Andrea Diana

Vitamin A deficiency produces spatial learning and memory impairment in rats

Vitamin A and its derivatives (retinoids) play important roles in many physiological processes. The recent finding of high levels of cellular retinol-binding protein type 1 immunoreactivity, cellular retinoic acid-binding protein type 1 immunoreactivity and the presence of nuclear retinoid receptors in the central nervous system of adult rodents suggests that retinoids may carry out important roles in the adult brain. In consideration of the role of the hippocampus in spatial learning and memory we evaluated the effect of vitamin A deprivation in adult rats on these functions. Following 12 weeks of vitamin A-free diet, rats were trained to acquire a radial-arm maze task. Results show that this diet induced a severe deficit in the spatial learning and memory task. The cognitive impairment was fully restored when vitamin A was replaced in the diet. We also found a significant decrease in hippocampal acetylcholine release induced by scopolamine, assessed using microdialysis technique, and a reduction in the size of hippocampal nuclei of CA1 region in vitamin-deficient rats, compared to rats fed with a vitamin A-sufficient diet. These results demonstrate that vitamin A has a critical role in the learning and memory processes linked to a proper hippocampal functioning.

Homogeneous longitudinal profiles and synchronous fluctuations of mitochondrial transmembrane potential.

This study reports for the first time (a) the longitudinal profile of the transmembrane potential (mΔψ) of single mitochondria using a Nernstian fluorescent probe and (b) the distribution of mΔψ fluctuations of mitochondria undergoing permanent depolarization. Our findings show that (1) mitochondria in different energetic conditions coexist in the same cell, (2) mΔψ is rather homogeneous along the entire length of single mitochondria, (3) mΔψ is not influenced by the surrounding cytoplasmic environment and (4) mΔψ fluctuations occur simultaneously in groups of mitochondria connected in a network. Taken together, these findings provide further evidence for a functional relationship between mitochondrial arrangement and energetic condition.

Neuroprotective and anti-inflammatory properties of a novel non-thiazolidinedione PPARγ agonist in vitro and in MPTP-treated mice.

Peroxisome proliferator-activated receptor (PPAR)γ is a potential pharmacological target for disease-modification in Parkinsons disease (PD), mainly acting by modulating the neuroinflammatory response. However, currently available agonists thiazolidinediones (TZDs) present limitations due to safety concerns. We evaluated a novel thiobarbituric-like compound MDG548, which acts as a functional PPARγ agonist displaying higher and selective binding affinity as compared to TZDs. Neuroprotection by MDG548 was tested in vitro and in a mouse MPTP model of PD, and neuroinflammation was investigated as a putative underlying mechanism. Viability assay on rat cortical neurons showed lack of cytotoxic effect in the dose-range of 100 nM-10 μM, which was therefore used for testing in vitro protection against H2O2 and MPP+ neurotoxicity. MDG548 dose-dependently increased cell viability of rat cortical neurons co-treated with H2O2 or pre-exposed to MDG548 prior to H2O2. Moreover, MDG548 induced neuroprotection in MPP+-treated PC12 cells. NF-kB activation was investigated to assess anti-inflammatory activity. MDG548 dose-dependently decreased NF-kB activation induced by LPS (100 ng/100ml) in HEK-Blue-hTLR4 cells. Given the supposed cancer risk of other PPARγ agonists, Ames test for genotoxicity was performed in Salmonella typhimurium TA100 and TA98 strains, showing that MDG548 was not genotoxic. In vivo, BL/6J mice were treated with MPTP (20mg/kg i.p. once/day for 4 days) in association with saline or MDG548 (2, 5, 10 mg/kg i.p.). Stereological counting showed that MDG548 prevented the MPTP-induced reduction in TH-positive cells in the substantia nigra compacta (SNc) at all doses tested. Moreover, MDG548 reduced reactive microglia and iNOS induction in the SNc. MDG548, being a non-TZD compound with high PPARγ affinity, void of genotoxicity, and with in vitro as well as in vivo neuroprotective properties, provides a promising alternative in the search for safer PPARγ agonists to be tested as potential disease-modifying drugs in PD.

Characterization of depolarization and repolarization phases of mitochondrial membrane potential fluctuations induced by tetramethylrhodamine methyl ester photoactivation

Depolarization and repolarization phases (D and R phases, respectively) of mitochondrial potential fluctuations induced by photoactivation of the fluorescent probe tetramethylrhodamine methyl ester (TMRM) were analyzed separately and investigated using specific inhibitors and substrates. The frequency of R phases was significantly inhibited by oligomycin and aurovertin (mitochondrial ATP synthase inhibitors), rotenone (mitochondrial complex I inhibitor) and iodoacetic acid (inhibitor of the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase). Succinic acid (mitochondrial complex II substrate, given in the permeable form of dimethyl ester) abolished the rotenone‐induced inhibition of R phases. Taken together, these findings indicate that the activity of both respiratory chain and ATP synthase were required for the recovery of the mitochondrial potential. The frequency of D phases prevailed over that of R phases in all experimental conditions, resulting in a progressive depolarization of mitochondria accompanied by NAD(P)H oxidation and Ca2+ influx. D phases were not blocked by cyclosporin A (inhibitor of the permeability transition pore) or o‐phenyl‐EGTA (a Ca2+ chelator), suggesting that the permeability transition pore was not involved in mitochondrial potential fluctuations.

Intra- and Intercellular Distribution of Mitochondrial Probes and Changes after Treatment with MDR Modulators

Fluorescent probes are currently used to evaluate the mitochondrial transmembrane potential in situ. However, in parallel experiments using the probes JC‐1 and TMRM in different cell types (human astrocytes, HEp‐2, Vero, KB, and HeLa cells), we found that the distribution of JC‐1 and TMRM is highly variable not only in different cell types but also in different cells of the same cell type, a condition that has never been documented until our work. This phenomenon depends on a hidden, widespread multidrug resistance (MDR) phenotype that can be recognized only by comparative assays with MDR inhibitors (progesterone, verapamil, and cyclosporin A) and represents a serious risk of error in the evaluation of the mitochondrial potential.

Nuclear patterns of apoptotic and developing neurons of superior cervical ganglion of newborn rat

Superior cervical ganglion (SCG) neurons of female rats aged 3, 5 and 7 days revealed conspicuous nuclear changes in neurons undergoing postnatal cell death. Several qualitative and quantitative data such as nuclear size and shape, the presence of atypical chromocenters and chromatin textural features discriminated well neurons candidate to degeneration and those advancing in the direction of adult maturation. At least on morphological grounds, postnatal death of SCG neurons appears to be of apoptotic type. The sequence of nuclear events observed enables the recognition of the early stages of involution which prelude neuron degeneration.

Tulipaline A: structure-activity aspects as a nematicide and V-ATPase inhibitor.

Carbonyl groups are known to form covalent adducts with endogenous proteins, but so far, their nematicidal mechanism of action of has been overlooked. The nematicidal activity of ten lactones was tested in vitro against the root knot nematodes Meloidogyne incognita and Meloidogynearenaria. In particular, the saturated lactones α-methylene-γ-butyrolactone or tulipaline A (1) and γ-butyrolactone (3) were active against M. incognita with an EC50/48h of 19.3±10.0 and 40.0±16.2mg/L respectively. Moreover the α, β-unsaturated lactone 5,6-dihydro-2H-pyran-2-one (2) exhibited the strongest nematicidal activity against the two species with EC50/48h 14.5±5.3 and 21.2±9.7mg/L respectively. Here we propose that the toxic effects of lactones and aldehydes on M.incognita and M. arenaria might be a consequence of their vacuolar-type H(+)-ATPase (V-ATPase) inhibition activity; in fact α-methylene-γ-butyrolactone (1) and salicylaldehyde (12) produced an increased pH in lysosomal-like organelles on HeLa human cell line and this alteration was most likely related to a V-ATPase impairment.

Nuclear pattern recognition by two-parameter texture analysis

The present paper describes a simple procedure for the analysis of chromatin texture. High-resolution digitized images of nuclei are first standardized to render gray values invariant to staining and illumination conditions. Subsequently, the nucleus is subdivided by a square grid into 0.4 x 0.4 microns2 quadrats and standard deviations of gray values within each quadrat are estimated. Finally, the overall mean and standard deviation of quadrat standard deviations are calculated. These values may be considered as pure descriptors of the nuclear texture, as they represent the distribution of chromatin changes, disregarding any absolute densitometric and morphometric feature. Using the above descriptors it is possible to recognize at least seven chromatin patterns in a mixed population of developing and degenerating neurons. Results are visually verified by mapping the original pictures at the corresponding bivariate plot points. Comparison with the Markovian texture analysis is discussed.

Comparison of two commercial enzyme-linked immunosorbent assays for cerebrospinal fluid measurement of amyloid β1-42 and total tau

Amyloid β1–42 (Aβ1–42), total tau (t-tau), and phosphorylated tau (p-tau) are the main cerebrospinal fluid (CSF) biomarkers for early diagnosis of Alzheimer’s disease (AD). Detection of AD is critically important in view of the growing number of potential new drugs that may influence the course of the disease in its early phases. However, cut-off levels for these CSF biomarkers have not yet been established. Variability in absolute concentrations of AD biomarkers is high among studies and significant differences were noticed even within the same datasets. Variability in biomarkers levels in these assays may be due to many aspects of operating procedures. Standardization of pre-analytical and analytical procedures in collection, treatment, and storage of CSF samples is crucial because differences in sample handling can drastically influence results. Multicenter studies showed that usage of ELISA kits from different manufacturers also affects outcome. So far only very few studies tested the efficiency of ELISA kits produced by different vendors. In this study, the performance of Innogenetics (Gent, Belgium) and Invitrogen (Camarillo, CA, USA) ELISA kits for t-tau and Aβ1–42 was tested. Passing-Bablok analysis showed significant differences between Invitrogen and Innogenetics ELISA methods, making it impossible to use them interchangeably.

Phosphorylation pattern of tau associated with distinct changes of the growth cone cytoskeleton.

That both axons and dendrites grow at specialized terminations called growth cones was recognized already by Santiago Ramon y Cajal (Ramon y Cajal 1890). After inventing the technique of tissue culture, Ross G. Harrison confirmed Cajal’s inference that growth cones represent the elongating tips of axons and first described a living growth cone that is moving by local extension and retraction (Harrison 1907).