Velia D'Agata

Oxidative stress, mitochondrial dysfunction and cellular stress response in Friedreich's ataxia

There is significant evidence that the pathogenesis of several neurodegenerative diseases, including Parkinsons disease, Alzheimers disease, Friedreichs ataxia (FRDA), multiple sclerosis and amyotrophic lateral sclerosis, may involve the generation of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) associated with mitochondrial dysfunction. The mitochondrial genome may play an essential role in the pathogenesis of these diseases, and evidence for mitochondria being a site of damage in neurodegenerative disorders is based in part on observed decreases in the respiratory chain complex activities in Parkinsons, Alzheimers, and Huntingtons disease. Such defects in respiratory complex activities, possibly associated with oxidant/antioxidant imbalance, are thought to underlie defects in energy metabolism and induce cellular degeneration. The precise sequence of events in FRDA pathogenesis is uncertain. The impaired intramitochondrial metabolism with increased free iron levels and a defective mitochondrial respiratory chain, associated with increased free radical generation and oxidative damage, may be considered possible mechanisms that compromise cell viability. Recent evidence suggests that frataxin might detoxify ROS via activation of glutathione peroxidase and elevation of thiols, and in addition, that decreased expression of frataxin protein is associated with FRDA. Many approaches have been undertaken to understand FRDA, but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to FRDA pathogenesis. Brains of FRDA patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat shock response, which is one form of stress response. Heat shock proteins are proteins serving as molecular chaperones involved in the protection of cells from various forms of stress. In the central nervous system, heat shock protein (HSP) synthesis is induced not only after hyperthermia, but also following alterations in the intracellular redox environment. The major neurodegenerative diseases, Alzheimers disease (AD), Parkinsons disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Huntingtons disease (HD) and FRDA are all associated with the presence of abnormal proteins. Among the various HSPs, HSP32, also known as heme oxygenase I (HO-1), has received considerable attention, as it has been recently demonstrated that HO-1 induction, by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. This may open up new perspectives in medicine, as molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. In particular, manipulation of endogenous cellular defense mechanisms, such as the heat shock response, through nutritional antioxidants, pharmacological compounds or gene transduction, may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration.

Memory-specific temporal profiles of gene expression in the hippocampus

Many experiments in the past have demonstrated the requirement of de novo gene expression during the long-term retention of learning and memory. Although previous studies implicated individual genes or genetic pathways in learning and memory, they did not uncover the collective behaviors or patterns of the genes. We have used genome-scale screening to analyze gene expression during spatial learning of rats in the Morris water maze. Our results show distinct temporal gene expression profiles associated with learning and memory. Exogenous administration of one peptide whose sustained increase during memory retention was implicated by microarray analysis, fibroblast growth factor (FGF)-18, improved spatial learning behavior, suggesting that pharmacological modulation of pathways and targets identified may allow new therapeutic approaches for improving learning and memory. Results of this study also suggest that while learning and physical activity involve common groups of genes, the behavior of learning and memory emerges from unique patterns of gene expression across time.

The Major Green Tea Polyphenol, (-)-Epigallocatechin-3-Gallate, Induces Heme Oxygenase in Rat Neurons and Acts as an Effective Neuroprotective Agent against Oxidative Stress

Background: Oxidative stress induced by hyperglycemia is a key factor in the pathogenesis of diabetic complications, such as neuropathy. Recently, green tea catechins have received much attention, as they can facilitate a number of antioxidative mechanisms and improve glycemic control. Objective: The aim of this study was to investigate the cytoprotective effects of (-)-epigallocatechin-3-gallate (EGCG) against oxidative stress damage in a cell line of rat neurons. The role of heme oxygenase 1 (HO-1) induction by EGCG and the transcriptional mechanisms involved were also evaluated. Methods: Immortalized rat neurons (H 19-7) were exposed to various concentrations of EGCG (10–200 µM). After treatments (6 or 24 hours), cells were harvested for the determination of heme oxygenase activity, mRNA levels, and protein expression. Nuclear levels of Nrf2, a transcriptional factor involved in HO-1 activation, were also measured. Neurons were pretreated for 12 hours with EGCG 50 µM or EGCG 50 µM + zinc protoporphyrin IX 10 µM and then exposed for 2 hours to 50 mµ/mL glucose-oxidase before cell viability was determined. Results: In cultured neurons, elevated expression of HO-1 mRNA and protein were detected after 6 hours of incubation with 25–100 µM EGCG, and its induction relates with the activation of Nrf2. Interestingly, pre-incubation (12 hours) with EGCG 50 µM resulted in an enhanced cellular resistance to glucose oxidase–mediated oxidative damage; this cytoprotective effect was considerably attenuated by zinc protoporphyrin IX, an inhibitor of heme oxygenase activity. Conclusions: In this study, we demonstrated that EGCG, the major green tea catechin, induced HO-1 expression in cultured neurons, possibly by activation of the transcription factor Nrf2, and by this mechanism was able to protect against oxidative stress–induced cell death.

Gene expression profiles during long-term memory consolidation

Changes in gene expression have been postulated to occur during long‐term memory (LTM). We used high‐density cDNA microarrays to assess changes in gene expression 24 h after rabbit eye blink conditioning. Paired animals were presented with a 400 ms, 1000 Hz, 82 dB tone conditioned stimulus that coterminated with a 100 ms, 60 Hz, 2 mA electrical pulse unconditioned stimulus. Unpaired animals received the same conditioned and unconditioned stimuli but presented in an explicitly unpaired manner. Differences in expression levels between paired and unpaired animals in the hippocampus and cerebellar lobule HVI, two regions activated during eye blink conditioning, indicated the involvement of novel genes as well as the participation of previously implicated genes. Patterns of gene expression were validated by in situ hybridization. Surprisingly, the data suggest that an underlying mechanism of LTM involves widespread decreased, rather than increased, gene expression. These results demonstrate the feasibility and utility of a cDNA microarray system as a tool for dissecting the molecular mechanisms of associative memory.

Gene Expression Profiles in a Transgenic Animal Model of Fragile X Syndrome

Fragile X syndrome is the most common inherited form of mental retardation. Although this syndrome originates from the absence of the RNA-binding protein FMRP, the molecular mechanisms underlying the cognitive deficits are unknown. The expression pattern of 6789 genes was studied in the brains of wild-type and FMR1 knockout mice, a fragile X syndrome animal model that has been associated with cognitive deficits. Differential expression of more than two-fold was observed for the brain mRNA levels of 73 genes. Differential expression of nine of these genes was confirmed by real-time quantitative reverse transcription-polymerase chain reaction and by in situ hybridization. In addition to corroborating the microarray data, the in situ hybridization analysis showed distinct spatial distribution patterns of microtubule-associated protein 2 and amyloid beta precursor protein. A number of differentially expressed genes associated with the fragile X syndrome phenotype have been previously involved in other memory or cognitive disorders.

Functional and molecular expression of PACAP/VIP receptors in the rat retina

Receptor binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP), positively coupled to adenylate cyclase, have been previously described in the retina of different mammalian species. In the present study, we determined the mRNA expression of PACAP/VIP receptor variants in the rat retina and investigated their coupling to phospholipase C in addition to adenylate cyclase. The two forms of PACAP, PACAP27 and PACAP38, induced a dose-dependent (1-100 nM) increase of cAMP and [3H]inositol monophosphate levels, whereas VIP stimulated, with lower potency and efficacy, cAMP formation only. Reverse transcription-PCR analysis in the rat retina detected both type-I (PACAP-R and PACAP-HOP splice variants) and type-II (VIP-I and -2) receptor-mRNAs. These data indicate that PACAP and VIP may interact with multiple receptor subtypes and activate one (VIP) or two (PACAP) signal transduction mechanisms in the rat retina.

Tissue‐specific and Developmental Expression of Pituitary Adenylate Cyclase‐activating Polypeptide (PACAP) Receptors in Rat Brain

The two forms of pituitary adenylate cyclase‐activating polypeptide, PACAP27 and PACAP38, are novel members of the vasoactive intestinal peptide (VIP)/secretin/glucagon family of peptides. PACAP receptors that are positively coupled to adenylate cyclase and phospholipase C have been recently identified. We examined the expression of PACAP receptors in the rat cortex, hippocampus, cerebellum and hypothalamus during postnatal development. Functional studies revealed PACAP stimulation of cAMP formation in all the brain areas examined and [3H]inositol monophosphate ([3H]insP) accumulation only in the cerebellum and hypothalamus. Throughout development, the efficacy of PACAP in stimulating cAMP formation slightly increased in the cortex and hypothalamus and decreased in the hippocampus and cerebellum; PACAP stimulation of [3H]lnsP formation decreased in the cerebellum and remained steady in the hypothalamus. The effects of PACAP27 and PACAP38 on cAMP levels and inositol phospholipid hydrolysis were dose‐dependent between 1 and 100 nM. In the same brain areas, treatment with VIP increased cAMP formation at doses greater than 100 nM and failed to affect [3H]lnsP content, thus suggesting the existence of type‐l PACAP receptors. The reverse transcription polymerase chain reaction (RT‐PCR) was used to analyse the mRNA expression of type‐l PACAP receptor splice variants. PACAP receptor gene expression in the central nervous system was regulated in a developmental‐ and tissue‐specific manner. The PACAP‐R transcript was detected in all the brain areas examined whereas PACAP‐R‐hop mRNA occurred only in the cerebellum and hypothalamus. The different expression profiles and functional properties of PACAP receptors in the developing rat brain suggest an involvement of PACAP in histogenesis, maturation and neurotransmission.

PACAP and VIP prevent apoptosis in schwannoma cells

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are structurally endogenous peptides showing rich profile of biological activities. These peptides bind specific membrane receptors belonging to the superfamily of G protein-coupled receptors, the PAC1 and VPAC type receptors. Although these receptors have been identified in oligodendrocytes progenitors cells, to date the effects of PACAP and VIP in Schwann cells are still unknown. In the present study we investigated the expression of these neuropeptides as well as their receptors in a schwannoma cell line. RT-PCR and western blot analysis demonstrated that both PAC1 and VPAC2 receptors, but also PACAP peptide were expressed. To study the physiological effects mediated by PAC1/VPAC receptors, we evaluated their role in preventing apoptotic cell death induced by serum deprivation. Treatment with 100 nM PACAP38 and 100 nM VIP increased survival of serum-deprived schwannoma cells. Anti-apoptotic effects of these peptides were correlated to changes in BCL2 and BAX gene expression. Our results suggested that both PACAP38 and VIP could act as trophic factors in Schwann cells.

Cloning and distribution of the rat parkin mRNA.

We have isolated by RT-PCR and sequenced a partial cDNA coding for the rat homolog of parkin, a gene mutated in autosomal recessive juvenile parkinsonism. The 1.46 kb rat cDNA clone contains a 1376 bp coding sequence that shares strong similarity with the human parkin cDNA. RT-PCR and in situ hybridization revealed widespread expression of parkin in the rat brain and the periphery. The availability of the rat parkin cDNA and the initial elucidation of its distribution should facilitate further research on the pathophysiological role of parkin in the nervous system.

Metabotropic Glutamate Receptors and Neuronal Toxicity

Specific glutamate receptors coupled to polyphosphoinositide (PPI) hydrolysis have been described in brain slices, cultured neurons, and astrocytes, and in amphibian oocytes injected with rat brain mRNA (Sladeczek et al., 1985; Nicoletti et al., 1986a,b; Sugiyama et al., 1987). In most of the systems, metabotropic receptors are activated by lS,3R-aminocyclopentandicarboxylic acid (ACPD), quisqualate, ibotenate, and L-glutamate, but not by α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA), kainate, and N-methyl-D-aspartate (NMDA) (Nicoletti et al., 1986a; Schoepp and Johnson, 1988; 1989; Palmer et al., 1989). Trans-ACPD has been described as the most selective agonist of metabotropic receptors (Palmer et al., 1989), although it is less potent than quisqualate in stimulating inositolphosphate formation. In brain slices, stimulation of PPI hydrolysis by metabotropic receptor agonists is extremely high at the earlier stages of postnatal development (within the first 2 weeks after birth) and progressively declines during maturation (Nicoletti et al., 1986a). In adult tissue, the activation of metabotropic receptors is amplified in response to deafferentation (Nicoletti et al., 1987), as well as after induction of long-term potentiation (Aronica et al., 1991) or electrical kindling (Iadarola et al, 1986; Akiyama et al., 1987). Hence, it is likely that metabotropic receptors contribute to the synaptic events involved in the regulation of neuronal plasticity. However, based on the toxic effects of quisqualate in hippocampal slices (Garthwaite and Garthwaite, 1989) and cultured cortical neurons (Patel et al., 1990), a role for metabotropic receptors in the mechanism of neuronal degeneration has been suggested. We have addressed this problem in primary cultures of cerebellar neurons.