mDA Neurons Research Articles

Circular RNAs regulate neuron size and migration of midbrain dopamine neurons during development

Midbrain dopamine (mDA) neurons play an essential role in cognitive and motor behaviours and are linked to different brain disorders. However, the molecular mechanisms underlying their development, and in particular the role of non-coding RNAs (ncRNAs), remain incompletely understood. Here, we establish the transcriptomic landscape and alternative splicing patterns of circular RNAs (circRNAs) at key developmental timepoints in mouse mDA neurons in vivo using fluorescence-activated cell sorting followed by short- and long-read RNA sequencing. In situ hybridisation shows expression of several circRNAs during early mDA neuron development and post-transcriptional silencing unveils roles for different circRNAs in regulating mDA neuron morphology. Finally, in utero electroporation and time-lapse imaging implicate circRmst, a circRNA with widespread morphological effects, in the migration of developing mDA neurons in vivo. Together, these data for the first time suggest a functional role for circRNAs in developing mDA neurons and characterise poorly defined aspects of mDA neuron development.

Multi-omics integration of scRNA-seq time series data predicts new intervention points for Parkinson’s disease

Parkinson’s disease (PD) is a complex neurodegenerative disorder without a cure. The onset of PD symptoms corresponds to 50% loss of midbrain dopaminergic (mDA) neurons, limiting early-stage understanding of PD. To shed light on early PD development, we study time series scRNA-seq datasets of mDA neurons obtained from patient-derived induced pluripotent stem cell differentiation. We develop a new data integration method based on Non-negative Matrix Tri-Factorization that integrates these datasets with molecular interaction networks, producing condition-specific “gene embeddings”. By mining these embeddings, we predict 193 PD-related genes that are largely supported (49.7%) in the literature and are specific to the investigated PINK1 mutation. Enrichment analysis in Kyoto Encyclopedia of Genes and Genomes pathways highlights 10 PD-related molecular mechanisms perturbed during early PD development. Finally, investigating the top 20 prioritized genes reveals 12 previously unrecognized genes associated with PD that represent interesting drug targets.

Transcriptomic atlas of midbrain dopamine neurons uncovers differential vulnerability in a Parkinsonism lesion model

Midbrain dopamine (mDA) neurons comprise diverse cells with unique innervation targets and functions. This is illustrated by the selective sensitivity of mDA neurons of the substantia nigra compacta (SNc) in patients with Parkinson’s disease, while those in the ventral tegmental area (VTA) are relatively spared. Here, we used single nuclei RNA sequencing (snRNA-seq) of approximately 70,000 mouse midbrain cells to build a high-resolution atlas of mouse mDA neuron diversity at the molecular level. The results showed that differences between mDA neuron groups could best be understood as a continuum without sharp differences between subtypes. Thus, we assigned mDA neurons to several ‘territories’ and ‘neighborhoods’ within a shifting gene expression landscape where boundaries are gradual rather than discrete. Based on the enriched gene expression patterns of these territories and neighborhoods, we were able to localize them in the adult mouse midbrain. Moreover, because the underlying mechanisms for the variable sensitivities of diverse mDA neurons to pathological insults are not well understood, we analyzed surviving neurons after partial 6-hydroxydopamine (6-OHDA) lesions to unravel gene expression patterns that correlate with mDA neuron vulnerability and resilience. Together, this atlas provides a basis for further studies on the neurophysiological role of mDA neurons in health and disease.

Transcriptomic atlas of midbrain dopamine neurons uncovers differential vulnerability in a Parkinsonism lesion model.

Midbrain dopamine (mDA) neurons comprise diverse cells with unique innervation targets and functions. This is illustrated by the selective sensitivity of mDA neurons of the substantia nigra compacta (SNc) in patients with Parkinson's disease, while those in the ventral tegmental area (VTA) are relatively spared. Here, we used single nuclei RNA sequencing (snRNA-seq) of approximately 70,000 mouse midbrain cells to build a high-resolution atlas of mouse mDA neuron diversity at the molecular level. The results showed that differences between mDA neuron groups could best be understood as a continuum without sharp differences between subtypes. Thus, we assigned mDA neurons to several 'territories' and 'neighborhoods' within a shifting gene expression landscape where boundaries are gradual rather than discrete. Based on the enriched gene expression patterns of these territories and neighborhoods, we were able to localize them in the adult mouse midbrain. Moreover, because the underlying mechanisms for the variable sensitivities of diverse mDA neurons to pathological insults are not well understood, we analyzed surviving neurons after partial 6-hydroxydopamine (6-OHDA) lesions to unravel gene expression patterns that correlate with mDA neuron vulnerability and resilience. Together, this atlas provides a basis for further studies on the neurophysiological role of mDA neurons in health and disease.

Structural insights into the recognition of the A/T-rich motif in target gene promoters by the LMX1a homeobox domain.

LIM homeodomain transcription factor 1-alpha (LMX1a) is a neuronal lineage-specific transcription activator that plays an essential role during the development of midbrain dopaminergic (mDA) neurons. LMX1a induces the expression of multiple key genes, which ultimately determine the morphology, physiology, and functional identity of mDA neurons. This function of LMX1a is dependent on its homeobox domain. Here, we determined the structures of the LMX1a homeobox domain in complex with the promoter sequences of the Wnt family member 1 (WNT1) or paired like homeodomain 3 (Pitx3) gene, respectively. The complex structures revealed that the LMX1a homeobox domain employed its α3 helix and an N-terminal loop to achieve specific target recognition. The N-terminal loop (loop1) interacted with the minor groove of the double-stranded DNA (dsDNA), whereas the third α-helix (α3) was tightly packed into the major groove of the dsDNA. Structure-based mutations in the α3 helix of the homeobox domain significantly reduced the binding affinity of LMX1a to dsDNA. Moreover, we identified a nonsyndromic hearing loss (NSHL)-related mutation, R199, which yielded a more flexible loop and disturbed the recognition in the minor groove of dsDNA, consistent with the molecular dynamics (MD) simulations. Furthermore, overexpression of Lmx1a promoted the differentiation of SH-SY5Y cells and upregulated the transcription of WNT1 and PITX3 genes. Hence, our work provides a detailed elucidation of the specific recognition between the LMX1a homeobox domain and its specific dsDNA targets, which represents valuable information for future investigations of the functional pathways that are controlled by LMX1a during mDA neuron development.

A dual-omics approach on the effects of fibroblast growth factor-2 (FGF-2) on ventral tegmental area dopaminergic neurons in response to alcohol consumption in mice.

Harmful alcohol consumption is a major socioeconomic burden to the health system, as it can be the cause of mortality of heavy alcohol drinkers. The dopaminergic (DAergic) system is thought to play an important role in the pathogenesis of alcohol drinking behaviour; however, its exact role remains elusive. Fibroblast growth factor 2 (FGF-2), a neurotrophic factor, associated with both the DAergic system and alcohol consumption, may play an important role in DAergic neuroadaptations during alcohol abuse. Within this study, we aimed to clarify the role of endogenous FGF-2 on the DAergic system and whether there is a possible link to alcohol consumption. We found that lack of FGF-2 reduces the alcohol intake of mice. Transcriptome analysis of DAergic neurons revealed that FGF-2 knockout (FGF-2 KO) shifts the molecular fingerprint of midbrain dopaminergic (mDA) neurons to DA subtypes of the ventral tegmental area (VTA). In line with this, proteomic changes predominantly appear also in the VTA. Interestingly, these changes led to an altered regulation of the FGF-2 signalling cascades and DAergic pathways in a region-specific manner, which was only marginally affected by voluntary alcohol consumption. Thus, lack of FGF-2 not only affects the gene expression but also the proteome of specific brain regions of mDA neurons. Our study provides new insights into the neuroadaptations of the DAergic system during alcohol abuse and, therefore, comprises novel targets for future pharmacological interventions.

Host-to-graft propagation of inoculated α-synuclein into transplanted human induced pluripotent stem cell-derived midbrain dopaminergic neurons

IntroductionCell therapeutic clinical trials using fetal mesencephalic tissue provided a proof-of-concept for regenerative therapy in patients with Parkinson's disease. Postmortem studies of patients with fetal grafts revealed that α-synuclein+ Lewy body (LB)-like inclusions emerged in long-term transplantation and might worsen clinical outcomes even if the grafts survived and innervated in the recipients. Various studies aimed at addressing whether host-derived α-synuclein could be transferred to the grafted neurons to assess α-synuclein+ inclusion appearance in the grafts. However, determining whether α-synuclein in the grafted neurons has been propagated from the host is difficult due to the intrinsic α-synuclein expression. MethodsWe induced midbrain dopaminergic (mDA) neurons from human induced pluripotent stem cells (hiPSCs) and transplanted them into the striatum of immunodeficient rats. The recombinant human α-synuclein preformed fibrils (PFFs) were inoculated into the cerebral cortex after transplantation of SNCA−/− hiPSC-derived mDA neural progenitors into the striatum of immunodeficient rats to evaluate the host-to-graft propagation of human α-synuclein PFFs. Additionally, we examined the incorporation of human α-synuclein PFFs into SNCA−/− hiPSC-derived mDA neurons using in vitro culture system. ResultsWe detected human α-synuclein-immunoreactivity in SNCA−/− hiPSC-derived mDA neurons that lacked endogenous α-synuclein expression in vitro. Additionally, we observed host-to-graft α-synuclein propagation into the grafted SNCA−/− hiPSC-derived mDA neurons. ConclusionWe have successfully proven that intracerebral inoculated α-synuclein PFFs are propagated and incorporated from the host into grafted SNCA−/− hiPSC-derived mDA neurons. Our results contribute toward the basic understanding of the molecular mechanisms related to LB-like α-synuclein deposit formation in grafted mDA neurons.

Secretome Analyses Identify FKBP4 as a GBA1-Associated Protein in CSF and iPS Cells from Parkinson's Disease Patients with GBA1 Mutations.

Mutations in the GBA1 gene increase the risk of developing Parkinson's disease (PD). However, most carriers of GBA1 mutations do not develop PD throughout their lives. The mechanisms of how GBA1 mutations contribute to PD pathogenesis remain unclear. Cerebrospinal fluid (CSF) is used for detecting pathological conditions of diseases, providing insights into the molecular mechanisms underlying neurodegenerative disorders. In this study, we utilized the proximity extension assay to examine the levels of metabolism-linked protein in the CSF from 17 PD patients carrying GBA1 mutations (GBA1-PD) and 17 idiopathic PD (iPD). The analysis of CSF secretome in GBA1-PD identified 11 significantly altered proteins, namely FKBP4, THOP1, GLRX, TXNDC5, GAL, SEMA3F, CRKL, APLP1, LRP11, CD164, and NPTXR. To investigate GBA1-associated CSF changes attributed to specific neuronal subtypes responsible for PD, we analyzed the cell culture supernatant from GBA1-PD-induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic (mDA) neurons. The secretome analysis of GBA1-PD iPSC-derived mDA neurons revealed that five differently regulated proteins overlapped with those identified in the CSF analysis: FKBP4, THOP1, GLRX, GAL, and CRKL. Reduced intracellular level of the top hit, FKPB4, was confirmed via Western Blot. In conclusion, our findings identify significantly altered CSF GBA1-PD-associated proteins with FKPB4 being firmly attributed to mDA neurons.

The impact of the mesoprefrontal dopaminergic system on the maturation of interneurons in the murine prefrontal cortex.

The prefrontal cortex (PFC) undergoes a protracted maturation process. This is true both for local interneurons and for innervation from midbrain dopaminergic (mDA) neurons. In the striatum, dopaminergic (DA) neurotransmission is required for the maturation of medium spiny neurons during a critical developmental period. To investigate whether DA innervation influences the maturation of interneurons in the PFC, we used a conditional knockout (cKO) mouse model in which innervation from mDA neurons to the mPFC (mesoprefrontal innnervation) is not established during development. In this mouse model, the maturation of parvalbumin (PV) and calbindin (CB) interneuron populations in the PFC is dysregulated during a critical period in adolescence with changes persisting into adulthood. PV interneurons are particularly vulnerable to lack of mesoprefrontal input, showing an inability to maintain adequate PV expression with a concomitant decrease in Gad1 expression levels. Interestingly, lack of mesoprefrontal innervation does not appear to induce compensatory changes such as upregulation of DA receptor expression in PFC neurons or increased innervation density of other neuromodulatory (serotonergic and noradrenergic) innervation. In conclusion, our study shows that adolescence is a sensitive period during which mesoprefrontal input plays a critical role in promoting the maturation of specific interneuron subgroups. The results of this study will help to understand how a dysregulated mesoprefrontal DA system contributes to the pathophysiology of neurodevelopmental disorders.

2,4-Dichlorophenoxyacetic Acid Induces Degeneration of mDA Neurons In Vitro.

Background: Parkinson's disease (PD) affects 1-2% of the population over the age of 60 and the majority of PD cases are sporadic, without any family history of the disease. Neuroinflammation driven by microglia has been shown to promote the progression of midbrain dopaminergic (mDA) neuron loss through the release of neurotoxic factors. Interestingly, the risk of developing PD is significantly higher in distinct occupations, such as farming and agriculture, and is linked to the use of pesticides and herbicides. Methods: The neurotoxic features of 2,4-Dichlorophenoxyacetic acid (2,4D) at concentrations of 10 µM and 1 mM were analyzed in two distinct E14 midbrain neuron culture systems and in primary microglia. Results: The application of 1 mM 2,4D resulted in mDA neuron loss in neuron-enriched cultures. Notably, 2,4D-induced neurotoxicity significantly increased in the presence of microglia in neuron-glia cultures, suggesting that microglia-mediated neurotoxicity could be one mechanism for progressive neuron loss in this in vitro setup. However, 2,4D alone was unable to trigger microglia reactivity. Conclusions: Taken together, we demonstrate that 2,4D is neurotoxic for mDA neurons and that the presence of glia cells enhances 2,4D-induced neuron death. These data support the role of 2,4D as a risk factor for the development and progression of PD and further suggest the involvement of microglia during 2,4D-induced mDA neuron loss.

Engrailed: Pathological and physiological effects of a multifunctional developmental gene.

Engrailed-1 (EN1) is a developmental gene that encodes En1, a highly conserved transcription factor involved in regionalization during early embryogenesis and in the later maintenance of normal neurons. After birth, EN1 still plays a role in the development and physiology of the body; for example, it exerts a protective effect on midbrain dopaminergic (mDA) neurons, and loss of EN1 causes mDA neurons in the ventral midbrain to gradually die approximately 6 weeks after birth, resulting in motor and nonmotor symptoms similar to those observed in Parkinson's disease. Notably, EN1 has been identified as a possible susceptibility gene for idiopathic Parkinson's disease in humans. EN1 is involved in the processes of wound-healing scar production and tissue and organ fibrosis. Additionally, EN1 can lead to tumorigenesis and thus provides a target for the treatment of some tumors. In this review, we summarize the effects of EN1 on embryonic organ development, describe the consequences of the deletion or overexpression of the EN1 gene, and discuss the pathways in which EN1 is involved. We hope to clarify the role of EN1 as a developmental gene and present potential therapeutic targets for diseases involving the EN1 gene.

A defined method for differentiating human iPSCs into midbrain dopaminergic progenitors that safely restore motor deficits in Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative condition that primarily affects motor functions; it is caused by the loss of midbrain dopaminergic (mDA) neurons. The therapeutic effects of transplanting human-induced pluripotent stem cell (iPSC)-derived mDA neural progenitor cells in animal PD models are known and are being evaluated in an ongoing clinical trial. However, However, improvements in the safety and efficiency of differentiation-inducing methods are crucial for providing a larger scale of cell therapy studies. This study aimed to investigate the usefulness of dopaminergic progenitor cells derived from human iPSCs by our previously reported method, which promotes differentiation and neuronal maturation by treating iPSCs with three inhibitors at the start of induction. Healthy subject-derived iPS cells were induced into mDA progenitor cells by the CTraS-mediated method we previously reported, and their proprieties and dopaminergic differentiation efficiency were examined in vitro. Then, the induced mDA progenitors were transplanted into 6-hydroxydopamine-lesioned PD model mice, and their efficacy in improving motor function, cell viability, and differentiation ability in vivo was evaluated for 16 weeks. Approximately ≥80% of cells induced by this method without sorting expressed mDA progenitor markers and differentiated primarily into A9 dopaminergic neurons in vitro. After transplantation in 6-hydroxydopamine-lesioned PD model mice, more than 90% of the engrafted cells differentiated into the lineage of mDA neurons, and approximately 15% developed into mature mDA neurons without tumour formation. The grafted PD model mice also demonstrated significantly improved motor functions. This study suggests that the differentiation protocol for the preparation of mDA progenitors is a promising option for cell therapy in patients with PD.

Phenotypic Profiling of Human Stem Cell-Derived Midbrain Dopaminergic Neurons.

Parkinson's disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro PD cellular models lack complexity and do not take multiple phenotypes into account. Phenotypic profiling in human induced pluripotent stem cell (iPSC)-derived mDA neurons can address these shortcomings by simultaneously measuring a range of neuronal phenotypes in a PD-relevant cell type in parallel. Here, we describe a protocol to obtain and analyze phenotypic profiles from commercially available human mDA neurons. A neuron-specific fluorescent staining panel is used to visualize the nuclear, α-synuclein, Tyrosine hydroxylase (TH), and Microtubule-associated protein 2 (MAP2) related phenotypes. The described phenotypic profiling protocol is scalable as it uses 384-well plates, automatic liquid handling and high-throughput microscopy. The utility of the protocol is exemplified using healthy donor mDA neurons and mDA neurons carrying the PD-linked G2019S mutation in the Leucine-rich repeat kinase 2 (LRRK2) gene. Both cell lines were treated with the LRRK2 kinase inhibitor PFE-360 and phenotypic changes were measured. Additionally, we demonstrate how multidimensional phenotypic profiles can be analyzed using clustering or machine learning-driven supervised classification methods. The described protocol will particularly interest researchers working on neuronal disease modeling or studying chemical compound effects in human neurons.

Expression and role of nicotinic acetylcholine receptors during midbrain dopaminergic neuron differentiation from human induced pluripotent stem cells.

Nicotinic acetylcholine receptors (nAChRs) expressed in midbrain dopaminergic (mDA) neurons modulate mDA neuronal activity. However, their expression patterns and functional roles during mDA neuronal development remain unknown. Here, we profiled the expression and function of nAChR subtypes during mDA neuron differentiation from human induced pluripotent stem cells (hiPSCs). Midbrain dopaminergic neurons were differentiated from hiPSCs using a recently developed proprietary method that replicates midbrain development. The expression patterns of developmental marker proteins were monitored during mDA neuronal differentiation using immunohistochemical analysis. Gene expression of nAChR subtypes was analyzed by reverse transcription polymerase chain reaction. Pharmacological nAChR agonists and antagonists were used to reveal the role of the α6 nAChR subunit in the differentiation of mDA neurons from hiPSCs. CHRNA4 expression was detected at the mDA neural progenitor stage, whereas CHRNA6 expression began during the mDA neuronal stage. CHRNA7 was expressed throughout the differentiation process, including in the undifferentiated hiPSCs. We also found that LMO3, a gene expressed in a subset of substantia nigra pars compacta (SNC) DA neurons in the midbrain, showed increased expression following nicotine treatment in a concentration-dependent manner. Additionally, 5-iodo A85380, a selective α6 nAChR agonist, also increased LMO3 expression in hiPSC-derived mDA neurons, and this increase was suppressed by simultaneous treatment with bPiDi, a selective α6 nAChR antagonist. Our findings suggest that stimulating the α6 nAChR subunit on hiPSC-derived mDA neurons may induce neuronal maturation that is biased toward SNC DA neurons.

The Crucial Roles of Pitx3 in Midbrain Dopaminergic Neuron Development and Parkinson's Disease-Associated Neurodegeneration.

The degeneration of midbrain dopaminergic (mDA) neurons, particularly in the substantia nigra pars compacta (SNc), is one of the most prominent pathological hallmarks of Parkinson's disease (PD). To uncover the pathogenic mechanisms of mDA neuronal death during PD may provide therapeutic targets to prevent mDA neuronal loss and slow down the disease's progression. Paired-like homeodomain transcription factor 3 (Pitx3) is selectively expressed in the mDA neurons as early as embryonic day 11.5 and plays a critical role in mDA neuron terminal differentiation and subset specification. Moreover, Pitx3-deficient mice exhibit some canonical PD-related features, including the profound loss of SNc mDA neurons, a dramatic decrease in striatal dopamine (DA) levels, and motor abnormalities. However, the precise role of Pitx3 in progressive PD and how this gene contributes to mDA neuronal specification during early stages remains unclear. In this review, we updated the latest findings on Pitx3 by summarizing the crosstalk between Pitx3 and its associated transcription factors in mDA neuron development. We further explored the potential benefits of Pitx3 as a therapeutic target for PD in the future. To better understand the transcriptional network of Pitx3 in mDA neuron development may provide insights into Pitx3-related clinical drug-targeting research and therapeutic approaches.

Therapeutic improvement of Parkinson’s disease transplantation therapy through modulating the immune system.

Abstract We report clinically the implantation of patient-derived midbrain dopaminergic progenitor cells (mDAPs), differentiated in vitrofrom autologous iPSCs, in a patient with idiopathic Parkinson's disease (PD). The patient-specific mDAPs were produced under Good Manufacturing Practice conditions and characterized as having the phenotypic properties of substantia nigra pars compacta neurons; testing in a humanized mouse model indicated an absence of immunogenicity to these cells. The cells were implanted into the putamen (left hemisphere followed by right hemisphere, 6 months apart) of a patient with PD, without the need for immunosuppression. Clinical measures of symptoms of PD after surgery stabilized or improved at 18 to 24 months after implantation. Although our previous data (Schweitzer*, Park* et al., NEJM, 382(20) p1926–1932, 2020) showed that using autologous mDAPs can avoid graft rejection, cell survival and functional recovery in the first patient as assessed by 18F-DOPA PET scan analysis and clinical motor assessments was modest, suggesting that survival of mDA neurons may have been suboptimal even in autologous implantation. We have developed a safer and more effective treatment by not only increasing mDA neuron survival by simultaneously controlling the causative immune response, but also by modulating the problematic proliferative property, which is a characteristic of progenitor cells after implantation. In conclusion, our new study suggests that initial, specific mDA neuron survival in these grafts is strongly affected by the host innate immune response, while long-term graft survival is subject to the adaptive immune response, elucidating a two-phase involvement of the immune system for graft survival. This work was supported by NIH grants (NS070577 and OD024622), the Parkinson’s Cell Therapy Research Fund at McLean Hospital and Massachusetts General Hospital, and the Masson Family Endowed Scholar in Neurosurgery, and the George A. Lopez, MD Endowed Chair in Neurosurgery.

Development, wiring and function of dopamine neuron subtypes.

The midbrain dopamine (mDA) system is composed of molecularly and functionally distinct neuron subtypes that mediate specific behaviours and are linked to various brain diseases. Considerable progress has been made in identifying mDA neuron subtypes, and recent work has begun to unveil how these neuronal subtypes develop and organize into functional brain structures. This progress is important for further understanding the disparate physiological functions of mDA neurons and their selective vulnerability in disease, and will ultimately accelerate therapy development. This Review discusses recent advances in our understanding of molecularly defined mDA neuron subtypes and their circuits, ranging from early developmental events, such as neuron migration and axon guidance, to their wiringand function, and future implications for therapeutic strategies.

Single-cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation.

Stem cell technologies provide new opportunities for modeling cells in health and disease and for regenerative medicine. In both cases, developmental knowledge and defining the molecular properties and quality of the cell types is essential. In this study, we identify developmental factors important for the differentiation of human embryonic stem cells (hESCs) into functional midbrain dopaminergic (mDA) neurons. We found that laminin-511, and dual canonical and non-canonical WNT activation followed by GSK3β inhibition plus FGF8b, improved midbrain patterning. In addition, neurogenesis and differentiation were enhanced by activation of liver X receptors and inhibition of fibroblast growth factor signaling. Moreover, single-cell RNA-sequencing analysis revealed a developmental dynamics similar to that of the endogenous human ventral midbrain and the emergence of high-quality molecularly defined midbrain cell types, including mDA neurons. Our study identifies novel factors important for human midbrain development and opens the door for a future application of molecularly defined hESC-derived cell types in Parkinson disease.

Human iPSC-derived midbrain organoids functionally integrate into striatum circuits and restore motor function in a mouse model of Parkinson's disease.

Rationale: Parkinson's disease (PD) is a prevalent neurodegenerative disorder that is characterized by degeneration of dopaminergic neurons (DA) at the substantia nigra pas compacta (SNpc). Cell therapy has been proposed as a potential treatment option for PD, with the aim of replenishing the lost DA neurons and restoring motor function. Fetal ventral mesencephalon tissues (fVM) and stem cell-derived DA precursors cultured in 2-dimentional (2-D) culture conditions have shown promising therapeutic outcomes in animal models and clinical trials. Recently, human induced pluripotent stem cells (hiPSC)-derived human midbrain organoids (hMOs) cultured in 3-dimentional (3-D) culture conditions have emerged as a novel source of graft that combines the strengths of fVM tissues and 2-D DA cells. Methods: 3-D hMOs were induced from three distinct hiPSC lines. hMOs at various stages of differentiation were transplanted as tissue pieces into the striatum of naïve immunodeficient mouse brains, with the aim of identifying the most suitable stage of hMOs for cellular therapy. The hMOs at Day 15 were determined to be the most appropriate stage and were transplanted into a PD mouse model to assess cell survival, differentiation, and axonal innervation in vivo. Behavioral tests were conducted to evaluate functional restoration following hMO treatment and to compare the therapeutic effects between 2-D and 3-D cultures. Rabies virus were introduced to identify the host presynaptic input onto the transplanted cells. Results: hMOs showed a relatively homogeneous cell composition, mostly consisting of dopaminergic cells of midbrain lineage. Analysis conducted 12 weeks post-transplantation of day 15 hMOs revealed that 14.11% of the engrafted cells expressed TH+ and over 90% of these cells were co-labeled with GIRK2+, indicating the survival and maturation of A9 mDA neurons in the striatum of PD mice. Transplantation of hMOs led to a reversal of motor function and establishment of bidirectional connections with natural brain target regions, without any incidence of tumor formation or graft overgrowth. Conclusion: The findings of this study highlight the potential of hMOs as safe and efficacious donor graft sources for cell therapy to treat PD.

Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson’s disease in midbrain dopaminergic neurons

Mutations in the SNCA gene cause autosomal dominant Parkinson’s disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small β-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34–41 post differentiation. Once midbrain identity fully developed, at day 48–62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.