iPSC-Derived Microglia as a Model to Study Inflammation in Idiopathic Parkinson's Disease.

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

The Contribution of Microglia to Neuroinflammation in Parkinson’s Disease

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

Differentiation of neuroblastoma cells as a model for investigation into latent infection of HSV-1

U neuroznanosti i virologiji Herpes simpleks virusa tipa 1 postoji težnja ka idealnom in vitro modelnom sustavu u istraživanjima koja se bave proučavanjem latentne infekcije HSV-1 virusa. Mnoge studije su se usredotočile na diferencijaciju stanica neuroblastoma u stanice nalik neuronima. Diferencijacijom pomoću retinoične kiseline i neurotrofnog faktora BDNF mogu se dobiti stanice promijenjene morfologije i biokemije. Homogena kultura takvih diferenciranih stanica rutinski se koristi u raznim istraživanjima, kao na primjer kod istraživanja neurodegenerativnih bolesti kao što su Parkinsonova i Alzheimerova bolest. Cilj ovog diplomskog rada jest optimizirati protokol diferencijacije stanične kulture neuroblastoma SH-SY5Y u homogenu kulturu stanica nalik neuronima, kako bi mogli proučavati uspostavu latencije i reaktivacije HSV-1 virusa. Diferencijacija je provedena tretmanom stanica s retinoičnom kiselinom i neurotrofnim faktorom, a sam proces diferencijacije je praćen kroz detekciju ekspresije dva proteina karakteristična za stanice neurona, NeuN i MAP-2. Nakon diferencijacije stanice su imale morfološke karakteristike neuralnih stanica, a također je došlo do povećanja ekspresije MAP-2 neuralnog markera. Rezultati ukazuju da se pomoću retinoične kiseline i BDNF-a može potaknuti diferencija stanica neuroblastoma te je, uz dodatna istraživanja i optimizacije protokola, moguće dobiti homogenu kulturu stanica nalik neuronima potrebnih za in vitro istraživanje latentne infekcije Herpes simpleks virusa tipa 1.In neuroscience and the virology of Herpes simplex virus type 1 there is a great need for the ideal in vitro model system for researching HSV-1 latency infection. A lot of studies are focused on differentiating neuroblastoma cell line SH-SY5Y to neuron-like cells. With help of retionic acid and BDNF factor, cells can differentiate and change their morphology and biochemistry. Completely homogenous cell culture can be used in a variety of research, and also there have been findings that those cells are used in researching neurodegenerative diseases such as Parkinson and Alzheimer's disease. Main goal of this Master's Thesis is to optimize the differentiation protocol to get homogenous cell culture for studying latency and reactivation of HSV-1. Differentiation was carried out by using retinoic acid and neurotrofic factor, and the process of differentiation is monitored by detecting the expression of two characteristic neuronal proteins, NeuN and MAP-2. After the treatment cells had morphological characteristics of neurons and the expression of MAP-2 neuronal marker was increased. The results show that retinoic acid and BDNF can induce differentiation of SH-SY5Y neuroblastoma cell line and also, with additional research and protocol optimization, it is possible to get homogenous neuron-like cell culture that can be used for in vitro research of HSV-1 latent infection

Differentiation of neuroblastoma cells as a model for investigation into latent infection of HSV-1

U neuroznanosti i virologiji Herpes simpleks virusa tipa 1 postoji težnja ka idealnom in vitro modelnom sustavu u istraživanjima koja se bave proučavanjem latentne infekcije HSV-1 virusa. Mnoge studije su se usredotočile na diferencijaciju stanica neuroblastoma u stanice nalik neuronima. Diferencijacijom pomoću retinoične kiseline i neurotrofnog faktora BDNF mogu se dobiti stanice promijenjene morfologije i biokemije. Homogena kultura takvih diferenciranih stanica rutinski se koristi u raznim istraživanjima, kao na primjer kod istraživanja neurodegenerativnih bolesti kao što su Parkinsonova i Alzheimerova bolest. Cilj ovog diplomskog rada jest optimizirati protokol diferencijacije stanične kulture neuroblastoma SH-SY5Y u homogenu kulturu stanica nalik neuronima, kako bi mogli proučavati uspostavu latencije i reaktivacije HSV-1 virusa. Diferencijacija je provedena tretmanom stanica s retinoičnom kiselinom i neurotrofnim faktorom, a sam proces diferencijacije je praćen kroz detekciju ekspresije dva proteina karakteristična za stanice neurona, NeuN i MAP-2. Nakon diferencijacije stanice su imale morfološke karakteristike neuralnih stanica, a također je došlo do povećanja ekspresije MAP-2 neuralnog markera. Rezultati ukazuju da se pomoću retinoične kiseline i BDNF-a može potaknuti diferencija stanica neuroblastoma te je, uz dodatna istraživanja i optimizacije protokola, moguće dobiti homogenu kulturu stanica nalik neuronima potrebnih za in vitro istraživanje latentne infekcije Herpes simpleks virusa tipa 1.In neuroscience and the virology of Herpes simplex virus type 1 there is a great need for the ideal in vitro model system for researching HSV-1 latency infection. A lot of studies are focused on differentiating neuroblastoma cell line SH-SY5Y to neuron-like cells. With help of retionic acid and BDNF factor, cells can differentiate and change their morphology and biochemistry. Completely homogenous cell culture can be used in a variety of research, and also there have been findings that those cells are used in researching neurodegenerative diseases such as Parkinson and Alzheimer's disease. Main goal of this Master's Thesis is to optimize the differentiation protocol to get homogenous cell culture for studying latency and reactivation of HSV-1. Differentiation was carried out by using retinoic acid and neurotrofic factor, and the process of differentiation is monitored by detecting the expression of two characteristic neuronal proteins, NeuN and MAP-2. After the treatment cells had morphological characteristics of neurons and the expression of MAP-2 neuronal marker was increased. The results show that retinoic acid and BDNF can induce differentiation of SH-SY5Y neuroblastoma cell line and also, with additional research and protocol optimization, it is possible to get homogenous neuron-like cell culture that can be used for in vitro research of HSV-1 latent infection

INVESTIGATING NEUROINFLAMMATION IN SPORADIC AND LRRK2-ASSOCIATED PARKINSON'S DISEASE

Inflammatory responses are evolutionarily conserved reactions to pathogens, injury, or any form of a serious perturbation of a human organism. These mechanisms evolved together with us and, although capable of somewhat adapting, innate responses are gravely impacted by prolonged human lifespan. Better sanitary measures, health systems, food and medicine supply have prolonged human life expectancy to ~72 years. Aging is characterized by prolonged, chronic (often low-grade) inflammation. With tissue and cellular defense mechanisms becoming dysfunctional over time, this inflammation becomes detrimental and destructive to the human body. Aging is a major risk factor for Parkinson’s disease (PD), a movement disorder characterized by the loss of dopaminergic neurons. Even though the disease is predominantly idiopathic, genetic cases are contributing to a better understanding of the underlying cellular and neuropathological mechanisms. In comparison to neuronal demise, the contribution of microglia (the immune cells of the brain) to PD is relatively understudied. Initially studied in PD patient-derived post-mortem tissue, novel in vitro technologies, such as induced pluripotent stem cells (iPSCs), are permitting the generation of specific cell types of interest in order to study disease mechanisms. We derived microglia cells from iPSCs of patients and healthy or isogenic controls to explore (shared) pathological immune responses in LRRK2-PD and idiopathic PD. Our findings suggest a significant involvement of microglia cells in the pathogenesis of PD and highlight potential therapeutic targets in alleviating overactive immune responses

Differentiation of neuroblastoma cells as a model for investigation into latent infection of HSV-1

U neuroznanosti i virologiji Herpes simpleks virusa tipa 1 postoji težnja ka idealnom in vitro modelnom sustavu u istraživanjima koja se bave proučavanjem latentne infekcije HSV-1 virusa. Mnoge studije su se usredotočile na diferencijaciju stanica neuroblastoma u stanice nalik neuronima. Diferencijacijom pomoću retinoične kiseline i neurotrofnog faktora BDNF mogu se dobiti stanice promijenjene morfologije i biokemije. Homogena kultura takvih diferenciranih stanica rutinski se koristi u raznim istraživanjima, kao na primjer kod istraživanja neurodegenerativnih bolesti kao što su Parkinsonova i Alzheimerova bolest. Cilj ovog diplomskog rada jest optimizirati protokol diferencijacije stanične kulture neuroblastoma SH-SY5Y u homogenu kulturu stanica nalik neuronima, kako bi mogli proučavati uspostavu latencije i reaktivacije HSV-1 virusa. Diferencijacija je provedena tretmanom stanica s retinoičnom kiselinom i neurotrofnim faktorom, a sam proces diferencijacije je praćen kroz detekciju ekspresije dva proteina karakteristična za stanice neurona, NeuN i MAP-2. Nakon diferencijacije stanice su imale morfološke karakteristike neuralnih stanica, a također je došlo do povećanja ekspresije MAP-2 neuralnog markera. Rezultati ukazuju da se pomoću retinoične kiseline i BDNF-a može potaknuti diferencija stanica neuroblastoma te je, uz dodatna istraživanja i optimizacije protokola, moguće dobiti homogenu kulturu stanica nalik neuronima potrebnih za in vitro istraživanje latentne infekcije Herpes simpleks virusa tipa 1.In neuroscience and the virology of Herpes simplex virus type 1 there is a great need for the ideal in vitro model system for researching HSV-1 latency infection. A lot of studies are focused on differentiating neuroblastoma cell line SH-SY5Y to neuron-like cells. With help of retionic acid and BDNF factor, cells can differentiate and change their morphology and biochemistry. Completely homogenous cell culture can be used in a variety of research, and also there have been findings that those cells are used in researching neurodegenerative diseases such as Parkinson and Alzheimer's disease. Main goal of this Master's Thesis is to optimize the differentiation protocol to get homogenous cell culture for studying latency and reactivation of HSV-1. Differentiation was carried out by using retinoic acid and neurotrofic factor, and the process of differentiation is monitored by detecting the expression of two characteristic neuronal proteins, NeuN and MAP-2. After the treatment cells had morphological characteristics of neurons and the expression of MAP-2 neuronal marker was increased. The results show that retinoic acid and BDNF can induce differentiation of SH-SY5Y neuroblastoma cell line and also, with additional research and protocol optimization, it is possible to get homogenous neuron-like cell culture that can be used for in vitro research of HSV-1 latent infection

The Contribution of Microglia to Neuroinflammation in Parkinson's Disease.

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

Comparison of two protocols for the generation of iPSC-derived human astrocytes

Abstract Background Astrocytes have recently gained attention as key contributors to the pathogenesis of neurodegenerative disorders including Parkinson’s disease. To investigate human astrocytes in vitro, numerous differentiation protocols have been developed. However, the properties of the resulting glia are inconsistent, which complicates the selection of an appropriate method for a given research question. Thus, we compared two approaches for the generation of iPSC-derived astrocytes. We phenotyped glia that were obtained employing a widely used long, serum-free (“LSF”) method against an in-house established short, serum-containing (“SSC”) protocol which allows for the generation of astrocytes and midbrain neurons from the same precursor cells. Results We employed high-content confocal imaging and RNA sequencing to characterize the cultures. The astrocytes generated with the LSF or SSC protocols differed considerably in their properties: while the former cells were more labor-intense in their generation (5 vs 2 months), they were also more mature. This notion was strengthened by data resulting from cell type deconvolution analysis that was applied to bulk transcriptomes from the cultures to assess their similarity with human postmortem astrocytes. Conclusions Overall, our analyses highlight the need to consider the advantages and disadvantages of a given differentiation protocol, when designing functional or drug discovery studies involving iPSC-derived astrocytes

Mitochondrial damage-associated inflammation highlights biomarkers in PRKN/PINK1 parkinsonism

There is increasing evidence for a role of inflammation in Parkinson’s disease. Recent research in murine models suggests that parkin and PINK1 deficiency leads to impaired mitophagy, which causes the release of mitochondrial DNA (mtDNA), thereby triggering inflammation. Specifically, the CGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) pathway mitigates activation of the innate immune system, quantifiable as increased interleukin-6 (IL6) levels. However, the role of IL6 and circulating cell-free mtDNA in unaffected and affected individuals harbouring mutations in PRKN/PINK1 and idiopathic Parkinson’s disease patients remain elusive. We investigated IL6, C-reactive protein, and circulating cell-free mtDNA in serum of 245 participants in two cohorts from tertiary movement disorder centres. We performed a hypothesis-driven rank-based statistical approach adjusting for multiple testing. We detected (i) elevated IL6 levels in patients with biallelic PRKN/PINK1 mutations compared to healthy control subjects in a German cohort, supporting the concept of a role for inflammation in PRKN/PINK1-linked Parkinson’s disease. In addition, the comparison of patients with biallelic and heterozygous mutations in PRKN/PINK1 suggests a gene dosage effect. The differences in IL6 levels were validated in a second independent Italian cohort; (ii) a correlation between IL6 levels and disease duration in carriers of PRKN/PINK1 mutations, while no such association was observed for idiopathic Parkinson’s disease patients. These results highlight the potential of IL6 as progression marker in Parkinson’s disease due to PRKN/PINK1 mutations; (iii) increased circulating cell-free mtDNA serum levels in both patients with biallelic or with heterozygous PRKN/PINK1 mutations compared to idiopathic Parkinson’s disease, which is in line with previous findings in murine models. By contrast, circulating cell-free mtDNA concentrations in unaffected heterozygous carriers of PRKN/PINK1 mutations were comparable to control levels; and (iv) that circulating cell-free mtDNA levels have good predictive potential to discriminate between idiopathic Parkinson’s disease and Parkinson’s disease linked to heterozygous PRKN/PINK1 mutations, providing functional evidence for a role of heterozygous mutations in PRKN or PINK1 as Parkinson’s disease risk factor. Taken together, our study further implicates inflammation due to impaired mitophagy and subsequent mtDNA release in the pathogenesis of PRKN/PINK1-linked Parkinson’s disease. In individuals carrying mutations in PRKN/PINK1, IL6 and circulating cell-free mtDNA levels may serve as markers of Parkinson’s disease state and progression, respectively. Finally, our study suggests that targeting the immune system with anti-inflammatory medication holds the potential to influence the disease course of Parkinson’s disease, at least in this subset of patients

Additional file 1 of Comparison of two protocols for the generation of iPSC-derived human astrocytes

Additional file 1