JNK and PI3K signal pathway did not inhibited neuronal morphology
The cortical neurons were extracted from 18th day embryonic mice and related intracellular signaling pathways were pharmacologically inhibited at 7-14 days (Figure 1A). Gradient screening was employed to determine the minimum inhibitory concentrations (MICs) for each inhibitor. On the 7th day of neuron culture, inhibitors were added basing on their MICs, and neuronal proteins were extracted after one week of treatment. The MICs of PKA inhibitors H89 and PKI, JNK inhibitors JNK-IN-8 and SP6000125, PI3K inhibitors GDC-0941 and LY294002 were 1 μM, 10 μM, 0.3 μM, 10 μM, 0.3 μM and 3 μM, respectively (Figure S1). Immunofluorescence of the DIV 15 neurons showed that PKA inhibitors H89 and PKI significantly decreased the size of soma, the number of primary dendrites and total neurite length (Figure 1B, F-H, P<0.05). However, the size of the neuronal soma, the number of primary dendrites and neuron length did not change markedly in the JNK and PI3K pathway (Figure C-H, P>0.05). Excessive changes in the growth and development of neurons irreversibly disrupt the signaling pathway and affect the maintenance of the whole cell homeostasis. Thus, only JNK and PI3K pathway were used to study synapse formation in further experiments, in order to avoid such factors interfering the accuracy of experimental results.
JNK pathway interfere pre-/post-synaptic specialization, and PI3K signal only perturb post-synaptic puncta density in the cultured neurons
The immunofluorescence was performed on pre- and post-synaptic markers to investigate potential intracellular signaling pathways governing synapse formation between neurons (Figure 2A). Inhibiting JNK pathway observably decreased synapse density and puncta size (Figure 2B and 2F, P<0.05). The PI3K inhibitor LY294002 had no effect on synapse density and size. However, GDC-0941, another PI3K inhibitor, caused an obvious decrease in synapse density and size (Figure 2A, 2B and 2F, P<0.05). Furthermore, the roles of the JNK pathway induced presynaptic VGluT1 and VGAT were verified by SP6000125 and JNK-IN-8 (Figure 2C and 2D). Presynaptic VGluT1 and VGAT were severally utilized as substitutes for excitatory and inhibitory synapses. Interestingly, only the JNK antagonist SP6000125 notably lessened the size of VGluT1 puncta (Figure 2G and 2H, P<0.05). JNK blocker SP6000125 and JNK-IN-8 also signally suppressed the post-synaptic specializations for the decrease of PSD95 puncta density and size (Figure 2E and 2I, P<0.05). Neither the densities of excitatory nor inhibitor pre-synaptic specialization was impressed by PI3K inhibitor (Figure 2C and 2D, P>0.05). Only the PI3K inhibitor LY294002 did not significantly reduce the size of the VGAT (Figure 2G and 2H, P>0.05). In contrast, PI3K inhibitors LY294002 and GDC-0941 observably intercepted post-synaptic specialization (Figure 2E and 2I, P<0.05). The canonical PKA pathway was identified as essential components for assembly of both pre- and post-synaptic specializations (Figure S2). Either pre- or post-synaptic specialization depended on JNK and PKA signals, but the PI3K pathway is merely particularly important for post-synaptic specialization (Figure 2 L-P).
Inhibition of the JNK and PI3K pathways impairs neuronal network activity and development
MEAs non-invasively and continuously record neuronal population activity, including spikes and bursts, through extracellular electrodes positioned at spatially distinct locations throughout primary cultures (Figure 3A). Cultured neuronal networks gradually mature from DIV 21 in vitro, at which the neuronal network exhibits steady and regular synchronous NBs activities. These NBs are essential not only for network communication, but also for the growth and maturation of the neural network. To inquiry the impact of different signaling pathways on the maturity of the neuronal network and neuronal activity, the electrophysiological signals of DIV 21 were recorded by MEAs. Suppression of JNK and PI3K signal increased the network MFR (Figure 3B-E, P<0.05) and network burst frequency (NBF, Figure 3F, P<0.05). JNK pathway inhibitors SP6000125 and JNK-IN-8 markedly decreased network burst duration (NBD, Figure 3C and G, P<0.05). Moreover, the inhibition of the PI3K pathway did not affect NBD (Figure 3D and G, P>0.05). Electrophysiology showed that inhibitors of JNK and PI3K also had a noteworthy implication on the number of spikes per NBs (Figure 3H). Therefore, the maturation of neuronal networks and the firing patterns was adjusted by JNK and PI3K pathway.
Electrophysiological data was continuously recorded from DIV 14 to DIV 49. During neurodevelopment, the MFR of JNK inhibitors SP600025 and JNK-IN-8 began to decrease after reaching the peak at 21 days and showed a significant decrease compared with the DMSO starting from the fifth week (Figure 4D, P<0.05). Similarly, the NBF showed a similar trend to MFR after inhibiting JNK pathway (Figure 4A-E). Additionally, the NBD of JNK inhibitors SP600025 and JNK-IN-8 presented a prominent decrease from the third to the fifth week (Figure 4F, P<0.05). Other network parameters did not markedly differ from the DMSO (Figure 4G-I, P>0.05). The JNK signaling pathway memorably contributes to the development of synchronized bursting within the neuronal network. Finally, the inhibitors LY294002 and GDC-0941 exhibited no effect on MFR of neuronal network development (Figure 4A-D). Furthermore, inhibitors LY294002 and GDC-0941 had higher NBF at 2-4 weeks (Figure 4E, P<0.05), but their difference in NBD were not statistically significant (Figure 4F, P>0.05). Inhibitors LY294002 and GDC-0941 showed a prominent aggrandizement in NBD at 4-7 weeks (Figure 4F, P<0.05). Unexpectedly, the inhibition of the PI3K signaling pathway observably reduced the synchrony of neuronal networks after 5-7 weeks (Figure 5I, P<0.05). The emphasis of the PI3K pathway in network development and maintenance of network synchrony was further verified.
Inhibition of JNK and PI3K pathways altered transcriptomic profile
Molecular changes in neuronal network phenotypic alterations resulting from inhibition of the JNK and PI3K-AKT pathways were also explored. RNA sequencing (RNA-seq) was conducted at DIV 15. Differentially expressed genes (DEGs) [FDR < 0.05, |log2FC| > log2 (2)] were detected in all treatment networks via DESeq2, as compared to dimethyl sulfoxide (DMSO, as control, Figure S4A). The transcriptional profiles' principal component analysis (PCA) showed distinct biological replicate clustering for each treatment (Figure S4B). Quantification of DEGs display that a series of genes were down-/up-regulated in treated cultures compared to the control. DEGs identified in all treatments were remarkably comparable and mostly correlated to axon dendrite and synapse according to Gene Ontology (GO) annotation evaluated for cellular components (CC) (Figure 6A-B). GO annotation of DEGs identified from inhibitors SP6000125 and JNK-IN-8 networks exhibited substantial similarity in their homologous biological processes (BPs), particularly during system development (Figure 6C-D). GSEA-GO analysis showed that SP6000125 was in connection with axons and myelination, synaptic transmission, synaptic vesicle exocytosis and postsynaptic membrane (Figure S5A). However, the main function of JNK-IN-8 in neurons primarily involves mitochondrial BPs (Figure S5B). Thence, altered network development might be driven predominantly by the JNK pathway. Also, the BPs associated with inhibitors LY294002 and GDC-0941 were similar, mainly about axon ensheathment and system development (Figure 6E-F). Detected DEGs were related to alike molecular functions (MF), including ion binding and protein binding (Figure 6E-F). However, GO annotation assessed for CC suggested that the PI3K-AKT pathway was connected with axon and myelin sheath (Figure 6E-F). GSEA-GO analysis showed that the PI3K-AKT pathway was relevant to voltage-gated ion channel activity and neuronal synaptic transmission (Figure S6). Therefore, PI3K-AKT pathway commanded the neuronal structures and function.
What’s more, 251 genes shared commonalities among different JNK inhibitors (Figure S7A). Remarkably, these 251 genes demonstrated an enrichment of GO terms associated with myelination and axon function (Figure S7C). 150 genes associated with PI3K pathway were also identified (Figure S7B), and they correlative were development, axon and myelination (Figure S7D). Consequently, pathway related genes represented parallel functions in harmonizing neuronal structures and activity, notably enrich genes, directly influencing neuronal and synaptic function.
Inhibition of JNK and PI3K pathways declined the expression of postsynaptic membrane receptors
Excitatory and inhibitory receptors are important in the spontaneous network activity and functional maturation of neuronal. Hence, on the 14th day, the excitatory receptors alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) were identified, along with the protein expression of the inhibitory receptors gamma-aminobutyric acid type A receptors (GABAARs). The JNK pathway inhibitor JNK-IN-8 significantly inhibited the protein expression of excitatory receptors AMPAR and NMDAR1 (Figure 7A, P<0.05). Similarly, the JNK pathway inhibitor SP6000125 also dramatically suppressed the expression of AMPAR and NMDAR1 (Figure 7B, P<0.01). Surprisingly, S6000125 also prominently inhibited the excitatory receptor GABAAR (Figure 7B, P<0.01). Inhibiting PI3K pathway with GDC-0941 and LY294002 led to conspicuous impairment in the excitatory receptor GABAAR (Figure 7C-D, P<0.05), but exhibited no arresting impact on inhibitory receptor proteins. Consequently, JNK and PI3K pathways exert an influence not only on synapse formation but also in post-synaptic membrane receptor types.