Autaptic cultures of human induced neurons as a versatile platform for studying synaptic function and neuronal morphology

Recently developed technology to differentiate induced pluripotent stem cells (iPSCs) into human induced neurons (iNs) provides an exciting opportunity to study the function of human neurons. However, functional characterisations of iNs have been hampered by the reliance on mass culturing protocols which do not allow assessment of synaptic release characteristics and neuronal morphology at the individual cell level with quantitative precision. Here, we have developed for the first time a protocol to generate autaptic cultures of iPSC-derived iNs. We show that our method efficiently generates mature, autaptic iNs with robust spontaneous and action potential-driven synaptic transmission. The synaptic responses are sensitive to modulation by metabotropic receptor agonists as well as potentiation by acute phorbol ester application. Finally, we demonstrate loss of evoked and spontaneous release by Unc13A knockdown. This culture system provides a versatile platform allowing for quantitative and integrative assessment of morphophysiological and molecular parameters underlying human synaptic transmission

Optogenetic acidification of synaptic vesicles and lysosomes

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes

PI3K/AKT Signaling Pathway Is Essential for Survival of Induced Pluripotent Stem Cells.

Apoptosis is a highly conserved biochemical mechanism which is tightly controlled in cells. It contributes to maintenance of tissue homeostasis and normally eliminates highly proliferative cells with malignant properties. Induced pluripotent stem cells (iPSCs) have recently been described with significant functional and morphological similarities to embryonic stem cells. Human iPSCs are of great hope for regenerative medicine due to their broad potential to differentiate into specialized cell types in culture. They may be useful for exploring disease mechanisms and may provide the basis for future cell-based replacement therapies. However, there is only poor insight into iPSCs cell signaling as the regulation of apoptosis. In this study, we focused our attention on the apoptotic response of Alzheimer fibroblast-derived iPSCs and two other Alzheimer free iPSCs to five biologically relevant kinase inhibitors as well as to the death ligand TRAIL. To our knowledge, we are the first to report that the relatively high basal apoptotic rate of iPSCs is strongly suppressed by the pancaspase inhibitor QVD-Oph, thus underlining the dependency on proapoptotic caspase cascades. Furthermore, wortmannin, an inhibitor of phosphoinositid-3 kinase / Akt signaling (PI3K-AKT), dramatically and rapidly induced apoptosis in iPSCs. In contrast, parental fibroblasts as well as iPSC-derived neuronal cells were not responsive. The resulting condensation and fragmentation of DNA and decrease of the membrane potential are typical features of apoptosis. Comparable effects were observed with an AKT inhibitor (MK-2206). Wortmannin resulted in disappearance of phosphorylated AKT and activation of the main effector caspase-3 in iPSCs. These results clearly demonstrate for the first time that PI3K-AKT represents a highly essential survival signaling pathway in iPSCs. The findings provide improved understanding on the underlying mechanisms of apoptosis regulation in iPSCs

RIM-binding protein 2 regulates release probability by fine-tuning calcium channel localization at murine hippocampal synapses

The tight spatial coupling of synaptic vesicles and voltage-gated Ca2+ channels (CaVs) ensures efficient action potential-triggered neurotransmitter release from presynaptic active zones (AZs). Rab-interacting molecule-binding proteins (RIM-BPs) interact with Ca2+ channels and via RIM with other components of the release machinery. Although human RIM-BPs have been implicated in autism spectrum disorders, little is known about the role of mammalian RIM-BPs in synaptic transmission. We investigated RIM-BP2-deficient murine hippocampal neurons in cultures and slices. Short-term facilitation is significantly enhanced in both model systems. Detailed analysis in culture revealed a reduction in initial release probability, which presumably underlies the increased short-term facilitation. Superresolution microscopy revealed an impairment in CaV2.1 clustering at AZs, which likely alters Ca2+ nanodomains at release sites and thereby affects release probability. Additional deletion of RIM-BP1 does not exacerbate the phenotype, indicating that RIM-BP2 is the dominating RIM-BP isoform at these synapses

Wortmannin significantly induced apoptosis in BIHi001-A and BIHi004-A iPSC lines.

<p>(A,E) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in iPSCs treated for 24 h with 10ng/ml TRAIL, 4 μM L-779,450, 4 μM BMS, 50 nM MLN-8237 (AKA-I), 4 μM MK-2206, 4 μM wortmannin (Wort.). (B,F) Whole cell cycle analysis after treatment with small molecules mentioned in A with respect to the amount of different phases (sub-G1, G1, S, G2/M) in percent. (C,D) Histogram examples of cells treated with BMS, AKA-I or wortmannin as compared to controls (DMSO). Sub-G1 cell populations are indicated (sG1). (G) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in orginal fibroblasts (HFF and NHDF) treated for 24 h with 10ng/ml TRAIL, 4 μM L-779,450, 4 μM BMS, 50 nM MLN-8237 (AKA-I), 4 μM MK-2206, 4 μM wortmannin (Wort.). Insets: Histogram examples of cells treated with AKA-I or wortmannin compared to controls (DMSO). Sub-G1 cell populations are indicated (sG1). (H,I) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in two iPSC-derived neurons treated for 24 h with 10ng/ml TRAIL, 4 μM L-779,450, 4 μM BMS, 50 nM MLN-8237 (AKA-I), 4 μM MK-2206, 4 μM wortmannin (Wort.). Insets: Histogram examples of cells treated with, BMS, AKA-I or wortmannin compared to controls (DMSO). Means and SDs are shown of three independent experiments in triplicates. Statistical significance (*; p < 0.05) is indicated for comparison of control cells and wortmannin-treated cells.</p

Western blot analysis of key proteins of apoptosis in wortmannin treated AD-iPSCs.

<p>Protein lysates were analyzed in AD-iPSCs after treatment with 4 μM wortmannin for different times. On the last lane of the blot at the right we subjected protein lysate of dead cells (DC) raised after 24 h in the supernatant to follow the basal apoptosis. Fourty μg of protein each were separated by SDS-PAGE (12%). Western blot analysis was used to monitor the expression of phosphorylated AKT (p-AKT, at serine 473) and total AKT, phosphorylated BAD and total BAD and p53. The anti-human Caspase-3 antibody used recognizes only the cleaved active form of caspase-3. The anti-human LC3A antibody recognizes both isomers, LC3I and LC3II. Furthermore we used anti-human BAX, BAK, BCL-2, BCL-X_L and PUMA antibodies, against members of the Bcl-2 family. Beta-actin and Coomassie blue staining were used to confirm similar protein loading across samples.</p

Wortmannin induced apoptosis in iPSCs causes nuclear condensation and fragmentation detected by Hoechst-33258 staining.

<p>(A) Phase contrast of untreated AD-iPSCs with sharp edges of round colonies (a) untreated AD-iPSC clones show mitotic cells, which can be seen by as intense blue (b-c). Phase contrast of wortmannin treated AD-iPSCs with frayed edges 1 h after treatment with 4μM wortmannin (d). A high proportion of cells showed clear indication of apoptosis by nuclear condensation and fragmentation, particularly pronounced at the edge of the colonies of AD-iPSCs (d-f) (examples indicated by arrows). B: Magnified images of untreated AD-iPSCs marked as mitotic cells (a), diffuse blue nucleus (b), sharp edge of untreated colony (c), magnified images of apoptotic cells with DNA condensation, DNA fragmentation (d-g). Changes in the morphology of the colonies one hour after treatment with wortmannin, and after 24 h complete detachment of the AD-iPSCs have been observed (h-i).</p

Comparative analysis of proteins between AD-iPSCs and AD-iPSC-derived neurons after wortmannin treatment.

<p>Protein lysates of AD-iPSCs and iPSC-derived neurons (INC) were analyzed after treatment with 4μM wortmannin for different times. Fourty μg of protein each were separated by SDS-PAGE (12%). (A) Western blot analysis was used to monitor the expression of total and phosphorylated Akt (p-Akt, at serine 473). The anti-human LC3A recognizes both isoforms LC3I and LC3II and could also show phosphorylated LC3II. (B) iPSCs compared to iPSC-derived neurons and original fibroblasts. The anti-human Caspase-3 used recognizes only the cleaved active form of caspase-3. Beta-tubulin and Coomassie blue staining were used to confirm similar protein loading across samples.</p

Decrease of membrane potential and increase of ROS production upon wortmannin treatment in AD-iPSCs.

<p>(A) Decreased membrane potential of mitochondria was determined by flow cytometry after TMRM⁺ staining in AD-iPSCs. Cells were treated with different concentrations of wortmannin (1 μM, 2 μM, 4 μM) for different times (1 h, 2 h, 24 h). Treated cells (open graphs) were compared to untreated controls (gray). (B) The quantitative data represent mean values of triplicate experiments +/-SD. (C) The production of ROS was determined after H2DCFDA staining in AD-iPSCs treated with two different concentrations of wortmannin (2–4 μM) or 4 μM of MK-2206 at two different time points (2h, 6h), by flow cytometry. Treated cells (open graphs) were compared to untreated controls (gray). (D) Quantitative data represent the median values of treated cells compared to control cells. ROS production was depicted as fold-increase, the control was set to 1. Two independent experiments with triplicates revealed comparable results</p