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Results for “Protein Aggregation, Pathological”

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PubMed2001

Thiolutin, an inhibitor of HUVEC adhesion to vitronectin, reduces paxillin in HUVECs and suppresses tumor cell-induced angiogenesis.

Recent studies have shown that integrin alpha v beta 3, a receptor for vitronectin, plays an important role in tumor-induced angiogenesis and tumor growth and that antagonists of alpha v beta 3 inhibit angiogenic processes including endothelial cell adhesion and migration. On the other hand, most inhibitors of integrin alpha v beta 3 are peptide antagonists that include the Arg-Gly-Asp (RGD) motif. We therefore reasoned that non-peptide inhibitors of endothelial cell adhesion to vitronectin might be useful for inhibition of tumor angiogenesis in vivo. We screened for low-molecular-weight natural products able to inhibit adhesion of human umbilical vein endothelial cells (HUVECs) to vitronectin, and pyrrothine group compounds including aureothricin, thioaurin and thiolutin were isolated from microbial culture broths. Of these compounds, thiolutin inhibited adhesion of HUVECs to vitronectin the most effectively (IC(50), 0.83 microM). In vivo experiments showed that thiolutin significantly suppressed angiogenesis induced by tumor cells (S-180), a pathological form of neovascularization, in a mouse dorsal air sac assay system. To explore the mechanism of inhibition of HUVEC adhesion to vitronectin by thiolutin, we examined the effect of this agent on intracellular cell adhesion signaling. We found that the amount of paxillin in HUVECs was significantly reduced by thiolutin treatment, while those of other focal adhesion proteins including vinculin and focal adhesion kinase (FAK) were not. Metabolic labeling experiments showed that thiolutin enhanced degradation of paxillin in HUVECs. Protease inhibitors (MG115 and E64-D) decreased the rate of degradation of the paxillin induced by thiolutin and partially restored thiolutin-induced inhibition of HUVEC adhesion to vitronectin. Based on these findings, we concluded that thiolutin, an inhibitor of HUVEC adhesion to vitronectin, reduces the paxillin level in HUVECs and suppresses tumor cell-induced angiogenesis in vivo.

Animals↗

PubMed2004

Mishandling of the therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils.

PURPOSE: Some therapeutic peptides exhibit amyloidogenic properties that cause insolubility and cytotoxicity against neuronal cells in vitro. Here, we characterize the conformational change in monomeric therapeutic peptide to its fibrillar aggregate in order to prevent amyloidogenic formation during clinical application. METHODS: Therapeutic peptides including glucagon, porcine secretin, and salmon calcitonin were dissolved in acidic solution at concentrations ranging from 1 mg/ml to 80 mg/ml and then aged at 37 degrees C. Amyloidogenic properties were assessed by circular dichroism (CD), electron microscopy (EM), staining with beta-sheet-specific dyes, and size-exclusion chromatography (SEC). Cytotoxic characteristics were determined concomitantly. RESULTS: By aging at 2.5 mg/ml or higher for 24 h, monomeric glucagon was converted to fibrillar aggregates consisting of a beta-sheet-rich structure with multimeric states of glucagon. Although no aggregation was observed by aging at the clinical concentration of 1 mg/ml for 1 day, 30-day aging resulted in the generation of fibrillar aggregates. The addition of anti-glucagon serum significantly inhibited fibrillar conversion of monomeric glucagon. Glucagon fibrils induced significant cell death and activated an apoptotic enzyme, caspase-3, in PC12 cells and NIH-3T3 cells. Caspase inhibitors attenuated this toxicity in a dose-dependent manner, indicating the involvement of apoptotic signaling pathways in the fibrillar formation of glucagon. On the contrary to glucagon, salmon calcitonin exhibited aggregation at a much higher concentration of 40 mg/ml and secretin showed no aggregation at the concentration as high as 75 mg/ml. CONCLUSIONS: These results indicated that glucagon was self-associated by its beta-sheet-rich intermolecular structure during the aging process under concentrated conditions to induce fibrillar aggregates. Glucagon has the same amyloidogenic propensities as pathologically related peptides such as beta-amyloid (Abeta)1-42 and prion protein fragment (PrP)106-126 including conformational change to a beta-sheet-rich structure and cytotoxic effects by activating caspases. These findings suggest that inappropriate preparation and application of therapeutic glucagon may cause undesirable insoluble products and side effects such as amyloidosis in clinical application.

Amyloid↗

PubMed2005

Solvent and mutation effects on the nucleation of amyloid beta-protein folding.

Experimental evidence suggests that the folding and aggregation of the amyloid beta-protein (Abeta) into oligomers is a key pathogenetic event in Alzheimer's disease. Inhibiting the pathologic folding and oligomerization of Abeta could be effective in the prevention and treatment of Alzheimer's disease. Here, using all-atom molecular dynamics simulations in explicit solvent, we probe the initial stages of folding of a decapeptide segment of Abeta, Abeta(21-30), shown experimentally to nucleate the folding process. In addition, we examine the folding of a homologous decapeptide containing an amino acid substitution linked to hereditary cerebral hemorrhage with amyloidosis-Dutch type, [Gln-22]Abeta(21-30). We find that: (i) when the decapeptide is in water, hydrophobic interactions and transient salt bridges between Lys-28 and either Glu-22 or Asp-23 are important in the formation of a loop in the Val-24-Lys-28 region of the wild-type decapeptide; (ii) in the presence of salt ions, salt bridges play a more prominent role in the stabilization of the loop; (iii) in water with a reduced density, the decapeptide forms a helix, indicating the sensitivity of folding to different aqueous environments; and (iv) the "Dutch" peptide in water, in contrast to the wild-type peptide, fails to form a long-lived Val-24-Lys-28 loop, suggesting that loop stability is a critical factor in determining whether Abeta folds into pathologic structures.

Amyloid beta-Peptides↗

PubMed1999

Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network.

A major activity of molecular chaperones is to prevent aggregation and refold misfolded proteins. However, when allowed to form, protein aggregates are refolded poorly by most chaperones. We show here that the sequential action of two Escherichia coli chaperone systems, ClpB and DnaK-DnaJ-GrpE, can efficiently solubilize excess amounts of protein aggregates and refold them into active proteins. Measurements of aggregate turbidity, Congo red, and 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid binding, and of the disaggregation/refolding kinetics by using a specific ClpB inhibitor, suggest a mechanism where (i) ClpB directly binds protein aggregates, ATP induces structural changes in ClpB, which (ii) increase hydrophobic exposure of the aggregates and (iii) allow DnaK-DnaJ-GrpE to bind and mediate dissociation and refolding of solubilized polypeptides into native proteins. This efficient mechanism, whereby chaperones can catalytically solubilize and refold a wide variety of large and stable protein aggregates, is a major addition to the molecular arsenal of the cell to cope with protein damage induced by stress or pathological states.

Bacterial Proteins↗

PubMed2002

Increased ability of erythrocytes to aggregate in spontaneously hypertensive rats.

The development of hypertension is accompanied by changes in the rheological properties of blood, particularly by increased red blood cell (RBC) aggregation leading to further pathological complications. However, it is not clear whether these changes in aggregation are caused only by increased concentrations of plasma adhesion proteins or if alterations in RBC membranes are also involved. The aim of the present study was to determine if RBC aggregability is altered during hypertension and if these changes correlate with alterations in RBC membrane protein concentrations. Aggregability changes were evaluated by comparing fibrinogen (Fb)-induced aggregation of RBCs from spontaneously hypertensive rats (SHR) with RBCs from age matched normotensive Wistar Kyoto (WKY) rats. ANOVA showed a significant increase in dose-dependent Fb-induced aggregation of RBCs in the SHR group. Analysis of Coomassie-stained gels of RBC membrane proteins using SDS-PAGE showed a significant increase in the amount of a protein at 110 kD in the SHR group. These results show that increased RBC aggregability is accompanied by alterations in RBC membrane protein composition during hypertension development.

Animals↗

PubMed2002

A candidate molecular mechanism for the association of an intronic polymorphism of FE65 with resistance to very late onset dementia of the Alzheimer type.

Late onset dementias of the Alzheimer type may be coupled to intrinsic aging processes. Their major pathological hallmarks are the deposition of aggregates of beta amyloid (Abeta) peptides, proteolytic products from internal portions of the Abeta precursor protein, betaPP. Susceptibility appears to be modulated by polymorphic alleles at multiple loci. Most of these putative assignments, however, have been controversial. It is therefore essential to provide evidence of a plausible biological basis for each such association. Here, we show such evidence for the case of a biallelic polymorphism of the FE65 intron 13. FE65 is an adaptor protein that tightly binds to the cytoplasmic tail of betaPP. Increasing evidence indicates that this binding plays a critical role in a signaling pathway. Our results reveal that a protective (minor) allele alters the splicing of the terminal exon by selection of an alternative acceptor site, resulting in an isoform, FE65a2, with an altered C-terminal region lacking part of a betaPP binding site. Pull down assays confirmed that the FE65a2 isoform binds to betaPP less efficiently, suggesting that an attenuated binding of FE65 with betaPP is, in part, responsible for resistance to the very late onset disease. Sequence analysis of the FE65 of mice, non-human primates and man revealed that the susceptibility allele, which codes for strong binding of the FE65 protein with betaPP, was favored by natural selection leading to our lineage. That allele may contribute to very late onset form of Alzheimer disease when we are aged.

Alternative Splicing↗

PubMed2005

Proteinaceous intracellular inclusions in neurodegenerative disorders.

Neurodegenerative disorders are characterized by accumulation of "toxic", pathologic proteins in brain cells. Mutations in genes coding these proteins result in conformational disturbances of the protein structure and their accumulation and aggregation manifesting at the level of light microscope as various intracellular inclusions. This paper is an attempt of approach cellular mechanisms underlying neurodegenerative disorders with special attention to mechanisms of protein elimination.

Animals↗

PubMed2000

Alpha-synuclein is not a requisite component of synaptic boutons in the adult human central nervous system.

It is increasingly clear that the normal protein alpha-synuclein is in some manner closely associated with presynaptic components of select neuronal types within the adult human central nervous system (CNS) and, in addition, that in its pathologically altered state alpha-synuclein aggregates selectively in the form of filamentous inclusion bodies during certain progressive neurodegenerative disorders, such as familial and sporadic Parkinson's disease. By having the antibody AFshp raised specifically to alpha-synuclein to label Parkinson disease-specific Lewy bodies and Lewy neurites as well as synaptic boutons containing the unaltered protein, an initial attempt is made to map the overall distribution pattern and describe the staining behavior of the immunoreactive punctae in select regions of the prosencephalon. Neocortical immunolabeling is most prominent in the prodigious, but incompletely myelinated, association fields and faintest in the heavily myelinated primary motor and primary sensory fields, with the premotor and first order sensory association areas occupying an intermediate position. Of the thalamic grays evaluated, those containing powerfully myelinated fiber tracts (e.g. centrum medianum, habenular complex) show the weakest immunolabeling, whereas, less sturdily myelinated structures are highly immunoreactive. The fact that the immunostaining spectrum for normal alpha-synuclein is so broad, together with the fact that some thalamic sites actually are immunonegative leads to the following conclusions (1) alpha-synuclein, although present in the synaptic boutons of many nerve cells in the adult human CNS, is by no means ubiquitous there, and (2) neuronal types lacking the normal protein cannot generate the Parkinson's disease-specific filamentous pathology.

Adult↗

PubMed2004

Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain.

In many species, the Sox2 transcription factor is a marker of the nervous system from the beginning of its development, and we have previously shown that Sox2 is expressed in embryonic neural stem cells. It is also expressed in, and is essential for, totipotent inner cell mass stem cells and other multipotent cell lineages, and its ablation causes early embryonic lethality. To investigate the role of Sox2 in the nervous system, we generated different mouse mutant alleles: a null allele (Sox2beta-geo 'knock-in'), and a regulatory mutant allele (Sox2DeltaENH), in which a neural cell-specific enhancer is deleted. Sox2 is expressed in embryonic early neural precursors of the ventricular zone and, in the adult, in ependyma (a descendant of the ventricular zone). It is also expressed in the vast majority of dividing precursors in the neurogenic regions, and in a small proportion of differentiated neurones, particularly in the thalamus, striatum and septum. Compound Sox2(beta-geo/DeltaENH) heterozygotes show important cerebral malformations, with parenchymal loss and ventricle enlargement, and L-dopa-rescuable circling behaviour and epilepsy. We observed striking abnormalities in neurones; degeneration and cytoplasmic protein aggregates, a feature common to diverse human neurodegenerative diseases, are observed in thalamus, striatum and septum. Furthermore, ependymal cells show ciliary loss and pathological lipid inclusions. Finally, precursor cell proliferation and the generation of new neurones in adult neurogenic regions are greatly decreased, and GFAP/nestin-positive hippocampal cells, which include the earliest neurogenic precursors, are strikingly diminished. These findings highlight a crucial and unexpected role for Sox2 in the maintenance of neurones in selected brain areas, and suggest a contribution of neural cell proliferative defects to the pathological phenotype.

Alleles↗

PubMed2001

Mechanisms of amyloid beta protein-induced modification in ion transport systems: implications for neurodegenerative diseases.

1. Alzheimer's disease (AD) is a neurodegenerative disorder that affects the cognitive function of the brain. Pathological changes in AD are characterized by the formation of amyloid plaques and neurofibrillary tangles as well as extensive neuronal loss. Abnormal proteolytic processing of amyloid precursor protein (APP) is the central step that leads to formation of amyloid plaque, neurofibrillary tangles, and neuronal loss. 2. The plaques, which accumulate extracellularly in the brain, are composed of aggregates and cause direct neurotoxic effects and/or increase neuronal vulnerability to excitotoxic insults. The aggregates consist of soluble pathologic amyloid beta peptides AbetaP[1-42] and AbetaP[1-43] and soluble nonpathologic AbetaP[1-40]. Both APP and AbetaP interact with ion transport systems. AbetaP induces a wide range of effects as the result of activating a cascade of mechanisms. 3. The major mechanisms proposed for AbetaP-induced cytotoxicity involve the loss of Ca2+ homeostasis and the generation of reactive oxygen species (ROS). The changes in Ca2+ homeostasis could be the result of (1) changes in endogenous ion transport systems, e.g. Ca2+ and K+ channels and Na+/K+-ATPase, and membrane receptor proteins, such as ligand-driven ion channels and G-protein-driven releases of second messengers, and (2) formation of heterogeneous ion channels. 4. The consequences of changes in Ca2+-homeostasis-induced generation of ROS are (a) direct modification of intrinsic ion transport systems and their regulatory mechanisms, and (b) indirect effects on ion transport systems via peroxidation of phospholipids in the membrane, inhibition of phosphorylation, and reduction of ATP levels and cytoplasmic pH. 5. We propose that in AD, AbetaP with its different conformations alters cell regulation by modifying several ion transport systems and also by forming heterogeneous ion channels. The changes in membrane transport systems are proposed as early steps in impairing neuronal function preceding plaque formation. We conclude that these changes damage the membrane by compromising its integrity and increasing its ion permeability. This mechanism of membrane damage is not only central for AD but also may explain other malfunctioned protein-processing-related pathologies.

Amyloid beta-Peptides↗

PubMed2001

Accumulation of C-terminally truncated tau protein associated with vulnerability of the perforant pathway in early stages of neurofibrillary pathology in Alzheimer's disease.

Neurofibrillary pathology is a characteristic hallmark of Alzheimer's disease that is closely correlated with cognitive decline. We have analysed the density and distribution of neurofibrillary tangles (NFTs) that are immunoreactive with the monoclonal antibody (mAb) 423 in a prospectively analysed population of Alzheimer's disease (AD) cases and age-matched controls. NFTs were examined in allocortical and isocortical areas and correlated with Braak pathological stage and clinical severity of dementia. The mAb 423 was used as it recognises a C-terminally truncated tau fragment that is a major constituent of NFTs. Our results show that extracellular NFTs and, to a lesser extent, intracellular NFTs, correlated significantly with both Braak stages and the clinical index of severity. Furthermore, a differential distribution of the two types of tangles indicates that layer II of the entorhinal cortex and the transentorhinal area are particularly vulnerable to neurofibrillary degeneration. These areas serve as a point of connection between isocortex and hippocampus. Our findings, therefore, suggest that the perforant pathway may be substantially affected by the accumulation of truncated tau protein in AD and that this represents a neuropathological predictor for the clinical severity of dementia. When neurofibrillary pathology was examined by combined labelling with mAbs 423 and Alz-50 and the dye thiazin red, we were able to demonstrate various stages of tau aggregation. The different stages may represent a sequence of conformational changes that tau proteins undergo during tangle formation in the allocortex during the early development of dementia in AD.

Aged↗

PubMed2006

1. Membrane origin for autophagy.

Autophagy is a degradative transport route conserved among all eukaryotic organisms. During starvation, cytoplasmic components are randomly sequestered into large double-membrane vesicles called autophagosomes and delivered into the lysosome/vacuole where they are destroyed. Cells are able to modulate autophagy in response to their needs, and under certain circumstances, cargoes, such as aberrant protein aggregates, organelles, and bacteria can be selectively and exclusively incorporated into autophagosomes. As a result, this pathway plays an active role in many physiological processes, and it is induced in numerous pathological situations because of its ability to rapidly eliminate unwanted structures. Despite the advances in understanding the functions of autophagy and the identification of several factors, named Atg proteins that mediate it, the mechanism that leads to autophagosome formation is still a mystery. A major challenge in unveiling this process arises from the fact that the origin and the transport mode of the lipids employed to compose these structures is unknown. This compendium will review and analyze the current data about the possible membrane source(s) with a particular emphasis on the yeast Saccharomyces cerevisiae, the leading model organism for the study of autophagosome biogenesis, and on mammalian cells. The information acquired investigating the pathogens that subvert autophagy in order to replicate in the host cells will also be discussed because it could provide important hints for solving this mystery.

Animals↗

PubMed2000

Heat-induced gelation of globular proteins: part 3. Molecular studies on low pH beta-lactoglobulin gels.

Heat-set gels and aggregates from beta-lactoglobulin (beta-Lg), one of the major globular proteins from milk, have been studied on a molecular distance scale using negative-staining transmission electron microscopy (TEM), wide-angle X-ray diffraction (WAXD), and Fourier transform infrared spectroscopy (FTIR). The microscopy showed long linear aggregates forming in solutions at pH 2 (and sometimes 2.5) after prolonged heating. While there appeared to be no differences in aggregates formed under these conditions in H(2)O as compared with D(2)O, at all other pH and pD values, and in the presence of added salt, much shorter linear aggregates were formed. These became slightly more extended the further the pH was removed from pI. Wide-angle X-ray diffraction (WAXD) showed a diffuse beta-sheet halo at 2θ=19 degrees in patterns for both dried native and aggregated protein (irrespective of pH) with only a small change (sharpening) of this feature on heat treatment. Solution FTIR spectra, measured at pD=2, 2.5, 3, and 7, during heating, indicated shoulder development at 1612 cm(-1) in the carbonyl-stretching Amide I region diagnostic of a modest increase in intermolecular beta-sheet. In terms of the shoulder size, no distinctions could be made between acid and neutral aggregate structures. At all pHs, beta-lactoglobulin showed only limited secondary and tertiary structural changes in aggregation, in contrast to previous studies of insulin aggregation, where highly ordered crystalline fibrils were indicated. The current work has implications both in structural studies of food biopolymers and in ongoing studies of pathological protein self-assembly in disease states, such as spongiform encephalopathies.

Animals↗

PubMed2000

PABP2 polyalanine tract expansion causes intranuclear inclusions in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy is caused by expansion of a (GCG)n trinucleotide repeat in the poly(A) binding protein 2 (PABP2) gene. The pathological hallmark of oculopharyngeal muscular dystrophy is the accumulation of intranuclear inclusions in muscle fibers. To test whether the polyalanine expansion of PABP2 directly leads to the formation of the nuclear aggregates, both normal and expanded PABP2 cDNAs were expressed in COS-7 cells. We find that expression of mutated PABP2 protein is sufficient for its accumulation as intranuclear inclusions.

Humans↗

PubMedDate not supplied

Abeta deposition does not cause the aggregation of endogenous tau in transgenic mice.

In Alzheimer disease, the extracellular deposition of beta-amyloid (Abeta) in the brain is accompanied by the intracellular accumulation of aggregated forms of hyperphosphorylated tau. In developing animal models of AD, the authors and others have been able to reproduce extracellular amyloid pathology in the brains of mice by expressing mutant amyloid precursor proteins (APP). The co-expression of APP with mutant presenilin leads to a dramatic acceleration in Abeta deposition, leading to very high amyloid burdens in mice. In the current study, the authors have examined whether the brains of mice with high burdens of amyloid deposition also contain aggregated forms of tau, using a cellulose acetate filter trap assay. Although discrete accumulations of phosphorylated tau immunoreactivity were apparent in neurites proximal to cored deposits of Abeta, little if any of this tau was in a SDS-resistant state of aggregation. By contrast, the brains of AD patients contained large amounts of aggregated tau. Overall, this study demonstrates that, in mice, deposition of Abeta does not cause endogenous tau to aggregate.

Alzheimer Disease↗

PubMed2003

Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death.

Converging evidence suggests that the accumulation of cerebral amyloid beta-protein (Abeta) in Alzheimer's disease (AD) reflects an imbalance between the production and degradation of this self-aggregating peptide. Upregulation of proteases that degrade Abeta thus represents a novel therapeutic approach to lowering steady-state Abeta levels, but the consequences of sustained upregulation in vivo have not been studied. Here we show that transgenic overexpression of insulin-degrading enzyme (IDE) or neprilysin (NEP) in neurons significantly reduces brain Abeta levels, retards or completely prevents amyloid plaque formation and its associated cytopathology, and rescues the premature lethality present in amyloid precursor protein (APP) transgenic mice. Our findings demonstrate that chronic upregulation of Abeta-degrading proteases represents an efficacious therapeutic approach to combating Alzheimer-type pathology in vivo.

Amyloid beta-Peptides↗

PubMed1994

Correlative light and electron microscopy studies of PrP localisation in 87V scrapie.

The transmissible neurodegenerative diseases, of which scrapie is the archetype, are caused by unconventional infectious agents. Prion protein (PrP), a widespread host coded, cell surface sialoglycoprotein, is thought to be an essential or, controversially, sole component of these agents. During infection, disease specific accumulations of PrP may be observed in immunostained brain sections of mice infected with the 87V scrapie strain as amyloid plaques or as diffuse or granular foci within the neuropil. Using serial light and electron microscopical preparations we determined immunocytochemically that infection specific PrP is present in amyloid fibrils, and accumulates on the plasmalemma of neurites at the periphery of plaques and in the neuropil, irrespective of the morphological form of PrP accumulation when viewed by light microscopy. In some brain areas with dense granular PrP expression complete disruption of neuropil with loss of neurites was associated with fibrils lying free in expanded extracellular space. These results suggest that normal PrP may be converted to its pathological form at the neuronal plasmalemma or in the extracellular space and, furthermore, that amyloid fibrils are formed following the accumulation and aggregation of subunit proteins at these sites.

Animals↗

PubMed1999

Bcl-2 and Bax protein expression in human myopathies.

Expression of the apoptosis-related proteins Bcl-2 and Bax was analysed by means of immunohistochemistry in muscle biopsies from 13 patients suffering from different muscular diseases (inclusion body myositis n=4, polymyositis n=3, Becker muscular dystrophy n=4, necrotizing myopathy n=2, and controls n=4), in an attempt to learn about the role of these proteins in human muscle diseases associated with muscle fiber necrosis and regeneration. Increased Bcl-2 and Bax immunoreactivity was observed as fine granular precipitates in the cytoplasm or as subsarcolemmal aggregates in pathological cases. Increased Bcl-2 and Bax immunoreactivity was detected in some necrotic fibers, regenerating fibers, ring fibers and some apparently normal muscle fibers. In addition, increased Bcl-2 and Bax immunoreactivity was observed in the periphery of rimmed vacuoles and in muscle fibers with abnormal mitochondria in patients suffering from inclusion body myositis. Double-labelling immunohistochemistry disclosed co-localization of both proteins in about 50% of Bcl-2-immunoreactive fibers. Finally, double-labelling immunohistochemistry using N-CAM antibodies revealed that some Bax-positive fibers were regenerating fibers. Since increased Bax immunoreactivity was not restricted to necrotic muscle fibers, the present results suggest that over-expression of this protein in human myopathies is probably independent of the process of cell death.

Adolescent↗