Treating Parkinson's: A walnut-sized mystery deep in the brain

In the previous article, "In the dye factory that produces noble purple, the antidote for schizophrenia was born", we talked about how the dye industry, which originated from anti-malarial research, developed into the modern pharmaceutical industry. In this process, three anti-schizophrenia drugs emerged: chlorpromazine (CPZ), reserpine, and haloperidol. Research on them all points to a very important neurotransmitter in the brain - dopamine.

Written by Yang Ben (Northwestern University, USA)

Before the 1950s, there were many theories about mental illness, but none of them were reliable. It was not until the 1950s that the three antipsychotic drugs mentioned in the previous article and some hallucinogenic drugs synthesized by dye factories appeared. Research on them led to new theories of schizophrenia, among which the "serotonin theory" became the mainstream at the time [1].

The serotonin theory holds that the main cause of mental illness is the imbalance of the neurotransmitter serotonin. The most important evidence for this theory is the discovery by Bernard Brodie, an academic giant at the time and the "father of modern chemical pharmacology", that the antipsychotic drug reserpine can clear serotonin from the brain (see the previous article for details).

At that time, Avid Carlsson (2000 Nobel Prize winner in Physiology or Medicine) who was visiting Brodie's laboratory also participated in the study of reserpine. Because dopamine and norepinephrine are similar in structure to serotonin, Carlsson asked whether reserpine would also clear dopamine and norepinephrine. But the strong Brodie felt that he had solved the mechanism of reserpine in treating schizophrenia and there was no need to study it further. Later, Carlsson returned to Sweden and continued to study reserpine on his own. As a result, he found that his hypothesis was correct. Reserpine can really clear dopamine and norepinephrine!

But at the time, dopamine was not a well-known neurotransmitter—in fact, whether it was a neurotransmitter at all, or just a product of the synthesis of norepinephrine, was still a hot topic of debate (Figure 1). Even the idea that nerve cells communicated by releasing chemicals called neurotransmitters rather than electrical signals was only just beginning to be accepted in the community. No one cared what dopamine was.

Figure 1. The biosynthetic pathways of dopamine and norepinephrine. Image from Wikipedia, translated by the author. (Click to see larger image)

Perhaps dopamine is important, too? If dopamine is an important target for reserpine, then replenishing dopamine could reverse the effects of reserpine, and dopamine could play an important role in schizophrenia.

Carlsson decided to design an experiment to test his idea.

Mice obviously won't tell us whether they are experiencing hallucinations or other symptoms of schizophrenia (but the latest research suggests they may). However, reserpine is primarily used as a sedative, so we can verify the effects of reserpine and dopamine by observing whether the mice are sedated (immobile) or awake (moving).

If the sedative effect of reserpine is caused by the clearing of dopamine, can injecting dopamine into mice resist sedation and make them move again?

Dopamine cannot cross the blood-brain barrier, so Carlsson chose to inject dopamine's metabolite, levodopa (see Figure 1). As a result, levodopa made the animals excited again from sedation in mice and rabbits treated with reserpine [2]. Then, Carlsson proved that levodopa was indeed metabolized into dopamine in the brain, rather than norepinephrine and serotonin [3]. Later, Carlsson's students found that in several brain regions (including the basal ganglia), the dopamine content was much higher than norepinephrine [4].

A series of evidences finally proved that dopamine is not only a metabolic intermediate, but also an independent neurotransmitter. Therefore, Carlsson proposed that schizophrenia may not only be caused by serotonin, but also by the abnormal function of three monoamine neurotransmitters: dopamine, serotonin and norepinephrine. He also proposed for the first time that dopamine may also directly regulate movement in addition to schizophrenia [5].

Although Carlsson was studying schizophrenia, the logical chain of "dopamine-induced movement" he deduced inadvertently opened a door to the study of another brain disease: Parkinson's disease.

Coincidentally, Parkinson's research eventually fed back into schizophrenia research and ultimately hatched the "dopamine theory" of schizophrenia.

Parkinson's disease

In the 1960s, when research on schizophrenia was underway, important progress was also made in research on Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (AD). Dr. James Parkinson first described Parkinson's symptoms in 1817, so the disease was named after him.

The main motor symptoms of Parkinson's disease include tremor, rigidity, akinesia/bradykinesia, and postural disturbance. Historically, surrealist artist Salvador Dali, boxing champion Muhammad Ali, actress Katharine Hepburn, and the well-known Chen Jingrun and Ba Jin have all suffered from Parkinson's disease. In the United States, two important Parkinson's disease funds also originated from two patients: The Michael J. Fox Foundation for Parkinson's Research, which was established after actor Michael J. Fox was diagnosed with Parkinson's disease, and the Morris K. Udall Centers of Excellence in Parkinson's Disease Research of the NIH, which was established in memory of Congressman Morris Udall who suffered from Parkinson's disease.

People generally believe that Parkinson's disease is a movement disorder and Alzheimer's disease is a memory disorder, but in fact both diseases have a very wide range of effects on the human body. In addition to movement and memory, they also affect emotions, perception and other aspects. It's just that motor impairment and memory impairment are the more obvious main symptoms of the two.

Parkinson's motor symptoms are caused by the death of dopamine cells in the substantia nigra pars compacta (SNc) of the midbrain. Dopamine cells are nerve cells that synthesize and release dopamine. They secrete melanin, so when dissected, you can see that this small area where dopamine cells are concentrated is black, so it is called "substantia nigra" (Figure 2-3).

Figure 2. Coronal section of the midbrain of the human brain. The black part is the dopamine cells in the substantia nigra pars compacta, and the ventral side (i.e., below the black part in the picture) is the substantia nigra pars reticulata (SNr, sometimes also called the sparse part pars diffusa, the cell density is relatively sparse compared to the compact part). | Author provided

Figure 3. Comparing the midbrain sections of healthy people and Parkinson's patients, it can be seen that the substantia nigra cells in Parkinson's patients are reduced or disappeared. 丨Photo provided by the author

The role of dopamine

Why does the death of substantia nigra dopamine cells lead to the motor symptoms of Parkinson's disease?

In 1960, Austrian scientist Oleh Hornykiewicz and his postdoctoral fellow Herbert Ehringer found that dopamine in a brain region called the striatum in Parkinson's patients was significantly reduced or disappeared [6]. Hornykiewicz then proposed for the first time that striatal dopamine may come from the projection of dopamine cells in the substantia nigra of the midbrain to the striatum [7] (What is projection? See Tip 1). In other words, the substantia nigra of Parkinson's patients is reduced/disappeared, and dopamine cannot be secreted, so naturally no dopamine is transported to the striatum.

Later studies found that the striatum plays an important role in the brain's regulation of movement. At the same time, antipsychotic drugs that block dopamine are known to cause side effects similar to Parkinson's symptoms (see previous article for details) - all these clues combined mean that dopamine may play an important role in both Parkinson's disease and schizophrenia.

Tip 1: Projections of nerve cells

Figure 4. Synaptic structure (click to see larger image). Image from GeneTex website, translated and annotated by the author.

There is a huge morphological difference between nerve cells and other cells: in addition to the cell body, nerve cells also have "hands" and "feet" extending out. The "hands" are axons, and the "feet" are dendrites.

A nerve cell usually has only one "hand", but when it reaches out, it will branch out, like multiple "fingers", and can reach far away to communicate with other nerve cells. For example, the dopamine cells in the substantia nigra of the midbrain extend their "hands" all the way to the striatum of the forebrain, which is called a projection pathway. The "hands" of dopamine cells will form many branches in the striatum, communicate with a large number of striatal cells, and regulate the activity of striatal cells by releasing dopamine (like blowing bubbles, see the previous article for details).

The "feet" of nerve cells receive neurotransmitter signals released by the "hands" of other nerve cells, just like tree roots go deep into the soil to absorb water and nutrients. Usually, the "hands" of a nerve cell will communicate with the "feet" of other nerve cells and the body. This place of communication is the circle in the picture, which was named synapse by Sir Charles Sherrington, one of the founders of modern neuroscience. The word synapse comes from the Greek word sunapsis, which means "contact point", and was recommended to Sherrington by the British classical scholar AW Verrall.

Inspired by Carlsson’s research, Hornykiewicz proposed using L-dopa to treat Parkinson’s disease. However, the mainstream view at the time was that Parkinson’s disease was also caused by a lack of serotonin. Hornykiewicz spent a year convincing neurologist Walther Birkmayer to inject L-dopa into Parkinson’s patients—but the neurologist did not believe Hornykiewicz’s theory at all and also believed that Parkinson’s disease was caused by a lack of serotonin [8].

However, Birkmayer was soon slapped in the face. He could not resist Hornykiewicz's persuasion and injected L-dopa into Parkinson's patients. The patients, who were already bedridden and unable to move, stood up and walked like normal people after the injection, just like the sedated mice and rabbits in Carlsson's experiment. This ultimately proved that L-dopa can indeed treat Parkinson's disease[9].

Almost at the same time, similar news came from Japan and Canada that dopamine disappeared in the striatum of Parkinson's patients, and injecting levodopa into the patients could improve their symptoms [10-11].

Later, after the dosage improvement by Dr. George Cotzias[12-13], levodopa was finally approved for the treatment of Parkinson's disease. Cotzias was awarded the Lasker Clinical Medicine Award in 1969. Currently, levodopa is still the main and most effective drug for the treatment of Parkinson's disease.

Hornykiewicz's absence from the 2000 Nobel Prize in Physiology or Medicine (two of the three winners that year, Carlsson and Greengard, were both researchers on dopamine) caused strong dissatisfaction in the academic community, and 250 neuroscientists and doctors jointly wrote a letter to the Nobel Prize Committee to condemn the event.[14]

Hornykiewicz died on May 26, 2020.

At the 13th International Parkinson's Conference in 1999, Hornykiewicz shared five scientific research suggestions with young scientists. The second one was: "When your mentor guides you, you should listen carefully and respectfully; but when you go back to continue your experiments, you should do what you think is right, even if it is different from your mentor's idea." [15]

“Listen attentively and respectfully when your supervisor and teachers give you advice; but after that, you should pursue an experimental plan that you think is the best, even if it differs from their opinions.”

——Oleh Hornykiewicz

Figure 5. Oleh Hornykiewicz (1926-2020)

Systemically sensitive spiny neurons

Now we know that dopamine cells in the substantia nigra of the midbrain project to the striatum, where they release dopamine, regulating striatal cell activity and thus movement. Once dopamine is lost in the striatum, Parkinson's disease can occur. So how is all this regulated?

To find out, we need to understand what the neurons in the striatum look like.

95% of the cells in the striatum are spiny projection neurons (SPNs, also called medium spiny neurons, MSNs in the past, but the name medium is not very accurate, so now they are mostly called SPNs). The "thorns" are where synapses are formed (Figure 6). This means that this type of neuron has many, many information receiving points throughout its body.

Figure 6. After the fluorescent protein is added to the striatal spiny projection neurons SPN, it can be seen that there are many "thorn-like" protrusions on its "feet" (marked by the numbers in the right picture) [16]. (Note: The right picture is the part in the yellow box in the left picture)

The striatum is so called because the projections from the cerebral cortex form fiber bundles (many nerve cell "hands" tied together into bundles), which form stripes in anatomy. These stripes are also called Wilson's pencils because neurologist SA Kinnier Wilson (1878-1937) described them as "pencil-like" nerve fiber bundles (Figure 7).

The synapse formed by the "hands" of the cerebral cortical neurons and the "feet" of the striatal spiny projection neurons SPN is at the top of this "thorn-like" place, that is, the "thorn" is where the cortex and striatum communicate.

Figure 7. The stripes of the striatum (arrows), also known as Wilson's pencils or the pencil fibers of Wilson[17]. (Click to see a larger image)

The movement of our body is regulated by the striatum and several other brain areas, which together form the basal ganglia (literally translated as basal ganglia, but it is not a ganglion, so the author believes that the translation of basal ganglia is more accurate).

Among them, the striatum is the entrance to the basal ganglia, receiving instructions from the cerebral cortex (for example, I want to move my eyeballs); the substantia nigra pars reticulata (SNr, Figure 2) mentioned above is the main exit of the basal ganglia, which outputs the signals processed by the basal ganglia to the brain area responsible for movement in the brainstem, directly regulating movement (for example, controlling the four muscles around the eyeball through the oculomotor nerve), or transmitting them back to the motor cortex of the brain through the thalamus to regulate movement (Figure 8).

Figure 8. The structure of the mouse basal ganglia. The blue spiny projection neurons in the striatum directly project to the substantia nigra (SNr) and the medial globus pallidus (GPm, m stands for medial, now mostly called GPi, i stands for internal), so this cell is also called direct pathway SPNs (dSPNs), and the blue arrows indicate the direct projection pathway; the red spiny projection neurons first project to the globus pallidus (GP, now mostly called the lateral globus pallidus GPe, e stands for external), then to the subthalamic nucleus (STN), and finally indirectly reach the substantia nigra (SNr) and the medial globus pallidus (GPm), so this cell is also called indirect pathway SPNs (iSPNs), and the red arrows indicate the indirect projection pathway [18]. (Click to see the larger image)

Classic Go/NoGo Model

By the end of the 1960s, scientists probably knew that the basal ganglia were involved in regulating movement, but the specific pathways were still unclear. Mahlon DeLong, who was a postdoctoral fellow at the NIH at the time, and his colleagues inserted electrodes into different brain regions of the basal ganglia to record cell activity and its effects on movement, and ultimately mapped out the specific pathways by which the basal ganglia regulated movement.

There are two projection pathways from the entrance of the basal ganglia to the exit of the basal ganglia: the direct pathway (blue arrow in Figure 8) and the indirect pathway (red arrow in Figure 8).

The direct pathway is also called "Go" (movement), and the indirect pathway is also called "NoGo" (immobility, or static). This is because, in the entire structure, except for the subthalamic nucleus (STN) which is an excitatory cell, all other nuclei, including the striatum and substantia nigra pars reticulata, are inhibitory cells.

Inhibitory cells release the inhibitory neurotransmitter GABA (γ-aminobutyric acid), which inhibits the activity of downstream cells and makes them "calm down"; while the subthalamic nucleus releases the excitatory neurotransmitter glutamate, which activates downstream cells and makes them "move".

At this point, we can see the subtlety of the nervous system:

There are inhibitory cells at both ends of the direct pathway. The cells downstream of the pathway should inhibit the cells downstream of it, but its function is inhibited by the upstream cells, "inhibition-inhibition", negative and negative equal positive, which results in promoting movement (Go).

Similarly, the indirect pathway is inhibition-inhibition-activation-inhibition (red arrows in Figure 8: striatum-globus pallidus-subthalamic nucleus-substantia nigra reticulata/globus pallidus medialis). There are three inhibitions, but the final result is still negative (inhibition), resulting in inhibition of movement (NoGo).

The Go/NoGo model is also known as the classic model of the basal ganglia.

Figure 9. Schematic diagram of the structure and function of the mouse basal ganglia. The black box is the basal ganglia nucleus; red represents excitatory neurons/projections, which release glutamate; blue represents inhibitory neurons/projections, which release GABA; and yellow represents dopamine cells/projections, which release dopamine. (Drawn by the author)

Eleven years ago, scientists used a new optogenetic technique (a technique that uses light to regulate the activity of nerve cells) to specifically activate the direct pathway cells in the striatum, which indeed promoted the movement of mice (Go); and specifically activated the indirect pathway cells in the striatum, which indeed inhibited the movement of mice (NoGo), ultimately proving that the Go/NoGo model is generally correct [19]. However, the latest research suggests that the model still needs to be revised (to be discussed in the next article).

Deep Brain Stimulation

In the late 1970s, Barry Kidston, a graduate student in chemistry at George Washington University, secretly synthesized the opium drug MPPP (commonly known as "synthetic heroin") by reading literature and sold it on campus and on the streets. However, his MPPP was not pure and was mixed with another product, MPTP (Figure 10). As a result, a large number of "customers" in their twenties developed Parkinson's disease at a young age.

Later, J. William Langston of the National Institutes of Health (NIH) of the United States determined that MPTP specifically kills dopamine cells in the substantia nigra of the midbrain, causing Parkinson's disease. This is unfortunate for those children who take drugs, but for researchers, MPTP has brought a very important animal model of Parkinson's disease - that is, injecting animals with MPTP to induce Parkinson's disease. Readers interested in this history can refer to the book The Case of the Frozen Addicts co-authored by Langston and Jon Palfreman (the author has not read it and cannot comment on the content of the book).

Figure 10. Chemically synthesized drug MPPP[20]. (Click to see larger image)

Using the MPTP animal model, DeLong found that the subthalamic nucleus (STN)—the only nucleus in the entire basal ganglia with a large number of excitatory neurons—discharges abnormally in Parkinson's disease, and its removal can improve Parkinson's symptoms[21].

At almost the same time, Alim Louis Benabid, a French neurosurgeon, made an unexpected discovery. When he was performing brain surgery on a patient with multiple tremors, he inserted electrodes into different areas of the thalamus for low-frequency stimulation as usual to determine whether the stimulation site was accurate. However, out of curiosity, he increased the stimulation frequency to nearly 100 Hz and unexpectedly found that the patient no longer trembled. Later, Benabid read DeLong's research on the removal of the subthalamic nucleus and tried to give Parkinson's patients high-frequency stimulation of the subthalamic nucleus. He eventually established another important Parkinson's disease treatment besides L-dopa - Deep Brain Stimulation (DBS) [22].

In recognition of DeLong's research on direct/indirect pathways and his contribution to the classic Go/NoGo model, and Benabid's development of deep brain stimulation for Parkinson's disease, the 2014 Lasker Clinical Medicine Award was awarded to the two. They subsequently won the Breakthrough Prize in Life Sciences in 2014 and 2015, respectively.

Figure 11. Mahlon DeLong and Alim Louis Benabid shared the 2014 Lasker Clinical Medicine Award.

Tip 2

The Breakthrough Prize is currently the highest-prize science award, with each winner receiving $3 million. It was established with donations from several wealthy people, including the Google founder and his wife, the Facebook founder and his wife, and Ma Huateng. In the field of life sciences, a separate Parkinson's and neurodegenerative disease award was also established. I estimate that this may be related to Google founder Sergey Brin, whose mother suffers from Parkinson's disease and whose family carries a mutation in the Parkinson's disease-causing gene LRRK2.

To be continued

In the latest Marvel movie "Black Widow", the behavior of the Black Widow agents is controlled by the spy organization "Red Room". The "scientific explanation" given by the movie is that the Red Room controls their basal ganglia brain area, which is the basal ganglia nucleus we introduced today. However, the example of the pig holding its breath used in the movie is not quite accurate, because breathing is controlled by several nuclei in the brain stem, not the basal ganglia. The basal ganglia mainly controls the gross movements related to Parkinson's symptoms, such as stable standing, walking, drinking water from a cup, etc., as well as some fine movements, motor skill acquisition, and habit formation, such as playing the piano and skating.

I am currently studying how basal ganglia cells regulate the acquisition of motor skills. Whenever I see my daughter struggling to practice a new piano piece or a new skating move, I wonder if I can artificially activate the relevant basal ganglia cells to help my daughter consolidate her piano and skating skills if I know the mechanism of motor skill acquisition? In this way, she doesn't have to practice every day - she doesn't even have to learn these moves. I just need to activate the relevant nerve cells and implant motor memory directly into her.

Figure 12. In the movie The Matrix (1999), programmers loaded judo fighting moves directly into Neo’s brain.

Of course, these ideas may seem a bit sci-fi now, but they are not impossible in the future. Like all scientific knowledge and technology, neuroscience can be used to help humanity or to destroy humanity. The idea of ​​the "Red Room" controlling the Black Widow agent was once thought of by a neuroscientist, Curtis C. Bell. He discovered a special type of synaptic pulse timing-dependent plasticity in the cerebellum [23]. Due to this concern, he gave up scientific research and launched an initiative requesting neuroscientists not to participate in research that is anti-human rights and anti-international law. The proposal and signature webpage are:

http://www.tinyurl.com/neuroscientistpledge

We now know that the striatum can regulate movement through two pathways, a direct pathway that promotes movement (Go) and an indirect pathway that inhibits movement (NoGo). We also know that abnormal discharges in the subthalamic nucleus (STN) in the basal ganglia are associated with Parkinson's disease.

But that’s not all.

Why are there two pathways for dopamine, and why is it so complicated? How does dopamine in the striatum regulate movement by regulating neuronal activity? And what does this have to do with schizophrenia?

We will analyze the answer next time.

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