Antidepressants Stimulate Hippocampal Neurogenesis by Inhibiting p21 Expression in the Subgranular Zone of the Hipppocampus

The relationships among hippocampal neurogenesis, depression and the mechanism of action of antidepressant drugs have generated a considerable amount of controversy. The cyclin-dependent kinase (Cdk) inhibitor p21Cip1 (p21) plays a crucial role in restraining cellular proliferation and maintaining cellular quiescence. Using in vivo and in vitro approaches the present study shows that p21 is expressed in the subgranular zone of the dentate gyrus of the hippocampus in early neuronal progenitors and in immature neurons, but not in mature neurons or astroglia. In vitro, proliferation is higher in neuronal progenitor cells derived from p21-/- mice compared to cells derived from wild-type mice. Proliferation is increased in neuronal progenitor cells after suppression of p21 using lentivirus expressing short hairpin RNA against p21. In vivo, chronic treatment with the non-selective antidepressant imipramine as well as the norepinephrine-selective reuptake inhibitor desipramine or the serotonin-selective reuptake inhibitor fluoxetine all decrease p21 expression, and this was associated with increased neurogenesis. Chronic antidepressant treatment did not affect the expression of other Cdk inhibitors. Untreated p21-/- mice exhibit a higher degree of baseline neurogenesis and decreased immobility in the forced swim test. Although chronic imipramine treatment increased neurogenesis and reduced immobility in the forced swim test in wild-type mice, it reduced neurogenesis and increased immobility in p21-/- mice. These results demonstrate the unique role of p21 in the control of neurogenesis, and support the hypothesis that different classes of reuptake inhibitor-type antidepressant drugs all stimulate hippocampal neurogenesis by inhibiting p21 expression

Safety Profile of Gutless Adenovirus Vectors Delivered into the Normal Brain Parenchyma: Implications for a Glioma Phase 1 Clinical Trial

Abstract Adenoviral vectors (Ads) have been evaluated in clinical trials for glioma. However, systemic immunity against the vectors can hamper therapeutic efficacy. We demonstrated that combined immunostimulation and cytotoxic gene therapy provides long-term survival in preclinical glioma models. Because helper-dependent high-capacity Ads (HC-Ads) elicit sustained transgene expression, in the presence of antiadenoviral immunity, we engineered HC-Ads encoding conditional cytotoxic herpes simplex type 1 thymidine kinase and immunostimulatory cytokine Fms-like tyrosine kinase ligand-3 under the control of the TetOn system. Escalating doses of combined HC-Ads (1?108, 1?109, and 1?1010 viral particles [VP]) were delivered into the rat brain. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points after vector delivery. Histopathological analysis did not reveal any evidence of toxicity or long-term inflammation at the lower doses tested. Vector genomes were restricted to the injection site. Serum chemistry did not uncover adverse systemic side effects at any of the doses tested. Taken together, our data indicate that doses of up to 1?109 VP of each HC-Ad can be safely administered into the normal brain. This comprehensive toxicity and biodistribution study will lay the foundations for implementation of a phase 1 clinical trial for GBM using HC-Ads.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98452/1/hgtb%2E2012%2E060.pd

Comparative effects of the CRF agonist, ovine CRF, and antagonist, astressin, on homecage behavior patterns and defense in the mouse

Thesis (M.A.)--University of Hawaii at Manoa, 2005.Includes bibliographical references (leaves 34-45).52 leaves, bound ill. 29 cmCorticotropin-releasing factor (CRF) has been implicated in physiological and behavioral responsivity to stress and emotions including fear and anxiety. Baseline CRF elevation has been linked to depression and anxiety disorders and its effects include disruptions in sleep patterns and feeding behaviors in animals. This study attempted to characterize the effects of CRF agonists and antagonists over time by evaluating the behavior patterns of CD-I mice in their homecage for a period of 3 hours following injection of the preferentially binding CRFI receptor agonist, ovine CRF, or the CRF receptor antagonist, astressin. Furthermore, anti-predator responses in a new pharmacologically validated anxiety model, the Rat Exposure Test (RET), were used to assess the defensive behaviors of mice following administration of these CRF compounds. Intracerebroventricular administration of ovine CRF interrupted sleep patterns over the 3 hour time period but also suppressed active behaviors including eating, drinking, grooming, rearing, and locomotor activity during the first hour of testing. Crouching or freezing was enhanced following both low and high doses of ovine CRF. Ovine CRF also produced an anxlOgemc response in the RET, decreasing locomotor activity and contact time while increasing freezing and avoidance behaviors. As expected, the CRF antagonist, astressin, did not disrupt behavior patterns in the homecage, since baseline anxiety levels were presumably low under this test condition. However, contrary to expectation, astressin also failed to produce an anxiolytic effect in the RET. This result provides a parallel to others which claim that the anxiolytic efficacy of astressin can only be observed following a pre-stress condition elevating CRF levels prior to infusion. Analysis combining ovine CRF followed by astressin may provide information on the efficacy of the latter in reducing elevated CRF levels. Further analysis of selective CRF1/CRF2 agonists and antagonists may reveal differential roles individual CRF receptor subtypes play in modulating defensive behaviors

Activation of central CRF receptor 1 by cortagine results in enhanced passive coping with a naturalistic threat in mice

CRF receptor subtype 1 (CRF1), abundantly expressed in the central nervous system, has been implicated in defensive behavior in rodents. Pharmacological activation of CRF1 by peptidic agonists results in enhancement of anxiety-like behavior. However, receptor specificity of commonly used agonists was confounded by significant affinity to other receptors and widely used laboratory tests of experimental anxiety suffer from artificial aversive stimulation (e.g. electric shock), and limited measures of anxiety-like behavior. We used the recently developed, CRF1-selective agonist cortagine in a mouse model of defensive behaviors under semi-natural conditions, the Rat Exposure Test (RET). Cortagine was injected bilaterally into the cerebral ventricles (i.c.v.) of male C57Bl/6J mice, 20 min before exposure to a rat in specifically designed box that evokes a wide variety of defensive behaviors such as active/passive avoidance, freezing, risk assessment, and burying. Pre-injection of the CRF receptor antagonist acidic astressin was used to test for receptor specificity of the observed cortagine effects. A control experiment with no rat present was performed to test for baseline effects of cortagine in the exposure setup. Cortagine dose-dependently enhanced passive avoidance and freezing while burying was decreased. CRF receptor antagonism reliably blocked the effects of cortagine. Our results confirm previous findings of anxiogenic-like effects of cortagine, and demonstrate the usefulness of the RET in investigating differential pattering of drug-induced anxiety-like behavior in mice. In conclusion, our results suggest that CRF1 activation in forebrain areas promotes passive coping with the natural threat presented in the RET

A comprehensive multiomics approach toward understanding the relationship between aging and dementia.

Because age is the greatest risk factor for sporadic Alzheimer's disease (AD), phenotypic screens based upon old age-associated brain toxicities were used to develop the potent neurotrophic drug J147. Since certain aspects of aging may be primary cause of AD, we hypothesized that J147 would be effective against AD-associated pathology in rapidly aging SAMP8 mice and could be used to identify some of the molecular contributions of aging to AD. An inclusive and integrative multiomics approach was used to investigate protein and gene expression, metabolite levels, and cognition in old and young SAMP8 mice. J147 reduced cognitive deficits in old SAMP8 mice, while restoring multiple molecular markers associated with human AD, vascular pathology, impaired synaptic function, and inflammation to those approaching the young phenotype. The extensive assays used in this study identified a subset of molecular changes associated with aging that may be necessary for the development of AD

p21 is expressed in hippocampal NPC of a neuronal lineage.

<p>Cultured neuronal progenitors were stained for p21 (intranucelar, red) and various neuronal markers. <b>A</b>) co-localization of p21 and SOX2 (green, intranuclear, double-positive nuclei appear yellow; <b>B</b>) co-localization of p21 and nestin (green, cytoplasmic); In differentiating NPC, p21 co-localizes with <b>C</b>) DCX (green, cytoplasmic); and with <b>D</b>) Tuj-1 (green, cytoplasmic), but not with <b>E</b>) GFAP (green, cytoplasmic). Numbers indicate the percentage of p21-positive cells among the total number of cells expressing a respective marker. Counting was performed in triplicate (>500 cells per sample). Data are presented as mean ± SEM.</p

Increased proliferation of NPC derived from p21-/- mice.

<p><b>A</b>) The spheres derived from WT and p21-/- hippocampi 7 days after plating. Note bigger size and more cellularity in p21-/- spheres; <b>B</b>) Seven days after seeding, the NPC spheres were collected, dispersed and the number of cells was counted; <b>C</b>) Sphere self-renewal analysis. NPC spheres were dispersed and cells were seeded at 0.5 cell/well into 96-well plates. The percent of cells forming new spheres was determined after 10 days; <b>D</b>) The percent of Ki67-positive cells co-expressing nestin or SOX2. Counting was performed in triplicate (>500 cells per sample). Data are presented as a mean ± SEM, *, p<0.05, **, p<0.01; <b>E</b>) Representative confocal images of proliferating NPC derived from WT and p21-/- hippocampi and stained for Ki67 (intranucelar, red) and nestin (cytoplasmic, green) or SOX2 (intranuclear, green). p21-/- NPC exhibit more Ki67-positive cells; <b>F</b>) Proliferating neuronal precursors derived from WT and p21-/- hippocampi and stained for Ki67 (intranucelar, red) and p21 (intranuclear, green). Note that WT NPC cells positive for Ki67 are negative for p21; <b>G</b>) Double-labeling analysis of WT NPC with a color-coded fluorogram. The confocal image shows the absence of co-localization between p21 (green) and Ki67 (red).</p

Chronic treatment with antidepressants suppressed p21 expression and increased neuronal proliferation in the SGZ of hippocampus.

<p><b>A</b>) Western blot analysis of p21 and other Cdk inhibitors in hippocampus of mice chronically treated with normal saline (NS), imipramine (IP), desipramine (DMI) or fluoxetine (FL). For each sample hippocampi from 5 mice/group were pooled; <b>B</b>) Quantitative analysis of three independent experiments. The intensity of p21 bands was measured, normalized to the loading control (β actin) and the ratios were corrected to NS control to quantify relative changes; <b>C</b>) Number of BrdU⁺ cells. Non-parametric Kruskal-Wallis test [(H3,N = 20) = 11.75, p<0.008]. <b>D</b>) Number of BrdU⁺/DCX⁺ cells. One way ANOVA followed by Dunnett's post-hoc test [(F3,16) = 3.88, p = 0.029 for all groups] in the SGZ. For each sample hippocampi from 5 mice/group were analyzed. Data are presented as a mean ± SEM.</p

Conditional suppression of p21 enhances proliferation of WT NPC.

<p><b>A</b>) One thousand WT NPC cells were kept in proliferating conditions for 4 days. Cells were cultured in 24-well plates in triplicates. The mean number of cells at the time of collection; <b>B</b>) Cells were fixed and stained for Ki67 or p21 . The graph depicts the percentage of p21- or Ki67-positive cells in each culture. Counting was performed in triplicates (>500 cells per sample). Data are presented as a mean ± SEM, *, p<0.05. Two independent experiments were conducted, and representative results are shown; <b>C</b>) Representative confocal images of NPC treated with either control lentivirus (control) or lentivirus expressing p21 shRNA. The cells were stained for Ki67 (intranucelar, red) and p21 (intranuclear, green). Note the increased number of Ki67-positive and the decreased number of p21-positive cells after p21 shRNa treatment.</p

p21 is expressed in neuronal progenitors in the SGZ of the hippocampus.

<p><b>A</b>) The confocal image shows that p21 (red) is expressed in the SGZ of the dentate gyrus and is not expressed in mature neurons (NeuN, green) in the GCL. Here, and in other confocal images, nuclei are stained with the DNA specific dye DAPI (blue). p21-positive cells appear pink. p21 is expressed mostly in nuclei; however, some cytoplasmic staining also is observed. SGZ, subgranular zone; GCL, granular cell layer; ML, molecular layer; <b>B–E</b>) Confocal images depicting p21 co-localization with neuronal markers in the SGZ of the hippocampus. p21-positive nuclei line the SGZ and appear light blue. Upper panel-low magnification (x20), lower panel–higher magnification (x63). <b>B</b>) p21 (green) is co-localized with SOX2 (intranuclear, red). Cells that express both proteins appear yellow; cells that expressed SOX2 only appear pink; <b>C</b>) p21 (green) is co-localized with nestin (cytoplasmic, red); <b>D</b>) p21 (green) is co-localized with DCX (cytoplasmic, red); <b>E</b>) p21 (green) is not co-localized with GFAP (cytoplasmic, pink). Some of p21 positive cells are marked with arrows.</p