Defining the molecular changes in the striatum of PD brains is necessary to understand the disorder and design of new therapeutic approaches. Microarrays have been used in other papers to analyze the striatum in mouse models of PD.6-8,39-42
However, differing toxins, doses, and time points in these studies complicate the identification of a reliable profile specific for PD in mice. We designed an approach using two neurotoxins, MPTP and METH, which each induce Parkinsonism in mice but have distinct mechanisms of action. Changes in common to both neurotoxins may represent a more confident molecular signature of PD, while changes specific to each neurotoxin may reflect their individual toxicology. While it is not entirely known whether the 7 day time point is best for measuring the differences between the two drugs, a significant loss of DA in the target organ, the striatum, is observed in both the MPTP and METH models of PD.
To gain deeper insights into the cellular response of the two neurotoxins, we assayed transcript and protein levels for both agents using microarrays and global quantitative proteomics. These techniques identified a number of significantly up- and downregulated transcripts and proteins in response to MPTP and METH. Some of the significantly regulated proteins may represent a protein group containing several isoforms with the common peptide detected. The current approach cannot pinpoint exactly which isoform(s) contributed to the observed changes, and orthogonal approaches will be necessary to validate the differences. The regulated genes/proteins may be attractive candidates for biomarker development in PD.
High correlations were found between proteins only or between transcripts only in response to MPTP and METH, suggesting that the general cellular response to both neurotoxins is similar. Somewhat surprisingly though, a low correlation was observed between the proteomics and microarray data in each of the neurotoxin models. Only two genes (Gpx4 and Gfap) overlapped between the regulated gene lists of proteomics and microarrays. The low concordance between microarray and proteomic data could be due to multiple factors, including translational and post-translational regulations. It is also possible that the differences in the quantification accuracy and the ability to detect low abundance genes for the two technologies play a major role in the observed low concordance. Most genes detected by global proteomics are relatively abundant because of the dynamic range limitation of current proteomic technologies;43
however, our results demonstrate the ability to detect as low as ~30% abundance changes exemplified by the Pcp4 protein. Low percentage changes in protein abundances could mean even smaller mRNA changes, which may not be detectable.
Another possibility for the low correlation may be the different mechanisms of the two neurotoxins. For example, we observed nearly a 6-fold decrease in protein abundances for aldehyde dehydrogenase gene Aldh1a1, a gene specifically expressed in dopamine neurons,44
in MPTP-treated mice but only a 3-fold decrease in METH-treated mice. MPTP causes cell death in neurons that project to the striatum, while METH suspends cell function and DA production without killing the cells. This difference may account for the more severe decrease in protein observed in the MPTP-treated mice.
Our investigation also evaluated proteins to better understand the relationship between transcript and protein levels in the striatum following the loss of dopaminergic afferents. A total of 1614 proteins were confidently identified, and very good consistency was found between replicates and neurotoxins. Differentially regulated proteins in response to drug treatment were also similar.
Interestingly, the Gfap protein was found to be upregulated in response to both MPTP and METH treatment, congruent with the microarray results. There were a number of other proteins that showed a similar response to both toxins. One protein with increased levels was spermidine synthase (Srm), which enhances Af3
-induced neurotoxicity through increased free-radical levels.45
As mentioned before, the Aldh1a1 protein was downregulated in response to both neurotoxins and is of particular interest because decreasing Aldh1a1 in human lens epithelial cells using siRNA increases the susceptibility of the cells to oxidative damage and apoptosis.46
To explore the relationship between transcript and protein abundance, we examined the absolute intensity for transcript expression across all experiments and compared them to the protein abundance levels for the 1200 genes in common between the MPTP and METH experiments. Although there were individual cases in which as much as a 26-fold abundance difference could be found between protein and transcript levels, a significant correlation (p < 10−21) between proteins and transcripts was found, suggesting that transcript levels can be used as a general indicator of protein abundance.
Our results also identified a number of proteins functionally associated with apoptosis and cell death. In general, downregulation of antiapoptotic proteins and upregulation of pro-apoptotic proteins were observed, although apoptosis is a process in which cellular localization may play as much of a role as the expression level of these factors.47
Increased apoptosis and cell death factors are likely the consequence of mitochondrial dysfunction and oxidative damage. Indeed, many of the proteins involved in mitochondrial dysfunction and oxidative damage are also suggested to play a role in apoptosis. For example, proteins Gpx4 and Gstm5, which function primarily as antioxidants also have a protective effect against apoptosis.33,34
Both proteins were downregulated. In addition, antiapoptotic mitochondrial heat-shock protein 10 kDa (Hspe1) had significant reduced protein abundances in both mouse models, again suggesting reduced protection against cell death. Conversely, the pro-apoptotic protein cytochrome C1 (Cyc1) was upregulated in MPTP-treated mice, also as a result of mitochondrial dysfunction. Consistent with these observations, oxidative stress and mitochondrial dysfunction are implicated as major contributors to Parkinsonism in both MPTP-48
A number of proteins () previously implicated in pro-apoptotic activity were observed with increased protein or transcript abundances in both mouse models. For instance, the classic glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (Gapdh) has been identified as a general mediator of one or more apoptotic cascades and promotes Lewy body formation.51
Cyc1 and Gapdh were only observed with significant abundance increases in MPTP-treated mice, suggesting a potentially higher level of oxidative damage and cell death. The upregulation of the calpain-2 (Capn2) protein in both MPTP- and METH-treated mice is indicative of increased endoplasmic reticulum (ER) stress. Capn2 protein is known to be pro-apoptotic through activation of caspase-12.52
Increased Capn2 expression and neuronal death in MPTP-treated mice has been previously observed.53
Importantly, microarray and proteomic measurements seem to be complementary by providing two different sets of regulated genes. The combined regulated gene and protein list provides a much clearer picture of the biological changes occurring in the striatum following the two drug treatments. Overall, our data provide clear evidence on mitochondrial dysfunction, increased oxidative stress and damage, misregulated protein degradation, increased apoptosis and cell death, and the potential activation of the astrocytic response. While we have focused on aspects of neurotoxicity, other protein or gene expression changes may be related to the loss of dopamine signaling in the striatum. Many of the novel proteins and genes may represent interesting targets for therapeutic intervention or further mechanistic studies. The discovery of common changes helped illuminate pathways relevant to PD, but pathways specific for each toxin were also uncovered. The use of transcript and protein profiling to identify the molecular changes occurring in toxins with similar pathophysiological end effects may be a general approach for differentiating the molecular pathology of disease models from agent-specific effects.
Our data shows that understanding mRNA changes is not enough, and further investigation using multimodal analysis will provide a clearer picture of what is happening in the mouse brain. Because the current AMT method may fail to identify post-translational modifications, subsequent proteomic analyses will use newer technology to include potential modifications into its AMT tag database.54
In addition, a time-course study will illuminate the evolving changes to the neurotoxins in the mouse brain, while a chronic drug-delivery system might better replicate damage caused by drug abuse in humans.