ABSTRACT
Human pluripotent stem cell (hPSC)-derived neural cultures have attracted interest for modeling epilepsy and seizure-like activity in vitro. Clinical and experimental evidence have shown that the multifunctional inflammatory cytokine interleukin (IL)-6 plays a significant role in epilepsy. However, the role of IL-6 in neuronal networks remains unclear. In this study, we modelled seizure-like activity in hPSC-derived cortical neurons using kainic acid (KA) and explored the effects of IL-6 and its counterpart, hyper-IL-6 (H-IL-6), a fusion protein consisting of IL-6 and its soluble receptor, IL-6R. In the seizure-like model, functionally mature neuronal networks responded to KA induction with an increased bursting phenotype at the single electrode level, while network level bursts decreased. The IL-6 receptors, IL6R and gp130, were expressed in hPSC-derived cortical neurons, and the gene expression of IL6R increased during maturation. Furthermore, the expression of IL-6R increased not only after IL-6 and H-IL-6 treatment but also after KA treatment. Stimulation with IL-6 or H-IL-6 was not toxic to the neurons and cytokine pretreatment did not independently modulate neuronal network activity or KA-induced seizures. Furthermore, the increased expression of IL-6R in response to IL-6, H-IL-6 and KA implies that neurons can respond through both classical and trans-signaling pathways. Acute treatment with IL-6 and H-IL-6 did not alter functional activity, suggesting that IL-6 does not affect the induction or modulation of newly induced seizures in healthy cultures. Overall, we propose this model as a useful tool to study seizure-like activity in neuronal networks in vitro.
Subject(s)
Epilepsy , Pluripotent Stem Cells , Cytokines/metabolism , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Kainic Acid/metabolism , Kainic Acid/toxicity , Neurons/metabolism , Pluripotent Stem Cells/metabolism , Seizures/chemically inducedABSTRACT
Epilepsies are a group of neurological disorders characterised by recurrent epileptic seizures. Seizures, defined as abnormal transient discharges of neuronal activity, can affect the entire brain circuitry or remain more focal in the specific brain regions and neuronal networks. Human pluripotent stem cell (hPSC)-derived neurons are a promising option for modelling epilepsies, but as such, they do not model groups of connected neuronal networks or focal seizures. Our solution is a Modular Platform for Epilepsy Modelling In Vitro (MEMO), a lab-on-chip device, in which three hPSC-derived networks are separated by a novel microfluidic cell culture device that allows controlled network-to-network axonal connections through microtunnels. In this study, we show that the neuronal networks formed a functional circuitry that was successfully cultured in MEMO for up to 98 days. The spontaneous neuronal network activities were monitored with an integrated custom-made microelectrode array (MEA). The networks developed spontaneous burst activity that was synchronous both within and between the axonally connected networks, i.e. mimicking both local and circuitry functionality of the brain. A convulsant, kainic acid, increased bursts only in the specifically treated networks. The activity reduction by an anticonvulsant, phenytoin, was also localised to treated networks. Therefore, modelling focal seizures in human neuronal networks is now possible with the developed chip.
Subject(s)
Biosensing Techniques , Epilepsy , Brain , Humans , Lab-On-A-Chip Devices , Nerve Net , Neurons , SeizuresABSTRACT
Microelectrode array (MEA) is a tool used for recording bioelectric signals from electrically active cells in vitro. In this paper, ion beam assisted electron beam deposition (IBAD) has been used for depositing indium tin oxide (ITO) and titanium nitride (TiN) thin films which are applied as transparent track and electrode materials in MEAs. In the first version, both tracks and electrodes were made of ITO to guarantee full transparency and thus optimal imaging capability. In the second version, very thin (20 nm) ITO electrodes were coated with a thin (40 nm) TiN layer to decrease the impedance of Ø30 µm electrodes to one third (1200 kΩ 320 kΩ) while maintaining (partial) transparency. The third version was also composed of transparent ITO tracks, but the measurement properties were optimized by using thick (200 nm) opaque TiN electrodes. In addition to the impedance, the optical transmission and electric noise levels of all three versions were characterized and the functionality of the MEAs was successfully demonstrated using human pluripotent stem cell-derived neuronal cells. To understand more thoroughly the factors contributing to the impedance, MEAs with higher IBAD ITO thickness as well as commercial sputter-deposited and highly conductive ITO were fabricated for comparison. Even if the sheet-resistance of our IBAD ITO thin films is very high compared to the sputtered one, the impedances of the MEAs of each ITO grade were found to be practically equal (e.g., 300-370 kΩ for Ø30 µm electrodes with 40 nm TiN coating). This implies that the increased resistance of the tracks, either caused by lower thickness or lower conductivity, has hardly any contribution to the impedance of the MEA electrodes. The impedance is almost completely defined by the double-layer interface between the electrode top layer and the medium including cells.
ABSTRACT
BACKGROUND: Many lines of evidence suggest that accumulation of aggregated alpha-synuclein (αSYN) in the Parkinson's disease (PD) brain causes infiltration of T cells. However, in which ways the stationary brain cells interact with the T cells remain elusive. Here, we identify astrocytes as potential antigen-presenting cells capable of activating T cells in the PD brain. Astrocytes are a major component of the nervous system, and accumulating data indicate that astrocytes can play a central role during PD progression. METHODS: To investigate the role of astrocytes in antigen presentation and T-cell activation in the PD brain, we analyzed post mortem brain tissue from PD patients and controls. Moreover, we studied the capacity of cultured human astrocytes and adult human microglia to act as professional antigen-presenting cells following exposure to preformed αSYN fibrils. RESULTS: Our analysis of post mortem brain tissue demonstrated that PD patients express high levels of MHC-II, which correlated with the load of pathological, phosphorylated αSYN. Interestingly, a very high proportion of the MHC-II co-localized with astrocytic markers. Importantly, we found both perivascular and infiltrated CD4+ T cells to be surrounded by MHC-II expressing astrocytes, confirming an astrocyte T cell cross-talk in the PD brain. Moreover, we showed that αSYN accumulation in cultured human astrocytes triggered surface expression of co-stimulatory molecules critical for T-cell activation, while cultured human microglia displayed very poor antigen presentation capacity. Notably, intercellular transfer of αSYN/MHC-II deposits occurred between astrocytes via tunneling nanotubes, indicating spreading of inflammation in addition to toxic protein aggregates. CONCLUSIONS: In conclusion, our data from histology and cell culture studies suggest an important role for astrocytes in antigen presentation and T-cell activation in the PD brain, highlighting astrocytes as a promising therapeutic target in the context of chronic inflammation.
Subject(s)
Antigen-Presenting Cells/metabolism , Astrocytes/metabolism , Brain/metabolism , Microglia/metabolism , Parkinson Disease/metabolism , Aged , Aged, 80 and over , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/pathology , Astrocytes/immunology , Astrocytes/pathology , Brain/immunology , Brain/pathology , Cells, Cultured , Female , Humans , Male , Microglia/immunology , Microglia/pathology , Middle Aged , Parkinson Disease/immunology , Parkinson Disease/pathologyABSTRACT
OBJECTIVE: To determine the frequency of mutations known to cause autosomal dominant Parkinson disease (PD) in a series with more than 10% of Sweden's estimated number of PD patients. METHODS: The Swedish Parkinson Disease Genetics Network was formed as a national multicenter consortium of clinical researchers who together have access to DNA from a total of 2,206 PD patients; 85.4% were from population-based studies. Samples were analyzed centrally for known pathogenic mutations in SNCA (duplications/triplications, p.Ala30Pro, p.Ala53Thr) and LRRK2 (p.Asn1437His, p.Arg1441His, p.Tyr1699Cys, p.Gly2019Ser, p.Ile2020Thr). We compared the frequency of these mutations in Swedish patients with published PD series and the gnomAD database. RESULTS: A family history of PD in first- and/or second-degree relatives was reported by 21.6% of participants. Twelve patients (0.54%) carried LRRK2 p.(Gly2019Ser) mutations, one patient (0.045%) an SNCA duplication. The frequency of LRRK2 p.(Gly2019Ser) carriers was 0.11% in a matched Swedish control cohort and a similar 0.098% in total gnomAD, but there was a marked difference between ethnicities in gnomAD, with 42-fold higher frequency among Ashkenazi Jews than all others combined. CONCLUSIONS: In relative terms, the LRRK2 p.(Gly2019Ser) variant is the most frequent mutation among Swedish or international PD patients, and in gnomAD. SNCA duplications were the second most common of the mutations examined. In absolute terms, however, these known pathogenic variants in dominant PD genes are generally very rare and can only explain a minute fraction of familial aggregation of PD. Additional genetic and environmental mechanisms may explain the frequent co-occurrence of PD in close relatives.