system played an important role in neurodegeneration. It is
known that with aging we begin to develop immune complexes
to brain components.48 Some have proposed that this
is the function of immune suppression commonly seen with
aging, to reduce the immune attack. Studies of Alzheimer’s
patients have shown elevated cytokines IL-1ß, IL-2, IL-6,
S-100 protein, tumor necrosis factor alpha (TNF-alpha),
and significant isolated autoantibodies to GM1.49,50 One
also sees elevated levels of PGD2 and thromboxane B2,
both inflammatory cytokines, in Alzheimer’s disease.51
Autoantibodies have also been described in amyotrophic
lateral sclerosis as well.52
This immune attack on neurons produces a state of
chronic inflammation that generates a constant high level of
free radicals.53 As the damage continues, the p53 gene is
activated, leading to apoptosis.54 Short of actual destruction
of the neurons, a reduction in mitochondrial energy generation
leads to increased free radical production. In addition,
low energy levels make the neurons infinitely more sensitive
to the excitotoxic effects of glutamate and aspartate.
55,56 In fact, in the face of low energy production, even
normal levels of glutamate can kill neurons. Glutamate, in
turn, stimulates the microglia, the CNS immune cell, to
produce even more cytokines, and to release the two excitotoxins
glutamate and quinolinic acid.57 This viscous cycle
leads to eventual neuronal cell death.
As we see, glutamate itself can act as the trigger for
microglial activation leading to the release of numerous
inflammatory cytokines, or some other event may trigger
the process, such as a viral infection, Lyme disease organism
invasion, or even heavy metal exposure. One factor that
may lead to autoimmunity is the prolonged assault of the
brain’s cellular components to oxidative stress. Oxidation
of the proteins, which alters their structure, can lead to
autoimmunity.
Recent studies have shown that activation of the transcription
factor NF kappa B plays a major role in neurodegeneration.
This transcription factor stimulates the production
of various cytokines including IL-1ß, IL-2, IL-6 and
TNF-alpha, all of which are increased in neurodegenerative
diseases.58,59,60 Oxidative stress is a common trigger for NF
kappa B activation.
Beta-amyloid has been shown to activate microglia by
way of protein kinase C.61 Studies indicate that beta-amyloid
production is increased in the face of activated
microglia and that the presence of beta-amyloid is sufficient
to maintain chronic brain inflammation. Microglia themselves
contain enough glutamate to elicit excitotoxicity.
They can also precipitate excitotoxicity by stimulating the
release of arachidonic acid.62 Microglia also contain considerable
amounts of the excitotoxin quinolinic acid, which
can be released during activation.63 Quinolenic acid is a
metabolic product of serotonin metabolism.
Animal studies have shown that mice with autoimmune
disorders have a more rapid decline in aged-related
learning than normal animals.64
ADVANCED GLYCATION END PRODUCTS
One of the consequences of a high dietary intake of
glucose, and especially fructose, is the glycation of numerous
proteins in the cell.65 When proteins are glycated, that
is, combined with sugar molecules, they become significantly
more vulnerable to free radical damage and produce
advance glycation end products (AGEs) which can interfere
with tyrosine and dopa utilization. Elevated levels of AGEs
have been found in Alzheimer’s, Parkinson’s disease, and
ALS.66,67 This is especially so in Parkinson’s disease
because of the early fall in cellular glutathione levels.
The problem of large amounts of AGEs is that they signal
glia cells to produce superoxide and nitric oxide,68 a
combination that leads to the production of the powerful peroxynitrite
radical. Cytokines are also potent stimulators of
inducible nitric oxide, and hence peroxynitrite production.
PEROXYNITRITE
As stated, when nitric oxide combines with superoxide it
produces peroxynitrite. This free radical is unusual in that it is
resistant to most of the common antioxidants, such as vitamin
C, vitamin E and the carotenoids.69 The most powerful scavenger
of peroxynitrite is glutathione. When glutathione levels
are low, as is seen in Parkinson’s disease as well as
Alzheimer’s dementia and ALS, neurons become significantly
more vulnerable. Peroxynitrite tends to concentrate in the
mitochondria, where it damages enzymes as well as DNA.70
These events dramatically interfere with the cell’s ability
to produce energy. Neurons and glia are very energy
dependent. Virtually every cellular process requires enormous
amounts of energy. The brain consumes 20% of the
blood’s oxygen and 25% of its glucose, even though it constitutes
only 2% of body weight. Even under deep anesthesia,
the brain’s metabolism is reduced only 50%.71
Several studies have demonstrated elevated peroxynitrite
levels in Alzheimer’s disease, ALS and Parkinson’s
disease.72 Damage by peroxynitrite is indicated by the accumulation
of nitrotyrosine.
4-HYDROXYNONENAL (4-HNE)
4-hydroxynonenal (4-HNE) is an aldehydic product of
lipid peroxidation. While malondialdhyde (MDA) is the most
abundant product of lipid peroxidation, 4-hydroxynonenal is
the most reactive with proteins. Interestingly, the distribution
of damage by peroxynitrite parallels that of 4-HNE.73
There is growing evidence that 4-HNE plays a major
role in several neurodegenerative disorders, including
19 JANA Vol. 5, No. 1 Winter 2002
Alzheimer’s dementia, Parkinson’s disease and ALS. In one
study of seven Alzheimer’s disease patients, 4-HNE was
found to be associated with all amyloid deposits and most
perivascular areas (89%).74
Another study found increased 4-HNE in several areas
of the brain in Alzheimer’s disease, reaching significant levels
in the amygdala, hippocampus and parahippocampus,
areas of primary damage in the disorder.75 Elevations of 4-
HNE have also been found in the ventricular fluid of
Alzheimer’s patients but not in age-matched controls.76
The distribution of 4-HNE appears to be dependent on
the presence of the APOE genotype. APOE4-possessing
subjects demonstrated primary accumulation of 4-HNE in
the cytoplasm of pyramidal neurons, while APOE3 genotypes
had both astrocytic and pyramidal cell distribution.77
APOE4 is strongly associated with Alzheimer’s disease as
well as a high risk of dementia pugilistica in boxers.78 It is
also known that individuals with APOE4 genotype have
impaired antioxidant enzymes, which may be the basis of
their increased incidence of neurodegenerative diseases.79
Injecting 4-HNE into the brain of rats causes a widespread
loss of neurons in the basal forebrain ipsilateral to
the injection and a 60 to 80% reduction in choline acetyltransferase
seven days post-injection.80 When FeCl2 is
given, it increases the levels of 4-HNE in the brain.
Similar elevation of 4-HNE has been demonstrated in
Parkinson’s disease.81 A study of seven brains of
Parkinson’s disease patients, demonstrated immunostaining
for 4-HNE in the striatum, but demonstrated the same findings
in only 9% of aged-matched controls.
Direct injection of 4-HNE into the substantia nigra of
mice caused a dose-dependent depletion of glutathione in
the brainstem.82 Glutathione levels fall early in Parkinson’s
disease. 4-HNE has also been shown to rapidly inactivate
glutathione reductase, needed to convert oxidized glutathione
to its reduced form.83
One of the best correlations with cognitive function in
Alzheimer’s disease is the synaptic concentration in the
brain.84 4-HNE has been shown to conjugate to synaptic
proteins and to impair transport of both glucose and glutamate.
Both result in a significant decrease in cellular production
of ATP.85
HOMOCYSTEINE AND OXIDIZED
CHOLESTEROL
While the cardiovascular system has gotten most of the
attention as regards homocysteine and cholesterol, the nervous
system is also vulnerable to its effects. Both LDL and
HDL exist in the brain, with LDL acting as a transporter of
cholesterol and phospholipids in the CNS. Receptors for
LDL have been located on microvessels, astrocytes,
microglia, and neurons.86 Like LDL in the plasma, brain
LDL and HDL can become oxidized, especially in the presence
of increased catalytic iron.87
It has been shown that oxidized LDL in the striatum
enters the neuron and can induce cell death. The mechanism
of neuronal injury is closely connected to excitotoxicity
since glutamate-blocking drugs, such as MK-801, protect
the neuron from oxidized LDL-mediated cell death.88
Highly oxidized HDL in the brain has also been shown to
increase oxidative stress in astrocytes, microglia, and neurons,
causing death in the latter.89 When oxidized lipoproteins
exist in the presence of glutamate and/or amyloid, neuronal
killing is enhanced.90 Oxidized LDL is toxic to motor
neuron cells, possibly linking it to amyotrophic lateral sclerosis.
91 Antioxidants, just as in the case of plasma lipoproteins,
reduce oxidized LDL and HDL neurotoxicity.92
Homocysteine has been strongly associated with cardiovascular
disease, even though the mechanism has not
been fully elucidated. Less well appreciated is the connection
between elevated levels of homocysteine and neurodegeneration.
Several recent studies have shown a strong correlation
between homocysteine levels and incidence of
Alzheimer’s disease.93,94 Rarely discussed is the fact that
homocysteine is an excitotoxin, as are homocysteic and
homocysteine sulphinic acid,95 two of its metabolic breakdown
products.
These excitotoxins act at the N-methyl-D-aspartate
(NMDA) receptor, triggering the entry of excessive
amounts of calcium into the neuron, leading to numerous
destructive reactions including the generation of peroxynitrite,
4-HNE, hydroxyl and peroxyl radicals, and activation
of the eicosanoid cascade. Whether lowering homocysteine
levels will reduce Alzheimer’s disease is as yet unknown.
We do know that folate, pyridoxine, and antioxidant vitamin
deficiencies are common in Alzheimer’s disease
patients.96,97 Several studies have shown low levels of vitamin
B12 as well.97
It is important to appreciate that the classical hematological
signs of B12 deficiency, macrocytosis and hypersegmented
neutrophils, are usually absent in these patients.98
While homocysteine levels were found to be consistently
elevated in Alzheimer’s patients, nutritional deficiency was
not confirmed using retinol binding protein (RBP). This
indicates an impaired cobalamin delivery to the tissues,
which explains the observed discrepancy between normal
serum levels of cobalamin and folate and low tissue metabolic
products found frequently in the elderly.
One recent study throws the homocysteine theory into
question.99 Centenarians living in two northern Italian
provinces were examined for blood levels of homocysteine,
folate and B12. They examined centenarians who were cognitively
normal, cognitively impaired, and those with a
diagnosis of Alzheimer’s disease. Elevated homocysteine
levels were found in 77% of normal, 100% of cognitively
Winter 2002 Vol. 5, No. 1 JANA 20
impaired, and 82% of Alzheimer’s patients. Demented centenarians
had the lowest folate levels. Low B12 and B6 levels
were found in 33% and 66% respectively of all centenarians
regardless of cognitive status.
There are several explanations for these negative findings.
First, the study was based on blood levels of the vitamins,
and as we have seen, there is little correlation
between blood levels and tissue levels of these three vitamins.
As for the lower level of homocysteine seen in the
centenarian Alzheimer patients, perhaps it was secondary to
metabolic burnout, something we see in the case of glutamate
as well.
Homocysteine is known to elicit apoptosis quite rapidly
when hippocampal neurons are exposed to this amino
acid.100 The mechanism includes DNA strand breaks with
activation of poly-ADP-ribose polymerase (PARP) which
depletes nicotinamide adenine dinucleotide) (NAD). This
in turn leads to mitochondrial dysfunction, oxidative stress
and caspase activation. In essence it markedly enhances the
vulnerability of hippocampal neurons to excitotoxic and
oxidative injury.
ENERGY PRODUCTION, EXCITOTOXICITY. AND
FREE RADICALS
There is an intimate connection between energy production,
excitotoxicity, and free radicals. It has been known
for some time that impaired mitochondrial energy production
can lead to dramatic increases in free radical production,
and that reduced neuronal energy production significantly
increases the neuron’s sensitivity to excitotoxicity.101
In fact, under such conditions even normal concentrations
of extracellular glutamate can trigger excitotoxic reactions.
Further, an increase in free radical production, either as
reactive oxygen or reactive nitrogen species, increases the
release of glutamate from the astrocyte. Glutamate in turn
increases free radical production, which further reduces
energy production. This vicious cycle continues until the
p53 gene is activated and apoptosis ensues. The neurons are
destroyed by necrosis as well. When elevated iron levels are
present, as it does in all neurodegenerative disorders, free
radical generation reactions are accelerated.
Another result of increased free radical generation is its
effect on the blood-brain barrier system. This gatekeeper
normally prevents or slows the passage of harmful molecules
into the brain’s environment. Unfortunately, the system
is not perfect. Recent studies have shown that free radicals
can open the barrier, allowing harmful compounds
inside, including the excitotoxins glutamate and aspartate.
102 Evidence also indicates that the barrier contains
glutamate receptors and that glutamate itself can open the
barrier.103 This means that elevated levels of blood glutamate
can open the barrier, further elevating the level of this
powerful excitotoxin in the extracellular space.
Related to energy deficits in neurodegeneration is the
finding that the glucose transporter is impaired in
Alzheimer’s disease secondary to alteration in the brain’s
vasculature.104 In addition, glutamate itself can impair glucose
entry into the brain.
So we see that there is an intimate connection between
glutamate, free radicals, energy production, and the widespread
destruction of neurons. Because neurons differ in the
types of neurotransmitter receptors present on their membranes,
some will be more sensitive than others.. This
accounts for the difference in pathological presentation.
Reduced energy production has been demonstrated in
all four major neurodegenerative disorders: Alzheimer’s
disease, Parkinson’s disease, Huntington’s disease and
amyotrophic lateral sclerosis. In each instance, specific
enzyme deficiencies are present. In Parkinson’s disease,
complex I is deficient.105 In Alzheimer’s disease complex I,
IV and pyruvate dehydrogenase complex are all deficient.
106,107
MAGNESIUM
Magnesium plays a special role in excitotoxicity and
free radical generation. The magnesium receptor is located
near the calcium channel on neurons possessing the NMDA
receptor, which regulates calcium entry into the neuron. As
we have seen, excessive intracellular calcium can trigger
destructive reactions involving nitric oxide synthase induction,
arachidonic acid release from the membrane with
eicosanoid activation, and changes in mitochondrial function.
Magnesium can block calcium entry as long as the neuron
is not firing. It has been demonstrated that low magnesium
levels greatly enhance the neuron’s sensitivity to glutamate,
again, where even normal levels can be excitotoxic.108
Other studies indicate that magnesium plays a major
role in preventing free radical accumulation.109 Cells isolated
in a low magnesium environment not only generate more
free radicals but are twice as sensitive to free-radicalinduced
cell death as cells with normal magnesium levels.110
In addition, hypomagnesmia lowers the cell’s glutathione
level and increases its cytokine level, which, as we have
seen, can inhibit glutamate uptake, thereby increasing free
radical generation that leads to excitotoxic neuron death.
Both experimental and clinical studies have demonstrated
significant neuroprotective effects of elevated magnesium
levels.111,112 An additional benefit is that magnesium
also inhibits the entry of oxidized lipids into the
endothelial cells of blood vessels.