Subthreshold Concentrations of Melatonin and Galantamine Improves Pathological AD-Hallmarks in Hippocampal Organotypic Cultures.

Melatonin is a neurohormone whose levels are significantly reduced or absent in Alzheimer's disease (AD) patients. In these patients, acetylcholinesterase inhibitors (AChEI) are the major drug class used for their treatment; however, they present unwanted cholinergic side effects and have provided limited efficacy in clinic. Because combination therapy is being extensively used to treat different pathological diseases such as cancer or acquired immune deficiency syndrome, we posed this study to evaluate if melatonin in combination with an AChEI, galantamine, could provide beneficial properties in a novel in vitro model of AD. Thus, we subjected organotypic hippocampal cultures (OHCs) to subtoxic concentrations of ß-amyloid (0.5 µM ßA) plus okadaic acid (1 nM OA), for 4 days. This treatment increased by 95 % cell death, which was mainly apoptotic as shown by positive TUNEL staining. In addition, the combination of ßA/OA increased Thioflavin S aggregates, hyperphosphorylation of Tau, oxidative stress (increased DCFDA fluorescence), and neuroinflammation (increased IL-1ß and TNFα). Under these experimental conditions, melatonin (1-1000 nM) and galantamine (10-1000 nM), co-incubated with the toxic stimuli, caused a concentration-dependent neuroprotection; maximal neuroprotective effect was achieved at 1 µM of melatonin and galantamine. Most effective was the finding that combination of sub-effective concentrations of melatonin (1 nM) and galantamine (10 nM) provided a synergic anti-apoptotic effect and reduction of most of the AD-related pathological hallmarks observed in the ßA/OA model. Therefore, we suggest that supplementation of melatonin in combination with lower doses of AChEIs could be an interesting strategy for AD patients.

Small synthetic hyaluronan disaccharides afford neuroprotection in brain ischemia-related models.

High molecular weight (HMW) glycosaminoglycanes of the extracellular matrix have been implicated in tissue repair. The aim of this study was to evaluate if small synthetic hyaluronan disaccharides with different degrees of sulfation (methyl 2-acetamido-2-deoxy-3-O-(ß-d-glucopyranosyluronic acid)-O-sulfo-α-d-glucopyranoside, sodium salt (di0S), methyl 2-acetamido-2-deoxy-3-O-(ß-d-glucopyranosyluronic acid)-6-di-O-sulfo-α-d-glucopyranoside, disodium salt (di6S) and methyl 2-acetamido-2-deoxy-3-O-(ß-d-glucopyranosyluronic acid)-4,6-di-O-sulfo-α-d-glucopyranoside, trisodium salt (di4,6S)) could improve cell survival in in vitro and in vivo brain ischemia-related models. Rat hippocampal slices subjected to oxygen and glucose deprivation and a photothrombotic stroke model in mice were used. The three hyaluran disaccharides, incubated during the oxygen and glucose deprivation (15min) and re-oxygenation periods (120min), reduced cell death of hippocampal slices measured as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, being the most potent di4,6S; in contrast, high molecular hyaluronan was ineffective. The protective actions of di4,6S against oxygen and glucose deprivation were related to activation of the PI3K/Akt survival pathway, reduction of p65 translocation to the nucleus, inhibition of inducible nitric oxide oxidase induction and reactive oxygen species production, and to an increase in glutathione levels. Administered 1h post-stroke, di4,6S reduced cerebral infarct size and improved motor activity in the beam walk test. In conclusion, di4,6S affords neuroprotection in in vitro and in vivo models of ischemic neuronal damage. Our results suggest that its neuroprotective effect could be exerted through its capability to reduce oxidative stress during ischemia. Its small molecular size makes it a more potential druggable drug to target the brain as compared with its HMW parent compound hyaluronan.

Trigeminothalamic barrelette neurons: natural structural side asymmetries and sensory input-dependent plasticity in adult rats.

In the rodent trigeminal principal nucleus (Pr5) the barrelette thalamic-projecting neurons relay information from individual whiskers to corresponding contralateral thalamic barreloids. Here we investigated the presence of lateral asymmetries in the dendritic trees of these neurons, and the morphometric changes resulting from input-dependent plasticity in young adult rats. After retrograde labeling with dextran amines from the thalamus, neurons were digitally reconstructed with Neurolucida, and metrically and topologically analyzed with NeuroExplorer. The most unexpected and remarkable result was the observation of side-to-side asymmetries in the barrelette neurons of control rats. These asymmetries more significantly involved the number of low-grade trees and the total dendritic length, which were greater on the left side. Chronic global input loss resulting from infraorbital nerve (IoN) transection, or loss of active touch resulting from whisker clipping in the right neutralized, or even reversed, the observed lateral differences. While results after IoN transection have to be interpreted in the context of partial neuron death in this model, profound bilateral changes were found after haptic loss, which is achieved without inflicting any nerve damage. After whisker trimming, neurons on the left side closely resembled neurons on the right in controls, the natural dendritic length asymmetry being reversed mainly by a shortening of the left trees and a more moderate elongation of the right trees. These results demonstrate that dendritic morphometry is both side- and input-dependent, and that unilateral manipulation of the sensory periphery leads to bilateral morphometric changes in second order neurons of the whisker-barrel system. The presence of anatomical asymmetries in neural structures involved in early stages of somatosensory processing could help explain the expression of sensory input-dependent behavioral asymmetries.

Differential growth of axons from sensory and motor neurons through a regenerative electrode: a stereological, retrograde tracer, and functional study in the rat.

Polyimide regenerative electrodes (RE) constitute a promising neural interface to selectively stimulate regenerating fibers in injured nerves. The characteristics of the regeneration through an implanted RE, however, are only beginning to be established. It was recently shown that the number of myelinated fibers distal to the implant reached control values 7 months postimplant; however, the functional recovery remained substantially below normal [J Biomed Mater Res 60 (2002) 517]. In this study we sought to determine the magnitude, and possible selectivity, of axonal regeneration through the RE by counting sensory and motor neurons that were retrogradely labeled from double tracer deposits in the sciatic nerve. Adult rats had their right sciatic nerves transected, and the stumps were placed in silicone tubes; some simply were filled with saline (Tube group), and others held a RE in its center (RE group). Simultaneously, the proximal stump was exposed to Diamidino Yellow. Two months later the nerves were bilaterally excised distal to the implant, and exposed to Fast Blue. Electrophysiological recordings, and skin nociceptive responses confirmed previous findings of partial functional recovery. In controls, an average of 20,000 and 3080 neurons were labeled in L4-L5 dorsal root ganglia (with minor contributions from L3 and/or L6), and in the ventral horn of the lumbar spinal cord, respectively. In the regenerating side, 35% of the DRG neurons were double-labeled, without differences between groups. In contrast, only 7.5% of motoneurons were double-labeled in the RE group, vs. 21% in the Tube group. Moreover, smaller ganglion cells regenerated better than large neurons by a significant 13.8%. These results indicate that the RE is not an obstacle for the re-growth of sensory fibers, but partially hinders fiber regeneration from motoneurons. They also suggest that fine fibers may be at an advantage over large ones to regenerate through the RE.

Protein variants of skeletal muscle regulatory myosin light chain isoforms: prevalence in mammals, generation and transitions during muscle remodelling.

The regulatory myosin light chains (rMLCs) of mammalian skeletal muscle display protein diversity arising from the existence of different isotypes and protein phosphorylation. Two-dimensional electrophoresis and immunoblotting allowed us to identify three variants of the slow and fast rMLC isoforms (designated LC2s, LC2s1, LC2s2, and LC2f, LC2f1, LC2f2, respectively, from less to more acidic). This study aimed to characterize their prevalence among different species and muscle types, the mechanism(s) of their generation and their transitions during fast-to-slow fibre type switching. In vitro dephosphorylation and back-phosphorylation experiments and mass spectrometric analysis of tryptic digests indicate that: (1) both acidic variants, within each isoform, contain a phosphorylated peptide, (2) all variants of each isoform share identical tryptic peptides, (3) only one phosphopeptide is present per isoform, and (4) the intermediate-acidic variants of both isoforms contain the same peptide in their phosphorylated and non-phosphorylated forms. The data indicate that the triad pattern of variants results from two partially superimposed doublets of phosphorylated/non-phosphorylated pairs. Continuous, low-frequency electrical stimulation of rat extensor digitorum longus muscle changed the relative proportions of variants within each isoform towards those of the soleus. It is suggested that the doublets of phosphorylated/non-phosphorylated pairs are involved in rMLC exchange during sarcomere remodelling.