Malka Cohen-Armon

Antiphospholipid antibodies permeabilize and depolarize brain synaptoneurosomes

Antiphospholipid antibodies (aPL) are associated with neurological diseases such as stroke, migraine, epilepsy and dementia and are thus associated with both vascular and non-vascular neurological disease. We have therefore examined the possibility that these antibodies interact directly with neuronal tissue by studying the electrophysiological effects of aPL on a brain synaptosoneurosome preparation. IgG from patients with high levels of aPL and neurological involvement was purified by protein-G affinity chromatography as was control IgG pooled from ten sera with low levels of aPL. Synaptoneurosomes were purified from perfused rat brain stem. IgG from the patient with the highest level of aPL at a concentration equivalent to 1:5 serum dilution caused significant depolarization of the synaptoneurosomes as determined by accumulation of the lipophylic cation [3H]-tetraphenylphosphonium. IgG from this patient as well as IgG from two elderly patients with high levels of aPL were subsequently shown to permeabilize the synaptosomes to labeled nicotinamide adenine dinucleotide (NAD) and pertussis toxin-ADP-ribose transferase (PTX-A protein) as assayed by labeled ADP-ribosylation of G-proteins in the membranes. No such effects were seen with the control IgG. aPL may thus have the potential to disrupt neuronal function by direct action on nerve terminals. These results may explain some of the non-thromboembolic CNS manifestations of the antiphospholipid syndrome.

PolyADP-Ribosylation Is Involved in Neurotrophic Activity

PolyADP-ribosylation is a transient posttranslational modification of proteins, mainly catalyzed by poly(ADP-ribose)polymerase-1 (PARP-1). This highly conserved nuclear protein is activated rapidly in response to DNA nick formation and promotes a fast DNA repair. Here, we examine a possible association between polyADP-ribosylation and the activity of neurotrophins and neuroprotective peptides taking part in life-or-death decisions in mammalian neurons. The presented results indicate an alternative mode of PARP-1 activation in the absence of DNA damage by neurotrophin-induced signaling mechanisms. PARP-1 was activated in rat cerebral cortical neurons briefly exposed to NGF-related nerve growth factors and to the neuroprotective peptides NAP (the peptide NAPVSIPQ, derived from the activity-dependent neuroprotective protein ADNP) and ADNF-9 (the peptide SALLRSIPA, derived from the activity-dependent neurotrophic factor ADNF) In addition, polyADP-ribosylation was involved in the neurotrophic activity of NGF-induced and NAP-induced neurite outgrowth in differentiating pheochromocytoma 12 cells as well as in the neuroprotective activity of NAP in neurons treated with the Alzheimers disease neurotoxin β-amyloid. A fast loosening of the highly condensed chromatin structure by polyADP-ribosylation of histone H1, which renders DNA accessible to transcription and repair, may underlie the role of polyADP-ribosylation in neurotrophic activity.

PolyADP‐ribosylation is required for long‐term memory formation in mammals

PolyADP‐ribosylation is a post‐translational modification of nuclear proteins, catalyzed by polyADP‐ribose polymerases (PARPs). In the nucleus, polyADP‐ribosylation catalyzed by PARP‐1 alters protein–protein and protein–DNA interactions, and is implicated in chromatin remodeling, DNA transcription, and repair. Previous results linked the activation of PARP‐1 with long‐term memory formation during learning in the marine mollusk Aplysia ( Science 2004, 304:1820–1822). Furthermore, PARP‐1 was highly activated in mammalian cerebral neurons treated with neurotrophins and neurotrophic peptides promoting neurite outgrowth and synaptic plasticity. Here, we examine the possibility that PARP‐1 activation is required for memory formation during learning in mammals. Mice were tested in two learning paradigms, object recognition and fear conditioning. PolyADP‐ribosylation of PARP‐1 and histone H1 were detected in their cerebral cortex and hippocampus immediately after their training session. Moreover, in both behavioral paradigms, suppression of PARP activity in the CNS during learning impaired their long‐term memory formation, without damaging their short‐term memory. These findings implicate PARP‐1 activation in molecular processes underlying long‐term memory formation during learning.

A selective eradication of human nonhereditary breast cancer cells by phenanthridine-derived polyADP-ribose polymerase inhibitors

IntroductionPARP-1 (polyADP-ribose polymerase-1) is known to be activated in response to DNA damage, and activated PARP-1 promotes DNA repair. However, a recently disclosed alternative mechanism of PARP-1 activation by phosphorylated externally regulated kinase (ERK) implicates PARP-1 in a vast number of signal-transduction networks in the cell. Here, PARP-1 activation was examined for its possible effects on cell proliferation in both normal and malignant cells.MethodsIn vitro (cell cultures) and in vivo (xenotransplants) experiments were performed.ResultsPhenanthridine-derived PARP inhibitors interfered with cell proliferation by causing G2/M arrest in both normal (human epithelial cells MCF10A and mouse embryonic fibroblasts) and human breast cancer cells MCF-7 and MDA231. However, whereas the normal cells were only transiently arrested, G2/M arrest in the malignant breast cancer cells was permanent and was accompanied by a massive cell death. In accordance, treatment with a phenanthridine-derived PARP inhibitor prevented the development of MCF-7 and MDA231 xenotransplants in female nude mice. Quiescent cells (neurons and cardiomyocytes) are not impaired by these PARP inhibitors.ConclusionsThese results outline a new therapeutic approach for a selective eradication of abundant nonhereditary human breast cancers.

Activation of Go-proteins by Membrane Depolarization Traced by in Situ Photoaffinity Labeling of Gαo-proteins with [α32P]GTP-azidoanilide

Evidence for depolarization-induced activation of G-proteins in membranes of rat brain synaptoneurosomes has been previously reported (Cohen-Armon, M., and Sokolovsky, M. (1991)J. Biol. Chem. 266, 2595–2605; Cohen-Armon, M., and Sokolovsky, M. (1993) J. Biol. Chem. 268, 9824–9838). In the present work we identify the activated G-proteins as Go-proteins by tracing their depolarization-inducedin situ photoaffinity labeling with [α32P]GTP-azidoanilide (GTPAA). Labeled GTPAA was introduced into transiently permeabilized rat brain-stem synaptoneurosomes. The resealed synaptoneurosomes, while being UV-irradiated, were depolarized. Relative to synaptoneurosomes at resting potential, the covalent binding of [α32P]GTPAA to Gαo1- and Gαo3-proteins, but not to Gαo2- isoforms, was enhanced by 5- to 7-fold in depolarized synaptoneurosomes, thereby implying an accelerated exchange of GDP for [α32P]GTPAA. Their depolarization-induced photoaffinity labeling was independent of stimulation of Go-protein-coupled receptors and could be reversed by membrane repolarization, thus excluding induction by transmitters release. It was, however, dependent on depolarization-induced activation of the voltage-gated sodium channels (VGSC), regardless of Na+ current. The α subunit of VGSC was cross-linked and co-immunoprecipitated with Gαo-proteins in depolarized brain-stem and cortical synaptoneurosomes. VGSC α subunit most efficiently cross-linked with guanosine 5′-O-2-thiodiphosphate-bound rather than to guanosine 5′-O-(3-thiotriphosphate)-bound Gαo-proteins in isolated synaptoneurosomal membranes. These findings support a possible involvement of VGSC in depolarization-induced activation of Go-proteins.

Ca2+-induced PARP-1 activation and ANF expression are coupled events in cardiomyocytes.

The nuclear protein PARP-1 [poly(ADP-ribose) polymerase-1] is activated in cardiomyocytes exposed to hypoxia causing DNA breaks. Unlike this stress-induced PARP-1 activation, our results provide evidence for Ca(2+)-induced PARP-1 activation in contracting newborn cardiomyocytes treated with growth factors and hormones that increased their contraction rate, induced intracellular Ca(2+) mobilization and its rhythmical and transient translocation into the nucleus. Furthermore, activated PARP-1 up-regulated the activity of phosphorylated ERK (extracellular-signal-regulated kinase) in the nucleus, promoting expression of the Elk1 target gene c-fos. Up-regulation of the transcription factor c-Fos/GATA-4 promoted ANF (atrial natriuretic factor) expression. Given that expression of ANF is known to be implicated in morphological changes, growth and development of cardiomyocytes, these results outline a PARP-1-dependent signal transduction mechanism that links contraction rate and Ca(2+) mobilization with the expression of genes underlying morphological changes in cardiomyocytes.

Cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells.

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent γ-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered γ-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

Exclusive destruction of mitotic spindles in human cancer cells

We identified target proteins modified by phenanthrenes that cause exclusive eradication of human cancer cells. The cytotoxic activity of the phenanthrenes in a variety of human cancer cells is attributed by these findings to post translational modifications of NuMA and kinesins HSET/kifC1 and kif18A. Their activity prevented the binding of NuMA to α-tubulin and kinesins in human cancer cells, and caused aberrant spindles. The most efficient cytotoxic activity of the phenanthridine PJ34, caused significantly smaller aberrant spindles with disrupted spindle poles and scattered extra-centrosomes and chromosomes. Concomitantly, PJ34 induced tumor growth arrest of human malignant tumors developed in athymic nude mice, indicating the relevance of its activity for cancer therapy.

A PARP1-ERK2 synergism is required for the induction of LTP.

Unexpectedly, a post-translational modification of DNA-binding proteins, initiating the cell response to single-strand DNA damage, was also required for long-term memory acquisition in a variety of learning paradigms. Our findings disclose a molecular mechanism based on PARP1-Erk synergism, which may underlie this phenomenon. A stimulation induced PARP1 binding to phosphorylated Erk2 in the chromatin of cerebral neurons caused Erk-induced PARP1 activation, rendering transcription factors and promoters of immediate early genes (IEG) accessible to PARP1-bound phosphorylated Erk2. Thus, Erk-induced PARP1 activation mediated IEG expression implicated in long-term memory. PARP1 inhibition, silencing, or genetic deletion abrogated stimulation-induced Erk-recruitment to IEG promoters, gene expression and LTP generation in hippocampal CA3-CA1-connections. Moreover, a predominant binding of PARP1 to single-strand DNA breaks, occluding its Erk binding sites, suppressed IEG expression and prevented the generation of LTP. These findings outline a PARP1-dependent mechanism required for LTP generation, which may be implicated in long-term memory acquisition and in its deterioration in senescence.

PARP1-Erk synergism in proliferating cells

A synergism between PARP1 and phosphorylated Erk mediating IEG (immediate early gene) expression has been recently reported in cerebral neurons and cardiomyocytes. Stimulation induced PARP-Erk synergism was required for IEG expression underlying synaptic plasticity and long-term memory acquisition during learning. It was similarly required for cardiomyocytes development. Here, we identified this mechanism in Erk-induced gene expression promoting proliferation. This mechanism can be targeted in malignant cells.