Publications by authors named "Chi Wang Ip"

31 Publications

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia.

J Vis Exp 2020 09 12(163). Epub 2020 Sep 12.

Department of Neurology, University Hospital of Würzburg;

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do not present a motor phenotype resembling the clinical aspects of the human disease. Pharmacological mouse models allow for a more direct study of the pathomechanisms and their effect on the behavioral phenotype. Osmotic pumps connected to brain cannulas open up the possibility of creating pharmacological mouse models via local and chronic drug delivery. For the hereditary movement disorder of rapid-onset dystonia-parkinsonism, the loss-of-function mutation in the α3-subunit of the Na/K-ATPase can be simulated by a highly specific blockade via the glycoside ouabain. In order to locally block the α3-subunit in the basal ganglia and the cerebellum, which are the two brain structures believed to be heavily involved in the pathogenesis of rapid-onset dystonia-parkinsonism, a bilateral cannula is stereotaxically implanted into the striatum and an additional single cannula is introduced into the cerebellum. The cannulas are connected via vinyl tubing to two osmotic pumps, which are subcutaneously implanted on the back of the animals and allow for the chronic and precise delivery of ouabain. The pharmacological mouse model for rapid-onset dystonia-parkinsonism carries the additional advantage of recapitulating the clinical and pathological features of asymptomatic and symptomatic mutation carriers. Just like mutation carriers of rapid-onset dystonia parkinsonism, the ouabain-perfused mice develop dystonia-like movements only after additional exposure to stress. We demonstrate a mild stress paradigm and introduce two modified scoring systems for the assessment of a motor phenotype.
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http://dx.doi.org/10.3791/61635DOI Listing
September 2020

[Genetic testing for Parkinson's disease: indication and practical implementation].

Fortschr Neurol Psychiatr 2020 Sep 28;88(9):601-608. Epub 2020 Jun 28.

Klinik für Neurologie, Universitätsklinik Essen.

More than 20 years have passed since the first description of a monogenic cause of Parkinson's disease. Despite the tremendous advances of genetic testing these techniques are rarely used in Parkinson's disease. However, genetic tests in patients with Parkinson's syndrome will play an important role in the future. This is not only to ensure the diagnosis of Parkinson's patients with a young onset and / or a positive family history, but also in the context of personalised medicine with new therapeutic options. In the following we would like to give an overview of the basics of genetic testing, the legal requirements, the procedure for genetic testing and an outlook into the future for hereditary Parkinson's diseases.
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http://dx.doi.org/10.1055/a-1155-6389DOI Listing
September 2020

Striatal dopaminergic dysregulation and dystonia-like movements induced by sensorimotor stress in a pharmacological mouse model of rapid-onset dystonia-parkinsonism.

Exp Neurol 2020 01 8;323:113109. Epub 2019 Nov 8.

Department of Neurology, University Hospital of Würzburg, Josef-Schneider-Straße 11, 97080 Würzburg, Germany. Electronic address:

Rapid-onset dystonia-parkinsonism (RDP) is a rare form of hereditary dystonia caused by loss-of-function mutations of the Na/K-ATPase α3 isoform (ATP1α3). An acute onset of generalized dystonia and parkinsonism after exposure to stress and an incomplete disease penetrance is described in RDP, thereby suggesting a gene-environmental interaction in individuals with a genetic predisposition for dystonia development. Dystonia is considered a central motor network disease and in line with this concept, alterations in cerebellar neuronal firing have been described in RDP mouse models, but the pathogenic role of the basal ganglia remains unclear. We have mimicked RDP pharmacologically by simultaneous perfusion of the selective ATP1α3-blocker ouabain into the striatum and cerebellum of mice, followed by repeated exposure to mild motor stress. Ouabain-perfused RDP mice developed dystonia-like movements, which were exacerbated by exposure to sensorimotor stress. Compared to control mice, ouabain perfusion of the striatum led to dendritic spine loss of medium spiny neurons in addition to loss of cholinergic and GABAergic interneurons in the striatum. High-pressure liquid chromatography analyses revealed significant dopamine (DA) depletion and increased DA and serotonergic turnover, while qPCR analyses displayed reduction of glutamatergic receptors. Adding stress to the ouabain-predisposed brain, however, resulted in an elevation of the striatal DA metabolism back to the level of control animals. Our results indicate an ouabain-induced basal ganglia and cerebellar motor network dysfunction characterized by structural and neurochemical alterations of striatal dopaminergic, cholinergic and glutamatergic pathways that represent a motor endophenotype of RDP mutation carriers. Challenging the motor circuit by sensorimotor stress causes exacerbation of dystonia-like movements tightly linked to a hyperdopaminergic state in the striatum. Our observations support a gene-environment interaction or "second-hit" hypothesis in the symptomatogenesis of RDP.
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http://dx.doi.org/10.1016/j.expneurol.2019.113109DOI Listing
January 2020

Generation and Analysis of Viral Vector-Mediated Rodent Models for Parkinson's Disease.

Methods Mol Biol 2019 ;1948:271-286

Department of Neurology, University Hospital of Würzburg, Würzburg, Germany.

The use of viral vector-mediated α-synuclein-overexpressing rodent models for Parkinson's disease has become increasingly accepted to study associated pathology and to use as a platform for therapeutic efficacy testing. We here describe methods to generate such models and how to analyze them by behavioral assessments, histological investigations, and examination of nigrostriatal dopaminergic function.
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http://dx.doi.org/10.1007/978-1-4939-9124-2_21DOI Listing
July 2019

Management of delirium in Parkinson's disease.

J Neural Transm (Vienna) 2019 07 6;126(7):905-912. Epub 2019 Feb 6.

Department of Neurology, Charité-Universitätsmedizin Berlin, Hindenburgdamm 30, 12200, Berlin, Germany.

Delirium is an acute and fluctuating disturbance of attention and awareness. Pre-existing cognitive disturbances or dementia are the most significant risk factors for developing delirium and precipitating factors such as drug treatment, infections, trauma, or surgery may trigger delirium. Patients with Parkinson's disease (PD) are at an increased risk for delirium which may be underdiagnosed due to phenomenological overlap between delirium and chronic neuropsychiatric features of PD or side effects of dopaminergic medication. Prognosis of delirium is detrimental in many cases including permanent cognitive decline, motor impairment, and increased mortality. Management of delirium comprises of pharmacological and non-pharmacological measures. Pharmacotherapy is aimed at treating medical precipitating factors such as infections, pain, and sleep deprivation. Adjustments of anti-parkinsonian medication are recommended to prevent or treat delirium, but no hard evidence in this respect is available from controlled studies. Administration of neuroleptics and other psychoactive drugs in the treatment of delirium is controversially discussed and should be reserved for patients with severe agitation or distressing psychosis. Non-pharmacological interventions to prevent or palliate delirium are based on withdrawing precipitating or distressing factors, and to provide sensory, emotional and environmental support. Appropriate instruments to detect and assess delirium in PD are needed, and efforts are warranted to improve understanding and treatment of this severe and common disorder.
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http://dx.doi.org/10.1007/s00702-019-01980-7DOI Listing
July 2019

[Parkinson Disease and Pain - diagnostic and therapeutic approaches to a challenging non-motor symptom].

Fortschr Neurol Psychiatr 2018 09 17;86(S 01):S48-S58. Epub 2018 Jul 17.

Universitätsklinikum Ulm, RKU, Klinik für Neurologie.

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http://dx.doi.org/10.1055/a-0590-4464DOI Listing
September 2018

Reply to "Can STN DBS protect both nigral somata and innervation of the striatum?"

Ann Neurol 2017 11 26;82(5):856. Epub 2017 Oct 26.

Department of Neurology, University Hospital of Würzburg, Würzburg, Germany.

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http://dx.doi.org/10.1002/ana.25066DOI Listing
November 2017

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment.

J Vis Exp 2017 09 1(127). Epub 2017 Sep 1.

Department of Neurology, University Hospital of Würzburg.

In pre-clinical Parkinson's disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.
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http://dx.doi.org/10.3791/56103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614405PMC
September 2017

Subthalamic nucleus deep brain stimulation is neuroprotective in the A53T α-synuclein Parkinson's disease rat model.

Ann Neurol 2017 Jun 9;81(6):825-836. Epub 2017 Jun 9.

Department of Neurology, University Hospital of Würzburg, Würzburg, Germany.

Objective: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is a highly effective symptomatic therapy for motor deficits in Parkinson's disease (PD). An additional, disease-modifying effect has been suspected from studies in toxin-based PD animal models, but these models do not reflect the molecular pathology and progressive nature of PD that would be required to evaluate a disease-modifying action. Defining a disease-modifying effect could radically change the way in which DBS is used in PD.

Methods: We applied STN-DBS in an adeno-associated virus (AAV) 1/2-driven human mutated A53T α-synuclein (aSyn)-overexpressing PD rat model (AAV1/2-A53T-aSyn). Rats were injected unilaterally, in the substantia nigra (SN), with AAV1/2-A53T-aSyn or control vector. Three weeks later, after behavioral and nigrostriatal dopaminergic deficits had developed, rats underwent STN-DBS electrode implantation ipsilateral to the vector-injected SN. Stimulation lasted for 3 weeks. Control groups remained OFF stimulation. Animals were sacrificed at 6 weeks.

Results: Motor performance in the single pellet reaching task was impaired in the AAV1/2-A53T-aSyn-injected stim-OFF group, 6 weeks after AAV1/2-A53T-aSyn injection, compared to preoperative levels (-82%; p < 0.01). Deficits were reversed in AAV1/2-A53T-aSyn, stim-ON rats after 3 weeks of active stimulation, compared to the AAV1/2-A53T-aSyn stim-OFF rats (an increase of ∼400%; p < 0.05), demonstrating a beneficial effect of DBS. This motor improvement was maintained when the stimulation was turned off and was accompanied by a higher number of tyrosine hydroxylase SN neurons (increase of ∼29%), compared to AAV1/2-A53T-aSyn stim-OFF rats (p < 0.05).

Interpretation: Our data support the putative neuroprotective and disease-modifying effect of STN-DBS in a mechanistically relevant model of PD. Ann Neurol 2017;81:825-836.
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http://dx.doi.org/10.1002/ana.24947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519923PMC
June 2017

Pathogenic inflammation in the CNS of mice carrying human PLP1 mutations.

Hum Mol Genet 2016 11;25(21):4686-4702

Department of Neurology, Section of Developmental Neurobiology, University Hospital Wuerzburg, D-97080 Wuerzburg, Germany.

Progressive forms of multiple sclerosis lead to chronic disability, substantial decline in quality of life and reduced longevity. It is often suggested that they occur independently of inflammation. Here we investigated the disease progression in mouse models carrying PLP1 point mutations previously found in patients displaying clinical features of multiple sclerosis. These mouse models show loss-of-function of PLP1 associated with neuroinflammation; the latter leading to clinically relevant axonal degeneration, neuronal loss and brain atrophy as demonstrated by inactivation of the recombination activating gene 1. Moreover, these pathological hallmarks were substantially amplified when we attenuated immune regulation by inactivation of the programmed cell death-1 gene. Our observations support the view that primary oligodendroglial abnormalities can evoke pathogenically relevant neuroinflammation that drives neurodegeneration, as observed in some forms of multiple sclerosis but also in other, genetically-mediated neurodegenerative disorders of the human nervous system. As many potent immunomodulatory drugs have emerged during the last years, it is tempting to consider immunomodulation as a treatment option not only for multiple sclerosis, but also for so far non-treatable, genetically-mediated disorders of the nervous system accompanied by pathogenic neuroinflammation.
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http://dx.doi.org/10.1093/hmg/ddw296DOI Listing
November 2016

AAV1/2-induced overexpression of A53T-α-synuclein in the substantia nigra results in degeneration of the nigrostriatal system with Lewy-like pathology and motor impairment: a new mouse model for Parkinson's disease.

Acta Neuropathol Commun 2017 Feb 1;5(1):11. Epub 2017 Feb 1.

Krembil Research Institute, Toronto Western Hospital, University Health Network, 60 Leonard Avenue, 8KD402, Toronto, ON, M5T 2S8, Canada.

α-Synuclein is a protein implicated in the etiopathogenesis of Parkinson's disease (PD). AAV1/2-driven overexpression of human mutated A53T-α-synuclein in rat and monkey substantia nigra (SN) induces degeneration of nigral dopaminergic neurons and decreases striatal dopamine and tyrosine hydroxylase (TH). Given certain advantages of the mouse, especially it being amendable to genetic manipulation, translating the AAV1/2-A53T α-synuclein model to mice would be of significant value. AAV1/2-A53T α-synuclein or AAV1/2 empty vector (EV) at a concentration of 5.16 x 10 gp/ml were unilaterally injected into the right SN of male adult C57BL/6 mice. Post-mortem examinations included immunohistochemistry to analyze nigral α-synuclein, Ser129 phosphorylated α-synuclein and TH expression, striatal dopamine transporter (DAT) levels by autoradiography and dopamine levels by high performance liquid chromatography. At 10 weeks, in AAV1/2-A53T α-synuclein mice there was a 33% reduction in TH+ dopaminergic nigral neurons (P < 0.001), 29% deficit in striatal DAT binding (P < 0.05), 38% and 33% reductions in dopamine (P < 0.001) and DOPAC (P < 0.01) levels and a 60% increase in dopamine turnover (homovanilic acid/dopamine ratio; P < 0.001). Immunofluorescence showed that the AAV1/2-A53T α-synuclein injected mice had widespread nigral and striatal expression of vector-delivered A53T-α-synuclein. Concurrent staining with human PD SN samples using gold standard histological methodology for Lewy pathology detection by proteinase K digestion and application of specific antibody raised against human Lewy body α-synuclein (LB509) and Ser129 phosphorylated α-synuclein (81A) revealed insoluble α-synuclein aggregates in AAV1/2-A53T α-synuclein mice resembling Lewy-like neurites and bodies. In the cylinder test, we observed significant paw use asymmetry in the AAV1/2-A53T α-synuclein group when compared to EV controls at 5 and 9 weeks post injection (P < 0.001; P < 0.05). These data show that unilateral injection of AAV1/2-A53T α-synuclein into the mouse SN leads to persistent motor deficits, neurodegeneration of the nigrostriatal dopaminergic system and development of Lewy-like pathology, thereby reflecting clinical and pathological hallmarks of human PD.
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http://dx.doi.org/10.1186/s40478-017-0416-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286802PMC
February 2017

Tor1a+/- mice develop dystonia-like movements via a striatal dopaminergic dysregulation triggered by peripheral nerve injury.

Acta Neuropathol Commun 2016 10 3;4(1):108. Epub 2016 Oct 3.

Department of Neurology, University Hospital of Würzburg, University of Würzburg, Josef-Schneider-Strasse 11, 97080, Würzburg, Germany.

Isolated generalized dystonia is a central motor network disorder characterized by twisted movements or postures. The most frequent genetic cause is a GAG deletion in the Tor1a (DYT1) gene encoding torsinA with a reduced penetrance of 30-40 % suggesting additional genetic or environmental modifiers. Development of dystonia-like movements after a standardized peripheral nerve crush lesion in wild type (wt) and Tor1a+/- mice, that express 50 % torsinA only, was assessed by scoring of hindlimb movements during tail suspension, by rotarod testing and by computer-assisted gait analysis. Western blot analysis was performed for dopamine transporter (DAT), D1 and D2 receptors from striatal and quantitative RT-PCR analysis for DAT from midbrain dissections. Autoradiography was used to assess the functional DAT binding in striatum. Striatal dopamine and its metabolites were analyzed by high performance liquid chromatography. After nerve crush injury, we found abnormal posturing in the lesioned hindlimb of both mutant and wt mice indicating the profound influence of the nerve lesion (15x vs. 12x relative to control) resembling human peripheral pseudodystonia. In mutant mice the phenotypic abnormalities were increased by about 40 % (p < 0.05). This was accompanied by complex alterations of striatal dopamine homeostasis. Pharmacological blockade of dopamine synthesis reduced severity of dystonia-like movements, whereas treatment with L-Dopa aggravated these but only in mutant mice suggesting a DYT1 related central component relevant to the development of abnormal involuntary movements. Our findings suggest that upon peripheral nerve injury reduced torsinA concentration and environmental stressors may act in concert in causing the central motor network dysfunction of DYT1 dystonia.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5048687PMC
http://dx.doi.org/10.1186/s40478-016-0375-7DOI Listing
October 2016

Lymphocytes reduce nigrostriatal deficits in the 6-hydroxydopamine mouse model of Parkinson's disease.

J Neural Transm (Vienna) 2015 Dec 20;122(12):1633-43. Epub 2015 Aug 20.

Department of Neurology, University of Wuerzburg, Josef-Schneider-Strasse 11, 97080, Würzburg, Germany.

Neuroinflammation is a well-known neuropathological feature of Parkinson's disease (PD), but it remains controversial whether it is causal or consequential to neurodegeneration. While the role of microglia in the pathogenesis has been thoroughly investigated in human and different rodent models, data concerning the impact of the adaptive immune system on the pathogenesis of PD are still rare, although lymphocyte populations were found in brain tissue of PD patients and have been implicated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated neurodegeneration in mice. To test the hypothesis that the adaptive immune system contributes to the progression of PD in the murine 6-hydroxydopamine (6-OHDA) model, we performed unilateral 6-OHDA injection into the medial forebrain bundle and compared wild-type mice with recombination activating gene-1 deficient mice (RAG-1(-/-)), that lack mature lymphocytes. After 6-OHDA injection, immune-deficient mice moved significantly slower and less often than wild-type mice. Rotarod analysis displayed a shorter latency to fall in RAG-1(-/-) mice. Immunohistochemical analysis in wild-type mice demonstrated a higher CD8+ T cell density in the ipsilesional striatum compared to sham-operated animals. Cell counts of tyrosine hydroxylase positive dopaminergic neurons of the substantia nigra in immune compromised mice were significantly reduced compared to wild-type mice. Wild type bone marrow reconstitution into RAG-1(-/-) recipients rescued the clinical deterioration as well as the neurodegeneration in RAG-1(-/-) deficient recipients ameliorated clinical symptoms and neurodegeneration after 6-OHDA treatment. Our data indicate that lymphocytes reduce the clinical and neuropathological impact of 6-OHDA lesioning and thus may play a protective role in this toxic mouse model of PD.
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http://dx.doi.org/10.1007/s00702-015-1444-yDOI Listing
December 2015

CD8⁺ T cell help is required for efficient induction of EAE in Lewis rats.

J Neuroimmunol 2013 Jul 7;260(1-2):17-27. Epub 2013 May 7.

Institute for Virology and Immunobiology, University of Würzburg, Versbacher Strasse 7, 97078 Würzburg, Germany.

The role of CD8⁺ T cells in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is still unclear. We describe here significantly reduced disease activity of EAE both in Lewis rats depleted of CD8⁺ T cells by monoclonal antibodies and CD8 knockout rats, which was accompanied by reduced leukocyte infiltration into the spinal cord. We detected myelin basic protein (MBP)-specific CD8⁺ T cells in peripheral lymphoid organs of CD8-depleted animals which, however, failed to differentiate into interferon-γ-producing effector cells. Our results indicate that CD8⁺ T cells interact with myelin-specific CD8⁺ T cells early in EAE enabling them to differentiate into pathogenic effector cells.
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http://dx.doi.org/10.1016/j.jneuroim.2013.04.014DOI Listing
July 2013

Immune cells perturb axons and impair neuronal survival in a mouse model of infantile neuronal ceroid lipofuscinosis.

Brain 2013 Apr 12;136(Pt 4):1083-101. Epub 2013 Mar 12.

Department of Neurology, Developmental Neurobiology, Josef-Schneider-Str. 11, D-97080 Wuerzburg, Germany.

The neuronal ceroid lipofuscinoses are fatal neurodegenerative disorders in which the visual system is affected early in disease progression. A typical accompanying feature is neuroinflammation, the pathogenic impact of which is presently obscure. Here we investigated the role of inflammatory cells in palmitoyl protein thioesterase 1-deficient (Ppt1(-/-)) mice, a model of infantile neuronal ceroid lipofuscinosis (CLN1 disease, infantile), predominantly focusing on the visual system. We detected an early infiltration of CD8+ T-lymphocytes and observed activation of microglia/macrophage-like cells. To analyse the pathogenic impact of lymphocytes, we crossbred Ppt1(-/-) mice with mutants lacking lymphocytes (Rag1(-/-)), and scored axonal transport, axonal perturbation and neuronal survival. This lack of lymphocytes led to a significant amelioration of disease phenotypes, not only in the retino-tectal system, but also in other regions of the central nervous system. Finally, reconstitution experiments revealed a crucial role of CD8+ T-lymphocytes in pathogenesis. Our study provides novel pathomechanistic insights that may be crucial for developing treatment strategies.
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http://dx.doi.org/10.1093/brain/awt020DOI Listing
April 2013

Neuroinflammation by cytotoxic T-lymphocytes impairs retrograde axonal transport in an oligodendrocyte mutant mouse.

PLoS One 2012 8;7(8):e42554. Epub 2012 Aug 8.

Department of Neurology, Section of Developmental Neurobiology, University of Würzburg, Würzburg, Germany.

Mice overexpressing proteolipid protein (PLP) develop a leukodystrophy-like disease involving cytotoxic, CD8+ T-lymphocytes. Here we show that these cytotoxic T-lymphocytes perturb retrograde axonal transport. Using fluorogold stereotactically injected into the colliculus superior, we found that PLP overexpression in oligodendrocytes led to significantly reduced retrograde axonal transport in retina ganglion cell axons. We also observed an accumulation of mitochondria in the juxtaparanodal axonal swellings, indicative for a disturbed axonal transport. PLP overexpression in the absence of T-lymphocytes rescued retrograde axonal transport defects and abolished axonal swellings. Bone marrow transfer from wildtype mice, but not from perforin- or granzyme B-deficient mutants, into lymphocyte-deficient PLP mutant mice led again to impaired axonal transport and the formation of axonal swellings, which are predominantly located at the juxtaparanodal region. This demonstrates that the adaptive immune system, including cytotoxic T-lymphocytes which release perforin and granzyme B, are necessary to perturb axonal integrity in the PLP-transgenic disease model. Based on our observations, so far not attended molecular and cellular players belonging to the immune system should be considered to understand pathogenesis in inherited myelin disorders with progressive axonal damage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042554PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3414455PMC
February 2013

Ectopic T-cell specificity and absence of perforin and granzyme B alleviate neural damage in oligodendrocyte mutant mice.

Am J Pathol 2010 Feb 30;176(2):549-55. Epub 2009 Dec 30.

Department of Neurology, Section of Developmental Neurobiology, University of Wuerzburg, Josef-Schneider Str. 11, D-97080 Wuerzburg, Germany.

In transgenic mice overexpressing the major myelin protein of the central nervous system, proteolipid protein, CD8+ T-lymphocytes mediate the primarily genetically caused myelin and axon damage. In the present study, we investigated the cellular and molecular mechanisms underlying this immune-related neural injury. At first, we investigated whether T-cell receptors (TCRs) are involved in these processes. For this purpose, we transferred bone marrow from mutants carrying TCRs with an ectopic specificity to ovalbumin into myelin mutant mice that also lacked normal intrinsic T-cells. T-lymphocytes with ovalbumin-specific TCRs entered the mutant central nervous system to a similar extent as T-lymphocytes from wild-type mice. However, as revealed by histology, electron microscopy and axon- and myelin-related immunocytochemistry, these T-cells did not cause neural damage in the myelin mutants, reflecting the need for specific antigen recognition by cytotoxic CD8+ T-cells. By chimerization with bone marrow from perforin- or granzyme B (Gzmb)-deficient mice, we demonstrated that absence of these cytotoxic molecules resulted in reduced neural damage in myelin mutant mice. Our study strongly suggests that pathogenetically relevant immune reactions in proteolipid protein-overexpressing mice are TCR-dependent and mediated by the classical components of CD8+ T-cell cytotoxicity, perforin, and Gzmb. These findings have high relevance with regard to our understanding of the pathogenesis of disorders primarily caused by genetically mediated oligodendropathy.
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http://dx.doi.org/10.2353/ajpath.2010.090722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808063PMC
February 2010

Collateral neuronal apoptosis in CNS gray matter during an oligodendrocyte-directed CD8(+) T cell attack.

Glia 2010 Mar;58(4):469-80

Department of Neurology, University of Würzburg, Germany.

Demyelination and death of oligodendrocytes accompanied by transection of neurites and neuronal apoptosis are pathological hallmarks of cortical and subcortical gray matter lesions in demyelinating viral and autoimmune inflammatory CNS disorders. In these disorders, leukocortical lesions, containing the perikarya of most efferent neurons, display pronounced infiltration by CD8(+) T cells of putative specificity for oligodendrocyte- and myelin-related antigens. Hence, neuronal apoptosis in gray matter lesions may be a collateral effect of an oligodendrocyte-directed attack by CD8(+) T cells. To challenge this hypothesis, we transferred activated antigen-specific CD8(+) T cells (OT-I T cells) into acute coronal brain slices from mice selectively expressing ovalbumin as a cytosolic neo-self-antigen in oligodendrocytes (ODC-OVA mice). We studied mechanisms and kinetics of oligodendroglial and neuronal apoptosis in the neocortex and hippocampus, using multicolor staining for different cell types and activated caspase-3. Within the gray matter, a single OT-I T cell caused simultaneous caspase-3 activation in about 30 ODCs and 10 neurons within 6 h in a strictly antigen-dependent manner. Experiments with OT-I T cells genetically deficient for perforin or the granzyme B-cluster and with blocking anti-FasL antibodies as well as proinflammatory cytokines revealed, that collateral apoptosis of neurons was likely due to a spillover of perforin and granzyme(s) from the OT-I T cell itself or the immunological synapse that it selectively formed with antigen-presenting oligodendrocytes. Collateral neuronal apoptosis could contribute to substantial neuronal loss in gray matter lesions and cause persistent neurological impairment in both acute and chronic gray matter lesions in various inflammatory CNS disorders.
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http://dx.doi.org/10.1002/glia.20938DOI Listing
March 2010

Accelerated course of experimental autoimmune encephalomyelitis in PD-1-deficient central nervous system myelin mutants.

Am J Pathol 2009 Jun 14;174(6):2290-9. Epub 2009 May 14.

Clinical Research Group for MS and Neuroimmunology, Department of Neurology, University of Wuerzburg, Josef Schneider Strasse 11, 97080 Wuerzburg, Germany.

It is assumed that the onset and course of autoimmune inflammatory central nervous system (CNS) disorders (eg, multiple sclerosis) are influenced by factors that afflict immune regulation as well as CNS vulnerability. We challenged this concept experimentally by investigating how genetic alterations that affect myelin (primary oligodendrocyte damage in PLPtg mice) and/or T-cell regulation (deficiency of PD-1) influence both the onset and course of an experimental autoimmune CNS inflammatory disease [MOG(35-55)-induced experimental autoimmune encephalomyelitis (EAE)]. We observed that double pathology was associated with a significantly earlier onset of disease, a slight increase in the neurological score, an increase in the number of infiltrating cells, and enhanced axonal degeneration compared with wild-type mice and the respective, single mutant controls. Double-mutant PLPtg/PD-1(-/-) mice showed an increased production of interferon-gamma by CNS immune cells at the peak of disease. Neither PD-1 deficiency nor oligodendropathy led to detectable spread of antigenic MHC class I- or class II-restricted epitopes during EAE. However, absence of PD-1 clearly increased the propensity of T lymphocytes to expand, and the number of clonal expansions reliably reflected the severity of the EAE disease course. Our data show that the interplay between immune dysregulation and myelinopathy results in a stable exacerbation of actively induced autoimmune CNS inflammation, suggesting that the combination of several pathological issues contributes significantly to disease susceptibility or relapses in human disease.
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http://dx.doi.org/10.2353/ajpath.2009.081012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2684193PMC
June 2009

PD-1 regulates neural damage in oligodendroglia-induced inflammation.

PLoS One 2009 6;4(2):e4405. Epub 2009 Feb 6.

Department of Neurology, University of Wuerzburg, Wuerzburg, Germany.

We investigated the impact of immune regulatory mechanisms involved in the modulation of the recently presented, CD8+ lymphocyte mediated immune response in a mouse model of oligodendropathy-induced inflammation (PLPtg-mutants). The focus was on the role of the co-inhibitory molecule PD-1, a CD28-related receptor expressed on activated T- and B-lymphocytes associated with immune homeostasis and autoimmunity. PLPtg/PD-1-deficient double mutants and the corresponding bone marrow chimeras were generated and analysed using immunohistochemistry, light- and electron microscopy, with particular emphasis on immune-cell number and neural damage. In addition, the immune cells in both the CNS and the peripheral immune system were investigated by IFN-gamma elispot assays and spectratype analysis. We found that mice with combined pathology exhibited significantly increased numbers of CD4+ and CD8+ T-lymphocytes in the CNS. Lack of PD-1 substantially aggravated the pathological phenotype of the PLPtg mutants compared to genuine PLPtg mutants, whereas the PD-1 deletion alone did not cause alterations in the CNS. CNS T-lymphocytes in PLPtg/PD-1-/- double mutants exhibited massive clonal expansions. Furthermore, PD-1 deficiency was associated with a significantly higher propensity of CNS but not peripheral CD8+ T-cells to secrete proinflammatory cytokines. PD-1 could be identified as a crucial player of tissue homeostasis and immune-mediated damage in a model of oligodendropathy-induced inflammation. Alterations of this regulatory pathway lead to overt neuroinflammation of high pathogenetic impact. Our finding may have implications for understanding the mechanisms leading to the high clinical variability of polygenic or even monogenic disorders of the nervous system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0004405PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2635015PMC
March 2009

Tacrolimus (FK506) causes disease aggravation in models for inherited peripheral myelinopathies.

Neurobiol Dis 2009 Feb 5;33(2):207-12. Epub 2008 Nov 5.

Department of Neurology, Developmental Neurobiology, University of Wuerzburg, Josef-Schneider-Str. 11, D-97080 Wuerzburg, Germany.

Mice hetero- or homozygously deficient for myelin protein zero (P0+/-, P0-/- mice) are models for distinct forms of inherited de- or dysmyelinating neuropathies, respectively. P0+/- mice show a demyelinating neuropathy with a pathogenetic implication of CD8+ T-lymphocytes and macrophages, while P0-/- mice show dysmyelination with axonal loss. It was, therefore, of interest to treat both mutants with FK506 (Tacrolimus), an agent with immunosuppressive and neuroprotective properties. Treatment of P0+/- mice led to an aggravation of demyelination, without affecting nervous CD8+ T-lymphocytes, but reducing splenic CD4+ cells. Treatment of P0-/- mice resulted in a substantial increase of the dysmyelination-related axon loss. Treatment of wildtype mice did not cause pathological changes in peripheral nerves. Our study shows that FK506 may not be suitable for the treatment of the human nerve disorders. Furthermore, when used as an immunosuppressant, the drug may generate detrimental neurological side effects in patients with an additional hereditary neuropathy.
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http://dx.doi.org/10.1016/j.nbd.2008.10.008DOI Listing
February 2009

The co-inhibitory molecule PD-1 modulates disease severity in a model for an inherited, demyelinating neuropathy.

Neurobiol Dis 2009 Jan 15;33(1):96-103. Epub 2008 Oct 15.

Department of Neurology, University of Wuerzburg, Wuerzburg.

We have previously shown that mice heterozygously deficient for P0 are characterized by a late onset myelin disorder implicating CD8+ T-lymphocytes and macrophages. We now investigated the impact of the co-inhibitory molecule "programmed death" (PD)-1 (CD279), a CD28-related receptor expressed on activated T- and B-lymphocytes on the pathogenic phenotype of CD8+ T-lymphocytes in the P0 myelin mutants. PD-1 deficiency in P0+/- mice leads to a stronger increase of CD8+ T-lymphocytes and a substantially aggravated histological phenotype in the PNS compared to P0+/- mice expressing PD-1. Correspondingly, functional down-stream features, such as electrophysiological parameters, walking coordination and mechano-sensation are more affected than in PD-1-expressing myelin mutants. Our study demonstrates that a monogenic nerve disorder can be substantially modified by immune-controlling mechanisms. Thus, understanding the implication of disease-modifiers in inherited demyelination could be of pivotal interest for limiting the detrimental impact of primarily genetically-mediated myelin disorders by fostering immuno-regulatory pathways.
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http://dx.doi.org/10.1016/j.nbd.2008.09.021DOI Listing
January 2009

Transient widespread blood-brain barrier alterations after cerebral photothrombosis as revealed by gadofluorine M-enhanced magnetic resonance imaging.

J Cereb Blood Flow Metab 2009 Feb 29;29(2):331-41. Epub 2008 Oct 29.

Department of Neurology, University of Wuerzburg, Wuerzburg, Germany.

Magnetic resonance imaging (MRI) is a powerful tool to assess brain lesions, but currently available contrast agents are limited in the assessment of cellular and functional alterations. By use of the novel MRI contrast agent gadofluorine M (Gf) we report on imaging of transient and widespread changes of blood-brain barrier (BBB) properties as a consequence of focal photothrombotic brain lesions in rats. After i.v. application, Gf led to bright contrast in the lesions, but also the entire ipsilateral cortex on T1-weighted MRI. In contrast, enhancement after application of gadolinium diethylenetriamine-pentaacetic acid (Gd-DTPA), a common clinical indicator of BBB leakage was restricted to the lesions. Remote Gf enhancement was restricted in time to the first 24 h after photothrombosis and corresponded to a transient breakdown of the BBB as revealed by extravasation of the dye Evans blue. In conclusion, our study shows that Gf can visualize subtle disturbances of the BBB in three dimensions not detectable by Gd-DTPA. Upon entry into the central nervous system Gf most likely is locally trapped by interactions with extracellular matrix proteins. The unique properties of Gf hold promise as a more sensitive contrast agent for monitoring BBB disturbances in neurologic disorders, which appear more widespread than anticipated previously.
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http://dx.doi.org/10.1038/jcbfm.2008.129DOI Listing
February 2009

Increased number of microglia in the brain of severe combined immunodeficient (SCID) mice.

Histochem Cell Biol 2008 Oct 4;130(4):693-7. Epub 2008 Jul 4.

Department of Anatomy, Faculty of Medicine and Health Sciences, UAE University, Box 17666, Al Ain, United Arab Emirates.

To assess the in vivo influence of the systemic immune system upon microglia, six defined brain regions of adult SCID mice (n = 10) lacking functional T- and B-lymphocytes have been analyzed by NDPase histochemistry, morphometry and immunohistochemistry. Despite absence of neuropathology and lack of microglial activation, microglial numerical density was significantly increased in SCID mice. Elevation was most marked in the cerebellar granular layer (by 32.6%; 95% confidence interval: 9.9-58.7%), followed by the fimbria hippocampi and the molecular layer of hippocampal CA1/CA3 region. These data need to be taken into account when using SCID mice as a model for microglial reaction in immunodeficient mice.
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http://dx.doi.org/10.1007/s00418-008-0463-2DOI Listing
October 2008

Origin of CD11b+ macrophage-like cells in the CNS of PLP-overexpressing mice: low influx of haematogenous macrophages and unchanged blood-brain-barrier in the optic nerve.

Mol Cell Neurosci 2008 Aug 2;38(4):489-94. Epub 2008 May 2.

Department of Neurology, Developmental Neurobiology, University of Wuerzburg, Josef-Schneider Str. 11, D-97080 Wuerzburg, Germany.

We have recently reported that overexpression of proteolipid protein in oligodendrocytes leads to a pathologically relevant increase of both CD8+ T-lymphocytes and CD11b+ cells in the CNS. We now focussed on the origin of the CD11b+ cells in the optic nerve, a well established structure for the analysis of the mutant, using bone marrow chimeric mice. Although there is an age-related increase in CD11b+ cells in the myelinated part of the optic nerve of the mutants, the percentage of infiltrating cells was not increased, but enhanced proliferation was detectable. In the non-myelinated optic nerve head, the rate of infiltrating CD11b+ cells and albumin extravasation was high in both genotypes. However, albumin extravasation was also high in the rostral myelinated part, where CD11b+ cell influx was low. Our study demonstrates an intrinsic origin of CD11b+ cells in the presence of an unchanged blood-brain-barrier in a CNS myelin mutant.
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http://dx.doi.org/10.1016/j.mcn.2008.04.009DOI Listing
August 2008

Monocyte chemoattractant protein-1 is a pathogenic component in a model for a hereditary peripheral neuropathy.

Mol Cell Neurosci 2008 Feb;37(2):359-66

Department of Neurology, Developmental Neurobiology, University of Wuerzburg, Wuerzburg, Germany.

Macrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in models for inherited demyelinating neuropathies. It is presently unknown which factors link the Schwann cell-based myelin mutation to the activation of endoneurial macrophages. Here we identified the chemokine monocyte chemoattractant protein-1 (MCP-1) as a first and crucial factor upregulated in Schwann cells of mice heterozygously deficient for the myelin protein zero. The chemokine could be identified as an important mediator of macrophage immigration into peripheral nerves. Furthermore, a 50% reduction of chemokine expression by crossbreeding with MCP-1-deficient mice reduced the increase in macrophage numbers in the mutant nerves and lead to a robust amelioration of pathology. Surprisingly, the complete absence of MCP-1 aggravated the disease. Our findings show that reducing but not eliminating chemokine expression can rescue genetically caused demyelination that may be an interesting target in treating demyelinating diseases of the peripheral nervous system.
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http://dx.doi.org/10.1016/j.mcn.2007.10.012DOI Listing
February 2008

Antigen therapy of experimental autoimmune encephalomyelitis selectively induces apoptosis of pathogenic T cells.

J Neuroimmunol 2007 Feb 2;183(1-2):146-50. Epub 2007 Jan 2.

Institute for Virology and Immunobiology, University of Würzburg, Versbacher Strasse 7, 97078 Würzburg, and Department of Neurology, St. Josef-Hospital, University of Bochum, Germany.

Administration of high-dose myelin antigen induces massive T cell apoptosis in experimental autoimmune encephalomyelitis (EAE) but the nature of the target cells remains elusive. Here we have used a cell line established in eGFP-transgenic Lewis rats to distinguish between pathogenic and bystander T cells in adoptive transfer EAE. Intravenous application of gpMBP strongly reduced the amount of encephalitogenic cells in spinal cord and spleen while the number of the other T cells remained constant. This could be attributed to their differential sensitivity to apoptosis. Thus, antigen therapy selectively targets pathogenic T cells and should therefore limit potential adverse effects.
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http://dx.doi.org/10.1016/j.jneuroim.2006.11.031DOI Listing
February 2007

Sialoadhesin deficiency ameliorates myelin degeneration and axonopathic changes in the CNS of PLP overexpressing mice.

Neurobiol Dis 2007 Jan 24;25(1):105-11. Epub 2006 Oct 24.

Department of Neurology, Section of Developmental Neurobiology, University of Wuerzburg, Josef-Schneider Str. 11, D-97080 Wuerzburg, Germany.

PLP overexpressing mice display demyelination and axonopathic changes, accompanied by an elevation of CD8+ T-lymphocytes and CD11b+ macrophages in the CNS. By crossbreeding these mutants with RAG-1-deficient mice lacking mature lymphocytes, we could recently demonstrate a pathogenetic impact of the CD8+ cells. In the present study, we investigated the pathogenetic impact of CD11b+ macrophages by crossbreeding the myelin mutants with knockout mice deficient for the macrophage-restricted adhesion molecule sialoadhesin (Sn). In the wild-type mice, Sn is barely detectable on CD11b+ cells, whereas in the myelin mutants, almost all CD11b+ cells express Sn. In the double mutants, upregulation of CD8+ T-cells and CD11b+ macrophages is reduced and pathological alterations are ameliorated. These data indicate that in a primarily genetically caused myelin disorder of the CNS macrophages expressing Sn partially mediate pathogenesis. These findings may have substantial impact on treatment strategies for leukodystrophic disorders and some forms of multiple sclerosis.
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http://dx.doi.org/10.1016/j.nbd.2006.08.023DOI Listing
January 2007

Polyclonal expansion of regulatory T cells interferes with effector cell migration in a model of multiple sclerosis.

Brain 2006 Oct 18;129(Pt 10):2635-47. Epub 2006 Aug 18.

Institute for Virology and Immunobiology, University of Würzburg, Germany.

Recruitment of naturally occurring CD4+ CD25+ regulatory T (T(reg)) cells is a highly promising approach for the treatment of experimental autoimmune encephalomyelitis (EAE), a widely used model of multiple sclerosis. Here, we studied the in vivo interaction of T(reg) cells, induced by the monoclonal anti-CD28 antibody JJ316, with encephalitogenic T cell lines established from eGFP-transgenic rats. By tracking these fluorescent cells using flow cytometry and confocal microscopy, we found that the activation and expansion of T(reg) cells inhibited infiltration of the CNS by pathogenic T cells. Interference with effector cell migration occured within the secondary lymphoid organs, since the early therapeutic effects were achieved despite the absence of T(reg) cells in the spinal cord. However, the delayed homing to the CNS seen after prophylactic JJ316 administration indicates that T(reg) cells may play an additional role within the target tissue. In addition, the blood-brain barrier remained largely intact after JJ316 treatment, the secretion of T(H)2 cytokines was augmented and the encephalitogenic T cells exhibited a reduced secretion of IFN-gamma. This in turn resulted in a reduced expression of the chemokine receptor CXCR-3 on effector T cells which may interfere with their capacity to infiltrate the CNS. Importantly, these effects were not achieved by direct action of JJ316 on the encephalitogenic cells. Our data rather suggest that polyclonal activation of T(reg) cells in the secondary lymphoid organs is instrumental in preventing the pathological transmigration of encephalitogenic T cells into the CNS. We anticipate that these results may help to better understand the role of T(reg) cells in controlling autoimmunity in the CNS.
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http://dx.doi.org/10.1093/brain/awl213DOI Listing
October 2006

Immune cells contribute to myelin degeneration and axonopathic changes in mice overexpressing proteolipid protein in oligodendrocytes.

J Neurosci 2006 Aug;26(31):8206-16

Department of Neurology, University of Wuerzburg, D-97080 Wuerzburg, Germany.

Overexpression of the major myelin protein of the CNS, proteolipid protein (PLP), leads to late-onset degeneration of myelin and pathological changes in axons. Based on the observation that in white matter tracts of these mutants both CD8+ T-lymphocytes and CD11b+ macrophage-like cells are numerically elevated, we tested the hypothesis that these cells are pathologically involved in the primarily genetically caused neuropathy. Using flow cytometry of mutant brains, CD8+ cells could be identified as activated effector cells, and confocal microscopy revealed a close association of the T-cells with MHC-I+ (major histocompatibility complex class I positive) oligodendrocytes. Crossbreeding the myelin mutants with mice deficient in the recombination activating gene-1 (RAG-1) lacking mature T- and B-lymphocytes led to a reduction of the number of CD11b+ cells and to a substantial alleviation of pathological changes. In accordance with these findings, magnetic resonance imaging revealed less ventricular enlargement in the double mutants, partially because of more preserved corpora callosa. To investigate the role of CD8+ versus CD4+ T-lymphocytes, we reconstituted the myelin-RAG-1 double mutants with bone marrow from either CD8-negative (CD4+) or CD4-negative (CD8+) mice. The severe ventricular enlargement was only found when the double mutants were reconstituted with bone marrow from CD8+ mice, suggesting that the CD8+ lymphocytes play a critical role in the immune-related component of myelin degeneration in the mutants. These findings provide strong evidence that a primary glial damage can cause secondary immune reactions of pathological significance as it has been suggested for some forms of multiple sclerosis and other leukodystrophies.
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http://dx.doi.org/10.1523/JNEUROSCI.1921-06.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6673777PMC
August 2006