Francisco Javier Miana-Mena

Optimal methods to characterize the G93A mouse model of ALS

In the present study, we used the SOD1 (G93A) mutant transgenic mice as a model of amyotrophic lateral sclerosis (ALS). This model is widely used as a laboratory tool to study experimental treatments in vivo for ALS to investigate new therapeutic strategies for this neurodegenerative disease. Such studies require the objective quantification of different parameters while mice develop the disease. We have applied a battery of different and specific tests: scoring of motor deficits by a trained observer, weighing, survival measure, hanging wire test, rotarod task and electromyography, most of them commonly used to evaluate G93A animals. We have critically compared these methods, showing the significant influence of gender on the onset of symptoms, and the optimal moment to apply each test. These results should be taken into account in future therapeutic assays on this ALS model.

Neuronal activity-dependent membrane traffic at the neuromuscular junction

During development and also in adulthood, synaptic connections are modulated by neuronal activity. To follow such modifications in vivo, new genetic tools are designed. The nontoxic C-terminal fragment of tetanus toxin (TTC) fused to a reporter gene such as LacZ retains the retrograde and transsynaptic transport abilities of the holotoxin itself. In this work, the hybrid protein is injected intramuscularly to analyze in vivo the mechanisms of intracellular and transneuronal traffics at the neuromuscular junction (NMJ). Traffic on both sides of the synapse are strongly dependent on presynaptic neural cell activity. In muscle, a directional membrane traffic concentrates β-galactosidase-TTC hybrid protein into the NMJ postsynaptic side. In neurons, the probe is sorted across the cell to dendrites and subsequently to an interconnected neuron. Such fusion protein, sensitive to presynaptic neuronal activity, would be extremely useful to analyze morphological changes and plasticity at the NMJ.

Melatonin and Structurally-Related Compounds Protect Synaptosomal Membranes from Free Radical Damage

Since biological membranes are composed of lipids and proteins we tested the in vitro antioxidant properties of several indoleamines from the tryptophan metabolic pathway in the pineal gland against oxidative damage to lipids and proteins of synaptosomes isolated from the rat brain. Free radicals were generated by incubation with 0.1 mM FeCl3, and 0.1 mM ascorbic acid. Levels of malondialdehyde (MDA) plus 4-hydroxyalkenal (4-HDA), and carbonyl content in the proteins were measured as indices of oxidative damage to lipids and proteins, respectively. Pinoline was the most powerful antioxidant evaluated, with melatonin, N-acetylserotonin, 5-hydroxytryptophan, 5-methoxytryptamine, 5-methoxytryptophol, and tryptoline also acting as antioxidants.

Sex, fiber‐type, and age dependent in vitro proliferation of mouse muscle satellite cells

During postnatal growth and after muscle injury, satellite cells proliferate and differentiate into myotubes to form and repair musculature. Comparison of studies on satellite cell proliferation and differentiation characteristics is confounded by the heterogeneity of the experimental conditions used. To examine the influence of sex, age, and fiber‐type origin on in vitro properties of satellite cells derived from postnatal muscles, fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles were extracted from male and female mice of 1 week to 3 months of age. Myoblast proliferation and myogenic regulatory factor (MRF) expression was measured from cultures of freshly isolated satellite cells. Higher proliferation rate and elevated Myod1 expression was found in male EDL and SOL derived cells compared with females at age of 40, 60, and 120 days, whereas inverse tendency for cell proliferation was apparent in EDL of juvenile (7‐day‐old) pups. Myogenin and Mrf4 transcripts were generally elevated in males of 40 and 60 days of age and in female EDL of juveniles. However, these differentiation markers did not significantly correlate with proliferation rate at all ages. Pax7, whose overexpression can block myogenesis, was up‐regulated especially in 40‐day‐old females where MRF expression was low. These results indicate that gender, postnatal age, and muscle fiber origin affect proliferation and muscle transcription factor expression in vitro. The results also support the view that satellite cells originating from slow and fast muscles are intrinsically different and warrant further studies on the effect of cell origin for therapeutic approaches. J. Cell. Biochem. 112: 2825–2836, 2011.

Melatonin reduces protein and lipid oxidative damage induced by homocysteine in rat brain homogenates

Numerous data indicate that hyperhomocysteinemia is a risk factor for cardio‐ and cerebrovascular diseases. At least in part, homocysteine (HCY) impairs cerebrovascular function because it generates large numbers of free radicals. Since melatonin is a well‐known antioxidant, which reduces oxidative stress and decreases HCY concentrations in plasma, the aim of this study was to investigate the effect of melatonin in preventing HCY‐induced protein and lipid oxidation in rat brain homogenates. Brain homogenates were obtained from Sprague–Dawley rats and were incubated with or without HCY (0.01–5 mM) or melatonin (0.01–3 mM). Carbonyl content of proteins, and malondialdehyde (MDA) and 4‐hydroxyalkenals (4‐HDA) concentrations in the brain homogenates were used as an index of protein and lipid oxidation, respectively. Under the experimental conditions used, the addition of HCY (0.01–5 mM) to the homogenates enhanced carbonyl protein and MDA+4‐HDA formation. Melatonin reduced, in a concentration‐dependent manner, protein and lipid oxidation due to HCY in the brain homogenates. These data suggest that preserving proteins from oxidative insults is an additional mechanism by which melatonin may act as an agent in potentially decreasing cardiovascular and cerebrovascular diseases related to hyperhomocysteinemia. J. Cell. Biochem. 102: 729–735, 2007.

A Non-Viral Vector for Targeting Gene Therapy to Motoneurons in the CNS

Gene therapy vectors that can be targeted to motoneuronal cells are required in the field of neurodegenerative diseases. We propose the use of the atoxic fragment C of tetanus toxin (TTC) as biological activity carrier to the motoneurons. Naked DNA encoding β-galactosidase-TTC hybrid protein was used to transfect muscle cells in vivo, resulting in a selective gene transfer of the enzymatic activity to the CNS. In the muscle, level expression of β-galactosidase was readily detectable 24 h after injection, reaching a maximum after 4 days and gradually decreasing thereafter. Labelling in the hypoglossal motoneurons and motor cortex was observed from 4 days after injection. In this paper, we show that TTC works as an enzymatic activity carrier to the CNS when muscle cells are transfected in vivo. We have also shown that the presence of TTC does not have any influence on the expression of the transfected gene. Both these results warrant further studies of TTC as a means of treating motoneuron diseases in the field of gene therapy.

Levels of membrane fluidity in the spinal cord and the brain in an animal model of amyotrophic lateral sclerosis

A mutant form of the copper/zinc superoxide dismutase (SOD1) protein is found in some patients with amyotrophic lateral sclerosis (ALS). Alteration of the activity of this antioxidant enzyme leads to an oxidative stress imbalance, which damages the structure of lipids and proteins in the CNS. Using fluorescence spectroscopy, we monitored membrane fluidity in the spinal cord and the brain in a widely used animal model of ALS, the SODG93A mouse, which develops symptoms similar to ALS with an accelerated course. Our results show that the membrane fluidity of the spinal cord in this animal model significantly decreased in symptomatic animals compared with age-matched littermate controls. To the best of our knowledge, this is the first report showing that membrane fluidity is affected in the spinal cord of a SODG93A animal model of ALS. Changes in membrane fluidity likely contribute substantially to alterations in cell membrane functions in the nervous tissue from SODG93A mice.

Melatonin Protects Against Taurolithocholic-Induced Oxidative Stress in Rat Liver

Cholestasis, encountered in a variety of clinical disorders, is characterized by intracellular accumulation of toxic bile acids in the liver. Furthermore, oxidative stress plays an important role in the pathogenesis of bile acids. Taurolithocholic acid (TLC) was revealed in previous studies as the most pro‐oxidative bile acid. Melatonin, a well‐known antioxidant, is a safe and widely used therapeutic agent. Herein, we investigated the hepatoprotective role of melatonin on lipid and protein oxidation induced by TLC alone and in combination with FeCl3 and ascorbic acid in rat liver homogenates and hepatic membranes. The lipid peroxidation products, malondialdehyde and 4‐hydroxyalkenals (MDA + 4‐HDA), and carbonyl levels were quantified as indices of oxidative damage to hepatic lipids and proteins, respectively. In the current study, the rise in MDA + 4‐HDA levels induced by TLC was inhibited by melatonin in a concentration‐dependent manner in both liver homogenates and in hepatic membranes. Melatonin also had protective effects against structural damage to proteins induced by TLC in membranes. These results suggest that the indoleamine melatonin may potentially act as a protective agent in the therapy of those diseases that involve bile acid toxicity. J. Cell. Biochem. 110: 1219–1225, 2010. Published 2010 Wiley‐Liss, Inc.

Antiapoptotic activity maintenance of Brain Derived Neurotrophic Factor and the C fragment of the tetanus toxin genetic fusion protein

Neurotrophic factors have been widely suggested as a treatment for multiple diseases including motorneuron pathologies, like Amyotrophic Lateral Sclerosis. However, clinical trials in which growth factors have been systematically administered to Amyotrophic Lateral Sclerosis patients have not been effective, owing in part to the short half-life of these factors and their low concentrations at target sites. A possible strategy is the use of the atoxic C fragment of the tetanus toxin as a neurotrophic factor carrier to the motorneurons. The activity of trophic factors should be tested because their genetic fusion to proteins could alter their folding and conformation, thus undermining their neuroprotective properties. For this purpose, in this paper we explored the Brain Derived Neurotrophic Factor (BDNF) activity maintenance after genetic fusion with the C fragment of the tetanus toxin. We demonstrated that BDNF fused with the C fragment of the tetanus toxin induces the neuronal survival Akt kinase pathway in mouse cortical culture neurons and maintains its antiapoptotic neuronal activity in Neuro2A cells.

Active behavior of abdominal wall muscles: Experimental results and numerical model formulation

In the present study a computational finite element technique is proposed to simulate the mechanical response of muscles in the abdominal wall. This technique considers the active behavior of the tissue taking into account both collagen and muscle fiber directions. In an attempt to obtain the computational response as close as possible to real muscles, the parameters needed to adjust the mathematical formulation were determined from in vitro experimental tests. Experiments were conducted on male New Zealand White rabbits (2047±34g) and the active properties of three different muscles: Rectus Abdominis, External Oblique and multi-layered samples formed by three muscles (External Oblique, Internal Oblique, and Transversus Abdominis) were characterized. The parameters obtained for each muscle were incorporated into a finite strain formulation to simulate active behavior of muscles incorporating the anisotropy of the tissue. The results show the potential of the model to predict the anisotropic behavior of the tissue associated to fibers and how this influences on the strain, stress and generated force during an isometric contraction.