The world according to GARP

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Transportation of material inside of cells is a critical aspect of normal cellular functioning. Any disruption to this activity can cause significant problems.

An interesting aspect of recent genetic analysis work in Parkinson’s has been the number of genetic risk factors for the condition that are associated with cellular transportation activity.

Recently, researchers have discovered that one particular Parkinson’s-associated protein – LRRK2 – interacts with a cellular transport protein complex called GARP.

In today’s post, we will discuss what LRRK2 and GARP do and why their interaction is important.

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John Irving. Source: Achievement

John Irving is not everyone’s cup of tea, but I quite like his books (A Prayer for Owen Meany being my favourite).

In his fourth book (The World According to Garp), Irving wrote about about the life of T. S. Garp. Born out of wedlock to a feminist leader, Garp grows up to be a struggling writer and freestyle wrestler. But it is his interactions with his wife and his mother’s friends & acquaintances that really make Garp’s unusual life a good read.

A young Robin Williams played Garp in the 1982 film adaptation of the book:

But what does this have to do with Parkinson’s?

Well, recently in the field of Parkinson’s research, the interactions of different kind of GARP have made for good reading.

What do you mean?

Earlier this year, this report was published:

Title: The Parkinson’s Disease Protein LRRK2 Interacts with the GARP Complex to Promote Retrograde Transport to the trans-Golgi Network.
Authors: Beilina A, Bonet-Ponce L, Kumaran R, Kordich JJ, Ishida M, Mamais A, Kaganovich A, Saez-Atienzar S, Gershlick DC, Roosen DA, Pellegrini L, Malkov V, Fell MJ, Harvey K, Bonifacino JS, Moore DJ, Cookson MR.
Journal: Cell Rep. 2020 May 5;31(5):107614.
PMID: 32375042                  (This report is OPEN ACCESS if you would like to read it)

In this report, the researchers were interested in determining and better undertstanding which proteins the Parkinson’s-associated protein LRRK2 was interacting with.

What is LRRK2?

Leucine-rich repeat kinase 2 (or LRRK2 – pronounced ‘lark 2’) – also known as ‘Dardarin (from the Basque word “dardara” which means “trembling”) – is an enzyme that has many functions within a cell – from supporting efforts to move things around inside the cell to helping to keep the power on (involved with mitochondrial function).

Fig-2-LRRK2-involvement-in-cellular-mechanisms-Several-data-posit-that-LRRK2-through

The many jobs of LRRK2. Source: Researchgate

The LRRK2 gene is made up of many different regions. Each of those regions is involved with the different functions of the eventual protein. As you can see in the image below, the regions of the LRRK2 gene have a variety of different functions:

image5

The regions and associated functions of the LRRK2 gene. Source: Intechopen

Genetic errors or variations within the LRRK2 gene are recognised as being some of the most common with regards to increasing ones risk of developing Parkinson’s (LRRK2 variants are present in approximately 1-2% of all cases of Parkinson’s).

image1

The structure of Lrrk2 and where various mutations lie. Source: Intech

As the image above suggests, mutations in the PARK8 gene are also associated with Crohn’s disease (Click here and here for more on this) – though that mutation is in a different location to those associated with Parkinson’s. And one particularly common Parkinson’s-associated LRRK2 mutation – called G2019S – is also associated with increased risk of certain types of cancer, especially for hormone-related cancer and breast cancer in women – Click here to read more about this. If you have a G2019S mutation, no reason to panic – but it is good to be aware of this association and have regular check ups.

The G2019S mutation (the name designates its location on the gene) is the most common LRRK2 mutation. In some populations of people it can be found in 40% of people with Parkinson’s (Click here to read more about this). But what is interesting about this mutation is that it gives rise to a LRRK2 enzyme that is hyperactive.

LRRK2 protein. Source: Youtube

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RECAP #1:  LRRK2 is a Parkinson’s-associated gene (functional region of DNA). The LRRK2 gene provides instructions for producing a multi-functional protein. When tiny errors occur in the LRRK2 gene, a hyperactive form of the LRRK2 protein results, which is believed to disrupt normal cellular function leading to the cell death associated with Parkinson’s.

Researchers are interested in characterising the interactions of LRRK2 with other proteins so that they can better understand the mechanisms by which neurodegeneration might be occurring in LRRK2-associated Parkinson’s.

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Ok, LRRK2. Got it. So the researchers were exploring the interactions of LRRK2 with other proteins. What did they find?

They reported that LRRK2 interacts with a protein called VPS52.

What is special about VPS52?

It is involved with transportation of material inside of cells.

How does it do that?

It is a subunit of the Golgi-associated retrograde protein (or GARP) complex.

Golgi-associated retrograde protein?!? What does that even mean?

GARP is a complex or collection of proteins. There are four proteins that form the subunits of GARP, and they are Vps51, −52, −53 and -54.

GARP is a membrane-tethered complex – which means that it is attached – to the surface of the trans-Golgi network. It plays an important role in transporting material from endosomes to the trans-Golgi network.

Mmmm, interesting. Two questions: # 1. what are endosomes?!?  And # 2. what is the trans-Golgi network?!?

Answer #1.

On a relatively continual basis, small parts of a cell’s outer surface (or membrane) are being brought inside the cell. This is a process called endocytosis.

It occurs when the cell needs to consume resources from the outside world in order to find what it requires to function and survive. As a section of cell membrane is brought into the cell, it forms what is called a vesicle (which is a term used to refer to small spherical bags of stuff inside cells). Given the process by which these outer membrane vesicles are formed, they is referred to as endosomes (sometimes it is also called a vacuole).

Endocytosis. Source: Socratic

Once the endosome is inside the cell and detached from the rest of the membrane, the contents of the endosome can be broken down by various enzymes into basic components that the cell can use to make useful things like proteins.

Which brings us to:

Answer #2.

Protein production in cells occurs in the endoplasmic reticulum (or ER), which is a network of tubules connected to the nucleus. RNA comes out of the nucleus, providing the instructions for making all kinds of proteins. Once the basic components of those proteins are made, they are then transported to the Golgi apparatus which is responsible for modifying them, sorting them and and packing them into vesicles. These vesicles can then be transported to wherever they are needed in the cell or taken to the cell membrane where the contents can released into the extra-cellular space.

ergolgi

The ER to Golgi pathway. Source: Welkescience

Ok, so endosomes bring stuff into the cell from the outside world, and the golgi is involved with preparing proteins to do their jobs. But how are these two things connected? And what does GARP have to do with it?

Conveyance of cargo among structures inside of cells is key to normal functioning. The small bags called vesicles that we mentioned above get shifted around inside of cells, serving different functions. But an important aspect of this activity is the tethering of vesicles to their targets.

And this is where GARP gets involved.

The GARP complex is specifically involved with tethering endosomes to the golgi network and helping to bring these vesicles into the golgi (which aids in supplying the golgi with material that it needs, such as sphingolipids).

GARP. Source: Cell

Any kind of disruption to the activity of GARP can cause accumulation of untethered vesicles that become scattered throughout the interior of cells.

And this occurs in “wobbler mice”, which carry a naturally occuring genetic mutation in the Vps54 gene (Click here to read more about this). As these mice develop, they experience a weakening of muscles and loss of motor neurons – similar to what is observed in motor neurons disease (aka ALS).

Curiously, no genetic variations in the Vps54 gene has been associated with ALS.

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RECAP #2:  GARP is a complex of multiple proteins that forms to play an important role in the tethering of small bags of material (vesicles) to the golgi complex (the structure of the cell where proteins are prepared for their jobs).

Mice that have a genetic mutation in proteins associated with the GARP complex exhibit accumulation of untethered vesicles and neurodegeneration (similar to ALS).

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Ok, so the researchers were found that LRRK2 interacts with GARP. What else did they find?

The investigators found that LRRK2 is involved not only in the localisation of VPS52, but also stablises the interaction between GARP and another protein – Syntaxin-6 – which is also involved in tethering of endosomes to the trans-Golgi network. In this fashion, LRRK2 plays a role in regulating this transportion process.

The researchers also found that the loss of either LRRK2 or VPS52 led to less efficient transport to the trans-Golgi network.

You said above that genetic mutations in LRRK2 are associated with Parkinson’s and cause a hyperactive form of the protein. What happens to GARP when LRRK2 is hyperactive?

When the researchers looked at transport of endosomes to the trans-Golgi network in cells with the ‘hyperactive’ G2019S mutant version of LRRK2, they found that transportation was increased.

And this was a good thing?

It is a good question, and the investigators wanted to check this as well. To better understand the biology, they used C. elegans.

What are C. elegans?

Caenorhabditis elegans (or simply C. elegans) are transparent nematode – also known as roundworms. They are about 1 mm in length, and they have very well characterised nervous systems.

Absolutely useless pub quiz fact: C. elegans have 302 neurons and 56 glial cells in total, which communicate through approximately 6400 chemical synapses, 900 gap junctions, and 1500 neuromuscular junctions (like I said, they are well characterised!).

Caenorhabditis elegans – cute huh? Source: Nematode

Given their well characterised nervous systems, C. elegans provide a useful tool for studying biology. They are easy to grow/maintain, they have an overall life span of 2-3 weeks, and researchers have developed a wide range of tools that allow for genetic manipulation to address specific questions.

The investigators exploring LRRK2 and GARP engineered C. elegans that carry the G2019S mutant version of LRRK2 in their dopamine neurons. These C. elegans display a progressive loss of their dopamine neurons.

Interestingly, when the researchers reduced levels of GARP in the G2019S-LRRK2 C. elegans, they witnessed an exaggeration of the loss of dopamine neurons, which indicated to the researchers that functional GARP reduces the toxicity of mutant LRRK2.

The scientists are yet to confirm if the manipulation of GARP on mutant LRRK2 toxicity in C. elegans also applies to human cells, but they noted in the discussion section of their report that additional genes associated with Parkinson’s (such as VPS35, DNAJC13, and PLA2G6) are all involved in the sorting and transportation of cargo from the endosomes to the trans-Golgi network, raising the possibility that this aspect of cellular activity could be involved with some cases of Parkinson’s.

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RECAP #3:  LRRK2 is involved in the localisation of VPS52. It is also required for the stable interaction between GARP and associated proteins (such as Syntaxin-6) which are involved in tethering of vesicles to the trans-Golgi network.

Mutant LRRK2 increases transport to the trans-Golgi network. Reducing GARP levels results in an intensification of mutant LRRK2-associated toxicity.

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This GARP stuff is all very interesting, but is there any therapeutic aspect to this research?

In their study, the researchers demonstrated that they were able to reverse the mutant LRRK2-induced trafficking defects in cells using a LRRK2 inhibitor.

What is a LRRK2 inhibitor?

Givent that Parkinson’s-associated genetic variants in LRRK2 (like G2019S) can cause a hyperactive form of the protein, biotech companies have been trying to develop inhibitors of the protein in an effort to tone down the hyperactivity – lowering the activity of the mutant protein back to normal and hopefully slowing the progression of Parkinson’s.

The best example of this is a company called Denali Therapeutics.

Denali has recently formed a major collaboration with the pharmaceutical company Biogen to co-develop and co-commercialise small molecule inhibitors of LRRK2 for Parkinson’s (Click here to read the press release).

We have discussed this news in a previous SoPD post (Click here to read more about this).

It is interesting to note though that the administration of a LRRK2 inhibitor was able to reverse the mutant LRRK2-induced trafficking defects in cells.

So what does it all mean?

LRRK2 is a protein that has received a lot of attention since it was first associated with Parkinson’s back in 2004 (Click here and here to read more about this). There are over 2,300 research reports on the Pubmed search engine with the key words “Parkinson’s & LRRK2”. We have learnt a great deal about this protein since then, and clinical trials targeting it were initiated in 2017 (Click here to read more about that).

It is a protein that appears to have many functions, and as a result there are many in the research community who believe that rather than modulating LRRK2, we should be targeting the proteins it interacts with. This will hopefully provide a more precise approach, with fewer potential side effects. Thus, there is a lot of research focused on identifying the protein interactions of LRRK2.

In a recent research paper, scientists reported that LRRK2 is binding to and stablising the GARP complex which is involved with the transportation of material inside of cells. This is an interesting discovery as a number of other Parkinson’s-associated protein are also active in this area of cellular activity, and it may be pointing towards a common mechanism underlying the pathology of the condition.

You only grow by coming to the end of something and by beginning something else.”
The World According to Garp

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