Neural connectivity predicts spreading of alpha-synuclein pathology in fibril-injected mouse models: Involvement of retrograde and anterograde axonal propagation.

In Parkinson's disease, some of the first alpha-synuclein aggregates appear in the olfactory system and the dorsal motor nucleus of the vagus nerve before spreading to connected brain regions. We previously demonstrated that injection of alpha-synuclein fibrils unilaterally into the olfactory bulb of wild type mice leads to widespread synucleinopathy in brain regions directly and indirectly connected to the injection site, consistently, over the course of periods longer than 6 months. Our previously reported observations support the idea that alpha-synuclein inclusions propagates between brain region through neuronal networks. In the present study, we further defined the pattern of propagation of alpha-synuclein inclusions and developed a mathematical model based on known mouse brain connectivity. Using this model, we first predicted the pattern of alpha-synuclein inclusions propagation following an injection of fibrils into the olfactory bulb. We then analyzed the fitting of these predictions to our published histological data. Our results demonstrate that the pattern of propagation we observed in vivo is consistent with axonal transport of alpha-synuclein aggregate seeds, followed by transsynaptic transmission. By contrast, simple diffusion of alpha-synuclein fits very poorly our in vivo data. We also found that the spread of alpha-synuclein inclusions appeared to primarily follow neural connections retrogradely until 9 months after injection into the olfactory bulb. Thereafter, the pattern of spreading was consistent with anterograde propagation mathematical models. Finally, we applied our mathematical model to a different, previously published, dataset involving alpha-synuclein fibril injections into the striatum, instead of the olfactory bulb. We found that the mathematical model accurately predicts the reported progressive increase in alpha-synuclein neuropathology also in that paradigm. In conclusion, our findings support that the progressive spread of alpha-synuclein inclusions after injection of protein fibrils follows neural networks in the mouse connectome

A new approach to disease-modifying drug trials in Parkinson's disease

Translating new findings in the laboratory into therapies for patients is a slow and expensive process. The development of therapies for neurodegenerative diseases is further complicated by the difficulty in determining whether the drug truly retards the slow degenerative process or provides only symptomatic benefit. In this issue, Aviles-Olmos et al. describe a first in Parkinson's disease (PD) patient study using a drug previously approved for diabetes treatment. In addition to suggesting that the drug may indeed be disease modifying in PD, their innovative approach suggests there may be more rapid and inexpensive avenues for testing novel therapies in PD

Membrane Interaction of α-Synuclein in Different Aggregation States

Aggregated α-synuclein in Lewy bodies is one of the hallmarks of Parkinson's disease (PD). Earlier observations of α-synuclein aggregates in neurons grafted into brains of PD patients suggested cell-to-cell transfer of α-synuclein and a prion-like mechanism. This prompted the current investigation of whether α-synuclein passes over model phospholipid bilayers. We generated giant unilamellar vesicles (GUVs) containing a small amount of a lipid-conjugated red emitting dye (rhodamine B) and varied the membrane charge by using different molar ratios of DOPC and DOPS or cardiolipin. We then used confocal fluorescence microscopy to examine how monomer, fibril as well as on-pathway α-synuclein species labeled with a green emitting fluorophore (Alexa488) interacted with the phospholipid bilayers of the GUV. We defined conditions that yielded reproducible aggregation kinetics under basal conditions and with none or moderate shaking. We found that on-pathway α-synuclein species and equilibrium amyloid aggregates, but not α-synuclein monomers, bound to lipid membranes. α-Synuclein was particularly strongly associated with GUVs containing the anionic lipids cardiolipin or DOPS, whereas it did not associate with GUVs containing only zwitterionic DOPC. We found that α-synuclein progressively aggregated at the surface of the GUVs, typically in distinct domains rather than uniformly covering the membrane, and that both lipid and protein were incorporated in the aggregates. Importantly, we never observed transport of α-synuclein over the GUV bilayer. This suggests that α-synuclein transport over membranes requires additional molecular players and that it might rely on active transport

Effects on differentiation of embryonic ventral midbrain progenitors by Lmx1a, MSX1, Ngn2, and Pitx3.

Neurons derived from neural stem cells could potentially be used for cell therapy in neurodegenerative disorders, such as Parkinson's disease. To achieve controlled differentiation of neural stem cells, we expressed transcription factors involved in the development of midbrain dopaminergic neurons in rat and human neural progenitors. Using retroviral-mediated transgene delivery, we overexpressed Lmx1a (LIM homeobox transcription factor 1, alpha), Msx1 (msh homeobox homolog 1), Ngn2 (neurogenin 2), or Pitx3 (paired-like homeodomain transcription factor 3) in neurospheres derived from embryonic day 14.5 rat ventral mesencephalic progenitors. We also expressed either Lmx1a or Msx1 in the human embryonic midbrain-derived progenitor cell line NGC-407. Rat cells transduced with Ngn2 exited the cell cycle and expressed the neuronal marker microtubule-associated protein 2 and catecholamine-neuron protein vesicular monoamine transporter 2. Interestingly, Pitx3 downregulated the expression of SOX2 (SRY-box containing gene 2) and Nestin, altered cell morphology, but never induced neuronal or glial differentiation. Ngn2 exhibited a strong neuron-inducing effect. In contrast, few Lmx1a-transduced cells matured into neurons, and Msx1 overexpression promoted oligodendrogenesis rather than neuronal differentiation. Importantly, none of these four genes, alone or in combination, enhanced differentiation of rat neural stem cells into dopaminergic neurons. Notably, the overexpression of Lmx1a, but not Msx1, in human neural progenitors increased the yield of tyrosine hydroxylase-immunoreactive cells by threefold. Together, we demonstrate that induced overexpression of transcription factor genes has profound and specific effects on the differentiation of rat and human midbrain progenitors, although few dopamine neurons are generated

Growth Factors and Feeder Cells Promote Differentiation of Human Embryonic Stem Cells into Dopaminergic Neurons: A Novel Role for Fibroblast Growth Factor-20

Human embryonic stem cells (hESCs) are a potential source of dopaminergic neurons for treatment of patients with Parkinson's disease (PD). Dopaminergic neurons can be derived from hESCs and display a characteristic midbrain phenotype. Once transplanted, they can induce partial behavioral recovery in animal models of PD. However, the potential research field faces several challenges that need to be overcome before clinical application of hESCs in a transplantation therapy in PD can be considered. These include low survival of the hESC-derived, grafted dopaminergic neurons after transplantation; unclear functional integration of the grafted neurons in the host brain; and, the risk of teratoma/tumor formation from the transplanted cells. This review is focused on our recent efforts to improve the survival of hESC-dervied dopaminergic neurons. In a recent study, we examined the effect of fibroblast growth factor (FGF)-20 in the differentiation of hESCs into dopaminergic neurons. We supplemented cultures of hESCs with FGF-20 during differentiation on PA6 mouse stromal cells for 3 weeks. When we added FGF-20 the yield of neurons expressing tyrosine hydroxylase increased. We demonstrated that at least part of the effect is contributed by enhanced cell differentiation towards the dopaminergic phenotype as well as reduced cell death. We compare our results with those obtained in other published protocols using different sets of growth factors. Taken together, our data indicate that FGF-20 has potent effects to generate large number of dopaminergic neurons derived from hESCs, which may be useful for hESC-based therapy in PD

Neurogenin2 Directs Granule Neuroblast Production and Amplification while NeuroD1 Specifies Neuronal Fate during Hippocampal Neurogenesis

The specification and differentiation of dentate gyrus granule neurons in the hippocampus require temporally and spatially coordinated actions of both intrinsic and extrinsic molecules. The basic helix-loop-helix transcription factor Neurogenin2 (Ngn2) and NeuroD1 are key regulators in these processes. Based on existing classification, we analyzed the molecular events occurring during hippocampal neurogenesis, primarily focusing on juvenile animals. We found that Ngn2 is transiently expressed by late type-2a amplifying progenitors. The Ngn2 progenies mature into hippocampal granule neurons. Interestingly, the loss of Ngn2 at early stages of development leads to a robust reduction in neurogenesis, but does not disturb granule neuron maturation per se. We found that the role of Ngn2 is to maintain progenitors in an undifferentiated state, allowing them to amplify prior to their maturation into granule neurons upon NeuroD1 induction. When we overexpressed Ngn2 and NeuroD1 in vivo, we found NeuroD1 to exhibit a more pronounced neuron-inductive effect, leading to granule neuron commitment, than that displayed by Ngn2. Finally, we observed that all markers expressed during the transcriptional control of hippocampal neurogenesis in rodents are also present in the human hippocampus. Taken together, we demonstrate a critical role of for Ngn2 and NeuroD1 in controlling neuronal commitment and hippocampal granule neuroblast formation, both during embryonic development and in post-natal hippocampal granule neurogenesis

Both apoptosis and necrosis occur early after intracerebral grafting of ventral mesencephalic tissue: a role for protease activation.

Neural transplantation is an experimental treatment for Parkinson's disease. Widespread clinical application of the grafting technique is hampered by a relatively poor survival (around 10%) of implanted embryonic dopamine neurones. Earlier animal studies have indicated that a large proportion of the grafted cells die during graft tissue preparation and within the first few days after intracerebral implantation. The present study was designed to reveal the prevalence of cell death in rat intrastriatal grafts at 90 min, 1, 3, 6 and 42 days after implantation. We examined apoptotic cell death using semi-thin and paraffin sections stained with methylene blue and an antibody against activated caspase 3, respectively. We identified abundant apoptotic cell death up to 3 days after transplantation. In addition, we studied calpain activation using an antibody specific for calpain-cleaved fodrin. We report a peak in calpain activity 90 min after grafting. Surprisingly, we did not observe any significant difference in the number of dopaminergic neurones over time. The present results imply that grafted cells may be victims of either an early necrotic or a later apoptotic cell death and that there is substantial cell death as early as 90 min after implantation

Are Stem Cell-Based Therapies for Parkinson's Disease Ready for the Clinic in 2016?

Recent news of an impending clinical cell transplantation trial in Parkinson's disease using parthenogenetic stem cells as a source of donor tissue have raised hopes in the patient community and sparked discussion in the research community. Based on discussions held by a global collaborative initiative on translation of stem cell therapy in Parkinson's disease, we have identified a set of key questions that we believe should be addressed ahead of every clinical stem cell-based transplantation trial in this disorder. In this article, we first provide a short history of cell therapy in Parkinson's disease and briefly describe the current state-of-art regarding human stem cell-derived dopamine neurons for use in any patient trial. With this background information as a foundation, we then discuss each of the key questions in relation to the upcoming therapeutic trial and critically assess if the time is ripe for clinical translation of parthenogenetic stem cell technology in Parkinson's disease.Some of the work discussed in this commentary was supported by grants from the EU FP 7 programme including TRANSEURO and NeuroStemCellRepair, and the Swedish Research Council. RAB is also supported by an NIHR grant of a Biomedical Research Centre to Addenbrooke’s Hospital and the University of Cambridge.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by IOP Press

The Biological and Ethical Basis of the Use of Human Embryonic Stem Cells for In Vitro Test Systems or Cell Therapy

Human embryonic stem cells (hESC) are now routinely cultured in many laboratories, and differentiation protocols are available to generate a large variety of cell types. In an ongoing ethical debate opinions of different groups are based on varying sets of religious, historical, cultural and scientific arguments as well as on widely differing levels of general information. We here give an overview of the biological background for non-specialists, and address all issues of the current stem cell debate that are of concern in different cultures and states. Thirty-five chapters address embryo definition, potential killing and the beginning of human life, in addition to matters of human dignity, patenting, commercialisation, and potential alternatives for the future, such as induced pluripotent (reprogrammed) stem cells. All arguments are compiled in a synopsis, and compromise solutions, e.g. for the definition of the beginning of personhood and for assigning dignity to embryos, are suggested. Until recently, the major application of hESC was thought to be transplantation of cells derived from hESC for therapeutic use. We discuss here that the most likely immediate uses will rather be in vitro test systems and disease models. Major and minor pharmaceutical companies have entered this field, and the European Union is sponsoring academic research into hESC-based innovative test systems. This development is supported by new testing strategies in Europe and the USA focussing on human cell-based in vitro systems for safety evaluations, and shifting the focus of toxicology away from classical animal experiments towards a more mechanistic understanding.JRC.I.3-In-vitro method

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