Previously
Parkinson’s disease (PD) is a neurodegenerative disease that causes the loss of dopamine-producing neurons (DANs) in the substantia nigra pars compacta (SNpc). The main clinical symptoms of PD are rigidity, tremor and slowness of movement. Current treatments for PD provide temporary symptomatic relieve, but they do not change the progressive course of disease. One experimental therapy aiming at changing disease progression is the surgical replacement of DANs. Cell replacement using donor human fetal ventral midbrain (VM) tissue has previously allowed some PD patients to progressively withdraw their medication. These studies provided proof-of-principle for clinical cell replacement, yet it was not without shortcomings. In addition of ethical issues associated with the use of fetal tissue, different donors were necessary. This made it difficult to collect enough cells and introduced variability in cell quality and composition, which contributed to variable clinical benefit and in some cases, resulted in complications, such as uncontrolled involuntary movements called dyskinesias.

Currently
Human pluripotent stem cells (hPSCs) have become the preferred source of DANs as we can grow them in the laboratory in a consistent and standardized manner.  In the last years, DANs derived stem cells have been used to treat PD patients in first-of-kind clinical trials initiated in Japan, USA, Sweden and China. However, safety or efficacy results are not yet available. During the duration of the clinical trials, our knowledge of DANs has continued to increase and there is much more that can be done to further fine-tune hPSCs and make the right kind of DANs. We know that DANs in the VM comprise not only 2 large anatomical areas, the SNpc and the ventral tegmental area (VTA), but within these two areas they can be further subdivided anatomically, and they also have different molecular flavors. This has been recently proven to be important as different subtypes of DANs have distinct connections and physiological functions as well as different vulnerabilities in PD.

Not all dopaminergic neurons are the same
A way to understand the heterogeneity of DANs is to study them at a single cell/nucleus resolution. Recent advancements in single-cell transcriptomic technologies have made it possible to appreciate an emerging diversity in otherwise apparently similar neuronal types. These technologies define an important attribute, the transcriptional phenotype, which describes the genetic makeup of each single cell and allows categorizing DANs beyond broad anatomical regions.

In our studies
We performed a detailed analysis of the transcriptome of DANs in both embryonic and adult tissues at the single-cell/nuclei level. To understand adult DANs subtypes, we focused on the neurons that produce the messenger RNA (mRNA) for the critical enzyme, tyrosine hydroxylase (TH+), which is required for the synthesis of the neurotransmitter dopamine. We observed 3 major classes of the TH enriched cells, which were defined by the presence of SOX6, CALB1 and GAD2 mRNAs. SOX6 and CALB1 have been used to molecularly differentiate cells either from SNpc or VTA respectively. We now discern a new class of human adult TH+ neurons that was previously described in mice, which produce mRNAs important to synthesize the neurotransmitters dopamine and GABA (i.e. TH+ and GAD2+). In addition, we noticed various DANs in the periaqueductal gray (PAG) that produce different mRNAs for other chemical transmitters called neuropeptides. Their different abundance suggests distinct roles of DANs in pain modulation or reward. Within the SOX6 class of DANs, we found two previously described subpopulations, AGTR1+, shown to be more susceptible in PD, and GFRA2+, as well as a new subtype that we called LPL+. By understanding the heterogeneity of adult human mDA neurons, their connections, and their changes in PD, it may be possible to unravel the mechanisms making DAN subtypes of the SOX6 subgroup more vulnerable to PD than other DANs.   

Lastly, to understand the development of DANs in ventral midbrain, we analyzed transcriptomics data of the VM between post conception week 5 and 15, a period comprising the birth and initial maturation of DANs. Compared to a previous study, we detected a greater molecular diversity in all cell categories, including neural progenitors, glial progenitors, neuronal progenitors, immature neurons and DANs. In addition, we described transcriptional changes along the development of DANs as well as in neighbor neurons or in sibling neurons migrating to an adjacent structure, the subthalamic nucleus. More importantly, by defining the genes enriched in specific cells during DAN development, we now have a precise roadmap of genes that can be used to follow, guide and/or enrich stem cell preparations in the specific subtype of DANs required for cell replacement therapy. This may allow to reduce possible off-target cell effects by non-therapeutic cells and to improve the precision of cell replacement therapy.

In sum, our work highlights the heterogeneity of mDA neurons in the adult brain and defines critical steps of the developmental cascade leading to their generation. This enables a more precise generation of specific mDA neurons subtypes for diverse applications such as in vitro modeling of PD, drug discovery and cell replacement therapy for PD.

Read more on single cell brain atlas efforts here:
Braun, E. et al. (2023) ‘Comprehensive cell atlas of the first-trimester developing human brain’. Science, 382 (6667). doi:10.1126/science.adf1226.

Siletti, K. et al. (2023) ‘Transcriptomic diversity of cell types across the adult human brain’, Science, 382(6667). doi:10.1126/science.add7046.

Kawai Lee is a PhD student from Ernest Arenas Group in the Division of Molecular Neurobiology, Karolinska Institute, Stockholm, Sweden. She presented her work as part of a guided poster tour at the WPC 2023 in Barcelona.

Ideas and opinions expressed in this post reflect that of the author solely. They do not reflect the opinions or positions of the World Parkinson Coalition®