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, 14 (13), 1709-25

Cell Type-Specific Gene Expression of Midbrain Dopaminergic Neurons Reveals Molecules Involved in Their Vulnerability and Protection

Affiliations

Cell Type-Specific Gene Expression of Midbrain Dopaminergic Neurons Reveals Molecules Involved in Their Vulnerability and Protection

Chee Yeun Chung et al. Hum Mol Genet.

Abstract

Molecular differences between dopamine (DA) neurons may explain why the mesostriatal DA neurons in the A9 region preferentially degenerate in Parkinson's disease (PD) and toxic models, whereas the adjacent A10 region mesolimbic and mesocortical DA neurons are relatively spared. To characterize innate physiological differences between A9 and A10 DA neurons, we determined gene expression profiles in these neurons in the adult mouse by laser capture microdissection, microarray analysis and real-time PCR. We found 42 genes relatively elevated in A9 DA neurons, whereas 61 genes were elevated in A10 DA neurons [> 2-fold; false discovery rate (FDR) < 1%]. Genes of interest for further functional analysis were selected by criteria of (i) fold differences in gene expression, (ii) real-time PCR validation and (iii) potential roles in neurotoxic or protective biochemical pathways. Three A9-elevated molecules [G-protein coupled inwardly rectifying K channel 2 (GIRK2), adenine nucleotide translocator 2 (ANT-2) and the growth factor IGF-1] and three A10-elevated peptides (GRP, CGRP and PACAP) were further examined in both alpha-synuclein overexpressing PC12 (PC12-alphaSyn) cells and rat primary ventral mesencephalic (VM) cultures exposed to MPP+ neurotoxicity. GIRK2-positive DA neurons were more vulnerable to MPP+ toxicity and overexpression of GIRK2 increased the vulnerability of PC12-alphaSyn cells to the toxin. Blocking of ANT decreased vulnerability to MPP+ in both cell culture systems. Exposing cells to IGF-1, GRP and PACAP decreased vulnerability of both cell types to MPP+, whereas CGRP protected PC12-alphaSyn cells but not primary VM DA neurons. These results indicate that certain differentially expressed molecules in A9 and A10 DA neurons may play key roles in their relative vulnerability to toxins and PD.

Conflict of interest statement

Conflict of Interest statement. None declared.

Figures

Figure 1
Figure 1. LCM and microarray on A9 and A10 DA neurons
(A) Coronal section of the mouse midbrain after quick TH immunostaining. A9 DA neurons are located in the SN pars reticulata (SNr) and the lateral part of SN pars compacta (SNc) marked by a red dotted line. A10 DA neurons are located in the medial part of the ventral tegmental area (VTA), the nucleus PN and the interfascicular nucleus (IF) marked by a green dotted line. (B–D) LCM of the midbrain DA neurons. Selection of DA neurons was guided by quick TH immunostaining. (B) TH-positive neurons in SNc before laser capture. (C) The TH-positive cells were targeted for laser capture with a 7.5 µm laser diameter. (D) Captured cells on the thermoplastic film were visualized before processing for RNA extraction. (E) Validation of GIRK2 and calbindin D28K mRNA levels in LCM samples by real-time PCR. The transcript level of GIRK2 was 2.78-fold (SEM ± 0.94) higher in A9 and that of calbindin was 2.90-fold (SEM ± 0.64) higher in A10 DA neurons. (F and G) Gene expression profiles of A9 and A10 DA neurons determined by micro-array analysis. There is a high reproducibility between A9 (F) and A10 (G) microarray replicates. (H) Differential gene expression between A9 and A10 replicates showed genes with differential expression. All genes were plotted on a log scale and represent a comparison between microdissected samples of A9 and A10. Five A9 replicates are plotted against six A10 replicates to determine the differential gene expression between the groups. The distance from the midline infers increasing levels of differential gene expression. All data were normalized using the probe level Robust Multi-Chip Analysis (RMA) algorithm.
Figure 2
Figure 2. Functional profiles of microarray data
A9- and A10-elevated genes were categorized based on biological functions and cellular components by Onto-Express (36). Genes with significant differences (FDR < 1%) were distributed into different categories of metabolisms (A), cellular components in cytoplasm (B) and transport mechanisms (C).
Figure 3
Figure 3. Altered vulnerability of PC12-αSyn cells and primary VM cultures by A9-elevated molecules
(A–C) Effects of GIRK2 overexpression in PC12-αSyn cells. Cells were transduced with either eGFP or GIRK2-expressing lentiviruses at an MOI of 5 and higher GIRK2 expression was achieved in GIRK2-transduced cells (B) when compared with untransduced cells (A). Red staining represents GIRK2 and cell nuclei were visualized by DAPI staining (blue). There was higher MPP+ toxicity in GIRK2 overexpressing PC12-αSyn cells (1 and 5 mm) when compared with eGFP-expressing control cells (C). Cell viability was measured by LDH release, which was significantly increased in GIRK2 overexpressing cells when compared with control eGFP-expressing cells (*P < 0.05 between groups). (D–E) Differential vulnerability of primary VM cells to MPP+. (D) Immunostaining of primary VM cultures demonstrated TH(+), GIRK2(+) and TH(+)/ GIRK2(+) cells. (E) TH(+)/GIRK2(+) or TH(+)/GIRK2(−) cells were counted after MPP+ treatment and were presented as percentage of total TH(+) neurons of non-treated control conditions. TH(+)/GIRK2(+) cells were more vulnerable to MPP+ than TH(+)/GIRK2(−) cells [*P < 0.05 in comparison with the control (no MPP+) condition]. (F–I) The effects of an ANT blocker, BA and IGF-1 in PC12 αSyn cells (F and H) and in primary VM cultures (G and I). In PC12 αSyn cells (F and H), cells were pretreated with BA (F) and IGF-1 (H) at concentrations indicated under the bar graph for 2 h prior to treatment with 1 mm MPP+ for 24 h. The levels of LDH release were presented as percentage of control group without treatment. LDH releases were significantly reduced by addition of these protective molecules [*P < 0.01 in comparison with the group of MPP+ only treatment (black bar graph); Student’s t-test]. In primary VM cultures (G and I), the ANT blocker (G) and IGF-1 (I) were added to the cultures at 5 days in vitro 2 h prior treatment with 10 µm MPP+. After 48 h, TH(+)/GIRK2(+) and TH(+)/GIRK2(−) neurons were counted and presented as percentage of total TH(+) neurons of control (no MPP+) conditions [*P < 0.05 in the comparison with the group of MPP+ only treatment (black bar graph)]. Student’s t-test was used to obtain statistical significance.
Figure 4
Figure 4. Altered vulnerability of PC12-αSyn cells and primary VM cultures by A10-elevated molecules
In PC12 cells (A, C and E), cells were pretreated with GRP (A), CGRP (C) and PACAP (E) with concentrations indicated under the bar graph for 2 h prior to 1 mm MPP+ treatment for 24 h. The levels of LDH release were presented as percentage of control group without treatment. Significant dose-dependent decreases in LDH release were detected in these experiments indicating neuroprotective effects of these peptides from toxic insult [*P < 0.01 in the comparison with the group of MPP+ only treatment (black bar graph); Student’s t-test]. In primary VM cultures (B, D and F), GRP (B), CGRP (D) and PACAP (E) were added to the cells at 5 days in vitro 2 h prior to treatment with 10 µm MPP+. After 48 h, TH(+)/GIRK2(+) and TH(+)/GIRK2(−) neurons were counted and presented as percentage of total TH(+) neurons of control (no MPP+) conditions [*P < 0.05 in the comparison with the group of MPP+ only treatment (black bar graph)]. Student’s t-test was used to obtain statistical significance.

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