We’re re-branding: It’s now called PARPinson’s

A new research report has been published this week which may point not only towards a new understanding of the biology of Parkinson’s, but also to potentially novel therapies which are clinically available. These exciting new findings involve a DNA repair mechanism called ‘poly ADP ribose polymerase’ (or simply PARP) and a process of cell death called Parthanatos. Biotech companies have developed PARP inhibitors which have been reported to rescue models of Parkinson’s. With a bit of tweaking, this class of drugs could potentially be re-purposed for Parkinson’s. In today’s post, we will look at what PARP is, explain how PARP inhibitors work, review what previous PD research has been conducted on this topic, evaluate the new report, and consider what it means for the Parkinson’s community (Spoiler alert: this will be a long post!).

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Source: Quotefancy

Ah, the good old days!

Remember them. Way back before Netflix. When life was sooo much easier.

You know what I’m talking about.

Back when biology was simple. Remember when DNA gave rise to RNA and RNA gave rise to protein, and that was it. Simpler times they were. Now, everything is so much more complicated. We have all manner of ‘regulatory RNA’, epigentics, splice variants, and let’s not get started on the labyrinthian world of protein folding.

Oh, how I long for the good old days.

Back when a cell could only die one of two ways: apoptosis (a carefully controlled programmed manner of death) and necrosis (cell death by injury):

Source: Researchgate

Now life is too complicated and complex beyond reason or imagination.

Let’s just take the example of cell death that I mentioned above: over the past decade, the Nomenclature Committee on Cell Death (or NCCD – I kid you not there is actually a committee for this) has written up guidelines for the definition/interpretation of ‘cell death’. And as part of that effort they have decided that there are now at least 12 (yes, 12) different ways a cell can die:

Source: Nature

For those of who are interested in reading more about all of these different kinds of cell death, click here to read NCCD committee’s most recent recommendations which were updated this year (2018). Some riveting betime reading.

Which form of cell death applies to Parkinson’s?

Now that’s a really good question!

One that has been studied and the source of debate for a very long time.

To be fair, we don’t really know. But fascinating new research published this week suggests that the Parthanatos pathway could be involved in the cell death associated with Parkinson’s.

What is Parthanatos?

First proposed in 2009 (Source), Parthanatos (which is derived from the Greek Θάνατος or Thanatos – literally the personification of death in Greek mythology) is a form of cell death that involves very specific biological pathways in a cell.

Thanatos. Source: Wikipedia

The Parthanatos form of cell death is characterised by two main features:

• the excessive production of poly-ADP ribose polymer
• the release of a protein called ‘apoptosis-inducing factor’ from the mitochondria

What is poly-ADP ribose polymer?

Poly-ADP ribose (or simply PAR) is part of a DNA repair mechanism in cells.

When a cell is put under stress, there will quite often be damage to the DNA in that cell.

Different forms of stress that lead to DNA damage. Source: Sierraoncology

And given the importance of DNA, mother nature has very wisely devised several very clever systems of DNA repair to maintain the integrity of this precious molecule.

One of those DNA repair mechanisms involves a protein called PARP.

And what is PARP?

Poly (ADP-ribose) polymerase (or PARP) is a family of proteins involved in a number of cellular processes such as DNA repair, genomic stability, and programmed cell death. There are currently 17 different types of PARP.

The research discussed in today’s post will be largely focused on PARP1, but for simplicity sake I will only refer to it as PARP.

The main function of PARP in cells is to detect DNA damage and initiate a response.

When damaged DNA is detected, PARP will bind to the DNA and begin to synthesise chains of poly-ADP ribose (or PAR which we mentioned above). These tendrils of PAR serve as a signalling mechanism for DNA-repairing enzymes, making them aware of the damage and recruiting their help to fix it.

PARP initiating DNA repair mechanisms. Source: bpsbioscience

This is a very good system if the DNA damage is not too bad and if PARP activity is kept under strict control. But problems start to arise when PARP gets a bit carried away and becomes over activated, causing an accumulation of PAR.

You see, if there is too much PAR floating around with nothing to do, it will start to cause trouble.

What kind of trouble?

One of the ways PAR can wreak havoc is by spilling out from nucleus of the cell (where it is usually found) and into the surrounding internal world of the cell. Once outside of the cell nucleus, PAR will head over to places it shouldn’t be, such as the mitochondria.

What are mitochondria?

Mitochondria are the power stations of each cell. They help to keep the lights on. Without them, the party is over and the cell dies.

Mitochondria and their location in the cell. Source: NCBI

Mitochondria can also have a powerful influence of cell survival in other ways.

Theses small bean shaped objects not only provide energy for the cell, but they can also release signalling proteins which can instruct the cell what to do.

For example, when PAR reaches the mitochondria it will bind to a protein called apoptosis-inducing factor, which is a protein that (as the ‘label-on-the-can’ suggests) induces apoptosis, a type of programmed cell death.

Source: Sciencedirect

PAR entering the mitochondria causes apoptosis-inducing factor to be released from mitochondria, and once unleashed apoptosis-inducing factor makes its way to the nucleus where it will start to activate regions of DNA that are involved with shutting down the cell (Click here to read more about this).

Left unchecked, this process will rapidly lead to the cell dying.

And this is Parthanatos in a nut shell (increase in PAR levels, causing apoptosis-inducing factor to be released from mitochondria, resulting in cell death).

What does PARP or Parthanatos have to do with Parkinson’s though?

In 1998, this research report was published:

Title: Inhibition of poly(ADP-ribose) polymerase: reduction of ischemic injury and attenuation of N-methyl-D-aspartate-induced neurotransmitter dysregulation.
Authors: Lo EH, Bosque-Hamilton P, Meng W.
Journal: Stroke. 1998 Apr;29(4):830-6.
PMID: 9550519                       (This report is OPEN ACCESS if you would like to read it)

In this study, the researchers noticed that PARP levels increase dramatically in the brain after an ischemic injury (stroke), and they wondered whether blocking PARP would help or hinder the recovery. They modelled stroke in two groups of rats (one group was treated with the PARP inhibitor 3-AB and the other group were used as controls), and they analysed their brains 24 hours after the induction of stroke. The treated group were given the PARP inhibitor immediately after the stroke was induced.

The investigators found that treating the animals with the PARP inhibitor significantly reduced the area of damage in the brain (the reduction was over 50%).

And this finding got other researchers asking whether PARP could be involved in other neurological conditions, including Parkinson’s.

Which led to this report being published in 1999:

Title: Poly(ADP-ribose) polymerase activation mediates 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism.
Authors: Mandir AS, Przedborski S, Jackson-Lewis V, Wang ZQ, Simbulan-Rosenthal CM, Smulson ME, Hoffman BE, Guastella DB, Dawson VL, Dawson TM.
Journal: Proc Natl Acad Sci U S A. 1999 May 11;96(10):5774-5779.
PMID: 10318960                  (This report is OPEN ACCESS if you would like to read it)

The researchers found that shorty after treating mice with a neurotoxin (MPTP) which kills dopamine neurons, the dopamine neurons in the brain would produce high levels of PARP (in the image below, the dopamine neurons on the substantia nigra pars compacta (SNpc) are visible because the protein PARP has been stained red):

Source: PMC

The researchers next genetically engineered some mice that do not produce PARP and they conducted an experiment in which the no-PARP mice and normal mice were treated with the MPTP neurotoxin. The investigators found that while the normal mice lost more than half of their dopamine neurons, the neurotoxin had little (if any) effect on the dopamine neurons in the no-PARP mice.

Next, these researchers wanted to better understand the process by which this PARP-associated cell death was occuring, and that research lead to this report:

Title: A novel in vivo post-translational modification of p53 by PARP-1 in MPTP-induced parkinsonism.
Authors: Mandir AS, Simbulan-Rosenthal CM, Poitras MF, Lumpkin JR, Dawson VL, Smulson ME, Dawson TM.
Journal: J Neurochem. 2002 Oct;83(1):186-92.
PMID: 12358742                (This report is OPEN ACCESS if you would like to read it)

In this study, the investigators found that following the neurotoxin treatment, PARP was interacting with another protein called p53.

What is p53?

p53 (or TP53) is a protein that has three major functions:

1. controlling cell division
2. DNA repair
3. Apoptosis (or cell death).

p53 performs these functions as a transcriptional activator (that is a protein that binds to DNA and helps produce RNA (the process of transcription)  – see our previous post explaining this).

p53 protein structure, bound to DNA (in gold). Source: Wikipedia

In regulating the cell division, p53 prevents cells from dividing too much and in this role it is known as a tumour suppression – it suppresses the emergence of cancerous tumours. Genetic mutations in the p53 gene can result in run away cell division, and (surprise!) as many as 50% of all human tumours contain mutations in the p53 gene.

Cancer vs no cancer. Source: Khan Academy

With regards to DNA repair, p53 is sometimes called “the guardian of the genome” as it prevents mutations and helps to conserve stability in the genome. This function also serves to prevent the development of cancer, by helping to repair potentially cancer causing mutations. In this role, p53 is known as a tumour suppressor – a mutation in the p53 gene results in a loss of this tumor suppression.

And finally, in cell death, p53 plays a critical role in telling a cell when to die. And (continuing with the cancer theme), if there is a mutation in the p53 gene, fewer cells will be told to die – increasing the risk of cancer occurring. And in this role p53 is known as a tumour suppression. But in the absence of a mutation in the p53 gene or PARKIN to reduce the activity of the gene, p53 levels will gradually rise and instruct the cell to initiate apoptosis.

What does PARP do when it interacts with p53?

When PARP interacts with p53, it stablises the p53 protein – which is no good.

p53 usually has a very short life span, and by stablising p53, PARP is probably seeking its help in DNA repair. But this stablisation is also increasing the risk that p53 will encourage cells to die. And the researchers suggested that it could be this influence of PARP on p53 that may underlie the mechanisms of neurotoxin-induced cell death.

This finding was made before Parthanatos had been discovered, and it highlights another potential mechanism by which over active PARP could be detrimental to cells.

And the finding that PARP over-activation is involved in cell death has been replicated by other independent research groups. In addition, some of those groups have extended the research by looking at drugs that can inhibit PARP in models of Parkinson’s.

Such as this study:

Title: Neuroprotective effects of a novel poly(ADP-ribose) polymerase-1 inhibitor, 2-[3-[4-(4-chlorophenyl)-1-piperazinyl] propyl]-4(3H)-quinazolinone (FR255595), in an in vitro model of cell death and in mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson’s disease.
Authors: Iwashita A, Yamazaki S, Mihara K, Hattori K, Yamamoto H, Ishida J, Matsuoka N, Mutoh S.
Journal: J Pharmacol Exp Ther. 2004 Jun;309(3):1067-78.
PMID: 14985416               (This report is OPEN ACCESS if you would like to read it)

In this study, the researchers noted that over activation of PARP can lead to severe, irreversible depletion of the levels of NAD and ATP.

What are NAD and ATP pools?

Nicotinamide adenine dinucleotide (or NAD) is a protein that plays a very critical role in a wide range of cellular reactions. Importantly, it is essential for the continued production of energy by the mitochondria in cells.

Mitochondria convert nutrients from food into Adenosine Triphosphate (or ATP). ATP is the fuel which cells run on, and NAD is required for the production of ATP.

We have discussed NAD in a previous post on the SoPD website (Click here to read that post), and it is critical for the normal functioning of cells. Severely reducing levels of NAD can be very bad news for cells.

But here is the thing: NAD is also required for PARP to do its DNA repair job.

You see when PARP binds to DNA it uses NAD to produce the chains of PAR that we were talking about above.

The role of NAD in PARP DNA repair. Source: Bpsbioscience

So if PARP becomes over activated in a cell, it can quickly reduce the supply of NAD, causing further trouble for cells.

The researchers in this particular report wanted to determine if blocking PARP activity could help to rescue a neurotoxin (MPTP) model of Parkinson’s, and restore NAD/ATP levels. They used a PARP inhibitor called FR261529, which was originally developed by Fujisawa Pharmaceutical Co., Ltd (which is now part of Astellas Pharma), but this drug is no longer being developed.

They injected mice with the PARP inhibitor 1 hour before giving them the neurotoxin and they found that it exerted a powerful neuroprotective effect and restored levels of NAD, which lead the researchers to conclude that PARP inhibition “could be an attractive candidate for several neurodegenerative disorders, including PD“.

This same research group expanded on these results – publishing a second research report just 4 months later (Click here to read that research report).

And other research groups have also replicated these results (Click here, here, and here to read some examples).

Interesting. But these are all models of Parkinson’s. Is PARP actully relevant in people with Parkinson’s?

Well, about the same time as these PARP inhibitor reports were being published, researcher also reported increased levels of PARP in the dopamine neurons in the postmortem brain of people with Parkinson’s (Click here to read more about this). In addition, there have been reports suggesting that genetic mutations in the PARP genes may actually protect people from developing Parkinson’s (Click here and here to read more about this).

Some research groups have also found that inhibition of PARP reduces alpha synuclein levels (Click here to read more about this).

And then this week, this research report was published:

Title: Poly (ADP-ribose) drives pathologic alpha-synuclein neurodegeneration in Parkinson’s disease
Authors: Kam TI, Mao X, Park H, Chou SC, Karuppagounder SS, Umanah GE, Yun SP, Brahmachari S, Panicker N, Chen R, Andrabi SA, Qi C, Poirier GG, Pletnikova O, Troncoso JC, Bekris LM, Leverenz JB, Pantelyat A, Ko HS, Rosenthal LS, Dawson TM, Dawson VL.
Journal: Science, 2018 Nov 2;362(6414). pii: eaat8407.
PMID: 30385548

In this study the researchers treated neurons grown in cell culture to preformed alpha synuclein fibrils.

What is alpha synclein fibrils?

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

By treating neurons grown in cell culture with preformed alpha synuclein fibrils, the researchers noted an increase in levels of both PARP and PAR, as well as an increase in cell death. When they treated these cells with PARP inhibitors, they found that the levels of PARP, PAR, and cell death all dropped dramatically.

Genetically removing PARP from the cells (using CRISPR gene editing technology) also protected neurons from the toxic effects of preformed alpha synuclein fibrils. These (and other experiments) suggested that preformed alpha synuclein fibrils were primarily killing neurons via parthanatos.

To confirm this result, the investigators next injected preformed alpha synuclein fibrils into both normal mice and mice that were genetically engineered to have no PARP.

And guess what?

Six months after being injected with preformed alpha synuclein fibrils, the dopamine neuron population in the normal mice was reduced by approximately 50%, but the mice with no PARP exhibited no dopamine cell loss. The researchers repeated this experiment with two groups of normal mice, but they treated one group with PARP inhibitor drugs. Again, after 6 months the PARP inhibitor treated mice exhibited little if any cell loss compared to their untreated counterparts (who suffered 50% dopamine neuron loss).

Now, because the rise in levels of PARP were associated with an increase in levels of PAR (when cells were treated with preformed alpha synuclein fibrils), the researchers asked what effect PAR itself may be having.

When they exposed normal human alpha synuclein protein to PAR in a solution, the investigators reported a marked acceleration of protein aggregation. In addition, when they looked at the brains of mice injected with preformed alpha synuclein fibrils, they found that 20% of alpha synuclein was bound to PAR.

On top of this result, exposing both normal cells and cells with no PARP to increased levels of PAR resulted in increased aggregation of alpha synuclein – which suggested to the researchers that it is PAR and not PARP that directly increases levels of alpha synuclein aggregation. By treating the cells with PARP inhibitors, the levels of PAR were also significantly reduced, which in turn resulted in less alpha synuclein aggregation.

This increase in PAR-induced alpha synuclein aggregation resulted in an raised levels of cell death in neurons grown in culture. And in the absence of alpha synuclein, they found that PAR was not toxic (even at very high levels).

Further investigations suggested that exposing preformed alpha synuclein fibrils to PAR was causing them to adopt an even more toxic state. And this demonstrated itself further when the researchers injected either PAR exposed preformed alpha synuclein fibrils or just normal preformed alpha synuclein fibrils into mice: the mice injected with PAR exposed preformed alpha synuclein fibrils had a more rapid loss of dopamine neurons and behavioural/motor problems.

Whoa! But has anyone ever looked in humans? Are there increased levels of PAR in humans?

Yes, this study was published a few years back:

Title: Parthanatos mediates AIMP2-activated age-dependent dopaminergic neuronal loss.
Authors: Lee Y, Karuppagounder SS, Shin JH, Lee YI, Ko HS, Swing D, Jiang H, Kang SU, Lee BD, Kang HC, Kim D, Tessarollo L, Dawson VL, Dawson TM.
Journal: Nat Neurosci. 2013 Oct;16(10):1392-400.
PMID: 23974709                (This report is OPEN ACCESS if you would like to read it)

In this study, the researchers found that PAR levels were increased in the midbrain of people with Parkinson’s (compared to healthy control samples). This analysis was conducted on post mortem sections of midbrain – and the midbrain is where the bulk of the dopamine neurons reside in the human brain.

In the report from this week which we are reviewing today, the researchers also looked at PAR levels in people with Parkinson’s. For their analysis, the investigators used samples collected from two independent groups of people with Parkinson’s (collectively over 100 individuals with PD and 61 controls), and they found increased levels of PAR in the cerebrospinal fluid (this is the fluid that the brain sits in). Intriguingly, one of those cohorts actually demonstrates an association between PAR levels in the cerebrospinal fluid and disease duration – meaning, the longer each person has had Parkinson’s, the higher their levels of PAR.

The researchers summarised their report with the following conclusions:

1. Alpha synuclein is killing cells by activating PARP (via the parthanatos cell death pathway)
2. PARP activation leads to increased levels of PAR which accelerate alpha synuclein aggregation (a feed-forward cycle)
3. PAR levels are increased in the brains of people with Parkinson’s

Given all of these results, the investigators suggested that clinically available PARP inhibitors should be considered for clinical testing in Parkinson’s

Wow! Has a PARP inhibitor ever been tested in Parkinson’s?

Not that I am aware of,… but we can assume that this is going to change very shortly.

PARP inhibitors are currently used in the treatment of certain cancers.

PARP inhibition in cancer. Source: Parp-inhibitors

The most common treatment for many cancers is chemotherapy, which functions by causing fatal DNA damage in cancer tumor cells. Key DNA repair pathways (like PARP), however, are hyperactive in a lot of cancers which results in increased resistance to chemotherapy treatment. But by inhibiting the DNA repair mechanism – by blocking PARP – researchers can potentiate the effect of chemotherapy, and increase the chances of killing off the tumor cells.

And this has resulted in the development of numerous PARP inhibitors by multiple pharmaceutical companies for clinical use in cancer.

Repurposing this class of drugs for Parkinson’s will be difficult, however, as most of the available PARP inhibitors have very poor penetration of the brain. They all have a lot of trouble getting across the blood-brain-barrier – the protective membrane surrounding our brains. And simply using higher doses of these drugs to increase levels actually entering the brain is a non-starter, due to the side effects associated with the PARP inhibitors.

Side effects include nausea (generally in 50% of cases), fatigue (33%), anemia (low hemoglobin levels in the blood), vomiting, and neutropenia (low level of neutrophils, white blood cells important to fighting off infections).

Thus, we will need to wait for compounds that do access the brain before such clinical trials can begin. Ideally, if those drugs could easily access the brain, perhaps we would be able to lower the dose and reduce the chances of negative side effects.

But the good news is that this was an area of VERY active interest for the research field even before this new report was published – Click here to read a rather comprehensive review (written in early 2017) of repurposing PARP inhibitors for the therapy of non‐oncological conditions.

So what does it all mean?

This is Profs Ted and Valina Dawson:

Source: Hopkinsmedicine

They are the husband and wife team at John Hopkins University behind not only much of the research presented in today’s post, but also providers of a lot of the research supporting other experimental therapeutic approaches for Parkinson’s (including Nilotinib and Exenatide). In addition, they were the first to define the Parthanatos cell death pathway (Source).

Two champions of the cause who deserve a special mention here.

Their labs have recently published a research report (which we have reviewed in today’s post) that suggests that the aggregated form of the Parkinson’s associated protein alpha synuclein may be killing cells via the over activation of an enzyme called PARP. PARP over activation leads to increased levels of a protein called PAR, which firstly causes cells to die by the parthanatos cell death pathway, but also accelerates alpha synuclein aggregation by causing futher aggregation – resulting in a feed-forward cycle.

They have also found that treating models of Parkinson’s with PARP inhibitors reduces cell death and the accumulation of alpha synuclein, providing support for the re-purposing of this class of drugs use in Parkinson’s. As mentioned above, this alternative use of these drugs may be initially hampered by the inability of the compounds to enter the brain, but the results of the Dawson’s tireless efforts have provided yet another potential therapy for Parkinson’s and we can now expect a great deal of follow up work focused on this approach.

I don’t like giving opinions on this website, but I find this new report on PARP inhibition in models of Parkinson’s to be hugely exciting. The culmination of a long sequence of dedicated research, making a fascinating and easy story to tell. And it will be very interesting to see how this situation develops.

You can expect to hear more on the SoPD website regarding this topic as things progress.

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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.

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