Survival Of mDA Neurons Research Articles

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.

Panning for gold: Purifying mesencephalic dopaminergic progenitors differentiated from human pluripotent stem cells.

In a recently published study, Xu et al. used two surface markers, CLSTN2 and PTPRO, to generate highly purified donor dopaminergic neurons and achieved stable and predictable therapeutic outcomes by transplantation into the brain of PD animal models (Xu et al., 2022).

The netrin-1 receptor DCC promotes the survival of a subpopulation of midbrain dopaminergic neurons: Relevance for ageing and Parkinson's disease.

Mechanisms that determine the survival of midbrain dopaminergic (mDA) neurons in the adult central nervous system (CNS) are not fully understood. Netrins are a family of secreted proteins that are essential for normal neural development. In the mature CNS, mDA neurons express particularly high levels of netrin‐1 and its receptor Deleted in Colorectal Cancer (DCC). Recent findings indicate that overexpressing netrin‐1 protects mDA neurons in animal models of Parkinson’s disease (PD), with a proposed pro‐apoptotic dependence function for DCC that triggers cell death in the absence of a ligand. Here, we sought to determine if DCC expression influences mDA neuron survival in young adult and ageing mice. To circumvent the perinatal lethality of DCC null mice, we selectively deleted DCC from mDA neurons utilizing DATcre/loxP gene‐targeting and examined neuronal survival in adult and aged animals. Reduced numbers of mDA neurons were detected in the substantia nigra pars compacta (SNc) of young adult DATcre/DCCfl/fl mice, with further reduction in aged DATcre/DCCfl/fl animals. In contrast to young adults, aged mice also exhibited a gene dosage effect, with fewer SNc mDA neurons in DCC heterozygotes (DATcre/DCCfl/wt). Notably, loss of mDA neurons in the SN was not uniform. Neuronal loss in the SN was limited to ventral tier mDA neurons, while mDA neurons in the dorsal tier of the SN, which resist degeneration in PD, were spared from the effect of DCC deletion in both young and aged mice. In the ventral tegmental area (VTA), young adult mice with conditional deletion of DCC had normal mDA neuronal numbers, while significant loss occurred in aged DATcre/DCCfl/fl and DATcre/DCCfl/wt mice compared to age‐matched wild‐type mice. Our results indicate that expression of DCC is required for the survival of subpopulations of mDA neurons and may be relevant to the degenerative processes in PD.

Spatiotemporal expression of sonic hedgehog signalling molecules in the embryonic mesencephalic dopaminergic neurons

Midbrain dopaminergic neurons (mDA) play an important role in controlling the voluntary motor movement, reward, and emotion-based behaviour. Differentiation of mDA neurons from progenitors depends on several secreted proteins, such as sonic hedgehog (SHH). The present study attempted to elucidate the possible role(s) of some SHH signaling components (Ptch1, Gli1, Gli2 and Gli3) in the spatiotemporal development of mDA neurons along the rostrocaudal axis of the midbrain and their possible roles in differentiation and survival of mDA neurons and the significance of using in vitro models for studying the development of mDA neurons. At E12 and E14, only Ptch1 and Gli1 were expressed in ventrolateral midbrain domains. All examined SHH signalling molecules were not detected in mDA area. Whereas, in MN9D cells, many SHH signalling molecules were expressed and co-localized with the dopaminergic marker; tyrosine hydroxylase (TH), and their expression were upregulated with SHH treatment of the MN9D cells. These results suggest that mDA neurons differentiation and survival might be independent of SHH in the late developmental stages (E12-18). Besides, MN9D cell line is not the ideal in vitro model for investigating the differentiation of mDA and hence, the ventral midbrain primary culture might be favored over MN9D line.

Lipopolysaccharide-Induced Microglia Activation Promotes the Survival of Midbrain Dopaminergic Neurons In Vitro

Microglia are the resident immune cells of the central nervous system (CNS) and respond to a variety of endogenous and exogenous stimuli in order to restore cell and tissue homeostasis. Lipopolysaccharide (LPS) is one of these exogenous stimuli, constitutes a major component of the outer membrane of Gram-negative bacteria, and binds to the microglial pattern recognition receptor Toll-like receptor 4 (TLR4). LPS-induced microglia activation is believed to promote neurodegeneration by release of neurotoxic factors such as interleukin-1β, tumor necrosis factor α, or nitric oxide. In the present study, we investigated whether the physical presence of microglia is required to promote neurotoxicity and whether microglia-derived factors are essential. Interestingly, we observed that dopaminergic (mDA) neuron survival was only affected in mixed neuron-glia cultures containing microglia but not in neuron-enriched cultures. Moreover, we clearly demonstrate that microglia-conditioned medium (MCM) after LPS treatment increased mDA neuron survival, process numbers as well as process length. The observed protective effects of MCM was rather caused by microglia-derived factors and only partially dependent on the increase in reactive astrocytes. These results indicate that LPS-induced microglia activation does not necessarily have detrimental effects on mDA neurons and further support the hypothesis that activated microglia support neuron survival by release of neurotrophic and neuroprotective factors.

Dopamine neuron loss by selective deletion of autophagy-related gene 5 is not exacerbated by MPTP toxicity in midbrain

Parkinson’s disease (PD) is a progressive neurological disease, one of the pathological characteristics is a gradual loss of midbrain dopaminergic (mDA) neurons in the substantia nigra pars compacta (SNpc). In animals, PD-like symptoms can be induced by genetic mutations or by neurotoxins such as 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). It has been reported that deletion of autophagy-related gene 5 (Atg5) in the brain can disrupt neural function and is accompanied by the accumulation of cytoplasmic inclusions. However, the exact role of autophagy in PD etiology has not fully been asserted. In this study, we used tyrosine hydroxylase (TH)-Cre mice to generate conditional knockouts (CKO) with the specific deletion of Atg5 in mDA neurons, and found that adult Atg5 CKO mice contained ubiquitin- and p62-positive inclusions and fewer TH-positive mDA neurons compared with wild-type controls. Interestingly, MPTP-induced loss of mDA neurons was not observed in Atg5 CKO mice. Thus, Atg5-associated autophagy is required for the survival of mDA neurons, and may be involved in MPTP-induced neuronal degeneration.

Niche-derived laminin-511 promotes midbrain dopaminergic neuron survival and differentiation through YAP.

Parkinson's disease (PD) is a neurodegenerative disorder in which the loss of dopaminergic neurons in the midbrain (mDA neurons) causes progressive loss of motor control and function. Using embryonic and mDA neurons, midbrain tissue from mice, and differentiated human neural stem cells, we investigated the mechanisms controlling the survival of mDA neurons. We found that the extracellular matrix protein laminin-511 (LM511) promoted the survival and differentiation of mDA neurons. LM511 bound to integrin α3β1 and activated the transcriptional cofactor YAP. LM511-YAP signaling enhanced cell survival by inducing the expression of the microRNA miR-130a, which suppressed the synthesis of the cell death-associated protein PTEN. In addition, LM511-YAP signaling increased the expression of transcription factors critical for mDA identity, such as LMX1A and PITX3, and prevented the loss of mDA neurons in response to oxidative stress, a finding that warrants further investigation to assess therapeutic potential for PD patients. We propose that by enhancing LM511-YAP signaling, it may be possible to prevent mDA neuron degeneration in PD or enhance the survival of mDA neurons in cell replacement therapies.

Expression patterns of key Sonic Hedgehog signaling pathway components in the developing and adult mouse midbrain and in the MN9D cell line

The temporal dynamic expression of Sonic Hedgehog (SHH) and signaling during early midbrain dopaminergic (mDA) neuron development is one of the key players in establishing mDA progenitor diversity. However, whether SHH signaling is also required during later developmental stages and in mature mDA neurons is less understood. We study the expression of SHH receptors Ptch1 and Gas1 (growth arrest-specific 1) and of the transcription factors Gli1, Gli2 and Gli3 in mouse midbrain during embryonic development [embryonic day (E) 12.5 onwards)], in newborn and adult mice using in situ hybridization and immunohistochemistry. Moreover, we examine the expression and regulation of dopaminergic neuronal progenitor markers, midbrain dopaminergic neuronal markers and markers of the SHH signaling pathway in undifferentiated and butyric acid-treated (differentiated) MN9D cells in the presence or absence of exogenous SHH in vitro by RT-PCR, immunoblotting and immunocytochemistry. Gli1 was expressed in the lateral mesencephalic domains, whereas Gli2 and Gli3 were expressed dorsolaterally and complemented by ventrolateral expression of Ptch1. Co-localization with tyrosine hydroxylase could not be observed. GAS1 was exclusively expressed in the dorsal mesencephalon at E11.5 and co-localized with Ki67. In contrast, MN9D cells expressed all the genes investigated and treatment of the cells with butyric acid significantly upregulated their expression. The results suggest that SHH is only indirectly involved in the differentiation and survival of mDA neurons and that the MN9D cell line is a valuable model for investigating early development but not the differentiation and survival of mDA neurons.

Targeting bone morphogenetic protein signalling in midbrain dopaminergic neurons as a therapeutic approach in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease, characterized by the degeneration of midbrain dopaminergic (mDA) neurons and their axons, and aggregation of α-synuclein, which leads to motor and late-stage cognitive impairments. As the motor symptoms of PD are caused by the degeneration of a specific population of mDA neurons, PD lends itself to neurotrophic factor therapy. The goal of this therapy is to apply a neurotrophic factor that can slow down, halt or even reverse the progressive degeneration of mDA neurons. While the best known neurotrophic factors are members of the glial cell line-derived neurotrophic factor (GDNF) family, their lack of clinical efficacy to date means that it is important to continue to study other neurotrophic factors. Bone morphogenetic proteins (BMPs) are naturally secreted proteins that play critical roles during nervous system development and in the adult brain. In this review, we provide an overview of the BMP ligands, BMP receptors (BMPRs) and their intracellular signalling effectors, the Smad proteins. We review the available evidence that BMP–Smad signalling pathways play an endogenous role in mDA neuronal survival in vivo, before outlining how exogenous application of BMPs exerts potent effects on mDA neuron survival and axon growth in vitro and in vivo. We discuss the molecular mechanisms that mediate these effects, before highlighting the potential of targeting the downstream effectors of BMP–Smad signalling as a novel neuroprotective approach to slow or stop the degeneration of mDA neurons in PD.

Purification of functional human ES and iPSC-derived midbrain dopaminergic progenitors using LRTM1.

Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (mDA) neurons for cell replacement therapy for Parkinson's disease (PD). However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. To eliminate these unwanted cells, cell sorting using antibodies for specific markers such as CORIN or ALCAM has been developed, but neither marker is specific for ventral midbrain. Here we employ a double selection strategy for cells expressing both CORIN and LMX1A::GFP, and report a cell surface marker to enrich mDA progenitors, LRTM1. When transplanted into 6-OHDA-lesioned rats, human iPSC-derived LRTM1+ cells survive and differentiate into mDA neurons in vivo, resulting in a significant improvement in motor behaviour without tumour formation. In addition, there was marked survival of mDA neurons following transplantation of LRTM1+ cells into the brain of an MPTP-treated monkey. Thus, LRTM1 may provide a tool for efficient and safe cell therapy for PD patients.

Deletion of autophagy-related gene 7 in dopaminergic neurons prevents their loss induced by MPTP

Parkinson’s disease (PD) is a neurodegenerative disease caused by a gradual loss of midbrain dopaminergic (mDA) neurons in the substantia nigra pars compacta (SNpc) during aging. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is one of the neurotoxins used widely to induce PD-like symptoms in PD animal models, including rodents and non-human primates. It has been reported that deletion of autophagy-related gene 7 (Atg7) in the brain results in a reduction of mDA neurons in adulthood. In this study, we used tyrosine hydroxylase (TH)-Cre mice to generate conditional knockout (CKO) mice with the specific deletion of Atg7 in mDA neurons. Consistent with previous reports, adult Atg7 CKO mice contained fewer TH-positive mDA neurons compared with wild-type (WT) controls. TH-expressing neurons containing puncta-like structures with p62 and ubiquitin immunoreactivity were observed in the midbrain of Atg7 CKO mice but were not detected in control mice. However, MPTP-induced loss of mDA neurons was not observed in Atg7 CKO mice. Our results indicate that Atg7-involved autophagy is required not only for the survival of mDA neurons in the mouse brain, but also for MPTP-induced mDA neuron degeneration.

Neuroprotective Transcription Factors in Animal Models of Parkinson Disease.

A number of transcription factors, including En1/2, Foxa1/2, Lmx1a/b, Nurr1, Otx2, and Pitx3, with key roles in midbrain dopaminergic (mDA) neuron development, also regulate adult mDA neuron survival and physiology. Mouse models with targeted disruption of some of these genes display several features reminiscent of Parkinson disease (PD), in particular the selective and progressive loss of mDA neurons in the substantia nigra pars compacta (SNpc). The characterization of these animal models has provided valuable insights into various mechanisms of PD pathogenesis. Therefore, the dissection of the mechanisms and survival signalling pathways engaged by these transcription factors to protect mDA neuron from degeneration can suggest novel therapeutic strategies. The work on En1/2-mediated neuroprotection also highlights the potential of protein transduction technology for neuroprotective approaches in PD.

Conditional Expression of Parkinson's Disease-Related Mutant -Synuclein in the Midbrain Dopaminergic Neurons Causes Progressive Neurodegeneration and Degradation of Transcription Factor Nuclear Receptor Related 1

α-Synuclein (α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson's disease (PD). However, only a few studies on α-syn have been performed in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We also systematically examined the subcellular abnormalities that appeared in the mDA neurons of mutant mice and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and the impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that overexpression of α-syn promoted a proteasome-dependent degradation of nuclear receptor-related 1 protein (Nurr1), whereas inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.

Decreased NURR1 and PITX3 gene expression in Chinese patients with Parkinson’s disease

The preferential degeneration of midbrain dopaminergic (mDA) neurons in the substantia nigra pars compacta is the pathological hallmark in Parkinson's disease (PD). The transcription factors NURR1 and PITX3 have been shown to play a crucial role in the maturation and survival of mDA neurons, and both of them were potential susceptibility genes for PD. To determine whether NURR1 and PITX3 gene expression are altered in Chinese patients with PD, we measured their gene expression in human peripheral blood lymphocytes (PBL) in 255 patients with PD and 211 age- and gender-matched healthy controls by quantitative real-time PCR technique. We found that both NURR1 and PITX3 gene expression in PBL were significantly decreased in patients with PD as compared with controls (P < 0.0001). Lower levels of NURR1 and PITX3 gene expression were significantly associated with the increased risk for PD in male and older subjects, respectively. Our results provide useful information that the NURR1 and PITX3 gene expression is decreased in the PBL of Chinese patients with PD, indicating their possible systemic involvement in PD.

Vesicular monoamine transporter 2 and dopamine transporter are molecular targets of Pitx3 in the ventral midbrain dopamine neurons

Midbrain dopamine (mDA) neurons play critical roles in the regulation of voluntary movement and their dysfunction is associated with Parkinson's disease. Pitx3 has been implicated in the proper development of mDA neurons in the substantia nigra pars compacta, which are selectively lost in Parkinson's disease. However, the basic mechanisms underlying its role in mDA neuron development and/or survival are poorly understood. Toward this goal, we sought to identify downstream target genes of Pitx3 by comparing gene expression profiles in mDA neurons of wild-type and Pitx3-deficient aphakia mice. This global gene expression analysis revealed many potential target genes of Pitx3; in particular, the expression of vesicular monoamine transporter 2 and dopamine transporter, responsible for dopamine storage and reuptake, respectively, is greatly reduced in mDA neurons by Pitx3 ablation. In addition, gain-of-function analyses and chromatin immunoprecipitation strongly indicate that Pitx3 may directly activate transcription of vesicular monoamine transporter 2 and dopamine transporter genes, critically contributing to neurotransmission and/or survival of mDA neurons. As the two genes have been known to be regulated by Nurr1, another key dopaminergic transcription factor, we propose that Pitx3 and Nurr1 may coordinately regulate mDA specification and survival, at least in part, through a merging and overlapping downstream pathway.