Kamath T, Abdulraouf A, Burris SJ, Langlieb J, Gazestani V, Nadaf NM, Balderrama K, Vanderburg C, Macosko EZ. Single-cell genomic profiling of human dopamine neurons identifies a population that selectively degenerates in Parkinson's disease. Nat Neurosci. 2022 May;25(5):588-595. Epub 2022 May 5 PubMed.

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  1. This article opens a new window for work on the root causes of Parkinson’s disease and a glimpse of how this may differ from Alzheimer’s disease. The authors used advanced snRNA-sequencing and related methods on a special population of PD brain samples and normative control samples. They identified the transcriptomic identities of the dopamine-containing substantia nigra neurons most vulnerable in PD. These express particular patterns of transcription factors, and can be identified for the most part across species, with mice lacking some of the markers conserved across macaques and humans.

    Crucially, the authors used spatial-seq to enable them to view the transcriptionally vulnerable neurons in anatomical maps of the substantial nigra. Because of this multimodal approach, they were able to achieve the so-far-rare goal of relating their transcriptional identification of the vulnerable neurons to the actual locations of the vulnerable neurons in the brain locus examined (here, substantial nigra). The remarkable result is that they have identified as the most transcriptionally vulnerable cell group the very most ventral zone originally earmarked as most vulnerable by classical neurochemical anatomical studies in postmortem PD patients, and scored along with other nigral zones in terms of a sequential vulnerability in the progression of PD. 

    The most vulnerable region, in the “ventral tier” of the substantial nigra, is identifiable by expression of Aldh1A1, which is a clear marker of the ventral tier from rodent to humans. The tanscriptomic identifier is AGTR1, alongside the nigrosome marker pattern of low Calbindin. The authors give strong evidence that these features are the crucial ones, making it likely that they actually are true identifiers of a cell type—what is called an endogenous or cell-autonomous feature.

    These are crucial facts that offer great potential for understanding and treating PD. This is because the ventral tier dopamine-containing neurons of the substantial nigra receive preferential input from the striosomal compartment of the striatum. This compartment has been linked to mood-related problems that frequently occur early on in PD, and it has molecularly specialized dopaminergic characteristics itself. 

    The early and strong vulnerability of this set of nigral dopamine-containing neurons could be a key to understanding the molecular and phenotypic expression of PD. It now will be possible to build a systems-neuroscience strategy to combat and defeat PD, and it will be possible then to design therapies including the use of enhancers and novel transport methods for therapeutic delivery.

    It is a great accomplishment to reach the level of cell-specific localization of the genes most clearly associated with PD. The authors' comparison with the case of AD is also illuminating—here, there is a local reference, compared with more global involvement in the case of advanced Alzheimer’s disease.

    View all comments by Ann M. Graybiel

  2. This paper is an impressive application of two types of single-cell analysis, especially when considering the additional flow sorting step to enrich for dopamine neurons in figure 1. That the general dorsal/ventral gradient of calbindin vs. SOX6 is replicated is reassuring, and the identification of susceptible subtypes that are marked by SOX6 and AGTR1 adds another dimension to what we know about DA neurons sensitivity.

    One area that I think needs some discussion is the genetic enrichment analyses. It is consistent with other recent enrichment analyses that suggest dopamine neurons account for some of the genetic risk of PD (Bressan et al., 2021). That said, some of the presented genes for PD in Kamath et al.’s analysis are uncertain, e.g. UCHL1, GUGYF2, HTRA2, EIF4G1, etc., hence how well the enrichment scores would work on a smaller gene set would be important to understand.

    Additionally, it was very surprising to see such a strong effect of LRRK2, especially in contrast to other recent surveys that indicated stronger expression in microglia and oligodendrocyte precursor cells in human substantia nigra (Wang et al., 2022). It would therefore be important to concatenate multiple nigral datasets using different single nuclear/cell RNA-Seq approaches to try to understand which results are most consistent between sample series.

    References:

    Bressan E, Reed X, Bansal V, Hutchins E, Cobb MM, Webb MG, Alsop E, Grenn FP, Illarionova A, Savytska S, Violich I, Broeer S, Fernandes N, Sivakumar R, Beilina A, Billingsley K, Berghausen J, Pantazis CB, Meechoovet B, Reiman R, Courtright-Lim A, Logemann A, Antone J, Barch M, Kitchen R, Li Y, Dalgard CL, The American Genome Center, Rizzu PR, Hernandez DG, Hjelm BE, Nalls M, Gibbs JR, Finkbeiner S, Cookson MR, Van Keuren-Jensen K, Craig DW, Singleton AB, Heutink P, Blauwendraat C. The Foundational data initiative for Parkinson’s disease (FOUNDIN-PD): enabling efficient translation from genetic maps to mechanism. BioRxiv, June 3, 2021 bioRxiv

    Wang Q, Wang M, Choi I, Ho L, Farrell K, Beaumont KG, Sebra R, Crary JF, Davis DA, Sun X, Zhang B, Yue Z. Single-cell transcriptomic atlas of the human substantia nigra in Parkinson’s disease. bioRxiv. March 30, 2022 bioRxiv

    View all comments by Mark Cookson

  3. Preferential vulnerability is one of the major mysteries of neurodegenerative diseases. This exciting study takes a hard look at identifying preferentially vulnerable dopamine neuron subtypes in the midbrain and provides intriguing new insights on possible subtypes of pars compacta dopamine neurons.

    The study raises several important questions:

    The substantia nigra pars compacta dopamine (SNpc) neurons analyzed are all vulnerable to PD, although to different degrees. The most resistant dopamine neurons in the midbrain, the VTA dopamine neurons, do not seem to have been included. Does this kick the can down the road? What makes SNpc dopamine neurons more vulnerable than VTA dopamine neurons?

    Do the 10 nominated SNpc dopamine neuron subtypes truly represent replicable, discrete subtypes rather than various presentations of the population spectrum of pars compacta neurons? The putative subtype clusters appear closely linked and interwoven rather than clearly separated. Considering the evolving “art and science of clustering,” this view might evolve as we learn more about their validity and the influence of covariates in larger cohorts.

    Generally, much larger sample sizes will help to solidify these intriguing clues. Caveats are the limited number of eight control midbrains, six of which were female, and the huge age range they represent, which did not seem to match the PD cases very well.

    This is an exciting time for brain research. Single-nucleus transcriptomics opens the awesome opportunity to map the circuit board of Parkinson’s with single-cell and single base-pair resolution. The PD5D Consortium at Harvard, the Broad Institute, and the University of Wisconsin—part of the ASAP Collaborative Research Network—have launched the ambitious initiative to map a Parkinson Cell Atlas in five dimensions based on spatial and single-nucleus multiomics of a million brain cells with the ultimate goal to map, simulate, and help to correct the flow of genetic information from patient genomes to brain cells in brain space.

    View all comments by Clemens Scherzer

  4. Neighboring but functionally distinct dopaminergic neurons are vulnerable or resistant to neurodegeneration during Parkinson’s disease. Over the last 20 years, we and others have used cell-type-specific analyses to characterize healthy neurons and to propose causal pathways of degeneration.

    These pathways include intrinsic oxidative stress, where, for example, live-cell imaging revealed that intraneuronal oxidation levels are greater in vulnerable dopaminergic neurons than in disease-resistant dopaminergic neurons (Guzman et al., 2010). Vesicular trafficking and mitochondrial pathways were highlighted when RNA was collected from populations of vulnerable and disease-resistant dopaminergic neurons (Chung et al., 2005). Image-based analyses demonstrated several converging pathways as healthy ventral-tier dopaminergic neurons age that could be consistent with changes in mitochondrial DNA over time (Chung et al., 2005; Dölle et al., 2016; Collier et al., 2011). 

    Beyond dopaminergic neuron subtypes, image-based analysis of human postmortem tissue revealed lipid accumulation in nigral dopaminergic neurons and microglia but not astrocytes (Brekk et al., 2020). In the same study, we found correlations between triglyceride and GPNMB levels in nigral tissue (Brekk et al., 2020). 

    During this period, we avoided molecular analyses of degenerating neurons because we felt that causality could not be measured in individual cells adapting in an asynchronous, protracted manner to a disease driver, ultimately leading to a collective loss of circuit function once the threshold of synaptic loss has been passed (Figure 1) (Engelender et al., 2017). 

    In this new work, Tushar Kamath, Abdulraouf Abdulraouf and colleagues provide an excellent open-access catalog of transcripts from different types of aged dopaminergic neurons isolated from the brains of individuals without neurological symptoms (Kamath et al., 2022). The single-cell data is broadly consistent with previous studies and methods (Mendez et al., 2005; Kadkhodaei et al., 2009; Thompson et al., 2005), while providing greater resolution. The data instantly fills gaps for drug discovery. For example, the initial steps of drug and biomarker discovery can be helped if a molecular target is known to be expressed by the target cell population.

    However, we continue to believe that causality cannot be interpreted from the transcriptome of degenerating patient neurons. The expression of genes associated with PD risk by ventral tier dopaminergic neurons is an important step when considering how to use “GWAS hits,” but we expect these genes to be expressed in different combinations across many different cell types of the body and under different dysregulated states. This concept needs experimental determination, but hints at underlying physiological processes in vulnerable neurons that drive expression of these genes under periods of neuronal challenge (“biomarkers of a dysregulated state”).

    Kamath et al. also propose to use the single-cell transcriptional data to improve in-vitro differentiation protocols for disease modeling. This idea seems like a bit of a stretch given that the cellular environment and functional state of the neurons in the aged brain are so strikingly different than cell culture, be it as a two- or three-dimensional culture. Even cells cultured over a couple of years are unlikely to reach toward the aged end of the structure and function spectrum that would be reflected in their transcriptome.  

    Figure 1. Transcriptional profiles within a population of aged, vulnerable neurons undergoing asynchronous neurodegeneration. (A,B) Melanized neurons of the young (A) or aged (B) ventral midbrain are a population of dopaminergic neurons that are vulnerable to neurodegeneration in Parkinson’s disease (PD). (C) Fewer melanized neurons are a characteristic of the ventral midbrain from a PD patient. (D) Hypothetical transcriptional diversity of the vulnerable neurons in the young healthy brain shows a largely homeostatic profile (green; most cell-cell transcriptional variability is caused by asynchronous, physiological responses to functional challenges). (E) Neurons begin to show dysregulated transcriptional profiles during aging that represent a significant and potentially lethal challenge to an individual cell but too few cells are lost to compromise the functional threshold of synaptic loss and cause symptoms unless the challenge persists over a protracted period (yellow-red). (F) The hypothetical transcriptional diversity in the PD patient population of degenerating neurons is again asynchronous and shows greater cell-cell variation and dysregulation as the cells reroute transcriptional activity to adapt and survive the disease driver.

    References:

    Guzman JN, Sanchez-Padilla J, Wokosin D, Kondapalli J, Ilijic E, Schumacker PT, Surmeier DJ. Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature. 2010 Dec 2;468(7324):696-700. PubMed.

    Chung CY, Seo H, Sonntag KC, Brooks A, Lin L, Isacson O. Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection. Hum Mol Genet. 2005 Jul 1;14(13):1709-25. PubMed.

    Dölle C, Flønes I, Nido GS, Miletic H, Osuagwu N, Kristoffersen S, Lilleng PK, Larsen JP, Tysnes OB, Haugarvoll K, Bindoff LA, Tzoulis C. Defective mitochondrial DNA homeostasis in the substantia nigra in Parkinson disease. Nat Commun. 2016 Nov 22;7:13548. PubMed.

    Collier TJ, Kanaan NM, Kordower JH. Ageing as a primary risk factor for Parkinson's disease: evidence from studies of non-human primates. Nat Rev Neurosci. 2011 Jun;12(6):359-66. PubMed.

    Brekk OR, Honey JR, Lee S, Hallett PJ, Isacson O. Cell type-specific lipid storage changes in Parkinson's disease patient brains are recapitulated by experimental glycolipid disturbance. Proc Natl Acad Sci U S A. 2020 Nov 3;117(44):27646-27654. Epub 2020 Oct 15 PubMed.

    Engelender S, Isacson O. The Threshold Theory for Parkinson's Disease. Trends Neurosci. 2017 Jan;40(1):4-14. Epub 2016 Nov 25 PubMed.

    Kamath T, Abdulraouf A, Burris SJ, Langlieb J, Gazestani V, Nadaf NM, Balderrama K, Vanderburg C, Macosko EZ. Single-cell genomic profiling of human dopamine neurons identifies a population that selectively degenerates in Parkinson's disease. Nat Neurosci. 2022 May;25(5):588-595. Epub 2022 May 5 PubMed.

    Mendez I, Sanchez-Pernaute R, Cooper O, Viñuela A, Ferrari D, Björklund L, Dagher A, Isacson O. Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson's disease. Brain. 2005 Jul;128(Pt 7):1498-510. PubMed.

    Kadkhodaei B, Ito T, Joodmardi E, Mattsson B, Rouillard C, Carta M, Muramatsu S, Sumi-Ichinose C, Nomura T, Metzger D, Chambon P, Lindqvist E, Larsson NG, Olson L, Björklund A, Ichinose H, Perlmann T. Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons. J Neurosci. 2009 Dec 16;29(50):15923-32. PubMed.

    Thompson L, Barraud P, Andersson E, Kirik D, Björklund A. Identification of dopaminergic neurons of nigral and ventral tegmental area subtypes in grafts of fetal ventral mesencephalon based on cell morphology, protein expression, and efferent projections. J Neurosci. 2005 Jul 6;25(27):6467-77. PubMed.

    View all comments by Oliver Cooper

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  1. Single-Cell Sleuthing Nabs Neurons Prone to Perish in Parkinson’s