Recently a really interesting research report was published that presented several rather amazing findings.
The researchers forced dopamine-producing cells in a rodent brain to start making a protein called neuromelanin and by doing this, they witnessed the occurence of Parkinson’s-like features (motor issues, Lewy body-like structures, and cell death).
The report also suggested a method by which this outcome could be reduced or rescued.
But the amazing part is that neuromelanin was previously considered to be protective and this new finding suggests we may need to rethink that idea.
In today’s post, we will discuss what neuromelanin is, what this new report found, and how this new knowledge could be useful in the context of Parkinson’s.
Prof Heiko Braak. Source – Memim.com
This is Prof Heiko Braak.
Many years ago, he sat down and examined hundreds of postmortem brains from people with Parkinson’s.
He had collected brains from people who passed away at different stages of the condition, and was looking for any kind of pattern that might explain where and how the disease starts. His research led to what is referred to as the “Braak staging” model of Parkinson’s – a six step explanation of how the condition spreads up from the brain stem (the top of the spinal cord) and into the rest of the brain (Click here and here to read more about this).
The Braak stages of PD. Source: Nature
Braak found that certain populations of cells in the brain were more vulnerable to Parkinson’s than others, such as the dopamine neurons in a region called the substantia nigra, the noradrenergic neurons of the locus coeruleus, and the neurons of the dorsal motor nucleus of the vagus (don’t worry about what any of those names actually mean, I’m just trying to sound smart and make you think that I know what I’m taking about).
One feature that all of these populations of neurons all share in common – in addition to vulnerability to Parkinson’s – is the production of pigment called neuromelanin.
What is neuromelanin?
Neuromelanin is the brain-version of a pigment called melanin, which is found in the skin, eyes, and hair. It is the substance that gives skin & eyes their colour. Dark-skinned people have more melanin in their skin than light-skinned people.
In the brain, certain types of cells, such as the dopamine neurons, produce neuromelanin.
Neuromelanin (the brown patches) in dopamine neurons. Source: Schatz
Neuromelanin appears in large quantities in the human brain, in much lesser amounts in some of the non-human primates, and is almost absent from the brain in many lower species (like mice and rats).
And dopamine neurons in the human brain produce so much neuromelanin that you can visualise it with your bare eye. As you can see in the image below, the Parkinsonian brain has less dark pigmented cells (in the substantia nigra region of the midbrain). As dopamine neurons – which are affected in Parkinson’s – are lost, so too is the dark pigment of neuromelanin.
The dark pigmented dopamine neurons in the substantia nigra are reduced in the Parkinsonian brain (right). Source:Memorangapp
Interresting. How is neuromelanin made?
The mechanism of neuromelanin synthesis are poorly understood, but it is generally agreed that melanin can be made via two methods:
- By the oxidation of dopamine
- By an enzymatic reaction
Sorry I asked. What does any of that actually mean?
So, melanin can firstly be made by the oxidation of dopamine.
What is oxidation?
Oxidation is the loss of electrons from a molecule, which in turn destabilises that particular molecule. Think of iron rusting. Rust is the oxidation of iron – in the presence of oxygen and water, iron molecules will lose electrons over time. Given enough time, this results in the complete break down of objects made of iron.
Rusting iron. Source: Thoughtco
The exact same thing happens in biology. Molecules in your body are constantly going through a similar process of oxidation – losing electrons and becoming unstable.
And this also applies to the protein dopamine.
Once dopamine is made, it is proned to oxidation, and when it is oxidized, dopamine is converted into aminochrome, which undergoes further changes before forming the dark pigment neuromelanin (Click here for a review of this process).
The production of neuromelanin from dopamine. Source: Gale
Ok, and the second way of making melanin? The enzymatic pathway?
Yes, there is a second way by which melanin can be generated and this is the method that is used outside the brain, in your skin and hair for example. This process involves the oxidation of Levodopa to the melanin precursor DOPAquinone. An enzyme called tyrosinase is responsible for this process.
Is tyrosinase present in the brain?
It has been reported that tyrosinase is present in the human brain:
Title: Tyrosinase-like activity in normal human substantia nigra.
Authors: Miranda, M., Botti, D., Bonfigli, A., Ventura, T. & Arcadi, A.
Journal: Gen. Pharmacol. 15, 541–544 (1984).
In this study, tyrosinase-like activity was found in the substantia nigra of healthy postmortem brain tissue. And this result was replicated by an independent research group (Click here to read more about this).
Interesting. So if tyrosinase is present in the substantia nigra region – which is affected by Parkinson’s – could tyrosinase be involved with Parkinson’s?
That is an interesting question.
One that has just recently been addressed:
Title: Brain tyrosinase overexpression implicates age-dependent neuromelanin production in Parkinson’s disease pathogenesis
Authors: Carballo-Carbajal I, Laguna A, Romero-Giménez J, Cuadros T, Bové J, Martinez-Vicente M, Parent A, Gonzalez-Sepulveda M, Peñuelas N, Torra A, Rodríguez-Galván B, Ballabio A, Hasegawa T, Bortolozzi A, Gelpi E, Vila M.
Journal: Nature Communications 2019 Mar 7;10(1):973.
PMID: 30846695 (This report is OPEN ACCESS if you would like to read it)
In this study, the researchers wanted to explore the consequences of inducing high levels of tyrosinase in the dopamine neurons of rats. They achieved this using a carefully engineered virus, in which the disease-causing components of the virus had been removed and the instructions for making tyrosinase were inserted. This made the virus a very effective biological delivery system of tyrosinase. Once the virus infected cells, those cells would start producing high levels of tyrosinase.
Remarkably, 2 months after delivering the virus to the dopamine neurons on one side of the rodent brain, the researchers could clearly visualise dark pigmentation in the dopamine neurons on the injected side of the brain (see the circled area in panel C of the image below). It was only present in the dopamine-producing neurons, and it looked very similar to the neuromelanin found in the dopamine neurons in the human brain.
By 2 months post virus delivery, the levels of neuromelanin in the rat dopamine neurons reached levels equivalent to those found in the substantia nigra of an elderly human (~80 years old), according to postmortem analysis.
But the cells kept producing neuromelanin, and by 4 months, the levels of neuromelanin were equivalent to those found in post-mortem dopamine neurons from people with Parkinson’s. And it was from this moment that the investigators observed something very interesting: a progressive, age-dependent loss of the neuromelanin producing dopamine neurons!
In the bar graph below, the black bars represent the number of dopamine (TH-positive) neurons on the injected side of the rodent brain. Note the gradual reduction over time.
And this loss of dopamine neurons was accompanied by impairments to motor behaviour in various tests, AND the accumulation of extracellular neuromelanin (that is, neuromelanin outside of cells – as cells died, they released their neuromelanin into the surrounding environment of the brain. And this last feature was associated with activation of microglia.
What does that mean? Activation of microglia?
Your brain has many different types of cells. Everyone is aware of neurons – the prima donna cells involved with passing messages around the brain – but there are other equally important support/helper cells. Microglia are one of these helper cells. 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
Microglia are constantly monitoring for trouble, and when they detect a problem they will literally be the judge and executioner in deciding if a sick or damaged cell lives or dies. The microglia will change shape as it becomes activated and it will start to release messenger proteins instructing the surrounding cells what to do. Microglia will also alert the immune system, informing it of any problem.
In this current study, the researchers found that microglia were activated when the dying dopamine neurons started releasing neuromelanin, and this has been shown before (Click here to read more about this).
Interesting. Did the researchers find anything else?
Yes they did.
And this is where the story takes an interesting twist.
You see, in addition to neurodegeneration, the researchers also observed Lewy body-like structures.
What are Lewy bodies?
Lewy bodies are dense circular clusters of protein that are characteristically found in specific regions of the brain in people with Parkinson’s (Click here for more on Lewy bodies).
A cartoon of a neuron, with the Lewy body indicated within the cell body. Source: Alzheimer’s news
The clustered (or aggregated) protein, however, is not limited to just the Lewy bodies. In the affected areas of the Parkinsonian brain, aggregated protein can be seen in the branches (or neurites; see black arrow in the image below) of cells. In the image below, the aggregated protein has been stained brown on a section of brain from a person with Parkinson’s.
Examples of Lewy neurites (stained in brown; indicated by arrows) from a human brain. Source: Wikimedia
So they researchers found Lewy bodies in the neuromelanin-producing rat dopamine neurons?
Yes. And the Lewy body-like structures could be labelled with many of the appropriate markers of a Lewy body (such as alpha synuclein, Ubiquitin, and p62).
A rodent dopamine neuron with a Lewy body-like structure, sitting in neuromelanin. Source: Nature
But here are the important details regarding these Lewy body-like structures:
- They were completely restricted to neuromelanin-producing dopamine neurons
- Their number peaked at 2 month post virus delivery
- The Lewy body-like structures were substantially reduced by 4 months post virus delivery,…once the neurodegeneration began
The researchers found that the Lewy body-like structures were most present at 2 months after the delivery of the tyrosinase producing virus. This was before the neurodegeneration actually started.
In addition, the neurons containing Lewy body-like structures were the ones that preferentially degenerated in these animals.
And this actually agrees with what Prof Heiko Braak originally found in the postmortem brains of people who passed away with Parkinson’s: the number of Lewy bodies in the dopamine neurons of Parkinsonian brains at advanced stages of the condition were much lower than those observed in the earlier stages of PD (Click here to read more about this).
So Lewy bodies are associated with cell death?
I’m not sure.
These results fly in the face of results from other research groups which found the opposite result:
Title: Contribution of somal Lewy bodies to neuronal death
Authors: Tompkins MM, Hill WD
Journal: Brain Res. 1997 Nov 14;775(1-2):24-9
In this study, the researchers wanted to determine if signs of cell death were more common in dopamine neurons that contained Lewy bodies than dopamine neurons without Lewy bodies. They had previously perfected a staining protocol that allowed them to identify cells in the early stages of apoptosis (or programmed cell death), and they now wanted to use that protocol to assess the role of Lewy bodies.
They stained sections of brain from four postmortem cases (2 cases of Alzheimer’s/Parkinson’s and 2 cases of Dementia with Lewy bodies). Over 1200 neurons were assessed per brain, and what the investigators found was that the total number of neurons that were displaying signs of apoptosis was much greater in the dopamine neurons without Lewy bodies.
That is to say, the majority of dopamine neurons undergoing apoptotic cell death did not appear to contain Lewy bodies (in fact, one of the cases had NO Lewy body-containing dopamine neurons undergoing apoptotic cell death – Lewy bodies were present in the specimen, but there was no sign of cell death in those Lewy bodies-containing cells). This finding led the researchers to conclude that some dopamine cells may be dying before Lewy bodies have a chance to form, which would suggest that the presence of a Lewy bodies does not predispose a neuron to cell death.
And this result was replicated by another independent research group:
Title: Lewy pathology is not the first sign of degeneration in vulnerable neurons in Parkinson disease.
Authors: Milber JM, Noorigian JV, Morley JF, Petrovitch H, White L, Ross GW, Duda JE.
Journal: Neurology. 2012 Dec 11;79(24):2307-14.
PMID: 23152586 (This article is OPEN ACCESS if you would like to read it)
The researchers who conducted this study examined the extent of dopamine neuron dysfunction and degeneration among postmortem sections of brain from 17 healthy controls, 33 with incidental Lewy body disease, and 13 cases of Parkinson’s (with a mean disease duration of 8.3 years). While the density of dopamine neurons (as measured by their total number) was observed to decrease as the Lewy body burden became more severe, a significantly high percentage of dopamine cells were found to be dysfunctional or dying without any Lewy bodies present inside those cells. These results suggest that significant neurodegeneration and cellular dysfunction precede the appearance of Lewy bodies in dopamine neurons,… which basically challenges the idea that Lewy bodies are playing a pathogenic role of Parkinson’s.
And this phenomenon of cell death occurring before protein aggregation does not appear to be specific to Parkinson’s – similar results have been observed in Huntington’s disease (Click here to read more about this).
So, it is fair to say that we are not sure about the role of Lewy bodies. The results of this new study is an interesting finding though.
That’s interesting. So what did they conclude from the study?
Hang on a second, they haven’t finished yet.
The interesting twist in this tale is only just beginning to twist.
One of the proteins that aggregates in Parkinson’s (and forms parts of the Lewy body we discussed about) is alpha synuclein. We have spoke at length about alpha synuclein on this website (Click here to read a recent SoPD post), as it is believed to be one of the chief villans in Parkinson’s.
Alpha synuclein protein. Source: Wikipedia
And given it’s role of “public enemy number one” in Parkinson’s, the researchers conducting the neuromelanin study decided to test whether it was having a role in the formation of Lewy body-like structures in the neuromelanin-producing dopamine neurons.
They did this by using rats that have been genetically engineered not to produce any alpha synuclein protein. These rats are happy and normal enough, and the absense of alpha synuclein does not appear to cause any issues. So the investigators decided to inject these rats with the ‘tyrosinase-producing virus’ and watch to see what happened.
They may have been expecting to see the absence of Lewy body-like structures and no neurodegeneration, but…
The Lewy body-like structures were still present.
Note the p62 (red) blobs on the right hand side of the image below, which do not have any alpha synuclein (green) colouration. ‘aSynKO’ refers to alpha synuclein knock-out – meaning that alpha synuclein has been removed from the DNA.
IN ADDITION, the absence of alpha synuclein protein had no impact on the neurodegeneration of the dopamine neurons – the same number of dopamine (TH-positive) cells died in both normal (‘wild-type’ or WT) rats as the alpha synuclein knock-out (aSyn KO) rats.
This finding suggested that alpha synuclein was not contributing to the neuromelanin-linked cell death in these animals.
Ooohh. Interesting. So summing up?
No, not yet. There is still more!
Given the appearance of Lewy body-like structures in the neuromelanin-producing cells, the researchers wondered whether the recycling/waste disposal system of the cell had been disrupted. Was the build up of clustered protein the result of a failure of the cell to remove old, used protein? Had this vital process become inhibited by the build up of neuromelanin in some way?
Their analysis suggested that it was.
The continuous buildup of neuromelanin ultimately exhausted the waste disposal system (the autophagic capacity) of the dopamine neurons, resulting in a general failure of proteostasis – the maintanence of a correct balance of proteins in a cell – and subsequently the degeneration of neuromelanin-laden neurons.
So if we reduce neuromelanin levels do you think we could slow do this failure and loss of cells?
This is exactly what the researchers asked.
They tested this idea by introducing high levels of a protein called transcription factor EB (or TFEB) in neuromelanin-producing dopamine neurons. TFEB is a master regulator of the recycling/waste disposal system (aka autophagy) – click here for a review of TFEB.
By introducing high levels of TFEB in neuromelanin-producing dopamine neurons, the researchers found that TFEB decreased levels of neuromelanin, markedly reduced the formation of Lewy body-like structures, rescued the loss of dopamine neurons, and improved the performance of the rats in behavioural tests of motor ability.
In the bar graph below, note that raising TFEB levels in rodents injected with the tyrosinase virus (hTyr) results in no significant difference in the number of dopamine (TH-positive) neurons between the injected and control side of the brain (far right column).
Wow! Where can I get me some of that TFEB stuff?
As far as I am aware there are no clinically available activators of TFEB (and I’d be happy to be corrected on this). There have been screening studies, evaluating large libraries of compounds (Click here to read one example).
But of particular interest to our discussion here is this report:
Title: Effects of ambroxol on the autophagy-lysosome pathway and mitochondria in primary cortical neurons
Authors: Magalhaes J, Gegg ME, Migdalska-Richards A, Schapira AH.
Journal: Sci Rep. 2018 Jan 23;8(1):1385.
PMID: 29362387 (This report is OPEN ACCESS if you would like to read it)
In this study, the researchers (who are also behind the RAPSODI study we discussed in a previous post – click here to read that post) treated mouse cortical neurons in cell culture with a respiratory medication called Ambroxol and they noted a significant increase in TFEB being activated and shifting to the nucleus of the cells (where it could activate additional waste disposal pathways).
Increase in TFEB in the nucleus after Ambroxol (AMBX) treatment. Source: Nature
What is Ambroxol?
Ambroxol is a commonly used treatment for respiratory diseases (the respiratory system being the lungs and related components required for breathing). Ambroxol promotes the clearance of mucus and eases coughing. It also has anti-inflammatory properties, reducing redness in a sore throat. It is the active ingredient of products like Mucosolvan, Mucobrox, and Mucol.
Ambroxol. Source: Skinflint
Researchers have previously reported beneficial effects of ambroxol treatment in models of Parkinson’s, which ultimately lead to a clinical trial.
We are currently awaiting the results of that clinical trial, which is named AiM-PD – Ambroxol in Disease Modification in Parkinson Disease). It was is a phase IIA prospective, single-centre, open label clinical trial to evaluate the safety, tolerability and pharmacodynamic effects of Ambroxol in Parkinson’s (Click here to read more about this trial).
This trial, which is funded by the Cure Parkinson’s Trust and the Van Andel Research Institute (USA), has been conducted at the Royal Free Hospital in London (UK). The study has involved 20 people with Parkinson’s self-administering Ambroxol (in 60 mg per tablet) over a 6 month time frame. The participants were given 5 escalating doses of the drug for the first few weeks of the study (from 60 mg three times per day, gradually building up to 420 mg three times a day after the first month of the study). It will be interesting to see the results of this study later this year.
For those interested in reading more about it, click here for an interesting OPEN ACCESS review of waste disposal enhancing agentsin the context of Parkinson’s.
Is this the first time anyone has ever investigated tyrosinase in the context of Parkinson’s?
Another independent research group published this study more than 10 years ago:
Title: Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson’s disease.
Authors: Greggio E, Bergantino E, Carter D, Ahmad R, Costin GE, Hearing VJ, Clarimon J, Singleton A, Eerola J, Hellström O, Tienari PJ, Miller DW, Beilina A, Bubacco L, Cookson MR.
Journal: J Neurochem. 2005 Apr;93(1):246-56.
PMID: 15773923 (This report is OPEN ACCESS if you would like to read it)
In this study, the researchers found that tyrosinase is present at very low levels in the postmortem human brain, but they also induced high levels of tyrosinase in cells in culture and found that the treatment made the cells more susceptibility to oxidative stress. They also conducted an anaysis of DNA and found no association between genetic variations in the region of DNA responsible for the production of tyrosinase and increased/decreased risk of Parkinson’s.
But other research groups have reported additional interesting connections to Parkinson’s, such as this report:
Title: Parkin protects against tyrosinase‐mediated dopamine neurotoxicity by suppressing stress‐activated protein kinase pathways
Authors: Hasegawa T, Treis A, Patenge N, Fiesel FC, Springer W, Kahle PJ.
Journal: J Neurochem. 2008 Jun;105(5):1700-15.
PMID: 18248610 (This report is OPEN ACCESS if you would like to read it)
In this study, the researchers reported that high levels of tyrosinase resulted in increased rates of cell death (apoptosis) in cell cultures. They also reported that the Parkinson’s-associated protein PARKIN could reduce the negative effects of high levels of tyrosinase (Click here to read a previous SoPD post about PARKIN).
So neuromelanin is bad then?
This is the curious part of this story.
Neuromelanin was originally believed to have a protective function.
It was reported that neuromelanin was very good at soaking up inorganic and organic toxins in dopamine neurons (Click here to read more about this). But now this idea appears to be shifting. And as neuromelanin increases with age in the human brain, there may be a threshold at which too much of a good thing, becomes a bad thing.
It will be very interesting to see this research independently replicated and extended by others.
So what does it all mean?
Researchers have published an interesting new study that investigates the role of the pigment neuromelanin in the development of Parkinson’s. Their results suggest that it may have a strong influence.
In a recent SoPD post, we discussed a new evolutionary theory of Parkinson’s. It suggests that some parts of the human brain (such as the region containing the substantia nigra dopamine neurons) have not expanded over evolutionary time as much as other areas of the brain (such as the larger cortical areas of our brain – click here to read that post). This results in an imbalance, because some populations of cells – like the dopamine neurons – have many connections in the newly expanded areas, and this forces them to work harder to do their function. In addition, as we live longer this puts even more pressure on those cells.
Now add to that mix the idea that has been discussed in today’s post: as we age certain cells in our brain accumulate neuromelanin. If that neuromelanin has a threshold, beyond which it becomes toxic, this is only going to add further pressure on an already stretched system… and hey-presto, Parkinson’s.
I find this idea quite appealing from the standpoint of late-onset (after 50 years of age) Parkinson’s.
But, while it may explain the loss of certain populations of cells in Parkinson’s which contain high levels of neuromelanin (such as the dopamine neurons in the substantia nigra, the noradrenergic neurons of the locus coeruleus, and the neurons of the dorsal motor nucleus of the vagus – all of these regions are affected by Parkinson’s), it does not explain the loss of other populations of cells in the brain that do not contain neuromelanin (such as the serotonergic neurons of the raphe nucleus and the cholinergic neurons in the nucleus basalis of Meynert – which are also are affected by Parkinson’s). Thus, while I am intrigued by this new research, I am containing my enthusiasm until I see independent replication and extension of the work.
If the results are reproduced, however, it could provide nice support for experimental treatments like Ambroxol which are targetting the recycling/waste disposal system of the cell. If boosting the clearance of rubbish from a cell can help make the cell function better and reduce the chances of the cell dying, this could represent a method of slowing down/stopping the progression of Parkinson’s.
While I don’t want to raise expectations regarding the Ambroxol clinical trial results, from an academic stand-point it will be interesting to see what they suggest.
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.
The banner for today’s post was sourced from thefourthangelsbowl