Complementation of dopaminergic signaling by Pitx3-GDNF synergy induces dopamine secretion by multipotent Ntera2 cells.

Human teratocarcinoma cell line Ntera2 (NT2) expresses dopamine signals and has shown its safe profile for clinical applications. Attempts to restore complete dopaminergic (DAergic) phenotype enabling these cells to secrete dopamine have not been fully successful so far. We applied a blend of gene transfer techniques and a defined medium to convert NT2 cells to fully DAergic. The cells were primarily engineered to overexpress the Pitx3 gene product and then cultured in a growth medium supplemented with knockout serum and retinoic acid to form embroid bodies (EBs). Trypsinization of EB colonies produced single cells ready for differentiation. Neuronal/DAergic induction was promoted by applying conditioned medium taken from engineered human astrocytomas over-secreting glial cell-derived neurotrophic factor (GDNF). Immunocytochemistry, reverse-transcription and real-time polymerase chain reaction analyses confirmed significantly induced expression of molecules involved in dopamine signaling and metabolism including tyrosine hydroxylase, Nurr1, dopamine transporter, and aromatic acid decarboxylase. High-performance liquid chromatography analysis indicated release of dopamine only from a class of fully differentiated cells expressing Pitx3 and exposed to GDNF. In addition, Pitx3 and GDNF additively promoted in vitro neuroprotection against Parkinsonian toxin. One month after transplantation to the striatum of 6-OHDA-leasioned rats, differentiated NT2 cells survived and induced significant increase in striatal volume. Besides, cell implantation improved motor coordination in Parkinson's disease (PD) rat models. Our findings highlight the importance of Pitx3-GDNF interplay in dopamine signaling and indicate that our strategy might be useful for the restoration of DAergic fate of NT2 cells to make them clinically applicable toward cell replacement therapy of PD.

Geldanamycin Reduces Aß-Associated Anxiety and Depression, Concurrent with Autophagy Provocation.

Neurodegenerative disorders are generally characterized by abnormal aggregation and deposition of specific proteins. Amyloid beta (Aß)-associated neurodegenerative disorder is characterized by an oxidative damage that, in turn, leads to some behavioral changes before the establishment of dementia such as depression and anxiety. In the current study, we investigated the effect of heat shock protein 90 inhibitor geldanamycin (GA) administration 24 h before Aß injection. In our experiment, 7 days after Aß injection, elevated plus maze and forced swimming test were conducted to assess anxiety and depression-like behaviors. Levels of autophagy markers and malondialdehyde (MDA) and also activity of catalase in the hippocampus of rats were evaluated. Our behavioral analyses demonstrated that GA pretreatment can significantly decrease anxiety- and depression-like behaviors in Aß-injected rats. Also, levels of autophagy markers including Atg12, Atg7, and LC3-II increased, while MDA level decreased and the activity of catalase increased in rats pretreated with GA compared to Aß-injected rats. Thus, we assumed that GA, at least in part, ameliorated Aß-mediated anxiety and depression by inducing autophagy and improving antioxidant defense system.

Chitosan prevents oxidative stress-induced amyloid beta formation and cytotoxicity in NT2 neurons: involvement of transcription factors Nrf2 and NF-kappaB.

Increased oxidative stress is a widely accepted factor in the development and progression of Alzheimer's disease. Here, we introduce chitosan, an antioxidant oligosaccharide, as a protective agent against H(2)O(2)/FeSO(4)-induced cell death in the NT2 neural cell line. Chitosan not only protects the neurons against cell death, as measured by MTT and caspase-3 activity, but also decreases amyloid beta formation. NT2 neurons can be used to elucidate the relationship between oxidative stress and Abeta formation. We induced Abeta formation through oxidative stress in NT2 neurons and studied the effect of chitosan. We demonstrate that chitosan can be neuroprotective by suppressing Abeta formation. We further show that chitosan exerts its protective effect by up-regulation of HO-1, gamma-GCS, Hsp-70, and Nrf2, while it inhibits activation of caspase-3 and NF-kappaB. Chitosan or chitosan derivatives have potential value as neuroprotective agents, particularly with regard to oxidative stress.

Purification and partial characterization of coxsackievirus B3 2A protease expressed in Escherichia coli.

Reported here is the overexpression, purification and partial characterization of recombinant coxsakievirus B3 2A protease (CVB3 2Apro) from bacterial cells transformed with a plasmid containing the CVB3 2Apro cDNA sequences. The structural investigation showed that the protein contains mostly beta-strand elements and requires Zn2+ ions as a structural component which appeared to be inhibitory if added exogenously. The purified enzyme activity was optimal at 4 degrees C and had a short half-life at physiological temperature. This feature can be the result of the presence of a high content of beta-structure and also hydrophobic residues in its structure.

Nifedipine suppresses morphine-induced thermal hyperalgesia: evidence for the role of corticosterone.

It has been shown that systemic administration of morphine induced a hyperalgesic response at an extremely low dose. We have examined the effect of nifedipine, as a calcium channel blocker, on morphine-induced hyperalgesia in intact and adrenalectomized rats and on hypothalamic-pituitary-adrenal axis activity induced by ultra-low dose of morphine. To determine the effect of nifedipine on hyperalgesic effect of morphine, nifedipine (2 mg/kg i.p. and 10 microg i.t.) that had no nociceptive effect, was injected concomitant with morphine (1 microg/kg i.p. and 0.01 microg i.t. respectively). The tail-flick test was used to assess the nociceptive threshold, before and 30, 60, 120, 180, 240 and 300 min after drug administration. The data showed that low dose morphine systemic administration could produce hyperalgesic effect in adrenalectomized rats equivalent to sham-operated animals while intrathecal injection of morphine only elicited hyperalgesia in sham-operated animals. Nifedipine could block morphine-induced hyperalgesia in sham and adrenalectomized rats and even a mild analgesic effect was observed in the adrenalectomized group which was reversed by corticosterone replacement. Systemic administration of low dose morphine produced significant increase in plasma level of corticosterone. Nifedipine has an inhibitory effect on morphine-induced corticosterone secretion. Thus, the data indicate that dihydropyridine calcium channels are involved in ultra-low dose morphine-induced hyperalgesia and that both the pattern of morphine hyperalgesia and the blockage of it by nifedipine are modulated by manipulation of the hypothalamic pituitary adrenal axis.