Mariarosa A. B. Melone

Molecular pathways involved in neural in vitro differentiation of marrow stromal stem cells

In recent years several reports have claimed to demonstrate trans‐differentiation, namely that stem cells have been derived from a given tissue and have differentiated into phenotypes characteristic of different tissues following transplantation or in vitro treatment. For example, the mesenchymal stem cells, also referred to as marrow stromal stem cells (MSCs), present in bone marrow, have been induced to differentiate into neurons. We decided to investigate this phenomenon more in depth by a molecular and morphological follow‐up. We analyzed the biochemical pathways that are currently induced to trigger neuron‐like commitment and maturation of MSCs. Our studies suggest that: (i) the increase in cAMP, induced to differentiate MSCs, activates the classical PKA pathway and not through the exchange protein directly activated by cAMP (EPAC), a guanine nucleotide exchange factor for the small GTPase Rap1 and Rap2; (ii) MEK–ERK signaling could contribute to neural commitment and differentiation; (iii) CaM KII activity seems dispensable for neuron differentiation. On the contrary, its inhibition could contribute to rescuing differentiating cells from death. Our research also indicates that the currently used in vitro differentiation protocols, while they allow the early steps of neural differentiation to take place, are not able to further sustain this process.

Modulation of cytokine production in activated human monocytes by somatostatin

The immunosuppressor effects of the widely distributed neuropeptide somatostatin were examined on purified peripheral blood human monocytes. Somatostatin, at concentrations thought to be physiologic (10(-10)-10(-7) M), regulated monocyte/macrophage responses to (LPS) stimulation, as reflected by interleukin production. In particular, somatostatin had direct inhibitory effects on TNF-alpha, IL-1 beta, and IL-6 secretion by LPS-activated monocytes, while the decrease on IL-8 synthesis was modulated mainly by the action of somatostatin on TNF-alpha and IL-1 beta. In fact, the addition of these two inflammatory cytokines to the monocyte culture medium was able to induce IL-8 expression, as demonstrated by mRNA analysis, also in presence of the neuropeptide. Although somatostatin affected IL-8 production in an indirect way, it suppressed directly the chemotactic response of neutrophils to IL-8. Finally, somatostatin downregulation of monocyte activation was confirmed by the decrease of HLA-DR expression on cell plasma membranes (52% versus 33%). Our results confirm that somatostatin exerts preferential effects on the suppression of immunoreactions by modulating cytokine production and activity.

Differentiation and apoptosis of neuroblastoma cells: role of N-myc gene product.

To clarify the role and function of the N‐myc product in cell differentiation and apoptosis, we used the antisense oligonucleotide technique to inhibit N‐myc gene expression in neuroblastoma cells with different phenotypes: intermediate (I) and neuronal (N), or Schwann‐glia (S), respectively. The results suggest that N‐myc operates along different pathways. Inhibiting N‐myc gene expression either results in suppression of cell proliferation or in induction of differentiation and/or apoptosis. J. Cell. Biochem. 73:97–105, 1999.

17‐B estradiol elicits an autocrine leiomyoma cell proliferation: Evidence for a stimulation of protein kinase‐dependent pathway

The mechanism by which estradiol (E2) acts on cell proliferation is still unclear. In this paper, we report the results of a series of experiments in an attempt to elucidate the effector pathway(s) involved in coupling the E2 receptors binding to cellular growth response in leiomyoma cells (LSMC). Under conditions of E2‐dependent growth, E2 treatment of LSMC triggers rapid and transient activation of the MAP‐kinase pathway. Interestingly, we demonstrate that the early downstream signal transduction events determined by E2‐stimulation in quiescent LSMC, including the rapid protein tyrosine phosphorylation of a subset of intracellular proteins, such GAP, PI‐3‐K, and PLCγ, and the concomitant activation of ancillary protein kinases, are related to E2‐induced PDGF secretion. Moreover, we identify the PDGF, alone or in association with other growth factors, as the main growth factor involved in the proliferation response of LSMC to E2 stimulation. The addition of neutralizing antibodies anti‐PDGF was able to inhibit the mitogenic activity present in LSMC conditioned media samples. On the other hand, E2 did not affect the constitutive expression as well as the ligand affinity of PDGF receptors on LSMC plasmamembrane. Cell treatment with the antiestrogen ICI 182780 correlate both with a perturbation of E2‐induced transductional circuit and with the disappearance of the mitogenic factor, PDGF, in LSMC conditioned media; the latter therefore, represents the main autocrine mediator of cell growth modulation, upregulated by E2 and down‐regulated by antiestrogenic compound. Our experiments suggest that growth factor secretion is an initial and integral part of the signaling events mediated by the estradiol receptors, not related, at least in part, to E2 transcriptional modulation.

RAGE-TXNIP axis is required for S100B-promoted Schwann cell migration, fibronectin expression and cytokine secretion.

During peripheral nerve injury, Schwann cells (SCs) adopt a migratory phenotype and remodel the extracellular matrix and provide a supportive activity for neuron regeneration. SCs synthesize neurotrophic factors and cytokines that are crucial for the repair of the injured nerve. The receptor for advanced glycation end products (RAGE) and its ligand S100B, which are secreted by SCs, are required for the repair of the injured peripheral nerve in vivo. However, the precise intracellular pathways involved have not been completely elucidated. Here, we show that RAGE-induced S100B secretion involves the recruitment of S100B in lipid rafts and caveolae. Moreover, we demonstrate for the first time that RAGE induces the expression of thioredoxin interacting protein (TXNIP) in SCs and the injured sciatic nerve in vivo. TXNIP is involved in the activation of p38 MAPK, CREB and NFκB in SCs. TXNIP silencing partially inhibits RAGE-induced SC migration and completely abolishes RAGE-induced fibronectin and IL-1β expression. Our results support a model in which TXNIP mediates in part RAGE-induced SC migration and is required for the expression of provisional ECM and pro-inflammatory IL-1β. We provide new insight on the role of the SC RAGE–TXNIP axis in the repair of injured peripheral nerves.

RAGE recycles at the plasma membrane in S100B secretory vesicles and promotes Schwann cells morphological changes

RAGE is a multiligand receptor of the immunoglobulin superfamily involved in regeneration of injured peripheral nerve and cell motility. RAGE is implicated in the development of various chronic diseases, such as neurodegenerative disorders, inflammatory responses, and diabetic complications. The correlation between RAGE endocytic trafficking and RAGE function is still uninvestigated. S100B is one of the ligands of RAGE. The molecular mechanisms responsible of S100B translocation in exocytic vesicles are still poorly investigated. In the present study we elucidate the role of RAGE endocytic trafficking in promoting S100B secretion in Schwann cells. Here we show that RAGE‐induced secretion of S100B requires phosphorylated caveolin1‐dependent endocytosis of RAGE. Endocytosis of RAGE in response to ligand binding promotes the fusion of endosomes with S100B‐positive secretory vesicles. Src promotes the fusion of endosomes with S100B‐secretory vesicles. Inhibition of src induces RAGE degradation. RAGE‐mediated src activation induces cav1 phosphorylation and relocalization in the perinuclear compartment. RAGE signaling and recycling are required for S100‐induced Schwann cells morphological changes and are inhibited by high‐glucose, suggesting a possible link between diabetes and peripheral nerve injury. Indeed, high glucose inhibits RAGE‐mediated src activation. Src inhibition blocks RAGE recycling, S100B secretion, and morphological changes. In summary, we identified a novel pathway of vesicular trafficking required for the amplification of RAGE signaling and cytoskeleton dynamics that is potentially involved in the regeneration of injured peripheral nerve. J. Cell. Physiol. 217: 60–71, 2008.

Skeletal muscle metabolism in physiology and in cancer disease.

Skeletal muscle is a tissue of high demand and it accounts for most of daily energy consumption. The classical concept of energy metabolism in skeletal muscle has been profoundly modified on the basis of studies showing the influence of additional factors (i.e., uncoupling proteins (UCPs) and peroxisome proliferator activated receptors (PPARs)) controlling parameters, such as substrate availability, cellular enzymes, carrier proteins, and proton leak, able to affect glycolysis, nutrient oxidation, and protein degradation. This extremely balanced system is greatly altered by cancer disease that can induce muscle cachexia with significant deleterious consequences and results in muscle wasting and weakness, delaying or preventing ambulation, and rehabilitation in catabolic patients. J. Cell. Biochem. 90: 170–186, 2003.

Systemic Delivery of Recombinant Brain Derived Neurotrophic Factor (BDNF) in the R6/2 Mouse Model of Huntington’s Disease

Loss of huntingtin-mediated BDNF gene transcription has been shown to occur in HD and thus contribute to the degeneration of the striatum. Several studies have indicated that an increase in BDNF levels is associated with neuroprotection and amelioration of neurological signs in animal models of HD. In a recent study, an increase in BDNF mRNA and protein levels was recorded in mice administered recombinant BDNF peripherally. Chronic, indwelling osmotic mini-pumps containing either recombinant BDNF or saline were surgically placed in R6/2 or wild-type mice from 4 weeks of age until euthanasia. Neurological evaluation (paw clasping, rotarod performance, locomotor activity in an open field) was performed. After transcardial perfusion, histological and immunohistochemical studies were performed. We found that BDNF- treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the vehicle treated ones. Primary outcome measures such as brain volume, striatal atrophy, size and morphology of striatal neurons, neuronal intranuclear inclusions and microglial reaction confirmed a neuroprotective effect of the compound. BDNF was effective in increasing significantly the levels of activated CREB and of BDNF the striatal spiny neurons. Moreover, systemically administered BDNF increased the synthesis of BDNF as demonstrated by RT-PCR, and this might account for the beneficial effects observed in this model.

Huntingtons Disease: New Frontiers for Molecular and Cell Therapy

Huntingtons disease (HD) is an incurable, adult-onset, dominantly inherited neurodegenerative disease, caused by a CAG expansion in the 5 coding region of the gene HD [encoding huntingtin (htt), which is ubiquitously expressed in all tissues]. The disease progresses inexorably with devastating clinical effects on motor, cognitive and psychological functions; death occurring approximately 18 years from the time of onset. These clinical symptoms primarily relate to the progressive death of medium-spiny GABA-ergic neurons of the striatum and in the deep layers of the cortex; during the later stages of the disease, the degeneration extends to a variety of brain regions, including the hypothalamus and hippocampus. The mechanism by which mutant htt leads to neuronal cell death and the question of why striatal neurons are targeted both remain to be further investigated. Certainly htt is required for cell survival and impairment of wild-type htt function can be involved in neurodegeneration, but considerable evidence also shows that trinucleotide repeat expansion into glutamine (polyQ domain) endows the protein with a newly acquired toxic activity. The increasing availability of HD animal models have allowed not only to investigate the function of htt, but also to screen and test potential therapeutic drugs in the promising area of neurotherapeutics. So, thorough analysis of these molecular and biochemical events, assessing the validity of candidate mechanisms, provides a means to identify effective therapeutic strategies for cellular repair. Here, the rationale and efficacy of different therapies are compared and alternative therapies are reviewed including intrastriatal transplantation of human fetal striatal tissue to support the cell replacement strategy in HD. Since functional restoration through neuronal replacement probably could be combined with neuroprotective strategies for optimum clinical benefit, in vivo and ex vivo gene therapy for delivery of neuroprotective growth factor molecules are also considered.

tBid induces alterations of mitochondrial fatty acid oxidation flux by malonyl-CoA-independent inhibition of carnitine palmitoyltransferase-1

Recent studies suggest a close relationship between cell metabolism and apoptosis. We have evaluated changes in lipid metabolism on permeabilized hepatocytes treated with truncated Bid (tBid) in the presence of caspase inhibitors and exogenous cytochrome c. The measurement of β-oxidation flux by labeled palmitate demonstrates that tBid inhibits β-oxidation, thereby resulting in the accumulation of palmitoyl-coenzyme A (CoA) and depletion of acetyl-carnitine and acylcarnitines, which is pathognomonic for inhibition of carnitine palmitoyltransferase-1 (CPT-1). We also show that tBid decreases CPT-1 activity by a mechanism independent of both malonyl-CoA, the key inhibitory molecule of CPT-1, and Bak and/or Bax, but dependent on cardiolipin decrease. Overexpression of Bcl-2, which is able to interact with CPT-1, counteracts the effects exerted by tBid on β-oxidation. The unexpected role of tBid in the regulation of lipid β-oxidation suggests a model in which tBid-induced metabolic decline leads to the accumulation of toxic lipid metabolites such as palmitoyl-CoA, which might become participants in the apoptotic pathway.