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. 2012 Sep 13;33(1):75–84. doi: 10.1007/s10571-012-9873-8

Comparison of Alpha-Synuclein Immunoreactivity in the Hippocampus Between the Adult and Aged Beagle Dogs

Ji Hyeon Ahn 1, Joon Ha Park 2, Bing Chun Yan 2, Jae-Chul Lee 2, Jung Hoon Choi 3, Choong Hyun Lee 4, Ki-Yeon Yoo 5, In Koo Hwang 6, Jin Sang Kim 1, Hyung-Cheul Shin 7,✉, Moo-Ho Won 2,✉
PMCID: PMC11498021  PMID: 22972205

Abstract

Alpha-synuclein (α-syn), as a neuroprotein, is expressed in neural tissue, and it is related to a synaptic transmission and neuronal plasticity. In this study, we compared the distribution and immunoreactivity of α-syn and related gliosis in hippocampus between young adult (2–3 years) and aged (10–12 years) beagle dogs. In both groups, α-syn immunoreactivity was detected in neuropil of all the hippocampal sub-regions, but not in neuronal somata. In the aged hippocampus, α-syn immunoreactivity was apparently increased in mossy fibers compared to that in the adult dog. In addition, α-syn protein level was markedly increased in the aged hippocampus. On the other hand, GFAP and Iba-1 immunoreactivity in astrocytes and microglia, respectively, were increased in all the hippocampal sub-regions of the aged group compared to that in the adult group: especially, their immunoreactivity was apparently increased around mossy fibers. In addition, in this study, we could not find any expression of α-syn in astrocytes and microglia. These results indicate that α-syn immunoreactivity apparently increases in the aged hippocampus and that GFAP and Iba-1 immunoreactivity are also apparently increased at the regions with increased α-syn immunoreactivity. This increase in α-syn expression might be a feature of normal aging.

Keywords: Aging, Neuroprotein, Hippocampal sub-regions, Mossy fibers, Nerve terminals and fibers, Gliosis

Introduction

Aging is a biological process that is associated with structural, functional changes, and cellular damage (Berry et al. 2008; Modi and Kanungo 2010; Okabayashi and Kimura 2007; Paramanik and Thakur 2010). Cellular dysfunctions in the aged brain are closely related to increased oxidative stress and inflammatory-related factors as well as protein aggregation, and they are associated with the development and progression of neurodegenerative diseases (Ross and Poirier 2004). In the central nervous system (CNS), the brain is vulnerable to oxidative damage due to its high oxygen utilization and high concentrations of easily oxidizable polyunsaturated fatty acids (Balu et al. 2005). Among brain regions, the hippocampus is particularly vulnerable and sensitive to the aging process (He et al. 2008; Kudo et al. 2005; Siwak-Tapp et al. 2008; Yamada et al. 2007; Yu et al. 2011).

Alpha-synuclein (α-syn) is a 140-amino acid synuclein protein, and the synuclein family consists of three known proteins: α-syn, β-syn, and γ-syn (George 2002). α-Syn as a neuroprotein is expressed primarily in neural tissue, particularly in the neocortex, hippocampus, and striatum (Abeliovich et al. 2000). Although the normal functions of α-syn are still being defined, several studies have shown that α-syn is related to transcriptive regulatory role in the nuclei (Kontopoulos et al. 2006). In addition, α-syn localized at presynaptic nerve terminals and fibers is related to a synaptic transmission, neuronal plasticity, and lipid transport (Totterdell et al. 2004).

In pathological situations, the aggregation of α-syn is distinctively increased in Parkinson’s disease, dementia with Lewy bodies, and Alzheimer’s disease (Eller and Williams 2011; Rampello et al. 2004). An abnormal accumulation of α-syn in the CNS with some neurodegenerative diseases is particularly of clinical relevance, because it might provide the pathological basis for CNS dysfunction (Probst et al. 2008).

Recently, it was reported that glia and α-syn could affect each other. An amount of α-syn is released from neurons under stress into the extracellular space and stimulates glia (Croisier and Graeber 2006; Lee et al. 2010a, b). In addition, it is well known that gliosis plays an important role as a primary target of normal aging as well as degenerative disease process.

Although there are many studies that have demonstrated the expression of α-syn in the brain and cultured cells, most of these studies have mainly focused on α-syn expression with neurodegenerative diseases. Few studies regarding changes of α-syn in the hippocampus of the aged dog, which is a good animal model of aging (Cotman and Head 2008; Sarasa and Pesini 2009), have been reported. The hippocampal complex is a typical old cortex including hippocampus proper and hippocampal dentate gyrus. The dentate gyrus gives rise to mossy fibers to enter the adjacent hippocampal CA3 region (Frotscher et al. 2006; Toni et al. 2008). Therefore, in this study, we compared the immunoreactivity of α-syn and related gliosis in the sub-regions of the hippocampus between the adult and aged beagle dogs.

Materials and Methods

Experimental Animals

Clinically and neurologically normal male beagle dogs were used at 2–3 years (young adult dogs, n = 12) and 10–12 years (aged dogs, n = 12) of age, with normal values in blood count, chemistry, gas analysis, and serum electrolytes. These animals did not show any clinical and other signs in neural disorders. The procedure for handling and caring for the animals adhered to the guidelines, which are in compliance with the current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85 23, 1985, revised 1996).

Tissue Processing

For histochemical analysis, young adult and aged dogs (n = 7 in each group) were anesthetized with zoletil 50 (8 mg/kg) and xylazine (2 mg/kg) mixture, and perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4 % paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The hippocampus was removed and post-fixed in the same fixative for 12 h. The hippocampal tissues were cryoprotected by infiltration with 30 % sucrose overnight. Thereafter, the frozen tissues were serially sectioned on a cryostat (Leica, Wetzlar, Germany) into 30 μm thickness, and the sections were then collected into 6-well plates containing PBS.

Immunohistochemistry

The sections were sequentially treated with 0.3 % H2O2 and 10 % normal goat serum. They were then incubated with diluted sheep anti-α-syn (diluted 1:1000, Abcam, Cambridge, MA), mouse anti-GFAP (diluted 1:800, Chemicon, Temecula, CA), and rabbit anti-Iba-1 (diluted 1:800, Wako, Richmond, VA), respectively, and subsequently exposed to biotinylated rabbit anti-sheep, goat anti-rabbit or horse anti-mouse IgG and streptavidin peroxidase complex (1:200, Vector, Burlingame, CA). They were then visualized by staining with 3,3′-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) in 0.1 M Tris–HCl buffer (pH 7.2) and mounted on gelatin-coated slides. The sections were mounted in Canada balsam (Kanto) following dehydration. In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of primary antibody. The negative control test resulted in the absence of immunoreactivity in all structures. In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of the primary antibodies. The negative control test was conducted in all groups.

Double Immunofluorescence Staining

The sections were processed by double immunofluorescence staining. Double immunofluorescence staining was performed using diluted sheep anti-α-syn (diluted 1:1500, Abcam, Cambridge, MA), mouse anti-GFAP (diluted 1:800, Chemicon, Temecula, CA), or rabbit anti-Iba-1 (diluted 1:800, Wako, Richmond, VA), respectively. The sections were incubated in the mixture of antisera overnight at room temperature. After washing three times for 10 min with PBS, they were incubated in a mixture of both FITC-conjugated donkey anti-sheep IgG (1:500; Jackson ImmunoResearch) and Cy3-conjugated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch, West Grove, PA) or Cy3-conjugated goat anti-rabbit IgG (1:500; Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature. The immunoreactions were observed under the confocal MS (LSM510 META NLO, Carl Zeiss, Germany).

Western Blot Analysis

To confirm change in α-syn, GFAP and Iba-1 levels in the hippocampus derived from the adult and aged dogs, the animals (n = 5 in each group) were killed and used for the western blot analysis. In brief, the tissues were homogenized in 50 mM PBS (pH 7.4) containing EGTA (pH 8.0), 0.2 % NP-40, 10 mM EDTA (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM PMSF, and 1 mM DTT. After centrifugation, the protein level in the supernatants was determined using a Micro BCA protein assay kit (Pierce Chemical, Rockford, IL, USA). Aliquots containing 20 μg of total protein were boiled in a loading buffer containing 150 mM Tris (pH 6.8), 3 mM DTT, 6 % SDS, 0.3 % bromophenol blue, and 30 % glycerol. The aliquots were then loaded onto a polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Pall Crop, East Hills, NY, USA). To reduce background staining, the membranes were incubated with 5 % non-fat dry milk in PBS containing 0.1 % Tween 20, followed by incubation with sheep anti-α-syn (diluted 1:1000, Abcam, Cambridge, MA), rabbit anti-Iba-1 (diluted 1:1000, Wako, Richmond, VA), and mouse anti-GFAP (diluted 1:1000, Chemicon, Temecula, CA), peroxidase-conjugated donkey anti-sheep, rabbit or mouse IgG (Sigma, St. Louis, MO, USA) and ECL kit (Pierce Chemical).

Data Analysis

Ten sections per animal were randomly selected from the corresponding areas of the hippocampal sub-regions in order to quantitatively analyze α-syn immunoreactivity. The images were calibrated into an array of 512 × 512 pixels corresponding to a tissue area of 1,200 × 1,200 μm (20× primary magnification). Data represented as percent of the adult dog. Images of α-syn-immunoreactive structures were obtained through an AxioM1 light microscope (Carl Zeiss, Göttingen, Germany) equipped with a digital camera (Axiocam, Carl Zeiss) connected to a PC monitor. Each pixel resolution was 256 gray levels. The staining intensity of all α-syn-immunoreactive structures was evaluated on the basis of a relative optical density (ROD), which was obtained after the transformation of the mean gray level using the formula: ROD = log(256/mean gray level). The ROD of the complete field was measured, and the brightness and contrast of each image file were calibrated using Adobe Photoshop version 8.0 and then analyzed using NIH Image 1.59 software. Values of background staining were obtained and subtracted from the immunoreactive intensities.

In addition, ten sections per animal were selected to quantitatively analyze Iba-1 and GFAP immunoreactivity. Digital images of the hippocampal sub-regions were captured using an AxioM1 light microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Carl Zeiss, Germany) connected to a PC monitor. Semi-quantification of the immunostaining intensities was evaluated with digital image analysis software (MetaMorph 4.01, Universal Imaging Corp.). The level of the immunoreactivity was scaled as −, ±, +, or ++, representing no staining (gray scale value: ≥200), weakly positive (gray scale value: 150–199), moderate (gray scale value: 100–149), or strong (gray scale value: ≤99), respectively.

The result of western blot analysis was scanned, and densitometric analysis for the quantification of the bands was done using Scion Image software (Scion Corp., Frederick, MD), which was used to count ROD: A ratio of the ROD was calibrated as percentage, with the adult group designated as 100 %.

Statistical Analysis

Every statistical analysis was carried out using GraphPad Prism 4.0 (GraphPad Software, USA). Data are expressed as the mean ± SEM. The data were evaluated by Student’s t test. Statistical significance was considered at P < 0.05.

Results

α-Syn Immunoreactivity

In both the adult and aged groups, α-syn immunoreactivity was easily detected in neuropil of all the hippocampal sub-regions (Fig. 1a, b): α-syn immunoreactivity was not detected in the cell bodies and dendrites of neurons.

Fig. 1.

Fig. 1

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α-Syn immunohistochemistry in the hippocampus proper (CA1–3 region) and dentate gyrus (DG) of the adult (a, c, e, g) and aged (b, d, f, h) dogs. In the aged dog, α-syn immunoreactivity was markedly increased in the mossy fibers (asterisks) compared to that in the adult group. GCL granule cell layer, ML molecular layer, PL polymorphic layer, SO stratum oriens, SP stratum pyramidale, SR stratum radiatum. Bar 800 μm (a, b), 200 μm (c–h)

In the CA1 region of the aged group, α-syn immunoreactivity was not changed compared to that in the adult group (Fig. 1c, d). In the CA2/3 region of the aged group, α-syn immunoreactivity was apparently increased in mossy fibers (Fig. 1e, f). In the dentate gyrus of the aged group, α-syn immunoreactivity was also markedly increased in mossy fibers in the polymorphic layer (Fig. 1g, h).

α-Syn Protein Levels

In western blot study, α-syn protein was detected in the adult hippocampus, and, in the aged group, α-syn protein was markedly increased (Fig. 2).

Fig. 2.

Fig. 2

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Western blot analysis of α-syn protein level in the hippocampus derived from the adult and aged dogs. The ROD of immunoblot bands is demonstrated as percent values (n = 5 per group; *P < 0.05 vs. the adult group). Data are presented as the mean ± SEM

GFAP Immunoreactivity

In the hippocampal CA1 region of the adult group, weak GFAP positive astrocytes were detected in all the layers (Fig. 3a, c): they showed small cytoplasm with thread-like processes. In the CA1 region of the aged group, GFAP immunoreactivity in astrocytes was slightly increased compared to that in the adult group (Table 1; Fig. 3b, d). In the CA2/3 of the aged group, GFAP immunoreactivity was apparently increased in all the layers compared to the adult group (Table 1; Fig. 3e, f): their cell bodies and processes were hypertrophied. In the DG of the aged, GFAP immunoreactivity in astrocytes was distinctively increased only in the polymorphic layer (Table 1; Fig. 3g, h).

Fig. 3.

Fig. 3

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GFAP immunohistochemistry in the hippocampus proper (CA1–3 region) and the dentate gyrus (DG) of the adult (a, c, e, g) and aged (b, d, f, h) dogs. In the aged dog, GFAP immunoreactivity was markedly increased in the stratum radiatum (SR, asterisk) in the CA2/3 region and in the polymorphic layer (PL, asterisk) in the dentate gyrus compared to that in the adult group (d, f, h). GCL granule cell layer, ML molecular layer, SO stratum oriens, SP stratum pyramidale. Bar 800 μm (a, b), 200 μm (c–h)

Table 1.

Semi-quantifications of the immunoreactivity of GFAP and Iba-1 in the hippocampal sub-regions of the adult and aged dogs

Antibody list Hippocampal sub-regions Category Groups
Adult Aged
GFAP CA1 CSO ± +
CSP ± ±
CSR ± +
CA2/3 CSO + ++
CSP + ++
CSR + ++
DG CML ± +
CGCL ± ±
CPL + ++
Iba-1 CA1 CSO + ++
CSP + ++
CSR + ++
CA2/3 CSO + ++
CSP + ++
CSR + ++
DG CML + ++
CGCL + +
CPL + ++
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Immunoreactivity is scaled as ±, +, or ++, representing weakly positive, moderate, or strong, respectively

CSP cells in stratum pyramidale, CSO cells in stratum oriens, CSR cells in stratum radiatum, CML cells in molecular layer, CGCL cells in granule cell layers, CPL cells in polymorphic layer

Iba-1 Immunoreactivity

Iba-1-immunoreactive microglia were detected in all the hippocampal sub-regions in the adult and aged groups (Fig. 4). In the CA1 region of the aged group, Iba-1 immunoreactivity in microglia was increased compared to that in the adult group (Table 1; Fig. 4b, d). In the CA2/3 region of the aged group, Iba-1 immunoreactivity was also increased (Table 1; Fig. 4e, f). Especially, Iba-1-immunoreactive microglia around the mossy fibers showed hypertrophied processes compared to that in the adult group (Fig. 4f). In the dentate gyrus of both the groups, many Iba-1-immunoreactive microglia were distributed in the polymorphic layer (Fig. 4g, h). In the aged group, Iba-1 immunoreactivity was apparently increased in the polymorphic layer compared to that in the adult group (Table 1; Fig. 4h).

Fig. 4.

Fig. 4

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Iba-1 immunohistochemistry in the hippocampus of the adult (a, c, e, g) and aged (b, d, f, h) dogs. In the aged dog, Iba-1 immunoreactivity was markedly increased in the stratum radiatum (SR, asterisk) in the CA2/3 region and in the polymorphic layer (PL, asterisk) of the dentate gyrus compared to that in the adult group (d, f, h). GCL granule cell layer, ML molecular layer, SO stratum oriens, SP stratum pyramidale. Bar 800 μm (a, b), 200 μm (c–h)

Double Immunofluorescence Staining of α-syn/GFAP and α-syn/Iba-1

In the aged hippocampus, α-syn, GFAP, and Iba-1 immunoreactivity were apparently increased in the mossy fibers in the CA2/3 region and in the polymorphic layer in the dentate gyrus. We found that α-syn+ structures were not co-localized with GFAP+ astrocytes and Iba-1+ microglia (data not shown) in the CA2/3 region and in the polymorphic layer in the dentate gyrus (Fig. 5).

Fig. 5.

Fig. 5

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Double immunofluorescence staining for α-syn (green) and GFAP (red) and merged images in the CA3 region and dentate gyrus (DG) of the adult (a–f) and aged (g–l) dogs. In the aged dog, α-syn immunoreactivity is markedly increased in the mossy fibers compared to that in the adult group; however, the α-syn immunoreactivity is not co-localized with GFAP immunoreactivity (arrows). GCL granule cell layer, ML molecular layer, PL polymorphic layer, SO stratum oriens, SP stratum pyramidale, SR stratum radiatum. Bar 200 μm (Color figure online)

Discussion

It has been reported that age-related increases in α-syn in the substantia nigra are non-aggregated and strongly associated with age-related decreases in tyrosine hydroxylase in humans and monkeys (Chu and Kordower 2007). In this study, we examined α-syn immunoreactivity and its protein levels in the hippocampus in the adult and aged beagle dogs to compare the distribution and change of α-syn between the adult and aged.

In the aged hippocampus, α-syn immunoreactivity in the CA1 region was not markedly changed compared to the adult dog; however, α-syn immunoreactivity was dramatically increased in the mossy fibers in the aged group. However, the expression pattern of α-syn in the aged hippocampus was non-aggregated profile (cloud-like neuropil staining) that is distinctively different from pathological features such as aggregation and inclusion of α-syn. This result is supported by previous studies, which showed that α-syn levels were increased in the brain of the senescence-accelerated mouse (Alvarez-Garcia et al. 2006; Caballero et al. 2008). In addition, it was reported that a dot-like granular or neuritic pattern of accumulated α-syn was observed in the stratum radiatum of the CA2/3 region and in the molecular layer of the dentate gyrus of a mouse model of dementia with Lewy bodies, whereas non-aggregated pattern was observed in the control group (Lim et al. (2011). On the contrary, it was reported that α-syn level was significantly decreased, but mRNA expression was not changed in the hippocampus of aged Wistar rats compared to that in the adult group (Adamczyk et al. 2005).

It is well known that aggregated α-syn is a main component of Lewy bodies which are considered as one of the neuropathological characters of Parkinson’s disease and Alzheimer’s disease (Eller and Williams 2011; Jellinger 2004; Rampello et al. 2004; Yokota et al. 2007). It was reported that there was an age-related increase of the abnormal deposition of cellular protein due to an age-related decline in the activity of proteasomes which degrade damaged or ubiquitinated proteins (Tai and Schuman 2008). In addition, it was reported that α-syn was aggregated at presynaptic terminals, impacted on synaptic dysfunction, and caused neurodegeneration (Kramer and Schulz-Schaeffer 2007). Therefore, it is likely that the increase of non-aggregated α-syn in the hippocampus of the aged dog might be an age-related change in normal condition.

In this study, GFAP (an astrocytes marker) and Iba-1 (a microglia marker) immunoreactivity were markedly increased in the stratum radiatum of the CA2/3 region and in the polymorphic layer of the dentate gyrus in the aged dogs, which is consistent with the α-syn increased regions. However, we could not observe that α-syn-immunoreactive structures were co-localized with GFAP-immunoreactive astrocytes or Iba-1-immunoreactive microglia in the CA2/3 region and in the polymorphic layer in the dentate gyrus, but they were surrounded with glia. It was reported that the activation of α-syn enhanced expression of α-syn in astrocytes and proinflammatory cytokines in astrocytic cell cultures and that they caused neurodegeneration in the A53T transgenic mouse (Gu et al. 2010; Klegeris et al. 2006). In addition, it was reported that astrogliosis triggered a number of proinflammatory cytokines and chemokines and this inflammatory microenvironment might contribute to neuroinflammatory response (Klegeris et al. 2006; Lee et al. 2010a).

Microglia, which is major immune cells in the CNS, can be activated by α-syn directly as well as indirectly by the activation of astrocytes (Halliday and Stevens 2011; Lee et al. 2010a; Su et al. 2009). It was reported that the overexpression of mutant human α-syn enhanced microgliosis (Su et al. 2009). In addition, it was reported that aggregated α-syn activated microglia with a production of reactive oxygen species which are closely related to neurodegeneration in Parkinson’s disease (Zhang et al. 2005). Therefore, it could be postulated that an increased non-aggregated α-syn expression with activated astrocytes and microglia in the hippocampus in the aged dog might be related to α-syn-induced inflammatory changes as well as an activation of glia.

In brief, α-syn immunoreactivity was markedly increased in the mossy fibers in the aged dog hippocampus, and GFAP and Iba-1 immunoreactivity were apparently increased in the regions with an increase of α-syn.

Acknowledgments

The authors would like to thank Mr. Seung Uk Lee for their technical help in this study. This work was supported by and by the Technology Innovation Program funded by the Ministry of Knowledge Economy (MKE, Korea), and by the Regional Core Research Program funded by the Korea Ministry of Education, Science and Technology (Medical & Bio-material Research Center).

Footnotes

Ji Hyeon Ahn and Joon Ha Park contributed equally to this study.

Contributor Information

Hyung-Cheul Shin, Phone: +82-33-248-2580, Email: hcshin@hallym.ac.kr.

Moo-Ho Won, Phone: +82-33-250-8891, FAX: +82-33-256-1614, Email: mhwon@kangwon.ac.kr.

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