In this study, we used LCM and genomic profiling to characterize the innate physiological differences between A9 and A10 DA midbrain neurons. Despite an overall high similarity in the gene expression profiles of these related DA neurons, several genes from different biological categories showed cell type-specific expression patterns. We examined some of these differentially expressed molecules using PC12-αSyn cells and primary VM cultures exposed to the PD-related neurotoxin, MPP+ Results from these bioassays showed that neuropeptides (GRP, CGRP and PACAP) expressed predominantly in A10 DA neurons protected against MPP+ Of genes elevated in A9 DA neurons, growth factors such as IGF-1 also decreased the vulnerability, whereas ANT-2 and GIRK2 appeared to increase cell toxicity. These results suggest that the study of genes with differential expression levels between A9 and A10 midbrain DA neurons can provide insights into specific neuroprotective and/or degenerative responses.
We propose at least two possible mechanisms whereby differential gene expression in A9 DA neurons could alter the vulnerability to neurotoxins. First, certain molecules may by themselves confer increased susceptibility in these neurons when their expression levels are relatively elevated. Elevated expression of such molecules may then decrease the threshold to extrinsic and intrinsic factors leading to cell-type specific degeneration (31
). For example, GIRK2 and ANT-2 may render A9 DA neurons more vulnerable because of their pathophysiological actions on the membrane potential and on the mitochondrial permeability transition, respectively (discussed subsequently). Or pro-apoptotic molecules, such as caspase 7 and Bim (38
) in A9 DA neurons (), may increase susceptibility of these neurons in pathological conditions. A second possibility is that A9 DA neurons may be more functionally dependent upon molecules with higher expression and therefore more vulnerable to fluctuation in their levels. In such cases, any insult, genetic polymorphisms or an age-dependent decrease in expression levels (45
) that reduces the physiological functions of these molecules may be neurotoxic. An example of this class of A9-elevated molecule might be IGF-1, a known neuroprotective factor (46
GIRK controls the neuronal membrane excitability by selectively permitting the flux of K+
ions near the resting membrane potential (48
). Among four isoforms of GIRK, only GIRK2 is exclusively expressed in vulnerable DA neurons (26
). GIRK2 generates a slow IPSP in DA neurons via activation of D2
receptors and controls the membrane excitability of DA neurons. A potential role of GIRK2 in A9 DA neuron pathology in PD has been revealed in weaver
mice, which have a spontaneous mutation in the GIRK2 gene and display PD-like patterns of DA neuron degeneration (28
). Mutation in GIRK2 decreases the channel selectivity for K+
, leading to the destabilization of the cell membrane (49
). Our data suggest that elevated expression levels of GIRK2 may also contribute to the increased vulnerability of A9 DA neurons.
ANT is thought to play a role in the transport of ADP and ATP across the mitochondrial inner membrane (43
). In addition, it is involved in the formation of the mitochondrial permeability transition pore (mPTP), a non-specific pore that is an important mediator of apoptosis (43
). The mPTP opens in response to stimuli including reactive oxygen species and inhibitors of the electron transport chain, including MPP+
). In our study, blocking the function of ANT-2 by BA decreased the vulnerability of both PC12-αSyn cells and primary VM culture neurons to MPP+
. This demonstrates that inhibition of ANT-2 is neuroprotective and, therefore, elevated ANT-2 expression levels in A9 DA neurons could confer increased susceptibility of these neurons to oxidative stress and toxins such as MPP+
. It is interesting to note the toxic effect of BA at higher doses (). It is unlikely that the toxic effect is due to non-specific inhibition of other proteins by BA as the doses used in our experiments are much lower than what is conventionally used in the field (44
). However, it could be that the unusual dose–response curve of BA may depend on ANT’s dual functions. Under pathological conditions, ANT is involved in forming the mPTP, which leads to cell death (44
). Thus, blocking the mPTP-forming function of ANT would be protective to cells. However, under physiological conditions, ANT imports ADP from the cytosol and exports ATP synthesized by mitochondria back to the cytosol to be used as an energy source. Therefore, inhibition of ANT’s physiological function could then be toxic (52
). At the MPP+
concentration used in our experiment, some ANT molecules may be involved in mPTP formation, but the remainder may still function as ADP/ATP carriers. At higher doses, this normal physiological function of ANT could be inhibited, thus depleting the cellular energy source and deleteriously affecting cell survival.
IGF-1 is neuroprotective in brain hypoxia–ischemia (53
), axotomy (55
), age-induced hippocampal death (56
) and glutamate-induced motor neuron death (57
). It protects embryonic DA neurons from apoptosis (58
) and dopaminergic cells from toxin-induced cell death in vitro
). In addition, IGF-1 or the N-terminal tripeptide of IGF-1 protected DA neurons and improved functional deficits in 6-OHDA treated rats (46
). Consistent with these findings, application of IGF-1 to the medium also reduced the MPP+
toxicity in PC12-αSyn cells and A9-like DA neurons of primary VM cultures in our study. This supports the notion that IGF-1 is an important neuroprotective factor which A9 DA neurons may depend on. Interestingly, plasma IGF-1 levels decrease with age (60
) and a causal relationship between age-dependent decrease in IGF-1 and reduced cognitive brain function has been proposed in many studies (61
). Taken together, the reduction of IGF-1 due to aging may contribute to increased vulnerability of A9 DA neurons in PD.
In our study, A10 DA neurons showed elevated transcription levels of several neuropeptides. We selected GRP, CGRP and PACAP to examine their roles in protecting DA cells from MPP+
-induced toxicity. GRP is a neuroendocrine peptide known to act primarily in the enteric and central nervous systems, where it regulates diverse functions, from satiety and smooth muscle contraction to the release of other gastrointestinal hormones (62
). It has also been studied extensively in the context of cancer cells where it plays a role as an autocrine growth factor (63
). Although the presence of GRP in SN was reported many years ago (65
), its function in DA neurons has not been described. In this study, our microarray data raised the possibility that GRP might be protective to DA neurons and we show here for the first time that GRP could reduce the toxic responses of both PC12-αSyn cells and primary mesencephalic DA neurons against MPP+
. We thus suggest that GRP may contribute to the reduced susceptibility of A10 DA neurons in PD.
CGRP exerts multiple biological actions in the central nervous, gastrointestinal and cardiovascular systems (66
). CGRP also influences the differentiation of immature DA neurons in primary VM cultures by inducing neurite out-growth and increasing DA uptake per neuron, but not their normal rate of survival (68
). In our study, CGRP reduced mitochondrial toxicity of PC12-αSyn cells. In primary VM cultures, it increased the susceptibility of A10-like DA neurons to the toxin in a dose-dependent manner, whereas it had no effect on the survival of A9-like DA neurons. These data raise the possibility that CGRP may not be a neuroprotective factor for DA neurons. However, it should be noted that primary VM cultures represent not adult but immature neurons. Given that there was a positive effect observed in PC12-αSyn cells, one cannot exclude a potential protective effect of CGRP in adult DA neurons in vivo
PACAP belongs to the family of peptides containing secretin, glucagons and vasoactive intestinal peptide (VIP) (69
). It is thought to act as a neurotrophic factor during development and as a neuroprotective factor against various insults (71
). PACAP is also neurotrophic for TH-positive neurons in primary VM culture (73
). In our in vitro
assays, PACAP reduced the toxic responses of both the PC12-αSyn cells and the DA neurons of primary VM cultures to MPP+
. In addition, the results from the primary VM cultures demonstrated that A9-like DA neurons were more responsive to the effects of PACAP than A10-like DA neurons. This result is further substantiated by a recent study in which injection of PACAP into the SN protected DA neurons and improved behavioral deficits in a rat model of PD (73
). Another neuropeptide from the same class, VIP is also expressed higher in A10 DA neurons () and its neurotrophic and neuroprotective effects in DA neurons against MPP+
have also been reported in a mouse model of PD (75
). These data indicate that some A10-elevated molecules may contribute to the reduced vulnerability of A10 DA neurons, suggesting that these factors may be applied to protect A9 DA neurons. Interestingly, several neuropeptides, including PACAP and VIP, are known to be transported through the blood brain barrier via transmembrane diffusion (76
), thus increasing their potential to be utilized in therapies for PD.
The microarray analyses also revealed that genes encoding energy-related metabolism and mitochondrial proteins are highly expressed in A9 DA neurons (Supplementary Material, Table S1
). This is particularly interesting as mitochondrial dysfunction is thought to contribute to the etiology of PD (77
). Elevated expression levels of these genes in A9 DA neurons are consistent with the notion that this neuronal population is highly energy (ATP)-dependent. Given the role of mitochondria in cellular energy metabolism, A9 DA neurons may be particularly susceptible to toxins such as MPP+
and rotenone (78
) and to mutant α-synuclein or parkin which have been reported to cause mitochondrial dysfunction (79
Another group of genes that are elevated in A9 DA neurons are genes related to vesicle-mediated transport, including RAB1, RAB3C, RAB6, RAB11A, RAB14, vacuolar protein sorting 35 and very low density lipoprotein receptor (Supplementary Material, Table S1
). Efficient DA sequestration into vesicles protects DA neurons from the deleterious effects of DA oxidation (82
). As A9 DA neurons have higher levels of the DA transporter than A10 or hypothalamic (A11, A13–A15) DA neurons (83
), vesicle-mediated transport may be a more active and critical physiological process in this neuronal population. Interestingly, vesicle-mediated transport genes have recently been recognized as susceptibility factors in PD (45
). In a genome-wide yeast screen for modifiers of α-synuclein-induced toxicity, modifiers were most prominently clustered in the vesicle-mediated transport and lipid metabolism categories (87
). Furthermore, several vesicle-mediated transport genes, including several RAB genes, were found within genomic linkage regions for PD (88
), and many vesicular transport genes were downregulated after age 40 in a recent study describing aging-dependent changes in human frontal cortex transcriptional profiles (45
). This suggests that defective vesicular transport may contribute to the increased susceptibility of the aged patient population to neurodegenerative diseases. Taken together, A9 DA neurons may be particularly vulnerable to genetic or environmental factors that diminish the function of the vesicle-recycling machinery.
A recent paper published after the completion of our study described gene expression differences between catecholaminergic neurons in the rat (89
). Although the study was done in rat tissue using a different microarray platform (14 800 element cDNA array), many of the genes the authors reported are consistent with the expression patterns seen in the mouse midbrain DA neuron microarray analysis reported here (Affymetrix oligonucleotide array with 22 000 probes). In addition to the microarray results, our study also includes the quantitative validation by real-time PCR and functional analyses, which illustrate that these differences in phenotypic gene expression may be relevant to neurotoxic responses.
In summary, we used LCM, microarray analysis and real-time PCR to determine gene expression profiles of A9 and A10 midbrain DA neurons and have begun to screen some of the molecules using in vitro bioassays. These data may offer opportunities for further in vivo modeling of neuroprotective and neurotoxic responses in midbrain DA neurons. Such scientific work may ultimately provide clues to pathogenetic mechanisms involved in PD and delineate neuroprotective and therapeutic interventions against this disease.