Vitamin E Deficiency Induces Axonal Degeneration in Mouse ... - J-Stage

J Nutr Sci Vitaminol, 58, 377–383, 2012

Vitamin E Deficiency Induces Axonal Degeneration in Mouse Hippocampal Neurons Koji Fukui1,6, Hiroaki Kawakami1, Tatsuki Honjo1, Reiko Ogasawara1, Hirokatsu Takatsu2,6, Tadashi Shinkai3, Tatsuro Koike4 and Shiro Urano5,6 1

Physiological Chemistry Laboratory, 3 Homeostatic Regulation Laboratory, and 5 Biochemistry Laboratory, Department of Bioscience and Engineering, College of Systems Engineering and Sciences, Shibaura Institute of Technology, Fukasaku 307, Minuma-ku, Saitama 337–8570, Japan 2 Malaysia Japan Higher Education Program, Kolej Kemahiran Tinggi MARA Beranang, Lot 2333, Jalan Kajang-Seremban, 43700 Beranang, Selangor Darul Ehsan, Malaysia 4 Molecular Neurobiology Laboratory, Graduate School of Life Science, Hokkaido University, Kita-ku N10W8, Sapporo 060–0810, Japan 6 Life Support Technology Research Center, Research Organization for Advanced Engineering, Shibaura Institute of Technology, Saitama 337–8570, Japan (Received April 17, 2012)

Summary Several lines of evidence demonstrate the relationship between vitamin E deficiency and cognitive dysfunction in rodent models, but little is known about the underlying mechanisms. In this study, we found axonal injury in the hippocampal CA1 region of vitamin E-deficient and normal old mice using immunohistochemical assay. The number of cells in the hippocampal CA1 region of vitamin E-deficient mice and normal old mice was significantly lower than in normal young mice. It is well known that collapsin response mediator protein (CRMP)-2 plays a crucial role in the maintenance of axonal conditions. The expressions of CRMP-2 in the cerebral cortex and hippocampus of vitamin E-deficient mice were significantly lower than both the regions of normal ones. In normal old mice, the expression of CRMP-2 in the cerebral cortex was significantly lower than in the normal ones. In addition, the appearance of microtubule-associated protein (MAP)–light chain 3 (LC3), a major index of autophagy, was higher in the cerebral cortex and hippocampus of vitamin E-deficient mice than in normal young and old mice. These results indicate that axonal degeneration is induced in living tissues, but not cultured cells, and that changes in CRMP-2 and MAP-LC3 may underlie vitamin E-deficiency-related axonal degeneration. Key Words vitamin E, aging, axon, CRMP-2, autophagy Vitamin E is a common natural lipophilic vitamin. One pivotal function of vitamin E is as an antioxidant (1), and vitamin E plays a role as a radical scavenger (2–4). Several lines of evidence demonstrate that vitamin E deficiency increases the risk of development and progression of serious diseases in human and animal models (5–7). For example, a vitamin E-deficient diet increased phospho-tau protein in mice expressing the human apolipoprotein E4 (5), and vitamin E-deficient rats exhibited hypersecretion of corticosterone, due to impairment of glucocorticosteroid receptors in the CA1 region of the hippocampus (6). Howard et al. showed that severe a-tocopherol deficiency induced lethal myopathy in animal models (7). It is well known that vitamin E deficiency induces cognitive dysfunction in rodent models (8, 9). In a previous study, we demonstrated that vitamin E-deficient rats exhibited cognitive dysfunction in several maze tasks (10, 11). We have been studying the mechanisms of cognitive dysfunction in this animal model, and have found evidence of apoptosis and b-amyloid-like proteins in the hipocampal CA1 region (12), indicating that cogE-mail: [email protected]

nitive dysfunction in vitamin E-deficient animals may be connected with injury to hippocampal neurons. However, by the time that these signs of neuronal injury are evident, it is difficult to recover cognitive function, because the neurons are severely damaged or have died. It is therefore necessary to find markers that can identify neuronal change before the induction of cell death. To address this problem we focused on the axons and dendrites, as they play an important role in neurotransmission (13, 14). In the axonal transport system, axons are involved in carrying several substances including neurotransmitters and other proteins. Furthermore, a large number of synapses exist on the distal ends of axons. If there are changes in axonal function, dysfunction of neurotransmission may be induced in some neurons. Urano et al. reported that a large number of synaptic vesicles had accumulated in synapses of aged rats and young rats exposed to reactive oxygen species (15). Previously, we reported that treatment of cerebellar granule neurons with a low concentration of hydrogen peroxide induced axon and dendrite degeneration, but not cell death (16, 17). However, these data were from cultured models, and axonal condition in living tissues is not yet understood.

377

378

Fukui K et al.

The purpose of this study was to determine whether vitamin E-deficient mice exhibit axonal degeneration or not. We focused on the hippocampal CA1 region, which plays an important role in cognitive function. We measured collapsin response mediator protein (CRMP)-2 to determine the mechanisms of morphological change in hippocampal neurons. CRMP-2, which is part of a family of cytoplasmic proteins in the brain, plays important roles in axon guidance (14, 18, 19). In this study, we found axonal degeneration and a change in CRMP-2 expression in the hippocampal neurons of vitamin E-deficient mice. Our data illustrate that axonal degeneration in vitamin E-deficient mice may be one of the early processes underlying cognitive dysfunction. Materials and Methods Animals and reagents. Normal mice were a wild-type C57BL/6 mouse strain obtained from Tokyo Metropolitan Institute of Gerontology (Tokyo, Japan). Young normal mice were 6 mo of age, and old normal mice were 24 mo of age. Vitamin E-deficient mice obtained from Japan SLC, Inc. (Hamamatsu, Japan) were fed a vitamin E-deficient diet from 4 wk to 6 mo of age. Experiments were performed on vitamin E-deficient mice at 6 mo of age. The vitamin E-deficient diet (AIN-76A) was purchased from Funabashi Farm Co., Ltd. (Chiba, Japan). All other chemical agents were obtained from either Wako Pure Chemicals Industries, Ltd. (Osaka, Japan) or Sigma-Aldrich Corporation (St. Louis, MO, USA). All other reagents were purchased from Sigma-Aldrich. All animal experiments were performed with the approval of the Animal Protection and Ethics Committee of the Shibaura Institute of Technology. Immobilization and slices. The mice were anesthetized with chloroform, and a laparotomy was performed. The mice were bled from the heart and immobilized by a 4% paraformaldehyde (PFA) solution (Merck KGaA, Darmstadt, Germany). The brain was surgically removed and fixed with 4% PFA solution for 48 h. A paraffin solution penetrated the fixed samples, and block samples were made for slices. The block samples were set on a microtome (Rotary microtome HM330, Thermo Fisher Scientific Inc., Waltham, MA) and sliced with a thickness of 10 mm. The slice samples were plated on an egg albumin-coated cover glass and used for immunohistochemical analysis. Immunohistochemical analysis. To quantify axonal condition in the mouse hippocampal region the continuous slice samples were stained using Bodian’s method with some modifications (20). The slice samples were stained with 1% silver protein solution (#43970, Alfa Aesar, Ward Hill, MA) at 37˚C for 20 h and were incubated with 0.5% HAuCl4 solution for 40 min. For mixing colors, the slice samples were incubated with 2% (COOH)2·2H2O solution for 30 min. Finally, the samples were fixed with 5% Na2S2O3 solution for 5 min and were dehydrated each in a different concentration of ethanol solution. To quantify nucleus condition, slice samples that were near a section of the Bodian’s stained samples were stained with Mayer’s hematoxylin for 5 min and

1% eosin Y solution for 1 min (the hematoxylin-eosin, H-E stain). Photomicrographs of the stained slices were taken on an Olympus IX81 phase-contrast microscope (Olympus, Tokyo, Japan) equipped with a DP72 digital camera (Olympus), stored, and then processed on a personal computer. Bodian’s and H-E stain experiments were performed at least three times using different blocks. Axonal condition. Axonal degeneration was evaluated by monitoring morphological hallmarks of neurite degeneration such as beading, accumulation and fragmentation, as described previously with some modifications (14, 21, 22). Photomicrographs of the hippocampal CA1 region on the Bodian’s stained slices were analyzed to determine axonal condition. At least 20 continuous sections of the stained slices were evaluated for beading formation in each mouse group. The number of cells per slice and the area of cell body per cell were determined using photomicrographs of the H-E stained slices. At least, 20 continuous slices that were near sections of the Bodian’s stained samples were evaluated in each mouse group. The cell count was independently confirmed by a student who was not involved in this study. Western blotting. All samples were homogenized in phosphate buffered saline and used in Western blotting as described previously, with some modifications (17). The lysates were centrifuged and protein content was determined using a Bio-Rad protein assay (#5000006JA, Bio-Rad Japan, Tokyo, Japan) according to the manufacturer’s procedure. Protein extracts (15 mg) were separated on 10% SDS-polyacrylamide gels and transferred to Immobilon transfer membranes (PVDF; Merck). The membranes were washed and incubated in blocking solution (Tris-HCl-buffered saline, pH 7.6 (TBS), containing 0.1% Tween 20 and 3% bovine serum albumin) for 1 h at room temperature (R/T). The membranes were washed in TBS containing 0.1% Tween 20, and then treated with anti-human CRMP-2 (C4G) mouse IgG monoclonal antibody (#11096, ImmunoBiological Laboratories Co., Ltd., Gunma, Japan) at 1 : 400 dilution overnight at 4˚C. Anti-mouse IgG HRP antibody (Promega Corp., Madison, WI, USA) was used as a secondary antibody at 1 : 4,000 dilution for 1 h at R/T. In the microtubule-associated protein (MAP)–light chain 3 (LC3) assay, the protein extracts (15 mg) were separated on 15% SDS-polyacrylamide gels. The transferred membranes were treated with anti-human LC3 (APG8B) (N-term) rabbit IgG polyclonal antibody (#AP1802a, Abgent, Inc., CA, USA) at 1 : 400 dilution overnight at 4˚C. Anti-rabbit IgG HRP antibody (Promega) was used as a secondary antibody at 1 : 4,000 dilution for 1 h at R/T. Both Western blotting experiments were performed at least three times. All chemiluminescent signals were generated by incubation with the detection reagents (ECL Prime Western Blotting Analysis Reagent; GE Healthcare UK Ltd., Buckinghamshire, UK) according to the manufacturer’s procedure. For normalization of each band of CRMP-2 or LC3, the

379

Vitamin E Deficient Induce Axonal Degeneration

(A) Normal

V.E-def.

Old

(B) Normal

V.E-def.

(D) 200

50 ** ** 25

0

Area [µm2] / Cell

Number of Cells / Slices

(C)

Old

175

150 Normal V.E-def.

Old

Normal V.E-def.

Old

Fig. 1. A, B: Photomicrographs showing axonal degeneration in the hippocampal CA1 region of vitamin E-deficient (V.Edef, n53), normal young (Normal, n53) and normal old (Old, n53) mice. The slice samples were stained using the Bodian (A) and hematoxylin-eosin (B) methods. Scale bar represents 20 mm. Enlarged boxes represent the regions defined in the black box of each phase-contrast photomicrograph, and scale bar represents 5 mm. C, D: The number (C) and average area (D) of cells in the slice sample. Each bar represents the mean of three independent experiments, with at least, three independent mice sliced samples being used per experiment. Error bars represent standard deviation. Data were analyzed using Student’s t-tests. ** indicates p,0.01 (vs Normal).

380

Fukui K et al.

(B)

0

0 Cortex

Hip

Cortex

Old

*

V.E-def.

** **

Normal

2

Upper Lower

50

Normal

Relative intensity [CRMP-2 / β-Actin]

4

Upper band Lower band

100

Old

Normal V.E-def. Old

6

V.E-def.

*

Relative intensity / Total CRMP-2 [%]

(A)

Hip

membranes were reprobed with anti-b actin antibodies (#ab8226, Abcam Plc., Cambridge, UK). The relative intensities of CRMP-2 or LC3 were determined using LAS-3000 (FUJIFILM Corp., Tokyo, Japan). Statistical analysis. Data are plotted as the mean6SE of results of three independent experiments for each mouse group (vitamin E-deficient; normal old; normal young). Data were analyzed using Student’s t-test, with findings of p,0.05 considered significant. Results Axonal degeneration in hippocampal neurons A large number of axonal beadings were found in hippocampal CA1 region of vitamin E-deficient and normal old mice (Fig. 1A). The number of beadings on vitamin E-deficient mice was larger than that on the normal old mice. The hippocampal CA1 region was morphologically similar in all three mouse groups (Fig. 1B). The number of cells per slice in the hippocampal CA1 region was lower in vitamin E-deficient and normal old mice than in normal young mice (p,0.01) (Fig. 1C). However, the average of cell body area was similar in all three mouse groups (p.0.9) (Fig. 1D). CRMP-2 in the cerebral cortex and hippocampus Usually, two original bands of CRMP-2 (64- and 72-kDa) were detected for a mouse brain homogenate sample on the data sheet of IBL Co., Ltd. We present the summed intensities of these two bands as total CRMP-2 expression. The total CRMP-2 expression in the cerebral cortex was lower in vitamin E-deficient and normal old mice than in normal young mice (p,0.01) (Fig. 2A). Furthermore, the total CRMP-2 expression in the cerebral cortex was lower in vitamin E-deficient mice than in normal old mice (p,0.05) (Fig. 2A). The total CRMP-2 expression in the hippocampus was lower in vitamin E-deficient mice than in normal young mice (p,0.05). The balance of the two original CRMP-2 bands in the cerebral cortex and the hippocampus of normal old mice was different to that in normal young mice and vitamin E-deficient mice (Fig. 2B).

Relative intensity [MAP-LC3 / β-Actin]

Fig 2. CRMP-2 expression in vitamin E-deficient (V.E-def, n54), normal young (Normal, n53) and normal old (Old, n53) mice. A: The ratio of total CRMP-2 band intensity to b-actin band intensity in the cerebral cortex (Cortex) and hippocampus (Hip). B: The contribution of upper (black) and lower (white) band intensity to total CRMP-2 band intensity. Each column represents the mean of three independent experiments. Data were analyzed using Student’s t-tests. * indicates p,0.05 and ** indicates p,0.01 (vs Normal).

2

Normal V.E-def. Old

** *

1

0

** Cortex

* Hip

Fig 3. MAP-LC3 expression in vitamin E-deficient (V.Edef, n55), normal young (Normal, n53) and normal old (Old, n54) mice. Data indicate the ratio of MAPLC3 band intensity and b-actin band intensity in the cerebral cortex (Cortex) and hippocampus (Hip). Each column represents the mean of three independent experiments. Data were analyzed using Student’s t-test. * indicates p,0.05 and ** indicates p,0.01 (vs Normal).

MAP-LC3 expression The relative intensity of MAP-LC3 in the cerebral cortex and the hippocampus was higher in vitamin E-deficient mice than in young and old normal mice (p,0.05) (Fig. 3). The expression of MAP-LC3 in the cerebral cortex and the hippocampus was lower in normal old mice than in normal young mice (p,0.05). Discussion In this study we focused on identifying early markers of neuronal damage in a vitamin E-deficient mouse model. We found axonal degeneration not only in cultured models but also in brain slices. Hippocampal neurons of vitamin E-deficient and normal old mice exhibited axonal degeneration, but no morphological changes in the cell bodies. These results indicate that axonal degeneration was one of the early events that occurred before induction of cell death. Axonal degeneration in the hippocampal CA1 region of vitamin E-deficient and normal old mice It is well known that beading formation is a major hallmark of neurite degeneration (21, 22). Furthermore, some reports suggest the significance of the relation


between axonal degeneration and neurodegenerative disorders (23, 24). Kambe et al. reported a relationship between axonal degeneration and hyperphosphorylation of tau (23), and accumulation of phospho-tau is implicated in Alzheimer’s disease (AD). The most typical pathological symptom of AD is cognitive dysfunction. In this study, we found axonal degeneration in the hippocampal region of vitamin E-deficient and normal old mice. Previously, we found b-amyloid-like substances in the hippocampal CA1 region of vitamin E-deficient and normal old rats (12). It is possible that axonal degeneration occurs in hippocampal neurons at an early stage in AD patients. The absence of morphological changes in the cell bodies of hippocampal neurons, including accumulation or aggregation of nuclei and shrinking or swelling of the cell bodies, indicates that the axonal degeneration we observed in vitamin E-deficient and normal old mice was an early event that occurred before the induction of cell death. It is well known that the hippocampal CA1 region plays a pivotal role in cognitive function (25, 26), and it is possible that axonal degeneration of the hippocampal CA1 region of vitamin E-deficient and old mice is one of the mechanisms underlying cognitive dysfunction in these models. Decreased expression of total CRMP-2 and increased the ratio of upper band of the CRMP-2 in vitamin E-deficient and old mice To identify the mechanisms of axonal degeneration in the hippocampal CA1 region of vitamin E-deficient and normal old mice, we measured CRMP-2 using Western blot analysis. CRMP-2 plays a pivotal role in neurite functions (14, 18, 19), binding to tubulin and connecting with the microtubule assembly. Changes in CRMP-2 may be expected to influence the axonal transport system. Overexpression of CRMP-2 in hippocampal neurons induces multiple axons (18). In this study, vitamin E-deficient mice exhibited decreased total CRMP-2 expression in the cerebral cortex and the hippocampus. However, the total expression of CRMP-2 in the hippocampus was not significantly different between young and old normal mice. It is well known that the hippocampus plays a pivotal role in the central nervous system, and recently, several lines of evidence have demonstrated that neurogenesis in adult animal models occurs in the hippocampus (27, 28). For the above-mentioned reasons, it is suggested that there may be a small difference in total CRMP-2 expression in the hippocampus between young and old normal mice. However, in the hippocampus of normal old mice, the ratio of the upper band of CRMP-2 expression was remarkably increased. Unfortunately, we could not identify the meaning of each band of CRMP-2. However, several studies have reported the expression of multiple bands of CRMP-2 (29–33). Yuasa-Kawada et al. (29) described the possibility of the existence of two CRMP-2 subtypes. These two proteins, which were termed CRMP-2A and -2B, have opposite effects. CRMP-2B exists in axons and dendrites and induces axon branching and a reduction of axon length, while

381

CRMP-2A exists only in axons and blocks the effects of CRMP-2B. They suggest that the two CRMP-2 subtypes contribute to the maintenance of axonal condition during remodeling of microtubule organization. Castegna et al. (30) also reported that multiple spots of CRMP-2 were detected in AD brains using two-dimensional electrophoresis analysis. They identified the multiple spots of CRMP-2 was oxidative modifications. Furthermore, CRMP-2 has multiple phosphorylation sites (Thr509, Thi514, Ser518, Ser522) and can be phosphorylated by glycogen synthase kinase 3b (GSK3b) and cyclindependent kinase 5 (Cdk5) (31–34). Phosphorylation sites of CRMP-2 have crucial roles in neurite outgrowth (32–34). CRMP-2 antibody recognized predominantly two bands on western blotting, and the two bands of CRMP-2 were also detected by pCRMP-2 antibody (31). In our study, we also detected two bands of CRMP-2 on Western blot analysis. We speculate that the lower band of CRMP-2 may be the major original band, and other may be a phosphorylation form. Another possibility is we detected two different phosphorylation forms of CRMP-2. Anyway, the ratio of two bands of CRMP-2 may play an important role in the maintenance of neurite functions. Changes in this ratio may be connected with induction of beading formation. However, further investigation is needed to determine the relation between this ratio and axonal degeneration in vitamin E-deficient and normal old mice. We are continuing to study the meaning of changes in the balance of CRMP-2 in vitamin E-deficient and normal old mice. Increased expression of MAP-LC3 in the cerebral cortex and hippocampus of vitamin E-deficient mice In order to determine if the axonal transport system was damaged in vitamin E-deficient and normal old mice we assessed autophagy using Western blot analysis. Contrary to expectation, expression of MAP-LC3 was increased in the cerebral cortex and hippocampus of vitamin E-deficient mice and decreased in the cerebral cortex and hippocampus of normal old mice. MAP-LC3 is one of the major autophagy-related proteins and participates in autophagosome formation (35). Autophagy activity decreases with aging (36, 37). In vitamin E-deficient mice autophagy may activate to recover intracellular components that have been damaged by vitamin E deficiency. However, it does not catch up with the recovery system, resulting in the gradual appearance of beading formation in hippocampal axons. Furthermore, deficiency of vitamin E accelerates oxidative damage in the brain. Vitamin E-deficient rats have lower activity of antioxidant enzymes and increased lipid peroxidation in the brain (10, 11). It is possible that enhancement of oxidative damage of vitamin E-deficient models affects autophagy. However, further investigation is needed to determine the relation between vitamin E-deficient derived oxidative damage and changes in autophagy function. Conclusions These results indicate that axonal beading occurs in hippocampal neurons of vitamin E-deficient and normal

382

Fukui K et al.

old mice, extending our previous findings from cultured models (16, 17). It is possible that axonal degeneration relates to changes in the CRMP-2 expression. Several lines of evidence demonstrate the relation between CRMP-2 and AD (30, 31, 38). For example, Cole et al. reported that hyper-phosphorylation of CRMP-2 is an early event in AD progression (31), and CRMP-2 is significantly oxidized in the AD brain (30). These data strongly suggest that changes in CRMP-2 may be relevant to AD progression. In the near future, we hope to examine the relation between axonal degeneration and age-related and AD-derived cognitive dysfunction. Conflict of interest The authors confirm that we have no conflicts of interest to declare. Acknowledgments We would like to thank Keisuke Ushiki, Shibaura Institute of Technology, for his technical assistance to this project.

10)

11)

12)

13)

14)

15)

Declaration of Interest Section This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT)-Supported Program for the Strategic Research Foundation at Private Universities. This study was also supported by a grant-in-aid for Project Research from the Shibaura Institute of Technology (Tokyo, Japan). References 1) Sies H. 1997. Oxidative stress: oxidants and antioxidants. Exp Physiol 82: 291–295. 2) Sies H, Stahl W, Sundquist AR. 1992. Antioxidant functions of vitamins. Vitamins E and C, beta-carotene, and other carotenoids. Ann NY Acad Sci 669: 7–20. 3) Niki E, Noguchi N. 2004. Dynamics of antioxidant action of vitamin E. Acc Chem Res 37: 45–51. 4) Samhan-Arias AK, Tyurina YY, Kagan VE. 2011. Lipid antioxidants: free radical scavenging versus regulation of enzymatic lipid peroxidation. J Clin Biochem Nutr 48: 91–95. 5) Chan A, Rogers E, Shea TB. 2009. Dietary deficiency in folate and vitamin E under conditions of oxidative stress increases phosphor-tau levels: potentiation by ApoE4 and alleviation by S-adenosylmethionine. J Alzheimers Dis 17: 483–487. 6) Kobayashi N, Machida T, Takahashi T, Takatsu H, Shinkai T, Abe K, Urano S. 2009. Elevation by oxidative stress and aging of hypothalamic-pituitary-adrenal activity in rats and its prevention by vitamin E. J Clin Biochem Nutr 45: 207–213. 7) Howard AC, McNeil AK, McNeil PL. 2011. Promotion of plasma membrane repair by vitamin E. Nat Commun 2: 597. 8) Nishida Y, Yokota T, Takahashi T, Uchihara T, Jishage K, Mizusawa H. 2006. Deletion of vitamin E enhances phenotype of Alzheimer disease model mouse. Biochem Biophys Res Commun 350: 530–536. 9) Koscik RL, Lai HJ, Laxova A, Zaremba KM, Kosorok MR, Douglas JA, Rock MJ, Splaingard ML, Ferrell PM. 2005. Preventing early, prolonged vitamin E deficiency: an opportunity for better cognitive outcomes via early diag-

16)

17)

18)

19)

20)

21)

22)

23)

24)

25)

nosis through neonatal screening. J Pediatr 147 (Suppl): S51–56. Fukui K, Onodera K, Shinkai T, Suzuki S, Urano S. 2001. Impairment of learning and memory in rats caused by oxidative stress and aging, and changes in antioxidative defense systems. Ann NY Acad Sci 928: 168–175. Fukui K, Omoi N, Hayasaka T, Shinkai T, Suzuki S, Abe K, Urano S. 2002. Cognitive impairment of rats caused by oxidative stress and aging, and its prevention by vitamin E. Ann NY Acad Sci 959: 275–284. Fukui K, Takatsu H, Shinkai T, Suzuki S, Abe K, Urano S. 2005. Appearance of amyloid beta-like substances and delayed-type apoptosis in rat hippocampus CA1 region through aging and oxidative stress. J Alzheimers Dis 8: 299–309. Kelly TA, Katagiri Y, Vartanian KB, Kumar P, Chen LL, Rosoff WJ, Urbach JS, Geller HM. 2010. Localized alteration of microtubule polymerization in response to guidance cues. J Neurosci Res 88: 3024–3033. Touma E, Kato S, Fukui K, Koike T. 2007. Calpain-mediated cleavage of collapsin response mediator protein (CRMP)-2 during neurite degeneration in mice. Eur J Neurosci 26: 3368–3381. Urano S, Sato Y, Otonari T, Makabe S, Suzuki S, Ogata M, Endo T. 1998. Aging and oxidative stress in neurodegeneration. Biofactors 7: 103–112. Fukui K, Takatsu H, Koike T, Urano S. 2011. Hydrogen peroxide induces neurite degeneration: Prevention by tocotrienols. Free Radic Res 45: 681–691. Fukui K, Ushiki K, Takatsu H, Koike T, Urano S. 2012. Tocotrienols prevent hydrogen peroxide-induced axon and dendrite degeneration in cerebellar granule cells. Free Radic Res 46: 184–193. Inagaki N, Chihara K, Arimura N, Menager C, Kawano Y, Matsuo N, Nishimura T, Amano M, Kaibuchi K. 2001. CRMP-2 induces axons in cultured hippocampal neurons. Nat Neurosci 4: 781–782. Arimura N, Menager C, Fukata Y, Kaibuchi K. 2004. Role of CRMP-2 in neuronal polarity. J Neurosci 26: 801–809. Artico M, Cavallotti C, Cavallotti D. 2002. Adrenergic nerve fibres and mast cells: correlation in rat thymus. Immunol Lett 84: 69–76. Yang Y, Kawataki T, Fukui K, Koike T. 2007. Cellular Zn21 chelators cause “dying-back” neurite degeneration associated with energy impairment. J Neurosci Res 85: 2844–2855. Ikegami K, Koike T. 2004. Non-apoptotic neurite degeneration in apoptotic neuronal death: pivotal role of mitochondrial function in neurites. Neuroscience 122: 81–93. Kambe T, Motoi Y, Inoue R, Kojima N, Tada N, Kimura T, Sahara N, Yamashita S, Mizoroki T, Takashima A, Shimada K, Ishiguro K, Mizuma H, Onoe H, Mizuno Y, Hattori N. 2011. Differential regional distribution of phosphorylated tau and synapse loss in the nucleus accumbens in tauopathy model mice. Neurobiol Dis 42: 404–414. Oyama F, Miyazaki H, Sakamoto N, Becquet C, Machida Y, Kaneko K, Uchikawa C, Suzuki T, Kurosawa M, Ikeda T, Tamaoka A, Sakurai T, Nukina N. 2006. Sodium channel beta4 subunit: down-regulation and possible involvement in neuritic degeneration in Huntington’s disease transgenic mice. J Neurochem 98: 518–529. Rushaidhi M, Jing Y, Kennard JT, Collie ND, Williams JM,


26)

27)

28)

29)

30)

31)

Zhang H, Liu P. 2012. Aging affects l-arginine and its metabolites in memory-associated brain structures at the tissue and synaptoneurosome levels. Neuroscience 209: 21–31. Zagaar M, Alhaider I, Dao A, Levine A, Alkarawi A, Alzubaidy M, Alkadhi K. 2012. The beneficial effects of regular exercise on cognition in REM sleep deprivation: behavioral, electrophysiological and molecular evidence. Neurobiol Dis 45: 1153–1162. Rizzi S, Bianchi P, Guidi S, Ciani E, Bartesaghi R. 2011. Impact of environmental enrichment on neurogenesis in the dentate gyrus during the early postnatal period. Brain Res 1415: 23–33. Eriksson PS, Perfilieva E, Björk-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH. 1998. Neurogenesis in the adult human hippocampus. Nat Med 4: 1313–1317. Yuasa-Kawada J, Suzuki R, Kano F, Ohkawara T, Murata M, Noba M. 2003. Axonal morphogenesis controlled bu antagonistic roles of two CRMP subtypes in microtubule organization. Eur J Neurosci 17: 2329–2343. Castegna A, Aksenov M, Thongboonkerd V, Klein JB, Pierce WM, Booze R, Markesbery WR, Butterfield DA. 2002. Proteomic identification of oxidavely modificated proteins in Alzheimer’s disease brain. Part II: dihydropyrimidinase-related protein 2, alpha-enolase and heart shock cognate. J Neurochem 82: 1524–1532. Cole AR, Noble W, van Aalten L, Plattner F, Meimaridou R, Hogan D, Tavlor M, LaFrancois J, Gunn-Moore F, Verkhratsky A, Oddo S, LaFeria F, Giese KP, Dinelev KT, Duff K, Richardson JC, Yan SD, Hanger DP, Allan SM, Sutherland C. 2007. Collapsin response mediator protein-2 hyperphosphorylation is an early event in Alzheimer’s disease progression. J Neurochem 103:

383

1132–1144. 32) Cole AR, Knebel A, Morrice NA, Robertson LA, Irvine AJ, Connolly CN, Sutherland C. 2004. GSK-3 phosphorylation of the Alzheimer’s epitope within collapsin response mediator protein regulates axon elongation in primary neurons. J Biol Chem 279: 50176–50180. 33) Uchida Y, Ohshima T, Sasaki Y, Suzuki H, Yanai S, Yamashita N, Nakamura F, Takei K, Ihara Y, Mikoshiba K, Kolattukudy P, Honnorat J, Goshima Y. 2005. Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3b phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer’s disease. Genes Cells 10: 165–179. 34) Yoshimura T, Kawano Y, Arimura N, Kawabata S, Kikuchi A, Kaibuchi K. 2005. GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity. Cell 120: 137–149. 35) Yang Y, Fukui K, Koike T, Zheng X. 2007. Induction of autophagy in neurite degeneration of mouse superior cervical ganglion neurons. Eur J Neurosci 26: 2979–2988. 36) Cuervo AM. 2008. Autophagy and aging keeping that old bloom working. Trends Genet 24: 604–612. 37) Donati A, Cavallini G, Bergamini E. 2012. Effects of aging, antiaging calorie restriction and in vivo stimulation of autophagy on the urinary excretion of 8OHdG in male Sprague-Dawley rats. Age (Dordr), doi: 10.1007/ s11357-011-9346-x. 38) Yoshida H, Watanabe A, Ihara Y. 1998. Collapsin response mediator protein-2 is associated with neurofibrillary tangles Alzheimer’s disease. J Biol Chem 273: 9761–9768.