Sebastiano Cavallaro

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.

Gene expression profiles of heme oxygenase isoforms in the rat brain.

In the last decade the heme oxygenase (HO) system has been strongly highlighted for its potential significance in maintaining cellular homeostasis. Nevertheless the physiological relevance of the three isoforms cloned to date, HO-1, HO-2 and HO-3, and their reciprocal interrelation have been poorly understood. In the brain the HO system has been reported to be very active and its modulation seems to play a crucial role in the pathogenesis of neurodegenerative disorders. To discriminate the regional and cellular distribution of HO isoforms in the CNS, we have developed a real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) protocol. With this highly sensitive methodology we have assessed for the first time the expression of all known HO isoform mRNAs in different rat brain areas. Although they presented a highly dissimilar range of expression, with HO-2>HO-1>HO-3, all three HO isoform transcripts demonstrated high level of expression in the cerebellum and the hippocampus, showing in a different scale, a strikingly parallel distribution gradient. We have also quantified the expression of HO mRNAs in primary culture of cortical neurons and type I astrocytes. While HO-1 and HO-2 were detected in both cellular types, HO-3 transcript was uniquely found in astrocytes. To further investigate the regional brain expression of this elusive and poorly studied isoform, we have performed in situ hybridization using an HO-3 specific riboprobe. HO-3 mRNA was expressed mainly in hippocampus, cerebellum and cortex. The initial elucidation of HO isoforms distribution should facilitate further research on their pathophysiological role in the nervous system.

Time domains of neuronal Ca2+ signaling and associative memory: steps through a calexcitin, ryanodine receptor, K+ channel cascade

Synaptic changes that underlie associative learning and memory begin with temporally related activity of two or more independent synaptic inputs to common postsynaptic targets. In turn, temporally related molecular events regulate cytosolic Ca2+ during progressively longer-lasting time domains. Associative learning behaviors of living animals have been correlated with changes of neuronal voltage-dependent K+ currents, protein kinase C-mediated phosphorylation and synthesis of the Ca2+ and GTP-binding protein, calexcitin (CE),and increased expression of the Ca2+-releasing ryanodine receptor (type II). These molecular events, some of which have been found to be dysfunctional in Alzheimers disease, provide means of altering dendritic excitability and thus synaptic efficacy during induction, consolidation and storage of associative memory. Apparently, such stages of behavioral learning correspond to sequential differences of Ca2+ signaling that could occur in spatially segregated dendritic compartments distributed across brain structures, such as the hippocampus.

Anticancer oral therapy: Emerging related issues

The use of oral anticancer drugs has shown a steady increase. Most patients prefer anticancer oral therapy to intravenous treatment primarily for the convenience of a home-based therapy, although they require that the efficacy of oral therapy must be equivalent and toxicity not superior than those expected with the intravenous treatment. A better patient compliance, drug tolerability, convenience and possible better efficacy for oral therapy as compared to intravenous emerge as the major reasons to use oral anticancer agents among oncologists. Inter- and intra-individual pharmacokinetic variations in the bioavailability of oral anticancer drugs may be more relevant than for intravenous agents. Compliance is particularly important for oral therapy because it determines the dose-intensity of the treatment and ultimately treatment efficacy and toxicity. Patient stands as the most important determinant of compliance. Possible measures for an active and safe administration of oral therapy include a careful preliminary medical evaluation and selection of patients based on possible barriers to an adequate compliance, pharmacologic issues, patient-focused education, an improvement of the accessibility to healthcare service, as well as the development of home-care nursing symptom-focused interventions. Current evidences show similar quality of life profile between oral and intravenous treatments, although anticancer oral therapy seems to be more convenient in terms of administration and reduced time lost for work or other activities. Regarding cost-effectiveness, current evidences are in favor of oral therapy, mainly due to reduced need of visits and/or day in hospital for the administration of the drug and/or the management of adverse events.

Pathways and genes differentially expressed in the motor cortex of patients with sporadic amyotrophic lateral sclerosis

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal disorder caused by the progressive degeneration of motoneurons in brain and spinal cord. Despite identification of disease-linked mutations, the diversity of processes involved and the ambiguity of their relative importance in ALS pathogenesis still represent a major impediment to disease models as a basis for effective therapies. Moreover, the human motor cortex, although critical to ALS pathology and physiologically altered in most forms of the disease, has not been screened systematically for therapeutic targets.ResultsBy whole-genome expression profiling and stringent significance tests we identify genes and gene groups de-regulated in the motor cortex of patients with sporadic ALS, and interpret the role of individual candidate genes in a framework of differentially expressed pathways. Our findings emphasize the importance of defense responses and cytoskeletal, mitochondrial and proteasomal dysfunction, reflect reduced neuronal maintenance and vesicle trafficking, and implicate impaired ion homeostasis and glycolysis in ALS pathogenesis. Additionally, we compared our dataset with publicly available data for the SALS spinal cord, and show a high correlation of changes linked to the diseased state in the SALS motor cortex. In an analogous comparison with data for the Alzheimers disease hippocampus we demonstrate a low correlation of global changes and a moderate correlation for changes specifically linked to the SALS diseased state.ConclusionGene and sample numbers investigated allow pathway- and gene-based analyses by established error-correction methods, drawing a molecular portrait of the ALS motor cortex that faithfully represents many known disease features and uncovers several novel aspects of ALS pathology. Contrary to expectations for a tissue under oxidative stress, nuclear-encoded mitochondrial genes are uniformly down-regulated. Moreover, the down-regulation of mitochondrial and glycolytic genes implies a combined reduction of mitochondrial and cytoplasmic energy supply, with a possible role in the death of ALS motoneurons. Identifying candidate genes exclusively expressed in non-neuronal cells, we also highlight the importance of these cells in disease development in the motor cortex. Notably, some pathways and candidate genes identified by this study are direct or indirect targets of medication already applied to unrelated illnesses and point the way towards the rapid development of effective symptomatic ALS therapies.

Spatial learning induced changes in expression of the ryanodine type II receptor in the rat hippocampus

Calcium signaling critical to neural functions is mediated through Ca2+ channels localized on both the plasma membrane and intracellular organelles such as endoplasmic reticulum. Whereas Ca2+ influx occurs via the voltage‐ or/and ligand‐sensitive Ca2+ channels, Ca2+ release from intracellular stores that amplifies further the Ca2+ signal is thought to be involved in more profound and lasting changes in neurons. The ryanodine receptor, one of the two major intracellular Ca2+ channels, has been an important target for studying Ca2+ signaling in brain functions, including learning and memory, due to its characteristic Ca2+‐induced Ca2+ release. In this study, we report regional and cellular distributions of the type‐2 ryanodine receptor (RyR2) mRNA in the rat brain, and effects of spatial learning on RyR2 gene expression at mRNA and protein levels in the rat hippocampus. Using in situ hybridization, reverse transcription polymerase chain reaction, and ribonuclease protection assays, significant increases in RyR2 mRNA were found in the hippocampus of rats trained in an intensive water maze task. With immunoprecipitation and immunoblotting, protein levels of RyR2 were also demonstrated to be increased in the microsomal fractions prepared from hippocampi of trained rats. These results suggest that RyR2, and hence the RyR2‐mediated Ca2+ signals, may be involved in memory processing after spatial learning. The increases in RyR2 mRNA and protein at 12 and 24 h after training could contribute to more permanent changes such as structural modifications during long‐term memory storage. Zhao, W., Meiri, N., Xu, H., Cavallaro, S., Quattrone, A., Zhang, L., Alkon, D. A. Spatial learning induced changes in expression of the ryanodine type II receptor in the rat hippocampus. FASEB J. 14, 290–300 (2000)

Functional and molecular diversity of PACAP/VIP receptors in cortical neurons and type I astrocytes

In the present study we determined the mRNA‐expression of pituitary adenylate cyclase activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) receptors in primary cultures of rat cortical neurons and type I astrocytes, and investigated the effects of PACAP38 on adenylyl cyclase, inositol phospholipid hydrolysis and intracellular calcium homeostasis. PACAP38 elicited a concentration‐dependent (1 n m–100 nm) increase in inositol phosphate levels and [Ca2+]i in neurons but not in type I astrocytes. The PACAP‐induced increase of intracellular calcium concentration, [Ca2+]i, was characterized by a spike, compatible with inositol trisphosphate (IP3) ‐induced calcium mobilization from intracellular stores, and a plateau phase, sustained by activation of capacitative calcium entry triggered by depletion of IP3‐sensitive calcium stores. In the absence of extracellular calcium, only the spike phase was present while the plateau phase was clearly reduced. In addition, thapsigargin pretreatment abolished the PACAP38‐induced [Ca2+]i rise. Treatment with 1 μm VIP did not affect [Ca2+]i in either neurons or type I astrocytes, clearly indicating the coupling of PAC1–HOP subtype to phospholipase‐C in neurons. In addition, as previously reported, PACAP38 stimulated cAMP formation in both neurons and type I astrocytes. Using the reverse transcription polymerase chain reaction, we found mRNA‐expression of PAC1 (PACAP – HOP variant) and VPAC2 in neurons, PAC1 (PACAP – R variant), VPAC1 and VPAC2 in astrocytes. These data indicate both a functional and molecular diversity of PACAP and VIP receptors in these cell types and support the view that the PAC1‐HOP variant may be responsible for phospholipase‐C activation and [Ca2+]i elevation in cortical neurons.

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.