Lorenzo Magrassi

Gene therapy of experimental brain tumors using neural progenitor cells

Glioblastomas, the most frequent and malignant of primary brain tumors, have a very poor prognosis. Gene therapy of glioblastomas is limited by the short survival of viral vectors and by their difficulty in reaching glioblastoma cells infiltrating the brain parenchyma. Neural stem/progenitor cells can be engineered to produce therapeutic molecules and have the potential to overcome these limitations because they may travel along the white matter, like neoplastic cells, and engraft stably into the brain. Retrovirus-mediated transfer of the gene for interleukin-4 is an effective treatment for rat brain glioblastomas. Here, we transferred the gene for interleukin-4 into C57BL6J mouse primary neural progenitor cells and injected those cells into established syngeneic brain glioblastomas. This led to the survival of most tumor-bearing mice. We obtained similar results by implanting immortalized neural progenitor cells derived from Sprague-Dawley rats into C6 glioblastomas. We also documented by magnetic resonance imaging the progressive disappearance of large tumors, and detected 5-bromodeoxyuridine-labeled progenitor cells several weeks after the injection. These findings support a new approach for gene therapy of brain tumors, based on the grafting of neural stem cells producing therapeutic molecules.

Flavobacteria as intracellular symbionts in cockroaches

Animal cells are the sole habitat for a variety of bacteria. Molecular sequence data have been used to position a number of these intracellular microorganisms in the overall scheme of eubacterial evolution. Most of them have been classified as proteobacteria or chlamydiae. Here we present molecular evidence placing an intracellular symbiont among the flavobacteria-bacteroides. This microorganism inhabits specialized cells in the cockroach fat body and has been described as a mutualistic endosymbiont of uncertain phylogenetic position. The small subunit ribosomal DNA of these bacteria was analysed after polymerase chain reaction amplification to investigate their phylogeny. The endosymbionts of five species of cockroaches were found to make up a coherent group with no close relatives within the eubacterial phylum defined by the flavobacteria. In addition, the relationships among the endosymbionts, as revealed by DNA sequence data, appeared to be congruent with the host taxonomic relationships. Based on the host fossil record, a tentative calibration of the nucleotide substitution rate for the cockroach flavobacteria gave results congruent with those obtained for the aphid endosymbiotic proteobacteria.

The establishment of intracellular symbiosis in an ancestor of cockroaches and termites

All cockroaches examined so far have been found to harbour a bacterial endosymbiont in specialized cells of the fat body, whereas Mastotermes darwiniensis is the only termite currently known to intracellular symbiont. The localization and mode of transmission of these bacteria are surprisingly similar, but so far no data have been published on their phylogenetic relationships. To address this issue, molecular sequence data were obtained from the genes encoding the small subunit ribosomal RNA of the M. darwiniensis endosymbiont, and compared with those obtained from endosymbionts of seven species of cockroaches. Molecular phylogenetic analysis unambiguously placed all these bacteria among the flavobacteria-bacteroides, indicating that the endosymbiont of M. darwiniensis is the sister group to the cockroach endosymbionts examined. Additionally, nucleotide divergence between the endosymbionts appears to be congruent with the palaeontological data on the hosts’s evolution. These results support previous claims that the original infection occurred in an ancestor common to cockroaches and termites. A loss of endosymbionts should subsequently have occurred in all termite lineages, except that which gave rise to M. darwiniensis.

Different types of cerebellar GABAergic interneurons originate from a common pool of multipotent progenitor cells

Different cerebellar phenotypes are generated according to a precise spatiotemporal schedule, in which projection neurons precede local interneurons. Glutamatergic neurons develop from the rhombic lip, whereas GABAergic neurons originate from the ventricular neuroepithelium. Progenitors in these germinal layers are committed toward specific phenotypes already at early ontogenetic stages. GABAergic interneurons are thought to derive from a subset of ventricular zone cells, which migrate in the white matter and proliferate up to postnatal life. During this period, different interneuron categories are produced according to an inside-out sequence, from the deep nuclei to the molecular layer (we show here that nuclear interneurons are also born during late embryonic and early postnatal days, after glutamatergic and GABAergic projection neurons). To ask whether distinct interneuron phenotypes share common precursors or derive from multiple fate-restricted progenitors, we examined the behavior of embryonic and postnatal rat cerebellar cells heterotopically/heterochronically transplanted to syngenic hosts. In all conditions, donor cells achieved a high degree of integration in the cerebellar cortex and deep nuclei and acquired GABAergic interneuron phenotypes appropriate for the host age and engraftment site. Therefore, contrary to other cerebellar types, which derive from dedicated precursors, GABAergic interneurons are produced by a common pool of progenitors, which maintain their full developmental potentialities up to late ontogenetic stages and adopt mature identities in response to local instructive cues. In this way, the numbers and types of inhibitory interneurons can be set by spatiotemporally patterned signals to match the functional requirements of developing cerebellar circuits.

Shc signaling in differentiating neural progenitor cells

Previously we found that the availability of ShcA adapter is maximal in neural stem cells but that it is absent in mature neurons. Here we report that ShcC, unlike ShcA, is not present in neural stem/progenitor cells, but is expressed after cessation of their division and becomes selectively enriched in mature neurons. Analyses of its activity in differentiating neural stem/progenitor cells revealed that ShcC positively affects their viability and neuronal maturation via recruitment of the PI3K-Akt-Bad pathway and persistent activation of the MAPK pathway. We suggest that the switch from ShcA to ShcC modifies the responsiveness of neural stem/progenitor cells to extracellular stimuli, generating proliferation (with ShcA) or survival/differentiation (with ShcC).

Members of the JAK/STAT proteins are expressed and regulated during development in the mammalian forebrain.

The presence and activation of members of the Janus Kinases/ Signal Transducers and Activator of Transcription proteins in response to specific cytokines is currently the focus of intense investigation in the hematopoietic system. Although some evidence suggests that cytokines might play an important role in brain development and brain pathologies, very limited information is available on the presence of the JAK/ STAT proteins in the Central Nervous System. Here we provide Western blot and immunohystochemistry data on the presence of Jak2 in vivo in the immature brain, its expression being greater in early stages of the embryonic life and gradually diminishing towards adulthood. Conversely, Jak1 was found expressed at a lower level compared to Jak2 and not modulated during brain maturation. Western blot data also show that specific members of the STAT family, the cytoplasmic substrates of the Janus Kinases, are present in vivo and that the extent of their expression is modulated differently at various stages. In particular, Stat6 protein levels were markedly attenuated at advanced stages of differentiation, as well as in the adult brain, with respect to early embryonic life. On the contrary, Stat3 levels did not vary. Analysis of Stat1 and Stat5 proteins showed a more complex expression pattern. These data indicate that members of the JAK/ STAT proteins are present and modulated in vivo in the embryonic and postnatal brain, therefore supporting their role in the modulation of gene expression during the different stages of brain maturation. J. Neurosci. Res. 54:320–330, 1998.

Laminar Fate and Phenotype Specification of Cerebellar GABAergic Interneurons

In most CNS regions, the variety of inhibitory interneurons originates from separate pools of progenitors residing in discrete germinal domains, where they become committed to specific phenotypes and positions during their last mitosis. We show here that GABAergic interneurons of the rodent cerebellum are generated through a different mechanism. Progenitors for these interneurons delaminate from the ventricular neuroepithelium of the embryonic cerebellar primordium and continue to proliferate in the prospective white matter during late embryonic and postnatal development. Young postmitotic interneurons do not migrate immediately to their final destination, but remain in the prospective white matter for several days. The different interneuron categories are produced according to a continuous inside-out positional sequence, and cell identity and laminar placement in the cerebellar cortex are temporally related to birth date. However, terminal commitment does not occur while precursors are still proliferating, and postmitotic cells heterochronically transplanted to developing cerebella consistently adopt host-specific phenotypes and positions. However, solid grafts of prospective white matter implanted into the adult cerebellum, when interneuron genesis has ceased, produce interneuron types characteristic of the donor age. Therefore, specification of cerebellar GABAergic interneurons occurs through a hitherto unknown process, in which postmitotic neurons maintain broad developmental potentialities and their phenotypic choices are dictated by instructive cues provided by the microenvironment of the prospective white matter. Whereas in most CNS regions the repertoire of inhibitory interneurons is produced by recruiting precursors from different origins, in the cerebellum it is achieved by creating phenotypic diversity from a single source.

REST Controls Self-Renewal and Tumorigenic Competence of Human Glioblastoma Cells

The Repressor Element 1 Silencing Transcription factor (REST/NRSF) is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM) specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets.

CELL DEATH IN THE OLFACTORY EPITHELIUM

In the nervous system of vertebrates the olfactory epithelium presents unique cytological characteristics. In the olfactory mucosa, olfactory neurons die and are replaced from undifferentiated neuroblasts over the entire life span of the animal. It remains unclear whether these neurons die as a result of a direct insult from the environment or in fulfillment of a physiological program of cell death. We have studied the distribution and the characteristics of cell death in the olfactory epithelium of normal, adult rats. The olfactory epithelium contains pycnotic bodies resembling those described for thymocytes undergoing terminal apoptotic changes. These appear at all levels in the epithelium, under both light and electron microscopes and can also be demonstrated after vital staining with acridine orange. Chromatin condensation into large blocks, often located at the nuclear periphery, is a morphological hallmark of the nuclei of mature olfactory neurons, which also present an increase in electron density of the cytoplasm. After non-radioactive in situ labeling of fragmented DNA, the nuclei of olfactory neurons are positive. Under the same reaction conditions (mild protease digestion), most of the nuclei of the supporting and basal cells are negative. In vivo incorporation of 5-bromouridine, a marker of RNA synthesis, is also lower in olfactory neurons than in basal and supporting cells. These findings suggest that olfactory neurons are committed very early to physiological cell death.

Interaction of the transplanted olfactory placode with the optic stalk and the diencephalon in Xenopus laevis embryos

The effect of olfactory placode transplantation on the differentiation of the optic vesicle and stalk has been studied in Xenopus laevis embryos. Host embryos (stages 23-24) received the transplant of two olfactory placodes from same-stage donors in place of a partially or totally removed optic vesicle. All tadpoles were sacrificed at stages 47-50. The host tadpoles were subdivided in three groups according to the amount of optic vesicle removed. In the first group all of the optic vesicle was removed. At sacrifice a lobar mass of nervous tissue, continuous with and protruding from the diencephalic wall, was penetrated by the olfactory nerves from the transplanted placodes and a well-defined glomerular layer was present at the entrance zone of the olfactory nerves. The lobar protrusion contained a normal ventricular cavity, connected by a foramen to the third ventricle. The ventricle was lined by a mitotically active ependymal layer. In the second group the host embryos were deprived of two-thirds of the optic vesicle. In these animals we observed the development of a round mass of nervous tissue connected by a peduncle to the diencephalic wall. A ventricle lined by ependyma was present in the formation; however, the cavity was not continuous with the third ventricle. Olfactory nerves from the transplanted placodes penetrated the rostral portion of the nervous mass and formed a distinct glomerular layer. In the third group of embryos only one-third of the optic vesicle was removed. These animals developed irregularly shaped structures where ocular tissue and nervous tissue coexisted. The termination of the supranumerary olfactory nerves and the glomerular layer were restricted to the non-ocular nervous tissue. The interpretation of the histogenetic phenomena determining the protrusions is difficult and further studies are needed. It seems highly probable, however, that the fate of such determined structures as the optic peduncle and vesicle can be influenced by the olfactory input, supporting the hypothesis that the ectopically directed olfactory fibres interfere with the differentiation of the optic structures.