Raffaella Pacchiana

Do Astrocytes Collaborate with Neurons in Spreading the “Infectious” Aβ and Tau Drivers of Alzheimer’s Disease?

Evidence has begun emerging for the “contagious” and destructive Aβ42 (amyloid-beta42) oligomers and phosphorylated Tau oligomers as drivers of sporadic Alzheimer’s disease (AD), which advances along a pathway starting from the brainstem or entorhinal cortex and leading to cognition-related upper cerebral cortex regions. Seemingly, Aβ42 oligomers trigger the events generating the neurotoxic Tau oligomers, which may even by themselves spread the characteristic AD neuropathology. It has been assumed that only neurons make and spread these toxic drivers, whereas their associated astrocytes are just janitorial bystanders/scavengers. But this view is likely to radically change since normal human astrocytes freshly isolated from adult cerebral cortex can be induced by exogenous Aβ25-35, an Aβ42 proxy, to make and secrete increased amounts of endogenous Aβ42. Thus, it would seem that the steady slow progression of AD neuropathology along specific cognition-relevant brain networks is driven by both Aβ42 and phosphorylated Tau oligomers that are variously released from increasing numbers of “contagion-stricken” members of tightly coupled neuron–astrocyte teams. Hence, we surmise that stopping the oversecretion and spread of the two kinds of “contagious” oligomers by such team members, perhaps via a specific CaSR (Ca2+-sensing receptor) antagonist like NPS 2143, might effectively treat AD.

The Aβ Peptides-Activated Calcium-Sensing Receptor Stimulates the Production and Secretion of Vascular Endothelial Growth Factor-A by Normoxic Adult Human Cortical Astrocytes

The excess vascular endothelial growth factor (VEGF) produced in the Alzheimer’s disease (AD) brain can harm neurons, blood vessels, and other components of the neurovascular units (NVUs). But could astrocytes partaking in networks of astrocyte-neuron teams and connected to blood vessels of NVUs contribute to VEGF production? We have shown with cultured cerebral cortical normal (i.e., untransformed) adult human astrocytes (NAHAs) that exogenous amyloid-β peptides (Aβs) stimulate the astrocytes to make and secrete large amounts of Aβs and nitric oxide by a mechanism mediated through the calcium-sensing receptor (CaSR). Here, we report that exogenous Aβs stimulate the NAHAs to produce and secrete even VEGF-A through a CaSR-mediated mechanism. This is indicated by the ability of Aβs to specifically bind the CaSR, and the capability of a CaSR activator, the “calcimimetic” NPS R-568, to imitate, and of the CaSR antagonist, “calcilytic” NPS 2143, to inhibit, the Aβs stimulation of VEGF-A production and secretion by the NAHAs. Thus, Aβs that accumulate in the AD brain may make the astrocytes that envelop and functionally collaborate with neurons into multi-agent AD-driving “machines” via a CaSR signaling mechanism(s). These observations suggest the possibility that CaSR allosteric antagonists such as NPS 2143 might impede AD progression.

The calcium-sensing receptor: a novel Alzheimer's disease crucial target?

Alzheimers disease (AD) is the most common human neurodegenerative ailment, the most prevalent (>95%) late-onset type of which has a still uncertain etiology. The progressive decline of cognitive functions, dementia, and physical disabilities of AD is caused by synaptic losses that progressively disconnect key neuronal networks in crucial brain areas, like the hippocampus and temporoparietal cortex, and critically impair language, sensory processing, memory, and conscious thought. ADs two main hallmarks are fibrillar amyloid-β (fAβ) plaques in extracellular spaces and intracellular accumulation of fAβ peptides and neurofibrillary tangles (NFTs). It is still undecided whether either or both these AD hallmarks cause or result from the disease. Recently, the dysregulation of calcium homeostasis has been advanced as a novel cause of AD. In this case, a suitable candidate of AD driver would be the Aβ peptides-binding/activated calcium-sensing receptor (CaSR), whose intracellular signalling is triggered by Aβ peptides. In this review, we briefly discuss CaSRs roles in normal adult human astrocytes (NAHAs) and their possible impacts on AD.

Mutant p53 proteins alter cancer cell secretome and tumour microenvironment: Involvement in cancer invasion and metastasis.

An ever-increasing number of studies highlight the role of mutant p53 proteins in the alteration of cancer cell secretome and in the modification of tumour microenvironment, sustaining an invasive phenotype of cancer cell. The knowledge of the molecular mechanisms underlying the interplay between mutant p53 proteins and the microenvironment is becoming fundamental for the identification of both efficient anticancer therapeutic strategies and novel serum biomarkers. In this review, we summarize the novel findings concerning the regulation of secreted molecules by cancer cells bearing mutant TP53 gene. In particular, we highlight data from available literature, suggesting that mutant p53 proteins are able to (i) alter the secretion of enzymes involved in the modulation of extracellular matrix components; (ii) alter the secretion of inflammatory cytokines; (iii) increase the extracellular acidification; and (iv) regulate the crosstalk between cancer and stromal cells.

Calcium-Sensing Receptors of Human Astrocyte-Neuron Teams: Amyloid-β-Driven Mediators and Therapeutic Targets of Alzheimer's Disease.

It is generally assumed that the neuropathology of sporadic (late-onset or nonfamilial) Alzheimer’s disease (AD) is driven by the overproduction and spreading of first Amyloid-βx-42 (Aβ42) and later hyperphosphorylated (hp)-Tau oligomeric “infectious seeds”. Hitherto, only neurons were held to make and spread both oligomer types; astrocytes would just remove debris. However, we have recently shown that exogenous fibrillar or soluble Aβ peptides specifically bind and activate the Ca2+-sensing receptors (CaSRs) of untransformed human cortical adult astrocytes and postnatal neurons cultured in vitro driving them to produce, accrue, and secrete surplus endogenous Aβ42. While the Aβ-exposed neurons start dying, astrocytes survive and keep oversecreting Aβ42, nitric oxide (NO), and vascular endothelial growth factor (VEGF)-A. Thus astrocytes help neurons’ demise. Moreover, we have found that a highly selective allosteric CaSR agonist (“calcimimetic”), NPS R-568, mimics the just mentioned neurotoxic actions triggered by Aβ●CaSR signaling. Contrariwise, and most important, NPS 2143, a highly selective allosteric CaSR antagonist (“calcilytic”), fully suppresses all the Aβ●CaSR signaling-driven noxious actions. Altogether our findings suggest that the progression of AD neuropathology is promoted by unceasingly repeating cycles of accruing exogenous Aβ42 oligomers interacting with the CaSRs of swelling numbers of astrocyte-neuron teams thereby recruiting them to overrelease additional Aβ42 oligomers, VEGF-A, and NO. Calcilytics would beneficially break such Aβ/CaSR-driven vicious cycles and hence halt or at least slow the otherwise unstoppable spreading of AD neuropathology

Combining immunofluorescence with in situ proximity ligation assay: a novel imaging approach to monitor protein-protein interactions in relation to subcellular localization.

Abstract The in situ Proximity Ligation Assay (PLA) is suited for visualizing protein–protein interactions and post-translational protein modifications in both tissue sections and in vitro cell cultures. Accurate identification and quantification of protein–protein interactions are critical for in vitro cell analysis, especially when studying the dynamic involvement of proteins in various processes, including cell proliferation, differentiation, and apoptosis. Here, we monitored the interactions between protein kinase-Cζ (PKCζ) and Bcl10 protein in untreated and etoposide (VP-16)-treated C4-I cells by means of a new combined morphological approach and validated it by taking stock of our previous proteomic and biochemical work (Chiarini et al. in J Proteome Res 11:3996–4012, 2012). We first analyzed the colocalization of PKCζ and Bcl10 proteins through classical immunofluorescent colocalization analysis. On the basis of these results, we developed a novel imaging approach combining immunofluorescence (IF) techniques with in situ PLA to identify the PKCζ·Bcl10 complexes at the level of a specific subcellular compartment, i.e., the nuclear envelope (NE). By this means, we could show that the amount of PKCζ·Bcl10 complexes localized at the NE of C4-I cells during proliferation or after treatment with VP-16 closely corresponded to our previous purely biochemical results. Hence, the present findings demonstrate that the combination of in situ PLA with classical IF detection is a novel powerful analytical tool allowing to morphologically demonstrate new specific protein–protein interactions at level of subcellular organelles, the complexes functions of which can next be clarified through proteomic/biochemical approaches.

The antioxidant uncoupling protein 2 stimulates hnRNPA2/B1, GLUT1 and PKM2 expression and sensitizes pancreas cancer cells to glycolysis inhibition

Several evidence indicate that metabolic alterations play a pivotal role in cancer development. Here, we report that the mitochondrial uncoupling protein 2 (UCP2) sustains the metabolic shift from mitochondrial oxidative phosphorylation (mtOXPHOS) to glycolysis in pancreas cancer cells. Indeed, we show that UCP2 sensitizes pancreas cancer cells to the treatment with the glycolytic inhibitor 2-deoxy-D-glucose. Through a bidimensional electrophoresis analysis, we identify 19 protein species differentially expressed after treatment with the UCP2 inhibitor genipin and, by bioinformatic analyses, we show that these proteins are mainly involved in metabolic processes. In particular, we demonstrate that the antioxidant UCP2 induces the expression of hnRNPA2/B1, which is involved in the regulation of both GLUT1 and PKM2 mRNAs, and of lactate dehydrogenase (LDH) increasing the secretion of L-lactic acid. We further demonstrate that the radical scavenger N-acetyl-L-cysteine reverts hnRNPA2/B1 and PKM2 inhibition by genipin indicating a role for reactive oxygen species in the metabolic reprogramming of cancer cells mediated by UCP2. We also observe an UCP2-dependent decrease in mtOXPHOS complex I (NADH dehydrogenase), complex IV (cytochrome c oxidase), complex V (ATPase) and in mitochondrial oxygen consumption, suggesting a role for UCP2 in the counteraction of pancreatic cancer cellular respiration. All these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation with the concomitant metabolic shift from mtOXPHOS to the glycolytic pathway.

Trichostatin A alters cytoskeleton and energy metabolism of pancreatic adenocarcinoma cells: an in depth proteomic study

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal of all human cancers with a high mortality rate. Resistance to conventional treatments and chemotherapeutics is a typical feature of PDAC. To investigate the causes of drug resistance it is essential to deeply investigate the mechanism of action of chemotherapeutics. In this study, we performed an in depth shotgun proteomic approach using the label‐free proteomic SWATH‐MS analysis to investigate novel insights of the mechanism of action of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) in PDAC cells. This proteomic analysis in PaCa44 cells and data elaboration of TSA‐regulated proteins by bioinformatics showed an overall up‐regulation of cytokeratins and other proteins related to the cytoskeleton organization, keratinization, and apoptotic cell death. On the contrary, a large amount of the down‐regulated proteins by TSA treatment belongs to the cellular energetic metabolism and to the machinery of protein synthesis, such as ribosomal proteins, determining synergistic cell growth inhibition by the combined treatment of TSA and the glycolytic inhibitor 2‐deoxy‐d‐glucose in a panel of PDAC cell lines. Data are available via ProteomeXchange with identifier PXD007801.

UCP2 inhibition induces ROS/Akt/mTOR axis: role of GAPDH nuclear translocation in genipin/everolimus anticancer synergism

Abstract Several studies indicate that mitochondrial uncoupling protein 2 (UCP2) plays a pivotal role in cancer development by decreasing reactive oxygen species (ROS) produced by mitochondrial metabolism and by sustaining chemoresistance to a plethora of anticancer drugs. Here, we demonstrate that inhibition of UCP2 triggers Akt/mTOR pathway in a ROS‐dependent mechanism in pancreatic adenocarcinoma cells. This event reduces the antiproliferative outcome of UCP2 inhibition by genipin, creating the conditions for the synergistic counteraction of cancer cell growth with the mTOR inhibitor everolimus. Inhibition of pancreatic adenocarcinoma cell growth and induction of apoptosis by genipin and everolimus treatment are functionally related to nuclear translocation of the cytosolic glycolytic enzyme glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). The synthetic compound (S)‐benzyl‐2‐amino‐2‐(S)‐3‐bromo‐4,5‐dihydroisoxazol‐5‐yl‐acetate (AXP3009), which binds GAPDH at its redox‐sensitive Cys152, restores cell viability affected by the combined treatment with genipin and everolimus, suggesting a role for ROS production in the nuclear translocation of GAPDH. Caspase‐mediated apoptosis by genipin and everolimus is further potentiated by the autophagy inhibitor 3‐methyladenine revealing a protective role for Beclin1‐mediated autophagy induced by the treatment. Mice xenograft of pancreatic adenocarcinoma further confirmed the antiproliferative outcome of drug combination without toxic effects for animals. Tumor masses from mice injected with UCP2 and mTOR inhibitors revealed a strong reduction in tumor volume and number of mitosis associated with a marked GAPDH nuclear positivity. Altogether, these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation and support the combined inhibition of UCP2 and of Akt/mTOR pathway as a novel therapeutic strategy in the treatment of pancreatic adenocarcinoma.

Autocrine mechanisms of cancer chemoresistance

An ever-increasing number of studies highlight the role of cancer secretome in the modification of tumour microenvironment and in the acquisition of cancer cell resistance to therapeutic drugs. The knowledge of the mechanisms underlying the relationship between cancer cell-secreted factors and chemoresistance is becoming fundamental for the identification of novel anticancer therapeutic strategies overcoming drug resistance and novel prognostic secreted biomarkers. In this review, we summarize the novel findings concerning the regulation of secreted molecules by cancer cells compromising drug sensitivity. In particular, we highlight data from available literature describing the involvement of cancer cell-secreted molecules determining chemoresistance in an autocrine manner, including: i) growth factors; ii) glycoproteins; iii) inflammatory cytokines; iv) enzymes and chaperones; and v) tumor-derived exosomes.