Chung, C.Y. et al. Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection. Hum. Mol. Genet. 14, 1709-1725

Harvard University, Cambridge, Massachusetts, United States
Human Molecular Genetics (Impact Factor: 6.39). 08/2005; 14(13):1709-25. DOI: 10.1093/hmg/ddi178
Source: PubMed


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.

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    • "We opted for a 96.96 dynamic array to evaluate 96 cells, each for the expression of 96 key genes, an approach used previously to discriminate stem cell identities (Buganim et al., 2012; Guo et al., 2010). We selected 96 gene candidates based on reported differential expression between SNc and VTA (Chung et al., 2005; Greene et al., 2005; Grimm et al., 2004), with a positive bias toward genes with validated midbrain mRNA expression as shown by in situ hybridization in public databases (Table S1). In addition, we evaluated the expression of housekeeping genes (Actb, Gapdh, and Hprt), genes linked to PD (Atp13a2, Lrrk2, Park2, Park7, Pink1, and Snca), as well as validated DA neuronal markers (Ddc, Th, Slc6a3, and Slc18a2). "
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    ABSTRACT: Effective approaches to neuropsychiatric disorders require detailed understanding of the cellular composition and circuitry of the complex mammalian brain. Here, we present a paradigm for deconstructing the diversity of neurons defined by a specific neurotransmitter using a microfluidic dynamic array to simultaneously evaluate the expression of 96 genes in single neurons. With this approach, we successfully identified multiple molecularly distinct dopamine neuron subtypes and localized them in the adult mouse brain. To validate the anatomical and functional correlates of molecular diversity, we provide evidence that one Vip+ subtype, located in the periaqueductal region, has a discrete projection field within the extended amygdala. Another Aldh1a1+ subtype, located in the substantia nigra, is especially vulnerable in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. Overall, this rapid, cost-effective approach enables the identification and classification of multiple dopamine neuron subtypes, with distinct molecular, anatomical, and functional properties.
    Cell Reports 11/2014; 9(3):930-43. DOI:10.1016/j.celrep.2014.10.008 · 8.36 Impact Factor
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    • "The human SN/VTA presents high complexity and is composed of several dopaminergic neuronal subpopulations that differ in their neurodevelopmental origin (Zecevic and Verney 1995; Puelles and Verney 1998; Verney et al. 2001), genetic and neurochemical profile (Haber et al. 1995; Grimm et al. 2004; Thuret et al. 2004; Chung et al. 2005; Luk et al. 2013), projection sites (van Domburg and ten Donkelaar 1991; Damier et al. 1999a; Nieuwenhuys et al. 2008), and susceptibility to disease (Gibb and Lees 1991; Damier et al. 1999b; Hauser et al. 2005; approximately all SN/VTA neuronal populations located rostral to the exit of the 3 rd cranial nerve and to the transition between the parvocellular and magnocellular parts of the red nucleus (see for anatomical details van Domburg and ten Donkelaar 1991; Damier et al. 1999a; Nieuwenhuys et al. 2008). In addition, detailed neuropathological studies in Parkinson's disease have shown that diencephalic SN/VTA dopaminergic neurons are significantly more resilient to neuronal death than the mesencephalic ones (Damier et al. 1999b), thus supporting the validity of using developmental criteria to define SN/VTA sub-regions for neuropathological studies in the adult brain. "
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    ABSTRACT: Previous work from our laboratory showed deficits in tyrosine hydroxylase protein expression within the substantia nigra/ventral tegmental area (SN/VTA) in schizophrenia. However, little is known about the nature and specific location of these deficits within the SN/VTA. The present study had two aims: (1) test if tyrosine hydroxylase deficits could be explained as the result of neuronal loss; (2) assess if deficits in tyrosine hydroxylase are sub-region specific within the SN/VTA, and thus, could affect specific dopaminergic pathways. To achieve these objectives: (1) we obtained estimates of the number of dopaminergic neurons, total number of neurons, and their ratio in matched SN/VTA schizophrenia and control samples; (2) we performed a qualitative assessment in SN/VTA schizophrenia and control matched samples that were processed simultaneously for tyrosine hydroxylase immunohistochemistry. We did not find any significant differences in the total number of neurons, dopaminergic neurons, or their ratio. Our qualitative study of TH expression showed a conspicuous decrease in labeling of neuronal processes and cell bodies within the SN/VTA, which was sub-region specific. Dorsal diencephalic dopaminergic populations of the SN/VTA presented the most conspicuous decrease in TH labeling. These data support the existence of pathway-specific dopaminergic deficits that would affect the dopamine input to the cortex without significant neuronal loss. Interestingly, these findings support earlier reports of decreases in tyrosine hydroxylase labeling in the target areas for this dopaminergic input in the prefrontal and entorhinal cortex. Finally, our findings support that tyrosine hydroxylase deficits could contribute to the hypodopaminergic state observed in cortical areas in schizophrenia. PMID: 25269834
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    • "Although considerable progress has been made in understanding key events determining the generation of all mDA neurons, remarkably little is known of how distinct subpopulations are specified during embryogenesis. Previous characterization has revealed molecular differences between SNc and VTA neurons, but it has remained unclear at what developmental stage SNc and VTA neurons first become molecularly distinct (Chung et al., 2005; Wolfart et al., 2001). Recently, it was shown that the transcription factor Otx2 is involved in controlling the subtype identity of VTA neurons. "
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    ABSTRACT: Distinct midbrain dopamine (mDA) neuron subtypes are found in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA), but it is mainly SNc neurons that degenerate in Parkinson's disease. Interest in how mDA neurons develop has been stimulated by the potential use of stem cells in therapy or disease modeling. However, very little is known about how specific dopaminergic subtypes are generated. Here, we show that the expression profiles of the transcription factors Sox6, Otx2, and Nolz1 define subpopulations of mDA neurons already at the neural progenitor cell stage. After cell-cycle exit, Sox6 selectively localizes to SNc neurons, while Otx2 and Nolz1 are expressed in a subset of VTA neurons. Importantly, Sox6 ablation leads to decreased expression of SNc markers and a corresponding increase in VTA markers, while Otx2 ablation has the opposite effect. Moreover, deletion of Sox6 affects striatal innervation and dopamine levels. We also find reduced Sox6 levels in Parkinson's disease patients. These findings identify Sox6 as a determinant of SNc neuron development and should facilitate the engineering of relevant mDA neurons for cell therapy and disease modeling.
    Cell Reports 08/2014; 8(4). DOI:10.1016/j.celrep.2014.07.016 · 8.36 Impact Factor
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