Roberta Nardacci

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.

Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV‐1 envelope

Syncytia arising from the fusion of cells expressing the HIV‐1‐encoded Env gene with cells expressing the CD4/CXCR4 complex undergo apoptosis following the nuclear translocation of mammalian target of rapamycin (mTOR), mTOR‐mediated phosphorylation of p53 on Ser15 (p53S15), p53‐dependent upregulation of Bax and activation of the mitochondrial death pathway. p53S15 phosphorylation is only detected in syncytia in which nuclear fusion (karyogamy) has occurred. Karyogamy is secondary to a transient upregulation of cyclin B and a mitotic prophase‐like dismantling of the nuclear envelope. Inhibition of cyclin‐dependent kinase‐1 (Cdk1) prevents karyogamy, mTOR activation, p53S15 phosphorylation and apoptosis. Neutralization of p53 fails to prevent karyogamy, yet suppresses apoptosis. Peripheral blood mononuclear cells from HIV‐1‐infected patients exhibit an increase in cyclin B and mTOR expression, correlating with p53S15 phosphorylation and viral load. Cdk1 inhibition prevents the death of syncytia elicited by HIV‐1 infection of primary CD4 lymphoblasts. Thus, HIV‐1 elicits a pro‐apoptotic signal transduction pathway relying on the sequential action of cyclin B–Cdk1, mTOR and p53.

Mechanisms of apoptosis induction by the HIV-1 envelope

The envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) can induce apoptosis by a cornucopia of distinct mechanisms. A soluble Env derivative, gp120, can kill cells through signals that are transmitted by chemokine receptors such as CXCR4. Cell surface-bound Env (gp120/gp41), as present on the plasma membrane of HIV-1-infected cells, can kill uninfected bystander cells expressing CD4 and CXCR4 (or similar chemokine receptors, depending on the Env variant) by at least three different mechanisms. First, a transient interaction involving the exchange of lipids between the two interacting cells (‘the kiss of death’) may lead to the selective death of single CD4-expressing target cells. Second, fusion of the interacting cells may lead to the formation of syncytia which then succumb to apoptosis in a complex pathway involving the activation of several kinases (cyclin-dependent kinase-1, Cdk1; checkpoint kinase-2, Chk2; mammalian target of rapamycin, mTOR; p38 mitogen-activated protein kinase, p38 MAPK; inhibitor of NF-κB kinase, IKK), as well as the activation of several transcription factors (NF-κB, p53), finally resulting in the activation of the mitochondrial pathway of apoptosis. Third, if the Env-expressing cell is at an early stage of imminent apoptosis, its fusion with a CD4-expressing target cell can precipitate the death of both cells, through a process that may be considered as contagious apoptosis and which does not involve Cdk1, mTOR, p38 nor p53, yet does involve mitochondria. Activation of some of the above- mentioned lethal signal transducers have been detected in patients’ tissues, suggesting that HIV-1 may indeed trigger apoptosis through molecules whose implication in Env-induced killing has initially been discovered in vitro.

NF-κB and p53 Are the Dominant Apoptosis-inducing Transcription Factors Elicited by the HIV-1 Envelope

The coculture of cells expressing the HIV-1 envelope glycoprotein complex (Env) with cells expressing CD4 results into cell fusion, deregulated mitosis, and subsequent cell death. Here, we show that NF-κB, p53, and AP1 are activated in Env-elicited apoptosis. The nuclear factor κB (NF-κB) super repressor had an antimitotic and antiapoptotic effect and prevented the Env-elicited phosphorylation of p53 on serine 15 and 46, as well as the activation of AP1. Transfection with dominant-negative p53 abolished apoptosis and AP1 activation. Signs of NF-κB and p53 activation were also detected in lymph node biopsies from HIV-1–infected individuals. Microarrays revealed that most (85%) of the transcriptional effects of HIV-1 Env were blocked by the p53 inhibitor pifithrin-α. Macroarrays led to the identification of several Env-elicited, p53-dependent proapoptotic transcripts, in particular Puma, a proapoptotic “BH3-only” protein from the Bcl-2 family known to activate Bax/Bak. Down modulation of Puma by antisense oligonucleotides, as well as RNA interference of Bax and Bak, prevented Env-induced apoptosis. HIV-1–infected primary lymphoblasts up-regulated Puma in vitro. Moreover, circulating CD4+ lymphocytes from untreated, HIV-1–infected donors contained enhanced amounts of Puma protein, and these elevated Puma levels dropped upon antiretroviral therapy. Altogether, these data indicate that NF-κB and p53 cooperate as the dominant proapoptotic transcription factors participating in HIV-1 infection.

AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1

Damaged mitochondria are eliminated by mitophagy, a selective form of autophagy whose dysfunction associates with neurodegenerative diseases. PINK1, PARKIN and p62/SQTMS1 have been shown to regulate mitophagy, leaving hitherto ill-defined the contribution by key players in ‘general’ autophagy. In basal conditions, a pool of AMBRA1 – an upstream autophagy regulator and a PARKIN interactor – is present at the mitochondria, where its pro-autophagic activity is inhibited by Bcl-2. Here we show that, upon mitophagy induction, AMBRA1 binds the autophagosome adapter LC3 through a LIR (LC3 interacting region) motif, this interaction being crucial for regulating both canonical PARKIN-dependent and -independent mitochondrial clearance. Moreover, forcing AMBRA1 localization to the outer mitochondrial membrane unleashes a massive PARKIN- and p62-independent but LC3-dependent mitophagy. These results highlight a novel role for AMBRA1 as a powerful mitophagy regulator, through both canonical or noncanonical pathways.

Critical Involvement of the ATM-Dependent DNA Damage Response in the Apoptotic Demise of HIV-1-Elicited Syncytia

DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein), as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.

Immunocytochemical localization of d-amino acid oxidase in rat brain

Abstractd-amino acid oxidase (d-AAO) is a peroxisomal flavoenzyme, the physiological substrate and the precise function of which are still unclear. We have investigated D-AAO distribution in rat brain, by immunocytochemistry, with an affinity-purified polyclonal antibody. Immunoreactivity occurred in both neuronal and glial cells, albeit at different densities. Glial immunostaning was strongest in the caudal brainstem and cerebellar cortex, particularly in astrocytes, Golgi-Bergmann glia, and tanycytes. Hindbrain neurons were generally more immunoreactive than those in the forebrain. Immunopositive forebrain cell populations included mitral cells in the olfactory bulb, cortical and hippocampal neurons, ventral pallidum, and septal, reticular thalamic, and paraventricular hypothalamic nuclei. Within the positive regions, not all the neuronal populations were equally immunoreactive; for example, in the thalamus, only the reticular and anterodorsal nuclei showed intense labelling. In the hindbrain, immunopositivity was virtually ubiquitous, and was especially strong in the reticular formation, pontine, ventral and dorsal cochlear, vestibular, cranial motor nuclei, deep cerebellar nuclei, and the cerebellar cortex, especially in Golgi and Purkinje cells.

Early alterations in gene expression and cell morphology in a mouse model of Huntington’s disease

Several mouse models for Huntingtons disease (HD) have been produced to date. Based on differences in strain, promoter, construct, and number of glutamines, these models have provided a broad spectrum of neurological symptoms, ranging from simple increases in aggressiveness with no signs of neuropathology, to tremors and seizures in absence of degeneration, to neurological symptoms in the presence of gliosis and TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling) positivity, and finally to selective striatal damage associated with electrophysiological and behavioral abnormalities. We decided to analyze the morphology of striatum and hippocampus from a mouse transgenic line obtained by microinjection of exon 1 from the HD gene after introduction of a very high number of CAG repeat units. We found a massive darkening and compacting of striatal and hippocampal neurons in affected mice, associated with a lower degree of more classical apoptotic cell condensation. We then explored whether this morphology could be explained with alterations in gene expression by hybridizing normal and affected total brain RNA to a panel of 588 known mouse cDNAs. We show that some genes are significantly and consistently up‐regulated and that others are down‐regulated in the affected brains. Here we discuss the possible significance of these alterations in neuronal morphology and gene expression.

Fenretinide induces autophagic cell death in caspase-defective breast cancer cells

The elimination of tumour cells by apoptosis is the main mechanism of action of chemotherapeutic drugs. More recently, autophagic cell death has been shown to trigger a nonapoptotic cell death program in cancer cells displying functional defects of caspases. Fenretinide (FenR), a synthetic derivative of retinoic acid, promotes growth inhibition and induces apoptosis in a wide range of tumour cell types. The present study was designed to evaluate the ability of fenretinide to induce caspase-independent cell death and to this aim we used the human mammary carcinoma cell line MCF-7, lacking functional caspase-3 activity. We demonstrated that in these cells fenretinide is able to trigger an autophagic cell death pathway. In particular we found that fenretinide treatment resulted in the increase in Beclin 1 expression, the conversion of the soluble form of LC3 to the autophagic vesicle-associated form LC3-II and its shift from diffuse to punctate staining and finally the increase in lysosomes/autophagosomes. By contrast, caspase-3 reconstituted MCF-7 cell line showed apoptotic cell death features in response to fenretinide treatment. These data strongly suggest that fenretinide does not invariably elicit an apoptotic response but it is able to induce autophagy when apoptotic pathway is deregulated. The understanding of the molecular mechanisms involved in fenretinide action is important for the future design of therapies employing this retinoid in breast cancer treatment.