B. METHODS
Ⅲ. RESULTS
A. Lithium increased BDNF mRNA expression after chronic treatment in brain and spinal cord.
Li+ concentration in plasma
The Li+ concentration was 0.48±0.02 mmol/L (n=9). In humans, the Li+ therapeutic concentration range is 0.8~1.2 mmol/L.
Li+ increased BDNF mRNA expression in brain and spinal cord.
The major aim of this study was to carry out a detailed analysis of BDNF mRNA levels following chronic Li+ treatment. BDNF mRNA expression was determined by SYBR real-time PCR. Li+ treatment increased BDNF mRNA in the cerebral cortex (Fig. 1A), hippocampal formation (Fig. 1B), striatum (Fig. 1C), midbrain (Fig. 1D), but not in the cerebellum (Fig. 1E). The BDNF mRNA expression increased in the spinal cord (divided into three parts: cervical, thoracic, and lumbar) following Li+ treatment (Fig. 2). These results mean that chronic Li+ treatment increases BDNF expression level in most of the CNS and it plays a central role in cell survival.
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Fig. 1. Li+ increases BDNF mRNA expression in various brain areas in adult rat. Real Time PCR analysis of BDNF mRNA in the cerebral cortex (A), hippocampal formation (B), striatum (C), midbrain (D), and cerebellum (E) in control rats and rats treated with Li+ for 3 weeks, mean ±S.E.M.(n = 5~9), *, significant differences compared with control, p<0.05.
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Fig. 2. Li+ increases expression of BDNF mRNA in the spinal cord of adult rat. Real Time PCR analysis of BDNF mRNA in the cervical, thoracic, and lumbar spinal cord of adult rat treated with Li+ for 3 weeks , mean ±S.E.M.(n = 5~9), *, significant differences compared with control, p<0.05.
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mRNA and protein of BDNF were exclusively localized in ventral motor neurons of lumbar segment in a lithium-sensitive manner.
Whether chronic Li+ treatment would have an influence on the expression of BDNF mRNA (Fig 3, a and b) and the protein of BDNF (Fig 3, c and d) was examined. mRNA and the protein of BDNF were selectively expressed in the motor neuron of the ventral horn in the mouse treated with Li+. These results indicate that mRNA and the protein of BDNF were exclusively localized in ventral motor neurons of lumbar segments in a lithium-sensitive manner.
B. Li+ had an effect on the protection neurons from degenerative death in the ALS animal model
Li+ attenuated degeneration of spinal motor neurons in the ALS animal model.
Next, it was examined whether protection from neuronal death in neurodegenerative diseases could be exerted by increased BDNF due to chronic Li+ treatment. ALS is one of the neurodegenerative diseases in which motor neurons die progressively and skeletal muscles denervate. ALS animal models, G93A mice, 16-weeks-old, fed chow containing Li+ (~200mg/kg/d) for three weeks were used. Then the lumbar regions of the spinal cords were obtained and stained with cresyl violet (Fig. 4A) and MAP-2/ChAT (Fig. 4B). In the G93A mice (Fig. 4A-b and B-b), most motor neurons died and axons degenerated, but in the G93A mouse group treated with lithium (Fig. 4A-c and B-c) motor neurons and axons were protected from degenerative death. The BDNF protein level and its downstream signal,
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phosphorylation of Akt (Ser473 and Thr308), were examined to confirm that BDNF was responsible for this protection. As can be seen in Fig. 5, Li+ increased the BDNF protein level as well as the BDNF mRNA level. Also, increased BDNF started the survival signaling pathway as indicated by the increasing phosphorylation of Akt.
In the ALS animal model, the number of TH positive cells in the SN was decreased.
It has been reported that the cell numbers of dopaminergic neurons in the SN of transgenic ALS mice decreased. To examine if Li+ would protect the dopaminergic neurons in ALS mice, loss of the dopaminergic neurons was first verified by counting cells immunoreactive to tyrosine hydroxylase (TH) antibody using a stereology system (CAST, Visopharm) method in 16-week old male G93A mice (Table 1). The mean total number of TH positive cells in the unilateral SN of the mice was estimated to be 4,500 (coefficient of variation (CV)=0.22) (with a mean coefficient of error (CE) of 0.09). It decreased as much as 28% from that of the littermate control mice (Table 1). In this study, the CE2/CV2 ratio is 0.2, verifying a low methodological bias in the precision of the estimated neuronal number (West, 1999). In ALS, the progress of cell death occurs in dopaminergic neurons in the SN as well as in spinal motor neurons. In addition, it is presumed that Li+ increased the BDNF level in the SN, because of Li+ protected cell death in this region.
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Fig. 3. Li+ increases mRNA and protein expression of BDNF in the lumbar spinal motor neurons of adult rat. Fluorescence (a and b, in situ hybridization with anti-sense BDNF cRNA probe ) and bright-field (c and d, immunocytochemistry with a BDNF antibody) photomicrographs of the lumbar ventral sections in control rats (a and c) and rats treated with Li+ for 3 weeks (b and d).
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Fig. 4. Li+ attenuates degeneration of spinal motor neurons in ALS mice. (A) Bright-field (A, cresyl vilotet staining) and fluorescence (B, double immunocytochemistry of MAP-2 (green) and ChAT (red) antibodies] in 16 week-old control littermate mouse (a), G93A transgenic mice (b), and G93A transgenic mouse treated with Li+ (~200 mg/kg/d) starting from 8 weeks of age (c).
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Fig. 5. Li+ increases BDNF expression and Akt phosphorylation in the lumber spinal cord of adult mice. (A) Western blot analysis of BDNF, p-Akt(Ser473), and p-Akt(Thr308) in the lumbar spinal cord of B6SJL mice treated with vehicle control (CTRL) or Li+ for 3 weeks. (B) Scheme of cell survival signaling pathway by growth factor.
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Table 1. The mean total number of TH positive cells in the unilateral SN of the 16 weeks old male G93A mouse. Table (A), Histogram (B). mean ±S.E.M.(n = 5), *, significant differences compared with control, p<0.05.
１６ Li+ prevents apoptotic cell death.
In this study, the mechanisms that could influence the protection of dopaminergic neurons after chronic lithium administration were investigated. Cell death due to apoptosis was first determined in the male 16 week-old G93A mice. Apoptotic signals are caspase-9 (Fig. 6A, wild type 100±50.077%, G93A 382.40±64.091%, G93A+Li 145.94±10.985%), caspase-8 (Fig. 6B, wild type 100±33.823%, G93A 1411.08±465.512%, G93A+Li 633.63±192.976%), and caspase-3 (Fig. 6C, wild type 100±82.456%, G93A 697.25±146.999%, G93A+Li 154.26±94.755%). These signals were increased in the transgenic mice but Li+ decreased the apoptotic signals (Fig. 6D). This could be one of the modes of neuron cell death protection by Li+ in the SN.
Li+ cannot reduce the ROS level.
It was then investigated as to whether lithium could inhibit oxidative stress (Fig. 7). In the familiar ALS animal model, lower motor neurons died from oxidative stress.
Immunohistochemistry with nitrotyrosine antibody staining was used to see if dopaminergic neuronal death in the SN of the 12 week-old male mouse is involved with oxidative stress, and if Li+ reduces the ROS level. The result proved that the ROS level was increased in the G93A mouse, and that lithium (administration for 28 days) did not reduce the ROS level.
This means that lithium did not provide protection against neuronal cell death through the ROS inhibition mechanism. The protection effect was attributed to the anti-apoptotic mechanism.
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Fig. 6 . Li+ prevents apoptotic neuronal cell death. (A) Fluorescence photomicrographs (a, e,and i, immunocytochemistry of caspase-9; b,f, and j, immunocytochemistry of NeuN; c,g, and k, DAPI ; d, h and l, triple merge ) in 16 week-old control littermate mouse (a, b, c and d), G93A transgenic mice (e, f, g and h), and G93A transgenic mouse treated with Li+ (~200 mg/kg/d) starting from 8 weeks of age (I, j, k and l). (B) Fluorescence photomicrographs (a, e,and i, immunocytochemistry of caspase-8; b,f, and j, immunocytochemistry of NeuN; c,g, and k, DAPI ; d, h and l, triple merge ) in 16 week-old control littermate mouse (a, b, c and d), G93A transgenic mice (e, f, g and h), and G93A transgenic mouse treated with Li+ (~200 mg/kg/d) starting from 8 weeks of age (I, j, k and l). (C) Fluorescence photomicrographs (a, e,and i, immunocytochemistry of caspase-3; b,f, and j, immunocytochemistry of NeuN; c,g, and k, DAPI ; d, h and l, triple merge ) in 16 week-old control littermate mouse (a, b, c and d), G93A transgenic mice (e, f, g and h), and G93A transgenic mouse treated with Li+ (~200 mg/kg/d) starting from 8 weeks of age (I, j, k and l). (D) Fluorescence intensity quantitation of caspase-9 (a), -8 (b), and-3 (c). mean ±S.E.M.(n = 5~9), *, significant differences
compared with control, p<0.05.
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Fig. 7. Li+ cannot reduce ROS level and cannot prevent ROS-mediated cell death.
Bright-field photomicrographs (immunocytochemistry of nitrotyrosine) in 12 week-old control littermate mouse (a), G93A transgenic mice (b), and G93A transgenic mouse treated with Li+ (~200 mg/kg/d) starting from 8 weeks of age (c).
２１ Li+ increases BDNF mRNA level
Chronic lithium treatment increase BDNF expression. Our animal model increased BDNF mRNA level also. We carried out fluorescence in situ hybridization (FISH) for BDNF mRNA expression (Fig. 8), lithium treatment group (for 28 days) was increased fluorescent intensity about 44% more than transgenic G93A group (wild type, 100±12.548; wild type+
Li+ ,192.38±11.612; G93A, 62.63±2.975; G93A+ Li+ ,97.47±11.065%). BDNF plays a central role in cell survival and activity dependent neural plasticity. This increasing BDNF level might exert protecting dopaminergic neurons from dying.
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Fig. 8. Li+ increases expression of BDNF mRNA in the substantia nigra in ALS animal model. (A) Fluorescence photomicrographs (a, e,and i, in situ hybridization of BDNF mRNA; b,f, and j, immunocytochemistry of TH; c,g, and k, DAPI ; d, h and l, triple merge ) in 12 week-old control littermate mouse (a, b, c and d), control littermate mouse treated with Li+ (~200 mg/kg/d) starting from 8 weeks of age (e, f, g and h), G93A transgenic mouse (I, j, k and l). and G93A transgenic mouse treated with Li+ (m,n,o and p). (B) Fluorescence intensity quantitation of BDNF mRNA. mean ±S.E.M.(n = 5~9), *, significant differences compared with control, p<0.05.
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Ⅳ. DISCUSSION
In the present study, treatment with Li+ for 21 days increased the BDNF expression in the cerebral cortex, hippocampal formation, striatum, midbrain, and spinal cord of the test subjects. However, acute treatment did not increase BDNF expression in any of the regions examined (data not shown). These results indicate that therapeutically more relevant, chronic treatment with Li+ enhances BDNF expression even at subtherapeutic concentrations (average 0.48mM). A variety of antidepressant modalities, when administered chronically, are known to have the characteristic neuroplastic effect of upregulation of BDNF in the hippocampus and frontal cortex (Duman, 2002;Nibuya et al., 1995). The present study indicates that Li+ shares the same neuroplastic effect as antidepressants. In addition, chronic administration of Li+ upregulates BDNF expression in other brain regions that are involved in neurodegenerative diseases.
It is suggested that Li+ may exert neuroprotective actions by increasing BDNF expression in specific regions of the CNS. However, our result seems to be inconsistent with other studies showing decreased BDNF level in the frontal cortex (Angelucci et al., 2003) by long term treatment with Li+.
There have been studies demonstrating increase in BDNF expression in the hippocampus by chronic Li+ treatment in animals (Drevets, 1999;Einat et al., 2003;Fukumoto et al., 2001;Jacobsen and Mork, 2004).
There has been evidence of enhancement of synaptic plasticity by lithium as evidenced by increase in long term potentiation (LTP) in the hippocampal subregions by subchronic (Shim et al., 2007) and chronic lithium administration (Son et al., 2003). Son et al (2003)
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reported the enhancement of LTP and the concomitant increase in BDNF level in the dentate gyrus (DG) after a 4- week Li+ treatment. These findings, plus the present results, suggest that an increase in BDNF expression may contribute to Li+-induced enhancement of synaptic plasticity.
Pharmacological activation of the cAMP-CREB pathway leads to an increase in hippocampal neurogenesis (Nakagawa et al., 2002;Williams et al., 2001;Zhu et al., 2004). It has been demonstrated that chronic Li+ treatment increases neurogenesis in the hippocampus with accompanying increases in cyptoprotective or neurotrophic molecules such as Bcl-2 and BDNF (Chen et al., 2000;Son et al., 2003).
The detailed mechanism of upregulation of BDNF expression in the specific brain regions is not fully understood. However, there are observations that cAMP responsive element binding protein (CREB) activity is modulated by Li+. The CREB binding activity and phosphorylated CREB levels are enhanced by the therapeutically relevant concentration of Li+ in cultured cerebellar granule cells (Ozaki and Chuang, 1997) and in the brain of the rat (Ozaki and Chuang, 1997). Phosphorylated CREB levels are increased in the rat hippocampus and frontal cortex by chronic Li+ administration (Einat et al., 2003). CREB is a common target of diverse signal transduction pathways including Ca2+ -calmodulin-dependent kinase, protein kinase C, ribosomal S6 kinase, and protein kinase A (Duman, 2002;Finkbeiner, 2000), and BDNF expression is modulated by CREB activated by the Ca2+
signal (Shieh and Ghosh, 1999).
This result means that Li+ exerts protection from neuronal cell death. In nitrotyrosine immunohistochemistry, the ROS level did not differ between G93A mice and G93A mice with Li+ treatment, and in both groups the ROS level was higher than the control littermate
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group. But in the caspase-9, 8 and 3 immunohistochemistry of 16-week old G93A mice, the fluorescent intensity of the Li+ administration group was lower than its G93A group. These data show that Li+ can protect neurons from apoptosis but it cannot provide protection from oxidative stress. Therefore, Li+ protects neurons of the SN through the mechanism of the anti-apoptotic pathway, suggesting that Li+ can be used for neurodegenerative diseases.
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Ⅴ. CONCLUSION
1. Chronic (3 weeks) administration of Li+ increases mRNA expression of BDNF in the brain and the spinal cord in adult rat.
2. Chronic administration of Li+ induces BDNF expression and AKT phosphorylation in the spinal cord of adult mouse.
3. Chronic administration of Li+ protects motor neurons in spinal cord and doparminergic neurons in SN from neurodegenerative cell death.
4. These protecting effects engage increased BDNF level by Li+ treatment, block the apoptotic signals.
The present study suggests that chronic treatment with Li+ promotes neuronal survival and regeneration through upregulation of BDNF in certain brain areas (e.g. SN) including the lumbar spinal motor neurons.
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