Biomedical subjects

U di Porzio

Publications and source records attributed to U di Porzio.

At least 19 recordsLinked to original sources

PubMed2001

Ontogeny of AMPA receptor gene expression in the developing rat midbrain and striatum.

AMPA receptors mediate most of the fast excitatory synaptic transmission in the mammalian CNS. Their ontogeny during embryonic (E) and postnatal (P) development is still poorly understood. We have studied the expression of the genes encoding for AMPA glutamate receptor subunits (GlurA, GlurB, GlurC and GlurD) in the rat ventral mesencephalon (MES) and striatum (STR) and in fetal midbrain primary cultures. Each receptor subunit shows unique area- and temporal-expression pattern. In MES, GluRA, GlurB and GlurC mRNA are detectable from the earliest embryonic stage studied (E13) and raise thereafter between E15 and E17, to plateau at E19 to adult values. Differently, GlurD mRNA increases throughout embryonic and postnatal development reaching its highest levels in the adult MES. The pattern of AMPA proteins corresponded to the mRNA levels for all subunits. In the STR, GlurA gene expression increases between E15 and E19, GlurB mRNA levels are sustained from the first embryonic stages analyzed (E15) until E19 and gradually decrease thereafter toward adult levels, GlurC gene expression increases gradually throughout ontogeny to reach its highest levels in the adult. STR GlurD transcripts remain at constant levels in all stages studied. In embryonic MES primary cultures, every subunit show a characteristic expression profile similar to that observed in vivo. They all decrease significantly during the second week in vitro. Thus, all the AMPA receptor subunit transcripts appear independently regulated during development, probably depending on the tissue-specific environment, which seems preserved in MES cultures.

Animals↗

PubMed2001

Neurofilament homeostasis and motoneurone degeneration.

Neurofilament disorganisation is a hallmark of various neurodegenerative diseases. We review here current knowledge of neurofilament structure, gene expression and function. Neurofilament involvement in motoneurone neurological diseases is discussed in view of recent data from transgenic and spontaneous mouse mutants. In the mammalian neurone, the three neurofilament subunits are assembled into intermediate filaments as obligate heteropolymers. The subunits are expressed differentially during development and adult life according to the cell type and its physiological state. In addition to the well-established role of neurofilaments in the control of axonal calibre, there is increasing evidence that neurofilaments can interact with other cytoskeletal components and can modulate the axoplasmic flow. Although the extent to which neurofilament abnormalities contribute to the pathogenesis in human diseases remains unknown, emerging evidence suggests that disorganised neurofilaments can provoke degeneration and death of neurones. BioEssays 23:24-33, 2001.

Animals↗

PubMed2001

Regionalized neurofilament accumulation and motoneuron degeneration are linked phenotypes in wobbler neuromuscular disease.

Abnormal neurofilament aggregates are pathological hall-mark of most neurodegenerative diseases, although their pathogenic role remains unclear. Increased expression of medium neurofilament (NFM) is an early molecular marker of wobbler mouse, an animal model of motoneuron disease. In the wr/wr, a vacuolar neuronal degeneration (VND) starts at 15 days postnatally, selectively in cervical spinal cord and brain stem motoneurons. Here we show that nfm gene hyperexpression is restricted to the aforementioned motoneurons and is specific for wr mutation. NF proteins accumulate in wr/wr before VND. wr/+ mice, which are asymptomatic, show intermediate NF accumulation between wr/wr and +/+ littermates, suggesting a gene dosage dependence of the wobbler pathology. Altogether our data indicate that NF hyperexpression and regionalized motoneuron degeneration are linked to the wr mutation, although with a still unknown relationship to the mutant gene activity.

Animals↗

PubMed2000

Epigenetic cues in midbrain dopaminergic neuron development.

Midbrain dopaminergic (DA) neurons subserve complex and varied neural functions in vertebrate CNS. Their progenitors give rise to DA neurons by the action of two extracellular inducers, Sonic Hedgehog and FGF8. After this first commitment, the function of selectively activated transcription factors, like the orphan steroid nuclear receptor Nurr1, is required for DA final determination. Subsequently, DA function is selectively modulated by specific interaction with the developing striatal target tissue. Committed and determined DA neurons express the key genes involved in DA neurotransmission at different times in development. Synthesis and intracellular accumulation of DA is achieved shortly after expression of Nurr1, while 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.

Animals↗

PubMed1999

Neuronal and glial properties coexist in a novel mouse CNS immortalized cell line.

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 c-AMP. 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, neuron-specific 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.

Animals↗

PubMed1999

Multiplex semi-quantitative reverse transcriptase-polymerase chain reaction of low abundance neuronal mRNAs.

The sequential use of reverse transcriptase and the polymerase chain reaction (RT-PCR) has provided molecular biology research with an exquisitely sensitive and fast technique for studying gene expression. This method is particularly useful to study transcripts in the nervous system, which are on average present at low levels and the amount of tissue or cells to be analyzed is often limited. Here, we describe a RT-PCR assay which allows the simultaneous detection and semi-quantitation of several transcripts (multiplex). Multiple PCR primer pairs are used to detect different target transcripts in a single reaction, together with a pair of primers able to amplify the hypoxantine-phosphoribosyl-transferase (HPRT), a gene constitutively expressed at low levels throughout the nervous system. HPRT levels remain constant also during neurogenesis and it is thus apt to be used in developmental neurobiology. This internal standard is the mRNA of reference to evaluate sample variation in RT and PCR reactions and to monitor the degradation and recovery of RNAs. Normalization with respect to HPRT cDNA allows to estimate the relative abundance of each target mRNA.

Animals↗

PubMed1996

Epigenetic factors and midbrain dopaminergic neurone development.

In the mammalian brain dopamine systems play a central role in the control of movement, hormone release, emotional balance and reward. Alteration of dopaminergic neurotransmission is involved in Parkinson's disease and other movement disorders, as well as in some psychotic syndromes. This review summarises recent findings, which shed some light on signals and cellular interactions involved in the specification and maturation of the dopaminergic function during neurogenesis. In particular we will focus on three major issues: (1) the differentiation of dopaminergic neurones triggered by direct contact with the midbrain floor plate cells through the action of sonic hedgehog; (2) the neurotrophic factors acting on dopaminergic neurones; and (3) the role of target striatal cells on the survival and the axonal growth of developing or grafted dopaminergic neurones.

Animals↗

PubMed1996

Early upregulation of medium neurofilament gene expression in developing spinal cord of the wobbler mouse mutant.

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 E13) 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 individuals 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.

Animals↗

PubMed1996

Dopamine transporter gene expression in rat mesencephalic dopaminergic neurons is increased by direct interaction with target striatal cells in vitro.

By using a semi-quantitative reverse transcriptase-PCR assay (RT-PCR) we have analyzed dopamine transporter (DAT), tyrosine hydroxylase (TH) and synaptic vesicle monoamine transporter (VMAT2) gene expression in rat mesencephalic (MES) primary cultures. Consistent with previous data obtained during rat MES ontogeny, the onset of DAT transcription in vitro is delayed in embryonic day (E)13, but not in E16, MES neurons when compared to that of TH and VMAT2. In co-culture, the addition of target striatal cells (STR) to E13 MES selectively increases DAT mRNA level in DA neurons during the first 3 days in vitro; cortical cells are ineffective. On the contrary, DAT gene does not appear up-regulated in E16 MES co-cultured with target STR cells, indicating that MES DA neurons respond to STR stimulation only at defined developmental stages. Up-regulation of DAT mRNA level by STR in E13 MES seems to require direct cell interactions since target cells do not exert their effect on DAT transcription when are separated from MES cells by a porous barrier, which only allows diffusion of soluble molecules. Thus maturation of DA neurotransmission in vitro appears to follow a developmental program which can be specifically modulated by their target STR cells.

Animals↗

PubMed1995

Acetylcholine esterase and peripherin mRNA level decrease in wobbler mouse.

Homozygote wobbler mice develop motoneurone degeneration. Throughout development the expression of choline acetyltransferase, of trkC receptor and F3 adhesion molecule genes is similar in wobbler and wild-type spinal cord. Acetylcholinesterase mRNA level instead is decreased to about 50% with respect to wild-type values in one forth of P5 and P10 wobbler progeny, putative wr/wr individuals; at P21 its expression is equally highly reduced in known homozygotes and it is reduced to 35% of normal values in about one half of the progeny, putative heterozygotes. Thus, similarly to medium neurofilament gene over-expression, reduced acetylcholinesterase gene expression is an early molecular marker for the wobbler mutation before onset of the illness.

Acetylcholinesterase↗

PubMed1994

Target cells modulate dopamine transporter gene expression during brain development.

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.

Animals↗

PubMed1993

Two functionally different glutamate receptors of the kainate subtype in embryonic rat mesencephalic cells.

The amino acid glutamate is a widespread excitatory neurotransmitter in the brain. It activates cation-selective channels expressed by nearly every neuron and by glial cells; also various second messenger cascades. Little is known about the ontogeny of glutamate neurotransmission during neurogenesis. We have analyzed the development and differentiation of excitatory amino acid responses and Na+ channels in cells dissociated from embryonic rat ventral mesencephalon and striatum as well as cortex and cerebellum using fluorescent voltage-sensitive oxonol dyes and flow cytometry. Analysis of fluorescence distribution revealed complex profiles under resting conditions which changed in a characteristic manner over the period studied (Embryonic (E) Days 12-20). The response to the Na+ channel agonist veratridine appeared at E12/13 in the mesencephalon. At E13 L-glutamate and kainate evoked changes in membrane potential interpreted as cellular hyperpolarization. At E15 some cells still responded by hyperpolarizing but an equal number began to depolarize. By E18 most cells depolarized. Both hyper- and depolarizations were eliminated by a specific antagonist at kainate receptors (6-cyano-7-nitroquinoxyline-2,3-dione) and by resuspending the cells in Na(+)-free medium. Both responses exhibited a concentration dependency with higher doses evoking stronger effects. In contrast, there was little effect of veratridine in the striatum at E15-E16, and the response to kainate or L-glutamate was predominantly depolarizing during the same embryonic period, with little or no effect until E18. These data show that in the developing CNS, sodium channel responses as well as excitatory aminoacid neurotransmitter responses first become functional in the mesencephalon and subsequently in the striatum, thus suggesting an anatomical gradient of expression. Our results also show that glutamate receptor-coupled functions vary with embryonic age and with regional distribution, suggesting possible roles of glutamate in early CNS embryogenesis, as morphogens or modulators of synaptic plasticity.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1993

Excitatory amino acid response in cultured rat striatal neurons results in a developmentally regulated cGMP formation.

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 amino 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.

Amino Acids↗

PubMed1991

Dopamine synthesis precedes dopamine uptake in embryonic rat mesencephalic neurons.

We have measured [3H]dopamine ([3H]DA) uptake and tyrosine hydroxylase-immunopositive immunostaining in cells acutely dissociated from the embryonic ventral mesencephalon (MSC). DA and its metabolites as well as catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) activities were determined in homogenates taken from the MSC and striatum (STR). In the embryonic ventral MSC measurable DA and tyrosine hydroxylase (TH) immunostaining were present as early as embryonic day (E) 12.5. At E14 the number of TH+ neurons was about 50% of the values at E18. In the MSC, DA concentration increased sharply at E16 and reached a plateau before birth that was 10-fold lower than adult values. In the STR, DA was first detected at E16, suggesting that DA fibers reach the STR at this embryonic stage. High-affinity DA uptake appeared in the MSC only at E16, concomitantly with the arrival of DA fibers in the STR, increased sharply between E16 and E18, and reached a plateau before birth. This uptake mechanism was not selective for catecholamine uptake inhibitors. Thus, DA synthesis in the MSC preceded the onset of high-affinity uptake mechanism, which could be correlated to the beginning of striatal DA innervation. Measurable MAO and COMT activities were detected as early as E13 (MSC) and E15 (STR), but not DA metabolites, which appeared later. We conclude that the high-affinity DA uptake mechanism in MSC DA neurons develops coincident with the arrival of DA fibers to the STR. The sharp increase of DA uptake between E16 and E18 is due only in part to an increase in the number of TH+ cells. These results support the hypothesis that in vivo the target STR neurons regulate the maturation of MSC DA cells.

Animals↗

PubMed1990

The effect of fetal mesencephalon implants on primate MPTP-induced parkinsonism. Histochemical and behavioral studies.

Parkinsonism or hemiparkinsonism was induced by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in four rhesus monkeys, which then received homologous fetal mesencephalon implants into the caudate nuclei. Cavities were prepared in the medial caudate nucleus 2 to 5 weeks before the fetal grafts were implanted. Control studies were conducted in unoperated MPTP-treated animals. Significant behavioral improvement, which occurred within weeks of implantation of fetal mesencephalon, was sustained for up to 7 months. No recovery was seen in the unoperated control animals. Histological examination revealed numerous surviving tyrosine hydroxylase (TH)-immunoreactive cell bodies. In addition to the graft, abundant TH-immunoreactive fibers were observed in the host caudate nucleus ventral to the region of the implanted and the nonimplanted cavities. Since TH-immunoreactive cell bodies of the substantia nigra compacta (A-9 cells) were destroyed by MPTP treatment and the ventral tegmental area (A-10) remained intact, it is concluded that sprouting of remaining host dopaminergic fibers occurs. These newly formed fibers appeared to emanate from the mesolimbic projection to the striatum. It is likely that the newly sprouted dopaminergic fibers account for the motor improvement elicited by precavitation and fetal mesencephalon implantation. These results suggest that the mechanism of recovery of parkinsonian primates after implantation of fetal dopaminergic tissue into the caudate nucleus is by stimulation of sprouting from host neurons. They also suggest that, with identification of the factors responsible for the formation of the new dopaminergic neuronal processes and with further development, tissue implantation may be an effective therapy for Parkinson's disease in humans.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1989

MPTP treatment combined with ethanol or acetaldehyde selectively destroys dopaminergic neurons in mouse substantia nigra.

We have previously reported that ethanol and acetaldehyde potentiate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice, enhancing dopamine (DA) depletion in the striatum. The present study was designed to determine whether such enhancement of neurotoxicity was specific for the nigro-striatal DA pathway. In 5-week-old mice acetaldehyde treatment did not enhance DA depletion seen 7 days after MPTP treatment. In 8-week-old animals, however, acetaldehyde or ethanol given with MPTP decreased striatal DA content to about 10% of controls, whereas the depletion was to 43% of controls when MPTP was given alone. In acetaldehyde or ethanol and MPTP-treated mice, changes in DA levels were observed only in the striatum. DA contents in the hypothalamus, olfactory bulb and frontal cortex were similar to that in controls. Contents of norepinephrine and serotonin in striatum, hypothalamus, olfactory bulb and cerebral cortex were not affected by any of the treatments. Three months after MPTP alone, striatal DA recovered to 74% of controls in 8-week-old mice, whereas no recovery occurred in acetaldehyde and MPTP-treated mice. Moreover, both tyrosine hydroxylase (TH) immunocytochemistry and Cresyl violet staining showed an extensive and selective cell loss in the pars compacta of the substantia nigra (SNc) of the mice treated with acetaldehyde or ethanol and MPTP, whereas MPTP alone caused only a limited cell degeneration.

Acetaldehyde↗

PubMed1989

Acetaldehyde directly enhances MPP+ neurotoxicity and delays its elimination from the striatum.

We have previously shown that ethanol and acetaldehyde (ACE) potentiate MPTP toxicity in mice, selectively enhancing dopamine (DA) depletion in the striatum and markedly increasing loss of DA neurons in the substantia nigra. Several months after these combined treatments there is no evidence of any recovery. In the present study, we measured the accumulation of the MPTP toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) in both striatum and whole brain, after MPTP alone or after combined treatments with ethanol or acetaldehyde, in order to determine whether this enhancement of toxicity is caused by changes in the MPTP metabolism. We also investigated whether acetaldehyde interfered with the conversion of MPTP to MPP+ by glial cells in vitro and studied its effects on the MPP+ uptake and spontaneous release from mesencephalic DA neurons or striatal astrocytes in primary cell cultures from E13 mouse embryos. The results from the in vivo experiments indicated that relatively low doses of ethanol or acetaldehyde potentiate directly MPP+ toxicity, apparently without interfering with its pharmacokinetics. However when higher doses of these drugs were administered, they also decreased MPP+ clearance from the striatum. ACE also increased initial MPTP accumulation in the whole brain but failed to enhance MPP+ levels, thus indicating that ACE effect is not related to MPTP metabolism. In vitro studies confirmed that ACE does not modify MPTP metabolism in striatal or mesencephalic astrocytes in culture. In mesencephalic neuronal cultures ACE does not change the levels of MPP+ uptake (MPP+ is accumulated in putative DA neurons in vitro with a mechanism similar to that of the DA high affinity uptake) nor its spontaneous release. These results indicate that the slower MPP+ clearance from the stratum after ACE is not related to a direct effect of ACE on DA neurons or astrocytes.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1988

Evolution of neurotransmitter receptor systems.

The presence of hormones, neurotransmitters, their receptors and biosynthetic and degradative enzymes is clearly not only associated with the present and the recent past but with the past several hundred million years. Evidence is mounting which indicates substantial conservation of protein structure and function of these receptors and enzymes over these tremendous periods of time. These findings indicate that the evolution and development of the nervous system was not dependent upon the formation of new or better transmitter substances, receptor proteins, transducers and effector proteins but involved better utilization of these highly developed elements in creating advanced and refined circuitry. This is not a new concept; it is one that is now substantiated by increasingly sophisticated studies. In a 1953 article discussing chemical aspects of evolution (Danielli, 1953) Danielli quotes Medawar, "... endocrine evolution is not an evolution of hormones but an evolution of the uses to which they are put; an evolution not, to put it crudely, of chemical formulae but of reactivities, reaction patterns and tissue competences." To also quote Danielli, "In terms of comparative biochemistry, one must ask to what extent the evolution of these reactivities, reaction patterns and competences is conditional upon the evolution of methods of synthesis of new proteins, etc., and to what extent the proteins, etc., are always within the synthetic competence of an organism. In the latter case evolution is the history of changing uses of molecules, and not of changing synthetic abilities." (Danielli, 1953). Figure 4 outlines a phylogenetic tree together with an indication of where evidence exists for both the enzymes that determine the biosynthesis and metabolism of the cholinergic and adrenergic transmitters and their specific cholinergic and adrenergic receptors. This figure illustrates a number of important points. For example, the evidence appears to show that the transmitters and their associated enzymes existed for a substantial period before their respective receptor proteins. While the transmitters and enzymes appear to exist in single cellular organisms, there is no solid evidence for the presence of adrenergic or cholinergic receptors until multicellular organisms where the receptors appear to be clearly associated with specific cellular and neuronal communication (Fig. 4). One can only speculate as to the possible role for acetylcholine and the catecholamine in single cell organisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗