Oxidative damage prevention in human skin and sensory neurons by a salicylic acid derivative.

BACKGROUND: Increased protein carbonylation is a hallmark of oxidative stress, protein homeostasis dysregulation and aging in the nervous system and skin. Sensory neurons interact with skin cells and are involved in skin homeostasis. We have previously reported that the 5-octanoyl salicylic acid (C8-SA), a salicylic acid derivative, increased C. elegans lifespan and delayed the accumulation of carbonylated proteins, through the stimulation of autophagy. OBJECTIVES: In this study we aimed to investigate if C8-SA protects human sensory neurons and human skin from extrinsic oxidative stressors as an approach to delay skin aging. METHODS: In vitro reconstituted human epidermis innervated with hiPSc-derived human sensory neurons, as well as ex vivo human organotypic full skin models were used. The fully differentiated sensory neurons were pretreated with C8-SA before oxidative stress induction. Skin explants were maintained in culture and treated topically with C8-SA before the application of urban pollutants. Carbonylated proteins were detected using amino-oxy functionalized fluorophores and quantified. Chaperone mediated autophagy was monitored with LAMP2A immunofluorescence. Inflammation, ROS detoxification and autophagy were assessed by RT-PCR. RESULTS: C8-SA prevented the accumulation of carbonylated proteins, both in human sensory neurons and skin explants. C8-SA stimulated chaperone-mediated autophagy and modulated NRF2 antioxidant response genes, as well as catalase enzymatic activity. CONCLUSIONS: C8-SA acts at two levels to protect skin against oxidative stress: 1) it prevents protein oxidation by stimulating endogenous antioxidant defense and 2) it increases the clearance of oxidized proteins by stimulating chaperone-mediated autophagy. These results suggest that C8-SA maintains skin health in urban polluted environments.

Pimavanserin Promotes Trophic Factor Release and Protects Cultured Primary Dopaminergic Neurons Exposed to MPP+ in a GDNF-Dependent Manner.

Neurodegeneration and impaired neural development are a common feature of many neuropsychiatric disorders. Second-generation antipsychotics (SGAs) and certain atypical antidepressants display neuroprotective effects. Though these drugs interact with many molecular targets, a common shared attribute is high antagonist potency at 5-HT2A receptors. Pimavanserin is a selective 5-HT2A inverse agonist/antagonist that was recently FDA approved for treating hallucinations and delusions associated with Parkinson's disease. Unlike SGAs, pimavanserin lacks activity at other targets like dopamine, histamine, muscarinic, and adrenergic receptors. To investigate whether selective 5-HT2A inverse agonists have neuroprotective properties, pimavanserin and another selective 5-HT2A inverse agonist, M100907, were applied to primary cultures of dopaminergic neurons treated with 1-methyl-4-phenylpyridinium (MPP+). Both pimavanserin and M100907 protected dopaminergic neurons against MPP+-induced cell death. The neuroprotective effects of pimavanserin required signaling through the extracellular signal-regulated kinase 1/2 pathway, restored mitochondrial function, and reduced oxidative stress. Further investigation showed that pimavanserin promotes the release of brain-derived neurotrophic factor and glial-derived neurotrophic factor (GDNF) and that the neuroprotective effects of pimavanserin were blocked by antibodies to GDNF but not by anti-tyrosine receptor kinase B receptor antibodies. Thus, pimavanserin induces release of neurotrophic factors and protects dopaminergic neurons against MPP+ toxicity in a GDNF-dependent manner.

Neuroprotective activity of stiripentol with a possible involvement of voltage-dependent calcium and sodium channels.

A growing body of data has shown that recurrent epileptic seizures may be caused by an excessive release of the excitatory neurotransmitter glutamate in the brain. Glutamatergic overstimulation results in massive neuronal influxes of calcium and sodium through N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainic acid glutamate subtype receptors and also through voltage-gated calcium and sodium channels. These persistent and abnormal sodium and calcium entry points have deleterious consequences (neurotoxicity) for neuronal function. The therapeutic value of an antiepileptic drug would include not only control of seizure activity but also protection of neuronal tissue. The present study examines the in vitro neuroprotective effects of stiripentol, an antiepileptic compound with γ-aminobutyric acidergic properties, on neuronal-astroglial cultures from rat cerebral cortex exposed to oxygen-glucose deprivation (OGD) or to glutamate (40 µM for 20 min), two in vitro models of brain injury. In addition, the affinity of stiripentol for the different glutamate receptor subtypes and the interaction with the cell influx of Na(+) and of Ca(2+) enhanced by veratridine and NMDA, respectively, are assessed. Stiripentol (10-100 µM) included in the culture medium during OGD or with glutamate significantly increased the number of surviving neurons relative to controls. Stiripentol displayed no binding affinity for different subtypes of glutamate receptors (IC50 >100 µM) but significantly blocked the entry of Na(+) and Ca(2+) activated by veratridine and NMDA, respectively. These results suggest that Na(+) and Ca(2+) channels could contribute to the neuroprotective properties of sitiripentol.

Combining two repurposed drugs as a promising approach for Alzheimer's disease therapy.

Alzheimer disease (AD) represents a major medical problem where mono-therapeutic interventions demonstrated only a limited efficacy so far. We explored the possibility of developing a combinational therapy that might prevent the degradation of neuronal and endothelial structures in this disease. We argued that the distorted balance between excitatory (glutamate) and inhibitory (GABA/glycine) systems constitutes a therapeutic target for such intervention. We found that a combination of two approved drugs - acamprosate and baclofen - synergistically protected neurons and endothelial structures in vitro against amyloid-beta (Aß) oligomers. The neuroprotective effects of these drugs were mediated by modulation of targets in GABA/glycinergic and glutamatergic pathways. In vivo, the combination alleviated cognitive deficits in the acute Aß25-35 peptide injection model and in the mouse mutant APP transgenic model. Several patterns altered in AD were also synergistically normalised. Our results open up the possibility for a promising therapeutic approach for AD by combining repurposed drugs.

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.

Moclobemide attenuates anoxia and glutamate-induced neuronal damage in vitro independently of interaction with glutamate receptor subtypes.

Recent data suggested the existence of a bidirectional relation between depression and neurodegenerative diseases resulting from cerebral ischemia injury. Glutamate, a major excitatory neurotransmitter, has long been recognised to play a key role in the pathophysiology of anoxia or ischemia, due to its excessive accumulation in the extracellular space and the subsequent activation of its receptors. A characteristic response to glutamate is the increase in cytosolic Na(+) and Ca(2+) levels which is due mainly to influx from the extracellular space, with a consequent cell swelling and oxidative metabolism dysfunction. The present study examined the in vitro effects of the antidepressant and type-A monoamine oxidase inhibitor, moclobemide, in neuronal-astroglial cultures from rat cerebral cortex exposed to anoxia (for 5 and 7 h) or to glutamate (2 mM for 6 h), two in vitro models of brain ischemia. In addition, the affinity of moclobemide for the different glutamate receptor subtypes and an interaction with the cell influx of Na(+) and of Ca(2+) enhanced by veratridine and K(+) excess, respectively, were evaluated. Moclobemide (10-100 microM) included in the culture medium during anoxia or with glutamate significantly increased in a concentration-dependent manner the amount of surviving neurons compared to controls. Moclobemide displayed no binding affinity for the different glutamate receptor subtypes (IC(50)>100 microM) and did not block up to 300 microM the entry of Na(+) and of Ca(2+) activated by veratridine and K(+), respectively. These results suggest that the neuroprotective properties of moclobemide imply neither the glutamate neurotransmission nor the Na(+) and Ca(2+) channels.

The use of streptolysin o for the treatment of scars, adhesions and fibrosis: initial investigations using murine models of scleroderma.

Diseases and conditions involving the deposition of excessive amounts of collagen include scleroderma, fibrosis, and scar and surgical adhesion formation. Diseases such as scleroderma may result from acute and chronic inflammation, disturbances in the normal parenchymal area, and activation of fibroblasts. ML-05, a modified form of the hemolytic and cytotoxic bacterial toxin, streptolysin O, is being developed for the treatment of such collagen-related disorders. At sublytic concentrations in vitro, ML-05 was shown to activate CD44 expression. This may modulate production of collagen, hyaluronate, and their associated enzymes to allow a restoration of normal extracellular matrices within tissues. More importantly, ML-05 appeared to decrease skin collagen levels in two in vivo models of collagen disorders, the tight skin mouse (Tsk) model of scleroderma, and the bleomycin-induced mouse skin fibrosis model. In the Tsk model, levels of hydroxyproline (a measure of total collagen) decreased by 25% in the Tsk+ML-05 treatment group relative to the Tsk+saline control group over a 3-month period. In the bleomycin-induced skin fibrosis study, hydroxyproline levels decreased from 15-22% over a 6-week period in a bleomycin-induced ML-05 treatment group (relative to levels in a bleomycin-induced, untreated control group). Hydroxyproline levels in samples from this treatment group were only slightly greater than levels in an uninduced control group at 8 weeks. Thus, ML-05 treatment appeared to reduce collagen levels in two separate mouse skin fibrosis models, one genetically based and the other chemically induced.