Para-Substituted α-Phenyl-N-tert-butyl Nitrones: Spin-Trapping, Redox and Neuroprotective Properties.

In this work, a series of para-substituted α-phenyl-N-tert-butyl nitrones (PBN) were studied. Their radical-trapping properties were evaluated by electron paramagnetic resonance, with 4-CF3-PBN being the fastest derivative to trap the hydroxymethyl radical (•CH2OH). The redox properties of the nitrones were further investigated by cyclic voltammetry, and 4-CF3-PBN was the easiest to reduce and the hardest to oxidize. This is due to the presence of the electron-withdrawing CF3 group. Very good correlations between the Hammett constants (σp) of the substituents and both spin-trapping rates and redox potentials were observed. These correlations were further supported by computationally determined ionization potentials and atom charge densities. Finally, the neuroprotective effect of these derivatives was studied using two different in vitro models of cell death on primary cortical neurons injured by glutamate exposure or on glial cells exposed to t BuOOH. Trends between the protection afforded by the nitrones and their lipophilicity were observed. 4-CF3-PBN was the most potent agent against t BuOOH-induced oxidative stress on glial cells, while 4-Me2N-PBN showed potency in both models.

Substituted α-Phenyl and α-Naphthlyl-N-tert-butyl Nitrones: Synthesis, Spin-Trapping, and Neuroprotection Evaluation.

New derivatives of α-phenyl-N-tert-butyl nitrone (PBN) bearing a hydroxyl, an acetate, or an acetamide substituent on the N-tert-butyl moiety and para-substituted phenyl or naphthlyl moieties were synthesized. Their ability to trap hydroxymethyl radical was evaluated by electron paramagnetic resonance spectroscopy. The presence of two electron-withdrawing substituents on both sides of the nitronyl function improves the spin-trapping properties, with 4-HOOC-PBN-CH2OAc and 4-HOOC-PBN-CH2NHAc being ∼4× more reactive than PBN. The electrochemical properties of the derivatives were further investigated by cyclic voltammetry and showed that the redox potentials of the nitrones are largely influenced by the nature of the substituents both on the aromatic ring and on the N-tert-butyl function. The acetamide derivatives PBN-CH2NHAc, 4-AcNHCH2-PBN-CH2NHAc, and 4-MeO-PBN-CH2NHAc were the easiest to oxidize. A computational approach was used to rationalize the effect of functionalization on the free energies of nitrone reactivity with hydroxymethyl radical as well as on the electron affinity and ionization potential. Finally, the neuroprotection of the derivatives was evaluated in an in vitro model of cellular injury on cortical neurons. Five derivatives showed good protection at very low concentrations (0.1-10 µM), with PBN-CH2NHAc and 4-HOOC-PBN being the two most promising agents.

Necrosis, apoptosis, necroptosis, three modes of action of dopaminergic neuron neurotoxins.

Most of the Parkinson's disease (PD) cases are sporadic, although several genes are directly related to PD. Several pathways are central in PD pathogenesis: protein aggregation linked to proteasomal impairments, mitochondrial dysfunctions and impairment in dopamine (DA) release. Here we studied the close crossing of mitochondrial dysfunction and aggregation of α-synuclein (α-syn) and in the extension in the dopaminergic neuronal death. Here, using rat primary cultures of mesencephalic neurons, we induced the mitochondrial impairments using "DA-toxins" (MPP+, 6OHDA, rotenone). We showed that the DA-Toxins induced dopaminergic cell death through different pathways: caspase-dependent cell death for 6OHDA; MPP+ stimulated caspase-independent cell death, and rotenone activated both pathways. In addition, a decrease in energy production and/or a development of oxidative stress were observed and were linked to α-syn aggregation with generation of Lewy body-like inclusions (found inside and outside the dopaminergic neurons). We demonstrated that any of induced mitochondrial disturbances and processes of death led to α-syn protein aggregation and finally to cell death. Our study depicts the cell death mechanisms taking place in in vitro models of Parkinson's disease and how mitochondrial dysfunctions is at the cross road of the pathologies of this disease.

Glutamate protects neuromuscular junctions from deleterious effects of ß-amyloid peptide and conversely: an in vitro study in a nerve-muscle coculture.

Murine models of Alzheimer's disease with elevated levels of amyloid-ß (Aß) peptide present motor axon defects and neuronal death. Aß1-42 accumulation is observed in motor neurons and spinal cords of sporadic and familial cases of amyotrophic lateral sclerosis (ALS). Motor neurons are highly susceptible to glutamate, which has a role in ALS neuronal degeneration. The current study investigates the link between Aß and glutamate in this neurodegenerative process. Primary rat nerve and human muscle cocultures were intoxicated with glutamate or Aß. Neuromuscular junction (NMJ) mean size and neurite length were evaluated. The role of N-methyl-D-aspartate receptor (NMDAR) was investigated by using MK801. Glutamate and Aß production were evaluated in culture supernatant. The current study shows that NMJs are highly sensitive to Aß peptide, that the toxic pathway involves glutamate and NMDAR, and that glutamate and Aß act in an interlinked manner. Some motor diseases (e.g., ALS), therefore, could be considered from a new point of view related to these balance disturbances.

Operational dissection of ß-amyloid cytopathic effects on cultured neurons.

Alzheimer disease (AD) affects mainly people over the age of 65 years, suffering from different clinical symptoms such as progressive decline in memory, thinking, language, and learning capacity. The toxic role of ß-amyloid peptide (Aß) has now shifted from insoluble Aß fibrils to smaller, soluble oligomeric Aß aggregates. The urgent need for efficient new therapies is high; robust models dissecting the physiopathological aspects of the disease are needed. We present here a model allowing study of four cytopathic effects of Aß oligomers (AßO): oxidative stress, loss of synapses, disorganization of the neurite network, and cellular death. By generating a solution of AßO and playing on the concentration of and time of exposure to AßO, we have shown that it was possible to reproduce early effects (oxidative stress) and the long-term development of structural alterations (death of neurons). We have shown that 1) all toxic events were linked to AßO according to a specific timing and pathway and 2) AßO were probably the key intermediates in AD pathogenesis. The present model, using Aß peptide solution containing AßO, reproduced essential neuropathological features of AD; the effects involved were similar whatever the kind of neurons tested (cortical vs. hippocampal). By using a single system, it was possible to embrace all toxic mechanisms at defined times and concentrations, to study each involved pathway, and to study the effects of new molecules on the different neurotoxic pathways responsible for development of AD.

A new long term in vitro model of myelination.

Besides in vivo models, co-cultures systems making use of Rat dorsal root ganglion explants/Schwann cells (SC) are widely used to essentially study myelination in vitro. In the case of animal models of demyelinating diseases, it is expected to reproduce a pathological process; conversely the co-cultures are primarily developed to study the myelination process and in the aim to use them to replace animals in experiences of myelin destruction or functional disturbances. We describe (in terms of protein expression kinetic) a new in vitro model of sensory neurons/SC co-cultures presenting the following advantages: both sensory neurons and SC originate from the same individual; sensory neurons and SC being dissociated, they can be co-cultured in monolayer, allowing an easier microscope observation; the co-culture can be maintained in a serum-free medium for at less three months, allowing kinetic studies of myelin formation both at a molecular and cellular level. Optimizing culture conditions permits to use 96-well culture plates; image analyses conducted with an automatic image analyzer allows rapid, accurate and quantitative expression of results. Finally, this system was proved by measuring the apparition of myelin protein to mimic in vitro the physiological process of in vivo myelination.