Brain. On. Fire.

Inflammation is part of the immune system’s response to damage or infection. It is a very natural process that our bodies undergo when we come into harms way. Researchers at the University of Queensland, have recently demonstrated something interesting about the inflammation associated with Parkinson’s: by inhibiting a very specific part of the inflammatory process, they can reduce the spread of Parkinson’s associated alpha synuclein pathology in models of PD. And they have developed a drug – called MCC950 – that specifically targets that component of the inflammation process which they are now seeking to test in clinical trials. In today’s post, we will discuss what inflammation is, review this new research, and consider what it could all mean for the Parkinson’s community.

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Spot the unhealthy cell – exhibiting signs of stress (yellow). Source: Gettyimages

No silly preamble today – this is going to be a very long post, so we’re diving straight in:

When cells in your body are stressed or sick, they begin to release messenger proteins which inform the rest of your body that something is wrong.

When enough cells release these messenger proteins, it can cause inflammation.

What is inflammation?

Inflammation is a vital part of the immune system’s response to trouble. It is the body’s way of communicating to the immune system that something is wrong and activating it so that it can help deal with the situation.

By releasing the messenger proteins, injured/sick cells kick off a process that results in multiple types of immune cells entering the troubled area of the body and undertaking very specific tasks.

The inflammatory process. Source: Trainingcor

The strength of the immune response depends on the volume of the signal arising from those released messenger proteins.

And the level of messenger proteins being released partly depends on multi-protein structures called inflammasomes.

What are inflammasomes?

Inflammasomes are multi-protein formations that are present inside of cells in your body. They detect pathogenic agents or stressors that have found their way inside of cells, and once these inflammasomes detect something that should not be there, they activate the release of highly pro-inflammatory messenger proteins (called cytokines) such as interleukin-1b (IL-1b) and IL-18.

These cytokines are released into the world outside of the cell and alert the immune system that something is not quite right.

Source: Youtube

There are difference types of inflammasomes and they vary based on what activates them. For example, the presence of RNA from a particular virus may activate one type of inflammasome, while a certain toxin will cause the assembly of a different inflammasome.

For the purpose of keeping things simple in today’s post, we are going to focus on one of the most well characterised inflammasomes.

It is called the NLRP3 inflammasome:

The NLRP3 inflammasome. Source: Twitter

The name of this particular inflammasome is derived from one of the three core components: a protein called NLRP3.

What is NLRP3?

NLRP3 is an abbreviation that is a lot easier to say than “Nucleotide-binding domain, Leucine-Rich-containing family, Pyrin domain-containing-3“.

As I mentioned above, inflammasomes are multi-protein formations. Critically, inflammasomes are made up of three primary components:

• a sensor protein
• an adaptor protein
• a zymogen procaspase-1 (don’t panic about how complicated this last one sounds just yet, we’ll come back to it in a second!).

NLRP3 is a sensor protein.

It act as a “pattern-recognition receptor“. That is to say, NLRP3 is a protein that recognises a particular pattern in the shape or structure of certain ‘activators’ (think, a virus or stressor). These activator patterns are called pathogen-associated molecular patterns (or PAMPs). There are many different forms of sensors, which help to recognise all the various patterns of troublesome agents.

Activation of NLRP3 inflammasome. Source: Nature

When NLRP3 protein is first produced by a cell, it floats around in an inactive state waiting for PAMPs to interact with it and activate it.

NLRP3 can actually be activated by a wide range of agents, including:

• low intracellular potassium concentrations
• Viruses (such as influenza A and Hepatitis C)
• Bacteria (including neisseria gonorrhoeae)
• Bacterial toxins (such as nigericin and maitotoxin)
• Inorganic particles (think titanium dioxide, silicon dioxide, and asbestos)
• Crystallized molecules (like cholesterol crystals and urate crystals in atherosclerosis and gout, respectively)

(The last two groups could result in lysosomal damage/dysfunction – which we see in some cases of Parkinson’s)

Once activated, the NLRP3 protein will start binding to other activated NLRP3 proteins and this is the beginning of the formation of a NLRP3 inflammasome.

And this is where the second component of the inflammasome comes into the picture: the adaptor protein

What is the adaptor protein?

In the NLRP3 inflammasome, there is a protein called PYCARD, which is sometimes referred to as ASC (or “Apoptosis-associated speck-like protein containing a CARD”). For simplicity sake, for the rest of this post, we will refer to the adaptor protein in the NLRP3 inflammasome as ASC.

An interesting feature of the adaptor protein ASC has recently been shown to act in a prion-like fashion (Click here to read more about this). Emerging evidence suggests that ASC is also released from inflammasome‐activated cells as ‘ASC specks’. These ASC specks accumulate in inflamed tissues, where they can continue to encourage the production of mature cytokines. There is also evidence of interactions with other proteins (more on this near the bottom of the post).

When the NLRP3 protein becomes activated and starts binding to other activated NLRP3 proteins, ASC will bind to it and this process attracts the zymogen procaspase-1.

Ok, now can I panic? That sounds really complicated. What is the zymogen procaspase-1?

A zymogen is simply an inactive precursor of an enzyme, and procaspase-1 is the precursor to the enzyme caspase-1.

And what is caspase-1?

Caspase-1 is the enzyme that causes the release of the proinflammatory messenger protein (the cytokines that we mentioned above), IL-1b and IL-18.

So the take home message here is:

By forming an inflammasome, NLRP3, ASC, and procaspase-1 cause the activation of caspase-1 which in turn results in the release of proinflammatory cytokines.

Clear as mud?

If not, watch this video (it does a better job of explaining it all than I do):

Over activation of inflammasomes is associated with several autoinflammatory and autoimmune conditions, and there are numerous efforts being made to develop therapies that target inflammasomes (as we shall see below). And the consequences of over activation of inflammasomes is serious.

Recently we discussed all of the different kinds of ways a cell can die (Click here to read that post), and inflammasomes cause a form of cell death termed pyroptosis. Pyroptosis differs from other forms of cell death in that it requires the activation of the enzyme caspase-1, and (as we discussed above) caspase-1 is activated during pyroptosis by inflammasomes.

For those interested, click here to read a brief review of evidence of pyroptosis in neurological conditions.

But how is NLRP3 associated with Parkinson’s?

So way back in 2013, this research report was published:

Title: Triggering of inflammasome by aggregated α-synuclein, an inflammatory response in synucleinopathies.
Authors: Codolo G, Plotegher N, Pozzobon T, Brucale M, Tessari I, Bubacco L, de Bernard M.
Journal: PLoS One. 2013;8(1):e55375.
PMID: 23383169                   (This report is OPEN ACCESS if you would like to read it)

In this study, the researchers wanted to investigate how the Parkinson’s associated protein alpha synuclein could cause inflammation.

What is alpha synuclein?

Alpha synuclein is one of the most abundant proteins in our brains – making up about 1% of all the proteins floating around in each neuron in your head – and it is a very well studied protein (with over 9700 research reports listed on the Pubmed search engine with the key words ‘alpha synuclein’).

When alpha synuclein protein is produced by a cell, it normally referred as an ‘unfolded protein’, in that is does not really have a defined structure. When it is first produced, alpha synuclein will look something like this:

Alpha synuclein. Source: Wikipedia

In this form, alpha synuclein is considered a monomer – which is a single molecule, just one copy of the protein. It is capable of binding to other molecules, and when it binds to other alpha synuclein proteins, they form what is called an oligomer (a collection of monomers). And these oligomers can have different structures.

In Parkinson’s, alpha synuclein also binds (or aggregates) to form what are called ‘fibrils’.

Microscopic images of monomers, oligomers and fibrils. Source: Brain

And it is believed that these oligomer and fibril forms of alpha synuclein protein may go on to produce the Lewy bodies that characterise the Parkinsonian brain.

Parkinson’s associated alpha synuclein. Source: Nature

In the 2013 study, the researchers exposed blood cells (monocytes, a type of white blood cell) to monomeric and fibrillar versions of alpha synuclein, and they found that both forms of the protein promote the production of the pro-inflammatory cytokine IL-1b. But interestingly, only cells exposed to the fibrillar form of alpha synuclein released the mature form of IL-1b.

The investigators also demonstrated that this fibrillar alpha synuclein induced release of IL-1b required NLRP3 inflammasome activation, which appears to have been brought on by lysosomal dysfunction (the failure of phagocytosis of fribrillar alpha synuclein) followed by increased production of pro-oxidative agents being released into the cell.

Taken together, these results suggested to the researchers that fibrillar alpha synuclein could act as a trigger for causing a strong inflammatory effect in Parkinson’s.

And these results have been expanded upon by other research groups (Click here and here to read some examples)

More recently, an analysis of postmortem brain tissue from people who passed away with Parkinson’s has been conducted:

Title: NLRP3 expression in mesencephalic neurons and characterization of a rare NLRP3 polymorphism associated with decreased risk of Parkinson’s disease.
Authors: von Herrmann KM, Salas LA, Martinez EM, Young AL, Howard JM, Feldman MS, Christensen BC, Wilkins OM, Lee SL, Hickey WF, Havrda MC.
Journal: NPJ Parkinsons Dis. 2018 Aug 15;4:24.
PMID: 30131971                  (This report is OPEN ACCESS if you would like to read it)

In this study, the researchers wanted to characterise NLRP3 activity in the Parkinsonian brain, so they collected postmortem samples from 17 PD brains and 11 control brains. They found that the PD brains had higher levels of NLRP3 RNA than the control brains. When they looked specifically at the dopamine neurons in particular, the investigators found elevated levels of NLRP3 in these cells, AND that labelling of NLRP3 protein overlapped with labelling of alpha synuclein protein, suggesting a close relationship.

The researchers also looked at the DNA of people with and without Parkinson’s (402 individuals with PD and 182 controls), and they found that genetic variations in the NLRP3 gene (which provides instructions for making the NLRP3 protein) were associated with a significantly reduced risk of developing Parkinson’s. Based on all of these results, the scientists concluded that inflammasome activity may impact the progression of Parkinson’s.

And then very recently, this report was published:

Title: Inflammasome inhibition prevents α-synuclein pathology and dopaminergic neurodegeneration in mice
Authors: Gordon R, Albornoz EA, Christie DC, Langley MR, Kumar V, Mantovani S, Robertson AAB, Butler MS, Rowe DB, O’Neill LA, Kanthasamy AG, Schroder K, Cooper MA, Woodruff TM.
Journal: Sci Transl Med. 2018 Oct 31;10(465)
PMID: 30381407

In this study, the researchers began by assessing postmortem human brains for signs of inflammasome activation. They used samples from 5 late-stage PD brains and 5 control brains, and they found evidence of increased cleaved caspase 1 and ASC in the PD brains. When they looked at which cell types were producing the markers of inflammasomes in the brains, they found that the resident immune cells – microglia – were activated and had increased levels of NLRP3 and ASC.

Next the investigators looked at blood samples collected from 21 people with Parkinson’s and they found further evidence of inflammasome activation, suggesting that a ‘systemic’ (body-wide) increase in inflammasome activity may be occurring in Parkinson’s.

The researchers then shifted their attention to animal models of PD, to determine if inflammasome activation is also occurring. Across multiple models (neurotoxin-based, genetic, and alpha-synuclein preformed fibrils), they found evidence of NLRP3 inflammasome activation.

Given that fribrils of alpha synuclein can cause NLRP3 activation (see above), the researchers in this current study wanted to evaluate the response of microglial cells to preformed fibrils of alpha synuclein protein. They collected microglia cells from normal mice and mice that were genetically engineered to have no NLRP3. They grew these cells as separate cultures and then exposed the cells to preformed fibrils of alpha synuclein protein.

They found that even when normal microglia were exposed to preformed fibrils of alpha synuclein protein, they would start to form characteristic ASC specks and release IL-1b 24 hours later. When microglia with no NLRP3 were exposed to preformed fibrils of alpha synuclein protein there was no release of IL-1b or formation of ASC specks (even at 24 hours after treatment). Interestingly, the alpha synuclein exposure did not cause pyroptosis in the microglia from normal mice.

Given the lack of response in microglia with no NLRP3 protein, the investigators next exposed the normal microglia to a drug called MCC950.

What is MCC950?

MCC950 is a potent small-molecule inhibitor of NLRP3.

Some of the researchers involved in this current study, were also part of a previous study which identified MCC950.

Here is that report:

Title: A small molecule inhibitior of the NLRP3 inflammasome is a potential therapeutic for inflammatory diseases
Authors: Coll RC, Robertson AA, Chae JJ, Higgins SC, Muñoz-Planillo R, Inserra MC, Vetter I, Dungan LS, Monks BG, Stutz A, Croker DE, Butler MS, Haneklaus M, Sutton CE, Núñez G, Latz E, Kastner DL, Mills KH, Masters SL, Schroder K, Cooper MA, O’Neill LA.
Journal: Nat Med. 2015 Mar;21(3):248-55.
PMID: 25686105                    (This report is OPEN ACCESS if you would like to read it)

In 2001, a set of diarylsulfonylurea-containing compounds were identified as potent inhibitors of IL-1b. In this more recent study, the researchers characterised of those compounds (named MCC950). They found that pre-treating cells with MCC950 inhibited ASC speck formation in pro-inflammatory situations, but curiously they also found that MCC950 does not prevent inflammasome formation (by directly blocking NLRP3 oligomerization or NLRP3-ASC interactions). The exact mechanims is still to be determined, but MCC950 was found to be a potent inhibitor of the NLRP3 inflammasome.

In the more recent study, when the investigators exposed normal microglia to preformed fibrils of alpha synuclein protein and treated those cells with MCC950, they witnessed a significant reduction in NLRP3 inflammasome activation. Even at very low doses, MCC950 was able to block the release of IL-1b.

As a result of this result, the researchers next turned their attention to models of Parkinson’s. Three models in fact. They tested MCC950 in a neurotoxin model (6-OHDA), a genetic model (MitoPark), and an alpha synuclein model of PD (pre-formed fibrils).

A lab mouse. Source: USNews

Not only did the reasearchers find that MCC950 entered the brain (crossing the blood brain barrier is one of the great challenges of any PD-oriented treatment), but it also improved motor features and reduced the level of dopamine cell loss in all three of these models of Parkinson’s. In addition, MCC950 was found to be a potent inhibitor of inflammation in the brain, reducing IL-1b, Caspase-1, and ASC levels dramatically.

Wow! This is really interesting. So summing up. What does it all mean?

No wait. We’re not finished yet.

We haven’t got to the really interesting part.

Que?

The researchers were interested to have a look at what effect MCC950 treatment may have on the spread of alpha synuclein pathology in the pre-formed fibrils model of Parkinson’s. The analysed the brains of mice 8 months after the fibrils had been injected and they found signs of alpha synuclein in regions beyond the dopamine system in mice that were NOT treated with MCC950 (these regions included the cerebral cortex).

In mice that were treated with MCC950 (following the delivery of pre-formed alpha synuclein fibrils), however, there was a very different picture: While there was no difference in the total amount of alpha synuclein being produced in the brain, there was a marked reduction in the number of alpha synclein aggregates.

These results led the researchers to conclude that that chronic NLRP3 activation contributes to the propagation of pathology seen in the pre-formed fibrils model, and pharmacological inhibition of NLRP3 using MCC950 can effectively reduce this pathological process.

And if nothing I have written here today makes any sense, the Australian researchers behind this study have kindly provided this video explaining it all:

Wow again! Are there any clinical trials of MCC950?

This drug is being developed by a company named Inflazome.

And they are hoping to start a Phase I clinical trials of MCC950 in early 2019. Phase I clinical trials are usually short in nature with the goal of determining whether the drug is safe in humans, at a range of doses. Once safety and dosing has been determined, the compound can move on to a Phase II trial which will provide an assessment of efficacy.

Is Inflazome the only biotech developing NLRP3 inhibitors?

No, Inflazome is definitely not the only biotech developing NLRP3 inhibitors. This is fast becoming a very busy area of research activity for pharmaceutical companies.

And there is also Jecure Therapeutics (although it is fair to say that this biotech is primarily looking at liver inflammation and the treatment of non-alcoholic steatohepatitis (NASH) and liver fibrosis).

Another biotech firm in this area is IFM Therapeutics (we previously mentioned them in the STING antagonists – another class of anti-inflammatory drugs being developed that could be useful in PD – Click here to read that post).

IFM Therapeutics was bought by the pharmaceutical company Bristol-Myers Squibb in 2017, and while they are continuing to develop antagonists of the STING pathway, their primary drug candidate being prepared for clinical testing is an NLRP3 antagonist (Click here to read more about this).

And on top of these examples, there are additional companies working in this space. We should see a lot of NLRP3 inhibitors going to clinical testing in the near future for a wide variety of inflammatory conditions.

Ok, so what do we do while we wait for this drug to get to the clinic?

So one thing readers should NOT do is to start playing/experimenting with broad anti-inflammatories.

There are a lot of different anti-inflammatories widely available, but they have very different mechanism of action and over use (or abuse) of those drugs can have nasty side effects.

What about Ibuprofen?

There has been a lot of evidence to suggest that the anti-inflammatory Ibuprofen can reduce ones risk of developing Parkinson’s (Click here to read an example of the research), and this has resulted in a great deal of interest in this readily available anti-inflammatory medication.

Source: Healthline

The problem is that over use of ibuprofen can lead to complications, in particular gastric bleeding.

Health individuals who take high doses of ibuprofen on a regular basis are 3x more likely to experience gastric bleeding than those who do not take the drug. And significant gastric bleeding can be found in otherwise healthy people as early as three days after starting an ibuprofen regimen (Click here to read more about this).

Thus, long term use of anti-inflammatory medication like ibuprofen is simply not an option. It would be an “out of the frying pan and into the fire” scenerio.

Are there any natural inhibitors of NLRP3?

There has been research published suggesting that certain readily available compounds have inhibitory activities on components of the NLRP3 pathway.

For example:

• EGCG (via green tea – not via supplement) has NLRP3 inhibiting effects – Click here and here for examples of the research, and click here to read a recent SoPD post about EGCG.
• Curcumin – Reduced IL-1beta levels and Caspase-1 inhibition (Click here, here and here for examples of the research)
• Resveratrol – Reduced IL-1beta levels and Caspase-1 inhibition (Click here, here and here for examples of the research)
• Sulforaphane – Reduced IL-1beta levels and Caspase-1 inhibition (Click here, here and here for examples of the research)
• Quercetin – Reduced IL-1beta levels and Caspase-1 inhibition (Click here, here and here for examples of the research)

For those interested in further reading on this topic, I can recommend a recent review of the research behind these (and other) natural inhibitors of components of the NLRP3 pathway – Click here to read that review.

In addition, some of the compounds currently going through clinical trials for Parkinson’s may also have NLRP3 inhibiting effects. For example, the c-ABL inhibitor, Dasatinib have been shown to reduce levels of NLRP3 inflammasome activation (Click here to read more about this). It would be interesting to determine if Nilotinib has similar properties. In addition, GLP-1 agonists and Dipeptidyl peptidase-4 inhibitors appear to also have some NLRP3 inhibiting effects (Click here and here to read more about this).

Interesting. Is this inflammasome stuff specific to Parkinson’s?

It does not appear to be.

At the end of last year, this very interesting report was published:

Title: Microglia-derived ASC specks cross-seed amyloid-β in Alzheimer’s disease.
Authors: Venegas C, Kumar S, Franklin BS, Dierkes T, Brinkschulte R, Tejera D, Vieira-Saecker A, Schwartz S, Santarelli F, Kummer MP, Griep A, Gelpi E, Beilharz M, Riedel D, Golenbock DT, Geyer M, Walter J, Latz E, Heneka MT.
Journal: Nature. 2017 Dec 20;552(7685):355-361.
PMID: 29293211

In this study, the researchers found that ASC specks are released from cells and they latch on to the Alzheimer’s associated protein beta amyloid, and this can drive the assembly of the plaque aggregates that are found in the Alzheimer’s brain.

 

The researchers found that by genetically removing ASC specks from mouse models of Alzheimer’s, they could dramatically reduce the levels of beta amyloid plaque formation. They also found ASC protein inside beta amyloid plaques in samples of postmortem brains from people who passed away with Alzheimer’s, even in an early-stage patient with mild cognitive impairment (it would be very interesting to do a thorough analysis of Lewy bodies in Parkinson’s to determine if ASC specks are found in them). And it should be said, that some of the researchers involved in this study are working with the biotech firm IFM Therapeutics (mentioned above) – so watch this space!

For those readers interested in learning more about this study, there was a useful editorial in the journal Nature on this report (Click here to read that) and an interesting discussion on the Alzforum website about it as well (Click here to read that).

So why doesn’t everyone who has inflammation develop Alzheimer’s then?

We are not sure.

And all of this research needs to be replicated independently before we conclude too much. But it may be that inflammation is not an initiator of a particular neurodegenerative condition, but rather a fascilitator.

And as we were discussing in the previous post (Click here to read that post), research in Parkinson’s supports this idea:

Title: Serum immune markers and disease progression in an incident Parkinson’s disease cohort (ICICLE-PD)
Authors: Williams-Gray CH, Wijeyekoon R, Yarnall AJ, Lawson RA, Breen DP, Evans JR, Cummins GA, Duncan GW, Khoo TK, Burn DJ, Barker RA; ICICLE-PD study group.
Journal: Mov Disord. 2016 Jul;31(7):995-1003.
PMID: 26999434                     (This report is OPEN ACCESS if you would like to read it)

In this study, researchers in multiple Parkinson’s research centres across the UK collected blood samples from 230 people with Parkinson’s (and 93 control subjects). They next looked for markers of inflammation – inflammation is the result of an immune response to damage or infection – in serum. Serum is the component of blood when both blood cells and clotting factors have been removed.

The investigators found that markers of inflammation were higher in serum from people with Parkinson’s than controls. And when they dug deeper into the data and focused on the PD group, the researchers found that high levels of “proinflammatory” marker (and low levels of “anti‐inflammatory” markers) were associated with more rapid motor decline (as measured by the UPDRS) in the Parkinson’s population over the 36 months period of the study (blood and clinical assessments were carried out at multiple times over those 36 months). They also found that higher “proinflammatory” levels were associated with lower cognitive scores at all of the assessment time points.

Thus, it may be that inflammation is a fascilitor of the disease process, and hopefully by reducing levels of inflammation we will be able to slow down the progression of the condition.

So what does it all mean?

Australian researchers have very recently demonstrated that components of the NLRP3 inflammasome pathway are elevated in the Parkinsonian brain. They have also presented data using multiple models of Parkinson’s, that suggest inhibiting the NLRP3 inflammasome pathway could slow the aggregation of the Parkinson’s associated protein alpha synuclein (which may be involved with causing the inflammation).

We often talk about the three core requirements of any ‘curative’ treatment for Parkinson’s (Click here to learn more about this). The first of those core requirement is a disease halting mechanism. If MCC950 is found to be effective in humans that slowing the spread of alpha synuclein pathology, this would potentially be a disease slowing mechanism. It will be very interesting to see this being tested over the next few years (both in the lab and in clinical trials).

And it will be interesting to see how different sub-types of Parkinson’s respond to NLRP3 inhibitors. For example, such a treatment could be of particular useful in PARK2/PARKIN-associated Parkinson’s as the absense of PARKIN appears to increase the impact of the NLRP3 inflammasome (Click here and here to read more about this).

Ok, long post.

I think I’ll stop here.

Interesting topic though, no?

 

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EDITOR’S NOTE: The information provided by the SoPD website is for information and educational purposes only. Under no circumstances should it ever be considered medical or actionable advice. It is provided by research scientists, not medical practitioners. Any actions taken – based on what has been read on the website – are the sole responsibility of the reader. Any actions being contemplated by readers should firstly be discussed with a qualified healthcare professional who is aware of your medical history. While some of the information discussed in this post may cause concern, please speak with your medical physician before attempting any change in an existing treatment regime.

In addition, many of the companies mentioned in this post are publicly traded companies. That said, the material presented on this page should under no circumstances be considered financial advice. Any actions taken by the reader based on reading this material is the sole responsibility of the reader. None of the companies have requested that this material be produced, nor has the author had any contact with any of the companies or associated parties. This post has been produced for educational purposes only.

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