| 1. |
- Aldrin-Kirk, Patrick, et al.
(författare)
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Novel AAV-Based Rat Model of Forebrain Synucleinopathy Shows Extensive Pathologies and Progressive Loss of Cholinergic Interneurons.
- 2014
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:7
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Tidskriftsartikel (refereegranskat)abstract
- Synucleinopathies, characterized by intracellular aggregation of α-synuclein protein, share a number of features in pathology and disease progression. However, the vulnerable cell population differs significantly between the disorders, despite being caused by the same protein. While the vulnerability of dopamine cells in the substantia nigra to α-synuclein over-expression, and its link to Parkinson's disease, is well studied, animal models recapitulating the cortical degeneration in dementia with Lewy-bodies (DLB) are much less mature. The aim of this study was to develop a first rat model of widespread progressive synucleinopathy throughout the forebrain using adeno-associated viral (AAV) vector mediated gene delivery. Through bilateral injection of an AAV6 vector expressing human wild-type α-synuclein into the forebrain of neonatal rats, we were able to achieve widespread, robust α-synuclein expression with preferential expression in the frontal cortex. These animals displayed a progressive emergence of hyper-locomotion and dysregulated response to the dopaminergic agonist apomorphine. The animals receiving the α-synuclein vector displayed significant α-synuclein pathology including intra-cellular inclusion bodies, axonal pathology and elevated levels of phosphorylated α-synuclein, accompanied by significant loss of cortical neurons and a progressive reduction in both cortical and striatal ChAT positive interneurons. Furthermore, we found evidence of α-synuclein sequestered by IBA-1 positive microglia, which was coupled with a distinct change in morphology. In areas of most prominent pathology, the total α-synuclein levels were increased to, on average, two-fold, which is similar to the levels observed in patients with SNCA gene triplication, associated with cortical Lewy body pathology. This study provides a novel rat model of progressive cortical synucleinopathy, showing for the first time that cholinergic interneurons are vulnerable to α-synuclein over-expression. This animal model provides a powerful new tool for studies of neuronal degeneration in conditions of widespread cortical α-synuclein pathology, such as DLB, as well an attractive model for the exploration of novel biomarkers.
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| 2. |
- Anisimov, Sergey, et al.
(författare)
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Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells.
- 2011
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Ingår i: Cellular & Molecular Biology Letters. - : Walter de Gruyter GmbH. - 1689-1392. ; 16:1, s. 79-88
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Tidskriftsartikel (refereegranskat)abstract
- The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early, intermediate and late passages using a custom DNA microarray platform (NeuroStem 2.0 Chip). The microarray data was validated using RT-PCR and virtual SAGE analysis. Our comparative gene expression study identified a limited number of molecular targets potentially involved in the ability of human neonatal foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates.
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| 3. |
- Fedele, Valentina, et al.
(författare)
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Neurogenesis In The R6/2 Mouse Model Of Huntington'S Disease Is Impaired At The Level Of Neurod1
- 2011
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Ingår i: Neuroscience. - : Elsevier BV. - 1873-7544 .- 0306-4522. ; 173, s. 76-81
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Tidskriftsartikel (refereegranskat)abstract
- Adult neurogenesis is impaired in the hippocampus of transgenic R6 mouse models of Huntington's disease (HD). The phenotypes of R6 transgenic mice mimic several symptoms and signs of the disease (Li et al., 2005). They exhibit neurological and endocrine changes resembling some symptoms seen in humans. The reduction in neurogenesis is only apparent in the dentate gyrus as the number of newborn neurons in the subventricular zone, and olfactory bulb, is normal in R6 mice. The mechanism(s) underlying the reduction in hippocampal neurogenesis is still not fully understood. Here we show that the number of neuroblasts, but not granule neuron progenitors, is greatly reduced in 11-week old transgenic mice compared with wild-type (WT) controls. We demonstrate that NeuroD1 expression is reduced in the hippocampus. This is coupled to a decreased expression of downstream markers doublecortin and calretinin in maturing neurons. Taken together, our results suggest that mutant huntingtin (Htt) causes alterations of proteins expression in hippocampal progenitors, which might contribute to cognitive deficits in Huntington's disease. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
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| 4. |
- Hansen, Christian, et al.
(författare)
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A novel α-synuclein-GFP mouse model displays progressive motor impairment, olfactory dysfunction and accumulation of α-synuclein-GFP.
- 2013
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Ingår i: Neurobiology of Disease. - : Elsevier BV. - 0969-9961. ; 56C:April,30, s. 145-155
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Tidskriftsartikel (refereegranskat)abstract
- Compelling evidence suggests that accumulation and aggregation of alpha-synuclein (α-syn) contribute to the pathogenesis of Parkinson's disease (PD). Here, we describe a novel Bacterial Artificial Chromosome (BAC) transgenic model, in which we have expressed wild-type human α-syn fused to green fluorescent protein (GFP), under control of the mouse α-syn promoter. We observed a widespread and high expression of α-syn-GFP in multiple brain regions, including the dopaminergic neurons of the substantia nigra pars compacta (SNpc) and the ventral tegmental area, the olfactory bulb as well as in neocortical neurons. With increasing age, transgenic mice exhibited reductions in amphetamine-induced locomotor activity in the open field, impaired rotarod performance and a reduced striatal dopamine release, as measured by amperometry. In addition, they progressively developed deficits in an odor discrimination test. Western blot analysis revealed that α-syn-GFP and phospho-α-syn levels increased in multiple brain regions, as the mice grew older. Further, we observed, by immunohistochemical staining for phospho-α-syn and in vivo by two-photon microscopy, the formation of α-syn aggregates as the mice aged. The latter illustrates that the model can be used to track α-syn aggregation in vivo. In summary, this novel BAC α-syn-GFP model mimics a unique set of aspects of PD progression combined with the possibility of tracking α-syn aggregation in neocortex of living mice. Therefore, this α-syn-GFP-mouse model can provide a powerful tool that will facilitate the study of α-syn biology and its involvement in PD pathogenesis.
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| 5. |
- Hansen, Christian, et al.
(författare)
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alpha-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells
- 2011
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Ingår i: Journal of Clinical Investigation. - 0021-9738. ; 121:2, s. 715-725
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Tidskriftsartikel (refereegranskat)abstract
- Post-mortem analyses of brains from patients with Parkinson disease who received fetal mesencephalic transplants show that alpha-synuclein-containing (alpha-syn-containing) Lewy bodies gradually appear in grafted neurons. Here, we explored whether intercellular transfer of alpha-syn from host to graft, followed by seeding of alpha-syn aggregation in recipient neurons, can contribute to this phenomenon. We assessed alpha-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 alpha-syn resulted in a gradual increase in double-labeled cells. Importantly, alpha-syn-GFP derived from 1 neuroblastoma cell line localized to red fluorescent aggregates in other cells expressing DsRed-alpha-syn, suggesting a seeding effect of transmitted alpha-syn. Extracellular alpha-syn was taken up by cells through endocytosis and interacted with intracellular alpha-syn. Next, following intracortical injection of recombinant alpha-syn in rats, we found neuronal uptake was attenuated by coinjection of an endocytosis inhibitor. Finally, we demonstrated in vivo transfer of alpha-syn between host cells and grafted dopaminergic neurons in mice overexpressing human alpha-syn. In summary, intercellularly transferred alpha-syn interacts with cytoplasmic alpha-syn and can propagate alpha-syn pathology. These results suggest that alpha-syn propagation is a key element in the progression of Parkinson disease pathology.
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| 6. |
- Holmqvist, Staffan, et al.
(författare)
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Direct evidence of Parkinson pathology spread from the gastrointestinal tract to the brain in rats.
- 2014
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Ingår i: Acta Neuropathologica. - : Springer Science and Business Media LLC. - 1432-0533 .- 0001-6322. ; 128:6, s. 805-820
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Tidskriftsartikel (refereegranskat)abstract
- The cellular hallmarks of Parkinson's disease (PD) are the loss of nigral dopaminergic neurons and the formation of α-synuclein-enriched Lewy bodies and Lewy neurites in the remaining neurons. Based on the topographic distribution of Lewy bodies established after autopsy of brains from PD patients, Braak and coworkers hypothesized that Lewy pathology primes in the enteric nervous system and spreads to the brain, suggesting an active retrograde transport of α-synuclein (the key protein component in Lewy bodies), via the vagal nerve. This hypothesis, however, has not been tested experimentally thus far. Here, we use a human PD brain lysate containing different forms of α-synuclein (monomeric, oligomeric and fibrillar), and recombinant α-synuclein in an in vivo animal model to test this hypothesis. We demonstrate that α-synuclein present in the human PD brain lysate and distinct recombinant α-synuclein forms are transported via the vagal nerve and reach the dorsal motor nucleus of the vagus in the brainstem in a time-dependent manner after injection into the intestinal wall. Using live cell imaging in a differentiated neuroblastoma cell line, we determine that both slow and fast components of axonal transport are involved in the transport of aggregated α-synuclein. In conclusion, we here provide the first experimental evidence that different α-synuclein forms can propagate from the gut to the brain, and that microtubule-associated transport is involved in the translocation of aggregated α-synuclein in neurons.
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| 7. |
- Kurowska, Zuzanna, et al.
(författare)
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Intracellular Nogo-A facilitates initiation of neurite formation in mouse midbrain neurons in vitro.
- 2014
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Ingår i: Neuroscience. - : Elsevier BV. - 1873-7544 .- 0306-4522. ; 256, s. 456-466
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Tidskriftsartikel (refereegranskat)abstract
- Nogo-A is a transmembrane protein originally discovered in myelin, produced by postnatal central nervous system (CNS) oligodendrocytes. Nogo-A induces growth cone collapse and inhibition of axonal growth in the injured adult CNS. In the intact CNS, Nogo-A functions as a negative regulator of growth and plasticity. Nogo-A is also expressed by certain neurons. Neuronal Nogo-A depresses long-term potentiation in hippocampus and modulates neurite adhesion and fasciculation during development in mice. Here we show that Nogo-A is present in neurons derived from human midbrain (LUHMES cell line), as well as in embryonic and postnatal mouse midbrain (dopaminergic) neurons. In LUHMES cells, Nogo-A was upregulated 3-fold upon differentiation and neurite extension. Nogo-A was localized intracellularly in differentiated LUHMES cells. Cultured midbrain (dopaminergic) neurons from Nogo-A knock-out mice exhibited decreased numbers of neurites and branches when compared with neurons from wild type mice. However, this phenotype was not observed when the cultures from wild type mice were treated with an antibody neutralizing plasma membrane Nogo-A. In vivo, neither the regeneration of nigrostriatal tyrosine hydroxylase fibers, nor the survival of nigral dopaminergic neurons after partial 6-hydroxydopamine lesions were affected by Nogo-A deletion. These results indicate that during maturation of cultured midbrain (dopaminergic) neurons, intracellular Nogo-A supports neurite growth initiation and branch formation. Abbreaviations: 6-OHDA, (6-hydroxydopamine); CNS, (central nervous system); DAB, (3,3'-Diaminobenzidine); DAPI, (4',6-diamidino-2-phenylindole); KO, (knock-out); LTP, (long term potentiation); LUHMES cells (Lund University Human Mesencephalon cells); PFA, (paraformaldehyde); SDS-PAGE, (sodium dodecyl sulfate polyacrylamide gel electrophoresis); TH, (tyrosine hydroxylase); WT, (wild type).
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| 8. |
- Morizane, Asuka, et al.
(författare)
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A simple method for large-scale generation of dopamine neurons from human embryonic stem cells.
- 2010
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Ingår i: Journal of Neuroscience Research. - : Wiley. - 1097-4547 .- 0360-4012. ; 88:16, s. 3467-3478
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Tidskriftsartikel (refereegranskat)abstract
- Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are potentially valuable in drug screening and as a possible source of donor tissue for transplantation in Parkinson's disease. However, existing culture protocols that promote the differentiation of DA neurons from hESCs are complex, involving multiple steps and having unreliable results between cultures. Here we report a simple and highly reproducible culture protocol that induces expandable DA neuron progenitors from hESCs in attached cultures. We found that the hESC-derived neuronal progenitors retain their full capacity to generate DA neurons after repeated passaging in the presence of basic fibroblast growth factor (bFGF) and medium conditioned with PA6 stromal cells. Using immunocytochemistry and RT-PCR, we found that the differentiated DA neurons exhibit a midbrain phenotype and express, e.g., Aldh1a, Ptx3, Nurr1, and Lmx1a. Using HPLC, we monitored their production of DA. We then demonstrated that the expanded progenitors are possible to cryopreserve without loosing the dopaminergic phenotype. With our protocol, we obtained large and homogeneous populations of dopaminergic progenitors and neurons. We conclude that our protocol can be used to generate human DA neurons suitable for the study of disease mechanisms, toxicology, drug screening, and intracerebral transplantation. © 2010 Wiley-Liss, Inc.
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| 9. |
- Murmu, Reena, et al.
(författare)
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Dendritic Spine Instability Leads to Progressive Neocortical Spine Loss in a Mouse Model of Huntington's Disease.
- 2013
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Ingår i: The Journal of Neuroscience. - 1529-2401. ; 33:32, s. 12997-13009
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Tidskriftsartikel (refereegranskat)abstract
- In Huntington's disease (HD), cognitive symptoms and cellular dysfunction precede the onset of classical motor symptoms and neuronal death in the striatum and cortex by almost a decade. This suggests that the early cognitive deficits may be due to a cellular dysfunction rather than being a consequence of neuronal loss. Abnormalities in dendritic spines are described in HD patients and in HD animal models. Available evidence indicates that altered spine and synaptic plasticity could underlie the motor as well as cognitive symptoms in HD. However, the exact kinetics of spine alterations and plasticity in HD remain unknown. We used long-term two-photon imaging through a cranial window, to track individual dendritic spines in a mouse model of HD (R6/2) as the disease progressed. In vivo imaging over a period of 6 weeks revealed a steady decrease in the density and survival of dendritic spines on cortical neurons of R6/2 mice compared with control littermates. Interestingly, we also observed increased spine formation in R6/2 mice throughout the disease. However, the probability that newly formed spines stabilized and transformed into persistent spines was greatly reduced compared with controls. In cultured neurons we found that mutant huntingtin causes a loss, in particular of mature spines. Furthermore, in R6/2 mice, aggregates of mutant huntingtin associate with dendritic spines. Alterations in dendritic spine dynamics, survival, and density in R6/2 mice were evident before the onset of motor symptoms, suggesting that decreased stability of the cortical synaptic circuitry underlies the early symptoms in HD.
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| 10. |
- Roybon, Laurent, et al.
(författare)
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GABAergic Differentiation Induced by Mash1 Is Compromised by the bHLH Proteins Neurogenin2, NeuroD1, and NeuroD2.
- 2010
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Ingår i: Cerebral Cortex. - : Oxford University Press (OUP). - 1460-2199 .- 1047-3211. ; 20, s. 1234-1244
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Tidskriftsartikel (refereegranskat)abstract
- During forebrain development, Mash1 directs gamma-aminobutyric acid (GABA)ergic neuron differentiation ventrally in the ganglionic eminences. Repression of Mash1 in the cortex is necessary to prevent the formation of GABAergic interneurons. Negative regulation of Mash1 has been attributed to members of the Neurogenin family; the genetic ablation of Neurogenin2 (Ngn2) leads to the derepression of Mash1 and the formation of ectopic GABAergic neurons in the cortex. We have developed an in vitro system to clarify the importance of NeuroD proteins in the Mash1 regulatory pathway. Using a neurosphere culture system, we show that the downstream effectors of the Ngn2 pathway NeuroD1 and NeuroD2 can abrogate GABAergic differentiation directed by Mash1. The ectopic expression of either of these genes in Mash1-expressing cells derived from the lateral ganglionic eminence, independently downregulate Mash1 expression without affecting expression of distal less homeodomain genes. This results in a complete loss of the GABAergic phenotype. Moreover, we demonstrate that ectopic expression of Mash1 in cortical progenitors is sufficient to phenocopy the loss of Ngn2 and strongly enhances ectopic GABAergic differentiation. Collectively, our results define the compensatory and cross-regulatory mechanisms that exist among basic helix-loop-helix transcription factors during neuronal fate specification.
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