In this study, we examined the effect of α-syn overexpression in mice on gene expression in the SNpc. Our data showed that α-syn overexpression caused alterations in a small number of genes early in the pathological process, before any cellular or behavioral changes were apparent in the transgenic mice. Alterations in gene expression were more pronounced at a later stage of disease when pathological changes were apparent, and these late changes affected different genes than those modified early. These late gene expression changes were focused around transcription-related processes.Our analysis also suggested that gender modified the alterations of gene expression in this model.
We found striking differences between early changes in gene expression and those observed after pathological changes had clearly developed in the α-syn model. Prior to the onset of pathological changes, gene expression changes in the α-syn transgenic mice were modest. By conservative statistical criteria, we found only 29 genes to be altered. In contrast, 179 genes were altered in older transgenic mice. The larger gene list from the nine-month-old mice analysis reflects in part a reduction in variability among the animals, which presumably is a result of a more stable state of α-syn-related pathology at this later stage. When limiting our data sets by false discovery rates, we found no genes altered at both early and late time periods. Only nine genes were found altered in transgenic mice at both time periods when we evaluated our less stringent gene lists not limited by false discovery rate. Two of these nine genes are implicated with cell cycle regulation – calmodulin 1 (Rasmussen and Means, 1989
) and cyclin D2 (Jena et al., 2002
). The other seven genes do not seem to share any particular biological processes, with their functions still largely unknown.
The most striking result of our study is that many of the genes altered later in the disease model were directly related to transcriptional regulation, nuclear function, and DNA binding. These data support the view that transcriptional dysregulation is an important consequence of α-syn overexpression and, therefore, may be critical to the pathogenesis of synucleinopathies. Alpha-syn has been identified within cell nuclei and has been shown to associate with histones in vitro
(Goers et al., 2003
; Maroteaux et al., 1988
). Studies by Kontopoulos et al. (2006)
have provided direct evidence that α-syn can inhibit histone acetylation in both mammalian cell culture models and transgenic Drosophila
. The effect of α-syn on histone acetylation appears critical to α-syn-induced toxicity, as treatment with histone deacetylase inhibitors rescues against α-syn-induced toxicity (Kontopoulos et al., 2006
). The present study confirms the transcriptional effect of α-syn in a mouse model for PD.
It is important to recognize that microarray studies do not directly assess transcription. The parameter measured is the abundance of specific mRNAs, which can be affected not only by transcription but also by alterations in RNA half-lives. While we cannot exclude an effect of α-syn on the stability of some transcripts, the large number of alterations as well as the observed changes in mRNAs for many components of the transcriptional process suggests that transcriptional dysregulation is the predominant effect underlying the changes.
Another significant finding is the confirmation in this mouse model that gender influences gene expression changes induced by α-syn overexpression. This analysis was prompted by the recent observation that gender has a marked effect on gene expression in human dopaminergic SN neurons, influencing the patterns of gene expression in both normal brain and the response to PD (Cantuti-Castelvetri et al., 2007
). Other microarray studies have also revealed gender differences in gene expression in normal human brain (Vawter et al., 2004
), but there is little additional data available on the interaction of gender and disease state on neuronal gene expression in either humans or animal models. The importance of gender is emphasized by the fact that the incidence of PD is nearly twice that in men as in women (Baldereschi et al., 2000
; Van Den Eeden et al., 2003
). Since our study was originally designed with gender-matched animal groups, we were able to examine the effect of gender directly, although the number of animals in the subgroup analysis is necessarily smaller and lacks statistical power compared to the primary analysis. Nevertheless, we found substantial gender-based differences in the response to α-syn overexpression. We observed that α-syn caused more gene changes among female mice, and most altered genes were different between genders. At present it is not known whether mice exhibit gender-based differences in vulnerability to α-syn toxicity, yet in other animal PD models, males have exhibited increased susceptibility to MPTP (Dluzen and McDermott, 2000
; Freyaldenhoven et al., 1996
; Miller et al., 1998
) and 6-hydroxydopamine (Murray et al., 2003
; Tamas et al., 2005
). Our findings suggest that issues of gender should be carefully considered in the design of experimental studies using animal models of PD.
The mouse model used here was selected because it demonstrates progressive dopaminergic dysfunction with a motor phenotype (Masliah et al., 2000
), but the nature of the model imposes some limitations on our study. Because the mouse SNpc is much more densely packed with cells than in the human SNpc, we dissected the entire SNpc region for this study, and not individual neurons. Thus, the transcriptional changes may reflect alterations in glia as well as neurons. It is also important to note that this transgenic α-syn mouse does not fully recapitulate all pathological aspects of PD. This mouse does show evidence of dopaminergic dysfunction and mild motor impairment, but it does not show loss of dopaminergic nigral neurons (Masliah et al., 2000
). Another pathological difference is that α-syn inclusions are much more widespread in Masliah mouse brains than are Lewy bodies found in PD brains. Therefore, many of the expression changes that we saw may be more broadly applicable to other synucleinopathies, such as dementia with Lewy Bodies. All other published α-syn transgenic mice also fail to show dopaminergic neuron loss, yet these models vary in the appearance of α-syn-positive inclusions and severity of motor disability (Hashimoto et al., 2003
; Maries et al., 2003
). Transgenic mice expressing the A53T α-syn mutant show more severe motor impairments (Giasson et al., 2002
; Lee et al., 2002
), and some of the other transgenic mice have α-syn inclusions more typical of α-syn aggregates found in PD (Kahle et al., 2000
; Lee et al., 2002
While there are accepted principles for microarray analyses, different approaches to normalization and selection may lead to different outcomes regarding which genes are defined as differentially expressed (Hoffmann et al., 2002
). Significance Analysis of Microarrays (SAM) is currently the method most commonly used that corrects for the large number of comparisons inherent in microarray experiments by estimating false discovery rate (Efron and Tibshirani, 2002
; Larsson et al., 2005
; Tusher et al., 2001
). We limited false positives by excluding those genes whose q values were > 20%. However, by using this conservative statistical method, we likely eliminated some genes that are truly altered between wildtype and transgenic animals. Indeed, by QPCR we did confirm alterations in two genes identified by less stringent criteria, Tiparp and cyclin D2, yet both of these genes had high q values in the three-month animals and were thus eliminated from the more stringent three-month list. Attempts to validate directly individual genes introduce additional issues, especially with regard to the selection of genes for normalization. Although genes such as beta-actin and GAPDH have often been used for this purpose, expression of such “housekeeping” genes can vary, especially in neurodegenerative disease (Gutala and Reddy, 2004
; Radonic et al., 2004
; Vandesompele et al., 2002
). Indeed, our array data does show that GAPDH levels are altered in the nine-month-old α-syn transgenic mice. We chose pyruvate decarboxylase and hypoxanthine phosphoribosyltransferase, both of which were unchanged in our microarray lists, to verify that similar results were obtained regardless of the gene chosen for normalization.
Given these considerations, we have decided to publish two sets of gene lists in this paper: 1) the three-month and nine-month tables ( & ) restricted by q values in addition to fold-change and p value criteria; and 2) the supplementary tables (Supp. Tables 1
) restricted only by fold-change and p value criteria. In addition, the full gene array dataset from this study is publicly available on the Array Express site and permits further analysis and comparison with other studies.
In conclusion, we have explored the molecular changes induced by α-syn at a transcriptional level by comparing α-syn transgenic mice to wildtype mice by microarray analysis. Significant transcriptional changes occur in the SNpc of older transgenic mice, and these changes are influenced by gender. A large proportion of the altered genes are involved in transcriptional regulation. The next step is to evaluate whether some of the differentially expressed genes are protective or, alternatively, can magnify α-syn toxicity in cellular and animal models. This study reinforces the concept that targeting transcriptional dysregulation as a mechanism may be a useful therapeutic approach for PD.