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Biomedical subjects

D E Bredesen

Publications and source records attributed to D E Bredesen.

At least 19 recordsLinked to original sources
PubMed2002

A ligand-receptor pair that triggers a non-apoptotic form of programmed cell death.

Several receptors that mediate apoptosis have been identified, such as Fas and tumor necrosis factor receptor I. Studies of the signal transduction pathways utilized by these receptors have played an important role in the understanding of apoptosis. Here we report the first ligand-receptor pair-the neuropeptide substance P and its receptor, neurokinin-1 receptor (NK(1)R)-that mediates an alternative, non-apoptotic form of programmed cell death. This pair is widely distributed in the central and peripheral nervous systems, and has been implicated in pain mediation and depression, among other effects. Here we demonstrate that substance P induces a non-apoptotic form of programmed cell death in hippocampal, striatal, and cortical neurons. This cell death requires gene expression, displays a non-apoptotic morphology, and is independent of caspase activation. The same form of cell death is induced by substance P in NK(1)R-transfected human embryonic kidney cells. These results argue that NK(1)R activates a death pathway different than apoptosis, and provide a signal transduction system by which to study an alternative, non-apoptotic cell death program.

Animals↗
PubMed2001

Caspase 7 can cleave tumor necrosis factor receptor-I (p60) at a non-consensus motif, in vitro.

Ligand binding to tumor necrosis factor receptor-I (TNFRI) can promote cell survival or activate the apoptotic caspase cascade. Cytoplasmic interaction of TNFRI with TRAF2 and RIP allows for the activation of JNK and NFkappaB pathways. Alternatively, a carboxy terminal death domain protein interaction motif can recruit TRADD, which then recruits FADD/MORT1, and finally procaspase 8. Aggregation of these components form a death inducing signaling complex, leading to the cleavage and activation of caspase 8. We have found that during apoptosis human TNFRI protein is lost in a caspase-dependent manner. The cytoplasmic tail of human TNFRI was found to be susceptible to caspase cleavage but not by caspase 8. Instead, the downstream executioner caspase 7 was the only caspase capable of cleaving TNFRI, in vitro. Identification and characterization of the cleavage site revealed a derivative of the classic EXD motif that incorporates a glutamate (E) in the P1 position. Using several criteria to establish that caspase activity was responsible for cleavage at this site, we confirmed that caspase 7 can cleave at a GELE motif. Mutation of the cleavage site prevented the apoptosis-associated cleavage of TNFRI. This ability of caspase 7 to cleave at a non-EXD or -DXXD motif suggests that the specificity of caspases may be broader than is currently held.

Amino Acid Sequence↗
PubMed2001

Effect of overexpression of BCL-2 on cellular oxidative damage, nitric oxide production, antioxidant defenses, and the proteasome.

Bcl-2 is a gene family involved in the suppression of apoptosis in response to a wide range of cellular insults. Multiple papers have suggested a link between Bcl-2 and oxidative damage/antioxidant protection. We therefore examined parameters of antioxidant defense and oxidative damage in two different cell lines, NT-2/D1 (NT-2) and SK-N-MC, overexpressing Bcl-2 as compared with vector-only controls. Bcl-2 transfectants of both cell lines were more resistant to H(2)O(2) and showed increases in GSH level and Cu/Zn-superoxide dismutase (SOD1) activity, but not in Mn-superoxide dismutase, glutathione peroxidase, or glutathione reductase activities. Catalase activity was increased in SK-N-MC cells. Overexpression of Bcl-2 did not significantly decrease levels of oxidative DNA damage (measured as 8-hydroxyguanine) or lipid peroxidation, but it decreased levels of 3-nitrotyrosine in both cell lines and protein carbonyls in SK-N-MC cells only. It also increased proteasome activity in both cell lines. We conclude that Bcl-2 raises cellular antioxidant defense status, but this is not necessarily reflected in decreased levels of oxidative damage to DNA and lipids. The ability of Bcl-2 overexpression to decrease 3-nitrotyrosine levels suggests that it may decrease formation of peroxynitrite or other reactive nitrogen species; this was confirmed as decreased production of NO(2)(-)/NO(3)(-) in the transfected cells and a fall in the level of nNOS protein.

Antioxidants↗
PubMed2001

Mining DNA microarray data using a novel approach based on graph theory.

The recent demonstration that biochemical pathways from diverse organisms are arranged in scale-free, rather than random, systems [Jeong et al., Nature 407 (2000) 651-654], emphasizes the importance of developing methods for the identification of biochemical nexuses--the nodes within biochemical pathways that serve as the major input/output hubs, and therefore represent potentially important targets for modulation. Here we describe a bioinformatics approach that identifies candidate nexuses for biochemical pathways without requiring functional gene annotation; we also provide proof-of-principle experiments to support this technique. This approach, called Nexxus, may lead to the identification of new signal transduction pathways and targets for drug design.

Apoptosis↗
PubMed2001

Neurodegenerative disease and cancer: two sides of a coin?

The mechanisms underlying disruption of physiologic cell-death programs are slowly being revealed. Information gleaned from genetic profiles soon may allow physicians to delay onset of many silent, age-related diseases. While development of actual cures could prove elusive, simply postponing clinical onset would have an impressive effect on the goal of healthy aging.

Aging↗
PubMed2001

Suppression of murine collagen-induced arthritis by targeted apoptosis of synovial neovasculature.

Because angiogenesis plays a major role in the perpetuation of inflammatory arthritis, we explored a method for selectively targeting and destroying new synovial blood vessels. Mice with collagen-induced arthritis were injected intravenously with phage expressing an RGD motif. In addition, the RGD peptide (RGD-4C) was covalently linked to a proapoptotic heptapeptide dimer, D(KLAKLAK)2, and was systemically administered to mice with collagen-induced arthritis. A phage displaying an RGD-containing cyclic peptide (RGD-4C) that binds selectively to the alpha(v)beta3 and alpha(v)beta5 integrins accumulated in inflamed synovium but not in normal synovium. Homing of RGD-4C phage to inflamed synovium was inhibited by co-administration of soluble RGD-4C. Intravenous injections of the RGD-4C-D(KLAKLAK)2 chimeric peptide significantly decreased clinical arthritis and increased apoptosis of synovial blood vessels, whereas treatment with vehicle or uncoupled mixture of the RGD-4C and the untargeted proapoptotic peptide had no effect. Targeted apoptosis of synovial neovasculature can induce apoptosis and suppress clinical arthritis. This form of therapy has potential utility in the treatment of inflammatory arthritis.

Animals↗
PubMed2001

A monoclonal antibody that induces neuronal apoptosis binds a metastasis marker.

The cell surface molecules controlling apoptosis in cortical neurons are largely unknown. A monoclonal antibody was derived that induces cultured neocortical neurons to undergo apoptosis. A Fab fragment of the antibody, however, lacked the ability to induce cell death. The antigen was purified, and characterized by compositional analysis, fast atom bombardment (FAB) mass spectrometry, sequential exoglycosidase treatments, methylation analysis, and (1)H-nuclear magnetic resonance spectroscopy, proving to be isoglobotetraosylceramide (IsoGb4). IsoGb4 has been shown previously to be a metastasis marker, antibodies against which block metastases in a mammary adenocarcinoma model (S. A. Carlsen et al., Cancer Res., 53: 2906-2911, 1993). Addition of the purified antigen to cells lacking this glycolipid demonstrated that it is capable of functioning as a portable apoptosis-transducing molecule. Intracellular ceramide levels were increased after the treatment with the apoptosis-inducing antibody, but the membrane sphingomyelin level remained unchanged. Fumonisin B1 inhibited both the ceramide increase and the apoptosis induced via IsoGb4, which indicated that the ceramide synthase pathway is likely to be involved in apoptosis induction by IsoGb4.

Animals↗
PubMed2001

Double-stranded RNA-dependent protein kinase, PKR, binds preferentially to Huntington's disease (HD) transcripts and is activated in HD tissue.

Fourteen neurological diseases have been associated with the expansion of trinucleotide repeat regions. These diseases have been categorized into those that give rise to the translation of toxic polyglutamine proteins and those that are untranslated. Thus far, compelling evidence has not surfaced for the inclusion of a model in which a common mechanism may participate in the pathobiology of both translated and untranslated trinucleotide diseases. In these studies we show that a double-stranded RNA-binding protein, PKR, which has previously been linked to virally-induced and stress-mediated apoptosis, preferentially binds mutant huntingtin RNA transcripts immobilized on streptavidin columns that have been incubated with human brain extracts. These studies also show, by immunodetection in tissue slices, that PKR is present in its activated form in both human Huntington autopsy material and brain tissue derived from Huntington yeast artificial chromosome transgenic mice. The increased immunolocalization of the activated kinase is more pronounced in areas most affected by the disease and, coupled with the RNA binding results, suggests a role for PKR activation in the disease process.

Animals↗
PubMed2001

Coupling endoplasmic reticulum stress to the cell death program. Mechanism of caspase activation.

The endoplasmic reticulum (ER) is the site of assembly of polypeptide chains destined for secretion or routing into various subcellular compartments. It also regulates cellular responses to stress and intracellular Ca(2+) levels. A variety of toxic insults can result in ER stress that ultimately leads to apoptosis. Apoptosis is initiated by the activation of members of the caspase family and serves as a central mechanism in the cell death process. The present study was carried out to determine the role of caspases in triggering ER stress-induced cell death. Treatment of cells with ER stress inducers such as brefeldin-A or thapsigargin induces the expression of caspase-12 protein and also leads to translocation of cytosolic caspase-7 to the ER surface. Caspase-12, like most other members of the caspase family, requires cleavage of the prodomain to activate its proapoptotic form. Caspase-7 associates with caspase-12 and cleaves the prodomain to generate active caspase-12, resulting in increased cell death. We propose that any cellular insult that causes prolonged ER stress may induce apoptosis through caspase-7-mediated caspase-12 activation. The data underscore the involvement of ER and caspases associated with it in the ER stress-induced apoptotic process.

Animals↗
PubMed2001

De novo-designed peptide transforms Golgi-specific lipids into Golgi-like nanotubules.

Cellular organelles, such as the Golgi apparatus and the endoplasmic reticulum, adopt characteristic structures depending on their function. While the tubular shapes of these structures result from complex protein-lipid interactions that are not fully understood, some fundamental machinery must be required. We show here that a de novo-designed 18-mer amphipathic alpha-helical peptide, Hel 13-5, transforms spherical liposomes made from a Golgi-specific phospholipid mixture into nanotubules on the scale of and resembling the shape of the nanotubules that form the Golgi apparatus. Furthermore, we show that that the size and the shape of such nanotubules depend on lipid composition and peptide properties such as length and the ratio of hydrophobic to hydrophilic amino acids. Although the question of precisely how nature engineers organellar membranes remains unknown, our simple novel system provides a basic set of tools to begin addressing this question.

Circular Dichroism↗
PubMed2001

APAP, a sequence-pattern recognition approach identifies substance P as a potential apoptotic peptide.

We have previously described a novel cancer chemotherapeutic approach based on the induction of apoptosis in targeted cells by homing pro-apoptotic peptides. In order to improve this approach we developed a computational method (approach for detecting potential apoptotic peptides, APAP) to detect short PAPs, based on the prediction of the helical content of peptides, the hydrophobic moment, and the isoelectric point. PAPs are toxic against bacteria and mitochondria, but not against mammalian cells when applied extracellularly. Among other peptides, substance P was identified as a PAP and subsequently demonstrated to be a pro-apoptotic peptide experimentally. APAP thus provides a method to detect and ultimately improve pro-apoptotic peptides for chemotherapy.

Amino Acid Sequence↗
PubMed2001

The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation.

The expression of DCC (deleted in colorectal cancer) is often markedly reduced in colorectal and other cancers. However, the rarity of point mutations identified in DCC coding sequences and the lack of a tumor predisposition phenotype in DCC hemizygous mice have raised questions about its role as a tumor suppressor. DCC also mediates axon guidance and functions as a dependence receptor; such receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand. We now show that DCC drives cell death independently of both the mitochondria-dependent pathway and the death receptor/caspase-8 pathway. Moreover, we demonstrate that DCC interacts with both caspase-3 and caspase-9 and drives the activation of caspase-3 through caspase-9 without a requirement for cytochrome c or Apaf-1. Hence, DCC defines an additional pathway for the apoptosome-independent caspase activation.

Apoptosis↗
PubMed2000

An alternative, nonapoptotic form of programmed cell death.

The term apoptosis often has been used interchangeably with the term programmed cell death. Here we describe a form of programmed cell death that is distinct from apoptosis by the criteria of morphology, biochemistry, and response to apoptosis inhibitors. Morphologically, this alternative form of programmed cell death appears during development and in some cases of neurodegeneration. Despite its lack of response to caspase inhibitors and Bcl-x(L), we show that this form of cell death is driven by an alternative caspase-9 activity that is Apaf-1-independent. Characterization of this alternative form of programmed cell death should lead to new insight into cell death programs and their roles in development and degeneration.

Apoptosis↗
PubMed2000

The RET proto-oncogene induces apoptosis: a novel mechanism for Hirschsprung disease.

The RET (rearranged during transfection) proto-oncogene encodes a tyrosine kinase receptor involved in both multiple endocrine neoplasia type 2 (MEN 2), an inherited cancer syndrome, and Hirschsprung disease (HSCR), a developmental defect of enteric neurons. We report here that the expression of RET receptor induces apoptosis. This pro-apoptotic effect of RET is inhibited in the presence of its ligand glial cell line-derived neurotrophic factor (GDNF). Furthermore, we present evidence that RET induces apoptosis via its own cleavage by caspases, a phenomenon allowing the liberation/exposure of a pro-apoptotic domain of RET. In addition, we report that Hirschsprung-associated RET mutations impair GDNF control of RET pro-apoptotic activity. These results indicate that HSCR may result from apoptosis of RET-expressing enteric neuroblasts.

Apoptosis↗
PubMed2000

Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells.

Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a site-directed htt mutant that is resistant to caspase-3 cleavage at positions 513 and 530 and to caspase-6 cleavage at position 586. In contrast to cleavable htt, caspase-resistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease.

Amino Acid Sequence↗
PubMed2000

Dimerization-dependent block of the proapoptotic effect of p75(NTR).

The biochemical mechanism by which neurons become dependent on neurotrophins for survival is unknown. We found previously that the common neurotrophin receptor, p75(NTR), is a mediator of neurotrophin dependence and that this effect requires a novel type of domain dubbed a neurotrophin dependence domain. We report here that, in contrast to other proapoptotic receptors such as Fas, apoptosis induction by p75(NTR) requires monomerization, with dimerization inhibiting the effect. Blocking the proapoptotic effect of the monomer by dimerization requires a distinct domain that lies at the carboxyterminus of p75(NTR). These results define a novel type of domain required for inhibiting apoptosis induction by p75(NTR).

Apoptosis↗
PubMed2000

A second cytotoxic proteolytic peptide derived from amyloid beta-protein precursor.

The amyloid beta-protein precursor gives rise to the amyloid beta-protein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of Alzheimer disease. Here we show that amyloid beta-protein precursor was proteolytically cleaved by caspases in the C terminus to generate a second unrelated peptide, called C31. The resultant C31 peptide was a potent inducer of apoptosis. Both caspase-cleaved amyloid beta-protein precursor and activated caspase-9 were present in brains of Alzheimer disease patients but not in control brains. These findings indicate the possibility that caspase cleavage of amyloid beta-protein precursor with the generation of C31 may be involved in the neuronal death associated with Alzheimer disease.

Alzheimer Disease↗