747
Comments, Opinions, and Reviews
Suppression of Protein Synthesis in
the Reperfused Brain
Gary S. Krause, MD, and Brian R. Tiffany, MD
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Background: Brain ischemia and reperfusion produce profound protein synthesis alterations, the extent
and persistence of which are dependent on the nature of the ischemia, the brain region, the cell layer
within a region, and the particular proteins studied. After transient ischemia, most brain regions recover
their protein synthesis capability; however, recovery in the selectively vulnerable areas is poor. It is
unknown whether this phenomenon itself provokes or is a consequence of the process of neuronal death.
Summary of Review: Protein synthesis suppression during ischemia is due to energy depletion, but this
is quickly reversed upon recirculation. Reperfusion does not appear to damage DNA or transcription
mechanisms, although there are changes in the profile of transcripts being made. Similarly, purified
ribosomes isolated from reperfused brains can make the normal repertoire of proteins and heat-shock
proteins. However, during early reperfusion, newly synthesized messenger RNAs appear to accumulate in
the nucleus; this alteration in RNA handling could reflect disruption at any of several steps, including
posttranscriptional processing, nuclear pore transport, cytoskeletal binding, or formation of the translation initiation complex. Another mechanism that may be responsible for protein synthesis suppression
during late reperfusion is progressive membrane destruction, with consequent shifts in the concentration
of ions crucial for ribosomal function.
Conclusions: Protein synthesis suppression after ischemia likely involves a progression of multiple
mechanisms during reperfusion. Although the recent work reviewed here offers new insight into the
potential mechanisms disrupting protein synthesis, detailed understanding will require further investigation. (Stroke 1993;24:747-756)
KEY WoRDs * cerebral ischemia * proteins * reperfusion
rotein synthesis can be regarded as an essential
prerequisite for long-term survival, especially
after insults such as tissue ischemia and reperfusion. Proteins have diverse roles in the cell, including
enzymatic reactions governing anabolism, catabolism,
transport, and storage, as well as intercellular signaling
and cellular structure. Therefore, significant changes in
the cell's ability to produce and maintain proteins would
be expected to have considerable effect on the cell's
capacity to survive. Depression of protein production
has long been noted to occur in the brain after ischemia
and reperfusion. It is not known whether this phenomenon is a contributing factor to neuronal death or just a
component of the process of cell death. We will review
data suggesting that the suppression of protein synthesis
during ischemia and reperfusion is a result of three
different processes: 1) during the ischemic phase, the
lack of adenosine triphosphate (ATP) and guanosine
triphosphate (GTP) precludes protein synthesis; 2) durP
From the Department of Emergency Medicine, Wayne State
University School of Medicine, Detroit, Mich.
B.R.T. is supported in part by a research fellowship grant from
the Emergency Medicine Foundation and the Eli Lilly Corporation. G.S.K. is supported in part by United States Public Health
Service grant NS-24819.
Address for correspondence: Gary S. Krause, MD, Department
of Emergency Medicine, Wayne State University, 4201 St. Antoine, Detroit, MI 48201.
Received June 26, 1992; final revision received January 25, 1993;
accepted January 27, 1993.
See Editorial Comment, page 755
ing the early reperfusion phase, there is a block in the
initiation of translation; and 3) during late reperfusion,
lipid peroxidation has destroyed the cell membrane to
the extent that it can no longer maintain the appropriate
intracellular ionic milieu needed for ribosome function.
Postischemic suppression of protein synthesis was first
reported in 1971 by Kleihues and Hossman.' Cats were
given `Glabeled amino acids intravenously during reperfusion and the protein extracted from brain homogenates
30 minutes later. A 30% decrease in label incorporation
was observed after 4 hours of reperfusion following 30
minutes of ischemia. These investigators subsequently
made qualitative observations on autoradiographs that
most brain cells survived the ischemic insult and were
synthesizing protein, with the exception of a few cortical
areas and the dentate gyrus that failed to incorporate
label.2 Other investigators have expanded on these findings and have demonstrated that the postischemic suppression of brain protein synthesis is severe and prolonged. Cooper et al measured [14C]phenylalanine
incorporation by in vitro translation using the postmitochondrial supernatant from rat brain homogenates.3 Incorporation was normal in the homogenates derived
from rats after 15 minutes of ischemia but had fallen 80%
after 15 minutes of reperfusion. They concluded that the
reduction of protein synthesis was a consequence of
reperfusion. Nowak et a14 found that in vitro amino acid
incorporation by the postmitochondrial fraction pre-
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pared from gerbil brain homogenates was normal after 30
minutes of ischemia without reperfusion. However, after
10 minutes of recirculation, protein synthesis had decreased 90%; it had recovered to 60% of baseline by 6
hours. Moreover, they showed that as little as 3 minutes
of ischemia triggered protein synthesis suppression during reperfusion. The degree of suppression was dependent on the duration of the ischemia for ischemic periods
of less than 5 minutes; longer ischemic times did not
result in any additional suppression. Recovery of protein
synthesis was not affected by the duration of ischemia in
this model. In contrast, Dienel et a15 found that increasing the duration of ischemia delays the recovery of in situ
protein synthesis in the surviving areas.
The brain's response to ischemia/reperfusion is not
regionally homogeneous; the cortex, hippocampus, and
caudate show severe and prolonged suppression of
protein synthesis, whereas the brain stem and midbrain
structures are relatively unscathed.5-11 Araki et al,10
using a bilateral carotid occlusion model in the gerbil,
demonstrated regional differences in L-[methyl-'4C]methionine incorporation and corroborated the existence
of an ischemic threshold for suppression of protein
synthesis. They found that 1 minute of ischemia did not
produce any reperfusion suppression of protein synthesis. However, after 2 minutes of ischemia there was a
severe but reversible suppression, and 3 minutes of
ischemia produced a severe loss of protein synthesis in
the neocortex, striatum, the whole hippocampus, and
the thalamus that slowly recovered during the following
5-24 hours; the CA1 region of the hippocampus never
recovered its ability to incorporate label. These areas
are also the ones most susceptible to delayed neuronal
death after ischemia,12 indicating that a prolonged
deficit in postischemic protein synthesis correlates with
the selective vulnerability of these areas to ischemia and
reperfusion."1
Individual proteins respond in different ways to ischemia/reperfusion. After reperfusion, immunohistochemical staining of constitutively expressed brain proteins such as tubulin, neuron-specific enolase, and
brain-specific creatine kinase show accelerated loss,
which is especially prominent in the selectively vulnerable areas.13 This presumably results from decreased
synthesis because in general, the rate of protein degradation is diminished during reperfusion.1114 Yoshimi et
al found no change in microtubule-associated protein 2
but increased clathrin immunoreactivity in the gerbil
hippocampus 3 hours after 5 minutes of ischemia.15
Other investigators have shown that brain ischemia and
reperfusion leads to increased translation of other proteins such as the proto-oncogene products c-FOS16 and
c-JUN.17 The latter two proteins are of special interest
because of their potential involvement in cellular repair
systems.18
Proteins of the c-FOS family bind to proteins of the
c-JUN family to form the AP-1 complex that binds with
high affinity and specificity to DNA promoter sequences upstream of target genes.19 AP-1-responsive
genes are thought to have important roles in the mechanisms of cellular proliferation and differentiation19 and
may play a role in membrane repair. Wessel et al,20
using in situ hybridization, studied c-fos and c-jun
transcripts in the rat brain after 20 minutes of transient
ischemia.20 There was strong expression of both genes
within 30 minutes of reperfusion in the dentate gyrus;
however, expression in the CA1 region of the hippocampus was delayed 1-2 hours and showed a biphasic
response. The initial moderate peak had returned to
baseline by 6 hours but was followed by a weaker
response
at 24 hours of reperfusion. Nowak et a121
demonstrated a strong early c-fos response in the dentate gyrus but only modest transcription in the CA1 and
CA3 regions. Uemura et al,22 using immunocytochemistry, found early c-FOS protein synthesis in gerbil
dentate gyrus, whereas there was only minimal response
in the CA1 layer. In contrast, Jorgensen et al observed
delay in both transcription (peaking at 48 hours of
reperfusion) of c-fos mRNA23 and translation of c-FOS
protein24 in the selectively vulnerable CA1 layer of the
rat hippocampus.
There may be an important relation between induction of c-fos and the translation of important stress
proteins.25 Heat-shock proteins appear to facilitate correct folding of proteins26-28 and ribosomal RNA
(rRNA)29 and have been proposed to be involved in
membrane synthesis.30 The gene for one of the major
heat-shock proteins, HSP-70, has, in addition to the
"Pelham box"26 promoter characteristic of all heatshock genes, a site that binds a promoter (serum
response element-2) in common with c fos.31 Heatshock messages3233 and proteins34-37 are expressed in
the brain during reperfusion. After an ischemic insult,
the selectively vulnerable neurons in the hippocampus
and cortex transcribe large quantities of hsp-70 messenger RNA (mRNA),32 but translation is considerably
decreased compared with surviving areas of the hippocampus (i.e., the dentate gyrus).35,37 Thus, the selectively vulnerable brain neurons with delayed expression
of c-fos correspond to those with poor translation of
hsp-70 transcripts.
Although the patterns of postischemic protein synthesis suppression in the brain are well described, the
mechanisms that cause it are not. Therefore, we will
now turn to an examination of the components of the
cellular protein transcription-translation system. Protein synthesis is a complex process that requires 1)
high-energy phosphates, 2) intact DNA, 3) intact transcription machinery, 4) processing and transport of
mRNA from the site of transcription to the site of
translation, and 5) intact translation machinery. Each of
these steps is a potential site of disruption after ischemia and reperfusion.
Because protein synthesis is an energy-requiring activity, depletion of ATP/GTP stores is responsible for its
cessation during ischemia.38 Nowak et a139 showed that the
postmitochondrial supernatant isolated from ischemic
brain would incorporate amino acids into polypeptides if a
source of high-energy phosphates were added. However,
high-energy phosphate depletion cannot account for the
prolonged suppression of protein synthesis seen during
reperfusion. Levels of ATP, phosphocreatine, and GTP,
which are essentially zero after 5 minutes of ischemia, are
normal by 15 minutes of recirculation.440 42
Brain DNA does not accrue any significant damage by
either endonucleolytic or free radical mechanisms during cardiac arrest or reperfusion. No evidence was
found of base damage or strand scission in genomic or
mitochondrial DNA after 20 minutes of ischemia followed by up to 8 hours of reperfusion.43,44Furthermore,
Krause and Tiffany Reperfusion Brain Protein Synthesis
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neither thymine glycols nor thymine dimers, known
transcription terminators formed by free radical mechanisms, were found in the same model.45
Total RNA synthesis is normal during reperfusion,46
although individual mRNAs show variability. Lindvall
et al,47 using in situ hybridization, found that brainderived neurotrophic factor and nerve growth factor
transcripts were increased in the dentate gyrus during
reperfusion, but neurotrophin-3 mRNA levels were
reduced in the dentate gyrus and in the CA1 and CA2
regions of the hippocampus. Xie et al,48 using in situ
hybridization, noted that transcription of structural
rRNA and mRNA for cytochrome c oxidase did not
show any changes, and transcripts for ,3-actin increased,
for up to 72 hours of reperfusion in the CA1 sector of
the gerbil hippocampus, although the cells were dying
by morphologic criteria. Expression of hsp-70, c-fos, and
ubiquitin mRNAs is increased after transient ischemia.2'33 Although there are changes in the relative
expression of different classes of mRNA, they do not
account for the observed changes in protein synthesis.
For example, during reperfusion, cells of the CA1 sector
of the hippocampus express increased amounts of
hsp-70 mRNA with no accumulation of HSP-70 protein.32,49 Because eukaryotic mRNA has a half-life
estimated at several hours50 and there is no evidence of
a generalized increase in mRNA degradation during
ischemia and reperfusion,3'51 this suggests a dissociation
of transcription and translation occurring at a step
beyond transcription.
There are some observations that suggest the newly
synthesized mRNA is not reaching the cytoplasm after
ischemia and reperfusion. Autoradiograms of pulselabeled RNA in gerbils reperfused after 5-minute forebrain ischemia showed accumulation of newly synthesized RNA in the nucleus, with little appearing in the
cytoplasm.52 Moreover, Matsumoto et al demonstrated
normal levels of newly synthesized mRNA in the nucleus and mitochondria during reperfusion but decreased new mRNA in the microsomal and ribosomal
fractions.46 Similarly, Maruno et al showed apparent
retention of RNA in the nucleus during reperfusion
using acridine orange staining.53 This indicates that a
block may occur in mRNA transport from nucleus to
cytoplasm, and a population of messages is being retained in the nucleus. Because the newly formed mRNA
(heterogenous nuclear RNA [hnRNA]) must undergo
posttranscriptional processing, nucleocytoplasmic transport, and delivery to the ribosomes and each of these
steps plays a role in the normal regulation of gene
expression,54 they are potential sites of disruption following ischemia and reperfusion.
Following synthesis by RNA polymerase II, the 5'
terminus of the hnRNA is capped by the formation of
7-methylguanosine (m7G), and then methylation of one
or more of the terminal riboses. Specific proteins in the
cytoplasm bind to this cap and facilitate assembly of the
translation complex composed of mRNA, ribosomes,
and initiation factor proteins.55 After 5' capping, most
but not all eukaryotic hnRNAs are terminated on the 3'
end by a polyadenylated tail [poly(A) + 100-200 nucleotides in length that increases the stability of the
message and appears to act as a signal for transport to
the cytoplasm.56
749
Once the ends of the hnRNA have been modified, it
enters the splicing pathway, where the introns (sequences of bases that are not going to be translated but
are interspersed between the coding sequences) are
removed and the transcript brought to its mature sequence and length.57-59 Intron-containing mRNAs are
tightly bound to the nuclear matrix,6061 most likely by
the splicing system, which seems to be an integral part
of matrix structure.60 Transit through the splicing pathway appears to be a prerequisite for transport of at least
some transcripts out of the nucleus; transfection of a
single complementary DNA (cDNA) gene results in
normal transcription, processing, and transport of the
messenger to the cytoplasm if and only if there is at least
one intron contained in the gene.62
Translocation of messenger RNA from nucleus to
cytoplasm can be divided into three distinct steps: 1)
release of the mature particle from the nuclear matrix
after posttranscriptional processing, 2) transport through
the pore complex, and 3) binding of the mRNA particle
to the cytoplasmic cytoskeleton. Release of mature
mRNA from the nuclear matrix requires ATP and DNA
topoisomerase II and is the rate-limiting step in mRNA
transport out of the nucleus.63 Mechanical disruption of
the nuclear membrane does not increase mRNA efflux;
immature (intron-containing) mRNA remains bound.64
There is evidence of at least two different systems to
export mRNA to the cytoplasm: one for those mRNAs
with the poly(A) + tail and another for those mRNAs
without the polyadenylated tail.
Poly(A) + mRNA departs the nucleus through the
nuclear pore,65 the site of most molecular transport
across the nuclear envelope.f5 Nuclear pores contain an
aqueous channel with a 10-nm functional diameter,
which allows free diffusion of molecules under 5 kd67;
larger molecules, such as ribonucleoproteins, must undergo active transport by these channels.68 The nuclear
lamina is a network of intermediate filament proteins
tightly associated with the inner nuclear membrane that
provides the structural framework on which the nuclear
pores are organized.69 Its presence is important to nuclear pore function since antibodies against lamin B
inhibit mRNA efflux from intact nuclei.70 Transport of
poly(A)+ mRNA requires hydrolysis of ATP or GTP71
and is mediated by a nucleoside triphosphatase (NTPase)72 that is associated with the inner nuclear membrane
and the nuclear lamina.70'73 The NTPase is stimulated in
situ by poly(A) + mRNA but not by poly(A) mRNA,74'75 an effect that is mediated by a pore-associated poly(A) + binding protein.54,76 Protein kinase and
protein phosphatase activities are closely associated with
the nuclear pore complex and appear to play a regulatory
role.76'77 Phosphorylation of the binding protein enhances its affinity for poly(A)+ mRNA.78 Phorbol esters,79 epidermal growth factor,80 insulin,8' and a number
of cytosolic proteins77 influence the rate of mRNA efflux
and appear to exert their effect via phosphorylation/
dephosphorylation of the carrier protein.
The issue of poly(A)- mRNA transport may be an
important one since the brain expresses the largest
number of poly(A)- mRNAs of any tissue (-50%).82
Poly(A) - mRNA transport is not influenced by some of
the regulatory mechanisms that govern poly(A) + transport.80 For example, poly(A) - does not stimulate the
NTPase involved in transport, and the presence of ATP
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is not required for poly(A)- mRNA efflux.83 A highly
conserved palindromic sequence on the 3' end of
poly(A)- transcripts appears to be necessary for their
transport.84 A stem-loop structure formed by the palindromic sequence may serve as a transport signal analogous to the poly(A) tract of poly(A)+ mRNA. Schroder et a185 showed that although duplex fragments of
poly(A)- mRNA were rapidly transported out of resealed nuclear envelope vesicles, single-stranded
poly(A)- fragments were not transported, and duplex
fragment efflux was inhibited by an RNA helicase
inhibitor. Although it is conceivable that poly(A)mRNA could be transported out of the nucleus by a
mechanism other than nuclear pores, no such system
has yet been demonstrated.
After translocation across the nuclear membrane, the
mRNA must be transported to the ribosomes for translation. Most cytoplasmic mRNA remains attached to the
cytoskeleton,86,87 and considerable evidence suggests that
translation may be dependent on this association; nearly
all of the mRNA actively engaged in protein synthesis is
associated with the cytoskeleton.88 90 Cytochalasin D,
which binds to actin and causes disruption of the microfilament network, releases poly(A)+ mRNA from the
cytoskeleton in a dose-dependent fashion and inhibits
protein synthesis.87 Additionally, initiation of viral protein synthesis and inhibition of host protein synthesis
coincides with attachment of viral mRNA to the cytoskeleton.91-94 Association with the cytoskeleton is not sufficient for translation; some cytoskeletal-bound mRNAs
are not translated.95
Blobel96 has proposed a "gene gating" hypothesis
suggesting that mRNA may in fact be transported in a
directed fashion along defined intranuclear "tracks"
that extend from specific genes toward a single or small
subset of nuclear pores. This view is supported by the
known asymmetrical distribution of the components of
the splicing system in the nucleus97 and the report by
Lawrence et a198 of strikingly localized, curvilinear
tracks of specific transcripts observed using in situ
hybridization. This hypothesis could also be used to
explain the observed subcellular localization of specific
mRNA messages.99-101 Thus, the mRNA transport system can be thought of as an "assembly line" that carries
newly minted hnRNA from the site of transcription
through the posttranscriptional processing system to
specific sites in the cytoplasm, where it is translated.
Dysfunction of either processing of hnRNA or any
aspect of the RNA transport systems could explain the
apparent gross retention of mRNA within the nucleus
and the decreased synthesis of some populations of
proteins following an ischemic insult. For example,
when Drosophila cells are subjected to hyperthermiainduced heat shock, there is a transient, generalized
block of the splicing system that persists for hours after
the insult,102 and unspliced messages accumulate in the
nucleus.103 Non-intron-containing transcripts are not
affected because they do not require splicing, but there
is some evidence suggesting that translation of these
transcripts is dependent on the sequence of the 5'
leader.104,105 In addition, hyperthermia-induced heat
shock causes collapse of the cytoskeleton,106,107 and
heat-shock protein synthesis is necessary for reformation of the cytoskeleton.'08 Disruption and reformation
of polyribosomes was coincident with the changes in the
cytoskeletal organization.109 It remains to be determined whether the heat-shock-like response that follows ischemia and reperfusion parallels the hyperthermia-induced heat-shock case.
Translation of mRNA into protein requires the presence of functional ribosomal subunits, translation factors, aminoacyl-transfer RNAs (tRNAs), amino acids,
an energy supply, and an appropriate ionic environment. Ischemia and reperfusion appears to significantly
alter some but not all of these components. For example, ischemia does not alter the intracellular levels of
amino acids.1"0 Levels of aminoacyl-tRNAs, required
for chain elongation, fall to no more than 64% of
control values during reperfusion,7 and the energy
charge of the cell rapidly normalizes upon return of
circulation. Ribosomes can tolerate prolonged periods
of ischemia without apparent functional impairment.
Nowak et a139 demonstrated stable in vitro incorporation of amino acids by brain polysomes after 1 hour of
decapitation ischemia. Marotta et all" reported that
human brain ribosomes obtained 2-6 hours postmortem could synthesize proteins in vitro. Dienel et a136
found that polysomes isolated from rat brains subjected
to 30 minutes of ischemia and 3 hours of reperfusion
qualitatively produced the same protein patterns, with
the addition of heat-shock proteins, on two-dimensional
polyacrylamide gel electrophoresis gels as did polysomes isolated from nonischemic rat brains. Our own
work1"2 demonstrated that the rate of in vitro translation by purified ribosomes was not inhibited after 20
minutes of cardiac arrest or by 2 or 8 hours of reperfusion, a time when protein synthesis is suppressed in vivo.
Although the above evidence argues against significant
damage to the ribosomes themselves, it is possible to
advance a parsimonious alternate explanation for both
the suppression of protein synthesis during reperfusion
and the apparent retention of mRNA in the nucleus.
Several lines of evidence are consistent with the hypothesis that protein synthesis may be suppressed by an
initiation inhibitor during early reperfusion, thereby deranging the "assembly line." Ribosomal sedimentation
profiles obtained from reperfused brains show a preponderance of ribosomal subunits, suggesting a block in the
formation of the initiation complex.3,36,38 In the study by
Cooper et al,3 using an in vitro translation system, the
addition of a chain initiation inhibitor (polyinosinic acid)
decreased protein synthesis 63% in control animals, as
expected. However, polyinosinic acid had no effect on in
vitro translation with the postmitochondrial supernatant
obtained after 15 minutes' reperfusion, indicating that
chain initiation was already maximally inhibited. By 45
minutes of reperfusion, this block in chain initiation was
gone. Several other investigators"13-116 have reported the
presence of a translation inhibitor in a wide variety of
models. The presence of such an inhibitor of initiation of
protein translation in the reperfused brain could cause
the accumulation of mRNAs in the nucleus as a result of
a backup in the normal flow of mRNA from the nucleus
to the cytoplasm.
The assembly of the initiation complex (Figure 1) is
an intricate process involving over 140 proteins and
calling for at least nine initiation factors in the eukaryotic cell (eIFs).117 Formation of the initiation complex
requires free 40S ribosomal subunits; however, under
normal
physiological conditions,
formation of inactive
Krause and Tiffany Reperfusion Brain Protein Synthesis
I~eF-3
elF-4C
Met-tRNA,
Temary Complex
GTP
_ 1elF-2
m7G -^_AUG (An
<>G~~~~~~~~~~~~~,-,,DP
r zI
elF4A, B4, 4F
e__
AUG ,,-(A).
GTP
2B
GT
l-2
ATP
ADP
m7G
A
(A),
eIF-5
4
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SeIF4D
*
m7G
elF-2-GDP
#U(A),
80s Iniiation Complex
FIGURE 1. Simplified diagram of the assembly of the 80S
initiation complex (see Reference 117).
80S ribosomes is favored. Binding of eIF-6 to the 60S
subunit and of eIF-3 plus eIF-4C to the 40S subunit act
to keep the subunits disassociated. Next, eIF-2 joins
GTP and the tRNA carrying the initial amino acid
(methionine in eukaryotic cells) into a ternary complex
(eIF-2 * GTP * Met-tRNAi), which then binds to the free
40S subunit. The mRNA to be translated then binds to
eIF-4F, which recognizes the m7G cap at the 5' end of
the mRNA. The eIF-4A, eIF-4B, and eIF-4F act in
concert to unwind the mRNA near the cap structure.
This is followed by the binding of the mRNA. eIF4A eIF-4B * eIF-4F complex to the 40S ternary complex. This complex migrates along the mRNA in the 5'
to 3' direction to locate the AUG start codon. Once the
appropriate match is made, eIF-5 triggers the hydrolysis
of the bound GTP molecule and the release of eIF2* GDP. The eIF-4D then brings about a conformational change such that the 60S subunit joins the 40S
subunit to complete the 80S initiation complex.
Translation is regulated at the initiation stage by
modulating the degree of phosphorylation of the initiation factors required for the formation of the ternary
complex and the recognition of mRNAs."18,"'9 The eIF-2
can be phosphorylated at Ser-51, and this causes a
generalized repression of protein synthesis. The eIF2. GDP complex, released after the start codon has
been located, binds to the recycling protein eIF-2B.
However, if eIF-2 is phosphorylated, the recycling complex cannot exchange the GDP for GTP. A 30% phosphorylation of eIF-2 is sufficient to severely inhibit all
protein synthesis. The extent of phosphorylation of
eIF-2
is
kinases
determined
by
(heme-regulated
at least two highly specific
inhibitor
[HRI]
and double-
751
stranded RNA-activated inhibitor [DRI]) and a phosphatase with broad specificity (type 2A). HRI activity is
stimulated by the phosphorylated form of HSP-90120 as
well as heavy metals (e.g., iron),12' polyunsaturated
fatty acids (e.g., arachidonate),122 and perhaps lipoperoxides.123 This pathway may provide a causal link between radical-mediated iron delocalization and lipid
peroxidation observed only during reperfusion and the
reperfusion-dependent suppression of protein synthesis. Insulin, which has been observed to salvage neurons
during postischemic reperfusion, decreases eIF-2 phosphorylation by an unknown mechanism.124
The cell appears to regulate which mRNAs are
translated by controlling their binding to eIF-4F,125
which is phosphorylated at multiple sites, with increased phosphorylation corresponding to increased
translation.126 Dephosphorylation of eIF-4F, such as
occurs with heat shock,127 inhibits translation. The
availability of eIF-4F appears to be limited in cells;
thus, by increasing the activity of eIF-4F, mRNAs that
compete poorly for the mRNA-specific initiation factors, such as proto-oncogenes, are better translated.128
Finally, during the later reperfusion period, there are
other mechanisms that could be responsible for continued suppression of protein synthesis. Substantial shifts in
potassium, magnesium, and calcium occur that could
effect translation. Ribosome activity is very sensitive to
concentrations of potassium and magnesium,129"130 both
of which are important for assembly of the ribosomal
subunits.13' Decreased Mg2+ concentrations result in
disaggregation of polysomes, increased missense errors,
and decreased protein elongation.130 Electron micrographs taken of the brain during reperfusion show evidence of disaggregation,' as do ribosome sedimentation
profiles.3,36,38 Normal pH132 and tissue concentrations
of Na+, K' and Ca 2+ are recovered early in reperfusion.133-35 However, a secondary loss of ionic homeostasis is seen by 8 hours of reperfusion following a 15minute cardiac arrest in dogs with concomitant loss of
30% of the lipid double-bond content and ultrastructural
evidence of large gaps in the neuron plasmalemma.136
The morphological progression of injury during reperfusion136'137 led to the hypothesis'38 that accelerated structural damage is a consequence of excessive generation of
oxygen radicals followed by lipid peroxidation.
Lipid peroxidation is a set of radical-mediated chemical reactions whereby the double bonds in unsaturated
fatty acid side chains are rearranged,139 altering the
composition, fluidity, and integrity of the membrane.140
The nature of the initiating species after brain ischemia
and reperfusion remains unclear despite intensive investigation,14'-'43 although it is well established that the
presence of a transition metal is required as a catalyst.
Iron is undoubtedly the biologically relevant transition
metal catalyst.144 The brain has abundant stores of oxidized (ferric) iron,145'146 especially in the selectively vulnerable areas.147 Most iron is in ferritin and transferrin,148 forms in which the iron is unable to act as a catalyst
for oxygen radical reactions. However, superoxide promotes reduction and release of (ferrous) iron from
ferritin,149 a reaction possible only during reperfusion.150
Membrane lipids are extensively peroxidized by irondependent radical reactions during reperfusion.147'5'-156
Sakamoto et al'55 demonstrated an increase in the in vitro
formation of N-tert-butyl-a-phenyl-nitrone (PBN) spin
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adducts during reperfusion after 10 or 20 minutes of
carotid occlusion in rats and interpreted this as representing a burst of free radical production. They found the PBN
adduct peaked at 5 minutes of reperfusion, and the peak
increased with increasing ischemic time. Furthermore,
they noted that products of lipid peroxidation were increased as early as 30 minutes after 10 minutes of ischemia, and the degree of lipid peroxidation increased
with ischemic time. Bromont et al154 found brain lipid
peroxide levels were selectively increased in the hippocampus, striatum, and cortex between 8 and 72 hours of
reperfusion after 30 minutes of four-vessel occlusion in
the rat. In our work, virtually all of the brain's iron could
be found in low-molecular-weight forms by 2 hours (the
earliest point examined) after a 15-minute cardiac
arrest in dogs.151 By 8 hours of reperfusion, there was
loss of 30% of the lipid double-bond content and
ultrastructural evidence of large gaps in the neuron
plasmalemma.136 At this point, the neurons were no
longer able to maintain ionic homeostasis.135
These biochemical effects can be observed directly
with the use of a histochemical stain. We have recently
used a rat model of 10-minute cardiac arrest and
resuscitation157 to conduct studies of cellular localization of reperfusion-induced lipid peroxidation.158 By 90
minutes after resuscitation, reperfusion-induced fluorescent products of the reaction between thiobarbituric
acid and lipid aldehydes formed by lipid peroxidation159
are found in the pyramidal neurons in the infragranular
layers of the cerebral cortex, including layer 5 and upper
layer 6, and in the pyramidal layer of Ammon's horn in
the hippocampus. Our observations are consistent with
the chemical observations of lipid peroxidation during
reperfusion and verify the study of Bromont et al154
showing that the selectively vulnerable neurons are
damaged specifically by lipid peroxidation during early
reperfusion. Specific vulnerability of these neurons to
damage by lipid peroxidation may be aggravated by
their deficiency in glutathione peroxidase,160 an enzyme
that detoxifies hydrogen peroxide and lipid hydroperoxides. As noted above, reactions that accompany lipid
peroxidation may be involved in early inhibition of
translation initiation and also lead to the later ionic
shifts by membrane destruction. This theoretical approach has the potential to consolidate the phenomena
of reperfusion radical reactions and suppression of
protein synthesis and requires further investigation.
Summary
We suggest that postischemic suppression of protein
synthesis in the brain has different etiologies depending
on the time examined. During ischemia, protein synthesis suppression is due to the lack of high-energy phosphates needed to support the energy-requiring synthetic
process. Although the high-energy phosphate levels
quickly return to normal during early reperfusion, initiation of radical-mediated iron delocalization and lipid
peroxidation at this time can lead to inhibition of the
initiation complex formation for translation, which is
reflected in a backup of mRNA handling and nuclear
retention of newly synthesized mRNA. Later in the
reperfusion period, lipid peroxidation eventually destroys the cell's membrane, and at this time it is likely
that protein synthesis is halted because the cell can no
longer maintain the requisite ionic milieu. There are
several other potential etiologies for the suppression of
protein synthesis, including 1) failure of or HSP-mediated inhibition of the splicing system, 2) failure of
transport at the nuclear pore, 3) degradation of the
cytoskeleton, and 4) inhibition of translation initiation
complex formation by as-yet-unknown mechanisms.
Further investigation can determine the contributions
of each of these possibilities to the suppression of
protein synthesis in the reperfused brain.
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Editorial Comment
The review by Krause and Tiffany provides a provocative summary of basic mechanisms that may be involved in protein synthesis deficits observed in brain
after ischemia. The following comments are offered to
provide further perspective, addressing first a general
question regarding the relevance of protein synthesis
deficits to postischemic pathology and then focusing on
specific issues that arise with respect to RNA processing, which constitutes the particular focus of these
authors.
As noted, prolonged protein synthesis deficits are
characteristic of neurons subjected to ischemic insults.
This is especially clear in the gerbil model, in which the
duration of detectable protein synthesis impairment is
Suppression of protein synthesis in the reperfused brain.
G S Krause and B R Tiffany
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Stroke. 1993;24:747-755
doi: 10.1161/01.STR.24.5.747
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