Multiple low-dose radiation-induced neuronal cysteine transporter expression and oxidative stress are rescued by N-acetylcysteine in neuronal SH-SY5Y cells.

Recently, several studies have demonstrated that low-dose radiation (LDR) therapy has positively impacts on the treatment of Alzheimer's disease (AD). LDR suppresses the production of pro-neuroinflammation molecules and improves cognitive function in AD. However, it is unclear whether direct exposure to LDR causes beneficial effects and what mechanism is involved in neuronal cells. In this study, we first determined the effect of high-dose radiation (HDR) alone on C6 cells and SH-SY5Y cells. We found that SH-SY5Y cells were more vulnerable than C6 cells to HDR. Moreover, in neuronal SH-SY5Y cells exposed to single or multiple LDR, N-type cells showed decreased cell viability with increasing radiation exposure time and frequency, but S-type cells were unaffected. Multiple LDR increased proapoptotic molecules such as p53, Bax and cleaved caspase-3, and decreased anti-apoptotic molecule (Bcl2). Multiple LDR also generated free radicals in neuronal SH-SY5Y cells. We detected a change in the expression of the neuronal cysteine transporter EAAC1. Pretreatment with N-acetylcysteine (NAC) rescued the increased in EAAC1 expression and the generation of ROS in neuronal SH-SY5Y cells after multiple LDR. Furthermore, we verified whether the increased in EAAC1 expression induces cell defense or cell death promotion signaling. We showed that transient overexpression of EAAC1 reduced the multiple LDR-induced p53 overexpression in neuronal SH-SY5Y cells. Our results indicate that neuronal cells can be injured by increased production of ROS not only by HDR but also by multiple LDR, which suggests that combination treatment with anti-free radical agents such as NAC may be useful in multiple LDR therapy.

Neuregulin 1/ErbB4/Akt signaling attenuates cytotoxicity mediated by the APP-CT31 fragment of amyloid precursor protein.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by neuronal and synaptic loss. The cytoplasmic tail of amyloid precursor protein (APP) undergoes sequential cleavage at a specific intracellular caspase site to generate the cytoplasmic terminal 31 (CT31) fragment. The APP-CT31 fragment is a potent inducer of apoptosis. The cytotoxicity of APP-CT31 in SH-SY5Y cells was evaluated by the lactate dehydrogenase (LDH) assay. TUNEL staining was used to detect apoptotic signals in SH-SY5Y cells and primary cortical neurons. The expression of apoptosis-related proteins, such as p53, PUMA (p53 up-regulated modulator of apoptosis), and cleaved was investigated by immunofluorescence analysis and Western blotting. In this study, we investigated the neuroprotective effect of neuregulin 1 (NRG1) against cytotoxicity induced by APP-CT31. Our data showed that CT31 induced cytotoxicity and apoptosis in SH-SY5Y cells and primary cortical neurons. NRG1 attenuated the neurotoxicity induced by the expression of APP-CT31. We also showed that APP-CT31 altered the expression of p53 and cleaved caspase 3. However, treatment with NRG1 rescued the APP-CT31-induced upregulation of p53 and cleaved caspase 3 expression. The protective effect of NRG1 was abrogated by inhibition of the ErbB4 receptor and Akt. These results indicate an important role of ErbB4/Akt signaling in NRG1-mediated neuroprotection, suggesting that endogenous NRG1/ErbB4 signaling represents a valuable therapeutic target in AD.

Neuregulin-1 inhibits CoCl2-induced upregulation of excitatory amino acid carrier 1 expression and oxidative stress in SH-SY5Y cells and the hippocampus of mice.

Excitatory amino acid carrier 1 (EAAC1) is an important subtype of excitatory amino acid transporters (EAATs) and is the route for neuronal cysteine uptake. CoCl2 is not only a hypoxia-mimetic reagent but also an oxidative stress inducer. Here, we found that CoCl2 induced significant EAAC1 overexpression in SH-SY5Y cells and the hippocampus of mice. Transient transfection of EAAC1 reduced CoCl2-induced cytotoxicity in SH-SY5Y cells. Based on this result, upregulation of EAAC1 expression by CoCl2 is thought to represent a compensatory response against oxidative stress in an acute hypoxic state. We further demonstrated that pretreatment with Neuregulin-1 (NRG1) rescued CoCl2-induced upregulation of EAAC1 and tau expression. NRG1 plays a protective role in the CoCl2-induced accumulation of reactive oxygen species (ROS) and reduction in antioxidative enzyme (SOD and GPx) activity. Moreover, NRG1 attenuated CoCl2-induced apoptosis and cell death. NRG1 inhibited the CoCl2-induced release of cleaved caspase-3 and reduction in Bcl-XL levels. Our novel finding suggests that NRG1 may play a protective role in hypoxia through the inhibition of oxidative stress and thereby maintain normal EAAC1 expression levels.

Early-life stress induces EAAC1 expression reduction and attention-deficit and depressive behaviors in adolescent rats.

Neonatal maternal separation (NMS), as an early-life stress (ELS), is a risk factor to develop emotional disorders. However, the exact mechanisms remain to be defined. In the present study, we investigated the mechanisms involved in developing emotional disorders caused by NMS. First, we confirmed that NMS provoked impulsive behavior, orienting and nonselective attention-deficit, abnormal grooming, and depressive-like behaviors in adolescence. Excitatory amino acid carrier 1 (EAAC1) is an excitatory amino acid transporter expressed specifically by neurons and is the route for the neuronal uptake of glutamate/aspartate/cysteine. Compared with that in the normal control group, EAAC1 expression was remarkably reduced in the ventral hippocampus and cerebral cortex in the NMS group. Additionally, EAAC1 expression was reduced in parvalbumin-positive hippocampal GABAergic neurons in the NMS group. We also found that EAAC1-knockout (EAAC1-/-) mice exhibited impulsive-like, nonselective attention-deficit, and depressive-like behaviors compared with WT mice in adolescence, characteristics similar to those of the NMS behavior phenotype. Taken together, our results revealed that ELS induced a reduction in EAAC1 expression, suggesting that reduced EAAC1 expression is involved in the pathophysiology of attention-deficit and depressive behaviors in adolescence caused by NMS.

Changes of gene expression of Gal3, Hsp27, Lcn2, and Timp1 in rat substantia nigra following medial forebrain bundle transection using a candidate gene microarray.

Neuroinflammation is an early event and important contributor to the pathobiology of neurodegenerative diseases. Neuroglia, especially microglia, are a major central nervous system population that can modulate neuroinflammation. To determine potential key molecules in this process, we employed microarray analysis in the substantia nigra (SN) following medial forebrain bundle (MFB) transection and analyzed the temporal expression profiles of candidate genes implicated in neuroglial activation and functional maturation. The DNA microarray analyzed, 8913 probes. Sixty nine genes were up-regulated and 11 genes were down-regulated at least twofold compared to normal control. Of the 80 genes, 23 were related to cell metabolism, 3 related to apoptosis, 27 related to immunity. Among them, 4 genes (Galectin 3, Heat shock protein 27, Lipocalin 2, Tissue inhibitory metalloproteinase 1) seemed to be related to the neuroglial function. The candidate genes were subjected to quantitative real-time PCR, Western blotting, and immunohistochemical approaches. Expression changes similar to the microarray were evident. In a double immunofluorescence assay, Galectin 3 almost completely co-localized with OX6-positive activated microglia, and Heat shock protein 27 mainly co-localized with glial fibrillary acidic protein (GFAP) positive astrocytes. Lipocalin 2, except for a few matches of GFAP positive astrocytes, did not co-localized with any of neuroglial markers. This is the first study to evaluate gene expression changes in the SN following MFB transection, which has been used as a parkinsonian animal model. Several candidate genes with potential roles in neuroglial activation and functional maturation were identified. The molecular significance of the candidate genes in neuroglial activation and neuroinflammation remains unclear.

Neuregulin-1 exerts protective effects against neurotoxicities induced by C-terminal fragments of APP via ErbB4 receptor.

Neuregulin-1 (NRG1) plays important roles in the development and plasticity of the brain, and it is also reported to have potent neuroprotective properties. We previously reported that NRG1 has neuroprotective actions against Swedish amyloid precursor protein-induced neurotoxicity. In addition to the amyloid beta peptide, other metabolites of amyloid precursor protein (APP) such as the C-terminal fragments of APP (APP-CTs) have been reported to possess cytotoxic effects in neuronal cells. In this study, we investigated whether NRG1 exerts neuroprotective effects against APP-CTs and attempted to determine its neuroprotective mechanisms. NRG1 attenuated the neurotoxicities induced by the expression of APP-CTs in neuronal cells. NRG1 also reduced the accumulation of reactive oxygen species and attenuated mitochondrial membrane potential loss induced by APP-CTs. In addition, NRG1 upregulated the expression of the anti-apoptotic protein Bcl-2. This effect was blocked by the inhibition of ErbB4, a key NRG1 receptor. Taken together, these results demonstrate the neuroprotective potential of NRG1 in Alzheimer's disease.

c-Fos immunoreactivity in the neurons of the lateral geniculate nucleus in albino rats by light exposure after dark rearing.

PURPOSE: To investigate the effect of dark rearing immediately after birth on the maturation of the visual relay neurons in the lateral geniculate nucleus. METHODS: Fifty neonatal rats were used. Neonates of the control groups were raised under a normal light/dark cycle. Neonates of the experiment groups were dark reared and isolated from light during the entire experimental period, then exposed to the sun light for 1 hour before sacrifice. RESULTS: In the control groups, the neurons in the dorsal lateral geniculate nucleus developed normally at each age tested. In the experiment groups, the cytoplasm of the large neurons in the dorsal lateral geniculate nucleus of 2-week-old rats contained small vesicles, and the cytoplasm of the large neurons of 4-week-old rats was converted into a vacuole-like space. Moreover, c-Fos immunoreactivity of the large neurons in the dorsal lateral geniculate nucleus in the experiment groups was significantly increased compared to that of the control groups. CONCLUSIONS: We suppose that the maturation of the neurons in the lateral geniculate nucleus might be influenced by light stimulation during the critical period. Furthermore, c-Fos could be a marker of the functional activity of the visual relay neurons of the lateral geniculate nucleus in albino rats.

Constitutive activation of signal transducers and activators of transcription 3 correlates with better prognosis, cell proliferation and hypoxia-inducible factor-1α in human gastric cancer.

OBJECTIVE: We aimed to investigate the biological significance of signal transducers and activators of transcription 3 (STAT3) in gastric carcinoma. METHODS: Immunohistochemistry was performed on tissue array slides containing 285 gastric carcinoma specimens. The relationship between the nuclear expression of phospho-Tyr705-STAT3 (pSTAT3), an active form of STAT3, and prognosis, clinicopathological factors, proliferation, cell cycle regulators, apoptosis regulators, or angiogenesis-related proteins was evaluated. RESULTS: In nonneoplastic gastric mucosa, pSTAT3 was observed primarily in the nuclei of cells in the proliferative zone and intestinal metaplasia. In gastric carcinomas, nuclear STAT3 activation was observed in 36% of cases and was positively correlated with the Ki-67 labeling index and earlier tumor stage, whereas it was inversely correlated with lymphatic metastasis and distant metastasis (p< 0.05). Moreover, survival analyses showed that pSTAT3 expression was an independent prognostic factor of good survival. In addition, the expression of nuclear pSTAT3 positively correlated with that of cyclin D1, p21, p27, hypoxia-inducible factor-1α, or vascular endothelial growth factor (p< 0.05). CONCLUSIONS: STAT3 activation is an early event in gastric tumorigenesis and significantly correlates with better prognosis, proliferation and angiogenesis. Thus, STAT3 activation may be a valuable prognostic variable and therapeutic target in gastric carcinoma.

Neuregulin-1 prevents amyloid ß-induced impairment of long-term potentiation in hippocampal slices via ErbB4.

Neuregulin-1 (NRG1) participates in numerous neurodevelopmental processes and plasticity of the brain. Despite this, little is known about its role in Alzheimer's disease (AD). Amyloid ß (Aß) peptide is generally believed to play a critical role in the pathogenesis of AD. The present study examined the effect of synthetic Aß1₋42 peptides on long-term potentiation (LTP) in the CA1 region of mice hippocampal slices, a cellular model of learning and memory. We found that application of a test dose of Aß1₋42 (200 nM) significantly inhibited the development of LTP without affecting basal synaptic transmission. Pretreatment with NRG1 effectively prevented Aß1₋42-induced impairment of LTP, an effect that was dose-dependent. This LTP-restoring action of NRG1 was almost completely abolished by blocking ErbB4, a key NRG1 receptor, suggesting that NRG1 acts through ErbB4 to exert its protective action on LTP. The present study thus provides the first demonstration that NRG1/ErbB4 protects against Aß-induced hippocampal LTP impairment, suggesting that NRG1 may be a promising candidate for the treatment of early-stage AD.

Impairment of nuclear factor-kappaB activation increased glutamate excitotoxicity in a motoneuron-neuroblastoma hybrid cell line expressing mutant (G93A) Cu/Zn-superoxide dismutase.

Mutations in the superoxide dismutase 1 (SOD1) gene are linked to glutamate excitotoxicity in familial amyotrophic lateral sclerosis (fALS), but the underlying mechanism remains unclear. We investigated whether nuclear factor-kappaB (NF-kappaB) activation is involved in glutamate excitotoxicity by using motor neuron-neuroblastoma hybrid cells that expressed a mutant (G93A) SOD1 (mtSOD1) or wild-type SOD1 (wtSOD1). MtSOD1 cells were more vulnerable to glutamate excitotoxicity than wtSOD1 cells and showed higher NF-kappaB activity, higher nuclear cRel expression, and lower nuclear RelA expression under basal conditions. Glutamate treatment increased NF-kappaB activation along with nuclear expressions of RelA and cRel in wtSOD1 cells but induced only weak nuclear RelA expression in mtSOD1 cells. Suppression of NF-kappaB activation using transfection of the superrepressive mutant form of IkappaBalpha (mIkappaBalpha) inhibited nuclear RelA expression in both types of SOD1 cells, which increased glutamate excitotoxicity in wtSOD1 cells but not in mtSOD1 cells. Furthermore, immunohistochemistry confirmed stronger RelA immunoreactivity in the nuclei of motor neurons of spinal cord in wild-type SOD1 transgenic mice than in those in SOD1 G93A transgenic mice. In addition, we found that glutamate treatment decreased XIAP expression and increased caspase-3 activity in mtSOD1 cells and mIkappaBalpha-overexpressing wtSOD1 cells. Our results suggest that glutamate excitotoxicity in motor neurons of SOD1-linked fALS is attributable, at least in part, to the impairment of IkappaBalpha-dependent RelA activation and subsequent apoptosis mediated by XIAP inhibition and caspase-3 activation.

Age-related changes in microvillar cells of rat olfactory epithelium.

The nature and function of microvillar cells (MVCs) of the mammalian olfactory epithelium (OE) are little understood. Previous studies have examined MVC morphology in the developing and mature OE, but not in the aged OE. The present study investigated the effect of aging on MVCs of the OE in male Sprague-Dawley rats using histological and immunohistochemical methods. OE of aged rats contained MVCs with marked hypertrophy and swollen end-feet, which reached the basement membrane. Such MVC features were not observed in the young OE. These MVC changes were more conspicuous in proximity to severely degenerated olfactory receptor neurons (ORNs) and supporting cells. The ratio of the number of MVCs to that of supporting cells increased with aging; however, MVCs in the aged OE were not proliferating cell nuclear antigen-immunoreactive. In addition, the total cell population was decreased in the aged OE. Thus, our results suggest that MVCs are non-neuronal and that they are more resistant to aging compared to ORNs and supporting cells.