Fiesel F.C.,Hertie Institute for Clinical Brain Research |
Voigt A.,RWTH Aachen |
Weber S.S.,Hertie Institute for Clinical Brain Research |
Van Den Haute C.,Laboratory for Neurobiology and Gene Therapy |
And 15 more authors.
EMBO Journal | Year: 2010
TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP-43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP-43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl-tubulin. HDAC6 levels were restored by re-expression of TDP-43, dependent on RNA binding and the C-terminal protein interaction domains. Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ-expanded ataxin-3 were found in TDP-43 silenced cells. In conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby contribute to pathogenesis. © 2010 European Molecular Biology Organization.
Brito-Armas J.M.,University of La Laguna |
Baekelandt V.,Laboratory for Neurobiology and Gene Therapy |
Castro-Hernandez J.R.,University of La Laguna |
Gonzalez-Hernandez T.,University of La Laguna |
And 2 more authors.
Histology and Histopathology | Year: 2013
Two hallmarks of Parkinson's disease (PD) are dopaminergic cell loss and the presence of cytoplasmic inclusions (Lewy bodies). Different point mutations in alpha-synuclein, the main constituent of Lewy bodies, have been identified in familial PD. Alpha-synuclein also constitutes one of the main components of Lewy bodies in sporadic cases of PD. Moreover, oxidant stress and generation of free radicals from both mitochondrial impairment and dopamine metabolism are considered to play critical roles in PD etiopathogenesis. Melatonin, a known potent antioxidant secreted by the pineal gland, may protect against the effect of several Parkinsonogenic compounds that are associated with progressive impairment of mitochondrial function and increased oxidative damage. However, the neuroprotective effect of melatonin has never been tested in the newly available genetic models of PD based on the viral expression of mutated alpha-synuclein. Lentiviral vectors encoding A30P mutant human alphasynuclein (lenti-A30P) were stereotactically injected into the right substantia nigra of adult male Sprague-Dawley rats and neuroprotection was examined by administration of melatonin or vehicle from two days before nigral administration of lenti-A30P until eight weeks after injection. It was found that lenti-A30P induced a significant TH+ cell-loss both in the medial and lateral substantia nigra versus the contrallateral side injected with lenti-eGFP. However, melatonin administration showed a total neuroprotective effect in both regions of the substantia nigra. In conclusion, the data here show that melatonin is neuroprotective against mutant alpha-synuclein-induced injury in the substantia nigra.
Van Camp N.,University of Antwerp |
Vreys R.,University of Antwerp |
Van Laere K.,University Hospital Leuven |
Lauwers E.,Laboratory for Neurobiology and Gene Therapy |
And 10 more authors.
Magnetic Resonance Materials in Physics, Biology and Medicine | Year: 2010
Object: In the present study, we aimed to evaluate the impact of neurodegeneration of the nigrostriatal tract in a rodent model of Parkinson's disease on the different MR contrasts (T2, T1, CBF and CBV) measured in the striatum. Material and methods: Animals were injected with 6-hydroxydopamine (6OHDA) in the substantia nigra resulting in massive loss of nigrostriatal neurons and hence dopamine depletion in the ipsilateral striatum. Using 7T MRI imaging, we have quantified T2, T1, CBF and CBV in the striata of 6OHDA and control rats. To validate the lesion size, behavioral testing, dopamine transporter μSPECT and tyrosine hydroxylase staining were performed. Results: No significant differences were demonstrated in the absolute MRI values between 6OHDA animals and controls; however, 6OHDA animals showed significant striatal asymmetry for all MRI parameters in contrast to controls. Conclusions: These PD-related asymmetry ratios might be the result of counteracting changes in both intact and affected striatum and allowed us to diagnose PD lesions. As lateralization is known to occur also in PD patients and might be expected in transgenic PD models as well, we propose that MR-derived asymmetry ratios in the striatum might be a useful tool for in vivo phenotyping of animal models of PD. © 2010 ESMRMB.
PubMed | Laboratory for Neurobiology and Gene Therapy, Laboratory for Biomolecular Modelling and RIKEN
Type: | Journal: Frontiers in molecular neuroscience | Year: 2014
Leucine-rich repeat kinase 2 (LRRK2) is a complex, multidomain protein which is considered a valuable target for potential disease-modifying therapeutic strategies for Parkinsons disease (PD). In mammalian cells and brain, LRRK2 is phosphorylated and treatment of cells with inhibitors of LRRK2 kinase activity can induce LRRK2 dephosphorylation at a cluster of serines including Ser910/935/955/973. It has been suggested that phosphorylation levels at these sites reflect LRRK2 kinase activity, however kinase-dead variants of LRRK2 or kinase activating variants do not display altered Ser935 phosphorylation levels compared to wild type. Furthermore, Ser910/935/955/973 are not autophosphorylation sites, therefore, it is unclear if inhibitor induced dephosphorylation depends on the activity of compounds on LRRK2 or on yet to be identified upstream kinases. Here we used a panel of 160 ATP competitive and cell permeable kinase inhibitors directed against all branches of the kinome and tested their activity on LRRK2 in vitro using a peptide-substrate-based kinase assay. In neuronal SH-SY5Y cells overexpressing LRRK2 we used compound-induced dephosphorylation of Ser935 as readout. In silico docking of selected compounds was performed using a modeled LRRK2 kinase structure. Receiver operating characteristic plots demonstrated that the obtained docking scores to the LRRK2 ATP binding site correlated with in vitro and cellular compound activity. We also found that in vitro potency showed a high degree of correlation to cellular compound induced LRRK2 dephosphorylation activity across multiple compound classes. Therefore, acute LRRK2 dephosphorylation at Ser935 in inhibitor treated cells involves a strong component of inhibitor activity on LRRK2 itself, without excluding a role for upstream kinases. Understanding the regulation of LRRK2 phosphorylation by kinase inhibitors aids our understanding of LRRK2 signaling and may lead to development of new classes of LRRK2 kinase inhibitors.
Vande Velde G.,Catholic University of Leuven |
Rangarajan J.R.,Catholic University of Leuven |
Toelen J.,Catholic University of Leuven |
Dresselaers T.,Catholic University of Leuven |
And 10 more authors.
Gene Therapy | Year: 2011
The development of in vivo imaging protocols to reliably track transplanted cells or to report on gene expression is critical for treatment monitoring in (pre)clinical cell and gene therapy protocols. Therefore, we evaluated the potential of lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) to express the magnetic resonance imaging (MRI) reporter gene ferritin in the rodent brain. First, we compared the induction of background MRI contrast for both vector systems in immune-deficient and immune-competent mice. LV injection resulted in hypointense (that is, dark) changes of T 2 /T 2 (spin-spin relaxation time)-weighted MRI contrast at the injection site, which can be partially explained by an inflammatory response against the vector injection. In contrast to LVs, AAV injection resulted in reduced background contrast. Moreover, AAV-mediated ferritin overexpression resulted in significantly enhanced contrast to background on T 2-weighted MRI. Although sensitivity associated with the ferritin reporter remains modest, AAVs seem to be the most promising vector system for in vivo MRI reporter gene imaging. © 2011 Macmillan Publishers Limited All rights reserved.
Van der Perren A.,Laboratory for Neurobiology and Gene Therapy |
Casteels C.,Leuven University Hospital |
Van Laere K.,Leuven University Hospital |
Gijsbers R.,Laboratory for Molecular Virology and Gene Therapy |
And 4 more authors.
Journal of Visualized Experiments | Year: 2016
In order to study the molecular pathways of Parkinson’s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic α-SYN mouse models develop gradual α-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. The substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson's disease. © 2016 Journal of Visualized Experiments.
PubMed | Laboratory for Neurobiology and Gene Therapy
Type: | Journal: BMC neuroscience | Year: 2010
Accurate and reproducible behavioral tests in animal models are of major importance in the development and evaluation of new therapies for central nervous system disease. In this study we investigated for the first time gait parameters of rat models for Parkinsons disease (PD), Huntingtons disease (HD) and stroke using the Catwalk method, a novel automated gait analysis test. Static and dynamic gait parameters were measured in all animal models, and these data were compared to readouts of established behavioral tests, such as the cylinder test in the PD and stroke rats and the rotarod tests for the HD group.Hemiparkinsonian rats were generated by unilateral injection of the neurotoxin 6-hydroxydopamine in the striatum or in the medial forebrain bundle. For Huntingtons disease, a transgenic rat model expressing a truncated huntingtin fragment with multiple CAG repeats was used. Thirdly, a stroke model was generated by a photothrombotic induced infarct in the right sensorimotor cortex. We found that multiple gait parameters were significantly altered in all three disease models compared to their respective controls. Behavioural deficits could be efficiently measured using the cylinder test in the PD and stroke animals, and in the case of the PD model, the deficits in gait essentially confirmed results obtained by the cylinder test. However, in the HD model and the stroke model the Catwalk analysis proved more sensitive than the rotarod test and also added new and more detailed information on specific gait parameters.The automated quantitative gait analysis test may be a useful tool to study both motor impairment and recovery associated with various neurological motor disorders.
PubMed | Laboratory for Neurobiology and Gene Therapy and Catholic University of Leuven
Type: | Journal: Journal of visualized experiments : JoVE | Year: 2016
In order to study the molecular pathways of Parkinsons disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic -SYN mouse models develop gradual -SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinsons disease.
PubMed | Laboratory for Neurobiology and Gene Therapy and Vrije Universiteit Brussel
Type: | Journal: Frontiers in behavioral neuroscience | Year: 2015
Parkinsons disease is a neurodegenerative disorder characterized by motor and non-motor disturbances. Various pathogenic pathways drive disease progression including oxidative stress, mitochondrial dysfunction, -synuclein aggregation and impairment of protein degradation systems. Dysfunction of the ubiquitin-proteasome system in the substantia nigra of Parkinsons disease patients is believed to be one of the causes of protein aggregation and cell death associated with this disorder. Lactacystin, a potent inhibitor of the proteasome, was previously delivered to the nigrostriatal pathway of rodents to model nigrostriatal degeneration. Although lactacystin-treated animals develop parkinsonian motor impairment, it is currently unknown whether they also develop non-motor symptoms characteristic of this disorder. In order to further describe the proteasome inhibition model of Parkinsons disease, we characterized the unilateral lactacystin model, performed by stereotaxic injection of the toxin in the substantia nigra of mice. We studied the degree of neurodegeneration and the behavioral phenotype 1 and 3 weeks after lactacystin lesion both in terms of motor impairment, as well as non-motor symptoms. We report that unilateral administration of 3 g lactacystin to the substantia nigra of mice leads to partial (~40%) dopaminergic cell loss and concurrent striatal dopamine depletion, accompanied by increased expression of Ser129-phosphorylated -synuclein. Behavioral characterization of the model revealed parkinsonian motor impairment, as well as signs of non-motor disturbances resembling early stage Parkinsons disease including sensitive and somatosensory deficits, anxiety-like behavior, and perseverative behavior. The consistent finding of good face validity, together with relevant construct validity, warrant a further evaluation of proteasome inhibition models of Parkinsons disease in pre-clinical research and validation of therapeutic targets.