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Biomedical subjects

Anders Björklund

Publications and source records attributed to Anders Björklund.

At least 19 recordsLinked to original sources
PubMed2007

In vitro characterization of a human neural progenitor cell coexpressing SSEA4 and CD133.

The stage-specific embryonic antigen 4 (SSEA4) is commonly used as a cell surface marker to identify the pluripotent human embryonic stem (ES) cells. Immunohistochemistry on human embryonic central nervous system revealed that SSEA4 is detectable in the early neuroepithelium, and its expression decreases as development proceeds. Flow cytometry analysis of forebrain-derived cells demonstrated that the SSEA4-expressing cells are enriched in the neural stem/progenitor cell fraction (CD133(+)), but are rarely codetected with the neural stem cell (NSC) marker CD15. Using a sphere-forming assay, we showed that both subfractions CD133(+)/SSEA4(+) and CD133(+)/CD15(+) isolated from the embryonic forebrain are enriched in neurosphere-initiating cells. In addition CD133, SSEA4, and CD15 expression is sustained in the expanded neurosphere cells and also mark subfractions of neurosphere-initiating cells. Therefore, we propose that SSEA4 associated with CD133 can be used for both the positive selection and the enrichment of neural stem/progenitor cells from human embryonic forebrain.

AC133 Antigen↗
PubMed2006

Neurogenin2 identifies a transplantable dopamine neuron precursor in the developing ventral mesencephalon.

In neural transplantation studies, there is an interest in identifying and isolating mesencephalic dopamine (mesDA) neuron precursors that have the capacity to differentiate into fully mature mesDA neurons after transplantation. We report here that in the developing ventral mesencephalon (VM) the proneural gene Neurogenin2 (Ngn2) is expressed exclusively in the part of the ventricular zone that gives rise to the migrating mesDA neuroblasts, but not in the differentiated mesDA neurons. From other studies, we know that Ngn2 is involved in the generation of mesDA neurons and that the development of mesDA neurons is severely compromised in Ngn2-null mutant mice. We show here that cells isolated by FACS from the developing VM of Ngn2-GFP knock-in mice are capable of generating mesDA neurons, both in vitro and after transplantation to the striatum of neonatal rats. All mesDA neuron precursors, but not the serotonergic or GABAergic neuron precursors, are contained in the Ngn2-GFP-expressing population. Moreover, all glial cells were generated from cells contained in the GFP-negative cell fraction. The results show that surviving mesDA neurons in VM grafts are derived from early postmitotic, probably Nurr1-expressing precursors before they have acquired their fully differentiated neuronal phenotype. The Ngn2-GFP reporter construct used here thus provides a tool for the identification of mesDA neuron precursors in the VM and selective isolation of transplantable mesDA neuron precursors for transplantation.

Aldehyde Dehydrogenase↗
PubMed2005

Graft placement and uneven pattern of reinnervation in the striatum is important for development of graft-induced dyskinesia.

In two recent double-blind clinical trials of fetal ventral mesencephalic cell transplants into the striatum in patients with Parkinson's disease (PD), a significant proportion of the grafted patients developed dyskinetic side effects, which were not seen in the sham operated patients. Comparison between dyskinetic and non-dyskinetic grafted patients in one of the trials suggested that an uneven pattern of striatal reinnervation might be the leading cause of the dyskinesias. Here, we studied the importance of graft placement for the development of dyskinesias in parkinsonian rats. Abnormal involuntary movements resembling peak-dose dyskinesias seen in PD patients were induced by daily injections of L-DOPA for 6 weeks. The dyskinetic animals received about 130.000 fetal ventral mesencephalic cells as single grafts placement in the rostral or the caudal aspect of the head of striatum. The results show that grafts placed in the caudal, but not the rostral, part are effective in reducing the L-DOPA-induced limb and orolingual dyskinesia, predominantly seen as hyperkinesia. The same grafts, however, also induced a new type of dyskinetic behavior after activation with amphetamine, which were not seen in non-grafted lesion controls. The severity of these abnormal involuntary movements was significantly correlated with a higher graft-derived dopaminergic reinnervation in the caudal aspect of the head of striatum relative to the rostral part. The results indicate that graft-induced dyskinesias in PD patients may be linked to single, small graft deposits that provide an uneven, patchy reinnervation of the putamen.

Animals↗
PubMed2005

Factors affecting the clinical outcome after neural transplantation in Parkinson's disease.

Intrastriatal grafts of embryonic mesencephalic tissue can survive in the brains of patients with Parkinson's disease, but the degree of symptomatic relief is highly variable and some cases develop troublesome dyskinesias. Here we explored, using clinical assessment and 18F-dopa and 11C-raclopride PET, factors which may influence the functional outcome after transplantation. We observed increased 18F-dopa uptake in the grafted putamen, signifying continued survival of the transplanted dopaminergic neurons, in parallel with a progressive reduction of 18F-dopa uptake in non-grafted regions for the whole patient group. The patients with the best functional outcome after transplantation exhibited no dopaminergic denervation in areas outside the grafted areas either preoperatively or at 1 or 2 years post-operatively. In contrast, patients with no or modest clinical benefit showed reduction of 18F-dopa in ventral striatum prior to or following transplantation, which may have limited graft-induced improvement. We obtained no evidence that dyskinesias were caused by abnormal dopamine (DA) release from the grafts. As has been observed for intrinsic dopaminergic neurons, there was a significant correlation between 18F-dopa uptake and methamphetamine-induced change of 11C-raclopride binding (as a measure of DA release) in the putamen containing the graft. Furthermore, we observed no correlation between 11C-raclopride binding in anterior, posterior or entire putamen under basal conditions or after methamphetamine, and dyskinesia severity scores in the contralateral side of the body. Withdrawal of immunosuppression at 29 months after transplantation caused no reduction of 18F-dopa uptake or worsening of UPDRS motor score, indicating continued survival and function of the graft. However, patients showed increased dyskinesia scores, which might have been caused either by growth of the graft or worsening of a low-grade inflammation around the graft. These findings indicate that poor outcome after transplantation is associated with progressive dopaminergic denervation in areas outside the grafts, a process which may have started already before surgery. Also, that the development of dyskinesias after transplantation is not associated with excessive DA release from the grafts. Finally, our data provide evidence that long-term immunosuppression can be withdrawn without interfering with graft survival or the motor recovery induced by transplantation.

Carbon Radioisotopes↗
PubMed2005

Increased fiber outgrowth from xeno-transplanted human embryonic dopaminergic neurons with co-implants of polymer-encapsulated genetically modified cells releasing glial cell line-derived neurotrophic factor.

We investigated whether a continuous supply of glial cell line-derived neurotrophic factor (GDNF) via encapsulated genetically modified cells can promote survival and fiber outgrowth from xenotransplanted human dopaminergic neurons. Cells genetically engineered to continuously secrete GDNF were confined in hollow fiber-based macrocapsules. Each hemiparkinsonian rat received either a single C2C12-hGDNF capsule (n=8) or a C2C12-control capsule (n=8) concomitantly with human embryonic ventral mesencephalic cell suspension transplants. Our results show that fiber outgrowth in the area between the capsule and the graft is more extensive in rats with GDNF-releasing capsules than in rats with control capsules. We suggest that continuous and safe delivery of GDNF to the brain could be a potential way to optimize neural transplantation as a therapy for Parkinson's disease.

Analysis of Variance↗
PubMed2005

Identification of dopaminergic neurons of nigral and ventral tegmental area subtypes in grafts of fetal ventral mesencephalon based on cell morphology, protein expression, and efferent projections.

Transplants of fetal ventral mesencephalic tissue are known to contain a mixture of two major dopamine (DA) neuron types: the A9 neurons of the substantia nigra pars compacta (SNpc) and the A10 neurons of the ventral tegmental area (VTA). Previous studies have suggested that these two DA neuron types may differ in their growth characteristics, but, because of technical limitations, it has so far been difficult to identify the two subtypes in fetal ventral mesencephalon (VM) grafts and trace their axonal projections. Here, we have made use of a transgenic mouse expressing green fluorescent protein (GFP) under the tyrosine hydroxylase promoter. The expression of the GFP reporter allowed for visualization of the grafted DA neurons and their axonal projections within the host brain. We show that the SNpc and VTA neuron subtypes in VM grafts can be identified on the basis of their morphology and location within the graft, and their expression of a G-protein-gated inwardly rectifying K+ channel subunit (Girk2) and calbindin, respectively, and also that the axonal projections of the two DA neuron types are markedly different. By retrograde axonal tracing, we show that dopaminergic innervation of the striatum is derived almost exclusively from the Girk2-positive SNpc cells, whereas the calbindin-positive VTA neurons project to the frontal cortex and probably also other forebrain areas. The results suggest the presence of axon guidance and target recognition mechanisms in the DA-denervated forebrain that can guide the growing axons to their appropriate targets and indicate that cell preparations used for cell replacement in Parkinson's disease will be therapeutically useful only if they contain cells capable of generating the correct nigral DA neuron phenotype.

Age Factors↗
PubMed2005

Reversal of dyskinesias in an animal model of Parkinson's disease by continuous L-DOPA delivery using rAAV vectors.

Dyskinesias are a major complication of long-term l-3,4-dihydroxyphenylalanine (L-DOPA) treatment in Parkinson's disease, and are believed to result from the intermittent and pulsatile supply of L-DOPA. Daily injections of L-DOPA can prime similar abnormal involuntary movements of the limb, orolingual and axial muscles in rats rendered parkinsonian by destruction of the nigrostriatal dopamine (DA) neurons. In this study we used 33 rats with severe nigrostriatal dopamine depletion and showed that in vivo gene transfer of the DA-synthetic enzymes tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) using recombinant adeno-associated virus vectors can provide a constant source of DOPA production locally in the striatum, at a level that is effective in reducing L-DOPA-induced dyskinesias by >85%, and reverse lesion-induced motor impairments. Furthermore, the abnormal expression of DeltaFosB, prodynorphin and preproenkephalin mRNA within the striatal projection neurons normally seen in dyskinetic animals was completely reversed by TH-GCH1 gene transfer. These findings form a strong basis for replacing, or supplementing, conventional systemic L-DOPA therapy by continuous intrastriatal DOPA using in vivo gene transfer in the treatment of patients with advanced Parkinson's disease.

Animals↗
PubMed2005

Cell transplantation in Parkinson's disease: how can we make it work?

Previous open-label clinical trials have provided proof of principle that intrastriatal transplants of fetal dopaminergic neurons can induce substantial and long-lasting functional benefits in patients with Parkinson's disease. However, in two recent NIH-sponsored double-blind trials, functional improvements were only marginal and the primary endpoints were not met. Severe off-phase dyskinesias were observed in a significant proportion of the transplanted patients, raising doubts about the viability of the cell-transplantation approach. Here, we discuss the problems raised by the NIH-sponsored trials and point to several shortcomings that might explain the overall poor outcome, and we identify several crucial issues that remain to be resolved to develop cell replacement into an effective and safe therapy.

Animals↗
PubMed2005

Functional properties and synaptic integration of genetically labelled dopaminergic neurons in intrastriatal grafts.

Intrastriatal grafts of fetal ventral mesencephalic tissue, rich in dopaminergic neurons, can reverse symptoms in Parkinson's disease. For development of effective cell replacement therapy, other sources of dopaminergic neurons, e.g. derived from stem cells, are needed. However, the electrophysiological properties grafted cells need to have in order to induce substantial functional recovery are poorly defined. It has not been possible to prospectively identify and record from dopaminergic neurons in fetal transplants. Here we used transgenic mice expressing green fluorescent protein under control of the rat tyrosine hydroxylase promoter for whole-cell patch-clamp recordings of endogenous and grafted dopaminergic neurons. We transplanted ventral mesencephalic tissue from E12.5 transgenic mice into striatum of neonatal rats with or without lesions of the nigrostriatal dopamine system. The transplanted cells exhibited intrinsic electrophysiological properties typical of substantia nigra dopaminergic neurons, i.e. broad action potentials, inward rectifying currents with characteristic 'sag', and spontaneous action potentials. The grafted dopaminergic neurons also received functional excitatory and inhibitory synaptic inputs from the host brain, as shown by the presence of both spontaneous and stimulation-evoked excitatory and inhibitory postsynaptic currents. Occurrence of spontaneous excitatory and inhibitory currents was lower, and of spontaneous action potentials was higher, in neurons placed in the dopamine-depleted striatum than of those in the intact striatum. Our findings define specific electrophysiological characteristics of transplanted fetal dopaminergic neurons, and we provide the first direct evidence of functional synaptic integration of these neurons into host neural circuitries.

Animals↗
PubMed2005

Isolation and characterization of neural precursor cells from the Sox1-GFP reporter mouse.

We have made use of a reporter mouse line in which enhanced green fluorescence protein (GFP) is inserted into the Sox1 locus. We show that the GFP reporter is coexpressed with the Sox1 protein as well as with other known markers for neural stem and progenitor cells, and can be used to identify and isolate these cells by fluorescence-activated cell sorting (FACS) from the developing or adult brain and from neurosphere cultures. All neurosphere-forming cells with the capacity for multipotency and self-renewal reside in the Sox1-GFP-expressing population. Thus, the Sox1-GFP reporter system is highly useful for identification, isolation and characterization of neural stem and progenitor cells, as well as for the validation of alternative means for isolating neural stem and progenitor cells. Further, transplantation experiments show that Sox1-GFP cells isolated from the foetal brain give rise to neurons and glia in vivo, and that many of the neurons display phenotypic characteristics appropriate for the developing brain region from which the Sox1-GFP precursors were derived. On the other hand, Sox1-GFP cells isolated from the adult subventricular zone or expanded neurosphere cultures gave rise almost exclusively to glial cells following transplantation. Thus, not all Sox1-GFP cells possess the same capacity for neuronal differentiation in vivo.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed2005

Cell therapy for Parkinson's disease: problems and prospects.

Cell replacement therapy in Parkinson's disease (PD) has so far been based on the use of primary dopaminergic (DA) neuroblasts obtained from the brain of aborted human fetuses. Clinical trials show that intrastriatal DA neuron transplants can give substantial symptomatic relief in advanced PD patients. Two recent NIH-sponsored placebo-controlled trials, however, have given disappointing results and highlighted a number of critical issues that need to be resolved in order to turn cell transplantation into an acceptable clinical therapy. First, graft survival and clinical outcome has so far been too variable, suggesting that DA neuron grafts may not be equally effective in all PD patients. Secondly, it has become clear that immune mechanisms leading to slowly developing inflammatory responses may compromise long-term graft survival and function. Third, the problems associated with the use of tissue from aborted fetuses make it necessary to develop alternative sources of cells for transplantation. Recent progress in the generation of DA neuroblasts from neural progenitors and embryonic stem cells suggest that these kinds of cell may offer more accessible, defined and standardized sources of cells for clinical transplantation in PD.

Cell Differentiation↗
PubMed2004

Striatal neuron differentiation from neurosphere-expanded progenitors depends on Gsh2 expression.

Neural stem and progenitor cells from the embryonic forebrain can be expanded under growth factor stimulation in vitro, either as free-floating aggregates called neurospheres or as attached monolayer cultures. We have previously shown that despite the maintenance of important regulatory genes such as Gsh2, in vitro expansion of cells from the lateral ganglion eminence (LGE) restricts their differentiation potential. Specifically, their ability to differentiate into striatal projection neurons is compromised. It is not clear whether this restriction is caused by loss of progenitors with the ability to generate striatal projection neurons or whether the restricted differentiation potential is caused by factors lacking during in vitro differentiation. To address this, we have set up an in vitro system, in which expanded LGE-derived cells are differentiated in coculture with primary cells isolated from different regions of the embryonic brain. We provide evidence that the primary cells supply the expanded cells with contact-mediated region-specific developmental cues. Neurosphere-expanded LGE progenitors can, when presented with these cues, differentiate into neurons with characteristics of striatal projection neurons. Furthermore, we show that the ability of the expanded LGE cells to respond to the developmental cues presented by the primary cells depends on the maintained expression of Gsh2 in the expanded cells.

Animals↗
PubMed2004

Overexpression of glial cell line-derived neurotrophic factor using a lentiviral vector induces time- and dose-dependent downregulation of tyrosine hydroxylase in the intact nigrostriatal dopamine system.

The effects of continuous glial cell line-derived neurotrophic factor (GDNF) overexpression in the intact nigrostriatal dopamine (DA) system was studied using recombinant lentiviral (rLV) vector delivery of GDNF to the striatum or substantia nigra (SN) in the rat. Intrastriatal delivery of rLV-GDNF resulted in significant overexpression of GDNF in the striatum (2-4 ng/mg tissue) and anterograde transport of GDNF protein to the SN. Striatal rLV-GDNF delivery initially induced an increase in DA turnover (1-6 weeks), accompanied by significant contralateral turning in response to amphetamine, suggesting an enhancement of the DA system on the injected side. Starting 6 weeks after continuous GDNF delivery, we observed a selective downregulation of tyrosine hydroxylase (TH) protein (approximately 70%) that was maintained until the end of the experiment (24 weeks). A similar effect was observed when rLV-GDNF was injected into the SN. The magnitude of TH downregulation was related to the level of GDNF expression and was most pronounced in animals in which the striatal GDNF level exceeded 0.7 ng/mg tissue. The decreased TH protein levels were associated with similar reductions in the in vitro TH enzyme activity (approximately 70%); however, in vivo L-3,4-dihydroxyphenylalanine production rate and DA tissue levels were maintained at normal levels. The results indicate that downregulation of TH protein reflects a compensatory effect in response to continuous GDNF stimulation of the DA neurons mediated by a combination of overactivity at the DA synapse and a direct GDNF-induced action on TH gene expression. This compensatory mechanism is proposed to maintain long-term DA neuron function within the normal range.

Animals↗
PubMed2004

Cell therapy in Parkinson's disease.

The clinical studies with intrastriatal transplants of fetal mesencephalic tissue in Parkinson's disease (PD) patients have provided proof-of-principle for the cell replacement strategy in this disorder. The grafted dopaminergic neurons can reinnervate the denervated striatum, restore regulated dopamine (DA) release and movement-related frontal cortical activation, and give rise to significant symptomatic relief. In the most successful cases, patients have been able to withdraw L-dopa treatment after transplantation and resume an independent life. However, there are currently several problems linked to the use of fetal tissue: 1) lack of sufficient amounts of tissue for transplantation in a large number of patients, 2) variability of functional outcome with some patients showing major improvement and others modest if any clinical benefit, and 3) occurrence of troublesome dyskinesias in a significant proportion of patients after transplantation. Thus, neural transplantation is still at an experimental stage in PD. For the development of a clinically useful cell therapy, we need to define better criteria for patient selection and how graft placement should be optimized in each patient. We also need to explore in more detail the importance for functional outcome of the dissection and cellular composition of the graft tissue as well as of immunological mechanisms. Strategies to prevent the development of dyskinesias after grafting have to be developed. Finally, we need to generate large numbers of viable DA neurons in preparations that are standardized and quality controlled. The stem cell technology may provide a virtually unlimited source of DA neurons, but several scientific issues need to be addressed before stem cell-based therapies can be tested in PD patients.

Animals↗
PubMed2003

Nigrostriatal alpha-synucleinopathy induced by viral vector-mediated overexpression of human alpha-synuclein: a new primate model of Parkinson's disease.

We used a high-titer recombinant adeno-associated virus (rAAV) vector to express WT or mutant human alpha-synuclein in the substantia nigra of adult marmosets. The alpha-synuclein protein was expressed in 90-95% of all nigral dopamine neurons and distributed by anterograde transport throughout their axonal and dendritic projections. The transduced neurons developed severe neuronal pathology, including alpha-synuclein-positive cytoplasmic inclusions and granular deposits; swollen, dystrophic, and fragmented neuritis; and shrunken and pyknotic, densely alpha-synuclein-positive perikarya. By 16 wk posttransduction, 30-60% of the tyrosine hydroxylase-positive neurons were lost, and the tyrosine hydroxylase-positive innervation of the caudate nucleus and putamen was reduced to a similar extent. The rAAV-alpha-synuclein-treated monkeys developed a type of motor impairment, i.e., head position bias, compatible with this magnitude of nigrostriatal damage. rAAV vector-mediated alpha-synuclein gene transfer provides a transgenic primate model of nigrostriatal alpha-synucleinopathy that is of particular interest because it develops slowly over time, like human Parkinson's disease (PD), and expresses neuropathological features (alpha-synuclein-positive inclusions and dystrophic neurites, in particular) that are similar to those seen in idiopathic PD. This model offers new opportunities for the study of pathogenetic mechanisms and exploration of new therapeutic targets of particular relevance to human PD.

Amphetamines↗