vitro. SHED cells might be considered as new candidates for the autologous transplantation of a wide variety of neurological diseases and neurotraumatic injuries.

Somatic cells can be reprogrammed to an ES-like state to create induced pluripotent stem cells (iPSCs) by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and cMyc. Here, we show that cellular microRNAs (miRNAs) regulate iPSC generation. Knock-down of key microRNA pathway proteins resulted in significant decreases in reprogramming efficiency. Three miRNA clusters, miR-17~92, miR-106b~25 and miR-106a~363, were shown to be highly induced during early reprogramming stages. Several miRNAs, including miR-93 and miR-106b, which have very similar seed regions, greatly enhanced iPSC induction and modulated mesenchymal-to-epithelial transition step in the initiation stage of reprogramming, and inhibiting these miRNAs significantly decreased reprogramming efficiency. Moreover, miR-iPSC clones reached the fully reprogrammed state. Further analysis revealed that Tgfbr2 and p21 are directly targeted by these miRNAs and that siRNA knock-down of both genes indeed enhanced iPSC induction. Here, for the first time, we demonstrate that miR-93 and its family members directly target TGF-ß receptor II to enhance iPSC generation. Overall, we demonstrate that miRNAs function in the reprogramming process and that iPSC induction efficiency can be greatly enhanced by modulating miRNA levels in cells.

Neuroblastoma, a cancer of the sympathetic nervous system, is the most common extracranial solid tumor in children. MYCN amplification and increased BDNF/TrkB signaling are features of high-risk tumors; yet, only ˜25% of malignant tumors display these features. Thus, the identification of additional biomarkers and therapeutic targets is essential. As aminoacylase 1 (ACY1), an amino acid deacetylase, is a putative tumor suppressor in small cell lung and renal cell carcinomas, we investigated whether it or the other family members aspartoacylase (ASPA, aminoacylase 2) or aminoacylase 3 (ACY3) could serve a similar function in neuroblastoma. Aminoacylase expression was examined in TrkB-positive, MYCN-amplified (SMS-KCNR and SK-N-BE) and TrkB-negative, non-MYCN-amplified (SK-N-AS, SK-N-SH, SH-SY5Y and SH-EP) neuroblastoma cell lines. Each aminoacylase exhibited distinct spatial localization (i.e., cytosolic ACY1, membrane-associated ASPA and nuclear ACY3). When SK-N-SH cells were treated with neural differentiation agents (e.g., retinoic acid and cAMP) in media containing 10% serum, ACY1 was the only aminoacylase whose expression was upregulated. ASPA was primarily expressed in SH-EP cells of a glial sublineage. ACY3 was more highly expressed in the TrkB-positive, MYCN-amplified lines. All three aminoacylases were expressed in normal human adrenal gland, a common site of neuroblastoma origin, but only ACY1 and ACY3 displayed detectable expression in primary neuroblastoma tumor. Bioinformatics data mining of Kaplan-Meier survival revealed that high ACY3 expression is correlated with poor prognosis, whereas low expression of ACY1 or ASPA is correlated with poor prognosis. These data suggest that aminoacylase expression is dysregulated in neuroblastoma.

Copyright © 2010 UICC.

Neural stem cells (NSCs), either isolated from fetal or adult human brain or derived from induced pluripotent stem cells, are now considered major candidates for in vitro generation of transplantable dopaminergic (DA) neurons and modeling of Parkinson's disease. It is generally thought that in vitro differentiation of neural stem cells into meso-diencephalic dopaminergic neurons, requires recapitulation of dopaminergic differentiation pathway normally occurring in the ventral mesencephalon during embryogenesis. This dopaminergic pathway is partially activated by a combination of the extracellular induction factors Sonic Hedgehog (Shh), Fibroblast Growth Factor 8 (FGF8) and Wnt1 that trigger specific intracellular transcription cascades. In vitro mimicking of these embryonic ventral mesencephalic conditions has been successful for dopaminergic differentiation of embryonic stem cells and ventral mesencephalic NSCs. Dopaminergic differentiation of non-mesencephalic NSCs (nmNSCs), however, is considered arduous. Here we examine whether Shh, FGF8 and Wnt1 can activate typical dopaminergic transcription factors, such as Lmx1a, Msx1 and Otx2 in nmNSCs. We found that Shh, FGF8 and Wnt1 induced the expression of Lmx1a and Otx2 in nmNSCs resulting in the differentiation of up to 39% of the nmNSCs into neurons expressing Pitx3. However, only a low number ( approximately 13%) of these cells became more DA-like neurons also expressing tyrosine hydroxylase (TH). The histone deacetylase (HDAC)-inhibitor trichostatin A combined with Shh, FGF8 and Wnt1 caused orchestrated induction of Lmx1a, Otx2, Msx1 plus the early DA transcription factor En1. Now significantly increased numbers of TH ( approximately 22%) and Pitx3 ( approximately 33%) neurons were observed. Most of these cells coexpressed the DA markers DAT and Vmat2. Taken together, we demonstrate that nmNSCs indeed can be differentiated towards DA-like neurons, but this differentiation is far from complete in comparison to ventral mesencephalic NSCs and embryonic stem cells; most likely, the nmNSCs lack the proper "primed" epigenetic state of these cells for DA differentiation facilitating the induction of DA specific transcription factors.

Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

A fast and simple model which uses lower animals on the evolutionary scale is beneficial for developing procedures for the reversal of neurobehavioral teratogenicity with neural stem cells. Here, we established a procedure for the derivation of chick neural stem cells, establishing embryonic day (E) 10 as optimal for progression to neuronal phenotypes. Cells were obtained from the embryonic cerebral hemispheres and incubated for 5-7 days in enriched medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) according to a procedure originally developed for mice. A small percentage of the cells survived, proliferated and formed nestin-positive neurospheres. After removal of the growth factors to allow differentiation (5 days), 74% of the cells differentiated into all major lineages of the nervous system, including neurons (Beta III tubulin-positive, 54% of the total number of differentiated cells), astrocytes (GFAP-positive, 26%), and oligodendrocytes (O4-positive, 20%). These findings demonstrate that the cells were indeed neural stem cells. Next, the cells were transplanted in two allograft chick models; (1) direct cerebral transplantation to 24-h-old chicks, followed by post-transplantation cell tracking at 24 h, 6 days and 14 days, and (2) intravenous transplantation to chick embryos on E13, followed by cell tracking on E19. With both methods, transplanted cells were found in the brain. The chick embryo provides a convenient, precisely-timed and unlimited supply of neural progenitors for therapy by transplantation, as well as constituting a fast and simple model in which to evaluate the ability of neural stem cell transplantation to repair neural damage, steps that are critical for progress toward therapeutic applications.

Copyright 2010 Elsevier Inc. All rights reserved.

Urodeles and fetal mammals are capable of impressive epimorphic regeneration in a variety of tissues, whereas the typical default response to injury in adult mammals consists of inflammation and scar tissue formation. One component of epimorphic regeneration is the recruitment of resident progenitor and stem cells to a site of injury. Bioactive molecules resulting from degradation of extracellular matrix (ECM) have been shown to recruit a variety of progenitor and stem cells in vitro in adult mammals. The ability to recruit multipotential cells to the site of injury by in vivo administration of chemotactic ECM degradation products in a mammalian model of digit amputation was investigated in the present study. Adult, 6- to 8-week-old C57/BL6 mice were subjected to midsecond phalanx amputation of the third digit of the right hind foot and either treated with chemotactic ECM degradation products or left untreated. At 14 days after amputation, mice treated with ECM degradation products showed an accumulation of heterogeneous cells that expressed markers of multipotency, including Sox2, Sca1, and Rex1 (Zfp42). Cells isolated from the site of amputation were capable of differentiation along neuroectodermal and mesodermal lineages, whereas cells isolated from control mice were capable of differentiation along only mesodermal lineages. The present findings demonstrate the recruitment of endogenous stem cells to a site of injury, and/or their generation/proliferation therein, in response to ECM degradation products.

Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential in brain, and are committed cells of the central nervous system. Recently, some reports have suggested the possibility of the NSCs to differentiate into non-CNS mesodermal derivatives, such as blood cells and skeletal muscle cells. Here we isolated NSCs as neurospheres from a neonatal mouse brain using serum replacement medium, and demonstrated that the stem cell population expressing pluripotent-related genes such as Oct-4, Sox-2, and Nanog possess multiple differentiation potentials to ectodermal, mesodermal, and endodermal lineages, that is, some neural cells, beating cardiomyocytes, adipocytes, and insulin-producing cells. The results of the present study partly provide further evidence for multiple differentiation properties of NSCs and suggest common characteristics between NSCs and other pluripotent stem cells.

Neuronal differentiation is a complex process that involves a plethora of regulatory steps. To identify transcription factors that influence neuronal differentiation we developed a high throughput screen using embryonic stem (ES) cells. Seven-hundred human transcription factor clones were stably introduced into mouse ES (mES) cells and screened for their ability to induce neuronal differentiation of mES cells. Twenty-four factors that are capable of inducing neuronal differentiation were identified, including four known effectors of neuronal differentiation, 11 factors with limited evidence of involvement in regulating neuronal differentiation, and nine novel factors. One transcription factor, Oct-2, was studied in detail and found to be a bifunctional regulator: It can either repress or induce neuronal differentiation, depending on the particular isoform. Ectopic expression experiments demonstrate that isoform Oct-2.4 represses neuronal differentiation, whereas Oct-2.2 activates neuron formation. Consistent with a role in neuronal differentiation, Oct-2.2 expression is induced during differentiation, and cells depleted of Oct-2 and its homolog Oct-1 have a reduced capacity to differentiate into neurons. Our results reveal a number of transcription factors potentially important for mammalian neuronal differentiation, and indicate that Oct-2 may serve as a binary switch to repress differentiation in precursor cells and induce neuronal differentiation later during neuronal development.

To develop transplantation of neural stem/progenitor cells (NSPCs) as a successful treatment of neurodegenerative disorders, the possible induction of an inflammatory response following implantation needs to be taken into consideration. Inflammatory cytokines can upregulate major histocompatibility complex (MHC) expression on transplanted cells, thereby rendering them more susceptible to graft rejection. Furthermore, cytokines also have a profound effect on cell differentiation, migration, and proliferation, which can greatly affect the outcome of transplantation. Here we studied the effect of three inflammatory cytokines, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6), from three different species (human, monkey, rat) on expression of MHC molecules and differentiation of two human NSPC lines derived from striatum and hippocampus. Human and monkey IFN-gamma strongly upregulate MHC expression in both NSPC lines in a dose-dependent manner, whereas rat IFN-gamma has an effect on MHC expression only in hippocampal cells. Furthermore, TNF-alpha, but not IL-6, upregulates MHC expression in both NSPC lines. Differentiation of NSPCs in the presence of cytokines showed that IFN-gamma increased the neuronal yield threefold in striatal NSPC cultures and increased the number of oligodendrocytes twofold in hippocampal NSPC cultures. Addition of TNF-alpha enhanced gliogenesis in both cell lines, whereas IL-6 stimulated neurogenesis. Human NSPC lines' response to cytokines is therefore species specific and also dependent on the NSPCs' region of origin. The successful translation of different cell lines from animal models to clinical trials could be substantially influenced by the species-specific regulation of MHC and differentiation as reported here. Disclosure of potential conflicts of interest is found at the end of this article.

In the present study, we examined the neuroprotective effects and mechanisms of implanted human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in ischemic stroke. hUC-MSCs were isolated from the endothelial/subendothelial layers of the human umbilical cord and cultured. Twenty days after the induction of in vitro neuronal differentiation, about 77.4% of the inoculated hUC-MSCs displayed morphological features of neurons and expressed neuronal cell markers like TU-20, Trk A, NeuN, and NF-M. However, functionally active neuronal type channels were not detected by electrophysiological examination. Before, during, or one day after in vitro neuronal differentiation, the hUC-MSCs produced granulocyte-colony stimulating factor, vascular endothelial growth factor, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor. In an in vivo study, implantation of the hUC-MSCs into the damaged hemisphere of immunosuppressed ischemic stroke rats improved neurobehavioral function and reduced infarct volume relative to control rats. Three weeks after implantation, most of the implanted hUC-MSCs were present in the damaged hemisphere; some of these cells expressed detectable levels of neuron-specific markers. Nestin expression in the hippocampus was increased in the hUC-MSC-implanted group relative to the control group. Since the hUC-MSCs were both morphologically differentiated into neuronal cells and able to produce neurotrophic factors, but had not become functionally active neuronal cells, the improvement in neurobehavioral function and the reduction of infarct volume might be related to the neuroprotective effects of hUC-MSCs rather than the formation of a new network between host neurons and the implanted hUC-MSCs.

N-Methyl-D-aspartate (NMDA) receptor (NMDAR) activation and downstream signaling are important for neuronal function. Activation of prosurvival Src family kinases and extracellular signal-regulated kinase (ERK) 1/2 is initiated by NMDAR activation, but the cellular organization of these kinases in relation to NMDARs is not entirely clear. We hypothesized that caveolin-1 scaffolds and coordinates protein complexes involved in NMDAR signaling and that this organization is necessary for neuronal preconditioning, whereby NMDAR activation protects neurons from subsequent ischemic cell death. We found that sublethal ischemia (SLI) or preconditioning via NMDA treatment of primary cortical neurons from neonatal rats or mice increases expression of phosphorylated (P) caveolin-1, P-Src, and P-ERK1/2. The NMDAR antagonist, MK801, or the Src inhibitor, PP2, attenuated SLI-induced preconditioning. NMDAR2B distributed to buoyant fractions and heavy fractions, partially colocalized with caveolin-1 and the membrane raft marker, cholera toxin B. Cultures of primary neurons treated with caveolin-1 small interfering RNA or from caveolin-1(-/-) mice lacked the NMDA-mediated increase in P-Src and P-ERK, as well as SLI- and NMDA-induced preconditioning. Adenovirally mediated expression of caveolin-1 in neurons from caveolin-1(-/-) mice restored NMDA-mediated enhancement of P-Src and P-ERK1/2, redistributed NMDAR2B to buoyant fractions, and enhanced NMDAR2B localization to membrane rafts. We conclude that caveolin-1, perhaps via its ability to scaffold key signaling components, is essential for NMDAR localization to neuronal membrane rafts, NMDAR/Src tyrosine kinase family/ERK signaling, and protection of neurons from ischemic injury and cell death.

Medulloblastomas are among the most common malignant brain tumors in childhood. They typically arise from neoplastic transformation of granule cell precursors in the cerebellum via deregulation of molecular pathways involved in normal cerebellar development. In a mouse model, we show here that impairment of the balance between proliferation and differentiation of granule cell precursors in the external granular layer of the developing cerebellum predisposes but is not sufficient to induce neoplastic transformation of these progenitor cells. Using array-based chromosomal comparative genomic hybridization, we show that genetic instability resulting from inactivation of the p53 pathway together with deregulation of proliferation induced by Rb loss eventually leads to neoplastic transformation of these cells by acquiring additional genetic mutations, mainly affecting N-Myc and Ptch2 genes. Moreover, we show that p53 loss influences molecular mechanisms that cannot be mimicked by the loss of either p19(ARF), p21, or ATM.

Retinoic acid (RA) is a signaling molecule that plays a pivotal role in major cellular processes and vertebrate development. RA action is mediated by specialized transcription factors, the nuclear RA receptors (RARs), which regulate the transcription of genes containing a RA-responsive element (RARE). Here we demonstrate that the genes for the RA-receptor RARbeta2 and the cytochrome P450 RA-specific hydrolase Cyp26a1 involved in RA catabolism are coordinately regulated by RA. We found that both RARbeta2 and Cyp26a1 genes are epigenetically silenced in the absence of DNA methylation in RAC65, a P19 embryocarcinoma cell line derivative carrying a dominant-negative RARalpha mutant and resistant to the growth-inhibitory and differentiation effects of RA. In response to RA, RARbeta2 transcription is epigenetically regulated by RARalpha. Similarly, we found that Cyp26a1 transcription is epigenetically regulated by RARbeta2. Knocking down RARbeta2 transcription by RNA interference in wild-type P19 cells, with an intact RARalpha, induced Cyp26a1 transcriptional repression in the absence of DNA methylation. Concomitantly, cells developed RA resistance and did not undergo RA-induced neuron differentiation. Apparently, RARalpha, RARbeta2 and Cyp26a1 are components of a RA-regulated gene network. Factors affecting an upstream gene of the network can trigger repressive chromatin changes -- which are propagated in a domino fashion - at downstream genes of the network. This study also shows that chromatin inactivity, and consequent transcriptional silencing, can be achieved in the absence of DNA methylation.

We looked at the possible interactions between astrocytes and neurones during reperfusion using an in vitro model of ischaemia-reperfusion injury, as a controlled environment that lends itself easily to manipulation of the numerous variables involved in such an insult. We constructed a chamber in which O2 can be lowered to a concentration of 1 microm and developed a primary cortical neuronal culture that is 99% pure and can survive to at least 10 days in vitro. We also established a novel system for the co-culture of astrocytes and neurones in order to study the communication between these cells in a manner that allows the complete separation of one cell type from another. Neurone cultures showed profound cell death following an ischaemic period of only 15 min. We co-cultured neurones that had been subjected to a 15-min ischaemic insult with either non-insulted astrocytes or astrocyte-conditioned medium during the reperfusion stage. Both astrocytes and astrocyte-conditioned medium enhanced neuronal survival. Our data also suggest that astrocyte-sourced neuronal glutathione synthesis may play a role in preventing neuronal death.

The Notch-Delta signaling pathway controls many conserved cell determination events. While the Notch end is fairly well characterized, the Delta end remains poorly understood. Mind bomb1 (MIB1) is one of two E3 ligases known to ubiquitinate Delta. We report here that a targeted mutation of Mib1 in mice results in embryonic lethality by E10.5. Mutants exhibit multiple defects due to their inability to modulate Notch signaling. As histopathology revealed a strong neurogenic phenotype, this study concentrates on characterizing the Mib1 mutant by analyzing Notch pathway components in embryonic neuroepithelium prior to developmental arrest. Premature neurons were observed to undergo apoptosis soon after differentiation. Aberrant neurogenesis is a direct consequence of lowered Hes1 and Hes5 expression resulting from the inability to generate Notch1 intracellular domain (NICD1). We conclude that MIB1 activity is required for S3 cleavage of the Notch1 receptor. These results have direct implications for manipulating the differentiation of neuronal stem cells and provide a putative target for the modulation of specific tumors.

Development of the nervous system is a complex process, involving coordinated regulation of diverse cellular processes including proliferation, differentiation and synaptogenesis. Disturbances to brain development such as pre- and perinatal hypoxia have been linked to behavioural and late onset of neurological disorders. This study examines the effect of hypoxia on neurite outgrowth in PC12 cells. Hypoxia not only caused a rapid induction of neurite outgrowth, but also synergistically enhanced nerve growth factor (NGF)-induced neurite outgrowth up to 24 h. Transactivation of TrkA receptors was ruled out since the TrkA inhibitor K252a did not block hypoxia-induced neurite outgrowth. Adenosine deaminase prevented hypoxia-induced neurite outgrowth indicating that the effect is mediated by adenosine. Use of the specific adenosine A2A receptor agonist CGS21680 and antagonist 8-3(chlorostyryl)caffeine demonstrated that activation of this receptor is critical for hypoxia-induced neurite outgrowth. Hypoxia-induced neurite outgrowth was blocked by the adenylate cyclase inhibitor, MDL-12,330A, indicating a role for activation of this enzyme in the pathway. Hypoxia was further shown to cause a decrease in growth-associated protein (GAP)-43 levels and a lack of induction of betaIII tubulin, in contrast to NGF treatment which resulted in increased cellular levels of both of these proteins. These findings suggest that hypoxia induces neurite outgrowth in PC12 cells via a pathway distinct from that activated by NGF. Thus, exposure to hypoxia at critical stages of development may contribute to aberrant neurite outgrowth and could be a factor in the pathogenesis of certain delayed developmental neurological disorders.

Olfactory sensory neurons (OSNs) can be sensitized to odorants by repeated exposure, suggesting that an animal's responsiveness to olfactory cues can be enhanced at the initial stage of detection. However, because OSNs undergo a regular cycle of apoptosis and replacement by ostensibly naive, precursor-derived neurons, the advantage of sensitization would be lost in the absence of a mechanism for odorant-enhanced survival of OSNs. Using recombinant adenoviruses in conjunction with surgical and electrophysiological techniques, we monitored OSN survival and function in vivo and find that odorant exposure selectively rescues populations of OSNs from apoptosis. We further demonstrate that odorant stimuli rescue OSNs in a cAMP-dependent manner by activating the MAPK/CREB-dependent transcriptional pathway, possibly as a result of expression of Bcl-2.

BACKGROUND:

Neural stem/precursor cells (NSCs) are of particular interest because of their potential application in cell therapy for brain damage. However, most brain injury cases are followed with neuroinflammatory stress, which affects the lineage selection of grafted NSCs by promoting astrocytogenesis, thus hampering the potential for neural replacement. The present study investigated the role of miR-17-92 in protecting against detrimental effects of neuroinflammation on NSC differentiation in cell therapy.

METHODS:

NSCs were treated with conditioned medium from lesioned astrocytes with/without neutralizing antibodies of leukemia inhibitory factor (LIF) or/and ciliary neurotrophic factor (CNTF), respectively. Afterward, the levels of p-STAT3 and p-JAK2 were determined by western blotting while expression of glial fibrillary acidic protein (GFAP) and ß-tubulin III was assessed by immunostaining. The activation of JAK-STAT pathway and cell differentiation were also evaluated after we overexpressed miR-17-92 in NSCs under different neuroinflammatory conditions. After the transplantation of miR-17-92-overexpressing NSCs into injured mouse cortex, PH3, nestin, GFAP, and NeuN were analyzed by immunostaining. In addition, motor coordination of mice was evaluated by rotarod test.

RESULTS:

Conditioned medium from lesioned astrocytes activated JAK-STAT pathway and facilitated astrocytic differentiation in NSCs while neutralizing antibodies of LIF and CNTF remarkably attenuated such effects. miR-17-92 cluster repressed the expression of multiple proteins including GP130, CNTFR, JAK2, and STAT3 in JAK-STAT pathway. Overexpression of miR-17-92 in NSCs systematically blocked the activation of JAK-STAT pathway mediated by LIF and CNTF, which facilitated neuronal differentiation in vitro. Furthermore, miR-17-92 increased neuronal generation of grafted NSCs and reduced astrogliosis, which resulted in the improvement of motor coordination of brain-injured mice.

CONCLUSIONS:

Our results suggest that miR-17-92 promotes neuronal differentiation of grafted NSCs under neuroinflammatory condition via inhibition of multiple proteins in JAK-STAT pathway.

AIMS:

Protein aggregation and oxidative stress are both key pathogenic processes in Parkinson's disease, although the mechanism by which misfolded proteins induce oxidative stress and neuronal death remains unknown. In this study, we describe how aggregation of alpha-synuclein (a-S) from its monomeric form to its soluble oligomeric state results in aberrant free radical production and neuronal toxicity.

RESULTS:

We first demonstrate excessive free radical production in a human induced pluripotent stem-derived a-S triplication model at basal levels and on application of picomolar doses of ß-sheet-rich a-S oligomers. We probed the effects of different structural species of a-S in wild-type rat neuronal cultures and show that both oligomeric and fibrillar forms of a-S are capable of generating free radical production, but that only the oligomeric form results in reduction of endogenous glutathione and subsequent neuronal toxicity. We dissected the mechanism of oligomer-induced free radical production and found that it was interestingly independent of several known cellular enzymatic sources.

INNOVATION:

The oligomer-induced reactive oxygen species (ROS) production was entirely dependent on the presence of free metal ions as addition of metal chelators was able to block oligomer-induced ROS production and prevent oligomer-induced neuronal death.

CONCLUSION:

Our findings further support the causative role of soluble amyloid oligomers in triggering neurodegeneration and shed light into the mechanisms by which these species cause neuronal damage, which, we show here, can be amenable to modulation through the use of metal chelation.

BACKGROUND:

Ethanol is a toxin responsible for the neurodevelopmental deficits of Fetal Alcohol Spectrum Disorders (FASD). Recent evidence suggests that ethanol modulates the protein expression of lineage specifier transcription factors Oct4 (Pou5f1) and Sox2 in early stages of mouse embryonic stem (ES) cell differentiation. We hypothesized that ethanol induced an imbalance in the expression of Oct4 and Sox2 in early differentiation, that dysregulated the expression of associated and target genes and signaling molecules and diverted cells from neuroectodermal (NE) formation.

METHODOLOGY/PRINCIPAL FINDINGS:

We showed modulation by ethanol of 33 genes during ES cell differentiation, using high throughput microfluidic dynamic array chips measuring 2,304 real time quantitative PCR assays. Based on the overall gene expression dynamics, ethanol drove cells along a differentiation trajectory away from NE fate. These ethanol-induced gene expression changes were observed as early as within 2 days of differentiation, and were independent of cell proliferation or apoptosis. Gene expression changes were correlated with fewer ßIII-tubulin positive cells of an immature neural progenitor phenotype, as well as a disrupted actin cytoskeleton were observed. Moreover, Tuba1a and Gapdh housekeeping genes were modulated by ethanol during differentiation and were replaced by a set of ribosomal genes with stable expression.

CONCLUSIONS/SIGNIFICANCE:

These findings provided an ethanol-response gene signature and pointed to the transcriptional dynamics underlying lineage imbalance that may be relevant to FASD phenotype.

BACKGROUND:

The A53T mutation in the a-synuclein gene causes autosomal-dominant Lewy body Parkinson's disease (PD). Cultured cell models have linked this mutation to increased cell macroautophagy, although evidence of enhanced macroautophagy in patients with this mutation has not been assessed.

OBJECTIVE:

To determine whether macroautophagy is increased by the A53T a-synuclein gene mutation in PD patients and cell models.

METHODS:

Formalin-fixed paraffin-embedded 10 µm-thick tissue sections from the substantia nigra and anterior cingulate cortex of two PD patients with the A53T a-synuclein gene mutation were compared with four sporadic PD cases and four controls obtained from the Sydney Brain Bank. Lewy bodies were isolated from frontal cortex of a case with late stage PD (recruited from South Australian Brain Bank). Immunohistochemistry was performed for a-synuclein and the macroautophagy markers autophagy-specific gene (ATG) 5, ATG6/Beclin1 and ATG8/LC3. SH-SY5Y cells were transfected with wild type or A53T mutant a-synuclein plasmids and observable changes in macroautophagy marker protein levels assessed using Western blotting.

RESULTS:

a-Synuclein immunoreactive neurites and dots were more numerous in patients with A53T mutations compared with late stage sporadic PD patients, and perinuclear cytoplasmic a-synuclein aggregates were observed in the a-synuclein A53T gene transfected SH-SY5Y cells compared to wild type transfections. All PD patients (with or without A53T mutations) had increased immunohistochemical evidence for macroautophagy compared with controls, and the levels of the ATG5 complex were equally increased in wild type and A53T a-synuclein gene transfected cells compared to controls.

CONCLUSION:

Despite increased a-synuclein accumulation with A53T mutations, macroautophagy is not increased above that observed in sporadic patients with PD or in cells transfected with wild type a-synuclein, suggesting that mutated a-synuclein protein is not removed by macroautophagy.

BACKGROUND:

Cell therapy is a potential therapeutic approach for several neurodegenetative disease, including Huntington Disease (HD). To evaluate the putative efficacy of cell therapy in HD, most studies have used excitotoxic animal models with only a few studies having been conducted in genetic animal models. Genetically modified animals should provide a more accurate representation of human HD, as they emulate the genetic basis of its etiology.

RESULTS:

In this study, we aimed to assess the therapeutic potential of a human striatal neural stem cell line (STROC05) implanted in the R6/2 transgenic mouse model of HD. As DARPP-32 GABAergic output neurons are predominately lost in HD, STROC05 cells were also pre-differentiated using purmorphamine, a hedgehog agonist, to yield a greater number of DARPP-32 cells. A bilateral injection of 4.5x105 cells of either undifferentiated or pre-differentiated DARPP-32 cells, however, did not affect outcome compared to a vehicle control injection. Both survival and neuronal differentiation remained poor with a mean of only 161 and 81 cells surviving in the undifferentiated and differentiated conditions respectively. Only a few cells expressed the neuronal marker Fox3.

CONCLUSIONS:

Although the rapid brain atrophy and short life-span of the R6/2 model constitute adverse conditions to detect potentially delayed treatment effects, significant technical hurdles, such as poor cell survival and differentiation, were also sub-optimal. Further consideration of these aspects is therefore needed in more enduring transgenic HD models to provide a definite assessment of this cell line's therapeutic relevance. However, a combination of treatments is likely needed to affect outcome in transgenic models of HD.

BACKGROUND:

EphB receptors and their ephrin-B ligands play an important role in nervous system development, as well as synapse formation and plasticity in the adult brain. Recent studies show that intrathecal treatment with EphB-receptor activator ephrinB2-Fc induced thermal hyperalgesia and mechanical allodynia in rat, indicating that ephrin-B2 in small dorsal root ganglia (DRG) neurons and EphB receptors in the spinal cord modulate pain processing. To examine the role of ephrin-B2 in peripheral pain pathways, we deleted ephrin-B2 in Nav1.8+ nociceptive sensory neurons with the Cre-loxP system. Sensory neuron numbers and terminals were examined using neuronal makers. Pain behavior in acute, inflammatory and neuropathic pain models was assessed in the ephrin-B2 conditional knockout (CKO) mice. We also investigated the c-Fos expression and NMDA receptor NR2B phosphorylation in ephrin-B2 CKO mice and littermate controls.

RESULTS:

The ephrin-B2 CKO mice were healthy with no sensory neuron loss. However, pain-related behavior was substantially altered. Although acute pain behavior and motor co-ordination were normal, inflammatory pain was attenuated in ephrin-B2 mutant mice. Complete Freund's adjuvant (CFA)-induced mechanical hyperalgesia was halved. Formalin-induced pain behavior was attenuated in the second phase, and this correlated with diminished tyrosine phosphorylation of N-methyl-D-aspartic acid (NMDA) receptor subunit NR2B in the dorsal horn. Thermal hyperalgesia and mechanical allodynia were significantly reduced in the Seltzer model of neuropathic pain.

CONCLUSIONS:

Presynaptic ephrin-B2 expression thus plays an important role in regulating inflammatory pain through the regulation of synaptic plasticity in the dorsal horn and is also involved in the pathogenesis of some types of neuropathic pain.

BACKGROUND:

The failure of regeneration after spinal cord injury (SCI) has been attributed to axonal demyelination and neuronal death. Cellular replacement and white matter regeneration are both necessary for SCI repair. In this study, we evaluated the co-transplantation of olfactory ensheathing cells (OEC) and embryonic stem (ES) cell-derived motor neurons (ESMN) on contused SCI.

METHODS:

OEC cultured from olfactory nerve rootlets and olfactory bulbs. ESMN was generated by exposing mouse ES cells to retinoic acid and sonic hedgehog. Thirty female rats were used to prepare SCI models in five groups. Control and medium-injected groups was subjected to induce lesion without cell transplantation. OEC or ESMN or both were transplanted into the site of the lesion in other groups.

RESULTS:

The purity of OEC culture was 95%. Motor neuron progenitor markers (Olig2, Nkx6.1 and Pax6) and motor neuron markers (Isl1, Isl2 and Hb9) were expressed. Histological analysis showed that significantly more (P<0.001) spinal tissue was spared in OEC, ESMN and OEC+ ESMN groups but the OEC+ ESMN group had a significantly greater percentage of spared tissue and myelination than other groups (P< 0.05). The numbers of ESMN in co-transplanted group were significantly higher than ESMN group (P<0.05). A significant (P<0.05) recovery of hindlimb function was observed in rats in the transplanted groups.

CONCLUSION:

We found that the co-transplantation of ESMN and OEC into an injured spinal cord has a synergistic effect, promoting neural regeneration, ESMN survival and partial functional recovery.