Maria Ribecco-Lutkiewicz

Differentiation of mouse Neuro 2A cells into dopamine neurons.

Neuro 2A (N2a) is a mouse neural crest-derived cell line that has been extensively used to study neuronal differentiation, axonal growth and signaling pathways. A convenient characteristic of these cells is their ability to differentiate into neurons within a few days. However, most differentiation methods reported for N2a cells do not provide information about the neuronal types obtained after each treatment. In this study, we evaluated the generation of N2a dopamine neurons following treatment with a number of factors known to induce neuronal differentiation. Our results showed that N2a cells express Nurr-related factor 1 (Nurr1) and produce low levels of tyrosine hydroxylase (TH) and dopamine. Both TH and dopamine levels were significantly enhanced in the presence of dibutyryl cyclic adenosine monophosphate (dbcAMP), as evidenced by Western blot, immunocytochemistry and high performance liquid chromatography (HPLC). In contrast to dbcAMP, other factors such as transforming growth factor beta1 (TGF beta 1), bone morphogenetic protein 4 (BMP4), glial cell-derived neurotrophic factor (GDNF) and retinoic acid (RA) did not increase TH expression. Further investigation confirmed that the effect of dbcAMP on production of TH-positive neurons was mediated through cyclic AMP (cAMP) responsive element binding protein (CREB) and it was antagonized by RA. Thus, although various treatments can be used to generate N2a neurons, only dbcAMP significantly enhanced the formation of dopamine neurons. Taken together, this study provided a simple and reliable method to generate dopamine neurons for rapid and efficient physiological and pharmacological assays.

Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: protective effects of coenzyme Q10.

Although glutamate excitotoxicity has long been implicated in neuronal cell death associated with a variety of neurological disorders, the molecular mechanisms underlying this process are not yet fully understood. In part, this is due to the lack of relevant experimental cell systems recapitulating the in vivo neuronal environment, mainly neuronal–glial interactions. To explore these mechanisms, we have analyzed the cytotoxic effects of glutamate on mixed cultures of NT2/N neurons and NT2/A astrocytes derived from human NT2/D1 cells. In these cultures, the neurons were resistant to glutamate alone (up to 2 mM for 24–48 hr), but they responded to a simultaneous exposure to 0.5 mM glutamate and 6 hr of hypoxia. Neuronal cell death occurred during subsequent periods of reoxygenation (>30% within 24 hr). This was associated with a marked decrease of intracellular ATP, a significant increase in reactive oxygen species (ROS) and downregulation of glutamate uptake by astrocytes. Thus, under energy failure and high levels of ROS production, only the neurons from these mixed cultures succumbed to glutamate neurotoxicity; the astrocytic cells remained unaffected by the treatment. Taken together, our data suggested that glutamate excitotoxicity might be due to the energy failure and oxidative stress affecting the properties of the NMDA glutamate receptors and causing impairment of glutamate transporters. Cells pretreated for 72 hr with 10 μg/ml of coenzyme Q10 (functions both as a ROS scavenger and co‐factor of mitochondrial electron transport), were protected, suggesting a useful role for coenzyme Q10 in treatments of neurological diseases associated with glutamate excitotoxicity. A model of the complex interactions between neurons and astrocytes in regulating glutamate metabolism is presented.

Astrocyte-secreted GDNF and glutathione antioxidant system protect neurons against 6OHDA cytotoxicity

In recent years, GDNF has emerged as a protective and restorative agent in several models of neurodegeneration; however, the exact molecular mechanisms responsible for these effects are not yet fully understood. Here we examined the effects of astrocytes secreting GDNF on neurons subjected to 6OHDA toxicity using in vitro neuron-astroglia co-cultures. Astrocytes were transduced with lentiviral vectors carrying the GDNF gene under the control of either human glial fibrillary acidic protein or cytomegalovirus promoters. The overexpression of GDNF, regardless of the promoter employed, had no obvious adverse effects on astroglia and the engineered cells stably produced and secreted GDNF for extended periods of time (> or =3 weeks). These astrocytes very effectively protected neurons against 6OHDA, in both mouse and human co-culture systems. The neuroprotective effects were mediated not only by GDNF, but also by the antioxidant GSH since its depletion reduced the level of GDNF protection. Furthermore, neurons and astrocytes expressed different components of GDNF signaling complex, suggesting that they might utilize separate pathways to mediate autocrine and paracrine effects of GDNF.

Epigenetic Modifications of SOX2 Enhancers, SRR1 and SRR2, Correlate With In Vitro Neural Differentiation

SOX2 is a key neurodevelopmental gene involved in maintaining the pluripotency of stem cells and proliferation of neural progenitors and astroglia. Two evolutionally conserved enhancers, SRR1 and SRR2, are involved in controlling SOX2 expression during neurodevelopment; however, the molecular mechanisms regulating their activity are not known. We have examined DNA methylation and histone H3 acetylation at both enhancers in NT2‐D1 progenitors, neurons and astrocytes, to establish the role of epigenetic mechanisms in cell‐type‐specific SOX2 expression. This study showed that 1) unmethylated DNA and acetylated histones at both enhancers correlated with a high level of SOX2 expression in proliferating neural progenitors and 2) reversible modifications of the SRR1 element were observed during gene reexpression in astrocytes, whereas permanent epigenetic marks on the SRR2 enhancer were seen in neurons where the gene was silenced. Taken together, these results are clear illustrations of cell‐type‐specific epigenomes and suggest mechanisms by which they may be created and maintained.

Regulation of MYPT1 stability by the E3 ubiquitin ligase SIAH2.

Myosin phosphatase target subunit 1 (MYPT1), together with catalytic subunit of type1 delta isoform (PP1cdelta) and a small 20-kDa regulatory unit (M20), form a heterotrimeric holoenzyme, myosin phosphatase (MP), which is responsible for regulating the extent of myosin light chain phosphorylation. Here we report the identification and characterization of a molecular interaction between Seven in absentia homolog 2 (SIAH2) and MYPT1 that resulted in the proteasomal degradation of the latter in mammalian cells, including neurons and glia. The interaction involved the substrate binding domain of SIAH2 (aa 116-324) and a central region of MYPT1 (aa 445-632) containing a degenerate consensus Siah-binding motif RLAYVAP (aa 493-499) evolutionally conserved from fish to humans. These findings suggest a novel mechanism whereby the ability of MP to modulate myosin light chain might be regulated by the degradation of its targeting subunit MYPT1 through the SIAH2-ubiquitin-proteasomal pathway. In this manner, the turnover of MYPT1 would serve to limit the duration and/or magnitude of MP activity required to achieve a desired physiological effect.

IDENTIFICATION OF ATAXIA-ASSOCIATED mtDNA MUTATIONS (m.4052T>C and m.9035T>C) AND EVALUATION OF THEIR PATHOGENICITY IN TRANSMITOCHONDRIAL CYBRIDS

The potential pathogenicity of two homoplasmic mtDNA point mutations, 9035T>C and 4452T>C, found in a family afflicted with maternally transmitted cognitive developmental delay, learning disability, and progressive ataxia was evaluated using transmitochondrial cybrids. We confirmed that the 4452T>C transition in tRNAMet represented a polymorphism; however, 9035T>C conversion in the ATP6 gene was responsible for a defective F0‐ATPase. Accordingly, mutant cybrids had a reduced oligomycin‐sensitive ATP hydrolyzing activity. They had less than half of the steady‐state content of ATP and nearly an 8‐fold higher basal level of reactive oxygen species (ROS). Mutant cybrids were unable to cope with additional insults, i.e., glucose deprivation or tertiary‐butyl hydroperoxide, and they succumbed to either apoptotic or necrotic cell death. Both of these outcomes were prevented by the antioxidants CoQ10 and vitamin E, suggesting that the abnormally high levels of ROS were the triggers of cell death. In conclusion, the principal metabolic defects, i.e., energy deficiency and ROS burden, resulted from the 9035T>C mutation and could be responsible for the development of clinical symptoms in this family. Furthermore, antioxidant therapy might prove helpful in the management of this disease. Muscle Nerve, 2009

Novel subtractive transcription-based amplification of mRNA (STAR) method and its application in search of rare and differentially expressed genes in AD brains

BackgroundAlzheimers disease (AD) is a complex disorder that involves multiple biological processes. Many genes implicated in these processes may be present in low abundance in the human brain. DNA microarray analysis identifies changed genes that are expressed at high or moderate levels. Complementary to this approach, we described here a novel technology designed specifically to isolate rare and novel genes previously undetectable by other methods. We have used this method to identify differentially expressed genes in brains affected by AD. Our method, termed S ubtractive T ranscription-based A mplification of mR NA (STAR), is a combination of subtractive RNA/DNA hybridization and RNA amplification, which allows the removal of non-differentially expressed transcripts and the linear amplification of the differentially expressed genes.ResultsUsing the STAR technology we have identified over 800 differentially expressed sequences in AD brains, both up- and down- regulated, compared to age-matched controls. Over 55% of the sequences represent genes of unknown function and roughly half of them were novel and rare discoveries in the human brain. The expression changes of nearly 80 unique genes were further confirmed by qRT-PCR and the association of additional genes with AD and/or neurodegeneration was established using an in-house literature mining tool (LitMiner).ConclusionThe STAR process significantly amplifies unique and rare sequences relative to abundant housekeeping genes and, as a consequence, identifies genes not previously linked to AD. This method also offers new opportunities to study the subtle changes in gene expression that potentially contribute to the development and/or progression of AD.

S/MAR-binding properties of Sox2 and its involvement in apoptosis of human NT2 neural precursors

DNA fragmentation in apoptosis, especially in lymphocytic cells, is initiated at scaffold/matrix attachment regions (S/MARs) and is preceded by the degradation of nuclear proteins. The present study was performed to establish whether the same mechanism occurred in human NT2 cells subjected to oxygen and glucose deprivation (OGD). We analyzed the integrity of c-myc S/MAR containing a base-unpairing region (BUR)-like element, which we established to be a binding site of the transcription factor Sox2. An accumulation of DNA breaks in close proximity to this element and a degradation of Sox2 were observed early in the OGD-induced apoptotic response. Identification of Sox2 as a novel c-myc BUR-binding protein was achieved through yeast one-hybrid screening and the Sox2/DNA interaction was confirmed by electrophoretic mobility shift assay and immunoprecipitation with Sox2 antibody. Our data support the notion that early proteolysis of unique BUR-binding proteins might represent a universal mechanism that renders these DNA sites vulnerable to endonucleolysis.

Therapeutic potential of amniotic fluid-derived cells for treating the injured nervous system

There is a need for improved therapy for acquired brain injury, which has proven resistant to treatment by numerous drugs in clinical trials and continues to represent one of the leading causes of disability worldwide. Research into cell-based therapies for the treatment of brain injury is growing rapidly, but the ideal cell source has yet to be determined. Subpopulations of cells found in amniotic fluid, which is readily obtained during routine amniocentesis, can be easily expanded in culture, have multipotent differentiation capacity, are non-tumourigenic, and avoid the ethical complications associated with embryonic stem cells, making them a promising cell source for therapeutic purposes. Beneficial effects of amniotic fluid cell transplantation have been reported in various models of nervous system injury. However, evidence that amniotic fluid cells can differentiate into mature, functional neurons in vivo and incorporate into the existing circuitry to replace lost or damaged neurons is lacking. The mechanisms by which amniotic fluid cells improve outcomes after experimental nervous system injury remain unclear. However, studies reporting the expression and release of neurotrophic, angiogenic, and immunomodulatory factors by amniotic fluid cells suggest they may provide neuroprotection and (or) stimulate endogenous repair and remodelling processes in the injured nervous system. In this paper, we address recent research related to the neuronal differentiation of amniotic fluid-derived cells, the therapeutic efficacy of these cells in animal models of nervous system injury, and the possible mechanisms mediating the positive outcomes achieved by amniotic fluid cell transplantation.

Development of BMP7-producing human cells, using a third generation lentiviral gene delivery system.

Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor β (TGF-β) superfamily, plays important roles in the development of various tissues and organs in mouse and human. In particular, BMP7 is critical for the formation of the nervous system and it is considered to have therapeutic potential in brain injury and stroke. One approach to make BMP7 more suitable for therapeutic purposes is the development of efficient vectors that allow the consistent, reliable and cost-effective production of the BMP7 protein. In this study, we developed an efficient BMP7 delivery system, using a third generation lentiviral vector to produce functional BMP7 protein. The lentiviral transduction of several human cell types, including human embryonic kidney 293 (HEK293) cells, amniotic fluid cells, NTera2 neurons (NT2-N) and primary neuronal cultures resulted in BMP7 expression. The production of BMP7 protein was achieved for at least 4 weeks post-transduction, as determined by enzyme-linked immunosorbent assay (ELISA). SMAD phosphorylation and neuronal differentiation assays verified the bioactivity and functionality of the lentiviral-based BMP7 protein, respectively. In addition, the intracerebroventricular injection of the lentivirus resulted in exogenous BMP7 expression in both neurons and astrocytes in the mouse brain. Taken together, this gene delivery system provides a reliable source of functional BMP7 protein for future in vitro and in vivo studies.