Stefano Giannetti

Early coding of reaching: frontal and parietal association connections of parieto-occipital cortex.

The ipsilateral association connections of the cortex of the dorsal part of the rostral bank of the parieto‐occipital sulcus and of the adjoining posterior part of the superior parietal lobule were studied by using different retrograde flourescent tracers. Fluoro‐Ruby, Fast blue and Diamidino yellow were injected into visual area V6A, and dorso‐caudal (PMdc, F2) and dorso‐rostral (PMdr, F7) premotor cortex, respectively. The parietal area of injection had been previously characterized physiologically in behaving monkeys, through a variety of oculomotor and visuomanual tasks. Area V6A is mainly linked by reciprocal projections to parietal areas 7m, MIP (medial intraparietal) and PEa, and, to a lesser extent, to frontal areas PMdr (rostral dorsal premotor cortex, F7) and PMdc (F2). All these areas project to that part of the dorsocaudal premotor cortex that has a direct access to primary motor cortex. V6A is also connected to area F5 and, to a lesser extent, to 7a, ventral (VIP) and lateral (LIP) intraparietal areas. This pattern of association connections may explain the presence of visually‐related and eye‐position signals in premotor cortex, as well as the influence of information concerning arm position and movement direction on V6A neural activity. Area V6A emerges as a potential ‘early’ node of the distributed network underlying visually‐guided reaching. In this network, reciprocal association connections probably impose, through re‐entrant signalling, a recursive property to the operations leading to the composition of eye and hand motor commands.

The neuroprotective and neurogenic effects of neuropeptide Y administration in an animal model of hippocampal neurodegeneration and temporal lobe epilepsy induced by trimethyltin

J. Neurochem. (2012) 122, 415–426.

A BMP7 variant inhibits the tumorigenic potential of glioblastoma stem-like cells

Glioblastoma multiforme (GBM) is among the most aggressive tumor types and is essentially an incurable malignancy characterized by resistance to chemo-, radio-, and immunotherapy. GBM is maintained by a hierarchical cell organization that includes stem-like, precursor, and differentiated cells. Recurrence and maintenance of the tumor is attributed to a small population of undifferentiated tumor-initiating cells, defined as glioblastoma stem-like cells (GSLCs). This cellular hierarchy offers a potential treatment to induce differentiation of GSLCs away from tumor initiation to a more benign phenotype or to a cell type more amenable to standard therapies. Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth and differentiation. In vitro, a BMP7 variant (BMP7v) decreased primary human GSLC proliferation, endothelial cord formation, and stem cell marker expression while enhancing neuronal and astrocyte differentiation marker expression. In subcutaneous and orthotopic GSLC xenografts, which closely reproduce the human disease, BMP7v decreased tumor growth and stem cell marker expression, while enhancing astrocyte and neuronal differentiation compared with control mice. In addition, BMP7v reduced brain invasion, angiogenesis, and associated mortality in the orthotopic model. Inducing differentiation of GSLCs and inhibiting angiogenesis with BMP7v provides a potentially powerful and novel approach to the treatment of GBM.

Combined PDK1 and CHK1 inhibition is required to kill glioblastoma stem-like cells in vitro and in vivo.

Glioblastoma (GBM) is the most common and deadly adult brain tumor. Despite aggressive surgery, radiation, and chemotherapy, the life expectancy of patients diagnosed with GBM is ∼14 months. The extremely aggressive nature of GBM results from glioblastoma stem-like cells (GSCs) that sustain GBM growth, survive intensive chemotherapy, and give rise to tumor recurrence. There is accumulating evidence revealing that GSC resilience is because of concomitant activation of multiple survival pathways. In order to decode the signal transduction networks responsible for the malignant properties of GSCs, we analyzed a collection of GSC lines using a dual, but complementary, experimental approach, that is, reverse-phase protein microarrays (RPPMs) and kinase inhibitor library screening. We treated GSCs in vitro with clinically relevant concentrations of temozolomide (TMZ) and performed RPPM to detect changes in phosphorylation patterns that could be associated with resistance. In addition, we screened GSCs in vitro with a library of protein and lipid kinase inhibitors to identify specific targets involved in GSC survival and proliferation. We show that GSCs are relatively insensitive to TMZ treatment in terms of pathway activation and, although displaying heterogeneous individual phospho-proteomic profiles, most GSCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. However, simultaneous multipathway inhibition by the staurosporin derivative UCN-01 results in remarkable inhibition of GSC growth in vitro. The activity of UCN-01 on GSCs was confirmed in two in vivo models of GBM growth. Finally, we used RPPM to study the molecular and functional effects of UCN-01 and demonstrated that the sensitivity to UCN-01 correlates with activation of survival signals mediated by PDK1 and the DNA damage response initiated by CHK1. Taken together, our results suggest that a combined inhibition of PDK1 and CHK1 represents a potentially effective therapeutic approach to reduce the growth of human GBM.

Organization of cortico-cortical associative projections in rats exposed to ethanol during early postnatal life

The fine organization of cortico-cortical associative projections was investigated in adult rats exposed to inhalation of ethanol during the first postnatal week. Ethanol-treated and control animals received cortical injections of biotinylated dextran amine combined with N-methyl-D-aspartic acid, in order to obtain a Golgi-like retrograde labeling of associative pyramidal neurons. The results obtained from the analysis of labeling can be summarized as follows: (a) there are fewer associative projection neurons in ethanol-treated than in normal animals; (b) the ratio between the number of supragranular and infragranular associative neurons is higher in ethanol-treated animals compared to controls; (c) the basal dendrites of pyramidal associative cells of layer 2/3 display a simplified dendritic branching in ethanol exposed cases as compared to controls; (d) the cluster analysis shows that normal dendrites can be clearly subdivided into different groups according to their geometric properties, whereas dendrites from animals exposed to ethanol follow less robust grouping criteria. These differences are discussed in consideration of the functional alterations that characterize the fetal alcohol syndrome.

Neurotrophic features of human adipose tissue-derived stromal cells: in vitro and in vivo studies.

Due to its abundance, easy retrieval, and plasticity characteristics, adipose-tissue-derived stromal cells (ATSCs) present unquestionable advantages over other adult-tissue-derived stem cells. Based on the in silico analysis of our previous data reporting the ATSC-specific expression profiles, the present study attempted to clarify and validate at the functional level the expression of the neurospecific genes expressed by ATSC both in vitro and in vivo. This allowed evidencing that ATSCs express neuro-specific trophins, metabolic genes, and neuroprotective molecules. They were in fact able to induce neurite outgrowth in vitro, along with tissue-specific commitment along the neural lineage and the expression of the TRKA neurotrophin receptor in vivo. Our observation adds useful information to recent evidence proposing these cells as a suitable tool for cell-based applications in neuroregenerative medicine.

Enhanced neurogenesis during trimethyltin-induced neurodegeneration in the hippocampus of the adult rat.

The occurrence of neurogenesis in the hippocampus of the adult rat during trimethyltin (TMT)-induced neurodegeneration was investigated using bromodeoxyuridine (BrdU). Fifteen days after TMT intoxication, BrdU-labeled cells were significantly more numerous in the hippocampus of treated animals, gradually decreasing towards the control value 21 days after intoxication in the dentate gyrus (DG), while in the CA3/hilus region BrdU-labeled cells were still more numerous in TMT-treated rats. In order to investigate the fate of newly-generated cells double labeling experiments using neuronal or glial markers were performed. Colocalization of the neuronal marker NeuN was detected in many BrdU-positive cells in the DG, while in the CA3/hilus region no colocalization of NeuN and BrdU could be observed. No colocalization of BrdU and the astroglial marker GFAP or the microglial marker OX-42 was detected either in the DG and or in the CA3/hilus region. The results indicate an enhancement of endogenous neurogenesis in the hippocampus during TMT-induced neurodegeneration, with the development of a subpopulation of regenerated cells into neurons in the DG, while in the CA3/hilus region the population of newly-generated cells should be regarded as undifferentiated.

Organization of cortico-cortical associative projections in a rat model of microgyria.

Microgyria was experimentally induced by focal freezing lesions of the frontal cortex in newborn rats. Adult microgyric animals received cortical injections of biotinylated dextran amine combined with NMDA, in order to obtain a Golgi-like retrograde labeling of cortico-cortical association neurons. Injections were performed either rostrally or caudally to the microgyric lesion. Results demonstrate that long-range association projections traveling across the zone of the microgyric lesion arise mainly from infragranular layers. In normal animals the same projections originate both from supragranular and infragranular layers. The analysis of single basal dendrites of layer 2/3 in microgyric animals demonstrates a simplified branching pattern, with a number of end points lower than in control animals. Potential implications for microgyria-associated epilepsy are discussed.

Chemical Compartmentation and Relationships between Calcium‐binding Protein Immunoreactivity and Layer‐specific Cortical and Caudate‐projecting Cells in the Anterior Intralaminar Nuclei of the Cat

Neurons projecting to the parietal cortex or striatum and neurons showing immunoreactivity for the calcium‐binding proteins parvalbumin and 28KD‐calbindin were examined in the anterior intralaminar nuclei (IL) of the cat. Retrograde tracing from deep or superficial parietal cortical layers or from the caudate nucleus was coupled with immunohistochemistry to determine which of these proteins were expressed in the projection neurons. It was found that IL neurons project to deep as well as to superficial layers of the parietal cortex, that IL‐cortical neurons could be differentiated into two populations according to their cortical projection pattern and their soma size, and that IL neurons projecting to the parietal cortex or to the striatum express 28KD calbindin immunoreactivity but not parvalbumin immunoreactivity. The distribution of immunoreactivity to 28KD calbindin and parvalbumin in the neuropil showed a consistent complementary distribution pattern in the IL. The compartments based on differential parvalbumin and 28KD calbindin expression may indicate the presence of functionally segregated units in IL.

Cerebellar input to the posterior parietal cortex in the rat.

The cerebello-thalamo-parietal projections were investigated in rats by means of a multiple anterograde-retrograde tracing technique. Retrograde fluorescent tracers were injected in different loci of the parietal cortex. Injected areas were verified cytoarchitectonically and confirmed by analyzing the retrograde thalamic labeling pattern obtained. Anterograde fluorescent tracers were placed in the intermediate and lateral deep cerebellar nuclei. The topographical overlap between cerebellar terminals and parietal-projecting thalamic neurons was analyzed. In the central lateral (CL) and ventrolateral (VL) thalamic nuclei, cells projecting to anterior somatosensory (S1) and posterior parietal (PPC) cortices were demonstrated to receive direct cerebellar input. In particular, two patterns of organization were revealed. In CL, the PPC- and S1-projecting neurons, both receiving cerebellar fibers, were intermingled. In VL, PPC, and S1-projecting neurons were instead segregated, and the areas containing labeled neurons received separate contingents of cerebellar fibers. These patterns suggest that in CL axons originating from the same or neighboring cerebellar neurons can terminate on both PPC- and S1-projecting neurons, while in VL cerebellar information is funneled to S1 and PPC through two segregated parallel pathways. The significance of the observed organization is discussed comparing findings in other species and in relation with electrophysiological and functional studies on cerebelloparietal interrelationships.