Targeted polyphosphatase expression alters mitochondrial metabolism and inhibits calcium-dependent cell death

Abstract

Polyphosphate (polyP) consists of tens to hundreds of phosphates, linked by ATP-like high-energy bonds. Although polyP is present in mammalian mitochondria, its physiological roles there are obscure. Here, we examine the involvement of polyP in mitochondrial energy metabolism and ion transport. We constructed a vector to express a mitochondrially targeted polyphosphatase, along with a GFP fluorescent tag. Specific reduction of mitochondrial polyP, by polyphosphatase expression, significantly modulates mitochondrial bioenergetics, as indicated by the reduction of inner membrane potential and increased NADH levels. Furthermore, reduction of polyP levels increases mitochondrial capacity to accumulate calcium and reduces the likelihood of the calcium-induced mitochondrial permeability transition, a central event in many types of necrotic cell death. This confers protection against cell death, including that induced by ␤-amyloid peptide, a pathogenic agent in Alzheimer's disease. These results demonstrate a crucial role played by polyP in mitochondrial function of mammalian cells. mitochondria ͉ permeability transition ͉ polyphosphate ͉ ␤-amyloid peptide ͉ necrosis T he chemical and physical properties of polyphosphate (polyP), including its high negative charge and its ability to form complexes with Ca 2ϩ and to form high energy bonds, underlie its potential to play an important role in cell metabolism. Significant amounts of polyP have been found in bacteria and in lower eukaryotes. In those organisms, it provides energy storage and a reserve pool of inorganic phosphate, participates in regulation of gene expression, protects cells from the toxicity of heavy metals by forming complexes with them, and participates in channel formation through assembly into complexes with Ca 2ϩ and polyhydroxybutyrate (PHB) (polyP/Ca 2ϩ /PHB complex) (1, 2) and possibly through interaction with channelforming proteins (3).PolyP has also been found in all higher eukaryotic organisms tested, where it is localized in various subcellular compartments, including mitochondria (4). Furthermore, mitochondrial polyP can form polyP/Ca 2ϩ /PHB complexes (5) with ion-conducting properties similar to those of native mitochondrial permeability transition pore (mPTP) (6). mPTP opening or formation in the mitochondrial inner membrane is believed to underlie the Ca 2ϩ -induced permeability transition (PT), a phenomenon that causes inner membrane depolarization and disruption of ATP synthesis and plays a central role during various types of necrotic and apoptotic cell death (7). The molecular composition of the conducting pathway of mPTP is currently not well defined.Recently, we have raised the possibility that, in vivo, the polyP/ Ca 2ϩ /PHB complex might comprise the ion-conducting part of the mPTP complex (6). If so, mitochondrial polyP should be essential for mPTP opening/formation. Here, we examine the involvement of polyP in normal mitochondrial function and in PT development during stress. To this end, we specifically reduced levels of mitocho...

Carbonic anhydrase inhibition selectively prevents amyloid β neurovascular mitochondrial toxicity

“…TMRM assay was performed using the probe in nonquenching mode conditions, as previously described in (Abramov, Fraley, et al., 2007). Cells were plated on 25‐mm optical borosilicate poly‐L‐lysine‐coated sterile glass covers (Thermo Fisher, Waltham, Massachusetts, USA) at a 70% confluence.…”

Phosphate Is Essential for Inhibition of the Mitochondrial Permeability Transition Pore by Cyclosporin A and by Cyclophilin D Ablation

“…the optimum for opening of the PTP, the probability of opening of which declines at both lower and higher matrix pH values (33). An additional possibility is that matrix P i gives rise to the formation of polyphosphate, which promotes the permeability transition (34). Because the inducing properties of P i are shared by As i and V i with the same concentration dependence, it will be interesting to test if the latter can substitute for P i in the formation of complexes with Ca 2ϩ and polyhydroxybutyrate, which mimic the ion-conductive properties of the PTP (35).…”

Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription

“…The method of polyP detection using DAPI was first described in the early 1980s. 21 Since that time, this procedure has been used in several studies (some examples are references: 6,8,13,16,[22][23][24][25] ). Emission fluorescence of DAPI shifts to longer wavelengths in the presence of polyP: this change is specific for polyP and is not produced by PPi, DNA, or other anions.…”