PACAP Protects Adult Neural Stem Cells from the Neurotoxic Effect of Ketamine Associated with Decreased Apoptosis, ER Stress and mTOR Pathway Activation

<div><p>Ketamine administration is a well-established approach to mimic experimentally some aspects of schizophrenia. Adult neurogenesis dysregulation is associated with psychiatric disorders, including schizophrenia. The potential role of neurogenesis in the ketamine-induced phenotype is largely unknown. Recent results from human genetic studies have shown the pituitary adenylate cyclase-activating polypeptide (PACAP) gene is a risk factor for schizophrenia. Its potential role on the regulation of neurogenesis in experimental model of schizophrenia remains to be investigated. We aimed to determine whether ketamine affects the viability of adult neural stem cells (NSC). We also investigated whether the detrimental effect mediated by ketamine could be counteracted by PACAP. NSCs were isolated from the subventricular zone of the mouse and exposed to ketamine with/without PACAP. After 24 hours, cell viability, potential involvement of apoptosis, endoplasmic reticulum (ER) stress, mTOR and AMPA pathway activation were assessed by quantitative RT-PCR and Western blot analysis. We show that ketamine impairs NSC viability in correlation with increased apoptosis, ER stress and mTOR activation. The results also suggest that the effect of ketamine occurs <i>via</i> AMPA receptor activation. Finally, we show that PACAP counteracted the decreased NSC viability induced by ketamine <i>via</i> the specific activation of the PAC-1 receptor subtype. Our study shows that the NSC viability may be negatively affected by ketamine with putative importance for the development of a schizophrenia phenotype in the ketamine induced animal model of schizophrenia. The neuroprotective effect via PAC-1 activation suggests a potentially novel pharmacological target for the treatment of schizophrenia, <i>via</i> neurogenesis normalization.</p></div

Ketamine impairs NSC viability <i>via</i> AMPA receptor activation.

<p>NSCs were plated as single cells and treated with 400 μM ketamine or pre-treated with 10 μM NBQX prior to 400 μM ketamine exposure. After 24 hour incubation, intracellular ATP levels were measured. Data are shown as mean ±SEM (n = 23–39). Kruskal-Wallis followed by Dunn’s test was used. Differences were considered significant at P<0.05. * denotes P<0.05 compared with control, # denotes P<0.05 compared to ketamine.</p

Ketamine decreases NSC viability in a dose-dependent manner.

<p>NSCs were plated as single cells treated with 50 μM, 200 μM, 400 μM and 1 mM ketamine. To measure cell viability, intracellular ATP levels were measured after 24 hours. Data are shown as mean ±SEM (n = 22–35). Kruskal-Wallis followed by Dunn’s test was used. Differences were considered significant at P <0.05. * denotes P<0.05 compared with control.</p

Ketamine-induced cell death is counteracted by PACAP <i>via</i> PAC-1 receptor activation.

<p>NSCs were plated as single cells. Cells were incubated with 400 μM ketamine and PACAP (100 nM) or agonist for PAC-1; Max-4 (30 nM). After 24 hour incubation, intracellular ATP levels were measured for cell viability. Data are shown as mean ±SEM (n = 13–39). Kruskal-Wallis followed by Dunn’s test was used. Differences were considered significant at P<0.05. * denotes P<0.05 compared with control, # denotes P<0.05 compared to 400 μM ketamine.</p

A schematic diagram showing a proposed signaling pathway for PACAP mediated suppression of ketamine induced neurotoxicity of NSCs <i>in vitro</i>.

<p>A schematic diagram showing a proposed signaling pathway for PACAP mediated suppression of ketamine induced neurotoxicity of NSCs <i>in vitro</i>.</p

PACAP counteracts ketamine induced mTOR activation.

<p>NSCs were plated as single cells and treated with 400 μM alone or with 100 nM PACAP and 400 μM ketamine. Cells were treated as indicated and after 1, 2, 6 and 24 hour incubation cells were harvested for Western blot experiments <b>(A)</b>. Cells were treated as indicated and incubated for 1 and 2 hours and harvested for Western blot experiments <b>(B)</b>. To obtain quantitative measurements p-mTOR were normalized against total-mTOR. Data are shown as mean ±SEM (A, n = 4–8; B, n = 3–5). Kruskal-Wallis followed by Dunn’s test or Fisher LSD test was used. Differences were considered significant at P<0.05. * denotes P<0.05 compared with control, # denotes P<0.05 compared to ketamine + 1 hour or ketamine + 2 hours.</p

qPCR validation of the newly discovered P6 duplication.

<p>Amplification plots for a female (green), a control male (purple) and a male with P6 dupl. (blue) are shown for markers RH38681 (A), sY1081 (B) and sY933 (C). D. Intensity signal plot (Log 2 ratio) for an individual with P6 dupl. showing that markers sY1081 and sY933 are positioned within the duplicated region, while RH38681 is located outside.</p

Distribution of CNV patterns that were significantly overrepresented in one haplogroup only.

<p>Each part of the figure shows the graphical representation of the first two eigenvectors after PCA analysis A. The figure shows PCA values for individuals with P3 dupl. significantly overrepresented in haplogroup E-M96. B. Individuals with b2/b3 del. significantly overrepresented in haplogroup NO-M214(xM175). C. Individuals with gr/gr del. (c8) significantly overrepresented in haplogroup D-M174. This CNV is also present in other haplogroups. D. Individuals with gr/gr dupl. (c9) + distal dupl. significantly overrepresented in haplogroup J-P256.</p

Distribution and frequency of CNV patterns significantly overrepresented within haplogroups.

<p>The table shows the distribution of CNV patterns among haplogroups for ten variants that showed overrepresentation in one or more haplogroups. Ambiguous individuals, for which haplotype determination was not possible, are shown in the table for completeness, but they were not included in the statistical analysis. The p-values after Pearson Chi-Square analysis, likelihood ratio and Fisher’s exact test are shown at the bottom of the table, together with the total amount of each CNV type. The % frequency is derived from CNV type observations divided by 1506 individuals for which haplogroup could be determined. The stars mark values that are significant with the standard residual indicated in parenthesis. (Ind.) individuals, (dupl) duplication, (del) deletion and (nd) not done.</p><p>Distribution and frequency of CNV patterns significantly overrepresented within haplogroups.</p

Additional file 1: Table S1. of A history of childhood trauma is associated with slower improvement rates: Findings from a one-year follow-up study of patients with a first-episode psychosis

Childhood trauma prevalence divided into gender. Additional file 1: Table S1 shows the prevalence of childhood trauma divided into gender. Females report more sexual and emotional abuse than males. Table S2. Childhood trauma prevalence divided into schizophrenia and affective psychoses. Additional file 1: Table S2 shows the prevalence of childhood trauma divided into schizophrenia and affective psychoses. Patients with schizophrenia report more often physical abuse and physical neglect compared to patients with an affective psychoses. (DOCX 17 kb