Microarray Studies in the Laser Capture Microdissected SNc after a Single Administration of MPTP in Common Marmosets
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ABSTRACT:
ORGANISM(S): Callithrix jacchus Homo sapiens
PROVIDER: GSE5176 | GEO | 2006/06/29
SECONDARY ACCESSION(S): PRJNA96591
REPOSITORIES: GEO
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Project description:We have previously detected cyclin D1-initiated and p53-related (PERP; p53 apoptosis-associated target) cell death in a striatal 6-hydroxydopamine (6-OHDA)-treated Parkinsonian mouse model by adaptor-tagged competitive PCR (ATAC-PCR) (Synapse 51, 279-286, 2004). In order to establish the mechanism of dopaminergic cell death in Parkinsonâs disease, various Parkinsonian animal models should be studied. Therefore, the time-course alteration of gene expression in the substantia nigra pars compacta was investigated after a single administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in common marmosets. In the MPTP model, gene expression of neither cyclin D1 nor PERP was altered. There were also few common alterations in gene expression between the MPTP and 6-OHDA models. This suggests that the mechanism of cell death may differ between these models, and extensive study in various Parkinsonian models should be performed to elucidate the mechanisms of dopaminergic cell death in Parkinsonâs disease. Experiment Overall Design: Materials and methods Experiment Overall Design: 1. Animals Experiment Overall Design: Five 3-year-old male common marmosets (Callithrix jacchus) weighing 350â400 g were used. A single marmoset was used as a vehicle-treated control, with the rest being administered MPTP. A microarray experiment and an immunohistochemical study were performed in the same animals. They were colonized in the Center for Life Science Resources in Kagoshima University, Japan. Animals were housed with free access to standard food in an air-conditioned room under a constant 12 h light/dark cycle (lights on at 7:00 a.m.) at a temperature of 25â27°C and 45â55% humidity. All efforts were made to minimize animal suffering, and to reduce the number of animals used. The present experiments were carried out after obtaining permission from the Committee of Animal Experimentation, Kagoshima University Graduate School of Medical and Dental Sciences, Japan. Experiment Overall Design: 2. MPTP administration Experiment Overall Design: A single dose of MPTP was administered intravenously (i.v.) to the animals in order to examine the time-course alteration in gene expression. As in our previous study, the dose of MPTP was 2.5 mg/kg (Nomoto et al., 1997). At this dose, animals exhibit immobility during an entire day, while doses greater than 2.5 mg/kg can induce death. MPTP hydrochloride (Sigma, St. Louis, MO, USA) was dissolved in 0.9% saline, and administered under intraperitoneal pentobarbital anesthesia (40 mg/kg). Under deep anesthesia with pentobarbital, the animals were decapitated either at 2 h, 6 h, 1 day or 14 days after MPTP administration. All experiments using MPTP were performed in accordance with safety guidelines (Przedborski et al., 2001). Experiment Overall Design: 3. Laser capture microdissection Experiment Overall Design: The midbrain of each marmoset was dissected on an ice-cold glass plate, and mounted on a specimen block with O.C.T. compound (Tissue-Tek, Sakura Finetechnical, Tokyo, Japan). The midbrains were frozen by covering them with very finely powdered dry ice. Sections (8 μm thickness) were cut on a cryostat (Microm HM500S, Carl Zeiss Japan, Tokyo, Japan), and mounted on ice-cold, membrane-coated glass slides (Carl Zeiss Japan). The membranes on the glass slides were coated with poly-L-lysine (P8920; Sigma). Immediately after mounting, slides were cooled on dry ice and stored at â80°C for less than 3 days. Slides were fixed in ethanol/acetate solution (19:1) at 4°C for 3 min, and washed with PBS at 4°C for 1 min. Slides were stained with 0.1% toluidine blue at room temperature for 10 s, washed with PBS at 4°C for 10 s, and then air-dried for 5 min. Immediately after staining, slides were processed for laser capture microdissection (LCM). Experiment Overall Design: A Leica AS LMD (Leica Microsystems, Wetzlar, Germany) was used for LCM. SNc sections corresponded to level A4.5 of the brain atlas (Stephan et al., 1980). These sections contained transverse sections of the oculomotor nerve. A strip of the unilateral SNc was dissected (Fig. 1). The SNcs from two slides were collected and placed in a PCR tube containing 10 μl extraction buffer of the PicoPure RNA Isolation Kit (Arcturus Bioscience, Mountain View, CA, USA). Experiment Overall Design: 4. Microarray experiments Experiment Overall Design: Total RNA was extracted by the PicoPure RNA Isolation Kit, and eluted in approximately 10 μl buffer, according to the manufacturerâs protocol. In order to obtain a sufficient amount of amplified RNA (aRNA) for the microarray experiments, total RNA was amplified using two rounds of in vitro transcription. The first round of amplification was performed using the RiboAmp HS RNA Amplification Kit (Arcturus). The second round of amplification used the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Palo Alto, CA, USA) and cyanine-3 or cyanine-5 labeling. The labeled aRNA was hybridized with Agilent whole human genome oligo microarray G4112A. In order to correct the buffer composition in the second round of amplification using the Low RNA Input Fluorescent Linear Amplification Kit, cDNA (11 μl) that was purified in the round 2 procedure by the RiboAmp HS RNA Amplification Kit, was added to the modified transcription buffer of the Agilent kit. Each sample contained 4 μl of 5 ï´ first strand buffer, 20 μl of 4 ï´ transcription buffer, 6 μl 0.1 M DTT, 8 μl NTP mix, 6.4 μl 50% PEG, 0.5 μl RNase inhibitor (RNase Out; Invitrogen, Carlsbad, CA, USA), 0.6 µl inorganic pyrophosphatase, 0.8 μl T7 RNA polymerase, 2.4 μl cyanine (PerkinElmer Life Sciences, Boston, MA, USA; cyanine-3 for controls and cyanine-5 for MPTP-treated marmosets), and 20.3 μl DW. Labeled aRNA was synthesized according to the instructions for the Agilent kit. Experiment Overall Design: An Agilent 2100 bioanalyzer and an RNA 6000 Pico LabChip Kit were used to examine the length distribution of labeled aRNAs. A preliminary experiment using the protocol found in the appendices of the HistoGene LCM Immunofluorescence Staining Kit (Arcturus) was performed to determine the degree of degradation of total RNA that occurred during tissue staining.
2008-06-13 | E-GEOD-5176 | biostudies-arrayexpress
Project description:Acupuncture stimulations at GB34 and LR3 inhibit the reduction of tyrosine hydroxylase in the nigrostriatal dopaminergic neurons in the parkinsonism animal models. Especially, behavioral tests showed that acupuncture stimulations improved the motor dysfunction in a previous study. The thalamus is a crucial area for the motor circuit and has been identified as one of the most markedly damaged areas in Parkinson’s disease (PD), so acupuncture stimulations might also have an effect on the thalamic damage. We investigated gene expression profile changes in the thalamic region of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism models after acupuncture at the acupoints GB34 and LR3 C57BL/6 mice were divided into four experimental groups; ① C: Control, ② M: MPTP-treatment only, ③ MA: MPTP- and acupuncture-treatment at acupoints GB34 and LR3, ④ MNA: MPTP- and acupuncture-treatment at non-acupoints. Total RNA was isolated from two brains' thalamic regions of each experimental group (4 experimental group × 2 samples of each experimental group = total 8 samples).
2012-01-18 | E-GEOD-35138 | biostudies-arrayexpress
Project description:Parkinson’s disease (PD) is one of the most common neurodegenerative disease caused by diminution of neurons in the substantia nigra (SN) which projects dopaminergic (DA) axons to the striatum and other target areas. Recently, accumulating data demonstrated the prospects of cell replacement therapy by using neural stem cell (NSCs) transplantation. However the mechanisms underlying the potential efficacy are not fully understood. To gain new insights into the mechanisms of 6-OHDA induced lesion and potential efficacy of hNSCs transplantation, Intrastriatal 6-Hydroxydopamine (6-OHDA) injected parkinsonian mice were unilaterally engrafted with undifferentiated human NSCs to striatum (ST). High-throughput quantitative proteomic approach was utilized to characterize the proteome profiles of PD related brain regions in these mice including SN, ST, olfactory bulb (OB) and subventricular zone (SVZ). The abundance of more than 5000 proteins with high confidence in each region was determined in this study which represents the most extensive proteomic study of PD mouse models to date. In addition to the disruption of the DA system, the quantitative analysis demonstrated the profound disturbance of SVZ proteome after 6-OHDA insult, in which the abundance of more than 20% proteins was significantly changed. After hNSCs engraftment, the proteome of SVZ was restored and the astrocytes in ST was greatly activated in companion with the increase in neurotrophic factors. Furthermore, bioinformatics analysis demonstrated that changes in proteome was not caused by the proliferation of hNSCs or their progeny, but rather by the reaction of endogenous cells. Overall, this study reveals that hNSCs benefits parkinsonian animals by eliciting endogenous responses in multiple brain regions, and discovers the unexpected role of SVZ cells in PD progress and treatment, providing new therapeutic targets.
2017-03-29 | PXD004152 | Pride
Project description:Parkinson's disease (PD) listed as the second most common neurodegenerative disease. 2-[[(1,1-Dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a novel nitrone derivative of tetramethylpyrazine, has shown benefits for the treatment both in vitro and in vivo, TBN protects and rescues dopaminergic neurons from MPP(+) and MPTP/6-OHDA-induced damage by reducing ROS and increasing cellular antioxidative defense capability. Here, we investigate the proteome changes in the 6-OHDA induced PD rat model and the rescue effects after treated by TBN.
2022-03-02 | PXD004812 | Pride
Project description:Parkinson’s disease (PD) is a representative neurodegenerative disease that is characterized the death of dopamine-generating cells in the substantia nigra (SN). Acupuncture stimulations at GB34 and LR3 resulted in the enhanced survival of dopaminergic neurons in the SN in the parkinsonism animal models. SN is known as an important player in brain function, in particular, movement. Behavioral tests showed that acupuncture stimulations improved the motor dysfunction in a previous study by almost 87.7%. This study investigated the changes of gene expression profiles in the SN regions of the relatively chronic parkinsonism model and demonstrated the genes that may be related to the inhibitory effect of acupuncture on the change in gene expression in the SN induced by an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication using a whole transcript array. We investigated gene expression profile changes in the substantia nigral region of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism models after acupuncture at the acupoints GB34 and LR3
2014-12-27 | GSE48293 | GEO
Project description:Cx30 deficiency induced acceleration of dopaminergic neuron death upon MPTP toxicity. Gene expressions in the striatum of wild type and Cx30 KO mice after MPTP treatment were different in the aspect of neuroprotective A2 response.
2018-06-01 | GSE113693 | GEO
Project description:The degenerative process in Parkinson’s disease (PD) causes a progressive loss of dopaminergic neurons (DaNs) in the nigrostriatal system. Resolving the differences in neuronal susceptibility warrants an amenable PD model that, in comparison to post-mortem human specimens, controls for environmental and genetic differences in PD pathogenesis. At present study, we generated a primate model of PD by carotid artery injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPTP and sampled substantia nigra and putamen of macaque for single cell sequencing analysis.
2023-11-19 | E-MTAB-13437 | biostudies-arrayexpress
Project description:Parkinson's disease pathogenesis proceeds through several phases, culminating in the loss of dopaminergic neurons of the substantia nigra (SN). Although the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of oxidative SN injury is frequently used to study degeneration of dopaminergic neurons in mice and non-human primates, an understanding of the temporal sequence of molecular events from inhibition of mitochondrial complex 1 to neuronal cell death is limited. Here, microarray analysis and integrative data mining were used to uncover pathways implicated in the progression of changes in dopaminergic neurons after MPTP administration. This approach enabled the identification of small, yet consistently significant, changes in gene expression within the SN of MPTP-treated animals. Such an analysis disclosed dysregulation of genes in three main areas related to neuronal function: cytoskeletal stability and maintenance, synaptic integrity, and cell cycle and apoptosis. The discovery and validation of these alterations provide molecular evidence for an evolving cascade of injury, dysfunction, and cell death. Keywords: Parkinson's disease; MPTP; gene expression; microarray; cytoskeleton; mouse
2006-05-12 | GSE4788 | GEO
Project description:Neuroinflammatory processes are a prominent contributor to the pathology of Parkinson’s disease (PD), characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and deposits of α-synuclein aggregates. MLKL-mediated cell necroptosis might occur in the onset of PD and lead to neuronal dopaminergic degeneration. However, the link between α-synuclein, neuroinflammatory processes, and neurodegeneration in PD remains unclear. Here, our in vitro study indicated that inhibition of MLKL exerted a protective effect against 6-OHDA- and TNF-α-induced neuronal cell death. Furthermore, we created a mouse model (Tg-Mlkl-/-) with typical progressive Parkinson traits by crossbreeding SNCA A53T transgenic mice with MLKL knockout mice. Tg-Mlkl-/ mice displayed dramatically improved motor symptoms and reduced hyperphosphorylated α-synuclein expression. More data suggested that MLKL deficiency protected dopaminergic neurons, blocked neuronal cell death, and attenuated neuroinflammation by inhibiting the activation of the microglia and astrocytes. Single-cell RNA-seq analysis revealed reduced microglial cells and damped neuron death in the SN of the Tg-Mlkl-/- mice. Subcluster analysis identified a unique cell type-specific transcriptome profiling in the MLKL deficiency mice. Thus, MLKL represents a critical therapeutic target for reducing neuroinflammation and preventing dopaminergic neuron degeneration.
2023-12-14 | GSE197679 | GEO
Project description:Engrailed homeoproteins are expressed in adult dopaminergic neurons of the substantia nigra. In Engrailed1 heterozygous mice, these neurons start dying at 6 weeks, are more sensitive to oxidative stress and progressively develop traits similar to those observed following an acute and strong oxidative stress inflected to wild-type neurons. These changes include DNA strand breaks and the modification (intensity and distribution) of several nuclear and nucleolar heterochromatin marks. Engrailed1 and Engrailed2 are biochemically equivalent transducing proteins previously used to antagonize dopaminergic neuron death in Engrailed heterozygous mice and in mouse models of Parkinson disease. Accordingly, we show that, following an acute oxidative stress, a single Engrailed2 injection restores all nuclear and nucleolar heterochromatin marks, decreases the number of DNA strand breaks and protects dopaminergic neurons against apoptosis. RNA-seq data for differentially expressed genes in the SNpc of En1+/- mice, En2 infused mice and 6-OHDA/En2 injection experiments.
2015-09-24 | E-GEOD-72321 | biostudies-arrayexpress