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author:("isaacson, Ole")
1.  Sustained Systemic Glucocerebrosidase Inhibition Induces Brain α-Synuclein Aggregation, Microglia and Complement C1q Activation in Mice 
Antioxidants & Redox Signaling  2015;23(6):550-564.
Abstract
Aims: Loss-of-function mutations in GBA1, which cause the autosomal recessive lysosomal storage disease, Gaucher disease (GD), are also a key genetic risk factor for the α-synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase and reductions in this enzyme result in the accumulation of the glycolipid substrates glucosylceramide and glucosylsphingosine. Deficits in autophagy and lysosomal degradation pathways likely contribute to the pathological accumulation of α-synuclein in PD. In this report we used conduritol-β-epoxide (CBE), a potent selective irreversible competitive inhibitor of glucocerebrosidase, to model reduced glucocerebrosidase activity in vivo, and tested whether sustained glucocerebrosidase inhibition in mice could induce neuropathological abnormalities including α-synucleinopathy, and neurodegeneration. Results: Our data demonstrate that daily systemic CBE treatment over 28 days caused accumulation of insoluble α-synuclein aggregates in the substantia nigra, and altered levels of proteins involved in the autophagy lysosomal system. These neuropathological changes were paralleled by widespread neuroinflammation, upregulation of complement C1q, abnormalities in synaptic, axonal transport and cytoskeletal proteins, and neurodegeneration. Innovation: A reduction in brain GCase activity has been linked to sporadic PD and normal aging, and may contribute to the susceptibility of vulnerable neurons to degeneration. This report demonstrates that systemic reduction of GCase activity using chemical inhibition, leads to neuropathological changes in the brain reminiscent of α-synucleinopathy. Conclusions: These data reveal a link between reduced glucocerebrosidase and the development of α-synucleinopathy and pathophysiological abnormalities in mice, and support the development of GCase therapeutics to reduce α-synucleinopathy in PD and related disorders. Antioxid. Redox Signal. 23, 550–564.
doi:10.1089/ars.2015.6307
PMCID: PMC4544823  PMID: 26094487
2.  Long-term dopamine transporter expression and normal cellular distribution of mitochondria in dopaminergic neuron transplants in Parkinson’s disease patients 
Cell reports  2014;7(6):1755-1761.
Summary
To determine the long-term health and function of transplanted dopamine neurons in Parkinson’s disease (PD) patients, the expression of dopamine transporters (DAT) and mitochondrial morphology was examined in human fetal midbrain cellular transplants. DAT was robustly expressed in transplanted dopamine neuron terminals in the reinnervated host putamen and caudate, for at least 14 years after transplantation. The transplanted dopamine neurons showed a healthy and non-atrophied morphology at all time points. Labeling of the mitochondrial outer membrane protein Tom20 and alpha-synuclein showed typical cellular pathology in the patients’ own substantia nigra, which was not observed in transplanted dopamine neurons. These results show that the vast majority of transplanted neurons remain healthy long-term in PD patients, consistent with the clinically maintained function of fetal dopamine neuron transplants for up to 15–18 years in patients. These findings are critically important for the rational development of stem cell-based dopamine neuronal replacement therapies for PD.
doi:10.1016/j.celrep.2014.05.027
PMCID: PMC4105701  PMID: 24910427
3.  Motor Impairments, Striatal Degeneration, and Altered Dopamine-Glutamate Interplay in Mice Lacking PSD-95 
Journal of neurogenetics  2014;28(0):98-111.
Excessive activation of the N-Methyl-D-Aspartate (NMDA) receptor and the neurotransmitter dopamine (DA) mediate neurotoxicity and neurodegeneration under many neurological conditions, including Huntington's disease (HD), an autosomal dominant neurodegenerative disease characterized by the preferential loss of medium spiny projection neurons (MSNs) in the striatum. PSD-95 is a major scaffolding protein in the postsynaptic density (PSD) of dendritic spines, where a classical role for PSD-95 is to stabilize glutamate receptors at sites of synaptic transmission. Our recent studies indicate that PSD-95 also interacts with the D1 DA receptor localized in spines and negatively regulates spine D1 signaling. Moreover, PSD-95 forms ternary protein complexes with D1 and NMDA receptors, and plays a role in limiting the reciprocal potentiation between both receptors from being escalated. These studies suggest a neuroprotective role for PSD-95. Here we show that mice lacking PSD-95, resulting from genetic deletion of the GK domain of PSD-95 (PSD-95-ΔGK mice), sporadically develop progressive neurological impairments characterized by hypolocomotion, limb clasping, and loss of DARPP-32-positive MSNs. Electrophysiological experiments indicated that NMDA receptors in mutant MSNs were overactive, suggested by larger, NMDA receptor-mediated miniature excitatory postsynaptic currents (EPSCs) and higher ratios of NMDA- to AMPA-mediated corticostriatal synaptic transmission. In addition, NMDA receptor currents in mutant cortical neurons were more sensitive to potentiation by the D1 receptor agonist SKF81297. Finally, repeated administration of the psychostimulant cocaine at a dose regimen not producing overt toxicity-related phenotypes in normal mice reliably converted asymptomatic mutant mice to clasping symptomatic mice. These results support the hypothesis that deletion of PSD-95 in mutant mice produces concomitant overactivation of both D1 and NMDA receptors that makes neurons more susceptible to NMDA excitotoxicity, causing neuronal damage and neurological impairments. Understanding PSD-95-dependent neuroprotective mechanisms may help elucidate processes underlying neurodegeneration in HD and other neurological disorders.
doi:10.3109/01677063.2014.892486
PMCID: PMC4406490  PMID: 24702501
4.  A Nurr1 Agonist Causes Neuroprotection in a Parkinson’s Disease Lesion Model Primed with the Toll-Like Receptor 3 dsRNA Inflammatory Stimulant Poly(I:C) 
PLoS ONE  2015;10(3):e0121072.
Dopaminergic neurons in the substantia nigra pars compacta (SNpc) are characterized by the expression of genes required for dopamine synthesis, handling and reuptake and the expression of these genes is largely controlled by nuclear receptor related 1 (Nurr1). Nurr1 is also expressed in astrocytes and microglia where it functions to mitigate the release of proinflammatory cytokines and neurotoxic factors. Given that Parkinson’s disease (PD) pathogenesis has been linked to both loss of Nurr1 expression in the SNpc and inflammation, increasing levels of Nurr1 maybe a promising therapeutic strategy. In this study a novel Nurr1 agonist, SA00025, was tested for both its efficiency to induce the transcription of dopaminergic target genes in vivo and prevent dopaminergic neuron degeneration in an inflammation exacerbated 6-OHDA-lesion model of PD. SA00025 (30mg/kg p.o.) entered the brain and modulated the expression of the dopaminergic phenotype genes TH, VMAT, DAT, AADC and the GDNF receptor gene c-Ret in the SN of naive rats. Daily gavage treatment with SA00025 (30mg/kg) for 32 days also induced partial neuroprotection of dopaminergic neurons and fibers in rats administered a priming injection of polyinosinic-polycytidylic acid (poly(I:C) and subsequent injection of 6-OHDA. The neuroprotective effects of SA00025 in this dopamine neuron degeneration model were associated with changes in microglial morphology indicative of a resting state and a decrease in microglial specific IBA-1 staining intensity in the SNpc. Astrocyte specific GFAP staining intensity and IL-6 levels were also reduced. We conclude that Nurr1 agonist treatment causes neuroprotective and anti-inflammatory effects in an inflammation exacerbated 6-OHDA lesion model of PD.
doi:10.1371/journal.pone.0121072
PMCID: PMC4376720  PMID: 25815475
5.  Progressive decline of glucocerebrosidase in aging and Parkinson's disease 
The principal risk factor for developing most adult onset neurodegenerative diseases is aging, with incidence rising significantly after age 50. Despite research efforts, the causes of Parkinson's disease (PD) remain unknown. As neurons age, they show signs of diminished lysosomal and mitochondrial function, including increased oxidative stress and accumulation of misfolded proteins, and these changes become exacerbated PD. We show that activity of the lysosomal hydrolase glucocerebrosidase gradually diminishes with age in the substantia nigra and putamen of healthy controls. This reduction is comparable to glucocerebrosidase activity in GBA1-mutation carrier PD patients. These data, demonstrate for the first time that an age-dependent reduction in glucocerebrosidase activity may lower the threshold for developing PD.
doi:10.1002/acn3.177
PMCID: PMC4402088  PMID: 25909088
6.  Future of Cell and Gene Therapies for Parkinson’s Disease 
Annals of neurology  2008;64(0 2):S122-S138.
The experimental field of restorative neurology continues to advance with implantation of cells or transfer of genes to treat patients with neurological disease. Both strategies have generated a consensus that demonstrates their capacity for structural and molecular brain modification in the adult brain. However, both approaches have yet to successfully address the complexities to make such novel therapeutic modalities work in the clinic. Prior experimental cell transplantation to patients with PD utilized dissected pieces of fetal midbrain tissue, containing mixtures of cells and neuronal types, as donor cells. Stem cell and progenitor cell biology provide new opportunities for selection and development of large batches of specific therapeutic cells. This may allow for cell composition analysis and dosing to optimize the benefit to an individual patient. The biotechnology used for cell and gene therapy for treatment of neurological disease may eventually be as advanced as today’s pharmaceutical drug-related design processes. Current gene therapy phase 1 safety trials for PD include the delivery of a growth factor (neurturin via the glial cell line–derived neurotrophic factor receptor) and a transmitter enzyme (glutamic acid decarboxylase and aromatic acid decarboxylase). Many new insights from cell biological and molecular studies provide opportunities to selectively express or suppress factors relevant to neuroprotection and improved function of neurons involved in PD. Future gene and cell therapies are likely to coexist with classic pharmacological therapies because their use can be tailored to individual patients’ underlying disease process and need for neuroprotective or restorative interventions.
doi:10.1002/ana.21473
PMCID: PMC3982797  PMID: 19127583
7.  Viral and Inflammatory Triggers of Neurodegenerative Diseases 
Science translational medicine  2012;4(121):121ps3.
Here, we synthesize research behind the emerging hypothesis that inflammation—which can result, for example, from viral infections—can initiate and propagate chronic neuronal dysfunction, an event that precedes the clinical onset of many neurodegenerative diseases. Therapeutic approaches that target immunological pathways in the prodromal phase of diseases might decrease the incidence of neurodegenerative disorders and increase the therapeutic window for neuroprotection.
doi:10.1126/scitranslmed.3003492
PMCID: PMC3982831  PMID: 22344685
8.  Clinical Translation of Stem Cells in Neurodegenerative Disorders 
Cell stem cell  2012;10(2):151-155.
Stem cells and their derivatives show tremendous potential for treating many disorders, including neurode-generative diseases. We discuss here the challenges and potential for the translation of stem-cell-based approaches into treatments for Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis.
doi:10.1016/j.stem.2012.01.009
PMCID: PMC3982832  PMID: 22305565
9.  Using stem cells and iPS cells to discover new treatments for Parkinson’s disease 
Parkinsonism & related disorders  2012;18(0 1):S14-S16.
SUMMARY
Fetal cell transplantation can improve the symptoms of Parkinson’s disease (PD) patients for more than a decade. In some patients, alpha-synuclein aggregates and Lewy bodies have been observed in the transplanted neurons without functional significance. Recently stem cells have emerged as an ethically acceptable source of cells for transplantation but, importantly, the type of stem cell matters. While the lineage restriction of adult neural stem cells limits their clinical applicability for patients with PD, human pluripotent stem cells provide an opportunity to replace specific types of degenerating neurons. Now, cellular reprogramming technology can provide patient-specific neurons for neural transplantation and problems with cell fate specification and safety are resolving. Induced pluripotent stem (iPS) cell-derived neurons are also a unique tool for interpreting the genetic basis for an individual’s risk of developing PD into clinically meaningful information. For example, clinical trials for neuroprotective molecules need to be tested in presymptomatic individuals when the neurons can still be protected. Patient-specific neural cells can also be used to identify an individual’s responsiveness to drugs and to understand the mechanisms of the disease. Along these avenues of investigation, stem cells are enabling research for new treatments in PD.
doi:10.1016/S1353-8020(11)70007-4
PMCID: PMC3982835  PMID: 22166414
10.  Enhanced ubiquitin-dependent degradation by Nedd4 protects against α-synuclein accumulation and toxicity in animal models of Parkinson's disease 
Neurobiology of Disease  2014;64(100):79-87.
Parkinson's disease is a neurodegenerative disorder, characterized by accumulation and misfolding of α-synuclein. Although the level of α-synuclein in neurons is fundamentally linked to the onset of neurodegeneration, multiple pathways have been implicated in its degradation, and it remains unclear which are the critical ubiquitination enzymes that protect against α-synuclein accumulation in vivo. The ubiquitin ligase Nedd4 targets α-synuclein to the endosomal–lysosomal pathway in cultured cells. Here we asked whether Nedd4-mediated degradation protects against α-synuclein-induced toxicity in the Drosophila and rodent models of Parkinson's disease. We show that overexpression of Nedd4 can rescue the degenerative phenotype from ectopic expression of α-synuclein in the Drosophila eye. Overexpressed Nedd4 in the Drosophila brain prevented the α-synuclein-induced locomotor defect whereas reduction in endogenous Nedd4 by RNAi led to worsening motor function and increased loss of dopaminergic neurons. Accordingly, AAV-mediated expression of wild-type but not the catalytically inactive Nedd4 decreased the α-synuclein-induced dopaminergic cell loss in the rat substantia nigra and reduced α-synuclein accumulation. Collectively, our data in two evolutionarily distant model organisms strongly suggest that Nedd4 is a modifier of α-synuclein pathobiology and thus a potential target for neuroprotective therapies.
Highlights
•Nedd4 rescues the α-synuclein-induced rough-eye phenotype.•Nedd4 prevents the α-synuclein-induced locomotor defect in the Drosophila brain.•Overexpressed Nedd4 promotes α-synuclein degradation in vivo.•Nedd4 knockdown increases the α-synuclein-induced loss of dopaminergic neurons.•Nedd4 decreases dopaminergic cell loss in the rat substantia nigra.
doi:10.1016/j.nbd.2013.12.011
PMCID: PMC3988924  PMID: 24388974
Lysosome; Ubiquitination; Protein-degradation; Endosomal-trafficking
11.  Alpha-synuclein overexpressing transgenic mice show internal organ pathology and autonomic deficits 
Neurobiology of Disease  2012;47(2):258-267.
While studying transgenic mice that overexpress human wildtype alpha-synuclein (Thy1-ASO, ASO) for typical brain alpha-synucleinopathy and central nervous system neuropathology, we observed progressive functional changes in the gastrointestinal and other peripheral organs. A more systematic study revealed that the gastrointestinal tract in ASO mice showed severe distension and blockage of the large intestine by 9–12 months of age. Functional assessments demonstrated a reduction in fecal water content and fecal pellet output, and increased whole gut transit time, in ASO mice compared to wildtype littermates, indicative of constipation, a symptom commonly reported by Parkinson's disease (PD) patients. Food intake was increased and body weight was decreased in 12 month old ASO mice, suggestive of metabolic abnormalities. Post-mortem histological analyses showed that human alpha-synuclein protein was robustly expressed in axonal fibers and in occasional cell bodies of the enteric nervous system, and in the heart of ASO mice. Accumulation of proteinase-K insoluble alpha-synuclein, reminiscent of neurodegenerative processes in PD was also observed. The functional and pathological changes we document here in ASO mice could relate to the autonomic deficits also seen in idiopathic and alpha-synuclein-mediated genetic forms of PD. These experimental data provide a foundation for therapeutic modeling of autonomic changes in PD and related alpha-synucleinopathies.
doi:10.1016/j.nbd.2012.04.009
PMCID: PMC3376017  PMID: 22549133
Alpha-synuclein; synucleinopathy; Parkinson's disease; constipation; gastrointestinal; autonomic; axonopathy
12.  Transcript expression levels of full-length alpha-synuclein and its three alternatively spliced variants in Parkinson’s disease brain regions and in a transgenic mouse model of alpha-synuclein overexpression 
Alternative splicing is a complex post-transcriptional process that can be regulated by cis-acting elements located within genomic non-coding regions. Recent studies have identified that polymorphic variations in non-coding regions of the α-synuclein gene (SNCA) locus are associated with an increased risk for developing Parkinson’s disease (PD). The underlying mechanism(s) for this susceptibility may involve changes in α-synuclein mRNA expression and alternative splicing. As a first step towards understanding the biology of α-synuclein splice variants in PD, we characterized the levels of the full-length SNCA-140 mRNA transcript and SNCA-126, -112, and -98 alternatively spliced variants in different neuronal regions from PD patients or transgenic mice overexpressing human α-synuclein (ASO). In human post-mortem tissue, α-synuclein spliced transcripts were expressed in a region-specific manner in cortex, substantia nigra, and cerebellum. We observed increased nigral SNCA-140 and SNCA-126 transcript levels in PD patients when compared to neurologically unaffected cases. Human α-synuclein splicing changes were also found to occur in a region-specific manner in ASO mice. Here, SNCA-126, -112, and -98 transcript levels did not increase proportionally with SNCA-140 levels, or parallel the region-specific mouse transcript ratios seen in wild-type (WT) littermates. While most transcripts were elevated in ASO mice when compared to WT mice, the most prominent increase was found in the ventral midbrain of 15-month-old ASO mice. These results demonstrate region-specific human α-synuclein transcript level abnormalities in PD patients and in a transgenic mouse model of α-synucleinopathy. This study is relevant to understanding the normal, adaptive, or pathological role(s) of α-synuclein splice variants.
doi:10.1016/j.mcn.2011.11.006
PMCID: PMC3340908  PMID: 22155155
Parkinson’s disease; alpha-synuclein; SNCA; alterative splicing; isoform; substantia nigra
13.  Familial Parkinson's disease iPSCs show cellular deficits in mitochondrial responses that can be pharmacologically rescued 
Science translational medicine  2012;4(141):141ra90.
Parkinson's disease (PD) is a common neurodegenerative disease caused by genetic and environmental factors. We analyzed induced pluripotent stem cell (iPSC)-derived neural cells from PD patients and presymptomatic individuals carrying mutations in the PINK1 and LRRK2 genes, and healthy control subjects. We measured several aspects of mitochondrial responses in the iPSC-derived neural cells including production of reactive oxygen species, mitochondrial respiration, proton leakage and intraneuronal movement of mitochondria. Cellular vulnerability associated with mitochondrial function in iPSC-derived neural cells from PD patients and at-risk individuals could be rescued with coenzyme Q10, rapamycin or the LRRK2 kinase inhibitor GW5074. Analysis of mitochondrial responses in iPSC-derived neural cells from PD patients carrying different mutations provides insights into convergence of cellular disease mechanisms between different familial forms of PD and highlights the importance of oxidative stress and mitochondrial dysfunction in PD.
doi:10.1126/scitranslmed.3003985
PMCID: PMC3462009  PMID: 22764206
14.  Creation of an Open-Access, Mutation-Defined Fibroblast Resource for Neurological Disease Research 
PLoS ONE  2012;7(8):e43099.
Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community.
doi:10.1371/journal.pone.0043099
PMCID: PMC3428297  PMID: 22952635
15.  Development of histocompatible primate induced pluripotent stem cells for neural transplantation 
Stem Cells (Dayton, Ohio)  2011;29(7):1052-1063.
Immune rejection and risk of tumor formation are perhaps the greatest hurdles in the field of stem cell transplantation. Here, we report the generation of several lines of induced pluripotent stem cells (iPSCs) from Cynomolgus macaque (CM) skin fibroblasts carrying specific major histocompatibility complex (MHC) haplotypes. In order to develop a collection of MHC-matched iPSCs, we genotyped the MHC locus of 25 CM by microsatellite PCR analysis. Using retroviral infection of dermal skin fibroblasts, we generated several CM-iPSC lines carrying different haplotypes. We characterized the immunological properties of CM-iPSCs and demonstrated that CM-iPSCs can be induced to differentiate in vitro along specific neuronal populations, such as midbrain dopaminergic (DA) neurons. Midbrain-like DA neurons generated from CM-iPSCs integrated into the striatum of a rodent model of Parkinson’s disease (PD) and promoted behavioral recovery. Importantly, neither tumor formation nor inflammatory reactions were observed in the transplanted animals up to six months after transplantation. We believe that the generation and characterization of such histocompatible iPSCs will allow the pre-clinical validation of safety and efficacy of iPSCs for neurodegenerative diseases and several other human conditions in the field of regenerative medicine.
doi:10.1002/stem.662
PMCID: PMC3340906  PMID: 21608081
Parkinson’s disease; stem cells; transplantation; non-human primates
16.  CD15, CD24, and CD29 Define a Surface Biomarker Code for Neural Lineage Differentiation of Stem Cells 
Stem Cells (Dayton, Ohio)  2009;27(12):2928-2940.
Identification and use of cell surface cluster of differentiation (CD) biomarkers have enabled much scientific and clinical progress. We identify a CD surface antigen code for the neural lineage based on combinatorial flow cytometric analysis of three distinct populations derived from human embryonic stem cells: (1) CD15+/CD29HI/CD24LO surface antigen expression defined neural stem cells; (2) CD15−/CD29HI/CD24LO revealed neural crest-like and mesenchymal phenotypes; and (3) CD15−/CD29LO/CD24HI selected neuroblasts and neurons. Fluorescence-activated cell sorting (FACS) for the CD15−/CD29LO/CD24HI profile reduced proliferative cell types in human embryonic stem cell differentiation. This eliminated tumor formation in vivo, resulting in pure neuronal grafts. In conclusion, combinatorial CD15/CD24/CD29 marker profiles define neural lineage development of neural stem cell, neural crest, and neuronal populations from human stem cells. We believe this set of biomarkers enables analysis and selection of neural cell types for developmental studies and pharmacological and therapeutic applications.
doi:10.1002/stem.211
PMCID: PMC3322476  PMID: 19725119
Stem cells; Neurons; Brain tumors; Flow cytometry; Transplantation; Surface antigens; Cell Therapy; Epithelial-mesenchymal transition
17.  The blood-brain barrier is intact after levodopa induced dyskinesias in parkinsonian primates – evidence from in vivo neuroimaging studies 
Neurobiology of Disease  2009;35(3):348-351.
It has been suggested, based on rodent studies, that levodopa (L-dopa) induced dyskinesia is associated with a disrupted blood-brain barrier (BBB). We have investigated BBB integrity with in vivo neuroimaging techniques in six 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesioned primates exhibiting L-dopa induced dyskinesia. Magnetic resonance imaging (MRI) performed before and after injection of Gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) revealed an intact BBB in the basal ganglia showing that L-dopa induced dyskinesia is not associated with a disrupted BBB in this model.
doi:10.1016/j.nbd.2009.05.018
PMCID: PMC3319396  PMID: 19501164
Levodopa; Dyskinesia; Primates; Blood-Brain Barrier; Imaging; Gadolinium-DTPA
18.  Differentiation of human ES and Parkinson’s disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid 
The cardinal motor symptoms of Parkinson’s disease (PD) are caused by the vulnerability to dysfunction and degeneration of ventral midbrain (VM) dopaminergic (DA) neurons. A major limitation for experimental studies of current ES/iPS cell differentiation protocols is the lack of VM DA neurons with a stable phenotype as defined by an expression marker code of FOXA2/TH/β-tubulin. Here we demonstrate a combination of three modifications that were required to produce VM DA neurons. Firstly, early and specific exposure to 10−8M (low dose) retinoic acid improved the regional identity of neural progenitor cells derived from human ES cells, PD or healthy subject-specific iPS cells. Secondly, a high activity form of human sonic hedgehog established a sizeable FOXA2+ neural progenitor cell population in vitro. Thirdly, early exposure to FGF8a, rather than Fgf8b, and WNT1 was required for robust differentiation of the FOXA2+ floor plate-like human neural progenitor cells into FOXA2+ DA neurons. FOXA2+ DA neurons were also generated when this protocol was adapted to feeder-free conditions. In summary, this new human ES and iPS cell differentiation protocol using FGF8a, WNT1, low dose retinoic acid and a high activity form of SHH can generate human VM DA neurons that are required for relevant new bioassays, drug discovery and cell based therapies for PD.
doi:10.1016/j.mcn.2010.06.017
PMCID: PMC2945816  PMID: 20603216
19.  Global gene expression profiling of somatic motor neuron populations with different vulnerability identify molecules and pathways of degeneration and protection 
Brain  2010;133(8):2313-2330.
Different somatic motor neuron subpopulations show a differential vulnerability to degeneration in diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and spinobulbar muscular atrophy. Studies in mutant superoxide dismutase 1 over-expressing amyotrophic lateral sclerosis model mice indicate that initiation of disease is intrinsic to motor neurons, while progression is promoted by astrocytes and microglia. Therefore, analysis of the normal transcriptional profile of motor neurons displaying differential vulnerability to degeneration in motor neuron disease could give important clues to the mechanisms of relative vulnerability. Global gene expression profiling of motor neurons isolated by laser capture microdissection from three anatomical nuclei of the normal rat, oculomotor/trochlear (cranial nerve 3/4), hypoglossal (cranial nerve 12) and lateral motor column of the cervical spinal cord, displaying differential vulnerability to degeneration in motor neuron disorders, identified enriched transcripts for each neuronal subpopulation. There were striking differences in the regulation of genes involved in endoplasmatic reticulum and mitochondrial function, ubiquitination, apoptosis regulation, nitrogen metabolism, calcium regulation, transport, growth and RNA processing; cellular pathways that have been implicated in motor neuron diseases. Confirmation of genes of immediate biological interest identified differential localization of insulin-like growth factor II, guanine deaminase, peripherin, early growth response 1, soluble guanylate cyclase 1A3 and placental growth factor protein. Furthermore, the cranial nerve 3/4-restricted genes insulin-like growth factor II and guanine deaminase protected spinal motor neurons from glutamate-induced toxicity (P < 0.001, ANOVA), indicating that our approach can identify factors that protect or make neurons more susceptible to degeneration.
doi:10.1093/brain/awq167
PMCID: PMC3139939  PMID: 20826431
motor neuron; SOD1G93A rat; microarray; hierarchical clustering; cranial nerves; cervical spinal cord; IGF-II
20.  The transcription factor orthodenticle homeobox 2 influences axonal projections and vulnerability of midbrain dopaminergic neurons 
Brain  2010;133(7):2022-2031.
Two adjacent groups of midbrain dopaminergic neurons, A9 (substantia nigra pars compacta) and A10 (ventral tegmental area), have distinct projections and exhibit differential vulnerability in Parkinson’s disease. Little is known about transcription factors that influence midbrain dopaminergic subgroup phenotypes or their potential role in disease. Here, we demonstrate elevated expression of the transcription factor orthodenticle homeobox 2 in A10 dopaminergic neurons of embryonic and adult mouse, primate and human midbrain. Overexpression of orthodenticle homeobox 2 using lentivirus increased levels of known A10 elevated genes, including neuropilin 1, neuropilin 2, slit2 and adenylyl cyclase-activating peptide in both MN9D cells and ventral mesencephalic cultures, whereas knockdown of endogenous orthodenticle homeobox 2 levels via short hairpin RNA reduced expression of these genes in ventral mesencephalic cultures. Lack of orthodenticle homeobox 2 in the ventral mesencephalon of orthodenticle homeobox 2 conditional knockout mice caused a reduction of midbrain dopaminergic neurons and selective loss of A10 dopaminergic projections. Orthodenticle homeobox 2 overexpression protected dopaminergic neurons in ventral mesencephalic cultures from Parkinson’s disease-relevant toxin, 1-methyl-4-phenylpyridinium, whereas downregulation of orthodenticle homeobox 2 using short hairpin RNA increased their susceptibility. These results show that orthodenticle homeobox 2 is important for establishing subgroup phenotypes of post-mitotic midbrain dopaminergic neurons and may alter neuronal vulnerability.
doi:10.1093/brain/awq142
PMCID: PMC2892944  PMID: 20573704
axon; protection; Parkinson’s disease; neuropeptides; transcription factor
21.  The Toll-like receptor-3 agonist poly(I:C) triggers nigrostriatal dopaminergic degeneration 
In Parkinson’s disease (PD), loss of striatal dopaminergic (DA) terminals and degeneration of DA neurons in the substantia nigra (SN) are associated with glial reactions. Such inflammatory processes are commonly considered an epiphenomenon of neuronal degeneration. However, there is increasing recognition of the role of neuroinflammation as an initiation factor of DA neuron degeneration. To investigate this issue, we established a new model of brain inflammation by injecting the Toll-like receptor 3 (TLR-3) agonist polyinosinic:polycytidylic acid [poly(I:C)] in the SN of adult rats. Poly(I:C) injection induced a sustained inflammatory reaction in the SN and in the dorsolateral striatum. Significant changes were detected in proteins relevant to synaptic transmission and axonal transport. In addition, cytoplasmic mislocalization of neuronal TDP-43 was observed. Poly(I:C) injection increased the susceptibility of midbrain DA neurons to a subsequent neurotoxic trigger (low dose 6-hydroxydopamine). Systemic delivery of IL-1 receptor antagonist (IL1-ra) protected SN DA neurons exposed to combined poly(I:C) induced inflammatory and neurotoxic oxidative stress.
These data indicate that viral-like neuroinflammation induces predegenerative changes in the DA system, which lowers the set point toward neuronal dysfunction and degeneration. New powerful neuroprotective therapies for PD might be considered by targeting critical inflammatory mechanisms, including cytokine-induced neurotoxicity.
doi:10.1523/JNEUROSCI.2400-10.2010
PMCID: PMC3075577  PMID: 21123556
Parkinson’s disease; neuroinflammation; neurodegeneration; Toll-like receptor; Interleukin 1; viral immunity
22.  Oct4-Induced Reprogramming Is Required for Adult Brain Neural Stem Cell Differentiation into Midbrain Dopaminergic Neurons 
PLoS ONE  2011;6(5):e19926.
Neural stem cells (NSCs) lose their competency to generate region-specific neuronal populations at an early stage during embryonic brain development. Here we investigated whether epigenetic modifications can reverse the regional restriction of mouse adult brain subventricular zone (SVZ) NSCs. Using a variety of chemicals that interfere with DNA methylation and histone acetylation, we showed that such epigenetic modifications increased neuronal differentiation but did not enable specific regional patterning, such as midbrain dopaminergic (DA) neuron generation. Only after Oct-4 overexpression did adult NSCs acquire a pluripotent state that allowed differentiation into midbrain DA neurons. DA neurons derived from Oct4-reprogrammed NSCs improved behavioural motor deficits in a rat model of Parkinson's disease (PD) upon intrastriatal transplantation. Here we report for the first time the successful differentiation of SVZ adult NSCs into functional region-specific midbrain DA neurons, by means of Oct-4 induced pluripotency.
doi:10.1371/journal.pone.0019926
PMCID: PMC3104995  PMID: 21655272
23.  Wnt1-lmx1a forms a novel autoregulatory loop and controls midbrain dopaminergic differentiation synergistically with the SHH-FoxA2 pathway 
Cell stem cell  2009;5(6):646-658.
Summary
Selective degeneration of midbrain dopaminergic (mDA) neurons is associated with Parkinson’s disease (PD), and thus an in-depth understanding of molecular pathways underlying mDA development will be crucial for optimal bioassays and cell replacement therapy for PD. In this study, we identified a novel Wnt1-Lmx1a autoregulatory loop during mDA differentiation of ES cells, and confirmed its in vivo presence during embryonic development. We found that the Wnt1-Lmx1a autoregulatory loop directly regulates Otx2 through the β-catenin complex and Nurr1 and Pitx3 through Lmx1a. We also found that Lmx1a and Lmx1b co-operatively regulate mDA differentiation with overlapping and cross-regulatory functions. Furthermore, co-activation of both Wnt1 and SHH pathways by exogenous expression of Lmx1a, Otx2 and FoxA2 synergistically enhanced the differentiation of ES cells to mDA neurons. Together with previous works, this study shows that two regulatory loops (Wnt1-Lmx1a and SHH-FoxA2) critically link extrinsic signals to cell-intrinsic factors and cooperatively regulate mDA neuron development.
doi:10.1016/j.stem.2009.09.015
PMCID: PMC2788512  PMID: 19951692
24.  Axon guidance and synaptic maintenance: preclinical markers for neurodegenerative disease and therapeutics 
Trends in neurosciences  2009;32(3):142-149.
Axon-guidance-pathway molecules are involved in connectivity and repair throughout life (beyond guiding brain wiring during fetal development). One study found that variations (single-nucleotide polymorphisms [SNPs]) in axon-guidance-pathway genes were predictive of three Parkinson’s disease (PD) outcomes (susceptibility, survival free of PD and age at onset of PD) in genome-wide association (GWA) datasets. The axon-guidance-pathway genes DCC, EPHB1, NTNG1, SEMA5A and SLIT3 were represented by SNPs predicting PD outcomes. Beyond GWA analyses, we also present relevant neurobiological roles of these axon-guidance-pathway molecules and consider mechanisms by which abnormal axon-guidance-molecule signaling can cause loss of connectivity and, ultimately, PD. Novel drugs and treatments could emerge from this new understanding.
doi:10.1016/j.tins.2008.11.006
PMCID: PMC2954610  PMID: 19162339
25.  Immature and Neurally Differentiated Mouse Embryonic Stem Cells Do Not Express a Functional Fas/Fas Ligand System 
Stem cells (Dayton, Ohio)  2007;25(10):2551-2558.
The potential of pluripotent embryonic stem (ES) cells to develop into functional cells or tissue provides an opportunity in the development of new therapies for many diseases including neurodegenerative disorders. The survival of implanted cells usually requires systemic immunosuppression, however, which severely compromises the host immune system, leading to complications in clinical transplantation. An optimal therapy would therefore be the induction of specific tolerance to the donor cells, while otherwise preserving functional immune responses. Fas ligand (FasL) is expressed in activated lymphocytes as well as cells in “immune-privileged” sites including the central nervous system. Its receptor, Fas, is expressed on various immune-reactive cell types, such as activated natural killer and T cells, monocytes, and polymorphic mononucleocytes, which can undergo apoptosis upon interaction with FasL. To render transplanted cells tolerant to host cellular immune responses, we genetically engineered mouse ES cells to express rat FasL (rFasL). The rFasL-expressing ES cells were analyzed for survival during in vitro neurodifferentiation and after transplantation to the rat brain without further immunosuppression. Although control transfected HEK-293T cells expressed functional rFasL, immature and differentiated mouse ES cells did not express the recombinant rFasL surface protein. Furthermore, there was no evidence for functional endogenous Fas and FasL expression on either ES cells or on neural cells after in vitro differentiation. Moreover, implanted rFasL-engineered ES cells did not survive in the rat brains in the absence of the immunosuppressive agent cyclosporine A. Our results indicate that immature and differentiated mouse ES cells do not express a functional Fas/FasL system.
doi:10.1634/stemcells.2006-0745
PMCID: PMC2951385  PMID: 17615270
Fas; Fas ligand; Fas/Fas ligand system; Embryonic stem cells; Neural differentiation; Immune response Immunosuppression; Brain; Transplantation

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