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High-risk neuroblastoma (HR-NB) still remains the most dangerous tumor in early childhood. For this reason, the identification of new therapeutic approaches is of fundamental importance. Recently, we combined the conventional pharmacological approach to NB, represented by cisplatin, with fendiline hydrochloride, an inhibitor of several transporters involved in multidrug resistance of cancer cells, which demonstrated an enhancement of the ability of cisplatin to induce apoptosis. In this work, we co-administrated acetazolamide, a carbonic anhydrase isoform IX (CAIX) inhibitor which was reported to increase chemotherapy efficacy in various cancer types, to the cisplatin/fendiline approach in SKNBE2 xenografts in NOD-SCID mice with the aim of identifying a novel and more effective treatment. We observed that the combination of the three drugs increases more than twelvefold the differences in the cytotoxic activity of cisplatin alone, leading to a remarkable decrease of the expression of malignancy markers. Our conclusion is that this approach, based on three FDA-approved drugs, may constitute an appropriate improvement of the pharmacological approach to HR-NB.

Certain proteases are involved in Alzheimer's disease (AD) and their erroneous control may contribute to the pathology onset and progression. In this study we evaluated the cerebral expression of eight proteases, involved in both AβPP processing and extracellular matrix remodeling. Among these proteases, ADAM10, ADAMTS1, Cathepsin D, and Meprin β show a significantly higher mRNAs expression in sporadic AD subjects versus controls, while ADAMTS1, Cathepsin D, and Meprin β show an increment also at the protein level. These data indicate that transcriptional events affecting brain proteases are activated in AD patients, suggesting a link between proteolysis and AD.

The processing of the amyloid-β protein precursor (AβPP) by β- and γ-secretases is a pivotal event in the genesis of Alzheimer's disease (AD). Besides familial mutations on the AβPP gene, or upon its overexpression, familial forms of AD are often caused by mutations or deletions in presenilin 1 (PSEN1) and 2 (PSEN2) genes: the catalytic components of the proteolytic enzyme γ-secretase (GS). The "amyloid hypothesis", modified over time, states that the aberrant processing of AβPP by GS induces the formation of specific neurotoxic soluble amyloid-β (Aβ) peptides which, in turn, cause neurodegeneration. This theory, however, has recently evidenced significant limitations and, in particular, the following issues are debated: 1) the concept and significance of presenilin's "gain of function" versus "loss of function"; and 2) the presence of several and various GS substrates, which interact with AβPP and may influence Aβ formation. The latter consideration is suggestive: despite the increasing number of GS substrates so far identified, their reciprocal interaction with AβPP itself, even in the AD field, is significantly unexplored. On the other hand, GS is also an important pharmacological target in the cancer field; inhibitors or GS activity are investigated in clinical trials for treating different tumors. Furthermore, the function of AβPP and PSENs in brain development and in neuronal migration is well known. In this review, we focused on a specific subset of GS substrates that directly interact with AβPP and are involved in its proteolysis and signaling, by evaluating their role in neurodegeneration and in cell motility or proliferation, as a possible connection between AD and cancer.

According to the “gain-of-toxicity mechanism”, neuronal loss during cerebral proteinopathies is caused by accumulation of aggregation-prone conformers of misfolded cellular proteins, although it is still debated which aggregation state actually corresponds to the neurotoxic entity. Autophagy, originally described as a variant of programmed cell death, is now emerging as a crucial mechanism for cell survival in response to a variety of cell stressors, including nutrient deprivation, damage of cytoplasmic organelles, or accumulation of misfolded proteins. Impairment of autophagic flux in neurons often associates with neurodegeneration during cerebral amyloidosis, suggesting a role in clearing neurons from aggregation-prone misfolded proteins. Thus, autophagy may represent a target for innovative therapies. In this work, we show that alterations of autophagy progression occur in neurons following in vitro exposure to the amyloidogenic and neurotoxic prion protein-derived peptide PrP90-231. We report that the increase of autophagic flux represents a strategy adopted by neurons to survive the intracellular accumulation of misfolded PrP90-231. In particular, PrP90-231 internalization in A1 murine mesencephalic neurons occurs in acidic structures, showing electron microscopy hallmarks of autophagosomes and autophagolysosomes. However, these structures do not undergo resolution and accumulate in cytosol, suggesting that, in the presence of PrP90-231, autophagy is activated but its progression is impaired; the inability to clear PrP90-231 via autophagy induces cytotoxicity, causing impairment of lysosomal integrity and cytosolic diffusion of hydrolytic enzymes. Conversely, the induction of autophagy by pharmacological blockade of mTOR kinase or trophic factor deprivation restored autophagy resolution, reducing intracellular PrP90-231 accumulation and neuronal death. Taken together, these data indicate that PrP90-231 internalization induces an autophagic defensive response in A1 neurons, although incomplete and insufficient to grant survival; the pharmacological enhancement of this process exerts neuroprotection favoring the clearing of the internalized peptide and could represents a promising neuroprotective tool for neurodegenerative proteinopathies.

Glia over-stimulation associates with amyloid deposition contributing to the progression of central nervous system neurodegenerative disorders. Here we analyze the molecular mechanisms mediating microglia-dependent neurotoxicity induced by prion protein (PrP)90-231, an amyloidogenic polypeptide corresponding to the protease-resistant portion of the pathological prion protein scrapie (PrPSc). PrP90-231 neurotoxicity is enhanced by the presence of microglia within neuronal culture, and associated to a rapid neuronal [Ca++] i increase. Indeed, while in "pure" cerebellar granule neuron cultures, PrP90-231 causes a delayed intracellular Ca++ entry mediated by the activation of NMDA receptors; when neuron and glia are co-cultured, a transient increase of [Ca++] i occurs within seconds after treatment in both granule neurons and glial cells, then followed by a delayed and sustained [Ca++] i raise, associated with the induction of the expression of inducible nitric oxide synthase and phagocytic NADPH oxidase. [Ca++] i fast increase in neurons is dependent on the activation of multiple pathways since it is not only inhibited by the blockade of voltage-gated channel activity and NMDA receptors but also prevented by the inhibition of nitric oxide and PGE2 release from glial cells. Thus, Ca++ homeostasis alteration, directly induced by PrP90-231 in cerebellar granule cells, requires the activation of NMDA receptors, but is greatly enhanced by soluble molecules released by activated glia. In glia-enriched cerebellar granule cultures, the activation of inducible nitric oxide (iNOS) and NADPH oxidase represents the main mechanism of toxicity since their pharmacological inhibition prevented PrP90-231 neurotoxicity, whereas NMDA blockade by D(-)-2-amino-5-phosphonopentanoic acid is ineffective; conversely, in pure cerebellar granule cultures, NMDA blockade but not iNOS inhibition strongly reduced PrP90-231 neurotoxicity. These data indicate that amyloidogenic peptides induce neurotoxic signals via both direct neuron interaction and glia activation through different mechanisms responsible of calcium homeostasis disruption in neurons and potentiating each other: the activation of excitotoxic pathways via NMDA receptors and the release of radical species that establish an oxidative milieu.

The aim of this review is to critically analyze promises and limitations of pharmacological inducers of autophagy against protein misfolding-associated neurodegeneration. Effective therapies against neurodegenerative disorders can be developed by regulating the “self-defense„ equipment of neurons, such as autophagy. Through the degradation and recycling of the intracellular content, autophagy promotes neuron survival in conditions of trophic factor deprivation, oxidative stress, mitochondrial and lysosomal damage, or accumulation of misfolded proteins. Autophagy involves the activation of self-digestive pathways, which is different for dynamics (macro, micro and chaperone-mediated autophagy), or degraded material (mitophagy, lysophagy, aggrephagy). All neurodegenerative disorders share common pathogenic mechanisms, including the impairment of autophagic flux, which causes the inability to remove the neurotoxic oligomers of misfolded proteins. Pharmacological activation of autophagy is typically achieved by blocking the kinase activity of mammalian target of rapamycin (mTOR) enzymatic complex 1 (mTORC1), removing its autophagy suppressor activity observed under physiological conditions; acting in this way, rapamycin provided the first proof of principle that pharmacological autophagy enhancement can induce neuroprotection through the facilitation of oligomers’ clearance. The demand for effective disease-modifying strategies against neurodegenerative disorders is currently stimulating the development of a wide number of novel molecules, as well as the re-evaluation of old drugs for their pro-autophagic potential.

Extracellular plaques of amyloid-? (A?) are the main histopathological signatures of Alzheimer's disease (AD). In vitro and in vivo analysis of amyloid deposits in AD revealed various N- and C-terminal variants, in addition biochemical studies showed that A? peptides isolated from AD brains were post-translationally modified. N-terminal deletions and pyroglutamylation enhance aggregation of A? into neurotoxic species and such peptides may initiate or nucleate the pathological deposition of A? into plaques. Among these, A?pE3-42 strongly affects cultured neuron and astrocyte survival. In the last years, a new molecular actor has appeared in AD pathophysiology: the cellular prion protein (PrPC). The role of this protein is still not completely disclosed, but its involvement in the complex pathway connecting A? to Fyn/Tau neuronal toxicity is now recognized. In particular, the trigger of the molecular cascade leading to synapses impairment and cellular death seems to be the direct interaction of A? oligomers (A?o) with PrPC. In this work we exploit the interactions between A? peptides and N-terminal truncated PrPC. In particular we study the effect of PrPc (90-231) on aggregation of A?1-42, A?pE3-42 and of their mixtures. To reach this goal we investigated the different interactions by NMR and we characterized the intermediate species by turbidimetry, circular dichroism, transmission electron microscopy, ThT and ANS fluorescence. To determine if PrPC is a mediator of A?-induced neurotoxicity, we studied the physiological response of cells to A?o, and the dysregulation of calcium homeostasis, both on glioblastoma cells where PrPC expression was inhibited or not

Cancer stem cells (CSCs) represent a small subpopulation of cells responsible for tumor formation and progression, drug resistance, tumor recurrence and metastasization. CSCs have been identified in many human tumors including osteosarcoma (OSA). CSC distinctive properties are the expression of stem cell markers, sustained growth, self-renewal and tumorigenicity. Here we report the isolation of stem-like cells from two canine OSA cultures, characterized by self-renewal, evaluated by sphere formation ability, differential marker expression, and in vitro proliferation when cultured in a medium containing EGF and bFGF. Current therapies for OSA increased survival time, but prognosis remains poor, due to the development of drug resistance and metastases. Chemotherapy shrinks the tumor mass but CSCs remain unaffected, leading to tumor recurrence. Metformin, a drug for type 2 diabetes, has been shown to possess antitumor properties affecting CSC survival in different human and animal cancers. Here we show that metformin has a significant antiproliferative effect on canine OSA stem-like cells, validating this in vitro model for further pre-clinical drug evaluations. In conclusion, our results demonstrate the feasibility of obtaining CSC-enriched cultures from primary canine OSA cells as a promising model for biological and pharmacological studies of canine and human OSAs.

// Alessandro Corsaro 1 , Adriana Bajetto 1 , Stefano Thellung 1, 2 , Giulia Begani 1 , Valentina Villa 1 , Mario Nizzari 1 , Alessandra Pattarozzi 1 , Agnese Solari 1 , Monica Gatti 1 , Aldo Pagano 3, 4 , Roberto Wurth 1 , Antonio Daga 4 , Federica Barbieri 1, 2 , Tullio Florio 1, 2 1 Sezione di Farmacologia, Dipartimento di Medicina Interna, University of Genova, Genova, Italy 2 Centro di Eccellenza per la Ricerca Biomedica (CEBR), University of Genova, Genova, Italy 3 Department of Experimental Medicine, University of Genova, Genova, Italy 4 IRCCS AOU San Martino - IST, Genova, Italy Correspondence to: Tullio Florio, email: tullio.florio@unige.it Keywords: prion protein, glioblastoma, cancer stem cells, in vivo tumorigenicity, GFAP Received: December 30, 2015 Accepted: May 04, 2016 Published: May 24, 2016 ABSTRACT Prion protein (PrP C ) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrP C is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrP C regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrP C in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrP C expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrP C expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrP C down-regulated cells. Moreover, PrP C down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways ( i.e. increased GFAP expression). Our results suggest that PrP C controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype.