# Progress and Prospects in Parkinson's Research/Magazine Section/Joining the Dots

JOINING THE DOTS
Evidence for and implications of endotoxin-induced inflammatory origins of Parkinson’s disease (PD)
by Rick Reverett
   This is a brief summary of an exciting new way of looking at PD that has been taking shape over the last five years and which holds promise of effective treatment via anti-inflammatory actions.

## Summary

I have been working on this for several months beginning with an interest in Helicbacter pylori There is no original research on my part here other than the attempt to connect “the dots” generated by true researchers. There are gaps, particularly where it is necessary to make a leap from animal model or test tube to human. There are no doubt big flaws that I have overlooked. But this theory answers more questions about PD than I would ever have imagined. See if you agree!

## Hypothesis

Parkinson’s patients are part of a subset of the population who show a hypersensitivity to the endotoxin lipopolysaccharide (LPS) resulting from neonatal exposure and which results in a chronic inflammatory state in the brain. This inflammatory state results in both long term damage to the substantia nigra (SN) and, perhaps more importantly, acute symptomology producing the typical Parkinson’s picture.
In addition to providing a plausible framework for the causes and effect leading to PD, this hypothesis explains many seemingly unrelated facts about the condition. Among them are:-
   the observed gender difference in PD;
   the finding of iron in the SN;
   the geographical limitation of damaged neurons to the SN;
   the nature of manganese induced parkinsonism;
   the sleep disturbances common in PD;
   the amplification of symptoms by stress;
   the elevated levels of cortisol common in PD;
   the role of mercury;
   the problem of mitochondrial function, etc.
In addition, numerous anecdotal reports by patients of temporary remission coinciding with use of drugs with known anti-inflammatory effects are explained by the hypothesis, as are similar reports and epidemological data related to non-prescription medicines such as green tea, turmeric, alpha-lipoic-acid, etc.
No other proposed hypothesis for the origins and course of Parkinson’s disease comes close to explaining all of the above. Nor does any offer the hope of such a quick test of the basic concept. Simply reproducing the anecdotal information referred to above would immediately verify the broader principle. Similarly, a move to clinical trials and even actual treatment by way of “off label” use of already approved drugs should allow speedy implementation.

## Inflammation as a cause of PD rather than an effect.

It has been known for a long time that inflammation in the brain was a feature of PD, but it was thought to be a secondary effect. Over the last few years more and more researchers are looking at the other side of the coin and producing evidence for a causal role, perhaps THE cause.
Inflammation in the brain is not the same as in the body. One critical feature is the activation of the defensive cells known as microglia. For reasons unknown, in some cases they don’t know when to stop. The result can be PD or Alzheimer’s.
Hirsch et al commented on the effects of neuroinflammation:-
   In Parkinson's disease (PD), post-mortem examination reveals a loss of dopaminergic (DA) neurons in the substantia nigra (SN) associated with a massive astrogliosis and the presence of activated microglial cells. Similarly, microglial activation has also been reported to be associated with the loss of DA neurons in animal models of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, annonacine and lipopolysaccharide (LPS). Recent evidence suggests that the disease may progress even when the initial cause of neuronal degeneration has disappeared, raising the possibility that toxic substances released by glial cells could be involved in the propagation of neuronal degeneration. Inhibition of the glial reaction and the inflammatory processes may thus represent a therapeutic target to reduce neuronal degeneration in PD.
McGeer et al concluded:-
   Classical dogma taught that the brain is immunologically privileged and does not mount an endogenous immune response. Immunohistochemical and molecular biological evidence accumulated over the past decade, however, has shown that the brain is capable of sustaining an immune response and that the result may be damaging to host cells. The brain, rather than being immunologically privileged, may be particularly vulnerable since neurons are postmitotic. They cannot divide. so that, once lost, they are not replaced. The evidence for a chronic inflammatory reaction in the brain is particularly strong in Alzheimer’s disease (AD), where it has been extensively studied, but there is also evidence suggesting that a local immune reaction occurs in affected regions of the brain in Parkinson’s disease (PD). The inflammation is silent because the brain has no pain fibres. Moreover, the local immune reaction does not involve the peripheral immune system. It occurs without antibodies and without significant involvement of T-cells. Instead, the reaction depends upon the synthesis of inflammatory components by local neurons and glia, and especially resident phagocytes—which, in the brain, are the microglia. The complement system, microglia, and inflammatory cytokines appear to play key roles.
   The complement is a phylogenetically primitive system, considerably predating the adaptive immune system, which developed in later vertebrates. It is thus not surprising that the system can be activated by molecules other than antibodies. One such molecule, which is found elevated in the substantia nigra in PD, is C-reactive protein. Once activated, the complement cascade produces anaphylatoxins that promote further inflammation, opsonizing components that mark material for phagocytosis and the membrane attack complex, which is directly lytic to cells. The membrane attack complex inserts itself into viable cell membranes, causing them to leak and produce cell death. It is intended to destroy foreign cells and viruses, but host cells are at significant risk of bystander lysis.
   The presence of complement proteins, including all components of the membrane attack complex, has been shown intracellularly on Lewy bodies and on oligodendroglia in the substantia nigra in PD and familial PD. Such oligodendroglia have been described as complement activated oligodendroglia. Staining for all the complement components is either absent or very weak in control substantia nigra.
   Microglia constitute about 10% of all glia. They are generally in the resting state in the normal adult brain. When activated, they upregulate or newly express a variety of receptors and other molecules involved in inflammation and phagocytosis. In the activated state, they also produce large amounts of superoxide anions and other potential neurotoxins. In culture, microglia have been shown to contribute to neurotoxicity, including that of dopaminergic cells. A profusion of reactive microglia is seen in the substantia nigra and striatum, not only in idiopathic PD, but also in familial PD, [6] as well as in the parkinsonism-dementia complex of Guam (EGM et al, unpublished data, 2001). The presence of many activated microglia in the substantia nigra of humans dying years after exposure to the toxin MPTP testifies to the fact that once the fire of inflammation is lit, it continues to burn long after the initiating event.
   Reactive microglia are also seen in the basal ganglia in 6-hydroxydopamine and MPTP animal models of PD, and there are several reports that anti-inflammatories inhibit dopaminergic neurotoxicity in such animal models.
   Microglia can be activated by products of the classical complement cascade, by various inflammatory cytokines, and by chromogranin A, which has been reported to occur in PD substantia nigra.
   Inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1, and interleukin-6, amplify and sustain inflammation and immune responses. In the periphery, they are thought to be primarily responsible for many of the clinical and pathological manifestations of such diseases as rheumatoid arthritis and inflammatory bowel syndrome. It is of some interest, therefore, that increased levels of interleukin-1ß, interleukin-6, and tumor necrosis factor-alpha have been found in the basal ganglia and CSF of PD patients. The increase in tumor necrosis factor-alpha was particularly dramatic, being 366% in tissue and 432% in CSF. Moreover, the presence of glial cells immunoreactive for tumor necrosis factor-alpha and/or interleukin-1ß has been reported in the substantia nigra of PD patients.
   The large increase in levels of the inflammatory cytokines and the profusion of reactive microlgia seen in the substantia nigra in PD suggests an intense inflammatory reaction… The inflammatory reaction in affected brain regions in these neurodegenerative diseases seems to be, at least by this measure, more intense than that in arthritic joints.
Those of you who have dealt with arthritis will be particularly impressed with that last statement, I am sure.

## Endotoxins as the central cause of inflammation and PD.

Endotoxin. Lipopolysaccharide (LPS). Both refer to a toxic building block in the cell wall of a certain class of bacteria. This class is referred to as “Gram-negative” because of the staining test used to identify them. A large family, it includes some familiar names: H pylori, E coli, Salmonella, etc.
The endotoxin (LPS) is unlike other bacterial toxins in that it is not released into the host system until the bacterium dies. It is a “poison pill” defense! Anything which kills a large colony can poison the host. Some species, such as H pylori, even greatly increase the production of LPS when under attack by antibiotics. But in the normal course of events there is a low, chronic drip of toxin into the system from the death of individual cells which the body detoxifies.
LPS triggers a powerful inflammatory response. In fact, it is used in research to do so. But in some cases it does much more. For unknown reasons LPS can trigger an often fatal cascade known as sepsis. The body severely over-reacts to the toxin, blood pressure drops, organs shut down, and the patient dies. Even the elimination of the invading bacteria fails to stop the cascade.
Under some circumstances LPS can cross the blood brain barrier and enter the brain where it activates the microglia and starts another runaway cascade – this one in slow motion. Years go by and neurons die one by one. The toxin may not even be present as the body attacks itself.
Castano et al studied the effect of LPS on DA cells in rats:-
   The pathogenesis of Parkinson's disease is still poorly understood. To address the hypothesis that immune-mediated events, such as microglial activation, may be involved in the dopaminergic neurodegeneration, we have studied the effect that intranigral injection of the immunostimulant lipopolysaccharide has on monoaminergic neurotransmitters in rats.
   Activation of microglial cells, visualized by immunohistochemistry with a specific monoclonal antibody was already obvious 2 days after injection. In relation to the biochemical parameters studied, we found a significant decrease of dopamine levels in both the substantia nigra and striatum up to at least 21 days after intranigral injection of lipopolysaccharide.
   This result was supported by the decrease in tyrosine hydroxylase activity and the loss of tyrosine hydroxylase-positive neuronal bodies, shown by immunohistochemistry. These alterations of the dopaminergic system did not reverse during the interval studied (21 days); conversely, the serotoninergic system suffered only transient damage. In addition, we found that the neurotoxic effect of lipopolysaccharide was not mediated by nitric oxide.
   Based on our results we suggest that the nigrostriatal dopaminergic system is susceptible to damage by inflammatory events and that these may be implicated in neurodegeneration processes such as Parkinson's disease.
And these fndings were corroborated by Li et al
   In order to investigate the neurotoxicity of lipopolysaccharide (LPS) on the dopaminergic neurons of substantia nigra and the pathogenesis of Parkinson disease, LPS was stereotaxically infused into substantia nigra (SN). At different dosages and different time points with 5 microg LPS, the damage of the dopaminergic neurons in SN was observed by using tyrosine-hydroxylase (TH)immunohistochemical staining.
   The results showed that 14 days after injection of 0.1 microg to 10 microg LPS into the rat SN, TH-positive (TH+) neurons in the SN were decreased by 5%, 15%, 20%, 45 %, 96% and 99% respectively. After injection of 5 microg LPS, as compared with the control groups, TH+ neurons began to decrease at 3rd day and obviously decrease at 14th day, only 5% of total cells,and almost disappeared 30 days later. The results suggested that LPS could induce the degeneration of dopaminergic neurons in the SN in a dose-and-ime-dependent manner.

## LPS and the central nervous system

The longterm damage to the brain by LPS exposure is a serious matter but from the perspective of PD a far more important question is “Are there acute effects that might produce some of the symptoms associated with PD?”
The reason this question is so important is this – if my symptoms only partially come from longterm damage to my brain and the remainder come from acute effects from the inflammatory response or other effects, then it might be possible to eliminate that portion of my symptoms by dealing with the underlying response.
Think that through. If we are wrong in thinking that PD is simply a damaged substantia nigra resulting in a dopamine shortage, then a great window opens. Any Parkie knows that PD is far more than simply a “movement disorder.” And research may not yet define just what LPS does in the brain but it is clear that it does have acute effects.
This was evaluated by Hsieh et al
   Inflammation and neuronal degeneration of the substantia nigra (SN) occur in Parkinson's disease (PD). We studied the effects of intranigral lipopolysaccharide (LPS) injection on adult Sprague-Dawley rats. Locomotor activity measurement, neurotransmitter determination and perfusion fixation for
   immunohistochemistry were done on the 7th day. Bilateral LPS injection increased
   locomotor activity 2- to 3-fold.
   In the SN, dopamine (DA) and serotonin (5-HT)decreased but the ratios ihydroxyphenylacetic acid (DOPAC)/DA, homovanillic acid (HVA)/DA and 5-hydroxyindole-acetic acid (5-HIAA)/5-HT increased. In the striatum, DA, DOPAC, HVA, 3-methoxytyramine and epinephrine decreased but HVA/DA d 5-HIAA/5HT ratios increased. Unilateral LPS decreased dopamineric neurons ipsilaterally but increased contralaterally. This study provides the first evidence of behavioral hyperactivity, epinephrine suppression and neuronal plasticity in the LPS model of PD.
And by Wilson et al
   The brain is not only immunologically active of its own accord, but also has complex peripheral immune interactions. Given the central role of cytokines in neuroimmmunoendocrine processes, it is hypothesized that these molecules influence cognition via diverse mechanisms. Peripheral cytokines penetrate the blood-brain barrier directly via active transport mechanisms or indirectly via vagal nerve stimulation. Peripheral administration of certain cytokines as biological response modifiers produces adverse cognitive effects in animals and humans.
   There is abundant evidence that inflammatory mechanisms within the
   central nervous system (CNS) contribute to cognitive impairment via cytokine-mediated interactions between neurons and glial cells. Cytokines mediate cellular mechanisms subserving cognition (e.g., cholinergic and dopaminergic pathways) and can modulate neuronal and glial cell function to facilitate neuronal regeneration or neurodegeneration. As such, there is a growing appreciation of the role of cytokine-mediated inflammatory processes in neurodegenerative diseases such as Alzheimer's disease and vascular dementia. Consistent with their involvement as mediators of bidirectional communication between the CNS and the peripheral immune system, cytokines play a key role in the hypothalamic-pituitary-adrenal axis activation seen in stress and depression. In addition, complex cognitive systems such as those that underlie religious beliefs, can modulate the effects of stress on the immune system. Indirect means by which peripheral or central cytokine dysregulation could affect cognition include impaired sleep regulation, micronutrient deficiency induced by appetite suppression, and an array of endocrine interactions. Given the multiple levels at which cytokines are capable of influencing cognition it is plausible that peripheral cytokine dysregulation with advancing age interacts
   with cognitive aging.

## PD and early exposure to LPS

With any explanation of PD, one of the first questions has to be why some get it and some don’t. Under the endotoxin theory the explanation is straight forward:
If one is exposed to LPS in the womb or shortly after birth the effects it lays the groundwork for PD as an adult.
Exposure in the womb is particularly likely to have this effect. Four results that have been noted are:-
   1) A lower density of dopaminergic neurons in the substantia nigra. We start out with a player’s “handicap.”
   2) A sensitivity to LPS that does not manifest until after puberty. The adult responds to exposures that a normal person would not.
   3) An alteration of the hypothalamus-pituitary-adrenal (HPA) axis that
   changes our response to stress. This raises our levels of cortisol and may account for the “Parkinson’s personality” – we tend to take
   responsibility as a means of controlling our stress.
   4) Increased inflammatory chemicals in the system.
Ling et al explored the long term effects of prenatal exposure to LPS:-
   We previously reported that injection of the Gram (-) bacteriotoxin,lipopolysaccharide (LPS), into gravid females at embryonic day 10.5 led to the birth of animals with fewer than normal dopamine (DA) neurons when assessed at postnatal days (P) 10 and 21. To determine if these changes continued into adulthood, we have now assessed animals at P120.
   As part of the previous studies, we also observed that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha) was elevated in the striatum, suggesting that these animals would be more susceptible to subsequent DA neurotoxin exposure. In order to test this hypothesis, we injected (at P99) 6-hydroxydopamine (6OHDA) or saline into animals exposed to LPS or saline prenatally.
   The results showed that animals exposed to prenatal LPS or postnatal 6OHDA alone had 33% and 46%,respectively, fewer DA neurons than controls, while the two toxins combined produced a less than additive 62% loss. Alterations in striatal DA were similar to, and significantly correlated with (r(2)=0.833) the DA cell losses. Prenatal LPS produced a 31% increase in striatal TNFalpha, and combined exposure with 6OHDA led to an 82% increase. We conclude that prenatal exposure to LPS produces a long-lived THir cell loss that is accompanied by an inflammatory state that leads to further DA neuron loss following subsequent neurotoxin exposure. The results suggest that individuals exposed to LPS prenatally, as might occur had their mother had bacterial vaginosis, would be at increased risk for Parkinson's disease.
So, at least in a rat, dopamine producing neurons were reduced by a third at birth, inflammatory TNFa was up 31%.