Parkinson's: Therapies

I am going to start this with a table just showing what I am going to cover in the green column and I will probably mention what is in the blue column. The red column is just there for anyone who wants to help someone with PD as some hings that can be done to help keep the person moving and healthy.

As you might expect, there is a timeline for what drugs to use at what stage in the progression of PD. Levodopa used to just be the treatment for most of the disease, but now it usually begins with MAO-B inhibitors alone, once these begin to fail, Amantadine, then COMT inhibitors or MAO-B inhibitors are used in combination with Levodopa. In late stage PD, dopamine agonists are used, often still with L.DOPA even though this is much less effective at this point. Anticholinergics are used to reduce hand tremor for much of PD but they are used late stage also to aid the dopamine agonists.

Monoamine oxidase-B inhibitors (MAOIs):

There are two types of MAO, A and B, A are more effective at metabolising serotonin and B are more effective at metabolising dopamine and noradrenalin. Drugs such as selegiline are selective for MAO-B and inhibit its action in the neuron and astrocytes to prevent the metabolism of dopamine into homovanillinic acid by covalently binding to the MAO-B enzymes to stop them catalysing reactions in both pathways needed for homovanillinic acid production. Selegiline enters the active site of MAO-B where it is converted into its active form but this form covalently binds to the active site of the enzyme during the catalytic phase preventing dopamine entering the active site. This action is irreversible so new MAO-B must be synthesised to break down dopamine so more dopamine is available to act on receptors. Some evidence shows MAOIs as antioxidants thus making them neuroprotective too which means it should slow the loss of dopaminergic neurons. This drug is usually used as a monotherapy to begin with and then combined with levodopa.

COMT inhibitors (COMTIs): 

I am going to talk about these before L.DOPA because they act on the same two pathways of dopamine metabolism as MAOIs but they act on a different enzyme, catechol-O methyl transferase, which also converts dopamine to homovanillinic acid. These are newer drugs, the most common of which is entacapone. COMT catalyses the transfer of S-adenosyl-L-methionine residues methyl group to a phenyl group in catechol structured molecules, such as dopamine. Entacapone selectively and reversibly inhibits COMT to stop the metabolism of dopamine. This means more dopamine is available and active at the synapse and it is active for longer. The idea of COMTIs and MAOIs is to counter the loss of dopaminergic neurons by increasing the dopamine available in the neurons that are left. I just mentioned that COMT acts on any catechol structure so not only does it act on other catecholamines like adrenaline but L.DOPA (the drug and the chemical that already exists in the body) also has a catechol structure. This precursor to dopamine is either converted to dopamine by decarboxylase or converted to 3-O-methyl DOPA by COMT, stopping this pathway is an extra effect of COMTIs because it means that L.DOPA (and the drug) is converted to dopamine not methyl DOPA. This is an added advantage of this drug and explains why it is more effectively used in combination with levodopa.

This image shows what I am talking about when I say COMT and MAO-B is used in both pathways to convert dopamine to homovanillinic acid, they are just used at different stages.

Levodopa (L.DOPA):

This is the most common and well known drug used to treat PD. The reason the precursor drug is used and dopamine not just given to patients is for one simple reason. Dopamine is a large molecule and the blood in the brain is almost completely sealed off from the brain itself. It is a phenomenon known as the blood brain barrier and it stops all but essential molecules entering the brain. Its evolutionary function is to stop pathogens and toxins etc... entering the brain and killing us. As I said, dopamine is to large to enter by diffusion and it is not an essential molecule because the brain makes its own dopamine so it has no transporter. However, L.DOPA is an essential molecule because the brain needs it to make dopamine, therefore it does have a transporter in the blood brain barrier so can enter easily.

There are some problems and I am going to point some out before going into the mechanism of action. Levodopa enters through the intestine no problem so is orally bioavailable, however because it is a dopamine precursor it is a monoamine and MAOs are abundant in the intestine walls, so 90% of levodopa is metabolised by MAOs. So 10% of the dose has entered the blood and of that 10% only 10% enters the brain because the other 90% is either metabolised in the blood or enters peripheral cells because dopamine is used throughout the body. This poses a further problem, the peripheral cells using dopamine are fine, so there is now excess dopamine in the periphery acting on receptors which causes vomiting and anorexia as well as many other side effects. The good news is that most dopamine receptors in the periphery are D2 receptors and a drug called carbidopa inhibits D2 receptors but is incapable of crossing the blood brain barrier, therefore it limits the peripheral effects of levodopa without compromising the central effects.

The mechanism of action is basically to put the precursor in the dopaminergic neurons and let the cell use it in its normal functioning. Levodopa enters the dopaminergic neurons in the substantia nigra that remain and is converted to dopamine via dopa decarboxylase. The overall effect being the same as the other drugs mentioned, compensate for the loss of neurons by increasing the output of those neurons that remain. If used too early it can lead to excess dopamine which is the believed cause of schizophrenia, so levodopa can cause psychosis but clozapine is an antipsychotic that inhibits D4 receptors, reversing the psychosis without effecting the treatment because motor movement is controlled by D1 and D2 receptors.

In the first image, amantadine is mentioned, many of you will have herd of amantadine and not know why and some of you will know that amantadine is an antiviral drug used to treat flu. It is used in PD because it increases the release of dopamine from vesicles in the synaptic cleft, because well no-one really knows and it acts on M2 proteins in flu viruses so there is no link there either but it works because science. It is useful in early stages but can be combined with L.DOPA which amazingly has showed to increase the effectiveness of levodopa to 79% of people responding to treatment up to late moderate stages. Statistically, it is significant at 0.05.

Dopamine agonists: 

During late stage PD all of the above become in effective because there is only so much you can increase the output of one neuron and eventually the neuron loss is so extensive that those that are left cannot increase their levels to counter that loss. So a different approach to just increasing the output of neurons is needed.

That approach is to activate the dopamine receptors in the striatum largely independently of whether the post synaptic membrane is being innervated by a dopaminergic neuron from the substantia nigra or whether that neuron has been lost. To do this, dopamine receptor agonists are needed, preferably ones specific to either D1, D2 or both (to reduce the chance of psychosis and to limit the areas the drug is activating. Earlier I said that dopamine cannot be used because it is too large to cross the blood brain barrier, agonists are just molecules that activate the receptors, they do not have to be the same or even very similar to dopamine. The agonists used are Bromocriptine which is selective for D1 and D2 and crosses the BBB and pergolide which is D2 selective and crosses the BBB. As you might expect they have similar side effects to levodopa due to the periphery but carbidopa will also help here. Their mechanism of action is just to mimic the action of dopamine in its absence.

Anticholinergics:

These can be used throughout PD for those with a hand tremor, benzhexol is very effective at reducing the hand tremor and they work by inhibiting the muscarinic receptors at the neuromuscular junctions. This is because acetylcholine release to the muscle at the NMJ is what initiates muscle movement. The lack of dopamine acting on D2 receptors in the in the striatum reduces the inhibition of unwanted movement which translates to increased acetylcholine which is enough to cause a tremor in the hand. Antimuscarinics block the muscarinic cholinergic receptors on the muscle to reduce the unwanted movement.

In late stage PD anticholinergics are useful because they inhibit cholinergic neuron activation in the striatum which opposes some dopaminergic action. This means a lower dopamine concentration is needed to elicit the same effect as the usual concentration of dopamine due to the lack of opposing influence of cholinergic neurons.

This diagram shows simply the mechanisms of the drugs above to just give a visual idea of what I am talking about. for the most part it is accurate but it is worth noting that COMTIs also do what MAOIs do and that MAO-B is mostly a neuronal enzyme so should be inside the neuron not in the extracellular matrix.

Deep brain stimulation:

I am not going to go into too much detail on this but in essence it causes action potentials in groups of neurons in the area of the electrode to cause a response, or it can hyperpolarise so in PD it potentially hyperpolarises the striatum to reduce the tremor and other movements. But honestly, so far there is no one clear or believed theory of how DBS works so I am not going to reel off  bunch of equally refutable hypotheses that may or may not be correct. Although, the results show that fully understood or not, it certainly works.

This will the most amazing thing you will have seen all day, probably the most amazing thing for quite a while. This is an amazing scientific and medical achievement not only because of how well it works but also the simplicity of use and that it is not just someone lying there with wires coming out of their head, which is sometimes peoples view of DBS but it is allowing people to have a relatively normal life. The difference really is staggering. Link just in case: https://www.youtube.com/watch?v=mO3C6iTpSGo

That is it for Parkinson's disease.

Parkinson's disease: Theories on the cause

In a general sense PD occurs due to a gradual and progressive loss of dopaminergic neurons specifially in the the substantia nigra which in turn causes a depigmentation of the area because the neurons here cause the area to appear black. The difference can be seen clearly with the naked eye. In addition to this, extensive gliosis occurs which is the infiltration of glial cells, mostly microglia into the area. This happens in areas of damage and neuron loss in a repair attempt and acts as a neural inflammatory response. These are the main hallmarks of PD as well as the always present Lewy bodies. These plaque balls exist inside the nucleus of the neuron (so are not the same as amyloid plaques which are extracelular). There are occlusions consisting of mainly alpha-synuclein and ubiquitin as well as other intracellular proteins that aggregate as the neuron begins to die.

Alpha-synuclein is not a very large protein, only 140 residues. It is generally unfolded, but normally randomly folds to a coil due to its hydrophobic domain. This occurs due to a lysine, threonine, lysine, glutamic acid, glycine, valine (KTKEGV) repeat in the N-terminus domain which leads to the formation of 2 alpha helices. These features suggest it is a membrane bound protein. The hydrophobic domain causes oligomerisation of multiple copies and converts the structure to anti-parallel beta sheet once aggregated. C-terminus has a serine that if phosphorylated, changes the hydrophobic nature and distribution of charge which contributes to its oligomerisation (this makes up a lot of each Lewy body. The significance of Lewy bodies suggests that it probably is not just general damage due to them leading to PD. So what is it?

It could be because alpha-synuclein regulates apoptosis via 3 other proteins: 14-3-3 which regulates cell viability, BAD which is pro-apoptosis and Bcl-2 which is anti-apoptosis. BAD will bind to Bcl-2 to inhibit it and allow apoptosis to begin. (apoptosis is controlled cell death, something your body does at least millions of times a day, but much less in the brain, in general it is safe and needed but unregulated apoptosis is not good).

If BAD is phosphorylated at a serine residue then 14-3-3 can bind to inhibit BAD, thus disinhibiting Bcl-2 to prevent apoptosis. Alpha-synuclein will interact with 14-3-3 to prevent it binding to BAD thus promoting apoptosis. However, this theory only explains PD if alpha-synuclein is being overexpressed. There is evidence that mutated alpha-synuclein is more likely to lead to PD due to incorrect folding. In addition, the aggregation of alpha-synuclein may interfere with ubiquitin which would lead to oxidative damage and therfore cell death, this may explain the presence of ubiquitin in Lewy bodies. Ubiquitin proteins have many roles so if this is the case then it could be many things. There is also the possibility of oxidative stress directly due to alpha-synuclein or oxidation that is independent of these mechanisms or the cause of these mechanisms. (e.g. oxidation could change the expression of alpha-synuclein).


Other potential cell death mechanisms:

Having already mentioned it lets talk about the theory of misfolding alpha-synuclein  before going into the details it is important to note that this theory gives an explanation as to how cell death spreads to other neurons. In the above theory the overexpression would have to occur in all neurons but not start until around 55 (this highly rules out just a genetic answer) but the misfolding theory suggests that the misfolded alpha-synuclein leaves the neuron in vesicles and enters other neurons via endocytosis. It acts like a virus in a way, infecting more cells which is why the disease progresses rather than alpha-synuclein just being overexpressed in all cells which would cause the patient to go from fine to late stage PD. So this theory so far is more attractive. The theory states that misfolding causes the protein to be non-functional and hyper-aggregate which is what causes the formation of Lewy bodies, which then transfer to nearby neurons.

Protein misfolding is not as uncommon as it sounds with an estimated 1 in 3 translated, folded proteins are incorrect, whether it be in folding or damaging events after folding there are many opportunities for misfolding. For the most part molecular chaperones called heat-shock proteins can refold proteins or mark them for proteolysis. Usually ubiquitin tags targeted proteins forming a polymer that can be degraded by 26S proteasome. The misfolding of synuclein can form pore structures for other Lewy bodies to exit and 'infect' other cells. In PD synuclein aggregates heavily which can make the ubiquitin-proteasome pathway redundant because it is too aggregated to enter the proteasome pore to be degraded but cells have another system in place to prevent these aggregations causing damage. It is called macroautophagy and is a process of an autophagosome engulfing the aggregated protein and entering the lysosome via endocytosis where the whole thing is degraded. Autophagosomes are large enough to internalise whole organelles so can deal with large aggregated proteins but in PD the synuclein aggregates to a point that neither process works which causes either necrosis or apoptosis, leaving Lewy bodies behind. Once in the ECM they attract microglia and inflammation which causes more damage and increases symptoms.

ubiquitin-proteasome pathway: I have spoken a little about this just but I want to go into ubiquitin. Ubiquitin has 3 enzymes, E1 which is an activating enzyme, E2, a conjugating enzyme and a ligating enzyme- E3 also called parkin protein. This protein catalyses the oligomerisation of ubiquitin and proteins that need degrading. There are a few missence mutations that can appear in parkin that lead to misfolding of the enzyme preventing it from interacting correctly with ubiquitin and change its solubility leading to progressive aggregation and toxicity. These mutations are often described as recessive disorders but increasing evidence suggests only one allele needs to be mutated to massively increase the risk of the PD. This certainly shows some evidence for it being at least a cause of PD, in addition, evidence suggests that an increase in age naturally decreases parkin solubility causing its aggregation, it would also mean that the synuclein aggregates cannot be degraded in the proteasome pathway because the E3 cannot ligate the ubiquitin. So this may be beginning to provide some insight into the formation of sporadic PD, something we still know very little about.

Oxidative stress and mitochondrial dysfunction: These are two separate mechanisms but very closely inter-related. Mitochondria are the primary cause for the generation of reactive oxygen species in cells. ROS are formed mainly through electron passage through complex I and III and the main ROS are superoxide radicals, which are one electron being added to oxygen during oxidative phosphorylation. For the most part superoxide dismutase converts it to hydrogen peroxide which is then detoxified by catalase. Unfortunately, is iron ions are present, hydrogen peroxide can be converted into hydroxyl radical due to a Fenton reaction and this molecule is highly reactive and causes massive damage in cells. Low ATP production corresponds to increased ROS level and it seems that postmortem the mitochondrial complex I has very low activity in the substantia nigra neurons suggesting a cause of PD to be a deficiency in mitochondrial complex I leading to ROS which cause cell death. It has also been found that the catalytic subunits of complex I contained oxidised proteins, correlating to decreased electron transfer, thus low ATP, increasing oxidation through complex I. this is an interesting loop and is possible, but it poses a chicken or the egg debate, how did the first oxidation occur to start this? Another question to ask is how PD mainly affects dopaminergic neurons when nearly all cells contain mitochondria.

An important thing to note is that mitochondria are more bacterial than they are eukaryotic, there is a substantial body of evidence that suggests that they are in fact prokaryotic organisms that entered eukaryotic cells millions of years ago and act in a symbiotic relationship because they produce ATP at a rapid rate and in huge numbers and they gain protection and nutrients from eukaryotic cells. This is supported by the fact their DNA is circular as in bacteria and mitochondria DNA only comes from your mother, not your father because they reproduce independently of the rest of the cell and carry their own DNA so the mitochondria from the egg reproduce rather than having genes from both parents mix to create it, which is the case with all other organelles.

Mitochondrial DNA encodes 13 proteins which are all subunits for ATP production mechanisms, it also encodes tRNA and rRNA for the protein synthesis of these genes. Mitochondria are not very big and much of the space is taken up for its function so this means that any ROS generated are always in close proximity to DNA. The close proximity accompanied by the lack of DNA protection (nucleotide excision repair, replaces damaged DNA in each cell at least 10,000 times a day but is not present in mitochondria) and no protective histones means mutations are possible and the presence of ROS makes mutations even more likely. The mutations in substantia nigra are often deletions and this type of mutation is not seen in the mitochondria in other cells like pyramidal cells, even in aged brains, suggesting that these deletions are specific to dopaminergic neurons which may then increase their susceptibility to oxidative stress, thus potentially explaining why dopaminergic neurons in the nigra are lost and not other neurons in other areas.

The final Parkinson's post will be on some of the treatments.

Apologies

Sorry for the hiatus, with bank holiday and working last week I have been very busy. I am working on the next Parkinson's post and because I will be back at uni very soon, I am going to endeavour to do one topic a week from now on, so only one or two posts but hopefully I will get through quite a few things.