Reuven Stein

Methylation of CpG sequences in eukaryotic DNA.

An understanding of the function of DNA methylation in eukaryotes will require a clearer, more precise, knowledge of the distribution of methylated bases in the eukaryotic genome. Although it has been known for some time that 5-methylcytosine (m’Cyt) is the only methylated base in eukaryotes, little is known about the sequence specificity of this modification. One important fact which has emerged from base analyses is that >90% of the msCyt residues are found in the sequence CpG [ 11. It was therefore of interest to determine what fraction of this dinucleotide sequence is methylated. The answer to this question would not only contribute to our knowledge of the distribution of msCyt but would also shed light on the factors which make up the recognition signal for eukaryotic methylation. In an attempt to answer this question we have developed a new experimental procedure for detecting msCyt in CpG sequences. This dinucleotide is found to be highly methylated in animal cells and may be the major determinant in the placement of methyl groups on DNA.

Aluminium in Alzheimer's disease: are we still at a crossroad?

Abstract.Aluminium, an environmentally abundant non-redox trivalent cation has long been implicated in the pathogenesis of Alzheimer’s disease (AD). However, the definite mechanism of aluminium toxicity in AD is not known. Evidence suggests that trace metal homeostasis plays a crucial role in the normal functioning of the brain, and any disturbance in it can exacerbate events associated with AD. The present paper reviews the scientific literature linking aluminium with AD. The focus is on aluminium levels in brain, region-specific and subcellular distribution, its relation to neurofibrillary tangles, amyloid beta, and other metals. A detailed mechanism of the role of aluminium in oxidative stress and cell death is highlighted. The importance of complex speciation chemistry of aluminium in relation to biology has been emphasized. The debatable role of aluminium in AD and the cross-talk between aluminium and genetic susceptibility are also discussed. Finally, it is concluded based on extensive literature that the neurotoxic effects of aluminium are beyond any doubt, and aluminium as a factor in AD cannot be discarded. However, whether aluminium is a sole factor in AD and whether it is a factor in all AD cases still needs to be understood.

Differential Interactions Between Beclin 1 and Bcl-2 Family Members

Autophagy, a cellular degradation system, promotes both cell death and survival. The interaction between Bcl-2 family proteins and Beclin 1, a Bcl-2 interacting protein that promotes autophagy, can mediate crosstalk between autophagy and apoptosis. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin 1. Our results show that Beclin 1 directly interacts with Bcl-2, Bcl-xL, Bcl-w and to a lesser extent with Mcl-1. Beclin 1 does not bind the pro-apoptotic Bcl-2 proteins. The interaction between Beclin 1 and the anti-apoptotic protein Bcl-xL was inhibited by BH3-only proteins, but not by multi-domain proteins. Sequence alignment and structural modeling suggest that Beclin 1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-xL. Mutation of the Beclin 1 amino acids predicted to mediate this interaction inhibited the association of Beclin 1 with Bcl-xL. Our results suggest that BH3 only proapoptotic Bcl-2 proteins may modulate the interactions between Bcl-xL and Beclin 1.

A cell type-preferred silencer element that controls the neural-specific expression of the SCG10 gene

SCG10 is a growth-associated protein that is expressed early in the development of neuronal derivatives of the neural crest. We describe here the isolation of the SCG10 chromosomal gene and the identification of regulatory regions that control its expression. The SCG10 transcription unit spans approximately 40 kb. Like other neural-specific genes, SCG10 contains multiple transcription initiation sites. The gene contains a constitutive enhancer-like element in the promoter-proximal region and a silencer located farther upstream. This silencer preferentially suppresses the activity of the enhancer in nonneuronal cells. Furthermore, the silencer is able to confer such preferential suppression upon a heterologous promoter in an orientation-independent manner. These data suggest that the expression of SCG10 in neuronal cells depends predominantly upon specific derepression.

The induction of a neural-specific gene, SCG10, by nerve growth factor in PC12 cells is transcriptional, protein synthesis dependent, and glucocorticoid inhibitable

We describe features of the regulation of a neural-specific gene, SCG10, which is induced by nerve growth factor (NGF) during the neuronal differentiation of the rat pheochromocytoma cell line PC12. Induction of SCG10 mRNA occurs within 12-24 hr of exposure to NGF, is sustained in the continued presence of the neurotrophic factor, and involves a mechanism that is, at least in part, transcriptional. Unlike the rapid, transient transcriptional activations of genes such as c-fos, SCG10 induction requires ongoing protein synthesis, suggesting the participation of a de novo synthesized regulatory protein in mediating the effects of NGF on this gene. Although c-fos itself may play this role, its induction is clearly insufficient to cause an induction of SCG10. NGF, FGF, and, to a lesser extent, phorbol esters induced SCG10, whereas EGF and dibutyryl cAMP did not. In these characteristics, SCG10 induction appears to constitute a reliable molecular index of the transcription-dependent neuronal differentiation induced by NGF. Glucocorticoids, which inhibit NGF-induced neurite outgrowth from normal primary chromaffin cells, partially blocked SCG10 induction in PC12 cells. A reciprocal pattern of regulation by NGF and glucocorticoids was observed for tyrosine hydroxylase mRNA. These data suggest that environmental signals such as NGF may act on specific genes, both positively and negatively, to control the choice of alternative fates by developing neural crest cells.

Inhibition of Drug‐Induced Apoptosis by Survival Factors in PC12 Cells

Abstract: Pheochromocytoma (PC12) cells have been shown to undergo apoptosis (programmed cell death) when deprived of serum and to be rescued by nerve growth factor, fibroblast growth factor, dibutyryl cyclic AMP, aurintricarboxylic acid, or exogenous expression of bcl‐2. We show here that the cytotoxic drugs cycloheximide, actinomycin D, colchicine, and EGTA also induce apoptosis in PC12 cells. These findings prompted us to investigate whether apoptosis induced by these drugs involves similar pathways in each case, and whether the factors preventing the apoptotic death of serum‐deprived PC12 cells can also protect the cells from apoptosis induced by the cytotoxic drugs. Nerve growth factor, dibutyryl cyclic AMP, and expression of bcl‐2 inhibited apoptosis induced by all four cytotoxic drugs. Fibroblast growth factor inhibited apoptosis induced by EGTA or colchicine. Aurintricarboxylic acid inhibited apoptosis induced by EGTA. These results suggest that apoptosis induced by treatments with the various drugs is mediated by different initiating pathways, all of which converge into a final, common pathway. Nerve growth factor, dibutyryl cyclic AMP, and bcl‐2 appear to affect the final common pathway, whereas fibroblast growth factor and aurincarboxylic acid appear to be more specific and affect only some of the pathways.

Dopamine-induced apoptosis is inhibited in PC12 cells expressing Bcl-2.

Abstract1. Degeneration of nigrostriatal dopaminergic neurons is the major pathogenic substrate of Parkinsons disease (PD). It is assumed that the lethal trigger is the accumulation of oxidative reactive species generated during metabolism of the natural neurotransmitter dopamine.2. We have recently shown that dopamine is capable of inducing programmed cell death (PCD) or apoptosis in cultured postmitotic chick sympathetic neurons and rat PC12 pheochromocytoma cells.3. The bcl-2 gene encodes a protein which blocks physiological PCD in many mammalian cells. In an attempt to elucidate further the mechanism of dopamine toxicity, we examined the potential protective effect of bcl-2 in PC12 cells which were transfected with the protooncogene.4. In our experiments, Bcl-2 producing cells showed a marked resistance to dopamine toxicity. The percentage of nuclear condensation and DNA fragmentation visualized by the end-labeling method following dopamine treatment was significantly lower in bcl-2 expressing cells. Bcl-2 did not protect PC12 cells against toxicity induced by exposure to dopamine-melanin. Extracts of PC12 cells containing Bcl-2 inhibited dopamine autooxidation and formation of dopamine-melanin. Furthermore, the presence of Bcl-2 protected cells from thiol imbalance and prevented thiol loss following exposure to dopamine.5. The protective effects of Bcl-2 against dopamine toxicity may be explained, in part, by its action as an antioxidant and by its interference in the production of toxic agents. The possible protection by Bcl-2 against neuronal degeneration caused by dopamine may play a role in the pathogenesis of PD andmay provide a new direction for the development of neuroprotective therapies.

Bcl-xS and Bax induce different apoptotic pathways in PC12 cells.

Apoptosis is regulated by the action of the Bcl-2 family of proteins, which includes anti- and pro-apoptotic members such as Bcl-xS and Bax. These proteins may differ from each other in structure, mechanism of action and interactions with anti-apoptotic signaling. The mechanism whereby Bax induces cell death has been studied in some cellular systems, but the mechanism of Bcl-xS-induced apoptosis is largely unknown. In this study we investigated and compared the apoptotic effects of Bcl-xS and Bax in the pheochromocytoma cell line, PC12 (a useful model system for studying neuronal apoptosis), and the extent to which they are protected by the survival factor, nerve growth factor (NGF). PC12 cells express endogenous Bcl-xS, Bax and Bcl-xL proteins. Subcellular fractionation revealed that Bax is presented mainly in the cytosolic and the heavy membrane fractions, Bcl-xS is present only in the cytosol, and the anti-apoptotic protein Bcl-xL is located mainly in the heavy membrane fraction. In contrast to the cytosolic localization of endogenous Bcl-xS, the exogenously overexpressed Bcl-xS is localized to the mitochondria. Overexpression of Bcl-xS or Bax induces cell death in the transfected cells. The cell death induced by overexpression of Bcl-xS was inhibited by co-expression of Bcl-xS with Bcl-2 or Bcl-xL, or by treatment with the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone (Z-VAD-FMK) or with NGF. The Bcl-2 mutants ΔC22, which lacks the transmembrane domain, and G145A (mI-3) were able to inhibit the death-inducing effect of Bcl-xS. These results therefore suggest that the apoptotic pathway induced by overexpression of Bcl-xS in PC12 cells can be controlled by Bcl-2 and Bcl-xL, is mediated by caspases, and can be inhibited by the NGF signaling pathway. The Bax-induced cell death was inhibited by co-expression of Bax with Bcl-2 or Bcl-xL, but was not inhibited by Z-VAD-FMK, NGF, or the Bcl-2 mI-3 or ΔC22 mutants. These results therefore suggest that Bax induces a caspase-independent cell death pathway which is blocked by Bcl-2 but not by the NGF signaling pathway. They further suggest that Bcl-xS and Bax induce different cell death pathways in PC12 cells.

Increased expression of the γ‐secretase components presenilin‐1 and nicastrin in activated astrocytes and microglia following traumatic brain injury

γ‐Secretase is an aspartyl protease composed of four proteins: presenilin (PS), nicastrin (Nct), APH1, and PEN2. These proteins assemble into a membrane complex that cleaves a variety of substrates within the transmembrane domain. The γ‐secretase cleavage products play an important role in various biological processes such as embryonic development and Alzheimers disease (AD). The major role of γ‐secretase in brain pathology has been linked to AD and to the production of the amyloid β‐peptide. However, little is known about the possible role of γ‐secretase following acute brain insult. Here we examined by immunostaining the expression patterns of two γ‐secretase components, PS1 and Nct, in three paradigms of brain insult in mice: closed head injury, intracerebroventricular injection of LPS, and brain stabbing. Our results show that in naïve and sham‐injured brains expression of PS1 and Nct is restricted mainly to neurons. However, following insult, the expression of both proteins is also observed in nonneuronal cells, consisting of activated astrocytes and microglia. Furthermore, the proteins are coexpressed within the same astrocytes and microglia, implying that these cells exhibit an enhanced γ‐secretase activity following brain damage. In view of the important role played by astrocytes and microglia in brain disorders, our findings suggest that γ‐secretase may participate in brain damage and repair processes by regulating astrocyte and microglia activation and/or function.

First evidence for helical transitions in supercoiled DNA by amyloid Beta Peptide (1-42) and aluminum: a new insight in understanding Alzheimer's disease.

Previously, we evidenced a B → Z helical change in Alzheimer’s brain genomic DNA, leading to a hypothesis that Alzheimer’s disease (AD) etiological factors such as aluminum (Al), amyloid β (Aβ) peptide, and Tau might play a role in modulating DNA topology. In the present study, we investigated the interaction of Al and Aβ with DNA. Our results show that Aβ(1–42) could induce a B → ψ (Psi) conformational change in pUC 18 supercoiled DNA (scDNA), Aβ(1–16) caused an altered B-form, whereas Al induced a complex B-C-A mixed conformation. Ethidium bromide binding and agarose gel electrophoresis studies revealed that Al uncoiled the DNA to a fully relaxed form, whereas Aβ(1–42) and Aβ(1–16) effected a partial uncoiling and also showed differential sensitivity toward chloroquine-induced topoisomer separation. Our findings show for the first time that Aβ and Al modulate both helicity and superhelicity in scDNA. A new hypothetical model explaining the potential toxicity of Aβ and Al in terms of their DNA binding properties leading to DNA conformational alteration is proposed.