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Abstract
Dorsal hippocampal regions are involved in memory and learning processes, while ventral areas are related to emotional and anxiety processes. Hippocampal dependent memory and behaviour alterations do not always come out in neurodegenerative diseases at the same time. In this study we have tested the hypothesis that dorsal and ventral dentate gyrus [DG] regions respond in a different manner to increased glycogen synthase kinase-3β [GSK3β] levels in GSK3β transgenic mice, a genetic model of neurodegeneration. Reactive astrocytosis indicate tissue stress in dorsal DG, while ventral area does not show that marker. These changes occurred with a significant reduction of total cell number and with a significantly higher level of cell death in dorsal area than in ventral one as measured by fractin-positive cells. Biochemistry analysis showed higher levels of phosphorylated GSK3β in those residues that inactivate the enzyme in hippocampal ventral areas compared with dorsal area suggesting that the observed susceptibility is in part due to different GSK3 regulation. Previous studies carried out with this animal model had demonstrated impairment in Morris Water Maze and Object recognition tests point out to dorsal hippocampal atrophy. Here, we show that two tests used to evaluate emotional status, the lightdark box and the novelty suppressed feeding test, suggest that GSK3β mice do not show any anxiety-related disorder. Thus, our results demonstrate that in vivo overexpression of GSK3β results in dorsal but not ventral hippocampal DG neurodegeneration and suggest that both areas do not behave in a similar manner in neurodegenerative processes.
Citation: Fuster-Matanzo A, Llorens-Martín M, de Barreda EG, Ávila J, Hernández F [2011] Different Susceptibility to Neurodegeneration of Dorsal and Ventral Hippocampal Dentate Gyrus: A Study with Transgenic Mice Overexpressing GSK3β. PLoS ONE 6[11]: e27262. //doi.org/10.1371/journal.pone.0027262
Editor: Cheng-Xin Gong, New York State Institute for Basic Research, United States of America
Received: July 8, 2011; Accepted: October 12, 2011; Published: November 3, 2011
Copyright: © 2011 Fuster-Matanzo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Spanish Comisión Interministerial de Ciencia y Tecnología [CICYT; SAF2010-15525, SAF 2006-02424], the Comunidad de Madrid [NEURODEGMODELS-CM, SAL/0202/2006], Fundación Centro de Investigaciones de Enfermedades Neurológicas [Fundación CIEN, PI 008-09], the Centro de Investigación Biomédica en Red on Neurodegeneration [CIBER] and by institutional grants from Fundación Botín and Fundación Ramón Areces. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
GSK3 is a kinase present in most tissues and is particularly abundant in the brain [1]. There are two isoforms of the enzyme termed GSK3α and GSK3β [1]. GSK3 is known to participate in multiple signaling pathways coupled to receptors for a variety of signaling molecules such as insulin or wnt among many others [2]. Aberrantly increased GSK3 activity is believed to play a key role in the pathogenesis of chronic metabolic disorders like type-II diabetes [3], as well as of CNS conditions such as bipolar mood disorder [4], schizophrenia [5], diseases like Huntington's disease [6], frontotemporal dementia with parkinsonism linked to chromosome 17 [7] and Alzheimer disease [8]. With regard to GSK3 and neurodegeneration, increased GSK3 activity has been reported to result in neuronal apoptosis and GSK3 inhibitors have been shown to exert antiapoptotic and neuroprotective effects in many different cell and mouse models [9], [10], [11]. Accordingly, potent and specific GSK3 inhibitors are currently under development [12], [13], [14].
Recent evidences have established that there are differences among dorsal and ventral hippocampal areas, at least in rodent [15]. All these differences are associated with functional specialization, as studies with lesions in dorsal or ventral hippocampus demonstrate [16]. Thus, dorsal regions are involved mainly in memory and learning processes, while ventral areas are related with anxiety, affective or emotional processes [17]. That regionalized processes correlate at genetic and cellular levels, showing that DG is not uniform and that there exist a regionalized specialization [15]. Those studies can be likely translated to human. Thus, the dorsal hippocampus corresponds to the posterior hippocampus in primates, while the ventral correspond to the anterior hippocampus in primates [15].
Here, we have first analyzed GSK3β levels in both DG areas in wild-type mice and explored the effect of GSK3β overexpression in both dorsal DG [dDG] and ventral DG [vDG] in a mouse model with increased GSK3β levels in those hippocampal areas [18]. This animal model exhibits a memory deficit [19], [20] and impaired synaptic plasticity [21]. We demonstrate that ventral hippocampus withstands a neurodegenerative signal as an increase in GSK3β levels better than dorsal hippocampus. In good agreement, evaluation of anxiety-related tests shows a normal behaviour.
Materials and Methods
Animals and tissue processing
Animal care.
Mice were obtained from the Centro de Biologıía Molecular and treated following the guidelines of Council of Europe Convention ETS123, recently revised as indicated in the Directive 86/609/EEC. Animal experiments were performed under protocols [P15/P16/P18/P22] approved by the Centro de Biología Molecular Severo Ochoa Institutional Animal Care and Utilization Committee [CEEA-CBM], Madrid, Spain.
GSK3β mice were generated as described previously [18]. Briefly, GSK3β mice were bred by crossing TetO mice [carrying the bi-direccional tet-responsive promoter followed by the GSK3β and β-galactosidase cDNAs, one in each direction] with CamKIIα-tTA mice. The dual transgenic mice were designated GSK3β, and they overexpress GSK3β in the cortex and hippocampus. Transgenic mice as well as wt mice [C57BL/6] were bred at the Centro de Biología Molecular Severo Ochoa [Madrid, Spain] and the mice were kept on a normal light-dark cycle [12 hours light/12 hours dark], with free access to food and water.
Tissue processing.
Animals were killed and the brain was removed, post-fixed overnight in 4% PFA and 30 µm sagittal sections were obtained on a cryostat.
Volumetric measurement of dentate gyrus atrophy
For volumetric measurement, thionine stained section areas of dentate gyrus were delineated and measured by means of Methamorph image-analysis system. The total volume [mm3] of each granule cell layer was achieved by integration of areas [mm2] with the distance between each sagital plane [mm]. The points for integration were 0.36 mm [Fig 104 of the atlas of Paxinos and Franklin [22]] and 3.00 mm [Fig 126 of the same atlas] with respect to the midline. In those sections were dentate gyrus is not divided in two areas, dorsal and ventral areas were measured taking into account the middle point.
Western blot analysis
Brains were quickly dissected on an ice-cold plate. Hippocampus was isolated and horizontally cut in two equals halves, the dorsal and the ventral hippocampus. Extracts for Western blot analysis were prepared by homogenizing the dorsal and ventral hippocampus from 2 months-old wildtype and transgenic animals in ice-cold extraction buffer consisting of 50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM sodium orthovanadate, 1 mM EDTA, a protease inhibitor cocktail [Roche] and 1 µM Okadaic acid [phosphatase inhibitor]. The samples were homogenized and protein content was determined by Bradford. Thirty micrograms of total protein were electrophoresed on 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane [Schleicher & Schuell, Keene, NH]. The experiments were performed using the following primary monoclonal antibodies: anti-GSK3β [1/1000] [BD Transduction Laboratories], anti-p21/9- GSK3α/β [1/500] [Cell Signalling], anti-Akt [1/1000] and anti-phospho-Akt [Ser473] [1/1000] [both from Cell Signalling] and anti-actin [1/5000] [Sigma]. The following anti-tau antibodies were used: PHF-1 [1/200, a kind gift from Dr. Davis] reacts with tau when serines 396 and 404 are phosphorylated [23] and 7.51 [1/200; a kind gift from Dr Wischik] which recognizes segments of the last two repeats within the microtubule binding domain of tau in a phosphorylation-independent manner [24]. The filters were incubated with the antibody at 4°C overnight in 5% nonfat dried milk. Secondary goat anti-mouse and anti-rabbit antibodies [1/1000; Invitrogen, San Diego, CA] and ECL detection reagents [Amersham Biosciences, Arlington Heights, IL] were used for immunodetection. Quantification was performed by densitometric scanning. The densitometry values were obtained in the linear range of detection with these antibodies. These values were normalized with respect to the values obtained with an anti-β-tubulin antibody to correct for total protein content.
Immunostaining
For GFAP immunohistochemistry, sections were rinsed in PBS and incubated in blocking solution [PBS/BSA/FBS/Tx-100] followed by an overnight incubation at 4° with the primary antibody: rabbit anti-GFAP [1/500] [Promega]. Then, sections were washed and incubated for 1 hour with the goat anti-rabbit biotinylated antibody [1400, Vector] and then another hour with an avidin-biotin-peroxidase complex complex [ABC, 1250, Vector]. The antibody staining was finally visualised with diaminobenzidine [DAB, 0.05%, Sigma]. Images were taken using an Axioskop 2 plus microscope and a CCD camera [Coolsnap FX color].
For immunofluorescence, immunostaining was carried out following a standard procedure. Sections were incubated with the primary antibody overnight at 4°C in a PB solution containing BSA 1% and TritonX-100 1%. The following antibodies were used: mouse anti-Myc, [1/100] [Hibridoma] and rabbit anti-fractin [1/500] [BD Pharmingen]. After washing with blocking solution 3 times, sections were incubated with donkey Alexa-conjugated secondary antibodies [anti-rabbit, anti-mouse Alexa-Fluor 488/555/633-conjugated] overnight at 4°C [11,000] [Molecular Probes, Millipore]. Finally, after washing with PB solution, sections were incubated with DAPI [1/5,000] [Calbiochem] for 10 minutes.
Cell counting
Fractin- and reactive GFAP-positive cells were quantified on a series of slices of dorsal and ventral dentate gyrus [DG] from 2 months-old wildtype and transgenic mice using an inverted Axiovert200 Zeiss fluorescence microscope. Astrocytes were considered reactive when they showed hypertrophy and a great number of shorter GFAP-positive processes. The number of positive cells for each marker was divided among each sectiońs DG volumen in order to obtain a cell density [cells/mm3]. The DG area of each section was estimated delineating de border of the granule cell layer on the same sections where cell numbers was estimated, by using DAPI staining and a 5X objective. Data are presented as mean cell density [cells/mm3]. All DG areas were measured using Image J software [ImageJ, v. 1.33, NIH, Bethesda, MD, USA, //rsb.info.nih.gov/ij].
Density of mature granule neurons [Number of cells/mm3] and myc-positive cells was analyzed through the application of a physical-dissector method developed for confocal microscopy [Zeiss LSM710], as described previously [25]. Briefly, the physical dissector was applied to sections stained with DAPI and myc, so all nuclei of mature neurons in the granule cell layer were counted [excluding those nuclei resembling erythrocytes, if any, and the immature cells, easily distinguishable because of its irregular nuclear profiles and highly condensed cromatin]. Data are presented as cell density [cells/mm3].
Novelty Suppressed Feeding test
Adult mice [three months] were weighed and food was removed from the cage, although water remained available ad libitum. Twenty-four hours later mice were transferred placed in a novel arena with in the center a pre-weighed quantity of food pellets. Each animal was placed in the corner of the testing area and the latency to chew a food pellet [about 2 g] located in the center of the arena, time spent feeding, and total food consumption were recorded over 10 min. All the experiments were performed between 13:00 and 18:00 h.
Light/Dark Choice Test
Exploration of the light/dark chamber was measured using the equipment from Med Associates Inc. The mouse is placed for 15 min in a box made of two compartments, one white and lit and the other dark. Two parameters were recorded, the percent of time spent in the dark compartment and the number of transitions between compartments [crossings].
Statistical analysis
Data were analyzed using two-way ANOVA in dorsal and ventral comparisons between wild-type and transgenic mice. To compare GSK-3 levels and its phosphorylated status in wild-type and GSK3β mice separately, we perform a t-test. All the analyses were performed using SPSS for Windows version 17.0.
Results
GSK3β levels in ventral and dorsal DG
To examine whether there exist dorso-ventral differences in GSK3β levels in wild-type mice, western blot analysis of dorsal hippocampus and ventral hippocampus homogenates were performed using antibodies that recognize inactive GSK-3β [phosphorylated at serine 9] and with an antibody that recognize GSK3β regardless of its phosphorylation state. Western blot determination of the phosphorylated form of GSK3β revealed an increase in the ratio phosphorylated/unphosphorylated forms in ventral area compared with dorsal area in wild-type mice [Figure 1]. The main kinase able to phosphorylate inhibitory GSK3 domains is AKT, thus we analyzed by western blot the hippocampal levels of the active form of AKT that results from phosphorylation on Ser473. Interestingly, and in good agreement with GSK3β findings, AKT is more active ventrally than dorsally. This resulted in a slight [although no statistically significant] decrease in phosphorylated tau, one of the main GSK3 substrates in the nervous system, as demonstrated by western blotting with the PHF-1 antibody. These data strongly suggest that some regional differences exist in the DG with a reduced ventral GSK3β activity compared with dorsal area.
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Figure 1. Western blots analysis of GSK3β, AKT and tau proteins in dDG and vDG from wild-type mice.
[A] Representative western blots showing GSK3β, phosphorylated GSK-3 [pSer9-GSK3β], AKT, phosphorylated AKT [pSer473-AKT], phosphorylated tau [pSer396/404-Tau], tau and actin in homogenates from dorsal and ventral hippocampus of wild-type mice. Hippocampal extracts were prepared from animals aged 2 months. [B] Histograms showing the densitometric quantification of samples shown in A. GSK3 and AKT ventral data are expressed in terms of the percentage of signal respect to dorsal levels. Histograms showing tau levels represent phosphorylated tau [PHF-1]/total Tau [7.51]. Solid bars, dorsal hippocampus; open bars, ventral hippocampus. *P