Gesine Paul

α-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells

Post-mortem analyses of brains from patients with Parkinson disease who received fetal mesencephalic transplants show that α-synuclein-containing (α-syn-containing) Lewy bodies gradually appear in grafted neurons. Here, we explored whether intercellular transfer of α-syn from host to graft, followed by seeding of α-syn aggregation in recipient neurons, can contribute to this phenomenon. We assessed α-syn cell-to-cell transfer using microscopy, flow cytometry, and high-content screening in several coculture model systems. Coculturing cells engineered to express either GFP- or DsRed-tagged α-syn resulted in a gradual increase in double-labeled cells. Importantly, α-syn-GFP derived from 1 neuroblastoma cell line localized to red fluorescent aggregates in other cells expressing DsRed-α-syn, suggesting a seeding effect of transmitted α-syn. Extracellular α-syn was taken up by cells through endocytosis and interacted with intracellular α-syn. Next, following intracortical injection of recombinant α-syn in rats, we found neuronal uptake was attenuated by coinjection of an endocytosis inhibitor. Finally, we demonstrated in vivo transfer of α-syn between host cells and grafted dopaminergic neurons in mice overexpressing human α-syn. In summary, intercellularly transferred α-syn interacts with cytoplasmic α-syn and can propagate α-syn pathology. These results suggest that α-syn propagation is a key element in the progression of Parkinson disease pathology.

Transplantation of Human Embryonic Stem Cell‐Derived Cells to a Rat Model of Parkinson's Disease: Effect of In Vitro Differentiation on Graft Survival and Teratoma Formation

Human embryonic stem cells (hESCs) have been proposed as a source of dopamine (DA) neurons for transplantation in Parkinsons disease (PD). We have investigated the effect of in vitro predifferentiation on in vivo survival and differentiation of hESCs implanted into the 6‐OHDA (6‐hydroxydopamine)‐lesion rat model of PD. The hESCs were cocultured with PA6 cells for 16, 20, or 23 days, leading to the in vitro differentiation into DA neurons. Grafted hESC‐derived cells survived well and expressed neuronal markers. However, very few exhibited a DA neuron phenotype. Reversal of lesion‐induced motor deficits was not observed. Rats grafted with hESCs predifferentiated in vitro for 16 days developed severe teratomas, whereas most rats grafted with hESCs predifferentiated for 20 and 23 days remained healthy until the end of the experiment. This indicates that prolonged in vitro differentiation of hESCs is essential for preventing formation of teratomas.

Behavioral characterization of a unilateral 6-OHDA-lesion model of Parkinson's disease in mice

Parkinsons disease (PD) is one of the most common neurodegenerative disorders. Several toxin-induced animals models simulate the motor deficits occurring in PD. Among them, the unilateral 6-hydroxydopamine (6-OHDA) model is frequently used in rats and has the advantage of presenting side-biased motor impairments. However, the behavioral consequences of a unilateral 6-OHDA-lesion have, so far, not been described in detail in mice. The aim of this study was to characterize mice with unilateral 6-OHDA-lesions placed in the median forebrain bundle using several motor behavioral tests in order to identify the most suitable predictor of nigral cell loss. Mice underwent various drug-induced (amphetamine- and apomorphine-induced rotation) and spontaneous motor tests (cylinder, rotarod, elevated body swing, and stride length test). The amphetamine-induced rotation test, the cylinder and the rotarod test were most sensitive and reliable in detecting loss of tyrosine hydroxylase-immunoreactive cells in the substantia nigra. This study demonstrates that substantial and stable unilateral 6-OHDA-induced lesions can be established in mice, and that these lesions can be functionally assessed using several different side-bias-based behavioral tests. This mouse model offers the opportunity to use transgenic mouse strains and study the interactions between genes of interest and toxins in relation to Parkinsons disease etiology in the future.

The Adult Human Brain Harbors Multipotent Perivascular Mesenchymal Stem Cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain.

The secretome of mesenchymal stem cells: potential implications for neuroregeneration.

Mesenchymal stem cells have shown regenerative properties in many tissues. This feature had originally been ascribed to their multipotency and thus their ability to differentiate into tissue-specific cells. However, many researchers consider the secretome of mesenchymal stem cells the most important player in the observed reparative effects of these cells. In this review, we specifically focus on the potential neuroregenerative effect of mesenchymal stem cells, summarize several possible mechanisms of neuroregeneration and list key factors mediating this effect. We illustrate examples of mesenchymal stem cell treatment in central nervous system disorders including stroke, neurodegenerative disorders (such as Parkinsons disease, Huntingtons disease, multiple system atrophy and cerebellar ataxia) and inflammatory disease (such as multiple sclerosis). We specifically highlight studies where mesenchymal stem cells have entered clinical trials.

Brain pericytes acquire a microglial phenotype after stroke.

Abstract Pericytes are located on the abluminal side of endothelial cells lining the microvasculature in all organs. They have been identified as multipotent progenitor cells in several tissues of the body including the human brain. New evidence suggests that pericytes contribute to tissue repair, but their role in the injured brain is largely unknown. Here, we investigate the role of pericytes in ischemic stroke. Using a pericyte-reporter mouse model, we provide unique evidence that regulator of G-protein signaling 5 expressing cells are activated pericytes that leave the blood vessel wall, proliferate and give rise to microglial cells after ischemic brain injury. Consistently, we show that activated pericytes express microglial markers in human stroke brain tissue. We demonstrate that human brain-derived pericytes adopt a microglial phenotype and upregulate mRNA specific for activated microglial cells under hypoxic conditions in vitro. Our study indicates that the vasculature is a novel source of inflammatory cells with a microglial phenotype in brain ischemia and hence identifies pericytes as an important new target for the development of future stroke therapies.

A partial lesion model of Parkinson's disease in mice - Characterization of a 6-OHDA-induced medial forebrain bundle lesion.

The most frequently used animal models for Parkinsons disease (PD) utilize unilateral injection of 6-hydroxydopamine (6-OHDA) in the medial forebrain bundle (MFB), which results in total denervation of the dopaminergic nigrostriatal pathway. However, neuroprotective interventions in PD require models resembling earlier stages of PD, where some dopaminergic cells and fibres remain. The aim of the present study was therefore to establish a MFB partial lesion model in mice. We tested four different 6-OHDA doses, and our results show a dose-dependent loss of nigral dopaminergic cells and striatal fibres that correlated with behavioural impairment in several behavioural tests. Specifically, doses of 0.7 μg and 1 μg of 6-OHDA induced a partial denervation of the nigrostriatal pathway, associated with a mild but quantifiable behavioural impairment. We identified the amphetamine-induced rotation, stepping, corridor and cylinder test to be sensitive enough to select partial lesion animals. Based on our data, we proposed a range of cut-off values for these different behavioural tests to select partial lesion mice. Using a statistical prediction model we identified two behavioural tests (the stepping test and amphetamine-induced rotation test) that with a high sensitivity and specificity predict the extent of nigral dopaminergic cell loss and select mice with a partial nigrostriatal lesion prior to further interventions. This model can serve as an important tool to study neuroprotective therapies for PD in mouse models, especially when the treatment targets the substantia nigra and/or the striatum.

Safety and tolerability of intracerebroventricular PDGF-BB in Parkinson's disease patients.

BACKGROUND. Recombinant human PDGF-BB (rhPDGF-BB) reduces Parkinsonian symptoms and increases dopamine transporter (DAT) binding in several animal models of Parkinsons disease (PD). Effects of rhPDGF-BB are the result of proliferation of ventricular wall progenitor cells and reversed by blocking mitosis. Based on these restorative effects, we assessed the safety and tolerability of intracerebroventricular (i.c.v.) rhPDGF-BB administration in individuals with PD. METHODS. We conducted a double-blind, randomized, placebo-controlled phase I/IIa study at two clinical centers in Sweden. Twelve patients with moderate PD received rhPDGF-BB via an implanted drug infusion pump and an investigational i.c.v. catheter. Patients were assigned to a dose cohort (0.2, 1.5, or 5 μg rhPDGF-BB per day) and then randomized to active treatment or placebo (3:1) for a 12-day treatment period. The primary objective was to assess safety and tolerability of i.c.v.-delivered rhPDGF-BB. Secondary outcome assessments included several clinical rating scales and changes in DAT binding. The follow-up period was 85 days. RESULTS. All patients completed the study. There were no unresolved adverse events. Serious adverse events occurred in three patients; however, these were unrelated to rhPDGF-BB administration. Secondary outcome parameters did not show dose-dependent changes in clinical rating scales, but there was a positive effect on DAT binding in the right putamen. CONCLUSION. At all doses tested, i.c.v. administration of rhPDGF-BB was well tolerated. Results support further clinical development of rhPDGF-BB for patients with PD. TRIAL REGISTRATION. Clinical Trials.gov NCT00866502. FUNDING. Newron Sweden AB (former NeuroNova AB) and Swedish Governmental Agency for Innovation Systems (VINNOVA).

Tyrosine hydroxylase expression is unstable in a human immortalized mesencephalic cell line--studies in vitro and after intracerebral grafting in vivo.

We have studied the stability of the dopaminergic phenotype in a conditionally immortalized human mesencephalic cell line, MESC2.10. Even though MESC2.10 cells exhibit features of dopaminergic neurons in vitro, none of the cells expressed tyrosine hydroxylase (TH) after transplantation into a rat model of Parkinsons disease. We examined whether this is caused by cell death or loss of transmitter phenotype. Cells were cultured in differentiation medium, then harvested and replated into the same medium where they continued to express TH, whereas replated cells fed medium lacking differentiation factors (dibutyryl cAMP and glial cell line-derived neurotrophic factor) did not. Interestingly, cultures grown in the absence of differentiation factors could regain TH expression once exposed to differentiation medium. Our data suggest that TH expression in vitro is inducible in neurons derived from the MESC2.10 cell line and that the dopaminergic phenotype of these cells in vivo might be unstable.

Stem cells: hype or hope?

Stem cells undergo self-renewal and differentiate into multiple lineages of mature cells. The identification of stem cells in diverse adult tissues and the findings that human embryonic stem cells can be proliferated and differentiated has kindled the imagination of both scientists and the public regarding future stem cell technology. These cells could constitute an unlimited supply of diverse cell types that can be used for cell transplantation or drug discovery. The new options raise several fundamental ethical issues. This review gives an overview of the scientific basis underlying the hope generated by stem cell research and discusses current ethical and funding regulations.