Biocast Episode 11 – Endotoxin and Dementia

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Episode 11 of The Biocast looks at some data on endotoxin and dementia.
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In this episode I’ll look at dementia, brain energy and endotoxin.This podcast is part of a series of podcasts looking at the biological effects of endotoxin, if you haven’t listened to the previous episodes you should do that first or this might be a little technical. I will give a brief summary so far:

Endotoxin is a part of the structure of some bacteria present in the digestive system, small amounts are released by the bacteria in normal metabolism and larger amounts are released when the bacteria die. When endotoxin is transported from the digestive system into the bloodstream and into other tissues there is an inflammatory response, possibly as the endotoxin structure is confused for a live pathogen, and this response can initiate may problematic processes possibly creating disease.

In two previous podcasts I’ve looked at how diet and other factors can alter gut bacteria and increase the transport of endotoxin into the plasma and tissue, from there it can be picked up by a number of factors and can be “sensed” by the proteins CD14 and TLR4 toll-like receptor 4. If, and when these proteins sense endotoxin they activate nuclear factor kappa B which initiates a long chain of inflammatory actions.


We have seen how endotoxin might cause diabetes, obesity and liver disease in the previous shows. In researching those I came across a number of interesting links to dementias and behavioural disorders and this show is going to look at dementias, particularly aspects of alzheimers and parkinsons.

A lot of what I’ll be looking at here is data on protein structures and the function of neurotransmitter dopamine, both of which are thought to be centrally involved in dementias. I will start out with standard some definitions and then I will move on to discussing the papers.

What is dementia?
Dementia is a syndrome – usually of a chronic or progressive nature – in which there is deterioration in cognitive function (i.e. the ability to process thought) beyond what might be expected from normal ageing. It affects memory, thinking, orientation, comprehension, calculation, learning capacity, language, and judgement. Consciousness is not affected.

Dementia is an overall term that describes a wide range of symptoms associated with a decline in memory or other thinking skills severe enough to reduce a person’s ability to perform everyday activities. Alzheimer’s disease accounts for 60 to 80 percent of cases. Vascular dementia, which occurs after a stroke, is the second most common dementia type.

Symptoms: Difficulty remembering recent conversations, names or events is often an early clinical symptom; apathy and depression are also often early symptoms. Later symptoms include impaired communication, poor judgment, disorientation, confusion, behavior changes and difficulty speaking, swallowing and walking.

Revised criteria and guidelines for diagnosing Alzheimer’s were published in 2011 recommending that Alzheimer’s be considered a slowly progressive brain disease that begins well before symptoms emerge.

Brain changes: Hallmark abnormalities are deposits of the protein fragment beta-amyloid (plaques) and twisted strands of the protein tau (tangles) as well as evidence of nerve cell damage and death in the brain.

As Parkinson’s disease progresses, it often results in a progressive dementia similar to dementia with Lewy bodies or Alzheimer’s.Lewy bodies are abnormal aggregates of protein that develop inside nerve cells in Parkinson’s disease (PD), Lewy body dementia, and some other disorders. They are identified under the microscope when histology is performed on the brain.

Symptoms: Problems with movement are common symptoms of the disease. If dementia develops, symptoms are often similar to dementia with Lewy bodies.
Brain changes: Alpha-synuclein clumps are likely to begin in an area deep in the brain called the substantia nigra. These clumps are thought to cause degeneration of the nerve cells that produce dopamine.

A-synuclein fibril
Fibre of a-synuclein, a-synuclein is a protein abundant in the human brain. It may help to regulate the release of dopamine.

Amyloids are aggregates of proteins that become folded into a shape that allows many copies of that protein to stick together. In the human body, amyloids are usually unhealthy developments, as these previously healthy proteins most often lose their normal physiological function and form large fibrils.

Lewy bodies are abnormal aggregates of protein that develop inside nerve cells in Parkinson’s disease (PD), Lewy body dementia, and some other disorders. They are identified under the microscope when histology is performed on the brain.
Low-dose endotoxemia and human neuropsychological functions

So the first paper that I have linked up is looking at the question of low-grade inflammation and the effect that has on things that might indicate age-related decline in cognitive function. The study looked at markers of inflammation as well as changes in emotional and cognitive parameters. This was done in humans, they performed an intravenous injection of E. coli endotoxin or saline solution for the control group. They ran tests of neuropsychological parameters prior to the injection and at 1.5 hours, six hours, and 24-hour it after.

The dose of endotoxin did not produce any significant changes in body temperature, cortisol, blood pressure or heart rate as would be expected with higher doses. The endotoxin did however increase tumour necrosis factor and interleukin-6. They determined that this low-dose endotoxaemia did not affect cognitive performance, but the effect on memory was related the cytokine increase. Tumour necrosis factor and interleukin-6 are inflammatory cytokines, that is, small proteins that affect cells, and the increase in these in response to the endotoxin was predictive of the decrease in memory. They ran memory tests to measure what they called explicit or decorative memory, and the basic distinction here seems to be that this is conscious memory.

The paper also notes that these negative effects on memory are independent of physical stress, and the HPA axis. The introduction notes the epidemiological studies suggesting low-grade inflammation and its correlation with age associated cognitive decline and Alzheimer’s disease. They also defined systemic low-grade inflammation as a 2 to threefold increase in inflammatory cytokines and acute-phase proteins. Acute phase proteins are modulated in the plasma in response to inflammation. The largest increases in interleukin-6 and tumour necrosis factor were at three hours post injection.
Systemic LPS Causes Chronic Neuroinflammation and Progressive Neurodegeneration

The next paper is looking at neuro inflammation and neurodegeneration in rodent models. They use conventional rodents and in other experiments to used TNF alpha knockout rodents. In one part of the experiment conventional mice were given a single intravenous dose of endotoxin at 5 mg per kilogram and then various samples were taken to assess tumour necrosis alpha. Tumour necrosis factor alpha messenger RNA increased 140 fold in the liver and peaked after 30 minutes. TNF alpha proteins in the increased 29 fold and peaked at 60 minutes. TNF alpha protein increased in the serum from 0 to 6.4 ng/mL after 60 minutes. In the brain both the messenger RNA and protein peaked after 60 minutes increasing over 7000% and over 650% respectively.

The study found that serum TNF alpha declined to the same level as controls by hour nine while at the same time TNF alpha protein levels in the liver were 18% of the peak by our nine, however in the brain TNF alpha remained elevated. One week after the endotoxin treatment the treated animals had almost 5 times as much TNF alpha in the brain as controls, in fact TFNa levels in brain remained very elevated even after 10 months, at around 80% of the peak levels reached at 1hr.

Further they tested endotoxin treatments on tumour necrosis factor knockout mice. These were mice that were genetically deficient in the genes for the TNF type I and the TNF type II receptors. They found that endotoxin increased TNF alpha messenger RNA and protein in the liver and serum of both conventional and knockout mice, but not in the brain of the knockout mice. The idea put forward is that the administration of endotoxin increases tumour necrosis factor alpha systemically, but the brain is protected from the endotoxin, inflammation in the brain then may be caused by TNF alpha sensors peripherally sensing increased TNF alpha and that leading to increasing TNF alpha in the brain.

Further experiments supported this idea, instead of injecting endotoxin they injected both types of mice with TNF alpha itself and saw very similar results. The authors explain that it’s indicated that the information is mediated via a type of cell located throughout the brain and spinal-cord called microglia, the paper discusses dopaminergic receptors also which are thought to be closely tied to some dementias.

They tested a marker of dopamine neurons in the substantia nigra, part of the brain that is very dense in microglia and believed to be very susceptible to inflammation. The marker was tyrosine hydroxylase immuno reactive cells, or THIR. Previous experiments had shown that endotoxin induced a decline of these cells, some evidence showed that endotoxin could cause a progressive loss of these cells in this part of the brain and that this evidence didn’t show up at four months post treatment but did show up at 7 to 10 months with a loss of 23% and then 47% respectively. This is thought to be as a result of cell death rather than selective down-regulation of the THIR cells specifically.

There is a chain of detection and inflammation going from endotoxin to tumour necrosis factor alpha. LPS->LPB>CD14>TLR4>NFKB>TNFa. One of the things that senses endotoxin is a protein called toll -like receptor four, toll -like receptor four initiates the NFKB pathway, which in turn initiates the production of inflammatory cytokines including TNF alpha. The next paper then takes a step back and looks at toll -like receptor four and neurodegeneration. (LBP is lipopolysaccharide binding protein mentioned in the previous episode)
Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway

The paper frames these responses in the context of evolutionary innate immunity. Within the central nervous system things like toll -like receptor four can be activated by pathogens acting as a sort of alarm system which leads to immune responses that would kill or detoxify pathogens. The paper explains that activation of the microglia is a feature of most neurodegenerative diseases including Parkinson’s, Alzheimer’s, multiple sclerosis, AIDS dementia, and ALS. It references some papers showing neurotoxicity from beta amyloid or HIV proteins in cell cultures and that inducible nitric oxide synthase seem to play a critical role in the dopaminergic related neurodegeneration in a rodent model of Parkinson’s. Amyloids are folded proteins in a certain shape that allows them to stick together, generally seen as unhealthy developments as they lose their normal physiological function, and form a larger structures called fibrils.
In one of the experiments they found that endotoxin induces loss of axons, oligodendrocyte’s, and microglia in central nervous system cell cultures from rat fore brains. Experiments indicated that the effect was mediated through the microglia as they express toll -like receptor four and CD14 which is also involved in sensing endotoxin. The oligodendrocytes expressed CD14 but not toll -like receptor four and experiments reported in this paper on rats found that toll -like receptor four was necessary for endotoxin induced neuronal injury and death.
Obesity is associated with hypothalamic injury in rodents and humans

Next is a paper looking at obesity and hypothalamic injury. the hypothalamus is a part of the brain that links the nervous system to the endocrine system. It’s thought to control body temperature, hunger ,thirst, fatigue, sleep, and circadian rhythms.

This paper talks about high-fat diets in rodent models. I’ve looked at this in previous podcasts, a high-fat diet in rodents will increase the permeability of the digestive system and change the balance of flora and this will lead to increased endotoxin in the plasma which initiates the detection and then the resulting inflammation.This paper tells us that unlike inflammation in peripheral tissues inflammation in the hypothalamus is elevated within 24 hours of exposure to the high-fat diet. In other experiments in other tissues the inflammation was seen after weeks or months.

The paper describes increased gliosis, a reactive change of the glial cells from the high fat diet in rodents and evidence of increased inflammatory signalling in a part of the hypothalamus in obese humans as measured by an MRI scan. In previous podcasts I’ve talked about the increase in energy intake in rodents on a high-fat diet that were free feeding versus rodents on a standard or relatively low fat diet, also free feeding, this might explain part of the mechanism there, as this part of the brain is also involved with leptin which is thought to be involved in appetite, satiety, and obesity.
Hypothalamic dysfunction in dementia.

The hypothalamus secretes corticotropin releasing hormone or CRH, this stimulates the secretion of adrenocorticotropic hormone or ACTH, this in turn acts on the adrenal cortex which produces among other things cortisol. So this paper looked at patients with Alzheimer’s, senile dementia of Alzheimer’s, and vascular dementia. it measured cortisol levels and performed a dexamethasone test. This is a standard test to assess the adrenal gland function by measuring how cortisol levels change in response to dexamethasone. It’s used to diagnose Cushing’s syndrome and was historically used for diagnosing depression. It was found that with a vascular dementia and a pathological response to the dexamethasone there was significantly more intellectual impairment, anxiety, fear, panic, and restlessness compared to patients with vascular dementia and a normal response to dexamethasone. Endotoxin at least acute administration’s seems to activate the production of ACTH until be involved here are a number of ways.
Endotoxin and the hypothalamo-pituitary-adrenal (HPA) axis.

The next paper looks at endotoxin’s effects on rodent brain slices.
The Bacterial Endotoxin Lipopolysaccharide Causes Rapid Inappropriate Excitation in Rat Cortex

So the paper tells us it is mounting evidence that inflammation and excitotoxicity might be involved in Alzheimer’s and dementia and multiple sclerosis. The authors exposed slices of rodent parietal cortical tissue to endotoxin and measurement neurotransmitters and adenosine. Noradrenaline and glutamate release was quickly and strongly triggered by the administration of endotoxin, the paper proposes that similar responses might be triggered due to immunity and may be associated with the conditions they mentioned previously.
Propagation of endotoxin-induced nasal mucosal inflammation to the olfactory bulb along the olfactory neural pathway

This next paper I’m going to look at is on a different vector for endotoxin induced brain inflammation. These experiments were done on mice where they received injections of endotoxin into the nasal cavity every other day and had samples taken at day one, three, seven, 14, and 21. The sensory neurons in the nasal cavity were damaged over time and the microglia were activated. The results indicated that endotoxin induced inflammation can be transmitted along the olfactory bulb and neural pathway.
Intranasal LPS-Mediated Parkinson’s Model Challenges the Pathogenesis of Nasal Cavity and Environmental Toxins

Another paper looks at intranasal endotoxin in a model of Parkinson’s disease. The paper talks about some of the previous chemical models for Parkinson’s being exposure to various toxins, and the various problems with those as models. It also talks about endotoxin producing some of the known factors Parkinson’s disease, being dopamine neurodegeneration and glial cell activation, though single intracranial injections had been insufficient to induce sufficient degeneration to provide a stable Parkinson’s disease model.

Humans are exposed to endotoxin in the air from very small suspended particles in the paper notes a recent idea that Parkinson’s disease might be primarily a disorder above the olfactory system in which the loss of the sense of smell precedes motor symptoms.

So the study looked model of Parkinson’s disease initiated by intranasal low-dose endotoxin every other day for five months in adult conventional mice. The endotoxin was dissolved in saline and dropped into the nasal cavity of the rodent when it was upside down. The various tests of neurological functions and behaviours for rodents and models include observation in an open field test, looking for spontaneous activity, and an adhesive removal testing which adhesive dots were placed on the limbs of the rodents and the time taken to remove the dots were recorded’s, and a maze test designed to assess memory and spatial learning. Physical samples from the animals were analysed also.

The open field test which was performed in the first, third and fifth months showed that endotoxin induced a progressive reduction in ambulatory motor activity, which is an indicator of Parkinson’s. Another indicator, that is progressive hypokinesia increased though not all those hypokinesia results were significant. The paper also reports that the model of Parkinson’s induced another feature of Parkinson’s disease that being chronic progressive Bradykinesia, meaning slowness of movement.

The adhesive removal test was performed in the fifth month, in this test they analyse the comparison of the times it takes to remove the adhesive from either fore limb. So this indicates a motor asymmetry. There was no significant difference in the control mice whereas the endotoxin group took significantly longer to remove the adhesive dots from the right side compared to the left side. In the analysis of the maze task no significant differences were seen between groups.

The measure used in some of these studies to indicate loss of dopaminergic neurons, a hallmark of Parkinson’s disease is tyrosine hydroxylase immunoreactive neurons and a severe loss of these was seen in the right substantia nigra of the endotoxin treated rodents, this is the part of the brain which contains dopamine producing neurons. Similar neurons in a region of the brain near to the substantia nigra, the ventral tegmental area were not damaged by the endotoxin administration.

These same markers of dopaminergic function were reduced in the olfactory bulb of the endotoxin treated rodents. The analysis for alpha synuclein show significant increase in the endotoxin group in both the brain and the olfactory bulb. Part of the brain, the striatum was tested for neuro transmitters and their metabolites. The levels of dopamine in endotoxin treated rodents was significantly lower and markers of dopamine turnover were significantly higher.

Glial activation is another marker of Parkinson’s and indicators of this went up in endotoxin treated rodents along with tumour necrosis factor alpha and interleukin beta.
Acute induction of anomalous and amyloidogenic blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide

This next paper looks at the increasing fibrin fibres and fibrinogen caused by endotoxin in blood. Fibrin and fibrinogen are involved in the coagulation of blood. Fibrinogen is primarily seen as a precursor to fibrin, high levels of either are associated with thrombosis, ischaemic disease, ischaemic stroke, venous embolism, inflammation and periodontal disease.

This paper showed that very small concentrations of endotoxin in whole blood or platelet poor plasma causes changes in fibrin fibres, possibly leading to an amyloid beta structure seen in a number of inflammatory and amyloid diseases.;jsessionid=D86115BFE0A47B7068A9BC8F96CD3EFC.f04t01
Progression of intestinal permeability changes and alpha-synuclein expression in a mouse model of Parkinson’s disease

The next paper is looking at Parkinson’s disease, specifically a model of Parkinson’s disease in mice. The paper tells us that increased permeability of the colon is associated with Parkinson’s disease and that there is a pathological expression a-synuclein in the colon. The authors tell us that these markers might be indicative of the initiation of Parkinson’s disease through gut derived endotoxin induced injury.

So they ran some experiments on rodents, groups were administered either endotoxin or saline solution. Tests of gut permeability and the rodents showed an increase in large intestine permeability in the endotoxin injected rodents which were returning to normal after five months. The changes in permeability in the endotoxin treated rodents was associated with change in a-syn immunoreactivity and pathological accumulation of a-syn in the colon somewhere to patients with PD. The paper also notes that there was neuro-degeneration of an area of the brain that is associated with Parkinson’s locomotive symptoms namely nigrostriatal degeneration, a dopaminergic structure connected to the substantia nigra.
Exposure to bacterial endotoxin generates a distinct strain of α-synuclein fibril

The next paper tells us that Parkinson’s, multiple system atrophy and dementia with Lewy bodies all have deposition of a-synuclein in neurons and glial cells, the patterns and cell type distribution differ in the conditions. The origin of a-synuclein depositions is unknown according to the paper. Viral and bacterial infections have been implicated with Parkinson’s and they mention an animal model using endotoxin has been shown to cause degeneration of dopaminergic function and motor problems.

They tested a-syn samples with and without endotoxin and found that the endotoxin treated samples produced different types of a-syn fibrils. These two structures were then injected into the brain of mice, they were injected into the striatum, this is part of the forebrain known to be involved with dopaminergic inputs, the reward system and thought to coordinate cognition, motor function, action planning, decision-making and so on. The striatum is thought to be central to Parkinson’s disease pathology. Both of these a-syn seeds induced different pathologies in the brains of the rodents. The endotoxin free fibrils induced a widespread and strong pathology throughout the brain by looking at phosphorylated a synuclein, the endotoxin positive fibril structures induced a widespread board weaker pathology by the measure of phosphorylated a synuclein, however these fibril seeds induced relatively strong accumulation of a-syn in the striatum and auditory cortex.The authors also measured distinct differences in the activation of real cells and cytokine genes such as TNF alpha.
Alzheimer’s may be caused by brain’s sticky defence against bugs

Amyloid-beta is a protein thought to cause the death of neurons in Alzheimer’s disease. This next paper looks at data suggesting that this protein is actually involved in protecting against fungal and bacterial infections, they shown various different models that amyloid-beta bind to microbial cell walls and inhibit the pathogen’s ability to adhere to the cells. They showed that a salmonella bacterial infection in the brains of mice would result in rapid accumulation of beta-amyloid, suggesting that this was a protective mechanism against the bacteria.

This salmonella bacterium is a gram-negative or endotoxin containing bacteria.
Can a bacterial endotoxin be a key factor in the kinetics of amyloid fibril formation?
Another paper shows that in cell cultures A-B fibrilogenesis was induced by endotoxin.
Protective role of methylene blue in Alzheimer’s disease via mitochondria and cytochrome c oxidase.

The next paper looks at a substance called methylene blue in regard to amyloid beta peptides and mitochondrial dysfunction. In a previous podcast I looked at nitric oxide and its interactions with red and near infrared light within the mitochondria. Nitric oxide will take up a position in the mitochondria within the cytochrome C oxidase enzyme and will block oxygen and oxidative metabolism. Endotoxin will raise nitric oxide, endotoxin will also raise serotonin, I will probably look at the effect of increased serotonin both systemically and specifically in the brain in future podcast, but here I will just mention that serotonin in the brain lowers oxidative metabolism and brain energy.
The relationships between endotoxins, nitric oxide and inflammatory cytokines in blood and intestinal tissues in experimental Trypanosoma brucei brucei infections.


Methylene blue is known to lower nitric oxide and has a number of antibiotic effects which may lead to lower serotonin. Methylene blue can have a positive effect on amyloid plaques and tangles of proteins associated with dementias. Alzheimer’s disease associated with a decrease in cytochrome oxidase activity and this can be reversed by methylene blue.

And the last paper I’ll look at today is on another cheap and ubiquitous chemical, namely aspirin.
Aspirin protects dopaminergic endotoxin neurons against lipopolysaccharide-induced neurotoxicity in primary midbrain cultures.

There are whole host of reasons why aspirin might be expected to lower the damage from endotoxin and at some point I do intend to go into all of its actions but the paper I’m looking at here was interested in protection from endotoxin induced damage to the dopaminergic neurons in cell cultures. So the relevance to dimensions as the association with these dopaminergic neurons. The cell cultures were treated with aspirin prior to being dosed with endotoxin. Aspirin protected the cultures by inhibiting nitric oxide, TNF alpha, and superoxide production in the microglial cells. Aspirin also increase the production of anti-inflammatory cytokines and both of these actions were thought to explain the previously observed protective effects of aspirin on the dopaminergic neurons.

That is the end of this podcast, next time I’m going to look at some related information around the themes of endotoxin, serotonin, nitric oxide and their influence behaviours and mood disorders.

Bacteria lurking in blood could be culprit in countless diseases
Low-dose endotoxemia and human neuropsychological functions
Systemic LPS Causes Chronic Neuroinflammation and Progressive Neurodegeneration
Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway
Obesity is associated with hypothalamic injury in rodents and humans
Hypothalamic dysfunction in dementia.
The Bacterial Endotoxin Lipopolysaccharide Causes Rapid Inappropriate Excitation in Rat Cortex
Propagation of endotoxin-induced nasal mucosal inflammation to the olfactory bulb along the olfactory neural pathway
Intranasal LPS-Mediated Parkinson’s Model Challenges the Pathogenesis of Nasal Cavity and Environmental Toxins
Acute induction of anomalous and amyloidogenic blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide;jsessionid=D86115BFE0A47B7068A9BC8F96CD3EFC.f04t01
Progression of intestinal permeability changes and alpha-synuclein expression in a mouse model of Parkinson’s disease
Exposure to bacterial endotoxin generates a distinct strain of α-synuclein fibril
Alzheimer’s may be caused by brain’s sticky defence against bugs
Does generalized hypo- oxygenation (hypoxia) allow endotoxin into the brain through the blood brain barrier, thus increasing the risk for Parkinson disease?
Protective role of methylene blue in Alzheimer’s disease via mitochondria and cytochrome c oxidase.
Immunity and emotions: Lipopolysaccharide increases defensive behaviours and potentiates despair in mice;jsessionid=D5FE7F28F6DA29C1C71E6360E4ED2732.f03t04
Long-term changes in blood–brain barrier permeability and white matter following prolonged systemic inflammation in early development in the rat
Inflammation selectively enhances amygdala activity to socially threatening images
Behavioral effects of lipopolysaccharide in rats: involvement of endogenous opioids.
Inflammation-Induced Anhedonia: Endotoxin Reduces Ventral Striatum Responses to Reward
Minocycline attenuates lipopolysaccharide (LPS)-induced neuroinflammation, sickness behavior, and anhedonia
Aspirin protects dopaminergic endotoxin neurons against lipopolysaccharide-induced neurotoxicity in primary midbrain cultures.

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