The cell culture system used in this study followed vendor (NeuCyte Inc.) recommended protocols for their standard SynFire co-culture platform. In short, human iPSC-derived glutamatergic and GABAergic neurons (at a ratio of 70:30) were co-cultured with primary human astrocytes at a ratio of 3:1 neurons:astrocytes (NeuCyte Inc.), as in previous studies (Bogguri et al., 2024; Lam et al., 2023; Soscia et al., 2020). The total seeding density was 3,125 cells/mm² based on vendor recommendation and previous studies conducted by our group (Bogguri et al., 2024) and others (Sasaki et al., 2019; Wang et al., 2023). Briefly, 6-well layout MEA devices (MEA200/30iR-ITO, Multi-channel Systems) were plasma-treated (PDC-001-HP, Harrick Plasma) for 3 min before being soaked in phosphate buffered saline with calcium and magnesium (PBS+; Sigma Aldrich) overnight at 30°C. The MEAs were washed with sterile DI water (4X) and then air dried before autoclaving at 121°C. Both MEA devices and 96 flat bottom-well plates were coated with 0.1% PEI (prepared in borate buffer) for overnight incubation at 37°C, washed with sterile DI water (4X), then coated with a 20 μg/mL of laminin solution in PBS without calcium and magnesium for 2 h at 37°C. The laminin solution was removed before cells were seeded. Purified and concentrated stocks of neuronal subtypes and astrocytes were thawed and diluted, and live cell counts were obtained using a Countess Automated Cell Counter. The appropriate volumes of live cell populations for each cell type were pooled to achieve cell type specific ratios, spun down at 250 g for 5 min, and resuspended in seeding media (NeuCyte Inc.) before deposition in the wells of the MEA device or 96-flat bottom-well plate (50 μL per well). Cultures were maintained in a humidified incubator (37°C, 5% CO₂). After 24 h, 150 μL of short-term media (NeuCyte Inc.) was added to each well, after which 50% of media was replaced every 2–3 days for culture maintenance. After 1 week in culture, media changes were performed using long-term media (NeuCyte Inc.) for the duration of the experiment. All media was used as supplied by the vendor with no alterations.

Recombinant human TNF-α (R&D systems) and IL-6 (R&D systems) were reconstituted to a stock concentration of 100 μg/mL using PBS with 0.1% w/v bovine serum albumin (BSA) and were stored at −20°C until use for experiments. At ~25 days in vitro (DIV), a 50% media exchange was conducted, adding TNF-α, IL-6, or both. A 2X working stock solution was prepared in culture media before added to the cultures for the final TNF-α (1 pg/mL, 25 pg/mL, 100 pg/mL, 10 ng/mL, or 100 ng/mL) or IL-6 (1 pg/mL, 10 pg/mL, 10 ng/mL, 50 ng/mL, or 100 ng/mL) concentration used in the study. All controls had fresh neuron media with an equivalent volume of 0.1% BSA in PBS to act as the vehicle control and to mimic the mechanical perturbations that result from dosing the cultures with the cytokines. Two days after the initial cytokine exposure, another 50% media exchange was conducted once more with the same 2X concentration of cytokine or media added for the respective treatment conditions. Cultures were exposed to cytokine(s) for a period of 7 days.

For recordings, the 6-well MEA device was placed within a 5% CO₂-regulated chamber on the heated stage (37°C) of a 256-channel MEA2100 recording system (Multichannel Systems). Following a 5-min equilibration time, electrophysiology activity was recorded for 30 min at a sampling frequency of 10 kHz and bandpass filtered between 4 and 4,000 Hz, as before by our group (Lam et al., 2019; Enright et al., 2020; Soscia et al., 2020; Soscia et al., 2017) and others (Novellino et al., 2011; Pastore et al., 2018). An action potential spike was defined by a lower limit threshold, set at 6.5x the standard deviation of baseline noise, for each electrode. Devices were recorded for 30 min once a week from 7 DIV and onwards to monitor the development and maturation of neural networks. Cytokine exposure experiments were conducted at ~25 DIV, a time point in which activity becomes stable (Enright et al., 2020; Lam et al., 2019). At this time point, baseline activity of the 6-well MEA device was recorded and then wells are randomly assigned for TNF-α, IL-6, or co-stimulation with TNF-α and IL-6. Following treatment with the cytokine(s), recordings were conducted within the first hour of exposure (i.e., 2 × 30 min recordings), and at 24 h, 48 h, and 7 days post-exposure.

As is previous studies (Enright et al., 2020; Lam et al., 2019; Soscia et al., 2017; Soscia et al., 2020), time-stamped data from each recording was exported as a HDF5 file and analyzed using an in-house custom R package. Burst detection parameters were defined based on previous publications by Charlesworth et al. (2015) and Chiappalone et al. (2005). Electrodes were included in the analysis if they recorded at least 10 spikes in the 30 min recording window. The burst parameters included: maximum beginning interspike interval (ISI) of 0.1 s, maximum end ISI of 0.2 s, minimum interburst interval (IBI) of 0.5 s, minimum burst duration of 0.05 s, and minimum number of spikes per burst of 6. Additional parameters included the removal of any electrodes with a mean burst duration greater than 5 s to eliminate potentially noisy electrodes (Bogguri et al., 2024). For electrodes within an array of a well that had no detectable spiking or bursting activity, a value of ‘0’ was determined. To minimize the effect of mechanical disturbance, attributed to pipetting in the cytokine solution, the mean (for a specific feature) prior to cytokine exposure (e.g., baseline) was calculated. Then, the mean following cytokine exposure was calculated and normalized to baseline activity. The values for the cytokine-treated wells (normalized to baseline) are expressed as a fold change relative to the average value from the 0 pg/mL cytokine concentration (normalized to baseline) at the same time point (e.g., age-matched) and used for further statistical analysis.

Coordinated activity between a pair of electrodes was calculated using SPIKE-distance as previously described (Kreuz et al., 2013). SPIKE-distance measures the dissimilarity between two spike trains as the average of the instantaneous dissimilarity between the two spike trains at different points of the recording. As in previous studies (Eisenman et al., 2015; Enright et al., 2020; Lam et al., 2019), spike train distances were subtracted from 1 to obtain a similarity or synchrony measure, such that a value of 1 represents perfect synchrony and a value of 0 denotes complete asynchrony. Additionally, values were normalized by the SPIKE-distance obtained on randomly generated spike trains to compensate for the documented bias of SPIKE distance to assign higher synchrony values to denser spike trains (Sihn and Kim, 2019). As before with spiking and bursting features, electrodes with a mean burst duration greater than 5s were eliminated to remove noisy electrodes from the SPIKE distance analysis. Less than 1% of electrodes across all experiments were removed by this criterion.

CyQuant™ Lactate Dehydrogenase (LDH) assay (Thermo Fisher Scientific) was performed according to the manufacturer’s instructions. Fresh culture supernatant was collected at 2 and 7 DIV and LDH activity was measured spectrophotometrically at 490 and 680 nm on the Synergy H1 multi-mode microplate reader (BioTek). The absorbance data for cytokine treatment was normalized to the untreated condition.

Cytokines were quantified using LEGENDplex™ Human Essential Immune Response panel, a multiplex fluorescence-encoded bead-based assay (BioLegend). Samples were prepared according to manufacturer’s instructions. Flow cytometry of the beads was performed using a FACS Aria Fusion (BD Biosciences), and data analyzed using BioLegend’s cloud-based analysis software¹.

Cultures were exposed to 25 pg/mL, 10 ng/mL, 100 ng/mL of TNF-α, or vehicle for 7 days before lysed using RLT buffer containing β-mercaptoethanol and RNA was isolated using the RNAeasy mini spin columns (Qiagen). Illumina Stranded mRNA Prep kit (Illumina) was used to prepare the sequencing libraries and sequencing was performed using an Illumina NextSeq 2000. The quality of sequencing data was checked using FastQC software². The reads were mapped to the human genome (hg38) using STAR and read counts per gene were determined using “featureCounts” from Rsubread package (Liao et al., 2014; Risso et al., 2014). Subsequently, differentially expressed genes were identified using the limma package with its voom method (Law et al., 2016; Ritchie et al., 2015). A gene was considered as significantly differentially expressed when its false discovery rate adjusted p-value was <0.05 and fold change was >1.5. Gene ontology (GO) analysis was performed using ToppGene (Chen et al., 2009). Heatmaps were generated using heatmap.2 function in “gplots” R package. Volcano plots were generated using Galaxy Europe³.

Data is expressed as mean ± standard error of the mean (SEM) for the number of replicates indicated, unless stated differently. Electrophysiological features were determined on a per-electrode basis (or electrode pair basis for synchrony), but statistical analysis was performed on a per-well basis by taking the overall mean from the active electrodes. For electrophysiology, LDH, and cytokine/chemokine experiments statistical significance was analyzed in GraphPad version 8 (GraphPad Software) using a one-way ANOVA or a mixed model repeated measures two-way ANOVA with Dunnett’s or Tukey’s post hoc analysis. For all experiments, statistical significance was determined by p-values <0.05. For bulk RNA-sequencing datasets statistical significance was determined as described above.

Elevated levels of TNF-α, IL-8, and IL-6, have been detected in hospitalized patients during the initial stages of SARS-CoV-2 infection, and TNF-α and IL-6, in particular, are significant predictors for increased severity and death (Del Valle et al., 2020). Here, we conducted a concentration-response study to assess how human iPSC-derived neural networks are modulated by prolonged exposure to TNF-α at clinically relevant concentrations. We tested concentrations of 1 and 25 pg/mL, which was in range with control patients (Del Valle et al., 2020; Hirzel et al., 2022; Schultheiss et al., 2022), and 100 pg/mL, which is in range of hospitalized COVID patients and patients suffering from symptoms of long-COVID (Torabi et al., 2020; Jarius et al., 2022; Ramezani et al., 2022; Schultheiss et al., 2022). We also evaluated 10 ng/mL and 100 ng/mL TNF-α, concentrations reported from previous in vitro studies using human iPSC-derived or neuroblastoma derived cells (Talley et al., 1995; Hyvärinen et al., 2019; Jayaraman et al., 2021; Saraf et al., 2021; Kerkering et al., 2023). Neurons and astrocytes were seeded at ratios outlined in the methods to model the cortical region of the brain (Defelipe and Farinas, 1992; Markram et al., 2004). Bulk RNA sequencing data confirmed the detection of neuronal markers, tubulin beta 3 (TUBB3) and RNA binding fox-1 homolog 3 (RBFOX3), and the astrocyte marker, glial fibrillary acidic protein (GFAP), from the co-culture system as shown in Supplementary Figure S1.

As in previous studies (Lam et al., 2019; Lam et al., 2022), the number of active electrodes became stable around 21 days in culture (Supplementary Figure S2) and mature neural and network activity, as indicated by coordinated spiking and bursting activity, is detected by day 25 (Figure 1A). Co-cultures on the MEA devices were selected at random and were treated with one of 6 concentrations of TNF-α (0 pg/mL, 1 pg/mL, 25 pg/mL, 100 pg/mL, 10 ng/mL, and 100 ng/mL). Thirty minute recordings were taken after 30 min, 60 min, 1, 2, and 7 days of exposure to TNF-α. Figure 1A displays representative raster plots of spiking and bursting patterns from three different TNF-α treatment concentrations (control, 25 pg/mL, and 100 ng/mL) across multiple days of exposure.

Mechanical disturbances caused by pipetting have been shown to affect neural network activity in culture (Lam et al., 2022). To minimize the effect of mechanical disturbance, time points for each feature were normalized to age-matched controls. When cultures were exposed to TNF-α at nanogram concentrations (e.g., 10 and 100 ng/mL), features of spiking activity (number of spikes, firing rate, and ISI) decreased at intermediate timepoints (24 and 48 h) but largely returned to control levels by 7 days of exposure (Figure 1B). Interestingly, at picogram concentrations that are within range of normal clinical levels (Del Valle et al., 2020; Hirzel et al., 2022; Schultheiss et al., 2022), ISI changes persisted throughout the length of the experiment (Figure 1B). For the 1 pg/mL condition, ISI trended down throughout the experiment, but did not become significantly decreased until day 7. For the 25 pg/mL condition, a significant decrease in ISI was observed 30 min post treatment, which remained throughout the 7 days of treatment (Figure 1B). No change in spiking activity was observed in the 100 pg/mL condition. For features of bursting activity (burst duration, bursts per minute, and total number of spikes in bursts) the presence of TNF-α, independent of concentration, decreased burst duration at early timepoints, which recovered by day 7 (Figure 1B). While we did observe a few combinations of timepoints and concentrations that showed statistically significant differences, overall the total number of spikes in bursts and bursts per minute were not affected by TNF-α treatment.

Next, we examined how TNF-α treatment affected the degree of coordination (or synchrony) in spiking activity between networks within the co-culture system. As previously described (Cadena et al., 2020; Enright et al., 2020; Lam et al., 2019; Lam et al., 2022), the SPIKE distance method (Kreuz et al., 2013) was used to score all possible electrode parings for a given 30 min recording within each MEA. A score of ‘0’ indicates that the pair of electrodes has no synchrony, while a score closer to ‘1’ indicates a high degree of synchrony. We observed a consistent unimodal distribution of synchrony scores across all timepoints and TNF-α concentrations (Figure 2A and Supplementary Figure S3). Similarly, no significant difference was observed when comparing the average synchrony score, which ranges from 0.25 to 0.35, across each concentration and time points (Figure 2B).

In addition to TNF-α, we also investigated the effect of IL-6 on neuron functionality, comparing a similar range of concentrations spanning control (0 and 1 pg/mL) (Han et al., 2020), hospitalized COVID patients and patients suffering from long-COVID (10 pg/mL) (Han et al., 2020; Donoso-Navarro et al., 2021; Ulhaq and Soraya, 2020; Schultheiss et al., 2022), along with concentrations shown to affect neural activity in culture (10, 50, and 100 ng/mL) (Vereyken et al., 2007; Nelson et al., 2002; D’Arcangelo et al., 2000; Kawasaki et al., 2008). Interestingly, in contrast to TNF-α, we did not observe a strong concentration-dependent effect on any of the measured electrophysiological features. We observed a general reduction in neural activity (number of spikes and firing rate) across all concentrations of IL-6, however significant differences were only observed sporadically at varying timepoints and concentrations, suggesting a broad, but minimal reduction in neural activity in response to IL-6 (Figure 3A). We found a more robust response to IL-6 exposure on the total number of spikes in bursts, with a significant reduction in at least two timepoints across all concentrations (Figure 3A). Similar to TNF-α, we did not observe a significant change in the distribution of synchrony scores (Supplementary Figure S4) or average synchrony score (Figure 3B) at any concentration or exposure time.

As TNF-α and IL-6 are both elevated in patients with long-COVID (Fernández-Castañeda et al., 2022; Schultheiss et al., 2022), we wanted to determine if the combination of both TNF-α and IL-6 exposure may lead to more significant effects on network activity. As we did not see a strong concentration-dependent effect with IL-6 exposure, we exposed the cultures to a combination of 10 ng/mL IL-6 [the lowest tested concentration that has been previously shown to affect neuron activity in culture (Vereyken et al., 2007; Nelson et al., 2002; D’Arcangelo et al., 2000; Kawasaki et al., 2008)] and either 25 pg/mL or 100 ng/mL TNF-α. These two concentrations of TNF-α were chosen as cultures exposed to 25 pg/mL did not show a significant change in firing rate (concentration-dependent effect) but did show changes to ISI and burst duration (concentration-independent effects), while cultures exposed to 100 ng/mL showed changes to both firing rate and ISI and burst duration.

As before, we observed a general reduction in neural activity and total number of spikes in bursts when cultures were exposed to 10 ng/mL IL-6, with significant decreases in these features at sporadic timepoints (Figure 4A). Similarly, we observed a significant decrease in ISI and burst duration in cultures exposed to both 25 pg/mL and 100 ng/mL TNF-α, but only detected a significant decrease in firing rate and total number of spikes in cultures exposed to 100 ng/mL at later timepoints (Figure 4A). When cultures were exposed to 25 pg/mL TNF-α in combination with 10 ng/mL IL-6, we observed no significant change in any electrophysiological feature at any timepoint, suggesting that TNF-α and IL-6 may have opposing effects on network activity (Figure 4A). However, when these systems were exposed to 100 ng/mL TNF-α with 10 ng/mL IL-6, the changes in electrophysiological features were nearly identical to that of just 100 ng/mL TNF-α, suggesting that TNF-α plays a dominant role at higher concentrations (Figure 4A). Finally, just as before, we observe no significant changes in the distribution of synchrony scores (Supplementary Figure S5) or average synchrony score (Figure 4B) at any exposure combination.

In order to determine if the changes to electrophysiological features was a consequence of cell death, we quantified extracellular LDH at 2- and 7-days of exposure for all TNF-α and IL-6 concentrations. We observed a significant increase in extracellular LDH at 7-days of exposure to 100 pg/mL, 10 ng/mL, and 100 ng/mL TNF-α, but not for lower concentrations or at 2-days of exposure (Figures 5A,C). No change in cell viability was observed at either 2- or 7-days of exposure to any concentration of IL-6 (Figure 5B). Finally, co-stimulation with IL-6 somewhat reduced the cytotoxic effects of prolonged exposure to high concentrations of TNF-α, as cultures exposed to 100 ng/mL of TNF-α in combination with 10 ng/mL IL-6 no longer show a significant increase in extracellular LDH at day 7 as compared to untreated controls (Figure 5C).

Due to the prolonged exposure times, we examined whether other cytokines secreted by the co-culture may have contributed to modulating neural activity. We compared cytokine secretion profiles of 13 key cytokines associated with the immune response (IL-4, IL-2, CXCL-10, IL-1β, IL-6, TNF-α, MCP-1, IL-17A, IL-10, IFN-γ, IL-12p70, TGF-β, and IL-8) following exposure to 25 pg/mL and 100 ng/mL TNF-α with and without co-stimulation with 10 ng/mL IL-6. At 2-days of exposure we found that 4 cytokines (IL-4, CXCL-10, IL-1β, and IL-10) showed significantly different secretion levels based on treatment condition (Figure 6A), while at 7-days of exposure we found that 8 cytokines had significantly different secretion levels (IL-4, IL-2, CXCL-10, IL-1β, IL-17A, IL-10, IFN-γ, IL-12p70, and IL-8; Figure 6). As expected, exposure conditions containing 100 ng/mL TNF-α and/or 10 ng/mL IL-6 had high concentrations of TNF-α and IL-6, respectively. Additionally, cultures exposed to 100 ng/mL TNF-α showed a significant increase in the concentration of IL-6 in the culture media, while cultures exposed to 10 ng/mL IL-6 did not show any change in TNF-α concentration (Supplementary Figure S5). Interestingly, we observed ~180 pg/mL of IL-6 in the conditioned media in the control conditions, while the average concentration of TNF-α was found to be at or less than the lower limit of detection for the assay (<2.5 pg/mL; Supplementary Figure S6).

Of the 4 cytokines that showed significantly different expression levels at 48 h (Figure 6A), increases in CXCL10 and IL-1β were primarily a response to treatment with 100 ng/mL TNF-α. Treatment with IL-6 significantly increased the levels of CXCL10 as compared to the corresponding untreated condition or 25 pg/mL TNF-α, however were unable to observe any potential further increase in CXCL10 concentration via co-stimulation of IL-6 with 100 ng/mL TNF-α as it was at or beyond the upper limit of detection. Co-stimulation with IL-6 did not change IL-1β concentration. Significant increases of IL-10 was primarily dependent on IL-6 treatment, as cultures treated with only TNF-α showed minimal changes in IL-10 concentration as compared to untreated control. Interestingly, increases in IL-4 seemed to be dependent on co-stimulation with both 100 ng/mL TNF-α and 10 ng/mL IL-6 as that conditioned showed not only a significant increase in IL-4 secretion as compared to the control, but also as compared to the 100 ng/mL TNF-α treatment condition. For each of these cytokines, the changes in cytokine levels at 7 days largely matched the changes seen at 48 h.

In addition to the four aforementioned cytokines, an additional four cytokines were significantly increased in the culture media following 7 days of TNF-α and IL-6 exposure (Figure 6B). Changes in IFN-γ and IL-17A showed similar patterns, with both cytokines showing significant increases in their concentration being primarily dependent on exposure to 100 ng/mL TNF-α. For both cytokines we observe a marginal (not statistically significant) decrease in concentration when co-stimulated with both TNF-α and IL-6. IL-12p70 appears dependent on the presence of a high concentration of both TNF-α and IL-6, as only the co-stimulated condition showed a significant increase over control. Finally, we observed a strong dependence of IL-8 concentration on high concentrations of TNF-α, as we observe a significant increase in IL-8 concentration in the 100 ng/mL TNF-α condition into the ng/mL range. Additionally, co-stimulation with IL-6 dramatically reduces IL-8 levels as compared with 100 ng/mL TNF-α treatment alone. Finally, there were three cytokines that did not show any changes in concentration (i.e., MCP-1) or were below the lower limit of detection (i.e., IL-2 and TGF-β) in any exposure condition (data not shown).

Next, we performed transcriptomic analysis of cultures treated with various concentrations of TNF-α to gain molecular insights into the strong concentration-dependent effect observed on electrophysiological features and the detected inflammatory cytokines. Differential gene expression (DEG) analysis identified that cultures treated with 10 and 100 ng/mL of TNF-α (but not 25 pg/mL) had altered gene expression profiles at 7 days of exposure (Figure 7A). Specifically, 1,045 genes were differentially expressed in 10 ng/mL TNF-α vs. control and 1,191 genes in 100 ng/mL TNF-α vs. control [DEGs, abs (log₂FC) > 0.5 and FDR < 0.05; Figure 7A]. As there were no significant alterations in the transcriptome of cultures treated with 25 pg/mL, it suggests that changes in neural functionality were not dependent on transcriptional regulation and expression of genes. For cultures exposed to nanogram concentrations of TNF-α, ontology enrichment analysis of DEGs identified GO biological processes for genes unique to 10 ng/mL TNF-α and were primarily associated with neuronal activity (Figure 7Bi). Specifically, this condition showed a downregulation of genes associated with ion channel activity, and an upregulation of genes involved in synaptic transmission (e.g., chemical synaptic transmission, glutamatergic synaptic transmission, and regulation of neurotransmitter receptor activity). Biological processes for genes unique to 100 ng/mL TNF-α included the downregulation of genes associated with receptor activity (e.g., integrin binding, collagen binding, MHC class II receptor activity, CD4 receptor binding) and the upregulation of genes for processes involved in an immune response (e.g., response to cytokine, type I interferon-mediated signaling pathway, respiratory burst, negative regulation of membrane potential, and glial cell-derived neurotropic factor receptor activity; Figure 7Bii). Biological processes with genes in common for both concentrations of TNF-α (Figure 7C) were associated with an increase in cytokine response or production (e.g., IL-12, type II interferon, and TNF superfamily), and cell death (e.g., regulation of programmed cell death, I-kappaB/NF-kappaB signaling). A decrease in the expression of genes associated with several cellular functions was also detected (e.g., neuron development, gliogenesis, chemotaxis, extracellular matrix organization, axon guidance, oligodendrocyte differentiation).

In Figure 8, we highlighted genes of interest that were highly upregulated or downregulated in both 10 and 100 ng/mL compared to 0 and 25 pg/mL of TNF-α. For cytokines (Figure 8A) many of the upregulated genes overlapped with the increased cytokine levels observed in the cytokine array (e.g., IL6, CXCL10, MCP-1/CCL2 and IL1B). The upregulation of genes in both the 10 and 100 ng/mL TNF-α treatment condition revealed the increase in specific receptors within the tumor necrosis factor receptor superfamily (e.g., TNFSF9, TNFSF10, TNFSF13B, and TNFSF14), and chemokines that bind to interleukin-8 (e.g., CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, and CX3CL) and CXCR3 chemokine (e.g., CXCL9, CXCL10, CXCL11, and CXCL13) receptors. Genes for growth factor activity were also upregulated (e.g., IL6, IL7, IL34, IL11, LIF, CSF1, CXCL2, and CLCF1). For gliogenesis (Figure 8B), genes that were downregulated in both the 10 and 100 ng/mL TNF-α suggest a shift in the state of astrocytes, in particular the role of astrocyte differentiation (e.g., SOX6, SOX8, SOX9, VIM, VAX1, DRD1, FGFR3, PLP1, NOTCH1 and NR2E1), and glial cell migration (e.g., VIM, NTN1, EFEMP1, ATP1B2, DOCK8, PTPRZ1, GLI3, FOXG1, and NR2E1). Additionally, in cultures exposed to nanogram concentrations of TNF-α we observe a decrease in genes involved with neural precursor cell proliferation (e.g., HAPLN4, SOX5, NFIA, PTPRZ1, GL13, FOXG1, NOTCH1, HAPLN1, and NR2E1), axonogenesis (e.g., VIM, NTN1, VAX1, ERBB2, FGFR3, NDP, OTX2, METRN, NKX2-1, PTPRZ1, GLI3, GOXG1, NOTCH1, and NR2E1), and genes related to abnormal neuron morphology (e.g., HAPLN4, CIM, NTN1, VAX1, ERBB2, DRD1, FGFR3, NDP, OTX2, ATP1B2, MYO6, NFIA, PLP1, ENPP2, IL33, GLI3, GOXG1, NOTCH1, KCNJ10, and NR2E1). For neuron development (Figure 8C), the top genes that were downregulated in both the 10 and 100 ng/mL TNF-α conditions were involved in Wnt signaling pathways (e.g., FZD1, FZD2, FZD4, FGFR2, FGFR3, NDP, LGR6, APOE, WLS, FOLR1, NOTCH1, and EDNRB), regulation of neurogenesis (e.g., FDZ4, VIM, SLIT2, LPAR3, VAX1, FGFR3, APOE, WLS, PTPRZ1, TNC, NOTCH1, NR2E1, SEMA3B), and axon guidance (e.g., LAMA3, COL4A6, SLIT2, VAX1, BMPR1B, NDP, OTX2, LGR6, NKX2-1, TNC, NOTCH1, SMEA3B).