p21cip1 restricts neuronal proliferation in the subgranular zone

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p21cip1 restricts neuronal proliferation in the subgranular zone of the dentate gyrus of the hippocampus:

p21 cip1 restricts neuronal proliferation in the subgranular zone of the dentate gyrus of the hippocampus Robert N. Pechnick *†, Svetlana Zonis ‡, Kolja Wawrowsky ‡, Jonathan Pourmorady ‡, and Vera Chesnokova ‡§ ‡ Department of Medicine, Division of Endocrinology, and *Department of Psychiatry and Behavioral Neurosciences, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048; and †Brain Research Institute, University of California, Los Angeles, CA 90024 PNAS January 29, 2008 vol. 105 no. 4 1358–1363

Hippocampus :

Hippocampus The hippocampus belongs to the limbic system and plays important roles in long-term memory and spatial navigation . It is located inside the medial temporal lobe beneath the cortical surface. Its curved shape reminded early anatomists of the horns of a ram ( Cornu Ammonis ), or a seahorse. The name was taken by the 16 th century anatomist Julius Caesar Aranzi from the Greek word for seahorse (Greek: ιππος , hippos = horse , καμπος , kampos = sea monster ).

Importance of hippocampus :

Importance of hippocampus Consolidation of new memories Processing emotions, especially emotional memory Navigation Spatial orientation

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Histology Subcomponents are--- a) Dentate gyrus b) Subiculum c) Sectors referred to as CA1, CA2, CA3, CA4. Department of Anatomy, JNMCH,AMU, ALIGARH 2010 Department of Anatomy JNMCH,AMU, ALIGARH 2010

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It’s a tri-laminar structure. Superficial molecular layer, consisting of nerve fibers and scattered small neurons The pyramidal layer, consisting of many large pyramid-shaped neurons The inner polymorphic layer, which is similar in structure to the polymorphic layer of the cortex seen elsewhere. Molecular layer Granular layer Polymorphic layer Molecular layer Pyramidal layer Polymorphic layer Department of Anatomy, JNMCH,AMU, ALIGARH 2010

Hippocampal Inputs and Outputs :

Hippocampal Inputs and Outputs The hippocampus has direct connections to the entorhinal cortex (via the subiculum) and the amygdala. Entorhinal cortex projects to the cingulate cortex, which has a connections to the temporal lobe cortex, orbital cortex, and olfactory bulb. Thus, all of these areas can be influenced by hippocampal output, primarily from CA1.

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Neurobiologists long believed that adult brains did not make new neurons, but now we know otherwise. In the early 1960s, Joseph Altman at MIT reported that new neurons were being produced in the brains of adult rats. Those findings were somewhat forgotten for the next 30 years. Recently, this work has been revived and advanced. Elizabeth Gould of Princeton University, one of this article’s authors (Gage) and others have reported the birth of new neurons-- neurogenesis --in the hippocampus of adult rats, monkeys and humans.

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Stem cells--primitive cells that are formed soon after fertilization and that can divide indefinitely, which can divide a limited number of times and give rise to cell types such as neurons and glia. Neurons continue to be born throughout life in the olfactory bulb , dentate gyrus of the hippocampus. (Very recent evidence indicates that some additional brain areas might also produce new brain cells.) These new neurons are derived from progenitor cells that reside in the brain’s subventricular zone(ventricles) or in a layer of the hippocampus called the subgranular zone.

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Investigators follow neurogenesis with tritiated-thymidine or bromodeoxyuridine. The compound incorporated into cells can be visualized under the microscope with autoradiographic or immunologic techniques for tritiated-thymidine or bromodeoxyuridine, respectively. These techniques show that progenitor cells in the subgranular zone produce progeny that migrate outward to the granule-cell layer and differentiate into neurons. In this way, these new granule cells join the population of existing neurons. These newly born cells mature in the granule-cell layer and send their dendrites outward, whereas their cell processes go inward and follow paths to other structures within the hippocampus, such as the CA3 cell fields. Department of Anatomy, JNMCH,AMU, ALIGARH 2010

Major Depression :

Major Depression Five (or more) of the following symptoms have been present during the same 2-week period and represent a change from previous functioning; at least one of the symptoms is either (1) Depressed mood or (2) Loss of interest or pleasure. (1) depressed mood most of the time, most every day (2) markedly diminished interest or pleasure in daily activities (3) significant weight loss when not dieting or weight gain, or appetite loss (4) insomnia or hypersomnia (5) psychomotor agitation or retardation (observable by others) (6) fatigue or loss of energy (7) feelings of worthlessness or excessive or inappropriate guilt (8) diminished ability to think or concentrate (9) recurrent thoughts of death (not just fear of dying), recurrent suicidal ideation without a specific plan, or a suicide attempt or a specific plan for committing suicide

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Various modes of stress can be used to induce depression in the rat or an individual. Studies in rats have shown that the hippocampus of stressed rats are on average smaller and microscopic examination reveals neuronal loss. Repair can be stimulated by many antidepressants.

p21 gene :

p21 gene p21WAF1/Cip1 belongs to the Cip /Kip family of cyclin kinase inhibitors (CKI) (p21Waf1/Cip1, p27Kip1, p57Kip1) p21Waf1/Cip1 was first described as a potent and universal inhibitor of cyclin -dependent kinases ( Cdks ). p21 functions as a checkpoint in the cell cycle by inhibiting cdks at the G1/S and G2/M interfaces.

In normal cells, p21 acts like a brake to block cell cycle progression in the event of DNA damage, preventing the cells from dividing and potentially becoming cancerous.:

In normal cells, p21 acts like a brake to block cell cycle progression in the event of DNA damage, preventing the cells from dividing and potentially becoming cancerous.

Abstract :

Abstract The subgranular zone (SGZ) of the dentate gyrus of the hippocampus is a brain region where robust neurogenesis continues throughout adulthood. Cyclin -dependent kinases (CDKs) have a primary role in controlling cell division and cellular proliferation. p21 cip1 (p21) is a CDK inhibitor that restrains cell cycle progression. Chronic treatment with the tricyclic antidepressant imipramine (10 mg/kg per day i.p . for 21 days)markedly decreased hippocampal p21 mRNA and protein levels . These results suggest that p21 restrains neurogenesis in the SGZ and imipramine -induced stimulation of neurogenesis might be a consequence of decreased p21 expression and the subsequent release of neuronal progenitor cells from the blockade of proliferation. Because many antidepressants stimulate neurogenesis, it is possible that their shared common mechanism of action is suppression of p21.

Introduction :

Introduction In the central nervous system, developing neurons are derived from quiescent multipotent or neural stem cells and progenitors (1). In the hippocampus, the neural progenitor cells are located in the SGZ of the DG, at the border between the hilus and the granular cell layer (GCL) (2, 3). Newborn cells proliferate in SGZ, migrate into the GCL, develop the morphological and functional properties of granule cell neurons , and become integrated into existing neuronal circuitry (4). This suggests an important role of intrinsic stimulatory and inhibitory factors in the regulation of proliferation of neuronal precursor cells. In mammalian cells, the control of cellular proliferation primarily is achieved in the G1 phase of the cell cycle. Cyclin dependent kinases (CDKs) tightly control the cell cycle process. Cell cycle progression is negatively regulated by two families of CDK inhibitors: Ink4/ARF type (p16, p15, p18, and p19) and Cip /Kip type (p21, p27, and p57).

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p21Cip1 (p21) acts in the G1phase of the cell cycle and delays or blocks the progression of the cell into the S phase (5). p21 maintains cell quiescence , and chronic activation of p21 can drive the cell into irreversible cell growth arrest and senescence (5, 6). Conversely, inhibition of p21 increases cellular proliferation (7). The induction of neurogenesis might be part of the molecular mechanisms underlying the therapeutic effects of antidepressant treatment (8, 9). All major classes of antidepressants stimulate neurogenesis in rodents (10–12) and nonhuman primates (13). The present study evaluated the role of the CDK inhibitor p21 in neurogenesis. The results suggest that antidepressants may stimulate SGZ neurogenesis by inhibiting p21 expression.

Materials and Methods :

Materials and Methods Experimental Animals. Two-month-old, male C57BL/6j mice were treated daily for 21 days with saline (0.9%) or imipramine hydrochloride (10 mg/kg per day i.p .; Sigma–Aldrich). Twenty-four hours after the last injection, the mice were subjected to BrdU injections and used for the behavioral experiment or killed for the other studies. Cdk 1atm1Tyj ( p21/) mice on 129S2 genetic background were obtained from The Jackson Laboratory. For real-time PCR, ELISA, and Western blot analyses, the mice were killed, the brains were removed and rapidly cooled in ice-cold saline, and the hippocampi were dissected out (55).

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Immunohistochemistry . Mice were anesthetized with isoflurane and perfused with paraformaldehyde (4%), and brain paraffin sections were processed as described previously (56). The slides were incubated with mouse anti-p21 monoclonal antibodies (BD Pharmingen ) and conjugated with Alexa Fluor 488 fluorescent dye (Molecular Probes). The antibody for NeuN ( Chemicon ) was conjugated with Alexa Fluor 568 fluorescent dye (Molecular Probes) and used to determine co-localization with p21 expression. DNA (nuclei) was stained with ToPro3 (Molecular Probes). Multi-parameter fluorescent microscopy and Leica Confocal Software were used to identify p21 intracellular localization and colocalization with the neuronal marker NeuN . For comparability, all images on confocal multi-image figure plates were adjusted with identical contrast and brightness settings.

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P 21 positive cells (red), neurons(green) and areas of colocalization blue

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Intranuclear p21 expression in the SGZ of the dentate gyrus. Double-labeling analysis with color-coded fluorogram (58) is shown. DNA (blue) p 21 (green)

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BrdU Immunochemistry. The entire left half of the brain was cut into 5-m sagittal sections and processed by using a BrdU Labeling and Detection Kit (Roche Applied Biosystems ). Sections were coded for blind observation. Unbiased random sampling from 0.36 to 0.6 mm lateral to the midline (55) was carried out. Every third section (of a total of 30 sections) was counted under a 100 objective, and the sum was multiplied by 3 to estimate the total number of BrdU -positive cells in the region. Cells were counted if they were in or touching the SGZ, and cells were excluded if they were more than two cell diameters from the GCL (8). Some sections derived from p21-null mice were double-labeled to detect DCX (Santa Cruz Biotechnology), and at least 20 BrdU -positive cells were examined by confocal microscopy to determine colocalization with DCX. Representative images of SGZ cells of p21/ mice coexpressing BrdU (green) and DCX (red). Cell nuclei were stained with the DNA-specific dye ToPro3 (blue).

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FACS Analysis . Hippocampi were dissociated with a Papain Dissociation System (Worthington Biochemical), and the cells were processed according to a Flow Cytometry Staining Protocol (Cell Signaling Technology). Fixed cells were incubated overnight with DCX antibodies (Cell Signaling Technology), washed, and treated with secondary antibodies ( Alexa Fluor 586 fluorescent dye, red), and samples were analyzed in a FACS Calibur system (Becton Dickinson). The cells were gated on the basis of forward/side scatter plot to eliminate the debris. DCX-positive and DCX-negative cells were sorted. For the double-staining experiments, both DCX-positive and DCX-negative cells were stained for p21 (BD Pharmingen ) for 2 h at 4°C, then stained with a secondary antibody ( Alexa Fluor 488 fluorescent dye, green), and the number of DCX/ p21-positive cells was determined. Control cells were stained with both secondary antibodies.

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Protein Isolation and Western Blot Analysis . Proteins were isolated ( Immunoprecipitation Kit; Roche Diagnostics), separated by SDS/PAGE, electroblotted onto membranes (Millipore), incubated overnight with primary antibodies, and then incubated with corresponding secondary antibodies as described (56). Immunoreactive bands were detected by the ECL immunodetection system. NeuN ( Chemicon ) and PCNA, DCX, and - actin (Santa Cruz Biotechnology) antibodies were used. For quantitative analysis blots were scanned by using an Epson V750 PRO and transferred to Adobe Photoshop Elements 3. Intensity analysis of the bands (all multiple bands simultaneously) was performed with ImageJ Software ( ImageJ ; W. S. Rosband , National Institutes of Health; http://rsb.info.nih.gov/ij )

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Chronic treatment with imipramine suppresses p21 expression in the hippocampus. ( A–H) Double-labeling analysis of the hippocampal dentate gyrus with color-coded fluorograms (51). Confocal images show double immunofluorescence labeling of hippocampal dentate gyrus after chronic treatment with saline ( A–D) or imipramine (E–H). p21-positive cells are red, and neurons (NeuN-labeled) are green. Cells where p21 and NeuN are colocalized are marked with a blue overlay. Colocalized pixels in the fluorograms are marked with blue ( D and H). The area imaged in the confocal micrographs is 250 250 m. SGZ, subgranular low; GCL; granular cell layer; ML, molecular layer. ( I) Real-time PCR of p21mRNAlevels in the hippocampi of mice after chronic treatment with saline (C) or imipramine (IP) (seven to eight mice per group). *, P 0.05. (J) p21 protein levels after chronic treatment with imipramine (IP) as measured by ELISA. P21 protein expression in normal saline-treated mice (C) was taken as 100%. Shown are results summarized from three independent experiments. For each experiment whole hippocampi from three mice per group were pooled. **, P 0.01.

Results :

Results p21 is expressed in neuroblasts and newly developing neurons in the SGZ of the hippocampus. P21 deletion increases proliferation of hippocampal neurons. Chronic treatment with imipramine decreases p 21 expression in the SGZ of the hippocampus. Chronic treatment with imipramine increases neurogenesis in the SGZ of the hippocampus and produces antidepressant-like activity in the forced swim test.

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