Towards cell replacement therapy in Parkinson’s disease. Proteoglycans and Nogo-A as modulators of axonal growth in midbrain dopaminergic neurons

Abstract

Parkinson’s disease (PD) is currently the second most common neurodegenerative disorder (after Alzheimer’s disease). PD is diagnosed on its motor symptoms, which include akinesia, bradykinesia, rigidity, postural imbalance and resting tremor. It is well established that the motor symptoms develop due to lack of dopamine in the striatum. This is caused by progressive degeneration of dopaminergic neurons (DNs) in the midbrain (substantia nigra pars compacta), which innervate the striatum. Although initially efficacious, the therapeutic effect of various anti-parkinsonian pharmacological treatments is limited to five to ten years. Hence, the new therapeutic strategies for PD are highly warranted.

The concept of cell replacement therapy in PD is based on the idea that the diminished dopamine levels in the striatum would be restored by the functional DNs, obtained from donated, electively aborted fetuses, and subsequently injected into the patients’ striatum. In several clinical trials, about 400 patients received such therapy worldwide. Although the therapeutic effects varied, some individuals experienced major motor improvement even for up to 16 years. Other PD patients did not respond so favorably and even developed adverse effects, i.e. graft-induced dyskinesia. The clinical trials are currently on hold.

In the first project, we have analyzed the survival and integration of the graft in a patient who underwent the transplantation in Lund, in 1987, as one of the first transplanted cases in the world. Clinically, the patient did not show the symptomatic relief in response to the transplantation. Examining the postmortem brain, we observed a very small surviving graft. In addition, we also detected signs of PD-like pathology in the transplanted DNs. Nonetheless, the graft survived in this brain for 22 years and such a long graft-life has never before been reported.

It is certain that embryonic tissue will not be routinely used as a cell source for transplantation therapy in PD. Yet, stem cells bear a great potential for future PD therapy. Recent publications show that DNs of the midbrain subtype could be differentiated from stem cells in a time-efficient manner. Moreover, those DNs could form well surviving tumor-free grafts which reverted the PD-like motor symptoms in animal models. Nonetheless, the differentiation of embryonic stem cells to DNs is still not fully understood and therefore difficult to control. Proteoglycans may be engaged in differentiation of DNs, as they govern the formation of the nervous system in developing vertebrate embryo. My second project aimed at defining the genes encoding proteoglycans and the enzymatic machinery fine-tuning their structure during the DN differentiation. From around 2000 proteoglycan-related genes, we identified two (neurocan and HS3ST5) that potentially could enhance the differentiation efficiency of DNs from stem cells. Our results can serve as a starting point for the further functional studies.

After transplantation, the grafted DNs have to survive, and also integrate with the host brain, i.e. grow neurites, form synapses and release dopamine in a regulated fashion. In the third project, we studied how Nogo-A protein affects the survival and neurite growth in DNs. Nogo-A is a strong growth-inhibitory protein in the brain and spinal cord. Interestingly, in recent years, the growth- and survival-supportive role of Nogo-A in various types of neurons has been shown. We have demonstrated here for the first time the Nogo-A roles in the substantia nigra pars compacta DNs.

A careful analysis of all data coming from initial clinical trials, studies on animal parkinsonian models, and the emergence of recent safe and effective dopaminergic differentiation protocols, collectively imply that the cell replacement approach in PD holds a great therapeutic potential. Understanding of the mechanisms governing the DNs differentiation, survival and neurite growth will help develop safe and efficient cell replacement therapies in PD. The work presented in this thesis will hopefully contribute to such advancement.

Abstract (Swedish)

Popular Abstract in English

Parkinson’s disease (PD) is the second most common, after Alzheimer’s disease, neurodegenerative disease in man. Stiffness in joints, shaky hands, and slowness of movements make it difficult for people with PD to complete normal daily tasks like buttoning up a shirt. Additionally, PD affects memory, mood, sleep and the function of the gastro-intestinal tract. Over time, the disease progresses gradually and worsens.

We still do not understand fully the mechanisms that cause PD, but what we do know is that the gradual loss of movement control is caused by successive death of specific cells – dopaminergic neurons, in a particular region of a brain known as (from Latin) the substantia nigra pars compacta. The dopaminergic neurons produce dopamine, a neurotransmitter, which regulates a large part of the movement-governing system in the brain. The current most common therapies for PD rely on restoration of the dopamine levels in this system. Usually, a patient takes pills of a medication called L-DOPA a few times per day and can control his or her body movements again. Although initially this therapy works, the dose of L-DOPA required to bring the relief gradually increases, and eventually this treatment must be discontinued due to troublesome side effects. Hence, the new therapies for PD are warranted.

One of the potential treatments for PD is based on the idea that dopamine can be provided by young dopaminergic neurons injected directly into the patients’ brain. Those neurons are taken from donated, electively aborted embryos, from the site where the substantia nigra would be formed. In several clinical trials, around 400 patients received this treatment worldwide, and although the therapeutic effects varied, some individuals experienced major improvement, even for up to 16 years, and could completely set aside L-DOPA. Other patients, however, did not experience any symptomatic relief and even developed some movement complications, so the clinical trials are currently on hold. Careful analysis of the clinical data from all of these trials, together with experiments, including ones in animal models of PD and in (dopaminergic) cell cultures, will deepen the understanding on how to improve cell replacement therapy in PD.

In my first project, we have analyzed the survival and integration of the graft in a patient who underwent the transplantation in Lund, in 1987, as one of the first transplanted cases in the world. Clinically, the patient did not show symptomatic relief in response to the transplantation. After examining the brain postmortem, we observed only a very small surviving graft. In addition, we saw signs of PD-like pathology in the transplanted neurons. Nonetheless, the graft survived in this brain for 22 years and such a long graft-life has never been reported elsewhere.

It is certain that in the future, embryonic tissue will not be routinely used as a cell source for transplantation therapy in PD. Instead, stem cells will be differentiated to dopaminergic neurons and subsequently injected into the brain. Although research in recent years has shown that embryonic stem cells can be a safe and an efficient source for obtaining transplants that survive and reverse the PD-like symptoms in animal models, the differentiation of embryonic stem cells to dopaminergic neurons is still not fully understood, and is therefore difficult to control. Proteoglycans (proteins with long sugar chains attached) may be engaged in differentiation of dopaminergic neurons, as they regulate the process of brain development in vertebrate embryos.

My second project aimed at defining the genes encoding proteoglycans and the enzymatic machinery fine-tuning their structure that is involved in the differentiation of dopaminergic neurons. From around 2000 proteoglycan-related genes, we identified two (neurocan and HS3ST5) that could potentially enhance the differentiation efficiency of dopaminergic neurons from stem cells. Our results may serve as a starting point for further functional studies.

In order to bring back, at least partially, the control of movement to an individual with PD, the grafted dopaminergic neurons have to survive, and also integrate with the neurons of the host brain, i.e. extend neurites, form synapses and release dopamine.

In my third project, we have been studying how a protein called Nogo-A affects dopaminergic cell survival and neurite growth. Nogo-A is a strong growth-stopping agent found within the brain and spinal cord (hence the name ‘no-go’). Interestingly, in recent years, some studies showed the growth- and survival-promoting role of Nogo-A in neurons. Our results supported this notion and we were first ones to suggest Nogo-A roles in dopaminergic neurons of the substantia nigra pars compacta. Perhaps upon grafting into the PD brain, the dopaminergic neurons stop producing Nogo-A and this may be the reason why many die or do not integrate with the host neurons following the operation?



The results of initial clinical trials, studies in animal parkinsonian models and recent safe and effective dopaminergic differentiation protocols, collectively imply that the cell replacement approach in PD holds great therapeutic potential. Nonetheless, in order to develop a safe and efficient cell replacement therapy in PD, the understanding of the mechanisms governing dopaminergic cell differentiation, survival and neurite growth is needed. I hope that my work will contribute to such positive development.

• author: Kurowska, Zuzanna
• supervisor: Jia-Yi Li; Patrik Brundin; Martin Schwab
• opponent: Kuhn, Hans-Georg, University of Gothenburg, Gothenburg, Sweden
• organization: Neural Plasticity and Repair (research group); MultiPark: Multidisciplinary research focused on Parkinson´s disease
• publishing date: 2012
• type: Thesis
• publication status: published
• subject: Neurosciences
• keywords: LUHMES cells, substantia nigra pars compacta, microarray, heparan sulphate 3-O-sulfotransferase, neurocan, midbrain dopaminergic neurons, grafting, embryonic stem cells, proteoglycans, glycosaminoglycans, Nogo-A, cell replacement therapy, Parkinson’s disease
• in: Lund University Faculty of Medicine Doctoral Dissertation Series
• volume: 2012:73
• pages: 106 pages
• defense location: Lund University, Wallenberg Neuroscience Center BMC A10, Segerfalksalen
• defense date: 2012-09-21 09:15:00
• ISSN: 1652-8220
• ISBN: 978-91-87189-36-4
• language: English
• LU publication?: yes
• id: 5c1b2d75-d3d3-4bee-bac2-9bd19056f68c (old id 3044164)
• date added to LUP: 2016-04-01 14:21:46
• date last changed: 2019-05-22 03:51:59