Introduction


The high prevalence, progressive and degenerative nature of Parkinson’s disease calls for therapeutic approaches to treat this disease. Due the idiopathic nature of PD and our still developing understanding of this disease there is currently no cure for this disorder. Nevertheless, over the years, researchers working in collaboration with clinicians have produced numerous ideas and implementations on treatment of Parkinson’s disease. This neurowiki will focus primarily on the classical pharmacological agents, such as L-DOPA, MAO B Inhibitors, and dopamine agonists to treat the symptoms of PD. In addition, the nonpharmacological management of PD will be briefly discussed. The last section of neurowiki will discuss in greater detail the on-going research in stem cell biology to generate dopaminergic neurons, which is possibly the main culprit of symptoms in PD patients. Both the embryonic stem (ES) cells, and induced pluripotential stem (iPS) cells will be discussed. In addition, a research article discussing astate of the art methodology to produce iPS cells free of viral integration and permanent genome changes will be discussed within the iPS cells section. It is important to note that pharmacological agents, and stem cell therapy are not the only approaches to Parkinson’s disease treatment. Sabrina Cheung discusses in her neurowiki different and intriguing PD treatment approaches, such as deep brain stimulation.

Table of Contents


1
Pharmacological Agents

1.1 L-DOPA

1.2 MAO B Inhibitors

1.3 Dopamine Agonists
2
Nonpharmacologic Management
3
Stem Cell Therapy

3.1 Embryonic Stem (ES) Cells

3.2 Induced Pluripotential Stem (iPS) Cells

3.3 Considerations and Controversies
4
References

Pharmacological Agents




L- DOPA

Historically, L-DOPA was the main pharmacological agent to treat patients with Parkinson’s disease.
pharmacology.jpg
Figure 1. Pharmacological Agents still play a vital role in PD treatment

Actually, even till present day, many physicians use this drug,
because it still remains as the most effective agent to treat PD.1,2 L-DOPA is a precursor molecule to the neurotransmitter dopamine.3 Parkinson’s disease involves the destruction of neurons in the substantia nigra, which leads to a deficit in the levels of dopamine. Thus, it is evident why L-DOPA is used to treat PD. In addition, this molecule has the appropriate structural properties to cross through the blood-brain-barrier.3
Clinicians often use benserazide or carbidopa in combination with L-DOPA administration.3 These two drugs inhibit the action of enzymes that would convert L-DOPA to dopamine in the peripheral tissues, thus allowing the L-DOPA molecules to reach the brain where they would implement their desired effects.3

Although the effectiveness of L-DOPA is unanimously recognized, there are several downsides associated with the use of this pharmaceutical agent. Patients using L-DOPA can develop side effects such as hypotention, nausea, and psychiatric symptoms.3 In addition, when PD patients use L-DOPA for prolonged periods of time, motor complications develop, such as dyskinesia.4 Some scientists advocate that L-DOPA may have neurotoxic effects because of its potential to form free radicals, which might injure the remaining DA neurons in substantia nigra.5

Monoamine oxidase (MAO) B Inhibitors

Monoamine oxidases are enzymes, which degrade monoamines, such as dopamine, serotonin, norepinephrine, etc. Essentially, MAO B inhibitors are a class of drugs that function by blocking the activity of monoamine oxidases, which causes less degradation of monoamines.3 Thus, neurotransmitters, such as dopamine remain for longer periods of time in the synapse. Selegeline, an example of a MAO B inhibitor, it decreases the breakdown and conversion of dopamine into dihydroxyphenylacetic acid (DOPAC) molecules and hydrogen peroxide.
MAO B inhibitors can be administered to PD patients prior to L-DOPA to delay the use of L-DOPA because of its complications with prolonged use.3

Dopamine Agonists

Dopamine agonists are another class of drugs which is used for PD treatment. However, these drugs directly act on dopamine receptors and mimic endogenous dopamine molecules.3 Dopamine agonists may have certain advantages over administering L-DOPA to PD patients.3 Just like MAO B inhibitors, they may be used to spare the need to use L-DOPA.3 In addition, dopamine agonists are not metabolised via the oxidative pathways, and therefore do not produce contribute to the production of free radicals.6 Lastly, some researchers claim that dopamine agonists may have antioxidative effects directly.7 However, these drugs may also produce side effects, such as hallucinations and nausea.3

Nonpharmacologic Management


Aside from the administration of pharmaceutical drugs to PD patients, there are other essential components of
Running-on-the-treadmill.jpg
Figure 2. Physical Exercise is an important component in PD treatment

treating Parkinson’s disease.8 It is important to provide appropriate education to patients and their family members on how to deal with this disorder. The diagnosis of PD can be frightening to many patients, which may lead to development of depression and anxiety.8
Patients with PD are encouraged to exercise regularly to promote mental and physical health, especially because of the progressive degeneration of motor function.8 Although exercise alone will not slow down the disease, orthopaedic effects of secondary nature, such as posture, and back pain may be improved.9
In addition to education and exercise, some PD patients may require speech therapy as dysarthria occurs frequently in manifestation of PD.8
Lastly, proper nutrition is important, especially for elderly patients with PD. Therefore, physicians recommend healthy and balanced diets, which would help avoid muscle and bone loss.8

Stem Cell Therapy


Embryonic Stem Cells

Figure1.jpg
Figure 3. Inner cell mass cells can be cultured and differented into mature cells

Embryonic stem cells are a group of inner mass cells in the blastocyst stage of development.10 In humans, when the sperm zygot meets the egg in the ampulla region of the fallopian tube, the sperm fertilizes the egg.10 The fertilized egg undergoes rapid divisions and eventually gives rise to a spherical cluster of cells producing the structure called the morula.11 The morula is composed of two different types of cells.11 The outer cells are the epilthelial trophoectoderm cells, while the inner cells are the inner mass cells.11 In the next developmental stage, the blastocyst stage, the inner mass cells migrate to one pole.10 These inner mass cells are the embryonic stem cells, which are pluripotential.12 As illustrated in figure 3, the pluripotential property of these cells allows them to differentiate essentially into any cell type, such as neurons, glial cells, pancreatic insulin-producing beta cells, etc.12 This important characteristic produced an overwhelming interest in the research community, which allows scientists to model diseases by creating specific tissues for disease modeling in vitro, whole organ engineering, and most importantly a potential therapeutic approach to treat diseases which involve the destruction and loss of cells, such as Parkinson’s Disease (PD), Type I Diabetes, spinal cord injuries, and etc.12 Regenerating new functional cells is especially important for neurodegenerative diseases, such as Parkinson’s Disease. Although treatments, such as pharmacological agents, deep brain stimulation (DBS), have been implemented in the past and present, it is very clear that an autonomous long-term treatment, such as cell therapy, produces a very attractive alternative.

The current approach in differentiating embryonic stem cells is by extracting them from the blastocyst and adding necessary growth factors in vitro.12 This forces the ES cells to mature into differentiated cells, such as dopaminergic neurons. The choice of the added growth factors will dictate the fate of stem cells.12

da_neurons_transplant.JPG
Figure 4. Mice with DA neurons (derived from Nurr1 ES cells) show motor improvements compared to control groups. (Kim, et al, 2002)

In 2002, a team of researchers from the National Institute of Neurological Disorders and Stroke in Maryland successfully transplanted dopaminergic (DA) neurons derived from ES cells into an animal model of Parkinson’s Disease.13 To model PD the scientists injected 6-hydroxy dopamine (6-OH-DA) into the striatum of mice, which results in destruction of dopaminergic neurons.13 The loss of dopaminergic neurons causes mice to exhibit Parkinsonian symptoms, including impairment of motor function. Following the 6-OH-DA administration, the researchers injected DA neurons derived from embryonic stem cells into the mice striatum.13 This group of mice was compared to the sham group, who also received the 6-OH-DA administration, but no transplant of DA neurons.13 The results of the experiment showed that transplanted neurons integrated and survived well into the new environment.13 In addition, the transplanted cells expressed key structural properties of DA neurons, such as calbindin, which is a calcium-binding protein, and tyrosine hydroxylase.13 However, it is crucial to analyse functional properties of transplanted cells, because the main aspect of interest is to investigate whether or not these cells could provide a therapeutic benefit to PD patients. Indeed, the transplanted neurons displayed appropriate electrophysiological properties, established functional synapses, and most importantly improved motor function, such as producing higher scores in mice on paw-reaching tasks.13 As illustrated in figure 4, the mice in the Nurr1 ES cell group, which represent mice that received dopaminergic neurons transplant, have statistically significant motor improvements compared to the control groups.13

Induced Pluripotential Stem (iPS) Cells

In the year of 2006, a Japanese researcher Shinya Yamanaka and his team changed the history of stem cell research. Yamanaka successfully converted mouse fibroblast cells into pluripotent stem cells.14 This discovery had an incredible significance because it bypassed the need for embryos as a source of stem cell and the need for immunosuppression. Yamanaka’s research protocol is extremely elegant. Originally, it involved using a viral vector to add four key transcription factor genes into the genome of the donor cells. The four factors are: Oct3/4, Sox2, Klf4, and c-Myc, which later became known as the “Yamanaka factors.” 14 When these genes were introduced into the mature mice fibroblast cells, the cells underwent reprogramming and become pluripotent stem cells.14 Similar to the embryonic stem cells, the iPS cells can also be differentiated into different cell types via the addition of appropriate signaling molecules.12 iPS cells can be differentiated into dopaminergic neurons, presenting a potential therapeutic approach to treat PD.

The iPS cells have sparked a tremendous amount of interest amongst the scientistsin the research community for their incredible potential. In fact, in 2009 iPS cell generation approach was nominated as “Method of the Year” by Nature.


However, although using iPS cells may seem as a promising therapy for PD as published in many research articles, there are important considerations and concerns that require attention and caution. One of the biggest criticisms amongst the researchers in the stem cell community is the viral integration problem.15 When retroviruses are used to add the four transcription factors into the donor cells, the added genes become permanently integrated into the genome. This alteration of DNA can affect normal cell behaviour and give rise to abnormal transcription and translation. It is important to note that one of the four reprogramming factors is c-Myc, which is an oncogene.15 Therefore, abnormal transcription of c-Myc can result in development of tumours.15

Appreciating the significance of the genome integration problem, Dr. Frank Soldner and his colleagues undertook on the challenge of generating iPS cells without permanently inserting the viral reprogramming factors. 15 In sum, they were able to convert human fibroblast cells into dopaminergic neurons free of exogenous genes.15 The level of complexity and the difficulty of this research definitely underscore our advancements in knowledge on stem cell biology.

It is worthy to note some of the important experimental features and key steps that Dr. Soldner and his team conducted throughout their experiment. Firstly, instead of working with animal models, the researchers decided to use fibroblast cells from five patients with idiopathic Parkinson’s Disease.15 In addition, instead of using the traditional approach of using retroviruses to add the four genes, these researchers used doxycycline (DOX)-inducible lentiviruses to introduce the transcription factors into the fibroblast cells.15 However, these lentiviral vectors possessed the ability to be removed with Cre-recombinase.15 Using this approach allowed the scientists to force the fibroblast cells to express the key transcription factors temporarily and reprogram into stem cells (iPSCs).15 However, the added genes are later removed, which leaves iPSCs free of reprogramming factors.15 These cells highly resemble human embryonic stem cells.15 Following the reprogramming procedure, the stem cells were differentiated into dopaminergic neurons via addition of necessary growth factors, such as FGF8 and FTF2, as well as sonic hedgehog (SHH).15 It was then necessary to confirm the appropriate cell fate by looking for characteristic properties of DA neurons, such as the presence of tyrosine hydroxylase enzyme.15
In conclusion, Dr. Soldner’s team provided a possible solution to the viral integration dilemma. And although there are still many unanswered questions, we are perhaps one step closer to implementing stem cells in treatment of degenerative diseases, such as Parkinson’s disease.

Issues and Controversies

At first glance it may seem that stem cell therapy provides an ideal and elegant solution to degenerative diseases, such as Parkinson’s disease. However, there are still numerous problems with both ES and iPS cells. ES cells were a topic of debate and speculation in the field of bioethics. Because these cells require embryos, many people suggest that research on ES cells is unethical.16 In addition, undifferentiated, or partly-differentiated ES cells form teratomas when transplanted to receiving animal.16 Lastly, even after transplant of mature cells derived from ES cells, recipient animal or human will require life-long immunosuppression, because the immune system will consider the transplanted cells as “foreign.”17

Biomedical research with iPS cells also faces challenges. More specifically, the rate of conversion of mature cells (i.e. skin cells) into stem cells is very inefficient, less than one percent.12 Also, iPS cell reprogramming relies mostly on using retroviruses to introduce the four transcription factors, which permanently alters the genome of cells. And although new approaches bypass this problem, these methods typically have even lower efficiencies.15

Thus, it is clearly evident that although stem cells provide an attractive possible therapeutic approach to diseases that involve cell loss, it is important to consider the risks and challenges associated with this choice of treatment.

References


  1. Silva, M.A., et al. Increased neostriatal dopamine activity after intraperitoneal administration of L-dopa: on the role of benserazide pretreatment. Synapse 27, 294-302 (1997).

  2. Lang A.E., and Lozano A.M. Medical progress: Parkinson’s disease. N Eng J Med 339, 1130-43 (1998).

  3. Munchau, A., and Bhatia, K.P. Pharmacological treatment of Parkinson’s disease. Postgrad Med J 76, 602-610 (2000).

  4. Lesser, R.D., et al. Analysis of the clinical problems in parkinsonism and the complications of long-term levodopa therapy. Neurology 29, 1253-1260 (1979).

  5. Basma, A.N., et al. L-dopa cytotoxicity to PC12 cells in culture is via its autooxidation. J Neurochem 64, 825-832 (1995).

  6. Mena, M.A., et al. Neurotoxicity of levodopa on cathecholamine-rich neurons. Mov Disord 7, 23-31 (1992).

  7. Yoshikawa, T., et al. Antioxidant properties of bromocriptine, a dopamine agonist. J Neurochem 62, 1034-1038 (1994).

  8. Tarsy, D., Hurtig, H.I., and Dashe J.F. Nonpharmacologic management of Parkinson disease. UpToDate. (2009).

  9. Hart, R.G., et al. Neuroprotection trials in Parkinson’s disease: systematic review. Mov Disord 24, 647 (2009).

  10. Sadler, T.W. Langman’s Medical Embryology, Ninth Edition. 31-43 (2003).

  11. Moore, K.L., Persaud, T.N, and Schmitt, W. The Developing Human: Clinically Oriented Embryology, 6th edition. 20-54 (2003).

  12. Cohen, D.E., and Melton, D. Turning straw into gold: directing cell fate for regenerative medicine. Nature 12, 243-252 (2011).

  13. Kim, J.H., et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 418, 50-56 (2002).

  14. Takahashi, K., and Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676 (2006).

  15. Soldner, F., et al. Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964-977 (2009).

  16. Denker, H.W. Potentiality of embryonic stem cells: an ethical problem even with alternative stem cell sources. J Med Ethics 32, 665-671 (2006).

  17. Odorico, J.S., Kaufman, D.S., and Thomson, J.A. Multilineage differentiation from human embryonic stem cell lines. Stem Cells 19, 193-204 (2001).