Assessment of Cell Death: Apoptosis, Necrosis, or In Between - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 22

Induction and Cleavage
of Poly(ADP-ribose)
Polymerase-1

The poly(ADP-ribose) polymerase (PARP)-1 family of ADP-
ribosyl-transferases are nuclear-localized enzymes that initiate the
repair of DNA strand breaks resulting from normal cellular stress
in virtually all eukaryotic cells. PARP-1 is a caspase-cleavage target
during apoptotic cell death, but is also involved in two caspase-
independent pathways of cell death. In one case, excessive alkylation
of DNA can lead to overactivation of PARP-1, resulting in rapid
depletion of NAD + (a necessary PARP co-factor), a halt in cellular
production of ATP, and the collapse of anabolic processes with
necrotic degeneration. In the second case, widespread DNA damage
(resulting from, for example, excitoxicity-mediated reactive oxygen
species) induces a translocation of PARP-1 to the cytoplasm where,
via a TRAF2/RIP1 pathway, causes AIF to translocate from the
inner mitochondrial membrane to the nucleus, inducing localized
DNA fragmentation, and localized nuclear condensation (as distin-
guished from apoptotic/chromatin-wide DNA fragmentation and
nuclear condensation) and death. This pathway appears to have
considerable overlap with or is identical to the so-called caspase-
independent PCD cell death pathway.
PARP-1 accounts for approximately 85% of cellular PARP
activity, and this member of the PARP family is targeted and pro-
teolytically cleaved not only by caspase-3 but by other proteases
such as calpains, cathepsins, and granzyme b in a variety of pathophy-
siological and cell death contexts. The subsequent cleavage pattern
of substrates such as PARP-1 can provide key circumstantial
evidence as to the identity of the responsible protease and, therefore,
of the particular cell death pathway involved.
Method . Cleavage of PARP-1 by caspase-3 will produce signature
fragments of 89 kD and 21 kD on a western blot, while necrotic
cell death involving cathepsin b activity will yield a 50-kD PARP-1
cleavage product. Following induction by genotoxic agents, PARPs
will target nuclear proteins, such as histones, and, using NAD + as a
substrate, covalently attach branched polymers (often hundreds)
of ADP-ribose moieties. There are a number of methods, many
commercially available, that can detect an increase in PARP-1 activity
and several employ specifi c anti-poly(ADP-ribose) primary antibodies.
These include target protein size-distribution analysis, immunological
detection of modifi ed proteins by western blotting, and immuno-
cytochemical analysis of cellular localization; an excellent yet
exhaustive treatment of these methods is provided in ( 54 ). To test
whether the PARP-1-mitochondrial pathway has been activated,
a specifi c and reliable marker is the immunological/histological
detection of AIF translocation to the nucleus. Activation of this
caspase-independent cell death pathway can be verifi ed and corrobo-
rated by exploiting the physical interaction that occurs between a
fraction of nuclear-localized AIF and activated PARP-1 and performing
co-immunoprecipitations or pull-downs ( 55 ).