Microglial implication in Parkinson's Disease: studying functional and morphological changes occurring in LRRK2 microglia during PD pathophysiology using a stem cell derived human model

[eng] Parkinson’s disease (PD) is an incurable neurodegenerative disease characterized by the loss of neuromelanin (NM)-containing dopamine neurons in Substantia Nigra pars compacta (SNpc) and accumulation of insoluble cytoplasmic protein inclusions known as Lewy bodies. Microglial activation, astrocyte reactivity and lymphocyte infiltration also occur in PD. Here, we hypothesize that PD is initiated years before the emergence of motor dysfunction in response to several mechanisms some of which triggered following microglia activation that impact negatively in neuronal survival. Taking advantage of our human iPSC-based model of PD, we first generated human Microglia-like cells (hMG) from LRRK2-PD and Control iPSCs and confirmed their identity by using specific microglial markers. We then carried out functional studies with pro-inflammatory stimuli such as LPS or NM, which revealed a higher motility, cytokine release and phagocytic activity of LRRK2-PD hMG compared to control hMG. In addition, we found that extracellular NM particles induced microglial activation and increases ROS production in LRRK2-PD microglia. The use of a corrected isogenic PD hMG reverted all previous phenotypes, confirming a LRRK2-dependent activation of hMG. Upon co-culture with LRRK2-PD hMG and in the presence of NM particles, Control Dopaminergic neurons (DAn) displayed morphological signs of neurodegeneration, such as short and few neurites as well as beaded necklace-like neurites, as well as increased neuronal loss. Thus, our findings indicate a critical role for neuromelanin-activated microglia in LRRK2-PD and may serve as a valid human cellular model to test compounds that can lower risk for PD or disease progression

Dissecting the non-neuronal cell contribution to Parkinson's disease pathogenesis using induced pluripotent stem cells

Parkinson's disease (PD) is an incurable age-linked neurodegenerative disease with characteristic movement impairments that are caused by the progressive loss of dopamine-containing neurons (DAn) within the substantia nigra pars compacta. It has been suggested that misfolded protein aggregates together with neuroinfammation and glial reactivity, may impact nerve cell function, leading to neurodegeneration and diseases, such as PD. However, not many studies have been able to examine the role of human glial cells in the pathogenesis of PD. With the advent of induced pluripotent stem cell (iPSC) technology, it is now possible to reprogram human somatic cells to pluripotency and to generate viable human patient-specifc DA neurons and glial cells, providing a tremendous opportunity for dissecting cellular and molecular pathological mechanisms occurring at early stages of PD. This reviews will report on recent work using human iPSC and 3D brain organoid models showing that iPSC technology can be used to recapitulate PD-relevant disease-associated phenotypes, including protein aggregation, cell death or loss of neurite complexity and defcient autophagic vacuoles clearance and focus on the recent co-culture systems that are revealing new insights into the complex interactions that occur between diferent brain cell types during neurodegeneration. Consequently, such advances are the key to improve our understanding of PD pathology and generate potential targets for new therapies aimed at curing PD patients

Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

Exposure to pesticides such as rotenone is a risk factor for Parkinson's disease. Dopaminergic neurons are especially sensitive to the toxicity of compounds that inhibit the mitochondrial respiratory chain such as rotenone and 1-methyl-4-phenylpyridinium (MPP+). However, there is scarce information on their effects on glia. To evaluate whether these neurotoxicants affect the immune response of glia, primary mouse mixed glial and microglial cultures were treated with interleukin (IL) 4 in the absence and presence of MPP+ or rotenone. Using qRTPCR or western blot, we determined the expression of anti-inflammatory markers, the CD200R1 microglial receptor and its ligand CD200, and genes regulating glycolysis and oxidative metabolism. ATP and lactate levels were additionally determined as an index of cell metabolism. Microglial phagocytosis was also evaluated. MPP+ and rotenone clearly abrogated the IL4-induced expression of anti-inflammatory markers in mixed glial cultures. CD200 and CD200R1 expression and microglia phagocytosis were also affected by the neurotoxicants. Changes in the mRNA expression of the molecules regulating glycolysis and oxidative metabolism, as well as in ATP levels and lactate release suggested that metabolic reprogramming in response to MPP+ and rotenone differs between microglial and mixed glial cultures. These findings support the hypothesis that parkinsonian neurotoxicants may impair brain immune response altering glial cell metabolism

Blocking IL-6 signaling prevents astrocyte-induced neurodegeneration in an iPSC-based model of Parkinson’s disease

Parkinson's disease (PD) is a neurodegenerative disease associated with progressive death of midbrain dopamine (DAn) neurons in the substantia nigra (SN). Since it has been proposed that patients with PD exhibit an overall proinflammatory state, and since astrocytes are key mediators of the inflammation response in the brain, here we sought to address whether astrocyte-mediated inflammatory signaling could contribute to PD neuropathology. For this purpose, we generated astrocytes from induced pluripotent stem cells (iPSCs) representing patients with PD and healthy controls. Transcriptomic analyses identified a unique inflammatory gene expression signature in PD astrocytes compared with controls. In particular, the proinflammatory cytokine IL-6 was found to be highly expressed and released by PD astrocytes and was found to induce toxicity in DAn. Mechanistically, neuronal cell death was mediated by IL-6 receptor (IL-6R) expressed in human PD neurons, leading to downstream activation of STAT3. Blockage of IL-6R by the addition of the FDA-approved anti-IL-6R antibody, Tocilizumab, prevented PD neuronal death. SN neurons overexpressing IL-6R and reactive astrocytes expressing IL-6 were detected in postmortem brain tissue of patients at early stages of PD. Our findings highlight the potential role of astrocyte-mediated inflammatory signaling in neuronal loss in PD and pave the way for the design of future therapeutics

Parkinson’s disease patient-specific neuronal networks carrying the LRRK2 G2019S mutation unveil early functional alterations that predate neurodegeneration

A deeper understanding of early disease mechanisms occurring in Parkinson's disease (PD) is needed to reveal restorative targets. Here we report that human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons (DAn) obtained from healthy individuals or patients harboring LRRK2 PD-causing mutation can create highly complex networks with evident signs of functional maturation over time. Compared to control neuronal networks, LRRK2 PD patients' networks displayed an elevated bursting behavior, in the absence of neurodegeneration. By combining functional calcium imaging, biophysical modeling, and DAn-lineage tracing, we found a decrease in DAn neurite density that triggered overall functional alterations in PD neuronal networks. Our data implicate early dysfunction as a prime focus that may contribute to the initiation of downstream degenerative pathways preceding DAn loss in PD, highlighting a potential window of opportunity for pre-symptomatic assessment of chronic degenerative diseases

Alterations in the development of a M2 phenotype in glial cells: rotenone in vitro model of Parkinson's disease

Parkinson’s disease is a chronic, progressive, disabling neurodegenerative disease of unknown cause. It turned out to be the second most common neurodegenerative disorder after Alzheimer Disease, and is characterized by motor and non-motor symptoms. As a multifactorial disease, it is characterized by dopaminergic degeneration, depositions of α-synuclein aggregates, and to run with an inflammatory process. The involvement of microglial cells on this chronic inflammation within the parenchyma is long accepted but its role is not fully understood. Both genetic and environmental factors have been postulated to play a role in the development of Parkinson’s disease. Epidemiological studies show that the exposition to certain toxins, among them rotenone, is a risk factor to develop this neurodegenerative disease. Although the neurotoxic effect of rotenone has been widely studied in experimental approaches, the effect of rotenone on glial cells remains poorly characterized. The present work was aimed to study whether rotenone induced the expression of M2 markers in glial cells, and also whether rotenone interfered with the development of a M2 phenotype induced by Interleukin-4 in glial cells, using primary glial cultures. The results obtained reveal that rotenone did not induce the expression of M2 markers in glial cells per se. However, it dramatically inhibited the development of a M2 phenotype induced by Interleukin-4 in mixed glial cell cultures. On the contrary, rotenone did not modify Interleukin-4 – induced M2 phenotype in microglial cell cultures, suggesting the existence of alternative mechanisms to obtain energy of microglial cell cultures. However, phagocytic activity was impaired in microglial cell cultures. The results obtained suggest that rotenone impairs the immune response of glial cells and that glial cell dysfunction due to a direct action of certain toxins on glial cells may contribute to the development of neurodegeneration in PD.Peer reviewe

Microglial implication in Parkinson's Disease: studying functional and morphological changes occurring in LRRK2 microglia during PD pathophysiology using a stem cell derived human model

Programa de Doctorat en Biomedicina[eng] Parkinson’s disease (PD) is an incurable neurodegenerative disease characterized by the loss of neuromelanin (NM)-containing dopamine neurons in Substantia Nigra pars compacta (SNpc) and accumulation of insoluble cytoplasmic protein inclusions known as Lewy bodies. Microglial activation, astrocyte reactivity and lymphocyte infiltration also occur in PD. Here, we hypothesize that PD is initiated years before the emergence of motor dysfunction in response to several mechanisms some of which triggered following microglia activation that impact negatively in neuronal survival. Taking advantage of our human iPSC-based model of PD, we first generated human Microglia-like cells (hMG) from LRRK2-PD and Control iPSCs and confirmed their identity by using specific microglial markers. We then carried out functional studies with pro-inflammatory stimuli such as LPS or NM, which revealed a higher motility, cytokine release and phagocytic activity of LRRK2-PD hMG compared to control hMG. In addition, we found that extracellular NM particles induced microglial activation and increases ROS production in LRRK2-PD microglia. The use of a corrected isogenic PD hMG reverted all previous phenotypes, confirming a LRRK2-dependent activation of hMG. Upon co-culture with LRRK2-PD hMG and in the presence of NM particles, Control Dopaminergic neurons (DAn) displayed morphological signs of neurodegeneration, such as short and few neurites as well as beaded necklace-like neurites, as well as increased neuronal loss. Thus, our findings indicate a critical role for neuromelanin-activated microglia in LRRK2-PD and may serve as a valid human cellular model to test compounds that can lower risk for PD or disease progression

Exposure to parkinsonian neurotoxins inhibits glial cells anti-inflammatory response

Trabajo presentado en el XIV European Meeting on Glial Cells in Health and Disease, celebrado en Oporto (Portugal), del 10 al 13 de julio de 2019Epidemiological studies show that the exposure to some pesticides, such as rotenone and paraquat -functional and structural analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) respectively-, is a risk factor for Parkinson’s disease. MPTP and its analogs are inhibitors of the mitochondrial respiratory chain and it has been suggested that mitochondrial dysfunction is involved in the induction of oxidative damage in dopaminergic neurons in parkinsonism. However, although the toxic effect of these compounds on dopaminergic neurons has been widely described using both in vivo and in vitro approaches, reports of their direct effect on glial cells are scarce. In a previous study, we showed that 1-methyl-4-phenylpyridinium (MPP+, the active metabolite of MPTP) and rotenone induce alterations in the response of glial cell cultures to a pro-inflammatory stimulus (RabanedaLombarte et al., 2019). The aim of the present work was to study the effect of these agents on the development of an anti-inflammatory response in glial cells. To this end, we treated mouse primary mixed glial and microglial cultures with IL4 in the absence and the presence of MPP+ and rotenone. We observed an inhibition of the expression of anti-inflammatory markers by glial cells mainly in MPP+-treated mixed glial cultures. In addition, both MPP+ and rotenone clearly impaired the anti-inflammatory response induced by IL4 on mixed glial cultures. MPP+ treatment also resulted in some alterations in the expression of IL4-induced anti-inflammatory markers in microglial cultures. IL4, MPP+ and rotenone had an inhibitory effect on microglial phagocytosis, but the effect was not potentiated when the cells were exposed to both kinds of stimuli. ATP production was increased in IL4- treated mixed glial cultures but not in microglial cultures, an effect that was inhibited by MPP+ and rotenone. This inhibition could be responsible for the MPP+ and rotenone-induced alterations observed in the response to IL4 mainly in mixed glial cultures. These results complement our previous observations on the impairment of a pro-inflammatory response in MPP+ and rotenone treated glial cells, and support the hypothesis that the exposure to agents that alter glial metabolism impair brain immune response, which may contribute to neuronal damage in Parkinson’s disease and other neurodegenerative disorders.Supported by Instituto de Salud Carlos III, Spain-FEDER funds, EU (PI15/00033). NRL is a recipient of a FPU contract from the Spanish Ministerio de Ciencia, Innovación y Universidades

Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

Exposure to pesticides such as rotenone is a risk factor for Parkinson’s disease. Dopaminergic neurons are especially sensitive to the toxicity of compounds that inhibit the mitochondrial respiratory chain such as rotenone and 1-methyl-4-phenylpyridinium (MPP+). However, there is scarce information on their effects on glia. To evaluate whether these neurotoxicants affect the immune response of glia, primary mouse mixed glial and microglial cultures were treated with interleukin (IL) 4 in the absence and presence of MPP+ or rotenone. Using qRTPCR or western blot, we determined the expression of anti-inflammatory markers, the CD200R1 microglial receptor and its ligand CD200, and genes regulating glycolysis and oxidative metabolism. ATP and lactate levels were additionally determined as an index of cell metabolism. Microglial phagocytosis was also evaluated. MPP+ and rotenone clearly abrogated the IL4-induced expression of anti-inflammatory markers in mixed glial cultures. CD200 and CD200R1 expression and microglia phagocytosis were also affected by the neurotoxicants. Changes in the mRNA expression of the molecules regulating glycolysis and oxidative metabolism, as well as in ATP levels and lactate release suggested that metabolic reprogramming in response to MPP+ and rotenone differs between microglial and mixed glial cultures. These findings support the hypothesis that parkinsonian neurotoxicants may impair brain immune response altering glial cell metabolism.This study was supported by grant PI15/00033 from the Instituto de Salud Carlos III (Spain) with joint financing by FEDER funds from the European Union. NR-L was recipient of a predoctoral FPU contract from the Spanish Ministerio de Ciencia, Innovación y Universidades (FPU13/05491).Peer reviewe