The authors have declared that no competing interests exist.
Conceived and designed the experiments: NMK TJC KSC CES FPM JWL. Performed the experiments: NMK TJC ACS TG ZRM KSC CES FPM JWL. Analyzed the data: NMK MEW ACS JWL. Contributed reagents/materials/analysis tools: NMK MEW JWL. Wrote the paper: NMK MEW JWL.
We hypothesized that the study of gene expression at 1, 2, 4, 6 and 16 weeks in the substantia nigra (SN) after intrastriatal 6-OHDA in the Sprague-Dawley rat (
The 6-hydroxydopamine (6-OHDA) model of Parkinson’s disease (PD) has been extensively characterized and employed to study the consequences of dopamine neuron loss and to screen therapeutics [
Using this model, we hypothesized that the study of progressive gene expression changes over time in the SN after 6-OHDA insult would identify protective (albeit insufficient) cellular responses that are engaged during the neurodegenerative process. Subsequent studies could then assess whether artificial induction or silencing of the observed gene expression changes could be exploited to protect against DA neuronal or axonal damage. In this way, we are not attempting to identify etiologic factors of PD, but rather endeavor to exploit the natural response of DA neurons to degenerative cues in order to identify new manipulatable targets to repair, stabilize, or interfere with the neurodegenerative process of SN DA neurons.
The current study examined the progressive gene expression changes over 16 weeks in the SN after an intrastriatal 6-OHDA lesion. We hypothesized that patterns of gene expression within the SN characterized by early, highly upregulated expression would be indicative of potential protective responses to a striatal 6-OHDA insult. We additionally hypothesized that the use of k-means cluster analysis to group transcripts based upon expression patterns would aid in the identification of previously unknown neuroprotective candidates. Our results demonstrated that intrastriatal 6-OHDA treatment upregulated several interrelated transcripts classified as members of the regeneration-associated gene (RAGs) family that segregated into one cluster. Upregulation of RAGs is historically associated with the response to axonal injury in the peripheral nerves [
Male, Sprague-Dawley rats (
Rats were anesthetized with equithesin (0.3ml/100 g of weight, i.p; chloral hydrate 42.5 mg/ml + sodium pentobarbital 9.72 mg/ml) and placed into a stereotaxic frame. Each rat received two injections of 6-OHDA HBr [Sigma-Aldrich, St. Louis, MO; 5 μg/μl, calculated as free base in 0.2% ascorbic acid and a 0.9% saline solution] at 2 μl per site. The two injection coordinates were (i) AP +1.6 mm, ML +2.4 mm, DV −4.2mm, (ii) MP +0.2 mm, ML +2.6 mm, DV −7.0mm. For all rats, the needle was zeroed at the skull directly above the injection site in order to target the DV coordinate. For each injection, the needle was lowered slowly to the injection site and 1 minute elapsed before injection commenced, 6-OHDA was injected at 0.5 μl/min and at the end of the injection the needle remained in place for an additional 4 minutes before retraction.
At the appropriate post-surgical time point, rats were anesthetized with pentobarbital (50mg/kg intraperitoneally), and decapitated. Brains were removed rapidly and submerged for 30 s in a 250ml beaker of isopentane chilled in powdered dry ice. The brains were then wrapped in foil and stored at -80°C until dissected. Frozen brains were slabbed on an inverted petri dish over a bed of crushed ice. Slabs containing the striatum were dissected using scalpels. A small portion (~ 2mm3) of the striatum from each side of the brain was separately reserved for confirmation of lesion status using HPLC (see below). Tissue from the substantia nigra (SN) was placed in 1 ml of trizol, (Life Technologies, Carlsbad, CA), homogenized by hand with a disposable plastic pestle, frozen on dry ice and stored at -80°C in preparation for RNA isolation
Striatal DA levels (i.e. 6-OHDA lesion status) were quantified by HPLC as described previously[
RNA extraction was performed using the RNA Clean and Concentrator kit (Zymo Research, Irvine, CA) and eluted into 15μl H2O. RNA quality was assessed using the RNA Nano 6000 Assay on an Agilent Bioanalyzer, (Santa Clara, CA). RNA quality was measured using the 10-point scale associated with the RNA Integrity Number (RIN). Only samples with RIN values ≥ 7 qualified for inclusion in microarray analyses. The mean and standard deviation of RIN values for all of the samples was 8.7 ± 0.71 (n = 33).
Isolated RNA from tissue samples (n = 33) were processed for microarray hybridization on the Rat Gene 1.0 ST Array at the Gene Expression Microarray Core of Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. 50-120ng of total RNA was converted to biotin–labeled sense-strand cDNA for hybridization using the Ambion WT Expression Kit (Life Technologies, Carlsbad, CA) combined with the GeneChip WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA). Chips were incubated at 45°C for 17 hours in the GeneChip Hybridization Oven 640, washed and stained in the Fluidics Station 450 (Affymetrix, Santa Clara, CA), and scanned using an Affymetrix Gene Chip Scanner 3000 7G (Affymetrix, Santa Clara, CA).
Only array images meeting all of the quality control measures defined by the Affymetrix Expression Control Program were included in this study. Specific quality control metrics included signal histogram, relative log expression signal, Pearson’s correlation, PM mean (average signal intensity of probes), and positive and negative area under the curve (AUC). Also measured were the expression values of spiked-in poly-A RNA controls, and values of spiked-in hybridization controls. Raw data are available on the NCBI Gene Expression Omnibus Repository (
Data processing and analysis were performed using R v3.0.2 (
To validate expression differences observed from microarray analysis, qPCR was performed to quantify levels of
The 94 probe sets representing 88 genes from Cluster-1 and their corresponding fold changes from WK1 (see
Animals were sacrificed 1 week after 6-OHDA (the peak of transcript expression in Cluster -1 and prior to significant loss of SN DA neurons). We utilized a combination of immunohistochemical (IHC) staining for TH and ISH for determining whether the highest fold-change Cluster-1 transcript,
Lesion status in the STR was quantified by the proportion of DA (ng/mg protein) from the ipsilateral (operated) to the contralateral (non-operated) side and converted to a percentage of the contralateral side. The 6-OHDA subjects that met exclusion criteria had mean DA levels of 0.8% ± 2.1% in the operated striatum (0.9 ng/mg protein) as compared to the intact side (119.1 ng/mg protein). Sham subjects had mean DA levels of 101.8% ±3.2% in the operated striatum (126.3 ng/mg protein) as compared to the non-operated striatum (124.1 ng/mg protein).
Using k-means cluster analysis we identified eight clusters of genes in the SN that covaried over time as a result of intrastriatal 6-OHDA lesion (
Eight clusters were identified through consensus clustering analysis. We hypothesized that a cluster of genes would be highly upregulated due to 6-OHDA administration that would fall-off over time due to ongoing DA neuron degeneration. Cluster-1 followed this hypothesized pattern.
The expression pattern of the non-RAG genes follows a “RAG-like” expression pattern suggesting that they may too be part of the RAG family.
25389 | 6.93 | 2.28x10-16 | ||
499660 | 19.16 | 2.26x10-13 | ||
60417 | 12.97 | 1.11x10-11 | ||
25112 | 3.31 | 5.79x10-10 | ||
Apoh | 287774 | 4.35 | 3.75x10-08 | |
84046 | 2.19 | 4.26x10-08 | ||
Foxp3 | 317382 | 2.68 | 2.31x10-07 | |
Arid5a | 316327 | 1.87 | 2.73x10-07 | |
Ucn | 29151 | 2.90 | 4.73x10-07 | |
Tnfrsf12a | 302965 | 2.10 | 9.80x10-07 | |
Tph1 | 24848 | 2.32 | 1.30x10-06 | |
113955 | 2.39 | 1.30x10-06 | ||
Xirp2 | 311098 | 1.77 | 1.31x10-06 | |
Mki67 | 291234 | 2.06 | 1.33x10-06 | |
Bcl2l11 | 64547 | 1.84 | 1.39x10-06 | |
Ccng1 | 25405 | 1.63 | 3.77x10-06 | |
Itgal | 308995 | 1.67 | 2.93x10-05 | |
Ipcef1/Oprm1 | 361474/25601 | 1.85 | 2.93x10-05 | |
C3 | 24232 | 2.07 | 2.93x10-05 | |
304608 | 1.92 | 3.45x10-05 |
Seven of the top twenty differentially expressed genes from the substantia nigra ipsilateral to intrastriatal injections (6-OHDA or vehicle) from Cluster-1 at 1 week post-surgery are part of the regeneration-associated (RAG) gene family (identified here in
3.074x10-4 | 1.687x10-2 | 3/28 | |
3.784x10-4 | 1.687x10-2 | 3/30 | |
4.593x10-4 | 1.687x10-2 | 3/32 | |
5.035x10-4 | 1.687x10-2 | 3/33 | |
6.524x10-4 | 1.748x10-2 | 3/36 | |
2.023x10-3 | 4.176x10-2 | 3/53 | |
2.250x10-3 | 4.176x10-2 | 3/55 | |
2.493x10-3 | 4.176x10-2 | 3/57 |
Significantly enriched pathways in Cluster-1 include the endoplasmic reticulum stress pathway which is highly associated with RAG transcripts. Pathways with a FDR adjusted p-value less than 0.05 were considered significant. The ratio is indicative of the number of pathway objects (denominator) included in Cluster-1 (numerator).
While the remaining seven clusters did not fit with our
The changes in expression levels for
The top 5 RAG transcripts from Cluster-1 were subjected to validation by qPCR. There was a high degree of agreement between the microarray and qPCR differential expression with the exception of Sprr1a. Differential expression of Sprr1a in the SN of 6-OHDA lesioned subjects was much greater when quantified by qPCR (80.3 fold) than originally observed by microarray (19.2 fold). This is likely due to saturation of the Sprr1a signal on the microarray chip, resulting in an underestimate of the differential upregulation.
Next, we sought to confirm our hypothesis that the upregulation of genes attempting to confer axoprotection (i.e. RAGs) in Cluster-1 occurred specifically in DA neurons of the SN. We therefore chose three transcripts from Cluster-1 to pursue further using a combination of ISH to identify transcript changes in the RAGs and IHC to identify SN DA neurons. We observed a robust upregulation of
The mRNA for Sprr1a (A and B), Gadd45a (C and D) and Sox11 (E and F) was identified using RNAscope
MetaCore network analysis demonstrated that transcripts from Cluster-1 were highly connected, with 62 of the transcripts showing evidence of interaction (
MetaCore Pathway analysis was conducted on the differential gene expression of Cluster-1 at 1 week post-surgery. The data were imported into Cytoscape for visualization. Diamonds represent transcription factors and ovals represent all other proteins. Arrows represent the direction of the interaction. The intensity of red coloring is indicative of the magnitude of upregulation. Values ranged from 1.23 (lightest pink) to 19.2 (darkest red). Of the 88 genes in Cluster-1, 62 had known interactions. The significant level of interaction among the transcripts along with the
In this study, we examined transcriptomic changes in the SN over 16 weeks after a unilateral, intrastriatal 6-OHDA lesion that results in progressive nigrostriatal DA axon degeneration followed by SN DA neuron loss [
The rapid induction and subsequent reduction in RAG and RAG-like expression over time indicates that these transcripts likely are (1) expressed in DA neurons of the SN and (2) their reduction over time is a result of the progressive degeneration of those DA neurons. Indeed, combing TH IHC with ISH for either
The specific RAG transcripts that we found upregulated in Cluster-1 include
Another member of the RAG family induced by axonal injury in nigrostriatal DA neurons is
Intriguingly, parkin overexpression induces
Other transcripts that behaved similar to established RAGs are less well characterized yet may play a significant role in the response to axonal damage in DA neurons. For instance,
Exactly what signals cause the upregulation of RAG and RAG-like transcripts requires further investigation. Interestingly, several transcription factors that are implicated in the ER stress response show significant pathway interaction as indicated by MetaCore analysis (
Notably, the SN RAG transcripts up-regulated in response to striatal 6-OHDA administration were not previously described in DA neurons. For many of these transcripts, this is the first time they were observed to be upregulated in response to CNS injury [
Finally, it should be restated here that while there is a wealth of data demonstrating differential gene expression across genes from all eight clusters, one must be cautious in drawing conclusions about the association of genes within any particular cluster without an
Recent discoveries regarding the early and robust nature of axonal degeneration in the DAergic nigrostriatal pathway that precedes overt DA neuron loss in the SN highlights the importance of targeting axonal degeneration in treating PD. In light of this recent observation [
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We would like to thank Dr. Scott E. Counts for his editorial advice on the manuscript and the Mattingly Family for the distinct privilege of allowing us the opportunity to work with Mr. Zachary R. Mattingly.