Maria Rosaria Bianco

Genetically modified pigs produced with a nonviral episomal vector

Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy.

A New Nerve Growth Factor-Mimetic Peptide Active on Neuropathic Pain in Rats

Analysis of the structure of nerve growth factor (NGF)-tyrosine kinase receptor A (TrkA) complex, site-directed mutagenesis studies and results from chemical modification of amino acid residues have identified loop 1, loop 4, and the N-terminal region of the NGF molecule as the most relevant for its biological activity. We synthesized several peptides mimicking the two loops (1 and 4) linked together with an appropriate spacer, with or without the N-terminal region. Two peptides named NL1L4 and L1L4 demonstrated good NGF agonist activity at a concentration as low as 3 μm. They induced differentiation of chick dorsal root ganglia and stimulated tyrosine phosphorylation of TrkA, but not TrkB, receptor. In addition L1L4 was able to induce differentiation of PC12 cells. More interestingly, the peptide with the highest “in vitro” activity (L1L4) was shown to reduce neuropathic behavior and restore neuronal function in a rat model of peripheral neuropathic pain, thereby suggesting a potential therapeutic role for this NGF-mimetic peptide.

Fibrillogenesis and cytotoxic activity of the amyloid-forming apomyoglobin mutant W7FW14F

The apomyoglobin mutant W7FW14F forms amyloid-like fibrils at physiological pH. We examined the kinetics of fibrillogenesis using three techniques: the time dependence of the fluorescence emission of thioflavin T and 1-anilino-8-naphthalenesulfonate, circular dichroism measurements, and electron microscopy. We found that in the early stage of fibril formation, non-native apomyoglobin molecules containing β-structure elements aggregate to form a nucleus. Subsequently, more molecules aggregate around the nucleus, thereby resulting in fibril elongation. We evaluated by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) the cytotoxicity of these aggregates at the early stage of fibril elongation versus mature fibrils and the wild-type protein. Similar to other amyloid-forming proteins, cell toxicity was not due to insoluble mature fibrils but rather to early pre-fibrillar aggregates. Propidium iodide uptake showed that cell toxicity is the result of altered membrane permeability. Phalloidin staining showed that membrane damage is not associated to an altered cell shape caused by changes in the cytoskeleton.

Intrathecal NGF Administration Reduces Reactive Astrocytosis and Changes Neurotrophin Receptors Expression Pattern in a Rat Model of Neuropathic Pain

Nerve growth factor (NGF), an essential peptide for sensory neurons, seems to have opposite effects when administered peripherally or directly to the central nervous system. We investigated the effects of 7-days intrathecal (i.t.) infusion of NGF on neuronal and glial spinal markers relevant to neuropathic behavior induced by chronic constriction injury (CCI) of the sciatic nerve. Allodynic and hyperalgesic behaviors were investigated by Von Frey and thermal Plantar tests, respectively. NGF-treated animals showed reduced allodynia and thermal hyperalgesia, compared to control animals. We evaluated on lumbar spinal cord the expression of microglial (ED-1), astrocytic (GFAP and S-100β), and C- and Aδ-fibers (SubP, IB-4 and Cb) markers. I.t. NGF treatment reduced reactive astrocytosis and the density of SubP, IB4 and Cb positive fibers in the dorsal horn of injured animals. Morphometric parameters of proximal sciatic nerve stump fibers and cells in DRG were also analyzed in CCI rats: myelin thickness was reduced and DRG neurons and satellite cells appeared hypertrophic. I.t. NGF treatment showed a beneficial effect in reversing these molecular and morphological alterations. Finally, we analyzed by immunohistochemistry the expression pattern of neurotrophin receptors TrkA, pTrkA, TrkB and p75NTR. Substantial alterations in neurotrophin receptors expression were observed in the spinal cord of CCI and NGF-treated animals. Our results indicate that i.t. NGF administration reverses the neuro-glial morphomolecular changes occurring in neuropathic animals paralleled by alterations in neurotrophin receptors ratio, and suggest that NGF is effective in restoring homeostatic conditions in the spinal cord and maintaining analgesia in neuropathic pain.

Neonatal separation stress reduces glial fibrillary acidic protein- and S100β-immunoreactive astrocytes in the rat medial precentral cortex

The interactions between the mother/parents and their offspring provides socioemotional input, which is essential for the establishment and maintenance of synaptic networks in prefrontal and limbic brain regions. Since glial cells are known to play an important role in developmental and experience‐driven synaptic plasticity, the effect of an early adverse emotional experience induced by maternal separation for 1 or 6 h on the expression of the glia specific proteins S100β and glial fibrillary acidic protein (GFAP) was quantitatively analyzed in anterior cingulate cortex, hippocampus, and precentral medial cortex. Three animal groups were analyzed at postnatal day 14: (i) separated for 1 h; (ii) separated for 6 h; (iii) undisturbed (control). Twenty‐four hours after stress exposure, the stressed brains showed significantly reduced numbers of S100β‐immunoreactive (ir) cells in the anterior cingulate cortex (6‐h stress) and in the precentral medial cortex (1‐ and 6‐h stress). Significantly reduced numbers of GFAP‐ir cells were observed only in the medial precentral cortex (1‐ and 6‐h stress); no significant changes were observed in the anterior cingulate cortex. No significant changes of the two glial markers were observed in the hippocampus. Double‐labeling experiments with GFAP and pCREB revealed pCREB labeling only in the hippocampus, where the stressed brains (1 and 6 h) displayed significantly reduced numbers of GFAP/pCREB‐ir glial cells. The observed downregulation of glia‐specific marker proteins is in line with our hypothesis that emotional experience can alter glia cell activation in the juvenile limbic system.

Gliosis alters expression and uptake of spinal glial amino acid transporters in a mouse neuropathic pain model.

Gliosis is strongly implicated in the development and maintenance of persistent pain states following chronic constriction injury of the sciatic nerve. Here we demonstrate that in the dorsal horn of the spinal cord, gliosis is accompanied by changes in glial amino acid transporters examined by immunoblot, immunohistochemistry and RT-PCR. Cytokines, proinflammatory mediators and microglia increase up to postoperative day (pd) 3 before decreasing on pd 7. Then, spinal glial fibrillary acidic protein increases on pd 7, lasting until pd 14 and later. Simultaneously, the expression of glial amino acid transporters for glycine and glutamate (GlyT1 and GLT1) is reduced on pd 7 and pd 14. Consistent with a reduced expression of GlyT1 and GLT1, high performance liquid chromatography reveals a net increase in the concentration of glutamate and glycine on pd 7 and pd 14 in tissue from the lumbar spinal cord of neuropathic mice. In this study we have confirmed that microglial activation precedes astrogliosis. Such a glial cytoskeletal rearrangement correlates with a marked decrease in glycine and glutamate transporters, which might, in turn, be responsible for the increased concentration of these neurotransmitters in the spinal cord. We speculate that these phenomena might contribute, via over-stimulation of NMDA receptors, to the changes in synaptic functioning that are responsible for the maintenance of persistent pain.

Reactive astrocytosis-induced perturbation of synaptic homeostasis is restored by nerve growth factor

Reactive gliosis has been implicated in both inflammatory and neurodegenerative diseases. However, mechanisms by which astrocytic activation affects synaptic efficacy have been poorly elucidated. We have used the spared nerve injury (SNI) of the sciatic nerve to induce reactive astrocytosis in the lumbar spinal cord and investigate its potential role in disrupting the neuro-glial circuitry. Analysis of spinal cord sections revealed that SNI was associated with an increase of microglial (Iba1) and astrocytic (GFAP) markers. These changes, indicative of reactive gliosis, were paralleled by (i) a decrease of glial amino acid transporters (GLT1 and GlyT1) and increased levels of (ii) neuronal glutamate transporter EAAC1, (iii) neuronal vesicular GABA transporter (vGAT) and (iv) the GABAergic neuron marker GAD65/67. Besides the increase of Glutamate/GABA ratio, indicative of the perturbation of synaptic circuitry homeostasis, the boost of glutamate also compromised glial function in neuroprotection by up-regulating the xCT subunit of the glutamate-cystine antiport system and reducing glutathione (GSH) production. Finally, this study also shows that all these structural changes were linked to an alteration of endogenous NGF metabolism, as demonstrated by the decrease of endogenous NGF expression levels and increased activity of the NGF-degrading metalloproteinases. All the changes displayed by SNI-animals were reversed by a 7-days i.t. administration of NGF or GM6001, a generic metalloproteinase inhibitor, as compared to vehicle (ACSF)-treated animals. All together, these data strongly support the correlation between reactive astrogliosis and mechanisms underlying the perturbation of the synaptic circuitry in the SNI model of peripheral nerve injury, and the essential role of NGF in restoring both synaptic homeostasis and the neuroprotective function of glia.

Discriminative behavioral assessment unveils remarkable reactive astrocytosis and early molecular correlates in basal ganglia of 3-nitropropionic acid subchronic treated rats.

Reactive astrocytosis seems to be strongly implicated in the development and maintenance of inflammatory and neurodegenerative disorders. We design a new toxic model treatment with 3-nitropropionic acid (3-NP), a mitochondrial complex II irreversible inhibitor, to induce in rats Huntingtons disease (HD) like syndrome, characterized by hindlimb dystonia, involuntary choreiform movements and reduced global activity. In an attempt to find out whether molecular and morphological changes in the neuro-glial network could be involved in the pathogenesis of this disease, we developed a protocol of subchronic intra-peritoneal 3-NP intoxication. Moreover we set up specific, highly discriminative, behavioral tests to detect very early mild motor disabilities in 3-NP treated rats. This treatment did not cause severe cell death. However, in the Caudate-Putamen (CPu) of all 3-NP treated animals we found a massive astrogliosis, revealed by increased GFAP levels, paralleled by changes of the glial glutamate transporter GLAST distribution. To these glial changes we detected a transcriptional upregulation of c-fos and Sub-P in the striatal medium spiny neurons (MSN). We propose that this model of 3-NP intoxication along with the designed set of behavioral analyses allow to unmask in a very early phase the motor deficits and the underlying morpho-molecular changes associated to the onset of motor disabilities in the HD-like syndrome. Therefore this model unveil the key role played by the different components of the tripartite synapse in the pathogenesis of the HD, a putative non-cell-autonomous disease.

Reactive astrocytosis and glial glutamate transporter clustering are early changes in a spinocerebellar ataxia type 1 transgenic mouse model

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeats within the coding sequence of the ataxin-1 protein. In the present study, we used a conditional transgenic mouse model of SCA1 to investigate very early molecular and morphological changes related to the behavioral phenotype. In mice with neural deficits detected by rotarod performance, and simultaneous spatial impairments in exploratory activity and uncoordinated gait, we observed both significant altered expression and patchy distribution of excitatory amino acids transporter 1. The molecular changes observed in astroglial compartments correlate with changes in synapse morphology; synapses have a dramatic reduction of the synaptic area external to the postsynaptic density. By contrast, Purkinje cells demonstrate preserved structure. In addition, severe reactive astrocytosis matches changes in the glial glutamate transporter and synapse morphology. We propose these morpho-molecular changes are the cause of altered synaptic transmission, which, in turn, determines the onset of the neurological symptoms by altering the synaptic transmission in the cerebellar cortex of transgenic animals. This model might be suitable for testing drugs that target activated glial cells in order to reduce CNS inflammation.

BB14, a Nerve Growth Factor (NGF)-like peptide shown to be effective in reducing reactive astrogliosis and restoring synaptic homeostasis in a rat model of peripheral nerve injury

Peptidomimetics hold a great promise as therapeutic agents for neurodegenerative disorders. We previously described a Nerve Growth Factor (NGF)-like peptide, now named BB14, which was found to act as a strong TrkA agonist and to be effective in the sciatic nerve injury model of neuropathic pain. In this report we present the effects of BB14 in reducing reactive astrocytosis and reverting neuroplastic changes of the glutamate/GABAergic circuitry in the lumbar spinal cord following spared nerve injury (SNI) of the sciatic nerve. Immunohistochemical analysis of spinal cord sections revealed that SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) responses, indicative of reactive gliosis. These changes were paralleled by (i) decreased glial aminoacid transporters (GLT1 and GlyT1) and increased levels of (ii) neuronal glutamate transporter EAAC1, (iii) neuronal vesicular GABA transporter (vGAT) and (iv) the GABAergic neuron marker GAD65/67. A remarkable increase of the Glutamate/GABA ratio and the reduction of glutathione (GSH) levels were also indicative of modifications of glial function in neuroprotection. All these molecular changes were found to be linked to an alteration of endogenous NGF metabolism, as demonstrated by decreased levels of mature NGF, increase of proNGF and increased activity of NGF-degrading methallo-proteinases (MMPs). Biochemical alterations and SNI-related neuropathic behavior, characterized by allodynia and hyperalgesia, were reversed by 7-days i.t. administration of the NGF-like peptide BB14, as well as by increasing endogenous NGF levels by i.t. infusion of GM6001, a MMPs inhibitor. All together, while confirming the correlation between reactive astrogliosis and perturbation of synaptic circuitry in the SNI model of peripheral nerve injury, these data strongly support the beneficial effect of BB14 in reducing reactive astrogliosis and restoring synaptic homeostasis under pathological conditions linked to alteration of NGF availability and signaling, thereby suggesting a potential role of BB14 as a therapeutic agent.