NEUROLOGICAL REVIEW

Mechanisms of Neurodegenerative Disorders

Part 2: Control of Cell Death
Benjamin Wolozin, MD, PhD; Christian Behl, PhD

R
ecent research into mechanisms of neurodegeneration in Alzheimer disease (AD), Par-
kinson disease, and other neurodegenerative disorders has lead to a dramatic increase
in our understanding of the mechanisms of cell death and neuroprotection. Apoptosis
is an active form of cell death that is carried out by proteins that are designed to kill
the cell. Necrosis tends to occur as a by-product of excessive oxidative stress, which can be in-
duced by agents such as b-amyloid, or excessive calcium influx induced by agents such as gluta-
mate. The neuron also has strong homeostatic mechanisms that can delay or prevent activation of
apoptosis and necrosis. The balance between neurotoxic and neuroprotective mechanisms deter-
mines whether a neuron lives or dies.

CELL DEATH AS NATURALLY nervous system to eliminate excess neu-

OCCURRING MECHANISM IN THE
DEVELOPMENT OF THE NERVOUS
SYSTEM AND DURING
NEURODEGENERATIVE EVENTS
Much of our knowledge of cell death
comes from studies of physiological
“programmed cell death.” The term pro-
grammed cell death refers to the physi-
ological suicide program that is critical
for the development and maintenance of
healthy tissues. There is another term,
“apoptosis,” that includes programmed
cell death and refers to the process of cell
suicide under any condition when car-
ried out by a cascade of executioner pro-
teases. The studies of programmed cell
death have identified many of the key
proteins that carry out apoptosis. How-
ever, as we will discuss later, we are learn-
ing that in neurons the boundary be-
tween apoptosis and necrosis is much less
distinct.
During the development of the ner-
vous system a large amount of neurons de-
generate owing to the competition for neu-
rotrophic molecules such as the nerve
growth factor. This is a paradigm of physi-
ological neuronal apoptosis that allows the
From the Department of Pharmacology, Loyola University Medical Center,
Maywood, Ill (Dr Wolozin); and the Max-Planck-Institute of Psychiatry,
Munich, Germany (Dr Behl).
rons. The importance of apoptosis for de-
velopment of the nervous system is exem-
plified by the phenotype of a knockout
mouse lacking caspase 3, the critical effec-
tor protease in apoptosis. This mouse has
deficient apoptosis and develops a brain that
is hypertrophic because of the extra neu-
rons that were not killed off by apoptosis.
See also page 793
The mechanism by which apoptosis most
commonly occurs in the nervous system
seems to differ from that occurring in the
immune system, where mechanisms of
apoptosis were first studied. Apoptosis in
the immune system commonly occurs
through engagement of death receptors,
such as Fas. Ligand binding leads to trim-
erization that activates a death domain on
the intracellular portion. The activated
death domain forms a large complex that
ultimately engages caspases inside the cells.
In contrast, in the nervous system apopto-
sis seems to occur most commonly through
loss of trophic signaling (Figure 1). Neu-
rons deprived of growth factors become
committed to apoptosis approximately 10
hours after trophic withdrawal. Thus, neu-
ronal apoptosis is commonly initiated by
a loss of signaling rather than a gain of sig-
naling, which is more common in the im-
mune system.

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 Neurons: Inhibition by Immune Cells: Activation
Receptor Tyrosine Kinases by Death Receptors
IGF-1 TNF-RI
NGF Receptor Death
Tyrosine Receptor
Kinase Cascade of
YP Other FADD/MORT 1
YP
Pro-Cas-3 Cas-3
Caspases Caspase 8
P13K
Pro-Cas-9 Cas-9
Apoptosis Substrates:
PKB
Cyt C APAF PARP
Actin
Presenilins
BAD-P BAD Huntingtin
Gelsolin
Bcl-2 PAK2
Cyt C Others
BAD
Bcl-2
APAF Bcl-2 (or Bcl-X) Mitochondria
Figure 1. Apoptotic pathways. In neurons, the main pathway regulating
apoptosis seems to be mediated by tyrosine kinases. In this pathway, growth
factors stimulate tyrosine kinases, which stimulate PI3 kinase, which
activates protein kinase B (PKB), which phosphorylates and inactivates BAD.
Loss of growth factor stimulation increases the level of phosphorylated-BAD
(BAD-P), which inactivates Bcl-2 and leads to activation of caspase 3 and,
thence, apoptosis. Immune cells, on the other hand, have receptors that can
directly activate caspases. Hence, apoptosis in immune cells can occur
rapidly after receptor activation. IGF-1 indicates insulinlike growth factor-1;
NGF, nerve growth factor; TNF-RI, tumor necrosis factor receptor; YP,
tyrosine phosphate; P13K, phosphoinositide-3 kinase; Cyt C, cytochrome C;
APAF, apoptosis-initiating factor; PARP, polyadenosine ribose phosphatase;
and PAK2, p21-activated kinase.
CASPASES AS EXECUTORS OF CELL DEATH
Caspases are the actual enzymes that carry out apopto-
sis. While there are many particular events that occur dur-
ing apoptosis, such as DNA cleavage, nuclear collapse,
or membrane ruffling, the only event that is universal to
all forms of apoptosis is caspase activation. Unfortu-
nately, caspase activation can be a complicated process
because there are at least 9 caspases, and many of these
caspases exist as inactive zymogens that must be cleaved
for activation. The most common caspases cited in the
literature are caspases 3, 8, and 9. Caspase 8 is activated
by binding to the signaling proteins, like FADD, that in-
teract with death receptors (Figure 1). Activated caspase
8 cleaves a series of other caspases that are sequentially
activated until caspase 3, the final executioner, is acti-
vated. It is caspase 3 that seems to carry out many of the
lethal cleavages that produce apoptosis. Caspase 3 can
also be activated by another mechanism in which de-
phosphorylated BAD binds to Bcl-2, which leads to re-
lease of cytochrome C and apoptosis activating factor,
activation of caspase 3, and apoptosis.
Growth factors prevent apoptosis by a pathway that
sequentially stimulates receptor tyrosine kinases, phos-
phoinositide 3 kinase and protein kinase B (also known
as Akt, Figure 1).1 Protein kinase B phosphorylates BAD,
which is a proapoptotic Bcl-2 homologue. Loss of growth
factor stimulation leads to dephosphorylation of BAD,
which binds to Bcl-2, inducing release of cytochrome C
and activation of caspase 3.2 Thus, caspases are prote-
ases that degrade many proteins critical for cell viability
and kill the neuron.
Because caspases are the proteins that actually kill the
cell, inhibitors of caspases may be of therapeutic value to
stop disease-related apoptotic processes. Viruses have de-
veloped physiological inhibitors of apoptosis (IAP), such
as p35 and CrmA, that suppress the host cell death re-
Fas
sponse to viral infections.3 The specificity of the IAP vary
among IAPs. CrmA blocks caspase 3, while p35 exhibits a
much broader spectrum of activity and inhibits caspase 1
to 4 and 7 to 10.3 Recent evidence points out that the mecha-
nisms used by IAPs are conserved among diverse species.
Inhibitors of apoptosis might also affect the signaling of the
stress-kinase pathways mediated by nuclear factor (NF)–kB
or JNK. Peptide inhibitors of apoptosis have also been de-
veloped. The most commonly used peptide inhibitor is the
caspase 3 inhibitor DEVD. Cell permeable analogs of DEVD,
such as DEVD-fluoromethylketone, have been shown to
block apoptosis induced by trophic deprivation in cell cul-
ture and ischemia in the brain. Recently, smaller peptide
inhibitors of apoptosis have also been developed, such as
the dimer bocaspartyl(OMe)-fluoromethylketone, which
also block apoptosis in culture and in ischemic neu-
rons. Very likely, more and more molecular players in
the induction and inhibition of apoptotic events will be
identified in the future. As our ability to inhibit apopto-
sis improves, particularly as nonpeptide inhibitors that
readily cross the blood-brain barrier are developed, the
potential applications to neurodegenerative disease will
also increase.
APOPTOSIS IN NEURODEGENERATION
Apoptosis occurs in many pathological situations in the
brain, including ischemia, Huntington disease, and AD.
In the ischemic brain, apoptotic neurons appear about 4
days after the injury. Although not the main form of cell
death in ischemia, the lag time between injury and apop-
tosis offers a window for therapeutic intervention to block
this mode of cell death. The role of apoptosis has not been
proven in Huntington disease; however, transgenic mice
overexpressing the mutant form of Huntingtin show ex-
tensive apoptosis in striatal neurons. The ability of the
mutant Huntingtin protein to induce apoptosis sug-
gests that apoptosis could play an important role in the
pathology of Huntington disease. Apoptosis has also been
implicated in AD. Neuropathologic studies of AD-
affected brain tissue show that the rate of apoptosis is in-
creased 30- to 50-fold over that of age matched con-
trols.4 Since apoptotic cells appeared to be cleared rapidly
by the body, rapid clearance of apoptotic neurons in the
AD-affected brain might lead to an underestimate of the
total amount of apoptosis occurring in the disease. How-
ever, the significance of the increased apoptosis is un-
clear because the apoptosis could be one of many harm-
ful processes occurring in the AD-affected brain.
PRESENILINS AND APOPTOSIS
One of the strongest lines of evidence implicating apop-
tosis in AD comes from the putative role of presenilins
in apoptosis. Presenilins are proteins that cause early-
onset familial AD.5 The mutations in presenilins that cause
familial AD have been shown to render neurons more vul-
nerable to apoptosis.6 The exact mechanism through
which presenilins affect apoptosis is unknown, but given
the pivotal role presenilins seem to play in protein pro-

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cessing, there are many possible mechanisms by which
abnormalities in presenilin function could affect apop-
tosis. The presenilins interact with Bcl-XL, Bcl-2,
b-catenin, and Notch, all of which regulate apoptosis and
cell death.7-10 The carboxy terminus of presenilin 2 (PS2)
is the region that binds to Bcl-XL, which could explain
why the carboxy terminal PS2 fragment, Alg-3, is neu-
roprotective.6,8 Because Bcl-XL plays a pivotal role in apop-
tosis, the interaction between presenilins and Bcl-XL could
account for much of the effects of presenilins on apop-
tosis. The interaction of PS1 with b-catenin might affect
presenilin-induced apoptosis because b-catenin also can
regulate apoptosis under some circumstances.7,9 Alter-
ing presenilin activity might be an important regulatory
step in the apoptotic process because both PS1 and PS2
have been shown to be substrates of caspases.8 Cleavage
of the presenilins might reflect a homeostatic, antiapop-
totic function, because the C-terminal fragment gener-
ated by the PS2–caspase 3 cleavage inhibits apoptosis.6,8
Thus, the AD-associated presenilin proteins are inti-
mately involved in the process of apoptosis.
The significant role of presenilins in apoptosis does
not imply that activation of apoptotic pathways drives
the pathophysiology of AD. Presenilins seem to regulate
the processing of multiple proteins that affect apoptosis
(such as Notch, b-catenin, Bcl-X, and amyloid precur-
sor protein). On the other hand, many general observa-
tions argue strongly against a primary role of apoptosis
in AD. Although cell death is an important feature of the
AD-affected brain, synaptic loss is thought to be the most
important feature of AD. In addition, most studies sug-
gest that the amyloid-b (Ab) peptide, whose accumula-
tion is thought to drive AD, causes toxicity by inducing
free radical production rather than caspase activation.11
Finally, although the mutations in presenilins increase
apoptosis, they also increase production of Ab42, which
is a form of Ab that is particularly pernicious because it
aggregates rapidly.5 Because the ability of presenilins to
increase Ab42 production is sufficient to explain famil-
ial AD, the increases in apoptosis associated with these
mutations might be an epiphenomenon. The increases
in apoptosis associated with mutant presenilins might oc-
cur as a by-product of the dual roles of presenilins in sig-
nal transduction (such as Notch and b-catenin signal-
ing) and protein processing.10,12 We have shown that
presenilins couple the processing of amyloid precursor
protein to signal transduction.12 Mutations in presenil-
ins that affect the processing of amyloid precursor pro-
tein would be likely to also affect signal transduction en-
zymes. Any change in the regulation of signal transduction
enzymes could easily affect apoptosis, even if this is not
the major reason that the mutations cause AD. Thus, given
all the questions about the potential role of apoptosis in
AD, caution argues against invoking a major role for apop-
tosis in AD.
NECROSIS IN NEURODEGENERATION
One of the major reasons that the importance of apop-
tosis in neurodegeneration is questioned is the overlap
between apoptosis and necrosis in neuronal biology. Ne-
crotic cells have swollen nuclei, swollen mitochondria,
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E2 IGF-1
NGF
Vitamin E Receptor
Lipophilic Antioxidant
Tyrosine Kinase Gs
ERα
ERβ
Adenylate cyclase
Ras IκB cAMP
Free Radical
Raf NF-κB
Scavenging MEK PKA
ERK1/2
NF-κB
Non-enzymatic
MAPK
CREB-P
Protective Mechanisms
Elk-P Nuclear
Substrates
Nucleus - Neuroprotective Programs
Figure 2. Neuroprotective pathways. Neurons also have protective pathways
that are mediated by transcription factors. Nuclear factor (NF)–kB is
arguably one of the most important endogenous neuroprotective
transcription factors. Activation of Elk (via receptor tyrosine kinases and the
extracellular regulated kinase) or activation of cyclic adenosine
monophosphate(cAMP) response element binding protein (via cAMP and
protein kinase A) also lead to neuroprotection. Estrogen also provides
neuroprotection by at least 2 routes. Like vitamin E, estrogen acts directly as
an antioxidant. In addition, estrogen can stimulate endogenous
neuroprotective pathways by binding to estrogen receptors, which are also
transcription factors. E2 indicates estrogen; IGF-1, insulinlike growth
factor-1; NGF, nerve growth factor; ERa, estrogen receptor a; Gs,
stimulatory GTP-binding regulatory protein; ERb, estrogen b; IkB, inhibitor
of NF-kB; PKA, protein kinase A; MEK, mitogen-activated
protein/extracellular regulated kinase (MAP/ERK); ERK1/2, extracellular
regulated kinases 1 and 2; MAPK, mitogen-activated kinase; CREB-P,
phosphorylated response element binding protein; and Elk-P,
phosphorylated Elk.
and loss of plasma membrane integrity. Necrosis is a pas-
sive form of cell death that typically results from injury
or from excess calcium influx during excitotoxicity. Ex-
citotoxicity occurs in multiple pathological situations in-
cluding ischemia, seizures, and head trauma. The divid-
ing line that separates necrosis from apoptosis has been
emphasized for years owing to the clear distinct fea-
tures that classifies both events. However, death in neu-
rons can be biphasic, beginning with necrosis and then
showing delayed apoptosis. Moreover, if apoptosis is
blocked, for instance by overexpressing the neuropro-
tective transcription factor NF-kB, the mode of cell death
often simply switches from apoptosis to necrosis.13 The
ability of neurons to switch from apoptotic death to ne-
crotic death raises the possibility that antiapoptotic treat-
ments, such as caspase inhibitors, will block apoptosis
but not prevent cell death. Moreover, in neurodegenera-
tive disorders such as AD, Ab is able to kill via multifac-
eted pathways, which raises the possibility that both types
of cell death contribute to the neurodegenerative events.
NEUROPROTECTION: EXOGENOUS
NEUROPROTECTION AND INTRACELLULAR
NEUROPROTECTION—BASIC RESEARCH AND
CLINICAL PERSPECTIVES
A variety of endogenous neuroprotective programs ex-
ist that protect nerve cells against degenerative insults
(Figure 2). Several such triggers of endogenous neu-
roprotective programs have been already identified. One
important endogenous neuroprotective agent is the tran-
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scription factor NF-kB.14 NF-kB is a protein that con-
tains 2 subunits and represents a family of homologous
proteins (Figure 2).14 NF-kB protects neuronal cells
against oxidative stress.15 Activated NF-kB can be found
in so-called early senile plaques consisting of low-
molecular-weight aggregates of AD that may indicate the
compensatory activation of self-defense genes in nerve
cells that encounter Ab-aggregates in the brain tissue.16
NF-kB may be induced by various stimuli including neu-
rotrophic factors such as nerve growth factor and insu-
linlike growth factor 1, which are potential neuropro-
tective agents. Insulinlike growth factor 1 has been
reported to protect cultivated hippocampal neurons
against Ab toxicity via a mechanism involving NF-kB and
the kinase Akt.17 Therefore, NF-kB turns out to be a cen-
tral modulator of neuroprotective mechanisms, which may
be activated by various trophic input.
In addition to NF-kB, other important proteins such
as Bcl-2 and Bcl-XL mediate endogenous neuroprotec-
tive programs. Bcl-2 and Bcl-XL are mitochondrial pro-
teins that inhibit activation of apoptosis by binding criti-
cal proteins such as apoptosis activating factor, which is
important for the activation of caspase 3 and induction
of chromatin condensation (Figure 1). Although Bcl-2
and Bcl-XL have not been used directly for preventing
apoptosis, a number of caspase inhibitors have been de-
veloped that can prevent apoptosis. Caspase 3 is in-
duced during stroke, so inhibiting its activity has been
hypothesized to reduce cell death. In fact, small caspase
inhibitors, such as boc-Asp-CH2F, reduce the amount
of apoptosis in the penumbra of area damaged by the
stroke. The difficulty is that in these types of paradigms,
preventing apoptosis or excitotoxicity does not prevent
cell death. Since, in stroke for instance, cell injury rather
than programmed cell death (apoptosis) is the primary
stimulus for death, blocking apoptosis causes neurons
to switch to a necrotic mode of cell death.
Antioxidants have proven to be among the best tools
identified to date for protecting neurons against toxicity
owing to oxidative stress. Antioxidants are powerful neu-
roprotectants and may prevent oxidative nerve cell death
in vitro and in vivo. The classical free radical scavenger
a-tocopherol (vitamin E) prevents Ab- and glutamate-
induced cell death. Recently, we identified the female sex
hormone estrogen as another phenolic antioxidant that may
serve as an antioxidant nerve cell shield.18 Vitamin E is also
one of the few agents that has been demonstrated to slow
the progression of AD in a clinical setting.19 Many anti-
oxidants suffer from poor central nervous system penetra-
tion, but newer antioxidants, based on the structures of
estrogen or melatonin, might overcome this limitation.20
Thus, antioxidants serve as a general neuroprotective shield,
which reduce the damage caused by production of free radi-
cals. Some potential limitations of antioxidants are not com-
monly discussed in the literature. Production of free radi-
cals is an important part of our immune defense system.
When a macrophage engulfs a bacterium, it kills by gen-
erating a burst of free radicals. Since antioxidants reduce
the amount of oxidation, it is possible that excessive use
of antioxidants might increase morbidity owing to infec-
tion in some patients. Thus, antioxidants need to be used
with caution.
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OUTLOOK
With increasing knowledge of specific mechanisms me-
diating cell death comes increasing hope of developing spe-
cific tools that can prevent neurodegeneration without in-
ducing unacceptable side effects. As in many parts of
medicine, different medications might be valuable in dif-
ferent clinical settings. For instance, inhibiting protein ag-
gregation might be useful as a preventive strategy; how-
ever, some patients will likely be initially seen with clinical
disease, despite the availability of preventive strategies. In
these cases, use of antioxidants, anti-inflammatory drugs,
apoptosis inhibitors, and/or other therapeutic modali-
ties, such as the neurotransmitter based strategies (which
were not covered in this review) might be indicated.
Accepted for publication January 31, 2000.
Correponding author: Benjamin Wolozin, MD, PhD,
Department of Pharmacology, Loyola University Medical
Center, Bldg 102, Room 3634, 2160 S First Street, May-
wood, IL 60153 (e-mail: bwolozi@luc.edu).
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