The inflammasome field is heating up

When a cell is sick or damaged it will send out signals alerting the immune system that something is wrong. If enough of these molecules are released, they will initate an “immune response” and this process is called inflammation. There is evidence in neurodegenerative conditions (like Parkinson’s and Alzheimer’s) that the inflammation process is involved, and inhibitors of particular aspects of inflammation are being developed as potential therapies for these conditions. Of particular interest are drugs targeting the NLRP3 inflammasome. In today’s post, we will discuss what the NLRP3 inflammasome is, look at new research identifying a novel NLRP3 inflammasome inhibitor, and provide an overview/update of where things are in the clinical testing of NLRP3 inflammasome inhibitors for Parkinson’s.

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Source: Science

One of the hottest areas of Parkinson’s research world is ‘inflammation’ (cheesy pun intended).

What is inflammation?

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

When enough of these messenger proteins are released that the immune system becomes activated, it can cause inflammation.

Inflammation is a critical 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 (called cytokines), 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 there are processes that can amplify the immune response.

One of those processes is 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

This is getting complicated. What is the adaptor protein?

Don’t panic.

If you are not interested in the biology, skip down to the first recap and read on from there.

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”). This is the adaptor protein. 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?

RECAP #1: Inflammation is a natural process that our bodies use to let the immune system know that something is wrong. By sending out tiny messenger proteins, damaged cells can activate the immune system to respond. Sometimes a stronger than normal immune response is required and this is where ‘inflammasomes’ can help to amplify the message being sent to the immune system.

 

So blocking the inflammasome could reduce inflammation?

Yes.

But how is any of this related to Parkinson’s?

So next time you are out to dinner with friends, try to drop the word ‘inflammaging’ into the conversation.

‘Inflammaging’?

It is the idea that as we ‘age’, an imbalance occurs in our ability to manage ‘inflammation’ (hence the combination of the words: inflamm-aging).

Source: PMC

The proposal is that as we get older there is a chronic low-grade inflammation, which may be influential in later cognitive issues or the development of neurodegenerative conditions like Alzheimer’s and Parkinson’s (Click here for a nice discussion on this concept of inflammaging)

Is there any evidence to support this idea?

There is an accumulating body of evidence supporting the idea of inflammaging (for example, click here, here and here to read some of the reports supporting the notion).

In addition, there has been a lot of preclinical work focused on targetting the inflammasome aspect of inflammation (as a potential approach to treating neurodegenerative conditions) which supports the inflammaging idea.

And the results have been interesting. For example, very recently, this research report was published:

Title: Systemic inflammation impairs microglial Aβ clearance through NLRP3 inflammasome.
Authors: Tejera D, Mercan D, Sanchez-Caro JM, Hanan M, Greenberg D, Soreq H, Latz E, Golenbock D, Heneka MT.
Journal: EMBO J. 2019 Sep 2;38(17):e101064.
PMID: 31359456                     (This report is OPEN ACCESS if your want to read it)

In this study, the researchers found that systemic (wide-spread or whole body) inflammation in mice affects microglia in an age‐dependent manner.

What are microglia?

Microglia are some of the helper cells in the brain – they act as the resident immune cells. When infection or damage occurs, the microglia become ‘activated’ and start cleaning up the area.

Different types of cells in the brain. Source: Dreamstime

The researchers reported that the response of microglia in aged mice was reduced compared to young mice (when the mice were challenged with systemic inflammation). But when the researchers repeated the experiment in genetically engineered mice which do not produce NLRP3 (a key component of the inflammasomes discussed above), the age-associated reduction was lost – the older mice were the same as the younger mice.

Next the researchers wanted to determine if this NLRP3 effect could influence a mouse model of Alzheimer’s, so they assessed mice that were genetically engineered to produce large amounts of the Alzheimer’s associated protein amyloid precursor protein (APP; bearing both the Swedish mutation and PSEN1 mutation). This genetic manipulation results in mice that display many of the hallmarks of Alzheimer’s, such as the build up of Alzheimer’s beta amyloid associated protein.

These mice usually start to exhibit the accumulation of beta amyloid protein (called plaques) after 5 months of age. So as you can see in the graph on the left side of the image below, at 5 months of age, the APP mice and APP mice with no NLRP3 have similar low levels of plaques (white and light grey columns). There is no difference between the two groups of mice.

Source: PMC

But by 15 months of age, the APP mice have much higher levels of plaques in their brains than the APP mice with no NLRP3 (dark grey and really dark grey columns in the left-hand side graph). In addition, the plaques that were present in the 15 month old APP mice with no NLRP3 were smaller than the APP mice with normal NLRP3 function.

This result suggested to the investigators that inflammation may be reducing the ability of microglia (over time with aging) to clear the accumulating beta amyloid protein in the APP mice. By blocking NLRP3 function, this situation appears to be reversed.

And the effect does not appear to be limited to beta amyloid. Very recently (again), these same researchers published this report:

Title: NLRP3 inflammasome activation drives tau pathology.
Authors: Ising C, Venegas C, Zhang S, Scheiblich H, Schmidt SV, Vieira-Saecker A, Schwartz S, Albasset S, McManus RM, Tejera D, Griep A, Santarelli F, Brosseron F, Opitz S, Stunden J, Merten M, Kayed R, Golenbock DT, Blum D, Latz E, Buée L, Heneka MT.
Journal: Nature. 2019 Nov 20. [Epub ahead of print]
PMID: 31748742

In this study, the scientists reported that the neurodegenerative protein Tau can activate the NLRP3 inflammasome. Tau is another protein that accumulates in conditions like Alzheimer’s and Parkinson’s (we discussed Tau in the previous SoPD post – click here to read that post).

The researchers also found that loss of NLRP3 function reduced tau aggregation, and prevented cognitive issues and neurodegeneration in a mouse that was genetically engineered to produce high levels of Tau. Importantly, they also demonstrated that pharmacological inhibition of the infammasome (with a drug that inhibits NLRP3) can reduced tau aggregation and the neurodegeneration observed in these mice.

Note here that some of the scientists involved in this and the previous report are associated with a company called IFM Therapeutics – more about this below.

RECAP #2: Blocking inflammasome function appears to reduce the pathological signs of neurodegeneration in models of Alzheimer’s. These effects can be achieved via treatment with drugs inhibiting components of the inflammasome (such as NLRP3).

 

This sounds great for Alzheimer’s, but what about Parkinson’s?

So late last year, 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 Parkinson’s brains and 5 control brains, and they found evidence of increased cleaved caspase 1 and ASC (hallmarks of inflammasome activation) in the Parkinsonian brains. When they looked at which cell types were producing the markers of inflammasomes in the PD 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.

This is really interesting. What did they do next?

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.

For those interested, this is a video of Dr Richard Gordon (University of Queensland – and one of the lead scientists on the MCC950 study) discussing this work:

RECAP #3: Evidence of inflammasomes activation in the Parkinsonian brain has been reported. Alpha synuclein can activate inflammasome, and blocking NLRP3 appears to beneficial in preclinical models of Parkinson’s. MCC950 is a NLRP3 inhibitor being developed for Parkinson’s.

 

Are there any clinical trials of MCC950?

This drug has been developed by a company named Inflazome.

And Inflazome has now started a Phase I clinical trials of an NLRP3-inhibitor called Inzomelid (a drug very similar to MCC950). Phase I clinical trials are usually short in nature with the goal of determining whether the drug is safe in healthy humans, at a range of doses. This Phase I study is expected to complete in January 2020 (Source), so we should learn about the results next year.

Once safety and dosing has been determined, the compound can move on to a Phase II trial which will assess safety and tolerability in the patient cohort of interest and hopefully provide some assessment of efficacy.

Is MCC950 the only inflammasome targetting molecule?

No, and this is where the silly title to this post comes into play: the inflammasome field really is heating up!

There are numerous biotech companies actively developing clinical programs around the NLRP3 target. For example, above we briefly mentioned IFM Therapeutics.

On April 1st of this year, the major pharmaceutical company Novartis acquired one clinical and two preclinical programs targeting the NLRP3 inflammasome from IFM Therapeutics.

The clinical program is for a drug called IFM-2427 which is a systemic antagonist of NLRP3 being targeted at an array of chronic inflammatory disorders (including gout, atherosclerosis and nonalcoholic steatohepatitis (NASH)). The two preclinical programs, however, include a gut-directed molecule for the treatment of inflammatory bowel disease but (more importantly) a brain-penetrant molecule which is being developed for targetting neurodegenerative conditions like Alzheimer’s and Parkinson’s (Source).

In addition, another inflammation focused biotech company called NodThera has very recently identified its lead NLRP3 targetting drug candidate, called NT-0167 which the company is now developing for clinical evaluation (Click here to read more about this).

And late last year, Genentech, a subsidiary of Swiss pharma giant Roche, bought up Jecure Therapeutics – a biotech firm with a portfolio of preclinical NLRP3 inhibitors aimed at various inflammatory conditions (it is fair to say that this biotech was primarily looking at liver inflammation, but perhaps a neuro-focused pharma like Roche will also explore potential neurodegenerative applications).

For those interested, click here to read more about the biotech efforts to develop NLRP3 inhibitors.

Very good. So summing up, what does it all mean?

Not just yet.

There is still more to discuss.

Que?

So (also) very recently this report was published:

Title: N,N’-Diacetyl-p-phenylenediamine restores microglial phagocytosis and improves cognitive defects in Alzheimer’s disease transgenic mice.
Authors: Park MH, Lee M, Nam G, Kim M, Kang J, Choi BJ, Jeong MS, Park KH, Han WH, Tak E, Kim MS, Lee J, Lin Y, Lee YH, Song IS, Choi MK, Lee JY, Jin HK, Bae JS, Lim MH.
Journal: Proc Natl Acad Sci U S A. 2019 Nov 4. [Epub ahead of print]
PMID: 31685616

In this study, the researchers reported that a tiny molecule called N,N’-Diacetyl-p-phenylenediamine (DAPPD) is an inhibitor of NLRP3.

What is DAPPD?

DAPPD is a molecule that resembles acetaminophen, which has been reported to be neuroprotective in models of Parkinson’s (Click here to read more about this).

Source: Alzforum

The researchers reported that activity of DAPPD reduced the cognitive issues observed in two different types of Alzheimer’s mouse models (APP/PS1 mice and 5×FAD mice). And it also reduced levels of beta amyloid accumulation. The mechanism of action (that is, how DAPPD achieves this effect) is not entirely clear, but it does lower various components of the NLRP3 inflammasome.

So DAPPD may be another yet avenue by which the NLRP3 inflammasome can be targetted.

Are there any naturally occurring NLRP3 inhibitors?

There is one molecule which appears to act inhibit NLRP3, which was (yet again) recently reported in this research report:

Title: Small molecule-driven NLRP3 inflammation inhibition via interplay between ubiquitination and autophagy: implications for Parkinson disease.
Authors: Han X, Sun S, Sun Y, Song Q, Zhu J, Song N, Chen M, Sun T, Xia M, Ding J, Lu M, Yao H, Hu G.
Journal: Autophagy. 2019 Nov;15(11):1860-1881.
PMID: 30966861

In this study, the researchers reported that the small molecule kaempferol protected mice against inflammation and alpha synuclein-induced neurodegeneration by inhibiting NLRP3 inflammasome activation.

What is kaempferol?

Kaempferol is a natural flavonol, a type of flavonoid, found in a variety of plants and plant-derived foods.

Kaempferol. Source: Wikipedia

The researchers found that kaempferol boosted waste disposal (or autophagy) in microglia, which led to reduced levels of NLRP3 protein. So kaempferol can not be considered a direct NLRP3/inflammasome inhibitor as it is not interacting with the protein, but rather indirectly.

Kaempferol has previously been reported to be neuroprotective in models of Parkinson’s (Click here and here to read several examples).

What are the best sources of Kaempferol?

According to Wikipedia, raw capers have 259mg/100g, while saffron has 205mg/100g. Canned capers 131mg/100g and raw arugula has 59mg/100g. From there things like raw kale, raw mustard greens, ginger, and raw common beans have between 25-50mg/100g.

For those interested in all of this NLRP3 research, click here to read a good recent review of the efforts to pharmacological inhibit the NLRP3 inflammasome.

So what does it all mean?

Phew, long post.

I have to admit that while writing this post it sometimes felt like it was never going to end (and I left out a lot). But such is the inflammasome field at the moment! There is a great deal of research being generated in this area and it will be interesting to watch how things develop in the clinical testing domain in the new year.

Specifically, we will get our first indications of whether pharmacological inhibition of NLRP3 is a safe and tolerated approach in humans. If it is, we will probably see additional biotech companies proposing their own forms of inflammasome blocking treatments. And it all of this occurs, we will hopefully see the initiation of a clinical trial evaluating NLRP3 inhibition in Parkinson’s.

Fingers crossed things go according to plan.

 

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