Sabrina Di Bartolomeo

Ambra1 regulates autophagy and development of the nervous system

Autophagy is a self-degradative process involved both in basal turnover of cellular components and in response to nutrient starvation or organelle damage in a wide range of eukaryotes. During autophagy, portions of the cytoplasm are sequestered by double-membraned vesicles called autophagosomes, and are degraded after fusion with lysosomes for subsequent recycling. In vertebrates, this process acts as a pro-survival or pro-death mechanism in different physiological and pathological conditions, such as neurodegeneration and cancer; however, the roles of autophagy during embryonic development are still largely uncharacterized. Beclin1 (Becn1; coiled-coil, myosin-like BCL2-interacting protein) is a principal regulator in autophagosome formation, and its deficiency results in early embryonic lethality. Here we show that Ambra1 (activating molecule in Beclin1-regulated autophagy), a large, previously unknown protein bearing a WD40 domain at its amino terminus, regulates autophagy and has a crucial role in embryogenesis. We found that Ambra1 is a positive regulator of the Becn1-dependent programme of autophagy, as revealed by its overexpression and by RNA interference experiments in vitro. Notably, Ambra1 functional deficiency in mouse embryos leads to severe neural tube defects associated with autophagy impairment, accumulation of ubiquitinated proteins, unbalanced cell proliferation and excessive apoptotic cell death. In addition to identifying a new and essential element regulating the autophagy programme, our results provide in vivo evidence supporting the existence of a complex interplay between autophagy, cell growth and cell death required for neural development in mammals.

The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy

When autophagy is induced, ULK1 phosphorylates AMBRA1, releasing the autophagy core complex from the cytoskeleton and allowing its relocalization to the ER membrane to nucleate autophagosome formation.

Ligand-independent CXCR2 Dimerization

Homo- and hetero-oligomerization have been reported for several G protein-coupled receptors (GPCRs). The CXCR2 is a GPCR that is activated, among the others, by the chemokines CXCL8 (interleukin-8) and CXCL2 (growth-related gene product β) to induce cell chemotaxis. We have investigated the oligomerization of CXCR2 receptors expressed in human embryonic kidney cells and generated a series of truncated mutants to determine whether they could negatively regulate the wild-type (wt) receptor functions. CXCR2 receptor oligomerization was also studied by coimmunoprecipitation of green fluorescent protein- and V5-tagged CXCR2. Truncated CXCR2 receptors retained their ability to form oligomers only if the region between the amino acids Ala-106 and Lys-163 was present. In contrast, all of the deletion mutants analyzed were able to form heterodimers with the wt CXCR2 receptor, albeit with different efficiency, competing for wt/wt dimer formation. The truncated CXCR2 mutants were not functional and, when coexpressed with wt CXCR2, interfered with receptor functions, impairing cell signaling and chemotaxis. When CXCR2 was expressed with the AMPA-type glutamate receptor GluR1, CXCR2 dimerization was again impaired in a dose-dependent way, and receptor functions were prejudiced. In contrast, CXCR1, a chemokine receptor that shares many similarities with CXCR2, did not dimerize alone or with CXCR2 and when coexpressed with CXCR2 did not impair receptor signaling and chemotaxis. The formation of CXCR2 dimers was also confirmed in cerebellar neuron cells. Taken together, we conclude from these studies that CXCR2 functions as a dimer and that truncated receptors negatively modulate receptor activities competing for the formation of wt/wt dimers.

AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation

Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene.

The Role of Autophagy During Development in Higher Eukaryotes

Autophagy is a lysosome‐mediated degradation pathway used by eukaryotes to recycle cytosolic components in both basal and stress conditions. Several genes have been described as regulators of autophagy, many of them being evolutionarily conserved from yeast to mammals. The study of autophagy‐defective model systems has made it possible to highlight the importance of correctly functioning autophagic machinery in the development of invertebrates as, for example, during the complex events of fly and worm metamorphosis. In vertebrates, on the other hand, autophagy defects can be lethal for the animal if the mutated gene is involved in the early stages of development, or can lead to severe phenotypes if the mutation affects later stages. However, in both lower and higher eukaryotes, autophagy seems to be crucial during embryogenesis by acting in tissue remodeling in parallel with apoptosis. An increase of autophagic cells is, in fact, observed in the embryonic stages characterized by massive cell elimination. Moreover, autophagic processes probably protect cells during metabolic stress and nutrient paucity that occur during tissue remodeling. In light of such evidence, it can be concluded that there is a close interplay between autophagy and the processes of cell death, proliferation and differentiation that determine the development of higher eukaryotes.

Autophagy induction impairs migration and invasion by reversing EMT in glioblastoma cells

Cell migration and invasion are highly regulated processes involved in both physiological and pathological conditions. Here we show that autophagy modulation regulates the migration and invasion capabilities of glioblastoma (GBM) cells. We observed that during autophagy occurrence, obtained by nutrient deprivation or by pharmacological inhibition of the mTOR complexes, GBM migration and chemokine‐mediated invasion were both impaired. We also observed that SNAIL and SLUG, two master regulators of the epithelial–mesenchymal transition (EMT process), were down‐regulated upon autophagy stimulation and, as a consequence, we found a transcriptional and translational up‐regulation of N‐ and R‐cadherins. Conversely, in BECLIN 1‐silenced GBM cells, an increased migration capability and an up‐regulation of SNAIL and SLUG was observed, with a resulting decrease in N‐ and R‐cadherin mRNAs. ATG5 and ATG7 down‐regulation also resulted in an increased migration and invasion of GBM cells combined to an up‐regulation of the two EMT regulators. Finally, experiments performed in primary GBM cells from patients largely confirmed the results obtained in established cell cultures.

Signalling pathways involved in the chemotactic activity of CXCL12 in cultured rat cerebellar neurons and CHP100 neuroepithelioma cells

We compared the signal transduction pathways activated by stromal cell-derived factor-1 (CXCL12) chemokine in two different cell systems: primary cultures of rat cerebellar granule neurons (CGN) and human neuroepithelioma CHP100 cells. Both cell types express functional CXC chemokine receptor 4 (CXCR4), which is coupled both to extracellular signal-regulated kinase (ERK) and Akt phosphorylation pathways. The activation of ERK shows different dependency on the phosphatidylinositol 3-kinase (PI3-K) pathway and different sensitivity to pertussis toxin (PTX) treatment, indicative of coupling to different G proteins in the two cell systems considered. We demonstrate that the inhibition of either the ERK kinase or the PI3-K pathways blocks the CXCL12 induced-chemotaxis in CHP100 cells; while only PI3-K activity is stringently necessary for CGN migration.

Reduced cathepsins B and D cause impaired autophagic degradation that can be almost completely restored by overexpression of these two proteases in Sap C-deficient fibroblasts

Saposin (Sap) C deficiency, a rare variant form of Gaucher disease, is due to mutations in the Sap C coding region of the prosaposin (PSAP) gene. Sap C is required as an activator of the lysosomal enzyme glucosylceramidase (GCase), which catalyzes glucosylceramide (GC) degradation. Deficit of either GCase or Sap C leads to the accumulation of undegraded GC and other lipids in lysosomes of monocyte/macrophage lineage. Recently, we reported that Sap C mutations affecting a cysteine residue result in increased autophagy. Here, we characterized the basis for the autophagic dysfunction. We analyzed Sap C-deficient and GCase-deficient fibroblasts and observed that autophagic disturbance was only associated with lack of Sap C. By a combined fluorescence microscopy and biochemical studies, we demonstrated that the accumulation of autophagosomes in Sap C-deficient fibroblasts is not due to enhanced autophagosome formation but to delayed degradation of autolysosomes caused, in part, to decreased amount and reduced enzymatic activity of cathepsins B and D. On the contrary, in GCase-deficient fibroblasts, the protein level and enzymatic activity of cathepsin D were comparable with control fibroblasts, whereas those of cathepsin B were almost doubled. Moreover, the enhanced expression of both these lysosomal proteases in Sap C-deficient fibroblasts resulted in close to functional autophagic degradation. Our data provide a novel example of altered autophagy as secondary event resulting from insufficient lysosomal function.

Apoptosis induced by N-hexanoylsphingosine in CHP-100 cells associates with accumulation of endogenous ceramide and is potentiated by inhibition of glucocerebroside synthesis.

We report that apoptosis induced by N-hexanoylsphingosine (C6-Cer) in CHP-100 human neuroepithelioma cells associates with accumulation of monohexosylsphingolipids produced not only by short-chain ceramide glycosylation but also through glycosylation of a ceramide pool endogenously produced. By high-performance thin layer chromatography on borate silica gel plates, newly formed monohexosylsphingolipids were identified as glucosylceramides (GluCer); however, accumulation of lactosylceramide or higher-order glycosphingolipids was not observed. GluCer accumulation was fully suppressed by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; moreover, while this inhibitor had no effect on cell viability when administered alone, it markedly potentiated the apoptotic effect of C6-Cer. These results provide evidence that activation of GluCer synthesis is an important mechanism through which CHP-100 cells attempt to escape ceramide-induced apoptosis.

Unleashing the Ambra1-Beclin 1 complex from dynein chains: Ulk1 sets Ambra1 free to induce autophagy

The Beclin 1-VPS34 complex plays a crucial role in the induction of the autophagic process by generating PtdIns(3)P-rich membranes, which act as platforms for ATG protein recruitment and autophagosome nucleation. Several cofactors, such as Ambra1, ATG14 and UVRAG, are necessary for Beclin 1 complex activity. However, the mechanism by which Beclin 1 complex activity is stimulated by autophagic stimuli has not yet been fully elucidated. Recently, we reported that autophagosome formation in mammalian cells is primed by Ambra1 release from the dynein motor complex. We found that Ambra1 specifically binds the dynein motor complex under normal conditions through a direct interaction with DLC1. When autophagy is induced, Ambra1-DLC1 are released from the dynein complex in an ULK1-dependent manner, and relocalize to the endoplasmic reticulum, thus enabling autophagosome nucleation. In addition, we found that both DLC1 downregulation and Ambra1 mutations in its DLC1-binding sites strongly enhance autophagosome formation. Ambra1 is therefore not only a cofactor of Beclin 1 in favoring its kinase-associated activity, but also a crucial upstream regulator of autophagy initiation.