The death of dopaminergic neurons in the substantia nigra is the defining feature of Parkinson’s disease, but not all neurons in the region suffer this fate. Why do some wither while others survive until the bitter end? A study published May 5 in Nature Neuroscience opens the door to answers. Working from a cutting-edge transcriptomic toolbox, researchers led by Evan Macosko at the Broad Institute of Harvard and MIT in Cambridge, Massachusetts, defined 10 transcriptionally distinct populations of dopaminergic neurons in the human substantia nigra pars compacta. One—marked by its expression of SOX6 and AGTR1—selectively succumbed to degeneration in people with PD and dementia with Lewy bodies (DLB). These cells reside in the ventral part of the SN, a region known for its susceptibility to neurodegeneration. What’s more, the cells expressed more PD risk genes than did any other dopaminergic neuron subtype, suggesting that neurodegeneration in these synucleinopathies is a cell-intrinsic process, as opposed to one inflicted by other cell types.
- Single-nucleus profiling uncovered 10 transcriptional subtypes of dopaminergic neuron in the human substantia nigra pars compacta.
- One, expressing SOX6 and AGTR1, was hit hardest in people with PD or DLB.
- This vulnerable subset expressed more PD risk genes than any other subtype of dopaminergic neuron.
“It is a great accomplishment to reach the level of cell-specific localization of the genes most clearly associated with PD,” wrote Ann Graybiel of the Massachusetts Institute of Technology. “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.”
That some dopaminergic (DA) neurons survive while others die over the course of Parkinson’s disease has been known for decades, yet pinning down the molecular mechanisms underlying this selective vulnerability is challenging (Yamada et al., 1990; Gibb and Lees, 1991; Lu et al., 2006; May 2009 news). Single-cell RNA-Sequencing techniques enable scientists to define the gene-expression profiles that distinguish hardy from fragile neurons. However, the diminutive size of the substantia nigra pars compacta, coupled with the throngs of cell types within and surrounding it, make teasing out DA neurons, let alone different subsets of them, a finicky endeavor. Previous single-cell transcriptomic studies have had to make do with vanishingly small numbers of dopaminergic neurons, making it hard to detect distinct subsets in the first place, much less robustly compare them across samples (Agarwal et al., 2020; Smajić et al., 2022).
To harvest many more nuclei from postmortem brain samples, co-first authors Tushar Kamath and Abdulraouf Abdulraouf and colleagues first fished for unique nuclear markers they could use to enrich for these nuclei. They ultimately pegged Nr4a2, a transcription factor only DA neurons express. Then they isolated nuclei from postmortem substantia nigra samples of eight people who had died without neurodegenerative disease. Selecting with Nr4a2, the researchers netted 5,684 DA neuron nuclei for RNA-Sequencing analysis—topping the haul from previous studies by 180-fold.
Fishing for DA Nuclei. Scientists carved out the substantia nigra pars compacta from postmortem brain, isolated the nuclei, and used the NR4A2 protein to snag those that came from DA neurons for transcriptomic profiling. [Courtesy of Kamath et al., Nature Neuroscience, 2022.]
Using LIGER, a computational method developed in the lab to compare transcriptomes from multiple samples, Kamath pulled 10 distinct transcriptional subsets from among the DA neuron herd. These subsets varied in their expression of two known DA developmental markers, SOX6 and CALB1, such that four subsets preferentially expressed SOX6, while six leaned toward CALB1.
The researchers identified 10 similar transcriptomic subsets of DA neurons within the macaque substantia nigra. Intriguingly, in mice and rats they detected all but one of these subsets—marked by its expression of CALB1 and GEM—suggesting that it occurs only in primates. The scientists are studying this subset further, on the hunch that it could be behind a nigral circuit with the cortex that has been seen in primates but not rodents. Macosko’s team is studying what this circuit might be doing, and whether it relates to neurodegenerative disease.
Exactly where within the substantia nigra were these different types of DA neuron? To find out, the researchers applied a technique called Slide-Seq to sections of the macaque substantia nigra, which is less than half as big the human SN but arranged similarly. The method works like this: Scientists first create an array of beads, each coated with a unique DNA sequence, or “barcode,” that enciphers the bead’s spatial location within the array. Then, the scientists apply a frozen slice of brain tissue to the array, so that RNA from within the tissue transfers onto the beads. By sequencing the RNA as well as the DNA barcode attached to each bead, researchers can discern the address of individual transcriptomes within the tissue section. In this way, the researchers were able to locate all 10 subsets of DA neurons within the macaque SNpc. Notably, one subset, marked by its expression of both SOX6 and AGTR1, resided entirely within the ventral segment of the substantia nigra, an area known to degenerate in PD.
Find My Subtype. RNA from regions (circle) of the macaque substantia nigra pars compacta (left) were transferred to bead arrays DNA-barcoded for spatial transcriptomics. Both ventral and dorsal halves of the SNpc contain DA neurons (top, black), but their subtypes were distributed differently across the SNpc (middle and bottom panels). The light blue ones are most prone to die in PD. [Courtesy of Kamath et al., Nature Neuroscience, 2022.]
To pinpoint which of these subsets might be most prone to perish in neurodegenerative disease, the researchers performed snRNA-Seq on substantia nigra samples from 10 people who had died with PD or DLB. Unsurprisingly, people with PD or DLB had a dearth of DA neurons relative to healthy donors. The AGTR1 subset was the most depleted, suggesting that this type of DA neuron is particularly vulnerable to neurodegeneration. In contrast, two other DA neuron subsets, both expressing CALB1, were relatively more preserved in people with PD/DLB.
A subset of microglia expressing GPNMB, a known marker of disease-associated microglia associated with AD, was expanded, as was a subset of astrocytes. The findings reflect overlapping neurodegenerative mechanisms in PD and DLB, which are both synucleinopathies but have important differences, as well. As Ernest Arenas of the Karolinska Institute in Stockholm noted in a commentary that accompanied the paper, people with DLB have cognitive symptoms, along with Aβ plaques and tau tangles, in addition to dopaminergic neuron loss and its attendant motor symptoms. “In this context, the analysis provides valuable information on common alterations in these two diseases, but some disease-specific alterations may be underrepresented and undetected. Future analysis of more samples from individuals with PD or DLB will help to clarify this point,” Arenas wrote.
To address the question of what made the AGTR1 subset of DA neurons so susceptible to the ravages of PD or DLB, the scientists searched the transcriptomes of nigral cell types for their expression of genes tied to PD risk. These included 26 genes for which rare familial PD variants had been identified, as well as myriad common variants that had been tied to PD risk in genome-wide association studies.
They found that, as a whole, DA neurons expressed more of these PD risk genes than did any other cell type. Among the DA neurons, the largest and only statistically significant enrichment of PD genetic risk genes was in the AGTR1 subtype.
Doomed to Degenerate? Transcriptional subsets of different cell types expressed different levels of genes tied to PD. Of the 10 dopaminergic subsets, only the AGTR1+ subset was significantly enriched for PD risk (top graph). Different PD risk genes were enriched in SOX6+AGTR1+ cells (red), SOX6+ cells (orange), or dopaminergic neurons more broadly (yellow). [Courtesy of Kamath et al., 2022.]
What are the cellular pathways active in the AGTR1+ DA neuron subset that might seal their fate in PD? This remains unclear, but the scientists have some suspects, including genes involved in cell stress and mitochondrial dysfunction that were ramped up in this vulnerable subtype relative to others. AGTR1 itself encodes the angiotensin receptor 1 and may play a role, Arenas noted in his commentary. Variants in the gene have been tied to PD risk in a Japanese population, and inhibitors of the renin-angiotensin system are known to boost motor function in people with PD (Saito et al., 2003; Reardon et al., 2000; Labandeira-García et al., 2014).
In contrast to dopaminergic neurons, glial cells did not express an overabundance of PD risk genes. Instead, these cells tended to express genes tied to AD risk. The authors interpreted these findings as evidence that neurodegeneration in PD is primarily a cell-intrinsic process, while in AD, neuronal death is more tied to the actions of other cell types including microglia.
To Macosko, this is the most important insight from the study. “The specific enrichment of genetic heritability is in the cells that die,” he said. “This suggests the disease is somewhat cell autonomous.”
Ole Isacson of Harvard Medical School in Boston took a different view. Noting that most PD is sporadic, meaning it arises from external stressors interacting with vulnerable dopaminergic neurons, he interprets the finding that mitochondrial and cell stress pathways were revved in this vulnerable subset as a sign that the cells are under duress. Because dopaminergic neurons of the SNpc are known to broil with quadruple the level of oxidative stress as their counterparts in nearby regions, the effects of that stress over a lifetime could build up to render the cells vulnerable to external insults such as environmental toxins and neuroinflammation, he believes. Isacson sees the proportional increase in GPNMB-expressing microglia as a sign of an ongoing inflammatory response to lipid stress in the SNpc (Brekk et al., 2020). His data, combined with studies indicating that TNF-α inhibitors reduce PD risk, implicates parallel processes of inherent stress within vulnerable dopaminergic neurons and rising neuroinflammatory insults that may reach a neurodegenerative "boiling point" with age (Hallett et al., 2019).
Mark Cookson at the National Institutes of Health in Bethesda, Maryland, agrees that the PD risk gene analysis needs further investigation, noting that several genes considered in the analysis have uncertain ties to PD. He was surprised to see such strong expression of LRRK2 in the vulnerable neurons, given that other single-cell transcriptomic studies have found this PD gene to be strongly expressed in microglia and oligodendrocyte precursor cells (e.g., Wang et al., 2022). “It would therefore be important to concatenate multiple nigral datasets … to try to understand which results are most consistent between sample series.”
“Preferential vulnerability is one of the major mysteries of neurodegenerative diseases,” commented Clemens Scherzer of Brigham and Women’s Hospital in Boston “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.”
However, Scherzer noted that while the study addresses differences among dopaminergic neurons within the SNpc, it does not explain why SNpc DA neurons are more vulnerable to PD than their counterparts in the neighboring ventral tegmental area. “The most resistant dopamine neurons in the midbrain, the VTA dopamine neurons, do not seem to have been included [in the study]. Does this kick the can down the road?” Scherzer asked (full comment below).
Macosko told Alzforum that the lab is currently investigating the transcriptomes of dopaminergic neurons located in different regions of the brain, and also in the peripheral nervous system. Some scientists believe that α-synuclein aggregation in PD starts outside of the brain, within neurons of the gut, where it hitches a ride into the CNS via the vagus nerve (Jul 2011 news). Comparing the gene-expression profiles of neurons posted along this proposed route might give researchers clues about the likelihood of this disease progression model, Macosko suggested.
He also hopes scientists will plumb the current dataset for therapeutic targets, design more accurate disease models, and even to differentiate the most needed types of dopaminergic neurons for cell replacement therapies.—Jessica Shugart
- Multiple Hits Explain Selective Loss of Dopamine Neurons in PD
- Parkinson's: It Started With a Gut Feeling
- Yamada T, McGeer PL, Baimbridge KG, McGeer EG. Relative sparing in Parkinson's disease of substantia nigra dopamine neurons containing calbindin-D28K. Brain Res. 1990 Sep 3;526(2):303-7. PubMed.
- Gibb WR, Lees AJ. Anatomy, pigmentation, ventral and dorsal subpopulations of the substantia nigra, and differential cell death in Parkinson's disease. J Neurol Neurosurg Psychiatry. 1991 May;54(5):388-96. PubMed.
- Lu L, Neff F, Fischer DA, Henze C, Hirsch EC, Oertel WH, Schlegel J, Hartmann A. Regional vulnerability of mesencephalic dopaminergic neurons prone to degenerate in Parkinson's disease: a post-mortem study in human control subjects. Neurobiol Dis. 2006 Aug;23(2):409-21. Epub 2006 Jun 6 PubMed.
- Agarwal D, Sandor C, Volpato V, Caffrey TM, Monzón-Sandoval J, Bowden R, Alegre-Abarrategui J, Wade-Martins R, Webber C. A single-cell atlas of the human substantia nigra reveals cell-specific pathways associated with neurological disorders. Nat Commun. 2020 Aug 21;11(1):4183. PubMed.
- Smajić S, Prada-Medina CA, Landoulsi Z, Ghelfi J, Delcambre S, Dietrich C, Jarazo J, Henck J, Balachandran S, Pachchek S, Morris CM, Antony P, Timmermann B, Sauer S, Pereira SL, Schwamborn JC, May P, Grünewald A, Spielmann M. Single-cell sequencing of human midbrain reveals glial activation and a Parkinson-specific neuronal state. Brain. 2022 Apr 29;145(3):964-978. PubMed.
- Saito S, Iida A, Sekine A, Kawauchi S, Higuchi S, Ogawa C, Nakamura Y. Catalog of 178 variations in the Japanese population among eight human genes encoding G protein-coupled receptors (GPCRs). J Hum Genet. 2003;48(9):461-468. Epub 2003 Aug 30 PubMed.
- Reardon KA, Mendelsohn FA, Chai SY, Horne MK. The angiotensin converting enzyme (ACE) inhibitor, perindopril, modifies the clinical features of Parkinson's disease. Aust N Z J Med. 2000 Feb;30(1):48-53. PubMed.
- Labandeira-García JL, Garrido-Gil P, Rodriguez-Pallares J, Valenzuela R, Borrajo A, Rodríguez-Perez AI. Brain renin-angiotensin system and dopaminergic cell vulnerability. Front Neuroanat. 2014;8:67. Epub 2014 Jul 8 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.
- Hallett PJ, Engelender S, Isacson O. Lipid and immune abnormalities causing age-dependent neurodegeneration and Parkinson's disease. J Neuroinflammation. 2019 Jul 22;16(1):153. PubMed.
- 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
- 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.
- Arenas E. Parkinson's disease in the single-cell era. Nat Neurosci. 2022 May;25(5):536-538. PubMed.