Re: Death by garbage

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--- Stobbs <greenmantoo@...> wrote:

> Www.world-science.net/

>

> See item at top of list on the left hand side...

>

Hi All,

See also the below, which is pdf-available for the Results section that is not

below.

Stroikin Y, Dalen H, Brunk UT, Terman A.

Testing the " garbage " accumulation theory of ageing: mitotic activity protects

cells

from death induced by inhibition of autophagy.

Biogerontology. 2005;6(1):39-47.

PMID: 15834662

Imperfect autophagic degradation of oxidatively damaged macromolecules and

organelles (so-called biological " garbage " ) is considered an important

contributor

to ageing and consequent death of postmitotic (non-dividing) cells, such as

neurons

and cardiac myocytes. In contrast, proliferating cells apparently escape

senescence

by a continuous dilution and repair of damaged structures during division.

Postmitotic ageing can be mimicked and studied in cultures of potentially

dividing

cells if their mitotic activity is inhibited. To test the " garbage accumulation "

theory of ageing, we compared survival of density-dependent growth-arrested

(confluent) and proliferating human fibroblasts and astrocytes following

inhibition

of autophagic sequestration with 3-methyladenine (3MA). Exposure of confluent

fibroblast cultures to 3MA for two weeks resulted in a significantly increased

proportion of dying cells compared to both untreated confluent cultures and

dividing

cells with 3MA-inhibited autophagy. Similar results were obtained when

autophagic

degradation was suppressed by the protease inhibitor leupeptin. In 3MA- or

leupeptin-exposed cultures, dying cells were overloaded with undegraded

autofluorescent material. The results support a key role of biological lysosomal

" garbage " accumulation in the triggering of ageing and death of postmitotic

cells,

as well as the anti-ageing role of cell division.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve & db=pubmed & dopt=Abstra\

ct & list_uids=15834662 & query_hl=36

Introduction

Despite increasing evidence implicating macro-

molecular damage by oxygen derived free radicals

in ageing (Harman 1956; Beckman and Ames

1998; Cadenas and Davies 2000) it remains unclear

why oxidatively damaged structures accumulate in

the presence of efficient renewing mechanisms,

which are responsible for a continuous degrada-

tion and re-synthesis of cellular components. The

most plausible explanation for the known pro-

gressive accumulation of oxidatively damaged

material, mainly occurring in long-lived postmi-

totic cells such as cardiac myocytes and neurons, is

that the removal of damaged structures by recy-

cling systems, including lysosomes, proteasomes

and cytosolic proteases, is inherently imperfect

(Terman 2001; Terman and Brunk 2004). This

explanation of age-related accumulation of dam-

aged structures seems more reasonable than

emphasising a role of erroneous synthesis of bio-

molecules as was suggested in the error catastro-

phe (Orgel 1973) and somatic mutation (Burnet

1973) theories of ageing. These theories per se

could not explain many manifestations of ageing

(Kirkwood 1989). Indeed, abnormally synthesised

structures would not accumulate and cause any

harm as long as they were reasonably well

removed.

Oxidatively modi & #64257;ed structures that accumulate

within ageing postmitotic cells include aberrant

proteins and other damaged macromolecules, and

defective mitochondria, as well as lipofuscin pig-

ment being an intralysosomal undegradable

polymeric and plastic-like material (Brunk and

Terman 2002a; Terman and Brunk 2004). Because

all these structures are functionally inefficient or

completely worthless, they can well be considered

as biological ‘‘garbage ’’or ‘‘waste ’’material

Terman 2001; Terman and Brunk 2004).

Although the importance of biological ‘‘waste ’’

accumulation in the progress of ageing has been

pointed out earlier (Sheldrake 1974; Hirsch 1978),

this idea has attracted little attention, most prob-

ably due to lack of knowledge regarding the nature

of ‘‘waste ’’products and their possible harmful

effects.

The accumulation of ‘‘waste ’’material is harm-

ful not only because it occupies a certain part of

the cells ’interior, apparently interfering with

intracellular transport, signalling and metabolic

processes, but also due to its toxic effects. In par-

ticular, there is extensive evidence for the toxicity

of protein oxidation products (Verbeke et al. 2001;

Goldberg 2003; Grune et al. 2003), and it was

found that damaged mitochondria of senescent

house & #64258;ies generate increased amounts of reactive

oxygen species (Sohal and Sohal 1991). Further-

more, lipofuscin deposition within cultured cells

increased their susceptibility to oxidant-induced

apoptosis (Terman et al. 1999a), while its accu-

mulation in cultured T-lymphocytes made them

progressively intolerant to activation (Gerland

et al. 2004). Most importantly, because heavy

lipofuscin loading of lysosomes appears to sup-

press autophagy and normal turnover of biomol-

ecules (Terman et al. 1999b), the accumulation of

garbage ’’would advance with age, inevitably

leading to cell death (Brunk and Terman 2002b).

The only possibility for cells to escape ageing

seems to involve dilution of undegradable struc-

tures through continuous mitotic activity. For

example, cell proliferation efficiently prevents

ageing of cultured cancer cells (Campisi 1996), as

well as of Hydra, a primitive animal whose cells

are all continuously replaced owing to differenti-

ating stem cells (ez 1998). To provide more

evidence in support of the role of biological

garbage ’’accumulation in ageing, and the role of

cell division in preventing it, we compared cell

survival following inhibition of autophagic deg-

radation in non-dividing (con & #64258;uent) and dividing

human & #64257;broblasts and astrocytes. We found that

suppression of autophagy using the sequestration

inhibitor 3-methyladenine (3MA), which acts by hin-

dering the activity of phosphatidylinositol-3-kinase

(Blommaart et al. 1997; Petiot et al. 2000), and the

lysosomal protease inhibitor leupeptin (Ivy et al.

1984) induced senescence-like alterations and en-

hanced cell death in growth-arrested cultures, but

not in cultures of proliferating cells.

.. . . Discussion

We found that prolonged inhibition of autophagy

(either at the sequestration step using 3MA or at

the stage of lysosomal degradation using leupep-

tin) dramatically increases death of non-dividing

(con & #64258;uent) cells and that this does not occur in

sparse cultures where cells divide. The role of

growth arrest in such effect of prolonged sup-

pression of autophagy is supported by reestab-

lishment of normal cell viability following release

from con & #64258;uency.

Increased cell death following inhibition of

autophagy, a major mechanism involved in the

removal of damaged cellular constituents, is con-

sistent with the idea that the advanced ‘‘garbage ’’

accumulation that progressively occurs within

postmitotic cells is an important determinant of

ageing and age-related cell death (Sheldrake 1974;

Terman 2001; Terman and Brunk 2004). More-

over, this hypothesis is supported by earlier & #64257;nd-

ings of progressive death of 3MA-exposed

neonatal postmitotic rat cardiac myocytes in cul-

ture (Terman et al. 2003). Altogether, our results

indicate that biological ‘‘garbage’’ is, indeed, del-

eterious for cells and & #64257;nally kills them if accumu-

lates in large enough quantities. Both normally

ageing cells (in which autophagy is inherently

imperfect) and those with pharmacologically

inhibited autophagy accumulate ‘‘waste ’’material

of a similar nature, including damaged mitochon-

dria and intralysosomal undegraded material. In

contrast to normal ageing, the accumulation of

‘‘garbage ’’following pharmacological inhibition

of autophagy occurs much faster and results in

dramatically increased cell death (compare 3MA-

or leupeptin-exposed con & #64258;uent cells with untreated

ones in Figure 2a –d).

There are good reasons to believe that death of

cells that are continually exposed to 3MA or leu-

peptin primarily occurs because of accumulation

of material excluded from autophagic degradation

and not because of other possible effects of the

drugs. First, the accumulation of undegraded

material is consistent with the effects of 3MA and

leupeptin as inhibitors of autophagy. Second, as

shown earlier (Terman et al. 2003), only a pro-

longed inhibition of autophagy substantially

decreased cell survival, the & #64257;nding being consistent

with gradual ‘‘garbage ’’accumulation, probably

& #64257;nally reaching a critical level incompatible with

maintenance of life. Third, as is evident from the

electron micrographs (Figure 4c, d) and & #64258;uores-

cence images (Figure 5d –f), dying cells, but not

surviving ones, were extremely overloaded with

undegraded material. Finally, and most impor-

tantly, cell division (con & #64257;rmed by the BrdU

incorporation assay, Figure 3) prevented cell

death induced by inhibition of autophagy in all

in vitro models tested. Cell division, especially

when occurring repetitively, obviously diminishes

the content of ‘‘waste ’’material by distributing it

between the daughter cells (Sheldrake 1974; Ter-

man 2001; Terman and Brunk 2004). From this

point of view, the rescue of cells by inducing

mitotic activity is a distinguishing feature of cell

death relevant to ‘‘garbage ’’accumulation. In this

special case, mitosis promotes cell survival despite

increased demands of dividing cells for ATP and

anabolic processes, which, theoretically, would

reduce adaptability.

Since cell proliferation is known to enhance

DNA damage (Ames and Gold 1990), the pre-

vention of cell death by mitotic activity is unlikely

to occur through effects on DNA damage or

repair, well-known determinants of the ageing

process (Campisi 2000). Our results, thus, support

the idea that not only DNA damage, but also the

accumulation of waste material per se is responsi-

ble for cellular aging. The accumulation of

lipofuscin and damaged mitochondria, however,

can indirectly lead to the enhancement of oxidative

stress and ensuing DNA damage (Brunk and

Terman 2002b).

The mechanisms of cell death caused by bio-

logical ‘‘garbage ’’accumulation are not com-

pletely understood. As discussed above, both

interference with the intracellular transport

mechanisms and enhancement of toxic effects,

are apparently involved in fatal disturbances of

normal cellular functions. As follows from

Figures 1, 2, 4 and 5, cells die either through

apoptosis or necrosis, although this distinction is

not always clear, as is the case for other diseased

conditions (Leist and Ja ¨a ¨ttela ¨2001). For exam-

ple, cells in Figure 4c, d show apoptotic-like

chromatin condensation and nuclear fragmenta-

tion, as well as necrotic-like cytoplasmic swelling.

Despite increased numbers of dense vacuoles

within dying cells, 3MA-or leupeptin-induced

cell death is not autophagic (Lockshin and

Zakeri 2004), since the process of autophagy is

suppressed.

Lipofuscin-loaded & #64257;broblasts were characterised

by less pronounced decrease of viability following

3MA exposure, as well as by less pronounced

protective effect of sub-cultivation compared to

cells with low lipofuscin content (Figures 1g –i and

2c). The observed lowered efficiency of 3MA in

inducing death of lipofuscin-loaded cells was

probably related to the fact that autophagy was

already partially inhibited by accumulated lipo-

fuscin (Terman et al. 1999b), and the remaining

autophagic capacity (which still could be inhibited

by 3MA) was thus relatively small. A decreased

proliferative potential of lipofuscin-rich & #64257;broblasts

(Figure 3) apparently explains why the otherwise

protective effect of sub-cultivation was weaker for

these cells than for & #64257;broblasts that were not loaded

with lipofuscin.

Overall, our results support the ‘‘garbage ’’

accumulation theory of ageing, which states that

due to inherent insufficiency of degradative pro-

cesses, postmitotic cells progressively accumulate

‘‘waste ’’products, such as damaged macromole-

cules, organelles and lipofuscin pigment. Progres-

sive accumulation of such biological ‘‘garbage ’’

seems to upset normal cellular functions, resulting

in decreased adaptability and & #64257;nally in cell death.

Cell division is apparently a natural anti-ageing

mechanism that, by simple dilution, effectively

decreases the content of biological ‘‘garbage ’’.

In long-lived species, such as humans, the

accumulation of biological ‘‘garbage’’ within

postmitotic cells occurs relatively slowly, appar-

ently due to efficient autophagy, proteasome

activity, DNA repair and other renewal mecha-

nisms, allowing some of these cells to survive

several decades. It would be attractive to extend

life of vitally important postmitotic cells, such as

cardiac myocytes and neurons, by stimulating

degradative processes and, thus, decreasing the

accumulation of ‘‘waste ’’material. The idea of

slowing ageing through enhancement of intracel-

lular degradation is consistent with the & #64257;ndings

that autophagy is up-regulated in calorie restricted

animals, known to have extended lifespans

(Bergamini et al. 2003), as well as in the long-lived

daf-2 C. elegans mutants (Melendez et al. 2003). In

addition, the anti-aging hormetic effect of repeated

mild heat shock on human & #64257;broblasts has been

found to be associated with increased proteasome

activity (Fonager et al. 2002). A more radical and

challenging anti-ageing strategy might involve

removal of already formed biological ‘‘garbage’’,

for instance, by transfection of cells with genes

coding for xenohydrolases, i. e. bacterial and fun-

gal enzymes capable of degrading lipofuscin

(de Grey 2002).

Al Pater, PhD; email: old542000@...

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