![]() Smajic, Semra ![]() Doctoral thesis (2022) For a very long time, the main focus in Parkinson’s disease (PD) research was the loss of neuromelanin-containing dopaminergic neurons from the substantia nigra (SN) of the midbrain - the key pathological ... [more ▼] For a very long time, the main focus in Parkinson’s disease (PD) research was the loss of neuromelanin-containing dopaminergic neurons from the substantia nigra (SN) of the midbrain - the key pathological feature of the disease. However, the association of neuronal vulnerability and neuromelanin presence has not been a common study subject. Recently, cells other than neurons also gained attention as mediators of PD pathogenesis. There are indications that glial cells undergo disease-related changes, however, the exact mechanisms remain unknown. In this thesis, I aimed to explore the contribution of every cell type of the midbrain to PD using single-nuclei RNA sequencing. Additionally, the goal was to explore their association to PD risk gene variants. As we identified microgliosis as a major mechanism in PD, we further extended our research to microglia. We sought to investigate the relation of microglia and neuromelanin. Thus, we aimed to, by means of immunohistochemical staining, imaging and laser-capture microdissection-based transcriptomics, elucidate this association on a single-cell level. This work resulted in the first midbrain single-cell atlas from idiopathic PD subjects and age- and sex-matched controls. We revealed SN-specific microgliosis with GPNMB upregulation, which also seemed to be specific to the idiopathic form of the disease. We further observed an accumulation of (extraneuronal) neuromelanin particles in Parkinson’s midbrain parenchyma, indicative of incomplete degradation. Moreover, we showed that GPNMB can be alleviated in microglia in contact with neuromelanin. Taken together, we provide evidence of a GPNMB-related microglial state as a disease mechanism specific to idiopathic PD, and highlight neuromelanin as an important player in microglia disease pathology. Further investigations are needed to understand whether the modulation of neuromelanin levels could be relevant in the context of PD therapy. [less ▲] Detailed reference viewed: 60 (15 UL)![]() Smajic, Semra ![]() ![]() in Brain : a journal of neurology (2022), 145(3), 964-978 Idiopathic Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, but the exact disease etiology remains largely unknown. To date, Parkinson's disease research has mainly ... [more ▼] Idiopathic Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, but the exact disease etiology remains largely unknown. To date, Parkinson's disease research has mainly focused on nigral dopaminergic neurons, although recent studies suggest disease-related changes also in non-neuronal cells and in midbrain regions beyond the substantia nigra. While there is some evidence for glial involvement in Parkinson's disease, the molecular mechanisms remain poorly understood. The aim of this study was to characterize the contribution of all cell types of the midbrain to Parkinson's disease pathology by single-nuclei RNA sequencing and to assess the cell type-specific risk for Parkinson's disease employing the latest genome-wide association study. We profiled >41 000 single-nuclei transcriptomes of postmortem midbrain from six idiopathic Parkinson's disease patients and five age-/sex-matched controls. To validate our findings in a spatial context, we utilized immunolabeling of the same tissues. Moreover, we analyzed Parkinson's disease-associated risk enrichment in genes with cell type-specific expression patterns. We discovered a neuronal cell cluster characterized by CADPS2 overexpression and low TH levels, which was exclusively present in IPD midbrains. Validation analyses in laser-microdissected neurons suggest that this cluster represents dysfunctional dopaminergic neurons. With regard to glial cells, we observed an increase in nigral microglia in Parkinson's disease patients. Moreover, nigral idiopathic Parkinson's disease microglia were more amoeboid, indicating an activated state. We also discovered a reduction in idiopathic Parkinson's disease oligodendrocyte numbers with the remaining cells being characterized by a stress-induced upregulation of S100B. Parkinson's disease risk variants were associated with glia- and neuron-specific gene expression patterns in idiopathic Parkinson's disease cases. Furthermore, astrocytes and microglia presented idiopathic Parkinson's disease-specific cell proliferation and dysregulation of genes related to unfolded protein response and cytokine signaling. While reactive patient astrocytes showed CD44 overexpression, idiopathic Parkinson's disease-microglia revealed a pro-inflammatory trajectory characterized by elevated levels of IL1B, GPNMB, and HSP90AA1. Taken together, we generated the first single-nuclei RNA sequencing dataset from the idiopathic Parkinson's disease midbrain, which highlights a disease-specific neuronal cell cluster as well as 'pan-glial' activation as a central mechanism in the pathology of the movement disorder. This finding warrants further research into inflammatory signaling and immunomodulatory treatments in Parkinson's disease. [less ▲] Detailed reference viewed: 124 (22 UL)![]() ; Smajic, Semra ![]() ![]() in Movement disorders : official journal of the Movement Disorder Society (2022) BACKGROUND: Mutations in the E3 ubiquitin ligase parkin cause autosomal recessive Parkinson's disease (PD). Together with PTEN-induced kinase 1 (PINK1), parkin regulates the clearance of dysfunctional ... [more ▼] BACKGROUND: Mutations in the E3 ubiquitin ligase parkin cause autosomal recessive Parkinson's disease (PD). Together with PTEN-induced kinase 1 (PINK1), parkin regulates the clearance of dysfunctional mitochondria. New mitochondria are generated through an interplay of nuclear- and mitochondrial-encoded proteins, and recent studies suggest that parkin influences this process at both levels. In addition, parkin was shown to prevent mitochondrial membrane permeability, impeding mitochondrial DNA (mtDNA) escape and subsequent neuroinflammation. However, parkin's regulatory roles independent of mitophagy are not well described in patient-derived neurons. OBJECTIVES: We sought to investigate parkin's role in preventing neuronal mtDNA dyshomeostasis, release, and glial activation at the endogenous level. METHODS: We generated induced pluripotent stem cell (iPSC)-derived midbrain neurons from PD patients with parkin (PRKN) mutations and healthy controls. Live-cell imaging, proteomic, mtDNA integrity, and gene expression analyses were employed to investigate mitochondrial biogenesis and genome maintenance. To assess neuroinflammation, we performed single-nuclei RNA sequencing in postmortem tissue and quantified interleukin expression in mtDNA/lipopolysaccharides (LPS)-treated iPSC-derived neuron-microglia co-cultures. RESULTS: Neurons from patients with PRKN mutations revealed deficits in the mitochondrial biogenesis pathway, resulting in mtDNA dyshomeostasis. Moreover, the energy sensor sirtuin 1, which controls mitochondrial biogenesis and clearance, was downregulated in parkin-deficient cells. Linking mtDNA disintegration to neuroinflammation, in postmortem midbrain with PRKN mutations, we confirmed mtDNA dyshomeostasis and detected an upregulation of microglia overexpressing proinflammatory cytokines. Finally, parkin-deficient neuron-microglia co-cultures elicited an enhanced immune response when exposed to mtDNA/LPS. CONCLUSIONS: Our findings suggest that parkin coregulates mitophagy, mitochondrial biogenesis, and mtDNA maintenance pathways, thereby protecting midbrain neurons from neuroinflammation and degeneration. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. [less ▲] Detailed reference viewed: 115 (10 UL)![]() Zagare, Alise ![]() ![]() ![]() in American Journal of Human Genetics (2022) Detailed reference viewed: 131 (12 UL)![]() ; Schymanski, Emma ![]() ![]() in Medizinische Genetik (2022), 34(2), 103--116 Detailed reference viewed: 62 (3 UL)![]() Badanjak, Katja ![]() ![]() ![]() in Frontiers in cell and developmental biology (2021), 9 Parkinson's disease (PD) is a neurodegenerative disease with unknown cause in the majority of patients, who are therefore considered "idiopathic" (IPD). PD predominantly affects dopaminergic neurons in ... [more ▼] Parkinson's disease (PD) is a neurodegenerative disease with unknown cause in the majority of patients, who are therefore considered "idiopathic" (IPD). PD predominantly affects dopaminergic neurons in the substantia nigra pars compacta (SNpc), yet the pathology is not limited to this cell type. Advancing age is considered the main risk factor for the development of IPD and greatly influences the function of microglia, the immune cells of the brain. With increasing age, microglia become dysfunctional and release pro-inflammatory factors into the extracellular space, which promote neuronal cell death. Accordingly, neuroinflammation has also been described as a feature of PD. So far, studies exploring inflammatory pathways in IPD patient samples have primarily focused on blood-derived immune cells or brain sections, but rarely investigated patient microglia in vitro. Accordingly, we decided to explore the contribution of microglia to IPD in a comparative manner using, both, iPSC-derived cultures and postmortem tissue. Our meta-analysis of published RNAseq datasets indicated an upregulation of IL10 and IL1B in nigral tissue from IPD patients. We observed increased expression levels of these cytokines in microglia compared to neurons using our single-cell midbrain atlas. Moreover, IL10 and IL1B were upregulated in IPD compared to control microglia. Next, to validate these findings in vitro, we generated IPD patient microglia from iPSCs using an established differentiation protocol. IPD microglia were more readily primed as indicated by elevated IL1B and IL10 gene expression and higher mRNA and protein levels of NLRP3 after LPS treatment. In addition, IPD microglia had higher phagocytic capacity under basal conditions-a phenotype that was further exacerbated upon stimulation with LPS, suggesting an aberrant microglial function. Our results demonstrate the significance of microglia as the key player in the neuroinflammation process in IPD. While our study highlights the importance of microglia-mediated inflammatory signaling in IPD, further investigations will be needed to explore particular disease mechanisms in these cells. [less ▲] Detailed reference viewed: 162 (20 UL)![]() Badanjak, Katja ![]() ![]() ![]() in International journal of molecular sciences (2021), 22(9), With the world's population ageing, the incidence of Parkinson's disease (PD) is on the rise. In recent years, inflammatory processes have emerged as prominent contributors to the pathology of PD. There ... [more ▼] With the world's population ageing, the incidence of Parkinson's disease (PD) is on the rise. In recent years, inflammatory processes have emerged as prominent contributors to the pathology of PD. There is great evidence that microglia have a significant neuroprotective role, and that impaired and over activated microglial phenotypes are present in brains of PD patients. Thereby, PD progression is potentially driven by a vicious cycle between dying neurons and microglia through the instigation of oxidative stress, mitophagy and autophagy dysfunctions, a-synuclein accumulation, and pro-inflammatory cytokine release. Hence, investigating the involvement of microglia is of great importance for future research and treatment of PD. The purpose of this review is to highlight recent findings concerning the microglia-neuronal interplay in PD with a focus on human postmortem immunohistochemistry and single-cell studies, their relation to animal and iPSC-derived models, newly emerging technologies, and the resulting potential of new anti-inflammatory therapies for PD. [less ▲] Detailed reference viewed: 87 (13 UL)![]() Smajic, Semra ![]() ![]() E-print/Working paper (2020) Parkinson’s disease (PD) etiology is associated with genetic and environmental factors that lead to a loss of dopaminergic neurons. However, the functional interpretation of PD-associated risk variants ... [more ▼] Parkinson’s disease (PD) etiology is associated with genetic and environmental factors that lead to a loss of dopaminergic neurons. However, the functional interpretation of PD-associated risk variants and how other midbrain cells contribute to this neurodegenerative process are poorly understood. Here, we profiled >41,000 single-nuclei transcriptomes of postmortem midbrain tissue from 6 idiopathic PD (IPD) patients and 5 matched controls. We show that PD-risk variants are associated with glia- and neuron-specific gene expression patterns. Furthermore, Microglia and astrocytes presented IPD-specific cell proliferation and dysregulation of genes related to unfolded protein response and cytokine signalling. IPD-microglia revealed a specific pro-inflammatory trajectory. Finally, we discovered a neuronal cell cluster exclusively present in IPD midbrains characterized by CADPS2 overexpression and a high proportion of cycling cells. We conclude that elevated CADPS2 expression is specific to dysfunctional dopaminergic neurons, which have lost their dopaminergic identity and unsuccessful attempt to re-enter the cell cycle. [less ▲] Detailed reference viewed: 66 (9 UL) |
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