Dale E. Bredesen

Anti-cancer activity of targeted pro-apoptotic peptides

We have designed short peptides composed of two functional domains, one a tumor blood vessel homing motif and the other a programmed cell death-inducing sequence, and synthesized them by simple peptide chemistry. The homing domain was designed to guide the peptide to targeted cells and allow its internalization. The pro-apoptotic domain was designed to be nontoxic outside cells, but toxic when internalized into targeted cells by the disruption of mitochondrial membranes. Although our prototypes contain only 21 and 26 residues, they were selectively toxic to angiogenic endothelial cells and showed anti-cancer activity in mice. This approach may yield new therapeutic agents.

Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis

A subset of individuals with familial amyotrophic lateral sclerosis (FALS) possesses dominantly inherited mutations in the gene that encodes copper-zinc superoxide dismutase (CuZnSOD). A4V and G93A, two of the mutant enzymes associated with FALS, were shown to catalyze the oxidation of a model substrate (spin trap 5,5′-dimethyl-1-pyrroline N-oxide) by hydrogen peroxide at a higher rate than that seen with the wild-type enzyme. Catalysis of this reaction by A4V and G93A was more sensitive to inhibition by the copper chelators diethyldithiocarbamate and penicillamine than was catalysis by wild-type CuZnSOD. The same two chelators reversed the apoptosis-inducing effect of mutant enzymes expressed in a neural cell line. These results suggest that oxidative reactions catalyzed by mutant CuZnSOD enzymes initiate the neuropathologic changes in FALS.

Pro-caspase-3 Is a Major Physiologic Target of Caspase-8

The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. We show here that in a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3 with activation rates (k cat/K m ) of 8.7 × 105 and 2.8 × 105 m −1 s−1, respectively. These rates are of sufficient magnitude to indicate direct processingin vivo. Differentially processed forms of caspase-3 that accumulate during its activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pathway to activation, pro-caspase-9, was without consequence. Taken together these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors.

Caspase Cleavage of Gene Products Associated with Triplet Expansion Disorders Generates Truncated Fragments Containing the Polyglutamine Tract

The neurodegenerative diseases Huntington disease, dentatorubropallidoluysian atrophy, spinocerebellar atrophy type 3, and spinal bulbar muscular atrophy are caused by expansion of a polyglutamine tract within their respective gene products. There is increasing evidence that generation of truncated proteins containing an expanded polyglutamine tract may be a key step in the pathogenesis of these disorders. We now report that, similar to huntingtin, atrophin-1, ataxin-3, and the androgen receptor are cleaved in apoptotic extracts. Furthermore, each of these proteins is cleaved by one or more purified caspases, cysteine proteases involved in apoptotic death. The CAG length does not modulate susceptibility to cleavage of any of the full-length proteins. Our results suggest that by generation of truncated polyglutamine-containing proteins, caspase cleavage may represent a common step in the pathogenesis of each of these neurodegenerative diseases.

A second cytotoxic proteolytic peptide derived from amyloid β-proteinprecursor

The amyloid β-protein precursor gives rise to the amyloid β-protein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of Alzheimer disease. Here we show that amyloid β-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 β-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 β-protein precursor with the generation of C31 may be involved in the neuronal death associated with Alzheimer disease.

The DCC gene product induces apoptosis by a mechanism requiring receptor proteolysis

The development of colonic carcinoma is associated with the mutation of a specific set of genes. One of these, DCC (deleted in colorectal cancer), is a candidate tumour-suppressor gene, and encodes a receptor for netrin-1, a molecule involved in axon guidance. Loss of DCC expression in tumours is not restricted to colon carcinoma, and, although there is no increase in the frequency of tumour formation in DCC hemizygous mice, re-establishment of DCC expression suppresses tumorigenicity,. However, the mechanism of action of DCC is unknown. Here we show that DCC induces apoptosis in the absence of ligand binding, but blocks apoptosis when engaged by netrin-1. Furthermore, DCC is a caspase substrate, and mutation of the site at which caspase-3 cleaves DCC suppresses the pro-apoptotic effect of DCC completely. These results indicate that DCC may function as a tumour-suppressor protein by inducing apoptosis in settings in which ligand is unavailable (for example, during metastasis or tumour growth beyond local blood supply) through functional caspase cascades by a mechanism that requires cleavage of DCC at Aspu20091,290.

Kennedy's disease: caspase cleavage of the androgen receptor is a crucial event in cytotoxicity.

Abstract : X‐linked spinal and bulbar muscular atrophy (SBMA), Kennedys disease, is a degenerative disease of the motor neurons that is associated with an increase in the number of CAG repeats encoding a polyglutamine stretch within the androgen receptor (AR). Recent work has demonstrated that the gene products associated with open reading frame triplet repeat expansions may be substrates for the cysteine protease cell death executioners, the caspases. However, the role that caspase cleavage plays in the cytotoxicity associated with expression of the disease‐associated alleles is unknown. Here, we report the first conclusive evidence that caspase cleavage is a critical step in cytotoxicity ; the expression of the AR with an expanded polyglutamine stretch enhances its ability to induce apoptosis when compared with the normal AR. The AR is cleaved by a caspase‐3 subfamily protease at Asp146, and this cleavage is increased during apoptosis. Cleavage of the AR at Asp146 is critical for the induction of apoptosis by AR, as mutation of the cleavage site blocks the ability of the AR to induce cell death. Further, mutation of the caspase cleavage site at Asp146 blocks the ability of the SBMA AR to form perinuclear aggregates. These studies define a fundamental role for caspase cleavage in the induction of neural cell death by proteins displaying expanded polyglutamine tracts, and therefore suggest a strategy that may be useful to treat neurodegenrative diseases associated with polyglutamine repeat expansions.

The protooncogene bcl-2 inhibits apoptosis in PC12 cells

Abstract: During development, many neuronal populations undergo a process of normal, programmed cell death, or apoptosis. Trophic factors regulate this process, but the mechanism by which they suppress apoptosis remains unclear. In the immune system, recent studies have implicated the protooncogene bcl‐2 in the lymphocyte survival response to growth factors. To determine whether a similar survival pathway exists in a neuroendocrine cell type, we have expressed bcl‐2 in the rat pheochromocytoma PC12 cell line and found that it abrogates the requirement for stimulation by growth factors to survive. bcl‐2 expression also substantially delays the onset of injury by the calcium ionophore A23187.

Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease

AbstractHuntingtons disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

INVITED COMMENTARY IS APOPTOSIS MEDIATED BY REACTIVE OXYGEN SPECIES

Apoptosis is a common mode of programmed cell death occurring during development as well as in many pathological conditions, in which the cell plays an active role in its own demise. Although the morphological and biochemical hallmarks of apoptosis are conserved across phyla and cell type, the mechanism(s) of apoptosis is unknown. However, data recently published demonstrate that expression of the anti-apoptotic gene bcl-2 decreases the net cellular generation of reactive oxygen species, and that reactive oxygen species serve as mediators of apoptosis in at least some cases.