Embryonic stem cells (ESC) are pluripotent and thus can differentiate into every cell type present in the body. Directed differentiation into motor neurons (MNs) has been described for pluripotent cells. Although neurotrophic factors promote neuronal survival, their role in neuronal commitment is elusive. Here, we developed double-transgenic lines of mouse ESC (mESC) that constitutively produce glial cell line-derived neurotrophic factor (GDNF) and also contain a GFP reporter, driven by HB9, which is expressed only by postmitotic MNs. After lentiviral transduction, ESC lines integrated and expressed the human GDNF (hGDNF) gene without altering pluripotency markers before differentiation. Further, GDNF-ESC showed significantly higher spontaneous release of this neurotrophin to the medium, when compared to controls. To study MN induction, control and GDNF cell lines were grown as embryoid bodies and stimulated with retinoic acid and Sonic Hedgehog. In GDNF-overexpressing cells, a significant increase of proliferative Olig2+ precursors, which are specified as spinal MNs, was found. Accordingly, GDNF increases the yield of cells with the pan motor neuronal markers HB9, monitored by GFP expression, and Isl1. At terminal differentiation, almost all differentiated neurons express phenotypic markers of MNs in GDNF cultures, with lower proportions in control cells. To test if the effects of GDNF were present at early differentiation stages, exogenous recombinant hGDNF was added to control ESC, also resulting in enhanced MN differentiation. This effect was abolished by the co-addition of neutralizing anti-GDNF antibodies, strongly suggesting that differentiating ESC are responsive to GDNF. Using the HB9::GFP reporter, MNs were selected for electrophysiological recordings. MNs differentiated from GDNF-ESC, compared to control MNs, showed greater electrophysiological maturation, characterized by increased numbers of evoked action potentials (APs), as well as by the appearance of rebound APs, sag inward rectification, spike frequency adaptation and spontaneous synaptic potentials. Upon challenge with kainate, GDNF-overexpressing cells are more resistant to excitotoxicity than control MNs. Together these data indicate that GDNF promotes proliferation of MN-committed precursors, promotes neuronal differentiation, enhances maturation, and confers neuroprotection. GDNF-expressing ESC can be useful in studies of development and disease.

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The functional preservation of nerve endings since the early stages of toxicity in a given damaging insult-either acute or chronic-by means of antioxidant and neuroprotective agents is a primary need to design therapeutic strategies for neurodegenerative disorders, with particular emphasis on those diseases with excitotoxic and depleted energy metabolism components. S-allylcysteine (SAC), a well-known antioxidant agent, was tested as a post-treatment in different in vitro and in vivo neurotoxic models. Quinolinic acid (QUIN) was used as a typical excitotoxic/pro-oxidant inducer, 3-nitropropionic acid (3-NP) was employed as a mitochondrial function inhibitor, and their combination (QUIN + 3-NP) was also evaluated in in vitro studies. For in vitro purposes, increasing concentrations of SAC (10-100 microM) were added to isolated brain synaptosomes at different times (1, 3 and 6 h) after the incubation with toxins (100 microM QUIN, 1 mM 3-NP or the combination of QUIN (21 microM) + 3-NP (166 microM). Thirty minutes later, lipid peroxidation (LP) and mitochondrial dysfunction (MD) were evaluated. For in vivo studies, SAC (100 mg/kg, i.p.) was given to QUIN- or 3-NP-striatally lesioned rats for 7 consecutive days (starting 120 min post-lesion). LP and MD were evaluated 7 days post-lesion in isolated striatal synaptosomes. Circling behavior was also assessed. Our results describe a differential pattern of protection achieved by SAC, mostly expressed in the 3-NP toxic model, in which nerve ending protection was found within the first hours (1 and 3) after the toxic insult started, supporting the concept that the ongoing oxidative damage and energy depletion can be treated during the first stages of neurotoxic events.

Cell rescue is a primary need during acute and chronic insults to the central nervous system. Functional preservation during the early stages of toxicity in a given degenerative event may represent a significant amelioration of detrimental processes linked to neuronal cell loss. Excitotoxicity and depleted cellular energy are toxic events leading to cell death in several neurodegenerative disorders. In this work, the effects of the well-known antioxidant and energy precursor, L: -carnitine (L: -CAR), were tested as a post-treatment in two neurotoxic models under in vitro and in vivo conditions. The experimental models tested included: (1) a typical excitotoxic and pro-oxidant inducer, quinolinic acid (QUIN); and (2) a mitochondrial energy inhibitor, 3-nitropropionic acid (3-NP). For in vitro studies, increasing concentrations of L: -CAR (10-1,000 microM) were added to the isolated brain synaptosomes at different times (1, 3 and 6 h) after the incubation with toxins (100 microM QUIN and 1 mM 3-NP), and 30 min later, lipid peroxidation (LP) and mitochondrial dysfunction (MD) were evaluated. For in vivo purposes, L: -CAR (100 mg/kg, i.p.) was given to rats either as a single administration 120 min after the intrastriatal infusion of QUIN (240 nmol/microl) or 3-NP (500 nmol/microl), or for 7 consecutive days (starting 120 min post-lesion). LP and MD were evaluated 4 h and 7 days post-lesions in isolated striatal synaptosomes. Our results show that, despite some variations depending on the toxic model tested, the time of exposure, or the biomarker evaluated, nerve ending protection can be mostly achieved by L: -CAR within the first hours after the toxic insults started, suggesting that targeting the ongoing oxidative damage and/or energy depletion during the first stages of neurotoxic events is essential to rescue nerve endings.