Legend: This immunofluorescence picture depicts a successfully transplanted neuron in a rhesus monkey brain. The neuron is marked in fluorescent green (GFP), and surrounding cells’ nuclei are shown in blue (DAPI). Tyrosine hydroxylase (TH) is stained in red. This enzyme catalyzes the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which can be processed into dopamine and it is used as a marker for dopamine-producing neurons. Adapted from: Vermilyea & Emborg, 2018.
One of the good side effects of the COVID19 quarantine reality has been the complete transition of science events and scientific communication into the online realm. Now, it is not required to travel around the world, actually not even leave your couch, to attend seminars on virtually any subject imaginable. One of these seminars caught my attention due to the enthusiasm from the relatively few people attending it and from the presenter, Dr. Marina Emborg, to introduce the exciting latest development on a therapy strategy for Parkinson’s disease (PD).
Dr. Emborg is based in the Primate Center of the University of Wisconsin-Madison, which is also one of the reasons I chose to write on this topic. I’m currently a PhD student at UW-Madison, and I was reflecting on how many amazing scientists and scientific discoveries are going on right next door. If it wasn’t for this weird pandemic setting of online seminars, I probably wouldn’t have heard about the elegant experiments and exciting results Dr. Emborg’s group is reporting. Hence, my foremost goal with this article is to share what I understood to be important research in the field of regenerative medicine, and also to give the reader a perspective about the long endeavor posed by scientific investigation in general.
Briefly, let’s understand what defines Parkinson’s disease (PD) and subsequently, what are the major challenges scientists have been facing in search of its cure.PD is relatively common, affecting one in 100 individuals older than ~65 years, making it the second most prevalent neurodegenerative disease in the US behind Alzheimer's disease. The pathological hallmark of PD is the loss of dopamine-producing neurons (dopaminergic neurons) within the substantia nigra, and their respective areas of projections. Dopamine is a neurotransmitter and a hormone that plays several roles in our body, including serving as a communication molecule exchanged between neurons. Substantia nigra is defined as a region within the midbrain where dopaminergic neurons are concentrated, and it is responsible for modulating motor movement (Figure 1). Hence, the initial PD’s symptoms to be noted involve impaired motor regulation. However, non-motor symptoms such as depression, anxiety, loss of sense of smell, gastrointestinal dysfunction, and cardiac complications appear to occur even decades before motor deregulation onset. Here’s Nature featured video with cool visuals that covers briefly the pathological implications in PD:
Figure1: Human brain depicted, with lines pointing to substantia nigra location, and the dopaminergic neuron's extensions represented in green by dopamine pathway. The affected areas, putamen, and caudate nucleus are also indicated.
https://parkinsonsdisease.net/basics/pathophysiology-what-is-it/
Besides severely impacting the quality of life, PD also impacts longevity since affected patients have 3 times higher mortality rate when compared to healthy age-matched individuals. The first line of treatment, a rather intuitive one, is to replace the lost dopamine with oral supplementation. Levodopa is a drug that contains a precursor for dopamine capable of crossing the blood-brain barrier, a protective membrane surrounding your brain so that only authorized molecules and components can safely penetrate. Unfortunately, as the disease progresses, the medication efficacy weakens until it doesn’t have any effect at all. Hence, scientists and doctors have been working on alternative strategies to attenuate and/or cure PD.
Since one specific cell type (dopaminergic neurons) is the root cause for the pathology, transplantation assays to replace the defective cells with brand new functional ones have been a strong candidate as an intervention for PD’s cure. In fact, Parkinson’s disease was the first neurological pathology for which transplants appeared to be a feasible option. In the early70’s, transplantations were performed using fetal stem cells and rats as model organisms to study the disease. In this experimental setting, scientists modeled the disease by administering to rats a synthetic compound called Oxidopamine, to selectively destroy the dopaminergic neurons. The lost cells would then be replaced with fetal rat mesencephalic tissue (roughly fetal brain cells), which contain precursor cells with the potential to form new substantia nigra. Besides fetal material, which impose limitations due to availability and ethical constraints as a therapy for human diseases, other tissues in the adult body can also synthesize dopamine (discussed further below) including a specific cell type found in the adrenal gland in the kidneys and in the heart’s carotid bodies, specialized chemoreceptor cells.
In the following decades, further studies explored the possibilities of introducing either fetal-derived cells or dopamine-producing cells into the brain to reverse dopamine loss. These initial studies provided some encouraging results by observing proper integration and survival of introduced cells, as well as improvement in the motor function of animal subjects. Eventually, the discovery of MPTP (n-methyl-4,phenyl-1,2,3,6,-tetrahydropyridine), a human neurotoxin that also targets dopaminergic neurons, allowed the development of new animal models with non-human primates. For those who want to deepen their understanding of the different mechanisms to mimic PD in animal models, they are thoroughly reviewed here (Jagmag et al., 2016; Vermilyea&Emborg, 2018).
By the 90s, transplantation with fetal material for human PD patients was conducted in open-label trials, which means both patients and doctors were informed about who received actual cells or just control treatment. Despite some promising results, several patients reported no beneficial effects or even experienced adverse effects. Besides, comparing results from most of these studies remained challenging due to the diversity of techniques and protocols: Which cell type would be more appropriate to replace lost neurons? How many cells should be transplanted? What are the conditions of cell delivery that are optimal for the survival and integration of the cells? In which area of the brain should the cells be introduced? Do transplant recipients develop an immune response against the “new” cells? Those are some of the critical questions pervading the field to this day.
In the early 2000s, randomized double-blind, sham control studies, the gold standard for clinical trials, were conducted with human ventral mesencephalic fetal neurons or nigral fetal grafts (fancy terms for the precursor brain cells found in the developing human fetus) as the transplantable material. In this type of experimental setting, neither doctors nor patients are informed about who’s actually getting the cell transplant, rather than just a control mock procedure. Despite ethical dilemmas, such controlled studies are necessary when there’s a possibility for a placebo effect that might significantly impact the interpretation of results. These reports showed that some patients benefited from the transplant, but again, the number of individuals with no improvement at all or experiencing adverse effects was still high enough to put a stop to clinical trials of fetal cell transplantation for PD. Concomitantly, scientists were also investigating new sources for dopaminergic cells.
A potential alternative source for dopamine-producing cells is actually pig brains. They have relatively large cephalic mass with a similar overall organization when compared to primates. But if you are thinking that transplanting pig cells to a human brain sounds dangerous, you are correct! Not only there is an increased risk of rejection by the immune system, but also it would impose a high risk for exposure to the porcine virus that might also infect human cells (I think we have had our share of the mixed pool of virus going on out there!). Nonetheless, a safety trial was conducted in which 12 subjects were implanted with embryonic porcine neural cells. The recipients were tested for viral infections and also received immunosuppressant drugs as part of the protocol to mitigate the aforementioned risks. However, no significant benefits were reported to compensate for such a risky procedure.
What about taking cells from our own bodies to replace dopamine-producing neurons? As mentioned above, other cell types in an adult organism can also produce and store dopamine. With that in mind, scientists tested taking cells from sympathetic ganglion tissue, which can be found on either side along the spinal cord. Although the motor performance of individuals who received the transplant did not improve, the graft did ameliorateLevodopa therapy effect on patients, perhaps functioning as a co-treatment strategy.
Carotid bodies and kidney’s adrenal medullary cells were also tested in independent studies. Carotid bodies are chemoreceptors found in our hearts that release dopamine at low oxygen states. Excitingly, the brain region where these cells would be transplanted also presents low oxygen levels, so maybe the transplanted cells would be triggered by the local environment to release dopamine. These clinical studies revealed some improvement of the motor condition in recipients, but lack of proper controls and no evidence for the integration of transplanted cells compromised the interpretation of these results. The authors even suggested that the beneficial effects could be a consequence of growth stimulated by the graft on local cells, rather than by the graft’s dopamine secretion. Adrenal glands are hormone-producing glands located in the kidneys. The adrenal medulla is found within those glands and is composed of special cells called chromaffin cells, which are packed around blood vessels through which they can release dopamine into circulation. In fact, these cells are considered simplified neurons since they share the same embryonic origins. An initial clinical trial with advanced PD subjects showed mild to moderate improvement of disease condition up to a year after transplantation. However, poor graft survival and failure to observe long-term benefits were again challenging this paradigm.
Notably, in some cases, beneficial effects from transplantation were reported even when there was no evidence of the successful integration of grafted cells. Based on the evidence, alternative mechanisms were proposed. Maybe the graft, though some indirect effect, could stimulate local neurons to produce more dopamine. Following that hint, a new line of studies focused on manipulating local neurons to produce more dopamine. An attractive target for that purpose is the glial cell-derived neurotrophic factor (GDNF), which is a protein secreted by brain cells known to enhance neuronal survival and dopamine storage. So now the problem is “just” delivering the factor to the proper region. The most creative strategy included genetically engineering baby hamster kidney fibroblasts to express the gene coding GDNF and encapsulating these cells in small plastic vesicles prior to implantation. A long-term non-human primate study, however, found that the technique elicited a serious immune reaction. An alternative strategy for GDNF delivery would be introducing a viral vector to “hijack” the cells into producing the factor. The viruses used for this are rendered innocuous and only retain their capacity to infect cells and introduce a sequence of interest into the host’s DNA (in this case, the gene coding for GDNF). However, as we know very well (yet another virus joke!), viruses can be unpredictable, so better find different strategies.
Hang tight! Science usually walks at a very slow pace, and every single contribution is yet another piece to complete the puzzle. While not necessarily achieving a cure, all this experimentation brought to light incredible techniques that even allowed porcine cells to be introduced in a human brain with no casualties (this blows my mind!) or baby hamster fibroblasts to be encapsulated by tiny plastic vehicles. Besides, the improvement of animal models to better mimic the human disease provided invaluable information on disease progression. Experimentation’s successes and failures further refined methods and protocols.
Fast forward to the current state of PD research puzzle. Scientists now know that the immune system, previously disregarded to brain biology, plays an important role in disease onset and progression, and immunomodulation can be used as a therapy strategy. Furthermore, in 1996, mutations in the alpha-synuclein (a-syn) gene were discovered in familial forms of PD, meaning the disease has a genetic component that can pass along generations. The precise functions of the protein a-synuclein, coded by that gene,is not fully elucidated quite yet, but evidence suggests it might be involved in neuronal membrane organization. The disease mutations in this gene cause the protein encoded by it to be misfolded and to form aggregates within the cells, which seem to be the primary cause of PD’s dopaminergic degradation. Those aggregates, termed Lewy bodies, are considered the pathological hallmark found in PD’s patients’brains and are now also the focus of intense research efforts as a therapeutic target.
Recent breakthroughs in regenerative medicine and stem cell biology have been crucial for the development of new methods to manipulate cellular differentiation, that is, the process through which a cell loses its “stemness” to become a specialized one. This brings us to Dr. Emborg’s lab and their efforts to further advance PD’s research.
The isolation of the first human embryonic stem cells (ESCs) (which also happened here in UW-Madison by Dr. Thomson’s lab in 1998), shed light to the potential of manipulating the fate of a stem or pluripotent cell, which is capable of differentiating into any cell type. Dr. Emborg studied the use of human ESCs, which were transplanted into the brain of 3 rhesus monkeys that had PD induced by MPTP treatment (the neurotoxin that destroys dopaminergic neurons). The cells were engineered to express a green fluorescent protein (GFP) marker so they could be distinguished from the host’s cells. Postmortem analysis at 3-months revealed that only one of the monkeys had retained live transplanted cells, and markers of immune response were detected. The authors emphasized how the immunological component might play a critical role in the survival of transplanted cells.
One way of significantly reducing the host’s immune response is to go from xenografts and allogeneic transplants to autologous transplants. Xenografts consist of transplanting material between distinct species. Allogeneic transplants occur between different individuals of the same species, whereas autologous transplant consists in taking cells from the same individual, manipulating the cells ex vivo and re-introducing them back in the organism. Logically, the immune response component will be significantly reduced as one moves from xenografts, to allogeneic transplant, and finally to autologous transplants. Dr. Emborg’s lab current efforts are focused on both allogeneic and autologous transplants using induced pluripotent stem cells (iPSCs), which consists in genetically manipulating certain cell types to “activate” their intrinsic capacity to differentiate into a completely distinct function. In this case, the group developed a protocol to differentiate fibroblasts (skin cells), into dopaminergic midbrain neurons. This multistep protocol involves approximately 1 month of cell culture, with the addition of several factors and different media so the cells are carefully “guided” into their new role as dopaminergic neurons. Each monkey can have their own iPSCs developed for transplantation (As someone who has some experience with cell culture, I can attest this is an incredible amount of work!). The idea is to develop personalized cell-based therapy for PD’s patients in the future.
By applying interdisciplinary efforts with physicists and medical engineers, the group also developed an elegant system for intracerebral delivery of biologic materials termed real-time intraoperative magnetic resonance imaging (RT-IMRI). This system couples the precise imaging by MRI, which is a pretty well-established medical imaging technique, with an apparatus containing a special thin transparent tube, called a canula, through which delicate material such as live cells can pass through. A mechanized pump is connected to a syringe and the pressure applied from the syringe to the cannula can be rigorously monitored in real-time while the material can be seen through the transparent cannula, which are particular advantages of this method. Before attempting this experiment with animals, the scientists conducted a careful evaluation of cell delivery and survival in culture plates and in a gel matrix that serves as a brain surrogate. Excitingly, results show the cells successfully survived the transplant procedure. For this pilot experiment, the iPSCs did not come from the same monkey that received the transplant, so markers of immune response were detected, as expected for an allogeneic transplant. The cells also expressed markers for early neural progenitors, which indicate they were properly following the expected differentiation pathway.
In the seminar, Dr. Emborg presented data on their ongoing studies using this technique to evaluate both allogeneic and autologous transplants, but in a long-term period. These animals should be evaluated for years post-transplant, and not only for cellular parameters but also for motor and behavior aspects. This is unpublished data of an ongoing study, so there isn’t much detail out there yet, but we are excited to continue to follow up with this story. The advancements resulting from Dr. Emborg and scientists combined efforts along the years are not only important for PD’s treatment, but also for other neurological diseases that could benefit from cell-based therapies.
Finally, during my reading and searching to write this post, I came across the Michael J. Fox Foundation for Parkinson’s Research. In case you are a bit disconnected from 80’s pop culture, Michael portrayed the famous Marty McFly from Back to the Future (1985), and in 1991, he was diagnosed with early-onset Parkinson’s disease at 29. In 2000, he created the research foundation, which is now the world's largest non-profit funder of Parkinson's drug development and it has made invaluable contributions to advancement in the field. I would also like to acknowledge the hardship faced by all patients and families that deal with Parkinson’s disease and also, share my gratitude to scientists and doctors who have been working hard to tackle this challenge.
Consulted references
Roitberg, B., Urbaniak, K. and Emborg, M., 2004. Cell transplantation for Parkinson's disease. Neurological research, 26(4), pp.355-362.
Swanson CR, Sesso SL, Emborg ME. Can we prevent parkinson's disease? Front Biosci (Landmark Ed). 2009 Jan 1;14:1642-60. doi: 10.2741/3331. PMID: 19273153.
Jagmag, S.A., Tripathi, N., Shukla, S.D., Maiti, S. and Khurana, S., 2016. Evaluation of models of Parkinson's disease. Frontiers in neuroscience, 9, p.503.
Nakao, N., Kakishita, K., Uematsu, Y., Yoshimasu, T., Bessho, T., Nakai, K., Naito, Y. and Itakura, T., 2001. Enhancement of the response to levodopa therapy after intrastriatal transplantation of autologous sympathetic neurons in patients with Parkinson disease. Journal of neurosurgery, 95(2), pp.275-284.
Meade, R.M., Fairlie, D.P. and Mason, J.M., 2019. Alpha-synuclein structure and Parkinson’s disease–lessons and emerging principles. Molecular neurodegeneration, 14(1), pp.1-14.
Emborg, M.E., Liu, Y., Xi, J., Zhang, X., Yin, Y., Lu, J., Joers, V., Swanson, C., Holden, J.E. and Zhang, S.C., 2013. Induced pluripotent stem cell-derived neural cells survive and mature in the nonhuman primate brain. Cell reports, 3(3), pp.646-650.
Emborg, M.E., Zhang, Z., Joers, V., Brunner, K., Bondarenko, V., Ohshima, S. and Zhang, S.C., 2013. Intracerebral transplantation of differentiated human embryonic stem cells to hemiparkinsonian monkeys. Cell transplantation, 22(5), pp.831-838.
Vermilyea, S.C. and Emborg, M.E., 2018. The role of nonhuman primate models in the development of cell-based therapies for Parkinson’s disease. Journal of Neural Transmission, 125(3), pp.365-384.
Vermilyea, S.C., Lu, J., Olsen, M., Guthrie, S., Tao, Y., Fekete, E.M., Riedel, M.K., Brunner, K., Boettcher, C., Bondarenko, V. and Brodsky, E., 2017. Real-time intraoperative MRI intracerebral delivery of induced pluripotent stem cell-derived neurons. Cell transplantation, 26(4), pp.613-624.
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