Byung Pil Cho

Sustained activation of Akt by melatonin contributes to the protection against kainic acid-induced neuronal death in hippocampus

Abstract:  In the present study, the underlying protective mechanism of melatonin on kainic acid (KA)‐induced excitotoxicity was examined in the hippocampus of mice. KA, administered intracerebroventricularly (i.c.v.), induced marked neuronal cell death with concurrent microglial activation and subsequent induction of inducible nitric oxide synthase (iNOS) in the hippocampus. Histopathological analysis demonstrated that melatonin (10 mg/kg), administered 1 hr prior to KA, attenuated KA‐induced death of pyramidal neurons in the CA3 region. Melatonin obviously suppressed KA‐induced microglial activation and consequent iNOS expression that were determined by increased immunoreactivities of microglial marker OX‐6 and iNOS, respectively. Increased phosphorylation of Akt in pyramidal neurons was observed as early as 2 hr after administration of melatonin. Further, melatonin resulted in increased expression of astroglial glial cell line‐derived neurotrophic factor (GDNF), which started to appear approximately 6 hr after administration of melatonin. The results of the present study demonstrate that melatonin exerts its neuroprotective action against KA‐induced excitotoxicity both through the activation of neuronal Akt and via the direct action on hippocampal neurons and through the increased expression of astroglial GDNF, which subsequently activates neuronal PI3K/Akt pathway. Therefore, the present study suggests that melatonin, pineal secretory product, is potentially useful in the treatment of acute brain pathologies associated with excitotoxic neuronal damage such as epilepsy, stroke, and traumatic brain injury.

Axotomy-induced dopaminergic neurodegeneration is accompanied with c-Jun phosphorylation and activation transcription factor 3 expression.

Accumulating evidence has shown that both phosphorylated c-Jun (pc-Jun) and activating transcription factor 3 (ATF3) were upregulated in a variety of tissue injuries and proposed to play an important role in cell death/survival. To elucidate the significance and functional role of these immediate-early genes during neuronal damage in the central nervous system, we examined temporal and spatial profiles of pc-Jun and ATF3 in dopaminergic neurons of the substantia nigra (SN) following transection of the medial forebrain bundle (MFB) in adult rats. Morphological characteristics of pc-Jun-positive dopaminergic neurons as well as microglial reaction in response to the axotomy-induced neurodegeneration were also investigated. Following MFB transection, both c-Jun phosphorylation and ATF3 were found in the nuclei of tyrosine hydroxylase-immunoreactive (TH-ir) neurons of the ipsilateral SN, but not in those of the contralateral SN. In the ipsilateral SN, the number of pc-Jun- and ATF3-positive nuclei was increased by 5-7 days post-lesion, and then progressively decreased probably due to the loss of neurons. Retrograde tracing with FluoroGold (FG) in hemi-axotomized rat brain demonstrated that none of the intact, unaxotomized (FG-ir) neurons was pc-Jun-positive, indicating phosphorylation of c-Jun occurs only in axotomized neurons. Concomitant co-localization of pc-Jun and ATF3 in the same TH-ir neuron was also demonstrated by triple immunofluorescence labeling. Many TH-ir neurons that underwent various steps of consecutive neurodegenerative changes retained pc-Jun in the condensed or fragmented nuclei. Moreover, numerous activated microglia, identified by both phagocytic (ED1) and MHC II (OX6) markers, closely apposed to these neurons throughout the entire neurodegenerative process, suggesting that they are actively phagocytosing dying neurons. Taken together, these results support the idea that pc-Jun and its putative dimeric partner ATF3 may be closely participating in axotomy-induced neurodegeneration.

Nerve injury alters profile of receptor-mediated Ca2+ channel modulation in vagal afferent neurons of rat nodose ganglia.

Although nerve injury is known to up- and down-regulate some metabotropic receptors in vagal afferent neurons of the nodose ganglia (NG), the functional significance has not been elucidated. In the present study, thus, we examined whether nerve injury affected receptor-mediated Ca2+ channel modulation in the NG neurons. In this regard, unilateral vagotomy was performed using male Sprague-Dawley rats. One week after vagotomy, Ca2+ currents were recorded using the whole-cell variant of patch-clamp technique in enzymatically dissociated NG neurons. In sham controls, norepinephrine (NE)-induced Ca2+ current inhibition was negligible. Following vagotomy, however, the NE responses were dramatically increased. This phenomenon was in accordance with up-regulation of alpha2A/B-adrenergic receptor mRNAs as quantified using real-time RT-PCR analysis. In addition, neuropeptide Y (NPY) and prostaglandin E2 responses were moderately augmented in vagotomized NG neurons. The altered NPY response appears to be caused by up-regulation of Y2 receptors negatively coupled to Ca2+ channels. In contrast, nerve injury significantly suppressed opioid (tested with DAMGO)-induced Ca2+ current inhibition with down-regulation of micro-receptors. Taken together, these results demonstrated for the first time that the profile of neurotransmitter-induced Ca2+ channel modulation is significantly altered in the NG neurons under pathophysiological state of nerve injury.

Co-localization of activating transcription factor 3 and phosphorylated c-Jun in axotomized facial motoneurons.

Activating transcription factor 3 (ATF3) and c-Jun play key roles in either cell death or cell survival, depending on the cellular background. To evaluate the functional significance of ATF3/c-Jun in the peripheral nervous system, we examined neuronal cell death, activation of ATF3/c-Jun, and microglial responses in facial motor nuclei up to 24 weeks after an extracranial facial nerve axotomy in adult rats. Following the axotomy, neuronal survival rate was progressively but significantly reduced to 79.1% at 16 weeks post-lesion (wpl) and to 65.2% at 24 wpl. ATF3 and phosphorylated c-Jun (pc-Jun) were detected in the majority of ipsilateral facial motoneurons with normal size and morphology during the early stage of degeneration (1-2 wpl). Thereafter, the number of facial motoneurons decreased gradually, and both ATF3 and pc-Jun were identified in degenerating neurons only. ATF3 and pc-Jun were co-localized in most cases. Additionally, a large number of activated microglia, recognized by OX6 (rat MHC II marker) and ED1 (phagocytic marker), gathered in the ipsilateral facial motor nuclei. Importantly, numerous OX6- and ED1-positive, phagocytic microglia closely surrounded and ingested pc-Jun-positive, degenerating neurons. Taken together, our results indicate that long-lasting co-localization of ATF3 and pc-Jun in axotomized facial motoneurons may be related to degenerative cascades provoked by an extracranial facial nerve axotomy.

Expression of GABAA receptor β2/3 subunits in the rat major pelvic ganglion

Several pharmacological and physiological studies have suggested that GABA(A) receptors (GABA(A) Rs) may exist in the rat major pelvic ganglion (MPG), a large coalescent pelvic ganglion that contains both sympathetic and parasympathetic components which innervates pelvic organs. However, the presence of GABA(A) R in the MPG has never been demonstrated directly by morphological studies. In the present study, we used immunohistochemistry to demonstrate the existence of GABA(A) R beta2/3 subunits for the first time in the rat MPG. We also analyzed the neurochemical properties of MPG neurons expressing GABA(A) R beta2/3 subunits. GABA(A) R beta2/3-immunoreactive (-IR) neurons occupied 27.4+/-7.0% of the whole neuronal population, and many of these (77.6%) were co-localized with tyrosine hydroxylase (TH). Likewise, most (86.5%) of TH-IR neurons were GABA(A) R beta2/3-positive. GABA(A) R beta2/3 subunits were also expressed in a few VIP- or NOS-IR neurons, the cholinergic or non-adrenergic, non-cholinergic (NANC) neurons. These results suggest that GABA(A) Rs are involved in the modulation of most sympathetic, noradrenergic neurons and also a subset of VIP and NOS neurons of the rat MPG.

Transplantation of human mesenchymal stem cells into the cisterna magna and its neuroprotective effects in a parkinsonian animal model

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases in the elderly, and therefore, the demand for effective therapies against PD has greatly increased. Therapeutic applications of stem cells have been considered to be one of the promising approaches in PD therapy. In the present study, the neuroprotective effects of hMSCs were evaluated in a parkinsonian rat model. The animal model was established by injecting 6-hydroxydopamine into the striatum of rats. Two weeks later, cultured hMSCs were transplanted into the cisterna magna. We subsequently identified changes in the expression of inflammatory cytokines, neurotrophic factors, microglial activation, and the survival rate of dopaminergic neurons in SNc. Behavioral recovery was also examined. The results indicated that hMSC transplantation increased the expression of anti-inflammatory cytokines as well as neurotrophic factors, and decreased the number of activated microglia. Compared to the sham-grafted group, relatively large numbers of TH-positive neurons were found in the ipsilateral SNc, and amphetamine-induced asymmetrical rotation was significantly reduced after hMSC transplantation. These findings suggest that hMSCs might be neuroprotective, probably through up-regulation of neurotrophic factors and anti-inflammatory cytokines, and this could have a functional impact in reversing PD symptoms.

Evidence for brain-derived neurotrophic factor-like neuropeptide in brain of the silk moth Bombyx mori during postembryonic periods

Brain-derived neurotrophic factor-like neuropeptide is produced in the brain of the silk moth, Bombyx mori. Immunocytochemical studies of brain and retrocerebral complex of larvae, prepupae, pupae and adults showed that four pairs of median neurosecretory cells and six pairs of lateral neurosecretory cells which had different immunoreactivities to BDNF peptide. Day-1 adult brains showed no evidence of neurons stained by anti-BDNF antibodies. Those reactivities, which were much stronger in median cells than in lateral cells, were the weakest in an earliest larval stage and a latest pupal stage but the strongest in late larval stage. Median neurosecretory cells projected their axons into the contralateral corpora allata by decussation in the median region, nerve corpora cardiaca (NCC) I, and nerve corpora allata (NCA) I, whereas lateral neurosecretory cells extended their axons to the ipsilateral corpora allata via NCC II and NCA I.

Effects of low- and high-frequency repetitive magnetic stimulation on neuronal cell proliferation and growth factor expression: A preliminary report.

Repetitive magnetic stimulation is a neuropsychiatric and neurorehabilitation tool that can be used to investigate the neurobiology of sensory and motor functions. Few studies have examined the effects of repetitive magnetic stimulation on the modulation of neurotrophic/growth factors and neuronal cells in vitro. Therefore, the current study examined the differential effects of repetitive magnetic stimulation on neuronal cell proliferation as well as various growth factor expression. Immortalized mouse neuroblastoma cells were used as the cell model in this study. Dishes of cultured cells were randomly divided into control, sham, low-frequency (0.5Hz, 1Tesla) and high-frequency (10Hz, 1Tesla) groups (n=4 dishes/group) and were stimulated for 3 days. Expression of neurotrophic/growth factors, Akt and Erk was investigated by Western blotting analysis 3 days after repetitive magnetic stimulation. Neuroblastoma cell proliferation was determined with a cell counting assay. There were differences in cell proliferation based on stimulus frequency. Low-frequency stimulation did not alter proliferation relative to the control, while high-frequency stimulation elevated proliferation relative to the control group. The expression levels of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) and platelet-derived growth factor (PDGF) were elevated in the high-frequency magnetic stimulation group. Akt and Erk expression was also significantly elevated in the high-frequency stimulation group, while low-frequency stimulation decreased the expression of Akt and Erk compared to the control. In conclusion, we determined that different frequency magnetic stimulation had an influence on neuronal cell proliferation via regulation of Akt and ERK signaling pathways and the expression of growth factors such as BDNF, GDNF, NT-3 and PDGF. These findings represent a promising opportunity to gain insight into how different frequencies of repetitive magnetic stimulation may mediate cell proliferation.

Adipose tissue-derived mesenchymal stem cells cultured at high cell density express brain-derived neurotrophic factor and exert neuroprotective effects in a 6-hydroxydopamine rat model of Parkinson’s disease

Mesenchymal stem cells (MSCs) secrete neurotrophic factors, and have been reported to improve functional outcomes in animal models of neurodegenerative diseases such as cerebral ischemia, stroke, spinal cord lesions, and Parkinson’s disease. Previously, we found that adipose tissue-derived mesenchymal stem cells (ASCs) cultured at high cell density (HD-ASCs) expressed interferon-beta (IFN-β). Here we demonstrate that ASCs expressing IFN-β also express brain-derived neurotrophic factor (BDNF). Growth rates of neuroblastoma cells (SK-N-BE(2)C) were increased when co-cultured with HD-ASCs or treated with concentrated medium obtained from HD-ASCs (HD-ASC-CM). The HD-ASC-CM induced AKT phosphorylation in SK-N-BE(2)C cells, and AKT inhibition by Ly294002 reduced cell viability of SK-N-BE(2)C cells. Additionally, a protective effect on SK-N-BE(2)C cells exposed to 6-hydroxydopamine (6-OHDA) was observed in the HD-ASC-CM or brain-derived neurotrophic factor (BDNF) treated cells. The protective effect of the HD-ASC-CM was neutralized by anti-BDNF antibody. In the 6-OHDA-induced Parkinson’s disease rat model, ASCs reduced amphetamine-induced rotations and a greater number of tyrosine hydroxylase (TH)-positive cells were observed in the HD-ASCs-injected group compared with sham controls and the low density cultured ASC-injected group. Moreover, the expression of BDNF, nerve growth factor (NGF), TH, and proliferating cell nuclear antigen (PCNA) in ipsilateral midbrain tissues including substantia nigra pars compacta (SNc) was increased by transplantation of HD-ASCs. These data indicate that HD-ASCs may induce neuroprotective effects through BDNF expression and subsequent increase of proliferation in neuronal cells both in vitro and in vivo.

Upregulated and prolonged differentiation potential of the ependymal cells lining the ventriculus terminalis in human fetuses

The ventriculus terminalis (VT) is a dilated cavity within the conus medullaris of the spinal cord. Although the VT was discovered in the mid-nineteenth century, little is known about its characteristics during development in human fetuses. Ependymal cells lining the cavities within the CNS retain high differentiation potential, and are believed to be responsible for the postnatal neurogenesis. To evaluate the differentiation capacity of the ependymal cells lining the VT during development, we examined glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) expression in the spinal cord of 18-24-week-old human fetuses. GFAP is a marker for the degree of ependymal cell differentiation in the human fetus, and PCNA is a well-known marker for cell division. Morphological characteristics of the VT were also examined. At the lower portion of the conus medullaris, the central canal abruptly expands dorsally to become the VT. Then the VT widens bilaterally while its anteroposterior diameter reduces gradually in a caudal direction. Finally, the VT becomes a narrow, transverse slit at the level of the lowermost conus medullaris. Compared with those lining the central canal, more numerous ependymal cells lining the VT showed more intensive GFAP and PCNA expression throughout all gestational ages examined. This suggests that, in the developing human spinal cord, ependymal cells lining the VT retain their differentiation potential, including a higher proliferative capacity, until a later stage of development than those lining the central canal.