In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

or by ␥-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body ␥-irradiation (8 Gy) mutant mice died rapidly from acute radiation toxicity to the small intestine. Mice-derived PARP ؊/؊ cells displayed a high sensitivity to MNU exposure: a G 2 ͞M arrest in mouse embryonic fibroblasts and a rapid apoptotic response and a p53 accumulation were observed in splenocytes. Altogether these results demonstrate that PARP is a survival factor playing an essential and positive role during DNA damage recovery.To protect their genome from the deleterious consequences of accumulation of unrepaired or misrepaired lesions, cells have developed an intricate DNA damage surveillance network. Through its function as a single-stranded breaks detector, poly(ADP-ribose) polymerase [PARP; NAD ϩ ADP-ribosyltransferase; NAD ϩ : poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30], a nuclear enzyme, participates to this basic process (1). PARP (113 kDa) has a modular organization (2): a N-terminal DNA-binding domain that acts as a molecular nick-sensor, encompassing two zinc-finger motifs (3) and a bipartite nuclear location signal (4), a central region bearing the auto-poly(ADP-ribosylation) sites which serves to regulate PARP-DNA interactions and a C-terminal catalytic domain involved in the nick-binding dependent poly(ADP-ribose) synthesis (5). The x-ray crystallographic structure of this domain has been recently solved revealing a surprising structural homology between the active site of PARP and that of bacterial mono-ADP-ribosylating toxins despite weak sequence homology (6).Although the physiological role of PARP is still much debated, recent molecular and genetic approaches including expression of either a dominant-negative mutant (7-10) or antisense (11) have clearly revealed the implication of PARP in the maintenance of the genomic integrity in the base excision repair pathway (7)(8)(9)(10)12). To elucidate its function we disrupted the mouse PARP gene by homologous recombination and exposed the PARP-deficient mice and derived cells to various genotoxins. MATERIALS AND METHODSGene Targeting in Embryonic Stem Cells and Generation of Mice. Mouse PARP was isolated from a 129SVJ strain genomic library. The targeting vector was constructed using a 9-kb EcoRI fragment extending from intron 2 to 7 by inserting PGK-neo (phosphoglycerate kinase promoter followed by the neo gene) in the BamHI site of the 4th exon and herpes simplex virus thymidine kinase followed by the TK gene (HSV-Tk) in the XhoI site outside the sequence of the targeting vector. Following electroporation, embryonic stem cells were selected in 200 g⅐ml Ϫ1 G418 and 2 mM of gancyclovir. A positive clone microinjected into C57BL͞6 blastocysts (13) gave rise to chimaeric offspring, which in turn were mated with C57BL͞6.

We have studied the apoptotic response of poly(ADPribose) polymerase (PARP)؊/؊ cells to different inducers and the consequences of the expression of an uncleavable mutant of PARP on the apoptotic process. The absence of PARP drastically increases the sensitivity of primary bone marrow PARP؊/؊ cells to apoptosis induced by an alkylating agent but not by a topoisomerase I inhibitor CPT-11 or by interleukin-3 removal. cDNA of wild type or of an uncleavable PARP mutant (D214A-PARP) has been introduced into PARP؊/؊ fibroblasts, which were exposed to anti-CD95 or an alkylating agent to induce apoptosis. The expression of D214A-PARP results in a significant delay of cell death upon CD95 stimulation. Morphological analysis shows a retarded cell shrinkage and nuclear condensation. Upon treatment with an alkylating agent, expression of wild-type PARP cDNA into PARP-deficient mouse embryonic fibroblasts results in the restoration of the cell viability, and the D214A-PARP mutant had no further effect on cell recovery. In conclusion, PARP؊/؊ cells are extremely sensitive to apoptosis induced by triggers (like alkylating agents), which activates the base excision repair pathway of DNA, and the cleavage of PARP during apoptosis facilitates cellular disassembly and ensures the completion and irreversibility of the process.Apoptosis or programmed cell death is a fundamental biological process that plays an important role in early development, cell homeostasis, and in diseases such as neurodegenerative disorders and cancer (1-3). Programmed cell death can occur in response to many stimuli such as genotoxic insult when DNA repair is saturated, removal of growth factors, or activation of the CD95 antigen by CD95 ligand or anti-CD95 antibodies. Morphologically it is characterized by the appearance of membrane blebbing, cell shrinkage, chromatin condensation, and DNA cleavage, and finally the cell is fragmented into membrane-bound apoptotic bodies. At the biochemical level, there is increasing evidence for a central role of the family of cysteine proteases, the caspases, in the pathway that mediates the highly ordered process leading to cell death (4). Caspases have been identified as the enzymes responsible for the proteolysis of key proteins to be selectively cleaved at the onset of apoptosis. It appears that the role of these proteases in cell suicide is to disable critical homeostatic and repair enzymes as well as key structural components. A discrete but increasing number of specific proteins appears to be targeted for proteolytic cleavage during apoptosis, including poly(ADP-ribose) polymerase (PARP, 1 EC 2.4.2.30), which was first described in Ref. 5. In the last years, cleavage of PARP has been used extensively as a marker of apoptosis. However, the reason for the cell to inactivate this protein during the execution phase of apoptosis is not fully understood.PARP is a nuclear zinc finger DNA-binding protein that detects and binds to DNA strand breaks. PARP has a modular organization comprising a NH 2 -terminal DNA-binding dom...

Poly (ADP-ribose) polymerase-1 is a nuclear DNAbinding protein that participates in the DNA base excision repair pathway in response to genotoxic stress in mammalian cells. Here we show that PARP-1-deficient cells are defective in NF-κB-dependent transcription activation, but not in its nuclear translocation, in response to TNF-α. Treating mice with lipopolysaccharide (LPS) resulted in the rapid activation of NF-κB in macrophages from PARP-1 ⍣/⍣ but not from PARP-1 -/-mice. PARP-1-deficient mice were extremely resistant to LPS-induced endotoxic shock. The molecular basis for this resistance relies on an almost complete abrogation of NF-κB-dependent accumulation of TNF-α in the serum and a downregulation of inducible nitric oxide synthase (iNOS), leading to decreased NO synthesis, which is the main source of free radical generation during inflammation. These results demonstrate a functional association in vivo between PARP-1 and NF-κB, with consequences for the transcriptional activation of NF-κB and a systemic inflammatory process.