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Olfactory deficits induce neurofilament hyperphosphorylation
Neuroscience Letters 506 (2012) 180–183
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Olfactory deficits induce neurofilament hyperphosphorylation Juan Hu, Xiang Wang, Dan Liu, Qun Wang, Ling-Qiang Zhu ∗ Department of Pathophysiology, Key Laboratory of Neurological Disease of Education Committee of China, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
a r t i c l e
i n f o
Article history: Received 27 June 2011 Received in revised form 9 September 2011 Accepted 29 October 2011 Keywords: Alzheimer’s disease Neurofilament proteins Hyperphosphorylation Olfactory bulbectomy Cnga2
a b s t r a c t Olfactory dysfunction, including structural abnormalities of the olfactory epithelium, the olfactory bulb and the central olfactory cortices is recognized as an early feature of Alzheimer disease (AD), the most prevalent neurodegenerative disease in aged population characterized by intracellular neurofibrillary tangles (NFTs). How olfactory deficits are linked with AD-like neuropathological changes is still unknown. Here, by using two anosmia animal models, bilateral olfactory bulbectomy (OBX) rats and Cnga2−/Y mice, which lack intact olfactory CNG channels, we found the immunoreactivity of phosphorylated neurofilament (NF) are highly increased in the neurites at both the hippocampus and the cortex. As hyperphosphorylated NF is one of the main components of NFTs, our study strongly suggested the underlying correlation of olfactory deficits with AD-like pathological impairments. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction AD is pathologically defined by the presence of amyloid- plaques, neurofibrillary tangles (NFTs) and neuron loss within the brain [19]. Clinically, AD is characterized by progressive memory deficits and the degree of dementia is highly correlated positively to the quantities of NFTs [3]. NFTs are mainly composed of aberrantly hyperphosphorylated cytoskeleton related proteins, such as tau and NF, the major cytoskeletal components of neurons which are abnormally wide spread in AD brains [17]. As a component of NFTs, NF proteins in their hyperphosphorylated form are also accumulated in the degenerated neurons in AD brains. NF proteins are the major cytoskeleton to maintain the normal architecture of the neurons and play a critical role in maintaining axonal caliber and morphological integrity of neurons. Multiple evidences suggest that phosphorylated NF proteins are abnormally deposited in AD brains [18,29,30], the abnormally hyperphosphorylated NF were found to be accumulated in NFTs [26] and their levels were elevated in the CSF [11]. In the olfactory, epithelium, dystrophic neurites that are immunoreactive for NF as well as deposits of -amyloid have been observed, and contribute to the olfactory dysfunction of AD [2]. The presence of smell loss indicated the pathological involvement of the olfactory pathways in the
Abbreviations: AD, Alzheimer’s disease; Cnga2, cyclic nucleotide-gated channel ␣2; NF, neurofilament; NFTs, neurofibrillary tangles; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; OBX, olfactory bulbectomy. ∗ Corresponding author. Tel.: +86 027 83692625; fax: +86 027 83693883. E-mail address: [email protected] (L.-Q. Zhu). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.10.076
formative stages of AD. Thus, olfactory deficits seen early in the course of this disease appear to be correlated with the decline in cognitive functions [9,21]. The olfactory system governs the basic olfactory sense function, which is the primary sense in mammals. Recently, the importance of the olfactory system in the brain functions had been extended to its role in the neural circuit, the basis of learning and memory [7,33,34]. Dysfunction of the olfactory system had been reported in many neurological diseases, such as AD [1]. It was suggested that olfactory deficits can be used as an early marker for AD and that inclusion of a criterion for olfactory decline may improve the detection of early AD [15]. Numerous structural and functional studies have observed both peripheral and central abnormalities in different parts of the olfactory system in AD patients, including the olfactory epithelium, olfactory bulb, entorhinal cortex, and hippocampus [14,27,28]. Previous studies have already demonstrated that abundant hyperphosphorylated tau protein were found in the olfactory system in all diagnosed AD cases, in two-thirds of limbic AD cases, as well in almost one-third of non-demented elders with Braak stage 2 [4]. These lines of evidence indicate the possible link between olfactory impairments with the AD-like pathology. However, the direct link is still missing. Here, by using two animal models with olfactory dysfunction, bilateral olfactory bulbectomized (OBX) rats and cyclic nucleotidegated channel subunit A2 (Cnga2) [6,36] knockout mice (Cnga2−/Y mice, failure with odorant-evoked responses to a variety of odorants), we found that (1) olfactory deficits could induce hyperphosphorylation of NF in the hippocampus and cortex; (2) the hyperphosphorylation of NF is mainly localized in the cell body in OBX rats but the neurites in the Cnga2−/Y mice.
J. Hu et al. / Neuroscience Letters 506 (2012) 180–183
181
2. Materials and methods
2.4. Immunohistochemistry
2.1. Animals
Immunohistochemistry was performed as described [32]. The OBX rats and Cnga2−/Y mice were deeply anesthetized to minimize the suffering of the animals. They were perfused through aorta with 150 ml 0.9% NaCl solution rapidly, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer containing 15% saturated picric acid (pH 7.4). The brains were removed from the skull and post-fixed in 4% PFA for 10 h at 4 ◦ C before it was coronally sliced into 30 m sections on a Vibratome (Leica, Nussloch, S100, TPI, Germany). Free floating sections were permeabilized with 0.3% H2 O2 and 0.5% triton X-100 in PBS for 15 min to block endogenous peroxidase and partially dissolve the nuclear membrane, and nonspecific sites were blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature. After washing with PBS, sections were then incubated for 48 h at 4 ◦ C with SMI34 antibodies (1:2000). The sections were subsequently incubated with biotinylated secondary antibodies (1:200) for 1 h at 37 ◦ C, and the immunoreaction was detected using avidinperoxidase conjugate (1:200) for 1 h at 37 ◦ C and visualized with the diaminobenzidine tetrachloride (DAB) system (0.05%). The images were observed using a microscope (Olympus BX60, Tokyo, Japan). The density of images was quantitatively evaluated by a Kontron Imaging System KS300 connected to a light microscope (Olympus BX60, Tokyo, Japan) with identical settings (×20 objective lens). Data were expressed as mean ± S.D. and all statistical analyses were carried out using SPSS 12.0 for Windows (SPSS Inc., Chicago, IL, USA). The Student’s t test was used to determine the differences between the groups.
The male SD rats (3 months old, 250 ± 20 g) used in this study were obtained from Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China. All rats were housed in standard cages with water and food ad libitum at stable temperature (25 ± 2 ◦ C) and humidity in a 12 h light:12 h dark cycle (lights on at 6:00 AM, off at 6:00 PM). All animal experiments were performed according to the guidelines outlined by the National Institutes of Health for the care and use of laboratory animals.
2.2. Surgery Rats were anaesthetized with 6% Chloral Hydrate (360 mg/kg, i.p.) and placed in a stereotaxic instrument (SR-6N, Tokyo Narishige, Japan) at a rat-skull position, with the incisor bar set 2 mm below the earbars. After the scalp was incised (5–8 mm), the skull was cleaned and two holes (2 mm in diameter) were drilled symmetrically over the olfactory bulbs (8 mm anterior to the bregma and 2 mm laterally from the midline). Both olfactory bulbs were carefully aspirated under visual control through a blunt needle attached to a water pump, Sham-operated (SO) rats were treated similarly with exception of the bulbectomy as controls [24]. Two-month old Cnga2−/Y mice (Cnga2 is X-linked) which lacked intact olfactory CNG channels and their wild-type littermates were provided by Prof. Fu-Qiang Xu, from State Key Laboratory of Magnetic Resonance, Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, China.
3. Results and discussion 2.3. Western blotting The Western blotting was carried as described previously [35]. The rats were sacrificed at 2 months after the OBX surgery. The hippocampus and cortex of OBX rats and Cnga2−/Y mice were quickly dissected and homogenized using a Teflon glass homogenizer in ice-cold homogenate buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 10 mM NaF, 1 mM Na3 VO4 , 5 mM EDTA, 2 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 g/ml each of leupeptin, aprotinin, and pepstatin A. Three volumes of homogenate was dissolved in one volume of lysis buffer containing 200 mM Tris–HCl (pH 7.6), 8% SDS, 40% glycerol, boiled for 10 min and then centrifuged at 15,000 × g for 10 min. The protein concentration of the supernatants was measured by BCA kit according to manufacturer’s instructions. Dithiothreitol (DTT) was added to attain a final concentration 100 mM at last. Equal amounts of protein were separated by 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Hybond C-Super, Amersham Pharmacia Biotech, Amersham, UK). After blocking with 5% nonfat milk dissolved in TBS (50 mM Tris–HCl, pH 7.6, 150 mM NaCl) for 30 min and probed with anti-SMI34 (1:5000) and DM1A (1:1000) for overnight at 4 ◦ C. Monoclonal antibody SMI34 to phosphorylated NF was purchased from Sternberger Monoclonals Inc. (Baltimore, MD, USA). Monoclonal antibody DM1A to ␣-tubulin was purchased from Sigma (St. Louis, MO). Then the blots were incubated with anti-mouse IgG conjugated to IRDyeTM (800CW) (1:10,000) for 1 h at room temperature. The protein bands were visualized and the density was quantitatively analyzed with the Odyssey Infrared Imaging System (Licor Biosciences, Lincoln, NE, USA). The total level of phosphorylated NF (SMI34) was normalized against DM1A.
To explore whether olfactory dysfunction affects the phosphorylation level of NF, we first detected the immunoreactivity of SMI34 that recognized phosphorylated NF by Western blotting in OBX rats. We found that the immunoreactivity of SMI34 increased to 2.4 folds in the hippocampus (Fig. 1A and B) and 3.5 folds in cortex of OBX rats compared with the SO group (Fig. 1C and D), suggesting OBX increased NF phosphorylation level. By immunohistochemistry, we found that the enhanced p-NF staining is mainly localized in the cell bodies and inward neurites of the hippocampal CA3 neurons (Fig. 1E). In the cortex, not only the hyperphosphorylation of NF but also the aggregation of the phosphorylated NF is observed in the cell bodies accompanied with the disorder neurites organization (Fig. 1F). The above data suggest that OBX induced hyperphosphorylated neurofilaments are mainly localized in the soma. We then detected the phosphorylation of NF in the Cnga2−/Y mice to verify the effect of olfactory disorder on NF phosphorylation. We found that the immunoreactivity of SMI34 increased to 1.8 folds in the hippocampus (Fig. 2A and B) and 1.6 folds in the cortex (Fig. 2C and D) in Cnga2−/Y mice compared to wild-type littermates normalized against DM1A, suggesting the NF phosphorylation level is also elevated in these mice. By immunohistochemistry, the enhanced SMI34 staining was also mainly observed in dendrites of the hippocampal CA3 region (Fig. 2E) and cortex (Fig. 2F) in Cnga2−/Y mice compared to wild-type mice, indicating that Cnga2 knockout induced hyperphosphorylated NF are mainly localized in the dendrites. We proposed that the difference between the localization of hyperphosphorylated NF in OBX rats and Cnga2−/Y mice may be due to the different level of olfactory loss, for the OBX rats loss of all their olfactory while Cnga2−/Y mice causes only partial loss of their olfactory [13]. These data further confirmed the effect of olfactory dysfunction on the phosphorylation of NF.
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J. Hu et al. / Neuroscience Letters 506 (2012) 180–183
Fig. 1. OBX induces NF hyperphosphorylation in hippocampus and cortex in rats. (A and B) Western blotting and quantitative analyses: the hippocampal extracts were prepared 2 months after OBX surgery and the phosphorylation level of NF proteins in the sham-operated (SO) and OBX rats was normalized against DM1A. (C and D) Western blotting and quantitative analysis of NF phosphorylation level in the cortex of SO and OBX rats. (E and F) Immunohistochemical staining of NF phosphorylation in the SO and OBX rats. Left panel: scale bar = 100 m; right panel: scale bar = 25 m (the right panel were amplified from region as indicated by black rectangle in the left panel).**p < 0.01 vs SO rats (Mean ± SD, n = 3).
It had been believed for dozens of years that the Alzheimer neurofibrillary tangles, the hallmark lesion of this disease, were composed of NF based on immunocytochemical studies at light electron microscopic level with antibodies to neurofilaments [26]. However, whether and to what extent NF proteins are actually hyperphosphorylated in the AD brain has remained a puzzle. The phosphorylated NF appear to be localized in the cell body in OBX rats but the neurites in the Cnga2−/Y mice suggesting the importance of NF phosphorylation in its transport and interaction with other cytoskeletal proteins. Bilateral olfactory bulbectomized (OBX) in rats was first used as the animal model for depression, and was also been suggested to be an AD animal model for it displays deficits in spatial learning and memory [16,23], disturbance of cholinergic system, A deposition in the neocortex and hippocampus, apparent cytoarchitectonic disruption in the cortex temporal lobe, along with the loss of clarity of its layer boundaries resulted from dystrophy or even loss of pyramidal neurons, which are also seen in patients with AD [5,20,31]. Binding of odorants to olfactory receptors activates the cAMP signaling pathway resulting in depolarization of olfactory receptor neurons in the main olfactory epithelium [8,22]. In this study, by using these two lines of animal models, we found that the expression of phosphorylated NF in hippocampus and cortex are elevated in both two animal models, which indicated olfactory deficits may induce hyperphosphorylation of NF proteins. It is well known that the nerve fibers in the olfactory system can project anatomically to the entorhinal cortex, which
Fig. 2. Higher phosphorylation level of NF proteins was observed in hippocampus and cortex in Cnga2−/Y mice compared to wild-type males. (A and B) Western blotting and quantitative analyses: the hippocampal extracts were prepared and the phosphorylation level of NF proteins in the Cnga2−/Y mice (ko) and wild-type males (wt) was measured. (C and D) Western blotting and quantitative analyses of NF phosphorylation level in the cortex of the Cnga2−/Y mice and wild-type males. (E and F) Immunohistochemical staining of NF phosphorylation in the Cnga2−/Y mice (ko) and wild-type males (wt) was measured. Left panel: scale bar = 100 m; right panel: scale bar = 25 m (the right panel were amplified from region as indicated by black rectangle in the left panel). **p < 0.01 vs wt mice (Mean ± SD, n = 3).
then formed important circuits with hippocampus in the learning and memory [25]. Removal of the olfactory bulbs of rats or knockout Cnga2 channels lead to decreased performance on cognitive tasks not dependent on olfaction [12], an effect attributed, in part, to degenerative disruption of interconnections with higher brain regions, such as those between the olfactory and septohippocampal systems [10]. In summary, we have demonstrated in the present study that olfactory deficits induce hyperphosphorylation of NF in the OBX rats and Cnga2−/Y mice. Conflict of interest The authors declare that they have no competing financial interests or conflicts of interest. Acknowledgements The authors thank Prof. Fu-Qiang Xu from Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences for the Cnga2−/Y mice. This work was supported in part by grants from National Natural Science Foundation of China (NSFC) (No. 91132725, 30800342, 30971478) and the New Century Excellent Talents in University (NCET-10-0421) to Dr. Ling-Qiang Zhu. References [1] M.W. Albers, M.H. Tabert, D.P. Devanand, Olfactory dysfunction as a predictor of neurodegenerative disease, Curr. Neurol. Neurosci. Rep. 6 (2006) 379–386. [2] S.E. Arnold, G.S. Smutzer, J.Q. Trojanowski, P.J. Moberg, Cellular and molecular neuropathology of the olfactory epithelium and central olfactory pathways in Alzheimer’s disease and schizophrenia, Ann. N.Y. Acad. Sci. 855 (1998) 762–775.
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Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Olfactory deficits induce neurofilament hyperphosphorylation Juan Hu, Xiang Wang, Dan Liu, Qun Wang, Ling-Qiang Zhu ∗ Department of Pathophysiology, Key Laboratory of Neurological Disease of Education Committee of China, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
a r t i c l e
i n f o
Article history: Received 27 June 2011 Received in revised form 9 September 2011 Accepted 29 October 2011 Keywords: Alzheimer’s disease Neurofilament proteins Hyperphosphorylation Olfactory bulbectomy Cnga2
a b s t r a c t Olfactory dysfunction, including structural abnormalities of the olfactory epithelium, the olfactory bulb and the central olfactory cortices is recognized as an early feature of Alzheimer disease (AD), the most prevalent neurodegenerative disease in aged population characterized by intracellular neurofibrillary tangles (NFTs). How olfactory deficits are linked with AD-like neuropathological changes is still unknown. Here, by using two anosmia animal models, bilateral olfactory bulbectomy (OBX) rats and Cnga2−/Y mice, which lack intact olfactory CNG channels, we found the immunoreactivity of phosphorylated neurofilament (NF) are highly increased in the neurites at both the hippocampus and the cortex. As hyperphosphorylated NF is one of the main components of NFTs, our study strongly suggested the underlying correlation of olfactory deficits with AD-like pathological impairments. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction AD is pathologically defined by the presence of amyloid- plaques, neurofibrillary tangles (NFTs) and neuron loss within the brain [19]. Clinically, AD is characterized by progressive memory deficits and the degree of dementia is highly correlated positively to the quantities of NFTs [3]. NFTs are mainly composed of aberrantly hyperphosphorylated cytoskeleton related proteins, such as tau and NF, the major cytoskeletal components of neurons which are abnormally wide spread in AD brains [17]. As a component of NFTs, NF proteins in their hyperphosphorylated form are also accumulated in the degenerated neurons in AD brains. NF proteins are the major cytoskeleton to maintain the normal architecture of the neurons and play a critical role in maintaining axonal caliber and morphological integrity of neurons. Multiple evidences suggest that phosphorylated NF proteins are abnormally deposited in AD brains [18,29,30], the abnormally hyperphosphorylated NF were found to be accumulated in NFTs [26] and their levels were elevated in the CSF [11]. In the olfactory, epithelium, dystrophic neurites that are immunoreactive for NF as well as deposits of -amyloid have been observed, and contribute to the olfactory dysfunction of AD [2]. The presence of smell loss indicated the pathological involvement of the olfactory pathways in the
Abbreviations: AD, Alzheimer’s disease; Cnga2, cyclic nucleotide-gated channel ␣2; NF, neurofilament; NFTs, neurofibrillary tangles; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; OBX, olfactory bulbectomy. ∗ Corresponding author. Tel.: +86 027 83692625; fax: +86 027 83693883. E-mail address: [email protected] (L.-Q. Zhu). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.10.076
formative stages of AD. Thus, olfactory deficits seen early in the course of this disease appear to be correlated with the decline in cognitive functions [9,21]. The olfactory system governs the basic olfactory sense function, which is the primary sense in mammals. Recently, the importance of the olfactory system in the brain functions had been extended to its role in the neural circuit, the basis of learning and memory [7,33,34]. Dysfunction of the olfactory system had been reported in many neurological diseases, such as AD [1]. It was suggested that olfactory deficits can be used as an early marker for AD and that inclusion of a criterion for olfactory decline may improve the detection of early AD [15]. Numerous structural and functional studies have observed both peripheral and central abnormalities in different parts of the olfactory system in AD patients, including the olfactory epithelium, olfactory bulb, entorhinal cortex, and hippocampus [14,27,28]. Previous studies have already demonstrated that abundant hyperphosphorylated tau protein were found in the olfactory system in all diagnosed AD cases, in two-thirds of limbic AD cases, as well in almost one-third of non-demented elders with Braak stage 2 [4]. These lines of evidence indicate the possible link between olfactory impairments with the AD-like pathology. However, the direct link is still missing. Here, by using two animal models with olfactory dysfunction, bilateral olfactory bulbectomized (OBX) rats and cyclic nucleotidegated channel subunit A2 (Cnga2) [6,36] knockout mice (Cnga2−/Y mice, failure with odorant-evoked responses to a variety of odorants), we found that (1) olfactory deficits could induce hyperphosphorylation of NF in the hippocampus and cortex; (2) the hyperphosphorylation of NF is mainly localized in the cell body in OBX rats but the neurites in the Cnga2−/Y mice.
J. Hu et al. / Neuroscience Letters 506 (2012) 180–183
181
2. Materials and methods
2.4. Immunohistochemistry
2.1. Animals
Immunohistochemistry was performed as described [32]. The OBX rats and Cnga2−/Y mice were deeply anesthetized to minimize the suffering of the animals. They were perfused through aorta with 150 ml 0.9% NaCl solution rapidly, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer containing 15% saturated picric acid (pH 7.4). The brains were removed from the skull and post-fixed in 4% PFA for 10 h at 4 ◦ C before it was coronally sliced into 30 m sections on a Vibratome (Leica, Nussloch, S100, TPI, Germany). Free floating sections were permeabilized with 0.3% H2 O2 and 0.5% triton X-100 in PBS for 15 min to block endogenous peroxidase and partially dissolve the nuclear membrane, and nonspecific sites were blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature. After washing with PBS, sections were then incubated for 48 h at 4 ◦ C with SMI34 antibodies (1:2000). The sections were subsequently incubated with biotinylated secondary antibodies (1:200) for 1 h at 37 ◦ C, and the immunoreaction was detected using avidinperoxidase conjugate (1:200) for 1 h at 37 ◦ C and visualized with the diaminobenzidine tetrachloride (DAB) system (0.05%). The images were observed using a microscope (Olympus BX60, Tokyo, Japan). The density of images was quantitatively evaluated by a Kontron Imaging System KS300 connected to a light microscope (Olympus BX60, Tokyo, Japan) with identical settings (×20 objective lens). Data were expressed as mean ± S.D. and all statistical analyses were carried out using SPSS 12.0 for Windows (SPSS Inc., Chicago, IL, USA). The Student’s t test was used to determine the differences between the groups.
The male SD rats (3 months old, 250 ± 20 g) used in this study were obtained from Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China. All rats were housed in standard cages with water and food ad libitum at stable temperature (25 ± 2 ◦ C) and humidity in a 12 h light:12 h dark cycle (lights on at 6:00 AM, off at 6:00 PM). All animal experiments were performed according to the guidelines outlined by the National Institutes of Health for the care and use of laboratory animals.
2.2. Surgery Rats were anaesthetized with 6% Chloral Hydrate (360 mg/kg, i.p.) and placed in a stereotaxic instrument (SR-6N, Tokyo Narishige, Japan) at a rat-skull position, with the incisor bar set 2 mm below the earbars. After the scalp was incised (5–8 mm), the skull was cleaned and two holes (2 mm in diameter) were drilled symmetrically over the olfactory bulbs (8 mm anterior to the bregma and 2 mm laterally from the midline). Both olfactory bulbs were carefully aspirated under visual control through a blunt needle attached to a water pump, Sham-operated (SO) rats were treated similarly with exception of the bulbectomy as controls [24]. Two-month old Cnga2−/Y mice (Cnga2 is X-linked) which lacked intact olfactory CNG channels and their wild-type littermates were provided by Prof. Fu-Qiang Xu, from State Key Laboratory of Magnetic Resonance, Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, China.
3. Results and discussion 2.3. Western blotting The Western blotting was carried as described previously [35]. The rats were sacrificed at 2 months after the OBX surgery. The hippocampus and cortex of OBX rats and Cnga2−/Y mice were quickly dissected and homogenized using a Teflon glass homogenizer in ice-cold homogenate buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 10 mM NaF, 1 mM Na3 VO4 , 5 mM EDTA, 2 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 g/ml each of leupeptin, aprotinin, and pepstatin A. Three volumes of homogenate was dissolved in one volume of lysis buffer containing 200 mM Tris–HCl (pH 7.6), 8% SDS, 40% glycerol, boiled for 10 min and then centrifuged at 15,000 × g for 10 min. The protein concentration of the supernatants was measured by BCA kit according to manufacturer’s instructions. Dithiothreitol (DTT) was added to attain a final concentration 100 mM at last. Equal amounts of protein were separated by 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Hybond C-Super, Amersham Pharmacia Biotech, Amersham, UK). After blocking with 5% nonfat milk dissolved in TBS (50 mM Tris–HCl, pH 7.6, 150 mM NaCl) for 30 min and probed with anti-SMI34 (1:5000) and DM1A (1:1000) for overnight at 4 ◦ C. Monoclonal antibody SMI34 to phosphorylated NF was purchased from Sternberger Monoclonals Inc. (Baltimore, MD, USA). Monoclonal antibody DM1A to ␣-tubulin was purchased from Sigma (St. Louis, MO). Then the blots were incubated with anti-mouse IgG conjugated to IRDyeTM (800CW) (1:10,000) for 1 h at room temperature. The protein bands were visualized and the density was quantitatively analyzed with the Odyssey Infrared Imaging System (Licor Biosciences, Lincoln, NE, USA). The total level of phosphorylated NF (SMI34) was normalized against DM1A.
To explore whether olfactory dysfunction affects the phosphorylation level of NF, we first detected the immunoreactivity of SMI34 that recognized phosphorylated NF by Western blotting in OBX rats. We found that the immunoreactivity of SMI34 increased to 2.4 folds in the hippocampus (Fig. 1A and B) and 3.5 folds in cortex of OBX rats compared with the SO group (Fig. 1C and D), suggesting OBX increased NF phosphorylation level. By immunohistochemistry, we found that the enhanced p-NF staining is mainly localized in the cell bodies and inward neurites of the hippocampal CA3 neurons (Fig. 1E). In the cortex, not only the hyperphosphorylation of NF but also the aggregation of the phosphorylated NF is observed in the cell bodies accompanied with the disorder neurites organization (Fig. 1F). The above data suggest that OBX induced hyperphosphorylated neurofilaments are mainly localized in the soma. We then detected the phosphorylation of NF in the Cnga2−/Y mice to verify the effect of olfactory disorder on NF phosphorylation. We found that the immunoreactivity of SMI34 increased to 1.8 folds in the hippocampus (Fig. 2A and B) and 1.6 folds in the cortex (Fig. 2C and D) in Cnga2−/Y mice compared to wild-type littermates normalized against DM1A, suggesting the NF phosphorylation level is also elevated in these mice. By immunohistochemistry, the enhanced SMI34 staining was also mainly observed in dendrites of the hippocampal CA3 region (Fig. 2E) and cortex (Fig. 2F) in Cnga2−/Y mice compared to wild-type mice, indicating that Cnga2 knockout induced hyperphosphorylated NF are mainly localized in the dendrites. We proposed that the difference between the localization of hyperphosphorylated NF in OBX rats and Cnga2−/Y mice may be due to the different level of olfactory loss, for the OBX rats loss of all their olfactory while Cnga2−/Y mice causes only partial loss of their olfactory [13]. These data further confirmed the effect of olfactory dysfunction on the phosphorylation of NF.
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Fig. 1. OBX induces NF hyperphosphorylation in hippocampus and cortex in rats. (A and B) Western blotting and quantitative analyses: the hippocampal extracts were prepared 2 months after OBX surgery and the phosphorylation level of NF proteins in the sham-operated (SO) and OBX rats was normalized against DM1A. (C and D) Western blotting and quantitative analysis of NF phosphorylation level in the cortex of SO and OBX rats. (E and F) Immunohistochemical staining of NF phosphorylation in the SO and OBX rats. Left panel: scale bar = 100 m; right panel: scale bar = 25 m (the right panel were amplified from region as indicated by black rectangle in the left panel).**p < 0.01 vs SO rats (Mean ± SD, n = 3).
It had been believed for dozens of years that the Alzheimer neurofibrillary tangles, the hallmark lesion of this disease, were composed of NF based on immunocytochemical studies at light electron microscopic level with antibodies to neurofilaments [26]. However, whether and to what extent NF proteins are actually hyperphosphorylated in the AD brain has remained a puzzle. The phosphorylated NF appear to be localized in the cell body in OBX rats but the neurites in the Cnga2−/Y mice suggesting the importance of NF phosphorylation in its transport and interaction with other cytoskeletal proteins. Bilateral olfactory bulbectomized (OBX) in rats was first used as the animal model for depression, and was also been suggested to be an AD animal model for it displays deficits in spatial learning and memory [16,23], disturbance of cholinergic system, A deposition in the neocortex and hippocampus, apparent cytoarchitectonic disruption in the cortex temporal lobe, along with the loss of clarity of its layer boundaries resulted from dystrophy or even loss of pyramidal neurons, which are also seen in patients with AD [5,20,31]. Binding of odorants to olfactory receptors activates the cAMP signaling pathway resulting in depolarization of olfactory receptor neurons in the main olfactory epithelium [8,22]. In this study, by using these two lines of animal models, we found that the expression of phosphorylated NF in hippocampus and cortex are elevated in both two animal models, which indicated olfactory deficits may induce hyperphosphorylation of NF proteins. It is well known that the nerve fibers in the olfactory system can project anatomically to the entorhinal cortex, which
Fig. 2. Higher phosphorylation level of NF proteins was observed in hippocampus and cortex in Cnga2−/Y mice compared to wild-type males. (A and B) Western blotting and quantitative analyses: the hippocampal extracts were prepared and the phosphorylation level of NF proteins in the Cnga2−/Y mice (ko) and wild-type males (wt) was measured. (C and D) Western blotting and quantitative analyses of NF phosphorylation level in the cortex of the Cnga2−/Y mice and wild-type males. (E and F) Immunohistochemical staining of NF phosphorylation in the Cnga2−/Y mice (ko) and wild-type males (wt) was measured. Left panel: scale bar = 100 m; right panel: scale bar = 25 m (the right panel were amplified from region as indicated by black rectangle in the left panel). **p < 0.01 vs wt mice (Mean ± SD, n = 3).
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