U Di Porzio | Consiglio Nazionale delle Ricerche (CNR) (original) (raw)

Papers by U Di Porzio

PLoS ONE, 2013

Background: Proliferative vitreoretinopathy (PVR) is a destructive complication of retinal detach... more Background: Proliferative vitreoretinopathy (PVR) is a destructive complication of retinal detachment and vitreoretinal surgery which can lead to severe vision reduction by tractional retinal detachments. The purpose of this study was to determine the gene expression profile of epiretinal membranes (ERMs) associated with a PVR (PVR-ERM) and to compare it to the expression profile of less-aggressive secondary ERMs. Methodology/Principal Findings: A PCR-amplified complementary DNA (cDNA) library was constructed using the RNAs isolated from ERMs obtained during vitrectomy. The sequence from the 59 end was obtained for randomly selected clones and used to generate expressed sequence tags (ESTs). We obtained 1116 nonredundant clusters representing individual genes expressed in PVR-ERMs, and 799 clusters representing the genes expressed in secondary ERMs. The transcriptome of the PVR-ERMs was subdivided by functional subsets of genes related to metabolism, cell adhesion, cytoskeleton, signaling, and other functions, by FatiGo analysis. The genes highly expressed in PVR-ERMs were compared to those expressed in the secondary ERMs, and these were subdivided by cell adhesion, proliferation, and other functions. Querying 10 cell adhesionrelated genes against the STRING database yielded 70 possible physical relationships to other genes/proteins, which included an additional 60 genes that were not detected in the PVR-ERM library. Of these, soluble CD44 and soluble vascular cellular adhesion molecule-1 were significantly increased in the vitreous of patients with PVR. Conclusions/Significance: Our results support an earlier hypothesis that a PVR-ERM, even from genomic points of view, is an aberrant form of wound healing response. Genes preferentially expressed in PVR-ERMs may play an important role in the progression of PVR and could be served as therapeutic targets.

NeuroReport, May 9, 1994

We have analysed the expression of the dopamine transporter (DAT) gene and compared it with that ... more We have analysed the expression of the dopamine transporter (DAT) gene and compared it with that of tyrosine hydroxylase, neuronal GABA transporter and synaptic vesicle monoamine transporter genes during pre- and post-natal development of rat mesencephalic dopaminergic (DA) neurones. Our results show that DAT transcripts are not detectable until embryonic day (E) 15, whilst those of the other genes analysed are already present at E12. In vitro, the level of DAT gene transcription in mesencephalic E13 DA neurones is increased in coculture with target striatal cells. Thus striatal targets cells regulate, at the transcriptional level, a key step of dopaminergic neurotransmission during DA neurone development.

Homozygous wobbler mouse mutants develop a progressive paralysis due to spinal motoneuron degener... more Homozygous wobbler mouse mutants develop a progressive paralysis due to spinal motoneuron degeneration. To understand the molecular aspect underlying the genetic defect we have studied the embryonic (from El3) and postnatal expression of the three neurofilament and choline acetyltransferase genes in each member from several wild-type (wt) and wobbler (wr) progenies. There are no variations among wt littermates at all ages studied. In contrast, analyses of neurofilament mRNA reveals a 3-4-fold increase of medium neurofilament (NFM) mRNA in wobbler mice (wr/wr). The pattern of increased NFM mRNA during development, prior to the appearance of the wobbler phenotype, among littermates (from heterozygous carriers) conforms to a mendelian inheritance of a single gene defect 1:2:1 (wr/wr:wr/+:+/+). Light and heavy neurofilament mRNA levels are also increased later in development exclusively in those individualIs with high NFM mRNA values indicating that increase of the latter is associated with increase of the light and heavy subunit expression. Also NF proteins are increased. Expression of choline acetyltransferase gene is instead always comparable to normal control. Our study provides novel insights into the nature of the wobbler defect, strengthening the hypothesis that neurofilament accumulation plays a pivotal role in the etiopathogenesis of motoneuron degeneration.

A mes-c-myc A1 (A1) cell line was generated by retroviral infection of cultured embryonic mesence... more A mes-c-myc A1 (A1) cell line was generated by retroviral infection of cultured embryonic mesencephalic cells and selected by neomycin resistance. A1 cells cease to divide and undergo morphological differentiation after serum withdrawal or addition of cAMP. Proliferating or morphologically differentiated A1 cells are all positive for vimentin and nestin, a marker of neural precursor, and show neuronal markers such as microtubule-associated protein 1, neuronspecific enolase and peripherin, and the glial marker glial fibrillary acidic protein. Neuronal and glial markers coexist in single cells. Furthermore, A1 cells show presence of glutamic acid decarboxylase 67 mRNA and its embryonic form EP10 and accumulate the neurotransmitter GABA. Electrophysiological studies demonstrate that morphologically differentiated A1 cells display voltage-gated sodium and potassium channels in response to depolarizing stimuli. A1 cells thus represent a novel, bipotent neural cell line useful for studying CNS differentiation and plasticity, as well as the molecular mechanisms underlying development of GABAergic neurotransmission.

The relatively few dopaminergic (DA) neurons in the mammalian brain regulate many important neura... more The relatively few dopaminergic (DA) neurons in the mammalian brain regulate many important neural functions, including motor integration, neuroendocrine hormone release, cognition, emotive behaviors and reward. A number of laboratories, including ours, have contributed to unravel the mechanisms of DA phenotype induction and maturation and elucidated the role of epigenetic factors involved in specification, development and maintenance of midbrain dopaminergic functions. DA progenitors are first "committed" to give rise to DA neurons by the action of two secreted factors, Sonic hedgehog and fibroblast growth factor 8 (FGF8). Subsequently, the function of selectively activated transcription factors, Nurr1 and Ptx3, is required for the DA final determination. Further development of DA neurotransmission requires specific interactions with the developing target striatal cells, which modulate key DA functions, namely synthesis and uptake of the neurotransmitter. Committed and determined DA neurons express the key genes involved in DA neurotransmission at different times in development. In rodents, synthesis and intracellular accumulation of DA is achieved shortly after expression of Nurr1, while the onset of high affinity uptake, responsible for ending the neurotransmission, takes place after a few days. Cell contacts between the presynaptic DA neurons and target striatal neurons are apparently necessary for the fine modulation of DA function, in vivo and in vitro. Strikingly, the in situ maturation and phenotypic specialization of DA neurons grafted into the adult striatum/caudate-putamen parallels the normal development of committed fetal dopamine neurons during neurogenesis. The correct matching between the right presynaptic and postsynaptic neurons is required also for grafted DA cells.

Academia Letters, 2021

A fascinating hypothesis why music is inherent to the human species is that it was the first way ... more A fascinating hypothesis why music is inherent to the human species is that it was the first way of communication for hominins, preceding verbal language, thus confirming the hypothesis that our brain is really "beat(en)". There is evidence that music was part of hominin's life since the earliest times. The oldest musical instrument, a flute made from a bear femur found in Slovenia, dates back to about 40,000 years ago and may have belonged to both Neanderthals and Homo sapiens sapiens. Nevetheless group singing and dancing and more perishable musical instruments were likely part of hominin's life since much earlier. Recent theories hold that music had a common origin with language. While Darwin hypothesized that the development of the "musical" man was the result of the seduction processes between the two sexes, as in birds, modern ethnomusicology believes that human dance and song did not initially have aesthetic or reproductive purposes, instead, they were aimed at influencing reality, they had the purpose of averting, propitiating and/or evoking. From a neurobiologist's point of view, the study of interactions between music and the brain is suitable for studying how the human brain works and how different brain functions interact. Perception, action, cognition, emotion, learning, and memory are all involved in music listening and practicing. The brain is extraordinarily complex: in humans, it contains approximately eighty billion neurons, that can establish up to ten thousand connections (synapses) with other nerve cells and receive as many. The number of possible combinations is therefore enormous. Nerve cells acquire their own unique identities and establish their ordered and precise synaptic connections during development according to the genetic program and refinement of synaptic connection (pruning) also through apoptosis or programmed cell death of less active neurons.

Scientific Reports, 2015

Spine motility analysis has become the mainstay for investigating synaptic plasticity but is limi... more Spine motility analysis has become the mainstay for investigating synaptic plasticity but is limited in its versatility requiring complex, non automatized instrumentations. We describe an entropy-based method for determining the spatial distribution of dendritic spines that allows successful estimation of spine motility from still images. This method has the potential to extend the applicability of spine motility analysis to ex vivo preparations. Known since the time of Cajal, dendritic spines are an interesting subdomain present in most neurons, thought to play a role in synaptic plasticity and memory storage 1. In fact, interrogation of spine density (number of spines per unit length) has become one of the most widely used contemporary morphological correlates of plasticity in neurobiology 2,3. However, this approach contrasts with two-photon microscopy data demonstrating, in vivo, that synaptic plasticity may occur without modification of the total number of spines. Empirical data have now documented that it is the number of 'stable' spines over time rather than the total number of spines that is reliable in studying synaptic plasticity 4. Therefore, methods that measure spine motility/turnover promise better insight into the physiology of dendritic spines 5. Unfortunately, these methods require complex instrumentations and a technically demanding setup, which limit the possibility to extend their application to high throughput systems. This has resulted in a widespread failure to address spatial distribution of spines. Here, we introduce the notion that spatial distribution of spines along a dendrite can be represented as a bar code, carrying information about the local network, which, in turn, is related to the spine motility. We present a new method to indirectly assess changes in spine dynamics, based on measures of entropy from information theory, which takes into consideration the spatial distribution of the spines rather than their abundance. Entropy is a useful measure of the disorder and complexity in data series 6 , and similar quantities such as sample entropy, are better suited for short noisy time series 7. It should be noted that the length of data series may, in fact, represent an important limitation when the aim is to calculate the amount of information (entropy) stored in the data; in the case of dendritic spines, the length of the data series is limited by the finite size of the dendrites and therefore their analysis require appropriate algorithms. Entropy (H) is a measure of the amount of information (classically measured in bits of information) required to describe a system. A sequence of random data shows high entropy, whereas a stream of very uniform, repetitive data shows low entropy (since less information is needed for their description). Methods To explore if entropy can be a suitable approach to measure the distribution of dendritic spines, we used a transcranial two-photon imaging system to follow, in a time window of 30 min, identified spines of cortical neurons expressing GFP. Spine motility/turnover (we use these terms interchangeably within our text)was determined by measuring changes in spine length between consecutive time frames (see

Progress in clinical and biological research, 1982

Frontiers in Behavioral Neuroscience, 2015

Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role... more Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5, and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization. In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development.

Neuroscience Letters, 2013

Real time PCR for SERT mRNA quantification For quantitative real-time PCR, primer sets for rat SE... more Real time PCR for SERT mRNA quantification For quantitative real-time PCR, primer sets for rat SERT were designed to amplify 100-to-200-bp products, using PRIMER3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3). The following primer pairs were used for rat SERT: 5'-TCGCCTCCTACTACAACACC-3' and 5'-AGGAGTTCGTGCAGCTAGTC-3'. Total RNA was extracted from the HEK293T cells using TRIzol reagent (Invitrogen, Basel, Switzerland), including a RNase-free DNase step, according to the manufacturer's protocol. The yield and the integrity of RNA were determined by A 260 determinations and agarose gel electrophoresis respectively. First strand cDNA was generated from 2 µg of total RNA and Oligo (dT12-18)-primer with the M-MLV reverse transcription kit (Invitrogen) in a total volume of 20 µl. SYBR Green real time-PCR reactions were performed in 96-well plates using 7900HT Fast Real-Time PCR System (Applied Biosystem). 2 µl of each cDNA sample was amplified in triplicate, and every reaction included a negative control (having no template) to eliminate the possibility of contamination. Thermal cycling conditions comprised an initial step at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15s and 60°C for 1 min. Gene expression levels were quantified by the comparative threshold cycle (CT) method (Schmittgen and Livak, 2008) using hypoxanthine phosphoribosyltransferase (HPRT) as an internal control gene (Pernas-Alonso et al., 1999). The fractional number of PCR cycles CT required to obtain a given amount of amplified product in the exponential phase of amplification was determined for the gene of interest and for HPRT in each cDNA sample. The relative expression level of the gene of interest was then expressed as 2-ΔCT where ΔCT = CT gene of interest-CT HPRT.

International Journal of Developmental Neuroscience, 1990

The development of mesencephalic catecholaminergic neurons in the embryonic and fetal mouse was a... more The development of mesencephalic catecholaminergic neurons in the embryonic and fetal mouse was analysed in tissues fixed with 5% acrolein using polyclonal rabbit antibodies against tyrosine hydroxylase (TH), the first enzyme in catecholamine synthesis. The first TH positive cells were identified as early as day 8.5-9 of gestation and some expressed TH while apparently still migrating from the proliferative layer. The number of catecholamine cells increased dramatically by embryonic day 9.5-10; at gestation days 10.5-11 numerous TH positive cells bearing many neurites were localized in the ventral part of the mesencephalon but they were not yet separated into two different groups (A9 and A10). After 13 days of gestation two separate catecholaminergic groups could be visualized, although many TH positive cells with long neurites (putative dopaminergic neurons) could still be seen at the edges of the ventricle, and appeared to be moving towards the ventral mesencephalon. On the basis of these results the possibility that catecholamine cells that are produced early during the development of the midbrain may have neurotrophic and/or morphogenetic roles is discussed.

International Journal of Developmental Neuroscience, 2012

Progress in Brain Research, 1983

International Journal of Developmental Neuroscience, 1993

Glutamate and its analogues play a central role in excitatory neurotransmission throughout the br... more Glutamate and its analogues play a central role in excitatory neurotransmission throughout the brain. Their signal in the postsynaptic cells can be transduced by several second messengers. Here we show that in primary cultures of embryonic rat striatum, excitatory ammo acid receptor stimulation increases cyclic GMP intracellular concentration and the magnitude of this response depends upon the time in culture. Formation of cyclic GMP appears to be mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA type excitatory amino acid receptors, it is blocked by specific excitatory amino acid antagonists and requires extracellular Ca ++. The effect mediated via the NMDA receptor is also regulated by extracellular Mg++. These results show that excitatory amino acids make use of cyclic GMP for signal transduction in striatal neurons in vitro. We suggest that cyclic GMP may be an independent second messenger possibly important in the development of a defined population of striatal neurons.