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

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

Is axonal degeneration a key early event in Parkinson’s disease?

Author Posting. © The Author(s), 2016. This is the author's version of the work. It is posted here by permission of IOS Press for personal use, not for redistribution. The definitive version was published in Journal of Parkinson's Disease 6 (2016): 703-707, doi:10.3233/JPD-160881.Recent research suggests that in Parkinson’s disease the long, thin and unmyelinated axons of dopaminergic neurons degenerate early in the disease process. We organized a workshop entitled ‘Axonal Pathology in Parkinson’s disease’, on March 23rd, 2016, in Cleveland, Ohio with the goals of summarizing the state-of-the-art and defining key gaps in knowledge. A group of eight research leaders discussed new developments in clinical pathology, functional imaging, animal models, and mechanisms of degeneration including neuroinflammation, autophagy and axonal transport deficits. While the workshop focused on PD, comparisons were made to other neurological conditions where axonal degeneration is well recognized

Signs of Degeneration in 12-22-Year Old Grafts of Mesencephalic Dopamine Neurons in Patients with Parkinson's Disease

We demonstrate that grafted human fetal mesencephalic neurons can survive and extend axons for 22 years in the brain of a patient with Parkinson's disease (PD). In this patient, the overall survival and fiber outgrowth of the grafts were, however, relatively poor, which is consistent with the lack of significant clinical graft-induced benefit. We have compared the morphology of neurons in the 22-year old grafts with those in two younger grafts (16- and 12-year old), which were sequentially implanted in another PD patient. In the case with the 22-year-old transplant, a high proportion (up to 38%) of the grafted dopaminergic (pigment-granule containing) neurons do not express tyrosine hydroxylase and dopamine transporter and their perikarya appear atrophic. The proportion of pigmented neurons not expressing these markers is lower in the 12-16 year old grafts. Furthermore, in the 22-year-old graft, 49% of the pigmented neurons display alpha-synuclein immunoreactivity in the cell body and 1.2% of them contain Lewy bodies. In conclusion, our results show that grafted dopaminergic neurons can survive for more than two decades. However, over time an increasing proportion of grafted neurons exhibit signs of degeneration

Identification of Multiple QTLs Linked to Neuropathology in the Engrailed-1 Heterozygous Mouse Model of Parkinson's Disease

Motor symptoms in Parkinson's disease are attributed to degeneration of midbrain dopaminergic neurons (DNs). Heterozygosity for Engrailed-1 (En1), one of the key factors for programming and maintenance of DNs, results in a parkinsonian phenotype featuring progressive degeneration of DNs in substantia nigra pars compacta (SNpc), decreased striatal dopamine levels and swellings of nigro-striatal axons in the SwissOF1-En1+/- mouse strain. In contrast, C57Bl/6-En1+/- mice do not display this neurodegenerative phenotype, suggesting that susceptibility to En1 heterozygosity is genetically regulated. Our goal was to identify quantitative trait loci (QTLs) that regulate the susceptibility to PD-like neurodegenerative changes in response to loss of one En1 allele. We intercrossed SwissOF1-En1+/- and C57Bl/6 mice to obtain F2 mice with mixed genomes and analyzed number of DNs in SNpc and striatal axonal swellings in 120 F2-En1+/- 17 week-old male mice. Linkage analyses revealed 8 QTLs linked to number of DNs (p = 2.4e-09, variance explained = 74%), 7 QTLs linked to load of axonal swellings (p = 1.7e-12, variance explained = 80%) and 8 QTLs linked to size of axonal swellings (p = 7.0e-11, variance explained = 74%). These loci should be of prime interest for studies of susceptibility to Parkinson's disease-like damage in rodent disease models and considered in clinical association studies in PD