Ari Barzilai

ATM deficiency and oxidative stress: a new dimension of defective response to DNA damage

ATM is one of the sentries at the gate of genome stability. This multifunctional protein kinase orchestrates the intricate array of cellular responses to DNA double-strand breaks. Absence or inactivation of ATM leads to the pleiotropic genetic disorder ataxia-telangiectasia (A-T), whose hallmarks are neuronal degeneration, immunodeficiency, genomic instability, premature aging and cancer predisposition. Several features of the complex clinical and cellular phenotype of A-T are reminiscent of other syndromes involving neurodegeneration, premature aging or genomic instability. A common denominator of many of these conditions is the perturbation of the cellular balance of reactive oxygen species, which leads to constant oxidative stress. Of these disorders, ATM deficiency is one of the most extensively studied with regard to the genome instability-oxidative stress connection. This connection may provide new insights into the phenotypes associated with genetic deficiencies of DNA damage responses, and point to new strategies to alleviate some of their clinical symptoms.

Dopamine induces apoptosis-like cell death in cultured chick sympathetic neurons — A possible novel pathogenetic mechanism in Parkinson's disease

We report that exposure of cultured, postmitotic chick-embryo sympathetic neurons, to physiological concentrations of dopamine (0.1-1 mM) for 24 h initiates a cellular death process characteristic of apoptosis (= programmed-cell-death, PCD). Dopamine caused marked morphological alterations, mainly axonal disintegration and severe shrinkage and condensation of cell bodies. Flow-cytometric analysis of propidium-iodide-stained cell nuclei revealed the characteristic apoptotic nuclear fragmentation: increase in nuclear granularity and emergence of a large, distinct population of nuclei with reduced DNA content (subdiploid, apoptotic peak). These alterations were similar to changes induced by nerve growth factor (NGF) deprivation, a model of sympathetic neuronal PCD. Alterations were inhibited by the anti-oxidative agent DTT. Inappropriate, dopamine-induced activation of PCD might have a role in nigral neuronal degeneration in Parkinsons disease.

Immunosuppressive role of semaphorin‐3A on T cell proliferation is mediated by inhibition of actin cytoskeleton reorganization

Timely negative regulation of the immune system is critical to allow it to perform its duty while maintaining it under tight control to avoid overactivation. We previously reported that the neuronal receptor neuropilin‐1 (NP‐1) is expressed in human lymph nodes. However, the role of NP‐1 interaction with its physiological ligand semaphorin‐3A (Sema‐3A) on immune cells remains elusive. Here we show that Sema‐3A is expressed by activated DC and T cells, and that its secretion in DC/T cell cocultures is delayed. Sema‐3A/NP‐1 interaction down‐modulated T cell activation since addition of Sema‐3A in DC/T cell cocultures dramatically inhibited allogeneic T cell proliferation. More importantly, neutralization by blocking antibodies or by antagonist peptide of endogenous Sema‐3A produced by DC/T cell cocultures resulted in a 130% increase in T cell proliferation. Sema‐3A acted directly on T cells, since it could block anti‐CD3/CD28‐stimulated proliferation of T cells. Finally, immunomodulatory functions of Sema‐3A relied on the blockage of actin cytoskeleton reorganization, affecting TCR polarization and interfering with early TCR signal transduction events such as ZAP‐70 or focal adhesion kinase phosphorylation. Therefore, we propose that Sema‐3A secretion and the resulting NP‐1/Sema‐3A interaction are involved in a late negative feedback loop controlling DC‐induced T cell proliferation.

Molecular mechanisms of selective dopaminergic neuronal death in Parkinson's disease

Parkinsons disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Although PD has been heavily researched, the precise etiology of nigral cell loss is still unknown and, consequently, treatment is largely symptomatic rather than preventive. There are conflicting data regarding the mode of dopaminergic cell death in PD and, hence, this remains controversial. Several mutations in specific genes have recently been linked with hereditary forms of PD. Although none of these mutations are seen in idiopathic disease cases, the elucidation of these genetic defects sheds light on the nature of idiopathic PD. It is possible that dopaminergic neurogenesis also contributes to the etiology of idiopathic PD. In addition, intracellular as well as extracellular substances found in the SNc are believed to function as damaging pathogenetic factors. These factors, and the interactions among them, might hold the secret to the underlying causes of the selective death of dopaminergic neurons in PD.

The role of the DNA damage response in neuronal development, organization and maintenance.

The DNA damage response is a key factor in the maintenance of genome stability. As such, it is a central axis in sustaining cellular homeostasis in a variety of contexts: development, growth, differentiation, and maintenance of the normal life cycle of the cell. It is now clear that diverse mechanisms encompassing cell cycle regulation, repair pathways, many aspects of cellular metabolism, and cell death are inter-linked and act in consort in response to DNA damage. Defects in the DNA damage response in proliferating cells can lead to cancer while defects in neurons result in neurodegenerative pathologies. Neurons are highly differentiated, post-mitotic cells that cannot be replenished after disease or trauma. Their high metabolic activity that generates large amounts of reactive oxygen species with DNA damaging capacity and their intense transcriptional activity increase the potential for damage of their genomic DNA. Neurons ensure their longevity and functionality in the face of these threats by elaborate mechanisms that defend the integrity of their genome. This review focuses on the DNA damage response in neuronal cells and points to the importance of this elaborate network to the integrity of the nervous system from its early development and throughout the lifetime of the organism.

Dopamine–melanin induces apoptosis in PC12 cells; possible implications for the etiology of Parkinson's disease

The function of neuromelanin (NM), the oxidized dopamine (DA) polymer, within the DA-producing cells in the human and primate substantia nigra (SN), is still an enigma. Some studies show that the vulnerability of nigral neurons in Parkinsons disease is correlated to their toxic NM content, while others suggest that it contributes to cellular protection. We showed recently that DA, the endogenous nigral neurotransmitter, triggers apoptosis, an active program of cellular self-destruction, in neuronal cultures. In the present study, we exposed cells to synthetic dopamine-melanin (DA-M) and analysed the cellular and genetic changes. We found that exposure of PC12 cells to DA-M (0.5 mg/ml for 24 h) caused 50% cell death, as indicated by trypan blue exclusion assay and 3H-thymidine incorporation. Gel electrophoresis DNA analysis of PC12 cells treated with DA-M showed the typical apoptotic DNA ladder, indicating inter-nucleosomal DNA degradation. The DNA fragmentation also was visualized histochemically in situ by DNA end-labeling staining (the TUNEL method). The FeCl2 (0.05 mM) significantly increased DA-M toxicity, while desferrioxamine, an iron chelator, totally abolished the additive toxicity of iron. The contribution of oxidative stress in this model of DA-M-induced cell death was examined using various antioxidants. In contrast to DA, inhibition of DA-M toxicity antioxidants by reduced glutathione (GSH), N-acetyl cysteine, catalase and Zn/Cu superoxide dismutase (SOD) was very limited. In conclusion, we found that DA-M may induce typical apoptotic death in PC12 cells. Our findings support a possible role of NM in the vulnerability of the dopaminergic neural degeneration in Parkinsons disease. The differential protective effect by antioxidants against toxicity of DA and DA-M may have implications for future neuroprotective therapeutic approaches for this common neurological disorder.

The neurological phenotype of ataxia-telangiectasia: solving a persistent puzzle.

Human genomic instability syndromes affect the nervous system to different degrees of severity, attesting to the vulnerability of the CNS to perturbations of genomic integrity and the DNA damage response (DDR). Ataxia-telangiectasia (A-T) is a typical genomic instability syndrome whose major characteristic is progressive neuronal degeneration but is also associated with immunodeficiency, cancer predisposition and acute sensitivity to ionizing radiation and radiomimetic chemicals. A-T is caused by loss or inactivation of the ATM protein kinase, which mobilizes the complex, multi-branched cellular response to double strand breaks in the DNA by phosphorylating numerous DDR players. The link between ATMs function in the DDR and the neuronal demise in A-T has been questioned in the past. However, recent studies of the ATM-mediated DDR in neurons suggest that the neurological phenotype in A-T is indeed caused by deficiency in this function, similar to other features of the disease. Still, major issues concerning this phenotype remain open, including the presumed differences between the DDR in post-mitotic neurons and proliferating cells, the nature of the damage that accumulates in the DNA of ATM-deficient neurons under normal life conditions, the mode of death of ATM-deficient neurons, and the lack of a major neuronal phenotype in the mouse model of A-T. A-T remains a prototype disease for the study of the DDRs role in CNS development and maintenance.

Biochemical and Temporal Analysis of Events Associated with Apoptosis Induced by Lowering the Extracellular Potassium Concentration in Mouse Cerebellar Granule Neurons

Abstract: We analyzed biochemically and temporally the molecular events that occur in the programmed cell death of mouse cerebellar granule neurons deprived of high potassium levels. An hour after switching the neurons to a low extracellular K+ concentration ([K+]o), a significant part of the genomic DNA was already cleaved to high‐molecular‐weight fragments. This phenomenon was intensified with the progression of the death process. Addition of cycloheximide to the neurons 4 h after high [K+]o deprivation resulted in no cell loss and complete recovery of the damaged DNA. DNA margination and nuclear fragmentation as assessed by 4,6‐diaminodiphenyl‐2‐phenylindole staining were observable in a few cells beginning ∼4 h after the removal of high [K+]o and developed to nuclear condensation 4 h later. Six hours after high [K+]o deprivation, the DNA was fragmented into oligonucleosome‐sized fragments. Within 6 h after removal of the extracellular K+, 50% of the neurons were committed to die and lost their ability to be rescued by readministration of 25 mM [K+]o. Similar to high [K+]o deprivation, inhibition of RNA or protein synthesis failed to halt neuronal degeneration of a similar percentage of cells 6 h after the onset of the death process. Mitochondrial function steadily decreased after [K+]o removal. An ∼40% decrease in RNA and protein synthesis was detected by 6 h of [K+]o removal during the period of cell death commitment; rates continued to decline gradually thereafter. The temporal characteristics of the DNA damage and recovery, DNA cleavage to oligonucleosome‐sized fragments, and the reduction in mitochondrial activity—events that occurred within the critical time—may indicate that these processes have an important part in the mechanism that committed the neurons to die.

Activation of nuclear transcription factor kappa B (NF-κB) is essential for dopamine-induced apoptosis in PC12 cells

The etiology of Parkinsons disease is still unknown, though current investigations support the notion of the pivotal involvement of oxidative stress in the process of neurodegeneration in the substantia nigra (SN). In the present study, we investigated the molecular mechanisms underlying cellular response to a challenge by dopamine, one of the local oxidative stressors in the SN. Based on studies showing that nuclear factor kappa B (NF‐κB) is activated by oxidative stress, we studied the involvement of NF‐κB in the toxicity of PC12 cells following dopamine exposure. We found that dopamine (0.1–0.5 m m) treatment increased the phosphorylation of the IκB protein, the inhibitory subunit of NF‐κB in the cytoplasm. Immunoblot analysis demonstrated the presence of NF‐κB‐p65 protein in the nuclear fraction and its disappearance from the cytoplasmic fraction after 2 h of dopamine exposure. Dopamine‐induced NF‐κB activation was also evidenced by electromobility shift assay using radioactive labeled NF‐κB consensus DNA sequence. Cell‐permeable NF‐κB inhibitor SN‐50 rescued the cells from dopamine‐induced apoptosis and showed the importance of NF‐κB activation to the induction of apoptosis. Furthermore, flow cytometry assay demonstrated a higher level of translocated NF‐κB‐p65 in the apoptotic nuclei than in the unaffected nuclei. In conclusion, our findings suggest that NF‐κB activation is essential to dopamine‐induced apoptosis in PC12 cells and it may be involved in nigral neurodegeneration in patients with Parkinsons disease.

Parallel induction of ATM-dependent pro- and antiapoptotic signals in response to ionizing radiation in murine lymphoid tissue

The ATM protein kinase, functionally missing in patients with the human genetic disorder ataxia-telangiectasia, is a master regulator of the cellular network induced by DNA double-strand breaks. The ATM gene is also frequently mutated in sporadic cancers of lymphoid origin. Here, we applied a functional genomics approach that combined gene expression profiling and computational promoter analysis to obtain global dissection of the transcriptional response to ionizing radiation in murine lymphoid tissue. Cluster analysis revealed a prominent pattern characterizing dozens of genes whose response to irradiation was Atm-dependent. Computational analysis identified significant enrichment of the binding site signatures of NF-κB and p53 among promoters of these genes, pointing to the major role of these two transcription factors in mediating the Atm-dependent transcriptional response in the irradiated lymphoid tissue. Examination of the response showed that pro- and antiapoptotic signals were simultaneously induced, with the proapoptotic pathway mediated by p53 targets, and the prosurvival pathway by NF-κB targets. These findings further elucidate the molecular network induced by IR, point to novel putative NF-κB targets, and suggest a mechanistic model for cellular balancing between pro- and antiapoptotic signals induced by IR in lymphoid tissues, which has implications for cancer management. The emerging model suggests that restoring the p53-mediated apoptotic arm while blocking the NF-κB-mediated prosurvival arm could effectively increase the radiosensitivity of lymphoid tumors.