Molecular heterogeneity in the substantia nigra: A roadmap for understanding PD motor pathophysiology.

As the ability to capture single-cell expression profiles has grown in recent years, neuroscientists studying a wide gamut of brain regions have discovered remarkable heterogeneity within seemingly related populations (Saunders et al., 2018a; Zeisel et al., 2015). These "molecular subtypes" have been demonstrated even within brain nuclei expressing the same neurotransmitter (Saunders et al., 2018a; Poulin et al., 2020; Ren et al., 2019; Okaty et al., 2020). Recently, dopamine (DA) neurons of the substantia nigra pars compacta (SNc) and adjacent ventral tegmental area (VTA) have been revealed to be diverse not only when comparing between these two dopaminergic nuclei, but within them, and with the distribution of identified subtypes often agnostic to traditional neuroanatomical boundaries (Saunders et al., 2018a; Hook et al., 2018; Kramer et al., 2018; La Manno et al., 2016; Poulin et al., 2014; Tiklova et al., 2019; Poulin et al., 2018). Such molecularly defined subpopulations have been the subject of several recent studies. Investigations of these subtypes have ultimately unveiled many distinctive properties across several domains, such as their axonal projections and functional properties (Poulin et al., 2018; Wu et al., 2019; Pereira Luppi et al., 2021; Evans et al., 2017; Evans et al., 2020). These key differences between subtypes have begun to corroborate the biological relevance of DA neuron taxonomic schemes. We hypothesize that these putative molecular subtypes, with their distinctive circuits, could shed light on the wide variety of dopamine-related symptoms observed across several diseases including depression, chronic pain, addiction, and Parkinson's Disease. While it is difficult to reconcile how a single neurotransmitter can be involved in so many seemingly unrelated phenotypes, one solution could be the existence of several individual dopaminergic pathways serving different functions, with molecular subtypes serving as distinct nodes for these pathways. Indeed, this conceptual framework is already the dogma for anatomically distinct DA pathways, including the mesocortical, mesolimbic and mesostriatal pathways (Bjorklund & Dunnett, 2007). Here, we discuss our existing knowledge of DA neuron subtypes and attempt to provide a roadmap for how their distinctive properties can provide novel insights into the motor symptoms of Parkinson's disease (PD) (Fig. 1A). By exploring the differences between molecular subtypes and correlating this to their relative degeneration within the SNc, we may gain a deeper understanding of the cell-intrinsic mechanisms underlying why some DA neurons degenerate more than others in PD. Similarly, by mapping the inputs, projections, and functions of individual subtypes, we may better understand their individual roles in the circuit-level dysfunction of dopaminergic diseases.

Molecular and spatial transcriptomic classification of midbrain dopamine neurons and their alterations in a LRRK2G2019S model of Parkinson's disease.

Several studies have revealed that midbrain dopamine (DA) neurons, even within a single neuroanatomical area, display heterogeneous properties. In parallel, studies using single cell profiling techniques have begun to cluster DA neurons into subtypes based on their molecular signatures. Recent work has shown that molecularly defined DA subtypes within the substantia nigra (SNc) display distinctive anatomic and functional properties, and differential vulnerability in Parkinson's disease (PD). Based on these provocative results, a granular understanding of these putative subtypes and their alterations in PD models, is imperative. We developed an optimized pipeline for single-nuclear RNA sequencing (snRNA-seq) and generated a high-resolution hierarchically organized map revealing 20 molecularly distinct DA neuron subtypes belonging to three main families. We integrated this data with spatial MERFISH technology to map, with high definition, the location of these subtypes in the mouse midbrain, revealing heterogeneity even within neuroanatomical sub-structures. Finally, we demonstrate that in the preclinical LRRK2G2019S knock-in mouse model of PD, subtype organization and proportions are preserved. Transcriptional alterations occur in many subtypes including those localized to the ventral tier SNc, where differential expression is observed in synaptic pathways, which might account for previously described DA release deficits in this model. Our work provides an advancement of current taxonomic schemes of the mouse midbrain DA neuron subtypes, a high-resolution view of their spatial locations, and their alterations in a prodromal mouse model of PD.

Unique functional responses differentially map onto genetic subtypes of dopamine neurons.

Dopamine neurons are characterized by their response to unexpected rewards, but they also fire during movement and aversive stimuli. Dopamine neuron diversity has been observed based on molecular expression profiles; however, whether different functions map onto such genetic subtypes remains unclear. In this study, we established that three genetic dopamine neuron subtypes within the substantia nigra pars compacta, characterized by the expression of Slc17a6 (Vglut2), Calb1 and Anxa1, each have a unique set of responses to rewards, aversive stimuli and accelerations and decelerations, and these signaling patterns are highly correlated between somas and axons within subtypes. Remarkably, reward responses were almost entirely absent in the Anxa1+ subtype, which instead displayed acceleration-correlated signaling. Our findings establish a connection between functional and genetic dopamine neuron subtypes and demonstrate that molecular expression patterns can serve as a common framework to dissect dopaminergic functions.

Sox6 expression distinguishes dorsally and ventrally biased dopamine neurons in the substantia nigra with distinctive properties and embryonic origins.

Dopamine (DA) neurons in the ventral tier of the substantia nigra pars compacta (SNc) degenerate prominently in Parkinson's disease, while those in the dorsal tier are relatively spared. Defining the molecular, functional, and developmental characteristics of each SNc tier is crucial to understand their distinct susceptibility. We demonstrate that Sox6 expression distinguishes ventrally and dorsally biased DA neuron populations in the SNc. The Sox6+ population in the ventral SNc includes an Aldh1a1+ subset and is enriched in gene pathways that underpin vulnerability. Sox6+ neurons project to the dorsal striatum and show activity correlated with acceleration. Sox6- neurons project to the medial, ventral, and caudal striatum and respond to rewards. Moreover, we show that this adult division is encoded early in development. Overall, our work demonstrates a dual origin of the SNc that results in DA neuron cohorts with distinct molecular profiles, projections, and functions.

Classification of Midbrain Dopamine Neurons Using Single-Cell Gene Expression Profiling Approaches.

Dysfunctional dopamine (DA) signaling has been associated with a broad spectrum of neuropsychiatric disorders, prompting investigations into how midbrain DA neuron heterogeneity may underpin this variety of behavioral symptoms. Emerging literature indeed points to functional heterogeneity even within anatomically defined DA clusters. Recognizing the need for a systematic classification scheme, several groups have used single-cell profiling to catalog DA neurons based on their gene expression profiles. We aim here not only to synthesize points of congruence but also to highlight key differences between the molecular classification schemes derived from these studies. In doing so, we hope to provide a common framework that will facilitate investigations into the functions of DA neuron subtypes in the healthy and diseased brain.

Rotating Magnetic Nanoparticle Clusters as Microdevices for Drug Delivery.

BACKGROUND: Magnetic nanoparticles (MNPs) hold promise for enhancing delivery of therapeutic agents, either through direct binding or by functioning as miniature propellers. Fluid-filled conduits and reservoirs within the body offer avenues for MNP-enhanced drug delivery. MNP clusters can be rotated and moved across surfaces at clinically relevant distances in response to a rotating magnet. Limited data are available regarding issues affecting MNP delivery by this mechanism, such as adhesion to a cellular wall. Research reported here was initiated to better understand the fundamental principles important for successful implementation of rotational magnetic drug targeting (rMDT). METHODS: Translational movements of four different iron oxide MNPs were tested, in response to rotation (3 Hz) of a neodymium-boron-iron permanent magnet. MNP clusters moved along biomimetic channels of a custom-made acrylic tray, by surface walking. The effects of different distances and cellular coatings on MNP velocity were analyzed using videography. Dyes (as drug surrogates) and the drug etoposide were transported by rotating MNPs along channels over a 10 cm distance. RESULTS: MNP translational velocities could be predicted from magnetic separation times. Changes in distance or orientation from the magnet produced alterations in MNP velocities. Mean velocities of the fastest MNPs over HeLa, U251, U87, and E297 cells were 0.24 ± 0.02, 0.26 ± 0.02, 0.28 ± 0.01, and 0.18 ± 0.03 cm/sec, respectively. U138 cells showed marked MNP adherence and an 87.1% velocity reduction at 5.5 cm along the channel. Dye delivery helped visualize the effects of MNPs as microdevices for drug delivery. Dye delivery by MNP clusters was 21.7 times faster than by diffusion. MNPs successfully accelerated etoposide delivery, with retention of chemotherapeutic effect. CONCLUSION: The in vitro system described here facilitates side-by-side comparisons of drug delivery by rotating MNP clusters, on a human scale. Such microdevices have the potential for augmenting drug delivery in a variety of clinical settings, as proposed.