Ji Hyun Jeon

Axonal Neuropathy-associated TRPV4 Regulates Neurotrophic Factor-derived Axonal Growth

Background: Because genetic linkage studies identified mutations in TRPV4 in patients with peripheral neuropathies, the function of TRPV4 in peripheral neurons is questioned. Results: TRPV4 was found to promote neurotrophic factor-driven neuritogenesis. Conclusion: TRPV4 mediates neurotrophic factor-driven neuritogenesis in peripheral neurons. Significance: This explains molecular mechanisms underlying neuritogenesis and maintenance of peripheral nerves. Spinal muscular atrophy and hereditary motor and sensory neuropathies are characterized by muscle weakness and atrophy caused by the degenerations of peripheral motor and sensory nerves. Recent advances in genetics have resulted in the identification of missense mutations in TRPV4 in patients with these hereditary neuropathies. Neurodegeneration caused by Ca2+ overload due to the gain-of-function mutation of TRPV4 was suggested as the molecular mechanism for the neuropathies. Despite the importance of TRPV4 mutations in causing neuropathies, the precise role of TRPV4 in the sensory/motor neurons is unknown. Here, we report that TRPV4 mediates neurotrophic factor-derived neuritogenesis in developing peripheral neurons. TRPV4 was found to be highly expressed in sensory and spinal motor neurons in early development as well as in the adult, and the overexpression or chemical activation of TRPV4 was found to promote neuritogenesis in sensory neurons as well as PC12 cells, whereas its knockdown and pharmacologic inhibition had the opposite effect. More importantly, nerve growth factor or cAMP treatment up-regulated the expression of phospholipase A2 and TRPV4. Neurotrophic factor-derived neuritogenesis appears to be regulated by the phospholipase A2-mediated TRPV4 pathway. These findings show that TRPV4 mediates neurotrophic factor-induced neuritogenesis in developing peripheral nerves. Because neurotrophic factors are essential for the maintenance of peripheral nerves, these findings suggest that aberrant TRPV4 activity may lead to some types of pathology of sensory and motor nerves.

Anthocyanins from the seed coat of black soybean reduce retinal degeneration induced by N-methyl-N-nitrosourea☆

Anthocyanins are known to have antioxidant effects and thus may play an important role in preventing various degenerative diseases. In this study, we examined the effect of anthocyanins extracted from the seed coat of black soybean on an animal model of retinal degeneration (RD), a leading cause of photoreceptor cell death resulting in blindness. RD was induced in rats by an intraperitoneal injection of N-methyl-N-nitrosourea (MNU) (50mg/kg), a DNA-methylating agent that causes photoreceptor damage. Anthocyanins extracted from black soybean seed coat (50mg/kg) were daily administered, orally, for 1, 2, and 4 weeks after MNU injection. Electroretinographic (ERG) recordings and morphological analyses were performed. In control rats with MNU-induced retinal damage, the ERG recordings showed a gradual significant time-dependent reduction in both a- and b-wave amplitudes compared with those of normal animals. In the MNU-induced RD rats given anthocyanins for 4 weeks, ERG responses were significantly increased compared with untreated RD rats, more apparently in scotopic stimulation than in the photopic condition. However, in the MNU-injected rats given anthocyanins for 1 and 2 weeks, the increase in ERG responses was not significant. Morphologically, the outer nuclear layer, where photoreceptors reside, was well preserved in the anthocyanin-treated rat retinas throughout the experimental period. In addition, retinal injury, evaluated by immunolabeling with an antibody against glial fibrillary acidic protein, was markedly reduced in anthocyanin-treated retinas. These results demonstrate that anthocyanins extracted from black soybean seeds can protect retinal neurons from MNU-induced structural and functional damages, suggesting that anthocyanins from black soybean seed coat may be used as a useful supplement to modulate RD.

Changes in transcript and protein levels of calbindin D28k, calretinin and parvalbumin, and numbers of neuronal populations expressing these proteins in an ischemia model of rat retina

Excessive calcium is thought to be a critical step in various neurodegenerative processes including ischemia. Calbindin D28k (CB), calretinin (CR), and parvalbumin (PV), members of the EF-hand calcium-binding protein family, are thought to play a neuroprotective role in various pathologic conditions by serving as a buffer against excessive calcium. The expression of CB, PV and CR in the ischemic rat retina induced by increasing intraocular pressure was investigated at the transcript and protein levels, by means of the quantitative real-time reverse transcription-polymerase chain reaction, western blot and immunohistochemistry. The transcript and protein levels of CB, which is strongly expressed in the horizontal cells in both normal and affected retinas, were not changed significantly and the number of CB-expressing horizontal cells remained unchanged throughout the experimental period 8 weeks after ischemia/reperfusion injury. At both the transcript and protein levels, however, CR, which is strongly expressed in several types of amacrine, ganglion, and displaced amacrine cells in both normal and affected retinas, was decreased. CR-expressing ganglion cell number was particularly decreased in ischemic retinas. Similar to the CR, PV transcript and protein levels, and PV-expressing AII amacrine cell number were decreased. Interestingly, in ischemic retinas PV was transiently expressed in putative cone bipolar cell types possibly those that connect with AII amacrine cells via gap junctions. These results suggest that these three calcium binding proteins may play different neuroprotective roles in ischemic insult by their ability to buffer calcium in the rat retina.

Cyanidin-3-glucoside Extracted from Mulberry Fruit Can Reduce N-methyl-N-nitrosourea-Induced Retinal Degeneration in Rats

Abstract Purpose: To investigate the effect of cyanidin-3-O-glucoside (C3G) on a rat retinal degeneration (RD) model. Materials and methods: Experimental RD was induced in rats by the intraperitoneal injection of N-methyl-N-nitrosourea (MNU) at 50 mg/kg. C3G extracted from mulberry (Morus alba L.) fruit (50 mg/kg) was orally administered, daily for 1, 2 and 4 weeks after MNU injection. The effects of C3G administration on MNU-induced RD retinas were histologically and functionally assessed by hematoxylin and eosin staining and electroretinography (ERG), respectively. The degree of retinal injury in C3G-administered RD rats was evaluated by immunohistochemistry with an antibody against glial fibrillary acidic protein (GFAP). The preferential protective effect of C3G on scotopic vision was examined by western blot analysis. Results: Marked loss of photoreceptors in the outer nuclear layer (ONL) was observed in RD rats at 2 and 4 weeks after MNU injection, while the ONL in the MNU-induced RD rats given C3G was relatively well preserved. Immunohistochemistry with anti-GFAP showed that retinal injury was also reduced in the retinas of the rats given C3G. Functional assessment by using ERG recordings showed that scotopic ERG responses were significantly increased in RD rats given C3G for 4 weeks (p < 0.01) compared with that of untreated RD rats. In the RD rats given short-term C3G (for 1 and 2 weeks), the increase in ERG responses was not significant. In addition, western blot analysis showed that rhodopsin level in the C3G-administered RD retinas significantly increased compared to that in the non-administered RD retinas (p < 0.05), whereas red/green opsin level did not show any significant difference. Conclusions: Long-term administration of C3G extracted from mulberry fruit could structurally reduce photoreceptor damage and functionally improve scotopic visual functions in the RD rat model induced by MNU.

Presynaptic Localization and Possible Function of Calcium-Activated Chloride Channel Anoctamin 1 in the Mammalian Retina

Calcium (Ca2+)-activated chloride (Cl−) channels (CaCCs) play a role in the modulation of action potentials and synaptic responses in the somatodendritic regions of central neurons. In the vertebrate retina, large Ca2+-activated Cl− currents (ICl(Ca)) regulate synaptic transmission at photoreceptor terminals; however, the molecular identity of CaCCs that mediate ICl(Ca) remains unclear. The transmembrane protein, TMEM16A, also called anoctamin 1 (ANO1), has been recently validated as a CaCC and is widely expressed in various secretory epithelia and nervous tissues. Despite the fact that tmem16a was first cloned in the retina, there is little information on its cellular localization and function in the mammalian retina. In this study, we found that ANO1 was abundantly expressed as puncta in 2 synaptic layers. More specifically, ANO1 immunoreactivity was observed in the presynaptic terminals of various retinal neurons, including photoreceptors. ICl(Ca) was first detected in dissociated rod bipolar cells expressing ANO1. ICl(Ca) was abolished by treatment with the Ca2+ channel blocker Co2+, the L-type Ca2+ channel blocker nifedipine, and the Cl− channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and niflumic acid (NFA). More specifically, a recently discovered ANO1-selective inhibitor, T16Ainh-A01, and a neutralizing antibody against ANO1 inhibited ICl(Ca) in rod bipolar cells. Under a current-clamping mode, the suppression of ICl(Ca) by using NPPB and T16Ainh-A01 caused a prolonged Ca2+ spike-like depolarization evoked by current injection in dissociated rod bipolar cells. These results suggest that ANO1 confers ICl(Ca) in retinal neurons and acts as an intrinsic regulator of the presynaptic membrane potential during synaptic transmission.

Escitalopram block of hERG potassium channels

Escitalopram, a selective serotonin reuptake inhibitor, is the pharmacologically active S-enantiomer of the racemic mixture of RS-citalopram and is widely used in the treatment of depression. The effects of escitalopram and citalopram on the human ether-a-go-go-related gene (hERG) channels expressed in human embryonic kidney cells were investigated using voltage-clamp and Western blot analyses. Both drugs blocked hERG currents in a concentration-dependent manner with an IC50 value of 2.6 μM for escitalopram and an IC50 value of 3.2 μM for citalopram. The blocking of hERG by escitalopram was voltage-dependent, with a steep increase across the voltage range of channel activation. However, voltage independence was observed over the full range of activation. The blocking by escitalopram was frequency dependent. A rapid application of escitalopram induced a rapid and reversible blocking of the tail current of hERG. The extent of the blocking by escitalopram during the depolarizing pulse was less than that during the repolarizing pulse, suggesting that escitalopram has a high affinity for the open state of the hERG channel, with a relatively lower affinity for the inactivated state. Both escitalopram and citalopram produced a reduction of hERG channel protein trafficking to the plasma membrane but did not affect the short-term internalization of the hERG channel. These results suggest that escitalopram blocked hERG currents at a supratherapeutic concentration and that it did so by preferentially binding to both the open and the inactivated states of the channels and by inhibiting the trafficking of hERG channel protein to the plasma membrane.

Expression and immunohistochemical localization of TMEM16A/anoctamin 1, a calcium-activated chloride channel in the mouse cochlea

Sound transduction in the cochlea depends on the unique high concentrations of K+ in the endolymph. The production and maintenance of high K+ concentrations are accompanied by Cl- cycling. In this study, we report on an investigation of the expression and localization of TMEM16A/anoctamin 1 (ANO1), a recently cloned Ca2+-activated Cl- channel, in the mouse cochlea by Western blot and immunhistochemistry. The ANO1 protein was identified in the cochlea by Western blotting. The immunoreactivity was found in stria vascularis as a line and in the organ of Corti as three plaques. The cellular localization of ANO1 was examined by means of double-labeling experiments with anti-claudin 11, a marker for basal cells of the stria vascularis. The results demonstrated that ANO1 colocalized with claudin 11, indicating its expression in basal cells. We also examined ANO1 localization in the organ of Corti by double- and triple-labeling techniques with anti-myosin VI, a marker for hair cells, and anti-synaptophysin, a marker for olivocochlear efferent nerve endings under hair cells. The results clearly showed that ANO1 is colocalized with synaptophysin, but not with myosin VI, indicating that ANO1 is localized at medial olivocochlear efferent nerve endings under outer hair cells. These results suggest that ANO1 may be specifically involved in synaptic transmission from medial olivocochlear efferent nerve endings to outer hair cells in the organ of Corti, as well as Cl- cycling in basal cells of the stria vascularis.

Mitral and Tufted Cells Are Potential Cellular Targets of Nitration in the Olfactory Bulb of Aged Mice

Olfactory sensory function declines with age; though, the underlying molecular changes that occur in the olfactory bulb (OB) are relatively unknown. An important cellular signaling molecule involved in the processing, modulation, and formation of olfactory memories is nitric oxide (NO). However, excess NO can result in the production of peroxynitrite to cause oxidative and nitrosative stress. In this study, we assessed whether changes in the expression of 3-nitrotyrosine (3-NT), a neurochemical marker of peroxynitrite and thus oxidative damage, exists in the OB of young, adult, middle-aged, and aged mice. Our results demonstrate that OB 3-NT levels increase with age in normal C57BL/6 mice. Moreover, in aged mice, 3-NT immunoreactivity was found in some blood vessels and microglia throughout the OB. Notably, large and strongly immunoreactive puncta were found in mitral and tufted cells, and these were identified as lipofuscin granules. Additionally, we found many small-labeled puncta within the glomeruli of the glomerular layer and in the external plexiform layer, and these were localized to mitochondria and discrete segments of mitral and tufted dendritic plasma membranes. These results suggest that mitral and tufted cells are potential cellular targets of nitration, along with microglia and blood vessels, in the OB during aging.

Axonal Synapses Utilize Multiple Synaptic Ribbons in the Mammalian Retina

In the mammalian retina, bipolar cells and ganglion cells which stratify in sublamina a of the inner plexiform layer (IPL) show OFF responses to light stimuli while those that stratify in sublamina b show ON responses. This functional relationship between anatomy and physiology is a key principle of retinal organization. However, there are at least three types of retinal neurons, including intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells, which violate this principle. These cell types have light-driven ON responses, but their dendrites mainly stratify in sublamina a of the IPL, the OFF sublayer. Recent anatomical studies suggested that certain ON cone bipolar cells make axonal or ectopic synapses as they descend through sublamina a, thus providing ON input to cells which stratify in the OFF sublayer. Using immunoelectron microscopy with 3-dimensional reconstruction, we have identified axonal synapses of ON cone bipolar cells in the rabbit retina. Ten calbindin ON cone bipolar axons made en passant ribbon synapses onto amacrine or ganglion dendrites in sublamina a of the IPL. Compared to the ribbon synapses made by bipolar terminals, these axonal ribbon synapses were characterized by a broad postsynaptic element that appeared as a monad and by the presence of multiple short synaptic ribbons. These findings confirm that certain ON cone bipolar cells can provide ON input to amacrine and ganglion cells whose dendrites stratify in the OFF sublayer via axonal synapses. The monadic synapse with multiple ribbons may be a diagnostic feature of the ON cone bipolar axonal synapse in sublamina a. The presence of multiple ribbons and a broad postsynaptic density suggest these structures may be very efficient synapses. We also identified axonal inputs to ipRGCs with the architecture described above.

Anoctamin 1 expression in the mouse auditory brainstem

Calcium-activated chloride channels (CaCCs) are involved in numerous physiological functions, including the epithelial movement of fluid. Anoctamin 1 (ANO1) has recently been cloned and characterized as a CaCC and is known to be expressed in various secretory epithelia and in nervous tissues such as the dorsal root ganglia and retina. However, data regarding the expression, function, and cellular and subcellular localization of CaCCs in the brain are still limited. We investigated the distribution and expression patterns of ANO1 in adult mouse brain. Reverse transcriptase plus the polymerase chain reaction, Western blot, and immunohistochemical analyses demonstrated that ANO1 was widely distributed throughout the brain. Furthermore, ANO1 was strongly expressed in two auditory brainstem nuclei: the medial nucleus of the trapezoid body (MNTB) and the anteroventral cochlear nucleus (AVCN). Double-labeling experiments revealed that this ANO1 expression was exclusive to the presynaptic endings of both the MNTB and AVCN. ANO1 is thus mainly localized at presynaptic terminals in various brain regions, specifically in two auditory brainstem nuclei, the MNTB and AVCN, and might therefore contribute to the high-frequency synaptic transmission of auditory signals.