Striatal neurons show a low induction of early UPR markers, whereas later ER stress responses are upregulated.

<p>Early responses and in some cases late ones are increased by expression of Htt111Q. A,B) Levels of UPR markers after short term ER stress. ST<i>Hdh</i>^Q7/7 were compared to ST<i>Hdh</i>^Q111/111 and NIH 3T3 cells. Immunoblots show results of a representative experiment of 3. Vertical lines indicate removal of irrelevant lanes. C-H) Quantification of A,B. ST<i>Hdh</i>^Q7/7 cells do not activate properly an early stress response, mediated by PERK-P and its target eIF2α-P, induced by Tun (C, 10 µg/ml) or by MG-132 (D, 40 µM). In ST<i>Hdh</i>^Q111/111 cells, PERK-P and eIF2α-P are induced (C,D). Later ER stress responses are increased in ST<i>Hdh</i>^Q7/7 compared to NIH 3T3 cells (E-H). Htt111Q expression causes even more enhanced upregulation of the UPR markers in some cases. Values were normalized to β-actin levels as a loading control.</p

Regulation of phosphorylated eIF2α levels by inhibition of its dephosphorylation.

<p><b>A</b>) Guanabenz (Gz), at a relatively high concentration (100 µM), inhibits eIF2α dephosphorylation in untreated ST<i>Hdh</i>^Q7/7 cells and also in those treated with Tun (5 µg/ml) up to 7h; this is also true in ST<i>Hdh</i>^Q111/111 cells but only after very short treatments. *P = 0.02, **P = 0.01. EIF2α-P levels were normalized by total eIF2α. <b>B</b>) Similar to (A), but for cells treated for 24 h. After these long treatments Gz did not inhibit ER stress-induced eIF2α dephosphorylation, it increased CHOP levels. The values in the graphs are averages from 3-4 independent experiments±SE. *P<0.05, **P = 0.002. <b>C</b>) Gz showed a minor effect in rescuing ST<i>Hdh</i>^Q111/111 cells from UPR-induced cell death (Tun for 48 h). ***P =  0.0001.</p

Regulation of phosphorylated eIF2α levels by inhibition of its phosphorylation and rescue of ST<i>Hdh</i>^Q111/111 cells.

<p><b>A</b>) EIF2α phosphorylation in ST<i>Hdh</i>^Q111/111 cells is PERK-mediated. ST<i>Hdh</i>^Q111/111 cells left untreated or treated with the PERK inhibitor A4 (50 µM) or the PKR inhibitor PKRi (1 µM) for the indicated times. **P = 0.009. <b>B</b>) ER stress-mediated eIF2α phosphorylation is inhibited by A4 and not by PKRi. As in (A), but with ST<i>Hdh</i>^Q7/7 cells treated for different times with Tun. <b>C</b>) PKR-mediated eIF2α phosphorylation is inhibited by PKRi and not by A4. As in (B), but with cells treated for 7h with the PKR inducer poly-I:C (200 µg/ml). <b>D-E</b>) A4 rescued ST<i>Hdh</i>^Q111/111 cells from UPR-induced cell death (Tun for 48 h, D), whereas PKRi had no effect (E). ***P =  0.0001. <b>F</b>) Total protein synthesis levels are much increased in ST<i>Hdh</i>^Q111/111 cells after prolonged ER stress (Tun for 24h) and reduced by A4 (50 µM). **P<0.002 (3 repeat experiments).</p

High sensitivity of striatal neurons to ER stress, further aggravated by expression of pathogenic huntingtin.

<p><b>A-C</b>) Strong induction of GADD34 and CHOP upon prolonged ER stress in ST<i>Hdh</i>^Q7/7 cells and even stronger in ST<i>Hdh</i>^Q111/111 cells; (3 independent experiments ±SE). *P = 0.02, <b>*</b>*P = 0.01, ***P = 0.0002. Immunoblots of a representative experiment are shown in A. GAPDH levels served here as a loading control. <b>D</b>) Prolonged ER stress induced with Tun or MG-132 leads to extensive death of ST<i>Hdh</i>^Q7/7 cells, further aggravated in ST<i>Hdh</i>^Q111/111 cells, as measured by FACS analysis of cell cycle progression with propidium iodide (PI) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090803#pone.0090803.s002" target="_blank">Fig. S2</a>); (6 independent experiments ± SE). *P<0.05, **P = 0.01, ***P = 0.001.</p

Very low eIF2α-P levels in striatal cells, much increased by expression of Htt111Q.

<p><b>A</b>) Basal level of eIF2α-P in murine cell lines normalized by total eIF2α. Graph: average of 3 experiments ± SE<b>.</b> **P = 0.004, ***P  = 0.001. <b>B</b>) Immunofluorescence images of cells fixed, permeabilized and stained with rabbit anti-eIF2α-P and mouse anti-eIF2α followed by secondary antibodies. Bar = 10 µm. Image exposure time was kept constant to be able to compare protein levels in the different cell types. Levels relative to ST<i>Hdh</i>^Q7/7 levels were quantified from images from 3 experiments ± SE (>20 cells, ***P<0.001).</p

Highly Ordered Large-Scale Neuronal Networks of Individual Cells – Toward Single Cell to 3D Nanowire Intracellular Interfaces

The use of artificial, prepatterned neuronal networks in vitro is a promising approach for studying the development and dynamics of small neural systems in order to understand the basic functionality of neurons and later on of the brain. The present work presents a high fidelity and robust procedure for controlling neuronal growth on substrates such as silicon wafers and glass, enabling us to obtain mature and durable neural networks of individual cells at designed geometries. It offers several advantages compared to other related techniques that have been reported in recent years mainly because of its high yield and reproducibility. The procedure is based on surface chemistry that allows the formation of functional, tailormade neural architectures with a micrometer high-resolution partition, that has the ability to promote or repel cells attachment. The main achievements of this work are deemed to be the creation of a large scale neuronal network at low density down to individual cells, that develop intact typical neurites and synapses without any glia-supportive cells straight from the plating stage and with a relatively long term survival rate, up to 4 weeks. An important application of this method is its use on 3D nanopillars and 3D nanowire-device arrays, enabling not only the cell bodies, but also their neurites to be positioned directly on electrical devices and grow with registration to the recording elements underneath

Developmental TLR4 deficiency, but not pharmacological TLR4 antagonism, enhances retention of spatial reference memory.

<p>(<b>A</b>) TLR4^−/− (n = 19) and TLR4^+/+ (n = 24) mice were tested in probe trials at 24, 48, 72 and 96 hours following training for retention of spatial reference memory. Tests were done after all experimental groups exhibited loss of memory of the platform location. Mean distance from the platform was measured and used to indicate efficiency in locating the hidden platform. TLR4^−/− mice showed shorter mean distance from the platform at 24 and 48 hours after training compared with TLR4^+/+ mice, indicating a more accurate swim toward the platform quadrant (<b>B</b>) Mice (C57BL/6) were implanted with an osmotic pump containing either aCSF (n = 10) or a TLR4 antagonist (n = 10). The pump was connected via tubing to a cannula, which was positioned to the lateral ventricle. Following training in the MWM task, mice were tested in probe trials at 24 and 48 hours following training for retention of spatial reference memory. Both experimental groups exhibited similar performance during probe trials, as measured by mean distance from the platform.</p

Pharmacological TLR4 inhibition does not affect fear-learning and memory.

<p>(<b>A</b>) aCSF infused mice (n = 10) show similar association curves in the fear-conditioning paradigm compared with TLR4 antagonist infused mice (n = 10). (<b>B</b>) aCSF infused mice (n = 10) show similar freezing levels in the fear-conditioning paradigm compared with TLR4 antagonist infused mice (n = 10). (<b>C</b>) Average freezing during contextual fear. (<b>D</b>) aCSF infused mice (n = 10) show similar freezing in the fear-conditioning paradigm compared with TLR4 antagonist infused mice (n = 10) in the presence of tone.</p

CREB and p-CREB are upregulated in the hippocampus of TLR4^−/− mice.

<p>Brains from TLR4^+/+ (n = 8) and TLR4^−/− (n = 8) mice were dissected and hippocampi were removed. Tissues were then lysed, electrophoresed and immunoblotted against GluR1, CREB, ERK and their phosphorylated forms. Representative blots demonstrate that levels of CREB and pCREB were upregulated in TLR4^−/− mice compared to TLR4^+/+ mice, whereas GluR1, ERK and their phosphorylated forms were not changed. * p<0.05.</p

Developmental TLR4 deficiency impairs fear-learning and memory.

<p>(<b>A</b>) TLR4^−/− (n = 19) mice show impaired association curves in the fear-conditioning paradigm compared with TLR4^+/+ (n = 24) mice. (<b>B</b>) TLR4^−/− mice exhibit significantly impaired hippocampus dependent contextual fear compared to TLR4^+/+ mice, as measured by time freezing during 5 minutes of exposure to the original context. (<b>C</b>) Average freezing during contextual fear. (<b>D</b>) TLR4^−/− mice exhibit reduced freezing compared with TLR4^+/+ mice in the presence of tone, indicating impaired fear response.</p