Project description:Alpha synuclein (SNCA) has been linked to neurodegenerative diseases (synucleinopathies) that include Parkinson’s disease (PD). Although the primary neurodegeneration in PD involves nigrostriatal dopaminergic neurons, more extensive yet regionally selective neurodegeneration is observed in other synucleinopathies. Furthermore, SNCA is ubiquitously expressed in neurons and numerous neuronal systems are dysfunctional in PD. Therefore it is of interest to understand how overexpression of SNCA affects neuronal function in regions not directly targeted for neurodegeneration in PD. To gain a better understanding of the consequences of excessive SNCA expression on basal ganglia function, we performed transcriptome analysis of striatal tissue from male Thy1-aSyn-mice and wt littermates. The present study investigated the consequences of SNCA overexpression on cellular processes and functions in the striatum of mice overexpressing wild-type, human SNCA under the Thy1 promoter (Thy1-aSyn mice) by transcriptome analysis. The analysis revealed alterations in multiple biological processes in the striatum of Thy1-aSyn mice, including synaptic plasticity, signaling, transcription, apoptosis, and neurogenesis. The present study investigated the consequences of SNCA overexpression on cellular processes and functions in the striatum of mice overexpressing wild-type, human SNCA under the Thy1 promoter (Thy1-aSyn mice) by transcriptome analysis. The analysis revealed alterations in multiple biological processes in the striatum of Thy1-aSyn mice, including synaptic plasticity, signaling, transcription, apoptosis, and neurogenesis. Animal care was conducted in accordance with the U. S. Public Health Service Guide for the Care and Use of Laboratory Animals and procedures were approved by the University of California, Los Angeles (UCLA), I.A.C.U. Committee. Transgenic (tg) mice overexpressing human wt SNCA under the Thy-1 promoter (Thy1-aSyn) created previously in a mixed C57BL/6-DBA background were kept in this background by breeding mutant females with wt males. Only male mice were used in the study. The genotype of all tg and wt mice was verified by PCR analysis of tail DNA. Animals were maintained on a 12 hr light/dark cycle with free access to water and food. Six-month old male Thy1-aSyn and wt littermates were sacrificed by decapitation. For microarray analysis, whole striata from each hemisphere were immediately dissected and pooled for each brain. Tissue was permeated in RNAlater (Qiagen, Valencia, CA), frozen in liquid nitrogen, and stored at - 80º C until used for RNA preparation. For PCR verification of transcriptional changes and for protein extracts preparation, brains from 5 male Thy1-aSyn and 5 wt littermates were obtained as above but then the brains were placed in a metal brain mold with grooves to ensure reproducible cutting of thick brain slices. A first coronal cut was made with a razor blade to remove the frontal part of the brain (at bregma + 1mm, at the anterior level of striatum). The following 1mm coronal slice (including the striatum but no globus pallidus) was used to dissect out striatal tissue. A horizontal cut was made through the anterior commissures to exclude the nucleus accumbens. One cube of striatum was dissected out from each hemisphere, taking care not to include any corpus callosum, choroid plexus, or subventricular zone. Samples were stored at -80º C until further processing. Total RNA was extracted from striata of Thy1-aSyn and wt littermates (all males) with Trizol (Invitrogen, Carlsbad, CA), followed by a clean-up step with RNeasy columns (Qiagen) and RNA integrity check using a Bioanalyzer (Agilent Tech., Foster city, CA). RNA samples were pooled, one pool representing the six control wt mice and the other representing the six SNCA overexpressing animals. After synthesis, using Superscript (Invitrogen) and labeling using the ENZO labeling kit (Affymetrix Inc., Santa Clara, CA), cRNA probes were hybridized to mouse MOE-430A GeneChip arrays (Affymetrix) following the manufacturer’s protocol at the UCLA microarray core facility. Significant differential gene expression between pooled tg and wt samples was ascertained by estimation of signal log2 ratios (or fold changes in expression values), after quality control checks, data normalization and estimating expression values using the Affymetrix Microarray Suite 5.0 Software (MAS 5.0). After pairwise comparisons and filtering of this gene list using the following criteria: change p-value <0.005 for induce genes, change p-value > 0.995 for decreased genes, signal log2 ratio > 0.6 (> 1.52 fold change), excluding probes called absent in both groups, a list of differentially expressed genes was generated. We used various statistical softwares and databases to ascertain pathways affected by overexpression of SNCA that are associated with overrepresented genes in this gene list, including, the functional annotation tools accessible through DAVID (Database for Annotation, Visualization and Integrated Discovery, http://david.abcc.ncifcrf.gov )

Project description:Parkinsonâs disease (PD) and Dementia with Lewy bodies (DLB) are the two most common examples of synucleinopathies, i.e. human disorders that display intracellular deposition of the Alpha-synuclein (aSYN) protein. The two forms of aSYN deposits, intracellular Lewy bodies and Lewy neurites, are in the PD brain primarily found in the substantia nigra whereas in DLB brains they are diffusely spread in cortical and limbic areas. We are currently analyzing the protective effects on aSYN pathology by the molecular chaperone hsp70 in mice overexpressing wildtype human aSYN. Moreover, transgenic mice overexpressing hsp70 have been cross-bred with the aSYN mouse line. Intriguingly, the aSYN / hsp70 double transgenics display a pronounced reduction in biochemically assessed aSYN aggregation. Gene expression of these mice will not only provide insight into the disease mechanisms of aSYN pathology but its prevention by the overexpression of hsp70, leading to new potential avenues of treatment. Project 1: To investigate and compare alterations in gene expression profiles of brains from wild-type mice and mice overexpressing wildtype human aSYN, human hsp70 and aSYN / hsp70, respectively. The comparison of the expression profiles of the groups of mice: aSYN, Hsp70, aSYN/ Hsp70 and non-transgenic littermates will help to identify the genes that are involved in the mediation of aggregation toxicity of aSYN expression as well as the genes that may mediate an attenuation of aSYN pathology in hsp70-coexpressors. Project 1: Selection of animals Mice of 4 month old (n=6) of age of each background (aSYN, Hsp70, aSYN/ Hsp70 and non-transgenic littermates) will be used for RNA-extraction for a total of 24 mice. Preparation of samples For the proposed experiments, we will use the recommended procedures, as outlined by the consortium, for extracting and purifying RNA from whole brain homogenate. Briefly, RNA will be extracted by the trizol method and purified by using the RNeasy MinElute Cleanup (QIAGEN Inc., USA). RNA labeling and hybridization procedures will be carried out by the microarray consortium. SPECIAL NOTE: The samples will be sent upon availability. Gene Expression Microarray Analysis Six mice will be included in each group per timepoint to generate statistically testable data and limit the possible confound of individual mouse gene expression variations. The genome expression will be evaluated through the use of the GeneChipÂ® Mouse Genome 430 2.0 Array (Affymetrix) for each individual mouse constituting a total of 24 arrays for the proposed study. Gene expression data interpretation will be assisted by the Microarray Consortium Center bioinformatics core.

Dataset Information

RNA-Sequencing of carnosine treated wild-type BDF1 (WT) and transgenic Thy1-aSyn mice

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